Ovarian Cancer Lines Research Articles

Abstract 924: In vitro study of the potential antineoplastic effect of synthetic naphthoquinones in cisplatin-resistant ovarian cancer lineage

Abstract Background: Cancer development has been associated with alterations in polyamine biosynthesis and metabolism, which induce cell proliferation, angiogenesis, expression of genes related to tumor invasion and metastasis; whereas inhibit apoptosis. Based on the strong rational to develop novel polyamine depleting molecules, and adding the strategy to have substances that can control cancer through different cellular pathways aiming to bypass the acquisition of drug resistant phenotype by cancer cells; this work aimed to screen, in an ovarian cancer (OVCA) line, 40 novel rationally developed potential anti-cancer compounds, following rapid, high efficient, and low cost synthetic methodologies, then confirmed by spectroscopic techniques. OVCA is the most lethal gynecological malignancy, with high rates of chemoresistance and disease relapse; therefore, supporting the urge to generate novel anti-OVCA agents. Methods: Novel naphthoquinone-derived compounds were rationally designed to act through multiple cellular pathways aiming the avoidance of drug resistant phenotype acquisition by cancer cells, and were synthesized by rapid, efficient and low cost synthetic method. Drugs antineoplastic efficacy (AE) was accessed in OVCAR3, through the evaluation of cellular metabolic viability (CMV) (MTT method). Drugs structures are protected by patent. Cells were cultured in RPMI media supplemented with 10% (v/v) FBS, antibiotics and antifungics, in 5% CO2, until subconfluence; then, 1.5x105 cells/well were subcultured for 72h prior to treatment with drugs in different concentrations (10−4, 10−5, 10−6, 10−7, and 10−8 M). After 24h, CMV was assessed. Experiments in which the lineage was treated with cisplatin, doxorubicin or paclitaxel were run in parallel. The mean and standard-deviation of the absorbancies were used to calculate CMV and drugs IC50 (PrismaGraphPad version 5.1). Findings: We have screened the AE of 40 novel naphtoquinone-derived drugs in OVCAR3; five have decreased its CMV by, at least, 70%, namely: M8 (IC50 1.64x10−5M; CMV decrease of 90%); M10 (IC50 1.37x10−5M; CMV decrease of 96%); M14 (IC50 1.45x10−5M; CMV decrease of 85%); PIC10 (IC50 9.13x10−6M; CMV decrease of 95%); PIC20 (IC50 5.31x10−5M; CMV decrease of 70%). The IC50 for cisplatin, the gold therapy against OVCA was 3.87x10−5M and CMV decrease was 85%. Interpretation: We herein present novel drugs to treat OVCA; whereas PIC 10 is more potent that cisplatin, M8, M10, M14, PIC10 and PIC20 seem to have similar or higher antineoplastic efficacy in treating cisplatin-resistant OVCA. We strongly believe that the present pre-clinical research project is innovative, as it introduces novel anti-OVCA drugs, economically viable and socially important, as it might bring hope to put OVCA treatment in a perspective in which the disease control is a real possibility. 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 924. doi:1538-7445.AM2012-924

Abstract 3620: A multiplex quantitative immunofluorescence assay for DNA damage repair in response to cytotoxic treatment

Abstract The use of DNA damaging agents remains one of the most common approaches to cancer therapy. The type of damage caused by the agent will determine which of the five main DNA damage repair (DDR) processes will respond. Initially we are developing multiplex quantitative immunoflourescence assays (qIFAs) for biomarkers of active Homologous Recombination (HR) and Nucleotide Excision Repair (NER) pathway and plan to develop mutiplex assays for the other repair pathways in the near future. The first multiplex panel to quantify DDR response has been developed to include Nbs1 phosphorylated at Serine343 and γH2AX, both early markers of DNA double strand breaks as well as ERCC1, which plays a key role in NER. Quantitation of repair pathway activation after drug treatment builds upon our validated qIFA for γH2AX, which scores individual nuclei as positive or negative for the biomarker and quantifies the γH2AX response as the fraction of nuclear area that is occupied by γH2AX foci. In contrast, the multiplex qIFA was designed to quantify the global DNA repair protein response to a variety of cytotoxic, genotoxic and targeted agents administered singly and in combination. Initial evaluations were performed in vitro on a panel of cell lines, characterized for repair pathway defects: the BRCA1-null human ovarian cancer line UWB1.289 (ATCC# CRL-2945), its isogenic line with restored BRCA1 named UWB1.289+BRCA1 (ATCC# CRL-2946), and SKOV3 (BRCA1 WT). Both gemcitabine and cisplatin increased pNbs1 both in UWB1.289 and UWB1.289+BRCA1, but not in SKOV3. Co-expression of pNbs1 with γH2AX was observed but was not 100% concordant. Combination treatment with a topoisomerase 1 inhibitor and PARP1/2 inhibitor (the indenoisoquinoline NSC 724998 with ABT-888) increased nuclear ERCC1 only in UWB1.289, but not in the isogenic UWB1.289+BRCA1 line, or in the ovarian lines SKOV3 and IGROV1, or the human fibroblast line GM00637. Assessment of drug-induced DDR response in a panel of cell lines with known repair defects is planned with the expectation that this will provide a scientific foundation for interpreting qIFA results when the assay is used to analyze clinical biopsies, and will assist in the evaluation of combination therapy strategies, such as synthetic lethality, designed to take advantage of DNA repair defects. Funded by NCI Contract No. HHSN261200800001E. 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 3620. doi:1538-7445.AM2012-3620

Abstract LB-346: EGF receptor, Jaks and Stat3 crosstalk promote enhanced colony-forming, motility, migration, and invasive phenotype of cisplatin-resistant ovarian cancer cells

Abstract Majority of the ovarian cancer patients who initially respond to cisplatin therapy develop recurrent and resistant disease that is mechanistically not well understood. To define the biological presentations and the molecular phenotype of cisplatin resistant ovarian cancer, we subjected the sensitive A2780S ovarian cancer line to multiple rounds of cisplatin treatments, followed by recovery and derived the lines, A2780S/CP1 (S/CP1), A2780S/CP3 (S/CP3), and A2780S/CP5 (S/CP5), which are resistant to 1, 3, and 5 μM cisplatin, respectively. In vitro studies show the three resistant lines have increased colony-forming ability and altered morphology consistent with enhanced motility, migration, and invasiveness. The malignant phenotype progresses with increasing resistance and is associated with hyperactive epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase 1/2 and Janus kinases (Jaks), and aberrant Signal Transducer and Activator of Transcription (Stat) 3 activation. Elevated expression of survivin and vascular endothelial growth factor (VEGF), and enhanced matrix metalloproteinase activities are evident in the resistant cells, together with increased vimentin and snail expression, and E-cadherin downregulation, indicative of epithelial-mesenchymal transition (EMT). The inhibition of EGFR or Stat3 activity in the resistant cells repressed the enhanced colony-forming, motility, and migratory phenotype, downregulated survivin, VEGF and vimentin expression, and sensitized the resistant cells to cisplatin. In vivo studies using intra-peritoneal xenograft models in nude mice revealed the resistant lines have a high tumor incidence and formed small, cisplatin-resistant tumor nodules at several locations on the small-intestine and colon, compared to the sensitive A2780S line that exhibited low tumor incidence and formed exclusively ovarian tumors. Treatment with EGFR or Stat3 inhibitor sensitized the resistant tumor nodules to cisplatin. Studies together indicate hyperactive EGFR and Jak signaling predominantly through the induction of aberrantly-active Stat3 promote EMT during cisplatin resistance development. Collectively, these events serve as critical mediators of the invasive phenotype associated with cisplatin resistant ovarian cancer and hence, are targets for preventing the development of resistance and the recurrent disease during cisplatin therapy in ovarian cancer. 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-346. doi:1538-7445.AM2012-LB-346

Abstract LB-149: The OPCML tumor suppressor binds and negatively regulates the Axl receptor tyrosine kinase, sensitizing cells to foretinib in epithelial ovarian cancer (EOC)

Abstract OPCML (opioid binding protein/cell adhesion like molecule) is a tumour suppressor gene that is silenced almost by somatic methylation in more than 80% of EOC cases, with loss of heterozygosity (LOH) occurring in 49% of cases [1,2]. OPCML binds a specific repertoire of receptor tyrosine kinases (RTKs) consisting of HER2, HER4 EphA2, FGFR1 and FGFR3. OPCML switches RTKs from clathrin to non clathrin dependent endocytosis, resulting in polyubiquitination of the RTK and targeting to the proteasome for destruction with associated signalling and growth inhibition [3]. We show that Axl, a TAM family RTK that is frequently activated in ovarian cancer [4], also interacts with and is downregulated by OPCML. Axl is downregulated following transient and stable OPCML expression in ovarian cancer lines SKOV3 and PEO1, with reciprocally increased Axl expression following stable knockdown of physiological OPCML in normal ovarian surface epithelial line OSE-C2. We performed co-IP and mammalian 2-hybrid (M 2-H) analysis between Axl and OPCML and determined that the Ig and FNIII domains are crucial to this interaction. We previously reported a complete loss of function proline 95 to arginine mutation [2]. Transfection of OPCMLWT protein in OPCML deficient cells promotes intercellular adhesion and adhesion to Collagen I and Fibronectin matrix, in association with Axl downregulation, In contrast OPCMLP95R transfection did not repress Axl and did not promote intercellular or cell to matrix adhesion. M 2-H data demonstrated that OPCMLP95R expression abolishes the interaction seen between OPCMLWT and Axl. We explored the impact of OPCML expression on cell signalling response to Foretinib (GSK1363089), a potent inhibitor of Axl [5]; OPCML led to a 3-fold sensitization to the tyrosine kinase inhibitor. The finding that OPCML, a frequently inactivated ovarian cancer tumor suppressor negatively regulates Axl, a frequently activated RTK in ovarian cancer and sensitises Axl positive OPCML deficient cells to Foretinib defines a promising predictive and therapeutic role for OPCML in ovarian cancer.

Protein profiles in human ovarian cancer cell lines correspond to their metabolic activity and to metabolic profiles of respective tumor xenografts

Many solid tumors show a large variability in glycolytic activity and lactate accumulation, which has been correlated with different metastatic spread, radioresistance and patient survival. To investigate potential differences in protein profiles underlying these metabolic variances, the highly glycolytic human ovarian cancer cell line OC316 was investigated and compared with the less glycolytic line IGROV-1. Extracellular acidification and oxygen consumption were analyzed with an extracellular flux analyzer. Glycolysis-associated proteins, including specific membrane transporters, were quantified through in-cell western analyses. Metabolic properties of corresponding tumor xenografts were assessed via induced metabolic bioluminescence imaging. Extracellular flux analyses revealed elevated bioenergetics of OC316 cells. Hexokinase II, pyruvate kinase, pyruvate dehydrogenase E1 beta subunit and pyruvate dehydrogenase kinase 1, as well as the glucose transporter 1 and the monocarboxylate transporter 4, were overexpressed in these cells compared with IGROV-1. When generating tumor xenografts in SCID mice, cells maintained their glycolytic behavior, i.e. OC316 showed higher lactate concentrations than IGROV-1 tumors. In summary, a congruency between protein profiles and metabolic properties has been demonstrated in the human ovarian cancer lines investigated. Also, a perpetuation of glycolytic characteristics during the transition from in vitro to the in vivo situation has been documented. This model system could be useful for systematic studies on therapeutic intervention by manipulation of tumor glycolysis and associated pathways.

Synergistic cytotoxic effect of sulindac and pyrrolidine dithiocarbamate against ovarian cancer cells

Sulindac, a non-steroidal anti-inflammatory drug, suppresses carcinogenesis and inhibits growth of tumor cells. Pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, has been also identified as a potential anti-neoplastic agent. We hypothesized that combination of sulindac and PDTC could result in augmentation of cytotoxicity against ovarian cancer cells. The effect of sulindac and PDTC was examined on several ovarian cancer lines. Tumor cell viability was assessed using the MTT assay. Annexin-V/PI staining was used to detect apoptosis, cell cycle distribution was analyzed in FACS, and expression of cellular proteins was detected by Western blotting. Incubation of OVA-14, OVP-10 and CAOV-1 ovarian cancer cells with sulindac and PDTC resulted in significantly greater inhibition of cell viability compared to either compound alone. In a model of OVA-14 cells it was evident that this effect was not related to the expression of COX enzymes since both active (sulindac sulfide) and inactive (sulindac) in vitro compounds affected the growth of tumor cells to a similar extent and synergized in cytotoxicity with PDTC. Combination of sulindac and PDTC lead to G0 arrest and massive apoptosis in co-treated cultures. Western blotting analysis argued for induction of the mitochondrial apoptotic pathway. These data demonstrate the synergistic cytotoxic effect of sulindac and PDTC on ovarian cancer cells through apoptosis and cell cycle arrest and prompt to test the efficacy of this combination in animal models.

Hyperactive EGF receptor, Jaks and Stat3 signaling promote enhanced colony-forming ability, motility and migration of cisplatin-resistant ovarian cancer cells

We present evidence that the cisplatin-resistant human ovarian cancer lines, A2780S/CP1 (S/CP1), A2780S/CP3 (S/CP3), and A2780S/CP5 (S/CP5), derived by subjecting the sensitive A2780S ovarian cancer line to multiple rounds of cisplatin treatments followed by recovery and are resistant to 1, 3, and 5 μM cisplatin, respectively, have increased colony-forming ability and altered morphology that is consistent with enhanced motility, migration, and invasiveness in vitro. The malignant phenotype progresses with increasing resistance and is associated with hyperactive epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (Erk)1/2 and Janus kinases (Jaks), aberrant Signal Transducer and Activator of Transcription (Stat) 3 activation promoted by EGFR and Jaks, and epithelial-mesenchymal transition (EMT) in vitro. Survivin and FLIP anti-apoptotic factors, vascular endothelial growth factor (VEGF), and matrix metalloproteinase activities are also elevated in the resistant cells. Accordingly, the ectopic expression of constitutively-active Stat3C in the sensitive A2780S cells diminished cisplatin sensitivity. The inhibition of EGFR or Stat3 activity repressed Survivin, VEGF and Vimentin expression and the colony-forming potential, viability, motility, and migration of the resistant cells, and sensitized them to cisplatin. Analysis of human ovarian cancer patients’ tumor tissues shows aberrantly-active EGFR and Stat3 that in certain cases correlate with Vimentin over-expression. Intra-peritoneal mouse xenograft studies revealed, compared to the sensitive A2780S line that had low tumor incidence restricted to the ovary, a high tumor incidence for the resistant S/CP3 and S/CP5 lines that formed tumor nodules at several locations on the small-intestine and colon, and which responded poorly to cisplatin, but were sensitive to concurrent treatment with cisplatin and EGFR or Stat3 inhibitor. Hyperactive EGFR signaling through Stat3 and the Jak-Stat3 activity together promote ovarian cancer progression to cisplatin resistance and therefore represent targets for preventing the development of cisplatin resistance and the recurrent disease during cisplatin therapy in ovarian cancer.

Abstract 1532: Small-molecule Stat3 inhibitor sensitizes Cisplatin-resistant ovarian cancer cells to Cisplatin-induced apoptosis

Abstract Ovarian Cancer causes the highest number of gynecologic neoplasm related deaths in the United States. The disease and its progression to drug resistance remain poorly understood. While the aberrant activation of the Signal Transducer and Activator of Transcription (Stat) 3 protein is prevalent in ovarian cancer and is detected in chemotherapy resistance, its exact role in the development and maintenance of the drug resistance phenotype remains poorly understood. We sought to investigate the molecular mechanisms by which constitutive Stat3 activity may support Cisplatin resistance in ovarian cancer. Compared to the sensitive A2780S ovarian cancer line, which does not harbor aberrant Stat3 activity, the SKOV3 line harboring constitutively-active Stat3 was partially sensitive to Cisplatin and showed evidence of moderate apoptosis in response to Cisplatin treatment. Compared to the sensitive A2780S cells, the partly-resistant, SKOV3 cells over-express c-Myc, Survivin, and Bcl-xL, and the XIAP anti-apoptotic factor, as well as VEGF pro-angiogenic factor and the matrix metalloproteinases, MMP-2 and MMP-9. The inhibition of constitutively-active Stat3 in SKOV3 cells by the small-molecule inhibitor, S3I-201 suppressed the expression of the anti-apoptotic and the pro-angiogenic factors, and induced moderate degree of apoptosis. Significantly, treatment with S3I-201 enhanced the sensitivity of SKOV3 cells to Cisplatin. Furthermore, the combined treatment of SKOV3 cells with Cisplatin and S3I-201 strongly inhibited colony formation and cell migration in vitro. Our study together identifies potential mechanisms by which aberrant Stat3 activity might support Cisplatin resistance in ovarian cancer, raising the potential that the combination therapy with Stat3 inhibitors would be effective therapeutic and chemo-sensitizing modalities for ovarian cancer and the recurrent disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1532. doi:10.1158/1538-7445.AM2011-1532

Abstract 2708: Functional characterization of a fluorescent highly tumorigenic ovarian cancer line to test cellular therapy in experimental models

Abstract Introduction: Because current therapies for ovarian cancer have little impact on the long-term survival, there is a compelling need to develop innovative strategies. Our aim was to functionally characterize a fluorescent highly tumorigenic ovarian cancer line to test cellular therapy in combination with cytokines or chemotherapies in experimental models. Methods: Two fluorescent highly tumorigenic sub-lines (SKOV3-AF1 and SKOV3-AF2) were derived from the SKOV-3 ovarian cancer cell line. Peripheral blood mononuclear cells (PBMC)-mediated cytotoxicity in combination with IL-2 and IFNα-2b of SKOV3-AF1 and -AF2 was assayed by lactate dehydrogenase release. Sensitivity of SKOV3-AF2 cells to polyethylene glycol (PEG)-IFNα-2b or IL-2 was assayed in a xenograph nude mouse model. Histopathology was performed on tumors harvested from mice to determine necrosis and tumor infiltrating lymphocytes in the solid tumors. Results: SKOV3-AF1 and -AF2 sub-lines exhibit increased cytotoxicity mediated by PBMC, IL-2, and IFNα-2b compared to parental SKOV-3 cells. The average cytotoxicity was 59% for both SKOV3-AF1 and -AF2 compared to 28% for SKOV-3 parental cells (p<0.05). SKOV3-AF1 and -AF2 cells are more tumorigenic in vivo compared to SKOV-3 cells as indicated by tumor incidence (SKOV3 66%, SKOV3-AF1 100%, SKOV3-AF2 100%), time to sacrifice, tumor weight, and ascitic fluid production. In addition, the SKOV3-AF2 cells continue to fluoresce when harvested from the mice. SKOV3-AF2 tumor growth was inhibited by PEG-IFNα-2b but not low-dose IL-2. The average tumor weight was 0.96 g in the control treatment group that did not receive PEG-IFNα-2b versus 0.23 g for PEG-IFNα-2b (12,000 U per week); p < 0.05). There was no significant difference in any of the IL-2 treatment groups (0-4,000 U, thrice weekly). Both PEG-IFNα-2b and IL-2 were well tolerated by the mice as demonstrated by no significant weight loss during the treatment. Histopathology revealed that the tumors consisted of poorly differentiated surface epithelial carcinoma and there was no significant difference in the percent necrosis or TIL score in any of the treatment groups. Conclusions: Characterization of SKOV3-AF2 cells revealed that these cells are more susceptible to PBMC-mediated cytotoxicity than SKOV-3 cells, are highly tumorigenic in a xenograph model, and continue to fluoresce when harvested from mice. Our data suggests that the SKOV3-AF2 sub-line will be a useful tool to test cellular therapy for the treatment of ovarian cancer utilizing experimental models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2708. doi:10.1158/1538-7445.AM2011-2708

Functional Characterization of a Fluorescent Highly Tumorigenic Ovarian Cancer Line to Test Cellular Therapy in Experimental Models

The objective of this study was to functionally characterize a fluorescent highly tumorigenic ovarian cancer line to test cellular therapy in combination with cytokines or chemotherapies in experimental models. A fluorescent highly tumorigenic subline (SKOV3-AF2) was derived from the SKOV3 ovarian cancer cell line. Peripheral blood mononuclear cell (PBMC)-mediated cytotoxicity of SKOV3-AF2 in the presence of interleukin 2 (IL-2) and interferon α-2b (IFNα-2b) was assayed by lactate dehydrogenase release. Sensitivity of SKOV3-AF2 cells to polyethylene glycol-IFNα-2b and IL-2 was assayed in a xenograph nude mouse model. Histopathology was performed to determine necrosis and tumor-infiltrating lymphocytes in the solid tumors. Reverse transcriptase-polymerase chain reaction was used for gene expression analyses of E-cadherin and cysteine-rich 61 (CCN1). The SKOV3-AF2 subline exhibits increased cytotoxicity (up to 70%), mediated by PBMCs, IL-2, and IFNα-2b, compared with parental SKOV3-red fluorescent protein (RFP) cells. SKOV3-AF2 cells are more tumorigenic in vivo as indicated by tumor incidence, time to sacrifice, tumor weight, and ascitic fluid production. SKOV3-AF2 tumor growth was inhibited by polyethylene glycol-IFNα-2b but not low-dose IL-2. Histopathology revealed that the tumors consisted of poorly differentiated surface epithelial carcinoma. SKOV3-RFP, and -AF2 cell lines as well as -AF2 tumors expressed E-cadherin. SKOV3-AF2 derived tumors expressed CCN1; however, the SKOV3-RFP and SKOV3-AF2 cell lines did not. Characterization of SKOV3-AF2 cells revealed that it is more susceptible to PBMC-mediated cytotoxicity than SKOV3-RFP and highly tumorigenic in a xenograph model, and AF-2 tumors express genes that promote aggressive behavior. Collectively, our data suggest that the SKOV3-AF2 subline will be a useful tool to test cellular therapy for the treatment of ovarian cancer utilizing experimental models.

A randomized phase II study of paclitaxel/carboplatin with or without sorafenib in the first-line treatment of patients with stage III/IV epithelial ovarian cancer.

TPS257 Background: The majority of patients diagnosed with ovarian cancer have advanced disease at the time of diagnosis, and are incurable with surgical therapy. Systemic chemotherapy, following initial cytoreductive surgery, has markedly improved the treatment of patients with advanced ovarian cancer. Standard first-line combination chemotherapy includes a taxane and a platinum agent. Further improvement in the first-line treatment of ovarian cancer will likely depend on the successful incorporation of novel, targeted agents into therapy. Several angiogenesis inhibitors have shown activity against refractory ovarian cancer. Sorafenib efficiently blocks the VEGF receptor and demonstrates antitumor activity in murine xenografts using a human ovarian cancer line (SKOV3). This randomized phase II study compares the efficacy and toxicity of a standard paclitaxel/carboplatin regimen, either alone or with the addition of sorafenib, in the first-line treatment of patients with stage III/IV ovarian cancer following cytoreductive surgery. Randomization of 120 patients will allow detection of an improvement in 2 yr PFS from 45% to 67% (α=.10). Methods: Eligibility: All patients must have previously untreated, histologically confirmed, stage III or IV epithelial ovarian carcinoma, with no residual tumor nodule > 3 cm, no involvement of the bowel (i.e. invasion into bowel wall) and no intestinal obstruction after surgical cytoreduction. Trial Design: Following recovery from cytoreductive surgery patients are randomized (1:1) to receive treatment with paclitaxel/carboplatin ± sorafenib. Regimen A: paclitaxel 175mg/m2, 1-3 hr infusion, day 1; carboplatin AUC=6.0 IV day 1; sorafenib 400 mg bid orally, days 1-21. Regimen B: paclitaxel/carboplatin (same doses as Regimen A). Cycles are repeated every 21 days. Patients are reevaluated every 6 weeks; responding/stable patients continue treatment for 6 cycles. After 6 cycles, responding patients on Regimen B continue single agent sorafenib to complete a 12 month course. Current Enrollment: 55 of 120 patients are currently enrolled. No significant financial relationships to disclose.

Abstract 1683: ATF4 down-regulation potentiates L-asparaginase activity in ovarian cancer cell lines

Abstract The enzyme-drug L-asparaginase (L-ASP) has been used for four decades to treat acute lymphoblastic leukemia. However, its unique mechanism of action is still poorly understood, and its clinical efficacy has proven unpredictable. Those problems have prompted a continuing search for biomarkers that predict L-ASP response. We previously found that the expression of asparagine synthetase (ASNS) is strongly correlated with L-ASP activity in leukemia cell lines of the NCI-60 cell line panel. Unexpectedly, however, the correlation was also apparent in ovarian cancer lines, suggesting that L-ASP might be effective against a low-ASNS subset of ovarian cancers if salient characteristics of the cell lines reflect clinical ovarian tumors. Using siRNA to down-regulate ASNS by 5-fold produced a proportional ∼5-fold potentiation of L-ASP activity in some ovarian cancer lines (OVCAR-3 and OVCAR-4). Surprisingly, ASNS siRNAs yielded a striking >500-fold potentiation in other ovarian lines (OVCAR-8 and OVCAR-8/ADR). Taken together, those findings suggest that the L-ASP/ASNS correlation is causal but also support the hypothesis that L-ASP activity is modulated by additional molecules or pathways. To address that hypothesis and to further elucidate the L-ASP mechanism of action, we have initiated genome-wide siRNA library screening. Preliminary screens in the OVCAR-8 cell line using a 418-gene apoptosis library, a custom 392-gene metabolic library, and the MTS assay as an endpoint yielded Z′ factors >0.90, indicating a robust screen. RNAi-mediated down-regulation of apoptosis genes had little or no effect on L-ASP's anticancer activity. That was surprising because L-ASP has been reported to cause apoptosis through glutamine depletion. Knock-down of ATF4, a transcription factor involved in cell metabolism, did markedly potentiate L-ASP activity (>500-fold). Furthermore, ATF4 knock-down markedly enhanced L-ASP anticancer activity toward the highly resistant cell line, OVCAR-4. These findings suggest that ATF4 is an important mediator of L-ASP resistance and provide rationale for investigation of ATF4 as a predictive biomarker of L-ASP effect. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1683.

Abstract 1948: Pvt1-encoded microRNA miR-1204 in tumorigenesis of human ovarian and breast cancer

Abstract Amplification at chromosome 8q24 is one of the most frequent genomic abnormalities in human cancers and is associated with reduced survival duration in ovarian and breast cancers. Recently, microRNAs (miRNAs) at 8q24 have been identified in the PVT1 locus. Herein, we explored the biological functions of one miRNA, miR-1204, residing within the exon1B of PVT1. Mature hsa-mir-1204 is over-expressed in PVT1 amplified ovarian and breast cancer cell lines (HEY, OVCAR8, SUM-159PT and MDA-MB231). In vitro treatment with antagomir against hsa-mir-1204 inhibited cell proliferation and induced apoptosis in the four PVT1 amplified ovarian and breast cancer lines. Conversely, over-expression of miR-1204 promoted cell growth in PVT1 non-amplified ovarian and breast cancer lines (OV90 and MCF10A) and cooperated with c-Myc to potentiate Rat1A cell colony growth in soft agar assays. Since an inverse expression pattern is often present between miRNA and its target genes, we used computational approaches (miRanda, TargetScan, Pita) to identify distinct miR-1204 candidate target genes from two independent expression microarray datasets, one with significantly down-regulated genes by PVT1 copy number gain in 147 breast tumors and the other involving genes significantly repressed by miR-1204 overexpression and derepressed by silencing of miR-1204 in ovarian and breast cancer cells. Thirty putative target genes of miR-1204 were empirically verified by Q-RT-PCR method, and this group of genes was enriched with genes regulating cell death, cell growth and proliferation, cell cycle, cellular assembly and organization. Combined, these data suggest that human miR-1204 contributes to ovarian and breast tumor proliferation and survival and down-regulates distinct targets including multiple tumor suppressor genes and serves as a potential therapeutic target against tumors with 8q24 amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1948.

Abstract 591: Host cell DNA damage and inflammation responses determine oncolytic adenovirus efficacy in ovarian cancer

Abstract Introduction. Oncolytic adenoviruses show promise as a cancer treatment, by replicating selectively within malignant cells. Viral replication requires multiple cell cycle checkpoints to be overcome. Ovarian cancer sensitivity to viral cytotoxicity varies greatly, even between cells with similar infectivity and intracellular virion production. Viruses also generate acute inflammatory responses with production of cytokines, especially TNF-α. We investigated the role of cellular DNA damage in efficacy of adenovirus dl922-947 in ovarian cancer and also whether inhibition of TNF-α augments activity. Methods. Host DNA damage and repair pathways were assessed in ovarian cancer lines, TOV21G, IGROV1 and A2780CP, which cover three-log range of virus sensitivity. The ATR-chk1 pathway was inhibited using siRNA and UCN-01 treatment. In addition, inflammatory cytokine responses to dl922-947 were assessed in vitro and in xenograft models. TNF-α was inhibited using shRNA and monoclonal antibodies. Results. Oncolytic adenovirus cytotoxicity is associated with extensive genomic DNA damage. Following infection, sensitive, but not resistant, ovarian cancer cells activate cdc25a and over-replicate genomic DNA, creating extensive double-strand breaks. Inhibition of ATR-chk1 in resistant cells augments cdc25a activity and increases genomic DNA over-replication. This enhances cytotoxicity without increasing E1A expression or viral replication. In addition, dl922-947 induces transcription of TNF-α, IL-6 and IL-8 in ovarian cancer cells and xenograft-bearing mice prior to any viral gene expression and host NF-κB activation. Both RNAi-mediated TNF-α knockdown and anti-TNF-α monoclonal antibody treatment increase virus cytotoxicity in vitro and in IGROV1 intraperitoneal xenografts. The increased efficacy results from suppression of cIAP1 transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. Conclusions. A phase I trial of dl922-947 in women with relapsed ovarian cancer is planned for 2011. Host cell DNA damage is a key determinant of oncolytic adenoviral activity, whilst TNF-α acts as a survival factor in infected cells. Inhibiting both repair of DNA damage and TNF-α could improve anti-tumour activity in future clinical trials. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 591.

Abstract LB-320: Inhibition of TGFß1 improves the therapeutic efficacy of non-immunologic tumor cell based vaccine

Abstract Background and objectives of the study: Many cancers, including ovarian cancer, over-express transforming growth factor-beta (TGF-ß), which helps them evade immune control through a variety of mechanisms. We investigated whether the efficacy of tumor cells to induce an immune response, when used intact or as part of a dendritic cell (DC)-based vaccine can be enhanced by inhibiting their production of TGFß1 via anti-sense. Methodology: The TGFß1 expression by two ovarian cancer cell lines was silenced by transfection with a non-replicating lentivirus shRNA. Autologous PBMC were co-cultivated, in the presense of IL-2, with Mitomycin-C (MMC) treated cancer cells or mature dendritic DC that had been pulsed with tumor lysates. The in vitro sensitized lymphoid cells were evaluated by ELISPOT for production of IFN-γ and by flow cytometry. Preliminary data: Quanitative ELISA showed that TGFß1 production in the two ovarian cancer lines was signficantly inhibited by transfection with shRNA. In vitro sensitization of autologous PBMC with either tumor cells where the TGFß1 gene had been silenced or with DC that had been pulsed with homogenates of tumor cells with a silencd TGFß1 gene induced a stronger IFNγ release and increased the frequency of NK cells and DC while it decreased the population of Treg cells. We are presently investigating the anti-tumor response in a mouse melanoma model after vaccination with murine bone-marrow derived DC which have benn pulsed with TGFß silenced tumor lysates. Conclusions: The production, by many tumors, of TGFß1 interferes with their ability to induce an anti-tumor response. This is true both when intact tumor cells and DC pulsed with tumor homogenate are used as immunogen. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-320.

Abstract 1187: The role of PI3K as a biomarker

Abstract The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been implicated in tumorigenesis. Evidence over the past years suggests a pivotal role for the catalytic subunit, PIK3CA, in human cancers. Receptor tyrosine kinases (RTKs) on the cell surface are key regulators of PI3K activity, and RTK antagonists have demonstrated significant anticancer effects in animal models and in patients. However, among tumor harboring activating mutations of PIK3CA, the therapeutic value of RTK antagonists may be compromised. The mutation status of PIK3CA may, therefore, serve as a potential biomarker for both prognosis and response to RTK inhibitors. Somatic mutations of the pik3ca gene occur primarily in three hot spots, E542K, E545K (exon 9) and H1047R (exon 20). These mutations are detected in a broad spectrum of cancer indications including breast, colorectal and lung. This study was designed to explore the nature of PIK3CA mutations among numerous human cancer cell lines, utilizing mostly cells with the capacity to form tumors in mice. Primers corresponding to exon 9 and 20 of human pik3ca gene were designed. Genomic DNA was harvested from over 200 lines, derived from different human cancer indications. The pik3ca locus was sequenced and its WT or mutation status was determined. Of these cells, 8% presented mutations equally distributed between exon 9 and 20. The frequency of PIK3CA mutations was particularly high among colon cancer lines, with fewer incidences among lung, breast and ovarian cancer lines. Interestingly, 50% of the PIK3CA mutated lines presented a concurrent mutation of either KRAS or BRAF. The capacity of PIK3CA mutation-bearing cell lines to respond to RTK blockers was then evaluated, and the mechanism contributing to such phenomenon was further discussed. These results stress PI3K diagnosis value and its potential role as a valid biomarker for patient susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1187.

Abstract 4288: Targeted therapy against aldehyde dehydrogenase in ovarian cancer

Abstract OBJECTIVE. Aldehyde dehydrogenase-1 (ALDH1) expression characterizes a subpopulation of cells with enhanced tumor initiating and differentiating properties in some cancers. We have examined the association of ALDH1 with chemoresistance and whether downregulation of ALDH1 sensitizes cells to chemotherapy in models of ovarian cancer. METHODS. Microarray profiling was performed on SKOV3ip1 and the taxane-resistant SKOV3TRip2 cell lines. Primary ovarian cancer xenografts with and without cisplatin exposure were examined for selection of ALDH1-positive cells. Small interfering RNA (siRNA) was used to downregulate ALDH1 in vitro, and in vivo by incorporation into neutral DOPC liposomes, for evaluation of chemosensitization in an orthotopic model of ovarian cancer. RESULTS. Microarray analysis found 29 genes upregulated and 18 genes downregulated by more than 10-fold when comparing the taxane-resistant SKOV3TRip2 ovarian cancer line compared to its parental SKOV3ip1 line. Included among these was a 92.7-fold higher ALDH1 signature. Increased expression and activity of ALDH1 was confirmed by Western blot and the ALDEFLUOR assay (58% of cells ALDH1-active). In primary ovarian cancer xenografts in NOD-Scid mice, cisplatin treatment resulted in an increase in ALDH1-positive cells, from a baseline of 1% to 38% with therapy (p<0.001). ALDH1-positive cells were not limited to perivascular, hypoxic, or advancing edge regions of the tumor. SiRNA constructs downregulating expression of ALDH1 were identified, and reduced viability of SKOV3TRip2 cells in vitro by 49% (p<0.001). ALDH1 targeting also reduced the docetaxel IC50 from 178nM to 82nM. In the A2780cp20 cell line (a cisplatin-resistant cell line derived from A2780), ALDH1 siRNA alone reduced growth by just 16%, but sensitized cells to cisplatin with a reduction in IC50 from 5.1 to 2.0μM. In an in vivo orthotopic model of ovarian cancer, we treated mice with control siRNA, ALDH1-siRNA incorporated into DOPC liposomes, chemotherapy, or combined chemo/ALDH1-siRNA-DOPC. ALDH1 alone or docetaxel alone had minimal effect on SKOV3TRip2 tumor growth, but ALDH1-siRNA-DOPC plus docetaxel reduced growth by 89.8% compared to docetaxel/control siRNA (p=0.003). Similarly, in the A2780cp20 model, ALDH1-siRNA-DOPC alone or cisplatin had a nonsignificant reduction, while ALDH1-siRNA-DOPC plus cisplatin reduced tumor growth by 73.4% compared to cisplatin/control siRNA (p=0.013). CONCLUSIONS. ALDH1 expression is associated with taxane and cisplatin chemoresistance in ovarian cancer cell lines. ALDH1 expression can be induced by cisplatin treatment in vivo, and targeting ALDH1 sensitizes resistant cell lines to chemotherapy. This enzyme may be important for identification and targeting the chemoresistant population in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4288.

Differential Susceptibility of Human Cancer Cell Lines to Wild-Type Tanapoxvirus Infection~!2009-09-05~!2010-02-23~!2010-04-08~!

Tanapoxvirus (TPV) is a member of the genus Yatapoxvirus in the family Poxviridae and is endemic to equatorial Africa. This disease is restricted to human and non-human primates, producing a mild febrile illness characterized by a single or more rarely additional pock-like lesions on the extremities. While there are several studies elucidating the replication cycle and host range of TPV, there is currently no standardized investigation comparing the ability of TPV to successfully replicate in a variety of tumor cell lines. This study examined the cytopathic effect and calculated the efficiency of TPV replication in vitro using 14 different human cancer cell lines. TPV replicates efficiently in some human tumor cells, and is restricted in others when measured by viral titer at 7 days post infection. Results described here clearly demonstrate that TPV replication in one glioblastoma cell line (U-373), and one colorectal cancer cell line (HCT-116) is more productive than in owl monkey kidney cells (OMK). Replication in two renal cancer cell lines (ACHN and Caki-1) is also increased when compared to OMK. TPV infection produced the greatest change in cellular morphology in U-373 cells, and to a much lesser degree in the breast cancer cell lines T-47D and MCF-7, and in the ovarian cancer line SK-OV3. Negligible change was noted in glioblastoma line U-87, breast cancer line MDA-MB-435, osteosarcoma line HOS, melanoma line SK-MEL5, colorectal cancer line COLO205, and prostate cancer line PC3. The cell lines least permissive to TPV replication were the glioblastoma (U-87) and melanoma (SK-MEL5) cell lines.

Enhancement of cisplatin sensitivity by NSC109268 in budding yeast and human cancer cells is associated with inhibition of S-phase progression

NSC109268 has been described previously as inhibitor of proteasomal degradation and of phosphatase 2Calpha. In a yeast screen, we isolated NSC109268 as an agent altering sensitivity to DNA-damaging agents. We found that NSC109268 and the related compound NSC109272 enhance cellular sensitivity to cis- and transplatin but reduce sensitivity to nitrogen mustard. We explored if similar effects could be found in human cancer cells and if cell cycle analysis could hint at the underlying molecular mechanism. Haploid yeast cells were treated in suspension with platinum agents and nitrogen mustard alone or in combination with NSC compounds, and survival was measured by colony-formation assays. Sensitivity of ovarian and prostate cancer cells toward these treatments was evaluated using the MTS assay. Cell cycle progression was determined by flow cytometry. The enhancement of cisplatin sensitivity by NSC109268 found in yeast was confirmed in cisplatin-sensitive and cisplatin-resistant human ovarian cancer lines and in prostate cancer cells. In yeast and in human carcinoma cells, a correlation of enhanced sensitivity with delaying S-phase progression was revealed. The known activities of NSC109268 as proteasome or phosphatase inhibitor could explain the phenotype of S-phase delay by assuming a higher initial DNA damage load, inhibition of DNA translesion synthesis or extended checkpoint arrest.

Abstract B174: The effect of histone deacetylase inhibition on BRCA1 expression: A potential novel mechanism for enhancing platinum sensitivity in breast and ovarian cancer

Abstract Introduction: Targeting BRCA1 expression has been shown to sensitize Breast-Cancer 1 (BRCA1)-expressing tumor cells to platinum-based chemotherapies. The improved outcome of (BRCA1)-deficient breast and ovarian cancer is believed to be linked to the impaired ability to repair chemotherapy-induced DNA damage. However, therapeutically relevant agents that target BRCA1 expression have yet to be identified. In this study, we explore the effect of histone deacetylase inhibition (HDACi) on BRCA1 expression and platinum sensitivity in a breast and ovarian cancer cell line model. Experimental procedures: BRCA1 mRNA and protein expression was determined by Q-PCR and Western blot, respectively. The efficacy of the HDACi, M344, to potentiate the cytotoxicity of platinum-based chemotherapeutics was evaluated in a range of breast and ovarian tumor cell lines using the MTT cell viability assay and confirmed by flow cytometry. The effect on DNA damage was measured by immunofluorescence staining and flow cytometry for H2A.X foci, a hallmark for the presence of DNA double strand breaks. Results: Baseline BRCA1 mRNA and protein expression was variable in three breast (MCF7, T47D and HCC1937) and three ovarian (A2780s, A2780cp a cisplatin-resistant variant and OVCAR4) cancer cell lines. Expression of BRCA1 protein decreased in response to the addition of the HDACi, M344, in the four cell lines that express detectable levels of BRCA1 by Western blot analysis. BRCA1 mRNA levels decreased with the addition of this HDACi in all the breast and ovarian cancer lines evaluated. Treatment with M344 also increased the sensitivity of these tumor cells to cisplatin and carboplatin treatment in the cell lines with high BRCA1 expression. Furthermore, expression of BRCA1 protein and mRNA decreased in cells treated with the combination of HDACi and cisplatin. A2780s cells subjected to combination platinum and HDACi treatment demonstrated increased levels of DNA damage as assessed by the presence of phosphorylated H2A.X foci in comparison to either treatment alone. Conclusion: This study supports a novel mechanism of BRCA1 targeting using an HDACi resulting in enhanced platinum sensitivity in breast and ovarian cancer cells. Further studies are needed to determine if a HDACi/platinum combination has potential clinical relevance in a subset of breast and ovarian cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B174.