Abstract A42: TWIST1 as a driver of accelerated tumor growth and drug resistance: Potential novel pathways and therapeutics.
Authors: Roberts Cai M. , Tran Michelle A. , Finlay James , Shahin Sophia Allaf , Loeza Joana , Cisneros Leslie , Ye Emily , Dellinger Thanh , Glackin Carlotta A. 
Published: May 2016

Introduction: Epithelial ovarian cancer (EOC) is particularly deadly due to its fast rate of growth, dissemination, and chemoresistant recurrences. Novel therapies are urgently needed to limit metastasis and restore platinum sensitivity. We have investigated the role of TWIST1, a basic helix-loop-helix transcription factor, in these processes. TWIST1 is a well-documented regulator of metastasis from solid tumors, which has been linked to cancer stem cell phenotypes, drug resistance, and poor prognosis in several tumor types. Here we present in vitro and in vivo data supporting TWIST1's role in EOC growth and drug resistance, and propose a therapeutic modality to target TWIST1.

Methodology:

In vitro and in vivo studies: We created two cell lines, derived from the EOC line OVCAR8, that differ only in their TWIST1 expression. The cells were stably transfected with a G418-selectible vector containing either TWIST1 or an shRNA targeting TWIST1, under the control of the CMV viral promoter. Cells from each line were injected IP (intraperitoneally) into NSG mice, allowed to grow for 7 weeks, and tumor burden was evaluated at necropsy. In addition, RNA from each of these OVCAR8 cell lines (TWIST1 overexpressing versus knockdown) was isolated and used for RNA-seq analysis. Raw data was processed using the Galaxy online platform, and differentially expressed genes were further analyzed using Ingenuity Pathway Analysis. To determine the effect of TWIST1 on drug resistance, OVCAR8 cells with and without TWIST1 were treated with varying doses of cisplatin, and survival was quantified using colorimetric cytotoxicity assays.

Therapeutic Application: Since we and others have shown that TWIST1's transactivation/protein binding (WR) domain is required for TWIST1 function, we produced a fusion protein comprised of GFP and the WR domain, and used CoIP to evaluate its ability to competitively inhibit TWIST1 binding in HEK 293 cells.

Results: TWIST1 knockdown resulted in reduced tumor size and overall tumor burden in an NSG mouse model of EOC. Specifically, all mice receiving TWIST1-overexpressing cells developed primary ovarian tumors which were scored +4 on H&E by a veterinary pathologist, whereas only 50% of animals with TWIST1 knockdown developed ovarian tumors, and only 1 of 4 mice had a tumor that scored +4. In addition, fewer metastases were present in mice receiving TWIST knockdown cells, and no mice with TWIST1 knockdown tumors produced ascites. RNA-seq analysis of the TWIST overexpressing versus knockdown cell lines revealed marked differences in genes and pathways responsible for cell migration and cell survival, including upregulation of genes implicated in metastasis, DNA repair, and resistance to apoptosis in TWIST1 overexpressing cells. TWIST1 overexpressing cells also showed less cell death following cisplatin treatment. Finally, in our in vitro inhibitor tests, a GFP-WR domain fusion protein successfully reduced TWIST1 binding to its partner protein NFkB-p65 in HEK 293 cells.

Conclusions: The importance of TWIST1 for tumor growth has been demonstrated for multiple tumor types – including ovarian cancer, as presented here. Our RNA-seq analysis reveals a complex network of TWIST1-mediated factors implicated in cell movement, survival, and resistance to DNA damaging agents, and we are working to determine the most important of these pathways in EOC. Most importantly, we have shown that TWIST1 expression in EOC cells leads to greater tumor burden in a mouse model, and by knocking down TWIST1, we were able to prevent peritoneal metastases and reduce tumor size. Therefore, we believe that TWIST1 is an attractive therapeutic target in EOC, owing to its roles at the crossroads of metastasis, drug resistance, and tumor growth.

Citation Format: Cai M. Roberts, Michelle A. Tran, James Finlay, Sophia Allaf Shahin, Joana Loeza, Leslie Cisneros, Emily Ye, Thanh Dellinger, Carlotta A. Glackin. TWIST1 as a driver of accelerated tumor growth and drug resistance: Potential novel pathways and therapeutics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A42.