Annexin A2 Mediates Up-regulation of NF-κB, β-catenin, and Stem Cell in Response to Progastrin in Mice and HEK-293 Cells

Abstract

Background & Aims Prograstrin induces proliferation in colon crypts by activating p65NF-κ B and β-catenin. We investigated whether Annexin A2 (AnxA2), a progastrin receptor, activates NF-κB and β-catenin in vivo. Method ANXA2-null (ANXA2− /−) and wild-type (ANXA2+/+) mice were studied, along with clones of progastrin-responsive HEK-293 cells that stably expressed full-length progastrin (HEK-mGAS) or an empty-vector (HEK-C). Small interfering RNA was used to downregulate AnxA2, p65NF-κB, and β-catenin in cells. Results Proliferation and activation of p65 and β-catenin increased significantly in HEK-mGAS, compared with HEK-C clones. HEK-mGAS cells had a 2–4-fold increase in relative levels of c-Myc, COX-2, CyclinD1, DCAMKL+1, and CD44, compared with HEK-C clones. Down-regulation of AnxA2 in HEK-mGAS clones reduced activation of NF-κB and β-catenin, as well as levels of DCAMKL+1. Surprisingly, downregulation of β-catenin had no effect on activation of p65NF-κB, whereas down-regulation of p65 significantly reduced activation of β-catenin in HEK-mGAS clones. Loss of either p65 or β-catenin significantly reduced proliferation of HEK-mGAS clones, indicating that both factors are required for the proliferative effects of progastrin. Lengths of colon crypts and levels of p65, β-catenin, DCAMKL+1, and CD44 were significantly higher in ANXA2+/+ mice compared to corresponding values measured in either ANXA2− /− mice injected with progastrin or ANXA2+/+ and ANXA2− /− mice injected with saline. Conclusions AnxA2 expression is required for the biological effects of progastrin in vivo and in vitro, and mediates the stimulatory effect of progastrin on p65NF-κ, β-catenin, and the putative stem-cell markers DCAMKL+1 and CD44. AnxA2 might therefore mediate the hyperproliferative and co-carcinogenic effects of progastrin.

Autocrine Secretion of Progastrin Promotes the Survival and Self-Renewal of Colon Cancer Stem–like Cells

“…Progastrin overexpression and its proliferative and tumor-promoting effects in colorectal cancer have been well-documented (12,42,43) and previous studies linked progastrin production with the expression of CD133, DCAMKL1, or CD44 (17)(18)(19), markers that enrich but are not specific for CSCs (20)(21)(22)(23). Importantly, these studies did not provide functional data demonstrating a role for progastrin in regulating CSCs hallmarks such as selfrenewal.…”

“…We previously demonstrated that progastrin regulates the Wnt and Notch pathways in colorectal cancer (12,14), suggesting that it could affect the phenotype of colon CSCs, which rely on these pathways for their survival (15). Progastrin was shown to promote the proliferation of progenitor cells in the mouse colonic epithelium (16) and a link has been suggested between progastrin expression and populations of cells expressing CD133 (17), DCAMKL1 (18), or CD44 (19) in mouse colonic crypts and human cancer cell lines. Yet, expression of these markers is not restricted to CSCs (20)(21)(22)(23), and the functional role of progastrin on CSC self-renewal and tumor-initiating potential has not been documented.…”

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

“…The Dclk1+ cells grew as organoids in nude mice, suggesting Dclk1+ cells were pluripotent, a hallmark of stem cells (18). We and others reported a significant increase in the number and intensity of Dclk1+cells in hyper proliferating mouse colonic crypts, in response to potent growth factors (19) or colon carcinogenic agents, AOM ± DSS (20), suggesting a role of Dclk1+ cells, at position 4, in hyper proliferation and carcinogenesis of colonic crypt cells. However, it soon became clear that Dclk1 is also expressed in specialized cells, called tuft cells, in the mouse colonic crypts which are enriched in Cox1/Cox2, Villin and α-Tubulin, and believed to be derived from Lgr5+ actively cycling stem/progenitor cells (21).…”

“…PG peptide induces hyper proliferation of colonic crypts and increases colon-carcinogenesis by many fold, in response to AOM±DSS (19,20,(24)(25)(26)(27). Besides, PG is expressed by >80% of human colon cancer cell lines (hCCCs) and CRCs (28,29).…”

“…Experiments with mouse models of tumorogenesis have confirmed a critical role of Dclk1 in tumorigenesis, and as a CSC marker, as described below. It is important to note that overexpression of PG in either mature colonic epithelium (as in Fabp-PG mice) (19,(24)(25)(26) or in intestinal epithelial (IEC) cells (30), results in hyper proliferation of colonic crypts/cells, but does not cause neoplastic transformation (IEC-cells) or formation of colonic tumors (Fabp-PG mice) in the absence of AOM±DSS (19,(24)(25)(26)30). However, overexpression of PG in embryonic epithelial cells, allowed neoplastic transformation of the epithelial cells confirming hyper-sensitivity of multi-potent embryonic stem cells, unlike mature stem cells.…”