Multiple Ovarian Cancer Research Articles

Abstract MIP-063: DEFINING NOVEL PARACRINE FUNCTIONS FOR INHIBIN IN OVARIAN CANCER

Abstract BACKGROUND AND SIGNIFICANCE: Inhibins are members of the TGF-β superfamily loss of which in mice results in gonadal tumorigenesis. In humans, Inhibin is elevated in patients across several ovarian cancer subtypes including GCT's, mucinous, clear cell, to lesser extents in high grade serous and also in the stroma of Brenner tumors as reported by several others. Other cancers with elevated Inhibin include prostate and common cancers associated with ascites accumulation such as colon, gastric, and pancreatic. The Type III TGF-β receptor/ Betaglycan is the only high affinity receptor molecule that has been shown to directly bind Inhibin, that does not induce signaling, but is frequently lost in ovarian cancer as reported previously. The overall goal of our studies supported in part by the Rivkin Center for Ovarian Cancer were to 1) define the possible functional consequences of elevated Inhibin in cancers in the absence of Betaglycan, 2) determine whether elevated Inhibin may act as a tumor-promoter via a novel complement of receptors in the absence of Betaglycan and 3) to evaluate the impact of blocking inhibin using a neutralizing antibody on ovarian cancer growth and metastasis. RESULTS: We find that Inhibin (alpha subunit, INHA) is largely expressed by multiple ovarian cancer epithelial cells with Inhibin A significantly elevated in the ascites of ovarian cancer patients. Reducing autocrine production of Inhibin by silencing expression of the alpha subunit in epithelial cells has only modest effects on invitro cancer cell growth, migration and invasion either in the presence or absence of Betaglycan. However, most strikingly, we find that conditioned media from ovarian cancer cells with high Inhibin levels rapidly induce SMAD1/5 activation in a paracrine manner on endothelial cells. We find that both recombinant InhibinA and paracrine Inhibin are activators of endothelial cell signaling and angiogenesis both in -in vitro endothelial differentiation assays and in vivo in mice. Mechanistically, Inhibin utilizes an alternate endothelial receptor complement system, components of which are elevated in the ascites of ovarian cancer patients. Antibodies to both the receptors recognizing Inhibin and Inhibin itself suppress Inhibin induced endothelial cell differentiation and angiogenesis. Additionally, Inhibin is a more robust mediator of angiogenesis as compared to TGF-β or Activin and can synergize with VEGF to stimulate endothelial cell differentiation and angiogenesis. IMPLICATIONS: We have discovered a new mechanism and function for Inhibin that may be relevant for ovarian cancer subtypes and pathologies that present elevated Inhibin. While current anti-angiogenic therapies targeting VEGF show promise for the treatment of advanced ovarian cancers, significant side effects and toxicities exist. Our findings on the paracrine role of Inhibin that is normally at extremely low levels in postmenopausal women suggest that consideration of anti- Inhibin antibodies in conjunction with anti- VEGF therapies may hold promise in the future. These findings will be presented and discussed. Citation Format: Priyanka Singh, Laura Jenkins, Kathleen O'Connell, YingMiao Liu, Andy Nixon, and Mythreye Karthikeyan. DEFINING NOVEL PARACRINE FUNCTIONS FOR INHIBIN IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-063.

Abstract 3856: The atomic basis for paclitaxel sensitization following loss of CRMP2 phosphorylation in ovarian cancer cells

Abstract The chemotherapeutic agent paclitaxel increases cancer cell microtubule stability, induces mitotic arrest and cancer cell death and improves survival of cancer patients. However, in ovarian and other cancers, it only elicits a response in less than half of patients who receive it. We have previously shown that enhancing microtubule stability in cells prior to paclitaxel treatment, further increases paclitaxel-induced microtubule stability and cancer cell death (Ahmed et al., Cancer Cell, 2007;12:514 and Ahmed et al., Cancer Research 2011;71:5806). We now report that the microtubule-associated protein CRMP2, and FER kinase are expressed in almost half of high-grade serous ovarian cancers and that inhibiting the phosphorylation of CRMP2 by FER is sufficient for enhancing paclitaxel-induced microtubule stability and cytotoxicity in multiple ovarian cancer cell lines. We are also able propose a detailed mechanism underlying these observations. A combination of biochemistry, super-resolution fluorescence microscopy, total internal reflection fluorescence (TIRF) microscopy and electron microscopy reveals that CRMP2 induces microtubule nucleation, elongation, bundling and stability. Moreover, FER phosphorylates CRMP2 at 6 tyrosine residues, two of which (Tyr479 and Tyr499) are critical for modulating CRMP2 function; this agrees with site-directed mutagenesis in cancer cells which points to Tyr479 as a critical site for association between CRMP2 and microtubules. A comparison of the crystal structures of wild-type CRMP2 and Tyr479Glu/Tyr499Glu phosphomimetic mutants indicate that phosphorylation of these sites induces significant conformational changes that prevent CRMP2 from forming tetramers that are known to be critical for its microtubule bundling activity. This then leads us to propose a molecular model of how CRMP2 associates with microtubules, explaining how targeting CRMP2 phosphorylation can be exploited for enhancing the therapeutic efficacy of paclitaxel. Citation Format: Yiyan Zheng, Ritika Sethi, Frank von Delft, Ahmed Ashour Ahmed. The atomic basis for paclitaxel sensitization following loss of CRMP2 phosphorylation in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3856.

Abstract A81: Targeting of free fatty acid signaling in ovarian cancer may serve as a potential therapeutic approach.

Abstract Recent studies have revealed an association between adipocyte driven free fatty acids (FFA) and the aggressiveness of epithelial ovarian cancer (EOC). Adipocyte derived free fatty acids (FFA) seem to promote epithelial ovarian cancer (EOC) growth and progression by acting as mitochondrial fuel source to support the energy requirements of EOC cells. Apart from acting as a fuel source, FFA may also enhance tumor growth through other signaling pathways that can promote proliferation and regulate metabolism. Recently, a family of FFA activated G-protein coupled receptors (FFAR/GPCRs) was identified, including: FFAR1/GPR40, FFAR2/GPR42, FFAR3/GPR41, FFAR4/GPR120 and GPR84. Using RT-PCR, we have found that exposing EOC cells to adipocytes results in overexpression of FFAR1/GPR40 in multiple ovarian cancer cell lines (ID8, A2780, C200, OVCAR3 and SKOV3). In mRNA extracted from formalin-fixed paraffin embedded tumor specimens, approximately 80% of high-grade serous ovarian carcinomas exhibited increased FFAR1 expression. Additionally, analysis of TCGA database revealed that patients with tumors exhibiting increased mRNA expression of FFAR1/GPR40 had poor overall survival and progression free survival. We have also found that targeting FFAR1 using its specific antagonist GW1100 inhibited the proliferation of A2780, ID8, C200, OVCAR3 and SKOV3 cell lines and resulted in downstream inhibition of insulin secretion and the Akt signaling pathway. On the other hand, treatment of the same cells with the FFAR1 agonist ,CAY10587, had no effect on proliferation. Thus, we conclude that FFAR1 may play an important role in the regulation of FFA mediated EOC cell proliferation. Targeting of FFAR1/GPR40 may be an attractive strategy in EOC, particularly for EOC patients presenting with high adiposity. Citation Format: Adnan Munkarah, Suhail Hamid, Jasdeep Chhina, Ismail Mert, LaToya Jackson, Sharon Hensley-Alford, Dhananjay Chitale, Shailendra Giri, Ramandeep Rattan. Targeting of free fatty acid signaling in ovarian cancer may serve as a potential therapeutic approach. [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 A81.

Cytotoxicity of Hypo-osmolar Lavage Fluid on Ovarian Cancer Cells In vitro

Objective: Ovarian cancer is difficult to diagnose early and many patients present with advanced disease. The presence of exfoliated ovarian cancer cells in the peritoneal cavity after debulking surgery is a poor prognostic indicator. Sterilization of the peritoneum during surgery may have clinical benefit in reducing tumor burden. Several studies have evaluated osmotic cytotoxicity in gastrointestinal and genitourinary cancers with varied results. We studied the cytotoxic effect of lavage fluids of differing osmolarities against multiple ovarian cancer cell lines (SKOV3, OV90, and OVCAR3) in vitro. Methods: Cells were treated for either 10 minutes or 30 minutes with water, or 5 mOsm, 10 mOsm, 50 mOsm, 100 mOsm, 200 mOsm, 280 mOsm NaCl (dilutions in water), and PBS. After 24 hours, surviving cells were enumerated in a Coulter Counter. Results: All 3 ovarian cancer cell lines were lysed progressively as lavage osmolarity was reduced (p<0.01). For the OVCAR3 ovarian cancer cells, lavage for 30 min resulted in greater cell cytotoxicity for lavage with the water, 5, 10, 50, and 200 mOsm saline when compared to lavage for 10 min. For the OV90 cancer cell line, lavage for 30 min resulted in greater cell cytotoxicity for the water, 5, and 10 mOsm saline lavages. In the SKOV3 ovarian cancer cell line, only lavage for the 30 min water had cytotoxicity. Conclusions: Since ovarian cancer uniquely resides in the peritoneal cavity, this anatomic feature allows concentrated washing to directly target these cancer cells residing in the peritoneal cavity. Hypo-osmolar treatment was found to be most effective in lysing ovarian cancer cell lines in vitro.

FAM98A is a novel substrate of PRMT1 required for tumor cell migration, invasion, and colony formation.

Protein arginine methylation, which is mediated by a family of protein arginine methyltransferases (PRMTs), is associated with numerous fundamental cellular processes. Accumulating studies have revealed that the expression of multiple PRMTs promotes cancer progression. In this study, we examined the role of PRMT1 in ovarian cancer cells. PRMT1 is expressed in multiple ovarian cancer cells, and the depletion of its expression suppressed colony formation, in vivo proliferation, migration, and invasion. To gain insight into PRMT1-mediated cancer progression, we searched for novel substrates of PRMT1. We found that FAM98A, whose physiological function is unknown, was arginine-methylated by PRMT1. FAM98A is expressed in numerous ovarian cancer cell lines and is important for the malignant characteristics of ovarian cancer cells. Our results indicate the possible role of the PRMT1-FAM98A pathway in cancer progression.

Celecoxib enhances [sorafenib + sildenafil] lethality in cancer cells and reverts platinum chemotherapy resistance

The present studies sought to determine whether the lethality of the drug combination [sorafenib + sildenafil] could be enhanced by the anti-inflammatory agent celecoxib, using ovarian cancer and other tumor cell lines as models. Also, in a dose dependent fashion celecoxib enhanced [sorafenib + sildenafil] lethality in multiple ovarian cancer cell lines. In a dose dependent fashion celecoxib enhanced the ability of [sorafenib + sildenafil] to reduce expression of multiple chaperone proteins in parallel with lower levels of the drug efflux pumps ABCB1 and ABCG2. Over-expression of GRP78 and HSP27 maintained pump expression in the presence of drugs. Cell killing by the 3 drug combination was mediated by mitochondrial / caspase 9 –dependent apoptotic signaling and by RIP-1 / caspases 2 and 4 / AIF –dependent necroptotic signaling. Pre-treatment of intrinsically resistant primary ovarian cancer cells with [celecoxib + sorafenib + sildenafil] significantly enhanced tumor cell killing by a subsequent cisplatin exposure. Similar data were obtained in some cancer cell lines, but not all, using the related platinum containing drugs, oxaliplatin and carboplatin. As our prior publications have also validated in vivo the combinations of [celecoxib + sildenafil] and [sorafenib + sildenafil] as cytotoxic to multiple tumor cell types, combined with the present findings, we would argue that the combination of celecoxib/sorafenib/sildenafil should be explored in a new phase I trial in ovarian cancer.

Abstract POSTER-BIOL-1325: Ovarian cancer-associated CDK12 mutations disable its kinase activity and cause defects in homologous recombination

Abstract PURPOSE: The purpose of this study was to determine whether a) high-grade serous ovarian cancer-associated CDK12 kinase domain mutations affect catalytic activity, b) loss of CDK12 affects BRCA1 expression levels and Homologous Recombination (HR) in ovarian cancer, c) tumor-associated CDK12 mutations cause HR defects in ovarian cancer, and d) loss of CDK12 sensitizes ovarian cancer to chemotherapy and Poly(ADP-ribose) Polymerase [PARP] inhibitors. EXPERIMENTAL PROCEDURES: We compared the kinase activities of wild-type CDK12 and tumor-associated CDK12 kinase domain mutants using in-vitro kinase assays. To determine the impact of CDK12 activity on HR, we assessed RAD51 foci formation and HR using a DR-GFP construct. To analyze the effect of tumor-associated CDK12 mutations on HR, CDK12-depleted OVCAR-8 cells were reconstituted with either wild-type CDK12 or the tumor-associated CDK12 mutant (G909R), irradiated and stained for RAD51 foci. We performed clonogenic assays using ovarian cancer cell lines (OVCAR-8, OVCAR-5 and OVCAR-3) to measure sensitivity to DNA cross-linking agents (melphalan and cisplatin) and the PARP inhibitor, veliparib, in the presence or absence of CDK12 activity. CDK12 depletion was carried out using at least two independent short-interfering RNAs (siRNAs). RESULTS: We showed that many, but not all, ovarian cancer-associated CDK12 kinase domain mutations severely disrupted kinase activity. Moreover, we found that disabling CDK12 activity in ovarian cancer cells decreased BRCA1 levels and disrupted HR. Additionally, the tumor-associated CDK12 G909R mutant did not restore impaired RAD51 foci formation in CDK12-depleted OVCAR-8 cells. Finally we show that CDK12 depletion sensitized multiple ovarian cancer cells to the DNA cross-linking agents melphalan and cisplatin and to the PARP inhibitor veliparib. CONCLUSION: Ovarian cancer-associated CDK12 mutations disrupt kinase activity and cause defects in HR, suggesting that ovarian tumors harboring deleterious CDK12 mutations may predict sensitivity to chemotherapeutic agents, including PARP inhibitors. Citation Format: Poorval M. Joshi, Shari L. Sutor, Catherine J. Huntoon, Larry M. Karnitz. Ovarian cancer-associated CDK12 mutations disable its kinase activity and cause defects in homologous recombination [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-BIOL-1325.

Abstract 3994: Herpes virus microRNA expression and significance in serous ovarian cancer

Abstract Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality. Citation Format: Deep Pandya, Marisa Mariani, Mark McHugh, Mirko Andreoli, Steven Sieber, Shiquan He, Candice Dowell-Martino, Paul Fiedler, Giovanni Scambia, Cristiano Ferlini. Herpes virus microRNA expression and significance in serous ovarian cancer. [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 3994. doi:10.1158/1538-7445.AM2015-3994

Abstract 2384: CD151-integrin complexes suppress ovarian tumor growth by repressing slug-mediated emt and canonical wnt signaling

Abstract The majority of human ovarian cancer is diagnosed in the late, metastatic stages and is highly resistant to current chemotherapies. The molecular and cellular mechanisms underlying the malignancy of ovarian cancer remain poorly understood. We report a surprising functional link between CD151-α31 integrin complexes and the malignancy of serous ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in approximately 90% of metastatic lesions, while it remains detectable in &amp;gt;40% of primary ovarian tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses with multiple ovarian cancer cell lines show that knocking down CD151 or α3β1 integrin markedly enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated upon CD151 removal. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcription complexes. Taken together, our study demonstrates that CD151-α3β1 integrin complexes suppress ovarian cancer malignancy by repressing Slug-mediated EMT and canonical Wnt signaling. Citation Format: Xiuwei H. Yang. CD151-integrin complexes suppress ovarian tumor growth by repressing slug-mediated emt and canonical wnt signaling. [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 2384. doi:10.1158/1538-7445.AM2015-2384

CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27Kip1 as well as TP53-dependent transcriptional induction of p21Cip1 in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

A phase I trial of the safety and immunogenicity of a multi-epitope folate receptor alpha peptide vaccine used in combination with cyclophosphamide in subjects previously treated for breast or ovarian cancer.

e14028 Background: Folate receptor alpha (FRα) is overexpressed by multiple cancers, including breast and ovarian cancers. Endogenous T-cell immunity to each of five degenerate peptides from FRα (F...

Disulfiram’s Antineoplastic Effects on Ovarian Cancer

Objective: Aldehyde dehydrogenase (ALDH) enzymatic activity identifies ovarian cancer stem-like cells. We investigated the antineoplastic activity of the ALDH inhibitor Disulfiram on bulk ovarian cancer cells and CD133 + /ALDH + cancer stem-like cells. Study Design: Ovarian cancer cell lines, human ovarian surface epithelial cells, and mesenchymal stem cells were treated with increasing concentrations of Disulfiram and/or Cisplatin in vitro. Treated cells were assessed for viability or FACS-analyzed for either percentage of ovarian cancer stem-like cells or induction of apoptosis. Disulfiram’s impact on cancer stem-like cells was tested in vitro using tumor sphere formation assays and in vivo using tumor initiation assays with in vitro-treated A2780 cells in NSG mice. Finally, Disulfiram’s in vivo activity was assessed versus CD133 + /ALDH + cell-initiated tumor xenografts. Results: Disulfiram demonstrated antineoplastic activity against multiple ovarian cancer cell lines. While Disulfiram had limited in vitro toxicity against human ovarian surface epithelial cells or mesenchymal stem cells (IC50 of ~15 µM and >30 µM, respectively), its antineoplastic activity against cell lines was comparable to Cisplatin (IC50 ~1.5 μM). Disulfiram-mediated cell death was due, at least in part, to induction of apoptosis. Disulfiram activity was additive with chemotherapy. Disulfiram demonstrated selective depletion of CD44 + cells but not the CD133 + cancer stem-like cells. Disulfiram had no therapeutic impact on tumor initiation studies or in vivo therapy of whole cell line or stem cell-initiated tumor xenografts. Conclusions: In biologically relevant concentrations, Disulfiram has clear antineoplastic activity against ovarian cancer cells in vitro. Disulfiram selectively depleted CD44+ but not CD133+ ovarian cancer stem-like cells in vitro. However, Disulfiram had no significant activity in vivo. Thus, improved and more selective ALDH inhibitors may be required to target ovarian cancer stem cells.

Herpes virus microRNA expression and significance in serous ovarian cancer.

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality.

Histone h1.3 suppresses h19 noncoding RNA expression and cell growth of ovarian cancer cells.

Ovarian cancer is a deadly gynecologic malignancy for which novel biomarkers and therapeutic targets are imperative for improving survival. Previous studies have suggested the expression pattern of linker histone variants as potential biomarkers for ovarian cancer. To investigate the role of histone H1 in ovarian cancer cells, we characterize individual H1 variants and overexpress one of the major somatic H1 variants, H1.3, in the OVCAR-3 epithelial ovarian cancer cell line. We find that overexpression of H1.3 decreases the growth rate and colony formation of OVCAR-3 cells. We identify histone H1.3 as a specific repressor for the noncoding oncogene H19. Overexpression of H1.3 suppresses H19 expression, and knockdown of H1.3 increases its expression in multiple ovarian epithelial cancer cell lines. Furthermore, we demonstrate that histone H1.3 overexpression leads to increased occupancy of H1.3 at the H19 regulator region encompassing the imprinting control region (ICR), concomitant with increased DNA methylation and reduced occupancy of the insulator protein CTCF at the ICR. Finally, we demonstrate that H1.3 overexpression and H19 knockdown synergistically decrease the growth rate of ovarian cancer cells. Our findings suggest that H1.3 dramatically inhibits H19 expression, which contributes to the suppression of epithelial ovarian carcinogenesis.

The changing landscape of genetic testing for hereditary breast and ovarian cancer

It took Angelina Jolie’s announcement in 2013 to bring broad awareness of hereditary breast and ovarian cancer (HBOC) to the general public. However, it has been 25 years since MaryClaire King mapped BRCA1 to chromosome 17 through linkage analysis involving painstaking collection of a large number of families with multiple cases of early-onset breast cancer. Though familial “clusters” of breast cancer had been described since the early 1800s, the concept that cancer can result from inheritance of a single faulty gene first made its way into the scientific literature when Knudson published his famous “2-hit hypothesis” in 1971. Lynch et al were one of the first to describe the association of breast with ovarian cancer in a 1978 article entitled “Familial Association of Breast and Ovarian.” However, it was the pioneering work of Gardner and Stephens initiated in the mid-1940s identifying and following large Mormon kindreds in Utah that ultimately drove the search for the “breast cancer gene.” Despite the work of Knudson, Lynch, Gardner, and others, the research and clinical communities remained skeptical well into the early 1980s that breast cancer could be hereditary, as opposed to primarily the result of environmental and lifestyle factors. The race to isolate the BRCA1 gene was ultimately won by Mark Skolnick, founder of Myriad Genetic Laboratories, Inc, in 1994, followed closely by the cloning of BRCA2 in 1995. At that time, despite the evidence of increased incidence of ovarian cancer in BRCA1 and BRCA2 families, many still believed there was an ovarian cancer–only gene that was responsible for pedigrees segregating multiple ovarian cancer cases, but no breast cancer. However, further study confirmed that there was not an “OVCA1” gene, and that BRCA1 and BRCA2 accounted for most familial site-specific ovarian cancer. In addition, the failure to find a BRCA1 and BRCA2 mutation in many apparently classic HBOC families led to the search for “BRCA3.” Ultimately it was determined that there likely did not exist another single major hereditary cancer gene responsible for most BRCA1and BRCA2-negative families.

TRPM7 is required for ovarian cancer cell growth, migration and invasion

Our previous study demonstrated that the melastatin-related transient receptor potential channel 7 (TRPM7) was highly expressed in ovarian carcinomas and its overexpression was significantly associated with poor prognosis in ovarian cancer patients. However, the function of TRPM7 in ovarian cancer is mostly unknown. In this study, we examined the roles of TRPM7 in ovarian cancer cell proliferation, migration and invasion. We found that short hairpin RNA interference-mediated silence of TRPM7 significantly inhibited cell proliferation, colony formation, migration and invasion in multiple ovarian cancer cell lines. Mechanistic investigation revealed that silence of TRPM7 decreased phosphorylation levels of Akt, Src and p38 and increased filamentous actin and focal adhesion number in ovarian cancer cells. Thus, our results suggest that TRPM7 is required for proliferation, migration and invasion of ovarian cancer cells through regulating multiple signaling transduction pathways and the formation of focal adhesions.

Abstract LB-70: BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression

Abstract Functional loss of BRCA1 has been implicated in genomic instability and cancer progression. A recent study demonstrated that BRCA1 plays a role in cancer cell migration. Using a random cell motility assay, we showed that BRCA1 promotes ovarian cancer cell migration. A proteomics study of BRCA1 +/- cell lines and tissues identified Profilin1 (Pfn1) as one of the differentially expressed proteins. This was further confirmed by Western blot and IHC analyses with tissue samples and cell lines. Pfn1 is an actin cytoskeleton-associated protein which promotes breast cancer cell motility/invasion and dissemination when downregulated. To further study whether BRCA1 impacts cell migration through Pfn1, Pfn1 was silenced which revealed enhanced motility in multiple BRCA1-WT ovarian cancer cell lines. Furthermore, overexpression of Pfn1 in BRCA1 cell lines reduced random motility which further implicates the importance of Pfn1 in ovarian cancer motility. This is the first evaluation of both motility and the functional role of Pfn1 in ovarian cancer cells. While BRCA1 has been extensively studied in the context of DNA repair, there is emerging evidence that BRCA1 proteins may play a role in cell migration. The increased motility seen in wild-type cells compared to BRCA1-mutated cells may portend increased metastatic potential in wild-type cancers. Our findings demonstrate that 1) BRCA1 positively influences ovarian cancer cell motility, and 2) BRCA1 status determines Pfn1's role in ovarian cancer cell motility suggesting a possible novel mechanism association between Pfn1 and BRCA1 and ovarian cancer cell motility. Future studies should explore whether Pfn1 downregulation enhances the metastatic potential of ovarian cancer cells with intact BRCA1 function and whether Pfn1 may serve as a possible therapeutic target. Citation Format: David M. Gau, Jamie Lesnock, Thomas Krivak, Robert Edwards, Partha Roy. BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-70. doi:10.1158/1538-7445.AM2014-LB-70

Abstract 1543: Systematic functional assessment of driver mutations for individual ovarian cancers

Abstract Although epithelial ovarian cancer is an intractable cancer and the overall five-year survival rate is approximately 40 to 50%, clinically effective molecular-targeted therapeutics to treat ovarian cancer has not been available thus far. Recent progress of massively parallel sequencing technologies has been enabling us to identify substantial number of genomic aberrations including somatic mutations, indels and translocations in variable cancer types exemplified by epithelial ovarian cancer and non-small cell lung cancer. However, it is still very difficult to identify a ‘driver’ gene, which is functionally relevant for cancer growth, from hundreds of mutations that are usually found from a single cancer sample of an individual patient. An in vitro cultured cell line, which is originated from a clinical sample, can be utilized to assess functions of the genes that are identified to have somatic aberrations in the paired clinical tumor. We have been establishing multiple ovarian cancer cell lines derived from clinical samples and analyzing pairs of the cell lines and clinical samples with exon-capture sequencing. The genes, which are somatically mutated and shared in a pair of cell line and clinical sample, are targeted with RNA interference in the cell line, and subsequently the effect on cell proliferation and survival is examined. As such, a driver mutation for an individual cancer can be identified by a set of experimental perturbations. Identified driver mutations would be then validated with publicly available databases and with clinical archive samples. As a proof-of-principle, somatic mutations of PIK3CA and KRAS were detected as drivers to promote cell proliferation of a carcinosarcoma. We believe this approach can increase an opportunity to identify molecular targets in ovarian cancer therapeutics. Citation Format: Seiichi Mori, Takako Yokomizo, Takeshi Fujiwara, Osamu Goto, Haruko Iwase, Kazuyoshi Kato, Tokuichi Kawaguchi, Tetsuo Noda. Systematic functional assessment of driver mutations for individual ovarian cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1543. doi:10.1158/1538-7445.AM2014-1543

Anti-Galectin-3 Therapy: A New Chance for Multiple Myeloma and Ovarian Cancer?

Here we review the role of Galectins in the molecular pathogenesis of multiple myeloma and ovarian cancer, with a special focus on Glectin-3. Multiple myeloma is the second most common hematologic malignancy worldwide. Because the pathogenesis of multiple myeloma is still incompletely understood, there is no ultimately effective cure, and this cancer results fatal. Ovarian cancer is the most lethal gynecologic malignancy worldwide. Due to the lack of screening techniques for early detection, patients are mostly diagnosed with advanced disease, which results ultimately fatal. Multiple myeloma and ovarian cancer have different biologies, but they share a strong dependence on adhesion with extracellular matrix and other cells. Galectin-3 plays a key role in regulating such adhesive abilities of tumor cells. Here we discuss the outcomes and possible mechanism of action of a truncated, dominant negative form of Galectin-3, Galectin-3C, in these malignancies. Overall, we report that Galectin-3C is a promising new compound for effective adjuvant therapies in advanced, refractory multiple myeloma and ovarian cancer.

Tumorgrafts as In Vivo Surrogates for Women with Ovarian Cancer

Ovarian cancer has a high recurrence and mortality rate. A barrier to improved outcomes includes a lack of accurate models for preclinical testing of novel therapeutics. Clinically relevant, patient-derived tumorgraft models were generated from sequential patients and the first 168 engrafted models are described. Fresh ovarian, primary peritoneal, and fallopian tube carcinomas were collected at the time of debulking surgery and injected intraperitoneally into severe combined immunodeficient mice. Tumorgrafts demonstrated a 74% engraftment rate with microscopic fidelity of primary tumor characteristics. Low-passage tumorgrafts also showed comparable genomic aberrations with the corresponding primary tumor and exhibit gene set enrichment of multiple ovarian cancer molecular subtypes, similar to patient tumors. Importantly, each of these tumorgraft models is annotated with clinical data and for those that have been tested, response to platinum chemotherapy correlates with the source patient. Presented herein is the largest known living tumor bank of patient-derived, ovarian tumorgraft models that can be applied to the development of personalized cancer treatment.