Background and aims: Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play an important role in intestinal epithelium homeostasis. Insulin receptor substrate 2 (IRS2) is a poorly characterised component in this pathway.
Methods: Using complementary in vitro and in vivo human and murine models, expression (mRNA and protein levels), localisation (immunohistochemistry) and regulation of IRS2 were investigated in the normal intestine and colorectal tumours. In silico analysis of the human IRS2 promoter was performed together with reporter and chromatin immunoprecipitation assays.
Results: Significant IRS2 expression was detected in the intestine, with specific protein localisation in the villus region of the ileum and in the surface epithelium of the colon. In human HT29 and Caco2 cells, IRS2 mRNA levels increased with spontaneous and induced differentiation, together with CDX2 (caudal-related homeobox protein 2), P21 and KLF4 (Krüppel-like factor 4). Adenoviral infection with human CDX2 induced IRS2 expression in APC- (adenomatous polyposis coli) and β-catenin-mutated cells. On the other hand, IRS2 downregulation was observed in differentiated enterocytes after adenoviral infection with short hairpin CDX2 (shCDX2), in the intestine of CDX2 heterozygous mice and in colorectal tumours of ApcMin/+ and patients with familial adenomatous polyposis (FAP). The human IRS2 promoter region presents several CDX2-binding sites where CDX2 immunoprecipitated in vivo. IRS2 reporters were functionally activated via CDX2 and blocked via a dominant-negative CDX2 protein.
Conclusions: Combining gain- and loss-of-function approaches, an intriguing scenario is presented whereby IRS2 is significantly expressed in the apical intestinal compartment and is directly controlled by CDX2 in normal intestine and tumours.
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Funding A Moschetta is funded by a Start Up Grant 2005–1007 of the Italian Association for Cancer Research (AIRC, Milan, Italy), by the European Research Council Starting Independent Grant-IDEAS 2008, by the Italian Ministry of Health and Education (FIRB-RBID08C9N7), by the European Community's Seventh Framework Programme FP7/2007–2013 under Grant Agreement No 202272 (LipidomicNet) and by the University of Bari. A Morgano is a fellow of the G d’Annunzio University Oncology Program. MP is supported by the Rosario Samanin Fund. M-TV is supported by the Association pour la Recherche sur le Cancer, France.
Competing interests None.
See Commentary, p 1179
Ethics approval The Ethics Committees of Consorzio Mario Negri Sud (Santa Maria Imbaro, Chieti, Italy), University “G. D’Annunzio” (Chieti, Italy) and University Louis Pasteur (Strasbourg, France) approved the study protocols for ApcMin/+ mice, patients with FAP and CDX2+/− mice, respectively.
▸ Additional methods, figures and a table are published online only at http://gut.bmj.com/content/vol58/issue9
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