T-cell factor 4 functions as a tumor suppressor whose disruption modulates colon cell proliferation and tumorigenesis Melinda L. Angus-Hilla, Kathryn M. Elberta, Julio Hidalgoa, and Mario R. Capecchia,b,1 aDepartment of Human Genetics, and bThe Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112 Contributed by Mario R. Capecchi, February 10, 2011 (sent for review January 5, 2011) The Wnt/β-catenin pathway plays multiple and diverse roles in de- important for the switch from active proliferation to terminal velopment by regulating gene expression via T-cell factor/Lymphoid differentiation. In the colon, Tcf4 protein levels are lowest in the enhancer-binding factor (Tcf/Lef) DNA binding factors. Misregula- proliferative cells at the base of the crypt and higher in differen- tion of this pathway is thought to initiate colon adenoma formation. tiating cells that are migrating toward the lumen (this study and It is controversial whether Tcf4 (Tcf7L2) functions as an oncogene or ref. 15). Similar expression domains are characteristic of other factors implicated in promoting differentiation of the intestinal tumor suppressor gene in colon carcinogenesis. We show here that fi epithelium, including Caudal type homeobox 1 (Cdx1) and Apc Tcf4 haploinsuf ciency results in colon tumor formation in a mouse (16, 17). By contrast, nuclear β-catenin protein levels are in the tumor model that normally only develops small intestinal tumors. inverse pattern, with higher levels in the proliferative compart- Further, we show that loss of Tcf4 early in development and in adult ment at the base of the crypt and decreased levels in differenti- colon results in increased cell proliferation. These findings strongly ating cells, an expression pattern that correlates with cell suggest that Tcf4 normally modulates proliferation of the colonic proliferation (this study and ref. 15). epithelium and that disruption of Tcf4 activity increases prolifera- Taken together, the experimental evidence suggests two alter- tion, leading to colon tumorigenesis. Taken together, our in vivo native models. Tcf4 could be required for progenitor cell pro- studies favor a tumor suppressor function for Tcf4. liferation, and Tcf4 loss would result in a loss of the proliferative cell compartment. Alternatively, Tcf4 could be required for GENETICS colon cancer | mouse model | differentiation | transit amplifying cells | modulating proliferation, and Tcf4 loss would force continued proliferation. To test these models, we have chosen to reexamine stem cells Tcf4 function in the mouse. We have developed and characterized a unique Tcf4 allele that resembles the loss of function mutations he Wnt/β-catenin signaling pathway plays a critical role during found in human CRC. With this system, we have defined a role for Tembryonic development and is often exploited in cancer to Tcf4 function in controlling intestinal cell proliferation throughout promote cell growth. The majority of familial [familial adeno- the gut, both during embryogenesis and in adult colon epithelium. matous polyposis (FAP)] and sporadic colorectal cancers (CRCs) Further, we show that as in human CRC, Tcf4 haploinsufficiency in feature genetic mutations in Adenomatous polyposis coli (APC), combination with Apc mutation strongly enhances colon tumor which encodes a key component of the Wingless-type (Wnt) sig- formation. Taken together, our data support a tumor suppressor naling pathway (reviewed in ref. 1). In the absence of Wnt ligands, function for Tcf4 in colon neoplasia. β-catenin is phosphorylated and targeted for degradation by Apc. Colon tumors form upon homozygous loss of APC, which allows Results β for the accumulation of -catenin in the nucleus where it binds Model of Tcf4 Function. Fig. 1 illustrates a model for the role of and transactivates Tcf/Lef proteins (2, 3). A number of mouse Tcf4 in the maintenance of intestinal epithelium homeostasis models have been developed that contain mutations in Apc, and colon adenoma tumorigenesis. This model is based on the commonly associated with human colon cancer. Consistent with expression pattern of Tcf4 in the intestinal crypt/villus axis, the human FAP, mice carrying a specific mutation in the Apc gene Min role of Tcf4 in the formation of the intestinal epithelium during (Apc ) develop intestinal tumors, but unlike human syndromes, embryogenesis, and studies of the function of Tcf4 in colon ad- mice rarely develop tumors in the colon (4, 5). enoma tumors. The principle feature of the model is that low The Tcf/Lef transcription factors are cell type-specific down- β Tcf4 protein levels favor normal intestinal epithelial and ade- stream effectors of the Wnt/ -catenin signaling pathway. Each noma cell proliferation and thus qualify Tcf4 as a tumor sup- contains a DNA-binding high mobility group (HMG) box, as well β pressor, whereas high levels of Tcf4 protein promote epithelial as an N-terminal -catenin binding domain, and their transcrip- cell differentiation. The reason for presenting the model first is tional activity is dependent upon bound corepressors or coac- that it forms the framework for integrating the results we present tivators. The different Tcf/Lef family members have variant on the role of Tcf4 in normal and pathological intestinal etiology. protein structure outside of the β-catenin binding domain with variable exon composition. This likely contributes to the func- Tcf4Cre Effectively Marks the Tcf4 Lineage in Early Stages of tional diversity and nonredundant functions of Tcf/Lef proteins Development. To trace Tcf4 cell lineage in the mouse and to (reviewed in refs. 6, 7). generate an effective null allele of this locus, the first exon of Much of our understanding about Tcf/Lef function has come from the studies of model organisms. Data from Tcf4 mutant mice show a loss of proliferative cells, suggesting that Tcf4 is important for stem cell renewal in the small intestine and the general as- Author contributions: M.L.A.-H. and M.R.C. designed research; M.L.A.-H., K.M.E., and J.H. sumption that the formation of the Tcf4/β-catenin complex is performed research; M.L.A.-H., K.M.E., and M.R.C. analyzed data; and M.L.A.-H. and cancer-promoting (8). However, the biological function of Tcf4 M.R.C. wrote the paper. has recently undergone renewed interrogation, because it has been The authors declare no conflict of interest. found to be mutated in clear cell renal cell carcinoma (CCRCC), Freely available online through the PNAS open access option. gastric carcinoma, and breast cancer (9–11). Additionally, Tcf loss Data deposition: The data reported in this paper have been deposited in the Gene Ex- of function mutations have been found in primary CRCs and these pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE27522). mutations enhance cell growth in cell lines, suggesting that Tcf4 1To whom correspondence should be addressed. E-mail: [email protected]. may function as a tumor suppressor (12–14). edu. Finding that Tcf4 loss of function mutations result in increased This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cell growth raises the possibility that the level of Tcf4 protein is 1073/pnas.1102300108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1102300108 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Normal Tcf4 Mutant ATcf4 Null B Het Null Increased Tcf4 Tcf4 Tcf4 High Differentiation ApcMin Colon Tcf4 Het Tcf4Het Adenoma Colon Low Differentiation Tcf4 Het Tcf4 Null High Proliferation C Decreased Tcf4 D High Proliferation Tcf4Null Proliferation Tcf4NeoNull Tcf4 Fig. 1. Model of the role of Tcf4 during intestinal development and in tu- Lineage mor formation (in the text). Tcf4 was replaced with an EGFP-Cre recombinase fusion E F (Tcf4Cre; Fig. S1A). Importantly, this exon is also targeted with nonsense and frameshift mutation in human colorectal tumors (12, 13). To allow conditional disruption of Tcf4, we also gen- erated an exon 1 floxed allele (Tcf4Lox; Fig. S1B). The latter Δ allele was used to generate a separate null allele of Tcf4 (Tcf4 ) ChGA by breeding to the HprtCre mouse. Mouse embryos, homozygous Δ Δ for all three mutant alleles (Tcf4Cre/Cre, Tcf4Cre/Lox, and Tcf4 / ) GH show the same mutant phenotype, which is distinct from the published loss of function allele (Tcf4Hyg/Hyg; in the text). Fig. S1C shows Tcf4 lineage in a E14.5 embryo obtained by breeding Tcf4Cre mice with RosamTmG reporter mice, expressing membrane-targeted Tomato fluorescent protein (mTom) before Uea1 Cre-dependent recombination and membrane-targeted green I J fluorescent protein (mGFP) following Cre recombination (18). The figure illustrates the broad cell lineage contribution of Tcf4 during embryonic development, including in the intestinal epi- thelial lining. In newborn Tcf4Cre RosamTmG mice, we observe that intestinal and colonic epithelial cells show Tcf4 lineage (Fig. S1D). In 6-month-old Tcf4Cre RosamTmG mice, Tcf4 lineage is Ki-67 found in many organs (Fig. S2) (19). Fig. 2. Newborn Tcf4Null mice are not viable and show necrotic death of proliferative progenitors and differentiated epithelial cell types in the colon. Loss of Tcf4 Results in Necrosis Throughout the Small Intestine and Null Cre/Cre Cre/Δ Δ/Δ (A) Newborn Tcf4 mice die within 24 h of birth and lack a milk spot (white Colon. At birth, homozygous Tcf4 , Tcf4 , Tcf4 , and arrows). (B) Intestines isolated from Tcf4Null mice have a distended duode- Tcf4Cre/lox mice are not viable, lack milk in their stomachs, Het num (red arrows) and diffuse meconium (black arrows) compared with the ± Het and are on average 21 8% smaller by weight than their Tcf4 intestines of Tcf4 newborn mice. The effects of loss of Tcf4 during in- WT or Tcf4 littermates (Fig. 2A). Further, there is no Tcf4 protein testinal development were analyzed in both Tcf4Het and Tcf4Null mice.