Anti-DCLK1 monoclonal antibody, clone 7I5

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. It arises from loss of intestinal epithelial homeostasis and hyperproliferation of the crypt epithelium. In order to further understand the pathogenesis of CRC it is important to further understand the factors regulating intestinal epithelial proliferation and more specifically, regulation of the intestinal epithelial stem cell compartment. Here, we investigated the role of the RNA binding protein RBM3 in stem cell homeostasis in colorectal cancers. Using a doxycycline (Dox) inducible RBM3 overexpressing cell lines HCT 116 and DLD-1, we measured changes in side population (SP) cells that have high xenobiotic efflux capacity and increased capacity for self-renewal. In both cell lines, RBM3 induction showed significant increases in the percentage of side population cells. Additionally, we observed increases in spheroid formation and in cells expressing DCLK1, LGR5 and CD44(Hi). As theWnt/beta-catenin signaling pathway is important for both physiologic and cancer stem cells, we next investigated the effects of RBM3 overexpression on beta-catenin activity. RBM3 overexpression increased levels of nuclear beta-catenin as well as TCF/LEF transcriptional activity. In addition, there was inactivation of GSK3 beta leading to decreased beta-catenin phosphorylation. Pharmacologic inhibition of GSK3 beta using (20Z, 30E)-6-Bromoindirubin-30-oxime (BIO) also recapitulates the RBM3 induced beta-catenin activity. In conclusion, we see that RNA binding protein RBM3 induces stemness in colorectal cancer cells through a mechanism involving suppression of GSK3 beta activity thereby enhancing beta-catenin signaling. (C) 2015 Wiley Periodicals, Inc.

Introduction "Tuft" cells, also known as brush or caveolated cells, are characteristically fusiform shaped, with a distinct apical "tuft" of microvilli extending into the lumen. Double cortin-like kinase 1 (DCLK1) is a microtubule kinase and is a specific marker of intestinal tuft cells. DCLK1-positive tuft cells have been shown to play a key role in gastrointestinal chemosensation, inflammation, and neurotransmission. DCLK1 and Choline acetyltransferase (ChAT), the enzymes responsible for acetylcholine production, are reported to be coexpressed within the gastrointestinal tract. We designed this study to investigate the hypothesis that DCLK1 gene expression is altered in Hirschsprung's disease (HSCR). Materials and Methods HSCR tissue specimens ( n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus ( n = 6). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was undertaken to quantify DCLK1 gene expression, and immunolabeling of DCLK1-positive tuft cells was visualized using confocal microscopy. Results qRT-PCR analysis revealed significant downregulation of the DCLK1 gene in both aganglionic and ganglionic HSCR specimens compared with controls ( p < 0.05). Confocal microscopy revealed DCLK1-positive tuft cell expression within the colonic mucosa, with a reduction in expression in both aganglionic and ganglionic HSCR colon compared with controls. Conclusion DCLK1 is significantly downregulated in HSCR colon, suggesting a role for tuft cells in cholinergic neurotransmission of the distal colon. The marked decrease in DCLK1 expression within ganglionic specimens highlights the physiologically abnormal nature of this segment in HSCR patients.