Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance

Decreased expression of RNA interference machinery, Dicer and Drosha, is associated with poor outcome in ovarian cancer patients

The clinical and functional significance of RNA interference (RNAi) machinery, Dicer and Drosha, in ovarian cancer is not known and was examined. Dicer and Drosha expression was measured in ovarian cancer cell lines (n=8) and invasive epithelial ovarian cancer specimens (n=111) and correlated with clinical outcome. Validation was performed with previously published cohorts of ovarian, breast, and lung cancer patients. Anti-Galectin-3 siRNA and shRNA transfections were used for in vitro functional studies. Dicer and Drosha mRNA and protein levels were decreased in 37% to 63% of ovarian cancer cell lines and in 60% and 51% of human ovarian cancer specimens, respectively. Low Dicer was significantly associated with advanced tumor stage (p=0.007), and low Drosha with suboptimal surgical cytoreduction (p=0.02). Tumors with both high Dicer and Drosha were associated with increased median patient survival (>11 years vs. 2.66 years for other groups; p<0.001). In multivariate analysis, high Dicer (HR=0.48; p=0.02), high-grade histology (HR=2.46; p=0.03), and poor chemoresponse (HR=3.95; p<0.001) were identified as independent predictors of disease-specific survival. Findings of poor clinical outcome with low Dicer expression were validated in separate cohorts of cancer patients. Galectin-3 silencing with siRNA transfection was superior to shRNA in cell lines with low Dicer (78-95% vs. 4-8% compared to non-targeting sequences), and similar in cell lines with high Dicer. Our findings demonstrate the clinical and functional impact of RNAi machinery alterations in ovarian carcinoma and support the use of siRNA constructs that do not require endogenous Dicer and Drosha for therapeutic applications

Tumor-Selective Response to Antibody-Mediated Targeting of αvβ3 Integrin in Ovarian Cancer1

The αvβ3 integrin is expressed on proliferating endothelial cells and some cancer cells, but its expression on ovarian cancer cells and its potential as a therapeutic target are unknown. In this study, expression of the αvβ3 integrin on ovarian cancer cell lines and murine endothelial cells was tested, and the effect of a fully humanized monoclonal antibody against αvβ3, Abegrin (etaracizumab), on cell invasion, viability, tumor growth, and the Akt pathway were examined in vitro and in vivo. We found that etaracizumab recognizes αvβ3 on the ovarian cancer cell lines SKOV3ip1, HeyA8, and A2780ip2 (at low levels) but not on murine endothelial cells. Etaracizumab treatment decreased ovarian cancer proliferation and invasion. In vivo, tumor-bearing mice treated with etaracizumab alone gave variable results. There was no effect on A2780ip2 growth, but a 36% to 49% tumor weight reduction in the SKOV3ip1 and HeyA8 models was found (P < .05). However, combined etaracizumab and paclitaxel was superior to paclitaxel in the SKOV3ip1 and A2780ip2 models (by 51–73%, P < .001) but not in the HeyA8 model. Treatment with etaracizumab was then noted to decrease p-Akt and p-mTOR in SKOV3ip1, but not in HeyA8, which is Akt-independent. Tumors resected after therapy showed that etaracizumab treatment reduced the proliferating cell nuclear antigen index but not microvessel density. This study identifies tumor cell αvβ3 integrin as an attractive target and defines the Akt pathway as a predictor of response to function-blocking antibody

Therapeutic efficacy of a novel focal adhesion kinase inhibitor TAE226 in ovarian carcinoma.

Focal adhesion kinase (FAK) overexpression is frequently found in ovarian and other cancers and is predictive of poor clinical outcome. In the current study, we characterized the biological and therapeutic effects of a novel FAK inhibitor, TAE226. Taxane-sensitive (SKOV3ip1 and HeyA8) and taxane-resistant (HeyA8-MDR) cell lines were used for in vitro and in vivo therapy experiments using TAE226 alone and in combination with docetaxel. Assessment of cytotoxicity, cell proliferation [proliferating cell nuclear antigen (PCNA)], angiogenesis (CD31), and apoptosis (terminal nucleotidyl transferase-mediated nick end labeling) were done by immunohistochemistry and immunofluorescence. In vitro, TAE226 inhibited the phosphorylation of FAK at both Y397 and Y861 sites, inhibited cell growth in a time- and dose-dependent manner, and enhanced docetaxel-mediated growth inhibition by 10- and 20-fold in the taxane-sensitive and taxane-resistant cell lines, respectively. In vivo, FAK inhibition by TAE226 significantly reduced tumor burden in the HeyA8, SKOV3ip1, and HeyA8-MDR models (46-64%) compared with vehicle-treated controls. However, the greatest efficacy was observed with concomitant administration of TAE226 and docetaxel in all three models (85-97% reduction, all P values <0.01). In addition, TAE226 alone and in combination with chemotherapy significantly prolonged survival in tumor-bearing mice. Even in larger tumors, combination therapy with TAE226 and docetaxel resulted in tumor regression. The therapeutic efficacy was related to reduced pericyte coverage, induction of apoptosis of tumor-associated endothelial cells, and reduced microvessel density and tumor cell proliferation. The novel FAK inhibitor, TAE226, offers an attractive therapeutic approach in ovarian carcinoma