β-catenin/Tcf-4 inhibition after progastrin targeting reduces growth and drives differentiation of intestinal tumors

Background & Aims: Aberrant activation of the (beta-catenin/Tcf-4 transcriptional complex represents an initiating event for colorectal carcinogenesis, shifting the balance from differentiation toward proliferation in colonic crypts. Here, we assessed whether endogenous progastrin, encoded by a target gene of this complex, was in turn able to regulate beta-catenin/ Tcf-4 activity in adenomatous polyposis coli (APC)-mutated cells, and we analyzed the impact of topical progastrin depletion on intestinal tumor growth in vivo. Methods: Stable or transient RNA silencing of the GAST gene was induced in human tumor cells and in mice carrying a heterozygous Apc mutation (APC Delta 14), which overexpress progastrin but not amidated or glycine-extended gastrin. Results: Depletion of endogenous progastrin production strongly decreased intestinal tumor growth in vivo through a marked inhibition of constitutive beta-catenin/Tcf-4 activity in tumor cells. This effect was mediated by the de novo expression of the inhibitor of (beta-catenin and Tcf-4 (ICAT), resulting from a down-regulation of integrin-linked kinase in progastrin-depleted cells. Accordingly, ICAT down-regulation was correlated with progastrin overexpression and Tcf-4 target gene activation in human colorectal tumors, and ICAT repression was detected in the colon epithelium of tumor-prone, progastrin-overexpressing mice. In APC Delta 14 mice, small interfering RNA-mediated progastrin depletion not only reduced intestinal tumor size and numbers, but also increased goblet cell lineage differentiation and cell apoptosis in the remaining adenomas. Conclusions: Thus, depletion of endogenous progastrin inhibits the tumorigenicity of APC-mutated colorectal cancer cells in vivo by promoting ICAT expression, thereby counteracting Tcf-4 activity. Progastrin targeting strategies should provide an exciting prospect for the differentiation therapy of colorectal cancer.