Primary Ovarian Cancer Cultures Research Articles

Three-Dimensional Modelling of Ovarian Cancer: From Cell Lines to Organoids for Discovery and Personalized Medicine.

Ovarian cancer has the highest mortality of all of the gynecological malignancies. There are several distinct histotypes of this malignancy characterized by specific molecular events and clinical behavior. These histotypes have differing responses to platinum-based drugs that have been the mainstay of therapy for ovarian cancer for decades. For histotypes that initially respond to a chemotherapeutic regime of carboplatin and paclitaxel such as high-grade serous ovarian cancer, the development of chemoresistance is common and underpins incurable disease. Recent discoveries have led to the clinical use of PARP (poly ADP ribose polymerase) inhibitors for ovarian cancers defective in homologous recombination repair, as well as the anti-angiogenic bevacizumab. While predictive molecular testing involving identification of a genomic scar and/or the presence of germline or somatic BRCA1 or BRCA2 mutation are in clinical use to inform the likely success of a PARP inhibitor, no similar tests are available to identify women likely to respond to bevacizumab. Functional tests to predict patient response to any drug are, in fact, essentially absent from clinical care. New drugs are needed to treat ovarian cancer. In this review, we discuss applications to address the currently unmet need of developing physiologically relevant in vitro and ex vivo models of ovarian cancer for fundamental discovery science, and personalized medicine approaches. Traditional two-dimensional (2D) in vitro cell culture of ovarian cancer lacks critical cell-to-cell interactions afforded by culture in three-dimensions. Additionally, modelling interactions with the tumor microenvironment, including the surface of organs in the peritoneal cavity that support metastatic growth of ovarian cancer, will improve the power of these models. Being able to reliably grow primary tumoroid cultures of ovarian cancer will improve the ability to recapitulate tumor heterogeneity. Three-dimensional (3D) modelling systems, from cell lines to organoid or tumoroid cultures, represent enhanced starting points from which improved translational outcomes for women with ovarian cancer will emerge.

Experimental models in the study of pathogenesis and the development of treatments for ovarian cancer (systematic review)

A systematic review of modern methods of experimental study of ovarian cancer using traditional (immunocompetent, genetically engineered and immunodeficient) and non-traditional (that don’t belong to the mammals) animal models,established and primary cultures of human ovarian cancer, including three-dimensional organotypic spheroids (3D- models ex vivo) is presented. The prospects of the considered models for studying of the pathogenesis of various molecular-genetic and histological variants of ovarian cancer, as well as for developing methods of personalized treatment, are discussed. The limitations of modern animal models are indicated. The greatest attention is paid to studies on immunodeficient animals using xenografts based on established cultures of human ovarian cancer cells and on tumor tissue obtained directly from the patients (patient derived xenografts, PDX). The questions of various variants of xenograft transplantation with an emphasis on the problems of orthotopic transplantation of human ovarian cancer into immunodeficient mice and the relevance of methods for local humanization in heterotopic transplantation are considered. The most promising, from the point of the author’s view, approaches to studying the effectiveness of drug therapy for ovarian cancer in immunodeficient animal models are outlined. To prepare a systematic review, a literature search was carried out on the Scopus, Web of Science, Med Line, PubMed, Cyber Leninka, RSCI databases. The analysis used literature sources indexed in the Scopus and Web of Science databases (97%) and the RSCI. More than 60% of the works amount has been published over the past 5 years.

Abstract 1185: PRMT5 inhibition epigenetically regulates DNA damage response pathways in cancer cells and sensitizes to chemotherapy and PARP inhibition

Abstract Genetic defects of DNA damage response (DDR) pathways in cancer cells induce synthetic lethality with chemotherapy and PARP inhibitors. However, many patients with advanced cancers develop resistance to these therapies mainly due to circumvention and/or restoration of the inactivated DDR genes. In the present study, we demonstrate that pharmacological inhibition of PRMT5, the major type II protein arginine methyltransferase, epigenetically downregulates multiple genes involved in the DDR/DNA replication pathways, resulting in the sensitization of cancer cells to chemotherapy and PARP inhibition. Treatment with C220 or PRT543, potent and selective PRMT5 inhibitors, downregulates expression of DDR genes and/or proteins including BRCA1/2, ATM/ATR, CHK1/2, RAD51, POLD1/3, and PNKP in multiple types of cancer cells in vitro. Mechanistically, PRT543 promotes global alternative splicing (ΔPSI) changes, including intron retention and exon skipping. In particular, PRT543 significantly increases retained intron of POLD1 and PNKP genes, which are downstream of the spliceosome subunit SRSF1, a major protein substrate of PRMT5. Additionally, PRT543 increases intron retention of ATM and ATR genes. For BRCA1/2, CHK1/2 and RAD51, no changes in alternative splicing were observed, suggesting the involvement of other mechanisms such as the CLNS1A/H4R3me2s chromatin regulation pathway. The regulation of the DDR pathway by PRT543 is associated with increased DNA damage as determined by COMET and gamma H2AX analyses. Combining PRT543 with PARP inhibitors or DNA-alkylating agents demonstrates a potent synergistic interaction in both HR proficient and HR deficient cancer cell lines in vitro. Similar combination effects are observed in primary cultures of patient-derived breast cancer and high-grade serous ovarian cancer. Moreover, combination of PRT543 with PARP inhibition effectively inhibits the growth of HCC1569 CDX breast cancer in vivo. Further ex vivo and in vivo combination studies with PRT543 and PARP inhibitors or chemotherapy in genetically defined CDX and PDX models are ongoing. These studies not only reveal a novel mechanism of PRMT5 inhibition, but also suggest beneficial combination effects with other therapies, particularly in patients with tumors that are resistant to therapies that are dependent on DNA damage as their mechanism of action. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Koichi Ito, Jack Carter, Venkat Thodima, Youyou Zhang, Monisha Sivakumar, Mu Xu, Neha Bhagwat, Joseph Rager, Jacob Spruance, Lin Zhang, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition epigenetically regulates DNA damage response pathways in cancer cells and sensitizes to chemotherapy and PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1185.

55P Evaluation of MDM2-p53 antagonists, nutlin-3 and RG7388, combined with the PARPi rucaparib in primary cultures of ovarian cancer

55P Evaluation of MDM2-p53 antagonists, nutlin-3 and RG7388, combined with the PARPi rucaparib in primary cultures of ovarian cancer

Abstract 191: Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer

Abstract Introduction: The presence of an endothelial cell-free perfusion pathway, known as Vasculogenic Mimicry (VM), strongly correlates with reduced patient survival, however the mechanisms by which a tumor can create a self-generated irrigation system remain unknown. Currently there is no mechanism of VM formation reported in fluid conducting tubular structures. Herein, using a standardized in vitro model, we sought to understand the pathways required for VM formation. Methods: We utilized confocal microscopy and Imaris reconstruction to demonstrate that cancer cells can create an internal tubular network in cultured cancer cell lines, cancer spheres and primary cancer cultures of ovarian cancer. Matrigel and 3D matrices containing specific protein components allowed the deciphering of the extracellular matrix proteins involved in VM formation. Intercellular pathways were resolved using chemical inhibitors. Results: VM only occurs when cells are grown in 3D culture and only 30% of primary cancer cultures undergo this process. The presence of laminin 1 is sufficient to trigger the formation of tubular structures and this may involve the alpha2beta1 integrin and metalloproteases. Downstream FAK and PI3K/AKT intercellular pathways are required to form functional tubular networks. Microarray data elucidated the presence of the mRNA transcripts that were increased in cells that undergo VM. Discussion: We demonstrate that Laminin 1, a component of the extracellular matrix (ECM), can promote the process of VM, which further involves the downstream signaling of the potentially druggable targets of the PI3K pathway. As VM is associated with reduced patient survival, elucidation of the mechanisms of formation may deliver new cancer therapies. Funding: FONDECYT 1180241, CONICYT FONDAP 15130011, IMII P09/016F Citation Format: Andres Valdivia, Gabriel Mingo, Varina Aldana, Alejandra Sandoval, Alejandro Corvalan, Francisco Nualart, Gareth I. Owen. Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 191.

Progesterone reduces cell survival in primary cultures of endometrioid ovarian cancer

BackgroundOvarian cancer is the most lethal of all gynecologic malignancies. The relationship between sexual steroids receptors and ovarian cancer progression has been largely evaluated. The presence of progesterone receptors has been associated with an increase of a disease-free period and overall survival in patients with ovarian carcinoma. In the present study, primary cultures of ovarian carcinoma obtained from 35 patients diagnosed with epithelial ovarian cancer were evaluated for cell survival after treatment with 10− 8 M of 17β-estradiol, progesterone, testosterone and dihydrotestosterone.ResultsThe results were analyzed considering histological subtypes: low grade serous, high grade serous, endometrioid and mucinous carcinoma; clear cell carcinoma was not included due to failure in obtaining successful cultures of this subtype. A significant reduction of cell survival was observed after progesterone treatment in endometrioid ovarian carcinoma. Changes were not observed in low grade serous, high grade serous and mucinous carcinoma. The effect of progesterone was related to the presence of progesterone receptor (PR), a 43% reduction in the cell number was observed in PR (+) endometrioid ovarian carcinoma.ConclusionsThis study supports the importance of progesterone and the presence of progesterone receptor in the reduction of ovarian cancer progression in the endometrioid ovarian carcinoma.

Dual Src and MEK Inhibition Decreases Ovarian Cancer Growth and Targets Tumor Initiating Stem-Like Cells.

Purpose: Rational targeted therapies are needed for treatment of ovarian cancers. Signaling kinases Src and MAPK are activated in high-grade serous ovarian cancer (HGSOC). Here, we tested the frequency of activation of both kinases in HGSOC and the therapeutic potential of dual kinase inhibition.Experimental Design: MEK and Src activation was assayed in primary HGSOC from The Cancer Genome Atlas (TGGA). Effects of dual kinase inhibition were assayed on cell-cycle, apoptosis, gene, and proteomic analysis; cancer stem cells; and xenografts.Results: Both Src and MAPK are coactivated in 31% of HGSOC, and this associates with worse overall survival on multivariate analysis. Frequent dual kinase activation in HGSOC led us to assay the efficacy of combined Src and MEK inhibition. Treatment of established lines and primary ovarian cancer cultures with Src and MEK inhibitors saracatinib and selumetinib, respectively, showed target kinase inhibition and synergistic induction of apoptosis and cell-cycle arrest in vitro, and tumor inhibition in xenografts. Gene expression and proteomic analysis confirmed cell-cycle inhibition and autophagy. Dual therapy also potently inhibited tumor-initiating cells. Src and MAPK were both activated in tumor-initiating populations. Combination treatment followed by drug washout decreased sphere formation and ALDH1+ cells. In vivo, tumors dissociated after dual therapy showed a marked decrease in ALDH1 staining, sphere formation, and loss of tumor-initiating cells upon serial xenografting.Conclusions: Selumetinib added to saracatinib overcomes EGFR/HER2/ERBB2-mediated bypass activation of MEK/MAPK observed with saracatinib alone and targets tumor-initiating ovarian cancer populations, supporting further evaluation of combined Src-MEK inhibition in clinical trials. Clin Cancer Res; 24(19); 4874-86. ©2018 AACR.

Ovarian Cancers Harbor Defects in Nonhomologous End Joining Resulting in Resistance to Rucaparib.

Purpose: DNA damage defects are common in ovarian cancer and can be used to stratify treatment. Although most work has focused on homologous recombination (HR), DNA double-strand breaks are repaired primarily by nonhomologous end joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic instability and have been associated with the development of chemoresistance.Experimental Design: NHEJ was assessed in a panel of ovarian cancer cell lines and 47 primary ascetic-derived ovarian cancer cultures, by measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. mRNA and protein expression of components of NHEJ was determined using RT-qPCR and Western blotting. Cytotoxicities of cisplatin and the PARP inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR function was assessed using γH2AX/RAD51 foci assay.Results: NHEJ was defective (D) in four of six cell lines and 20 of 47 primary cultures. NHEJ function was independent of HR competence (C). NHEJD cultures were resistant to rucaparib (P = 0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD cultures were sensitive to rucaparib (compared with NHEJC/HRC P = 0.034, NHEJD/HRC P = 0.0002, and NHEJD/HRD P = 0.0045). The DNA-PK inhibitor, NU7441, induced resistance to rucaparib (P = 0.014) and HR function recovery in a BRCA1-defective cell line.Conclusions: This study has shown that NHEJ is defective in 40% of ovarian cancers, which is independent of HR function and associated with resistance to PARP inhibitors in ex vivo primary cultures. Clin Cancer Res; 23(8); 2050-60. ©2016 AACR.

Phosphatase and Tensin Homolog Is a Potential Target for Ovarian Cancer Sensitization to Cytotoxic Agents.

The phosphatase and tensin homolog (PTEN) tumor suppressor protein has been found to be inactivated or mutated in various human malignancies and to play a role in cisplatin and poly(ADP-ribose) polymerase inhibitor sensitivity. In this study, we assessed the association of PTEN loss with homologous recombination (HR) deficiency and increased chemosensitivity. The PTEN knockdown models were created using MISSION shRNA lentiviral transduction particles in cell lines derived from normal ovarian surface epithelium and a mixed endometrioid/clear-cell carcinoma. Sensitivity to common therapeutics was assessed using sulforhodamine B assay. Twenty-eight unselected primary epithelial ovarian cancer cultures derived from ascitic fluid collected at the time of surgery and matched genomic DNA were assessed for PTEN mutations using polymerase chain reaction amplification and Sanger sequencing and for mRNA expression using quantitative reverse transcription-polymerase chain reaction; HR was determined using γH2AX/RAD51 assay. The Cancer Genome Atlas data were analyzed using cBioPortal. In the carcinoma cell line, the PTEN knockdown enhanced sensitivity to cisplatin, rucaparib, doxorubicin, camptothecin, paclitaxel, and irradiation. In the primary ovarian cancer cultures, 2 point mutations were found (1105T>TG, 25L>L in 6 cultures and 1508G>GA, 159R>R in 4 cultures). The PTEN mRNA expression varied over 40-fold between the cultures, but did not correlate with HR status or in vitro sensitivity to cisplatin or rucaparib. The Cancer Genome Atlas data showed a rate of 8% alteration in PTEN and a trend toward improved survival in PTEN-mutated cases. These data indicate that although PTEN mutations in ovarian cancer are rare, PTEN inhibition results in therapeutic sensitization. Therefore, PTEN may be an important therapeutic target, in at least some cancers.

The effect of celecoxib on tumor growth in ovarian cancer cells and a genetically engineered mouse model of serous ovarian cancer.

Our objective was to evaluate the effect of the COX-2 inhibitor, celecoxib, on (1) proliferation and apoptosis in human ovarian cancer cell lines and primary cultures of ovarian cancer cells, and (2) inhibition of tumor growth in a genetically engineered mouse model of serous ovarian cancer under obese and non-obese conditions. Celecoxib inhibited cell proliferation in three ovarian cancer cell lines and five primary cultures of human ovarian cancer after 72 hours of exposure. Treatment with celecoxib resulted in G1 cell cycle arrest, induction of apoptosis, inhibition of cellular adhesion and invasion and reduction of expression of hTERT mRNA and COX-2 protein in all of the ovarian cancer cell lines. In the KpB mice fed a high fat diet (obese) and treated with celecoxib, tumor weight decreased by 66% when compared with control animals. Among KpB mice fed a low fat diet (non-obese), tumor weight decreased by 46% after treatment with celecoxib. In the ovarian tumors from obese and non-obese KpB mice, treatment with celecoxib as compared to control resulted in decreased proliferation, increased apoptosis and reduced COX-2 and MMP9 protein expression, as assessed by immunohistochemistry. Celecoxib strongly decreased the serum level of VEGF and blood vessel density in the tumors from the KpB ovarian cancer mouse model under obese and non-obese conditions. This work suggests that celecoxib may be a novel chemotherapeutic agent for ovarian cancer prevention and treatment and be potentially beneficial in both obese and non-obese women.

Pseudotyped αvβ6 integrin-targeted adenovirus vectors for ovarian cancer therapies.

Encouraging results from recent clinical trials are revitalizing the field of oncolytic virotherapies. Human adenovirus type 5 (HAdV-C5/Ad5) is a common vector for its ease of manipulation, high production titers and capacity to transduce multiple cell types. However, effective clinical applications are hindered by poor tumor-selectivity and vector neutralization. We generated Ad5/kn48 by pseudotyping Ad5 with the fiber knob domain from the less seroprevalent HAdV-D48 (Ad48). The vector was shown to utilize coxsackie and adenovirus receptor (CAR) but not CD46 for cell entry. A 20-amino acid peptide NAVPNLRGDLQVLAQKVART (A20) was inserted into the Ad5. Luc HI loop (Ad5.HI.A20) and Ad5/kn48 DG loop (Ad5/kn48.DG.A20) to target a prognostic cancer cell marker, αvβ6 integrin. Relative to the Ad5.Luc parent vector, Ad5.HI.A20, Ad5.KO1.HI.A20 (KO1, ablated CAR-binding) and Ad5/kn48.DG.A20 showed ∼ 160-, 270- and 180-fold increased transduction in BT-20 breast carcinoma cells (αvβ6high). Primary human epithelial ovarian cancer (EOC) cultures derived from clinical ascites provided a useful ex vivo model for intraperitoneal virotherapy. Ad5.HI.A20, Ad5.KO1.HI.A20 and Ad5/kn48.DG.A20 transduction was ∼ 70-, 60- and 16-fold increased relative to Ad5.Luc in EOC cells (αvβ6high), respectively. A20 vectors transduced EOC cells at up to ∼ 950-fold higher efficiency in the presence of neutralizing ovarian ascites, as compared to Ad5.Luc. Efficient transduction and enhanced cancer-selectivity via a non-native αvβ6-mediated route was demonstrated, even in the presence of pre-existing anti-Ad5 immunity. Consequently, αvβ6-targeted Ad vectors may represent a promising platform for local intraperitoneal treatment of ovarian cancer metastases.

Response of primary culture of human ovarian cancer cells to chemotherapy: In vitro individualized therapy.

This study focused on whether primary cultures of ovarian cancer (OC) cells established from ascites can be used to evaluate response to chemotherapeutic agents and if curcumin could enhance the efficacy of these agents. We established five primary cultures of ascitic cells from OC patients and treated them with curcumin, carboplatin, and paclitaxel singly and in combinations. The percentage of apoptotic cells was determined by flow cytometry. There was a wide variation in the response of individual primary cultures to treatment with the chemotherapeutic agents. Curcumin by itself was as good as carboplatin or paclitaxel in inducing apoptosis in the primary OC cells. Curcumin was not able to affect the carboplatin mediated cell killing. However, a combination of curcumin and paclitaxel was additive and was equally effective as a combination of carboplatin and paclitaxel. A combination of curcumin carboplatin, and paclitaxel was also found to be additive and, in fact, turned out to be the best combination that gave the highest percentage of apoptosis in vitro. This study highlights the fact that primary cultures of OC cells can be used to detect response to chemotherapeutic agents and help to individualize the treatment offered to OC patients.

The HMG-CoA reductase inhibitor, simvastatin, exhibits anti-metastatic and anti-tumorigenic effects in ovarian cancer

Ovarian cancer is the 5th leading cause of cancer death among women in the United States. The mevalonate pathway is thought to be a potential oncogenic pathway in the pathogenesis of ovarian cancer. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) inhibitor, is a widely used drug for inhibiting the synthesis of cholesterol and may also have anti-tumorigenic activity. Our goal was to evaluate the effects of simvastatin on ovarian cancer cell lines, primary cultures of ovarian cancer cells and in an orthotopic ovarian cancer mouse model. Simvastatin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, and caused cellular stress via reduction in the enzymatic activity of HMGCR and inhibition of the MAPK and mTOR pathways in ovarian cancer cells. Furthermore, simvastatin induced DNA damage and reduced cell adhesion and invasion. Simvastatin also exerted anti-proliferative effects on primary cell cultures of ovarian cancer. Treatment with simvastatin in an orthotopic mouse model reduced ovarian tumor growth, coincident with decreased Ki-67, HMGCR, phosphorylated-Akt and phosphorylated-p42/44 protein expression. Our findings demonstrate that simvastatin may have therapeutic benefit for ovarian cancer treatment and be worthy of further exploration in clinical trials.

Abstract POSTER-THER-1439: Sensitizing ovarian cancer cells to chemotherapy by interfering with pathways that are involved in the formation and maintenance of cancer stem cells

Abstract It has been suggested that tumor re-growth, as well as chemotherapy resistance and metastasis, are dependent on a small sub-population of cancer cells within the tumor that are thought to represent cancer stem cells (CSCs), i.e. self-renewing cells with pluripotent potential and tumor forming ability. We and others have shown that putative ovarian CSCs are in a transitional phase between epithelial and mesenchymal cell stages and that considerable plasticity exists between non-CSCs and CSCs (1). These transitions are based on epigenomic changes involving either epithelial-to-mesenchymal transition (EMT) or the reverse process mesenchymal-to-epithelial transition (MET). Here we report that primary ovarian cancer cells treated with the chemotherapeutic drug cisplatin undergo partial MET-like changes in culture and in xenograft tumors. These changes involve the upregulation of epithelial markers (e.g. E-cadherin) and the downregulation of mesenchymal markers (e.g. vimentin). Cisplatin-triggered MET was also observed using a luciferase reporter system that is responsive to MAP-Kinase activation. Concurrently with the induction of MET, cisplatin treatment of primary ovarian cancer cells enriched for CSCs that were positive for the stem cell markers Nanog, Oct4, and Sox2, indicating that the formation of CSC involves MET-like events. We therefore hypothesized that compounds that block MET counteract the trans-differentiation into chemoresistant CSCs and increase the efficacy of chemotherapy. Notably, MET initiates and is required for the nuclear reprogramming of fibroblasts into induced pluripotent stem (iPS) cells indicating similarities in the formation of iPS cells and CSCs. We therefore screened 30 compounds from a library of small molecules, which has previously been generated to study EMT/MET signaling in iPS cells. We identified compounds (e.g. compound GHDM1516) that blocked MET and the formation of CSCs in primary ovarian cancer cultures. Pre-treatment of cancer cells with GHDM1516 significantly increased cisplatin induced apoptosis/necrosis in CSCs as assessed by flow cytometry for Annexin V/7-AAD. In contrast, compounds that promoted EMT (e.g. compound SD83) acted synergistically with cisplatin and increased the number of CSCs and cisplatin-resistant cancer cells. Our data indicate that it might be possible to sensitize late stage tumors to chemotherapy by interfering with pathways that are involved in the formation and maintenance of CSCs. Our findings also suggest that similarities in iPS and CSC formation can be exploited to find new cancer treatments that are targeted towards CSCs. 1. Strauss, R., Z. Y. Li, Y. Liu, I. Beyer, J. Persson, P. Sova, T. Moller, S. Pesonen, A. Hemminki, P. Hamerlik, C. Drescher, N. Urban, J. Bartek, and A. Lieber. 2011. Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic heterogeneity and plasticity. PLoS One 6:e16186. Citation Format: Kamola Saydaminova, Robert Strauss, Charles Drescher , Andre Lieber. Sensitizing ovarian cancer cells to chemotherapy by interfering with pathways that are involved in the formation and maintenance of cancer stem cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1439.

Abstract 3469: Measuring antitumor effect of c-Myc-Max heterodimerization inhibitor 100258-F4 on ovarian cancer cells using cellometer image cytometry

Abstract Epithelial ovarian carcinoma is the most lethal gynecological cancer due to its silent onset and recurrence with resistance to chemotherapy. Overexpression of oncogene c-Myc is one of the most frequently encountered events present in ovarian carcinoma. Disrupting the function of c-Myc and its downstream target genes is a promising strategy for cancer therapy. In this work, we aimed to evaluate the potential effects of small-molecule c-Myc inhibitor, 10058-F4, on ovarian cancer cells and the underlying mechanisms by which 10058-F4 exerts its actions. Using Cellometer image cytometry for cell cycle and Annexin V apoptosis assays, flow cytometry, MTT assay, and colony formation, we found that 10058-F4 significantly inhibited cell proliferation of both SKOV3 and Hey ovarian cancer cells in a dose dependent manner through induction of apoptosis and cell cycle G1 arrest. Treatment with 10058-F4 reduced cellular ATP production and ROS levels in SKOV3 and Hey cells. Consistently, primary cultures of ovarian cancer treated with 10058-F4 showed induction of caspase-3 activity and inhibition of cell proliferation in 15 of 18 cases. These novel findings suggest that targeting c-Myc-Max heterodimerization could be a potential therapeutic strategy for ovarian cancer. Note: This abstract was not presented at the meeting. Citation Format: Leo Li-Ying Chan, Jiandong Wang, Xiaoli Ma, Fang Song, Weiyuan Zhang, Hannah M. Jones, Victoria L. Bae-Jump, Chunxiao Zhou. Measuring antitumor effect of c-Myc-Max heterodimerization inhibitor 100258-F4 on ovarian cancer cells using cellometer image cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3469. doi:10.1158/1538-7445.AM2015-3469

Abstract 1147: Differential expression of enzymes associated with glycine metabolism in ovarian cancer stem like cell

Abstract OBJECTIVES: The recent advances in understanding the relation between cancer and metabolism have highlighted the relevance of serine/glycine biosynthesis and one-carbon metabolism. The aim of this study was to evaluate the expression of enzymes associated with serine/glycine metabolism in ovarian cancer stem like cells (CSCs) and discuss their potential clinical implications. METHODS: We established primary ovarian cancer cell cultures from dissected fresh tumor tissue. To isolate CD117(+) and CD44(+) CSCs from primary ovarian cancer cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity. In the present study, we used Western blotting and immunohistochemistry to examine three serine-/glycine-metabolism-associated proteins (PHGDH, SHMT, and GLDC) in CSCs and ovarian cancer cases using tissue microarray (TMA). The expression of stemness gene (Nanog, Oct3/4, Sox2 and ABCG2) mRNA was determined by RT-PCR and protein expression was detected by Western blot analysis. RESULTS: GLDC, PHGDH and SHMT, associated with serine/ glycine metabolism, showed lower expression in the CSCs. TMA showed that the different expressions of GLDC, PHGDH and SHMT in platinum-sensitive and platinum-resistant ovarian cancer cases (p<0.004). GLDC mRNA was most frequently expressed in primary ovarian cancer culture cells. By Cox multivariate analysis, tumor SHMT1 negativity (HR: 2.132, 95% CI: 1.229-3.664, p = 0.007) was associated with short overall survival. CONCLUSIONS: Based on our findings, we concluded that the expression of serine-metabolism-associated proteins was related with resistance of ovarian cancer cells and patient's survival. Citation Format: So-Jin Shin, Hye-Won Chung, Hyun-Gyo Lee, Eun Som Choi, Chi-Heum Cho. Differential expression of enzymes associated with glycine metabolism in ovarian cancer stem like cell. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1147. doi:10.1158/1538-7445.AM2015-1147

458. Sensitizing Ovarian Cancer Cells To Chemotherapy By Interfering With Pathways That Are Involved in the Formation and Maintenance of Cancer Stem Cells

It has been suggested that tumor re-growth, as well as chemotherapy resistance and metastasis, are dependent on a small sub-population of cancer cells within the tumor that are thought to represent cancer stem cells (CSCs), i.e. self-renewing cells with pluripotent potential and tumor forming ability. We and others have shown that putative ovarian CSCs are in a transitional phase between epithelial and mesenchymal cell stages and that considerable plasticity exists between non-CSCs and CSCs. These transitions are based on epigenetic changes involving either epithelial-to-mesenchymal transition (EMT) or the reverse process mesenchymal-to-epithelial transition (MET). Here we report that primary ovarian cancer cells treated with the chemotherapeutic drug cisplatin undergo partial EMT/MET-like changes in culture and in xenograft tumors. Concurrently with the induction of partial EMT/MET, cisplatin treatment of primary ovarian cancer cells enriched for CSCs that were positive for the stem cell markers Nanog, Oct4, and Sox2, indicating that the formation of CSC involves partial EMT/MET-like events. In vivo cisplatin treatment of mice with xenograft tumors showed an increase in Nanog and E-cadherin expression as well as in MAPK signaling. Notably, MET initiates and is required for the nuclear reprogramming of fibroblasts into induced pluripotent stem (iPS) cells indicating similarities in the formation of iPS cells and CSCs. We therefore screened 30 compounds from a library of small molecules, which has previously been generated to study EMT/MET signaling in iPS cells. We identified compounds (e.g. compound GHDM1516) that blocked EMT and the formation of CSCs in primary ovarian cancer cultures. Co-treatment of cancer cells with GHDM1516 significantly increased cisplatin induced apoptosis/necrosis in CSCs as assessed by flow cytometry for Annexin V/7-AAD. In contrast, compounds that promoted MET (e.g. compound SD83) acted synergistically with cisplatin and increased the number of CSCs and cisplatin-resistant cancer cells. We are currently performing in vivo studies with a combination GHDM1516 and cisplatin in xenograft models of ovarian cancer. Furthermore, a “wound healing”, functional EMT assays showed the EMT inhibitory potential of GHDM1516 after treatment of ovarian cancer cells with other chemotherapy drugs such as Paclitaxel and Etoposide. Our findings suggest that similarities in iPS and CSC formation can be exploited to find new cancer treatments that are targeted towards CSCs. Our data indicate that it might be possible to sensitize late stage tumors to chemotherapy by interfering with pathways that are involved in the formation and maintenance of CSCs.

Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells.

Epithelial ovarian carcinoma is the most lethal gynecological cancer due to its silent onset and recurrence with resistance to chemotherapy. Overexpression of oncogene c-Myc is one of the most frequently encountered events present in ovarian carcinoma. Disrupting the function of c-Myc and its downstream target genes is a promising strategy for cancer therapy. Our objective was to evaluate the potential effects of small-molecule c-Myc inhibitor, 10058-F4, on ovarian carcinoma cells and the underlying mechanisms by which 10058-F4 exerts its actions. Using MTT assay, colony formation, flow cytometry and Annexin V FITC assays, we found that 10058-F4 significantly inhibited cell proliferation of both SKOV3 and Hey ovarian cancer cells in a dose dependent manner through induction of apoptosis and cell cycle G1 arrest. Treatment with 10058-F4 reduced cellular ATP production and ROS levels in SKOV3 and Hey cells. Consistently, primary cultures of ovarian cancer treated with 10058-F4 showed induction of caspase-3 activity and inhibition of cell proliferation in 15 of 18 cases. The response to 10058-F4 was independent the level of c-Myc protein over-expression in primary cultures of ovarian carcinoma. These novel findings suggest that the growth of ovarian cancer cells is dependent upon c-MYC activity and that targeting c-Myc-Max heterodimerization could be a potential therapeutic strategy for ovarian cancer.

The Use of Ovarian Cancer Cells from Patients Undergoing Surgery to Generate Primary Cultures Capable of Undergoing Functional Analysis

The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91%) cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model.

Abstract B9: PTEN expression level is important in determining sensitivity to cytotoxic agents in vitro, but is not representative of PTEN function observed in primary ovarian cancer cultures

Abstract Introduction: The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumour suppressor protein is a central negative regulator of the PI3K/AKT signalling cascade and induces apoptosis and suppresses cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. The role of PTEN in cancer however is still not fully understood. PTEN loss has been suggested to impair double strand break repair by homologous recombination and has therefore been suggested to improve sensitivity to PARP inhibitors. However, conflicting results suggest that PTEN loss is associated with cisplatin resistance development. In this study we assess the role of PTEN in ovarian cancer using experimental knockdown models and primary ovarian cancer cultures. Methods: PTEN knockdown models were created in a normal epithelial and clear cell carcinoma cell lines using MISSION®shRNA lentiviral transduction particles and grown in purimycin selection media. Sensitivity of the knockdown models to common therapies were assessed using SRB assay (these included cisplatin, paplitaxol, camptothecin, doxorubicin and irradiation). We screened 28 unselected primary epithelial ovarian cancer cultures derived from ascitic fluid collected at the time of surgery and matched genomic DNA for PTEN mutations in exons 2-5, 6-7 and 9-10 using PCR amplification and Sanger sequencing. We also assessed mRNA expression of PTEN in this panel of ovarian cancer cultures using qRT-PCR. Sensitivity to cisplatin was assessed in the primary ovarian cultures using SRB assay and HR status was determined using γH2AX/RAD51 immunofluorescence assay. Results: In normal epithelial cells, PTEN knock-down had no effect on cytotoxic agent sensitivity whilst in clear cell carcinoma cells, PTEN knockdown enhanced sensitivity to cisplatin, doxorubicin and irradiation. PTEN knockdown produced G1 arrest in both normal epithelial and clear cell carcinoma cells. The primary ovarian cancer cultures included 20 high grade serous, 6 mixed endometroid / clear cell, 1 clear cell and 1 mucinous carcinoma. Whilst two point mutations were detected in primary cultures (1105T>TG, 25L>L in 6 cultures and 1508G>GA, 159R>R in 4 cultures), no somatic mutations were observed in any of the PTEN coding sequences. The mutations found did not correlate with histological type or the level of PTEN mRNA expression. PTEN mRNA expression varied over 40 fold between the cultures. PTEN expression did not correlate with HR status or cisplatin sensitivity in primary ovarian cancer cultures. Discussion: These data indicate that PTEN knockdown results in therapeutic sensitisation. However, we have also shown that in primary ovarian cancers mutations in PTEN gene are rare and that PTEN mRNA expression does correlate with cisplatin sensitivity or HR status. Therefore the results of experimental PTEN knockdown models need to be interpreted with caution and PTEN inhibition needs to be further assessed in primary cultures to assess its role ovarian cancer. Citation Format: Aiste McCormick, Eleanor Earp, Charlotte Leeson, Michelle Dixon, Rachel ODonnell, Richard J. Edmondson. PTEN expression level is important in determining sensitivity to cytotoxic agents in vitro, but is not representative of PTEN function observed in primary ovarian cancer cultures. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B9.