Abstract 4220: Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis

Abstract

Abstract Introduction: Solid tumor cancers including breast cancer possess a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. These features render these cells as highly desirable albeit elusive therapeutic targets. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we establish the scientific rationale for targeting DCLK1 to inhibit breast cancer proliferation, tumor stem cell growth, self-renewal, EMT, circulating tumor cells (CTCa) and metastasis. Methods: Small interfering RNA (siRNA) against DCLK1 was transfected into breast cancer cell lines (MCF7 and MDAMB231 (triple negative breast cancer cell line)). For analyzing circulating tumor cells MDAMB231 cells are stably transfected with RFP (red fluorescence protein. Cell proliferation, colony formation, mammosphere formation assay (self-renewal), cell migration and invasion assays were assessed to evaluate the effect of DCLK1 knockdown on breast cancer cells self-renewal and metastatic potential. Isograft model was used to identify the role of DCLK1 knockdown on breast cancer growth and circulating tumor cells in vivo. The assays were analyzed utilizing experimental protocols for cell culture, clonogenic culture, circulating tumor cells by gradient centrifugation, immunohistochemistry, real-time PCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal tissue. DCLK1 siRNA inhibited the expression of DCLK1 mRNA and protein in breast cancer cells. Silencing DCLK1 decreased the proliferation (∼40%), colony formation (∼70%), and self-renewal (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to significantly less (40-80%) breast cancer cells migrated and invaded. Further, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were significantly (p<0.05) reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in isograft models resulted in reduced tumor growth (∼81%) and CTCs (∼98%) in vivo. Conclusions: DCLK1 expression is associated with breast cancer cell survival, self-renewal, metastasis and tumorigenesis. Silencing DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis, tumor growth and survival. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Vivian Taylor, William Berry, Randal May, Dongfeng Qu, Meghna Singh, Sripathi Sureban, Naushad Ali, Michael Bronze, Courtney Houchen. Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4220. doi:10.1158/1538-7445.AM2015-4220