Shiro Takamatsu

YAP1 Suppression by ZDHHC7 Is Associated with Ferroptosis Resistance and Poor Prognosis in Ovarian Clear Cell Carcinoma

Abstract Ovarian clear cell carcinoma (OCCC), which has unique clinical characteristics, arises from benign endometriotic cysts, forming an oxidative stress environment because of excess iron accumulation, and exhibits poor prognosis, particularly in advanced stages owing to resistance to conventional therapeutics. Ferroptosis is an iron-dependent form of programmed cell death induced by lipid peroxidation and controlled by Hippo signaling. We hypothesized that overcoming ferroptosis resistance is an attractive strategy because OCCC acquires oxidative stress resistance during its development and exhibits chemoresistant features indicative of ferroptosis resistance. This study aimed to determine whether OCCC is resistant to ferroptosis and clarify the mechanism underlying resistance. Unlike ovarian high-grade serous carcinoma cells, OCCC cells were exposed to oxidative stress. However, OCCC cells remained unaffected by lipid peroxidation. Cell viability assays revealed that OCCC cells exhibited resistance to the ferroptosis inducer erastin. Moreover, Samroc analysis showed that the Hippo signaling pathway was enriched in OCCC cell lines and clinical samples. Furthermore, patients with low expression of nuclear yes-associated protein 1 (YAP1) exhibited a significantly poor prognosis of OCCC. Moreover, YAP1 activation enhanced ferroptosis in OCCC cell lines. Furthermore, suppression of zinc finger DHHC-type palmitoyltransferase 7 (ZDHHC7) enhanced ferroptosis by activating YAP1 in OCCC cell lines. Mouse xenograft models demonstrated that ZDHHC7 inhibition suppressed tumor growth via YAP1 activation by erastin treatment. In conclusion, YAP1 activation regulated by ZDHHC7 enhanced ferroptosis in OCCC. Thus, overcoming ferroptosis resistance is a potential therapeutic strategy for OCCC.

Molecular classification of ovarian high-grade serous/endometrioid carcinomas through multi-omics analysis: JGOG3025-TR2 study

Considerable interobserver variability exists in diagnosis of ovarian high-grade endometrioid carcinoma (HGEC) and high-grade serous carcinoma (HGSC) due to histopathological similarities. While homologous recombination deficiency (HRD) correlates with drug sensitivity in HGSC, the molecular features of HGEC are unclear. Fresh-frozen samples from 15 ovarian HGECs and 274 ovarian HGSCs in the JGOG-TR2 cohort were submitted to targeted DNA sequencing, RNA sequencing, DNA methylation array, and SNP array. We additionally analyzed 555 ovarian HGSCs from TCGA-OV and 287 endometrial high-grade carcinomas from TCGA-UCEC. Unsupervised clustering using copy number signatures identified four distinct tumor groups (C1, C2, C3 and C4). C1 (n = 41) showed CCNE1 amplification and poor survival. C2 (n = 160) and C3 (n = 59) showed high BRCA1/2 alteration frequency with low and moderate ploidy, respectively. C4 (n = 22) was characterized by favorable outcome, higher HGEC proportion, no BRCA1/2 alteration or CCNE1 amplification, and low levels of HRD score, ploidy, intra-tumoral heterogeneity, cell proliferation rate, and WT1 gene expression. Notably, C4 exhibited a normal endometrium-like DNA methylation profile, thus, defined as "HGEC-type" tumors, which were also identified in TCGA-OV and TCGA-UCEC. Ovarian "HGEC-type" tumors present a non-HRD status, favorable prognosis, and endometrial differentiation, possibly constituting a subset of clinically diagnosed HGSCs.

Homologous Recombination Deficiency Unrelated to Platinum and PARP Inhibitor Response in Cell Line Libraries

AbstractWhile large publicly available cancer cell line databases are invaluable for preclinical drug discovery and biomarker development, the association between homologous recombination deficiency (HRD) and drug sensitivity in these resources remains unclear. In this study, we comprehensively analyzed molecular profiles and drug screening data from the Cancer Cell Line Encyclopedia. Unexpectedly, gene alterations in BRCA1/2 or homologous recombination-related genes, HRD scores, or mutational signature 3 were not positively correlated with sensitivity to platinum agents or PARP inhibitors. Rather, higher HRD scores and mutational signature 3 were significantly associated with resistance to these agents in multiple assays. These findings were consistent when analyzing exclusively breast and ovarian cancer cell lines and when using data from the COSMIC Cell Line Project. Collectively, the existing data from established cancer cell lines do not reflect the expected association between HRD status and drug response to platinum agents and PARP inhibitors in clinical tumors. This discrepancy may extend to other tumor characteristics, highlighting the importance of recognizing potential limitations in cell line data for researchers.

Prognostic relevance of HRDness gene expression signature in ovarian high-grade serous carcinoma; JGOG3025-TR2 study

This study aimed to evaluate the homologous recombination repair pathway deficiency (HRD) in ovarian high-grade serous carcinoma (HGSC). In the ovarian cancer data from The Cancer Genome Atlas, we identified genes differentially expressed between tumours with and without HRD genomic scars and named these genes "HRDness signature". We performed SNP array, RNA sequencing, and methylation array analyses on 274 HGSC tumours for which targeted sequencing of 51 genes and clinical data were available to generate JGOG3025-TR2 dataset. The HRDness signature was tested on external datasets, including the JGOG3025-TR2 cohort, by computational scoring and machine-learning prediction. High scores and positive predictions of the HRDness signature were significantly associated with BRCA alterations, genomic scar scores, and better survival. On the other hand, among cases with high scores and/or positive predictions, those with BRCA1 methylation showed poorer survival. In the JGOG3025-TR2 cohort, HRD status was significantly associated with the use of olaparib after relapse and progression-free survival after its initiation. The HRDness gene expression signature is associated with a good prognosis, while BRCA1 methylation is associated with a poor prognosis. The newly generated JGOG3025-TR2 dataset will be useful in future HGSC studies.

Homologous recombination deficiency status-based classification of high-grade serous ovarian carcinoma

AbstractHomologous recombination repair (HRR) pathway deficiency (HRD) is involved in the tumorigenesis and progression of high-grade serous ovarian carcinoma (HGSOC) as well as in the sensitivity to platinum chemotherapy drugs. In this study, we obtained data from The Cancer Genome Atlas (TCGA) on HGSOC and identified scores for the loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions, and calculated the HRD score. We then investigated the relationships among the score, genetic/epigenetic alterations in HRR-related genes, and the clinical data. We found that BRCA1/2 mutations were enriched in the group with HRD scores ≥63. Compared with the groups with scores ≤62, this group had a good prognosis; we thus considered HRD scores ≥63 to be the best cutoff point for identifying HRD cases in HGSOC. Classification of HGSOC cases by the HRD status revealed a better prognosis for HRD cases caused by genetic alterations (genetic HRD) than those caused by epigenetic changes and those caused by undetermined reasons (p = 0.0002). Among cases without macroscopic residual tumors after primary debulking surgery, 11 of 12 genetic HRD cases survived after the median observation period of 6.6 years, showing remarkably high survival rates (p = 0.0059). In conclusion, HGSOC can be classified into subtypes with different prognoses according to HRD status. This classification could be useful for personalized HGSOC treatment.

B7-H3 Suppresses Antitumor Immunity via the CCL2–CCR2–M2 Macrophage Axis and Contributes to Ovarian Cancer Progression

Abstract New approaches beyond PD-1/PD-L1 inhibition are required to target the immunologically diverse tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC). In this study, we explored the immunosuppressive effect of B7-H3 (CD276) via the CCL2–CCR2–M2 macrophage axis and its potential as a therapeutic target. Transcriptome analysis revealed that B7-H3 is highly expressed in PD-L1–low, nonimmunoreactive HGSOC tumors, and its expression negatively correlated with an IFNγ signature, which reflects the tumor immune reactivity. In syngeneic mouse models, B7-H3 (Cd276) knockout (KO) in tumor cells, but not in stromal cells, suppressed tumor progression, with a reduced number of M2 macrophages and an increased number of IFNγ+CD8+ T cells. CCL2 expression was downregulated in the B7-H3 KO tumor cell lines. Inhibition of the CCL2–CCR2 axis partly negated the effects of B7-H3 suppression on M2 macrophage migration and differentiation, and tumor progression. In patients with HGSOC, B7-H3 expression positively correlated with CCL2 expression and M2 macrophage abundance, and patients with B7-H3–high tumors had fewer tumoral IFNγ+CD8+ T cells and poorer prognosis than patients with B7-H3–low tumors. Thus, B7-H3 expression in tumor cells contributes to CCL2–CCR2–M2 macrophage axis–mediated immunosuppression and tumor progression. These findings provide new insights into the immunologic TME and could aid the development of new therapeutic approaches against the unfavorable HGSOC phenotype.

CXCL13-producing CD4+ T cells accumulate in the early phase of tertiary lymphoid structures in ovarian cancer

Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates whose formation might be caused by chronic inflammation states, such as cancer. However, how TLS are induced in the tumor microenvironment (TME) and how they affect patient survival are not well understood. We investigated TLS distribution in relation to tumor infiltrating lymphocytes (TILs) and related gene expression in high-grade serous ovarian cancer (HGSC) specimens. CXCL13 gene expression correlated with TLS presence and the infiltration of T cells and B cells, and it was a favorable prognostic factor for patients with HGSC. Coexistence of CD8+ T cells and B cell lineages in the TME significantly improved the prognosis of HGSC and was correlated with the presence of TLS. CXCL13 expression was predominantly coincident with CD4+ T cells in TLS and CD8+ T cells in TILs, and it shifted from CD4+ T cells to CD21+ follicular DCs as TLS matured. In a mouse ovarian cancer model, recombinant CXCL13 induced TLS and enhanced survival by the infiltration of CD8+ T cells. These results suggest that TLS formation was associated with CXCL13-producing CD4+ T cells and that TLS facilitated the coordinated antitumor response of cellular and humoral immunity in ovarian cancer.

Gain-of-Function Chromatin Remodeling Activity of Oncogenic FOXL2C134W Reprograms Glucocorticoid Receptor Occupancy to Drive Granulosa Cell Tumors

Abstract Adult type ovarian granulosa cell tumors (AGCT) are rare malignancies with the near universal c.C402G (p.Cys134Trp) somatic mutation in FOXL2, a forkhead box family transcription factor important for ovarian function. Relapsed AGCT is incurable, but the mechanism of the unique FOXL2 mutation could confer therapeutic vulnerabilities. To identify FOXL2C134W-dependent pharmacologic synergies, we created and characterized endogenous FOXL2 isogenic AGCT cells and an AGCT tumoroid biobank. A drug screen identified that glucocorticoids promote FOXL2C134W-dependent AGCT growth. Epigenetic investigation revealed that the Cys134Trp mutation exposes latent DNA sequence–specific chromatin remodeling activity in FOXL2. FOXL2C134W-dependent chromatin remodeling activity redirected glucocorticoid receptor chromatin occupancy to drive hyaluronan synthase 2 gene expression and increase extracellular hyaluronan secretion. Treatment of AGCT models with hyaluronidase reduced viability, and dexamethasone rescued this effect. Combinatorial drug–drug interaction experiments demonstrated that dexamethasone antagonizes the potency of paclitaxel, a chemotherapy agent frequently used in the treatment of AGCT. Thus, gain-of-function pioneering activity contributes to the oncogenic mechanism of FOXL2C134W and creates a potentially targetable synergy with glucocorticoid signaling. Significance: Glucocorticoids promote granulosa cell tumor growth via epigenetic coregulation with the disease driver FOXL2C134W, providing mechanistic insight into disease oncogenesis and uncovering a potential treatment strategy.