Abstract 1940: Short (S) isoform of cancer-stem-cell marker, DCLK1, is critically required to maintain proliferative/tumorigenic potential of colon cancer cells: identification of associated molecular pathways

DCLK1 (doublecortin-like-kinase-1) was recently identified as a specific marker for intestinal cancer-stem-cells (CSC) (Sureban et al, 2013; Nakanishi et al, 2013). Importantly, DCLK1 expression is significantly increased during colon-carcinogenesis (Gagliardi et al, 2012). We recently reported that 2 isoforms of DCLK1 (DCLK1-L/DCLK1-S) are transcribed by two different promoters (5′ and intron V), and that the L isoform (∼80KDa) is silenced by DNA methylation in human colon-adenocarcinomas, while the S-isoform (∼47KDa) is up-regulated by many fold in tumors; normal colonic cells mainly express the L-isoform (∼80KDa). Loss of DCLK1 expression in cancer cells/polyps was reported to result in loss of proliferation/polyp formation (Sureban et al 2011; Nakanishi et al, 2013). RNAi methods used so far, target both isoforms of DCLK1. In the current studies we used shRNA to specifically target S-isoform, in order to delineate biological role of cancer-specific S-isoform. Isogenic clones of colon cancer cells, expressing control shRNA (C-clones) or DCLK1-shRNA (KO clones), were generated. Western Blot analysis and RT-PCR analysis confirmed 80-90% knockdown compared to C-clones. Proliferation and Colonogenic/tumorigenic potential of KO clones was completely attenuated compared to C-clones, in vitro and in vivo. Our results confirmed that DCLK1-S is required for maintaining proliferative/tumorigenic potential of colon CSCs. Surprisingly, KO clones did not form spheroids in non-adherent cultures, while the C-clones formed spheroids within 4-6 days of seeding, suggesting DCLK1 may maintain stemness of colon cancer cells. To evaluate molecular pathways mediating effects of DCLK1-S, isogenic C and KO clones were subjected to next generation sequencing (RNAseq). 12 genes were up-regulated and 14 genes were down-regulated in KO vs C clones by >3fold; several others were changed by >1fold. Key genes involved in mitosis, EMT, cell adhesion, caspase-independent apoptosis, metabolism, and toll-like receptor signaling, were significantly down/up-regulated confirming that the S-isoform plays a critical role in CSC biology, by either directly or indirectly regulating several molecular pathways which likely support CSC phenotype. Conclusion. Our results show DCLK1-S is critically required for maintaining invasive/tumorigenic potential of colon cancer cells by directly or indirectly regulating several molecular pathways involved in maintaining non-differentiated/tumorigenic phenotype of CSCs. Thus DCLK1-S may serve as a diagnostic/prognostic marker and provide a useful cancer-specific target for eradicating colon CSCs. This work was supported by NIH Grants to PS (R01CA09795909 and R01CA09795909-S1) Citation Format: Malaney R. O9Connell, Shubhashish Sarkar, Pomila Singh. Short (S) isoform of cancer-stem-cell marker, DCLK1, is critically required to maintain proliferative/tumorigenic potential of colon cancer cells: identification of associated molecular pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1940. doi:10.1158/1538-7445.AM2014-1940