The Short Isoform Of Stem-Cell-Marker Dclk1(Dclk1-S) Is Strongly Up-Regulated During Adenoma-Carcinoma Sequence Of Coloncancer Disease: Role Of Autocrine/Endocrine Progastrin

Abstract We now know that colonic tumors increasingly express progastrins (PG) as they progress through hyperproliferative (Hp) / adenoma (Ad) / adenocarcinoma (AdCA) sequence. We and others have recently reported that autocrine PG up-regulates expression of stem-cell-markers DCLK1/CD44/LGR5 (Gastro, 2011; IJC, 2012). Two major isoforms of DCLK1 (DCLK1-S, DCLK1-L) have been reported in neuroprogenitor cells. The two isoforms were discovered to be similarly expressed in colonic epithelial cells. Regulatory effects of PG were examined on the two isoforms in normal/cancer cells. Specific primer sets were designed to amplify S and L transcripts using qRT-PCR. Colon cancer cells either expressed S (HCT-116) or both S and L forms (HT-29, DLD1). HEK293 cells were used as a model of non-transformed cells since they respond to PG, do not express autocrine PG, and are non-tumorogenic. PG significantly up-regulated both the transcripts in HEK293 cells. The L-isoform is transcribed by the 5’ promoter of the gene while S-isoform is transcribed by intron V promoter. Promoter-reporter constructs, specific to the two promoters, were generated to confirm promoter activation in response to PG in HEK293 cells. Colonic tumors obtained from consented patients at the time of colonoscopy, were immunohistochemically analyzed, using an antibody which recognizes both the forms of DCLK1; Hp/Ad/AdCA increasingly expressed DCLK1. Relative expression of S vs L transcripts was examined in stages I-III AdCAs, using a commercial cDNA plate. We learnt for the first time that S-isoform is increasingly up-regulated by 5-20-fold in stages I-III AdCAs, compared to that in surrounding normal tissues; the L-isoform, on the other hand, was only slightly elevated in most tumor samples. Thus even though PG can potentially up-regulate expression of both S and L isoforms, the L-isoform appears to be silenced in many tumors in a stage-dependent manner. This possibility was confirmed in established colon cancer cell lines, including HCT-116. The physiological significance of S vs L isoform, at different stages of colon cancer development, remains unknown. Our studies so far strongly suggest that DCLK1 expression is required for maintaining proliferation of colon cancer cells and that while normal colonic cells/non-transformed HEK293 cells mainly express the L-isoform, colonic tumors increasingly express the S-isoform, suggesting a differential role of these isoforms in the normal vs cancer cell biology. Thus over-expression of DCLK1-S/PG may serve as a robust diagnostic/prognostic marker, and provide a useful target for treating cancer-stem-cells, while sparing normal stem cells (which largely express the L-isoform and do not express PG). This work was supported by NIH Grant to PS (R01CA09795909). Citation Format: Malaney R. O'Connell, Shubhashish Sarkar, Gurinder Luthra, Suimin Qui, Aakash Gajjar, Carrie Maxwell, Pomila Singh. The short isoform of stem-cell-marker DCLK1(DCLK1-S) is strongly up-regulated during adenoma-carcinoma sequence of coloncancer disease: Role of autocrine/endocrine progastrin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2013-4891