Multiple Ovarian Cancer Research Articles

ZNF300P1 Encodes a lincRNA that regulates cell polarity and is epigenetically silenced in type II epithelial ovarian cancer

BackgroundWe previously identified that the CpG island-associated promoter of the novel lincRNA ZNF300P1 (also known as LOC134466) is frequently hypermethylated and silenced in ovarian cancer tissues. However, the function of ZNF300P1 was unknown. In this report we demonstrate that ZNF300P1 is involved in the regulation of key cell cycle and cell motility networks in human ovarian surface epithelial cells, and may play a role in promoting metastasis in ovarian cancer cells.MethodsWe applied methylated DNA immunoprecipitation on whole genome promoter tiling arrays and Sequenom assays to examine methylation status of ZNF300P1 in multiple ovarian cancer cell lines, as well as in normal ovarian and ovarian tumor tissues. Transcript profiling was used to investigate the effects of ZNF300P1 suppression in ovarian cancer cells. We utilized siRNA knockdown in normal ovarian surface epithelial cells and performed cellular proliferation, migration and adhesion assays to validate and explore the profiling results.ResultsWe demonstrate that ZNF300P1 is methylated in multiple ovarian cancer cell lines. Loss of ZNF300P1 results in decreased cell proliferation and colony formation. In addition, knockdown of the ZNF300P1 transcript results in aberrant and less persistent migration in wound healing assays due to a loss of cellular polarity. Using an ex vivo peritoneal adhesion assay, we also reveal a role for ZNF300P1 in the attachment of ovarian cancer cells to peritoneal membranes, indicating a potential function of ZNF300P1 expression in metastasis of ovarian cancer cells to sites within the peritoneal cavity.ConclusionOur findings further support ZNF300P1 as frequently methylated in ovarian cancer and reveal a novel function for ZNF300P1 lincRNA expression in regulating cell polarity, motility, and adhesion and loss of expression may contribute to the metastatic potential of ovarian cancer cells.

Abstract A137: Combinatorial therapy design in cancer as a directed attack on the hubs of a scale-free network.

Abstract Scale-free networks, characterized by a power-law degree distribution, are known to be resilient to random failures, yet vulnerable to directed attacks on their hubs (their most connected nodes). Here, we show that protein-protein interaction networks in cancer are scale-free, validate the effectiveness of hub attacks using published RNAi lethality screens conducted on human cancer cells, and propose a novel approach for combinatorial hub attacks. Methods: Proteins involved in 14 different cancer types were extracted from the KEGG database. For each cancer type, a network was constructed by incorporating the experimentally-validated physical interactions of the pertinent KEGG proteins from the BioGRID interaction network. To validate the presence of scale-free cancer networks, we fit the degree distribution of the full network to a power-law. Then, we quantified the degree distribution of the lethality proteins discovered from RNAi screens (conducted on human multiple myeloma and epithelial ovarian cancer cells) and compared it to the rest of the network. A previously-derived mathematical relationship between the destruction of the giant component (a metric of network integrity) and the fraction of hubs removed was applied to the cancer networks to investigate the utility of combination therapy. Results: In agreement with previous studies of many biological networks, we found that the 14 cancer types listed in KEGG all demonstrated scale-free behavior. We analyzed previously published, genome-wide RNAi lethality screens in two cancer cells, and found a significant enrichment (≈ 5-fold) of median degree-connectivity among lethal proteins compared to all the proteins in the network. As expected, for a scale-free network, the removal of < 5% of the proteins completely destroyed network integrity. In the case of basal-cell carcinoma, as few as 9 proteins were responsible for the integrity of the entire network. Also, the marginal utility of each successive hub knockout for network destruction remained relatively constant, consistent with a combinatorial approach in tackling cancer. Conclusion: In this work, we demonstrate that cancer networks are scale-free. The correlation between connectivity and lethality in the investigated cancer networks suggests that the number of neighbor-interactions of a protein may determine its potency as a drug target. We demonstrate a greedy algorithm for combinatorial therapy design based on the principle of simultaneous targeting of high-connectivity nodes. As the network of proteins responsible for toxicity in response to anticancer agents is also likely to be scale-free (and may be uncorrelated with cancer protein networks) the targeted destruction of cancer network hubs may in effect be a random attack on the toxicity nodes. This suggests that a combinatorial approach of targeting multiple proteins with high connectivity in cancer networks may be effective in creating an optimal therapeutic window. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A137. Citation Format: Andrew Chen, Arijit Chakravarty, Jerome Mettetal, Wen Chyi Shyu, Joseph Bolen, Santhosh Palani. Combinatorial therapy design in cancer as a directed attack on the hubs of a scale-free network. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A137.

Abstract A253: HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer.

Abstract We have developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group which selectively inhibits STAT3 activation, translocation, and DNA binding activity, without affecting the expression of other pSTATs. This new STAT3 inhibitor offers advantages when compared to other STAT3-targeting molecules currently available, under development or undergoing preclinical trials. Most notably, these compounds exhibit minimal toxicity toward normal cells and tissues while inducing apoptosis in ovarian cancer cells, and exhibit good oral bioavailability. Results show that selective cytotoxicity of the lead compound, HO-3867, towards cancer cells is multi-faceted. AutoDock molecular modeling indicates that the DAP compounds interact with STAT3 at the DNA-binding domain. Liquid chromatography / Mass spectroscopy analysis demonstrates greater bioabsorption and bioavailability of active (cytotoxic) metabolites in cancer cells when compared to normal cells. Differential activation of the Akt pathway was demonstrated using Akt siRNA in normal versus cancer cells. Knockdown of STAT3 expression using two different shRNAs shows the requirement of STAT3 for HO-3867-mediated apoptosis. In vivo testing shows that HO-3867 inhibits xenograft tumor growth without toxic side effects. In addition, HO-3867 significantly inhibited cell survival, migration/invasion and induced cell death in primary ovarian cancer cell populations isolated from ascites of patients with recurrent and advanced ovarian cancer. This study provides evidence of a safe, targeted STAT3 inhibitor (HO-3867) with demonstrated efficacy in treating multiple ovarian cancer models. This suggests significant translational potential of these compounds for the treatment of ovarian cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A253. Citation Format: Karuppaiyah Selvendiran, Kellie S. Rath, Shan Naidu, Pushpa Latha, Bid Hemant, Georgia A. McCann, Veronica Bravo, Peter Houghton, Gustavo Leone, Kálmán Hideg, Periannan Kuppusamy, David E. Cohn. HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A253.

The gep proto-oncogene Gα12 mediates LPA-stimulated activation of CREB in ovarian cancer cells

Lysophosphatidic acid (LPA) plays a critical role in the pathophysiology of ovarian cancers. Previous studies have shown that LPA stimulates the proliferation of ovarian cancer cells via Gα12. The present study utilizing Protein/DNA array analyses of LPA-stimulated HeyA8 cells in which the expression of Gα12 was silenced, demonstrates for the first time that Gα12-dependent mitogenic signaling by LPA involves the atypical activation cAMP-response element binding protein (CREB). Results indicate that the robust activation of CREB by LPA is an early event that can be monitored by the phosphorylation of SER133 of CREB as early as 3min. The findings that the expression of the constitutively activated mutant of Gα12 stimulates CREB even in the absence of LPA in multiple ovarian cancer cell lines confirm the direct role of Gα12 in the activation of CREB. This is further substantiated by the observation that the silencing of Gα12 drastically attenuates LPA-stimulated phosphorylation of CREB. Our results also establish that LPA–Gα12-dependent activation of CREB is through a cAMP-independent, but Ras–ERK-dependent mechanism. More significantly, our findings indicate that the expression of the dominant negative S133A mutant of CREB leads to a reduction in LPA-stimulated proliferation of HeyA8 ovarian cancer cells. Thus, results presented here demonstrate for the first time that CREB is a critical signaling node in LPA–LPAR and Gα12/gep proto-oncogene stimulated oncogenic signaling in ovarian cancer cells.

Death Domain-associated Protein DAXX Promotes Ovarian Cancer Development and Chemoresistance

The role of DAXX in ovarian cancer development and metastasis has not been investigated before now. Overexpression of DAXX enhanced ovarian cancer cell proliferation, colony formation, and migration, whereas Daxx depletion had the opposite effects. DAXX promotes ovarian cancer cell proliferation and chemoresistance. ModulatingDAXXmay be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers. Understanding the genes involved in apoptosis and DNA damage responses may improve therapeutic strategies for ovarian cancer. The death domain-associated protein DAXX can be either a pro-apoptotic or an anti-apoptotic factor, depending on the cell type and context. In this study, we found that DAXX was highly expressed in human ovarian surface epithelial tumors but not in granulosa cell tumors. In cultured ovarian cancer cells, DAXX interacted with promyelocytic leukemia protein (PML) and localized to subnuclear domains (so-called PML nuclear bodies). A role for DAXX in ovarian cancer cell proliferation, metastasis, and radio/chemoresistance was examined. Overexpression of DAXX enhanced multiple ovarian cancer cell lines' proliferation, colony formation, and migration, whereas Daxx depletion by RNA interference had the opposite effects. When transplanted into nude mice, ovarian cancer cells that overexpressed DAXX displayed enhanced tumorigenesis capability in vivo, whereas Daxx depletion inhibited tumor development. Importantly, Daxx induced tumorigenic transformation of normal ovarian surface epithelial cells. Daxx also protected ovarian cancer cells against x-irradiation- and chemotherapy-induced DNA damage by interacting with PML. Taken together, our results suggest that DAXX is a novel ovarian cancer oncogene that promotes ovarian cancer cell proliferation and chemoresistance in ovarian cancer cells. Thus, modulating DAXX-PML nuclear body activity may be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers.

Abstract 5475: Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage.

Abstract Background: Endoglin (ENG, CD105) is a protein markedly overexpressed in tumor-associated endothelial cells, and a target for anti-angiogenic therapy. Recently we have noted it to be overexpressed in chemoresistant ovarian cancer cells as well. Our objective was to evaluate the effects and mechanisms of targeting endoglin specifically in ovarian cancer cells. Methods: Endoglin expression was assessed in multiple ovarian cancer lines by Western blot, immunohistochemistry, and flow cytometry. Anti-endoglin siRNAs were used to downregulate expression in ES2 and HeyA8MDR. In vitro, the effects of endoglin-knockdown individually and with chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation and qPCR array for mediators of DNA damage and repair. In an orthotopic murine model, mice inoculated with ES2 or HeyA8MDR cell lines were administered chitosan-encapsulated anti-ENG siRNA or control siRNA with and without carboplatin. PCNA, γH2AX, and 53BP1 IHC and the TUNEL assay were performed to examine biologic effects of endoglin knockdown. Results: Endoglin is expressed to varying degrees by multiple ovarian cancer cell lines. Expression was on the cell surface, consistent with its recognized role as a cofactor with TGF-beta receptor, in only 5% of cells, with most other cells having cytoplasmic expression. In ES2 and HeyA8MDR cell lines, endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Endoglin downregulation led to a decrease in expression of several DNA repair genes, including NBN, NTHL1, BARD1, H2AFX and SIRT1, and an increase in expression of DDIT3 and PPP1R15A. BARD1-specific downregulation, in turn, led to a significant decrease in BRCA1 expression, likely through ubiqutinylation. In vivo, anti-endoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared to control (35-41% reduction, p<0.05). Combination treatment with anti-Endoglin siRNA and carboplatin was associated with even greater inhibitory effect compared to control (58-62% reduction, p<0.001). Conclusions: Targeting endoglin improves platinum sensitivity of ovarian cancer cells both in vivo and in vitro. Endoglin downregulation induces DNA damage at multiple levels, one of which is through decreased expression of BARD1 and BRCA1. Anti-endoglin therapies should be pursued further as their development would allow dual treatment of both tumor angiogenesis and an aggressive subset of chemoresistant tumor cells. Citation Format: Angela J. Ziebarth, Somaria Nowsheen, Adam D. Steg, Monjri M. Shah, Ashwini A. Katre, Zachary C. Dobbin, Anil K. Sood, Michael G. Conner, Eddy S. Yang, Charles N. Landen. Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2013-5475 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5619: Preclinical development of anti-SLC34A2 antibody drug conjugate as a therapeutic for non-small cell lung and ovarian cancers.

Abstract SLC34A2, also known as type II Na/Pi co-transporters NaPi2b or NaPi3b, is a member of solute carrier protein family SLC34. SLC34A2 is highly expressed on cancers of the lung, ovary and thyroid as well as on normal lung pneumocytes, and is a promising target for antigen-directed therapy. Antibody drug conjugates (ADCs) specify the delivery of a cytotoxic drug to cells expressing an accessible antigenic target. Here we have appended monomethyl auristatin E (MMAE) to an antibody recognizing SLC34A2. Anti-SLC34A2-vc-MMAE inhibits tumor growth in multiple ovarian and non-small cell lung cancer xenograft models that express SLC34A2. Anti-SLC34A2 antibody and its conjugate cross-react with both human and monkey, and toxicity studies in monkeys are conducted to identify both target- and MMAE-related effects. Anti-SLC34A2-vc-MMAE was well tolerated in monkeys at efficacious doses and produced dose-related bone marrow suppression and increased apoptosis and mitotic figures consistent with MMAE-related pharmacology. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue expression. Overall, our preclinical results suggest that the antibody drug conjugate targeting the SLC34A2 may provide an effective new therapy for the treatment of NSCLC and ovarian cancer. Citation Format: Kedan Lin, Crystal Zhang, Eric Harstad, Hajime Hiraragi, Willy Solis, Siao Ping Tsai, Keyang Xu, Maria Hristopoulos, Rayna Venook, Bonnee Rubinfeld, Paul Polakis. Preclinical development of anti-SLC34A2 antibody drug conjugate as a therapeutic for non-small cell lung and ovarian cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5619. doi:10.1158/1538-7445.AM2013-5619

Abstract 4200: Effects of microRNA-100 on cell proliferation and apoptosis is associated with dysregulation of Plk1 in hepatocellular carcinoma cells.

Abstract The microRNAs (miRNAs) are a family of small non-coding RNAs that negatively regulate gene expression by degrading mRNA and by inhibiting translation of protein. Numerous studies have demonstrated the critical roles of miRNA in physiological and pathological procedures, including cell proliferation, differentiation and apoptosis, and in carcinogenesis. Recent studies have particularly reported that miR-100 is aberrantly expressed in multiple cancers, including colorectal, lung, prostate, ovarian, thyroid, pancreatic, liver, breast, cervical and nasopharyngeal cancers. It has also been reported that reduction of miRNA-100 is associated with poor prognosis. But the effects of miRNA-100 on human hepatocellular carcinoma cells and its potential mechanisms are largely unknown. In the present study, we transfected miRNA-100 analogue into human hepatocellular carcinoma cell line HepG2 cell, employed quantitative RT-PCR, immunoblotting, immunofluorescence, flow cytometry and other approaches, and found that the expression of miRNA-100 was markedly lower but polo-like kinase 1 (Plk1) was higher in the HCC cell line and fetal liver, compared to those in the normal hepatocytes and adult liver, respectively. Moreover, increasing miRNA-100 expression in HepG2 cells inhibited cell proliferation, caused cell cycle arrest and promoted apoptosis. Mechanistic and correlation studies revealed that the anti-cancer effects of miRNA-100 were associated with downregulation of Plk1 expression at mRNA and protein levels. Factually, bioinformatic analysis has predicted that Plk1 could be a target of miRNA-100. In conclusion, our results have shown that miRNA-100 has a tumor suppressor function in human hepatocellular carcinoma cells, which may be associated with downregulation of Plk1, and implicate the potential application of miR-100 in connection with Plk1 for targeted gene therapy of hepatocellular carcinoma. Citation Format: Wuling Zhu, Binglin Fan, Hongge zhang, Xiaohui Ji, Wancai Yang, Wancai Yang. Effects of microRNA-100 on cell proliferation and apoptosis is associated with dysregulation of Plk1 in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4200. doi:10.1158/1538-7445.AM2013-4200

Abstract 4290: c-Myc and Ras differentially regulate mitochondrial apoptotic priming and chemosensitivity.

Abstract Successful treatment of most human cancers is dependent on tumor cell sensitivity to chemotherapy yet the basis for chemosensitivity is poorly understood. We have previously shown that a cancer's proximity to the apoptotic threshold, a property we call "apoptotic priming," governs its chemosensitivity in patients with multiple myeloma, ALL, AML and ovarian cancer. The genetic and molecular events that underlie the differential priming in tumors, however, are currently unknown. Utilizing in vitro ovarian, kidney and lung tumorigenesis models we find that oncogenes are powerful modulators of apoptotic priming and are consequently responsible for differential chemosensitivity. For example, forced expression of the c-Myc oncogene increases priming and chemosensitivity of immortalized normal epithelium. In contrast, Ras activation in isogenic cells dramatically decreases both priming and chemosensitivity. Our in vitro findings are validated by clinical observations. Apoptotic priming, an important determinant of patient outcomes, is modulated in contrasting manners by oncogenes. Citation Format: Kristopher A. Sarosiek, Triona Ni Chonghaile, Alison Karst, Luv Patel, Ronny Drapkin, Anthony Letai. c-Myc and Ras differentially regulate mitochondrial apoptotic priming and chemosensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4290. doi:10.1158/1538-7445.AM2013-4290

Abstract 2996: Systematic functional identification 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. We believe this approach can increase an opportunity to identify molecular targets in ovarian cancer therapeutics. Citation Format: Seiichi Mori, Takeshi Fujiwara, Takako Yokomizo, Haruko Iwase, Kazuyoshi Kato, Yukie Nakashima, Tokuichi Kawaguchi, Tetsuo Noda. Systematic functional identification of driver mutations for individual ovarian cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2996. doi:10.1158/1538-7445.AM2013-2996

Abstract 3215: Detecting low abundance therapeutic targets for ovarian cancer using engineered gold nanoparticles.

Abstract Gold nanoparticles (AuNPs) have been widely studied for use in disease therapeutics as targeting and imaging agents, drug delivery vehicles and as self-therapeutics. When AuNPs interact with the biological milieu they form a corona layer, which is predominantly composed of proteins. The outer “soft” layer is dynamic and here proteins are free to exchange over time. The inner “hard” layer on the other hand consists of proteins firmly bound to the NP surface. The Vromans effect predicts that given a limited surface area, low affinity, high abundance proteins, that first attach to the surface, are over time replaced by high affinity, low abundance proteins. Our aim was to enrich low abundance proteins on the AuNP surface to probe for differentially expressed proteins that are not detected by traditional methods of protein identification. We studied the binding of ovarian cell lysates (cancerous and non cancerous) to 10 nm sized positively and negatively charged AuNPs. The formation of corona around the AuNPs was characterized by dynamic light scattering and zeta potential measurements. The composition of the protein corona was identified using mass spectrometry. Proteins were also identified from the cell lysate pool to serve as a control to assess protein enrichment on NP surface. Using this approach, Hepatoma Derived Growth Factor (HDGF) was identified as a low abundance protein that was detected by mass spectrometry in cancer cells only after enrichment on the surface of positively charged AuNPs. HDGF is overexpressed in multiple ovarian cancer cells lines and knock down using siRNA shows decrease in cell proliferation, G2/M arrest and apoptosis. This suggests an important role of HDGF in the tumorigenicity of ovarian cancer, which could serve as an important therapeutic target for the disease. Citation Format: Karuna Giri, Rochelle Arvizo. Detecting low abundance therapeutic targets for ovarian cancer using engineered gold nanoparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3215. doi:10.1158/1538-7445.AM2013-3215

Abstract 3020: The gep protooncogene Gα12 mediates LPA-stimulated activation of CREB in ovarian cancer cells.

Abstract Lysophosphatidic acid (LPA), plays a critical role in the pathophysiology of ovarian cancers. Our previous studies have shown that LPA stimulates the proliferation of ovarian cancer cells through GNA12, the gep protooncogene Gα12. The present study is focused on identifying the Gα12-dependent mechanism through which LPA stimulates the proliferation of ovarian cancer cells. Using ovarian cancer cells in which the expression of Gα12 is silenced, we demonstrate here that Gα12-dependent mitogenic signaling by LPA involves the atypical activation of the transcription factor, cAMP-response element binding protein (CREB). Protein/DNA array analyses using LPA-stimulated Hey8 cells in which the expression of Gα12 was silenced indicated the potent but Gα12-dependent activation of CREB by LPA. After validating the array data with immunoblot analysis using antibodies specific to Ser133-phosphorylated active form of CREB, we demonstrate that the expression of the constitutively activated mutant of Gα12 stimulate the activation of CREB even in the absence of LPA whereas silencing of Gα12 abrogates such LPA-stimulated activation in multiple ovarian cancer cell lines. Furthermore, our results indicate that the robust activation of CREB by LPA is an early event, which can be monitored upon LPA stimulation from 3 minutes onwards. In addition, we establish that LPA/Gα12-dependent activation of CREB involves an atypical cAMP-independent mechanism involving Ras and ERK. More significantly, our findings indicate that the expression of the dominant negative mutant of CREB (S133A) leads to a reduction in LPA-stimulated increase in Cyclin A levels along with an attenuation of LPA-stimulated proliferation of these cells. Thus, these studies unravel a novel Gα12-dependent mechanism involving CREB through which LPA stimulates the proliferation of ovarian cancer cells. Citation Format: Ji Hee Ha, Danny Dhanasekaran, Jeremy Ward, Yoon Mi Yang, Sang Geon Kim, Lakshmi Varadarajalu. The gep protooncogene Gα12 mediates LPA-stimulated activation of CREB in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2013-3020

Abstract 953: Knockdown of kinases that regulate microtubule stability and centrosome function enhance paclitaxel sensitivity in multiple ovarian cancer cell lines.

Abstract Virtually all patients with newly diagnosed ovarian cancer are treated with a combination of paclitaxel and carboplatin. While 70% of ovarian cancers respond to carboplatin, more than half do not respond to the paclitaxel. Inhibition of kinases that modulate primary resistance to paclitaxel, could enhance response to therapy. To identify kinases that regulate sensitivity to paclitaxel, high throughput screening with a kinome wide siRNA library was performed in SKOv3 ovarian cancer cells. Among 45 hits, 14 target proteins (AATK, ACRBP, BMP2K, CHUK, EDN2, IKBKB, ILK, RAPGEF3, RAPGEF4, RFP, SIK2, STK24, STK39 and TBK1) regulated sensitivity to paclitaxel and were expressed or overexpressed in a fraction of ovarian cancers. siRNAs against each of these kinases were tested for the ability to enhance sensitivity to paclitaxel in each of 12 ovarian cancer cell lines that reflected the heterogeneity observed in ovarian cancers including mutations of TP53, BRCA1/2, KRAS, BRAF, PI3K and PTEN. Knockdown of 7 individual genes reduced the IC50 of paclitaxel in more than half of the cell lines by 25 to 80 percent. Knockdown of STK39 and NFKB pathway kinases IKBKB and TBK1 enhanced microtubule stability judged by microtubule fractionation assay. Other kinases are known to affect centrosome function (SIK2, ACRBP). Our studies have identified several proteins that regulate primary sensitivity to paclitaxel in ovarian cancer cells and that could serve as targets to enhance response to primary therapy. Citation Format: Hailing Yang, Xiaoyan Wang, Ahmed A. Ahmed, Zhen Lu, Robert C. Bast. Knockdown of kinases that regulate microtubule stability and centrosome function enhance paclitaxel sensitivity in multiple ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 953. doi:10.1158/1538-7445.AM2013-953

Network-based Survival Analysis Reveals Subnetwork Signatures for Predicting Outcomes of Ovarian Cancer Treatment

Cox regression is commonly used to predict the outcome by the time to an event of interest and in addition, identify relevant features for survival analysis in cancer genomics. Due to the high-dimensionality of high-throughput genomic data, existing Cox models trained on any particular dataset usually generalize poorly to other independent datasets. In this paper, we propose a network-based Cox regression model called Net-Cox and applied Net-Cox for a large-scale survival analysis across multiple ovarian cancer datasets. Net-Cox integrates gene network information into the Cox's proportional hazard model to explore the co-expression or functional relation among high-dimensional gene expression features in the gene network. Net-Cox was applied to analyze three independent gene expression datasets including the TCGA ovarian cancer dataset and two other public ovarian cancer datasets. Net-Cox with the network information from gene co-expression or functional relations identified highly consistent signature genes across the three datasets, and because of the better generalization across the datasets, Net-Cox also consistently improved the accuracy of survival prediction over the Cox models regularized by or . This study focused on analyzing the death and recurrence outcomes in the treatment of ovarian carcinoma to identify signature genes that can more reliably predict the events. The signature genes comprise dense protein-protein interaction subnetworks, enriched by extracellular matrix receptors and modulators or by nuclear signaling components downstream of extracellular signal-regulated kinases. In the laboratory validation of the signature genes, a tumor array experiment by protein staining on an independent patient cohort from Mayo Clinic showed that the protein expression of the signature gene FBN1 is a biomarker significantly associated with the early recurrence after 12 months of the treatment in the ovarian cancer patients who are initially sensitive to chemotherapy. Net-Cox toolbox is available at http://compbio.cs.umn.edu/Net-Cox/.

Endoglin (CD105) Contributes to Platinum Resistance and Is A Target for Tumor-Specific Therapy in Epithelial Ovarian Cancer

Endoglin (CD105) is a membranous protein overexpressed in tumor-associated endothelial cells, chemoresistant populations of ovarian cancer cells, and potentially stem cells. Our objective was to evaluate the effects and mechanisms of targeting endoglin in ovarian cancer. Global and membranous endoglin expression was evaluated in multiple ovarian cancer lines. In vitro, the effects of siRNA-mediated endoglin knockdown with and without chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation, and quantitative PCR. In an orthotopic mouse model, endoglin was targeted with chitosan-encapsulated siRNA with and without carboplatin. Endoglin expression was surprisingly predominantly cytoplasmic, with a small population of surface-positive cells. Endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Targeting endoglin downregulates expression of numerous DNA repair genes, including BARD1, H2AFX, NBN, NTHL1, and SIRT1. BARD1 was also associated with platinum resistance, and was induced by platinum exposure. In vivo, antiendoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared with control (35%-41% reduction, P < 0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared with control (58%-62% reduction, P < 0.001). Endoglin downregulation promotes apoptosis, induces significant DNA damage through modulation of numerous DNA repair genes, and improves platinum sensitivity both in vivo and in vitro. Antiendoglin therapy would allow dual treatment of both tumor angiogenesis and a subset of aggressive tumor cells expressing endoglin and is being actively pursued as therapy in ovarian cancer.

Abstract LB-441: Mutation in SLX4 gen in a multiple case breast cancer family

Abstract Fanconi Anemia (FA) is a genetically heterogeneous, rare autosomal recessive disorder characterized by development abnormalities, bone marrow failure, and childhood cancers. Over the past few years, a common genetic basis for FA and breast and/or ovarian cancer susceptibility has been uncovered. In particular, BRCA2 (FANCD1), BRIP1 (also known as BACH1 and FANCJ), PALB2 (FANCN), and RAD51C germ-line mutations have been found to lead both to FA (when biallelic) and to increased breast and/or ovarian cancer risk (when monoallelic). Recently, two studies demonstrated that biallelic germ-line mutations in SLX4, which encodes an endonuclease, cause a previously unknown FA subtype (FA-P). Therefore, SLX4 should be considered a bona fide candidate gene for breast cancer susceptibility, as suggested in both studies. We have screened 347 non-BRCA1/BRCA2 index cases from multiple case breast and/or ovarian cancer families for point mutations in the SLX4 gene. The whole coding sequence and flanking intronic regions were analyzed using High Resolution Melting (HRM) analysis followed by direct sequencing of abnormal melting curve samples. An SLX4 missense change in c.1114C&amp;gt;T (p.Arg372Trp) was detected in a family with four cases of breast cancer in two generations. This mutation segregates with disease within the family and is not present in the dbSNP (build 34). Polyphen-2 and SIFT in silico analysis tools predict a deleterious effect on protein structure and multiple alignment within orthologous proteins suggests a high degree of conservation. Previous studies in FA patients have identified two different splicing mutations (c.1163+2T&amp;gt;A and c.1163+3dupT) that cause in frame exon 5 skipping. The resulting protein (p.Arg317_Phe387del) lacks two repeated UBZ4 domains known to interact with other endonucleases, mismatch repair proteins, and ubiquitin. It has been suggested that UBZ4 may be involved in targeting SLX4 to sites of DNA damage by binding to ubiquitinated proteins. The missense change we have found introduces a highly hydrophobic amino acid within the same region, which could affect the folding of the UBZ4 domain or disrupt a specific protein-protein interaction. Our study supports a role for SLX4 in breast cancer susceptibility. Further studies performed in larger cohorts will be necessary to determine the prevalence of SLX4 mutations among breast cancer risk factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-441. doi:1538-7445.AM2012-LB-441

Abstract 4367: Contribution of malignant thrombocytosis to ovarian cancer proliferation and resistance to chemotherapy-induced-apoptosis through Platelet Derived Growth Factor Receptor-Alpha (PDGFRA) signaling

Abstract Background: Thrombocytosis at the time of ovarian cancer diagnosis is correlated with decreased interval to progression and decreased overall survival, but the mechanisms for these outcomes are poorly understood. We considered the possible role of Platelet-Derived Growth Factor (PDGF) signaling through PDGFRA as a mechanism for platelet-driven increases in tumor cell growth and progression. Methods: In vitro (co-culture experiments with multiple ovarian cancer cell lines and platelets) and in vivo (orthotopic mouse model of ovarian cancer) experiments were performed to assess the functional and biological effects of platelets on ovarian cancer with regard to invasion, proliferation, and apoptotic response to chemotherapy. In vitro assays queried whether PDGFRA may be implicated in the observed effects. Results: Co-culture assays of ovarian cancer cells with and without platelets showed increased ovarian cancer cell migration (p&amp;lt;0.05 for all cell lines) and proliferation (p&amp;lt;0.05 for all cell lines) in the presence of platelets. Co-culture assays including docetaxel showed ovarian cancer cells had a decreased apoptotic response in the presence of platelets (p&amp;lt;0.05 for all lines). Proliferative and anti-apoptotic effects did not require direct platelet-to-cell contact. Blocking PDGFRA using a monoclonal antibody and/or siRNA reversed, in part, the protective effects that platelets had in decreasing docetaxel-mediated apoptosis (p&amp;lt;0.05). In an orthotopic mouse model of ovarian cancer, platelet transfusions resulted in increased mean tumor weight (4.1g v 2.1g, p=0.03) and increased mean number of tumor nodules (62 v 20, p=0.004). These effects were abrogated by platelet pre-treatment with aspirin and by mouse treatment with aspirin. Immunohistochemistry of tumor specimens showed increased proliferation in the platelet transfused-mice compared to control (82.5% v 66.3%, p&amp;lt;0.001) and decreased apoptosis in transfused-mice compared to control (37.5% decrease, p=0.009). CD31 suggested greater blood vessel maturation in platelet-transfused mice; the ratio of lumen-containing to total vessels was greater in the platelet transfused mice (1.23 v 0.679, p=0.03). Immunofluorescence studies demonstrated platelet extravasation into tumor, confirming the potential for direct platelet-tumor cell interaction. Conclusions: Adverse cancer outcomes correlated to thrombocytosis are partially explained by PDGF-mediated taxane resistance in vitro. We confirmed in vivo that thrombocytosis contributes to increased aggressiveness of malignancy, suggesting that the clinical and mechanistic implications of malignant thrombocytosis warrant further study in order to identify clinically relevant biomarkers and therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4367. doi:1538-7445.AM2012-4367

Spanish registry and screening program for families at high risk of pancreatic adenocarcinoma.

192 Background: 5-10% of pancreatic cancer (PC) cases show familial aggregation. 20% of these correspond to syndromes: Peutz Jeghers, hereditary pancreatitis, familial multiple melanoma, breast and ovarian cancer (HBOC) and others. Familial pancreatic cancer (FPC) is defined as: ≥2 first degree relatives with PC and with no other known syndromes. FPC seems to have autosomal dominant inheritance, but the genetic cause is unknown. Methods: The objectives are: 1) To develop the first FPC Spanish Registry, connecting different groups interested in this disease 2) To study inheritance, phenotypic and molecular characteristics of the FPC, and families with early PC 3) To establish a strategy for early detection of PC in high-risk individuals and to implement it 4) To characterize preneoplastic lesions and diagnosed PC by monitoring high-risk individuals. 16 Spanish hospitals are participating. This study has two components: 1) Cohort to identify families with FPC and hereditary PC. Sources for the families are the PanGen-ES Study, a case-control study of PC which identifies families through a questionnaire on family history of cancer, and Genetic Counseling Units, 2) Cohort of high-risk families. The latter will be followed up by endoscopic ultrasound (EUS) and CT ± abdominal magnetic resonance imaging. In addition, circulating tumor cells (CTC) in peripheral blood will be determined. Results: The assessment of family history of the 421 cases included in the PanGen Study has identified 32 (7.6%) families with FPC and 52 patients with PC ≤ 50 years (12.4%). In addition, the 190 families presenting PC aggregation with other neoplasms are being further evaluated. At this time we have obtained clinical data and blood samples to carry out molecular studies of 23 individuals: 17 belonging to 3 families with FPC, and 6 members of 2 families with an HBOC with some cases of pancreatic cancer. 18 relatives at risk began a follow-up with EUS and CT, with no detection of any suspicious pancreatic lesion; also CTC have not been detected. Conclusions: This initiative will permit to know more about FPC and will serve to evaluate protocols and PC markers in screening the high-risk population, and promote connections with other international groups.

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad-) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad- cells. Similarly, proliferation of the 3+Ecad- cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3-Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad- subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad- cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.

Metformin and phenethyl isothiocyanate combined treatment in vitro is cytotoxic to ovarian cancer cultures.

BackgroundHigh mortality rates in ovarian cancer are largely a result of resistance to currently used chemotherapies. Expanding therapies with a variety of drugs has the potential to reduce this high mortality rate. Metformin and phenethyl isothiocyanate (PEITC) are both potentially useful in ovarian cancer, and they are particularly attractive because of their safety.MethodsCell proliferation of each drug and drug combination was evaluated by hemacytometry with Trypan blue exclusion or Sytox green staining for cell death. Levels of total and cleaved PARP were measured by Western blot. General cellular and mitochondrial reactive oxygen species were measured by flow cytometry and live cell confocal microscopy with the fluorescent dyes dihydroethidine and MitoSOX.ResultsIndividually, metformin and PEITC each show inhibition of cell growth in multiple ovarian cancer cell lines. Alone, PEITC was also able to induce apoptosis, whereas metformin was primarily growth inhibitory. Both total cellular and mitochondrial reactive oxygen species were increased when treated with either metformin or PEITC. The growth inhibitory effects of metformin were reversed by methyl succinate supplementation, suggesting complex I plays a role in metformin's anti-cancer mechanism. PEITC's anti-cancer effect was reversed by N-acetyl-cysteine supplementation, suggesting PEITC relies on reactive oxygen species generation to induce apoptosis. Metformin and PEITC together showed a synergistic effect on ovarian cancer cell lines, including the cisplatin resistant A2780cis.ConclusionsHere we show that when used in combination, these drugs are effective in both slowing cancer cell growth and killing ovarian cancer cells in vitro. Furthermore, the combination of these drugs remains effective in cisplatin resistant cell lines. Novel combinations such as metformin and PEITC show promise in expanding ovarian cancer therapies and overcoming the high incidence of cisplatin resistant cancers.