Abstract POSTER-THER-1426: Characterization and targeting of TWIST family proteins to prevent chemoresistance and metastasis in ovarian cancer

Abstract

Abstract INTRODUCTION/PURPOSE: The vast majority of epithelial ovarian cancer (EOC) patients present with advanced metastatic disease, but will respond well to first line chemotherapy consisting of a platinum drug and/or paclitaxel. Unfortunately, most of these patients will relapse with disease that is both metastatic and drug resistant, leading to a five-year survival rate under 20%. Therefore, novel therapies that can eliminate disseminated and chemoresistant cells are urgently needed. Two potential targets to supplement current treatment are TWIST1 and TWIST2, related basic helix-loop-helix transcription factors vital in development and reactivated in many cancers. TWIST proteins regulate epithelial to mesenchymal transition (EMT), the process underlying metastatic spread. Furthermore, recent studies from multiple groups have correlated mesenchymal characteristics with a cancer stem cell phenotype, and with chemoresistance to a variety of agents. However, transcription factors are difficult to target with small molecule drugs due to their nuclear localization. Therefore, we employ siRNA to target TWIST mRNA, in order to reduce metastasis and re-sensitize cancer cells to conventional chemotherapeutic agents. METHODS: We have designed and validated two siRNAs against TWIST. We have also developed two nanoparticle-based delivery systems for siRNA which would be translatable from the bench to the clinic. The first is YTZ3-15, an amphiphilic generation three poly(amidoamine) (PAMAM) dendrimer, the second, a polyethylenimine (PEI) coated mesoporous silica nanoparticle (MSNP). Several EOC cell lines varying in their TWIST1 and TWIST2 expression were treated siRNA delivered via YTZ3-15 or MSNPs. Fluorescent microscopy was used to evaluate siRNA uptake. Western blotting and qRT-PCR were used to assay TWIST knockdown. Sulphorhodamine B and MTT cytotoxicity assays were used to test chemosensitivity before and after TWIST knockdown. TWIST1 levels in clinical samples were evaluated using IHC. RESULTS: Fluorescent microscopy demonstrated that all tested cell lines efficiently take up PAMAM dendrimers and MSNPs, delivering their siRNA cargo to the cell interior. Tracking of punctate signals over time showed slow release from endosomes into the cytosol. Both YTZ3-15 and MSNP delivery of siRNA resulted in robust TWIST knockdown lasting at least one week post-transfection. Furthermore, TWIST knockdown sensitized cells to chemotherapy compared to cells treated with non-targeting control siRNA. In clinical samples, nuclear TWIST1 staining correlated with tumor stage, with early stage tumors showing cytoplasmic staining, and staining in surrounding cells rather than tumor cells themselves. CONCLUSIONS: TWIST family proteins are promising targets to address the compound problems of metastasis and acquired drug resistance in EOC. Addition of TWIST inhibitors, such as the siRNA-based technologies we have developed, to a traditional treatment regimen has the potential to cure currently intractable disease. These combined therapies will drastically improve survival and provide new hope for patients with recurrent ovarian cancer. Citation Format: Cai Roberts, Gina Lowe, James Finlay, Joana Loeza, Thanh Dellinger, Ernest Han, Jeff Zink, Fuyu Tamanoi, Carlotta Glackin. Characterization and targeting of TWIST family proteins to prevent chemoresistance and metastasis in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1426.