Doublecortin-like kinase 1 (DCLK1), is a tumor stem cell-specific marker, expressed in many cancers and highly correlated to cancer initiation, EMT and progression. We recently reported DCLK1's epigenetic dysregulation and specific overexpression in clear cell renal carcinoma (RCC). In this study, we assessed DCLK1's value as a therapeutic target in RCC progression.
Materials and Methods
RCC cell line Caki-2 was infected with Lentivirus to overexpress DCLK1. Moreover, DCLK1 gene expression was knocked down in ACHN and Caki-2 cell lines using small interfering RNA. Gene and protein expression levels were measured by real time RT-PCR and Western blotting respectively. Proliferation and drug resistance was determined by MTT assay. Cell cycle analysis and sorting of DCLK1+/DCLK1- cells were completed by FACS. Clonogenicity was evaluated using colony formation assay in matrigel.
Overexpression of DCLK1 increases Caki-2 cell proliferation, but despite DCLK1's microtubule localization, there was no evidence of altered cell cycle status. Aldehyde dehydrogenase (ALDH), one of only a few known stem cell markers in RCC, was highly upregulated (>5-fold) by DCLK1 overexpression and downregulated (2-fold) by knockdown of DCLK1 in RCC cells. FACS results also indicated the existence of small ALDH and DCLK1 double-positive populations in both Caki-2 (1.51%) and ACHN (0.54%) RCC cells. Furthermore, there was approximately 7% more ALDH+ cells in DCLK1 overexpressing Caki-2 cells compared to control. In further support of molecular hallmarks, we observed increased expression of HIF-1α and Vimentin mRNA and protein as well as enhanced clonogenic capacity of ACHN and Caki-2 RCC cell lines in matrigel at baseline and after 72 h treatment with 0.5 μM Sunitinib. Immunocytochemistry of the spheroids suggested that DCLK1 enhances colony formation ability by triggering expression of pluripotency factors NANOG and POU5F1/OCT4. Since DCLK1 is known to have an extracellular domain we performed FACS sorting to determine if we could isolate a viable DCLK1+ population from the ACHN cell line. ACHN-DCLK1+ cells had >1.5 fold higher relative colony number and larger sphere size in three-dimensional clonogenic assay compared to ACHN-DCLK1- cells. Finally, we performed MTT assays demonstrating that overexpression of DCLK1 caused Caki-2 cells to become resistant to Sunitinib, Everolimus, and Temsirolimus compared to controls after 48 h treatment. Converesely, knockdown of DCLK1 sensitized both ACHN and Caki-2 cells to Sunitinib and Sorafenib after 48 hours treatment.
DCLK1 modulates proliferation, stemness, tumorigenesis and resistance to FDA-approved drugs, and marks a population of stem-like cells in RCC. These findings suggest that targeting DCLK1 may be a novel primary or adjuvant therapeutic strategy in RCC.
Citation Format: Yang Ge, Nathaniel Weygant, Dongfeng Qu, Randal May, William L. Berry, Parthasarathy Chandrakesan, James J. Tomasek, Guangyu An, Courtney W. Houchen. Doublecortin-like kinase 1 marks cancer stem-like cells and modulates drug-resistance, self-renewal, and tumorigenesis in renal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3340.