Kentaro Nakayama

NAC1 Regulates PCK2 Expression and Activates Truncated Gluconeogenesis for Growth Advantage in Ovarian Cancer Cells

Nucleus accumbens-associated protein 1 (NAC1), a cancer-related transcriptional regulator, is overexpressed in several malignancies, including ovarian cancer. However, its role in ovarian carcinogenesis remains unclear. We aimed to investigate whether NAC1 contributes to metabolic adaptation in endometriosis-related ovarian neoplasms (ERONs) and elucidate its regulatory mechanisms. The clinical relationship between NAC1 and its potential downstream target, phosphoenolpyruvate carboxykinase isoform 2 (PCK2), was examined using immunohistochemical analysis of ovarian cancer specimens. A cell viability assay was performed to clarify the impact of PCK2 on ovarian cancer cell viability. Reporter and chromatin immunoprecipitation (ChIP) assays were conducted to evaluate transcriptional regulation by NAC1. Metabolomic profiling was performed to assess the functional impact of the NAC1–PCK2 axis. A positive correlation between NAC1 and PCK2 expression was observed, and co-expression was associated with poor long-term survival. Knockdown of PCK2 led to a significant reduction in cell viability, indicating that PCK2 is required for maintaining cell survival. Reporter and ChIP assays confirmed that NAC1 directly binds to the PCK2 promoter via the CATG motif. The metabolomic analysis demonstrated that NAC1 promotes truncated gluconeogenesis and de novo serine synthesis through PCK2 upregulation. These findings suggest that NAC1 contributes to ovarian cancer progression by promoting metabolic adaptation, highlighting the NAC1–PCK2 axis as a potential therapeutic target for ERONs.

NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms

NAC1, a transcription regulator protein associated with cancer, is highly expressed in several tumor types, including ovarian cancer. However, it remains unclear how NAC1 is involved in carcinogenesis. Our previous studies demonstrated that the knockdown of NAC1 in ovarian clear cell carcinoma (OCCC) cell lines induces apoptosis and restores their sensitivity to chemotherapy, suggesting NAC1 as a potential therapeutic target. The present study aimed to identify molecular pathways through which NAC1 is involved in the development of endometriosis-related ovarian neoplasms (ERONs). Immunohistochemistry was performed to clarify the relationship between NAC1 and the potential target protein ACOX2 in surgical specimens of ERONs. Reporter assays were conducted to determine the interaction of NAC1 with the specific cis-element on the ACOX2 promoter. Subsequently, a ChIP assay was performed to investigate the in vivo interaction of NAC1 with the ACOX2 promoter. There was an inverse relationship between NAC1 and ACOX2 expressions in the tumor specimens of ERONs. High NAC1/low ACOX2 expression was found to be a worse prognostic marker for patient survival. Reporter assays demonstrated that NAC1 negatively regulated the ACOX2 promoter via the proximal CATG site. ChIP assays confirmed in vivo binding of NAC1 to the promoter. The present study implicated that NAC1 may contribute to the development of ERONs as a transcriptional repressor by regulating ACOX2 expression via specific binding sites on the promoter, providing a novel insight into the NAC1/ACOX2 axis as a potential therapeutic target of this tumor type.

Exploring the Genetic and Clinical Landscape of Dedifferentiated Endometrioid Carcinoma

Dedifferentiated endometrioid carcinoma (DDEC) is rare, has a poor prognosis, and the genes responsible for dedifferentiation remain unclear. This study aimed to clarify the characteristics of DDEC in Japanese patients and develop treatment strategies. Eighteen DDEC cases were included; their clinicopathological features and prognoses were analyzed and compared to those of other histological subtypes. The samples were divided into well-differentiated and undifferentiated components; immunostaining and whole-exome sequencing (n = 3 cases) were conducted. The incidence of DDEC was 2.0% among endometrial cancers. The 5-year progression-free survival and the 5-year overall survival for DDEC was approximately 40% and 30%, respectively. Immunohistochemistry showed that 66.7% of patients were mismatch repair deficient. The rate of p53 mutations was higher than that reported in previous studies, and patients with p53 mutations in the undifferentiated components had a poor prognosis. Whole-exome sequencing revealed different gene mutations and mutation signatures between well-differentiated and undifferentiated components. New genetic mutations in undifferentiated regions were uncommon in all three cases. One case (case 1) exhibited homologous recombination deficiency, whereas the other two showed microsatellite instability-high and hypermutator phenotypes. Genetic analysis suggests that immune checkpoint and poly (ADP-ribose) polymerase inhibitors and drugs targeting the p53 pathway may be effective against DDEC.

Genomic Landscapes of Endometrioid and Mucinous Ovarian Cancers and Morphologically Similar Tumor Types

Abstract Endometrioid and mucinous ovarian carcinomas represent nearly a fifth of ovarian cancers, but their molecular characteristics and pathologic origins are poorly understood. To identify the genomic and epigenomic alterations characteristic of these ovarian cancer subtypes and evaluate links to morphologically similar tumors from other sites, we performed a combination of sequence, copy number, mutation signature, and rearrangement analyses from tumor samples and matched normal tissues of 133 patients, as well as methylation analyses of these tumors and tissues of 150 patients from The Cancer Genome Atlas. Genomic analyses included samples from patients with ovarian endometrioid (n = 44), ovarian mucinous (n = 43), uterine endometrioid (n = 15), and gastrointestinal mucinous carcinomas (n = 31), including mucinous carcinomas of the stomach, colon, and pancreas. In addition to identifying genes previously known to be involved in these tumors, we identified alterations in RAD51C, NOTCH4, SMARCA1/4, and JAK1 in ovarian endometrioid, ESR1 in uterine endometrioid, and SMARCA4 in ovarian mucinous carcinomas. Whole-genome sequencing revealed rearrangements involving PTEN, NF1, and NF2 in ovarian endometrioid carcinomas and NF1 and MED1 in ovarian mucinous carcinomas. The number of alterations, affected genes, and genome-wide methylation profiles were not distinguishable between ovarian and uterine endometrioid carcinomas, supporting the hypothesis that these tumors share a tissue of origin. In contrast, mutation and methylation patterns in ovarian mucinous carcinomas were different from gastrointestinal mucinous carcinomas. These analyses provide insights into the genomic landscapes and origins of mucinous and endometrioid ovarian carcinomas, providing new avenues for early clinical intervention and management of patients with these cancers. Significance: Integrative multi-omic analyses support a common tissue of origin between ovarian endometrioid and uterine endometrioid carcinomas but not between ovarian mucinous and gastric or pancreatic mucinous carcinomas.

Integrated Molecular and Functional Characterization of Cervical Small-Cell Neuroendocrine Carcinoma Using a 3D Organoid Model

Cervical small-cell neuroendocrine carcinoma (SCNEC) is a rare cervical cancer with high metastatic potential and is frequently associated with high-risk human papillomavirus (HPV) infection. Because of its low incidence, SCNEC remains understudied and treatment options are limited, posing major therapeutic challenges. This study aimed to characterize SCNEC at the molecular and functional levels to support more informed therapeutic strategies. Organoids and spheroids were generated from a cervical SCNEC biopsy, and a matched organoid-derived xenograft was established in immunodeficient mice. Model fidelity was evaluated by histopathology and immunohistochemistry. HPV status was assessed by p16 immunostaining and HPV18 PCR, and viral–host integration sites were inferred using whole-exome sequencing (WES) junction reads. WES was also used to compare shared somatic variants and copy-number alterations across the patient tumor, organoid, and xenograft. Drug responses were assessed in organoids and spheroids following exposure to a panel of chemotherapeutic agents and a targeted inhibitor. Organoids exhibited robust growth, morphologic maturation, and efficient recovery after cryopreservation. The organoids and matched xenografts faithfully recapitulated SCNEC, with preserved neuroendocrine differentiation (CD56, synaptophysin, and NSE positivity), a high Ki-67 proliferative index (>80%), and strong p16 expression. HPV18 status was conserved across the primary tumor, organoids, and xenografts, with an integration site at chr8 (8q24.21) associated with increased MYC expression. WES revealed strong cross-model concordance, including 26 shared somatic variants with a canonical PIK3CA hotspot mutation (p.E542K) and conserved oncogenic copy-number gains of PIK3CA, TERT, and MYC, as well as copy number loss of TP53. Functional assays showed dose-dependent loss of viability following exposure to conventional cytotoxic agents or an mTOR pathway inhibitor. This study presents the first integrated molecular and functional analyses of patient tumors and matched organoid and xenograft models in cervical SCNEC. These models offer robust resources for mechanistic studies and may enable precision therapeutic strategies for this rare malignancy.

A Comparative Analysis of Usual- and Gastric-Type Cervical Adenocarcinoma in a Japanese Population Reveals Distinct Clinicopathological and Molecular Features with Prognostic and Therapeutic Insights

Gastric-type cervical adenocarcinoma (GCA) is a rare and aggressive subtype of cervical adenocarcinoma. Despite its clinical significance, its molecular carcinogenesis and therapeutic targets remain poorly understood. This study aimed to compare the clinicopathological, immunohistochemical, and molecular profiles of GCA and usual-type cervical adenocarcinoma (UCA), exploring prognostic and therapeutic biomarkers in a Japanese population. A total of 110 cervical adenocarcinoma cases, including 16 GCA and 94 UCA cases, were retrospectively analyzed for clinicopathological features, and a panel of immunohistochemical markers was assessed. Sanger sequences were performed for the KRAS, PIK3CA, and BRAF genes, and survival and clinicopathological correlations were assessed using Kaplan–Meier and Cox regression analyses. GCA was significantly associated with more aggressive features than UCA, including lymph node involvement, advanced FIGO stages, increasing recurrence rate, and poor survival status. High ARID1B expression was observed in a subset of GCA cases and correlated with worse progression-free and overall survival. Additionally, PD-L1 expression was more frequent in GCA than UCA and was associated with unfavorable prognostic factors. Conversely, UCA cases showed strong p16 expression, supporting their HPV-driven pathogenesis. Molecular profiling revealed KRAS and PIK3CA mutations in both subtypes, while BRAF mutations were identified exclusively in GCA. These findings reveal distinct clinical and molecular profiles for both tumor types and underscore ARID1B and PD-L1 as predictive prognostic and therapeutic biomarkers in GCA, implicating the use of subtype-specific treatment strategies.

Histological and Genetic Diversity in Ovarian Mucinous Carcinomas: A Pilot Study

Tumor heterogeneity remains an ongoing challenge in the field of cancer therapy. Intratumor heterogeneity significantly complicates the diagnosis of cancer and presents challenging clinical problems due to resistance to drug therapy. This study aimed to elucidate the genetic changes histologically (mucinous cystadenoma (MCA), mucinous borderline tumor (MBT), and mucinous ovarian carcinoma (MOC)) in a portion of mucinous ovarian tumors within the same sample. Seven tumor samples obtained from different patients were used to evaluate the genetic mutations in each component. Intratumor genetic heterogeneity was observed in all patients; among them, BRAF (V600E) and p53 (T118I, P142S, T150I, and T170M) point mutations were observed in the MBT component, while KRAS (G12D and G13D) and PIK3CA (E545K) mutations were found in the MOC component. The current findings suggest that diverse genetic alterations occur in mucinous tumors, according to tumor histology. Tumor heterogeneity and genetic diversity in mucinous ovarian tumors might be the cause of treatment failure. Knowledge of intertumor heterogeneity may lead to an increased understanding of the tumor response to treatment.

Mucinous borderline ovarian tumors with BRAFV600E mutation may have low risk for progression to invasive carcinomas

Abstract Purpose Mucinous ovarian carcinomas (MOCs) are relatively rare. It has been proposed that a subset of mucinous cystadenomas (MCAs) may progress to mucinous borderline tumors (MBTs), and then to MOCs. KRAS is the predominantly mutated gene in MOC; however, other associated mutations and the mechanism underlying carcinogenesis in MOC remain unclear. Here, we assessed molecular genetic alterations in mucinous ovarian tumors and constructed mutation profiles. Methods Using the Sanger sequencing method, we assessed genetic mutations (KRAS, BRAF, TP53, and PIK3CA) in 16 cases of MOC, 10 cases of MBT, and 12 cases of MCA. Results Among MOC cases, the prevalence of G12D and G13D KRAS mutations was 43.8% (7/16). No MOC cases showed V600E BRAF and TP53 mutations. Among MBT cases, the prevalence of G12D KRAS mutation was 20.0% (2/10), those of TP53 and PIK3CA mutations were nil, and that of V600E BRAF mutation was 40% (4/10). None of the genetic mutations assessed were detected among MCA cases. Conclusion These results suggest that MBT with V600E BRAF mutation may rarely progress to MOC, while MBT with G12D or G13D KRAS mutation may more commonly progress to MOC.

High PD-1 expression level is associated with an unfavorable prognosis in patients with cervical adenocarcinoma

AbstractPurposeThe effectiveness of immunotherapy for cervical adenocarcinoma (CA) has not been demonstrated yet. Programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), and CD8 may be used as biomarkers of response to immune therapy in CA patients. In the present study, we aimed to investigate whether the expression levels of PD-1, PD-L1, and CD8 can predict the prognosis of patients with CA and their response to immune checkpoint inhibition therapy.MethodsIn the present study, the clinical stage for all 82 patients with cervical adenocarcinoma was classified according to the guidelines of the International Federation of Gynecology and Obstetrics (FIGO); there were 5, 48, 5, 14, 8, and 2 patients with stage IA, IB, IIA, IIB, IIIB, and IVB disease, respectively. The levels of PD-1, PD-L1, and CD8 were analyzed by the immunohistochemical analysis of the formalin-fixed paraffin-embedded tumor samples. The correlation between the expression levels and patient prognosis was analyzed using the Kaplan–Meier method and univariate and multivariate Cox proportional hazard regression models.ResultsWe observed a significant inverse correlation between the expression of PD-1 and CD8 (p = 0.001, chi-square test). We also found a significant inverse correlation between the expression of PD-L1 and CD8 (p = 0.027). The overall survival and progression-free survival rates were significantly worse in patients with positive PD-1 expression (p = 0.031;p = 0.087, respectively).ConclusionOur results suggest that a high PD-1 expression is associated with a poor prognosis in patients with CA. Further research is necessary to identify the molecular mechanisms that mediate this association.

Establishment of a Novel In Vitro and In Vivo Model to Understand Molecular Carcinogenesis of Endometriosis-Related Ovarian Neoplasms

The molecular mechanisms through which endometriosis-related ovarian neoplasms (ERONs) develop from benign endometrioma remain unclear. It is especially a long-standing mystery why ovarian endometrioma has the potential to develop into two representative histological subtypes: endometrioid ovarian carcinoma or clear cell ovarian carcinoma. This study aimed to investigate the molecular carcinogenesis of ERONs using newly developed in vitro and in vivo carcinogenesis models. Epithelial cells were isolated and purified from surgically removed benign endometrioma samples, followed by immortalization by overexpressing cyclinD1/CDK4 in combination with the human TERT gene. Immortalized cells were subjected to various molecular manipulations by combining knockout or overexpression of several candidate drivers, including ARID1A, KRAS, PIK3CA, AKT, and MYC, based on previous comprehensive genome-wide studies of ERONs. These cells were then inoculated into immunocompromised mice and evaluated for malignant transformation. Inoculated cells harboring a combination of three genetic alterations successfully developed tumors with malignant features in mice, whereas those with two genetic manipulations failed to do so. Especially, ARID1A gene knockout, combined with overexpressing the KRAS oncogenic mutant allele (or overexpressing AKT) and c-Myc overexpression led to efficient tumor formation. Of note, these three combinations of genetic alterations produced tumors that histologically represented typical clear cell carcinoma in SCID mice, while the same combination led to tumors with endometrioid histology in nude mice. A combination of ARID1A mutation, KRAS mutation or AKT activation, and c-Myc overexpression were confirmed to be the main candidate drivers for the development of ERONs, as suggested by comprehensive genetic analyses of ERONs. A tumor immune microenvironment involving B-cell signaling may contribute to the diverse histological phenotypes. The present model may help to clarify the molecular mechanisms of ERON carcinogenesis and understand their histological diversity and novel molecular targets.