High-grade Serous Ovarian Cancer Cell Research Articles

Sequential Targeting of PLK1 and PARP1 Reverses the Resistance to PARP Inhibitors and Enhances Platin-Based Chemotherapy in BRCA-Deficient High-Grade Serous Ovarian Cancer with KRAS Amplification.

Ovarian cancer (OC) accounts for approximately 4% of cancer deaths in women worldwide and is the deadliest gynecologic malignancy. High-grade serous ovarian cancer (HGSOC) is the most predominant ovarian cancer, in which BRCA1/2 gene mutation ranges from 3 to 27%. PARP inhibitors (PARPi) have shown promising results as a synthetically lethal therapeutic approach for BRCA mutant and recurrent OC in clinical use. However, emerging data indicate that BRCA-deficient cancers may be resistant to PARPi, and the mechanisms of this resistance remain elusive. We found that amplification of KRAS likely underlies PARPi resistance in BRCA2-deficient HGSOC. Our data suggest that PLK1 inhibition restores sensitivity to PARPi in HGSOC with KRAS amplification. The sequential combination of PLK1 inhibitor (PLK1i) and PARPi drastically reduces HGSOC cell survival and increases apoptosis. Furthermore, we were able to show that a sequential combination of PLK1i and PARPi enhanced the cellular apoptotic response to carboplatin-based chemotherapy in KRAS-amplified resistant HGSOC cells and 3D spheroids derived from recurrent ovarian cancer patients. Our results shed new light on the critical role of PLK1 in reversing PARPi resistance in KRAS-amplified HGSOC, and offer a new therapeutic strategy for this class of ovarian cancer patients where only limited options currently exist.

An experimental model for ovarian cancer: propagation of ovarian cancer initiating cells and generation of ovarian cancer organoids

BackgroundOvarian cancer (OC) is the most lethal gynecological cancer due to the recurrence of drug-resistance. Cancer initiating cells (CICs) are proposed to be responsible for the aggressiveness of OC. The rarity and difficulty of in vitro long-term cultivation of CICs challenge the development of CIC-targeting therapeutics. Reprogramming cancer cells into induced cancer initiating cell (iCICs) could be an approach to solve these. Several inducible CICs have been acquired by activating the expression of stemness genes in different cancer cells. However, few reports have demonstrated the feasibility in OC.MethodsPatients with primary OC receiving surgery were enrolled. Tumor tissue were collected, and OCT4, SOX2, and NANOG expressions were assessed by immunohistochemistry (IHC) staining to investigate the association of stemness markers with overall survival (OS). An high-grade serous ovarian cancer (HGSOC) cell line, OVCAR-3 was reprogrammed by transducing Yamanaka four factors OCT4, SOX2, KLF4 and MYC (OSKM) to establish an iOCIC model, iOVCAR-3-OSKM. CIC characteristics of iOVCAR-3-OSKM were evaluated by RT-PCR, sphere formation assay and animal experiments. Drug-resistance and migration ability were accessed by dye-efflux activity assay, MTT assay and migration assay. Gene profile was presented through RNA-sequencing. Lineage differentiation ability and organoid culture were determined by in vitro differentiation assays.ResultsIn OC patients, the co-expression of multiple stem-related transcription factors (OCT4, SOX2, and NANOG) was associated with worse OS. iOVCAR-3-OSKM cells generated by reprogramming successfully exhibited stemness characteristics with strong sphere-forming and tumorigenesis ability. iOVCAR-3-OSKM cells also showed malignant potential with higher drug resistance to chemodrug, Paclitaxel (PTX) and migration ability. iOVCAR-3-OSKM was maintainable and expandable on feeder-dependent culture condition, it also preserved ovarian lineage differentiation abilities, which could well differentiate into OC cells with CK-7 and CA125 expressions and develop into an organoid mimic poor prognostic OC histological feature.ConclusionsThe establishment of iOVCAR-3-OSKM not only allows us to fill the gap in the information on induced CICs in OC but also provides a potential strategy to develop personalized CICs and organoid models for treating OC in the near future.

Adaptive transcriptomic and immune infiltrate responses in the tumor immune microenvironment following neoadjuvant chemotherapy in high grade serous ovarian cancer reveal novel prognostic associations and activation of pro-tumorigenic pathways.

The high rate of ovarian cancer recurrence and chemoresistance necessitates further research into how chemotherapy affects the tumor immune microenvironment (TIME). While studies have shown that immune infiltrate increases following neoadjuvant (NACT) chemotherapy, there lacks a comprehensive understanding of chemotherapy-induced effects on immunotranscriptomics and cancer-related pathways and their relationship with immune infiltrate and patient responses. In this study, we performed NanoString nCounter® PanCancer IO360 analysis of 31 high grade serous ovarian cancer (HGSOC) patients with matched pre-treatment biopsy and post-NACT tumor. We observed increases in pro-tumorigenic and immunoregulatory pathways and immune infiltrate following NACT, with striking increases in a cohort of genes centered on the transcription factors ATF3 and EGR1. Using quantitative PCR, we analyzed several of the top upregulated genes in HGSOC cell lines, noting that two of them, ATF3 and AREG, were consistently upregulated with chemotherapy exposure and significantly increased in platinum resistant cells compared to their sensitive counterparts. Furthermore, we observed that pre-NACT immune infiltrate and pathway scores were not strikingly related to platinum free interval (PFI), but post-NACT immune infiltrate, pathway scores, and gene expression were. Finally, we found that higher levels of a cohort of proliferative and DNA damage-related genes was related to shorter PFI. This study underscores the complex alterations in the ovarian TIME following chemotherapy exposure and begins to untangle how immunologic factors are involved in mediating chemotherapy response, which will allow for the future development of novel immunologic therapies to combat chemoresistance.

SLCO4A1 expression is associated with activated inflammatory pathways in high-grade serous ovarian cancer.

Patients with high-grade serous ovarian cancer (HGSOC) have a very poor overall survival. Current therapeutic approaches do not bring benefit to all patients. Although genetic alterations and molecular mechanisms are well characterized, the molecular pathological conditions are poorly investigated. Solute carrier organic anion transporter family member 4A1 (SLCO4A1) encodes OATP4A1, which is an uptake membrane transporter of metabolic products. Its expression may influence various signaling pathways associated with the molecular pathophysiological conditions of HGSOC and consequently tumor progression. RNA sequencing of 33 patient-derived HGSOC cell lines showed that SLCO4A1 expression was diverse by individual tumors, which was further confirmed by RT-qPCR, Western blotting and immunohistochemistry. Gene Set Enrichment Analysis revealed that higher SLCO4A1 level was associated with inflammation-associated pathways including NOD-like receptor, adipocytokine, TALL1, CD40, NF-κB, and TNF-receptor 2 signaling cascades, while low SLCO4A1 expression was associated with the mitochondrial electron transport chain pathway. The overall gene expression pattern in all cell lines was specific to each patient and remained largely unchanged during tumor progression. In addition, genes encoding ABCC3 along with SLCO4A1-antisense RNA 1, were associated with higher expression of the SLCO4A1, indicating their possible involvement in inflammation-associated pathways that are downstream to the prostaglandin E2/cAMP axis. Taken together, increased SLCO4A1/OATP4A1 expression is associated with the upregulation of specific inflammatory pathways, while the decreased level is associated with mitochondrial dysfunction. These molecular pathophysiological conditions are tumor specific and should be taken into consideration by the development of therapies against HGSOC.

CCNE1 is a potential target of Metformin for tumor suppression of ovarian high-grade serous carcinoma

ABSTRACT High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer, accounting for 70%–80% of mortality. However, the treatment of HGSOC has improved little in the past few decades. Metformin is the first-line medication for the treatment of type 2 diabetes and has now gained more attention in cancer treatment. In this study, we sought to identify potential hub genes that metformin could target in the treatment of HGSOC. We downloaded GSE69428 and GSE69429 in the Gene Expression Omnibus database and performed the bioinformatics analysis. Subsequently, we analyzed the effect of Metformin in HGSOC through biological experiments. Molecular simulation docking was used to predict the interaction of Metformin and CCNE1. We chose CCNE1 for the study based on bioinformatics analysis, literature studies, and preliminary data. We evaluated that CCNE1 is overexpressed in HGSOC tissues and found that HGSOC cells with high CCNE1 expression increase sensitivity to Metformin treatment in the analysis of cell proliferation and anchorage-independent growth. Metformin could inhibit the expression of CCNE1, which is associated with the anti-proliferative effect of tumor cells. Moreover, Metformin could ameliorate the tumor growth in syngeneic orthotopic transplantation mouse models and xenograft tumorigenesis models. Furthermore, molecular simulation docking showed that Metformin may bind to CCNE1 protein, suggesting that CCNE1 could be a potential target for Metformin. Our data revealed that Metformin has antitumor effects on ovarian cancer and CCNE1 could be a potential target for Metformin.

Effects of Wee1 inhibitor adavosertib on patient-derived high-grade serous ovarian cancer cells are multiple and independent of homologous recombination status.

ObjectiveA major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.MethodsA comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.ResultsSeveral compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.ConclusionOur study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.

The disruption of the CCDC6 – PP4 axis induces a BRCAness like phenotype and sensitivity to PARP inhibitors in high-grade serous ovarian carcinoma

BackgroundTreatment with PARP inhibitors (PARPi) is primarily effective against high-grade serous ovarian cancers (HGSOC) with BRCA1/2 mutations or other deficiencies in homologous recombination (HR) repair mechanisms. However, resistance to PARPi frequently develops, mostly as a result of BRCA1/2 reversion mutations. The tumour suppressor CCDC6 is involved in HR repair by regulating the PP4c phosphatase activity on γH2AX. In this work, we reported that in ovarian cancer cells, a physical or functional loss of CCDC6 results synthetic lethal with the PARP-inhibitors drugs, by affecting the HR repair. We also unravelled a role for CCDC6 as predictive marker of PARPi sensitivity in ovarian cancer, and the impact of CCDC6 downregulation in overcoming PARPi resistance in these tumours.MethodsA panel of HGSOC cell lines (either BRCA-wild type or mutant) were treated with PARPi after CCDC6 was attenuated by silencing or by inhibiting USP7, a CCDC6-deubiquitinating enzyme, and the effects on cell survival were assessed. At the cellular and molecular levels, the processes underlying the CCDC6-dependent modification of drugs’ sensitivity were examined. Patient-derived xenografts (PDXs) were immunostained for CCDC6, and the expression of the protein was analysed statistically after digital or visual means.ResultsHGSOC cells acquired PARPi sensitivity after CCDC6 depletion. Notably, CCDC6 downregulation restored the PARPi sensitivity in newly generated or spontaneously resistant cells containing either wild type- or mutant-BRCA2. When in an un-phosphorylated state, the CCDC6 residue threonine 427 is crucial for effective CCDC6-PP4 complex formation and PP4 sequestration, which maintains high γH2AX levels and effective HR. Remarkably, the PP4-dependent control of HR repair is influenced by the CCDC6 constitutively phosphorylated mutant T427D or by the CCDC6 loss, favouring PARPi sensitivity. As a result, the PP4 regulatory component PP4R3α showed to be essential for both the activity of the PP4 complex and the CCDC6 dependent PARPi sensitivity. It's interesting to note that immunohistochemistry revealed an intense CCDC6 protein staining in olaparib-resistant HGSOC cells and PDXs.ConclusionsOur findings suggest that the physical loss or the functional impairment of CCDC6 enhances the PP4c complex activity, which causes BRCAness and PARPi sensitivity in HGSOC cells. Moreover, CCDC6 downregulation might overcome PARPi resistance in HGSOCs, thus supporting the potential of targeting CCDC6 by USP7 inhibitors to tackle PARPi resistance.

Linoleic Acid-Glucosamine Hybrid for Endogenous Iron-Activated Ferroptosis Therapy in High-Grade Serous Ovarian Cancer.

As the most common subtype in ovarian malignancies, high-grade serous ovarian cancer (HGSOC) made less therapeutic progress in past decades due to the lack of effective drug-able targets. Herein, an effective linoleic acid (LA) and glucosamine (GlcN) hybrid (LA-GlcN) was synthesized for the treatment of HGSOC. The GlcN was introduced to recognize the glucose transporter 1 (GLUT 1) overexpressed in tumor cells to enhance the uptake of LA-GlcN, and the unsaturated LA was employed to trigger ferroptosis by iron-dependent lipid peroxidation. Since the iron content of HGSOC was ∼5 and 2 times, respectively, higher than that of the normal ovarian cells and low-grade serous ovarian cancer cells, these excess irons make them a good target to enhance the ferroptosis of LA-GlcN. The in vitro study demonstrated that LA-GlcN could selectively kill HGSOC cells without affecting normal cells; the in vivo study revealed that LA-GlcN at the dose of 50 mg kg-1 achieved a comparable tumor inhibition as doxorubicin hydrochloride (4 mg kg-1) while the overall survival of mice was extended largely due to the low toxicity, and when the dose was increased to 100 mg kg-1, the therapeutic outcomes could be improved further. This dietary hybrid which targets the excess endogenous iron to activate ferroptosis represents a promising drug for HGSOC treatment.

Single-cell analysis of a high-grade serous ovarian cancer cell line reveals transcriptomic changes and cell subpopulations sensitive to epigenetic combination treatment.

Ovarian cancer (OC) is a lethal gynecological malignancy with a five-year survival rate of only 46%. Development of resistance to platinum-based chemotherapy is a common cause of high mortality rates among OC patients. Tumor and transcriptomic heterogeneity are drivers of platinum resistance in OC. Platinum-based chemotherapy enriches for ovarian cancer stem cells (OCSCs) that are chemoresistant and contribute to disease recurrence and relapse. Studies examining the effect of different treatments on subpopulations of HGSOC cell lines are limited. Having previously demonstrated that combined treatment with an enhancer of zeste homolog 2 inhibitor (EZH2i) and a RAC1 GTPase inhibitor (RAC1i) inhibited survival of OCSCs, we investigated EZH2i and RAC1i combination effects on HGSOC heterogeneity using single cell RNA sequencing. We demonstrated that RAC1i reduced expression of stemness and early secretory marker genes, increased expression of an intermediate secretory marker gene and induced inflammatory gene expression. Importantly, RAC1i alone and in combination with EZH2i significantly reduced oxidative phosphorylation and upregulated Sirtuin signaling pathways. Altogether, we demonstrated that combining a RAC1i with an EZH2i promoted differentiation of subpopulations of HGSOC cells, supporting the future development of epigenetic drug combinations as therapeutic approaches in OC.

The immune checkpoint controller C1q (C1QA/B/C) is a druggable oncologically relevant target: Proof-of-concept in vitro with an FDA-approved medicine, used as pioneer inhibitor of protein hydroxylation (273)

<b>Objectives:</b> C1q, a key initiator of immune reactions (adaptive, innate), is the single most important protein mediating tolerogenic clearance of apoptotic debris (‘self-waste') and functions in tissue homeostasis, e.g., angiogenesis. Cancer cells employ locally secreted C1q to secure growth and immune escape [Cancer Immunol. Res. 7.7, 1091-1195 (2019)]. In the tissue of high-grade serous ovarian cancer (HGSOC), expression of <i>C1</i>QA, or <i>C1QB,</i> or <i>C1QC</i> is a negative prognostic indicator [OSFPreprint 10.31219/osf.io/bcv9a]. C1q consists of ABC monomers, each comprising two domains, globule, and stem, the latter aligning six of these monomers into a ‘bouquet-of-tulips.' Every ABC monomer requires multiple Pro/Lys hydroxylations by specific 2-oxoacid utilizing dioxygenases (2OUDs) to form its stem as a triple helix identical to collagen. This collagenous stem (CS) mediates all C1q functions. Several experimental agents, read off the HAG mechanism that defines the principally possible inhibitors of 2OUDs (https://pubmed.ncbi.nlm.nih.gov/6281585/), are long known to prevent CS formation in C1q, to cause intracellular accumulation of underhydroxylated collagen and thus CS-defective C1q, and to block the secretion of any C1q that is dysfunctional due to CS mis-or unfolding [Biochem. J. 248, 625-33 (1987)]. We hypothesized that HGSOC cells generate their own C1q, which is rendered CS-defective and thus intracellularly retained by the Pro/Lys hydroxylation-inhibiting medicine deferiprone (DEF), discovered per the HAG mechanism. We aimed to Identify a medicine that disrupts all C1q support for malignancy. <b>Methods:</b> Cell line cultures (KURAMOCHI [HGSOC], ARK1 [uterine serous cancer, USC], MRC5 [normal fibroblasts, NF]); RNA-seq; multi-channel flow cytometry. <b>Results:</b> KURAMOCHI, which is unique among 47 HGSOC cell lines for displaying superior genetic similarity to HGSOC tumors [Nat. Commun. 4.1, 1-10 (2013)], synthesized C1q within 48 hours of plating, as detected intracellularly by the FITC-labeled monoclonal antibody JL-1 that specifically recognizes the CS domain of C1q. At 96 hours, the flow-cytometric JL-1 signal indicated further C1q increase by at least half and mirrored C1q kinetics in MRC5, a model of NF, the major source of C1q in non-malignant tissues. DEF, ranged at its concentrations in serum during multi-year treatment of iron overload conditions, e.g., transfusion-dependent thalassemia, caused all cell lines to accumulate JL-1 reactive C1q material, even at the residual 50 µM that patients on the FDA-approved DEF regimen may maintain for most or all of 24 hours [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154842]. After 96 hours on the drug, this intracellular accumulation exceeded the JL-1 signal of C1q within untreated controls by a factor of 3 in KURAMOCHI and by a factor of 10 in ARK1 (p<0.02), whereas JL-1 reactivity in treated MRC5 had, even at 150 µM, reverted to that of untreated MRC5. In KURAMOCHI, the accumulated JL-1 reactivity coincided with a 25-fold increase in active caspase 3 at 96 hours, a level of apoptotic activity reached in ARK1 after 48 hours already. In MRC5, active caspase-3 was suppressed at 96 hours on DEF. <b>Conclusions:</b> DEF at clinically relevant concentrations disrupts C1q synthesis selectively in HGSOC and USC cells and triggers their apoptotic self-destruction; however, NF remain unaffected. Our results encourage a DEF trial in HGSOC/USC patients and the further HAG mechanism-guided development of improved 2OUD inhibitors for oncological indications.

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

ScRNA seq can identify different cell populations in ovarian cancer Bulk RNA seq experiments (269)

ScRNA seq can identify different cell populations in ovarian cancer Bulk RNA seq experiments (269)

The Potential of Novel Lipid Agents for the Treatment of Chemotherapy-Resistant Human Epithelial Ovarian Cancer.

Simple SummaryDisease recurrence and chemotherapy resistance are the major causes of mortality for the majority of epithelial ovarian cancer (EOC) patients. Standard of care relies on cytotoxic drugs that induce a form of cell death called apoptosis. EOC cells can evolve to resist apoptosis. We developed drugs called glycosylated antitumor ether lipids (GAELs) that kill EOC cells by a mechanism that does not involve apoptosis. GAELs most likely induce cell death through a process called methuosis. Importantly, we showed that GAELs are effective at killing chemotherapy-resistant EOC cells in vitro and in vivo. Our work shows that the EOC community should begin to investigate methuosis-inducing agents as a novel therapeutic platform to treat chemotherapy-resistant EOC.Recurrent epithelial ovarian cancer (EOC) coincident with chemotherapy resistance remains the main contributor to patient mortality. There is an ongoing investigation to enhance patient progression-free and overall survival with novel chemotherapeutic delivery, such as the utilization of antiangiogenic medications, PARP inhibitors, or immune modulators. Our preclinical studies highlight a novel tool to combat chemotherapy-resistant human EOC. Glycosylated antitumor ether lipids (GAELs) are synthetic glycerolipids capable of killing established human epithelial cell lines from a wide variety of human cancers, including EOC cell lines representative of different EOC histotypes. Importantly, GAELs kill high-grade serous ovarian cancer (HGSOC) cells isolated from the ascites of chemotherapy-sensitive and chemotherapy-resistant patients grown as monolayers of spheroid cultures. In addition, GAELs were well tolerated by experimental animals (mice) and were capable of reducing tumor burden and blocking ascites formation in an OVCAR-3 xenograft model. Overall, GAELs show great promise as adjuvant therapy for EOC patients with or without chemotherapy resistance.

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.

Abstract 3456: Discrepancies in the metastatic potential of high-grade serous ovarian cancer cells representing disease progression when comparing two-dimensional assays versus three-dimensional organotypic culture model systems

Abstract Due to the lack of early symptoms and valid screening methods, most patients with the most prevalent histotype of ovarian cancer, high-grade serous ovarian cancer (HGSOC), are diagnosed at late stage, at which point metastasis has already occurred. The purpose of this study was to characterize the migratory, invasive, and adhesive abilities of HGSOC cells, representing different stages of disease evolution, in both two-dimensional (2D) and three-dimensional (3D) organotypic assays. Five HGSOC cell lines, collected at different stages of disease progression were used throughout this study; PEO1, PEO4, and PEO6 cells were obtained from a first patient, whereas PEO14 and PEO23 cells were isolated from a second patient. Both wound healing (WH) and Boyden chamber (BC) assays were performed to study the migratory capacity of each cell line, while invasion was assessed by adding a layer of extracellular matrix to the BC assays. Finally, 2D adhesion assays were performed on fibronectin coated plates. On the other hand, 3D organotypic models composed of a mixture of collagen I and fibroblasts, topped with a monolayer of mesothelial cells, were constructed to further study the metastatic potential of all five HGSOC cell lines; HGSOC were plated on top of the mesothelial cells, and their adhesive behavior and capacity to displace mesothelial cells was followed by fluorescence microscopy. In 2D studies, the migratory and invasive capacities of the HGSOC cells decreased along disease evolution in both cell line series. Moreover, a distinct migration pattern was observed; cell lines representing early-stage disease had a tendency to migrate as individual cells, while cells representing late stage disease migrated in clusters. Also in 2D conditions, adhesion rates were found to be similar between all cell lines. Noteworthy, these results obtained in 2D conditions were somewhat contradicted by the results obtained in the 3D organotypic models, as both late-state disease PEO6 and PEO23 cells adhered more rapidly to the mesothelial cell monolayer than their early-stage counterparts. Furthermore, cell lines previously found to be tumorigenic in vivo such as PEO4, PEO6, and PEO14, demonstrated to trigger higher rates of mesothelial cell displacement in the 3D organotypic model system. This mesothelial cell displacement was also observed after exposure of mesothelial cells to conditioned media derived from PEO4, PEO6, or PEO14 cultures. In conclusion, 2D in vitro assays demonstrated lower migration and invasion capacities at late- rather than early-stage disease, while the opposite was observed in the organotypic models. Mesothelial cell displacement was found to be associated with tumorigenicity in vivo and to be independent of physical interactions between cancer cells and mesothelial cells. Citation Format: Sabrina J. Ritch, Alicia A. Goyeneche, Abu S. Noman, Carlos M. Telleria. Discrepancies in the metastatic potential of high-grade serous ovarian cancer cells representing disease progression when comparing two-dimensional assays versus three-dimensional organotypic culture model systems [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 3456.

Abstract 1839: Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition

Abstract High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, the rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction of HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction of clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. Western blot analysis of underlying molecular mechanisms revealed activation by NFV of the three signaling arms of the unfolded protein response (UPR): PERK, IRE1alpha and ATF6 - in a similar manner to the classical endoplasmic reticulum (ER) stressor tunicamycin. NFV-mediated modulation of the UPR was accompanied by the inhibition of global protein synthesis as analyzed through a non-radioactive method by labelling the nascent polypeptides with puromycin. NFV maintained a proapoptotic environment through the UPR, evidenced by a dose- and time-dependent enhanced expression of ATF4, CHOP, increased Bax/Bcl2 ratio, and cleavage of executioner caspase-7. Enhanced gamma-H2AX suggested NFV-mediated induction of DNA damage, which was associated with decreased proliferation signals driven by the AKT and ERK pathways. NFV increased the level of autophagosome-associated protein LC3II in HGSOC cells of varying platinum sensitivities; however, the autophagic flux was not increased during co-treatment with NFV and the lysosomal inhibitor bafilomycin A1, suggesting a likely impairment of the lysosomes by NFV impeding autophagosome clearance. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Prisca Bustamante, Michael A. Lisio, Julia V. Burnier, Carlos M. Telleria. Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition [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 1839.

Abstract 3790: A study of evolutionary dynamics induced by carboplatin, olaparib and paclitaxel in high-grade serous ovarian cancer cell line models

Abstract Background High-grade serous ovarian cancer (HGSOC) is characterized by extensive intratumoral heterogeneity, which is associated with drug resistance. Our aim was to study the effects of drugs used in the treatment of HGSOC - carboplatin (C), paclitaxel (P) and olaparib (O) - on clonal dynamics in experimental models. Methods We tracked clonal evolution in PEO1, a BRCA2-mutant HGSOC cell line model, by transfecting an initial population of 1.714x106 cells with a barcode (BC) library (CloneTrackerXP). Barcoded populations of cells (18.73x106 cells/flask) were grown in HYPERflasks till confluence, and then replicates were exposed to C (3163 nM) for 5 weeks, P (6.32 nM) for 4 weeks, and O (28467 nM) for 5 weeks (as single agents), reducing the cell population to &amp;lt; 10% (controls kept till cell confluence in the absence of drugs). After that, drug pressure was removed, allowing cells to regrow fully. NGS was used to quantify BCs in the final cell populations and, to get information on the temporal dynamics, also in dead floating cells collected from the weekly culture media change. Heterogeneity of the cell populations was estimated using the Shannon diversity index (SDI). Results The SDI in the controls was 6.306 (95% Bootstrap CI 6.304-6.310) for C and 6.192 (95% Bootstrap CI 6.190-6.197) for P and O. After exposure to C, P and O, the SDI was reduced to 3.208, 2.520 and 4.753 (95% Bootstrap CI 3.209-3.211, 2.517-2.523, 4.750-4.759) respectively, using aggregated replicates (pairwise 2-sided Hutcheson t-test: p &amp;lt; 2.2e-16 pre-drug exposure vs C, P and O). The number of unique BCs in controls vs C, P and O were 1799 vs 545, 1463 vs 288 and 1463 vs 1270, respectively, demonstrating reduction of heterogeneity and change in frequency of clones following drug selection pressure. Cell populations exposed to C and O share some similar clone selection (approximately 60%). This is important as, although the degree of heterogeneity caused by individual drugs are different (C and P lead to less heterogeneous populations than O), there is some concordance between the remaining clones determined by DNA damaging/DNA damage response inhibitors compared to clone selection following exposure to antimicrotubule agents. The selection pattern of BCs among replicates of the different drugs was similar with conservation of BCs with the highest fitness, demonstrating that the initial conditions are determinants of selection under drug exposure. Conclusion Our work, for the first time, quantifies and shows that heterogeneity followed by O therapy is greater than by C and P. Furthermore, the emergent population of cells post-selection pressure was more similar in the C and O groups when compared to P-exposed cells, suggesting correlation in the mechanism of action of drugs and drug resistance/tolerance. Our results also indicate that evolutionary dynamics are largely deterministic and, therefore, can be predictable. Citation Format: Alvaro H. Ingles Russo Garces, Salvatore Milite, Javier Fernandez-Mateos, Bingjie Chen, Lisa Pickard, Adam Stewart, Sara Diaz Sanchez, Rachel Lau, Erica Oliveira, Susana Banerjee, Andrea Sottoriva, Udai Banerji. A study of evolutionary dynamics induced by carboplatin, olaparib and paclitaxel in high-grade serous ovarian cancer cell line models [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 3790.

Abstract 4066: Anticancer effects of the gold complex auranofin on high-grade serous ovarian cancer cells

Abstract High-grade serous ovarian cancer (HGSOC) is the 11th most common cancer-causing death in women. HGSOC is treated with platinum (Pt)-based chemotherapy, to which patients develop resistance over time, leaving them with very limited therapeutic options. Thus, new treatments are needed for these patients. In the present work, we provide evidence for the in vitro anti-HGSOC efficacy of the gold complex auranofin (AUF), a compound approved in the clinic in the 1980’s as an anti-rheumatic agent. AUF targets thioredoxin reductase 1 (TrxR1), which is overexpressed in many cancers, including ovarian cancer. Using the Kaplan-Meir plotter database we analyzed that high expression of TrxR1 predicted poor overall survival, suggesting that TrxR1 plays an important role in ovarian cancer progression. We show that AUF impairs the growth of HGSOC cells regardless of Pt sensitivity. HGSOC cells obtained from the same patient when it was clinically sensitive (PEO1) or resistant (PEO4) to Pt-based chemotherapy, were both equally sensitive to the growth inhibition induced by the gold complex in a concentration-dependent manner when using an MTT reduction assay. The toxicity of AUF against HGSOC cells was further studied exposing PEO1 cells to the compound and assessing cell number, cell viability, and cell cycle traverse. Results showed that AUF blocked cell proliferation, reduced viability, and caused accumulation of cells with hypo-diploid DNA content, suggesting a likely apoptosis-mediated cell death mechanism. We confirmed that AUF-triggered lethality was driven by apoptosis by measuring the accumulation of Annexin V positive/7-AAD negative cells via flow cytometry. We also found out that the lethality of AUF required the induction of oxidative stress because the effect was prevented by the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine. We further show that AUF-induced ROS production is associated with the generation of DNA damage as denoted by the increase in the phosphorylation of the Ser-139 residue of the histone variant H2AX by western blotting thus signaling a genotoxic endpoint. Finally, we generated evidence that the combination of AUF and the Pt agent cisplatin (CDDP) is more potent than either drug alone in inducing lethality in PEO4 cells isolated from a patient when Pt-resistant. In summary, we provide proof-of-principle that the TrxR1 inhibitor AUF impairs the survival capacity of HGSOC cells regardless of their Pt sensitivities, triggering a program of apoptosis induced by oxidative stress that is associated with DNA damage; such toxicity is enhanced when AUF is combined with CDDP. The novelty of this work for cancer research in general and HGSOC in particular is that, as AUF is clinically approved to treat arthritis-related conditions, the compound can be rapidly repositioned as a consolidation therapy following Pt-based standard of care and/or overcome Pt resistant disease. Citation Format: Farah H. Abdalbari, Alicia A. Goyeneche, Elvis M. Jaramillo, Siham Sabri, Carlos M. Telleria. Anticancer effects of the gold complex auranofin on high-grade serous ovarian cancer cells [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 4066.

Abstract 4071: Utilizing colforsin as a novel therapeutic for high grade serous ovarian cancer treatment

Abstract Epithelial ovarian cancer (EOC) is currently the deadliest gynecological cancer with approximately 20,000 new cases and 15,000 deaths per year in the United States. Ovarian cancer can be stratified into distinct subtypes of which high-grade serous ovarian cancer (HGSOC) comprises 90% of all cases. The majority of HGSOC patients are diagnosed with late-stage disease and have a poor prognosis with a 5-year survival rate of less than 30%. Thus, there is a critical need to develop novel therapeutic strategies to improve patient treatment and survival outcomes. Our lab has recently discovered that the small molecule colforsin has potent anti-cancer properties when used to treat HGSOC in vitro and in vivo. Colforsin is a water-soluble derivative of forskolin, and acts as an activator of cAMP signaling via direct binding and activation of adenylyl cyclase. It is currently in clinical use for treatment of conditions such as glaucoma, diabetes, and liver fibrosis. This makes colforsin a prime candidate for drug repurposing to treat ovarian cancer. Our data show that colforsin can induce cell cycle arrest and cell death in established HGSOC cell line models in vitro. We have also found that colforsin can inhibit tumor growth and tumor burden in an intraperitoneal model of HGSOC using HEYA8 cells. In addition, we have also found that colforsin acts synergistically with cisplatin to cause HGSOC cell death. Taken together, our data show that colforsin has potential as a new therapeutic tool to aid in HGSOC treatment in combination with existing therapies. Citation Format: Matthew Knarr, Hunter Reavis, Marilyn Mitchell, Ronny Drapkin. Utilizing colforsin as a novel therapeutic for high grade serous ovarian cancer treatment [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 4071.

Abstract 1551: miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel

Abstract Although a majority of women with high-grade serous ovarian cancer (HGSC) achieve a complete response to first-line platinum- and paclitaxel-based chemotherapy, around thirty percent of patients are identified as incomplete responders. Their diseases typically either does not respond or progresses during treatment (refractory) or recurs within 6 months of completing treatment (resistant), suggesting the inherit or intrinsic characteristics of the tumor. Currently, there are no biomarkers that can predict intrinsic resistance, which can avoid unnecessary treatment and toxicity. MicroRNAs (miRNAs) provide a novel master layer of regulation of gene expression. However, the molecular mechanisms by which miRNAs confer a chemoresistant phenotype in ovarian cancer have not been elucidated. To identify miRNAs that are associated with intrinsic chemoresistance in HGSC, Ion Torrent next-generation sequencing analysis of miRNAs was performed on microdissected chemosensitive and chemorefractory primary HGSC cases. Amongst all the miRNAs that were significantly down-regulated in chemorefractory cases, lower miR-625-3p expression was found to be associated with poorer overall and progression-free survivals. Further functional studies showed that overexpression of miR-625-3p significantly decreased cisplatin and paclitaxel resistance in ovarian cancer cells in vitro and in vivo. Online target prediction algorithms together with transcriptome profiling of HGSC cells transfected with miR-625-3p mimics or control mimics identified SSX2IP as a direct target of miR-625-3p, suggesting that SSX2IP may play a role in conferring cisplatin/paclitaxel resistance in HGSC by mediating the effect of miR-625-3p. SSX2IP is a microtubule anchoring and centriolar satellite protein, which plays a critical role in controlling microtubule length and orientation, and centrosome maturation. We demonstrated that HGSC cells transfected with SSX2IP had marked increases in microtubule masses, intracellular multivesicular body trafficking and exosomal cisplatin level; and a decrease in intracellular cisplatin level. In conclusion, down-regulation of miR-625-3p confers cisplatin/paclitaxel resistance in ovarian cancer cells via its novel direct target SSX2IP. SSX2IP alters the microtubule dynamics and subsequently mediates inter-organelle crosstalks between microtubules and multivesicular bodies, which facilitate exosomal export of cisplatin and paclitaxel of HGSC cells. This study is crucial for developing new predictive biomarkers for intrinsic chemoresistance, and new treatment strategies for HGSC based on upregulating miR-625-3p or downregulating SSX2IP expression in ovarian cancer cells, which will enhance cisplatin and paclitaxel sensitivity, and improve patient survival rates. Citation Format: Chi Lam Au Yeung, Tetsushi Tsuruga, Kay-Pong Yip, Sammy Ferri-Borgogno, Samuel Mok. miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel [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 1551.