Platinum-resistant Cell Lines Research Articles

Cell-intrinsic platinum response and associated genetic and gene expression signatures in ovarian cancer cell lines and isogenic models.

Ovarian cancers are still largely treated with platinum-based chemotherapy as the standard of care, yet few biomarkers of clinical response have had an impact on clinical decision making as of yet. Two particular challenges faced in mechanistically deciphering platinum responsiveness in ovarian cancer have been the suitability of cell line models for ovarian cancer subtypes and the availability of information on comparatively how sensitive ovarian cancer cell lines are to platinum. We performed one of the most comprehensive profiles to date on 36 ovarian cancer cell lines across over seven subtypes and integrated drug response and multiomic data to improve on our understanding of the best cell line models for platinum responsiveness in ovarian cancer. RNA-seq analysis of the 36 cell lines in a single batch experiment largely conforms with the currently accepted subtyping of ovarian cancers, further supporting other studies that have reclassified cell lines and demonstrate that commonly used cell lines are poor models of high-grade serous ovarian carcinoma. We performed drug dose response assays in the 32 of these cell lines for cisplatin and carboplatin, providing a quantitative database of IC 50 s for these drugs. Our results demonstrate that cell lines largely fall either well above or below the equivalent dose of the clinical maximally achievable dose (C max ) of each compound, allowing designation of cell lines as sensitive or resistant. We performed differential expression analysis for high-grade serous ovarian carcinoma cell lines to identify gene expression correlating with platinum-response. Further, we generated two platinum-resistant derivatives each for OVCAR3 and OVCAR4, as well as leveraged clinically-resistant PEO1/PEO4/PEO6 and PEA1/PEA2 isogenic models to perform differential expression analysis for seven total isogenic pairs of platinum resistant cell lines. While gene expression changes overall were heterogeneous and vast, common themes were innate immunity/STAT activation, epithelial to mesenchymal transition and stemness, and platinum influx/efflux regulators. In addition to gene expression analyses, we performed copy number signature analysis and orthogonal measures of homologous recombination deficiency (HRD) scar scores and copy number burden, which is the first report to our knowledge applying field-standard copy number signatures to ovarian cancer cell lines. We also examined markers and functional readouts of stemness that revealed that cell lines are poor models for examination of stemness contributions to platinum resistance, likely pointing to the fact that this is a transient state. Overall this study serves as a resource to determine the best cell lines to utilize for ovarian cancer research on certain subtypes and platinum response studies, as well as sparks new hypotheses for future study in ovarian cancer.

TCEB3 initiates ovarian cancer apoptosis by mediating ubiquitination and degradation of MCL-1.

Platinum resistance remains a major contributor to the poor prognosis of ovarian cancer. Anti-apoptotic protein myeloid cell leukemia-1 (MCL-1) has emerged as a promising target for overcoming drug resistance, but different cancer cells utilize distinct protein degradation pathways to alter MCL-1 level. We systematically investigated E3 ligases to identify novel candidates that mediate platinum resistance in ovarian cancer. Transcription Elongation Factor B (TCEB3) has been identified as a novel E3 ligase recognition subunit that targets MCL-1 in the cytoplasm during platinum treatment other than its traditional function of targeting the Pol II in the nuclear compartment. TCEB3 expression is downregulated in platinum-resistant cell lines and this low expression is associated with poor prognosis. The ubiquitination of MCL-1 induced by TCEB3 leads to cell death in ovarian cancer. Moreover, platinum treatment increased the cytoplasm proportion of TCEB3, and the cytoplasm localization of TCEB3 is important for its targeting of MCL-1. This study emphasizes the dual function of TCEB3 in homeostasis maintenance and in cell fate determination under different conditions, and provides a new insight into drug resistance in ovarian cancer.

Abstract B095: Tenascin C promotes ovarian cancer stem cells interactions with the niche

Abstract Background: Effective treatment of ovarian cancer (OC) is greatly impacted by advanced, late-stage diagnosis which usually presents with intra-peritoneal metastases, chemoresistance and high recurrence. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids disseminated in the peritoneal fluid. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components that stiffen the tumor stroma and promote the aberrant activation of oncogenic processes, including survival, invasion, and stemness. Tenascin C (TNC) is one of the molecules secreted in the ECM and correlates with migration and metastatic progression in a variety of carcinomas. However, the functional role of TNC in spheroid formation and in the maintenance of the platinum resistant ovarian cancer stem phenotype has not been investigated yet. We tested the hypothesis that the increased expression of TNC and its secretion in the ECM compartment promotes survival of OCSCs, supports OC spheroid formation and tumor progression. Methods: We compared TNC expression in OC cells grown as spheroids and monolayers and in chemoresistant vs sensitive OC cells by realtime-PCR and Western Blot (WB). TNC blockade by siRNA knockdown (KD) was evaluated in combination with carboplatin on spheroid assay, colony formation, and by qPCR of stemness related gene expression. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the TNC promoter. Results: OC cells grown as spheroids showed a significant increase in TNC expression compared to monolayers. Our results also demonstrated an increase in TNC expression levels in platinum resistant OC cell lines when compared to the platinum sensitive counterpart. A comparison of normal adjacent ovarian epithelium and primary OC tumors on a multitissue array revealed that TNC expression coincided with a pro-tumorigenic phenotype (n=88, P<0.001), correlating with poor outcome. TNC-KD in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. Mechanistically, b-catenin-KD decreased TNC expression levels and β-catenin coimmunoprecipitated with the TNC promoter, suggesting that TNC is a direct β-catenin target. Additionally, OC spheroids treated with the Wnt ligand WNT-3A showed increased TNC expression compared to control untreated cells, as demonstrated by WB. Importantly, the enhanced expression of TNC was followed by its secretion in the ECM compartment, as observed via immunofluorescence imaging, implying a direct role of TNC in the ECM-rearrangement which is responsible for CSC maintenance and spheroid formation. Conclusions: TNC is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that strategies aimed at interfering with TNC expression and/or secretion along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Amber Rogers, Maya Shebeshi, Morganna Gordon, Rula Atwani, Virginie Lazar, Salvatore Condello. Tenascin C promotes ovarian cancer stem cells interactions with the niche [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B095.

MYC drives platinum resistant SCLC that is overcome by the dual PI3K-HDAC inhibitor fimepinostat

BackgroundSmall cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with an appalling overall survival of less than 5% (Zimmerman et al. J Thor Oncol 14:768-83, 2019). Patients typically respond to front line platinum-based doublet chemotherapy, but almost universally relapse with drug resistant disease. Elevated MYC expression is common in SCLC and has been associated with platinum resistance. This study evaluates the capacity of MYC to drive platinum resistance and through screening identifies a drug capable of reducing MYC expression and overcoming resistance.MethodsElevated MYC expression following the acquisition of platinum resistance in vitro and in vivo was assessed. Moreover, the capacity of enforced MYC expression to drive platinum resistance was defined in SCLC cell lines and in a genetically engineered mouse model that expresses MYC specifically in lung tumors. High throughput drug screening was used to identify drugs able to kill MYC-expressing, platinum resistant cell lines. The capacity of this drug to treat SCLC was defined in vivo in both transplant models using cell lines and patient derived xenografts and in combination with platinum and etoposide chemotherapy in an autochthonous mouse model of platinum resistant SCLC.ResultsMYC expression is elevated following the acquisition of platinum resistance and constitutively high MYC expression drives platinum resistance in vitro and in vivo. We show that fimepinostat decreases MYC expression and that it is an effective single agent treatment for SCLC in vitro and in vivo. Indeed, fimepinostat is as effective as platinum-etoposide treatment in vivo. Importantly, when combined with platinum and etoposide, fimepinostat achieves a significant increase in survival.ConclusionsMYC is a potent driver of platinum resistance in SCLC that is effectively treated with fimepinostat.

Platinum-Resistant Ovarian Cancer Is Vulnerable to the cJUN-XRCC4 Pathway Inhibition.

DNA double-strand breaks (DSBs) caused by platinum drugs are dangerous lesions that kill cancer cells in chemotherapy. Repair of DSB by homologous recombination (HR) and nonhomologous end joining (NHEJ) is frequently associated with platinum resistance in ovarian cancer. While the role of the HR pathway and HR-targeting strategy in platinum resistance is well studied, dissecting and targeting NHEJ machinery to overcome platinum resistance in ovarian cancer remain largely unexplored. Here, through an NHEJ pathway-focused gene RNAi screen, we found that the knockdown of XRCC4 significantly sensitized cisplatin treatment in the platinum-resistant ovarian cancer cell lines. Moreover, upregulation of XRCC4 is observed in a panel of platinum-resistant cell lines relative to the parental cell lines, as well as in ovarian cancer patients with poor progression-free survival. Mechanistically, the increased sensitivity to cisplatin upon XRCC4 knockdown was caused by accumulated DNA damage. In cisplatin-resistant ovarian cancer, the JNK-cJUN complex, activated by cisplatin, translocated into the nucleus and promoted the transcription of XRCC4 to confer cisplatin resistance. Knockdown of XRCC4 or treatment of the JNK inhibitor led to the attenuation of cisplatin-resistant tumor growth in the xenograft mouse models. These data suggest targeting XRCC4 is a potential strategy for ovarian cisplatin resistance in ovarian cancer.

Platinum-resistance in epithelial ovarian cancer: an interplay of epithelial–mesenchymal transition interlinked with reprogrammed metabolism

BackgroundEpithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases.MethodsWe explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial–mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan–Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME).ResultsOur proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFβ1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFβ1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8+ T cells, macrophages, neutrophils, and dendritic cells in the TME.ConclusionsOur research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients.

Dinaciclib as an effective pan-cyclin dependent kinase inhibitor in platinum resistant ovarian cancer.

Ovarian cancer (OC) is amongst the most lethal of common cancers in women. Lacking in specific symptoms in the early stages, OC is predominantly diagnosed late when the disease has undergone metastatic spread and chemotherapy is relied on to prolong life. Platinum-based therapies are preferred and although many tumors respond initially, the emergence of platinum-resistance occurs in the majority of cases after which prognosis is very poor. Upregulation of DNA damage pathways is a common feature of platinum resistance in OC with cyclin dependent kinases (CDKs) serving as key regulators of this process and suggesting that CDK inhibitors (CDKis) could be effective tools in the treatment of platinum resistant and refractory OC. The aim of this study was to evaluate the efficacy of CDKis in platinum resistant OC models and serve as a predictor of potential clinical utility. The efficacy of CDKi, dinaciclib, was determined in wildtype and platinum resistant cell line pairs representing different OC subtypes. In addition, dinaciclib was evaluated in primary cells isolated from platinum-sensitive and platinum-refractory tumors to increase the clinical relevance of the study. Dinaciclib proved highly efficacious in OC cell lines and primary cells, which were over a thousand-fold more sensitive to the CDKi than to cisplatin. Furthermore, cisplatin resistance in these cells did not influence sensitivity to dinaciclib and the two drugs combined additively in both platinum-sensitive and platinum-resistant OC cells suggesting a potential role for pan-CDKis (CDKis targeting multiple CDKs), such as dinaciclib, in the treatment of advanced and platinum-resistant OC.

CUL4B increases platinum-based drug resistance in colorectal cancer through EMT: A study in its mechanism.

Platinum-based chemotherapy drugs play a very important role in the treatment of patients with advanced colorectal cancer, but the drug resistance of platinum-based chemotherapy drugs is an important topic that puzzles us. If we can find mechanisms of resistance, it will be revolutionary for us. We analysed the differential genes, core genes and their enrichment pathways in platinum-resistant and non-resistant patients through a public database. Platinum-resistant cell lines were cultured in vitro for in vitro colony and Transwell analysis. Tumorigenesis analysis of nude mice in vivo. Verify the function of core genes. Through differential gene and enrichment analysis, we found that CUL4B was the main factor affecting platinum drug resistance and EMT. Our hypothesis was further verified by in vitro drug-resistant and wild-type cell lines and in vivo tumorigenesis analysis of nude mice. CUL4B leads to platinum drug resistance in colorectal cancer by affecting tumour EMT.

Circulating exosomal TMEM205 protein expression for early prediction of platinum-resistance in high grade serous ovarian cancer (255)

Circulating exosomal TMEM205 protein expression for early prediction of platinum-resistance in high grade serous ovarian cancer (255)

A novel cell line panel reveals non-genetic mediators of platinum resistance and phenotypic diversity in high grade serous ovarian cancer

ObjectivesResistance to cancer therapy is an enduring challenge and accurate and reliable preclinical models are lacking. We interrogated this unmet need using high grade serous ovarian cancer (HGSC) as a disease model. MethodsWe created five in vitro and two in vivo platinum-resistant HGSC models and characterised the entire cell panel via whole genome sequencing, RNASeq and creation of intraperitoneal models. ResultsMutational signature analysis indicated that platinum-resistant cell lines evolved from a pre-existing ancestral clone but a unifying mutational cause for drug resistance was not identified. However, cisplatin-resistant and carboplatin-resistant cells evolved recurrent changes in gene expression that significantly overlapped with independent samples obtained from multiple patients with relapsed HGSC. Gene Ontology Biological Pathways (GOBP) related to the tumour microenvironment, particularly the extracellular matrix, were repeatedly enriched in cisplatin-resistant cells, carboplatin-resistant cells and also in human resistant/refractory samples. The majority of significantly over-represented GOBP however, evolved uniquely in either cisplatin- or carboplatin-resistant cell lines resulting in diverse intraperitoneal behaviours that reflect different clinical manifestations of relapsed human HGSC. ConclusionsOur clinically relevant and usable models reveal a key role for non-genetic factors in the evolution of chemotherapy resistance. Biological pathways relevant to the extracellular matrix were repeatedly expressed by resistant cancer cells in multiple settings. This suggests that recurrent gene expression changes provide a fitness advantage during platinum therapy and also that cancer cell-intrinsic mechanisms influence the tumour microenvironment during the evolution of drug resistance. Candidate genes and pathways identified here could reveal therapeutic opportunities in platinum-resistant HGSC.

Impairment of RAD17 Functions by miR-506-3p as a Novel Synthetic Lethal Approach Targeting DNA Repair Pathways in Ovarian Cancer.

Epithelial ovarian cancer (EOC) remains the most lethal gynecological cancer and development of chemo-resistance is a major factor in disease relapse. Homologous recombination (HR) is a critical pathway for DNA double strand break repair and its deficiency is associated to a better response to DNA damage-inducing agents. Strategies to inhibit HR-mediated DNA repair is a clinical need to improve patients’ outcome. MicroRNA (miRNAs) affect most of cellular processes including response to cancer treatment. We previously showed that miR-506-3p targets RAD51, an essential HR component. In this study we demonstrated that: i) another HR component, RAD17, is also a direct target of miR-506-3p and that it is involved in mediating miR-506-3p phenotypic effects; ii) the impairment of miR-506-3p binding to RAD17 3’ UTR reverted the miR-506-3p induced platinum sensitization; iii) miR-506-3p/RAD17 axis reduces the ability of EOC cell to sense DNA damage, abrogates the G2/M cell cycle checkpoint thus delaying the G2/M cell cycle arrest likely allowing the entry into mitosis of heavily DNA-damaged cells with a consequent mitotic catastrophe; iv) RAD17 expression, regulated by miR-506-3p, is synthetically lethal with inhibitors of cell cycle checkpoint kinases Chk1 and Wee1 in platinum resistant cell line. Overall miR-506-3p expression may recapitulate a BRCAness phenotype sensitizing EOC cells to chemotherapy and helping in selecting patients susceptible to DNA damaging drugs in combination with new small molecules targeting DNA-damage repair pathway.

Abstract 1129: Interrogating JAK/STAT signaling in ovarian cancer as a potential oncogenic driver and therapeutic target

Abstract Ovarian cancer is the 7th most common cancer in women. It is the most fatal of all female reproductive cancers, due to the 5-year overall survival of 10-30% and over half of patients being diagnosed with late-stage disease. Despite initial response to front line platinum- and taxol-based chemotherapies, over 70% of patients develop recurrent and chemotherapy resistant disease and nearly all of these women die of their disease. Recently, the use of targeted therapies such as bevacizumab and PARP inhibitors have been shown to improve progression-free survival. However, these regimens have thus far failed to improve overall survival in patients without specific genetic signatures, highlighting the urgent need for alternative therapies. We have identified lestaurtinib as a potent inhibitor of many ovarian cancer cell lines, including platinum and PARP inhibitor resistant models, and patient derived organoid models. RNA-sequencing following lestaurtinib treatment identified JAK/STAT signaling as one of the most profoundly downregulated pathways. Additionally, it was recently reported via single cell RNA sequencing of patient tumors that downstream mediators of the JAK/STAT pathway are among the most highly expressed genes in ovarian tumor cells. Further, we have discovered that STAT1 and STAT3 are constitutively activated in chemotherapy and PARP inhibitor resistant cell lines as reflected by high levels of Tyr701/705 and Ser727 phosphorylation suggesting induction of this pathway may drive resistance. Selective siRNA-mediated knockdown of STAT1/3 resulted in significant growth inhibition confirming their importance in maintaining cell viability. Surprisingly, we have identified profound differences in the ability of various JAK/STAT inhibitors to suppress ovarian cancer cell growth. For example, ruxolitinib, the only JAK/STAT inhibitor currently in clinical trials for ovarian cancer, failed to inhibit the growth sensitive, platinum resistant or PARP inhibitor resistant cell lines. Our preliminary data indicate that JAK/STAT inhibitor efficacy in preventing ovarian cancer cell growth is related to their ability to block specific phosphorylation events on STAT1/3 proteins. We believe that precise and selective inhibition of STAT1 and STAT3 signaling represents a robust, durable, and novel approach for the treatment of highly aggressive and resistant forms of ovarian cancer. Further we plan to elucidate the molecular mechanisms by which STAT1 and STAT3 function to support ovarian cancer cell viability and growth, in order to substantially advance our understanding of JAK/STAT signaling in ovarian cancer and to identify the most effective ways to pharmacologically inhibit JAK/STAT signaling in ovarian cancer cells. Outcomes from this work will have both immediate and long-term impacts for our patients and will lay the foundation for future biomarker-driven clinical trials. Citation Format: Esther Rodman, Michael Emch, Elizabeth Bruinsma, Xiaonan Hou, John Weroha, John Hawse. Interrogating JAK/STAT signaling in ovarian cancer as a potential oncogenic driver and therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1129.

Abstract 5408: Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in the management of high-grade serous ovarian cancer (HGSOC). However, most HGSOC ultimately develops resistance to PARPi, leading to unmet medical needs for better therapeutic strategies. ATR-mediated G2/M cell cycle checkpoints are important for cell survival and DNA repair, especially in HGSOC due to its dysfunctional p53-mediated G1/S checkpoint, making them an attractive therapeutic target. However, only modest monotherapy activity of ATR inhibitors (ATRis) has been reported in patients with HGSOC, highlighting the need for combination treatment. We conducted a high-throughput drug combination screen of ATRi (ceralasertib) with 1,912 drugs in two PARPi-resistant BRCA2-mutant PEO1 (PEO1/OlaR and PEO1/OlaJR) HGSOC cell lines. In this screen, 234 drugs showed synergistic cytotoxicity with ATRi, including 31 (13.2%) cell cycle checkpoint inhibitors, 27 (11.5%) chemotherapeutic drugs, and 13 (5.6%) PI3K/AKT pathway inhibitors. We prioritized the PI3K/AKT pathway inhibitors for ATRi combination because the PI3K/AKT pathway is upregulated in &amp;gt;70% of HGSOC and associated with an aggressive phenotype. Moreover, PI3K/AKT signaling is found to be activated in HGSOC cells with PARPi resistance. We separately validated that an ATRi (ceralasertib &amp;lt; 1 μM) in combination with an AKT inhibitor (AKTi; capivasertib &amp;lt; 10 μM) yielded additive/synergistic cytotoxic effects (combination index &amp;lt; 1) in various HGSOC cell lines, including acquired and de novo PARPi-resistant (PEO1/OlaR, PEO1/OlaJR, and PEO4) and platinum-resistant HGSOC cell lines (OVCAR3), by XTT and colony formation assays. With clinically attainable concentrations of ATRi (1 μM) and AKTi (10 μM), the combination treatment increased cell apoptosis compared to ATRi (increased 2.1 to 95.2-fold; P &amp;lt; 0.001) or AKTi alone (increased 3.3 to 92.2-fold; P &amp;lt; 0.001) in both PARPi-sensitive and -resistant HGSOC cells. ATRi+AKTi treatment also showed greater DNA damage as evidenced by increased percentage of cells with ≥ 5 γH2AX foci relative to ATRi (increased 1.1 to 7.6-fold; not significant in PEO1 and P &amp;lt; 0.001 in PARPi-resistant cells) or AKTi (increased 2.3 to 6.2-fold; P &amp;lt; 0.001) in both PARPi-sensitive and -resistant cells. Furthermore, ATRi+AKTi treatment augmented replication stress as measured by increased phospho-RPA+/γH2AX+ populations compared with ATRi (increased 1.7 to 4.7-fold; P &amp;lt; 0.001) or AKTi (increased 3.7 to 12.0-fold; P &amp;lt; 0.001). Overall, our results suggest that dual inhibition of ATR and AKT pathways results in greater cell death by increasing DNA damage and replication stress in HGSOC cells with PARPi resistance. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Nitasha Gupta, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5408.

Differential and divergent activity of insulin‑like growth factor binding protein 6 in platinum‑sensitive versus platinum‑resistant high‑grade serous ovarian carcinoma cell lines

Insulin-like growth factor binding protein 6 (IGFBP6) is a secreted protein with a controversial role in human malignancies, being downregulated in most types of human cancer, but upregulated in selected tumors. Ovarian cancer (OC) is a human malignancy characterized by IGFBP6 downregulation; however, the significance of its low expression during ovarian carcinogenesis is still poorly understood. In the present study, IGFBP6 expression and activation of its associated signaling pathway were evaluated in two matched OC cell lines derived from a high-grade serous OC before and after platinum resistance (PEA1 and PEA2 cells, respectively). A whole genome gene expression analysis was comparatively performed in both cell lines upon IGFBP6 stimulation using Illumina technology. IGFBP6 gene expression data from human OC cases were obtained from public datasets. Gene expression data from public datasets confirmed the downregulation of IGFBP6 in primary and metastatic OC tissues compared with in normal ovarian tissues. The comparative analysis of platinum-sensitive (PEA1) and platinum-resistant (PEA2) cell lines showed quantitative and qualitative differences in the activation of IGFBP6 signaling. Notably, IGFBP6 enhanced ERK1/2 phosphorylation only in PEA1 cells, and induced more evident and significant gene expression reprogramming in PEA1 cells compared with in PEA2 cells. Furthermore, the analysis of selected genes modulated by IGFBP6 (i.e., FOS, JUN, TNF, IL6, IL8 and EGR1) exhibited an inverse regulation in PEA1 versus PEA2 cells. In addition, selected hallmarks (TNFA_signaling_via_NFKB, TGF_beta_signaling, P53_pathway) and IL-6 signaling were positively regulated in PEA1 cells, whereas they were inhibited in PEA2 cells in response to IGFBP6. These data suggested that dysregulation of IGFBP6 signaling may serve a role in the progression of OC, and is likely associated with the development of platinum resistance.

Chronic stress induces platinum and Niraparib resistance in mouse models of ovarian cancer

Resistance to platinum and PARP inhibitors represents a major barrier to the long-term survival of ovarian cancer patients. We aim to explore the potential role of chronic stress in drug resistance in ovarian cancer. Leveraging four ovarian cancer with chronic stress (OCCS) mouse models, we explore the therapeutic efficacy of platinum, Niraparib, and Docetaxel treatment in vivo, and compare the efficacy of these anti-tumor drugs in vitro using cell viability assays. Comparing the transcriptional characteristics in RNA-Seq of OCCS mice with public databases, we analyze the molecular mechanism of chronic stress promoting drug resistance in ovarian cancer. We find that chronic stress is positively correlated with platinum-resistant recurrence in ovarian cancer patients. Chronic stress can induce platinum and Niraparib resistance of ovarian cancer, but it does not affect the therapeutic efficacy of Docetaxel treatment in vivo. And the platinum-resistant cell lines are not sensitive to these anti-tumor drugs, which is different from the result in vivo. Then, we identify several gene networks and their constituent genes that are most significantly associated with chronic stress and drug resistance in ovarian cancer, including the glycolysis pathway and DNA damage. This study develops Niraparib and platinum-resistant in vivo models, reflecting the ability of OCCS mice to reproduce different aspects of human ovarian cancer molecular mechanism, and provides a new theoretical basis for overcoming the double drug resistance of ovarian cancer.

Aberrant expression of TMEM205 signaling promotes platinum resistance in ovarian cancer: An implication for the antitumor potential of DAP compound

IntroductionTMEM205 is a novel transmembrane protein associated with platinum resistance (PR) in epithelial ovarian carcinoma (OC), however, the specific mechanisms associated with this resistance remain to be elucidated. MethodsTMEM205 expression was evaluated in platinum-sensitive (PS) versus platinum resistant (PR) ovarian cancer cell lines and patient serum/tissues. Exosomal efflux of platinum was evaluated with inductively coupled plasma mass spectrometry (ICP-MS) after pre-treatment with small molecule inhibitors (L-2663/L-2797) of TMEM205 prior to treatment with platinum. Cytotoxicity of combination treatment was confirmed in vitro and in an in vivo model. ResultsTMEM205 expression was 10–20 fold higher in PR compared to PS ovarian cancer cell lines, serum samples, and tissues. Co-localization with CD1B was confirmed by in-situ proximity ligation assay suggesting that TMEM205 may mediate PR via the exosomal pathway. Exosomal secretion was significantly increased 5–10 fold in PR cell lines after treatment with carboplatin compared to PS cell lines. Pre-treatment with L-2663 prior to carboplatin resulted in significantly increased intracellular concentration of fluorescently-labeled cisplatin and decreased exosomal efflux of platinum. Decreased cell survival and tumor growth in vitro and in vivo was observed when PR cells were treated with a combination of L-2663 with carboplatin compared to carboplatin alone. ConclusionTMEM205 appears to be involved in the development of PR in ovarian cancer through the exosomal efflux of platinum agents. This study provides pre-clinical evidence that TMEM205 could serve as a possible biomarker for PR as well as a therapeutic target in combination with platinum agents.

Predicting response to platinum and non-platinum drugs through bioluminescence resonance energy transfer (BRET) based bio-molecular interactions in platinum resistant epithelial ovarian cancer

Therapy induced rewiring of signalling networks often lead to acquirement of platinum-resistance, thereby necessitating the use of non-platinum agents as second-line treatment particularly for epithelial ovarian cancer (EOC). A prior subject-specific assessment can guide the choice of optimal non-platinum agent/s and possible targeted therapeutic/s. Assessment of protein-protein interactions are superior to simple cytotoxicity assays to determine therapeutic efficacy and associated molecular responses. Utilizing improved PIP3-AKT and ERK1/2 activation Bioluminescence Resonance Energy Transfer (BRET) sensors, we report chemotherapy-induced ERK1/2 activation predominantly in cisplatin-paclitaxel resistant EOC cells and increased activation of both ERK1/2 and AKT in malignant ascites derived cancer cells from platinum-resistant patients but not from treatment-naive or platinum-sensitive relapse patients. Further, majority of the non-platinum drugs except irinotecan increased ERK1/2 activation in platinum-taxol resistant cells as observed by live-cell BRET assessment which were associated with p90RSK1/2 and BAD activation along with upregulation of multidrug transporter gene ABCC1 and cell survival genes like cyclin D1 and Bcl2. Interestingly, only irinotecan was able to sensitize these resistant cells. Altogether, this first report of BRET based sensing of molecular pathway activations in platinum resistant cell lines and patient's derived cancer cells highlight the clinical potential of BRET sensors in management of therapy resistant cancer.

Identifying the role of transmembrane protein TMEM205 in promoting platinum resistance in ovarian cancer through exosomal efflux of platinum agents

<h3>Objectives:</h3> To identify the molecular mechanisms of TMEM205-mediated exosome secretion that leads to the development of chemoresistance in ovarian cancer and determine if targeted inhibition of TMEM205 with small molecule inhibitors can sensitize ovarian cancer cells to platinum-based therapy. <h3>Methods:</h3> The baseline expression of TMEM205 was determined on western blot analysis in two platinum-resistant cell lines (TR127 & PEOC4) compared to two platinum-sensitive cell lines (OVCAR4 & PEOC1). Ultracentrifugation was performed to isolate exosomes after treatment with carboplatin and the concentration observed using flow cytometry in both platinum sensitive and resistant cell lines. Exosomal efflux of carboplatin was determined on inductively coupled mass spectrometry (ICMS) in platinum sensitive versus platinum resistant cell lines after treatment with carboplatin. TMEM205 inhibition with small molecule inhibitors (L-2663 and L-2797) was confirmed on western blot analysis. Fluorescently-labeled cisplatin alone or in combination with L-2663 or L-2797 and immunocytochemistry was performed to visualize inhibition of platinum efflux in TR-127 cells. Cell viability of platinum sensitive and resistant cells treated with small molecule inhibitor was analyzed by SRB assay. <h3>Results:</h3> Increased expression of TMEM205 was observed in platinum-resistant cell lines compared to platinum-sensitive cell lines on western blot analysis and immunocytochemistry. Exosomal secretion was significantly increased in platinum-resistant cell lines after treatment with carboplatin compared to an untreated control (p0.001***). Exosomal efflux of carboplatin was significantly higher in the TR127 cell line compared to the OVCAR4 cell line (p0.05*). TMEM205 expression was decreased in TR127 and PEOC4 cell lines after treatment with L-2663/L-2797at 24 hours. Increased intracellular concentration of fluorescently-labeled cisplatin was observed when TR127 cells were treated with a combination of L-2663 + cisplatin or L-2797 + cisplatin compared to cisplatin alone on immunocytochemistry. Exosomal efflux of carboplatin was decreased in TR127 cells when treated with a combination of L-2663 + carboplatin compared to carboplatin alone. <h3>Conclusions:</h3> TMEM205 appears to be involved in the exosomal efflux of platinum agents in ovarian cancer and could serve as a possible therapeutic target in combination with platinum agents in platinum-resistant disease providing pre-clinical evidence as to the efficacy of targeting TMEM205 along with platinum treatmentin ovarian cancer.

Visudyne™: a new therapeutic strategy for the treatment of ovarian cancer

Objectives: Yes-associated protein (YAP) is a transcriptional coactivator regulated via the Hippo pathway that has been shown to be tumorigenic in ovarian cancer (OC). YAP has also been identified to be uniquely expressed within induced regulatory T cells (Tregs). A conditional knockout of YAP in T cells decreased Tregs within the tumor microenvironment and increased antitumor immune response in a subcutaneous mouse melanoma model. Visudyne™ is a liposomal derivative of verteporfin (VP), a known YAP inhibitor that is FDA approved for the treatment of adult macular degeneration. The objective of our study is to: 1) evaluate the effect of Visudyne™ on YAP and its transcriptional targets, 2) determine cytotoxic effects of Visudyne™ in platinum sensitive and resistant OC cell lines, and 3) to evaluate its effect on immune regulatory cells. Methods: Western blot analysis and immunofluorescence were performed to determine YAP expression and localization in R182 and MR182 OC cell lines using specific antibodies for YAP, phosphorylated YAP (pYAP), and connective tissue growth factor (CTGF), a transcriptional target of YAP. Cell proliferation was measured using Incucyte real-time imaging following treatment with increasing doses of cisplatin, VP and Visudyne™ in A2780 wildtype and platinum resistant OC cells. IC50 was calculated using GraphPad PRISM. Three C57/B6 mice were injected intraperitoneally (IP) with triple knockout p53mut/PTENmut/mCherry OC cells. Tumor progression was monitored by mCherry signal with fluorescence spectroscopy. Visudyne™ was administered IP at 2 mg/kg and 3 days later at 4 mg/kg. Flow cytometry was performed using antibodies for CD4, CD8, FOXP3, and interferon gamma to identify Treg and cytolytic T cells in peritoneal lavage and spleen of the control and Visudyne™ treated mice. Download : Download high-res image (329KB) Download : Download full-size image Results: Treatment of OC cells with 1uM Visudyne™ is associated with a significant decrease expression of total YAP, pYAP, and CTGF (Figure 1A). Immunofluorescence demonstrated loss of nuclear YAP and increased pYAP in the cytoplasm of R182 and MR182 cells after VP treatment (Figure 1B). Increasing doses of Visudyne™ significantly decreased cell growth in platinum resistant A2780 OC cells when compared to cells treated with cisplatin alone. In vivo treatment of tumor bearing mice with Visudyne™ is associated with a significant decrease of Treg CD4+/FOXP3+ cells in peritoneal lavage of treated mice compared to the control (85.5% vs. 92.9%, Figure IC). No changes were observed in the spleen in treated mice vs. control. Conclusions: We propose that Visudyne™ should be further investigated as a novel therapeutic agent for the treatment of OC. Its effect on immune function, specifically Tregs, suggests a promising effect either as a single agent or in combination with other checkpoint inhibitors in platinum sensitive and resistant OC.

Differentially expressed genes in platinum-resistant high-grade serous ovarian cancer

<h3>Objectives:</h3> The purpose of this study was to identify genes and pathways differentially expressed in platinum resistant high grade serous ovarian cancer (HGSOC) when compared to sensitive HGSOC. <h3>Methods:</h3> A total of 37 patients with HGSOC tissue samples underwent RNA sequencing performed by TEMPUS (N=37, 21 platinum sensitive, 16 resistant; 85% Stage III-IV; 58% received neoadjuvant chemotherapy). RNA gene expression data and significantly impacted pathways were analyzed using Advaita Bio's iPathwayGuide. Differentially expressed (DE) genes were identified using FDR of 0.05 and fold-change of 1.5. Genes from several impacted canonical metabolic pathways were validated by PCR against external data sets in a separate ovarian cancer sample group (n=15), platinum resistant ovarian cancer mouse tumor model, and wild-type sensitive and platinum resistant ovarian cancer cell lines. Relative gene expression was calculated using the comparative Ct method, also referred to as the "2 DDCT", using L27 as internal control gene. <h3>Results:</h3> We identified 177 differentially expressed (DE) genes out of a total of 16,607 genes (1.1%) with measured expression. 15 pathways were found to be significantly impacted. Of the 15 canonical pathways, all were up regulated in the resistant HGSOC and the majority of the most significantly altered (5/10) were related to metabolism (Retinol metabolism (p-value = 0.002); Tyrosine Metabolism (p-value = 0.005); Tryptophan Metabolism (p-value = 0.009); and Phenylalanine Metabolism (p-value = 0.012); CYP Drug Metabolism (p-value = 0.022)). A total of 3 separate genes from the CYP family and two from the Dopa Decarboxylase family of genes were validated against an external data set of human ovarian tissue samples, cell lines, mouse ovarian tumor model, and found to have similarly increased gene expression in the genes tested in the platinum resistant groups. Compilation of KEGG analysis and the common network genes revealed pathways associated with amino acid metabolism to be most significantly altered. <h3>Conclusions:</h3> We describe the identification of a unique transcriptomic profile associated with platinum resistance. Interestingly, the main pathways identified are related to metabolism, suggesting that the survival to chemotherapy demands a major metabolic adaptation. These findings also represent a first step towards the identification of biomarkers for the detection of chemo-resistant disease and metabolism-based drug targets specific for chemo-resistant tumors. Further validation of this model is required in order to determine its clinical value.