Abstract 2892: FOXD3 is a novel repressor of the expression of short isoform of DCLK1 (DCLK1-S) from IntronV(β)-promoter of hDCLK1-gene, and is epigenetically silenced in hCRCs: prognostic/diagnostic implications of FOXD3/DCLK1-S expression in hCRCs
Authors: O’Connell Malaney R. , Sarkar Shubhashish , Luthra Gurinder , Okugawa Yoshinaga , Toiyama Yuji , Ward Denise , Goel Ajay , Singh Pomila 
Published: July 2016

Abstract
DCLK1 is a specific marker of colon and pancreatic cancers in mice, and is elevated in human colon adenocarcinomas (hCRCs). Downregulation of DCLK1, results in loss of cancer stem-cell-markers and tumorogenic-potential of human colon cancer cells (hCCCs). We recently reported a novel finding that hCRCs express short-transcripts of DCLK1 (DCLK1-S) from an alternate promoter located within IntronV of DCLK1-gene, while normal human colons express the canonical long transcript (DCLK1-L) from 5’(α)-promoter. We and others have demonstrated that 5’(α)-promoter is hypermethylated in hCRCs, resulting in epigenetic silencing and loss of expression of DCLK1-L in hCRCs. Although 5’(α)-promoter is differentially methylated in normal human colons vs hCRCs, methylation status of IntronV(β)-promoter does not change. We therefore hypothesized that differential expression of DCLK1-S in normal colons vs hCRCs is perhaps due to differences in transcriptional activity of the promoter in normal vs cancer cells. To test our hypothesis, we used several in silico and molecular biology approaches, and report for the first time that FOXD3 is a potent transcriptional inhibitor of the IntronV(β)-promoter, resulting in the absence of DCLK1-S expression in normal human colons. Our results suggest that FOXD3 gene becomes methylated during colon carcinogenesis, causing loss of FOXD3 expression, and results in the expression of DCLK1-S in hCRCs. In order to examine pathophysiological relevance of the loss of FOXD3 and gain of DCLK1-S expression in hCRCs, we measured relative levels of FOXD3/DCLK1-S by qRT-PCR in a cohort of 92 CRC patients, in relation to overall survival and clinicopathological parameters. Patients expressing high-DCLK1-S/low-FOXD3 had significantly worse overall-survival compared to patients expressing low-DCLK1-S/high-FOXD3. High expression of DCLK1-S, in conjunction with low expression of FOXD3, was a stronger independent prognostic factor than expression of high levels of DCLK1-S alone. Conclusions. Based on our studies, we report for the first time that FOXD3 is a potent repressor of IntronV(β)-promoter of hDCLK1-gene in normal cells, and that loss of FOXD3 expression due to hypermethylation and silencing of FOXD3 gene during colon carcinogenesis, results in the expression of DCLK1-S in hCRCs, representing an important biomarker of hCRCs. Our findings also suggest a prognostic/diagnostic value of measuring relative expression levels of DCLK1-S/FOXD3 in tumors of CRC patients. It is speculated that loss of DCLK1-L and FOXD3 expression, associated with increased expression of DCLK1-S can be used as an early diagnostic marker of epigenetic changes, associated with colon carcinogenesis in humans.
Citation Format: Malaney R. O’Connell, Shubhashish Sarkar, Gurinder Luthra, Yoshinaga Okugawa, Yuji Toiyama, Denise Ward, Ajay Goel, Pomila Singh. FOXD3 is a novel repressor of the expression of short isoform of DCLK1 (DCLK1-S) from IntronV(β)-promoter of hDCLK1-gene, and is epigenetically silenced in hCRCs: prognostic/diagnostic implications of FOXD3/DCLK1-S expression in hCRCs. [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 2892.