Molecular Oncology

imPlatelet classifier: image‐converted RNA biomarker profiles enable blood‐based cancer diagnostics

Liquid biopsies offer a minimally invasive sample collection, outperforming traditional biopsies employed for cancer evaluation. The widely used material is blood, which is the source of tumor‐educated platelets. Here, we developed the imPlatelet classifier, which converts RNA‐sequenced platelet data into images in which each pixel corresponds to the expression level of a certain gene. Biological knowledge from the Kyoto Encyclopedia of Genes and Genomes was also implemented to improve accuracy. Images obtained from samples can then be compared against standard images for specific cancers to determine a diagnosis. We tested imPlatelet on a cohort of 401 non‐small cell lung cancer patients, 62 sarcoma patients, and 28 ovarian cancer patients. imPlatelet provided excellent discrimination between lung cancer cases and healthy controls, with accuracy equal to 1 in the independent dataset. When discriminating between noncancer cases and sarcoma or ovarian cancer patients, accuracy equaled 0.91 or 0.95, respectively, in the independent datasets. According to our knowledge, this is the first study implementing an image‐based deep‐learning approach combined with biological knowledge to classify human samples. The performance of imPlatelet considerably exceeds previously published methods and our own alternative attempts of sample discrimination. We show that the deep‐learning image‐based classifier accurately identifies cancer, even when a limited number of samples are available.

Prognostic relevance of longitudinal HGF levels in serum of patients with ovarian cancer

The pleiotropic protein hepatocyte growth factor (HGF) is the only known ligand of the tyrosine kinase mesenchymal–epithelial transition (cMET) receptor. The HGF/cMET pathway mediates invasion and migration of ovarian cancer cells, and upregulation of HGF/cMET pathway components has been associated with poor prognosis. This study investigated the clinical relevance of circulating HGF in serum of patients with ovarian cancer. Serum HGF (sHGF) was determined by enzyme‐linked immunosorbent assay in a total of 471 serum samples from 82 healthy controls and 113 patients with ovarian cancer (88.5% with ≥ FIGO III). Patient samples were collected at primary diagnosis and at four follow‐up time points throughout treatment and at disease recurrence. Patients with ovarian cancer showed elevated median sHGF levels at primary diagnosis, and sHGF levels transiently increased after surgery and normalized in the course of chemotherapy, even dropping below initial baseline. Higher levels of sHGF were an independent predictor for shorter overall survival (OS) (a) at primary diagnosis (HR = 0.41, 95% CI: 0.22–0.78, P = 0.006), (b) at longitudinal follow‐up time points (after surgery and before/during/after chemotherapy), (c) along the patients’ individual dynamics (HR = 0.21, 95% CI: 0.07–0.63, P = 0.005), and (d) among a subgroup analysis of patients with BRCA1/2 wild‐type ovarian cancer. This is the first study proposing sHGF as an independent prognostic biomarker for ovarian cancer at primary diagnosis and in the course of platinum‐based chemotherapy, irrespective of the postoperative residual disease after surgical debulking. sHGF could be implemented into clinical diagnostics as a CA125 auxiliary tumor marker for individualized prognosis stratification and sHGF‐guided therapy monitoring.

Long non‐coding RNA SNHG1 stimulates ovarian cancer progression by modulating expression of miR‐454 and ZEB1

Ovarian cancer (OC) is highly prevalent and is associated with high mortality rates due to metastasis and relapse. In this study, we assessed the role of long non‐coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in OC to gain further insight into mechanisms that contribute to its aggressiveness. We analyzed the correlation between SNHG1, miR‐454 and zinc finger E‐box‐binding homeobox 1 (ZEB1) using a dual‐luciferase reporter assay. Alterations in cell metastasis and invasiveness were observed using wound‐healing and Transwell invasion assays, respectively. Tumor xenografts allowed us to monitor liver metastasis of mice injected with A2780 cells. We found that SNHG1 is overexpressed in OC. Downregulation of SNHG1 promoted miR‐454 expression and reduced ZEB1 levels. In addition, knockdown of SNHG1, also reduced the aggressiveness of A2780 and SK‐OV3 cells. Furthermore, SNHG1 downregulation by siRNA hindered cell migration and invasion; however, this effect was reversed by co‐transfection of miR‐454 into A2780 and SK‐OV3 cells. Moreover, SNHG1 increased ZEB1 expression by downregulating miR‐454 and activated Akt signaling, thereby promoting epithelial‐mesenchymal transition and enhancing the invasiveness of OC cells. Tumor xenograft analyses confirmed that SNHG1 affects OC proliferation and metastasis in vivo. In summary, our data demonstrate that SNHG1 plays crucial roles in tumor progression and may be a useful maker for OC prognosis.

European Code Against Cancer, 5th edition – hormone replacement therapy, other common medical therapies and cancer

Several medical therapies modify the risk of developing certain cancers in an individual. The aim of this paper was to provide the scientific justification for the 5th edition of the European Code Against Cancer (ECAC5) recommendation on the use of hormone replacement therapy (HRT) and other drugs used at population scale, such as hormonal contraceptives and aspirin. HRT modifies the risk of developing certain cancers in an individual. Except for vaginal oestrogens, all forms of HRT are associated with an increased breast cancer risk; the risk of serous ovarian cancer and endometrial cancer may also be increased. Despite such an increase in cancer risk, HRT often remains the only option for the management of certain menopausal symptoms for the restoration of quality of life and mental health. Therefore, the ECAC5 recommends using HRT for bothersome menopausal symptoms only after a thorough discussion with a healthcare professional and limiting its use for as short a duration as possible. On review of up‐to‐date evidence for hormonal contraceptives and aspirin, the ECAC5 Working Group elected not to make a recommendation for the average‐risk general population regarding the use of these therapies.

Loss of primary cilia promotes EphA2 ‐mediated endothelial‐to‐mesenchymal transition in the ovarian tumor microenvironment

Endothelial‐to‐mesenchymal transition (EndMT) is closely associated with tumor progression. Endothelial cells (ECs) in the tumor microenvironment (TME) use EndMT programs to facilitate tumor progression; however, the underlying mechanisms in ovarian cancer are poorly understood. Here, we describe the involvement of primary cilia in EndMT of the ovarian TME. We showed that ECs from human ovarian tumors displayed robust EndMT and impaired cilia formation, as was also observed in ECs in response to ovarian cancer cell culture‐conditioned media (OV‐CM). Notably, ECs lacking primary cilia exhibited increased OV‐CM‐induced EndMT. Vascular abnormalities, such as enhanced cell migration and vessel permeability, were observed in vitro . Furthermore, in vivo experiments using endothelial‐specific kinesin family member 3A ( Kif3a )‐knockout mice showed enhanced EndMT in the ovarian TME. Mechanistically, we identified ephrin type‐A receptor 2 (EphA2) as a key regulator of EndMT. Upon OV‐CM treatment, EphA2 expression increased, and depletion of EphA2 in ECs decreased OV‐CM‐induced EndMT and vascular abnormalities. These results highlight that the loss of primary cilia and the consequent EphA2 activation are key mechanisms by which EndMT programs induce the acquisition of cancer‐associated fibroblast‐like cells in the ovarian TME, thereby promoting ovarian cancer progression.

A single gene mutation predicts response to immune checkpoint blockade in ovarian clear cell carcinoma

There is a lack of genetic biomarkers for predicting response to immune checkpoint blockade (ICB) therapy in cancer. The recent discovery that loss‐of‐function mutations in the gene encoding the protein phosphatase 2A (PP2A) scaffold protein PPP2R1A confer sensitivity to immune checkpoint blockade in ovarian clear cell carcinoma, therefore represents a breakthrough. Mechanistically, mutations in the PPP2R1A gene induce a strong interferon gamma response in tumor cells, which enhances infiltration of activated CD8+ T cells into the tumor. The activity of these T cells is then fortified by ICB. Furthermore, preclinical studies have shown that PP2A inhibition leads to the generation of neoantigens by disrupting RNA splicing, and PP2A inhibition can remodel the immune microenvironment of tumors to enhance responses to ICB. The finding that loss‐of‐function PPP2R1A mutations predict benefit from immunotherapy also suggests that pharmacological inhibition of PP2A may act synergistically with ICB therapy.

Targeting of PTP4A3 overexpression sensitises HGSOC cells towards chemotherapeutic drugs

Ovarian cancer (OC) has the highest mortality rate of all gynaecological malignancies, partly attributable to its propensity for chemotherapy resistance. The most common subtype of OC is serous, of which high‐grade serous ovarian cancer (HGSOC) is the most lethal subtype. Protein tyrosine phosphatase 4A3 (PTP4A3) overexpression is implicated in tumour cell invasion and metastasis by upregulating the PI3K/Akt/mTORC1 axis. Previously, we reported that PTP4A3 increased the survival of non‐serous OC cells by activating the autophagy pathway. Here, we investigated the impact of PTP4A3 on cell proliferation, autophagy and chemoresistance in HGSOC cells and whether targeting PTP4A3 in HGSOC cells that overexpress this phosphatase would sensitise them to existing chemotherapeutic drugs. Gene silencing of PTP4A3 resulted in the upregulation of compensatory mechanisms that overcame the loss of PTP4A3 expression, but this was mitigated by pan‐PTP4A inhibition with JMS‐053 in HGSOC cells. Moreover, shRNA‐mediated silencing of PTP4A3 sensitised HGSOC cells to clinically relevant chemotherapeutic drugs. Overall, we show that compensatory mechanisms from PTP4A1 and PTP4A2 can arise when specifically targeting PTP4A3 in HGSOC and that pan‐PTP4A inhibition can overcome those effects.

Tumor clusters with divergent inflammation and human retroelement expression determine the clinical outcome of patients with serous ovarian cancer

High‐grade serous ovarian carcinoma (HGSOC) associates with the worst patient outcome. Understanding the tumor environment in terms of quantifying endogenous retroviruses ( ERVs ) and LINE‐1 expression and their correlations with inflammation genes, checkpoint inhibitors and patient survival is needed. Analysis of 102 treatment‐naïve HGSOC and control tissues for ERVs , LINE‐1 , inflammation and immune checkpoints identified five clusters with diverse patient recurrence‐free survivals. One cluster termed Triple‐I with the best patient survival showed the highest number of tumor infiltrating lymphocytes along with 22 overexpressed genes, including CXCL9 and AIM2 . However, Triple‐I associated with the lowest ERV/LINE‐1 expression. The tumor cluster with the second‐best patient survival had both high ERV/LINE‐1 expression and inflammation. Multiplex‐immunohistochemistry revealed CD28 protein solely on immune cells, without co‐expression of the inhibitory CTLA4 receptor. The largest tumor cluster with high ERV/LINE‐1 expression but low inflammation showed a significant low gene expression of the dsRNA sensors MDA5 and RIG‐I supporting an aberrant block in IFN signaling. Our study represents an intrinsic ‘molecular and immunological snapshot’ of the HGSOC tumor environment important for understanding retroelements and inflammation for clinical relevance.

The anticancer effect of the HDAC inhibitor belinostat is enhanced by inhibitors of Bcl‐ x L or Mcl‐1 in ovarian cancer

Identifying innovative therapeutic strategies is crucial to improve clinical management of ovarian cancer. Previously, we showed that ovarian cancer cell apoptosis can be triggered by inhibiting the anti‐apoptotic proteins Bcl‐x L and Mcl‐1 and/or by inducing their pro‐apoptotic partners Bim, Puma, and Noxa. The expression of these pro‐apoptotic proteins can be hindered by excessive histone deacetylation, resulting from HDAC overexpression. This study aimed to evaluate whether belinostat, an FDA‐approved pan‐HDAC inhibitor, could increase Bim, Puma, and/or Noxa expression and induce ovarian cancer cell death, either alone or in combination with strategies targeting Bcl‐x L or Mcl‐1. Belinostat exerted a cytostatic effect and, at higher concentrations, an apoptotic effect in SKOV3 and IGROV1‐R10 ovarian cancer cells. It induced a concentration‐dependent increase in Bim, Puma, and Noxa protein expression, while partially repressing that of Bcl‐x L . Inhibition of Bcl‐x L sensitized both cell lines to belinostat, as did inhibition of Mcl‐1 in IGROV1‐R10 cells. Interestingly, belinostat's anticancer activity was also enhanced by inhibitors of Bcl‐x L or Mcl‐1 in patient‐derived tumor organoids. This study therefore positions belinostat‐based strategies as promising therapies for ovarian cancer.

Vertical inhibition of p110α/ AKT and N‐cadherin enhances treatment efficacy in PIK3CA ‐aberrated ovarian cancer cells

Phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha [ PIK3CA , encoding PI3Kalpha (also known as p110α)] is one of the most commonly aberrated genes in human cancers. In serous ovarian cancer, PIK3CA amplification is highly frequent but PIK3CA point mutation is rare. However, whether PIK3CA amplification and PIK3CA driver mutations have the same functional impact in the disease is unclear. Here, we report that both PIK3CA amplification and E545K mutation are tumorigenic. While the protein kinase B (AKT) signaling axis was activated in both E545K knock‐in cells and PIK3CA ‐overexpressing cells, the mitogen‐activated protein kinase 3/1 (ERK1/2) pathway was induced selectively by E545K mutation but not PIK3CA amplification. Intriguingly, AKT signaling in these PIK3CA ‐aberrated cells increased transcriptional coactivator YAP1 (YAP) Ser127 phosphorylation and thereby cytoplasmic YAP levels, which in turn increased cell migration through Ras‐related C3 botulinum toxin substrate 1 (RAC1) activation. In addition to the altered YAP signaling, AKT upregulated N‐cadherin expression, which also contributed to cell migration. Pharmacological inhibition of N‐cadherin reduced cell migratory potential. Importantly, co‐targeting N‐cadherin and p110α/AKT caused additive reduction in cell migration in vitro and metastases formation in vivo . Together, this study reveals the molecular pathways driven by the PIK3CA aberrations and the exploitable vulnerabilities in PIK3CA ‐aberrated serous ovarian cancer cells.

Ovarian cancer relies on the PDGFRβ –fibronectin axis for tumorsphere formation and metastatic spread

High‐grade serous ovarian cancer (HGSOC) is the deadliest gynecological malignancy. The most common form of metastatic spread of HGSOC is transcoelomic dissemination. In this process, detached cells from the primary tumor aggregate as tumorspheres and promote the accumulation of peritoneal ascites. This represents an early event in HGSOC development and is indicative of poor prognosis. In this study, based on tumorspheres isolated from ascitic liquid samples from HGSOC patients, ovarian cancer spheroid 3D cultures, and in vivo models, we describe a key signal for tumorsphere formation in HGSOC. We report that platelet‐derived growth factor receptor beta (PDGFRβ) is essential for fibronectin‐mediated cell clustering of ovarian cancer cells into tumorspheres. This effect is mediated by the kinase NUAK family SNF1‐like kinase 1 (NUAK1) and blocked by PDGFRβ pharmacological or genetic inhibition. In the absence of PDGFRβ, ovarian cancer cells can be provided with fibronectin by cancer‐associated fibroblasts to generate chimeric spheroids. This work provides new insights that uncover potential targets to prevent peritoneal dissemination, the main cause of advanced disease in HGSOC patients.

Peripheral blood leukocyte signatures as biomarkers in relapsed ovarian cancer patients receiving combined anti‐ CD 73/anti‐ PD ‐ L 1 immunotherapy in arm A of the NSGO ‐ OV ‐ UMB1 / ENGOT ‐ OV 30 trial

Immune checkpoint inhibitors have demonstrated limited efficacy in overcoming immunosuppression in patients with epithelial ovarian cancer (EOC). Although certain patients experience long‐term treatment benefit, reliable biomarkers for responder pre‐selection and the distinction of dominant immunosuppressive mechanisms have yet to be identified. Here, we used a 40‐marker suspension mass cytometry panel to comprehensively phenotype peripheral blood leukocytes sampled over time from patients with relapsed EOC who underwent combination oleclumab (anti‐CD73) and durvalumab (anti‐PD‐L1) immunotherapy in the NSGO‐OV‐UMB1/ENGOT‐OV30 trial. We found that survival duration was impacted by baseline abundances of total peripheral blood mononuclear cells. Longitudinal analyses revealed a significant increase in CD14 + CD16 − myeloid cells during treatment, with significant expansion of monocytic myeloid‐derived suppressor cells occurring in patients with shorter progression‐free survival, who additionally showed a continuous decrease in central memory T‐cell abundances. All patients demonstrated significant PD‐L1 upregulation over time on most T‐cell subsets. Higher CD73 and IDO1 expression on certain leukocytes at baseline significantly positively correlated with longer progression‐free survival. Overall, our study proposes potential biomarkers for EOC immunotherapy personalization and response monitoring; however, further validation in larger studies is needed.

Plasma‐activated media selectively induces apoptotic death via an orchestrated oxidative stress pathway in high‐grade serous ovarian cancer cells

High‐grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer. Due to a lack of an early detection test and overt symptoms, many patients are diagnosed at a late stage where metastasis makes treatment very challenging. Furthermore, the current standard treatment for HGSOC patients, consisting of debulking surgery and platinum‐taxane chemotherapy, reduces quality of life due to debilitating side‐effects. Sadly, 80–90% of patients diagnosed with advanced stage ovarian cancer will die due to treatment resistance. As such, novel therapeutic strategies for HGSOC that are both more effective and less toxic are urgently required. Here we describe the assessment of cold atmospheric pressure (CAP) gas discharge technology as a novel treatment strategy in pre‐clinical models of HGSOC. Plasma‐activated media (PAM) was generated using cell growth media. HGSOC cell lines, patient ascites cells and primary tissue explants were tested for their response to PAM via analysis of cell viability, cell death and oxidative stress assays. Our data show that PAM treatment can be more effective than standard carboplatin chemotherapy at selectively targeting ovarian cancer cells in primary patient samples. Further, we also observed PAM to induce apoptosis in HGSOC cancer cell lines via induction of oxidative stress and mitochondrial‐mediated apoptosis. These findings suggest that PAM is a viable therapeutic strategy to test in in vivo models of ovarian cancer, with a view to develop an intraperitoneal PAM‐based therapy for HGSOC patients. Our studies validate the ability of PAM to selectively target tumour tissue and ascites cells. This work supports the development of PAM towards in vivo validation and translation into clinical practice.

Interaction between membranous EBP50 and myosin 9 as a favorable prognostic factor in ovarian clear cell carcinoma

Ezrin‐radixin‐moesin‐binding phosphoprotein 50 (EBP50) is a scaffold protein that is required for epithelial polarity. Knockout (KO) of membranous EBP50 (Me‐EBP50) in ovarian clear cell carcinoma (OCCC) cells induced an epithelial–mesenchymal transition (EMT)‐like phenotype, along with decreased proliferation, accelerated migration capability, and induction of cancer stem cell (CSC)‐like properties. Shotgun proteomics analysis of proteins that co‐immunoprecipitated with EBP50 revealed that Me‐EBP50 strongly interacts with myosin 9 (MYH9). Specific inhibition of MYH9 with blebbistatin phenocopied Me‐EBP50 KO, and blebbistatin treatment potentiated the effects of Me‐EBP50 KO. In OCCC cells from clinical samples, Me‐EBP50 and MYH9 were co‐localized at the apical plasma membrane. Patients with a combination of Me‐EBP50‐high and MYH9‐high scores had the best prognosis for overall and progression‐free survival. Our data suggest that Me‐EBP50 has tumor‐suppressive effects through the establishment and maintenance of epithelial polarization. By contrast, loss of Me‐EBP50 expression induces EMT‐like phenotypes, probably due to MYH9 dysfunction; this results in increased cell mobility and enhanced CSC‐like properties, which in turn promote OCCC progression.

The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer

The selenoenzyme type I iodothyronine deiodinase (DIO1) catalyzes removal of iodine atoms from thyroid hormones. Although DIO1 action is reported to be disturbed in several malignancies, no work has been conducted in high‐grade serous ovarian carcinoma (HGSOC), the most lethal gynecologic cancer. We studied DIO1 expression in HGSOC patients [The Cancer Genome Atlas (TCGA) data and tumor tissues], human cell lines (ES‐2 and Kuramochi), normal Chinese hamster ovarian cells (CHO‐K1), and normal human fallopian tube cells (FT282 and FT109). To study its functional role, DIO1 was overexpressed, inhibited [by propylthiouracil (PTU)], or knocked down (KD), and cell count, proliferation, apoptosis, cell viability, and proteomics analysis were performed. Lower DIO1 levels were observed in HGSOC compared to normal cells and tissues. TCGA analyses confirmed that low DIO1 mRNA expression correlated with worse survival and therapy resistance in patients. Silencing or inhibiting the enzyme led to enhanced ovarian cancer proliferation, while an opposite effect was shown following DIO1 ectopic expression. Proteomics analysis in DIO1‐KD cells revealed global changes in proteins that facilitate tumor metabolism and progression. In conclusion, DIO1 expression and ovarian cancer progression are inversely correlated, highlighting a tumor suppressive role for this enzyme and its potential use as a biomarker in this disease.

Comparing self‐reported race and genetic ancestry for identifying potential differentially methylated sites in endometrial cancer: insights from African ancestry proportions using machine learning models

While the incidence of endometrial cancer is increasing among all US women, Black women face higher mortality rates. The reasons for this remain unclear. In this study, whole genome differential methylation analysis, along with state‐of‐the‐art computational methods such as the recursive feature elimination technique and supervised/unsupervised machine learning models, was used to identify 38 epigenetic signature genes (ESGs) and four core‐ESGs (cg19933311: TRPC5; cg09651654: APOBEC1; cg27299712: PLEKHG5; cg03150409: WHSC1) in endometrial tumors from Black and White women, incorporating genetic ancestry estimation. Methylation at two Core‐ESGs, namely APOBEC1 and PLEKHG5, showed statistically significant overall survival differences between the two ancestral groups (Likelihood ratio test; P value = 0.006). Moreover, our comprehensive ancestry‐based analysis revealed that tumors from women with high African ancestry exhibited increased hypomethylation compared to those with low African ancestry. These hypomethylated genes were enriched in drug metabolism pathways, indicating a potential link between genetic ancestry, epigenetic modifications, and pharmacogenomic responses. Combining ancestry, race, and disease type may help identify which patient groups will benefit most from these biomarkers for targeted treatments.

Multiplex single‐cell profiling of putative cancer stem cell markers ALDH1 , SOX9 , SOX2 , CD44 , CD133 and CD15 in endometrial cancer

The presence of cancer stem cells is linked to aggressive disease and higher risk of recurrence, and multiple markers have been proposed to detect cancer stem cells. However, a detailed evaluation of the expression patterns and the prognostic value of markers relevant for endometrial cancer is lacking. As organoid models are suggested to be enriched in cancer stem cells, such models may prove valuable to define tissue‐specific cancer stem cells. To address this, imaging mass cytometry and multiplex single‐cell analyses were performed on an endometrial cancer patient series including both tumor biopsies and corresponding patient‐derived organoids. An antibody panel focused on cancer stem cell markers was used to identify cancer stem cell phenotypes. Over 70% of epithelial cells in the tumor biopsies expressed at least one putative cancer stem cell marker. We identified distinct cancer cell phenotypes with heterogeneous expression within individual patients and between patient samples. Few differences in the distribution of cancer cell phenotypes were observed between tumor biopsies and corresponding organoids. Cells expressing aldehyde dehydrogenase 1 (ALDH1) were more prevalent in high‐grade tumors, while expression of CD44 was more prevalent in grade 1 tumors. Spatial analysis revealed significantly less interaction between ALDH1‐ and CD44‐expressing cells. Gene expression data was used to further investigate selected markers. CD44 gene expression was associated with a favorable prognosis and was further validated using immunohistochemistry. High expression of CD44 was significantly associated with better survival. The general high expression of proposed stem cell markers may indicate alternative roles for these in endometrial cancer.

PELP1 inhibition by SMIP34 reduces endometrial cancer progression via attenuation of ribosomal biogenesis

Endometrial carcinoma (ECa) is the fourth most common cancer among women. The oncogene PELP1 is frequently overexpressed in a variety of cancers, including ECa. We recently generated SMIP34, a small‐molecule inhibitor of PELP1 that suppresses PELP1 oncogenic signaling. In this study, we assessed the effectiveness of SMIP34 in treating ECa. Treatment of established and primary patient‐derived ECa cells with SMIP34 resulted in a significant reduction of cell viability, colony formation ability, and induction of apoptosis. RNA‐seq analyses showed that SMIP34‐regulated genes were negatively correlated with ribosome biogenesis and eukaryotic translation pathways. Mechanistic studies showed that the Rix complex, which is essential for ribosomal biogenesis, is disrupted upon SMIP34 binding to PELP1 . Biochemical assays confirmed that SMIP34 reduced ribosomal biogenesis and new protein synthesis. Further, SMIP34 enhanced the efficacy of mTOR inhibitors in reducing viability of ECa cells. SMIP34 is also effective in reducing cell viability in ECa organoids in vitro and explants ex vivo . Importantly, SMIP34 treatment resulted in a significant reduction of the growth of ECa xenografts. Collectively, these findings underscore the potential of SMIP34 in treating ECa.

ARID1A ‐deficient cells require HDAC6 for progression of endometrial carcinoma

AT‐rich interactive domain‐containing protein 1A (ARID1A) loss‐of‐function mutation accompanied by a loss of ARID1A protein expression is frequently observed in endometrial carcinomas. However, the molecular mechanisms linking these genetic changes to the altered pathways regulating tumour initiation, maintenance and/or progression remain poorly understood. Thus, the main aim of this study was to analyse the role of ARID1A loss of function in endometrial tumorigenesis. Here, using different endometrial in vitro and in vivo models, such as tumoral cell lines, 3D primary cultures and metastatic or genetically modified mouse models, we show that altered expression of ARID1A is not enough to initiate endometrial tumorigenesis. However, in an established endometrial cancer context, ARID1A loss of function accelerates tumoral progression and metastasis through the disruption of the G2/M cell cycle checkpoint and ATM/ATR‐mediated DNA damage checkpoints, increases epithelial cell proliferation rates and induces epithelial mesenchymal transition through the activation of histone deacetylase 6 (HDAC6). Next, we demonstrated that the inhibition of HDAC6 function, using the HDAC6‐specific inhibitor ACY1215 or by transfection with HDAC6 short hairpin RNA (shRNA), can reverse the migratory and invasive phenotype of ARID1A‐ knockdown cells. Further, we also show that inhibition of HDAC6 activity causes an apoptotic vulnerability to etoposide treatments in ARID1A‐ deficient cells. In summary, the findings exposed in this work indicate that the inhibition of HDAC6 activity is a potential therapeutic strategy for patients suffering from ARID1A‐mutant endometrial cancer diagnosed in advanced stages.

Therapeutic potential of the human endogenous retroviral envelope protein HEMO: a pan‐cancer analysis

Human endogenous retroviruses represent approximately 8% of our genome. Most of these sequences are defective except for a few genes such as the ancestral retroviral HEMO envelope gene (Human Endogenous MER34 ORF), recently characterized by our group. In this study, we characterized transcriptional activation of HEMO in primary tumors from The Cancer Genome Atlas (TCGA) and in metastatic tumors from a Gustave Roussy cohort. Pan‐cancer detection of the HEMO protein in a series of patient samples validated these results. Differential gene expression analysis in various TCGA datasets revealed a link between HEMO expression and activation of Wnt/β‐catenin signaling, in particular in endometrial cancer. Studies on cell models led us to propose that the Wnt/β‐catenin pathway could act as an upstream regulator of this retroviral endogenous sequence in tumor condition. Characterization of transcriptomic profiles of both HEMOLow and HEMOHigh tumors suggested that activation of HEMO is negatively associated with immune response signatures. Taken together, these results highlight that HEMO, as an endogenous retroviral envelope protein specifically expressed in tumors, represents a promising tumor biomarker and therapeutic target.

Pharmacologic inhibition of Akt in combination with chemotherapeutic agents effectively induces apoptosis in ovarian and endometrial cancer cell lines

The PI3K/Akt signaling pathway, the most frequently altered signaling system in human cancer, is a crucial inducer of dysregulated proliferation and neoplastic processes; however, few therapeutic strategies using PI3K/Akt inhibitors singly have been shown to be effective. The purpose of this paper was to underline the potential benefit of pharmacological modulation of the PI3K/Akt pathway when combined with specific chemotherapeutic regimens. We have studied the ability of NVP‐BEZ235 (PI3K/mTOR inhibitor) and AZD5363 (Akt inhibitor) in the sensitization of cancer cells to cisplatin and doxorubicin. Our results show that NVP‐BEZ235 sensitizes cells preferentially to cisplatin while AZD5363 sensitizes cells to doxorubicin. At equal concentrations (5 μ m ), both inhibitors reduce ribosomal protein S6 phosphorylation, but AZD5363 is more effective in reducing GSK3β phosphorylation as well as S6 phosphorylation. Additionally, AZD5363 is capable of inducing FOXO1 and p53 nuclear localization and reduces BAD phosphorylation, which is generally increased by cisplatin and doxorubicin. Finally, the combination of AZD5363 and doxorubicin induces apoptosis in cells and robustly reduces cell ability to clonally replicate, which underlines a potential cooperative effect of the studied compounds.

LncRNA‐ZXF1 stabilizes P21 expression in endometrioid endometrial carcinoma by inhibiting ubiquitination‐mediated degradation and regulating the miR‐378a‐3p/PCDHA3 axis

Long noncoding RNAs (lncRNAs) have a profound effect on biological processes in various malignancies. However, few studies have investigated their functions and specific mechanisms in endometrial cancer. In this study, we focused on the role and mechanism of lncRNA‐ZXF1 in endometrial cancer. Bioinformatics and in vitro and in vivo experiments were used to explore the expression and function of lncRNA‐ZXF1. We found that lncRNA‐ZXF1 altered the migration and invasion of endometrioid endometrial cancer (EEC) cells. Furthermore, our results suggest that lncRNA‐ZXF1 regulates EEC cell proliferation. This regulation may be achieved by the lncRNA‐ZXF1‐mediated alteration in the expression of P21 through two mechanisms. One is that lncRNA‐ZXF1 functions as a molecular sponge of miR‐378a‐3p to regulate PCDHA3 expression and then modulate the expression of P21. The other is that lncRNA‐ZXF1 inhibits CDC20‐mediated degradation of ubiquitination by directly binding to P21. To the best of our knowledge, this study is the first to explore lncRNA‐ZXF1 functioning as a tumor‐suppressing lncRNA in EEC. LncRNA‐ZXF1 may become therapeutic, diagnostic, and prognostic indicator in the future.

Cancer‐associated V‐ATPase induces delayed apoptosis of protumorigenic neutrophils

Tumors and neutrophils undergo an unexpected interaction, in which products released by tumor cells interact to support neutrophils that in turn support cancer growth, angiogenesis, and metastasis. A key protein that is highly expressed by cancer cells in tumors is the a2 isoform V‐ATPase (a2V). A peptide from a2V (a2NTD) is secreted specifically by cancer cells, but not normal cells, into the tumor microenvironment. This peptide reprograms neutrophils to promote angiogenesis, cancer cell invasiveness, and neutrophil recruitment. Here, we provide evidence that cancer‐associated a2V regulates the life span of protumorigenic neutrophils by influencing the intrinsic pathway of apoptosis. Immunohistochemical analysis of human cancer tissue sections collected from four different organs shows that levels of a2NTD and neutrophil counts are increased in cancer compared with normal tissues. Significant increases in neutrophil counts were present in both poorly and moderately differentiated tumors. In addition, there is a positive correlation between the number of neutrophils and a2NTD expression. Human neutrophils treated with recombinant a2NTD show significantly delayed apoptosis, and such prolonged survival was dependent on NF‐κB activation and ROS generation. Induction of antiapoptotic protein expression (Bcl‐xL and Bcl‐2A1) and decreased expression of proapoptotic proteins (Bax, Apaf‐1, caspase‐3, caspase‐6, and caspase‐7) were a hallmark of these treated neutrophils. Autocrine secretion of prosurvival cytokines of TNF‐α and IL‐8 by treated neutrophils prolongs their survival. Our findings highlight the important role of cancer‐associated a2V in regulating protumorigenic innate immunity, identifying a2V as a potential important target for cancer therapy.

Integrated genomic and proteomic profiling reveals insights into chemoradiation resistance in cervical cancer

Cervical cancer is highly prevalent in India, with most cases being diagnosed at advanced stages. Despite the standard concurrent chemoradiotherapy (CCRT), 30–40% of patients' experience treatment failure, underscoring the need for improved therapeutic strategies. Understanding resistance mechanisms and identifying predictive biomarkers are crucial to improve treatment efficacy and enable personalized medicine. We conducted a comprehensive genomic and proteomic analysis to identify molecular signatures associated with CCRT. We identified recurrent mutations in phosphatidylinositol 4,5‐bisphosphate 3‐kinase catalytic subunit alpha isoform ( PIK3CA ) and histone‐lysine N‐methyltransferase 2D ( KMT2D ), with mutation signature analysis revealing a prevalent DNA dC‐ > dU‐editing enzyme, APOBEC mutagenesis signature. Distinct genomic alterations, including epidermal growth factor receptor ( EGFR ) amplification and serine/threonine kinase 11 ( STK11 ) deletion, were exclusively observed in the chemoradiation‐resistant cohort. Proteomic analysis identified 73 significantly dysregulated proteins, with syntaxin‐3 (STX3), SERPINB7, lipopolysaccharide‐binding protein (LBP), EMILIN2, and ribosyldihydronicotinamide dehydrogenase (quinone) (NQO2) being the top five upregulated proteins. Integrative pathway analysis highlighted an active DNA repair pathway in the resistant cohort. This study presents the first proteogenomic profiling of cervical cancer in the Indian population, linking molecular alterations to CCRT response. STK11 and STX3 emerged as predictive biomarkers for poor response, whereas EGFR presents as a promising therapeutic target in the resistant group.

European Code Against Cancer, 5th edition – organised cancer screening programmes

The 5th edition of the European Code Against Cancer (ECAC5) recommends sustainable, organised screening programmes for: (a) colorectal cancer using biennial quantitative faecal immunochemical test (FIT) for individuals aged 50–74 years. As an alternative strategy, once‐only endoscopy may be considered within the same age range; (b) breast cancer using biennial digital mammography for women aged 50–69 years. Implementing this strategy for women aged 45–49 years and 70–74 years can be considered. Other screening strategies or additional examinations could be considered for women with high mammographic density; (c) cervical cancer using human papillomavirus (HPV) screening at intervals no shorter than 5 years for women aged 30–65 years. It is recommended to adapt policies according to vaccination status and screening history; and (d) lung cancer using annual low‐dose computed tomography (LDCT) for individuals considered to be at increased risk of lung cancer based on age, history of smoking or validated risk models, with biennial screening as an alternative. Screening should incorporate smoking cessation interventions.

Patient‐derived acellular ascites fluid affects drug responses in ovarian cancer cell lines through the activation of key signalling pathways

Malignant ascites is commonly produced in advanced epithelial ovarian cancer (EOC) and serves as unique microenvironment for tumour cells. Acellular ascites fluid (AAF) is rich in signalling molecules and has been proposed to play a role in the induction of chemoresistance. Through in vitro testing of drug sensitivity and by assessing intracellular phosphorylation status in response to mono‐ and combination treatment of five EOC cell lines after incubation with AAFs derived from 20 different patients, we investigated the chemoresistance‐inducing potential of ascites. We show that the addition of AAFs to the culture media of EOC cell lines has the potential to induce resistance to standard‐of‐care drugs (SCDs). We also show that AAFs induce time‐ and concentration‐dependent activation of downstream signalling to signal transducer and activator of transcription 3 (STAT3), and concomitantly altered phosphorylation of mitogen‐activated protein kinase kinase (MEK), phosphoinositide 3‐kinase (PI3K)–protein kinase B (AKT) and nuclear factor NF‐kappa‐B (NFκB). Antibodies targeting the interleukin‐6 receptor (IL6R) effectively blocked phosphorylation of STAT3 and STAT1. Treatments with SCDs were effective in reducing cell viability in only a third of 30 clinically relevant conditions examined, defined as combinations of drugs, different cell lines and AAFs. Combinations of SCDs and novel therapeutics such as trametinib, fludarabine or rapamycin were superior in another third. Notably, we could nominate effective treatment combinations in almost all conditions except in 4 out of 30 conditions, in which trametinib or fludarabine showed higher efficacy alone. Taken together, our study underscores the importance of the molecular characterisation of individual patients' AAFs and the impact on treatment resistance as providing clinically meaningful information for future precision treatment approaches in EOC.

Combined spatially resolved metabolomics and spatial transcriptomics reveal the mechanism of RACK1‐mediated fatty acid synthesis

Lipid metabolism is altered in rapidly proliferating cancer cells, where fatty acids (FAs) are utilized in the synthesis of sphingolipids and glycerophospholipids to produce cell membranes and signaling molecules. Receptor for activated C‐kinase 1 (RACK1; also known as small ribosomal subunit protein) is an intracellular scaffolding protein involved in signaling pathways. Whether such lipid metabolism is regulated by RACK1 is unknown. Here, integrated spatially resolved metabolomics and spatial transcriptomics revealed that accumulation of lipids in cervical cancer (CC) samples correlated with overexpression of RACK1, and RACK1 promoted lipid synthesis in CC cells. Chromatin immunoprecipitation verified binding of sterol regulatory element‐binding protein 1 (SREBP1) to acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FASN) promoters. RACK1 enhanced de novo FA synthesis by upregulating expression of sterol regulatory element binding transcription factor 1 (SREBP1) and lipogenic genes FASN and ACC1. Co‐immunoprecipitation and western blotting revealed that RACK1 interacted with protein kinase B (AKT) to activate the AKT/mammalian target of rapamycin (mTOR)/SREBP1 signaling pathway to promote FA synthesis. Cell proliferation and apoptosis experiments suggested that RACK1‐regulated FA synthesis is key in the progression of CC. Thus, RACK1 enhanced lipid synthesis through the AKT/mTOR/SREBP1 signaling pathway to promote the growth of CC cells. RACK1 may become a therapeutic target for CC.

Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti‐metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin‐ and KLK6‐based therapies, based on our previously developed mutants of the human amyloid β‐protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI‐3M (prostate and breast cancer) and APPI‐4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.

Endometrioid ovarian carcinoma landscape: pathological and molecular characterization

Endometrioid ovarian cancers (EOvC) are usually managed as serous tumors. In this study, we conducted a comprehensive molecular investigation to uncover the distinct biological characteristics of EOvC. This retrospective multicenter study involved patients from three European centers. We collected clinical data and formalin‐fixed paraffin‐embedded (FFPE) samples for analysis at the DNA level using panel‐based next‐generation sequencing and array‐comparative genomic hybridization. Additionally, we examined mRNA expression using NanoString nCounter® and protein expression through tissue microarray. We compared EOvC with other ovarian subtypes and uterine endometrioid tumors. Furthermore, we assessed the impact of molecular alterations on patient outcomes, including progression‐free survival (PFS) and overall survival (OS). Preliminary analysis of clinical data from 668 patients, including 86 (12.9%) EOvC, revealed more favorable prognosis for EOvC compared with serous ovarian carcinoma (5‐year OS of 60% versus 45%; P  = 0.001) driven by diagnosis at an earlier stage. Immunohistochemistry and copy number alteration (CNA) profiles of 43 cases with clinical data and FFPE samples available indicated that EOvC protein expression and CNA profiles were more similar to endometrioid endometrial tumors than to serous ovarian carcinomas. EOvC exhibited specific alterations, such as lower rates of PTEN loss, mutations in DNA repair genes, and P53 abnormalities. Survival analysis showed that patients with tumors harboring loss of PTEN expression had worse outcomes (median PFS 19.6 months vs. not reached; P  = 0.034). Gene expression profile analysis confirmed that EOvC differed from serous tumors. However, comparison to other rare subtypes of ovarian cancer suggested that the EOvC transcriptomic profile was close to that of ovarian clear cell carcinoma. Downregulation of genes involved in the PI3K pathway and DNA methylation was observed in EOvC. In conclusion, EOvC represents a distinct biological entity and should be regarded as such in the development of specific clinical approaches.

Tumor and germline testing with next generation sequencing in epithelial ovarian cancer: a prospective paired comparison using an 18‐gene panel

Genetic testing in epithelial ovarian cancer (EOC) in Ontario includes germline next‐generation sequencing (NGS) for 19 genes. Additionally, tumor tissue undergoes reflex NGS testing for BRCA1/2 to assess eligibility for PARPi. Although parallel testing confers advantages, this model duplicates healthcare resources. Here, we prospectively assessed the feasibility of tumor‐first multigene testing by comparing tumor tissue with germline testing of peripheral blood. An 18‐gene NGS panel was used to test tumor and germline DNA ( n  = 106 patients). In 26 patients, 27 tumor Tier I or II variants were identified, with 16/27 (59%) being germline pathogenic variants (PV) (13 BRCA1/2 ; 3 other genes) and 11/27 (41%) somatic variants (9 BRCA1/2 ; 2 other). In 51/106 patients, there were no tumor variants (excluding TP53 ), of which one patient had a germline BRCA1 copy number variant deletion in exon 12. Tumor‐first testing detected variant‐positive and variant‐negative germline cases in 105/106 patients (99.1%). Among 50 BRCA ‐negative patients, 14/50 (28%) were homologous recombination deficiency (HRD)‐positive. Therefore, we demonstrate that multigene NGS tumor‐testing is effective in identifying germline variants in EOC with a < 1% false‐negative rate.

Cell‐free DNA from ascites identifies clinically relevant variants and tumour evolution in patients with advanced ovarian cancer

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour‐enriched tissue samples. The use of cell‐free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next‐generation sequencing was performed on ascites‐derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites‐derived tumour cells ( n  = 5) and archived formalin‐fixed paraffin‐embedded (FFPE) tissue ( n  = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large‐scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy.

Systematic profiling of cancer‐fibroblast interactions reveals drug combinations in ovarian cancer

Ovarian cancer (OC) is a leading cause of death of gynecological cancers in women. Poor patient response to treatment highlights the need to better understand how the tumor microenvironment affects OC progression. Growing evidence indicates the crucial role of non‐cancerous components, such as cancer‐associated fibroblasts, in establishing a complex network of cellular and molecular interactions, influencing cancer progression and response to treatment. Therefore, in this study, we sought to characterize the impact of fibroblasts on OC cell behavior and drug response. Using both direct and indirect cell co‐culture systems, we observed distinct changes in cancer cell proliferation, morphology, and secretome in the presence of fibroblasts. Furthermore, an imaging‐based high‐throughput drug screen of 528 oncology compounds revealed multiple drugs that showed altered efficacy in the co‐culture conditions, demonstrating the role of fibroblasts in driving cancer cell resistance to treatment. Most importantly, our data identified the two drug combinations of Birinapant or Vorinostat with Carboplatin as promising treatments, exploiting the altered cancer cell phenotype in co‐cultures. These findings were supported by the increased sensitivity of ex vivo cultures to these combinations.

Regulation and tumor‐suppressive function of the miR‐379/miR‐656 (C14MC) cluster in cervical cancer

Cervical cancer (CC) is a key contributor to cancer‐related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR‐379/miR‐656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5‐aza‐2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation‐regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral‐based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post‐transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer‐related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3‐kinase (PI3K)– AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT , MYC , and cyclin E1 ( CCNE1 ) and the overexpression of p16 , p21 , and p27 . We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca 2+ levels, and lipid peroxidation rates, and inhibits epithelial–mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase‐3 ( PDK3 ) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR‐494‐mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor‐suppressive and methylation‐regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.

Ribosome biogenesis‐based predictive biomarkers in endocrine therapy (Anastrozole) combined with mTOR inhibitor (Vistusertib) in endometrial cancer: translational study from the VICTORIA trial in collaboration with the GINECO group

Resistance of advanced hormone‐dependent endometrial carcinoma to endocrine therapy remains a worldwide clinical issue. We recently reported that the combination of Vistusertib (V, mTOR inhibitor) and Anastrozole (A, aromatase inhibitor) improves the progression‐free rate compared to Anastrozole alone. However, a better patient selection based on biomarkers would improve patient outcome. We evaluate for the first time the usage of ribosome biogenesis (RiBi) factors as a source of innovative markers. Using 47 FFPE tumours (A n  = 18; V + A n  = 29), 32 blood samples (A n  = 13; V + A n  = 19) and 30 samples of total RNAs (A n  = 12; V + A n  = 18) from the VICTORIA clinical trial, we observed an association between RiBi‐associated markers and drug activity or prediction of treatment response. NOP10 and NHP2 mRNA levels were significantly higher in non‐responders compared to responders in the Vistusertib + Anastrozole arm ( P  = 0.0194 and P  = 0.0002 respectively; i.e. 8 weeks progression‐free survival as endpoint). This study provides RiBi‐based markers relevant for a better selection of patients with advanced endometrial carcinoma by predicting the response of endocrine therapy combined with mTOR inhibitor.

Sacituzumab govitecan, an antibody‐drug conjugate targeting trophoblast cell‐surface antigen 2, shows cytotoxic activity against poorly differentiated endometrial adenocarcinomas in vitro and in vivo

Endometrial cancer is the most common gynecologic malignancy in developed countries. The antibody–drug conjugate (ADC) sacituzumab govitecan (SG) targets trophoblast cell‐surface antigen‐2 (Trop‐2) – a cell‐surface glycoprotein highly expressed in many epithelial tumors – and delivers the active metabolite of irinotecan SN‐38 to Trop‐2‐positive tumor cells. We evaluated Trop‐2 expression in endometrial endometrioid carcinoma (EC) tissues and the activity of SG against primary poorly differentiated EC cell lines and xenografts. Trop‐2 expression was assessed in 143 formalin‐fixed–paraffin‐embedded tumors and seven primary tumor cell lines by immunohistochemistry and flow cytometry, respectively. Cell viability of primary tumor cell lines was assessed following exposure to SG, or control antibodies. Antibody‐dependent cell cytotoxicity (ADCC) against Trop‐2‐positive and Trop‐2‐negative EC cell lines was measured in vitro using 4‐h chromium release assays. A Trop‐2‐positive EC xenograft model was used to determine the in vivo activity of SG. Moderate‐to‐strong staining was detected in 84% (120/143) of EC samples, whereas 43% (3/7) of the primary EC cell lines tested overexpressed Trop‐2. EC cell lines overexpressing Trop‐2 were significantly more sensitive to SG compared to control ADC (P = 0.014 and P = 0.005). Both SG and the unconjugated parental antibody hRS7 mediated high ADCC against Trop‐2‐positive cell lines. Moreover, SG induced significant bystander killing of Trop‐2‐negative tumors cocultured with Trop‐2‐positive tumors. In the xenograft model, intravenous administration of SG twice weekly for three weeks was well tolerated and demonstrated impressive tumor growth inhibition against poorly differentiated, chemotherapy‐resistant EC xenografts (P = 0.011). In summary, SG is a novel ADC with remarkable preclinical activity against poorly differentiated EC cell lines overexpressing Trop‐2. These findings warrant future clinical trials.

Resistance prediction in high‐grade serous ovarian carcinoma with neoadjuvant chemotherapy using data‐independent acquisition proteomics and an ovary‐specific spectral library

High‐grade serous ovarian carcinoma (HGSOC) is the most common subtype of ovarian cancer with 5‐year survival rates below 40%. Neoadjuvant chemotherapy (NACT) followed by interval debulking surgery (IDS) is recommended for patients with advanced‐stage HGSOC unsuitable for primary debulking surgery (PDS). However, about 40% of patients receiving this treatment exhibited chemoresistance of uncertain molecular mechanisms and predictability. Here, we built a high‐quality ovary‐specific spectral library containing 130 735 peptides and 10 696 proteins on Orbitrap instruments. Compared to a published DIA pan‐human spectral library (DPHL), this spectral library provides 10% more ovary‐specific and 3% more ovary‐enriched proteins. This library was then applied to analyze data‐independent acquisition (DIA) data of tissue samples from an HGSOC cohort treated with NACT, leading to 10 070 quantified proteins, which is 9.73% more than that with DPHL. We further established a six‐protein classifier by parallel reaction monitoring (PRM) to effectively predict the resistance to additional chemotherapy after IDS (Log‐rank test, P  = 0.002). The classifier was validated with 57 patients from an independent clinical center ( P  = 0.014). Thus, we have developed an ovary‐specific spectral library for targeted proteome analysis, and propose a six‐protein classifier that could potentially predict chemoresistance in HGSOC patients after NACT‐IDS treatment.

Plasma membrane SK2 channel activity regulates migration and chemosensitivity of high‐grade serous ovarian cancer cells

No data are currently available on the functional role of small conductance Ca 2+ ‐activated K + channels (SKCa) in ovarian cancer. Here, we characterized the role of SK2 (KCa2.2) in ovarian cancer cell migration and chemosensitivity. Using the selective non‐cell‐permeant SK2 inhibitor Lei‐Dab7, we identified functional SK2 channels at the plasma membrane, regulating store‐operated Ca 2+ entry (SOCE) in both cell lines tested (COV504 and OVCAR3). Silencing KCNN2 with short interfering RNA (siRNA), or blocking SK2 activity with Lei‐Dab7, decreased cell migration. The more robust effect of KCNN2 knockdown compared to Lei‐Dab7 treatment suggested the involvement of functional intracellular SK2 channels in both cell lines. In cells treated with lysophosphatidic acid (LPA), an ovarian cancer biomarker of progression, SK2 channels are a key player of LPA pro‐migratory activity but their role in SOCE is abolished. Concerning chemotherapy, SK2 inhibition increased chemoresistance to Taxol® and low KCNN2 mRNA expression was associated with the worst prognosis for progression‐free survival in patients with serous ovarian cancer. The dual roles of SK2 mean that SK2 activators could be used as an adjuvant chemotherapy to potentiate treatment efficacy and SK2 inhibitors could be administrated as monotherapy to limit cancer cell dissemination.

Cis‐regulatory effect of HPV integration is constrained by host chromatin architecture in cervical cancers

Human papillomavirus (HPV) infections are the primary drivers of cervical cancers, and often HPV DNA gets integrated into the host genome. Although the oncogenic impact of HPV encoded genes is relatively well known, the cis‐regulatory effect of integrated HPV DNA on host chromatin structure and gene regulation remains less understood. We investigated genome‐wide patterns of HPV integrations and associated host gene expression changes in the context of host chromatin states and topologically associating domains (TADs). HPV integrations were significantly enriched in active chromatin regions and depleted in inactive ones. Interestingly, regardless of chromatin state, genomic regions flanking HPV integrations showed transcriptional upregulation. Nevertheless, upregulation (both local and long‐range) was mostly confined to TADs with integration, but not affecting adjacent TADs. Few TADs showed recurrent integrations associated with overexpression of oncogenes within them (e.g. MYC, PVT1, TP63 and ERBB2) regardless of proximity. Hi‐C and 4C‐seq analyses in cervical cancer cell line (HeLa) demonstrated chromatin looping interactions between integrated HPV and MYC/PVT1 regions (~ 500 kb apart), leading to allele‐specific overexpression. Based on these, we propose HPV integrations can trigger multimodal oncogenic activation to promote cancer progression.

Th17 cells target the metabolic miR ‐142‐5p–succinate dehydrogenase subunit C/D ( SDHC / SDHD ) axis, promoting invasiveness and progression of cervical cancers

During cervical carcinogenesis, T‐helper (Th)‐17 cells accumulate in the peripheral blood and tumor tissues of cancer patients. We previously demonstrated that Th17 cells are associated with therapy resistance as well as cervical cancer metastases and relapse; however, the underlying Th17‐driven mechanisms are not fully understood. Here, using microarrays, we found that Th17 cells induced an epithelial‐to‐mesenchymal transition (EMT) phenotype of cervical cancer cells and promoted migration and invasion of 2D cultures and 3D spheroids via induction of microRNA miR‐142‐5p. As the responsible mechanism, we identified the subunits C and D of the succinate dehydrogenase (SDH) complex as new targets of miR‐142‐5p and provided evidence that Th17–miR‐142‐5p‐dependent reduced expression of SDHC and SDHD mediated enhanced migration and invasion of cancer cells using small interfering RNAs (siRNAs) for SDHC and SDHD, and miR‐142‐5p inhibitors. Consistently, patients exhibited high levels of succinate in their serum associated with lymph node metastases and diminished expression of SDHD in patient biopsies correlated with increased numbers of Th17 cells. Correspondingly, a combination of weak or negative SDHD expression and a ratio of Th17/CD4 + T cells > 43.90% in situ was associated with reduced recurrence‐free survival. In summary, we unraveled a previously unknown molecular mechanism by which Th17 cells promote cervical cancer progression and suggest evaluation of Th17 cells as a potential target for immunotherapy in cervical cancer.

Circulating cell‐free HPV DNA is a strong marker for disease severity in cervical cancer

For cervical cancer (CC), circulating cell‐free HPV DNA (ccfHPV) may establish disease severity. Furthermore, HPV integration has been correlated to viral load and survival. In this study, pre‐treatment plasma from 139 CC cases (50 primary surgery patients, 22 primary surgery + adjuvant oncological therapy patients, and 67 primary oncological therapy patients) was collected (2018–2020). Furthermore, plasma from 25 cervical intraepithelial neoplasia grade 3 patients and 15 healthy women (negative controls) were collected. Two next‐generation sequencing (NGS) panels were used to establish ccfHPV presence and human papillomavirus type 16 (HPV16) integration status. ccfHPV was detected in four primary surgery (8.0%), eight primary surgery + adjuvant oncology (36.4%), and 54 primary oncology (80.6%) patients. For primary oncology patients with HPV16‐related cancer ( n  = 37), more ccfHPV neg than ccfHPV pos patients had HPV16 integration ( P  = 0.04), and in patients with HPV16 integration ( n  = 13), ccfHPV pos patients had higher disease stages than ccfHPV neg patients ( P  = 0.05). In summary, ccfHPV presence is related to disease severity and may add to the debated Sedlis criteria used for identifying patients for adjuvant oncological therapy. However, ccfHPV detection is influenced by HPV integration status and disease stage, and these factors need to be considered in ccfHPV neg patients.

BRCA1 promoter hypermethylation on circulating tumor DNA correlates with improved survival of patients with ovarian cancer

Methylation of the BRCA1 promoter is an epigenetic gene expression regulator and is frequently observed in ovarian cancer; however, conversion of methylation status is thought to drive disease recurrence. Therefore, longitudinal monitoring of methylation status by liquid biopsy in cell‐free DNA may be a predictive marker. In total, 135 plasma samples were collected from 69 ovarian cancer patients before and during systemic treatment. Our liquid biopsy assay could detect down to a single molecule of methylated DNA in a high background of normal DNA (0.03%) with perfect specificity in control samples. We found that 60% of the cancer patients exhibited BRCA1 promoter hypermethylation at one point, although 24% lost hypermethylation during treatment. Multivariate survival analyses indicate that relapses are independent events and that hypermethylation and methylation conversion are independently correlated to longer relapse‐free survival. We present a highly sensitive and specific methylation‐specific quantitative PCR‐based liquid biopsy assay. BRCA1 promoter hypermethylation is frequently found in ovarian cancer and is often reversed upon recurrence, indicating the selection of therapy‐resistant clones and unfavorable clinical outcome.

High‐grade serous peritoneal cancer follows a high stromal response signature and shows worse outcome than ovarian cancer

In the era of personalized medicine, where transition from organ‐based to individualized genetic diagnosis takes place, the tailoring of treatment in cancer becomes increasingly important. This is particularly true for high‐grade, advanced FIGO stage serous adenocarcinomas of the ovary (OC), fallopian tube (TC), and peritoneum (PC), which are currently all treated identically. We analyzed three independent patient cohorts using histopathologically classified diagnosis and various molecular approaches (transcriptomics, immunohistochemistry, next‐generation sequencing, fluorescent and chromogenic in situ hybridization). Using multivariate Cox regression model, we found that PC is more aggressive compared with advanced‐stage OC independent of residual disease as shown by an earlier relapse‐free survival in two large cohorts (HR: 2.63, CI: 1.59–4.37, P < 0.001, and HR: 1.66, CI: 1.04–2.63, P < 0.033). In line with these findings, transcriptomic data revealed differentially expressed gene signatures identifying PC as high stromal response tumors. The third independent cohort (n = 4054) showed a distinction between these cancer types for markers suggested to be predictive for chemotherapy drug response. Our findings add additional evidence that ovarian and peritoneal cancers are epidemiologically and molecularly distinct diseases. Moreover, our data also suggest consideration of the tumor‐sampling site for future diagnosis and treatment decisions.

In vivo anti‐V‐ATPase antibody treatment delays ovarian tumor growth by increasing antitumor immune responses

Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar‐ATPase (V‐ATPase) proton pumps encompass unique plasma membrane‐associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the in vivo therapeutic efficacy of an antibody (a2v‐mAB) targeting specific V‐ATPase‐‘V0a2’ surface isoform in controlling ovarian tumor growth. In vitro a2v‐mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. In vivo intraperitoneal a2v‐mAb treatment drastically delayed ovarian tumor growth with no measurable in vivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v‐mAb‐treated tumor tissues displayed high immune cell infiltration (M1‐macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN‐y, IL‐1α) compared to control. There was marked decrease in CA‐125‐positive cancer cells and an enhanced active caspase‐3 expression in a2v‐mAb‐treated tumors. RNA‐seq analysis of a2v‐mAb tumor tissues further revealed upregulation of apoptosis‐related and toll‐like receptor pathway‐related genes. Indirect coculture of a2v‐mAb‐treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN‐y, IL‐17, and IL‐12‐A in PBMCs, further validating the in vivo antitumor responses. In conclusion, V‐ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.

Epithelial/mesenchymal heterogeneity of high‐grade serous ovarian carcinoma samples correlates with miRNA let‐7 levels and predicts tumor growth and metastasis

Patient‐derived samples present an advantage over current cell line models of high‐grade serous ovarian cancer (HGSOC) that are not always reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we set out to characterize a panel of patient‐derived cells and determine their epithelial and mesenchymal characteristics. We analyzed RNA and protein expression levels in patient‐derived xenograft (PDX) models of HGSOC, and functionally characterized these models using flow cytometry, wound healing assays, invasion assays, and spheroid cultures. Besides in vitro work, we also evaluated the growth characteristics of PDX in vivo (orthotopic PDX). We found that all samples had hybrid characteristics, covering a spectrum from an epithelial‐to‐mesenchymal state. Samples with a stronger epithelial phenotype were more active in self‐renewal assays and more tumorigenic in orthotopic xenograft models as compared to samples with a stronger mesenchymal phenotype, which were more migratory and invasive. Additionally, we observed an inverse association between microRNA let‐7 (lethal‐7) expression and stemness, consistent with the loss of let‐7 being an important component of the cancer stem cell phenotype. We observed that lower let‐7 levels were associated with the epithelial state and a lower epithelial mesenchymal transition (EMT) score, more efficient spheroid and tumor formation, and increased sensitivity to platinum‐based chemotherapy. Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: Cells with lower let‐7 levels were more tumorigenic, but less migratory, and with a lower EMT score, than those with higher let‐7 levels. We conclude that let‐7 expression and epithelial/mesenchymal state are valuable predictors of HGSOC proliferation, in vitro self‐renewal, and tumor burden in vivo.

Upregulation of mesothelial genes in ovarian carcinoma cells is associated with an unfavorable clinical outcome and the promotion of cancer cell adhesion

A hallmark of ovarian high‐grade serous carcinoma (HGSC) is its early and massive peritoneal dissemination via the peritoneal fluid. It is generally believed that tumor cells must breach the mesothelium of peritoneal organs to adhere to the underlying extracellular matrix (ECM) and initiate metastatic growth. However, the molecular mechanisms underlying these processes are only partially understood. Here, we have analyzed 52 matched samples of spheroids and solid tumor masses (suspected primary lesions and metastases) from 10 patients by targeted sequencing of 21 loci previously proposed as targets of HGSC driver mutations. This analysis revealed very similar patterns of genetic alterations in all samples. One exception was FAT3 with a strong enrichment of mutations in metastases compared with presumed primary lesions in two cases. FAT3 is a putative tumor suppressor gene that codes for an atypical cadherin, pointing a potential role in peritoneal dissemination in a subgroup of HGSC patients. By contrast, transcriptome data revealed clear and consistent differences between tumor cell spheroids from ascites and metastatic lesions, which were mirrored by the in vitro adherence of ascites‐derived spheroids. The adhesion‐induced transcriptional alterations in metastases and adherent cells resembled epithelial–mesenchymal transition, but surprisingly also included the upregulation of a specific subset of mesothelial genes, such as calretinin (CALB2) and podoplanin (PDPN). Consistent with this finding, calretinin staining was also observed in subsets of tumor cells in HGSC metastases, particularly at the invasive tumor edges. Intriguingly, a high expression of either CALB2 or PDPN was strongly associated with a poor clinical outcome. siRNA‐mediated CALB2 silencing triggered the detachment of adherent HGSC cells in vitro and inhibited the adhesion of detached HGSC cells to collagen type I. Our data suggest that the acquisition of a mesenchymal–mesothelial phenotype contributes to cancer cell adhesion to the ECM of peritoneal organs and HGSC progression.

The RAD52 S346X variant reduces risk of developing breast cancer in carriers of pathogenic germline BRCA2 mutations

Women who carry pathogenic mutations in BRCA1 and BRCA2 have a lifetime risk of developing breast cancer of up to 80%. However, risk estimates vary in part due to genetic modifiers. We investigated the association of the RAD52 S346X variant as a modifier of the risk of developing breast and ovarian cancers in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. The RAD52 S346X allele was associated with a reduced risk of developing breast cancer in BRCA2 carriers [per‐allele hazard ratio (HR) = 0.69, 95% confidence interval (CI) 0.56–0.86; P = 0.0008] and to a lesser extent in BRCA1 carriers (per‐allele HR = 0.78, 95% CI 0.64–0.97, P = 0.02). We examined how this variant affected DNA repair. Using a reporter system that measures repair of DNA double‐strand breaks (DSBs) by single‐strand annealing (SSA), expression of hRAD52 suppressed the loss of this repair in Rad52−/− mouse embryonic stem cells. When hRAD52 S346X was expressed in these cells, there was a significantly reduced frequency of SSA. Interestingly, expression of hRAD52 S346X also reduced the stimulation of SSA observed upon depletion of BRCA2, demonstrating the reciprocal roles for RAD52 and BRCA2 in the control of DSB repair by SSA. From an immunofluorescence analysis, we observed little nuclear localization of the mutant protein as compared to the wild‐type; it is likely that the reduced nuclear levels of RAD52 S346X explain the diminished DSB repair by SSA. Altogether, we identified a genetic modifier that protects against breast cancer in women who carry pathogenic mutations in BRCA2 (P = 0.0008) and to a lesser extent BRCA1 (P = 0.02). This RAD52 mutation causes a reduction in DSB repair by SSA, suggesting that defects in RAD52‐dependent DSB repair are linked to reduced tumor risk in BRCA2‐mutation carriers.

Diphenhydramine increases the therapeutic window for platinum drugs by simultaneously sensitizing tumor cells and protecting normal cells

Platinum‐based compounds remain a well‐established chemotherapy for cancer treatment despite their adverse effects which substantially restrict the therapeutic windows of the drugs. Both the cell type‐specific toxicity and the clinical responsiveness of tumors have been associated with mechanisms that alter drug entry and export. We sought to identify pharmacological agents that promote cisplatin (CP) efficacy by augmenting the levels of drug‐induced DNA lesions in malignant cells and simultaneously protecting normal tissues from accumulating such damage and from functional loss. Formation and persistence of platination products in the DNA of individual nuclei were measured in drug‐exposed cell lines, in primary human tumor cells and in tissue sections using an immunocytochemical method. Using a mouse model of CP‐induced toxicity, the antihistaminic drug diphenhydramine (DIPH) and two methylated derivatives decreased DNA platination in normal tissues and also ameliorated nephrotoxicity, ototoxicity, and neurotoxicity. In addition, DIPH sensitized multiple cancer cell types, particularly ovarian cancer cells, to CP by increasing intracellular uptake, DNA platination, and/or apoptosis in cell lines and in patient‐derived primary tumor cells. Mechanistically, DIPH diminished transport capacity of CP efflux pumps MRP2, MRP3, and MRP5 particularly in its C2+C6 bimethylated form. Overall, we demonstrate that DIPH reduces side effects of platinum‐based chemotherapy and simultaneously inhibits key mechanisms of platinum resistance. We propose that measuring DNA platination after ex vivo exposure may predict the responsiveness of individual tumors to DIPH‐like modulators.

A SIRPα‐Fc fusion protein enhances the antitumor effect of oncolytic adenovirus against ovarian cancer

Oncolytic viruses armed with therapeutic transgenes of interest show great potential in cancer immunotherapy. Here, a novel oncolytic adenovirus carrying a signal regulatory protein‐α (SIRPα)‐IgG1 Fc fusion gene (termed SG635‐SF) was constructed, which could block the CD47 ‘don't eat me’ signal of cancer cells. A strong promoter sequence (CCAU) was chosen to control the expression of the SF fusion protein, and a 5/35 chimeric fiber was utilized to enhance the efficiency of infection. As a result, SG635‐SF was found to specifically proliferate in hTERT‐positive cancer cells and largely increased the abundance of the SF gene. The SF fusion protein was effectively detected, and CD47 was successfully blocked in SK‐OV3 and HO8910 ovarian cancer cells expressing high levels of CD47. Although the ability to induce cell cycle arrest and cell death was comparable to that of the control empty SG635 oncolytic adenovirus in vitro, the antitumor effect of SG635‐SF was significantly superior to that of SG635 in vivo. Furthermore, CD47 was largely blocked and macrophage infiltration distinctly increased in xenograft tissues of SK‐OV3 cells but not in those of CD47‐negative HepG2 cells, indicating that the enhanced antitumor effect of SG635‐SF was CD47‐dependent. Collectively, these findings highlight a potent antitumor effect of SG635‐SF in the treatment of CD47‐positive cancers.

MAP4K4 and WT1 mediate SOX6‐induced cellular senescence by synergistically activating the ATF2–TGFβ2–Smad2/3 signaling pathway in cervical cancer

SRY‐box transcription factor 6 (SOX6) is a member of the SOX gene family and inhibits the proliferation of cervical cancer cells by inducing cell cycle arrest. However, the final cell fate and significance of these cell‐cycle‐arrested cervical cancer cells induced by SOX6 remains unclear. Here, we report that SOX6 inhibits the proliferation of cervical cancer cells by inducing cellular senescence, which is mainly mediated by promoting transforming growth factor beta 2 (TGFB2) gene expression and subsequently activating the TGFβ2–Smad2/3–p53–p21WAF1/CIP1–Rb pathway. SOX6 promotes TGFB2 gene expression through the MAP4K4–MAPK (JNK/ERK/p38)–ATF2 and WT1–ATF2 pathways, which is dependent on its high‐mobility group (HMG) domain. In addition, the SOX6‐induced senescent cervical cancer cells are resistant to cisplatin treatment. ABT‐263 (navitoclax) and ABT‐199 (venetoclax), two classic senolytics, can specifically eliminate the SOX6‐induced senescent cervical cancer cells, and thus significantly improve the chemosensitivity of cisplatin‐resistant cervical cancer cells. This study uncovers that the MAP4K4/WT1–ATF2–TGFβ2 axis mediates SOX6‐induced cellular senescence, which is a promising therapeutic target in improving the chemosensitivity of cervical cancer.

CRISPR targeting of FOXL2 c. 402C >G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti‐tumoral compounds

Forkhead box L2 ( FOXL2 ) encodes a transcription factor essential for sex determination, and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9‐mediated strategy selectively targets the mutation without affecting the wild‐type allele. Granulosa cells lacking FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation and invasion. Furthermore, these modified cells are more susceptible to dasatinib and ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR‐modified granulosa tumor cells shift their expression profiles towards a wild‐type‐like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro‐apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing GCTs, highlighting its therapeutic promise for treating this rare ovarian cancer.

Deubiquitinase PSMD14 promotes ovarian cancer progression by decreasing enzymatic activity of PKM2

Dysregulation of deubiquitination has been reported to contribute to carcinogenesis. However, the function and mechanism of deubiquitinating enzyme 26S proteasome non‐ATPase regulatory subunit 14 (PSMD14) in the progression of ovarian cancer (OV), the deadliest gynecological cancer, still remains to be characterized. The present study demonstrated that PSMD14 was overexpressed in OV tissues and its higher levels correlated with a higher International Federation of Gynecology and Obstetrics (FIGO) stage in OV patients. A high level of PSMD14 expression was related to poor survival in OV patients. Knockdown and overexpression experiments elucidated that PSMD14 stimulated OV cell proliferation, invasion, and migration in vitro. Repression of PSMD14 suppressed OV tumor growth in vivo. PSMD14 inhibitor O‐phenanthroline (OPA) effectively attenuated malignant behaviors of OV cells in vitro and OV tumor growth in vivo. Mechanistically, we uncovered that PSMD14 was involved in post‐translational regulation of pyruvate kinase M2 isoform (PKM2). PSMD14 decreased K63‐linked ubiquitination on PKM2, downregulated the ratio of PKM2 tetramers to dimers and monomers, and subsequently diminished pyruvate kinase activity and induced nuclear translocation of PKM2, contributing to aerobic glycolysis in OV cells. Collectively, our findings highlight the potential roles of PSMD14 as a biomarker and therapeutic candidate for OV.

Molecular characteristics and tumorigenicity of ascites‐derived tumor cells: mitochondrial oxidative phosphorylation as a novel therapy target in ovarian cancer

Ovarian cancer disseminates primarily intraperitoneally. Detached tumor cell aggregates (spheroids) from the primary tumor are regarded as ‘metastatic units’ that exhibit a low sensitivity to classical chemotherapy, probably due to their unique molecular characteristics. We have analyzed the cellular composition of ascites from OvCa patients, using flow cytometry, and studied their behavior in vitro and in vivo . We conclude that ascites‐derived cultured cells from OvCa patients give rise to two subpopulations: adherent cells and non‐adherent cells. Here, we found that the AD population includes mainly CD90 + cells with highly proliferative rates in vitro but no tumorigenic potential in vivo , whereas the NAD population contains principally tumor cell spheroids (EpCAM + /CD24 + ) with low proliferative potential in vitro . Enriched tumor cell spheroids from the ascites of high‐grade serous OvCA patients, obtained using cell strainers, were highly tumorigenic in vivo and their metastatic spread pattern precisely resembled the tumor dissemination pattern found in the corresponding patients. Comparative transcriptome analyses from ascites‐derived tumor cell spheroids ( n  = 10) versus tumor samples from different metastatic sites ( n  = 30) revealed upregulation of genes involved in chemoresistance ( TGM1 , HSPAs, MT1s), cell adhesion and cell‐barrier integrity ( PKP3 , CLDNs, PPL ), and the oxidative phosphorylation process. Mitochondrial markers (mass and membrane potential) showed a reduced mitochondrial function in tumoroids from tumor tissue compared with ascites‐derived tumor spheroids in flow cytometry analysis. Interestingly, response to OXPHOS inhibition by metformin and IACS010759 in tumor spheroids correlated with the extent of mitochondrial membrane potential measured by fluorescence‐activated cell sorting. Our data contribute to a better understanding of the biology of ovarian cancer spheroids and identify the OXPHOS pathway as new potential treatment option in advanced ovarian cancer.

The levels of soluble cMET ectodomain in the blood of patients with ovarian cancer are an independent prognostic biomarker

The tyrosine kinase mesenchymal–epithelial transition (cMET) is typically overexpressed in up to 75% of patients with ovarian cancer, and cMET overexpression has been associated with poor prognosis. The proteolytic release of the soluble cMET (sMET) ectodomain by metalloproteases, a process called ectodomain shedding, reflects the malignant potential of tumour cells. sMET can be detected in the human circulation and has been proposed as biomarker in several cancers. However, the clinical relevance of sMET in ovarian cancer as blood‐based biomarker is unknown and was therefore investigated in this study. sMET levels were determined by enzyme‐linked immunosorbent assay in a set of 432 serum samples from 85 healthy controls and 86 patients with ovarian cancer (87% FIGO III/IV). Samples were collected at primary diagnosis, at four longitudinal follow‐up time points during the course of treatment and at disease recurrence. Although there was no significant difference between median sMET levels at primary diagnosis of ovarian cancervs. healthy controls, increased sMET levels at primary diagnosis were an independent predictor of shorter PFS (HR = 0.354, 95% CI: 0.130–0.968,P = 0.043) and shorter OS (HR = 0.217, 95% CI: 0.064–0.734,P = 0.014). In the follow‐up samples, sMET levels were prognostically most informative after the first three cycles of chemotherapy, with high sMET levels being an independent predictor of shorter PFS (HR = 0.245, 95% CI: 0.100–0.602,P = 0.002). This is the first study to suggest that sMET levels in the blood can be used as an independent prognostic biomarker for ovarian cancer. Patients at high risk of recurrence and with poor prognosis, as identified based on sMET levels in the blood, could potentially benefit from cMET‐directed therapies or other targeted regimes, such as PARP inhibitors or immunotherapy.

CircATRNL1 activates Smad4 signaling to inhibit angiogenesis and ovarian cancer metastasis via miR‐378

Ovarian cancer is one of the most frequent carcinomas in females, and the occurrence rate is still rising despite many advances made. The pathogenesis of ovarian cancer remains greatly unclear. Here, we investigated the mechanisms of ovarian cancer, with the focus on circATRNL1. Human ovarian cancer tissues and cell lines were used to examine levels of circATRNL1, miR‐378, Smad4, AKT, and other proliferation‐related and migration‐related proteins. Cellular assays were used to determine cancer cell proliferation, invasion, migration, apoptosis, and angiogenesis. We validated the interactions of circATRNL1/miR‐378 and miR‐378/Smad4, and a mouse tumor xenograft model was employed to assess the effect of circATRNL1 on tumor growth and metastasis in vivo. We found that circATRNL1 was decreased while miR‐378 was increased in human ovarian cancer tissues and cells. circATRNL1 bound to miR‐378 while miR‐378 directly targeted Smad4. Overexpression of circATRNL1 or knockdown of miR‐378 suppressed angiogenesis and ovarian cancer cell proliferation, invasion, and migration via decreasing proliferation‐ and migration‐related proteins via miR‐378 or Smad4, respectively. Overexpression of circATRNL1 restrained ovarian cancer growth and abdominal metastasis in vivo. Our findings indicate that circATRNL1 acts as a miR‐378 sponge to active Smad4 signaling and suppresses angiogenesis and ovarian cancer metastasis.

Tetraspanin 1 promotes endometriosis leading to ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) reportedly develops from endometriosis. However, the molecular mechanism underlying its malignant progression to OCCC remains elusive. This study aimed to identify an essential gene in the malignant transformation of endometriosis to OCCC. We performed RNA sequencing in formalin‐fixed, paraffin‐embedded (FFPE) tissues of endometriosis (n = 9), atypical endometriosis (AtyEm) (n = 18), adjacent endometriosis to OCCC (AdjEm) (n = 7), and OCCC (n = 17). We found that tetraspanin 1 (TSPAN1) mRNA level was significantly increased by 2.4‐ (DESeq2) and 3.4‐fold (edgeR) in AtyEm and by 80.7‐ (DESeq2) and 101‐fold (edgeR) in OCCC relative to endometriosis. We confirmed that TSPAN1 protein level was similarly overexpressed in OCCC tissues and cell lines. In immortalized endometriosis cell lines, TSPAN1 overexpression enhanced cell growth and invasion. Mechanistically, TSPAN1 triggered AMP‐activated protein kinase (AMPK) activity, promoting endometriosis and cell growth. Upregulated levels of TSPAN1 are considered an early event in the development of high‐risk endometriosis that could progress to ovarian cancer. Our study suggests the potential of TSPAN1 as a screening candidate for high‐risk endometriosis.

DNA repair as a shared hallmark in cancer and ageing

Increasing evidence demonstrates that DNA damage and genome instability play a crucial role in ageing. Mammalian cells have developed a wide range of complex and well‐orchestrated DNA repair pathways to respond to and resolve many different types of DNA lesions that occur from exogenous and endogenous sources. Defects in these repair pathways lead to accelerated or premature ageing syndromes and increase the likelihood of cancer development. Understanding the fundamental mechanisms of DNA repair will help develop novel strategies to treat ageing‐related diseases. Here, we revisit the processes involved in DNA damage repair and how these can contribute to diseases, including ageing and cancer. We also review recent mechanistic insights into DNA repair and discuss how these insights are being used to develop novel therapeutic strategies for treating human disease. We discuss the use of PARP inhibitors in the clinic for the treatment of breast and ovarian cancer and the challenges associated with acquired drug resistance. Finally, we discuss how DNA repair pathway‐targeted therapeutics are moving beyond PARP inhibition in the search for ever more innovative and efficacious cancer therapies.

SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK

Salt‐inducible kinase 2 (SIK2; also known as serine/threonine‐protein kinase SIK2) is overexpressed in several cancers and has been implicated in cancer progression. However, the mechanisms by which SIK2 regulates cancer cell motility, migration and metastasis in ovarian cancer have not been fully discovered. Here, we identify that SIK2 promotes ovarian cancer cell motility, migration and metastasis in vitro and in vivo. Mechanistically, SIK2 regulated cancer cell motility and migration by myosin light chain kinase, smooth muscle (MYLK)‐meditated phosphorylation of myosin light chain 2 (MYL2). SIK2 directly phosphorylated MYLK at Ser343 and activated its downstream effector MYL2, promoting ovarian cancer cell motility and metastasis. In addition, we found that adipocytes induced SIK2 phosphorylation at Ser358 and MYLK phosphorylation at Ser343, enhancing ovarian cancer cell motility. Moreover, SIK2 protein expression was positively correlated with the expression of MYLK‐pS343 in ovarian cancer cell lines and tissues. The co‐expression of SIK2 and MYLK‐pS343 was associated with reduced median overall survival in human ovarian cancer samples. Taken together, SIK2 positively regulates ovarian cancer motility, migration and metastasis, suggesting that SIK2 is a potential candidate for ovarian cancer treatment.

The STAT3‐miR‐223‐TGFBR3/HMGCS1 axis modulates the progression of cervical carcinoma

Cervical cancer is induced by persistent infections with high‐risk human papillomaviruses (HPVs), which produce the early protein 6 of HPVs (E6)/E7 protein that is involved in cell transformation by interacting with several oncoproteins or tumor suppressors. However, the role of noncoding RNA in mediating the pathogenesis of cervical cancer remains unclear. Here, we report that the novel signal transducer and activator of transcription 3 (STAT3)‐microRNA‐223‐3p (miR‐223)‐TGFBR3/HMGCS1 axis regulated by the E6 protein controls cervical carcinogenesis. miR‐223 was highly expressed in cervical tumor tissues, whereas TGFBR3 or HMGCS1 was significantly downregulated. miR‐223 targeted the 3′‐UTRs of TGFBR3 and HMGCS1 and suppressed their expression, leading to increased anchorage‐independent growth and cervical squamous cell carcinoma (CSCC) tumor growth in vitro and in vivo. The increased expression of miR‐223 was mediated by the transcription factor STAT3, whose activity was enhanced by E6 in the context of interleukin (IL)‐6 stimulation. In addition, exosomal miR‐223 derived from CSCC cells induced IL‐6 secretion by monocyte/macrophage in a coculture system in vitro, and IL‐6 secretion, in turn, led to enhanced STAT3 activity in CSSC cells, forming a positive feedback loop. Furthermore, modified miR‐223 inhibitor effectively suppressed tumor growth in cell line‐derived xenograft model, suggesting that miR‐223 is a potential promising therapeutic target in CSCC. In conclusion, our results demonstrate that the STAT3‐miR‐223‐HMGCS1/TGFBR3 axis functions as a key signaling pathway in cervical cancer progression and provides a new therapeutic target.

Periostin+cancer‐associated fibroblasts promote lymph node metastasis by impairing the lymphatic endothelial barriers in cervical squamous cell carcinoma

Lymph node metastasis (LNM), a critical prognostic determinant in cancer patients, is critically influenced by the presence of numerous heterogeneous cancer‐associated fibroblasts (CAFs) in the tumor microenvironment. However, the phenotypes and characteristics of the various pro‐metastatic CAF subsets in cervical squamous cell carcinoma (CSCC) remain unknown. Here, we describe a CAF subpopulation with elevated periostin expression (periostin+CAFs), located in the primary tumor sites and metastatic lymph nodes, that positively correlated with LNM and poor survival in CSCC patients. Mechanistically, periostin+CAFs impaired lymphatic endothelial barriers by activating the integrin‐FAK/Src‐VE‐cadherin signaling pathway in lymphatic endothelial cells and consequently enhanced metastatic dissemination. In contrast, inhibition of the FAK/Src signaling pathway alleviated periostin‐induced lymphatic endothelial barrier dysfunction and its related effects. Notably, periostin‐CAFs were incapable of impairing endothelial barrier integrity, which may explain the occurrence of CAF‐enriched cases without LNM. In conclusion, we identified a specific periostin+CAF subset that promotes LNM in CSCC, mainly by impairing the lymphatic endothelial barriers, thus providing the basis for potential stromal fibroblast‐targeted interventions that block CAF‐dependent metastasis.

Vaccination against HPV: boosting coverage and tackling misinformation

The availability of human papillomavirus (HPV) vaccines and screening tests has raised the possibility of globally eliminating cervical cancer, which is caused by HPV. Cervical cancer is a very common malignancy worldwide, especially among deprived women. High vaccination coverage is key to the containment and eventual elimination of the infection. Public HPV vaccination programmes in Italy and Denmark were swiftly established and are among the most successful worldwide. Still, in both countries, it has been challenging to achieve and maintain the recommended coverage of > 80% in girls. In a well‐studied Italian region, vaccination coverage in girls at age 15 years (World Health Organization's gold standard) reached 76% in 2015 but decreased to 69% in 2018, likely due to work overload in public immunization centres. In Denmark, doubts about safety and efficacy of the HPV vaccine generated a decline in coverage among girls age 12–17, from 80% in 2013 down to 37% in 2015, when remedial actions made it rise again. Insights from these two countries are shared to illustrate the importance of monitoring coverage in a digital vaccine registry and promptly reacting to misinformation about vaccination.

HPV‐related methylation‐based reclassification and risk stratification of cervical cancer

Human papillomavirus (HPV) is a clear etiology of cervical cancer (CC). However, the associations between HPV infection and DNA methylation have not been thoroughly investigated. Additionally, it remains unknown whether HPV‐related methylation signatures can identify subtypes of CC and stratify the prognosis of CC patients. DNA methylation profiles were obtained from The Cancer Genome Atlas to identify HPV‐related methylation sites. Unsupervised clustering analysis of HPV‐related methylation sites was performed to determine the different CC subtypes. CC patients were categorized into cluster 1 (Methylation‐H), cluster 2 (Methylation‐M), and cluster 3 (Methylation‐L). Compared to Methylation‐M and Methylation‐L, Methylation‐H exhibited a significantly improved overall survival (OS). Gene set enrichment analysis (GSEA) was conducted to investigate the functions that correlated with different CC subtypes. GSEA indicated that the hallmarks of tumors, including KRAS signaling, TNFα signaling via NF‐κB, inflammatory response, epithelial–mesenchymal transition, and interferon‐gamma response, were enriched in Methylation‐M and Methylation‐L. Based on mutation and copy number variation analyses, we found that aberrant mutations, amplifications, and deletions among the MYC, Notch, PI3K‐AKT, and RTK‐RAS pathways were most frequently detected in Methylation‐H. Additionally, mutations, amplifications, and deletions within the Hippo, PI3K‐AKT, and TGF‐β pathways were presented in Methylation‐M. Genes within the cell cycle, Notch, and Hippo pathways possessed aberrant mutations, amplifications, and deletions in Methylation‐L. Moreover, the analysis of tumor microenvironments revealed that Methylation‐H was characterized by a relatively low degree of immune cell infiltration. Finally, a prognostic signature based on six HPV‐related methylation sites was developed and validated. Our study revealed that CC patients could be classified into three heterogeneous clusters based on HPV‐related methylation signatures. Additionally, we derived a prognostic signature using six HPV‐related methylation sites that stratified the OS of patients with CC into high‐ and low‐risk groups.

Genomic characterization of small cell carcinomas of the uterine cervix

Small cell carcinoma (SCC) of the uterine cervix is a rare and aggressive form of neuroendocrine carcinoma, which resembles small cell lung cancer (SCLC) in its histology and poor survival rate. Here, we sought to define the genetic underpinning of SCCs of the uterine cervix and compare their mutational profiles with those of human papillomavirus (HPV)‐positive head and neck squamous cell carcinomas, HPV‐positive cervical carcinomas, and SCLCs using publicly available data. Using a combination of whole‐exome and targeted massively parallel sequencing, we found that the nine uterine cervix SCCs, which were HPV18‐positive (n = 8) or HPV16‐positive (n = 1), harbored a low mutation burden, few copy number alterations, and other than TP53 in two cases no recurrently mutated genes. The majority of mutations were likely passenger missense mutations, and only few affected previously described cancer‐related genes. Using RNA‐sequencing, we identified putative viral integration sites on 18q12.3 and on 8p22 in two SCCs of the uterine cervix. The overall nonsilent mutation rate of uterine cervix SCCs was significantly lower than that of SCLCs, HPV‐driven cervical adeno‐ and squamous cell carcinomas, or HPV‐positive head and neck squamous cell carcinomas. Unlike SCLCs, which are reported to harbor almost universal TP53 and RB1 mutations and a dominant tobacco smoke‐related signature 4, uterine cervix SCCs rarely harbored mutations affecting these genes (2/9, 22% TP53; 0% RB1) and displayed a dominant aging (67%) or APOBEC mutational signature (17%), akin to HPV‐driven cancers, including cervical adeno‐ and squamous cell carcinomas and head and neck squamous cell carcinomas. Taken together, in contrast to SCLCs, which are characterized by highly recurrent TP53 and RB1 alterations, uterine cervix SCCs were positive for HPV leading to inactivation of the suppressors p53 and RB, suggesting that these SCCs are convergent phenotypes.

Chemoradiotherapy‐induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse

Cervical cancer therapy is still a major clinical challenge, as patients substantially differ in their response to standard treatments, including chemoradiotherapy (CRT). During cervical carcinogenesis, T‐helper (Th)‐17 cells accumulate in the peripheral blood and tumor tissues of cancer patients and are associated with poor prognosis. In this prospective study, we find increased Th17 frequencies in the blood of patients after chemoradiotherapy and a post‐therapeutic ratio of Th17/CD4 + T cells > 8% was associated with early recurrence. Furthermore, Th17 cells promote resistance of cervical cancer cells toward CRT, which was dependent on the AKT signaling pathway. Consistently, patients with high Th17 frequencies in pretherapeutic biopsies exhibit lower response to primary CRT. This work reveals a key role of Th17 cells in CRT resistance and elevated Th17 frequencies in the blood after CRT correspond with early recurrence. Our results may help to explain individual treatment responses of cervical cancer patients and suggest evaluation of Th17 cells as a novel predictive biomarker for chemoradiotherapy responses and as a potential target for immunotherapy in cervical cancer.

ERBB2 mutations define a subgroup of endometrial carcinomas associated with high tumor mutational burden and the microsatellite instability‐high (MSI‐H) molecular subtype

Anti‐HER2 therapy is indicated for erb‐b2 receptor tyrosine kinase 2 ( ERBB2 )‐amplified/overexpressing endometrial carcinoma (EC). Mutations constitute another mode of ERBB2 activation, but only rare ERBB2 ‐mutated ECs have been reported. We sought to characterize the clinicopathologic and genetic features of ERBB2 ‐mutated EC. From an institutional cohort of 2638 ECs subjected to clinical tumor‐normal panel sequencing, 69 (2.6%) with pathogenic ERBB2 mutation(s) were identified, of which 11 were also ERBB2 ‐amplified. The most frequent ERBB2 hotspot mutations were V842I (38%) and R678Q (25%). ERBB2 mutations were clonal in 87% of evaluable cases. Immunohistochemistry revealed low HER2 protein expression in most ERBB2 ‐mutated ECs (0/1+ in 66%, 2+ in 27%); all 3+ tumors (7.3%) were also ERBB2 ‐amplified. Compared to ERBB2 ‐wildtype ECs (with or without ERBB2 amplification), ERBB2 ‐mutated/non‐amplified ECs were enriched for the microsatellite instability‐high (MSI‐H) and, to a lesser extent, DNA polymerase epsilon, catalytic subunit ( POLE ) molecular subtypes, and associated with high tumor mutational burden and low chromosomal instability. Survival outcomes were similar between patients with ERBB2 ‐mutated/non‐amplified versus wildtype EC, whereas ERBB2 amplification was associated with worse prognosis on univariate, but not multivariate, analyses. In conclusion, ERBB2 mutation defines a rare subgroup of ECs that is pathogenically distinct from ERBB2 ‐wildtype and ERBB2 ‐amplified ECs.

miR‐200a/b/‐429 downregulation is a candidate biomarker of tumor radioresistance and independent of hypoxia in locally advanced cervical cancer

Many patients with locally advanced cervical cancer experience recurrence within the radiation field after chemoradiotherapy. Biomarkers of tumor radioresistance are required to identify patients in need of intensified treatment. Here, the biomarker potential of miR‐200 family members was investigated in this disease. Also, involvement of tumor hypoxia in the radioresistance mechanism was determined, using a previously defined 6‐gene hypoxia classifier. miR‐200 expression was measured in pretreatment tumor biopsies of an explorative cohort (n = 90) and validation cohort 1 (n = 110) by RNA sequencing. Publicly available miR‐200 data of 79 patients were included for the validation of prognostic significance. A score based on expression of the miR‐200a/b/‐429 (miR‐200a, miR‐200b, and miR‐429) cluster showed prognostic significance in all cohorts. The score was significant in multivariate analysis of central pelvic recurrence. No association with distant recurrence or hypoxia status was found. Potential miRNA target genes were identified from gene expression profiles and showed enrichment of genes in extracellular matrix organization and cell adhesion. miR‐200a/b/‐429 overexpression had a pronounced radiosensitizing effect in tumor xenografts, whereas the effect was minor in vitro. In conclusion, miR‐200a/b/‐429 downregulation is a candidate biomarker of central pelvic recurrence and seems to predict cell adhesion‐mediated tumor radioresistance independent of clinical markers and hypoxia.