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Driving Intestinal Tumorigenesis

 

By: Carol A. Rouzer, VICB Communications
Published: December 21, 2017

 

New data reveal that a partial deficiency of p120-catenin promotes intestinal tumor formation, whereas total loss of the protein is lethal.

 

A defining characteristic of epithelial cells is the presence of adherens junctions that link adjacent cells to each other. Adherens junctions comprise a complex of proteins centered on the transmembrane protein E-cadherin. p120-Catenin (p120) binds to E-cadherin's cytoplasmic domain, retaining and stabilizing E-cadherin at the plasma membrane. In addition, β-catenin binds to E-cadherin and recruits α-catenin, which links the complex to actin filaments of the cytoskeleton (Figure 1). Considerable evidence suggests a role for E-cadherin as a suppressor of tumorigenesis and metastasis. In fact, mislocalization, downregulation, and/or total loss of all of the components of the adherens junction have been observed in many kinds of cancer. Now, Vanderbilt Institute of Chemical Biology member Albert Reynolds, along with his collaborators, Robert Coffey (Department of Medicine), Nancy Jenkins and Neal Copeland (Methodist Hospital Research Institute, Houston, TX), and Michael Payne (Chulalongkorn University, Bangkok), report new work on the role of p120 as an intestinal tumor suppressor. They reveal that loss of one allele of the gene that codes for p120 (Ctnnd1) is strongly tumor-promoting in the intestine, whereas loss of both alleles is lethal to cancer cells (S. P. Short et al. J. Clin. Invest., 2017, doi:10.1172/JCI77217).

 

 

FIGURE 1. Diagram of an E-cadherin complex in an epithelial cell adherens junction. E-cadherin is a transmembrane protein comprising 5 extracellular cadherin repeats connected to a transmembrane domain and an intracellular domain. p120 binds to E-cadherin just beneath the plasma membrane, helping to stabilize and retain it in the membrane. β-Catenin also binds to E-cadherin and serves to recruit α-catenin which, in turn, provides a link to the actin cytoskeleton. β-Catenin also serves as a transducer in the Wnt signaling pathway. Figure reproduced by permission from Macmillan, Ltd. from T. Lecuit and A. S. Yap (2015) Nat. Cell Biol., 17, 533. Copyright 2015.

 

 

The epithelium of the colon is a constantly regenerating monolayer of cells that covers a lawn of finger-like projections known as villi. Between the villi are invaginations, the crypts, where intestinal stem cells (ISCs) are located. ISCs give rise to progenitor cells that undergo 5 to 6 rounds of division and differentiation while moving upward in the crypt. The cells then migrate outward to the surface of the villus where, over the following 48 hours, they gradually progress upward. When they reach the villus tip, they undergo apoptosis and are shed (Figure 2). This process of division and differentiation is highly dependent on the Wnt signaling system (Figure 3). Notably, β-catenin, a key component of the adherens junction, also plays an important role in this pathway. Under the resting state, nearly all of the β-catenin in the cytosol is associated with adherens junctions. Any free protein is subject to phosphorylation, which targets it for degradation. Binding of Wnt to its receptor, however, blocks β-catenin phosphorylation, allowing it to accumulate and translocate to the nucleus. There, it interacts with the transcription factor TCF4, and the resulting complex stimulates transcription of Wnt target genes, leading to increased cellular proliferation. The importance of correct regulation of the Wnt pathway in colonic epithelium is evident by the fact that the protein adenomatous polyposis coli (APC), a component of the complex that phosphorylates β-catenin in resting cells, is a key tumor suppressor in this tissue. Indeed, deficiency of APC expression is found in approximately 80% of human colorectal cancers. In prior work, the Reynolds lab had created mouse models in which p120 expression could be selectively and only partially knocked out in the intestine. These models suggested that, like E-cadherin and APC, p120 also plays an important tumor suppressor role in that tissue. In their new work, they evaluated the importance of p120 in the context of tumors resulting from mutation or knockout of the gene encoding APC (Apc).

 

 


FIGURE 2. Diagrammatic representation of the intestinal epithelium. The surface of the intestine consists of a lawn of finger-like projections, the villi, separated by deep invaginations, the crypts. Intestinal stem cells (ISCs) are located in the crypts. They differentiate into highly proliferative progenitor cells that undergo 5 to 6 rounds of cell division as they move up the crypt. The cells then progress onto the surface of the villi, where they gradually move from the base to the tip over a period of about 48 hours. Upon reaching the tip, the cells undergo apoptosis and are shed. Figure reproduced by permission from Macmillan, Ltd. from T. Reya and H. Clevers (2005) Nature, 434, 843. Copyright 2005.

 

 

 

FIGURE 3. The Wnt signaling pathway. Under resting conditions, most β-catenin is found in adherens junctions that join epithelial cells together. Free β-catenin is phosphorylated by a complex containing Apc. Phosphorylation targets β-catenin for degradation. Binding of Wnt to its receptor LRP leads to blockade of β-catenin phosphorylation, allowing it to accumulate. The free β-catenin translocates to the nucleus where it binds to the TCF transcription factor and initiates transcription of Wnt pathway target genes. Figure reproduced by permission from Macmillan, Ltd. from T. Reya and H. Clevers (2005) Nature, 434, 843. Copyright 2005.

 

 

The investigators began by creating two different mouse models that combined knockout of Apc along with conditional knockout of Ctnnd1 in the intestine. The models differed by the specific mutation in the Apc gene. In all mouse models of APC-driven tumorigenesis, the mice bear a heterozygous mutation in Apc, since homozygous knockout is lethal. Intestinal tumors arise when the second, normal allele is lost through a process known as loss of heterozygosity. In both of their models, the mice developed multiple intestinal tumors in the presence of wild-type p120 expression levels. However, conditional partial knockout of p120 led to increased tumor numbers in both cases. Careful examination of tumor and normal tissues by immunofluorescence revealed that p120 expression in the tumors was reduced but not eliminated. Pockets of cells that did not express p120 at all were present, but only in normal epithelium. Additional studies looking at early time points following conditional knockout of p120 revealed the presence of some tumor cells totally lacking the protein. However, the foci disappeared after a few weeks. These findings suggested that loss of one Ctnnd1 allele, leading to a reduction in p120 levels, was strongly tumorigenic, whereas loss of both alleles was lethal to tumor, but not normal cells.


To better understand the apparent lethality of total p120 loss in tumor cells, the investigators created organoid cultures using adenomas derived from a mouse model of APC-driven tumorigenesis. In some organoids, they used gene silencing techniques to totally ablate p120 expression. In those organoids, they noted a switch from a cystic to a highly branched morphology accompanied by an increased rate of proliferation. Furthermore, the cells in p120-deficient tumors exhibited poor survival unless an inhibitor of Rho kinase was also included to prevent apoptosis. These findings suggested that loss of p120 markedly increases cell proliferation rates but at the expense of greater susceptibility to apoptosis.


The Sleeping Beauty (SB) transposon is a short stretch of DNA that can be randomly inserted into genomic DNA via a transposase. Depending upon its placement, it can either cause overexpression or premature truncation of transcription of a nearby gene. This system has been used to artificially generate gain- or loss-of-function mutations that lead to tumorigenesis in mice. Subsequent examination of the tumors reveals the location of the transposon, enabling the identification of the abnormally expressed genes. The result is a list of genes that may serve as drivers of tumorigenesis. The investigators used this approach to identify potential tumor drivers of intestinal cancer in mice bearing mutations in Apc, Kras, Smad4, or Tp53. All of these mutations render mice susceptible to intestinal tumorigenesis. The results demonstrated that in each of the models, components of the adherens junction play an important role as tumor suppressors. For example, of the 919 genes identified in the Apc mouse model, p120 haploinsufficiency ranked third in strength as a tumor driver. It placed 7th, 8th, and 47th respectively in the models of Kras-, Smad4-, and TP53-driven tumorigenesis. Rankings for α-catenin and E-cadherin were similarly high. In fact, the results showed that half of all tumors contained an inactivating mutation in at least one of these three genes. A search of colorectal cancers in the Human Genome Atlas demonstrated that, although the genes for p120 or α-catenin are not frequently mutated, a marked reduction in p120 expression is present in many tumors. Thus, the role of p120 as a tumor suppressor appears to also be relevant in human intestinal cancer.


The findings suggest that p120 is an obligate haploinsufficient tumor driver. In other words, loss of one Ctnnd1 allele promotes tumor formation, but loss of both alleles is lethal to cancer cells. The researchers speculate that the obligate nature of the haploinsufficiency may serve as a check against survival of highly aberrant cells. This may be particularly important in the case of the gene for α-catenin, which lies in close proximity to that of APC in the genome. This proximity of the two genes increases the chance that they would be lost together. In the case of APC, total loss is a first step towards tumorigenesis, but like p120, total loss of α-catenin is likely lethal, thereby blocking tumor formation. The investigators also propose that the lethality of total p120 loss in cancer cells might be exploited as a mechanism for cancer therapy; however, they acknowledge that the critical role of p120 in the adherence junction of all epithelia may limit its value as a therapeutic target.

 

 

 

ViewJournal of Clinical Investigation article: p120-Catenin is an obligate haploinsufficient tumor suppressor in intestinal neoplasia

 

 

 

 

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