Epigenetic changes and alternate promoter usage by human colon cancers for expressing DCLK1-isoforms: Clinical Implications

Abstract

DCLK1 specifically marks colon/pancreatic cancers in mice, and is expressed by human colon adenocarcinomas (hCRCs). Down-regulation of DCLK1 results in loss of cancer-stem-cells (CSCs), and inhibits spheroidal/xenograft growths from hCRC-cells. The 5′-promoter of DCLK1-gene is reportedly hypermethylated in hCRCs, resulting in loss of expression of DCLK1-transcripts, originating from 5′(α)-promoter (termed DCLK1-L, in here). However, in mouse colon-tumors, 5′-promoter of DCLK1-gene remains unchanged, and DCLK1-L, originating from 5′(α)-promoter, is expressed. We hypothesized that elevated levels of DCLK1-protein in hCRC-cells, may be transcribed/translated from an alternate-promoter. Several in silico and molecular biology approaches were used to test our hypothesis. We report for the first time that majority of hCRCs express short-transcripts of DCLK1 (termed DCLK1-S, in here) from an alternate β-promoter in IntronV of the gene, while normal-colons mainly express DCLK1-L from 5′(α)-promoter. We additionally report an important role of β-catenin and TCF4/LEF binding-sites for activating (α)-promoter, while activated NF-κBp65 (bound to NF-κB-cis-element), activates (β)-promoter in cancer-cells. DCLK1-S expression was examined in a cohort of 92 CRC patients; high-expressors had significantly worse overall-survival compared to low-expressors. Our novel findings’ regarding usage of alternate (β)-promoter by hCRCs, suggests that DCLK1-S may represent an important target for preventing/inhibiting colon-cancers, and for eliminating colon-CSCs.

Functional implication of Dclk1 and Dclk1-expressing cells in cancer

“…Thus, O'Connell and colleagues were able to reconcile the apparent contradictory findings of widespread epigenetic silencing of the Dclk1C gene on one hand, and the clear functional importance of the transcript in a broad variety of cancers on the other. 41 Accordingly, Dclk1 expression has been observed in neuroendocrine tumors of the mammary gland and the rectum, 42,43 consistent with the known expression of Dclk1 in neuronal and enteroendocrine cells, but no specific role for Dclk1 in these tumors has been suggested so far. Finally, the relevance of Dclk1 expression in esophageal cancer is presently unclear, 44 but the cells seem to correlate with an increased progression to cancer in a murine model and human patients.…”

Epigenetic regulation of human DCLK-1 gene during colon-carcinogenesis: clinical and mechanistic implications

“…We began examining possible use of an alternate promoter for expression of DCLK1 protein, to resolve the discrepancy between silencing of 5'promoter and presence of DCLK1 in hCRCs/ hCCCs. After extensive literature search and in silico analysis of hDCLK1 gene, we discovered that the previously suggested β promoter (11,45), was located in IntronV of the hDCLK1 gene, and had a canonical TATA box (9). The alternate β promoter was transcriptionally active in 80-90% of hCCCs and transformed (HEKmGAS) cells, but was inactive in non-transformed (HEK293) and normal colonic epithelial cells (9).…”

“…After extensive literature search and in silico analysis of hDCLK1 gene, we discovered that the previously suggested β promoter (11,45), was located in IntronV of the hDCLK1 gene, and had a canonical TATA box (9). The alternate β promoter was transcriptionally active in 80-90% of hCCCs and transformed (HEKmGAS) cells, but was inactive in non-transformed (HEK293) and normal colonic epithelial cells (9). A transcript originating from the alternate β promoter was confirmed to match isoform 2 in the NCBI data base (NM_001195415.1) (9).…”

“…The full length transcript originating from the 5' promoter is labeled as isoform 1 (NM_004734.4) in the NCBI data base. Unique primer sets which amplified transcripts from isoforms 1 or 2 were designed (9), and the expression of full length isoform 1 (~82 KDa) was confirmed in normal colonic mucosal samples of patients, while the shorter isoform 2 (47 KDa) was expressed at variable levels in colonic adenomas and adenocarcinomas from patients, at both RNA and protein levels (9). The use of α/β promoters for expressing L or S isoforms of DCLK1 by normal vs. cancer cells is diagrammatically shown in Figure 1.…”

Expression and prognostic significance of doublecortin‑like kinase 1 in patients with hepatocellular carcinoma

“…The present study identified a positive association between DCLK1 expression and tumor intrahepatic metastasis, while no association was observed between DCLK1 expression and sex, grade, hepatic venous metastasis, portal venous metastasis, bile duct invasion or cirrhosis. Previous studies demonstrated that DCLK1 expression was associated with T stage and lymphatic vessel involvement in colorectal cancer (35). Although DCLK1 expression has been studied in HCC, there is little data on the expression and survival significance of DCLK1 in patients with HCC.…”