Abstract
Introduction: Epithelial Ovarian Cancer (EOC) is a leading cause of death among women with gynecologic cancers. Most patients respond to primary treatment with platinum-based therapies, but eventually develop platinum-resistant metastatic disease. Novel treatment strategies to address both metastasis and platinum-resistance are therefore urgently needed. A promising target for such therapy is TWIST1, a basic helix-loop-helix transcription factor which is aberrantly reactivated in many cancers, and which acts as a master regulator of epithelial to mesenchymal transition (EMT), a process by which epithelial cancer cells gain the ability to migrate and metastasize. A growing body of literature suggests EMT as a chemoresistance mechanism, though little research has focused on the role of TWIST1 in chemoresistance, or as potential therapeutic target in EOC.
Purpose: To a. characterize the relationship between TWIST1 and platinum-resistance in EOC, and b. to develop dendrimer delivery of RNA-based TWIST1 inhibitors to prevent metastatic spread and platinum- resistance in EOC.
Methods: TWIST1 expression was evaluated via Western Blot in A2780 and A2780R cells, which are platinum-sensitive and platinum-resistant EOC cell lines, respectively. TWIST1 was knocked down via siRNA against TWIST1 complexed with poly(amidoamine) dendrimers, or using shRNA (without dendrimer). Cellular uptake of siRNA-dendrimer complexes was assayed via fluorescent microscopy of AlexaFluor tagged siRNA. Expression of TWIST1 was quantified by Western blotting and quantitative real-time RT-PCR. To assay chemoresistance, the platinum-resistant EOC cells, A2780R, were treated with logarithmically increasing doses of cisplatin (CDDP), and cytotoxicity assayed by sulforhodamine B staining, and compared with a platinum-sensitive A2870 EOC cells, as well as A2780R knockdown of TWIST1.
Lastly, TWIST1 expression was correlated with EOC progression, in twenty-two EOC patient samples (eighteen Stage III/IV, four Stage I/II, obtained at City of Hope) via immunohistochemistry (IHC) using anti-Twist polyclonal antibody (Glackin 14-2).
Results: TWIST1 was significantly expressed in A2780R cells, with little to no protein expression in A2780 cells. Development of siRNA-dendrimer complexes has resulted in effective, long-term TWIST1 silencing in EOC cells, with peak efficacy up to one week following transfection A2780 cells. TWIST1 knockdown in platinum-resistant EOC cells results in reversal of resistance, with sensitivity approaching that of parental, platinum-sensitive cells. A CDDP dose of 10 μM produced 40% cell death in resistant cells with TWIST1 knockdown, compared to 0% cell death in untreated resistant cells. By comparison, treatment of sensitive cells with the same dose yielded approximately 60% cell death.
TWIST1 expression in human EOC tissues by IHC shows strong nuclear expression in advanced stage III/IV cancers, compared to low nuclear expression in early stage I/II disease. Interestingly, stage I/II cancers displayed TWIST1 expression in stromal cells surrounding the tumor as opposed to strong nuclear expression in advanced stage samples, possibly as a result of an inflammatory response.
Conclusions: TWIST1 is a promising novel therapeutic target in advanced EOC given its role in metastasis and chemoresistance. It is predominantly expressed in advanced stage disease and platinum-resistant EOC cell lines. Long-term silencing of this transcription factor is feasible with dendrimer complexing, and results in significant reversal of platinum resistance in vitro. Preclinical mouse models to confirm these in vitro results will pave the way for clinical applications to silence EMT transcriptional suppressors and potentially reverse chemoresistance and prevent metastasis.
Citation Format: Cai Roberts, Ernest S. Han, Gina Lowe, James Finlay, Sharon Wilczynski, Lucy Ghoda, Carlotta Glackin, Thanh H. Dellinger. The role of TWIST1 in chemoresistance and metastasis in ovarian cancer – potential therapeutic applications. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B38.
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