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Metastasis causes poor prognosis and yet remains the least understood aspect of cancer. To identify key regulators of ovarian cancer metastasis that drive early colonization and are also essential for advanced metastasis, we have applied a novel, comprehensive approach of combining the analysis of an organotypic 3D culture model of early metastasis with ovarian cancer patient tumors. Using matched primary and metastatic tumors from 42 OC patients, we have compared the fully formed metastatic tumors with the primary tumor of the same patient. Since this is an end-point analysis and reveals little about the intermediated regulatory steps, we have also studied the changes occurring during early metastatic colonization as a result of the interactions with the metastatic microenvironment by using an organotypic 3D culture model of omental metastasis. By combining these two approaches, we have identified key master regulators of metastasis that are essential for early metastatic colonization and remain important in the fully formed metastatic tumor. We specifically focused on microRNAs because of their pleotropic effects through translational inhibition of multiple targets, and also our previous studies have demonstrated their important role in metastasis. Using this approach, we identified a novel microRNA miR-4454, downregulated in the metastasizing ovarian cancer cells through their specific paracrine interactions with fibroblasts in the metastatic microenvironment. The downregulation of miR-4454 promoted high-grade serous ovarian cancer cell migration, invasion, proliferation, and clonogenic growth as well as metastasis in xenografts. The targets of miR-4454 were identified by RNA-seq in high-grade serous ovarian cancer cells overexpressing the microRNA. SPARC and BAG5 were found to be the functional effectors of miR-4454. SPARC activated FAK signaling and promoted metastasis, while BAG5 inhibited apoptosis and helped the metastasizing cancer cells tide over adverse conditions in the microenvironment. Since miR-4454 is essential for early metastatic colonization as well as in advanced metastasis, targeting it is a promising approach to treat metastatic ovarian cancer. Citation Format: Subramanyam Dasari, Taruni Pandhiri, Tommaso Grassi, Daniel W. Visscher, Francesco Multinu, Komal Agarwal, Andrea Mariani, Viji Shridhar, Anirban K. Mitra. Paracrine interactions with microenvironmental fibroblasts promote ovarian cancer metastasis through downregulation of miR-4454 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B66.

Purpose: In humans, several distinctive cancers result from mutations that aberrantly activate hedgehog (HH) signal transduction. Here, we investigate the role of HH signaling in ovarian cancer. Experimental Design: We assessed the expression of different components of hedghehog pathway in primary tumor samples and cell lines. We used specific hedghehog pathway blocker to study the effect on clonal growth and proliferation of ovarian cancer cell both in vitro and in vivo. Results: We show that the up-regulation of several HH pathway components is a common feature of primary ovarian tumors and cell lines. However, expression of PATCHED1 (PTCH1), a direct transcriptional target of the HH pathway, is down-regulated in ovarian cancer in direct contrast to the expression observed in other adult solid tumors. Cyclopamine, a specific HH pathway inhibitor, inhibits the proliferation and clonal growth of ovarian tumor cells in vitro and arrests ovarian tumor growth in vivo. Expression of BMI-1, a polycomb gene, is down-regulated in ovarian cancer cells following cyclopamine treatment. Overexpression of PTCH1 phenocopied the effects of cyclopamine; it down-regulated BMI-1 and reduced clonal growth in ovarian cancer cell lines. Furthermore, knocking down BMI-1 using small interfering RNA also inhibited the clonal growth of all the ovarian cancer cell lines tested. Conclusions: In brief, the constitutive low-level expression of PTCH1 contributes to proliferation and clonal growth of ovarian cancer cells by an aberrant HH signal. Because the HH pathway can be inhibited by specific inhibitors, these findings point toward possible new treatments to inhibit ovarian cancer growth.

Resistance to chemotherapy is a major hurdle that results in the increased mortality associated with ovarian cancer (OvCa). New therapeutics that synergize with cis-carboplatin/paclitaxel in recurrent tumors will have an enormous impact in prolonging the survival of OvCa patients. Due to the enormous cost of designing and testing new drugs, we have adopted the drug repurposing approach and tested the effect of the antimalarial drug quinacrine (QC) in sensitizing drug resistant OvCa cells to carboplatin induced cytotoxicity and determined that it is autophagy dependent. We have discovered that QC, an autophagy modulator exhibited distinct anti-cancer properties in OvCa cell lines in contrast to other anti-malarial/autophagy inhibitor drugs such as hydroxyquinacrine, bafilomycin A and 3-MA. It is well established that autophagy can be a survival mechanism under stress in cancer or a death promoting phenomenon that is cell type and contextual dependent. Here we demonstrate that QC acts to promote chemotherapy induced cell death specifically more in resistant cells in an autophagy dependent manner. Our preliminary data both in vitro and in vivo indicate that QC induced autophagy downregulates p62 and upregulates cathepsin L levels to sensitize the chemoresistant cells to autophagic and caspase mediated cell death in a p53 independent manner. These unexpected findings led us to explore the mechanistic basis of QC-induced upregulation of the cell cycle inhibitor p21 in cells bearing mutant p53. We uncovered that irrespective of p53 status, QC induces p21 expression in an autophagy-dependent p62-Skp2 mediated ubiquitin pathway resulting in cell cycle arrest and cell death. Since QC is already in PhaseI Clinical trial, these studies may spur new treatment strategies to improve OvCa patient outcome and patient care. Citation Format: Debarshi Roy, Ashwani Khurana, Eleftheria Kalogera, Xuyang Wen, Susmita Mondal, Viji Shridhar. Can we repurpose anti-malarial drug quinacrine to treat ovarian cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B74.

High-grade serous (HGS) ovarian cancer is the most lethal form of epithelial ovarian cancer because it represents the most common histologic subtype and usually present at an advanced stage. Due to its advanced stage presentation, HGS ovarian cancer has been described as arising de novo from undefined precursor lesions that progress rapidly to advanced disease. To gain insights into clonal heterogeneity and progression to distant metastases, we analyzed single nucleotide variations (SNVs) from nine regions of carcinomas from four patients with HGS ovarian cancer. Three primary sites, three metastases from omentum, and three metastases from bowel were collected from each patient and subjected to RNA sequencing and SNV analysis. Although a modest number (23.62%) of SNVs are shared within the same anatomic site, only limited number (1.26%) of SNVs are shared among all 9 sites, suggesting a high degree of intratumor heterogeneity in these patients. Truncal mutations, such as TP53 mutations, are shared among all sites and observed at high allele frequencies. In addition, mutations in genes associated with epigenetic modifiers, such as SETD2, CHD8, KDM5A, SMARCA1 and ARID5B are observed as truncal mutations in these patients. Phylogenetic tree reconstruction of tumors from various sites suggests a concurrent ovarian and peritoneal metastases in one patient and peritoneal metastases preceding the ovarian carcinomas in three other patients. Finally, subclone inference from multi-region sequencing indicates the presence of multiple subclones at each site in these patients. Dominant subclones, present at metastases, are often ancestral clones in the clonal hierarchy, further providing evidence that peritoneal spreading occurs early in the clonal expansion. Collectively, these results provide evidence for early peritoneal spreading that often precedes ovarian carcinomas in HGS ovarian cancer. This abstract is also presented as Poster B44. Citation Format: Jeremy Chien, Viji Shridhar, Andrea Mariani. Evidence for extra-ovarian origin and peritoneal metastases that precede ovarian carcinomas in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR08.

FoxM1, a member of the Forkhead transfection factors, has been found to be overexpressed in many human cancers including ovarian cancer. Although FoxM1 is known to be downregulated by p53, the molecular mechanisms are not well characterized. In this study, we used the MDM2 inhibitor Nutlin-3 as a tool to modulate p53 expression and investigated the mechanism by which p53 regulates FoxM1 expression in ovarian cancer cells. We observed that Nutlin-3 upregulated p53 protein and downregulated FoxM1 mRNA and protein levels in ovarian cancer cell lines with wild type TP53, but not in cell lines with mutant TP53. This effect was at least partially blocked by cycloheximide or actinomycin D, indicating a transcription- and translation-dependent mechanism. Pulse-chase studies revealed that downregulation of FoxM1 by Nutlin-3 is due to enhanced FoxM1 mRNA decay. Knockdown of p53 using shRNAs abrogated the suppression of FoxM1 expression by Nutlin-3, indicating a p53-dependent mechanism. Furthermore, silencing of FoxM1 by shRNAs in ovarian cancer cells with or without wild-type TP53 led to a significant decrease in cell proliferation. The FoxM1 inhibitor, thiostrepton, induces cytotoxicity in ovarian cancer cell lines and enhances sensitivity to cisplatin in these cells. Thiostrepton downregulates FoxM1 expression in several ovarian cancer cell lines and enhances sensitivity to carboplatin in vivo. Finally, FoxM1 expression is elevated in nearly all (47/48) ovarian tumors. In summary, the present study provides novel evidence that p53 downregulates FoxM1 in ovarian cancer cells at least in part by destabilizing FoxM1 mRNA and that FoxM1 may be targeted for therapeutic benefits in ovarian cancer. Citation Format: Xuan Zhang, Lihua Cheng, Kay Minn, Magdalena Waggoner, Nikhil K. Parelkar, Iman Jokar, George A. Vielhauer, Rashna Madan, Andrew K. Godwin, Viji Shridhar,, Jeremy Chien. Targeting p53-FoxM1 axis in ovarian cancer [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-1409.