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PAX2 enhances proliferation and survival in normal mOSE cells.

Western blot analysis showing there is no T-antigen expression in RM tumors.

PAX2 is an essential transcription factor for development. Aberrant PAX2 expression in adult tissues is associated with carcinogenesis and experimental evidence shows that PAX2 generally exhibits oncogenic properties. Although PAX2 is not expressed in normal ovaries, it is highly expressed in low malignant potential and low-grade epithelial ovarian tumors, suggesting that PAX2 induction in ovarian surface epithelium (OSE) may contribute to transformation. Herein, we provide evidence that expression of PAX2 in normal murine OSE cells (mOSE) enhances their proliferation and survival and, with loss of p53, induces tumorigenicity. PAX2 expression in murine ovarian cancer cells enhanced or inhibited tumorigenicity, depending on the model system. In RM cells (mOSE transformed by K-RAS and c-MYC), PAX2 expression inhibited p53 and induced pERK1/2 and COX2, resulting in enhanced angiogenesis and decreased apoptosis of tumors arising from these cells. However, in a murine model of high-grade serous ovarian cancer (STOSE), PAX2 expression improved animal survival by reducing proliferation and metastasis, which correlated with increased Htra1 and decreased COX2. Thus, PAX2 may not be a classical oncogene or tumor suppressor but instead can act in either role by differential regulation of COX2 and/or HTRA1. Cancer Prev Res; 8(12); 1163–73. ©2015 AACR.

Expression of PAX2 in p53-null cells enhanced proliferation but not survival.

IHC analysis shows that PAX2 is expressed in the epithelium of the murine oviduct and uterus, but not in the ovary.

Supplementary Table 1: Primer pairs (forward [F] and reverse [R]) that were used to amplify murine genes. Supplementary Table 2: The primary antibodies that were used for western blot analysis in the current studies. Supplementary Table 3: The top 10 genes upregulated and downregulated by PAX2 in MO505 cells, as shown by microarray analysis, and their known relationships to ovarian tumorigenesis. Supplementary Table 4: Functional analysis of genes that were changed in M0505+PAX2 cells compared to M0505+WPI and M0505+PAX2+Cre and are involved in tumor growth and progression.

Supplementary Figure S1 shows clonogenic survival curves for ovarian cancer cell lines exposed to different genotoxic agents.

Supplementary Table S1 documents clinical and genetic characteristics of ovarian cancer cell lines.

This file contains supplementary figures and tables with their legends.

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines.Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561–73. ©2018 AACR.

Although initial treatment of ovarian cancer is successful, tumors typically relapse and become resistant to treatment. Because of poor infiltration of effector T cells, patients are mostly unresponsive to immunotherapy. Plasma gelsolin (pGSN) is transported by exosomes (small extracellular vesicle, sEV) and plays a key role in ovarian cancer chemoresistance, yet little is known about its role in immunosurveillance. Here, we report the immunomodulatory roles of sEV-pGSN in ovarian cancer chemoresistance. In chemosensitive conditions, secretion of sEV-pGSN was low, allowing for optimal CD8+ T-cell function. This resulted in increased T-cell secretion of IFNγ, which reduced intracellular glutathione (GSH) production and sensitized chemosensitive cells to cis-diaminedichloroplatinum (CDDP)-induced apoptosis. In chemoresistant conditions, increased secretion of sEV-pGSN by ovarian cancer cells induced apoptosis in CD8+ T cells. IFNγ secretion was therefore reduced, resulting in high GSH production and resistance to CDDP-induced death in ovarian cancer cells. These findings support our hypothesis that sEV-pGSN attenuates immunosurveillance and regulates GSH biosynthesis, a phenomenon that contributes to chemoresistance in ovarian cancer.Significance:These findings provide new insight into pGSN-mediated immune cell dysfunction in ovarian cancer chemoresistance and demonstrate how this dysfunction can be exploited to enhance immunotherapy.

Epithelial ovarian cancer is the most lethal gynecological malignancy, owing notably to its high rate of therapy-resistant recurrence in spite of good initial response to chemotherapy. Although poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promise for ovarian cancer treatment, extended therapy usually leads to acquired PARPi resistance. Here we explored a novel therapeutic option to counter this phenomenon, combining PARPi and inhibitors of nicotinamide phosphoribosyltransferase (NAMPT). Cell-based models of acquired PARPi resistance were created through an in vitro selection procedure. Using resistant cells, xenograft tumors were grown in immunodeficient mice, while organoid models were generated from primary patient tumor samples. Intrinsically PARPi-resistant cell lines were also selected for analysis. Our results show that treatment with NAMPT inhibitors effectively sensitized all in vitro models to PARPi. Adding nicotinamide mononucleotide, the resulting NAMPT metabolite, abrogated the therapy-induced cell growth inhibition, demonstrating the specificity of the synergy. Treatment with olaparib (PARPi) and daporinad (NAMPT inhibitor) depleted intracellular NAD+ , induced double-strand DNA breaks, and promoted apoptosis as monitored by caspase-3 cleavage. The two drugs were also synergistic in mouse xenograft models and clinically relevant patient-derived organoids. Therefore, in the context of PARPi resistance, NAMPT inhibition could offer a promising new option for ovarian cancer patients.

Background Resistance to chemotherapy continues to be a challenge when treating epithelial ovarian cancer (EOC), contributing to low patient survival rates. While CA125, the conventional EOC biomarker, has been useful in monitoring patients’ response to therapy, there are no biomarkers used to predict treatment response prior to chemotherapy. Previous work in vitro showed that plasma gelsolin (pGSN) is highly expressed in chemoresistant EOC cell lines, where it is secreted in small extracellular vesicles (sEVs). Whether sEVs from tumour cells are secreted into the circulation of EOC patients and could be used to predict patient chemoresponsiveness is yet to be determined. This study aims to identify if sEV-pGSN in the circulation could be a predictive biomarker for chemoresistance in EOC. Methods Sandwich ELISA was used to measure pGSN concentrations from plasma samples of 96 EOC patients (primarily high grade serous EOC). sEVs were isolated using ExoQuick ULTRA and characterized using western blot, nanoparticle tracking analysis, and electron microscopy after which pGSN was measured from the sEVs. Patients were stratified as platinum sensitive or resistant groups based on first progression free interval (PFI) of 6 or 12 months. Results Total circulating pGSN was significantly decreased and sEV-pGSN increased in patients with a PFI ≤ 12 months (chemoresistant) compared to those with a PFI > 12 months (chemosensitive). The ratio of total pGSN to sEV-pGSN further differentiated these groups and was a strong predictive marker for chemoresistance (sensitivity: 73.91%, specificity: 72.46%). Predetermined CA125 was not different between chemosensitive and chemoresistant groups and was not predictive of chemoresponsiveness prior to treatment. When CA125 was combined with the ratio of total pGSN/sEV-pGSN, it was a significant predictor of chemoresponsiveness, but the test performance was not as robust as the total pGSN/sEV-pGSN alone. Conclusions Total pGSN/sEV-pGSN was the best predictor of chemoresponsiveness prior to treatment, outperforming the individual biomarkers (CA125, total pGSN, and sEV-pGSN). This multianalyte predictor of chemoresponsiveness could help to inform physicians’ treatment and follow up plan at the time of EOC diagnosis, thus improving patients’ outcomes.

Although initial treatment of ovarian cancer is successful, tumors typically relapse and become resistant to treatment. Because of poor infiltration of effector T cells, patients are mostly unresponsive to immunotherapy. Plasma gelsolin (pGSN) is transported by exosomes (small extracellular vesicle, sEV) and plays a key role in ovarian cancer chemoresistance, yet little is known about its role in immunosurveillance. Here, we report the immunomodulatory roles of sEV-pGSN in ovarian cancer chemoresistance. In chemosensitive conditions, secretion of sEV-pGSN was low, allowing for optimal CD8+ T-cell function. This resulted in increased T-cell secretion of IFNγ, which reduced intracellular glutathione (GSH) production and sensitized chemosensitive cells to cis-diaminedichloroplatinum (CDDP)-induced apoptosis. In chemoresistant conditions, increased secretion of sEV-pGSN by ovarian cancer cells induced apoptosis in CD8+ T cells. IFNγ secretion was therefore reduced, resulting in high GSH production and resistance to CDDP-induced death in ovarian cancer cells. These findings support our hypothesis that sEV-pGSN attenuates immunosurveillance and regulates GSH biosynthesis, a phenomenon that contributes to chemoresistance in ovarian cancer. Significance: These findings provide new insight into pGSN-mediated immune cell dysfunction in ovarian cancer chemoresistance and demonstrate how this dysfunction can be exploited to enhance immunotherapy.

Background: Mitochondrial dynamics (e.g. fission/fusion) play an important role in controlling chemoresistance in representative gynecologic malignancies, ovarian and cervical cancer. Processing the long form of Optic atrophy (L-Opa)1 is a distinctive character of mitochondrial fragmentation, associated with chemosensitivity. Here, we examined the role of prohibitin (Phb)1 in increasing L-Opa1 processing via the regulating mitochondrial protease, Oma1 and its direct interaction with p-p53 (ser15) and pro-apoptotic Bcl-2 antagonist/killer (Bak) 1 in the signaling axis and if this phenomenon is associated with prognosis of patients.Methods: We compared Cisplatin (CDDP)-induced response of mitochondrial dynamics, molecular interaction among p-p53 (ser15)-Phb1-Bak, and chemoresponsiveness in paired chemosensitive and chemoresistant gynecologic cancer cells (ovarian and cervical cancer cell lines) using western blot, immunoprecipitation, sea horse, and immunofluorescence. Translational strategy with proximity ligation assessment in phb1-p-p53 (ser15) in human ovarian tumor sections further confirmed in vitro finding, associated with clinical outcome.Results: We report that: (1) Knock-down of Phb1 prevents Cisplatin (cis‐diamine-dichloroplatinum; CDDP) -induced changes in mitochondrial fragmentation and Oma1 mediated cleavage, and Opa1 processing; (2) In response to CDDP, Phb1 facilitates the p-p53 (ser15)-Phb1-Bak interaction in mitochondria in chemosensitive gynecologic cancer cells but not in chemoresistant cells; (3) Akt overexpression results in suppressed p-p53(Ser15)-Phb1 interaction and dysregulated mitochondrial dynamics, and (4) Consistent with in vitro findings, proximity ligation assessment (PLA) in human ovarian tumor sections demonstrated that p-p53(ser15)-Phb1-Bak interaction in mitochondria is associated with better chemoresponsiveness and clinical outcome of patients. Determining the molecular mechanisms by which Phb1 facilitates mitochondrial fragmentation and interacts with p53 may advance the current understanding of chemoresistance and pathogenesis of gynecologic cancer.Conclusion: Determining the key molecular mechanisms by which Phb1 facilitates the formation of p-p53 (ser15)-Bak-Phb1 and its involvement in the regulation of mitochondrial dynamics and apoptosis may ultimately contribute to the current understanding of molecular and cellular basis of chemoresistance in this gynecologic cancer.

Ovarian cancer (OVCA) is the most lethal gynaecological cancer with a 5-year survival rate less than 50%. Despite new therapeutic strategies, such as immune checkpoint blockers (ICBs), tumor recurrence and drug resistance remain key obstacles in achieving long-term therapeutic success. Therefore, there is an urgent need to understand the cellular mechanisms of immune dysregulation in chemoresistant OVCA in order to harness the host’s immune system to improve survival. The over-expression of plasma gelsolin (pGSN) mRNA is associated with a poorer prognosis in OVCA patients; however, its immuno-modulatory role has not been elucidated. In this study, for the first time, we report pGSN as an inhibitor of M1 macrophage anti-tumor functions in OVCA chemoresistance. Increased epithelial pGSN expression was associated with the loss of chemoresponsiveness and poor survival. While patients with increased M1 macrophage infiltration exhibited better survival due to nitric-oxide-induced ROS accumulation in OVCA cells, cohorts with poor survival had a higher infiltration of M2 macrophages. Interestingly, increased epithelial pGSN expression was significantly associated with the reduced survival benefits of infiltrated M1 macrophages, through apoptosis via increased caspase-3 activation and reduced production of iNOS and TNFα. Additionally, epithelial pGSN expression was an independent prognostic marker in predicting progression-free survival. These findings support our hypothesis that pGSN is a modulator of inflammation and confers chemoresistance in OVCA, in part by resetting the relative abundance and function of macrophage subtypes in the ovarian tumor microenvironment. Our findings raise the possibility that pGSN may be a potential therapeutic target for immune-mediated chemoresistance in OVCA.

Simple Summary Cancer drugs have the lowest success rate of approval in drug development programs. In order to address this, predictive preclinical assays that correctly reflect the clinical efficacy of a drug represent an urgent need in both clinical oncology and pharmaceutical research. To address this need, multiple tumor models have been developed, including tumor explant culture platforms, which are the only models that preserve the integrity of the tumor tissue. However, these models have not been fully characterized and multiple variables exist between studies. We investigated the effect of tissue size and culture vessel type on the survival of tumor explants by comparing micro-dissected tumor tissue with corresponding tumor slices. Our results show that the model geometry and culture vessel affect proliferation, apoptosis, and hypoxia in tissue cultures, and must be considered in designing tumor explant culture platforms. Abstract Predicting patient responses to anticancer drugs is a major challenge both at the drug development stage and during cancer treatment. Tumor explant culture platforms (TECPs) preserve the native tissue architecture and are well-suited for drug response assays. However, tissue longevity in these models is relatively low. Several methodologies have been developed to address this issue, although no study has compared their efficacy in a controlled fashion. We investigated the effect of two variables in TECPs, specifically, the tissue size and culture vessel on tissue survival using micro-dissected tumor tissue (MDT) and tissue slices which were cultured in microfluidic chips and plastic well plates. Tumor models were produced from ovarian and prostate cancer cell line xenografts and were matched in terms of the specimen, total volume of tissue, and respective volume of medium in each culture system. We examined morphology, viability, and hypoxia in the various tumor models. Our observations suggest that the viability and proliferative capacity of MDTs were not affected during the time course of the experiments. In contrast, tissue slices had reduced proliferation and showed increased cell death and hypoxia under both culture conditions. Tissue slices cultured in microfluidic devices had a lower degree of hypoxia compared to those in 96-well plates. Globally, our results show that tissue slices have lower survival rates compared to MDTs due to inherent diffusion limitations, and that microfluidic devices may decrease hypoxia in tumor models.

Ovarian cancer (OVCA) is the most lethal gynecological cancer, due predominantly to late presentation, high recurrence rate and common chemoresistance development. The expression of the actin-associated protein cytosolic gelsolin (GSN) regulates the gynecological cancer cell fate resulting in dysregulation in chemosensitivity. In this study, we report that elevated expression of plasma gelsolin (pGSN), a secreted isoform of GSN and expressed from the same GSN gene, correlates with poorer overall survival and relapse-free survival in patients with OVCA. In addition, it is highly expressed and secreted in chemoresistant OVCA cells than its chemosensitive counterparts. pGSN, secreted and transported via exosomes (Ex-pGSN), upregulates HIF1α–mediated pGSN expression in chemoresistant OVCA cells in an autocrine manner as well as confers cisplatin resistance in otherwise chemosensitive OVCA cells. These findings support our hypothesis that exosomal pGSN promotes OVCA cell survival through both autocrine and paracrine mechanisms that transform chemosensitive cells to resistant counterparts. Specifically, pGSN transported via exosomes is a determinant of chemoresistance in OVCA.

Ovarian cancer (OVCA) patients with suboptimal residual disease (RD) and advanced stages have poor survival. pGSN is an actin binding protein which protects OVCA cells from cisplatin-induced death. There is an urgent need to discover reliable biomarkers to optimize individualized treatment recommendations. 99 plasma samples with pre-determined CA125 were collected from OVCA patients and pGSN assayed using sandwich-based ELISA. Associations between CA125, pGSN and clinicopathological parameters were examined using Fisher’s exact test, T test and Kruskal Wallis Test. Univariate and multivariate Cox proportional hazard models were used to statistically analyze clinical outcomes. At 64 µg/ml, pGSN had sensitivity and specificity of 60% and 60% respectively, for the prediction of RD where as that of CA125 at 576.5 U/mL was 43.5% and 56.5% respectively. Patients with stage 1 tumor had increased levels of pre-operative pGSN compared to those with tumor stage >1 and healthy subjects (P = 0.005). At the value of 81 µg/mL, pGSN had a sensitivity and specificity of 75% and 78.4%, respectively for the detection of early stage OVCA. At the value of 0.133, the Indicator of Stage 1 OVCA (ISO1) provided a sensitivity of 100% at a specificity of 67% (AUC, 0.89; P P = 0.05) was an independent significant predictor of progression free survival (PFS) but not CA125 (HR, 0.68; CI, 0.41–1.13; P = 0.13). Pre-operative circulating pGSN is a favorable and independent biomarker for early disease detection, RD prediction and patients’ prognosis.

Senescence is a tumor suppression mechanism defined by stable proliferation arrest. Here we demonstrate that the known synthetic lethal interaction between poly(ADP-ribose) polymerase 1 inhibitors (PARPi) and DNA repair triggers p53-independent ovarian cancer cell senescence defined by senescence-associated phenotypic hallmarks including DNA-SCARS, inflammatory secretome, Bcl-XL-mediated apoptosis resistance, and proliferation restriction via Chk2 and p21 (CDKN1A). The concept of senescence as irreversible remains controversial and here we show that PARPi-senescent cells re-initiate proliferation upon drug withdrawal, potentially explaining the requirement for sustained PARPi therapy in the clinic. Importantly, PARPi-induced senescence renders ovarian and breast cancer cells transiently susceptible to second-phase synthetic lethal approaches targeting the senescence state using senolytic drugs. The combination of PARPi and a senolytic is effective in preclinical models of ovarian and breast cancer suggesting that coupling these synthetic lethalities provides a rational approach to their clinical use and may together be more effective in limiting resistance., Senescence induction is known to induce stable proliferation arrest. Here, the authors show that sustained PARP inhibition promotes a reversible p53-independent senescence, and that PARP inhibition is synthetic lethal when combined with senolytic agents in pre-clinical models of ovarian and breast cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Among the key challenges in developing effective therapeutics is the poor translation of preclinical models used in the drug discovery pipeline. This leaves drug attrition rates and costs at an unacceptably high level. Previous work has highlighted the discrepancies in therapeutic response between current in vitro and in vivo models. To address this, we conducted a comparison study to differentiate the carboplatin chemotherapy response across four different model systems including 2D monolayers, 3D spheroids, 3D ex vivo tumors and mouse xenograft models. We used six previously characterized EOC cell lines of varying chemosensitivity and performed viability assays for each model. In vivo results from the mouse model correlated with 2D response in 3/6 cell lines while they correlated with 3D spheroids and the ex vivo model in 4/6 and 5/5 cell lines, respectively. Our results emphasize the variability in therapeutic response across models and demonstrate that the carboplatin response in EOC cell lines cultured in a 3D ex vivo model correlates best with the in vivo response. These results highlight a more feasible, reliable, and cost-effective preclinical model with the highest translational potential for drug screening and prediction studies in EOC.

While aneuploidy is a main enabling characteristic of cancers, it also creates specific vulnerabilities. Here we demonstrate that Ran inhibition targets epithelial ovarian cancer (EOC) survival through its characteristic aneuploidy. We show that induction of aneuploidy in rare diploid EOC cell lines or normal cells renders them highly dependent on Ran. We also establish an inverse correlation between Ran and the tumor suppressor NR1D1 and reveal the critical role of Ran/NR1D1 axis in aneuploidy-associated endogenous DNA damage repair. Mechanistically, we show that Ran, through the maturation of miR4472, destabilizes the mRNA of NR1D1 impacting several DNA repair pathways. We showed that NR1D1 interacts with both PARP1 and BRCA1 leading to the inhibition of DNA repair. Concordantly, loss of Ran was associated with NR1D1 induction, accumulation of DNA damages, and lethality of aneuploid EOC cells. Our findings suggest a synthetic lethal strategy targeting aneuploid cells based on their dependency to Ran.

Cancer cell lines are amongst the most important pre-clinical models. In the context of epithelial ovarian cancer, a highly heterogeneous disease with diverse subtypes, it is paramount to study a wide panel of models in order to draw a representative picture of the disease. As this lethal gynaecological malignancy has seen little improvement in overall survival in the last decade, it is all the more pressing to support future research with robust and diverse study models. Here, we describe ten novel spontaneously immortalized patient-derived ovarian cancer cell lines, detailing their respective mutational profiles and gene/biomarker expression patterns, as well as their in vitro and in vivo growth characteristics. Eight of the cell lines were classified as high-grade serous, while two were determined to be of the rarer mucinous and clear cell subtypes, respectively. Each of the ten cell lines presents a panel of characteristics reflective of diverse clinically relevant phenomena, including chemotherapeutic resistance, metastatic potential, and subtype-associated mutations and gene/protein expression profiles. Importantly, four cell lines formed subcutaneous tumors in mice, a key characteristic for pre-clinical drug testing. Our work thus contributes significantly to the available models for the study of ovarian cancer, supplying additional tools to better understand this complex disease.

Although initial treatment of ovarian cancer is successful, tumors typically relapse and become resistant to treatment. Because of poor infiltration of effector T cells, patients are mostly unresponsive to immunotherapy. Plasma gelsolin (pGSN) is transported by exosomes (small extracellular vesicle, sEV) and plays a key role in ovarian cancer chemoresistance, yet little is known about its role in immunosurveillance. Here, we report the immunomodulatory roles of sEV-pGSN in ovarian cancer chemoresistance. In chemosensitive conditions, secretion of sEV-pGSN was low, allowing for optimal CD8

Ovarian cancer (OC) is one of the most intractable diseases, exhibiting tremendous molecular heterogeneity and lacking reliable methods for screening, resulting in late diagnosis and widespread peritoneal dissemination. Menopausal estrogen replacement therapy is a well‐recognized risk factor for OC, but little is known about how estrogen might contribute to this disease at the cellular level. This study identifies chemokine receptor CXCR7/ACKR3 as an estrogen‐responsive gene, whose expression is markedly enhanced by estrogen through direct recruitment of ERα and transcriptional active histone modifications in OC cells. The gene encoding CXCR7 chemokine ligand I‐TAC/CXCL11 was also upregulated by estrogen, resulting in Ser‐118 phosphorylation, activation, and recruitment of estrogen receptor ERα at the CXCR7 promoter locus for positive feedback regulation. Both CXCR7 and CXCL11, but not CXCR3 (also recognized to interact with CXCL11), were found to be significantly increased in stromal sections of microdissected tumors and positively correlated in mesenchymal subtype of OC. Estrogenic induction of mesenchymal markers SNAI1, SNAI2, and CDH2 expression, with a consequent increase in cancer cell migration, was shown to depend on CXCR7, indicating a key role for CXCR7 in mediating estrogen upregulation of mesenchymal markers to induce invasion of OC cells. These findings identify a feed‐forward mechanism that sustains activation of the CXCR7/CXCL11 axis under ERα control to induce the epithelial–mesenchymal transition pathway and metastatic behavior of OC cells. Such interplay underlies the complex gene profile heterogeneity of OC that promotes changes in tumor microenvironment and metastatic acquisition.

Simple Summary Hexokinase II (HKII) is a key glycolysis enzyme associated with tumorigenesis, but its molecular mechanism and pathophysiological role in chemoresistant ovarian cancer remain elusive. In this study, we delineate the novel mechanism showing that activated phosphorylated-p53 (P-p53 Ser15) is required for the regulation of HKII intracellular trafficking and metabolic regulation in chemosensitive ovarian cancer, but not in chemoresistant ovarian cancer harboring p53 mutation. We have observed that increased nuclear HKII-P-p53 (Ser15) interaction is likely associated with chemosensitivity and better survival outcomes in epithelial ovarian cell lines, human primary epithelial ovarian cancer cells, and tumor sections. Nuclear HKII-P-p53 (Ser15) interaction may function as a promising prognostic biomarker, enabling prediction of patients with poor prognosis for deciding better clinical strategies. Abstract In epithelial ovarian cancer (EOC), carboplatin/cisplatin-induced chemoresistance is a major hurdle to successful treatment. Aerobic glycolysis is a common characteristic of cancer. However, the role of glycolytic metabolism in chemoresistance and its impact on clinical outcomes in EOC are not clear. Here, we show a functional interaction between the key glycolytic enzyme hexokinase II (HKII) and activated P-p53 (Ser15) in the regulation of bioenergetics and chemosensitivity. Using translational approaches with proximity ligation assessment in cancer cells and human EOC tumor sections, we showed that nuclear HKII-P-p53 (Ser15) interaction is increased after chemotherapy, and functions as a determinant of chemoresponsiveness as a prognostic biomarker. We also demonstrated that p53 is required for the intracellular nuclear HKII trafficking in the control of glycolysis in EOC, associated with chemosensitivity. Mechanistically, cisplatin-induced P-p53 (Ser15) recruits HKII and apoptosis-inducing factor (AIF) in chemosensitive EOC cells, enabling their translocation from the mitochondria to the nucleus, eliciting AIF-induced apoptosis. Conversely, in p53-defective chemoresistant EOC cells, HKII and AIF are strongly bound in the mitochondria and, therefore, apoptosis is suppressed. Collectively, our findings implicate nuclear HKII-P-p53(Ser15) interaction in chemosensitivity and could provide an effective clinical strategy as a promising biomarker during platinum-based therapy.

There is an urgent need and strong clinical and pharmaceutical interest in developing assays that allow for the direct testing of therapeutic agents on primary tissues. Current technologies fail to provide the required sample longevity, throughput, and integration with standard clinically proven assays to make the approach viable. Here we report a microfluidic micro-histological platform that enables ex vivo culture of a large array of prostate and ovarian cancer micro-dissected tissue (MDT) followed by direct on-chip fixation and paraffination, a process we term paraffin-embedding lithography (PEL). The result is a high density MDT-Micro Array (MDTMA) compatible with standard clinical histopathology that can be used to analyse ex vivo tumor response or resistance to therapeutic agents. The cellular morphology and tissue architecture are preserved in MDTs throughout the 15 day culture period. We also demonstrate how this methodology can be used to study molecular pathways involved in cancer by performing in-depth characterization of biological and pharmacological mechanisms such as p65 nuclear translocation via TNF stimuli, and to predict the treatment outcome in the clinic via MDT response to taxane-based therapies.

Simple Summary Olaparib is an oral medication typically used to treat certain advanced ovarian and breast cancers with mutations in BRCA1 or BRCA2 genes. Mutations in these genes can increase the risk of developing breast, ovarian, and other types of cancer. Olaparib is the first clinically approved drug that specifically targets a vulnerability of cancers with these mutations. Genetic alterations in cancer tumors can affect response to treatment in cancer patients. Cancer models such as cell lines, which are cancer cells derived from patients and have been grown in the laboratory over time, can be used to identify these alterations which may contribute to sensitivity or resistance to treatment. We analyzed data from two independent groups of cancer cell lines and identified alterations in additional genes (PUM3, EEF1A1 and ELP4) that potentially increase sensitivity to olaparib. Further experimental and clinical investigations are required to validate our findings. Abstract The benefit of PARP inhibitor olaparib in relapsed and advanced high-grade serous ovarian carcinoma (HGSOC) is well established especially in BRCA1/2 mutation carriers. Identification of additional biomarkers can help expand the population of patients most likely to benefit from olaparib treatment. To identify candidate markers of olaparib response we analyzed genomic and in vitro olaparib response data from two independent groups of cancer cell lines. Using pan-cancer cell lines (n = 896) from the Genomics of Drug Sensitivity in Cancer database, we applied linear regression methods to identify statistically significant gene predictors of olaparib response based on mRNA expression. We then analyzed whole exome sequencing and mRNA gene expression data from our collection of 18 HGSOC cell lines previously classified as sensitive, intermediate, or resistant based on in vitro olaparib response for mutations, copy number variation and differential expression of candidate olaparib response genes. We identify genes previously associated with olaparib response (SLFN11, ABCB1), and discover novel candidate olaparib sensitivity genes with known functions including interaction with PARP1 (PUM3, EEF1A1) and involvement in homologous recombination DNA repair (ELP4). Further investigations at experimental and clinical levels are required to validate novel candidates, and ultimately determine their efficacy as potential biomarkers of olaparib sensitivity.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America, underscoring the need for the development of new therapeutic strategies for the management of this disease. Although many drugs are pre-clinically tested every year, only a few are selected to be evaluated in clinical trials, and only a small number of these are successfully incorporated into standard care. Inaccuracies with the initial in vitro drug testing may be responsible for some of these failures. Drug testing is often performed using 2D monolayer cultures or 3D spheroid models. Here, we investigate the impact that these different in vitro models have on the carboplatin response of four EOC cell lines, and in particular how different 3D models (polydimethylsiloxane-based microfluidic chips and ultra low attachment plates) influence drug sensitivity within the same cell line. Our results show that carboplatin responses were observed in both the 3D spheroid models tested using apoptosis/cell death markers by flow cytometry. Contrary to previously reported observations, these were not associated with a significant decrease in spheroid size. For the majority of the EOC cell lines (3 out of 4) a similar carboplatin response was observed when comparing both spheroid methods. Interestingly, two cell lines classified as resistant to carboplatin in 2D cultures became sensitive in the 3D models, and one sensitive cell line in 2D culture showed resistance in 3D spheroids. Our results highlight the challenges of choosing the appropriate pre-clinical models for drug testing.

// Lydia Edjekouane 1, 2 , Samira Benhadjeba 1, 2 , Maika Jangal 3 , Hubert Fleury 4 , Nicolas Gevry 3 , Euridice Carmona 4 , Andre Tremblay 1, 2, 5 1 Research Center, CHU Sainte-Justine, Montreal, Quebec, H3T 1C5 Canada 2 Department of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4 Canada 3 Department of Biology, Faculty of Sciences, University of Sherbrooke, Sherbrooke, Quebec, J1K 2R1 Canada 4 CHUM Research Center, Institut du cancer de Montreal, Montreal, Quebec, H2X 0A9 Canada 5 Department of Obstetrics & Gynecology, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4 Canada Correspondence to: Andre Tremblay, email: andre.tremblay.1@umontreal.ca Keywords: hyaluronidase, estrogen receptors, 3p21.3 cluster Received: April 19, 2016 Accepted: September 24, 2016 Published: October 13, 2016 ABSTRACT Chromosomal and genome abnormalities at the 3p21.3 locus are frequent events linked to epithelial cancers, including ovarian and breast cancers. Genes encoded in the 3p21.3 cluster include HYAL1, HYAL2 and HYAL3 members of hyaluronidases involved in the breakdown of hyaluronan, an abundant component of the vertebrate extracellular matrix. However, the transcriptional regulation of HYAL genes is poorly defined. Here, we identified the estrogen receptor ERα as a negative regulator of HYAL1 expression in breast cancer cells. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL1 gene expression in human breast tumors. ChIP-Seq analysis identified several ERα binding sites within the 3p21.3 locus, supporting the role of estrogen as an upstream signal that diversely regulates the expression of 3p21.3 genes at both proximal and distal locations. Of these, HYAL1 was repressed by estrogen through ERα binding to a consensus estrogen response element (ERE) located in the proximal promoter of HYAL1 and flanked by an Sp1 binding site, required to achieve optimal estrogen repression. The repressive chromatin mark H3K27me3 was increased at the proximal HYAL1 ERE but not at other EREs contained in the cluster, providing a mechanism to selectively downregulate HYAL1. The HYAL1 repression was also specific to ERα and not to ERβ, whose expression did not correlate with HYAL1 in human breast tumors. This study identifies HYAL1 as an ERα target gene and provides a functional framework for the direct effect of estrogen on 3p21.3 genes in breast cancer cells.

High-grade serous ovarian carcinoma (HGSOC) commonly responds to initial therapy, but this response is rarely durable. Understanding the cell fate decisions taken by HGSOC cells in response to treatment could guide new therapeutic opportunities. Here, we find that more than 90% of tissue-derived primary HGSOC cultures, reflecting the original disease, retain the capacity to undergo stress-induced cellular senescence and primarily undergo therapy-induced senescence (TIS) in response to first-line carboplatin/taxol chemotherapy. HGSOC-TIS displays senescence-associated hallmarks, including a stable proliferation arrest, increased p16INK4A expression, persistent DNA damage, an inflammatory secretome, and senolytic sensitivity, suggesting new avenues for selective pharmacological manipulation of these cells. Comparison of pre- and post-chemotherapy patient HGSOC tissue samples revealed changes in physio-pathological senescence biomarkers supporting the occurrence of post-treatment TIS. Whether cell senescence induced by cancer therapy is beneficial or detrimental to treatment outcomes remains unknown. We find that patients with stronger TIS biomarkers in post-chemotherapy tissues have a more favorale 5-year survival, suggesting that the induction of senescence in HGSOC cells accounts, at least in part, for beneficial responses to treatment. Given that HGSOC cells almost universally retain the capacity to undergo senescence and that senescence appears beneficial in this context, senescence-centric therapeutic avenues should be further explored. Citation Format: Michael Skulimowski, Llilians Calvo-Gonzales, Shuofei Cheng, Isabelle Clément, Lise Portelance, Yu Zhan, Euridice Carmona, Manon de Ladurantaye, Julie Lafontaine, Kurosh Rahimi, Diane Provencher, Anne-Marie Mes-Masson, Francis Rodier. Senescence is a central response to chemotherapy in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5833.

Introduction: Epithelial ovarian cancer (EOC) is still a deadly disease, with a 5-year overall survival of 45%. Current research focuses on the development of new targeted and personalized therapies to improve survival in these patients. The drug discovery pipeline is dependent on preclinical models, which have mostly focused on 2D systems because of time and cost efficiency. However, it is currently unclear whether they accurately reflect patient response and whether other in vitro models may better predict therapeutic response. Preliminary data from our laboratory suggest that the sensitivity to carboplatin chemotherapy varies between 2D and 3D in vitro models. We hypothesize that the 3D model will more closely reflect therapeutic response. The primary objective of this study is to characterize the sensitivity to carboplatin of our EOC cell lines in 2D monolayers and 3D spheroids and compare them to their in vivo response using our xenograft mouse models. Methods: We are injecting NOD-RAG mice with 7 EOC cell lines (TOV112D, TOV21G, OV4485, OV4453, OV1946, OV90, OV3133). At 200mm3, weekly carboplatin treatments (group1 = control, group 2= 25mg/kg, group 3= 50mg/kg, group 4= 75mg/kg) are given up to 6 cycles. Tumor volume and survival curves are used to categorize chemosensitivity between cell lines. Furthermore, the same cell lines are seeded in ultra-low attachment microplates to form spheroids over 48hr and are thereafter treated with 24hr of carboplatin. Flow cytometry analyses are done to classify cell lines. Results: The results for the 2D models using clonogenic assays (IC50) of the 7 cell lines have previously been published. Thus far, we have completed a first set of in vivo experiments for TOV21G showing a nonsignificant tumor growth suppression after 6 doses (no difference between the 3 treated and the nontreated group). Tumor growth suppression for OV90 was statistically significant with 50mg/kg and 75mg/kg of carboplatin after 6 doses. This demonstrates thus far that the most resistant cell line in 2D is more sensitive in an in vivo model. Conclusion: The results will help define the most reliable preclinical model with the highest translational potential to predict treatment response. Citation Format: Melica Nourmoussavi, Euridice Carmona, Anne-Marie Mes-Masson. Predictive treatment response models for epithelial ovarian cancer: Comparison of 2D, 3D, and in vivo models [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B80.

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines. Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561–73. ©2018 AACR.

Few cell line models of epithelial ovarian cancer (EOC) have been developed for the high-grade serous (HGS) subtype, which is the most common and lethal form of gynaecological cancer. Here we describe the establishment of six new EOC cell lines spontaneously derived from HGS tumors (TOV2978G, TOV3041G and TOV3291G) or ascites (OV866(2), OV4453 and OV4485). Exome sequencing revealed somatic TP53 mutations in five of the cell lines. One cell line has a novel BRCA1 splice-site mutation, and another, a recurrent BRCA2 nonsense mutation, both of germline origin. The novel BRCA1 mutation induced abnormal splicing, mRNA instability, resulting in the absence of BRCA1 protein. None of the cell lines harbor mutations in KRAS or BRAF, which are characteristic of other EOC subtypes. SNP arrays showed that all of the cell lines exhibited structural chromosomal abnormalities, copy number alterations and regions of loss of heterozygosity, consistent with those described for HGS. Four cell lines were able to produce 3D-spheroids, two exhibited anchorage-independent growth, and three (including the BRCA1 and BRCA2 mutated cell lines) formed tumors in SCID mice. These novel HGS EOC cell lines and their detailed characterization provide new research tools for investigating the most common and lethal form of EOC.

Anthracyclines such as daunorubicin are anticancer agents that are transported into cells and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines.

We have shown previously that sperm surface arylsulfatase A (ASA) of mouse, pig, and human is involved in sperm–egg zona pellucida (ZP) binding. By treating capacitated mouse sperm with A23187 to induce the acrosome reaction, we demonstrated by immunoblotting that ASA also existed in the acrosomal content and on the inner acrosomal membrane. Since mZP2 and mZP3 are known as sperm receptors, whereas mZP1 as a cross-linker of mZP2/mZP3, we determined whether purified ASA bound to mZP2 and mZP3 selectively. The three mZP glycoproteins were purified from solubilized ovarian ZP by size exclusion column chromatography. Immuno-dot blot analyses revealed that purified sperm ASA bound to mZP2 at the highest level followed by mZP3, whereas the binding of ASA to mZP1 was minimal. The results confirmed the physiological significance of sperm ASA in the ZP binding process. The binding of ASA to mZP2 and mZP3 was, however, not dependent on the active site pocket amino acids, Cys69, Lys123, and Lys302, which are pertinent to the capturing of an arylsulfate substrate, since ASA mutant with Ala substitution at these three residues still bound to mZP2 and mZP3. The availability of the active site pocket of ASA bound to the ZP suggested that ASA would still retain enzymatic activity, which might be important for subsequent sperm penetration through the ZP.