Jillian Pope

Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling

Objective Claudin-1 expression is increased and dysregulated in colorectal cancer and causally associates with the dedifferentiation of colonic epithelial cells, cancer progression and metastasis. Here, we have sought to determine the role claudin-1 plays in the regulation of intestinal epithelial homeostasis. Design We have used a novel villin-claudin-1 transgenic (Cl-1Tg) mouse as model (with intestinal claudin-1 overexpression). The effect of claudin-1 expression upon colonic epithelial differentiation, lineage commitment and Notch-signalling was determined using immunohistochemical, immunoblot and real-time PCR analysis. The frequently used mouse model of dextran sodium sulfate (DSS)-colitis was used to model inflammation, injury and repair. Results In Cl-1Tg mice, normal colonocyte differentiation programme was disrupted and goblet cell number and mucin-2 (muc-2) expressions were significantly downregulated while Notch- and ERK1/2-signalling were upregulated, compared with the wild type-littermates. Cl-1Tg mice were also susceptible to colonic inflammation and demonstrated impaired recovery and hyperproliferation following the DSS-colitis. Our data further show that claudin-1 regulates Notch-signalling through the regulation of matrix metalloproteinase-9 (MMP-9) and p-ERK signalling to regulate proliferation and differentiation. Conclusions Claudin-1 helps regulate intestinal epithelial homeostasis through the regulation of Notch-signalling. An upregulated claudin-1 expression induces MMP-9 and p-ERK signalling to activate Notch-signalling, which in turn inhibits the goblet cell differentiation. Decreased goblet cell number decreases muc-2 expression and thus enhances susceptibility to mucosal inflammation. Claudin-1 expression also induces colonic epithelial proliferation in a Notch-dependent manner. Our findings may help understand the role of claudin-1 in the regulation of inflammatory bowel diseases and CRC.

Caudal Homeobox Protein Cdx-2 Cooperates with Wnt Pathway to Regulate Claudin-1 Expression in Colon Cancer Cells

Dysregulation of tight junctions (TJs) is often associated with human diseases including carcinogenesis and recent studies support role of TJ integral proteins in the regulation of Epithelial-to-Mesenchymal Transition (EMT). In this regard, expression of claudin-1, a key constituent of TJs, is highly increased in colon cancer and is causally associated with the tumor growth and progression. However, mechanism/s underlying regulation of claudin-1 expression in intestinal epithelial cells remains poorly understood. In our studies, we have identified putative binding sites for intestinal transcription factors Cdx1, -2 and GATA4 in the 5′-flanking region of the claudin-1 gene. Our further studies using full length and/or deletion mutant constructs in two different human colon cancer cell lines, SW480 and HCT116, showed key role of Cdx1, Cdx2 and GATA4 in the regulation of claudin-1 mRNA expression. However, overexpression of Cdx2 had the most potent effect upon claudin-1 mRNA expression and promoter activity. Also, in colon cancer patient samples, we observed a significant and parallel correlation between claudin-1 and Cdx2 expressions. Chromatin immunoprecipitation (ChIP) assay confirmed the Cdx2 binding with claudin-1 promoter in vivo. Using Cdx2 deletion mutant constructs, we further mapped the Cdx2 C-terminus domain to be important in the regulation of claudin-1 promoter activity. Interestingly, co-expression of activated β-catenin further induced the Cdx2-dependent upregulation of claudin-1 promoter activity while expression of the dominant negative (dn)-TCF-4 abrogated this activation. Taken together, we conclude that homeodomain transcription factors Cdx1, Cdx2 and GATA4 regulate claudin-1 gene expression in human colon cancer cells. Moreover, a functional crosstalk between Wnt-signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins and GATA-proteins is demonstrated in the regulation of claudin-1 promoter-activation.

Claudin-1 overexpression in intestinal epithelial cells enhances susceptibility to adenamatous polyposis coli-mediated colon tumorigenesis

BackgroundThe tight junction protein Claudin-1, a claudin family member, has been implicated in several gastro-intestinal pathologies including inflammatory bowel disease (IBD) and colorectal cancer (CRC). In this regard, we have demonstrated that claudin-1 expression in colon cancer cells potentiates their tumorigenic ability while in vivo expression of claudin-1 in the intestinal epithelial cells (IECs) promotes Notch-activation, inhibits goblet cell differentiation and renders susceptibility to mucosal inflammation. Notably, a key role of inflammation in colon cancer progression is being appreciated. Therefore, we examined whether inflammation plays an important role in claudin-1-dependent upregulation of colon carcinogenesis.MethodsAPCmin mice were crossed with Villin-claudin-1 transgenic mice to generate APC-Cldn1 mice. H&E stained colon tissues were assessed for tumor number, size and histological grade. Additionally, microarray and qPCR analyses of colonic tumors were performed to assess molecular changes due to claudin-1 expression. APC-Cldn1 and APCmin controls were assessed for colonic permeability via rectal administration of FITC-dextran, and bacterial translocation via qPCR analysis of 16S rDNA.ResultsClaudin-1 overexpression in APCmin mice significantly increased (~4-fold) colonic tumor growth and size, and decreased survival. Furthermore, transcriptome analysis supported upregulated proliferation, and increased Wnt and Notch-signaling in APC-Cldn1 mice. APC-Cldn1 mice also demonstrated inhibition of mucosal defense genes while expression of pro-inflammatory genes was sharply upregulated, especially the IL-23/IL-17 signaling. We predict that increased Notch/Wnt-signaling underlie the early onset of adenoma formation in APC-Cldn1 mice. An increase in mucosal permeability due to the adenomas and the inherent barrier defect in these mice further facilitate bacterial translocation into the mucosa to induce inflammation, which in turn promote the tumorigenesis.ConclusionTaken together, these results confirm the role of claudin-1 as a promoter of colon tumorigenesis and further identify the role of the dysregulated antigen-tumor interaction and inflammation in claudin-1-dependent upregulation of colon tumorigenesis.

Claudin-7 expression induces mesenchymal to epithelial transformation (MET) to inhibit colon tumorigenesis.

In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase–extracellular signal–regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest that loss of claudin-7 potentiates EMT to promote colon cancer, in a manner dependent on Rab25.

Abstract 2963: Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development

Background: Inactivation of the TGF-β signaling occurs in 50-75% of all colorectal cancer cases. Down-regulation of Smad4, the central mediator of TGF-β signaling, occurs in >50% of stage III patients. How Smad4 contributes to normal intestinal homeostasis and functions as a tumor suppressor are incompletely understood. We reported that β-catenin mRNA expression is inhibited by BMP-mediated Smad4 signaling, and loss of Smad4 signaling results in biologically significant amplification of the Wnt/β-catenin signal. Here, we report on the consequences of Smad4 loss for intestinal homeostasis. Methods: We generated mice genetically engineered to undergo inducible, tissue-specific deletion of Smad4 within intestinal epithelial cells. K19-CreERT x Smad4 flx/flx and Lrig1-CreERT x Smad4 flx/flx mice were treated with vehicle or tamoxifen to induce recombination at the Smad4 locus in the intestinal stem cell compartment. Intestines from sacrificed mice were cleansed for enteroid isolation, Ussing chamber studies, RNA-seq analysis and preservation by formalin fixation and paraffin embedding (FFPE). We validated Smad4 status via immunohistochemistry and stained FFPE sections for different cell populations within the crypt. To evaluate inflammatory cytokines, we performed Luminex cytokine array analysis on colonic mucosa from control and tamoxifen treated animals. To determine the role for Smad4 in intestinal response to injury, we also treated control and tamoxifen treated animals with dextran sodium sulfate (DSS). We utilized RNA-Seq analysis to determine an intestinal Smad4 loss gene signature. Results: Smad4 deficient crypts exhibited an expansion of the Ki67 positive cells in the proliferative zone and fewer Carbonic anhydrase II staining cells suggesting fewer mature enterocytes. We found that loss of Smad4 in the intestinal crypt is accompanied by an accumulation of pericryptal CD3+ lymphocytes and F4/80+ macrophages and is associated with increased intestinal permeability as measured by low molecular weight FITC Dextran using the Ussing chamber. Cytokine analysis revealed Macrophage Inflammatory Protein 3a (MIP3a) to be up-regulated in Smad4 knockout mice. Guided by our RNA-Seq analysis, we observe increased levels of pErk in Smad4 deplete tissue. We also observed that mice with intestinal epithelial Smad4 depletion developed advanced mucinous adenocarcinoma after recovery from an inflammatory stimulus induced by DSS. Conclusions: These results suggest that Smad4 loss disrupts intestinal homeostasis by enabling persistently high levels of Erk signaling to interfere with cell maturation, thereby contributing to increased inflammation. We postulate that the increased inflammation observed in Smad4 knockout mice leads to a tumorigenic environment. Citation Format: Tanner J. Freeman, Jillian Pope, Josh Smith, Daniel Sharbel, Kay Washington, Xi Chen, Rupesh Chaturvedi, Keith Wilson, Noah Shroyer, Punita Dhawan, Anna Means, Natasha G. Deane, R. Daniel Beauchamp. Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2014-2963

Abstract 1334: Overexpression of claudin-1 induces Notch and Wnt signaling to regulate colon carcinogenesis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Under physiological conditions, colonic homeostasis is a carefully balanced interplay between stem cells, their progeny, differentiation and cell fate determination. Dysregulation of this harmony signifies susceptibility to neoplastic growth. Colonic epithelium undergoes continuous proliferation, differentiation, and apoptosis along the crypt (crypt bottom to top). Notch signaling is a key regulator of the absorptive/secretory lineage determination. Dysregulation of notch signaling disrupts the normal colonic differentiation and depletes the goblet cell population while immature colonocyte population increases. Importantly, emerging literature suggests that Notch- signaling helps regulate colon cancer growth and progression in association with Wnt signaling. We have previously demonstrated that claudin-1 expression is highly increased in colon cancer and is causally associated with the tumorigenic and metastatic abilities of colon cancer cells. We also reported an inverse relationship between claudin-1 expression and differentiation in colon cancer cells. Here, we show a significant decrease in the goblet cell population (as determined by PAS and Alcian Blue) and Muc-2 expression accompanied with an increase in Carbonic Anhydrase I (CAI) expression, a commonly used marker for colonocytes in Villin-claudin-1 transgenic (Cl-1TG) mice that overexpress claudin-1 in the intestinal epithelium. Further analysis to detect Ki-67 expression suggested increase in colonic proliferation in Cl-1TG mice vs. WT mice. Taken together, these data suggest a role for claudin-1 in the regulation of colonic epithelial differentiation. To further determine the role of claudin-1 overexpression in cancer progression, we crossed Cl-1TG mice with APCmin mice, most widely used mouse model of colon cancer. Notably, APCmin mice develop few colonic tumors (mean=0.8) and have a life span of ∼22.5-23 weeks. Importantly, we noted a ∼4fold increase in the colonic tumor burden in (mean=3.8 tumors) and decrease in life span in APCmin/Cl-1 mice vs. APCmin mice. Also, tumors in APCmin/Cl-1 mice were larger, exhibited high grade of dysplasia and increased nuclear β-catenin (activated Wnt signaling) compared to tumors from APCmin mice. Our further studies using SW480claudin-1 cells {SW480 cells stably overexpressing claudin-1} and SW620siRNA cells {SW620 cells with stable inhibition of claudin-1 expression} demonstrated activation of Notch and Wnt signaling in SW480 cells (APCmutant) overexpressing claudin-1 while inhibited on inhibition of claudin-1. Taken together, our data supported a novel role of claudin-1 in the regulation of Notch and Wnt-signaling and thus colon carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1334. doi:1538-7445.AM2012-1334

Abstract 337: Expression of claudin-7 in colon cancer cells induces mesenchymal to epithelial transition (MET) and inhibits tumorigenesis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The claudin family of proteins is integral to the structure and function of tight junction. However, studies have revealed roles of tight junction proteins including claudins in the regulation of proliferation, migration and epithelial differentiation in addition to their known role in epithelial barrier function. Recent studies support a key role of claudin-7 in the regulation of intestinal epithelial cell functioning and we have reported marked decreases in claudin-7 expression in colon cancer samples in a stage-specific manner. However, role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Objective: To determine the role of claudin-7 expression in the regulation of colon cancer growth and progression. Methods: Full-length human claudin-7 cDNA was stably expressed in SW620 colon cancer cells (no detectable endogenous claudin-7 expression). In parallel, claudin-7 expression was stably inhibited in HT-29 colon cancer cells by RNA-interference using anti-human claudin-7 siRNA. Effect upon cell morphology, proliferation, apoptosis and migration was examined. Colony-forming and invasion assays were used to examine effect upon tumorigenic and/or invasive abilities of the cell lines used. Effect of claudin-7 expression upon tumor number/growth was examined using in vivo injection of the cells in nude athymic mice. Results: Claudin-7 expression in the poorly differentiated SW620 cells induced epithelial differentiation as SW620claudin-7 cells acquired epithelial morphology, showed increased transepithelial resistance (4-fold), decreased permeability (5-fold) and proliferated less (3-fold) compared to SW620vector cells. Additionally, claudin-7 expression induced E-cadherin expression while expression of Vimentin as well as claudin-1, a known colon tumor promoter was markedly decreased. Assays using growth in soft-agar (3-fold) and cell invasion (3-fold) further showed significant decreases in the tumorigenic ability of SW620claudin-7 cells versus SW620vector cells. In contrast, claudin-7 knockdown in HT-29 cells led to a decrease in E-cadherin expression, and increased Vimentin expression. Furthermore, Claudin-7 knockdown cells formed more colonies on softagar (4-fold) and invaded more (3-fold) when subjected to transwell-based invasion assay. Most, importantly, when inoculated into the athymic (nude) mice, SW620claudin-7 cells produced fewer tumors and of smaller size compared to the SW620vector cells while claudin-7 knockdown cells produced more tumors compared to the control cells. Conclusion: Taken together, outcome from our study suggests a colon tumor suppressive role for claudin-7 and thus claudin-7 may represent potential marker for colon cancer detection and a potential target for therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 337. doi:1538-7445.AM2012-337

Abstract 4331: Characterization of targeted overexpression of Claudin-1 transgenic mice

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Disruption of cell-cell junctions and changes in the expression of junctional proteins is a critical event in the progression of cancer. We have previously reported a tumor stage specific increase in the expression of a tight junction protein, Claudin-1, in colorectal cancer and its association with tumor-growth and metastasis. Furthermore, our studies have shown that in colon cancer cells, claudin-1 expression is regulated by a complex interaction between APC, Smad4 and β-catenin, three important regulators of colorectal carcinogenesis. We wanted to investigate the consequence of overexpression of claudin -1 in vivo specifically in the colon. We have generated a transgenic mouse model in which Claudin-1 is expressed under the control of the villin promoter. The villin promoter is expressed in the adult intestinal epithelium and has been frequently used for intestinal-specific overexpression of several genes of interest. Western blot analysis demonstrated a robust expression of claudin-1 in the colon, small intestine and cecum of transgenic mice while low levels of expression were present in wild type mice. These results are confirmed with immunohistochemistry analysis where claudin-1 was largely expressed on the membrane in the same tissues. Colonic permeability was assayed using FITC-dextran, however, there was no significant difference between the flux of FITC-dextran in wild-type and transgenic mice. Real-Time PCR analysis for inflammatory cytokine expression showed a decrease in IL-10 mRNA in transgenic mice compared to that of wild-type mice. Further, treatment with Dextran Sodium Sulfate (DSS) to induce colitis showed a significant decrease in body weight of claudin-1 transgenic mice as compared to wild type mice on DSS. An increase in disease severity was observed in transgenic mice as determined by histology scoring, as well as a significant difference in the ratio of colon weight to colon length. Claudin-1 expression in transgenic mice is localized to the membrane, where it maintains its normal function. Further experiments with this model include combined treatment with DSS and AOM to examine the effect of Claudin -1 overexpression in inflammation-induced-tumorigenesis as well as performing crosses with sporadic models of tumorigenesis involving mutations in the APC gene, to examine the role of claudin-1 upregulation in tumor formation and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4331. doi:10.1158/1538-7445.AM2011-4331