Cancer Science

Tumor Microenvironments in Malignant Ovarian Germ Cell Tumors: MHC Class I Loss and T‐Cell Exhaustion in Dysgerminoma

ABSTRACT Malignant ovarian germ cell tumors are rare neoplasms primarily affecting young women. Although generally responsive to chemotherapy, long‐term adverse effects remain a concern. Improved understanding of the tumor microenvironment may reveal alternative therapeutic strategies; however, comprehensive analyses in malignant ovarian germ cell tumors remain limited. We analyzed 56 malignant ovarian germ cell tumor samples: 23 dysgerminomas, 14 yolk sac tumors, and 19 immature teratomas. Immune cell infiltration and immune checkpoint molecule expression were evaluated via immunohistochemistry, while immune‐related gene expression was assessed using transcriptomic profiling. In dysgerminoma, T‐cell exhaustion was characterized through multiplex immunofluorescence. Major histocompatibility complex class I expression was assessed across all subtypes. Dysgerminomas showed abundant CD4 + and CD8 + T‐cell, B‐cell, and M1 macrophage infiltration, with the presence of tertiary lymphoid structures, indicating an immunologically active “hot tumor” phenotype. Immune checkpoint molecules were upregulated, and transcriptomic analysis revealed enrichment of immunostimulatory and immunosuppressive pathways. Conversely, yolk sac tumors were dominated by M2 macrophages, while immature teratomas exhibited minimal immune cell infiltration, a “cold tumor” feature. In dysgerminomas with high programmed cell death protein 1 levels, most CD8 + T cells exhibited a nonexhausted phenotype. All tumor subtypes showed loss of major histocompatibility complex class I expression. These findings indicate that malignant ovarian germ cell tumor subtypes harbor distinct tumor microenvironments, with dysgerminoma characterized by abundant but potentially ineffective T‐cell responses owing to absent major histocompatibility complex class I–mediated antigen presentation. Therapeutic strategies restoring major histocompatibility complex class I expression may enhance T cell–based immunotherapy efficacy in dysgerminomas.

Long noncoding RNATUG1 promotes cisplatin resistance in ovarian cancer via upregulation of DNA polymerase eta

AbstractChemoresistance is a major cause of high mortality and poor survival in patients with ovarian cancer (OVCA). Understanding the mechanisms of chemoresistance is urgently required to develop effective therapeutic approaches to OVCA. Here, we show that expression of the long noncoding RNA, taurine upregulated gene 1 (TUG1), is markedly upregulated in samples from OVCA patients who developed resistance to primary platinum‐based therapy. Depletion of TUG1 increased sensitivity to cisplatin in the OVCA cell lines, SKOV3 and KURAMOCHI. Combination therapy of cisplatin with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system effectively relieved the tumor burden in xenograft mouse models. Mechanistically, TUG1 acts as a competing endogenous RNA by downregulating miR‐4687‐3p and miR‐6088, both of which target DNA polymerase eta (POLH), an enzyme required for translesion DNA synthesis. Overexpression of POLH reversed the effect of TUG1 depletion on cisplatin‐induced cytotoxicity. Our data suggest that TUG1 upregulation allows OVCA to tolerate DNA damage via upregulation of POLH; this provides a strong rationale for targeting TUG1 to overcome cisplatin resistance in OVCA.

DNA Methylation in Ovarian and Endometrial Cancers: Predictive and Mechanistic Roles in PARP Inhibitor and ICI Response

ABSTRACT Cancer treatment is shifting from an organ‐based approach to one driven by biological phenotypes, emphasizing the need to understand molecular mechanisms. DNA methylation plays a pivotal role in tumor biology, not only through gene silencing but also by inducing distinct behaviors beyond genetic mutations. In gynecologic cancers, molecular diagnostics, such as homologous recombination deficiency status guiding poly(ADP‐ribose) polymerase (PARP) inhibitor therapy in ovarian cancer and deficient mismatch repair/microsatellite instability‐high status informing immune checkpoint inhibitor (ICI) therapy in endometrial cancer have already been used in clinical practice. However, tumors with epigenetically driven functional deficiencies, such as BRCA1 promoter methylation in homologous recombination‐deficient ovarian cancers or MLH1 promoter methylation in deficient mismatch repair/microsatellite instability‐high endometrial cancers, often exhibit poorer prognoses and reduced therapeutic responses compared to their genetically mutated counterparts. Given the unique impact of DNA methylation, precise detection is crucial. Integrating methylation analysis into molecular classification could refine diagnostics—both by identifying mechanistic contributors to treatment response and by serving as predictive biomarkers for therapy selection—thereby optimizing patient management. This review explores the role of DNA methylation in modulating responses to PARP inhibitors and ICIs, highlights its promise as a biomarker in precision oncology, and outlines current developments and clinical challenges in BRCA1 and MLH1 methylation assays.

Allelic Imbalance and Chromothripsis Lead to Diversity in Japanese Tumor Genomes With Whole‐Genome Duplication

ABSTRACTWhole‐genome analyses have revealed that large‐scale structural variations (SVs) such as whole‐genome duplication (WGD) occur early in the development of many cancers. However, the diversity of chromosomal abnormalities within tumors before and after WGD remains poorly understood. Here, we analyzed various types of Japanese tumor genomes via whole‐genome sequencing and examined the diversity of WGD by focusing on large SVs at the chromosomal level. WGD was detected in 52% of cases, while the frequency of chromothripsis (CT) was 20%. Although aneuploidy via deletion of chromosome arms was common in many cancers, in rare ovarian cancers, all chromosomes were near‐haploidy before WGD. Minor allele analysis revealed that many non‐mutated ohnolog genes drifted down chromosome arms after WGD and returned to normal ploidy, but only 17p, including TP53, which is also an ohnolog, underwent loss of heterozygosity due to arm deletion before WGD in most cancers. TP53 mutations were frequently detected in WGD and CT‐positive tumors, and these SVs strongly correlated with homologous recombination deficiency scores. Furthermore, these tumors had many mutations that continued to generate neoantigens and resulted in worse survival outcomes. Diversity analysis of tumors with WGD will provide a new perspective on structural abnormalities in tumor genomes.

Ligand‐Based CAR‐T Cells Targeting EGFR Exhibit Favorable Antitumor Effects Against Gynecologic Malignancies

ABSTRACTEpidermal growth factor receptor (EGFR) has been reported to be overexpressed in gynecologic malignancies. However, the clinical efficacy of existing molecular EGFR‐targeted therapies against gynecologic malignancies has not been demonstrated. In this study, we investigated the antitumor effects of ligand‐based EGFR chimeric antigen receptor (CAR)‐T cells on gynecologic malignancies. First, we evaluated EGFR expression in patient samples using immunohistochemistry. EGFR positivity was observed in 41%, 82%, and 79% of ovarian, endometrial, and cervical cancer in patient samples, respectively. Second, we generated ligand‐based EGFR CAR‐T cells via piggyBac‐mediated gene transfer. EGFR CAR‐T cells were successfully generated with high CAR positivity and a high proportion of naïve/stem cell memory‐like T cells. Finally, we investigated the antitumor effects of EGFR CAR‐T cells on gynecologic malignancies. EGFR CAR‐T cells were co‐cultured with six EGFR‐positive gynecologic cancer cell lines. The growth of all six gynecologic cancer cell lines was significantly suppressed by EGFR CAR‐T cells compared to mock T cells. In in vivo studies, tumor‐bearing mice implanted with gynecologic cancer cell lines in their intraperitoneal cavity were administered EGFR CAR‐T cells, CD19 CAR‐T cells, or PBS intraperitoneally. Mice treated with EGFR CAR‐T cells displayed a significantly decreased tumor burden compared to those treated with either CD19 CAR‐T cells or PBS. Additionally, mice treated with EGFR CAR‐T cells had a significantly longer survival than the other groups. In summary, ligand‐based EGFR CAR‐T cells may be a promising therapy for various gynecologic malignancies.

BRCA1 Promoter Methylation in Ovarian Cancer: Clinical Relevance and a Novel Diagnostic Approach Using Fragment Analysis

ABSTRACTHomologous recombination deficiency (HRD) tests, including MyChoice CDx, are companion diagnostics for poly (ADP‐ribose) polymerase (PARP) inhibitors. BRCA1 promoter hypermethylation, a major HRD cause, may correlate with poorer prognosis. This study aimed to develop a simple, accurate method for detecting BRCA1 promoter hypermethylation and elucidate the characteristics of such cases. BRCA1 promoter methylation was analyzed using bisulfite sequencing (BIS‐seq) in high‐grade serous ovarian carcinoma specimens. We developed a newly developed BRCA1 methylation assay, BRCA1‐Fragment Analysis of Methylation (BRCA1‐FAM), which combines restriction enzyme digestion with fragment analysis. The accuracy of this assay was compared to the results of BIS‐seq. We evaluated the relationship between BRCA1 promoter hypermethylation and prognosis and examined its association with BRCA1 expression and loss of heterozygosity. BRCA1 mutations and promoter methylation were mutually exclusive in the analyzed cases, with methylation observed in 28.9% (22/76) of primary debulking surgery cases. The BRCA1‐FAM showed high sensitivity (91.3%) and specificity (100%) for detecting BRCA1 promoter hypermethylation, comparable to BIS‐seq. Cases with BRCA1 promoter hypermethylation had significantly poorer progression‐free survival (log‐rank test, p = 0.048). Among these cases, 86.4% displayed abnormal BRCA1 immunostaining, with lower frequencies of BRCA1 loss of heterozygosity compared to those of other groups. BRCA1 promoter hypermethylation is associated with poor prognosis, underscoring the importance of its identification for HRD stratification. BRCA1‐FAM is a simple and highly accurate method for evaluating BRCA1 promoter methylation. This approach may potentially enhance the precision of personalized therapies for ovarian cancer.

Risk Stratification Prediction of Endometrial Cancer Using Microstructural Mapping Based on Time‐Dependent Diffusion MRI

ABSTRACTTime‐dependent diffusion MRI (td‐dMRI) has potential in characterizing microstructural features; however, its value in imaging endometrioid endometrial adenocarcinoma (EEA) remains uncertain. Patients surgically confirmed with EEA were finally enrolled in our study. The td‐dMRI data were acquired using pulsed gradient spin echo sequence and oscillating gradient spin echo sequences. The microstructural markers, including cell diameter, intracellular volume fraction (Vin), cellularity, and extracellular diffusivity (Dex), were fitted with the imaging microstructural parameters using a limited spectrally edited diffusion (IMPULSED) model. The parameters were compared between low‐ and high‐risk groups and between low‐ and high‐proliferation groups. The diagnostic performance was evaluated using receiver‐operating characteristic curve and logistic regression analysis. Diameter, Dex, ADCPGSE, ADCN1, and ADCN2 were significantly low, whereas cellularity, ΔADC1 and ΔADC2 were significantly high in the high‐risk and high‐proliferation groups. Cellularity, ΔADC1, and ΔADC2 demonstrated excellent diagnostic efficacy in predicting both risk stratification and proliferation status. Cellularity was the only independent predictor for risk stratification, which exhibited a satisfactory positive correlation with cell density in histopathologic examination. The diagnostic potential of td‐dMRI‐based microstructural mapping was demonstrated to noninvasively probe the pathologic characteristics of patients with EEA in a clinical setting, which provided a valuable contribution to surgical guidance.

Insights From Nonsense‐Mediated mRNA Decay for Prognosis in Homologous Recombination‐Deficient Ovarian Cancer

ABSTRACTNot all ovarian cancer patients with homologous recombination deficiency, especially those with germline BRCA mutations, can benefit from platinum‐based and targeted therapy. Our study aimed to determine the value of nonsense‐mediated mRNA decay, which targeted these mutations. The retrospective analysis of 797 ovarian cancer patients was performed using two public cohorts and one in‐house cohort. We developed a prediction algorithm for nonsense‐mediated mRNA decay to discriminate between trigger and escape status, finding that escape status indicated a better prognosis. Subsequently, we analyzed differential gene expression and functional pathways between the two statuses and filtered 8 genes associated with the cell cycle. Then the optimized key gene model was built using integrated machine learning algorithms (mean AUC > 0.89), which had a higher independent prognostic value for ovarian cancer with germline BRCA variants or homologous recombination deficiency than the nonsense‐mediated mRNA decay algorithm. Furthermore, we classified patients into high‐ and low‐risk groups by the machine learning model and found that the low‐risk group had a better prognosis with higher drug response and immune levels of activated dendritic cells than the high‐risk controls. Our findings provide a perspective based on nonsense‐mediated mRNA decay and cell cycle pathways to distinguish subtypes of germline BRCA or homologous recombination deficiency.

Susceptibility of Brca1(L63X/+) rat to ovarian reserve dissipation by chemotherapeutic agents to breast cancer

AbstractBRCA1 is one of the causative genes for hereditary breast and ovarian cancer syndrome with a high risk of early‐onset breast cancer. Whereas olaparib (OLA), an inhibitor of poly‐ADP‐ribose polymerase, has been applied as adjuvant therapy to those cancer patients, its effect on ovarian reproductive function remains unelucidated. Recently, a rat model (MUT; Brca1(L63X/+) mutation) mimicking a human BRCA1 pathogenic variant has been established. Using this model, we evaluated the effects of OLA on ovarian reproductive function in comparison with the wild‐type (WT) rats. MUT showed a significantly reduced number of primordial follicles and subfertility in accordance with aging. Oxidative stress was significantly elevated in the young MUT granulosa cells (GCs) accompanied by increased mTOR but decreased PTEN signals. OLA administration in MUT further decreased primordial follicles, with gene set enrichment analysis, indicating upregulated DNA repair pathways. Furthermore, a combination of OLA and cyclophosphamide (CPA) induced empty primordial follicles, recognized as CPA‐induced severe ovarian toxicity. Whereas OLA + CPA caused greater reduction in primordial follicles both in MUT and WT in comparison with CPA alone, MUT ovaries were more susceptible to oxidative stress, potentially depleting primordial follicles via activation of GCs and inducing oocyte death due to accumulated DNA damage by OLA treatment. Our findings in this preclinical model underscore the importance of evaluating ovarian reserve prior to chemotherapy by performing reproductive consultation with female patients with BRCA1 pathogenic variants.

Loss of SMARCA4 induces sarcomatogenesis through epithelial–mesenchymal transition in ovarian carcinosarcoma

AbstractOvarian carcinosarcoma (OCS) is a rare and aggressive tumor, and the development of its sarcomatous component is believed to be due to epithelial–mesenchymal transition (EMT). The SWIch/sucrose nonfermentable chromatin remodeling factor (CRF) is closely related to EMT; however, the relationship between CRF and EMT in OCS remains unclear. In this study, we analyzed the protein expression of CRFs, including ARID1A and SMARCA4, and their downstream mRNA expression in 28 OCS cases, two fallopian tube CS cases, and one peritoneal CS case. ARID1A and SMARCA4 exhibited a histological type‐specific loss of protein expression in 5 of 11 (45%) endometrioid cases and all 5 serous/homologous OCS cases, respectively. The mRNA analysis suggested that sarcomatogenesis is induced by the transforming growth factor‐β and Hippo signaling pathways, both of which regulate YAP1. Immunostaining for YAP1 suggested YAP1‐associated sarcomatogenesis in the CRF‐retained group, whereas YAP1‐unassociated sarcomatogenesis was suggested in the CRF‐reduced group. High‐grade serous carcinoma cell line experiments showed that the transcriptome of the SMARCA4‐knockdown group showed lower expression of the epithelial gene CDH1 and higher expression of mesenchymal genes such as VIM, ZEB1, and SNAI1 than the control group. Moreover, cell adhesion disappeared and cell morphology changed to a spindle shape, indicating sarcomatogenesis. In conclusion, this study reveals a mechanism for sarcoma development in OCS and provides novel therapeutic possibilities.

Real‐world genome profiling in Japanese patients with pancreatic ductal adenocarcinoma focusing on HRD implications

AbstractPancreatic ductal adenocarcinoma (PDAC) poses significant challenges due to its high mortality, making it a critical area of research. This retrospective observational study aimed to analyze real‐world data from comprehensive genome profiling (CGP) of Japanese patients with PDAC, mainly focusing on differences in gene detection rates among panels and the implications for homologous recombination deficiency (HRD) status. This study enrolled 2568 patients with PDAC who had undergone CGP between June 2019 and December 2021 using data from the nationwide Center for Cancer Genomics and Advanced Therapeutics database. Two types of CGP assays (tissue and liquid biopsies) were compared and a higher detection rate of genetic abnormalities in tissue specimens was revealed. HRD‐related gene alterations were detected in 23% of patients, with BRCA1/2 mutations accounting for 0.9% and 2.9% of patients, respectively. Treatment outcome analysis indicated that patients with BRCA1/2 mutations had a longer time to treatment discontinuation with FOLFIRINOX than gemcitabine plus nab‐paclitaxel as first‐line therapy (9.3 vs. 5.6 months, p = 0.028). However, no significant differences were observed in the treatment response among the other HRD‐related genes. Logistic regression analysis identified younger age and family history of breast, prostate, and ovarian cancers as predictive factors for HRD‐related gene alterations. Despite the lack of progression‐free survival data and the inability to discriminate between germline and somatic mutations, this study provides valuable insights into the clinical implications of CGP in Japanese patients with PDAC. Further research is warranted to optimize panel selection and elucidate the efficacy of platinum‐based therapies depending on the HRD status.

Impacts of cytoplasmic p53 aggregates on the prognosis and the transcriptome in lung squamous cell carcinoma

AbstractThe tumor suppressor TP53 gene, the most frequently mutated gene in human cancers, produces the product tumor protein p53, which plays an essential role in DNA damage. p53 protein mutations may contribute to tumorigenesis by loss of tumor suppressive functions and malignancy of cancer cells via gain‐of‐oncogenic functions. We previously reported that mutant p53 proteins form aggregates and that cytoplasmic p53 aggregates were associated with poor prognosis in human ovarian cancer. However, the prognostic impact of p53 aggregation in other tumors including lung squamous cell carcinoma (SCC) is poorly understood. Here, we demonstrated that lung SCC cases with cytoplasmic p53 aggregates had a significantly poor clinical prognosis. Analysis via patient‐derived tumor organoids (PDOs) established from lung SCC patients and possessing cytoplasmic p53 aggregates showed that eliminating cytoplasmic p53 aggregates suppressed cell proliferation. RNA sequencing and transcriptome analysis of p53 aggregate‐harboring PDOs indicated multiple candidate pathways involved in p53 aggregate oncogenic functions. With lung SCC‐derived cell lines, we found that cytoplasmic p53 aggregates contributed to cisplatin resistance. This study thus shows that p53 aggregates are a predictor of poor prognosis in lung SCC and suggests that detecting p53 aggregates via p53 conventional immunohistochemical analysis may aid patient selection for platinum‐based therapy.

PGD: Shared gene linking polycystic ovary syndrome and endometrial cancer, influencing proliferation and migration through glycometabolism

AbstractThe relationship among polycystic ovary syndrome (PCOS), endometrial cancer (EC), and glycometabolism remains unclear. We explored shared genes between PCOS and EC, using bioinformatics to unveil their pathogenic connection and influence on EC prognosis. Gene Expression Omnibus datasets GSE226146 (PCOS) and GSE196033 (EC) were used. A protein–protein interaction (PPI) network was constructed to identify the central genes. Candidate markers were screened using dataset GSE54250. Differences in marker expression were confirmed in mouse PCOS and human EC tissues using RT‐PCR and immunohistochemistry. The effect of PGD on EC proliferation and migration was explored using Ki‐67 and Transwell assays. PGD's impact on the glycometabolic pathway within carbon metabolism was assessed by quantifying glucose content and lactic acid production. R software identified 31 common genes in GSE226146 and GSE196033. Gene Ontology functional classification revealed enrichment in the “purine nucleoside triphosphate metabolism process,” with key Kyoto Encyclopedia of Genes and Genomes pathways related to “carbon metabolism.” The PPI network identified 15 hub genes. HK2, NDUFS8, PHGDH, PGD, and SMAD3 were confirmed as candidate markers. The RT‐PCR analysis validated distinct HK2 and PGD expression patterns in mouse PCOS ovarian tissue and human EC tissue, as well as in normal and EC cells. Transfection experiments with Ishikawa cells further confirmed PGD's influence on cell proliferation and migration. Suppression of PGD expression impeded glycometabolism within the carbon metabolism of EC cells, suggesting PGD as a significant PCOS risk factor impacting EC proliferation and migration through modulation of single carbon metabolism. These findings highlight PGD's pivotal role in EC onset and prognosis.

Even though active recommendation for HPV vaccination has restarted, Japan's rates have not recovered

AbstractJapan has a particularly critical situation surrounding its collapsed HPV vaccination program for preventing HPV‐caused cervical cancers, a problem exacerbated by the lack of a national immunization database. We have determined the year‐to‐year HPV vaccination uptake by Japanese females and analyzed by birth fiscal year (FY) the monthly number of people receiving initial HPV vaccination. Our analysis covers the period from the start of public subsidies in 2010 to September 2023, using data provided by local governments. We calculated the cumulative number of monthly immunizations for those unimmunized as of April (the beginning of each vaccination year). The monthly number of initial HPV vaccinations was highest in August for every FY from FY 2010 to FY 2023; a second vaccination peak tended to occur in March when the vaccination year ended. The highest number of August vaccinations occurred in FY 2011, followed (in order) by 2012, 2021, 2022, 2023, and 2013. In Japan's ongoing catch‐up vaccination program for young women, the monthly number of vaccinations increased in August 2022 but then slowed the following year. After FY 2021, the cumulative vaccination coverage of subjects unvaccinated at the beginning of the vaccination year but subsequently covered by routine immunizations was slightly improved. FY 2021 was when the governmental recommendations for HPV vaccination were resumed. More recent vaccination rates are considerably lower than those in FY 2011–2012 when vaccinations were first fully endorsed. Paralyzing HPV vaccination hesitancy, which began in FY 2013, will linger in Japan in FY 2024.

FoxO1 promotes ovarian cancer by increasing transcription and METTL14‐mediated m6A modification of SMC4

AbstractThe transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells in vitro and in vivo. ChIP‐seq combined with GEPIA2 and Kaplan–Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its −1400/−1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase‐like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

Mitochondrial fission enhances IL‐6‐induced metastatic potential in ovarian cancer via ERK1/2 activation

AbstractOvarian cancer is a lethal gynecologic cancer mostly diagnosed in an advanced stage with an accumulation of ascites. Interleukin‐6 (IL‐6), a pro‐inflammatory cytokine is highly elevated in malignant ascites and plays a pleiotropic role in cancer progression. Mitochondria are dynamic organelles that undergo fission and fusion in response to external stimuli and dysregulation in their dynamics has been implicated in cancer progression and metastasis. Here, we investigate the effect of IL‐6 on mitochondrial dynamics in ovarian cancer cells (OVCs) and its impact on metastatic potential. Treatment with IL‐6 on ovarian cancer cell lines (SKOV3 and PA‐1) led to an elevation in the metastatic potential of OVCs. Interestingly, a positive association was observed between dynamin‐related protein 1 (Drp1), a regulator of mitochondrial fission, and IL‐6R in metastatic ovarian cancer tissues. Additionally, IL‐6 treatment on OVCs was linked to the activation of Drp1, with a notable increase in the ratio of the inhibitory form p‐Drp1(S637) to the active form p‐Drp1(S616), indicating enhanced mitochondrial fission. Moreover, IL‐6 treatment triggered the activation of ERK1/2, and inhibiting ERK1/2 mitigated IL‐6‐induced mitochondrial fission. Suppressing mitochondrial fission through siRNA transfection and a pharmacological inhibitor reduced the IL‐6‐induced migration and invasion of OVCs. This was further supported by 3D invasion assays using patient‐derived spheroids. Altogether, our study suggests the role of mitochondrial fission in the metastatic potential of OVCs induced by IL‐6. The inhibition of mitochondrial fission could be a potential therapeutic approach to suppress the metastasis of ovarian cancer.

Inhibition of palmitoyltransferase ZDHHC12 sensitizes ovarian cancer cells to cisplatin through ROS‐mediated mechanisms

AbstractPlatinum‐based therapies have revolutionized the treatment of high‐grade serous ovarian cancer (HGSOC). However, high rates of disease recurrence and progression remain a major clinical concern. Impaired mitochondrial function and dysregulated reactive oxygen species (ROS), hallmarks of cancer, hold potential as therapeutic targets for selectively sensitizing cisplatin treatment. Here, we uncover an oncogenic role of the palmitoyltransferase ZDHHC12 in regulating mitochondrial function and ROS homeostasis in HGSOC cells. Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer data revealed significantly elevated ZDHHC12 expression, demonstrating the strongest positive association with ROS pathways among all ZDHHC enzymes. Transcriptomic analysis of independent ovarian cancer datasets and the SNU119 cell model corroborated this association, highlighting a strong link between ZDHHC12 expression and signature pathways involving mitochondrial oxidative metabolism and ROS regulation. Knockdown of ZDHHC12 disrupted this association, leading to increased cellular complexity, ATP levels, mitochondrial activity, and both mitochondrial and cellular ROS. This dysregulation, achieved by the siRNA knockdown of ZDHHC12 or treatment with the general palmitoylation inhibitor 2BP or the fatty acid synthase inhibitor C75, significantly enhanced cisplatin cytotoxicity in 2D and 3D spheroid models of HGSOC through ROS‐mediated mechanisms. Markedly, ZDHHC12 inhibition significantly augmented the anti‐tumor activity of cisplatin in an ovarian cancer xenograft tumor model, as well as in an ascites‐derived organoid line of platinum‐resistant ovarian cancer. Our data suggest the potential of ZDHHC12 as a promising target to improve the outcome of HGSOCs in response to platinum‐based chemotherapy.

Altered tumor signature and T‐cell profile after chemotherapy reveal new therapeutic opportunities in high‐grade serous ovarian carcinoma

AbstractChemotherapy combined with debulking surgery is the standard treatment protocol for high‐grade serous ovarian carcinoma (HGSOC). Nonetheless, a significant number of patients encounter relapse due to the development of chemotherapy resistance. To better understand and address this resistance, we conducted a comprehensive study investigating the transcriptional alterations at the single‐cell resolution in tissue samples from patients with HGSOC, using single‐cell RNA sequencing and T‐cell receptor sequencing techniques. Our analyses unveiled notable changes in the tumor signatures after chemotherapy, including those associated with epithelial–mesenchymal transition and cell cycle arrest. Within the immune compartment, we observed alterations in the T‐cell profiles, characterized by naïve or pre‐exhausted populations following chemotherapy. This phenotypic change was further supported by the examination of adjoining T‐cell receptor clonotypes in paired longitudinal samples. These findings underscore the profound impact of chemotherapy on reshaping the tumor landscape and the immune microenvironment. This knowledge may provide clues for the development of future therapeutic strategies to combat treatment resistance in HGSOC.

LINC00629, a HOXB4‐downregulated long noncoding RNA, inhibits glycolysis and ovarian cancer progression by destabilizing c‐Myc

AbstractOvarian cancer (OC) cells typically reprogram their metabolism to promote rapid proliferation. However, the role of long noncoding RNAs (lncRNAs) in the metabolic reprogramming of ovarian cancer, especially in glucose metabolic reprogramming, remains largely unknown. LINC00629 has been reported in our previous study to promote osteosarcoma progression. Upregulated LINC00629 was found to enhance the growth‐suppressive effect of apigenin on oral squamous cell carcinoma. However, the precise function of LINC00629 in ovarian cancer development remains poorly understood. In this study, we found that LINC00629 was significantly downregulated in OC tissues and that low LINC00629 expression was associated with poor survival. Inhibition of LINC00629 was required for increased glycolysis activity and cell proliferation in ovarian cancer. In vivo, overexpression of LINC00629 dramatically inhibited tumor growth and lung metastasis. Mechanistically, LINC00629 interacted with and destabilized c‐Myc, leading to its ubiquitination and proteasome degradation, further resulting in increased expression of downstream glycolysis‐related genes and glucose metabolic reprogramming in OC. Interestingly, HOXB4 bound to the LINC00629 promoter and inhibited its transcription, indicating that LINC00629 is a transcriptional target of HOXB4. Collectively, these findings establish a direct role for LINC00629 in suppressing glucose metabolism, and HOXB4/LINC00629/c‐Myc might serve as a potential biomarker and an effective therapeutic strategy for OC cancer treatment.

BRCA1/2 reversion mutations in a pan‐cancer cohort

AbstractTumor sensitivity to platinum (Pt)‐based chemotherapy and poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors is increased by homologous recombination deficiency‐causing mutations; in particular, reversion mutations cause drug resistance by restoring protein function. Treatment response is predicted by breast cancer susceptibility gene 1/2 (BRCA1/2) mutations; however, BRCA1/2 reversion mutations have not been comprehensively studied in pan‐cancer cohorts. We aimed to characterize BRCA1/2 reversion mutations in a large pan‐cancer cohort of Japanese patients by retrospectively analyzing sequencing data for BRCA1/2 pathogenic/likely pathogenic mutations in 3738 patients with 32 cancer types. We identified somatic mutations in tumors or circulating cell‐free DNA that could restore the ORF of adverse alleles, including reversion mutations. We identified 12 (0.32%) patients with somatic BRCA1 (n = 3) and BRCA2 (n = 9) reversion mutations in breast (n = 4), ovarian/fallopian tube/peritoneal (n = 4), pancreatic (n = 2), prostate (n = 1), and gallbladder (n = 1) cancers. We identified 21 reversion events—BRCA1 (n = 3), BRCA2 (n = 18)—including eight pure deletions, one single‐nucleotide variant, six multinucleotide variants, and six deletion–insertions. Seven (33.3%) reversion deletions showed a microhomology length greater than 1 bp, suggesting microhomology‐mediated end‐join repair. Disease course data were obtained for all patients with reversion events: four patients acquired mutations after PARP‐inhibitor treatment failure, two showed somatic reversion mutations after disease progression, following Pt‐based treatment, five showed mutations after both treatments, one patient with pancreatic cancer and BRCA1 reversion mutations had no history of either treatment. Although reversion mutations commonly occur in BRCA‐associated cancers, our findings suggest that reversion mutations due to Pt‐chemotherapy might be correlated with BRCA1/2‐mediated tumorigenesis even in non‐BRCA‐associated histologies.

Adjuvant Chemotherapy for Endometrial Cancer (ACE) trial: A randomized phase II study for advanced endometrial carcinoma

AbstractThis study evaluated the feasibility and efficacy of three postoperative adjuvant chemotherapy regimens for endometrial cancer. Endometrioid cancer patients with intermediate‐risk stage I and II or high‐risk stage III and IV disease were randomly assigned to receive six cycles of either paclitaxel‐epirubicin‐carboplatin (TEC), paclitaxel‐anthracycline (doxorubicin)‐carboplatin (TAC), or dose‐dense paclitaxel‐carboplatin (ddTC). The primary end‐point was the completion rate (CRate) of six cycles of treatment. The secondary end‐points were progression‐free survival (PFS) and overall survival (OS). One hundred and one patients were treated as follows: 33 received TEC, 33 TAC, and 35 ddTC. The CRates for TEC, TAC, and ddTC were 94%, 64%, and 69%, respectively (P = .005). The TEC CRate was significantly higher than for the other two groups. However, the PFS and OS outcomes were not statistically different between the three groups. The 2‐year survival rates were 94%, 97%, and 97% for TEC, TAC, and ddTC, respectively. When compared to the current standard treatments for endometrial cancer, TEC is a promising candidate for a phase III trial based on its significantly superior CRate and equivalent PFS and OS. This study is registered with UMIN Clinical Trials Registry (UMIN000008911).

Transferrin Receptor Promotes Endometrial Cancer Proliferation by Activating the Iron‐Dependent PI3K/AKT/mTOR Signaling Pathway

ABSTRACTAberrant iron metabolism is frequently observed in cancers, including endometrial cancer (EC). However, the role of transferrin receptor (TFRC), a key regulator of iron metabolism, remains unclear in endometrial cancer. We found transferrin receptor expression was significantly upregulated in endometrial cancer tissues compared to adjacent nontumor tissues, and high transferrin receptor levels were associated with poor prognosis. Functional studies revealed that transferrin receptor knockdown impaired endometrial cancer cell proliferation in vitro and in vivo, while transferrin receptor overexpression enhanced endometrial cancer cell proliferation. Mechanistically, transferrin receptor activated the PI3K/AKT/mTOR signaling pathway, as its knockdown suppressed the pathway, and rapamycin, an mTOR inhibitor, reversed transferrin receptor‐induced pathway activation and proliferation. Modulation of the labile iron pool by ferric ammonium citrate (FAC) or deferoxamine (DFO) rescued transferrin receptor‐induced biological effects. Additionally, AURKA was identified as a regulator of transferrin receptor expression. These findings demonstrate the oncogenic role of transferrin receptor in endometrial cancer and suggest that targeting iron homeostasis and the PI3K/AKT/mTOR pathway may represent potential therapeutic strategies for endometrial cancer.

CXCL9 and CXCL13 shape endometrial cancer immune‐activated microenvironment via tertiary lymphoid structure formation

AbstractImmune checkpoint inhibitor (ICI) therapy has been successfully applied to various cancers; however, not all patients respond to ICI therapy. Tumors with an immune‐activated environment are highly responsive to ICIs. To identify the cells and molecules essential to the formation of an immune‐activated cancer microenvironment, we focused on the tertiary lymphoid structure (TLS) and performed histological and genomic analyses using endometrial cancer material. In the high immunogenic group, numerous TLSs were observed, and CXCL9 and CXCL13 expression was markedly increased. CXCL9‐positive antigen‐presenting and CXCL13‐positive follicular dendritic cells were distributed in the T‐ and B‐cell zones of TLSs, respectively. A group of molecules whose expression was upregulated along with CXCL9 and CXCL13 expression was strongly associated with cellular immunity. These results suggest that CXCL9‐expressing antigen‐presenting cells and CXCL13‐expressing follicular dendritic cells coordinately shape the immune‐activated microenvironment through TLS formation. The current findings will contribute to a better understanding of the mechanisms underlying the activated cancer immune microenvironment, thereby advancing the field of precision cancer medicine.

IGFBP2 Promotes Proliferation and Glycolysis of Endometrial Cancer by Regulating PKM2/HIF‐1α Axis

ABSTRACTEndometrial cancer (EC) is a worldwide gynecologic malignancies, with a remarking increase of incidence and mortality rates in recent years. Growing evidence indicates that glucose metabolism reprogramming is the most representative metabolic signature of tumor cells and exploring its modulatory function in EC development will promote identifying potential EC therapeutic targets. IGFBP2 is an insulin‐like growth factor binding protein which is closely associated with a variety of metabolic diseases. However, its biological role in EC and its effects on glucose metabolism remain unclear. In this study, we demonstrated that IGFBP2 was highly expressed in EC tissues and correlated with poor prognosis. Overexpression of IGFBP2 promoted proliferation and glycolysis in EC cells, whereas IGFBP2 knockdown had the opposite effect. Mechanistically, IGFBP2 directly interacted with PKM2, inducing weakened PKM2 protein degradation, and knockdown IGFBP2 expression prevented the translocation of PKM2 to the nucleus. Additionally, IGFBP2 expression was upregulated under the condition of hypoxia which directly regulated by transcriptional activation of HIF‐1α. Finally, the role of the IGFBP2/PKM2/HIF‐1α axis in EC tumor growth was confirmed in vivo using mouse xenograft models. Taken together, the current study identifies IGFBP2 as an upstream activator of PKM2‐driven proliferation and glycolysis in EC cells, providing a promising therapeutic target for EC.

Cholesterol biogenesis is a PTEN‐dependent actionable node for the treatment of endocrine therapy‐refractory cancers

AbstractPTEN and PIK3CA mutations are the most prevalent PI3K pathway alterations in prostate, breast, colorectal, and endometrial cancers. p110β becomes the prominent PI3K isoform upon PTEN loss. In this study, we aimed to understand the molecular mechanisms of PI3K dependence in the absence of PTEN. Using online bioinformatical tools, we examined two publicly available microarray datasets with aberrant PI3K activation. We found that the rate‐limiting enzyme of cholesterol biogenesis, SQLE, was significantly upregulated in p110β‐hyperactivated or PTEN‐deficient mouse prostate tumors. Concomitantly, the expression of cholesterol biosynthesis pathway enzymes was directly correlated with PI3K activation status in microarray datasets and diminished upon PTEN re‐expression in PTEN‐null prostate cancer cells. Particularly, PTEN re‐expression decreased SQLE protein levels in PTEN‐deficient prostate cancer cells. We performed targeted metabolomics and detected reduced levels of cholesteryl esters as well as free cholesterol upon PTEN re‐expression. Notably, PTEN‐null prostate and breast cancer cell lines were more sensitive to pharmacological intervention with the cholesterol pathway than PTEN‐replete cancer cells. Since steroid hormones use sterols as structural precursors, we studied whether cholesterol biosynthesis may be a metabolic vulnerability that enhances antihormone therapy in PTEN‐null castration‐resistant prostate cancer cells. Coinhibition of cholesterol biosynthesis and the androgen receptor enhanced their sensitivity. Moreover, PTEN suppression in endocrine therapy‐resistant luminal‐A breast cancer cells leads to an increase in SQLE expression and a corresponding sensitization to the inhibition of cholesterol synthesis. According to our data, targeting cholesterol biosynthesis in combination with the hormone receptor signaling axis can potentially treat hormone‐resistant prostate and breast cancers.

A Mendelian randomization study identified obesity as a causal risk factor of uterine endometrial cancer in Japanese

AbstractCausal inference is one of the challenges in epidemiologic studies. Gynecologic diseases have been reported to have association with obesity, however the causality remained controversial except for uterine endometrial cancer. We conducted two‐sample Mendelian randomization (MR) analysis using the large‐scale genome‐wide association study (GWAS) results of gynecologic diseases and body mass index (BMI) in the Japanese population to assess causal effect of BMI on gynecologic diseases. We first conducted GWAS of ovarian cancer, uterine endometrial cancer, uterine cervical cancer, endometriosis, and uterine fibroid (n = 647, 909, 538, 5236, and 645 cases, respectively, and 39 556 shared female controls), and BMI (81 610 males and non‐overlapping 23 924 females). We then applied two‐sample MR using 74 BMI‐associated variants as instrumental variables. We observed significant causal effect of increased BMI on uterine endometrial cancer (β = 0.735, P = .0010 in inverse variance‐weighted analysis), which is concordant with results of European studies. Causal effect of obesity was not apparent in the other gynecologic diseases tested. Our MR analyses provided strong evidence of the causal role of obesity in gynecologic diseases etiology, and suggested a possible preventive effect of intervention for obesity.

FGF13 enhances resistance to platinum drugs by regulating hCTR1 and ATP7A via a microtubule‐stabilizing effect

AbstractPlatinum‐based regimens are the most widely used chemotherapy regimens, but cancer cells often develop resistance, which impedes therapy outcome for patients. Previous studies have shown that fibroblast growth factor 13 (FGF13) is associated with resistance to platinum drugs in HeLa cells. However, the mechanism and universality of this effect have not been clarified. Here, we found that FGF13 was associated with poor platinum‐based chemotherapy outcomes in a variety of cancers, such as lung, endometrial, and cervical cancers, through bioinformatics analysis. We then found that FGF13 simultaneously regulates the expression and distribution of hCTR1 and ATP7A in cancer cells, causes reduced platinum influx, and promotes platinum sequestration and efflux upon cisplatin exposure. We subsequently observed that FGF13‐mediated platinum resistance requires the microtubule‐stabilizing effect of FGF13. Only overexpression of FGF13 with the ‐SMIYRQQQ‐ tubulin‐binding domain could induce the platinum resistance effect. This phenomenon was also observed in SK‐MES‐1 cells, KLE cells, and 5637 cells. Our research reveals the mechanism of FGF13‐induced platinum drug resistance and suggests that FGF13 can be a sensibilization target and prognostic biomarker for chemotherapy.

PLOD2 promotes colorectal cancer progression by stabilizing USP15 to activate the AKT/mTOR signaling pathway

AbstractProcollagen‐lysine, 2‐oxoglutarate 5‐dioxygenase 2 (PLOD2) has been reported as an oncogenic gene, affecting various malignant tumors, including endometrial carcinoma, osteosarcoma, and gastric cancer. These effects are mostly due to the enhanced deposition of collagen precursors. However, more studies need to be conducted on how its lysyl hydroxylase function affects cancers like colorectal carcinoma (CRC). Our present results showed that PLOD2 expression was elevated in CRC, and its higher expression was associated with poorer survival. Overexpression of PLOD2 also facilitated CRC proliferation, invasion, and metastasis in vitro and in vivo. In addition, PLOD2 interacted with USP15 by stabilizing it in the cytoplasm and then activated the phosphorylation of AKT/mTOR, thereby promoting CRC progression. Meanwhile, minoxidil was demonstrated to downregulate the expression of PLOD2 and suppress USP15, and the phosphorylation of AKT/mTOR. Our study reveals that PLOD2 plays an oncogenic role in colorectal carcinoma, upregulating USP15 and subsequently activating the AKT/mTOR pathway.

miR‐548ag functions as an oncogene by suppressing MOB1B in the development of obesity‐related endometrial cancer

AbstractObesity is a high‐risk factor in the development of endometrial cancer (EC). Our previous study observed that miR‐548ag was significantly overexpressed in the sera of obese individuals. Here, we report the function of miR‐548ag and its mechanism in promoting the obesity‐related progression of EC. The content of miR‐548ag was increased in the serum of obese EC individuals. Bioinformatics analysis indicated that the survival rate of EC patients with a higher expression of miR‐548ag was significantly reduced. The Mps One Binder Kinase Activator 1B (MOB1B, the core member of the Hippo signaling pathway) is a direct target gene of miR‐548ag, which is inversely correlated with the expression of miR‐548ag. The overexpression of miR‐548ag enhances the proliferation, invasion, and migration, and inhibits apoptosis by downregulating the expression of MOB1B, leading to the deactivation of the Hippo pathway in EC cell lines and contributing to tumor progression in vivo. Our study has established that miR‐548ag functions as an oncogene by suppressing MOB1B in the development of obesity‐related EC.

Frequent PIK3CA mutation in normal endometrial gland drives spheroid formation and may be involved in stem cell propagation

AbstractRecent studies reported the presence of oncogenic mutations in normal endometrial glands, but the biological significance remains unclear. The present study investigated the status of KRAS/PIK3CA driver mutations in normal endometrial glands as well as spheroids derived from single glands. The normal endometria of surgically removed uteri (n = 3) were divided into nine regions, and 40 endometrial single glands were isolated from each region. The DNAs of 10 glands in each region were extracted and subjected to Sanger sequencing for KRAS or PIK3CA driver mutations, while the remaining 30 glands were conferred to a long‐term spheroid culture, followed by Sanger sequencing. Immunohistochemical analyses of stem cell (Axin2, ALDH1A1, SOX9) markers were undertaken for spheroids. Sanger sequencing successfully detected oncogenic mutations of KRAS or PIK3CA in a single gland. Twenty‐five of the 270 glands (9.3%) had mutations in either KRAS or PIK3CA, and the mutation frequency in each endometrial region varied from 0% to 50%. The droplet digital PCR showed high mutation allele frequency (MAF) of PIK3CA mutation, suggestive of clonal expansion of mutated cells within a gland. Over 60% of the collected spheroids had PIK3CA mutations, but no KRAS mutations were detected. Immunohistochemically, spheroids were mainly composed of cells with stem cell marker expressions. High MAF of PIK3CA mutation in a single gland as well as frequent PIK3CA mutation in stem cell‐rich spheroids that originated from a single gland suggest the role of PIK3CA mutation in stem cell propagation. This information could improve our understanding of endometrial physiology as well as stem cell‐oriented endometrial regeneration and carcinogenesis.

Enhanced antitumor activity of combined lipid bubble ultrasound and anticancer drugs in gynecological cervical cancers

AbstractChemotherapy plays an important role in the treatment of patients with gynecological cancers. Delivering anticancer drugs effectively to tumor cells with just few side effects is key in cancer treatment. Lipid bubbles (LB) are compounds that increase the vascular permeability of the tumor under diagnostic ultrasound (US) exposure and enable the effective transport of drugs to tumor cells. The aim of our study was to establish a novel drug delivery technique for chemotherapy and to identify the most effective anticancer drugs for the bubble US‐mediated drug delivery system (BUS‐DDS) in gynecological cancer treatments. We constructed xenograft models using cervical cancer (HeLa) and uterine endometrial cancer (HEC1B) cell lines. Lipid bubbles were injected i.v., combined with either cisplatin (CDDP), pegylated liposomal doxorubicin (PLD), or bevacizumab, and US was applied to the tumor. We compared the enhanced chemotherapeutic effects of these drugs and determined the optimal drugs for BUS‐DDS. Tumor volume reduction of HeLa and HEC1B xenografts following cisplatin treatment was significantly enhanced by BUS‐DDS. Both CDDP and PLD significantly enhanced the antitumor effects of BUS‐DDS in HeLa tumors; however, volume reduction by BUS‐DDS was insignificant when combined with bevacizumab, a humanized anti‐vascular endothelial growth factor mAb. The BUS‐DDS did not cause any severe adverse events and significantly enhanced the antitumor effects of cytotoxic drugs. The effects of bevacizumab, which were not as dose‐dependent as those of the two drugs used prior, were minimal. Our data suggest that BUS‐DDS technology might help achieve “reinforced targeting” in the treatment of gynecological cancers.

The prognosis of endometrial cancers stratified with conventional risk factors and modified molecular classification

AbstractThis study aimed to validate the Proactive Molecular Risk Classifier for Endometrial Cancer, a modified version of The Cancer Genome Atlas, using data from 184 patients with endometrial cancer (median age: 57.5 years; median follow‐up period: 109 months) who had undergone radical surgery (including systemic lymphadenectomy) and subsequent adjuvant chemotherapy (patients with intermediate or high recurrence risk) from 2003 to 2015. Tissue microarrays were prepared from surgical specimens and classified using the conventional clinical risk classifier. Immunohistochemistry was used to detect mismatch repair proteins, L1 cell adhesion molecule, and p53. Direct sequencing was used to identify hotspot mutations in the polymerase‐epsilon gene. Forty‐five patients were identified as having high L1 cell adhesion molecule expression, 41 as low risk, 34 as mismatch repair‐deficient, 13 as polymerase‐epsilon gene‐mutated, five as having abnormal p53, and 46 as other. Patients were stratified into significantly different prognostic groups (p < 0.0001): favorable (low risk and polymerase‐epsilon gene‐mutated), intermediate (mismatch repair‐deficient and other), and unfavorable (high L1 cell adhesion molecule expression and abnormal p53) with 5‐year disease‐specific survival rates of 100%, 93.8%, and 75.1%, respectively (Kaplan–Meier method). The combination of conventional recurrent risk classification, sequencing for polymerase‐epsilon gene mutations and immunohistochemistry for L1 cell adhesion molecule, p53, and mismatch repair proteins can be used to determine the prognoses of patients with endometrial cancer.

The HOXC‐AS2/miR‐876‐5p/HKDC1 axis regulates endometrial cancer progression in a high glucose‐related tumor microenvironment

AbstractThe tumor microenvironment (TME) is related to chronic inflammation and is currently identified as a risk factor for the occurrence and development of endometrial cancer (EC). Pyroptosis is a new proinflammatory form of programmed cell death that plays a critical role in the progression of multiple diseases. However, the important role of pyroptosis in high‐glucose (HG)‐related EC and the underlying molecular mechanisms remain elusive. In the present study, transcriptome high‐throughput sequencing revealed significantly higher hexokinase domain‐containing 1 (HKDC1) expression in EC patients with diabetes than in EC patients with normal glucose. Mechanistically, HKDC1 regulates HG‐induced cell pyroptosis by modulating the production of reactive oxygen species and pyroptosis‐induced cytokine release in EC. In addition, HKDC1 regulates TME formation by enhancing glycolysis, promoting a metabolic advantage in lactate‐rich environments to further accelerate EC progression. Subsequently, miR‐876‐5p was predicted to target the HKDC1 mRNA, and HOXC‐AS2 was identified to potentially inhibit the miR‐876‐5p/HKDC1 axis in regulating cell pyroptosis in HG‐related EC. Collectively, we elucidated the regulatory role of the HOXC‐AS2/miR‐876‐5p/HKDC1 signal transduction axis in EC cell pyroptosis at the molecular level, which may provide an effective therapeutic target for patients with diabetes who are diagnosed with EC.

Growth arrest‐specific transcript 5 represses endometrial cancer development by promoting antitumor function of tumor‐associated macrophages

AbstractThe tumor‐suppressor role of long noncoding RNA (lncRNA) growth arrest‐specific transcript 5 (GAS5) has been proven in various types of cancer. However, the specific function of GAS5 in tumor‐associated macrophages (TAMs) of endometrial cancer (EC) is elusive. Quantitative PCR results showed that GAS5 expression decreased in EC tissues and primary TAMs from EC tumors. Tumor‐associated macrophage infiltration was significantly positively associated with the developmental stage of EC. Direct coculture of GAS5‐overexpressing TAMs and EC cells showed that GAS5 enhanced phagocytosis, antigen presentation, and activation of cytotoxic T cells, and repressed “Don’t eat me” signals between TAMs and EC cells. Tumor formation in immunodeficient mice showed that GAS5‐overexpressing macrophages could repress EC formation in vivo. GAS5 promoted M1 polarization by activating the microRNA‐21– phosphatase and tensin homolog (PTEN)–AKT signaling pathway and directly repressing the nuclear accumulation and phosphorylation of oncogenic yes‐associated protein 1 (YAP1) in TAMs. GAS5 inhibited the development of EC from both innate and adaptive immunity by transforming TAMs from a protumor to an antitumor phenotype. These antitumor effects of GAS5 on TAMs were mediated by the activation of the miR‐21‐PTEN‐AKT pathway and inhibition of YAP1.

ASH2L is involved in promotion of endometrial cancer progression via upregulation of PAX2 transcription

AbstractAbsent, small or homeotic 2‐like protein (ASH2L) is a core component of a multimeric histone methyltransferase complex that is involved in the maintenance of active transcription, participating in several cancers, however the biological function and molecular mechanism of ASH2L in endometrial cancer (ECa) are largely unknown. Endometrial cancer is a common malignant tumor in women and the incidence of this cancer is on the rise. Estrogen‐ERα signaling, as an oncogenic pathway, plays a crucial role in endometrial carcinogenesis. Therefore, further exploration of the molecular mechanisms around ERα‐mediated gene transcription in ECa would be helpful to the understanding of tumor development and to finding a new therapeutic target for ECa. Here, our study demonstrated that ASH2L was highly expressed in ECa samples, and higher expression of ASH2L was positively correlated with a poor prognosis. Moreover, we identified that ASH2L associated with ERα and that knockdown of ASH2L resulted in decreased expression of a subset of the estrogen‐induced target genes, including paired box 2 (PAX2), an oncogenic gene in ECa. ASH2L was recruited to cis‐regulatory elements in PAX2, thereby altering histone H3K4me3 and H3K27me3 levels, to enhance ERα‐mediated transactivation. Finally, depletion of ASH2L suppressed endometrial cancer cell proliferation and migration. Our findings suggest that ASH2L participates in the promotion of ECa progression, if not totally at least partially, via upregulation of PAX2 transcription.

Efficacy and safety of standard of care with/without bevacizumab for platinum‐resistant ovarian/fallopian tube/peritoneal cancer previously treated with bevacizumab: The Japanese Gynecologic Oncology Group study JGOG3023

AbstractWe investigated the efficacy and safety of further bevacizumab therapy in patients with platinum‐resistant ovarian cancer whose disease had progressed after bevacizumab plus chemotherapy. In this multicenter, open‐label, phase II trial (JGOG3023), patients were randomized 1:1 to a single‐agent chemotherapy alone (either pegylated liposomal doxorubicin [40 or 50 mg/m2 administered intravenously], topotecan [1.25 mg/m2 intravenously], paclitaxel [80 mg/m2 intravenously], or gemcitabine [1000 mg/m2 intravenously]) or single‐agent chemotherapy + bevacizumab (15 mg/m2 intravenously). The primary endpoint was investigator‐assessed progression‐free survival (PFS) according to RECIST version 1.1. Secondary endpoints were overall survival (OS), objective response rate (ORR), and response rate according to Gynecological Cancer Intergroup cancer antigen 125 criteria. In total, 103 patients were allocated to chemotherapy (n = 51) or chemotherapy + bevacizumab (n = 52). Median investigator‐assessed PFS was 3.1 and 4.0 mo in each group, respectively (hazard ratio [HR] = 0.54, 95% confidence interval [CI]: 0.32‐0.90, P = .0082). Median OS was 11.3 and 15.3 mo in each group, respectively (HR = 0.67, 95% CI: 0.38‐1.17, P = .1556). Respective ORRs were 13.7% and 25.0% (P = .0599) and response rates were 16.7% and 21.4% (P = .8273). The incidence of grade ≥3 treatment‐related AEs was 42.0% in the chemotherapy group and 54.9% in the chemotherapy + bevacizumab group; AEs were well tolerated, with only 2 and 12 events leading to discontinuation of therapy, respectively. Bevacizumab was effective beyond progressive disease and AEs were manageable. The observed improvement in PFS requires further verification.

Single‐Nucleus RNA Sequencing and Spatial Transcriptomics for Squamous Cell Carcinoma Arising From Ovarian Mature Teratoma

ABSTRACTSquamous cell carcinoma arising from mature teratoma (SCC‐MT) is a rare ovarian malignancy. The detailed molecular pathology of SCC‐MT is not well understood. Moreover, the prognosis of the patients remains poor because no standard treatment has been established. In this study, we performed single‐nucleus RNA sequencing and spatial transcriptomics using clinical SCC‐MT samples to identify novel therapeutic candidates. snRNA‐seq revealed three epithelial cell clusters, of which one was significantly associated with epidermis and keratinocyte development. Moreover, spatial transcriptomics revealed that the epithelial‐mesenchymal transition was significantly inhibited, and the MYC and E2F targets were significantly activated in cancer spots on specimen sections. We focused on KLF5, which was one of the upregulated genes in cancer cells, and performed a functional analysis using NOSCC‐1, a cell line derived from an SCC‐MT. KLF5 downregulation significantly decreased cell proliferation and increased apoptosis. Furthermore, we previously identified miR‐145‐5p as a downregulated miRNA in SCC‐MT. We demonstrated that miR‐145‐5p overexpression attenuated cell proliferation and decreased KLF5 expression. In conclusion, through multi‐omics analyses, we identified unique gene expression profiles of SCC‐MT and determined a role for KLF5 in SCC‐MT development. Therefore, KLF5‐related factors may be novel therapeutic targets, and further studies are needed to improve the diagnosis and treatment of SCC‐MT.

Secondary Lower‐Body Sarcomas in Cervical Cancer Survivors Following Surgery, Radiotherapy, or Both: A Population‐Based Study

ABSTRACT Secondary sarcomas in cervical cancer survivors are understudied. We investigated the incidence and subtypes of secondary sarcomas by treatment modalities, the prognosis of secondary sarcomas, and whether surgery plus radiotherapy increases angiosarcoma incidence. This population‐based retrospective cohort study analyzed Osaka Cancer Registry data on women aged 20–84 years diagnosed with cervical cancer (1980–2015), treated with surgery, radiotherapy, or both. Patients with distant metastases, survival time < 1 year, or missing data were excluded. We included 7591 patients with invasive cancer treated with surgery, 3882 with radiotherapy, and 4090 with surgery plus radiotherapy and 13,205 with carcinoma in situ treated with surgery. We assessed the first lower‐body sarcoma occurrence ≥ 1 year post‐diagnosis. Sarcomas developed in 6 patients treated with surgery (0 angiosarcomas), 10 with radiotherapy (1 angiosarcoma), and 19 with surgery plus radiotherapy (9 angiosarcomas). At 10 years, the cumulative incidence was 0.083% (95% confidence interval [CI], 0.024%–0.24%) for radiotherapy and 0.21% (95% CI, 0.10%–0.41%) for surgery plus radiotherapy, higher than that for surgery (invasive, 0.013%; in situ, 0.028%) ( p  < 0.001). Angiosarcoma incidence was higher with surgery plus radiotherapy (0.080%; 95% CI, 0.023%–0.23%) than with radiotherapy (0.028%; 95% CI, 0.003%–0.16%) ( p  = 0.029). Among patients diagnosed with sarcoma after radiotherapy or surgery plus radiotherapy, the 1‐year overall survival rate was 33.3% (95% CI, 19.6%–56.8%). Radiotherapy, alone or combined with surgery, increased secondary lower‐body sarcoma incidence compared with surgery. To our knowledge, this is the first population‐based study to suggest that surgery plus radiotherapy is associated with angiosarcoma in cervical cancer survivors.

Survival Benefit of Surgical Staging Before Radiotherapy in Locally Advanced Cervical Cancer: A Pooled Analysis

ABSTRACTLymph node metastasis significantly impacts prognosis and treatment in locally advanced cervical cancer (LACC). Surgical staging offers precise information on node involvement, though its survival benefit is debated. We pooled data from 9 cohort studies involving 2553 patients to evaluate the benefit of pre‐treatment surgical staging in patients with locally advanced cervical cancer. Fixed effects models or random effects models were used to calculate the pooled hazard ratios (HRs). The overall pooled results showed no difference in PFS (HR 0.94, 95% CI 0.73–1.22, p = 0.65) or OS (HR 1.00, 95% CI 0.74–1.35, p = 0.99) between the two approaches of lymph node staging. However, the subgroup analyses found the PFS superiority of surgical staging in patients with FIGO stage II (HR 0.68, 95% CI 0.49–0.95, p = 0.02). Additionally, for the patients with no evidence of lymph node metastasis on imaging, surgical staging was associated with significantly improved PFS (HR 0.69, 95% CI 0.56–0.86, p = 0.001) and OS (HR 0.56, 95% CI 0.36–0.87, p = 0.01). In the subgroup of patients with suspicious bulky nodes on imaging, lymph node debulking‐based surgical staging did not significantly improve either PFS (HR 0.97, 95% CI 0.72–1.31, p = 0.31) or OS (HR 1.16, 95% CI 0.68–1.99, p = 0.59) in comparison with imaging staging. Surgical staging may not be applicable to all patients with LACC. However, for the patients with FIGO II disease or those without suspicious lymph node involvement on imaging, node surgery staging could afford a survival benefit.Trial Registration: PROSPERO ID: CRD42024543768

CYP24A1 Binding to FUS Maintains Tumor Properties by Regulating the miR‐200c/ZEB1/EMT Axis

ABSTRACTThe active vitamin D‐degrading enzyme (CYP24A1) is commonly overexpressed in various types of cancer, which is associated with poor prognosis in cancer patients. Recent studies highlight the antagonism of CYP24A1 toward the anticancer role of active vitamin D. However, the impact of CYP24A1 on tumorigenesis and its underlying mechanisms largely remains unexplored. This study also found that high CYP24A1 mRNA expressions were associated with poor prognosis in ovarian cancer and lung adenocarcinoma (LUAD) patients. Moreover, we demonstrated that the overexpression of CYP24A1 accelerated the proliferation, migration, and invasion of ovarian cancer and LUAD cancer cells in vitro. Furthermore, knockdown of CYP24A1 displayed an anticancer effector both in vitro and in vivo. Mechanically, 87–297 amino acid motif of CYP24A1 bound specifically to FUS protein, consequentially reducing FUS affinity for miR‐200c. Considering FUS promotes gene silencing by binding to microRNA targets, a decrease in miR‐200c levels led to a notable activation of its target ZEB1, resulting in the promotion of the epithelial‐mesenchymal transition (EMT) process. In conclusion, FUS binding specifically by CYP24A1 impaired miR‐200c‐mediated ZEB1 silencing, thereby augmenting EMT progression and tumorigenesis. These findings elucidate a fundamental mechanism by which CYP24A1 operates as an oncogene, offering potential targets for therapeutic interventions in cancer treatment.

Microbial and Metabolic Disorders in Cervical Cancer: Structural Insights, Biomarkers, Mechanisms, and Therapeutic Strategies

ABSTRACT The development of cervical cancer is strongly associated with persistent high‐risk HPV infection. Microbiota and metabolomics offer new perspectives. This article focuses on the role of microbial dysbiosis and metabolic reprogramming in the development of cervical cancer, revealing its synergistic regulation of the tumor immune microenvironment and treatment resistance. Machine learning and multi‐omics have identified new biomarkers, while treatment strategies include microbiota modulation, metabolic targeting, and combination therapies. Despite limitations such as small sample size and unclear mechanisms, this review proposes a multi‐target precision medicine framework. In the future, we should focus on multi‐center and multi‐omics research and optimized clinical trials.

Downregulation of Chromosome 19 miRNA Cluster and the Tumor‐Suppressive Role of miR ‐517a‐3p in Choriocarcinoma

ABSTRACT Choriocarcinoma is a rare gynecologic malignancy. MicroRNAs, which are noncoding RNAs approximately 22 nucleotides in length, are known to regulate gene expression and play important roles in various cancers; however, their functions in choriocarcinoma remain largely unknown. This study aimed to identify disease‐specific microRNAs involved in choriocarcinoma development. Eleven cases of choriocarcinoma and five cases of complete hydatidiform mole treated at our institution were analyzed. Total RNA was extracted from trophoblast cells in formalin‐fixed, paraffin‐embedded specimens using laser capture microdissection, and microRNA sequencing was performed. The analysis revealed that 87 microRNAs were significantly upregulated, whereas 28 were downregulated in choriocarcinoma compared to complete hydatidiform mole. Notably, 13 of the 28 downregulated microRNAs belonged to the chromosome 19 microRNA cluster. In vitro experiments demonstrated that overexpression of miR‐517a‐3p, a representative member of this cluster, significantly suppressed cell proliferation, migration, and invasion in JEG‐3 and BeWo cell lines. Further transcriptome sequencing and computational analysis identified SRSF1 as a target gene of miR‐517a‐3p, which was validated by dual‐luciferase reporter assays. Knockdown of SRSF1 also led to significant reductions in proliferation, migration, and invasion, supporting its functional relevance. Immunohistochemical analysis confirmed that SRSF1 protein was highly expressed in choriocarcinoma tissues compared to complete hydatidiform mole. These findings indicate that downregulation of the chromosome 19 microRNA cluster is a characteristic feature of choriocarcinoma and that miR‐517a‐3p functions as a tumor suppressor by directly regulating SRSF1 expression.

Human papillomavirus vaccine to prevent CIN3 or worse (CIN3+): A nationwide case–control study in Japan

AbstractAn increase in cervical cancer incidence has been reported in Japan. The Ministry of Health, Labor, and Welfare of Japan has resumed the active recommendation of regular HPV vaccines in 2022. In Japan, the preventive effect of CIN3+ in the real world has not yet been demonstrated in age‐adjusted cohort or case–control studies. This study aimed to estimate the effect of the HPV vaccine against CIN3+ in Japanese women. This nationwide case–control study from April 2013 to March 2020 targeted women aged 20–26 years old at the time of cervical screening. We compared HPV vaccination exposure between those with abnormal and those with normal cytology. Abnormal cytology was classified into cervical intraepithelial neoplasia (CIN)1+, CIN2+, and CIN3+. We calculated the odds ratio (OR) and 95% confidence interval (CI) of the above endpoints and vaccination exposure using the conditional logistic regression model and estimated vaccine effectiveness using the formula (1 −OR) × 100. A total of 2790 cases and 13,990 controls (one‐to‐five matching) were eligible in 37 municipalities in Japan. In this study, 61 CIN3 (2.2%) and 10 squamous cell carcinomas (SCC) (0.4%) were found. The OR for CIN3+ versus controls was 0.14 (95% CI, 0.03–0.75), equating to a vaccine effectiveness of 86%. Of the 10 patients who had SCC none were vaccinated. This nationwide case–control study in Japan demonstrated a substantial risk reduction in CIN3+ among women who did versus those who did not receive HPV vaccination.

Extracellular vesicle‐encapsulated microRNA‐296‐3p from cancer‐associated fibroblasts promotes ovarian cancer development through regulation of the PTEN/AKT and SOCS6/STAT3 pathways

AbstractCancer‐associated fibroblasts (CAFs), as important components of the tumor microenvironment, can regulate intercellular communication and tumor development by secreting extracellular vesicles (EVs). However, the role of CAF‐derived EVs in ovarian cancer has not been fully elucidated. Here, using an EV‐microRNA sequencing analysis, we reveal specific overexpression of microRNA (miR)‐296‐3p in activated CAF‐derived EVs, which can be transferred to tumor cells to regulate the malignant phenotypes of ovarian cancer cells. Moreover, overexpression of miR‐296‐3p significantly promotes the proliferation, migration, invasion, and drug resistance of ovarian cancer cells in vitro, as well as tumor growth in vivo, while its inhibition has the opposite effects. Further mechanistic studies reveal that miR‐296‐3p promotes ovarian cancer progression by directly targeting PTEN and SOCS6 and activating AKT and STAT3 signaling pathways. Importantly, increased expression of miR‐296‐3p encapsulated in plasma EVs is closely correlated with tumorigenesis and chemoresistance in patients with ovarian cancer. Our results highlight the cancer‐promoting role of CAF‐derived EVs carrying miR‐296‐3p in ovarian cancer progression for the first time, and suggest that miR‐296‐3p encapsulated in CAF‐derived EVs could be a diagnostic biomarker and therapeutic target for ovarian cancer.

Unlocking Japan's cervical cancer prevention deadlock

Cervical cancer prevention in Japan, which had nearly halted due to misinformation, is now recovering through HPV vaccination and screening efforts. While significant progress has been made, much work remains, yet the recent improvements are highly encouraging.

Cervical mucus can be used for metabolite screening in cervical cancer

AbstractApproximately 660,000 women are diagnosed with cervical cancer annually. Current screening options such as cytology or human papillomavirus testing have limitations, creating a need to identify more effective ancillary biomarkers for triage. Here, we evaluated whether metabolomic analysis of cervical mucus metabolism could be used to identify biomarkers of cervical intraepithelial neoplasia (CIN) and cervical cancer. The case–control group consisted of 181 CIN, 69 squamous cell carcinoma (SCC) patients, and 48 healthy controls in the primary cohort. We undertook metabolomic analyses using ultra‐HPLC–tandem mass spectrometry. Univariate and multivariate analyses were carried out to profile metabolite characteristics, and receiver operating characteristic (ROC) analysis identified biomarker candidates. Five metabolites conferred the highest discriminatory power for SCC: oxidized glutathione (GSSG) (area under the ROC curve, 0.924; 95% confidence interval, 0.877–0.971), malic acid (0.914, 0.859–0.968), kynurenine (0.884, 0.823–0.945), GSSG/glutathione (GSH) (0.936, 0.892–0.979), and kynurenine/tryptophan (0.909, 0.856–0.961). Malic acid was the best marker for detection of CIN2 or worse (0.858, 0.793–0.922) and was a clinically useful metabolite. We confirmed the reproducibility of the results by validation cohort. Additionally, metabolomic analyses revealed eight pathways strongly associated with cervical neoplasia. Of these, only the tricarboxylic acid cycle was strongly associated with all CINs and cancer, indicating active energy production. Aberrant arginine metabolism by decreasing arginine and increasing citrulline might reduce tumor immunity. Changes in cysteine‐methionine and GSH pathways might drive the initiation and progression of cervical cancer. These results suggest that metabolic analysis can identify ancillary biomarkers and could improve our understanding of the pathophysiological mechanisms underlying cervical neoplasia.

Optimization of prediction methods for risk assessment of pathogenic germline variants in the Japanese population

AbstractPredicting pathogenic germline variants (PGVs) in breast cancer patients is important for selecting optimal therapeutics and implementing risk reduction strategies. However, PGV risk factors and the performance of prediction methods in the Japanese population remain unclear. We investigated clinicopathological risk factors using the Tyrer‐Cuzick (TC) breast cancer risk evaluation tool to predict BRCA PGVs in unselected Japanese breast cancer patients (n = 1,995). Eleven breast cancer susceptibility genes were analyzed using target‐capture sequencing in a previous study; the PGV prevalence in BRCA1, BRCA2, and PALB2 was 0.75%, 3.1%, and 0.45%, respectively. Significant associations were found between the presence of BRCA PGVs and early disease onset, number of familial cancer cases (up to third‐degree relatives), triple‐negative breast cancer patients under the age of 60, and ovarian cancer history (all P < .0001). In total, 816 patients (40.9%) satisfied the National Comprehensive Cancer Network (NCCN) guidelines for recommending multigene testing. The sensitivity and specificity of the NCCN criteria for discriminating PGV carriers from noncarriers were 71.3% and 60.7%, respectively. The TC model showed good discrimination for predicting BRCA PGVs (area under the curve, 0.75; 95% confidence interval, 0.69‐0.81). Furthermore, use of the TC model with an optimized cutoff of TC score ≥0.16% in addition to the NCCN guidelines improved the predictive efficiency for high‐risk groups (sensitivity, 77.2%; specificity, 54.8%; about 11 genes). Given the influence of ethnic differences on prediction, we consider that further studies are warranted to elucidate the role of environmental and genetic factors for realizing precise prediction.

Polarity switching of ovarian cancer cell clusters via SRC family kinase is involved in the peritoneal dissemination

AbstractPeritoneal dissemination is a predominant pattern of metastasis in patients with advanced ovarian cancer. Despite recent progress in the management strategy, peritoneal dissemination remains a determinant of poor ovarian cancer prognosis. Using various histological types of patient‐derived ovarian cancer organoids, the roles of the apicobasal polarity of ovarian cancer cell clusters in peritoneal dissemination were studied. First, it was found that both ovarian cancer tissues and ovarian organoids showed apicobasal polarity, where zonula occludens‐1 (ZO‐1) and integrin beta 4 (ITGB4) served as markers for apical and basal sides, respectively. The organoids in suspension culture, as a model of cancer cell cluster floating in ascites, showed apical‐out/basal‐in polarity status, while once embedded in extracellular matrix (ECM), the organoids switched their polarity to apical‐in/basal‐out. This polarity switch was accompanied by the SRC kinase family (SFK) phosphorylation and was inhibited by SFK inhibitors. SFK inhibitors abrogated the adherence of the organoids onto the ECM‐coated plastic surface. When the organoids were seeded on a mesothelial cell layer, they cleared and invaded mesothelial cells. In vivo, dasatinib, an SFK inhibitor, suppressed peritoneal dissemination of ovarian cancer organoids in immunodeficient mice. These results suggest SFK‐mediated polarity switching is involved in peritoneal metastasis. Polarity switching would be a potential therapeutic target for suppressing peritoneal dissemination in ovarian cancer.

Silybin suppresses ovarian cancer cell proliferation by inhibiting isocitrate dehydrogenase 1 activity

AbstractMetabolic reprogramming is a sign of malignant tumors, and targeting the metabolism of tumor cells has become a promising therapeutic approach. Here, we report that Silybin (a nontoxic flavonoid commonly used for liver protection) exhibits prominent anti‐tumor effects on human ovarian cancer cells. Treatment of an ovarian cancer cell line with Silybin interfered with glutamine metabolism and the tricarboxylic acid cycle. We applied the drug affinity responsive target stability approach to show that Silybin binds to isocitrate dehydrogenase 1 (IDH1). This combination leads to reduced phosphorylation of IDH1 and inhibits enzyme activity. IDH1 dysfunction significantly increases the ratio of NADP/NADPH in the cell, causing an increase in reactive oxygen species generation. Immunohistochemistry demonstrated that IDH1 was increased in ovarian cancer samples compared with normal para‐tumoral tissues. Xenograft murine experiments indicated that Silybin administered orally suppressed the growth of the tumor formed by ovarian cancer cells. In combination, our data strongly suggest that Silybin targets IDH1 in ovarian cancer cells and may be a novel treatment candidate.

Corrected human papillomavirus vaccination rates for each birth fiscal year in Japan

AbstractIn Japan, the serious adverse events after human papillomavirus (HPV) vaccination were widely reported in the media. The Ministry of Health, Labour and Welfare of Japan (MHLW) announced the suspension of the governmental recommendation of HPV vaccine in 2013, and the inoculation rate has since sharply declined. The estimated inoculation rate for each birth fiscal year (FY) announced by the MHLW and the actual numbers for each birth FY surveyed by local governments were very different. In particular, the cumulative vaccination rate of girls born in FY2000 was regarded to be as high as 42.9% by the Council of the MHLW. However, this estimation included a confusion. When the suspension of the governmental recommendation was announced in FY2013, the girls born in FY2000 turned 13 years old, the targeted starting age of the HPV vaccination. The vaccination rate of this generation is considered to be quite low. The numbers were recalculated in this study. This study revealed that the real vaccination rate is only 14.3%. Female individuals born in or after FY2000 have been confirmed to be exposed to the same cervical cancer risk as before the HPV vaccine was introduced in Japan.

Targeting the mutant PIK3CA gene by DNA‐alkylating pyrrole‐imidazole polyamide in cervical cancer

AbstractPIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole‐imidazole polyamide‐seco‐CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole‐imidazole polyamides. P3AE5K interacted with double‐stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K‐positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole‐imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.

Rapid generation of genetically engineered T cells for the treatment of virus‐related cancers

AbstractAdoptive transfer of T cell receptor (TCR)‐engineered T cells targeting viral epitopes represents a promising approach for treating virus‐related cancers. However, the efficient identification of epitopes for T cells and the corresponding TCR remains challenging. Here, we report a workflow permitting the rapid generation of human papillomavirus (HPV)‐specific TCR‐T cells. Six epitopes of viral proteins belonged to HPV16 or HPV18 were predicted to have high affinity to A11:01 according to bioinformatic analysis. Subsequently, CTL induction were performed with these six antigen peptides separately, and antigen‐specific T cells were sorted by FACS. TCR clonotypes of these virus‐specific T cells were determined using next‐generation sequencing. To improve the efficiency of TCRαβ pair validation, a lentiviral vector library containing 116 TCR constructs was generated that consisted of predominant TCRs according to TCR repertoire analysis. Later, TCR library transduced T cells were simulated with peptide pool‐pulsed antigen‐presenting cells, then CD137‐positive cells were sorted and subjected to TCR repertoire analysis. The top‐hit TCRs and corresponding antigen peptides were deduced and validated. Through this workflow, a TCR targeting the E692–101 of HPV16 was identified. These HPV16‐specific TCR‐T cells showed high activity towards HPV16‐positive human cervical cancer cells in vitro and efficiently repressed tumor growth in a murine model. This study provides a HPV16‐specific TCR fitted to the HLA‐A11:01 population, and exemplifies an efficient approach that can be applied in large‐scale screening of virus‐specific TCRs, further encouraging researchers to exploit the therapeutic potential of the TCR‐T cell technique in treating virus‐related cancers.

Aberrant expression of claudin‐6 contributes to malignant potentials and drug resistance of cervical adenocarcinoma

AbstractRecent studies have revealed that aberrant expression of tight junction (TJ) proteins is a hallmark of various solid tumors and it is recognized as a useful therapeutic target. Claudin‐6 (CLDN6), a member of the family of TJ transmembrane proteins, is an ideal therapeutic target because it is not expressed in human adult normal tissues. In this study, we found that CLDN6 is highly expressed in uterine cervical adenocarcinoma (ADC) and that high CLDN6 expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor. Shotgun proteome analysis revealed that cell‐cell adhesion‐related proteins and drug metabolism‐associated proteins (aldo‐keto reductase [AKR] family proteins) were significantly increased in CLDN6‐overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell‐cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin. Taken together, the results indicate that aberrant expression of CLDN6 enhances malignant potentials and drug resistance of cervical ADC, possibly due to increased cell‐cell adhesion properties and drug metabolism. Our findings provide an insight into a new therapeutic strategy, a CLDN6‐targeting therapy, against cervical ADC.

IRF4 Enhances Radiosensitivity of Cervical Cancer by Inhibiting the PI3K/Akt/mTOR Pathway to Regulate Autophagy

ABSTRACT Interferon regulatory factor 4 (IRF4), a critical member of the IRF transcription factor family, harbors an elusive biological role in cervical cancer. Through immunohistochemical staining and immunoblotting, CCK‐8 viability assays, EdU incorporation tests, clonogenic survival experiments, flow cytometric detection, transmission electron microscopy, immunofluorescence staining and heterotopic transplantation model, we discover that IRF4 expression was markedly decreased in cervical cancer tissues and cell lines compared to normal controls. Overexpression of IRF4 suppressed proliferation, migration, and invasion in both Siha and HeLa cells, while concurrently enhancing radiosensitivity. Mechanistically, IRF4 upregulated autophagy‐related proteins (LC3, Beclin‐1) and promoted autophagosome formation, while downregulating P62 by inhibiting the PI3K/Akt/mTOR pathway. In vivo studies demonstrated that IRF4 augmented the tumor response to radiation and further potentiated the effects when combined with rapamycin treatment, confirming its pivotal role in promoting radiosensitivity through PI3K/Akt/mTOR‐mediated autophagy. IRF4 emerges as a critical regulator of cervical cancer progression via modulation of autophagy and influences the tumor's response to radiotherapy. It holds promise as a potential therapeutic target to enhance cervical cancer radiosensitivity.

In‐Tumor CRISPR ‐Cas9 Knockout Screening and Novel Therapy Development for Malignant Transformation of Ovarian Teratoma

ABSTRACT Malignant transformation of mature cystic teratoma (MTMCT) of the ovary is a rare but aggressive malignancy for which no standardized chemotherapy or effective targeted therapies currently exist. To identify therapeutic vulnerabilities in MTMCT, we performed a genome‐wide CRISPR‐Cas9 knockout screen using the MTMCT‐derived NOSCC1 cell line. Two parallel selective pressures were applied: in vivo tumorigenicity in immunodeficient mice and cisplatin exposure in vitro. From this screen, 67 negatively selected genes were identified, among which SOD1 and NDUFB4 emerged as top candidates based on high basal expression levels and clinical relevance. Integration with spatial transcriptomic data from three independent MTMCT patient tumors further supported the prioritization of these targets. SOD1 was selected for further investigation due to the availability of known pharmacological inhibitors. Both siRNA‐mediated knockdown and small‐molecule inhibition of SOD1 using LCS‐1 significantly suppressed MTMCT cell proliferation in vitro by inducing oxidative stress and impairing cell cycle progression. This antiproliferative effect was reversed by co‐treatment with N ‐acetylcysteine, a reactive oxygen species scavenger. In vivo validation using patient‐derived xenograft models demonstrated that oral administration of LCS‐1 led to significant tumor growth suppression and increased expression of apoptotic and DNA damage markers, including cleaved caspase‐3 and γH2AX. These findings establish SOD1 as a critical vulnerability in MTMCT and provide preclinical evidence supporting redox modulation as a therapeutic strategy for this highly chemoresistant and understudied ovarian cancer subtype. Our integrative approach combining functional genomics, spatial transcriptomics, and pharmacologic validation offers a framework for the discovery of novel targets in rare gynecologic malignancies.

Tumor‐Associated Macrophage‐Derived CXCL1 Promotes Endometrial Cancer Progression Through the CXCR2/NF‐κB Pathway

ABSTRACT Advanced‐stage and metastatic endometrial cancer (EC) are characterized by markedly poor survival outcomes, necessitating a deeper understanding of the mechanisms driving disease advancement. Tumor‐associated macrophages (TAMs), particularly the M2 subtype, are instrumental in remodeling the tumor microenvironment and promoting EC proliferation, migration, and metastasis. However, the specific mediators responsible for TAM‐driven EC progression remain inadequately elucidated. In this study, we investigated the role of the C–X–C motif chemokine ligand 1 (CXCL1) and its receptor, chemokine receptor 2 (CXCR2), in TAM‐induced EC progression. CXCR2, as the primary receptor for CXCL1, activates the NF‐κB signaling pathway upon binding, thereby mediating the epithelial–mesenchymal transition process in EC cells and enhancing metastatic potential. This mechanism can be effectively inhibited by silencing CXCR2 or by employing the NF‐κB inhibitor BAY 11‐7082. Neutralizing CXCL1 markedly diminished the proliferative and migratory burdens imposed by TAMs on EC in subcutaneous xenograft EC models. Additionally, in EC tissue samples, CXCL1 and CXCR2 expression, as well as the extent of macrophage infiltration, exhibited a significant positive relationship with disease progression, suggesting an unfavorable prognosis. In conclusion, targeting the CXCL1/CXCR2 axis is a potential therapeutic approach for EC treatment.

Inhibition of squalene epoxidase linking with PI3K/AKT signaling pathway suppresses endometrial cancer

AbstractEndometrial cancer (EC) is a common malignant tumor that lacks any therapeutic target and, in many cases, recurrence is the leading ca use of morbidity and mortality in women. Widely known EC has a strongly positive correlation with abnormal lipid metabolism. Squalene epoxidase (SQLE), a crucial enzyme in the cholesterol synthesis pathway regulating lipid metabolic processes has been found to be associated with various cancers in recent years. Here, we focused on studying the role of SQLE in EC. Our study revealed that SQLE expression level was upregulated significantly in EC tissues. In vitro experiments showed that SQLE overexpression significantly promoted the proliferation, and inhibited cell apoptosis of EC cells, whereas SQLE knockdown or use of terbinafine showed the opposite results. Furthermore, we found out that the promotional effect of SQLE on the proliferation of EC cells might be achieved by activating the PI3K/AKT pathway. In vivo, studies confirmed that the knockdown of SQLE or terbinafine can observably inhibit tumor growth in nude mice. These results indicate that SQLE may promote the progression of EC by activating the PI3K/AKT pathway. Moreover, SQLE is a potential target for EC treatment and its inhibitor, terbinafine, has the potential to become a targeted drug for EC treatment.

Lenvatinib plus pembrolizumab in Japanese patients with endometrial cancer: Results from Study 309/KEYNOTE‐775

AbstractStudy 309/KEYNOTE‐775 is a phase 3 open‐label, randomized trial of lenvatinib plus pembrolizumab versus treatment of physician's choice (TPC) in patients with advanced endometrial cancer with progression after platinum‐based therapy. Primary endpoints of superiority for lenvatinib plus pembrolizumab were met for progression‐free survival (PFS) and overall survival (OS) in all‐comers (ie, regardless of mismatch repair [MMR] status) and patients with MMR proficiency (pMMR). We present results for the Japanese subset. Patients were randomized to oral lenvatinib 20 mg/day plus intravenous pembrolizumab 200 mg every 3 weeks (Q3W; up to 35 cycles of pembrolizumab) or TPC (intravenous doxorubicin 60 mg/m2 Q3W or paclitaxel 80 mg/m2 QW [3 weeks on/1 week off]). Primary endpoints were PFS by blinded independent central review per RECIST version 1.1 and OS. One hundred four patients were randomized in Japan (data cutoff, October 26, 2020; median follow‐up, 11.8 [range, 1.1–26.9] months). Hazard ratios (HRs) for PFS with lenvatinib plus pembrolizumab versus TPC were 1.04 (95% CI, 0.63–1.73) in patients with pMMR and 0.81 (0.50–1.31) in all‐comers. Hazard ratios for OS were 0.74 (0.41–1.34) with pMMR and 0.59 (0.33–1.04) for all‐comers. Adverse events were manageable and led to discontinuation of one/both study drugs in 36.5% of patients in the lenvatinib plus pembrolizumab group versus 7.8% in the TPC group. Similar to the global Study 309/KEYNOTE‐775 results, this analysis suggested favorable efficacy and manageable safety with lenvatinib plus pembrolizumab after platinum‐based chemotherapy in Japanese patients with advanced endometrial cancer and supports this combination as a new standard of care in this population.

Long noncoding RNA EIF1AX‐AS1 promotes endometrial cancer cell apoptosis by affecting EIF1AX mRNA stabilization

AbstractLong noncoding RNAs (lncRNAs) have been found to play an important role in the occurrence and development of endometrial carcinoma (EC). Here, using RNA sequencing analysis, we systemically screened and identified the lncRNA eukaryotic translation initiation factor 1A, X‐linked (EIF1AX)‐AS1, which is aberrantly downregulated in clinical EC tissues and closely correlated with tumor type. EIF1AX‐AS1 markedly inhibited EC cell proliferation and promoted apoptosis in vitro and in vivo. Mechanistically, EIF1AX‐AS1 interacts with EIF1AX mRNA and poly C binding protein 1 (PCBP1), which promote EIF1AX mRNA degradation. Intriguingly, by interacting with internal ribosome entry site‐related protein Y‐box binding protein 1 (YBX‐1), EIF1AX promotes c‐Myc translation through the internal ribosome entry site pathway. c‐Myc promotes EIF1AX transcription and thus forms a feed‐forward loop to regulate EC cell proliferation. Taken together, these data reveal new insights into the biology driving EC proliferation and highlights the potential of lncRNAs as biomarkers for prognosis and future therapeutic targets for cancer.

Single‐Cell and Multiomic Analysis Reveals Neutrophil Heterogeneity and Prognostic Value in Cervical Lesions

ABSTRACT Cervical cancer is a prevalent malignancy among women, yet the involvement of neutrophils in its tumor microenvironment remains insufficiently explored. This study utilized single‐cell RNA sequencing (scRNA‐seq) to delineate neutrophil subsets and elucidate their roles in disease progression and prognosis. Analysis of 20 cervical biopsy samples across different disease stages identified five neutrophil subsets (N0–N4), among which the N4 subset exhibited a marked increase during disease advancement. Spatial transcriptomics and tissue microarray analyses revealed that N4 neutrophils are enriched in tumor regions and are associated with genes implicated in proliferation, metastasis, and immune evasion. Functional characterization demonstrated that N4 promotes tumor progression via activation of the Wnt signaling pathway and extracellular matrix remodeling. A neutrophil infiltration‐based risk model was established and validated through multi‐omics approaches, highlighting its potential in prognostic prediction. These findings underscore the pivotal role of N4 neutrophils in cervical cancer and provide valuable insights for the development of targeted immunotherapies and personalized treatment strategies.

TGM2 Regulates Radiosensitivity via POGZ ‐Mediated Repair of DNA Double‐Strand Breaks in Cervical Cancer

ABSTRACT The high capacity of cancer cells for DNA damage repair constitutes a critical factor contributing to their radioresistance. Previous studies have demonstrated that aberrant expression of transglutaminase 2 (TGM2) is linked to treatment resistance. However, the role of TGM2 in cervical cancer radiosensitivity and its underlying mechanisms remain unclear. In this study, we found that TGM2 was significantly upregulated in radioresistant cervical cancer cells and tissues. TGM2 knockdown significantly enhanced the radiosensitivity of cervical cancer cells, while TGM2 overexpression conferred radioresistance. TGM2 depletion exacerbated ionizing radiation (IR)‐induced DNA double‐strand breaks (DSBs). Mechanistically, IR triggered the nuclear translocation of TGM2, where it physically interacted with POGO transposable element derived with ZNF domain protein (POGZ) and upregulated POGZ protein levels. TGM2 knockdown impaired BRCA1 recruitment to DSB sites, phenocopying POGZ depletion effects. Rescue experiments demonstrated that POGZ knockdown reversed the radioresistance and reduction in DNA DSBs caused by TGM2 overexpression. Subcutaneous xenograft mouse models further verified these findings and the regulatory role of TGM2 in cervical cancer radiosensitivity in vivo. Together, our results demonstrated that TGM2 regulates radiosensitivity by POGZ‐mediated DNA DSBs repair, providing a novel strategy for increasing cervical cancer radiosensitivity.

VRK1 Is a Novel Therapeutic Target for Small Cell Neuroendocrine Carcinoma of the Cervix

ABSTRACT Small cell neuroendocrine carcinoma of the cervix (SCNEC) is classified as a high‐grade neuroendocrine carcinoma with a worse prognosis than other major histological types of cervical cancer. Identifying novel therapeutic targets based on its molecular characteristics is highly desirable but challenging due to the rarity of SCNEC and the resulting lack of research resources. In this study, we identified vaccinia‐related kinase 1 (VRK1) as a potential therapeutic target for SCNEC. VRK1 was prioritized based on our previously reported proteomic analysis of patient‐derived organoids. Immunohistochemistry of patient samples consistently revealed high VRK1 expression in SCNEC, as opposed to its variable expression in other cervical carcinomas. Although VRK1 knockdown in SCNEC had only a limited effect on cell proliferation in two‐dimensional cultures, it significantly suppressed cell proliferation in three‐dimensional cultures and inhibited xenograft tumor growth in vivo. Gene set enrichment analysis of RNA‐sequencing data from mouse xenograft models demonstrated that VRK1 is associated with mitochondrial‐related pathways. Furthermore, under oxidative stress conditions, VRK1 knockdown resulted in a reduction of mitochondrial membrane potential, an indicator of mitochondrial integrity, and decreased expression of cytochrome c oxidase subunit IV (COX IV), a nuclear‐encoded subunit of cytochrome c oxidase, the terminal enzyme complex of the mitochondrial respiratory chain. These findings suggest that VRK1 knockdown indirectly impaired mitochondrial function. Collectively, these anti‐tumor effects highlight VRK1 as a promising therapeutic target for SCNEC.

Assessment for the timing of comprehensive genomic profiling tests in patients with advanced solid cancers

AbstractComprehensive genomic profiling (CGP) tests have been covered by public insurance in Japan for patients with advanced solid tumors who have completed or are completing standard treatments or do not have them. Therefore, genotype‐matched drug candidates are often unapproved or off‐label, and improving clinical trial access is critical, involving the appropriate timing of CGP tests. To address this issue, we analyzed the previous treatment data for 441 patients from an observational study on CGP tests discussed by the expert panel at Hokkaido University Hospital between August 2019 and May 2021. The median number of previous treatment lines was two; three or more lines accounted for 49%. Information on genotype‐matched therapies was provided to 277 (63%). Genotype‐matched clinical trials were ineligible because of an excess number of previous treatment lines or use of specific agents were found in 66 (15%) patients, with the highest proportion in breast and prostate cancers. Many patients met the exclusion criteria of one to two or more treatment lines across cancer types. In addition, previous use of specific agents was a frequent exclusion criterion for breast, prostate, colorectal, and ovarian cancers. The patients with tumor types with a low median number (two or fewer) of previous treatment lines, including most rare cancers, primary unknown cancers, and pancreatic cancers, had significantly fewer ineligible clinical trials. The earlier timing of CGP tests may improve access to genotype‐matched clinical trials, with their proportion varying by cancer type. Each relevant society needs to advocate the desirable timing of CGP testing nationwide.

An engineered Accum‐E7 protein‐based vaccine with dual anti‐cervical cancer activity

AbstractWorldwide prevalence of cervical cancer decreased significantly with the use of human papilloma virus (HPV)‐targeted prophylactic vaccines. However, these multivalent antiviral vaccines are inert against established tumors, which leave patients with surgical ablative options possibly resulting in long‐term reproductive complications and morbidity. In an attempt to bypass this unmet medical need, we designed a new E7 protein‐based vaccine formulation using Accum™, a technology platform designed to promote endosome‐to‐cytosol escape as a means to enhance protein accumulation in target cells. Prophylactic vaccination of immunocompetent mice using the Accum‐E7 vaccine (aE7) leads to complete protection from cervical cancer despite multiple challenges conducted with ascending C3.43 cellular doses (0.5‐, 1.0‐, and 2.0 × 106 cells). Moreover, the humoral response induced by aE7 was higher in magnitude compared with naked E7 protein vaccination and displayed potent inhibitory effects on C3.43 proliferation in vitro. When administered therapeutically to animals with pre‐established C3.43 or Tal3 tumors, the vaccine‐induced response synergized with multiple immune checkpoint blockers (anti‐PD‐1, anti‐CTLA4, and anti‐CD47) to effectively control tumor growth. Mechanistically, the observed therapeutic effect requires cross‐presenting dendritic cells as well as CD8 T cells predominantly, with a non‐negligible role played by both CD4+ and CD19+ lymphocytes. good laboratory practice (GLP) studies revealed that aE7 is immunogenic and well tolerated by immunocompetent mice with no observed adverse effects despite the use of a fourfold exceeding dose. In a nutshell, aE7 represents an ideal vaccine candidate for further clinical development as it uses a single engineered protein capable of exhibiting both prophylactic and therapeutic activity.

Homologous recombination inquiry through ovarian malignancy investigations: JGOG3025 Study

AbstractThe Cancer Genome Atlas (TCGA) network has clarified that ~50% of high‐grade serous ovarian cancers show homologous recombination deficiency (HRD). However, the frequency of HRD in Japanese patients with ovarian cancer remains unclear. We aimed to identify the frequency of HR‐associated gene mutations in Japanese patients with ovarian cancer. The JGOG3025 study is a multicenter collaborative prospective observational study involving 65 study sites throughout Japan. We recruited 996 patients who were clinically diagnosed with ovarian cancer before surgery from March 2017 to March 2019, and 701 patients were eligible according to the criteria. We used frozen tumor tissues to extract DNA and performed next‐generation sequencing for 51 targeted genes (including 29 HR‐associated genes) in 701 ovarian cancers (298 high‐grade serous cases, 189 clear cell cases, 135 endometrioid cases, 12 mucinous cases, 3 low‐grade serous cases, and 64 others). HRD was defined as positive when at least one HR‐associated gene was mutated. The frequencies of HRD and tumor BRCA1/2 mutations were 45.2% (317/701) and 18.5% (130/701), respectively, in the full analysis set. Next, we performed multivariate Cox proportional hazards regression analysis for progression‐free survival (PFS) and overall survival (OS). Advanced‐stage ovarian cancer patients with HRD had adjusted hazard ratios of 0.72 (95% CI, 0.55–0.94) and 0.57 (95% CI, 0.38–0.86) for PFS and OS, respectively, compared with those without HRD (p = 0.016 and 0.007). Our study demonstrated that mutations in HR‐associated genes were associated with prognosis. Further studies are needed to investigate the prognostic impact of each HR‐associated gene in ovarian cancer.

Spatial genomic diversity associated with APOBEC mutagenesis in squamous cell carcinoma arising from ovarian teratoma

AbstractAlthough the gross and microscopic features of squamous cell carcinoma arising from ovarian mature cystic teratoma (MCT‐SCC) vary from case to case, the spatial spreading of genomic alterations within the tumor remains unclear. To clarify the spatial genomic diversity in MCT‐SCCs, we performed whole‐exome sequencing by collecting 16 samples from histologically different parts of two MCT‐SCCs. Both cases showed histological diversity within the tumors (case 1: nonkeratinizing and keratinizing SCC and case 2: nonkeratinizing SCC and anaplastic carcinoma) and had different somatic mutation profiles by histological findings. Mutation signature analysis revealed a significantly enriched apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC) signature at all sites. Intriguingly, the spread of genomic alterations within the tumor and the clonal evolution patterns from nonmalignant epithelium to cancer sites differed between cases. TP53 mutation and copy number alterations were widespread at all sites, including the nonmalignant epithelium, in case 1. Keratinizing and nonkeratinizing SCCs were differentiated by the occurrence of unique somatic mutations from a common ancestral clone. In contrast, the nonmalignant epithelium showed almost no somatic mutations in case 2. TP53 mutation and the copy number alteration similarities were observed only in nonkeratinizing SCC samples. Nonkeratinizing SCC and anaplastic carcinoma shared almost no somatic mutations, suggesting that each locally and independently arose in the MCT. We demonstrated that two MCT‐SCCs with different histologic findings were highly heterogeneous tumors with clearly different clones associated with APOBEC‐mediated mutagenesis, suggesting the importance of evaluating intratumor histological and genetic heterogeneity among multiple sites of MCT‐SCC.

Identification of target cells of human papillomavirus 18 using squamocolumnar junction organoids

AbstractHuman papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high‐risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR‐GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR‐GFP vectors were transduced into patient‐derived squamocolumnar junction organoids, and the presence of GFP‐positive cells was evaluated. Single‐cell RNA sequencing of GFP‐positive and GFP‐negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP‐positive and GFP‐negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP‐positive cells, whereas keratinization and mitophagy/autophagy‐related pathways were upregulated in GFP‐negative cells. siRNA‐mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV‐related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18‐infected cells downregulated stem cell‐related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18‐infected cervical lesions.

Human papillomavirus vaccine impact on invasive cervical cancer in Japan: Preliminary results from cancer statistics and the MINT study

AbstractThe first prophylactic vaccine against human papillomavirus (HPV) 16 and HPV18 was licensed in Japan in 2009. HPV vaccine effectiveness against high‐grade cervical lesions has been demonstrated among young Japanese women, but evidence of its effects on invasive cervical cancer (ICC) is lacking. Using data from two different cancer registries, we compared recent trends of new ICC cases by age group using Poisson regression analysis. We also analyzed time trends in HPV16/18 prevalence among 1414 Japanese women aged <40 years newly diagnosed with ICC in the past decade. Based on the population‐based cancer registry, the incidence of ICC among young women aged 20–29 years showed a significant decline from 3.6 to 2.8 per 100 000 women‐years during 2016–2019, but no similar decline was observed for older age groups (p < 0.01). Similarly, using data from the gynecological cancer registry of the Japan Society of Obstetrics and Gynecology, the annual number of ICCs among women aged 20–29 years also decreased from 256 cases to 135 cases during 2011–2020 (p < 0.0001). Furthermore, a declining trend in HPV16/18 prevalence in ICC was observed only among women aged 20–29 years during 2017–2022 (90.5%–64.7%, p = 0.05; Cochran–Armitage trend test). This is the first report to suggest population‐level effects of HPV vaccination on ICC in Japan. Although the declining trend in HPV16/18 prevalence among young women with ICC supports a causal linkage between vaccination and results from cancer registries, further studies are warranted to confirm that our findings are attributable to vaccination.

Development of novel tracers for sentinel node identification in cervical cancer

AbstractIndocyanine green (ICG) with near‐infrared (NIR) fluorescence imaging is used for lymphatic mapping. However, binding of ICG to blood proteins like serum albumin can shorten its retention time in sentinel lymph nodes (SLNs). Here, we investigated the efficacy and safety of a new fluorescence tracer comprising phytate and liposome (LP)‐encapsulated ICG. Coadministration of phytate with LP containing phosphatidic acid promotes chelation mediated by Ca2+ in bodily fluids to enhance SLN retention. Uniformly sized LPs (100 nm) encapsulating ICG under conditions that minimized fluorescence self‐quenching during storage were produced. We analyzed the behavior of the new tracer (ICG‐phytate‐LP) and control tracers (ICG and ICG‐LP) in the lymphatic flow of mice in terms of lymph node retention time. We also tested lymphatic flow and safety in pigs that have a more human‐like lymphatic system. LPs encapsulating stabilized ICG were successfully prepared. Mixing LP with phytate in the presence of Ca2+ increased both the particle size and negative surface charge. In mice, ICG‐phytate‐LP had the best lymph node retention, with a fluorescence intensity ratio that increased over 6 h and then decreased slowly over the next 24 h. In pigs, administration of ICG and ICG‐phytate‐LP resulted in no death or weight loss. There were no obvious differences between blood test results for the ICG and ICG‐phytate‐LP groups, and the overall safety was good. ICG‐phytate‐LP may be a useful new tracer for gynecological cancers that require time for lymph node identification due to a retroperitoneal approach.

Genetically engineered mouse models for hereditary cancer syndromes

AbstractAdvances in molecular diagnostics have led to improved diagnosis and molecular understanding of hereditary cancers in the clinic. Improving the management, treatment, and potential prevention of cancers in carriers of predisposing mutations requires preclinical experimental models that reflect the key pathogenic features of the specific syndrome associated with the mutations. Numerous genetically engineered mouse (GEM) models of hereditary cancer have been developed. In this review, we describe the models of Lynch syndrome and hereditary breast and ovarian cancer syndrome, the two most common hereditary cancer predisposition syndromes. We focus on Lynch syndrome models as illustrative of the potential for using mouse models to devise improved approaches to prevention of cancer in a high‐risk population. GEM models are an invaluable tool for hereditary cancer models. Here, we review GEM models for some hereditary cancers and their potential use in cancer prevention studies.

Omental cancer‐associated fibroblast‐derived exosomes with low microRNA‐29c‐3p promote ovarian cancer peritoneal metastasis

AbstractOvarian cancer (OC) is characterized by frequent widespread peritoneal metastasis. Cancer‐associated fibroblasts (CAFs) represent a critical stromal component of metastatic niche and promote omentum metastasis in OC patients. However, the role of exosomes derived from omental CAFs in metastasis remains unclear. We isolated exosomes from primary omental normal fibroblasts (NFs) and CAFs from OC patients (NF‐Exo and CAF‐Exo, respectively) and assessed their effect on metastasis. In mice bearing orthotopic OC xenografts, CAF‐Exo treatment led to more rapid intraperitoneal tumor dissemination and shorter animal survival. Similar results were observed in mice undergoing intraperitoneal injection of tumor cells. Among the miRNAs downregulated in CAF‐Exo, miR‐29c‐3p in OC tissues was associated with metastasis and survival in patients. Moreover, increasing miR‐29c‐3p in CAF‐Exo significantly weakened the metastasis‐promoting effect of CAF‐Exo. Based on RNA sequencing, expression assays, and luciferase assays, matrix metalloproteinase 2 (MMP2) was identified as a direct target of miR‐29c‐3p. These results verify the significant contribution of exosomes from omental CAFs to OC peritoneal metastasis, which could be partially due to the relief of MMP2 expression inhibition mediated by low exosomal miR‐29c‐3p.

Inhibition of microRNA let‐7b expression by KDM2B promotes cancer progression by targeting EZH2 in ovarian cancer

AbstractMicroRNA let‐7b is a potent tumor suppressor and targets crucial oncogenes. Previous studies have shown that let‐7b expression is suppressed in ovarian cancer; however, the regulatory mechanisms of let‐7b in ovarian cancer are still not well defined. The cellular role and targets of let‐7b in ovarian cancer remain elusive. In the present study, we showed that histone demethylase, KDM2B, directly suppressed let‐7b expression by H3K36me2 demethylation. Moreover, let‐7b inhibited EZH2 expression in ovarian cancer cells. Based on these results we know that let‐7b antagonizes the enhancement of EZH2 expression caused by KDM2B overexpression, and its expression is negatively correlated with KDM2B and EZH2 expression. More importantly, proliferation, migration, and wound healing assays showed that let‐7b inhibited ovarian cancer cell proliferation and migration in vitro. Additionally, let‐7b overexpression neutralized KDM2B‐promoted cell proliferation and migration. Furthermore, downregulation of let‐7b increased the xenografted tumor volumes in nude mice that were transplanted with KDM2B‐silenced cells. EZH2 silencing reversed the tumor growth enhancement mediated by inhibition of let‐7b. Last, we show that let‐7b expression is suppressed in ovarian carcinomas and its expression is negatively associated with the clinicopathological features of ovarian cancer, including histological type, histological grade, International Federation of Gynecology and Obstetrics (FIGO) stage, and lymph node metastatic status. In conclusion, in ovarian cancer, let‐7b expression is epigenetically suppressed by high expression of KDM2B. The loss of let‐7b upregulates the expression of EZH2, which promotes ovarian cancer growth in vitro and in vivo.

Effect of murine double‐minute 2 inhibitors in preclinical models of advanced clear cell carcinomas originating from ovaries and kidneys

AbstractAdvanced clear cell carcinomas originating from both ovaries and kidneys with cancerous peritonitis have poor prognoses. Murine double‐minute 2 (MDM2) is a potential therapeutic target for clear cell ovarian carcinomas with WT TP53. Herein, we characterized the antiangiogenic and antitumor effects of the MDM2 inhibitors DS‐3032b and DS‐5272 in 6 clear cell ovarian carcinoma cell lines and 2 clear cell renal carcinoma cell lines, as well as in clear cell ovarian carcinomas s.c. xenograft and ID8 (murine ovarian cancer cells with WT TP53) cancer peritonitis mouse models. In clear cell ovarian carcinoma s.c. xenograft mouse models, DS‐3032b significantly reduced WT TP53 clear cell ovarian carcinoma‐ and clear cell renal carcinoma‐derived tumor volumes. In ID8 mouse models, DS‐5272 significantly inhibited ascites production, reduced body weight, and significantly improved overall survival. Additionally, DS‐5272 reduced the tumor burden of peritoneal dissemination and decreased CD31+ cells in a dose‐dependent manner. Furthermore, DS‐5272 significantly decreased vascular endothelial growth factor concentrations in both sera and ascites. Combined therapy with MDM2 inhibitors and everolimus showed synergistic, and dose‐reduction potential, for clear cell carcinoma treatment. Our findings suggest that MDM2 inhibitors represent promising molecular targeted therapy for clear cell carcinomas, thereby warranting further studies to evaluate the efficacy and safety of dual MDM2/mTOR inhibitors in clear cell carcinoma patients.

Novel monoclonal antibody against integrin α3 shows therapeutic potential for ovarian cancer

AbstractOvarian cancer has a high recurrence rate after platinum‐based chemotherapy. To improve the treatment of ovarian cancer and identify ovarian cancer‐specific antibodies, we immunized mice with the human ovarian carcinoma cell line, SKOV‐3, and generated hybridoma clones. Several rounds of screening yielded 30 monoclonal antibodies (mAbs) with no cross‐reactivity to normal cells. Among these mAbs, OV‐Ab 30‐7 was found to target integrin α3 and upregulate p53 and p21, while stimulating the apoptosis of cancer cells. We further found that binding of integrin α3 by OV‐Ab 30‐7 impaired laminin‐induced focal adhesion kinase phosphorylation. The mAb alone or in combination with carboplatin and paclitaxel inhibited tumor progression and prolonged survival of tumor‐bearing mice. Moreover, immunohistochemical staining of ovarian patient specimens revealed higher levels of integrin α3 in cancer cells compared with normal cells. By querying online clinical databases, we found that elevated ITGA3 expression in ovarian cancer is associated with poor prognosis. Taken together, our data suggest that the novel mAb, OV‐Ab 30‐7, may be considered as a potential therapeutic for ovarian cancer.

Clonal lineage from normal endometrium to ovarian clear cell carcinoma through ovarian endometriosis

AbstractClear cell carcinoma of the ovary is thought to arise from endometriosis. In addition, retrograde menstruation of shed endometrium is considered the origin of endometriosis. However, little evidence supports cellular continuity from uterine endometrium to clear cell carcinoma through endometriosis at the genomic level. Here, we performed multiregional whole‐exome sequencing to clarify clonal relationships among uterine endometrium, ovarian endometriosis and ovarian clear cell carcinoma in a 56‐year‐old patient. Many somatic mutations including cancer‐associated gene mutations in ARID1A, ATM, CDH4, NRAS and PIK3CA were shared among epithelium samples from uterine endometrium, endometriotic lesions distant from and adjacent to the carcinoma, and the carcinoma. The mutant allele frequencies of shared mutations increased from uterine endometrium to distant endometriosis, adjacent endometriosis, and carcinoma. Although a splice site mutation of ARID1A was shared among the four epithelium samples, a frameshift insertion in ARID1A was shared by adjacent endometriosis and carcinoma samples, suggesting that the biallelic mutations triggered malignant transformation. Somatic copy number alterations, including loss of heterozygosity events at PIK3CA and ATM, were identified only in adjacent endometriosis and carcinoma, suggesting that mutant allele‐specific imbalance is another key factor driving malignant transformation. By reconstructing a clonal evolution tree based on the somatic mutations, we showed that the epithelium samples were derived from a single ancestral clone. Although the study was limited to a single patient, the results from this illustrative case could suggest the possibility that epithelial cells of ovarian endometriosis and clear cell carcinoma were descendants of uterine endometrial epithelium.

Association of gBRCA1/2 mutation locations with ovarian cancer risk in Japanese patients from the CHARLOTTE study

AbstractWhether germline (g) breast cancer susceptibility gene (BRCA) mutations are located within or outside the ovarian cancer cluster region (OCCR) (1380‐4062 bp for gBRCA1, and between 3249‐5681 bp and 6645‐7471 bp for gBRCA2) may influence risk variations for ovarian cancers. This ad hoc analysis of the CHARLOTTE epidemiological study in Japan assessed the distribution of gBRCA1/2 mutations in patients with newly diagnosed ovarian cancer, and investigated an association between gBRCA1/2 mutation locations and ovarian cancer risk. Differences in patient background and clinical characteristics in subgroups stratified by gBRCA1/2 mutation locations were also evaluated. We analyzed the data of 93 patients (14.7%) from the CHARLOTTE study who were positive for gBRCA1/2 mutations. After excluding 16 cases with L63X founder mutation, 28 (65.1%) of gBRCA1 mutations were within the OCCR. Of 30 gBRCA2 mutations, 15 (50.0%) were within the OCCR. Of 27 patients (one patient excluded for unknown family history) with gBRCA1 mutations located in the OCCR, 11 (40.7%) had a family history of ovarian cancer; the proportion of patients with a family history of ovarian cancer and gBRCA1 mutations outside the OCCR was lower (13.3%). Sixty percent of patients with gBRCA1 mutations outside the OCCR had a family history of breast cancer; the proportion of patients with a family history of breast cancer and gBRCA1 mutations within the OCCR was relatively lower (33.3%). Understanding the mutation locations may contribute to more accurate risk assessments of susceptible individuals and early detection of ovarian cancer among gBRCA mutation carriers.

Nitric oxide affects cisplatin cytotoxicity oppositely in A2780 and A2780‐CDDP cells via the connexin32/gap junction

AbstractChemoresistance is a main obstacle in ovarian cancer therapy and new treatment strategies and further information regarding the mechanism of the medication cisplatin are urgently needed. Nitric oxide has a critical role in modulating the activity of chemotherapeutic drugs. Our previous work showed that connexin32 contributed to cisplatin resistance. However, whether nitric oxide is involved in connexin32‐mediated cisplatin resistance remains unknown. In this study, using A2780 and A2780 cisplatin‐resistant cells, we found that S‐nitroso‐N‐acetyl‐penicillamine, a nitric oxide donor, attenuated cisplatin toxicity by decreasing gap junctions in A2780 cells. Enhancement of gap junctions using retinoic acid reversed the effects of S‐nitroso‐N‐acetyl‐penicillamine on cisplatin toxicity. In A2780 cisplatin‐resistant cells, however, S‐nitroso‐N‐acetyl‐penicillamine enhanced cisplatin toxicity by decreasing connexin32 expression. Downregulation of connexin32 expression by small interfering RNA exacerbated the effects of S‐nitroso‐N‐acetyl‐penicillamine on cisplatin cytotoxicity and upregulation of connexin32 expression by pcDNA transfection reversed the effects of S‐nitroso‐N‐acetyl‐penicillamine on cisplatin cytotoxicity. Our study suggests for the first time that combining cisplatin with nitric oxide in clinical therapies for ovarian cancer should be avoided before cisplatin resistance emerges. The present study provides a productive area of further study for increasing the efficacy of cisplatin by combining cisplatin with the specific inhibitors or enhancers of nitric oxide in clinical treatment.

Folate‐appended cyclodextrin carrier targets ovarian cancer cells expressing the proton‐coupled folate transporter

AbstractFolate receptor alpha (FRα) is overexpressed in >80% of epithelial ovarian cancer (EOC). Accordingly, folate is attracting attention as a targeting ligand for EOC. For EOC patients, paclitaxel (PTX) is generally used as a first‐line chemotherapeutic agent in combination with platinum‐based drugs. Cyclodextrin (CyD) is a potential new formulation vehicle for PTX that could replace Cremophor‐EL, a traditional formulation vehicle that causes significant side effects, including neutropenia. Several years ago, folate‐appended β‐CyD (Fol‐c1‐β‐CyD) was developed as an FRα‐targeting drug carrier, but its efficacy as a treatment for EOC remains to be determined. In this study, we assessed the antitumor activity of PTX in Fol‐c1‐β‐CyD (PTX/Fol‐c1‐β‐CyD) in EOC‐derived cell lines. We found that PTX/Fol‐c1‐β‐CyD killed not only FRα‐expressing cells but also FRα‐negative cells. In the FRα‐negative A2780 cells, knockdown of proton‐coupled folate transporter (PCFT) significantly decreased the cytotoxicity of PTX/Fol‐c1‐β‐CyD, whereas knockdown of FRα did not. By contrast, knockdown of either FRα or proton‐coupled folate transporter (PCFT) decreased the cytotoxicity of PTX/Fol‐c1‐β‐CyD in FRα‐expressing SK‐OV‐3 cells. Furthermore, the cytotoxicity of PTX/Fol‐c1‐β‐CyD in A2780 cells was increased at acidic pH, and this increase was suppressed by PCFT inhibitor. In mice intraperitoneally inoculated with FRα‐expressing or PCFT‐expressing EOC cells, intraperitoneal administration of PTX/Fol‐c1‐β‐CyD significantly suppressed the growth of both types of EOC cells relative to PTX alone, without inducing a significant change in the neutrophil/white blood cell ratio. Our data suggest that Fol‐c1‐β‐CyD targets not only FRα but also PCFT, and can efficiently deliver anticancer drugs to EOC cells in the peritoneal cavity.

PDK2 leads to cisplatin resistance through suppression of mitochondrial function in ovarian clear cell carcinoma

AbstractOvarian clear cell carcinoma (CCC) exhibits an association with endometriosis, resistance to oxidative stress, and poor prognosis owing to its resistance to conventional platinum‐based chemotherapy. A greater understanding of the molecular characteristics and pathogenesis of ovarian cancer subtypes may facilitate the development of targeted therapeutic strategies, although the mechanism of drug resistance in ovarian CCC has yet to be determined. In this study, we assessed exome sequencing data to identify new therapeutic targets of mitochondrial function in ovarian CCC because of the central role of mitochondria in redox homeostasis. Copy number analyses revealed that chromosome 17q21‐24 (chr.17q21‐24) amplification was associated with recurrence in ovarian CCC. Cell viability assays identified an association between cisplatin resistance and chr.17q21‐24 amplification, and mitochondrion‐related genes were enriched in patients with chr.17q21‐24 amplification. Patients with high expression of pyruvate dehydrogenase kinase 2 (PDK2) had a worse prognosis than those with low PDK2 expression. Furthermore, inhibition of PDK2 synergistically enhanced cisplatin sensitivity by activating the electron transport chain and by increasing the production of mitochondrial reactive oxygen species. Mouse xenograft models showed that inhibition of PDK2 with cisplatin inhibited tumor growth. This evidence suggests that targeting mitochondrial metabolism and redox homeostasis is an attractive therapeutic strategy for improving drug sensitivity in ovarian CCC.

CD4/CD8 ratio is a prognostic factor in IgG nonresponders among peptide vaccine‐treated ovarian cancer patients

AbstractThe identification of useful biomarkers is an urgent issue in cancer treatment, particularly for immunotherapy, as only some patients experience benefits from this treatment. The early induction of the IgG response has been reported as a useful biomarker of favorable prognosis for cancer patients treated with a personalized peptide vaccination, but a portion of these patients (IgG nonresponders) fail to achieve an early induction of IgG response yet experience long‐term survival. It is thus necessary to identify other biomarkers of favorable prognosis among these patients. Here we report the usefulness of classical T‐cell markers (ie, the CD8 content and the CD4/CD8 ratio in peripheral blood) in IgG nonresponders among advanced or recurrent ovarian cancer patients treated with a personalized peptide vaccination. Among IgG nonresponders (n = 25), the overall survival (OS) of the increased‐CD8 group (n = 7) was significantly longer than that of the decreased‐CD8 group (n = 18; P = .018), and the OS of the patients with a decreased CD4/CD8 ratio (n = 10) was significantly longer than that of the patients with an increased ratio (n = 15; P = .0055). Thus, an increased content of CD8 and a decreased CD4/CD8 ratio are each favorable prognosis markers in IgG nonresponders treated with a personalized peptide vaccination.

CD70 antibody‐drug conjugate: A potential novel therapeutic agent for ovarian cancer

AbstractThis study aimed to investigate the cytotoxicity of a cluster of differentiation 70 antibody‐drug conjugate (CD70‐ADC) against ovarian cancer in in vitro and in vivo xenograft models. CD70 expression was assessed in clinical samples by immunohistochemical analysis. Western blotting and fluorescence‐activated cell sorting analyses were used to determine CD70 expression in the ovarian cancer cell lines A2780 and SKOV3, and in the cisplatin‐resistant ovarian cancer cell lines A2780cisR and SKOV3cisR. CD70 expression after cisplatin exposure was determined in A2780 cells transfected with mock‐ or nuclear factor (NF)‐κB‐p65‐small interfering RNA. We developed an ADC with an anti‐CD70 monoclonal antibody linked to monomethyl auristatin F and investigated its cytotoxic effect. We examined 63 ovarian cancer clinical samples; 43 (68.3%) of them expressed CD70. Among patients with advanced stage disease (n = 50), those who received neoadjuvant chemotherapy were more likely to exhibit high CD70 expression compared to those who did not (55.6% [15/27] vs 17.4% [4/23], P < .01). CD70 expression was confirmed in A2780cisR, SKOV3, and SKOV3cisR cells. Notably, CD70 expression was induced after cisplatin treatment in A2780 mock cells but not in A2780‐NF‐κB‐p65‐silenced cells. CD70‐ADC was cytotoxic to A2780cisR, SKOV3, and SKOV3cisR cells, with IC50 values ranging from 0.104 to 0.341 nmol/L. In A2780cisR and SKOV3cisR xenograft models, tumor growth in CD70‐ADC treated mice was significantly inhibited compared to that in the control‐ADC treated mice (A2780cisR: 32.0 vs 1639.0 mm3, P < .01; SKOV3cisR: 232.2 vs 584.9 mm3, P < .01). Platinum treatment induced CD70 expression in ovarian cancer cells. CD70‐ADC may have potential therapeutic implications in the treatment of CD70 expressing ovarian cancer.

CircMAN1A2_009 facilitates YBX1 nuclear localization to induce GLO1 activation for cervical adenocarcinoma cell growth

AbstractThe molecular mechanisms driving the development of cervical adenocarcinoma (CADC) and optimal patient management strategies remain elusive. In this study, we have identified circMAN1A2_009 as an oncogenic circular RNA (circRNA) in CADC. Clinically, circMAN1A2_009 showed significant upregulation in CADC tissues, with an impressive area under the curve value of 0.8075 for detecting CADC. Functional studies, involving both gain‐of‐function and loss‐of‐function experiments, revealed that circMAN1A2_009 suppressed reactive oxygen species accumulation and apoptosis, and boosted cell viability in CADC cells. Conversely, silencing circMAN1A2_009 reversed these effects. Further mechanistic investigations indicated that circMAN1A2_009 interacted with YBX1, facilitating the phosphorylation levels of YBX1 at serine 102 (p‐YBX1S102) and facilitating YBX1 nuclear localization through sequence 245–251. This interaction subsequently increased the activity of the glyoxalase 1 (GLO1) promoter, leading to the activation of GLO1 expression. Consistently, inhibition of either YBX1 or GLO1 mirrored the biological effects of circMAN1A2_009 in CADC cells. Additionally, knockdown of YBX1 or GLO1 partially reversed the oncogenic behaviors induced by circMAN1A2_009. In conclusion, our findings propose circMAN1A2_009 as a potential oncogene and a promising indicator for diagnosing and guiding therapy in CADC patients.

Prevalence and outcomes of germline pathogenic variants of homologous recombination repair genes in ovarian cancer

AbstractGermline pathogenic variants (PVs) are pivotal in gynecological oncology. We focused on the prevalence, clinicopathological features, and survival impact of homologous recombination repair (HRR) PVs in patients with epithelial ovarian cancer (EOC). This was a multicenter retrospective cohort study, and 1248 patients with EOC were registered. Eligible patients (n = 1112) underwent germline DNA analysis for 26 cancer predisposition genes, including nine HRR‐related genes, such as BRCA1/2, BRIP1, PALB2, RAD51C/D, and ATM. The associations between clinicopathological factors and HRR‐related PVs were examined. Kaplan–Meier and Cox regression analyses were conducted. Among 1091 analyzed patients, 153 (14.0%) carried PVs and 140 (12.8%) were HRR‐related. HRR‐PV‐positive status significantly correlated with serous carcinoma (22.9% vs. 4.8%, P < 0.0001) and advanced disease (18.5% vs. 5.9%, P < 0.0001). The HRR‐PV‐positive group exhibited higher prevalence of personal breast (12.9%) and familial breast/ovarian (29.2%) cancer history. HRR status independently improved overall survival in stage III/IV disease (P = 0.04) but not progression‐free survival. HRR‐related germline PVs exhibit distinct clinicopathological features with survival implications. Variants were significantly associated with serous carcinoma and advanced disease, underscoring the importance of genetic testing to develop individualized EOC treatment strategies. Considering the study period (2000–2019), the limited use of bevacizumab and poly (ADP‐ribose) polymerase inhibitors as maintenance therapy should be recognized.

Clinical features and impact of p53 status on sporadic mismatch repair deficiency and Lynch syndrome in uterine cancer

AbstractThe clinical features of sporadic mismatch repair deficiency (MMRd) and Lynch syndrome (LS) in Japanese patients with endometrial cancer (EC) were examined by evaluating the prevalence and prognostic factors of LS and sporadic MMRd in patients with EC. Targeted sequencing of five LS susceptibility genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) was carried out in 443 patients with EC who were pathologically diagnosed with EC at the National Cancer Center Hospital between 2011 and 2018. Pathogenic variants in these genes were detected in 16 patients (3.7%). Immunohistochemistry for MMR proteins was undertaken in 337 of the 433 (77.9%) EC patients, and 91 patients (27.0%) showed absent expression of at least one MMR protein. The 13 cases of LS with MMR protein loss (93.8%) showed a favorable prognosis with a 5‐year overall survival (OS) rate of 100%, although there was no statistically significant difference between this group and the sporadic MMRd group (p = 0.27). In the MMRd without LS group, the 5‐year OS rate was significantly worse in seven patients with an aberrant p53 expression pattern than in those with p53 WT (53.6% vs. 93.9%, log‐rank test; p = 0.0016). These results suggest that p53 abnormalities and pathogenic germline variants in MMR genes could be potential biomarkers for the molecular classification of EC with MMRd.

HSP90 promotes radioresistance of cervical cancer cells via reducing FBXO6‐mediated CD147 polyubiquitination

AbstractHSP90 inhibition might be a promising strategy to overcome the radioresistance of some cancers. In the current study, we further explored the mechanisms of HSP90 in regulating the radiosensitivity of cervical cancer cells. Bioinformatic analysis was performed based on data from TCGA‐CESC. Cellular and molecular studies were conducted using CaSki and SiHa and the derived radioresistant (RR) subclones. Through a proteomics screen, we identified HSP90 chaperones (both HSP90α and HSP90β) as CD147‐binding partners supporting its stabilization. Targeting HSP90 sensitized CaSki‐RR and SiHa‐RR cancer cells to irradiation partially through CD147 destabilization. Mechanistically, HSP90 interacts with FBXO6 and reduces FBXO6‐mediated proteasomal degradation of CD147. Enforced FBXO6 overexpression also sensitized CaSki‐RR and SiHa‐RR cancer cells to irradiation. These effects were enhanced using 17‐AAG treatment but were weakened by CD147 overexpression. Survival analysis further confirmed the association between high FBXO6 expression and favorable progression‐free survival among patients with cervical cancer. In conclusion, this study showed that HSP90 promotes radioresistance of cervical cancer cells partially via reducing FBXO6 mediated CD147 polyubiquitination. These findings help to explain why HSP90 inhibitor exerts radio‐sensitizing effects in cervical cancer.

Effectiveness of catch‐up and routine program of the 9‐valent vaccine on cervical cancer risk reduction in Japan

AbstractIn 2013, the national human papillomavirus (HPV) immunization program began. However, in June 2013, Japan's Ministry of Health, Labor and Welfare (MHLW) announced a “temporary” suspension of its recommendation for the human papillomavirus vaccine. Finally, in November 2021, the MHLW ended its suspension of the recommendation of the HPV vaccine. To address the 9‐year gap in HPV vaccinations the suspension had caused, the MHLW conducted a program of catch‐up vaccinations from April 2022 to March 2025. Finally, in April 2023, the 9‐valent HPV vaccine was approved for both the routine and catch‐up vaccination programs in Japan. In this study, we investigated the potential effects of the introduction of the 9‐valent vaccine on the increased risk of cervical cancer in females born after fiscal year (FY) 2000. We estimated the lifetime relative risk of cervical cancer incidence and death using the improved routine and catch‐up vaccination rates after the recent resumption of the governmental recommendation for women and girls to have the HPV vaccination. These relative risks were calculated using a lifetime risk of 1.000 for cervical cancer incidence and death for females born in FY 1993. We predicted that even if a 90% vaccination rate were to be achieved by FY 2024 with the 9‐valent vaccine among women born between FY 2000 and FY 2005, the risk would remain higher than for the vaccination generation. Therefore, for women born between FY 2000 and FY 2005, it will be necessary to significantly improve the cervical cancer screening rate to compensate for this increased risk.

miRNA levels are associated with body mass index in endometrial cancer and may have implications for therapy

AbstractEndometrial cancer (EC) is the most prevalent gynecological cancer in high‐income countries. Its incidence is skyrocketing due to the increase in risk factors such as obesity, which represents a true pandemic. This study aimed to evaluate microRNA (miRNA) expression in obesity‐related EC to identify potential associations between this specific cancer type and obesity. miRNA levels were analyzed in 84 EC patients stratified based on body mass index (BMI; ≥30 or <30) and nine noncancer women with obesity. The data were further tested in The Cancer Genome Atlas (TCGA) cohort, including 384 EC patients, 235 with BMI ≥30 and 149 with BMI <30. Prediction of miRNA targets and analysis of their expression were also performed to identify the potential epigenetic networks involved in obesity modulation. In the EC cohort, BMI ≥30 was significantly associated with 11 deregulated miRNAs. The topmost deregulated miRNAs were first analyzed in 84 EC samples by single miRNA assay and then tested in the TCGA dataset. This independent validation provided further confirmation about the significant difference of three miRNAs (miR‐199a‐5p, miR‐449a, miR‐449b‐5p) in normal‐weight EC patients versus EC patients with obesity, resulting significantly higher expressed in the latter. Moreover, the three miRNAs were significantly correlated with grade, histological type, and overall survival. Analysis of their target genes revealed that these miRNAs may regulate obesity‐related pathways. In conclusion, we identified specific miRNAs associated with BMI that are potentially involved in modulating obesity‐related pathways and that may provide novel implications for the clinical management of obese EC patients.

Prevalence of hereditary breast and ovarian cancer (HBOC) predisposition gene mutations among 882 HBOC high‐risk Chinese individuals

AbstractIdentification of deleterious variants in hereditary breast and ovarian cancer (HBOC) susceptibility genes allows for increased clinical surveillance and early detection, and could predict the response to poly (ADP‐ribose) polymerase (PARP) inhibitor in patients with advanced ovarian carcinomas. To determine the prevalence and clinical prediction factors for HBOC syndrome, 882 selected individuals underwent multigene panel testing for HBOC risk assessment during the period from January 2015 to March 2018. Overall, 176 deleterious mutations were observed in 19.50% (n = 172) of individuals. Twenty‐six of 176 mutations could not be retrieved in related public databases and were considered to be novel. Among patients with ovarian cancer, 115 deleterious mutations were identified in 429 patients (48.6%) with significant enrichment for a family history of breast or ovarian cancer syndrome (P < .05). In the breast cancer subgroup, 31 deleterious mutations were identified in 261 patients. Besides BRCA1 (8; 25.8%) and BRCA2 (11; 35.5%), the most frequently occurring genes, an additional 12 deleterious mutations (38.7%) were found in seven other susceptibility genes. Higher mutation incidence (57.9%) was observed in subjects with histories of breast and ovarian cancer. Our results highlighted the genetic heterogeneity of HBOC and the efficiency of a multigene panel in carrying out risk assessment.

WEE1 inhibitor and ataxia telangiectasia and RAD3‐related inhibitor trigger stimulator of interferon gene‐dependent immune response and enhance tumor treatment efficacy through programmed death‐ligand 1 blockade

AbstractWEE1 plays an important role in the regulation of cell cycle G2/M checkpoints and DNA damage response (DDR). Inhibition of WEE1 can increase the instability of the genome and have anti–tumor effects in some solid tumors. However, it has certain limitations for multiple cancer cells from different lineages. Therefore, we consider the use of synthetic lethal interactions to enhance the therapeutic effect. Our experiments proved that WEE1 inhibitor (WEE1i) can activate the ataxia telangiectasia and RAD3‐related (ATR) pathway and that blockage of ATR dramatically sensitized the WEE1i‐induced cell death. The tumor‐selective synthetic lethality between bioavailable WEE1 and ATR inhibitors led to tumor remission in vivo. Mechanistically, the combination promoted the accumulation of cytosolic double‐strand DNA, which subsequently activated the stimulator of the interferon gene (STING) pathway and induced the production of type I interferon and CD8+ T cells, thereby inducing anti–tumor immunity. Furthermore, our study found that immune checkpoint programmed death‐ligand 1 is upregulated by the combination therapy, and blocking PD‐L1 further enhances the effect of the combination therapy. In summary, as an immunomodulator, the combination of WEE1i with ATR inhibitor (ATRi) and immune checkpoint blockers provides a potential new approach for cancer treatment.

Loss of Krüppel‐like factor 9 facilitates stemness in ovarian cancer ascites‐derived multicellular spheroids via Notch1/slug signaling

AbstractThe ascites that develops in advanced OC, both at diagnosis and upon recurrence, is a rich source of multicellular spheroids/aggregates (MCSs/MCAs), which are the major seeds of tumor cell dissemination within the abdominal cavity. However, the molecular mechanism by which specific ascites‐derived tumor cells survive and metastasize remains largely unknown. In this study, we elucidated cancer stem cell (CSC) properties of ascites‐derived MCSs, concomitant with enhanced malignancy, induced EMT, and low KLF9 (Krüppel‐like factor 9) expression, compared with PTCs. KLF9 was also downregulated in OC cell line‐derived spheroids and the CD117+CD44+ subpopulation in MCSs. Functional experiments demonstrated that KLF9 negatively modulated stem‐like properties in OC cells. Mechanistic studies revealed that KLF9 reduced the transcriptional expression of Notch1 by directly binding to the Notch1 promoter, thereby inhibiting the function of slug in a CSL‐dependent manner. Clinically, expression of KLF9 was associated with histological grade and loss of KLF9 predicts poor prognosis in OC.

Novel LAMC2 fusion protein has tumor‐promoting properties in ovarian carcinoma

AbstractLaminins are heterotrimeric ECM proteins composed of α, β, and γ chains. The γ2 chain (Lm‐γ2) is a frequently expressed monomer and its expression is closely associated with cancer progression. Laminin‐γ2 contains an epidermal growth factor (EGF)‐like domain in its domain III (DIII or LEb). Matrix metalloproteinases can cleave off the DIII region of Lm‐γ2 that retains the ligand activity for EGF receptor (EGFR). Herein, we show that a novel short form of Lm‐γ2 (Lm‐γ2F) containing DIII is generated without requiring MMPs and chromosomal translocation between LAMC2 on chromosome 1 and NR6A1 gene locus on chromosome 9 in human ovarian cancer SKOV3 cells. Laminin‐γ2F is expressed as a truncated form lacking domains I and II, which are essential for its association with Lm‐α3 and ‐β3 chains of Lm‐332. Secreted Lm‐γ2F can act as an EGFR ligand activating the EGFR/AKT pathways more effectively than does the Lm‐γ2 chain, which in turn promotes proliferation, survival, and motility of ovarian cancer cells. LAMC2‐NR6A1 translocation was detected using in situ hybridization, and fusion transcripts were expressed in ovarian cancer cell tissues. Overexpression and suppression of fusion transcripts significantly increased and decreased the tumorigenic growth of cells in mouse models, respectively.To the best of our knowledge, this is the first report regarding a fusion gene of ECM showing that translocation of LAMC2 plays a crucial role in the malignant growth and progression of ovarian cancer cells and that the consequent product is a promising therapeutic target against ovarian cancers.

Prevalence and clinical characterization of BRCA1 and BRCA2 mutations in Korean patients with epithelial ovarian cancer

AbstractThis study was performed to investigate the prevalence, clinical characteristics, and treatment response according to BRCA1 and BRCA2 (BRCA) mutations in Korean patients with epithelial ovarian cancer (EOC). Two‐hundred and ninety‐eight Korean women diagnosed with high‐grade serous and/or endometrioid EOC from 2010 to 2015 were tested for germline and 86 specimens for somatic BRCA mutations, regardless of the family history. Clinical characteristics including survival outcomes were compared in patients with and without BRCA mutations (NCT02963688). A total of 43 different germline BRCA mutations were identified in 78 patients among 298 patients (26.2%). Somatic BRCA mutations were identified in 11 (12.8%) patients among patients without germline BRCA mutations. Haplotype analysis demonstrated no founder mutations in our Korean patient cohort. Insignificant differences in age at diagnosis, primary site, and residual disease after surgery were observed between patients with and without BRCA mutations. In multivariate analysis for overall survival (OS), the presence of BRCA mutation was significantly associated with OS (P = .049) in addition to platinum sensitivity (P < .001), indicating it is an independent prognostic factor for survival regardless of platinum sensitivity to first‐line chemotherapy. In addition, a higher response rate to subsequent chemotherapy after recurrence was observed in EOC patients with BRCA mutations resulting in better OS. In the current study, the prevalence of BRCA mutations in Korean patients with EOC was higher than previously reported in other ethnic groups. We demonstrated characteristics and treatment response in Korean EOC patients with BRCA mutations. These findings may provide valuable information to be considered in future clinical trials including Asian patients.

Clonal evidence for the development of neuroblastoma with extensive copy‐neutral loss of heterozygosity arising in a mature teratoma

AbstractMature teratomas are usually benign tumors that rarely undergo malignant transformation. We report an advanced neuroblastoma arising in a mature teratoma of the ovary. Whole‐exome sequencing identified extensive copy‐neutral loss of heterozygosity (LOH) in both neuroblastoma and teratoma elements, suggesting that the neuroblastoma evolved from the teratoma. In addition, several truncating germline heterozygous variants in tumor suppressor genes, including RBL2 and FBXW12, became homozygous as a result of LOH. Collectively, we speculate that extensive LOH in teratoma cells may force heterozygous germline variants to become homozygous, which, in turn, may contribute to the development of neuroblastoma with the acquisition of additional chromosomal changes.

VPS33B interacts with NESG1 to suppress cell growth and cisplatin chemoresistance in ovarian cancer

Abstract The pathogenesis and cisplatin chemoresistance of ovarian cancer (OC) are still unclear. Vacuolar protein sorting‐associated 33B (VPS33B) has not been reported in OC to date. In this study, immunohistochemistry was used to detect VPS33B protein expression between OC and ovarian tissues. MTT, EdU, colony formation, cell cycle, in vivo tumorigenesis, western blot, ChIP, EMSA, co‐immunoprecipitation (CoIP), qRT‐PCR, and microconfocal microscopy were used to explore the function and molecular mechanisms of VPS33B in OC cells. The results of the present study demonstrated that VPS33B protein expression was obviously reduced in OC compared with that in ovarian tissues. Overexpressed VPS33B suppressed cell cycle transition, cell growth, and chemoresistance to cisplatin in vitro and in vivo. Analysis of the mechanism indicated that overexpressed VPS33B regulated the epidermal growth factor receptor (EGFR)/PI3K/AKT/c‐Myc/p53/miR‐133a‐3p feedback loop and reduced the expression of the cell cycle factor CDK4. Nasopharyngeal epithelium‐specific protein 1 (NESG1) as a tumor suppressor not only interacted with VPS33B, but was also induced by VPS33B by the attenuation of PI3K/AKT/c‐Jun‐mediated transcription inhibition. Overexpressed NESG1 further suppressed cell growth by mediating VPS33B‐modulated signals in VPS33B‐overexpressing OC cells. Finally, NESG1 induced VPS33B expression by reducing the inhibition of PI3K/AKT/c‐Jun‐mediated transcription. Our study is the first to demonstrate that VPS33B serves as a tumor suppressor, and VPS33B can interact with NESG1 to suppress cell growth and promote cisplatin sensitivity by regulating the EGFR/PI3K/AKT/c‐Myc/p53/miR‐133a‐3p feedback loop in OC cells.

Biological significance of KRAS mutant allele expression in ovarian endometriosis

AbstractKRAS is the most frequently mutated in ovarian endometriosis. However, it is unclear whether the KRAS mutant allele's mRNA is expressed and plays a biological role in ovarian endometriosis. Here, we performed mutation‐specific RNA in situ hybridization to evaluate mutant allele expression of KRAS p.G12V, the most frequently detected mutation in ovarian endometriosis in our previous study, in formalin‐fixed paraffin‐embedded tissue (FFPE) samples of ovarian endometriosis, cancer cell lines, and ovarian cancers. First, we verified that mutant or wild‐type allele of KRAS were expressed in all 5 cancer cell lines and 9 ovarian cancer cases corresponding to the mutation status. Next, we applied this assay to 26 ovarian endometriosis cases, and observed mutant allele expression of KRAS p.G12V in 10 cases. Mutant or wild‐type allele of KRAS were expressed in line with mutation status in 12 available endometriosis cases for which KRAS gene sequence was determined. Comparison of clinical features between ovarian endometriosis with KRAS p.G12V mutant allele expression and with KRAS wild‐type showed that KRAS p.G12V mutant allele expression was significantly associated with inflammation in ovarian endometriosis. Finally, we assessed the spatial distribution of KRAS mutant allele expression in 5 endometriosis cases by performing multiregional sampling. Intratumor heterogeneity of KRAS mutant allele expression was observed in two endometriosis cases, whereas the spatial distribution of KRAS p.G12V mutation signals were diffuse and homogenous in ovarian cancer. In conclusion, evaluation of oncogene mutant expression will be useful for clarifying the biological significance of oncogene mutations in benign tumors.

Cystathionine‐gamma‐lyase overexpression in T cells enhances antitumor effect independently of cysteine autonomy

AbstractT cells could be engineered to overcome the aberrant metabolic milieu of solid tumors and tip the balance in favor of a long‐lasting clinical response. Here, we explored the therapeutic potential of stably overexpressing cystathionine‐gamma‐lyase (CTH, CSE, or cystathionase), a pivotal enzyme of the transsulfuration pathway, in antitumor CD8+ T cells with the initial aim to boost intrinsic cysteine metabolism. Using a mouse model of adoptive cell transfer (ACT), we found that CTH‐expressing T cells showed a superior control of tumor growth compared to control T cells. However, contrary to our hypothesis, this effect was not associated with increased T cell expansion in vivo or proliferation rescue in the absence of cysteine/cystine in vitro. Rather than impacting methionine or cysteine, ACT with CTH overexpression unexpectedly reduced glycine, serine, and proline concentration within the tumor interstitial fluid. Interestingly, in vitro tumor cell growth was mostly impacted by the combination of serine/proline or serine/glycine deprivation. These results suggest that metabolic gene engineering of T cells could be further investigated to locally modulate amino acid availability within the tumor environment while avoiding systemic toxicity.

Prognostic Biomarker of Fertility‐Preserving Hormonal Therapy Based on Multigene Panel Testing for Endometrial Cancer

ABSTRACT In this study, we identified prognostic biomarkers that predict treatment outcome in patients receiving fertility‐preserving high‐dose medroxyprogesterone acetate (MPA) therapy through comprehensive multigene panel testing. A total of 38 patients (20 atypical endometrial hyperplasia and 18 stage IA G1 without myometrial invasion) who received first‐line MPA therapy were enrolled. Genomic DNA was extracted from formalin‐fixed paraffin‐embedded samples, and PleSSision‐Rapid multigene panel testing was performed. Of the 38 patients, 31 (82%) achieved complete response (CR), 2 (5%) had stable disease (SD), and 5 (13%) had progressive disease (PD) following initial treatment. The median duration to achieve tumor disappearance was 7 months (range: 4–14 months). Following initial treatment, 25 of 32 patients (78%) experienced recurrence, with a median recurrence‐free survival (RFS) of 21 months (range: 2–84 months). The most frequently observed actionable gene mutations were PTEN (68.4%), CTNNB1 (55.2%), and PIK3CA (33.3%). Patients harboring PTEN mutations in EMG1 required a significantly longer duration to achieve tumor disappearance ( p  = 0.011). In addition, the presence of PIK3CA mutations in AEH was significantly associated with shorter RFS ( p  = 0.048). Molecular classification identified 34 patients (89%) with no specific molecular profile (NSMP), 1 patient (3%) with POLE mutation, and 3 patients (8%) with deficient mismatch repair (d‐MMR). Most patients undergoing MPA therapy were classified as having NSMP. Genetic alterations, specifically mutations in PTEN and PIK3CA , were significantly associated with treatment outcomes, highlighting their potential as prognostic biomarkers.

Diallyl trisulfide alleviates chemotherapy sensitivity of ovarian cancer via the AMPK/SIRT1/PGC1α pathway

AbstractPlatinum‐based chemotherapy promotes drug resistance in ovarian cancer. We investigated the antichemoresistance characteristics of diallyl trisulfide (DATS) in cisplatin‐resistant ovarian cancer cells, in vitro and in vivo. Previous preclinical studies have revealed that DATS regulates distinct hallmark cancer‐signaling pathways. The cell cycle pathway is the most investigated signaling pathway in DATS. Additionally, post‐DATS treatment has been found to promote proapoptotic capacity through the regulation of intrinsic and extrinsic apoptotic pathway components. In the present study, we found that treating cisplatin‐sensitive and cisplatin‐resistant ovarian cell lines with DATS inhibited their proliferation and reduced their IC50. It induced cell apoptosis and promoted oxidative phosphorylation through the regulation of the AMPK/SIRT1/PGC1α pathway, OXPHOS, and enhanced chemotherapy sensitivity. DATS treatment alleviated glutamine consumption in cisplatin‐resistant cells. Our findings highlight the role of DATS in overcoming drug resistance in ovarian cancer in vitro and in vivo. In addition, we elucidated the role of the AMPK/SIRT1/PGC1α signaling pathway as a potential target for the treatment of drug‐resistant ovarian cancer.

Japanese nationwide observational multicenter study of tumor BRCA1/2 variant testing in advanced ovarian cancer

AbstractThe association between germline BRCA1 and BRCA2 pathogenic variants (mutations: gBRCAm) and ovarian cancer risk is well established. Germline testing alone cannot detect somatic BRCA1/2 pathogenic variants (sBRCAm), which is calculated based on the proportion of tumor BRCAm (tBRCAm) from tumor samples and gBRCAm. Homologous recombination deficiency (HRD) results mainly from genetic/epigenetic alterations in homologous recombination repair‐related genes and can be evaluated by genomic instability status. In Japan, the prevalence of tBRCAm, sBRCAm, and HRD remains unclear. This multicenter, cross‐sectional, observational study, CHaRacterIzing the croSs‐secTional approach to invEstigate the prevaLence of tissue BRCA1/2 mutations in newLy diagnosEd advanced ovarian cancer patients (CHRISTELLE), evaluated the prevalence of tBRCAm, sBRCAm, and HRD in tumor specimens from newly diagnosed patients with ovarian cancer who underwent gBRCA testing. Of the 205 patients analyzed, 26.8% had a tBRCAm, including tBRCA1m (17.6%) and tBRCA2m (9.3%). The overall prevalence of tBRCAm, gBRCAm, sBRCAm, and HRD‐positive status was 26.8%, 21.5%, 6.3%, and 60.0%, respectively. The calculated sBRCAm/tBRCAm ratio was 23.6% (13/55), and the prevalence of gBRCA variant of uncertain significance was 3.9%. These results suggest gBRCA testing alone cannot clearly identify the best course of treatment, highlighting the importance of sBRCA testing in Japan. The present results also suggest that testing for tBRCA and HRD should be encouraged in advanced ovarian cancer patients to drive precision medicine.

Expression of long noncoding RNAs in cancer‐associated fibroblasts linked to patient survival in ovarian cancer

AbstractCancer‐associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment and are responsible for producing the desmoplastic reaction that is a poor prognostic factor in ovarian cancer. Long non‐coding RNAs (lncRNAs) have been shown to play important roles in cancer. However, very little is known about the role of lncRNAs in the tumor microenvironment. We aimed to identify lncRNAs expressed in ovarian CAFs that were associated with patient survival and used computational approaches to predict their function. Increased expression of 9 lncRNAs and decreased expression of 1 lncRNA in ovarian CAFs were found to be associated with poorer overall survival. A “guilt‐by‐association” approach was used to predict the function of these lncRNAs. In particular, MIR155HG was predicted to play a role in immune response. Further investigation revealed high MIR155HG expression to be associated with higher infiltrates of immune cell subsets. In conclusion, these data indicate expression on several lncRNAs in CAFs are associated with patient survival and are likely to play an important role in regulating CAF function.

Metformin inhibits the proliferation and invasion of ovarian cancer cells by suppressing tripartite motif‐containing 37‐induced tumor necrosis factor receptor‐associated factor 2 ubiquitination

AbstractOvarian cancer is the leading cause of death in gynecological malignancies worldwide. Our previous studies have proved that metformin inhibited the proliferation and invasion of ovarian cancer in vitro and in vivo. However, the underlying mechanisms have not been fully elucidated. Immunohistochemistry was carried out to detect the expression of tripartite motif‐containing 37 (TRIM37), Ki‐67, and MMP‐9 in ovarian cancer and normal tissues. The influence of TRIM37 on the proliferation and invasion of ovarian cancer cells was verified by the real‐time cellular analysis proliferation test, colony formation test, and Transwell assay. Western blot analysis and immunoprecipitation were used to detect the expression of the nuclear factor‐κB (NF‐κB) pathway and the interaction between TRIM37 and tumor necrosis factor receptor‐associated factor 2 (TRAF2). Ubiquitination detection was carried out to detect the ubiquitination level of TRAF2. The present study revealed that TRIM37 expression was significantly increased in ovarian cancer tissues compared with normal control tissues, and its overexpression was closely associated with proliferation and metastasis. Metformin inhibited the NF‐κB signaling pathway by downregulating TRIM37. Metformin also inhibited the ubiquitination of TRAF2 induced by TRIM37 overexpression. Metformin inhibits the proliferation and invasion of ovarian cancer cells by suppressing TRIM37‐induced TRAF2 ubiquitination.

H1.0 induces paclitaxel‐resistance genes expression in ovarian cancer cells by recruiting GCN5 and androgen receptor

AbstractMore than 90% of ovarian cancer deaths are due to relapse following development of chemoresistance. Our main objective is to better understand the molecular mechanism underlying paclitaxel resistance (taxol resistance, Txr) in ovarian cancer. Here, we observed that the linker histone H1.0 is upregulated in paclitaxel‐resistant ovarian cancer cells. Knockdown of H1.0 significantly downregulates the androgen receptor (AR) and sensitizes paclitaxel‐resistant SKOV3/Txr and 2774/Txr cell lines to paclitaxel. Conversely, ectopic expression of H1.0 upregulates AR and increases Txr in parental SKOV3 and MDAH2774 cells. Notably, H1.0 upregulation is associated with disease recurrence and poor survival in a subset of ovarian cancer subjects. Inhibition of PI3K significantly reduces H1.0 mRNA and protein levels in paclitaxel‐resistant cells, suggesting the involvement of the PI3K/AKT signaling pathway. Knockdown of H1.0 and AR also downregulates the Txr genes ABCB1 and ABCG2 in paclitaxel‐resistant cells. Our data show that H1.0 induces GCN5 expression and histone acetylation, thereby enhancing Txr gene transactivation. These findings suggest that Txr in ovarian cancer involves the PI3K/AKT pathway and leads to upregulation of histone H1.0, recruitment of GCN5 and AR, followed by upregulation of a subgroup of Txr genes that include ABCB1 and ABCG2. This study is the first report describing the relationship between histone H1.0 and GCN5 that cooperate to induce AR‐dependent Txr in ovarian cancer cells.

Mutation profiles in circulating cell‐free DNA predict acquired resistance to olaparib in high‐grade serous ovarian carcinoma

AbstractAlthough resistance to poly(ADP‐ribose) polymerase inhibitors (PARPi) has gradually become a major challenge in the maintenance therapy for high‐grade serous ovarian carcinoma (HGSOC), there are no universal indicators for resistance monitoring in patients. A key resistance mechanism to PARPi is the restoration of homologous recombination repair (HRR), including BRCA reversion mutations and changes in DNA damage repair proteins. To explore mutation profiles associated with PARPi resistance, we undertook targeted 42‐gene deep sequencing of circulating cell‐free DNA (cfDNA) extracted from HGSOC patients pre‐ and post‐treatment with olaparib maintenance therapy. We found that pathogenic germline mutations in the HRR pathway, including BRCA1/2, were strongly associated with improved clinical outcomes, and newly acquired MRE11A mutations significantly shortened the progression‐free survival (PFS) of patients. Furthermore, dynamic fluctuations of somatic mutation sites in CHEK2:p.K373E and CHEK2:p.R406H can be used for evaluating the therapeutic efficacy of patients. MRE11A:p.K464R might be a vital driving factor of olaparib resistance, as patients with newly acquired MRE11A:p.K464R in post‐treatment cfDNA had significantly shorter PFS than those without it. These findings provide potential noninvasive biomarkers for efficacy evaluation and resistance monitoring of olaparib treatment, and lay the foundation for developing combination treatment after olaparib resistance.

Comprehensive network analysis of dysregulated genes revealed MNX1‐AS1/hsa‐miR‐4697‐3p/HOXB13 axis in ovarian cancer chemotherapy response

AbstractPoor chemotherapy response is the main obstacle of ovarian cancer (OC) treatment. Platinum‐refractory and ‐resistant patients are associated with a worse outcome than platinum‐sensitive and partially sensitive patients, but the comprehensive similarities and differences among them are not yet clear. In this study, we analyzed the data of patients with different chemotherapy response in The Cancer Genome Atlas. We found a minority of altered genes were overlapped in refractory and resistant groups, as did the enriched pathways and Gene Ontology terms. We noticed that the neural signaling and drug metabolism enzymes were more significantly enriched and the protein–protein interaction supported these results. The transcription analysis highlighted PDX1 as the common and central transcription factor in both refractory and resistant groups. The competing endogenous RNA (ceRNA) network shared no common ceRNA pairs, indicating a major difference in noncoding RNA post‐transcriptional regulation. In the end, we validated the expression, regulation, binding, and effect on chemotherapy response for selected MNX1‐AS1/hsa‐miR‐4697‐3p/HOXB13 in OC cell lines. Our study offered a novel and comprehensive insight into chemotherapy response, and potential targets for improving chemotherapy response in OC.

PHOSPHATE exporter XPR1/SLC53A1 is required for the tumorigenicity of epithelial ovarian cancer

AbstractOvarian cancer is the fifth most common cause of cancer‐related death in women. Ovarian clear cell carcinoma (OCCC) is a chemotherapy‐resistant epithelial ovarian cancer with poor prognosis. As a basis for the development of therapeutic agents that could improve the prognosis of OCCC, we performed a screen for proteins critical for the tumorigenicity of OCCC using the CRISPR/Cas9 system. Here we show that knockdown of the phosphate exporter XPR1/SLC53A1 induces the growth arrest and apoptosis of OCCC cells in vitro. Moreover, we show that knockdown of XPR1/SLC53A1 inhibits the proliferation of OCCC cells xenografted into immunocompromised mice. These results suggest that XPR1/SLC53A1 plays a critical role in the tumorigenesis of OCCC cells. We speculate that XPR1/SLC53A1 might be a promising molecular target for the therapeutic treatment of OCCC.

Aberrant expression of junctional adhesion molecule‐A contributes to the malignancy of cervical adenocarcinoma by interaction with poliovirus receptor/CD155

AbstractRecent studies have shown that aberrant expression of tight junction proteins (TJP) contributes to malignant potential of various cancers. In the present study, we investigated the expression of junctional adhesion molecule‐A (JAM‐A), one of the transmembrane TJP, in uterine cervical adenocarcinoma and the significance of its expression for malignancy. Immunohistochemistry on human surgical specimens showed that JAM‐A was aberrantly expressed in neoplastic regions including adenocarcinoma in situ (AIS). Knockout of JAM‐A significantly suppressed cell proliferation and colony‐forming and migration abilities. We also showed that an antibody specific to an extracellular region of JAM‐A reduced cell proliferation ability and that loss of JAM‐A increased drug sensitivity of cervical adenocarcinoma cells. Based on a comprehensive proteome analysis, we found that poliovirus receptor (PVR/CD155) was regulated by JAM‐A and formed a physical interaction with JAM‐A. In human surgical specimens, PVR/CD155 expression was significantly correlated with some clinicopathological features and prognosis of cervical adenocarcinoma. Interestingly, most of the PVR/CD155‐positive cases expressed a high level of JAM‐A, and patients with the expression pattern of PVR/CD155 positive/JAM‐A high had significantly shorter periods of relapse‐free survival (P = .00964) and overall survival (P = .0204) than those for the other patients. Our observations suggest that aberrant expression of JAM‐A promotes malignancy of uterine cervical adenocarcinoma by regulation of PVR/CD155, and JAM‐A is therefore a potential therapeutic target for this malignancy.

Association of homozygous variants of STING1 with outcome in human cervical cancer

AbstractDNA‐sensing receptor Cyclic GMP–AMP Synthase (cGAS) and its downstream signaling effector STimulator of INterferon Genes (STING) have gained significant interest in the field of tumor immunology, as a dysfunctional cGAS‐STING pathway is associated with poor prognosis and worse response to immunotherapy. However, studies so far have not taken into account the polymorphic nature of the STING‐encoding STING1 gene. We hypothesized that the presence of allelic variance in STING1 would cause variation between individuals as to their susceptibility to cancer development, cancer progression, and potential response to (immuno)therapy. To start to address this, we defined the genetic landscapes of STING1 in cervical scrapings and investigated their corresponding clinical characteristics across a unique cohort of cervical cancer patients and compared them with independent control cohorts. Although we did not observe an enrichment of particular STING1 allelic variants in cervical cancer patients, we did find that the occurrence of homozygous variants HAQ/HAQ and R232H/R232H of STING1 were associated with both younger age of diagnosis and higher recurrence rate. These findings were accompanied by worse survival, despite comparable mRNA and protein levels of STING and numbers of infiltrated CD8+ T cells. Our findings suggest that patients with HAQ/HAQ and R232H/R232H genotypes may have a dysfunctional cGAS‐STING pathway that fails to promote efficient anticancer immunity. Interestingly, the occurrence of these genotypes coincided with homozygous presence of the V48V variant, which was found to be individually associated with worse outcome. Therefore, we propose V48V to be further evaluated as a novel prognostic marker for cervical cancer.

Human papillomavirus vaccine to prevent cervical intraepithelial neoplasia in Japan: A nationwide case‐control study

AbstractCervical cancer remains among the most common cancers in women worldwide and can be prevented by vaccination. The Ministry of Health, Labour and Welfare of Japan suspended active recommendation of regular human papillomavirus (HPV) vaccines in 2013 because of various symptoms including chronic pain and motor impairment. This nationwide case‐control study from April 2013 to March 2017 targeted women aged 20‐24 years old at cervical screening. We compared HPV vaccination exposure between those with abnormal and normal cytology. Abnormal cytology was classified based on the results of histological test and we calculated the odds ratio (OR) and 95% confidence interval (CI) of the above endpoints and vaccination exposure using the conditional logistic regression model and estimated vaccine effectiveness using the formula (1 – OR) × 100. A total of 2483 cases and 12 296 controls (one‐to‐five matching) were eligible in 31 municipalities in Japan. The distribution of histological abnormalities among cases was 797 CIN1 (including dysplasia) (32.1%), 165 CIN2 (6.7%), 44 CIN3 (1.8%), and eight squamous cell carcinoma (SCC) (0.3%). The OR of HPV vaccination compared with no vaccination for abnormal cytology, CIN1+, CIN2+, and CIN3+ versus controls was 0.42 (95% CI, 0.34‐0.50), 0.42 (95% CI, 0.31‐0.58), 0.25 (95% CI, 0.12‐0.54), and 0.19 (95% CI, 0.03‐1.15), respectively, equating to a vaccine effectiveness of 58.5%, 57.9%, 74.8%, and 80.9%, respectively. Eight patients had SCC, none was vaccinated. This nationwide case‐control study in Japan demonstrated a substantial risk reduction in abnormal cytology and CIN among women who did versus those who did not receive HPV vaccination.

lncRNA PART1 and MIR17HG as ΔNp63α direct targets regulate tumor progression of cervical squamous cell carcinoma

AbstractCervical cancer (CC) remains one of the leading causes of mortality of female cancers worldwide, with more than 90% being cervical squamous cell carcinoma (CSCC). ΔNp63α is the predominant isoform expressed in cervical epithelial tissues and exerts its antitumor function in CSCC. In this study, we have identified 39 long noncoding RNAs as ΔNp63α targets in CSCC through RNA sequencing and chromatin immunoprecipitation sequencing, in which we further confirmed and focused on the two tumor‐related long noncoding RNAs, PART1 (lncPART1) and MIR17HG (lncMIR17HG). Experiments from stable overexpression/knockdown cell lines revealed that lncPART1 and lncMIR17HG regulated cell proliferation, migration, and invasion. In vivo experiments further showed that lncPART1 suppresses tumor growth in CSCC‐derived tumors. Examinations of clinical tissues indicated that the expression of lncPART1 was positively correlated with ΔNp63α expression, while lncMIR17HG was negatively correlated with ΔNp63α expression, suggesting that ΔNp63α plays a central role via regulating its direct targets in the progression of CSCC. These findings provide novel insights in targeted therapy of cervical cancers.

Aldolase A promotes epithelial‐mesenchymal transition to increase malignant potentials of cervical adenocarcinoma

AbstractRecent studies have revealed that metabolic reprogramming is closely associated with epithelial‐mesenchymal transition (EMT) during cancer progression. Aldolase A (ALDOA) is a key glycolytic enzyme that is highly expressed in several types of cancer. In this study, we found that ALDOA is highly expressed in uterine cervical adenocarcinoma and that high ALDOA expression promotes EMT to increase malignant potentials, such as metastasis and invasiveness, in cervical adenocarcinoma cells. In human surgical specimens, ALDOA was highly expressed in cervical adenocarcinoma and high ALDOA expression was correlated with lymph node metastasis, lymphovascular infiltration, and short overall survival. Suppression of ALDOA expression significantly reduced cell growth, migration, and invasiveness of cervical cancer cells. Aldolase A expression was partially regulated by hypoxia‐inducible factor‐1α (HIF‐1α). Shotgun proteome analysis revealed that cell‐cell adhesion‐related proteins were significantly increased in ALDOA‐overexpressing cells. Interestingly, overexpression of ALDOA caused severe morphological changes, including a cuboidal‐to‐spindle shape shift and reduced microvilli formation, coincident with modulation of the expression of typical EMT‐related proteins. Overexpression of ALDOA increased migration and invasion in vitro. Furthermore, overexpression of ALDOA induced HIF‐1α, suggesting a positive feedback loop between ALDOA and HIF‐1α. In conclusion, ALDOA is overexpressed in cervical adenocarcinoma and contributes to malignant potentials of tumor cells through modulation of HIF‐1α signaling. The feedback loop between ALDOA and HIF‐1α could become a therapeutic target to improve the prognosis of this malignancy.

Human papillomavirus genotype contribution to cervical cancer and precancer: Implications for screening and vaccination in Japan

AbstractTo obtain baseline data for cervical cancer prevention in Japan, we analyzed human papillomavirus (HPV) data from 5045 Japanese women aged less than 40 years and diagnosed with cervical abnormalities at 21 hospitals during 2012‐2017. These included cervical intraepithelial neoplasia grade 1 (CIN1, n = 573), CIN2‐3 (n = 3219), adenocarcinoma in situ (AIS, n = 123), and invasive cervical cancer (ICC, n = 1130). The Roche Linear Array was used for HPV genotyping. The HPV type‐specific relative contributions (RCs) were estimated by adding multiple infections to single types in accordance with proportional weighting attributions. Based on the comparison of type‐specific RCs between CIN1 and CIN2‐3/AIS/ICC (CIN2+), RC ratios were calculated to estimate type‐specific risks for progression to CIN2+. Human papillomavirus DNA was detected in 85.5% of CIN1, 95.7% of CIN2‐3/AIS, and 91.2% of ICC. Multiple infections decreased with disease severity: 42.9% in CIN1, 40.4% in CIN2‐3/AIS, and 23.7% in ICC (P < .0001). The relative risk for progression to CIN2+ was highest for HPV16 (RC ratio 3.78, 95% confidence interval [CI] 3.01‐4.98), followed by HPV31 (2.51, 1.54‐5.24), HPV18 (2.43, 1.59‐4.32), HPV35 (1.56, 0.43‐8.36), HPV33 (1.01, 0.49‐3.31), HPV52 (0.99, 0.76‐1.33), and HPV58 (0.97, 0.75‐1.32). The relative risk of disease progression was 1.87 (95% CI, 1.71‐2.05) for HPV16/18/31/33/35/45/52/58, but only 0.17 (95% CI, 0.14‐0.22) for HPV39/51/56/59/66/68. Human papillomavirus 16/18/31/33/45/52/58/6/11 included in a 9‐valent vaccine contributed to 89.7% (95% CI, 88.7‐90.7) of CIN2‐3/AIS and 93.8% (95% CI, 92.4‐95.3) of ICC. In conclusion, our data support the Japanese guidelines that recommend discriminating HPV16/18/31/33/35/45/52/58 genotypes for CIN management. The 9‐valent vaccine is estimated to provide over 90% protection against ICC in young Japanese women.

Is the catch‐up program of HPV vaccination for Japanese adult women effective or not?

Performance of an ancillary test for cervical cancer that measures miRNAs and cytokines in serum and cervical mucus

AbstractCurrently, human papillomavirus tests and cytology are used to screen for cervical cancer. However, more accurate ancillary screening tests are needed. MicroRNAs (miRNAs) and cytokines are promising biomarkers that are aberrantly expressed in cervical cancer. Therefore, the potential of developing new screening markers based on the levels of miRNAs and cytokines in serum and local mucus samples from the same patients with cervical neoplasia was investigated. miRNA screening was performed by microarray and measurement using real‐time reverse‐transcriptase PCR. Cytokine were measured using multiplex bead assay, and changes in expressions were analyzed based on disease severity. As lesions progressed, miR‐20b‐5p, −155‐5p, −144‐3p, −451a, and −126‐3p expression levels were increased in mucus, and miR‐16‐5p, −223‐3p, and ‐451a expression levels were decreased in serum. Regarding cytokines, IL‐6, IL‐8, monocyte chemoattractant protein‐1, Eotaxin, interferon‐γ, and RANTES were increased, whereas granulocyte–colony‐stimulating factor (G‐CSF) was significantly decreased in mucus. miRNAs and cytokines in serum did not have high diagnostic accuracy. However, a combination of miR‐20b‐5p, ‐451a, ‐126‐3p, Eotaxin, as well as G‐CSF in mucus samples, had high diagnostic accuracy with an area under the receiver operating characteristic curve of 0.989 (0.979–0.999). Our results suggest that using mucus for this ancillary test is more beneficial than serum.

Overexpression of ARHGAP30 suppresses growth of cervical cancer cells by downregulating ribosome biogenesis

AbstractWe aimed to identify whether Rho GTPase activating proteins (RhoGAPs) were downregulated in cervical cancers and might be targeted to reduce the growth of cervical cancer using the GEO database and immunohistochemical analysis to identified changes in transcription and protein levels. We analyzed their proliferation, clone formation ability, and their growth as subcutaneous tumors in mice. To detect ARHGAP30 localization in cells, immunofluorescence assays were conducted. Mass spectrometry combined with immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co‐immunoprecipitation assays. Western‐blot and q‐PCR were applied to analyze candidate binding proteins that were associated with ribosome biogenesis. Puromycin incorporation assay was used to detect the global protein synthesis rate. We identified that ARHGAP30 was the only downregulated RhoGAP and was related to the survival of cervical cancer patients. Overexpression of ARHGAP30 in cervical cancer cells inhibited cell proliferation and migration. ARHGAP30 immunoprecipitated proteins were enriched in the ribosome biogenesis process. ARHGAP30 was located in the nucleous and interacted with nucleolin (NCL). Overexpression of ARHGAP30 inhibited rRNA synthesis and global protein synthesis. ARHGAP30 overexpression induced the ubiquitination of NCL and decreased its protein level in Hela cells. The function of ARHGAP30 on cervical cancer cell ribosome biogenesis and proliferation was independent of its RhoGAP activity as assessed with a RhoGAP‐deficient plasmid of ARHGAP30R55A. Overall, the findings revealed that ARHGAP30 was frequently downregulated and associated with shorter survival of cervical cancer patients. ARHGAP30 may suppress growth of cervical cancer by reducing ribosome biogenesis and protein synthesis through promoting ubiquitination of NCL.

A nationwide birth year‐by‐year analysis of effectiveness of HPV vaccine in Japan

AbstractIn Japan, the age‐adjusted incidence of cervical cancer has been increasing constantly and rapidly among younger women. We set out to accurately confirm the effectiveness of the HPV vaccine in Japan. Data were collected for women born in the fiscal year (FY) 1990 to 1997, who became eligible for their 20‐y‐old cervical cancer screening between the FY 2010 to 2017. The adjusted incidence of cervical intraepithelial neoplasia (CIN)1+ in women born in FY 1990 to 1993, that is those who reached the national vaccination target age prior to the introduction of publicly subsidized HPV vaccinations, referred here after as “the pre‐introduction generation”, was 1.42% (242/17 040). The incidence in the “vaccination generation” (women born in FY 1994 to 1997, that is those who were heavily vaccinated as a group when they were of the nationally targeted age of 13‐16) was 1.66% (135/8020). There was no significant difference between these incidence rates. However, our FY birth year‐by‐year analysis revealed that the incidence of CIN1+ was obviously lower than that predicted based on just the trend for CIN1+ seen in the pre‐introduction generation. Our analysis revealed that the incidence of CIN3+ was obviously lower in the vaccination generation than in the pre‐introduction generation (P = .0008). The incidence of CIN was already tending to increase in both the pre‐introduction and vaccination generations. The changes in CIN incidence by individual birth FY must be examined to accurately determine the actual effects of the HPV vaccine for reducing mild cervical lesions.

RPL34‐AS1–induced RPL34 inhibits cervical cancer cell tumorigenesis via the MDM2‐P53 pathway

AbstractRibosomal proteins (RPs) are important components of ribosomes and related to the occurrence and development of tumors. However, little is known about the effects of the RP network on cervical cancer (CC). In this study, we screened differentially expressed RPL34 in CC by high‐throughput quantitative proteome assay. We found that RPL34 acted as a tumor suppressor and was downregulated in CC and inhibited the proliferation, migration, and invasion abilities of CC cells. Next, we verified that RPL34 regulated the CC through the MDM2‐P53 pathway by using Act D medicine, MDM2 inhibitor, and a series of western blotting（WB）assays. Moreover, an antisense lncRNA, RPL34‐AS1, regulated the expression of RPL34 and participated in the tumorigenesis of CC. RPL34 can reverse the effect of RPL34‐AS1 in CC cells. Finally, by RNA‐binding protein immunoprecipitation (RIP) assay we found that eukaryotic initiation factor 4A3 (EIF4A3), which binds to RPL34‐AS1, regulated RPL34‐AS1 expression in CC. Therefore, our findings indicate that RPL34‐AS1–induced RPL34 inhibits CC cell proliferation, invasion, and metastasis through modulation of the MDM2‐P53 signaling pathway, which provides a meaningful target for the early diagnosis and treatment of CC.

A Bayesian network predicting survival of cervical cancer patients—Based on surveillance, epidemiology, and end results

AbstractThis study aimed to build a comprehensive model for predicting the overall survival (OS) of cervical cancer patients who received standard treatments and to build a series of new stages based on the International Federation of Gynecologists and Obstetricians (FIGO) stages for better such predictions. We collected the cervical cancer patients diagnosed since the year 2000 from the Surveillance, Epidemiology, and End Results (SEER) database. Cervical cancer patients who received radiotherapy or surgery were included. Log‐rank tests and Cox regression were used to identify potential factors of OS. Bayesian networks (BNs) were built to predict 3‐ and 5‐year survival. We also grouped the patients into new stages by clustering their 5‐year survival probabilities based on FIGO stage, age, and tumor differentiation. Cox regression suggested black ethnicity, adenocarcinoma, and single status as risks for poorer prognosis, in addition to age and stage. A total of 43,749 and 39,333 cases were finally eligible for the 3‐ and 5‐year BNs, respectively, with 11 variables included. Cluster analysis and Kaplan‐Meier curves indicated that it was best to divide the patients into nine modified stages. The BNs had excellent performance, with area under the curve and maximum accuracy of 0.855 and 0.804 for 3‐year survival, and 0.851 and 0.787 for 5‐year survival, respectively. Thus, BNs are excellent candidates for predicting cervical cancer survival. It is necessary to consider age and tumor differentiation when estimating the prognosis of cervical cancer using FIGO stages.

FOXP4 inhibits squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3‐dependent pathway

AbstractAlthough the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre‐existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4‐positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1‐derived HPV 16‐positive W12 cells via an ELF3‐dependent pathway. In organotypic raft cultures, FOXP4‐downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte‐derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3‐dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.

Pembrolizumab plus chemotherapy in Japanese patients with persistent, recurrent or metastatic cervical cancer: Results from KEYNOTE‐826

AbstractPembrolizumab plus chemotherapy with or without bevacizumab demonstrated prolonged progression‐free survival (PFS) and overall survival (OS) versus chemotherapy in patients with persistent, recurrent, or metastatic cervical cancer in the phase 3, randomized, double‐blind, placebo‐controlled KEYNOTE‐826 study. We report outcomes in patients enrolled in Japan. Patients received pembrolizumab 200 mg or placebo Q3W for up to 35 cycles plus chemotherapy (paclitaxel 175 mg/m2 + cisplatin 50 mg/m2 or carboplatin AUC 5) with or without bevacizumab 15 mg/kg. Dual primary endpoints were PFS per RECIST v1.1 by investigator assessment and OS in the global population; these were evaluated in patients with tumors with PD‐L1 combined positive score (CPS) ≥1, all‐comers, and PD‐L1 CPS ≥10. Fifty‐seven patients from Japan were randomized (pembrolizumab plus chemotherapy, n = 35; placebo plus chemotherapy, n = 22). Pembrolizumab plus chemotherapy improved PFS versus placebo plus chemotherapy in patients with PD‐L1 CPS ≥1 (n = 51; hazard ratio [HR; 95% CI], 0.36 [0.16–0.77]), all‐comers (n = 57; 0.45 [0.22–0.90]), and patients with PD‐L1 CPS ≥10 (n = 25; 0.36 [0.12–1.07]). HRs (95% CI) for OS were 0.38 (0.14–1.01), 0.41 (0.17–1.00), and 0.37 (0.10–1.30), respectively. Incidence of grade 3–5 AEs was 94% in the pembrolizumab group and 100% in the placebo group. Consistent with findings in the global KEYNOTE‐826 study, pembrolizumab plus chemotherapy with or without bevacizumab may prolong survival versus placebo plus chemotherapy with or without bevacizumab and had a manageable safety profile in Japanese patients with persistent, recurrent, or metastatic cervical cancer.

Scientific evaluation of alleged findings in HPV vaccines: Molecular mimicry and mouse models of vaccine‐induced disease

AbstractCervical cancer is caused by infections of the human papillomavirus (HPV), which can be prevented by vaccinations. In Japan, although about 3000 people die of cervical cancer annually, the HPV vaccination rate has remained extremely low in the eligible population since many Japanese have been concerned that “diverse symptoms,” such as chronic pain, movement disorders, and cognitive impairment, may occur as adverse reactions after HPV vaccination. The concern has been raised by media coverage of the ongoing HPV vaccine lawsuits, in which the plaintiffs complained of their symptoms caused by HPV vaccination. The claims have been based on the alleged pathogenic findings in research articles on HPV vaccines, summarized in the document prepared by the plaintiffs' attorneys. We critically evaluated these articles, in which the authors proposed the following findings/hypothesis: (i) molecular mimicry between HPV L1 and human proteins leads to the production of cross‐reactive antibodies; and (ii) HPV vaccine injection in mice causes damage in the brain, a mouse model for HPV vaccine associated neuro‐immunopathic syndrome (HANS). We found that these hypotheses were based mainly on the findings from a few research groups and that all the articles had flaws in the method, result, or discussion sections. Our current evaluation should help better understand the validity of the findings, which have been often misunderstood as the truth by the general public. We propose to accumulate high‐quality data on potential adverse events following HPV vaccination and to continue critically evaluating them.

Effectiveness of human papillomavirus vaccine against cervical precancer in Japan: Multivariate analyses adjusted for sexual activity

AbstractJapanese girls aged 12–16 years are offered free human papillomavirus (HPV) vaccination and cervical cancer screening is conducted with cytology and not HPV testing from the age of 20 years. So far, no study has analyzed the effect of HPV vaccination against cervical precancers considering HPV infection status and sexual activity. We aimed to analyze the vaccine effectiveness (VE) against HPV infection and cytological abnormalities, adjusted for sexual activity. This study comprised women aged 20–26 years who underwent cervical screening in Niigata. We obtained HPV vaccination status from municipal records and a questionnaire along with information concerning sexual activity. Of 5194 women registered for this study, final analyses included 3167 women in the vaccinated group (2821 vaccinated women prior to sexual debut) and 1386 women in the unvaccinated group. HPV 16/18 (0.2% vs 3.5%), 31/45/52 (3.4% vs 6.6%), and 31/33/45/52/58 (5.0% vs 9.3%) positive rates were significantly lower in the vaccinated group (P < 0.001). No women vaccinated before sexual debut had HPV 16/18‐related cytological abnormalities. VE for HPV 16/18 infection and high‐grade cytological abnormalities in women vaccinated prior to sexual debut were 95.8% (95% CI 81.9–99.0%; P < 0.001) and 78.3% (95% CI 11.3–94.7%; P = 0.033), respectively, in multivariate analyses adjusted for age and number of sexual partners. However, analyses of all vaccinated women did not show significant effectiveness against cytological abnormalities. Our results showed the effectiveness of HPV vaccine against high‐grade cervical cytological abnormalities and the importance of the vaccination before sexual debut.

Trends in cervical cancer incidence and mortality of young and middle adults in Japan

AbstractIn most high‐resource countries with organized screening programs, the incidence and mortality of cervical cancer is decreasing. Recent statistics have also revealed a reduction in invasive cervical cancer incidence as a result of national vaccination programs. Paradoxically, cervical cancer incidence has increased in Japan, particularly amongst women of reproductive age. This study aimed to examine the trends in cervical cancer incidence and mortality for young and middle adult women in Japan, by analyzing trends in 10‐year interval age‐groups. Cervical cancer incidence for young and middle adult women (ages 20‐59 years) was obtained from high‐quality population‐based cancer registries in three prefectures from 1985 to 2015. National cancer mortality data were obtained from published vital statistics from 1985 to 2019. Trends in crude and age‐standardized rates (ASR) were analyzed using Joinpoint regression. The cervical cancer incidence trend in 20‐59‐year‐old women combined significantly increased over the observation period. Both crude and ASR increased from 1985 to 2015 with an annual percent change (APC) of +1.6% (95% confidence interval, 1.1, 2.1) and +1.7% (1.2, 2.3), respectively. Similar increases were seen in ages 20‐29, 30‐39, and 40‐49 years with higher APCs especially in 20s and 30s. Both crude and ASR mortality significantly increased after the early 1990s in ages 20‐59 years combined. Based on the recognition that current cervical cancer control strategies in Japan have not been effective in reducing the cervical cancer burden in young and middle adults, promotion of screening and vaccination should be urgently strengthened.

Tisotumab vedotin in Japanese patients with recurrent/metastatic cervical cancer: Results from the innovaTV 206 study

AbstractNew treatments, particularly second‐line options, are needed to improve outcomes for patients with recurrent/metastatic cervical cancer (r/mCC). Tisotumab vedotin (TV) is an antibody–drug conjugate directed to tissue factor, a transmembrane protein commonly expressed in cancer cells, to deliver cytotoxic monomethyl auristatin E. This single‐arm, open‐label phase 1/2 trial evaluated the consistency of safety and efficacy outcomes of TV in Japanese patients with r/mCC to bridge the current findings with those reported in previous trials in non‐Japanese patients in the United States and Europe. In part 1 (dose escalation; N = 6), patients with advanced solid tumors received TV 1.5 or 2.0 mg/kg once every 3 weeks to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Part 2 (dose expansion; N = 17) evaluated the RP2D in r/mCC patients with 1–2 prior lines of therapy. In part 1, no dose‐limiting toxicities were observed, the MTD was not reached, and TV 2.0 mg/kg was established as the RP2D. In part 2, the most common treatment‐emergent adverse events were anemia (58.8%), nausea (58.8%), alopecia (47.1%), epistaxis (47.1%), and diarrhea (35.3%); adverse events of special interest were bleeding (76.5%), ocular events (35.3%), and peripheral neuropathy (17.6%), and were mostly grade 1/2. In part 2, confirmed objective response rate was 29.4%, median duration of response was 7.1 months, and median time to response was 1.2 months. In Japanese patients with r/mCC, TV demonstrated a manageable and tolerable safety, pharmacokinetics, and efficacy profile consistent with that observed in non‐Japanese patients.

Synergistic effect of the inhibitors of RAF/MEK and AXL on KRAS‐mutated ovarian cancer cells with high AXL expression

AbstractKRAS mutation is frequently seen in a subtype of ovarian cancer categorized as type 1. The KRAS‐MAPK pathway, which is closely involved in type 1 cancer progression, is under the regulation of receptor tyrosine kinases (RTKs). AXL, one of the RTKs, has been reported to be overexpressed in ovarian cancer and contributes to the poor prognosis. However, there is no useful target‐based agent against such gene profiles. We examined the combined effect of the dual RAF/MEK inhibitor CH5126766 and AXL inhibitor R428 on the growth of ovarian cancer HEY‐T30 and OVCAR‐5 cell lines, both of which bear KRAS mutation and express AXL at a high level, using the WST‐8 assay and the colony formation assay. The synergistic effect of the combination was evaluated by the combination index. The apoptotic cells were analyzed by flow cytometry. The expression of apoptotic proteins and the phosphorylation of MAPK and AKT pathway proteins were investigated by western blotting. We found that CH5126766 and R428 suppressed the phosphorylation of ERK and AKT, respectively, and their combination synergistically inhibited the growth of both cell lines with enhancement of apoptosis accompanied by the Bim upregulation. Combined treatment with CH5126766 and R428 is expected as the novel therapeutic option for KRAS‐mutated ovarian cancer with high expression of AXL.

Long‐term effectiveness of HPV vaccination against HPV infection in young Japanese women: Real‐world data

AbstractIn Japan, public funding for HPV vaccination began in 2010 for girls aged 13–16 years (birth cohort years 1994–1997) and women born in 1994 who turned 25 in 2019. We aimed to verify the long‐term effectiveness of the bivalent HPV vaccine in women aged 25 years. Subjects were women aged 25–26 years who underwent cervical cancer screening and HPV testing in Niigata from 2019 to 2020 (birth cohort years 1993–1994). Information on vaccination status and sexual behavior was obtained from a questionnaire and municipal records. We compared the HPV infection rates of the vaccinated and unvaccinated groups. Of the 429 registrants, 150 (35.0%) and 279 (65.0%) were vaccinated and unvaccinated, respectively. The average period from HPV vaccination to HPV testing was 102.7 months (8.6 years), with a median of 103 months (range 92–109 months). The HPV high‐risk infection rate was 21.3% (32/150) in the vaccinated group and 23.7% (66/279) in the unvaccinated group (P = 0.63). The HPV16/18 infection rate was 0% (0/150) in the vaccinated group and 5.4% (15/279) in the unvaccinated group, showing a significant difference (P = 0.0018), and the vaccine effectiveness was 100%. The cross‐protective type HPV31/45/52 infection rate in the vaccinated group was significantly lower than that in the unvaccinated group (3.3% vs. 10.0%, P = 0.013). There was no significant difference in the mean age at sexual debut and the number of previous sexual partners between the two groups. We have demonstrated the long‐term 9‐year effectiveness of the bivalent vaccine against HPV infection for the first time in Japan.

Human papillomavirus vaccine effectiveness by age at first vaccination among Japanese women

AbstractIn Japan, the National Immunization Program against human papillomavirus (HPV) targets girls aged 12‐16 years, and catch‐up vaccination is recommended for young women up to age 26 years. Because HPV infection rates increase soon after sexual debut, we evaluated HPV vaccine effectiveness by age at first vaccination. Along with vaccination history, HPV genotyping results from 5795 women younger than 40 years diagnosed with cervical intraepithelial neoplasia grade 2‐3 (CIN2‐3), adenocarcinoma in situ (AIS), or invasive cervical cancer were analyzed. The attribution of vaccine‐targeted types HPV16 or HPV18 to CIN2‐3/AIS was 47.0% for unvaccinated women (n = 4297), but 0.0%, 13.0%, 35.7%, and 39.6% for women vaccinated at ages 12‐15 years (n = 36), 16‐18 years (n = 23), 19–22 years (n = 14), and older than 22 years (n = 91), respectively, indicating the greater effectiveness of HPV vaccination among those initiating vaccination at age 18 years or younger (P < .001). This finding was supported by age at first sexual intercourse; among women with CIN2‐3/AIS, only 9.2% were sexually active by age 14 years, but the percentage quickly increased to 47.2% by age 16 and 77.1% by age 18. Additionally, the HPV16/18 prevalence in CIN2‐3/AIS was 0.0%, 12.5%, and 40.0% for women vaccinated before (n = 16), within 3 years (n = 8), and more than 3 years after (n = 15) first intercourse, respectively (P = .004). In conclusion, our data appear to support routine HPV vaccination for girls aged 12‐14 years and catch‐up vaccination for adolescents aged 18 years and younger in Japan.