Inactivation of Itf2 promotes intestinal tumorigenesis in ApcMin/+ mice

https://doi.org/10.1016/j.bbrc.2015.04.009Get rights and content

Highlights

  • Loss of Itf2 increases small intestinal tumor number in ApcMin/+ mice.

  • Disruption of Itf2 increases tumor size in the small intestine of ApcMin/+ mice.

  • Itf2 acts as a tumor suppressor gene of the intestinal tract in vivo.

Abstract

Deregulation of Wnt/β-catenin signaling following inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene is frequently found in colorectal cancer. We have previously shown that levels of ITF-2B, encoded by the β-catenin target gene ITF2 that is located on the tumor suppressor gene locus 18q21, are increased in colonic adenomas with deregulated β-catenin activity. However, during tumor progression ITF-2B levels are reduced, suggesting that ITF-2B interferes with tumor development. To investigate the role of ITF2 in intestinal tumorigenesis, we specifically inactivated Itf2 in the intestinal epithelium of ApcMin/+ mice. We found that genetic disruption of Itf2 on the ApcMin/+ background results in earlier death and a significant increase in tumor number and size in the small intestine. Based on these data Itf2 acts as a tumor suppressor gene of the intestinal tract that inhibits tumor initiation and growth.

Introduction

Colorectal cancer is a genetic disease that develops from adenomatous precursor lesions by the accumulation of multiple independent somatic alterations in oncogenes and tumor suppressor genes [1]. Inactivating mutations of the APC tumor suppressor gene are viewed as an early event in up to 80% of human sporadic colorectal cancers and have been implicated in the development of hundreds of adenomas mainly in the colon in hereditary familial adenomatous polyposis (FAP) [2], [3], [4]. Loss of APC function leads to deregulation of the Wnt/β-catenin signaling cascade, resulting in stabilization and nuclear translocation of the protein β-catenin (reviewed in Ref. [1]). Subsequently, β-catenin modulates the transcription of its target genes. The gene ITF2 alias TCF4 encoding the basic helix-loop-helix protein (bHLH) and transcription factor ITF-2B has been identified as a β-catenin target gene [5]. Previously, we have demonstrated that deregulated activity of the protein β-catenin induces the transcription of the ITF2 gene in colonic adenomas. However, during tumor progression, ITF-2B protein levels are frequently reduced due to loss of heterozygosity on chromosome 18q as well as deacetylation of the ITF2 promoter, suggesting that loss of ITF2 function is necessary for tumor development [6].

The ApcMin/+ mouse is a widely used mouse model for the study of colorectal carcinogenesis. ApcMin/+ mice carry an inactivating mutation in one allele of the Apc tumor suppressor gene [7]. Somatic loss of the remaining Apc wild-type allele leads to deregulation of the Wnt signaling pathway, resulting in the development of a multitude of tumors throughout the whole intestinal tract [8]. The majority of these neoplastic lesions is distributed to the small intestine and only few occur in the colon [9], [10], [11]. In ApcMin/+ mice all intestinal tumors are benign adenomas. Progression to adenocarcinoma is very rare and may occasionally be observed in older animals. ApcMin/+ mice die as a consequence of secondary effects of tumor growth, mainly intestinal bleeding and obstruction caused by tumors [9], [11].

The aim of the present study was to investigate the role of Itf2 in intestinal tumorigenesis in vivo. Therefore, we inactivated Itf2 specifically in the intestinal epithelium of ApcMin/+ mice and analyzed consequences for tumor formation.

Section snippets

Animals

Itf2fl/fl mice [12] were crossed with vil-Cre+ mice [13]. Itf2fl/fl;vil-Cre+ mice heterozygous for the vil-Cre transgene were bred into the ApcMin/+ background to obtain Itf2fl/fl;ApcMin/+ and Itf2fl/fl;vil-Cre+;ApcMin/+ mice, respectively. In all experiments, mice on the Itf2fl/fl background were used as controls. ApcMin/+ mice were purchased from the Jackson Laboratory. All mouse strains were maintained on a C57BL/6 background.

Mice were inspected on a daily basis and sacrificed when moribund.

Results

To study the effect of inactivation of the Itf2 gene on intestinal tumor formation, we crossed Itf2fl/fl mice with mice expressing Cre recombinase under the control of the intestinal epithelium specific Villin promoter ([7], [12], [13], [16]). Itf2fl/fl;vil-Cre+ (Itf2−/−) mice were viable, fertile and showed no increased morbidity and mortality. Detailed histological and immunohistochemical analysis of small intestine and colon revealed no changes in gut anatomy or in the number and

Discussion

ITF-2B is a widely expressed transcription factor that is involved in differentiation processes [17]. ITF2 expression is up-regulated in primary endometroid ovarian carcinomas due to deregulation of the Wnt/β-catenin signaling cascade and ITF-2B promotes neoplastic transformation of RK3E cells, an adenovirus E1A-immortalized epithelial cell line [5] In the past constitutive and inducible knockout mouse models revealed a crucial role for Itf2 in both B and T lymphocyte development [12], [18] and

Conflict of interest

None.

Acknowledgments

The authors gratefully acknowledge Ingrid Renner-Müller and Petra Renner for help with animal care and Felix Hiltwein for help with necropsy. This work was supported by a grant of the German Research Foundation (KO1826/6-1) to FTK.

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    These authors contributed equally to this work.

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