Epithelial-cadherin (E-cadherin) is a master organizer of the epithelial phenotype. Its function is regulated in part by p120-catenin (referred to herein as p120), a cytoplasmic binding partner that directly regulates cadherin stability. As it has been suggested that cadherins have a role in inflammatory bowel disease (IBD), we sought to investigate this further by assessing the effect of p120 deficiency in mouse small intestine and colon. p120 conditional KO mice were superficially normal at birth but declined rapidly and died within 21 days. Cellcell adhesion defects and inflammation led to progressive mucosal erosion and terminal bleeding, similar to what is observed in a dominant-negative cadherin mouse model of IBD. Additionally, selective loss of adherens junctions and accumulation of atypical COX-2-expressing neutrophils in p120-null areas of the colon were observed. To elucidate the mechanism, direct effects of p120 deficiency were assessed in vitro in a polarizing colon cancer cell line. Notably, transepithelial electrical resistance was dramatically reduced, neutrophil binding was increased 30 fold, and levels of COX-2, an enzyme associated with IBD, were markedly increased in neutrophils. Our data suggest that p120 loss disrupts the neonatal intestinal barrier and amplifies neutrophil engagement and that these changes lead to catastrophic inflammation during colonization of the neonatal gut with bacteria and other luminal antigens. Thus, we conclude that p120 has an essential role in barrier function and epithelial homeostasis and survival in the intestine.

p120 loss destabilizes E-cadherin and could therefore result in tumor and/or metastasis-promoting activities similar to those caused by E-cadherin downregulation. Previously, we reported that p120 is essential in the intestine for barrier function, epithelial homeostasis and survival. Conditional p120 ablation in the mouse intestine induced severe inflammatory bowel disease, but long-term cancer-related studies were impossible because none of the animals survived longer than 21 days. Here, we used a tamoxifen-inducible mouse model (Vil-Cre-ERT2;p120fl/fl) to limit the extent of p120 ablation and thereby enable long-term studies. Reducing p120 KO to ∼10% of the intestinal epithelium produced long-lived animals outwardly indistinguishable from controls. Effects of prolonged p120 absence were then evaluated at intervals spanning 2 to 18 months. At all time points, immunostaining revealed microdomains of p120-null epithelium interspersed with normal epithelium. Thus, stochastic p120 ablation is compatible with crypt progenitor cell function and permitted lifelong renewal of the p120-null cells. Consistent with previous observations, a barrier defect and frequent infiltration of neutrophils was observed, suggesting that focal p120 loss generates a microenvironment disposed to chronic inflammation. We report that 45% of these animals developed tumors within 18 months of tamoxifen induction. Interestingly, β-catenin was upregulated in the majority, but none of the tumors were p120 null. Although further work is required to directly establish mechanism, we conclude that limited p120 ablation can promote tumorigenesis by an indirect non-cell autonomous mechanism. Given that byproducts of inflammation are known to be highly mutagenic, we suggest that tumorigenesis in this model is ultimately driven by the lifelong inability to heal chronic wounds and the substantially increased rates of stochastic gene mutation in tissue microenvironments subjected to chronic inflammation. Indeed, although technical issues precluded direct identification of mutations, β-catenin upregulation in human colon cancer almost invariably reflects mutations in APC and/or β-catenin.

IntroductionThe vast majority of human colorectal cancer (CRC) is triggered by inactivation of the tumor suppressor adenomatous polyposis coli (APC) in one of several intestinal stem cells (ISCs) present in each intestinal crypt. The result is unscheduled translocation of β-catenin to the nucleus where tumorigenesis is initiated via interaction with the transcription factor TCF4 and unregulated transactivation of Wnt pathway target genes (e.g., AXIN2, MYC) (1-4). The ISC contributes several stem cell-restricted properties (e.g., self-renewal, mesenchymal characteristics) that are hijacked by the constitutively activated Wnt pathway and are essential for tumor initiation. Progression to malignancy involves subsequent genetic and epigenetic alterations, including mutations in genes such as KRAS, SMAD4, and TP53, which are frequently targeted in CRC, and a much larger number of less frequently mutated genes uncovered recently by whole genome sequencing. The transition to malignancy is often accompanied by the onset of metastasis, a phenomenon responsible for most cancer-related mortality and frequently linked to genetic and/or epigenetic dysregulation of the cell-cell adhesion receptor epithelial cadherin (E-cadherin) (5-8).E-cadherin is widely regarded as the principle organizer of the epithelial phenotype (9, 10). It functions as a classic tumor suppressor in diffuse gastric cancer (11) and lobular carcinoma of the breast (12), where germline mutations in the E-cadherin gene (CDH1) are responsible for familial inheritance of one mutant allele. Early loss of the second allele (i.e., loss of heterozygosity [LOH]) then plays a causal role in tumorigenesis (11)(12)(13). In the vast majority of epithelial cancers, however, E-cadherin is considered a metastasis suppressor because it is downregulated in advanced tumors and plays a causal role in the transition to metastasis (5,7,(14)(15)(16). Although factors underpinning the distinct differences in timing of E-cadherin loss between these groups are not understood, the former is frequently irreversible (e.g., gene mutation) while the latter often occurs by epithelial-to-mesenchymal transition (EMT) and is later reversed during colonization of distant sites (17).As with all classical cadherins, E-cadherin mediates specific adhesion of adjacent cells via homophilic interaction between extracellular domains (9). Strong E-cadherin-mediated adhesion, however, is absolutely dependent upon formation of a multimeric complex with 3 cytoplasmic binding partners, namely α-, β-, and p120-catenins (p120) (9,18). All members of the classical cadherin family, in fact, use these same core components to organize the actin cytoskeleton and coordinate signaling (9, 19). p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated wi...

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.