Ovarian Clear Cell Carcinoma Cell Research Articles

Involvement of ferroptosis in eribulin-induced cytotoxicity in ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is a unique clinicopathological subtype of epithelial ovarian cancer that is resistant to standard chemotherapy. Eribulin, a microtubule dynamics inhibitor of halichondrin class, has unique effects in the cancer microenvironment such as induction of epithelization and reduction in metastatic potential in breast cancer cells; however, nothing is known about the effect of eribulin and the detailed mechanisms in OCCC. This study aimed to investigate the involvement of ferroptosis and its mechanism in the antitumor activity of eribulin in OCCC cells and a mouse xenograft model. We found that eribulin-induced cell death was reduced by ferroptosis inhibitors; deferoxamine, an iron chelator and ferrostatin-1, a lipid peroxidation inhibitor. Eribulin increased the levels of intracellular iron, reactive oxygen species (ROS), and lipid peroxides, and increased the mitochondrial membrane potential. Eribulin downregulated the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), and superoxide dismutase (SOD) activity. The combination of eribulin and ML210, a glutathione peroxidase 4-inhibiting ferroptosis inducer, had a synergistic effect on ferroptosis. Taken together, our findings show firstly that eribulin triggers ferroptosis in OCCC and this effect occurs via the suppression of the Nrf2–HO-1 signaling pathway, SOD activity and the promotion of lipid peroxidation. These findings suggest that eribulin-induced ferroptosis is associated with its anti-tumor effect and also could be a potential therapeutic target in OCCC.

Moesin affects the plasma membrane expression and the immune checkpoint function of CD47 in human ovarian clear cell carcinoma

Among major histological subtypes of epithelial ovarian cancer, a higher incidence of ovarian clear cell carcinoma (OCCC) is observed in East Asian populations, particularly in Japan. Despite recent progress in the immune checkpoint inhibitors for a wide variety of cancer cell types, patients with OCCC exhibit considerably low response rates to these drugs. Hence, urgent efforts are needed to develop a novel immunotherapeutic approach for OCCC. CD47, a transmembrane protein, is overexpressed in almost all cancer cells and disrupts macrophage phagocytic activity in cancer cells. Ezrin-Radixin-Moesin (ERM) family member of proteins serve as scaffold proteins by crosslinking certain transmembrane proteins with the actin cytoskeleton, contributing to their plasma membrane localization. Here, we examined the role of ERM family in the plasma membrane localization and functionality of CD47 in OCCC cell lines derived from Japanese women. Confocal laser scanning microscopy analysis showed colocalization of CD47 with all three ERM in the plasma membrane of OCCC cells. RNA interference-mediated gene silencing of moesin, but not others, decreased the plasma membrane expression and immune checkpoint function of CD47, as determined by flow cytometry and in vitro phagocytosis assay using human macrophage-like cells, respectively. Interestingly, clinical database analysis indicated that moesin expression in OCCC was higher than that in other histological subtypes of ovarian cancers, and the expression of CD47 and moesin increased with the cancer stage. In conclusion, moesin is overexpressed in OCCC and may be the predominant scaffold protein responsible for CD47 plasma membrane localization and function in OCCC.

3'-Hydroxypterostilbene Potently Suppresses Tumor Growth via Inhibiting the Activation of the JAK2/STAT3 Pathway in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer (EOC) that is associated with higher interleukin-6 (IL-6) levels, and suppression of the Janus kinase 2/Signal transducer and activator of transription 3 (JAK2/STAT3) pathway may contribute to the suppression of this cancer. This study aims to compare the anti-cancer effect of pterostilbene (PSB) and 2'- and 3'-hydroxypterostilbene (2HPSB and 3HPSB, respectively) on the JAK2/STAT3 pathway. In vitro experiments with the OCCC cell line TOV21G and a xenograft nude mouse model are used to achieve the study aims. The results showed that 3HPSB has the greatest anti-proliferative and pro-apoptotic effects of the three compounds studied. Activation of the JAK2/STAT3 pathway and the nuclear translocation of STAT3 are effectively inhibited by 3HPSB and PSB. Both 3HPSB and PSB can effectively suppress tumor growth, which is mediated by the inhibition of JAK2/STAT3 phosphorylation. This is the first study to compare the efficacy of PSB, 3HPSB, and the newly identified compound 2HPSB regarding ovarian cancer. Moreover, targeting JAK2/STAT3 is shown to be a potentially effective strategy for OCCC treatment. This study is expected to provide new insights into the potential of the abovementioned phytochemicals for development as adjuvants for cancer treatment in the future.

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

Ezrin-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 knockout, and blebbistatin treatment potentiated the effects of Me-EBP50 knockout. 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. In 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.

Transcriptomic analyses of ovarian clear-cell carcinoma with concurrent endometriosis.

Endometriosis, a benign inflammatory disease whereby endometrial-like tissue grows outside the uterus, is a risk factor for endometriosis-associated ovarian cancers. In particular, ovarian endometriomas, cystic lesions of deeply invasive endometriosis, are considered the precursor lesion for ovarian clear-cell carcinoma (OCCC). To explore this transcriptomic landscape, OCCC from women with pathology-proven concurrent endometriosis (n = 4) were compared to benign endometriomas (n = 4) by bulk RNA and small-RNA sequencing. Analysis of protein-coding genes identified 2449 upregulated and 3131 downregulated protein-coding genes (DESeq2, P< 0.05, log2 fold-change > |1|) in OCCC with concurrent endometriosis compared to endometriomas. Gene set enrichment analysis showed upregulation of pathways involved in cell cycle regulation and DNA replication and downregulation of pathways involved in cytokine receptor signaling and matrisome. Comparison of pathway activation scores between the clinical samples and publicly-available datasets for OCCC cell lines revealed significant molecular similarities between OCCC with concurrent endometriosis and OVTOKO, OVISE, RMG1, OVMANA, TOV21G, IGROV1, and JHOC5 cell lines. Analysis of miRNAs revealed 64 upregulated and 61 downregulated mature miRNA molecules (DESeq2, P< 0.05, log2 fold-change > |1|). MiR-10a-5p represented over 21% of the miRNA molecules in OCCC with endometriosis and was significantly upregulated (NGS: log2fold change = 4.37, P = 2.43e-18; QPCR: 8.1-fold change, P< 0.05). Correlation between miR-10a expression level in OCCC cell lines and IC50 (50% inhibitory concentration) of carboplatin in vitro revealed a positive correlation (R2=0.93). MiR-10a overexpression in vitro resulted in a significant decrease in proliferation (n = 6; P< 0.05) compared to transfection with a non-targeting control miRNA. Similarly, the cell-cycle analysis revealed a significant shift in cells from S and G2 to G1 (n = 6; P< 0.0001). Bioinformatic analysis predicted that miR-10a-5p target genes that were downregulated in OCCC with endometriosis were involved in receptor signaling pathways, proliferation, and cell cycle progression. MiR-10a overexpression in vitro was correlated with decreased expression of predicted miR-10a target genes critical for proliferation, cell-cycle regulation, and cell survival including [SERPINE1 (3-fold downregulated; P< 0.05), CDK6 (2.4-fold downregulated; P< 0.05), and RAP2A (2-3-fold downregulated; P< 0.05)]. These studies in OCCC suggest that miR-10a-5p is an impactful, potentially oncogenic molecule, which warrants further studies.

Exploring the cellular and molecular differences between ovarian clear cell carcinoma and high-grade serous carcinoma using single-cell RNA sequencing and GEO gene expression signatures

The two most prevalent subtypes of epithelial ovarian carcinoma (EOC) are ovarian clear cell carcinoma (OCCC) and high-grade serous ovarian carcinoma (HGSC). Patients with OCCC have a poor prognosis than those with HGSC due to chemoresistance, implying the need for novel treatment target. In this study, we applied single-cell RNA sequencing (scRNA-seq) together with bulk RNA-seq data from the GEO (Gene Expression Omnibus) database (the GSE189553 dataset) to characterize and compare tumor heterogeneity and cell-level evolution between OCCC and HGSC samples. To begin, we found that the smaller proportion of an epithelial OCCC cell subset in the G2/M phase might explain OCCC chemoresistance. Second, we identified a possible pathogenic OCCC epithelial cell subcluster that overexpresses LEFTY1. Third, novel biomarkers separating OCCC from HGSC were discovered and subsequently validated on a wide scale using immunohistochemistry. Amine oxidase copper containing 1 (AOC1) was preferentially expressed in OCCC over HGSC, while S100 calcium-binding protein A2 (S100A2) was detected less frequently in OCCC than in HGSC. In addition, we discovered that metabolic pathways were enriched in the epithelial compartment of the OCCC samples. In vitro experiments verified that inhibition of oxidative phosphorylation or glycolysis pathways exerted direct antitumor effects on both OCCC and HGSC cells, while targeting glutamine metabolism or ferroptosis greatly attenuated chemosensitivity only in OCCC cells. Finally, to determine whether there were any variations in immune cell subsets between OCCC and HGSC, data from scRNA-seq and mass cytometry were pooled for analysis. In summary, our work provides the first holistic insights into the cellular and molecular distinctions between OCCC and HGSC and is a valuable source for discovering new targets to leverage in clinical treatments to improve the poor prognosis of patients with OCCC.

Mechanism of Cell Death by Combined Treatment with an xCT Inhibitor and Paclitaxel: An Alternative Therapeutic Strategy for Patients with Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian carcinoma that responds poorly to chemotherapy. Glutathione (GSH) is a primary antioxidant, which protects cells against reactive oxygen species (ROS). High levels of GSH are related to chemotherapeutic resistance. The glutamine/cystine transporter xCT is essential for intracellular GSH synthesis. However, whether xCT inhibition can overcome the resistance to chemotherapeutic agents in OCCC remains unclear. This study demonstrated that combined treatment with paclitaxel (PTX) and the xCT inhibitor sulfasalazine (SAS) significantly enhanced cytotoxicity more than the individual drugs did in OCCC cells. Treatment with PTX and SAS induced apoptosis more effectively than did individual drug treatments in the cells with significant generation of ROS. Moreover, combined treatment with PTX and SAS induced ferroptosis in the cells with low expression of glutathione peroxidase (GPx4), high levels of intracellular iron and significant lipid ROS accumulation. Therefore, our findings provide valuable information that the xCT inhibitor might be a promising therapeutic target for drug-resistant OCCC. The strategy of combined administration of PTX and SAS can potentially be used to treat OCCC and help to develop novel therapeutic methods.

Fibroblast Growth Factor 11 (FGF11) Promotes Progression and Cisplatin Resistance Through the HIF-1α/FGF11 Signaling Axis in Ovarian Clear Cell Carcinoma.

A poor prognosis is often associated with ovarian clear cell carcinoma (OCCC) due to its relative resistance to platinum-based chemotherapy. Although several studies have been launched to explore the pathogenesis of OCCC, the mechanism of chemoresistance has yet to be uncovered. Nanostring nCounter PanCancer Pathways Panel was performed to explore the expression profiles of OCCC tissues from patients showing different platinum sensitivity. Bioinformatic analysis was performed to select genes associated with chemoresistance and cell function assays, including colony formation, wound healing, transwell and flow cytometric analysis, were used to explore the role of the target gene in the progression of OCCC and resistance to cisplatin (DDP). Gene expression profiles and bioinformatic analysis verified that the expression of fibroblast growth factor 11 (FGF11) was significantly increased in platinum-resistant OCCC tissues and increased FGF11 expression was related to poorer survival. Downregulation of FGF11 inhibited the proliferation, migration, and invasion, reversing the DDP resistance of OCCC cells. Mechanically, FGF11 was regulated by hypoxia-inducible factor-1α (HIF-1α) to modulate the DDP sensitivity. FGF11 was highly expressed in platinum-resistant OCCC tissues, promoting progression and resistance to DDP through the HIF-1α/FGF11 signaling axis.

Selective Inhibition of L-type Amino Acid Transporter 1 Suppresses Cell Proliferation in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a histological type of ovarian cancer that is refractory to chemotherapy and has poor prognosis, which necessitates the development of novel treatment therapies. In this study, we focused on L-type amino acid transporter 1 (LAT1), which is involved in cancer growth, and investigated the effect of its selective inhibition on cell proliferation in OCCC. The inhibitory effect of nanvuranlat (JPH203), a LAT1 selective inhibitor, on the cellular uptake of [3H] leucine was evaluated using the OCCC cell line JHOC9, which expresses the LAT1 protein. In addition, the kinetics of cell proliferation and changes in phosphorylation of the mTOR pathway were analyzed. The correlation between LAT1 expression and progression-free survival (PFS) was evaluated using clinical specimens of OCCC. Nanvuranlat inhibited [3H] leucine intracellular uptake and cell proliferation in a dose-dependent manner in JHOC9 cells. In addition, it suppressed the activity of the mTOR signaling pathway, which is thought to inhibit cancer cell proliferation. LAT1 expression was most frequent in OCCC among clinical specimens of epithelial ovarian cancer. A correlation between LAT1 expression and PFS was observed in OCCC. LAT1 selective inhibition suppresses cell proliferation via the mTOR pathway by inhibiting leucine uptake in OCCC. This study illustrates the potential of using LAT1 selective inhibition as a treatment strategy for OCCC.

Treatment for ovarian clear cell carcinoma with combined inhibition of WEE1 and ATR

BackgroundStandard platinum-based therapy for ovarian cancer is inefficient against ovarian clear cell carcinoma (OCCC). OCCC is a distinct subtype of epithelial ovarian cancer. OCCC constitutes 25% of ovarian cancers in East Asia (Japan, Korea, China, Singapore) and 6–10% in Europe and North America. The cancer is characterized by frequent inactivation of ARID1A and 10% of cases of endometriosis progression to OCCC. The aim of this study was to identify drugs that are either FDA-approved or in clinical trials for the treatment of OCCC.ResultsHigh throughput screening of 166 compounds that are either FDA-approved, in clinical trials or are in pre-clinical studies identified several cytotoxic compounds against OCCC. ARID1A knockdown cells were more sensitive to inhibitors of either mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738) or MDM2 (RG7388) compared to control cells. Also, compounds targeting BH3 domain (AZD4320) and SRC (AZD0530) displayed preferential cytotoxicity against ARID1A mutant cell lines. In addition, WEE1 inhibitor (AZD1775) showed broad cytotoxicity toward OCCC cell lines, irrespective of ARID1A status.ConclusionsIn a selection of 166 compounds we showed that inhibitors of ATR and WEE1 were cytotoxic against a panel of OCCC cell lines. These two drugs are already in other clinical trials, making them ideal candidates for treatment of OCCC.

Tumor Suppressive Role of the PRELP Gene in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) has a poor prognosis, and its therapeutic strategy has not been established. PRELP is a leucine-rich repeat protein in the extracellular matrix of connective tissues. Although PRELP anchors the basement membrane to the connective tissue and is absent in most epithelial cancers, much remains unknown regarding its function as a regulator of ligand-mediated signaling pathways. Here, we obtained sets of differentially expressed genes by PRELP expression using OCCC cell lines. We found that more than 1000 genes were significantly altered by PRELP expression, particularly affecting the expression of a group of genes involved in the PI3K-AKT signaling pathway. Furthermore, we revealed the loss of active histone marks on the loci of the PRELP gene in patients with OCCC and how its forced expression inhibited cell proliferation. These findings suggest that PRELP is not only a molecule anchored in connective tissues but is also a signaling molecule acting in a tumor-suppressive manner. It can serve as the basis for early detection and novel therapeutic approaches for OCCC toward precision medicine.

MEX3A Mediates p53 Degradation to Suppress Ferroptosis and Facilitate Ovarian Cancer Tumorigenesis.

Degradation of p53 mediated by MEX3A drives ovarian cancer growth by circumventing p53 tumor suppressive functions, suggesting targeting MEX3A as a potential strategy for treating of ovarian cancer expressing WT p53.

Activity-based protein profiling reveals active serine proteases that drive malignancy of human ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.

3P Identification of novel biomarkers of response to ATR inhibitors in ARID1A mutant ovarian clear cell carcinoma

BackgroundOvarian clear cell carcinoma (OCCC) is characterised by a high prevalence of ARID1A mutations and chemotherapy resistance. We previously found that loss of ARID1A causes ATR inhibitor (ATRi) sensitivity, which led to the phase II ATARI clinical trial (NCT04065269). We aim to identify novel genetic determinants of ATRi response in ARID1A-mutant OCCC.MethodsA genome wide CRISPR knockout (CRISPRn) screen was performed in OCCC TOV21G cells. CRISPR prime gene-editing was used to introduce PPP2R1A p.R183 mutations. In vitro and in vivo ATRi sensitivity was assessed in PPP2R1A isogenic models. Cell cycle analysis was performed via flow cytometry. Phospho-proteomic profiling was performed using mass spectrometry.ResultsA CRISPRn screen in ARID1A mutant OCCC cells identified protein phosphatase 2 (PP2A) complex subunits as ATRi response genes. In OCCC, PPP2R1A missense mutations cause amino acid substitutions at residue p.R183. Characterisation of a cohort of OCCC primary tumours revealed a higher prevalence of structural subunit (PPP2R1A) mutations than previously reported (50%) which frequently co-occurred with ARID1A mutations. ARID1A-mutant OCCC cells with heterozygous PPP2R1A p.R183P or p.R183W mutations displayed enhanced ATRi sensitivity in vitro and in vivo. The most profound cell-cycle defect caused by PPP2R1A missense mutation was an ATRi-induced reduction in active S phase. ATRi exposure in PPP2R1A mutants increased 53BP1 bodies, a mark of residual DNA damage in mitosis. Phospho-proteomic profiling of PPP2R1A mutant OCCC cells revealed the selective increase in phosphorylation of Lysine Deficient Protein Kinase 1 (WNK1) following ATRi exposure, as well as increased phosphorylation of the WNK1 substrate Oxidative Stress Response Kinase 1 (OSR1). Depletion of WNK1 rescued ATRi sensitivity and S phase defects in PPP2R1A mutant cells suggesting a novel role for this kinase.ConclusionsThe ability of PPP2R1A missense mutations to enhance ATRi sensitivity in tumours cells with pre-existing ARID1A mutations suggests that the co-occurrence of these mutations may be better predictors of ATRi sensitivity than either mutation alone. Increased phosphorylation of WNK1 may drive ATRi sensitivity in PPP2R1A mutant cells.Legal entity responsible for the studyThe authors.FundingCancer Research UK.DisclosureS. Banerjee: Financial Interests, Institutional, Research Grant: AstraZeneca; Financial Interests, Institutional, Research Grant: GlaxoSmithKline; Financial Interests, Personal, Advisory Board: Amgen; Financial Interests, Personal, Advisory Board: Genmabs; Financial Interests, Personal, Advisory Board: GlaxoSmithKline; Financial Interests, Personal, Advisory Board: Immunogen; Financial Interests, Personal, Advisory Board: MSD; Financial Interests, Personal, Advisory Board: Merck Sereno; Financial Interests, Personal, Advisory Board: Mersana; Financial Interests, Personal, Advisory Board: Oxcerna; Financial Interests, Personal, Advisory Board: Seagen; Financial Interests, Personal, Advisory Board: Shattuk Labs; Financial Interests, Personal, Invited Speaker: Amgen; Financial Interests, Personal, Invited Speaker: AstraZeneca; Financial Interests, Personal, Invited Speaker: Immunogen; Financial Interests, Personal, Invited Speaker: MSD; Financial Interests, Personal, Invited Speaker: Mersana; Financial Interests, Personal, Invited Speaker: Pfizer; Financial Interests, Personal, Invited Speaker: Roche. C. Lord: Financial Interests, Institutional, Research Grant: AstraZeneca; Financial Interests, Institutional, Research Grant: Merck; Financial Interests, Institutional, Research Grant: KGaG; Financial Interests, Institutional, Research Grant: Artios; Financial Interests, Personal, Advisory Board: Syncona; Financial Interests, Personal, Advisory Board: Sun Pharma; Financial Interests, Personal, Advisory Board: Gerson Lehrman; Financial Interests, Personal, Advisory Board: Merck; Financial Interests, Personal, Advisory Board: Vertex; Financial Interests, Personal, Advisory Board: AstraZeneca; Financial Interests, Personal, Advisory Board: Dark Blue Therapeutics; Financial Interests, Personal, Stocks/Shares: Tango; Financial Interests, Personal, Stocks/Shares: Ovibio; Financial Interests, Personal, Stocks/Shares: Enedra Tx; Financial Interests, Personal, Stocks/Shares: Hysplex; Financial Interests, Personal, Stocks/Shares: Tesselate. All other authors have declared no conflicts of interest. BackgroundOvarian clear cell carcinoma (OCCC) is characterised by a high prevalence of ARID1A mutations and chemotherapy resistance. We previously found that loss of ARID1A causes ATR inhibitor (ATRi) sensitivity, which led to the phase II ATARI clinical trial (NCT04065269). We aim to identify novel genetic determinants of ATRi response in ARID1A-mutant OCCC. Ovarian clear cell carcinoma (OCCC) is characterised by a high prevalence of ARID1A mutations and chemotherapy resistance. We previously found that loss of ARID1A causes ATR inhibitor (ATRi) sensitivity, which led to the phase II ATARI clinical trial (NCT04065269). We aim to identify novel genetic determinants of ATRi response in ARID1A-mutant OCCC. MethodsA genome wide CRISPR knockout (CRISPRn) screen was performed in OCCC TOV21G cells. CRISPR prime gene-editing was used to introduce PPP2R1A p.R183 mutations. In vitro and in vivo ATRi sensitivity was assessed in PPP2R1A isogenic models. Cell cycle analysis was performed via flow cytometry. Phospho-proteomic profiling was performed using mass spectrometry. A genome wide CRISPR knockout (CRISPRn) screen was performed in OCCC TOV21G cells. CRISPR prime gene-editing was used to introduce PPP2R1A p.R183 mutations. In vitro and in vivo ATRi sensitivity was assessed in PPP2R1A isogenic models. Cell cycle analysis was performed via flow cytometry. Phospho-proteomic profiling was performed using mass spectrometry. ResultsA CRISPRn screen in ARID1A mutant OCCC cells identified protein phosphatase 2 (PP2A) complex subunits as ATRi response genes. In OCCC, PPP2R1A missense mutations cause amino acid substitutions at residue p.R183. Characterisation of a cohort of OCCC primary tumours revealed a higher prevalence of structural subunit (PPP2R1A) mutations than previously reported (50%) which frequently co-occurred with ARID1A mutations. ARID1A-mutant OCCC cells with heterozygous PPP2R1A p.R183P or p.R183W mutations displayed enhanced ATRi sensitivity in vitro and in vivo. The most profound cell-cycle defect caused by PPP2R1A missense mutation was an ATRi-induced reduction in active S phase. ATRi exposure in PPP2R1A mutants increased 53BP1 bodies, a mark of residual DNA damage in mitosis. Phospho-proteomic profiling of PPP2R1A mutant OCCC cells revealed the selective increase in phosphorylation of Lysine Deficient Protein Kinase 1 (WNK1) following ATRi exposure, as well as increased phosphorylation of the WNK1 substrate Oxidative Stress Response Kinase 1 (OSR1). Depletion of WNK1 rescued ATRi sensitivity and S phase defects in PPP2R1A mutant cells suggesting a novel role for this kinase. A CRISPRn screen in ARID1A mutant OCCC cells identified protein phosphatase 2 (PP2A) complex subunits as ATRi response genes. In OCCC, PPP2R1A missense mutations cause amino acid substitutions at residue p.R183. Characterisation of a cohort of OCCC primary tumours revealed a higher prevalence of structural subunit (PPP2R1A) mutations than previously reported (50%) which frequently co-occurred with ARID1A mutations. ARID1A-mutant OCCC cells with heterozygous PPP2R1A p.R183P or p.R183W mutations displayed enhanced ATRi sensitivity in vitro and in vivo. The most profound cell-cycle defect caused by PPP2R1A missense mutation was an ATRi-induced reduction in active S phase. ATRi exposure in PPP2R1A mutants increased 53BP1 bodies, a mark of residual DNA damage in mitosis. Phospho-proteomic profiling of PPP2R1A mutant OCCC cells revealed the selective increase in phosphorylation of Lysine Deficient Protein Kinase 1 (WNK1) following ATRi exposure, as well as increased phosphorylation of the WNK1 substrate Oxidative Stress Response Kinase 1 (OSR1). Depletion of WNK1 rescued ATRi sensitivity and S phase defects in PPP2R1A mutant cells suggesting a novel role for this kinase. ConclusionsThe ability of PPP2R1A missense mutations to enhance ATRi sensitivity in tumours cells with pre-existing ARID1A mutations suggests that the co-occurrence of these mutations may be better predictors of ATRi sensitivity than either mutation alone. Increased phosphorylation of WNK1 may drive ATRi sensitivity in PPP2R1A mutant cells. The ability of PPP2R1A missense mutations to enhance ATRi sensitivity in tumours cells with pre-existing ARID1A mutations suggests that the co-occurrence of these mutations may be better predictors of ATRi sensitivity than either mutation alone. Increased phosphorylation of WNK1 may drive ATRi sensitivity in PPP2R1A mutant cells.

Caveolin-1-ACE2 axis modulates xenobiotic metabolism-linked chemoresistance in ovarian clear cell carcinoma.

Among epithelial ovarian cancers, ovarian clear cell carcinoma (OCCC) remains markedly resistant to platinum-based chemotherapy, leading to poor clinical outcomes. In response to xenobiotic insults, caveolar platforms play crucial roles in modulating stress signaling responses in cancer cells. It has been hypothesized that caveolin-1 (Cav-1), a main component of the lipid raft, may regulate the response to platinum-based treatment in OCCC. The clinical transcriptomic evaluation demonstrated that high Cav-1 expression was positively associated with a favorable prognosis in patients with ovarian cancer. Cav-1 overexpression enhanced sensitivity to cisplatin (CDDP) treatment, whereas Cav-1 deficiency promoted chemoresistance in OCCC cells. Mechanistically, although Cav-1 counteracted angiotensin-converting enzyme 2 (ACE2) expression, ACE2 positively facilitated resistance to CDDP in OCCC cells. Furthermore, ACE2 restricted aryl hydrocarbon receptor expression and subsequent transcription of drug-metabolizing enzymes. Of note, ACE2 positively regulated the expression of the platinum-clearing enzyme CYP3A4. These findings suggest that the Cav-1-ACE2 axis modulates xenobiotic metabolism-linked chemoresistance in OCCC, predicting potential roles for the stress sentinel networks in oncogenic processes.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10565-022-09733-1.

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

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

CRISPR/Cas9 Screening for Identification of Genes Required for the Growth of Ovarian Clear Cell Carcinoma Cells.

Epithelial ovarian cancer is classified into four major histological subtypes: serous, clear cell, endometrioid and mucinous. Ovarian clear cell carcinoma (OCCC) responds poorly to conventional chemotherapies and shows poor prognosis. Thus, there is a need to develop new drugs for the treatment of OCCC. In this study, we performed CRISPR/Cas9 screens against OCCC cell lines and identified candidate genes important for their proliferation. We found that quite different genes are required for the growth of ARID1A and PIK3CA mutant and wild-type OCCC cell lines, respectively. Furthermore, we found that the epigenetic regulator KDM2A and the translation regulator PAIP1 may play important roles in the growth of ARID1A and PIK3CA mutant, but not wild-type, OCCC cells. The results of our CRISPR/Cas9 screening may be useful in elucidating the molecular mechanism of OCCC tumorigenesis and in developing OCCC-targeted drugs.

The splicing factor DHX38/PRP16 is required for ovarian clear cell carcinoma tumorigenesis, as revealed by a CRISPR-Cas9 screen.

Certain cancers, such as ovarian clear cell carcinoma (OCCC), display high levels of genetic variation between patients, making it difficult to develop effective therapies. In order to identify novel genes critical to OCCC growth, we carried out a comprehensive CRISPR‐Cas9 knockout screen against cell growth using an OCCC cell line and a normal ovarian surface epithelium cell line. We identified the gene encoding DHX38/PRP16, an ATP‐dependent RNA helicase involved in splicing, as critical for the growth and tumorigenesis of OCCC. DHX38/PRP16 knockdown in OCCC cells, but not normal cells, induces apoptosis and impairs OCCC tumorigenesis in a mouse model. Our results suggest that DHX38/PRP16 may play a role in OCCC tumorigenesis and could potentially be a promising therapeutic target.

Preclinical Activity of Plitidepsin Against Clear Cell Carcinoma of the Ovary

To evaluate the antitumor effects of Plitidepsin against clear cell carcinoma (CCC) of the ovary. The expression of eEF1A2 in ovarian cancer was assessed by immunohistochemistry. Using ovarian CCC cell lines, the antitumor effect of Plitidepsin was assessed both in vitro and in vivo. By over-expressing or knocking down the eEF1A2 expression, we investigated the role of eEF1A2 in the sensitivity of CCC cells to Plitidepsin. Immunoreactivity to eEF1A2 was observed in 76.2% of CCC, which was significantly higher than other histological subtypes of ovarian cancer. Plitidepsin exhibited significant antitumor activity toward chemonaive and chemoresistant CCC cells both in vitro and in vivo. Ectopic expression of eEF1A2 in CCC cells resulted in increased sensitivity to Plitidepsin. In contrast, eEF1A2 knockdown decreased sensitivity of CCC cells to plitidepsin. Plitidepsin, a novel anti-cancer agent that targets eEF1A2, may be a promising agent for treating ovarian CCC.

LncRNA GAS5 inhibits the proliferation and invasion of ovarian clear cell carcinoma via the miR-31-5p/ARID1A axis.

To investigate the role of the lncRNA growth arrest special 5 (GAS5) in ovarian clear cell carcinoma (OCCC), we measured the expression of GAS5 and miR-31-5p in OCCC tissue samples and OCCC cell lines using RT-qPCR. MTT and colony formation assays were used to measure cell viability and colony formation ability. Cell invasion was determined by Transwell assays. The binding between GAS5 and miR-31-5p as well as miR-31-5p and ARID1A was determined by dual-luciferase reporter assays. The ARID1A protein levels were detected using western blotting. Kaplan-Meier curves were used for the analysis of the 5-year survival rate of patients with OCCC. GAS5 and ARID1A levels were significantly decreased, while miR-31-5p levels were strongly elevated in the OCCC tissues and cell lines. Patients with lower GAS5/ARID1A levels had shorter overall survival times. Overexpression of GAS5 or inhibition of miR-31-5p suppressed cell viability and invasion of OCCC cells and upregulated the protein levels of ARID1A. Moreover, overexpression of miR-31-5p reversed the effects of overexpression of GAS5. Cotransfection with pcDNA3.1-GAS5 and miR-31-5p inhibitor led to the lowest cell viability and cell invasion rates. A dual-luciferase reporter assay was performed to confirm the target relationship between GAS5 and miR-31-5p, as well as between miR-31-5p and ARID1A. LncRNA GAS5 inhibited cell viability and invasion of OCCC through activation of ARID1A by sponging miR-31-5p.