Publications by authors named "Punita Dhawan"

42 Publications

Claudin-2 inhibits renal clear cell carcinoma progression by inhibiting YAP-activation.

J Exp Clin Cancer Res 2021 Feb 23;40(1):77. Epub 2021 Feb 23.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE, 68198-5870, USA.

Background: Claudin-2 expression is upregulated in multiple cancers and promotes cancer malignancy. Remarkably, the regulation of claudin-2 expression in kidney cell lines contrasts its reported regulation in other organs. However, claudin-2 role in renal clear cell carcinoma (RCC) remains unknown despite its predominant expression in the proximal tubular epithelium (PTE), the site of RCC origin.

Methods: Publicly available and independent patient databases were examined for claudin-2 association with RCC. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by Mass spectroscopy, immunoprecipitation and mutational studies, and functional evaluations.

Results: We show that the significant decrease in claudin-2 expression characterized PTE cells and Ex-vivo cultured mouse kidney subjected to dedifferentiation. Inhibition of claudin-2 was enough to induce mesenchymal plasticity and invasive mobility in these models. Further, a progressive loss of claudin-2 expression associated with the RCC progression and poor patient survival. Overexpression of claudin-2 in RCC-derived cancer cells inhibited tumorigenic abilities and xenograft tumor growth. These data supported a novel tumor-suppressive role of claudin-2 in RCC. Mechanistic insights further revealed that claudin-2 associates with YAP-protein and modulates its phosphorylation (S127) and nuclear expression. The tumor suppressive effects of claudin-2 expression were lost upon deletion of its PDZ-binding motif emphasizing the critical role of the PDZ-domain in claudin-2 interaction with YAP in regulating RCC malignancy.

Conclusions: Our results demonstrate a novel kidney specific tumor suppressive role for claudin-2 protein and further demonstrate that claudin-2 co-operates with the YAP signaling in regulating the RCC malignancy.
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http://dx.doi.org/10.1186/s13046-021-01870-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901196PMC
February 2021

Inflammatory bowel disease: Therapeutic limitations and prospective of the stem cell therapy.

World J Stem Cells 2020 Oct;12(10):1050-1066

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68118, United States.

Inflammatory bowel disease (IBD), consisting primarily of ulcerative colitis and Crohn's disease, is a group of debilitating auto-immune disorders, which also increases the risk of colitis-associated cancer. However, due to the chronic nature of the disease and inconsistent treatment outcomes of current anti-IBD drugs (, approximately 30% non-responders to anti-TNFα agents), and related serious side effects, about half of all IBD patients (in millions) turn to alternative treatment options. In this regard, mucosal healing is gaining acceptance as a measure of disease activity in IBD patients as recent studies have correlated the success of mucosal healing with improved prognosis. However, despite the increasing clinical realization of the significance of the concept of mucosal healing, its regulation and means of therapeutic targeting remain largely unclear. Here, stem-cell therapy, which uses hematopoietic stem cells or mesenchymal stem cells, remains a promising option. Stem cells are the pluripotent cells with ability to differentiate into the epithelial and/or immune-modulatory cells. The over-reaching concept is that the stem cells can migrate to the damaged areas of the intestine to provide curative help in the mucosal healing process. Moreover, by differentiating into the mature intestinal epithelial cells, the stem cells also help in restoring the barrier integrity of the intestinal lining and hence prevent the immunomodulatory induction, the root cause of the IBD. In this article, we elaborate upon the current status of the clinical management of IBD and potential role of the stem cell therapy in improving IBD therapy and patient's quality of life.
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http://dx.doi.org/10.4252/wjsc.v12.i10.1050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596447PMC
October 2020

MASTL: A novel therapeutic target for Cancer Malignancy.

Cancer Med 2020 09 21;9(17):6322-6329. Epub 2020 Jul 21.

VA Nebraska-Western Iowa Health Care System, Omaha, NE, USA.

Targeting mitotic kinases is an emerging anticancer approach with promising preclinical outcomes. Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl), is an important mitotic kinase that regulates mitotic progression of normal or transformed cells by blocking the activity of tumor suppressor protein phosphatase 2A (PP2A). MASTL upregulation has now been detected in multiple cancer types and associated with aggressive clinicopathological features. Apart, an aberrant MASTL activity has been implicated in oncogenic transformation through the development of chromosomal instability and alteration of key oncogenic signaling pathways. In this regard, recent publications have revealed potential role of MASTL in the regulation of AKT/mTOR and Wnt/β-catenin signaling pathways, which may be independent of its regulation of PP2A-B55 (PP2A holoenzyme containing a B55-family regulatory subunit). Taken together, MASTL kinase has emerged as a novel target for cancer therapeutics, and hence development of small molecule inhibitors of MASTL may significantly improve the clinical outcomes of cancer patients. In this article, we review the role of MASTL in cancer progression and the current gaps in this knowledge. We also discuss potential efficacy of MASTL expression for cancer diagnosis and therapy.
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http://dx.doi.org/10.1002/cam4.3141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476815PMC
September 2020

Claudin-1, A Double-Edged Sword in Cancer.

Int J Mol Sci 2020 Jan 15;21(2). Epub 2020 Jan 15.

Division of Translational Medicine, Research Branch, Sidra Medicine, Doha 26999, Qatar.

Claudins, a group of membrane proteins involved in the formation of tight junctions, are mainly found in endothelial or epithelial cells. These proteins have attracted much attention in recent years and have been implicated and studied in a multitude of diseases. Claudins not only regulate paracellular transepithelial/transendothelial transport but are also critical for cell growth and differentiation. Not only tissue-specific but the differential expression in malignant tumors is also the focus of claudin-related research. In addition to up- or down-regulation, claudin proteins also undergo delocalization, which plays a vital role in tumor invasion and aggressiveness. Claudin (CLDN)-1 is the most-studied claudin in cancers and to date, its role as either a tumor promoter or suppressor (or both) is not established. In some cancers, lower expression of CLDN-1 is shown to be associated with cancer progression and invasion, while in others, loss of CLDN-1 improves the patient survival. Another topic of discussion regarding the significance of CLDN-1 is its localization (nuclear or cytoplasmic vs perijunctional) in diseased states. This article reviews the evidence regarding CLDN-1 in cancers either as a tumor promoter or suppressor from the literature and we also review the literature regarding the pattern of CLDN-1 distribution in different cancers, focusing on whether this localization is associated with tumor aggressiveness. Furthermore, we utilized expression data from The Cancer Genome Atlas (TCGA) to investigate the association between CLDN-1 expression and overall survival (OS) in different cancer types. We also used TCGA data to compare CLDN-1 expression in normal and tumor tissues. Additionally, a pathway interaction analysis was performed to investigate the interaction of CLDN-1 with other proteins and as a future therapeutic target.
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http://dx.doi.org/10.3390/ijms21020569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013445PMC
January 2020

Curcumin use in ulcerative colitis: is it ready for prime time? A systematic review and meta-analysis of clinical trials.

Ann Gastroenterol 2020 Jan-Feb;33(1):53-58. Epub 2019 Nov 29.

Division of Gastroenterology and Hepatology, University of Utah School of Medicine, Salt Lake City, Utah (Douglas G. Adler), USA.

Background: Curcumin, an active ingredient of the Indian herb turmeric (Curcuma longa), has shown promising anti-inflammatory properties. Studies of its potential benefits in treating patients with ulcerative colitis (UC) are limited. We performed a systematic review and meta-analysis of human randomized placebo controlled trials to evaluate the efficacy of adjunctive therapy with curcumin in treating patients with UC.

Methods: We conducted a search of several databases (from January 2000 to September 2018). A random-effects model was used for analysis. We assessed heterogeneity between study-specific estimates using the Cochran Q statistical test, 95% prediction interval (PI) and I statistics. The outcomes assessed were the pooled odds of clinical response and remission as well as the endoscopic response.

Results: A total of 7 studies with 380 patients (curcumin n=188; placebo n=190) were included in the final analysis. The pooled odds ratio for clinical remission with curcumin use was 2.9 (95%CI 1.5-5.5, I=45, P=0.002), clinical response was 2.6 (95%CI 1.5-4.5, I=74%, P=0.001), and endoscopic response/remission was 2.3 (95%CI 1.2-4.6, I=35.5%, P=0.01).

Conclusions: Based on our study, combined mesalamine and curcumin therapy was associated with roughly threefold better odds of a clinical response compared to placebo, with minimal side effects. This response was statistically significant, albeit with heterogeneity, probably due to the different severity scoring indices, curcumin dosages and routes of drug delivery used.
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http://dx.doi.org/10.20524/aog.2019.0439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928475PMC
November 2019

Role of Claudin Proteins in Regulating Cancer Stem Cells and Chemoresistance-Potential Implication in Disease Prognosis and Therapy.

Int J Mol Sci 2019 Dec 20;21(1). Epub 2019 Dec 20.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA.

Claudins are cell-cell adhesion proteins, which are expressed in tight junctions (TJs), the most common apical cell-cell adhesion. Claudin proteins help to regulate defense and barrier functions, as well as differentiation and polarity in epithelial and endothelial cells. A series of studies have now reported dysregulation of claudin proteins in cancers. However, the precise mechanisms are still not well understood. Nonetheless, studies have clearly demonstrated a causal role of multiple claudins in the regulation of epithelial to mesenchymal transition (EMT), a key feature in the acquisition of a cancer stem cell phenotype in cancer cells. In addition, claudin proteins are known to modulate therapy resistance in cancer cells, a feature associated with cancer stem cells. In this review, we have focused primarily on highlighting the causal link between claudins, cancer stem cells, and therapy resistance. We have also contemplated the significance of claudins as novel targets in improving the efficacy of cancer therapy. Overall, this review provides a much-needed understanding of the emerging role of claudin proteins in cancer malignancy and therapeutic management.
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http://dx.doi.org/10.3390/ijms21010053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982342PMC
December 2019

Cocaine Induces Inflammatory Gut Milieu by Compromising the Mucosal Barrier Integrity and Altering the Gut Microbiota Colonization.

Sci Rep 2019 08 21;9(1):12187. Epub 2019 Aug 21.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198, USA.

Cocaine use disorder (CUD), a major health crisis, has traditionally been considered a complication of the CNS; however, it is also closely associated with malnourishment and deteriorating gut health. In light of emerging studies on the potential role of gut microbiota in neurological disorders, we sought to understand the causal association between CUD and gut dysbiosis. Using a comprehensive approach, we confirmed that cocaine administration in mice resulted in alterations of the gut microbiota. Furthermore, cocaine-mediated gut dysbiosis was associated with upregulation of proinflammatory mediators including NF-κB and IL-1β. In vivo and in vitro analyses confirmed that cocaine altered gut-barrier composition of the tight junction proteins while also impairing epithelial permeability by potentially involving the MAPK/ERK1/2 signaling. Taken together, our findings unravel a causal link between CUD, gut-barrier dysfunction and dysbiosis and set a stage for future development of supplemental strategies for the management of CUD-associated gut complications.
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http://dx.doi.org/10.1038/s41598-019-48428-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704112PMC
August 2019

Correction: Upregulated claudin-1 expression promotes colitis-associated cancer by promoting β-catenin phosphorylation and activation in Notch/p-AKT-dependent manner.

Oncogene 2019 Sep;38(38):6566

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

In Fig. 1 legend, should read as follows: Cld-1 intensifies colitis, leads to impaired recovery from DSS induction and triggers dysplasia: a Schematic illustration of the experimental protocol. Age-matched and sex-matched WT (n = 8) and Cld-1 Tg (n = 8) mice were treated with 2.5% DSS in drinking water for 7 days ad libitum (colitis group), followed by drinking water for 10 days (DSS recovery group). b Representative histological images of WT and Cld-1 Tg mice under DSS and DSS recovery protocol showing regenerative crypts in WT DSS Recovery and dysplastic crypts in Cld-1 Tg Recovery. c The mean changes in body weight of the WT and Cld-1 Tg mice after being fed with 2.5% DSS were measured every day until day 7 for the colitis group and day 10 for the DSS recovery group. The percentage of mice possessing dysplastic crypt in the recovery protocol among WT and Cld-1 Tg groups. Results are statistically significant at p < 0.05. Values sharing following symbols differ significantly, asterisk (*) compared with WT DSS Recovery.
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http://dx.doi.org/10.1038/s41388-019-0864-9DOI Listing
September 2019

Anti-Claudin-1 Conjugated to a Near-Infrared Fluorophore Targets Colon Cancer in PDOX Mouse Models.

J Surg Res 2019 10 9;242:145-150. Epub 2019 May 9.

Department of Surgery, University of California San Diego, La Jolla, California; Moores Cancer Center, University of California San Diego, La Jolla, California; Department of Surgery, VA San Diego Healthcare System, San Diego, California. Electronic address:

Introduction: Claudins are tight-junction proteins, which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated for development of colon cancer. We postulated that Claudin-1 may be a useful target in near-infrared imaging and fluorescence-guided surgery.

Methods: We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 human colon cancer cell lysates was performed. Animal imaging was performed with the LI-COR Pearl Trilogy Fluorescence Imaging System. A dose-response study was carried out with subcutaneous LS174T colon cancer cell line models. Increasing doses of Claudin-1-IRDye800CW via tail vein injection were administered to three groups of mice. Two groups of mice were used as controls (antibody alone, and dye alone). In vivo imaging was performed at 24, 48, and 72 h after administration of the conjugated dye. Orthotopic implantation of patient-derived tumors and cell lines was performed and peritoneal carcinomatosis models were created. After tumor growth, mice were administered Claudin-1-IRDye800CW and imaged in vivo 48 h later. The mice were euthanized and laparotomy was performed to assess internal organs and toxicity.

Results: Western blotting revealed that all colon cancer cell lysates expressed varying amounts of Claudin-1. All tumors demonstrated strong and specific fluorescence labeling at 800 nm, even with the lowest dose of 12.5 μg of Claudin-1-IRDye800CW.

Conclusions: Claudin-1 is a useful target for near-infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.
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http://dx.doi.org/10.1016/j.jss.2019.04.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144806PMC
October 2019

Upregulated claudin-1 expression promotes colitis-associated cancer by promoting β-catenin phosphorylation and activation in Notch/p-AKT-dependent manner.

Oncogene 2019 06 10;38(26):5321-5337. Epub 2019 Apr 10.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

In IBD patients, integration between a hyper-activated immune system and epithelial cell plasticity underlies colon cancer development. However, molecular regulation of such a circuity remains undefined. Claudin-1 (Cld-1), a tight-junction integral protein deregulation alters colonic epithelial cell (CEC) differentiation, and promotes colitis severity while impairing colitis-associated injury/repair. Tumorigenesis is a product of an unregulated wound-healing process and therefore we postulated that upregulated Cld-1 levels render IBD patients susceptible to the colitis-associated cancer (CAC). Villin Cld-1 mice are used to carryout overexpressed studies in mice. The role of deregulated Cld-1 expression in CAC and the underlying mechanism was determined using a well-constructed study scheme and mouse models of DSS colitis/recovery and CAC. Using an inclusive investigative scheme, we here report that upregulated Cld-1 expression promotes susceptibility to the CAC and its malignancy. Increased mucosal inflammation and defective epithelial homeostasis accompanied the increased CAC in Villin-Cld-1-Tg mice. We further found significantly increased levels of protumorigenic M2 macrophages and β-cateninSer552 (β-CatSer552) expression in the CAC in Cld-1Tg vs. WT mice. Mechanistic studies identified the role of PI3K/Akt signaling in Cld-1-dependent activation of the β-CatSer552, which, in turn, was dependent on proinflammatory signals. Our studies identify a critical role of Cld-1 in promoting susceptibility to CAC. Importantly, these effects of deregulated Cld-1 were not associated with altered tight junction integrity, but on its noncanonical role in regulating Notch/PI3K/Wnt/ β-CatSer552 signaling. Overall, outcome from our current studies identifies Cld-1 as potential prognostic biomarker for IBD severity and CAC, and a novel therapeutic target.
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http://dx.doi.org/10.1038/s41388-019-0795-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597297PMC
June 2019

SMAD4 Loss in Colorectal Cancer Patients Correlates with Recurrence, Loss of Immune Infiltrate, and Chemoresistance.

Clin Cancer Res 2019 03 26;25(6):1948-1956. Epub 2018 Dec 26.

Colorectal Service, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: SMAD4 has shown promise in identifying patients with colorectal cancer at high risk of recurrence or death. A discovery cohort and independent validation cohort were classified by SMAD4 status. SMAD4 status and immune infiltrate measurements were tested for association with recurrence-free survival (RFS). Patient-derived xenografts from SMAD4-deficient and SMAD4-retained tumors were used to examine chemoresistance.

Results: The discovery cohort consisted of 364 patients with stage I-IV colorectal cancer. Median age at diagnosis was 53 years. The cohort consisted of 61% left-sided tumors and 62% stage II/III patients. Median follow-up was 5.4 years (interquartile range, 2.3-8.2). SMAD4 loss, noted in 13% of tumors, was associated with higher tumor and nodal stage, adjuvant therapy use, fewer tumor-infiltrating lymphocytes (TIL), and lower peritumoral lymphocyte aggregate (PLA) scores (all < 0.04). SMAD4 loss was associated with worse RFS ( = 0.02). When stratified by SMAD4 and immune infiltrate status, patients with SMAD4 loss and low TIL or PLA had worse RFS ( = 0.002 and = 0.006, respectively). Among patients receiving 5-fluorouracil (5-FU)-based systemic chemotherapy, those with SMAD4 loss had a median RFS of 3.8 years compared with 13 years for patients with SMAD4 retained. In xenografted mice, the SMAD4-lost tumors displayed resistance to 5-FU. An independent cohort replicated our findings, in particular, the association of SMAD4 loss with decreased immune infiltrate, as well as worse disease-specific survival.

Conclusions: Our data show SMAD4 loss correlates with worse clinical outcome, resistance to chemotherapy, and decreased immune infiltrate, supporting its use as a prognostic marker in patients with colorectal cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421131PMC
March 2019

Tight junction proteins in gastrointestinal and liver disease.

Gut 2019 03 8;68(3):547-561. Epub 2018 Oct 8.

Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France.

Over the past two decades a growing body of evidence has demonstrated an important role of tight junction (TJ) proteins in the physiology and disease biology of GI and liver disease. On one side, TJ proteins exert their functional role as integral proteins of TJs in forming barriers in the gut and the liver. Furthermore, TJ proteins can also be expressed outside TJs where they play important functional roles in signalling, trafficking and regulation of gene expression. A hallmark of TJ proteins in disease biology is their functional role in epithelial-to-mesenchymal transition. A causative role of TJ proteins has been established in the pathogenesis of colorectal cancer and gastric cancer. Among the best characterised roles of TJ proteins in liver disease biology is their function as cell entry receptors for HCV-one of the most common causes of hepatocellular carcinoma. At the same time TJ proteins are emerging as targets for novel therapeutic approaches for GI and liver disease. Here we review our current knowledge of the role of TJ proteins in the pathogenesis of GI and liver disease biology and discuss their potential as therapeutic targets.
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http://dx.doi.org/10.1136/gutjnl-2018-316906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453741PMC
March 2019

MASTL induces Colon Cancer progression and Chemoresistance by promoting Wnt/β-catenin signaling.

Mol Cancer 2018 08 1;17(1):111. Epub 2018 Aug 1.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE-68022, USA.

Background: Chemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood.

Methods: We utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer.

Results: Here, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colon tissue (P < 0.001). Also, increased levels of MASTL associated with high-risk of the disease and poor prognosis. Further, the shRNA silencing of MASTL expression in colon cancer cells induced cell cycle arrest and apoptosis in vitro and inhibited xenograft-tumor growth in vivo. Mechanistic analysis revealed that MASTL expression facilitates colon cancer progression by promoting the β-catenin/Wnt signaling, the key signaling pathway implicated in colon carcinogenesis, and up-regulating anti-apoptotic proteins, Bcl-xL and Survivin. Further studies where colorectal cancer (CRC) cells were subjected to 5-fluorouracil (5FU) treatment revealed a sharp increase in MASTL expression upon chemotherapy, along with increases in Bcl-xL and Survivin expression. Most notably, inhibition of MASTL in these cells induced chemosensitivity to 5FU with downregulation of Survivin and Bcl-xL expression.

Conclusion: Overall, our data shed light on the heretofore-undescribed mechanistic role of MASTL in key oncogenic signaling pathway/s to regulate colon cancer progression and chemo-resistance that would tremendously help to overcome drug resistance in colon cancer treatment.
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http://dx.doi.org/10.1186/s12943-018-0848-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090950PMC
August 2018

HDAC-4 regulates claudin-2 expression in EGFR-ERK1/2 dependent manner to regulate colonic epithelial cell differentiation.

Oncotarget 2017 Oct 23;8(50):87718-87736. Epub 2017 Sep 23.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

In normal colon, claudin-2 expression is restricted to the crypt bottom containing the undifferentiated and proliferative colonocytes. Claudin-2 expression is also upregulated in colorectal cancer (CRC) and promotes carcinogenesis. However, cellular mechanism/s regulated by increased claudin-2 expression during the CRC and mechanism/s regulating this increase remain poorly understood. Epigenetic mechanisms help regulate expression of cancer-associated genes and inhibition of Histone Deacetylases (HDACs) induces cell cycle arrest and differentiation. Accordingly, based on a comprehensive and analysis we here report that Histone Deacetylases regulate claudin-2 expression in causal association with colonocyte dedifferentiation to promote CRC. Detailed differentiation analyses using colon cancer cells demonstrated inverse association between claudin-2 expression and epithelial differentiation. Genetic manipulation studies revealed the causal role of HDAC-4 in regulating claudin-2 expression during this process. Further analysis identified transcriptional regulation as the underlying mechanism, which was dependent on HDAC-4 dependent modulation of the EGFR-ERK1/2 signaling. Accordingly, colon tumors demonstrated marked upregulation of the HDAC-4/ERK1/2/Claudin-2 signaling. Taken together, we demonstrate a novel role for HDAC-4/EGFR/ERK1/2 signaling in regulating claudin-2 expression to modulate colonocyte differentiation. These findings are of clinical significance and highlight epigenetic regulation as potential mechanism to regulate claudin-2 expression during mucosal pathologies including CRC.
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http://dx.doi.org/10.18632/oncotarget.21190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675667PMC
October 2017

Obesity-induces Organ and Tissue Specific Tight Junction Restructuring and Barrier Deregulation by Claudin Switching.

Sci Rep 2017 07 11;7(1):5125. Epub 2017 Jul 11.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68198, USA.

Obesity increases susceptibility to multiple organ disorders, however, underlying mechanisms remain unclear. The subclinical inflammation assisted by obesity-induced gut permeability may underlie obesity-associated co-morbidities. Despite eminent clinical significance of the obesity led gut barrier abnormalities, its precise molecular regulation remains unclear. It is also unknown whether barrier deregulations, similar to the gut, characterize other vital organs in obese individuals. The claudin family of proteins is integral to the tight junction (TJ), the apical cell-cell adhesion and a key regulator of the epithelial barrier. Using comprehensive physiological and biochemical analysis of intestinal and renal tissues from high-fat diet fed mice, critical for maintaining metabolic homeostasis, this study demonstrates that profound TJ-restructuring by organ and tissue-specific claudin switching characterize obese organs. Protein expression and cellular distribution were examined. In-silico analysis further highlighted potential association of select claudins, modulated by the obesity, with signaling and metabolic pathways of pathological significance. In vitro studies using Leptin or DCA-treatment suggested causal significance of obesity-induced changes in tissue microenvironment in regulating barrier deregulations in tissue-specific manner. Overall, current findings advances our understanding of the molecular undertakings of obesity associated changes that help predispose to specific diseases and also identifies novel windows of preventive and/or therapeutic interventions.
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http://dx.doi.org/10.1038/s41598-017-04989-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505957PMC
July 2017

Claudin proteins, outside-in signaling, and carcinogenesis.

Pflugers Arch 2017 01 17;469(1):69-75. Epub 2016 Dec 17.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68198-5870, USA.

Environment affects an individual's development and disease risk which then suggest that the environmental cues must have ways of reaching to the cellular nuclei to orchestrate desired genetic changes. Polarized and differentiated epithelial cells join together by cell-cell adhesions to create a protective sheet which separates body's internal milieu from its environment, albeit in highly regulated manner. Among these cell-cell adhesions, a key role of tight junction, the apical cell-cell adhesion, in maintaining epithelial cell polarity and differentiation is well recognized. Moreover, significant changes in expression and cellular distribution of claudin proteins, integral component of the tight junction, characterize pathophysiological changes including neoplastic growth and progression. Studies have further confirmed existence of complex claudin-based interactomes and demonstrated that changes in such protein partnering can influence barrier integrity and communication between a cell and its environment to produce undesired outcome. Cell signaling is the process by which cells respond to their environment to make dynamic decisions to live, grow and proliferate, or die. Thus, pivotal role of the deregulated tight junction structure/function in influencing cellular signaling cascades to alter cellular phenotype can be envisaged, however, is not well understood. Needless to mention that advanced knowledge in this area can help improve therapeutic considerations and preventive measures. Here, we discuss potential role of the tight junction in the regulation of "outside-in" signaling to regulate cancer growth, with specific focus upon the claudin family of proteins.
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http://dx.doi.org/10.1007/s00424-016-1919-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166644PMC
January 2017

Claudin-1 promotes TNF-α-induced epithelial-mesenchymal transition and migration in colorectal adenocarcinoma cells.

Exp Cell Res 2016 Nov 11;349(1):119-127. Epub 2016 Oct 11.

From the Department of Veterans Affairs, University of Nebraska Medical Center, Omaha, NE 68022, United States; Departments of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68022, United States; Buffet Cancer Center, University of Nebraska Medical Center, Omaha, NE 68022, United States. Electronic address:

Epithelial-mesenchymal transition (EMT) is an important mechanism in cancer progression and malignancy including colorectal cancer (CRC). Importantly, inflammatory mediators are critical constituents of the local tumor environment and an intimate link between CRC progression and inflammation is now validated. We and others have reported key role of the deregulated claudin-1 expression in colon carcinogenesis including colitis-associated colon cancer (CAC). However, the causal association between claudin-1 expression and inflammation-induced colon cancer progression remains unclear. Here we demonstrate, TNF-α, a pro-inflammatory cytokine, regulates claudin-1 to modulate epithelial to mesenchymal transition (EMT) and migration in colon adenocarcinoma cells. Importantly, colon cancer cells cultured in the presence of TNF-α (10ng/ml), demonstrated a sharp decrease in E-cadherin expression and an increase in vimentin expression (versus control cells). Interestingly, TNF-α treatment also upregulated (and delocalized) claudin-1 expression in a time-dependent manner accompanied by increase in proliferation and wound healing. Furthermore, similar to our previous observation that claudin-1 overexpression in CRC cells induces ERK1/2 and Src- activation, signaling associated with colon cancer cell survival and transformation, TNF-α-treatment induced upregulation of phospho-ERK1/2 and -Src expression. The shRNA-mediated inhibition of claudin-1 expression largely abrogated the TNF-α-induced changes in EMT, proliferation, migration, p-Erk and p-Src expression. Taken together, our data demonstrate TNF-α mediated regulation of claudin-1 and tumorigenic abilities of colon cancer cells and highlights a key role of deregulated claudin-1 expression in inflammation-induced colorectal cancer growth and progression, through the regulation of the ERK and Src-signaling.
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http://dx.doi.org/10.1016/j.yexcr.2016.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166648PMC
November 2016

MUC4 is negatively regulated through the Wnt/β-catenin pathway via the Notch effector Hath1 in colorectal cancer.

Genes Cancer 2016 May;7(5-6):154-168

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center (UNMC), Omaha, NE, USA.

MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC.
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http://dx.doi.org/10.18632/genesandcancer.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979589PMC
May 2016

Mucins and Wnt/β-catenin signaling in gastrointestinal cancers: an unholy nexus.

Carcinogenesis 2016 Mar 13;37(3):223-32. Epub 2016 Jan 13.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA, Fred and Pamela Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA

The Wnt/β-catenin signaling pathway is indispensable for embryonic development, maintenance of adult tissue homeostasis and repair of epithelial injury. Unsurprisingly, aberrations in this pathway occur frequently in many cancers and often result in increased nuclear β-catenin. While mutations in key pathway members, such as β-catenin and adenomatous polyposis coli, are early and frequent occurrences in most colorectal cancers (CRC), mutations in canonical pathway members are rare in pancreatic ductal adenocarcinoma (PDAC). Instead, in the majority of PDACs, indirect mechanisms such as promoter methylation, increased ligand secretion and decreased pathway inhibitor secretion work in concert to promote aberrant cytosolic/nuclear localization of β-catenin. Concomitant with alterations in β-catenin localization, changes in mucin expression and localization have been documented in multiple malignancies. Indeed, numerous studies over the years suggest an intricate and mutually regulatory relationship between mucins (MUCs) and β-catenin. In the current review, we summarize several studies that describe the relationship between mucins and β-catenin in gastrointestinal malignancies, with particular emphasis upon colorectal and pancreatic cancer.
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http://dx.doi.org/10.1093/carcin/bgw005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014091PMC
March 2016

Cancer Stem Cell and Gastrointestinal Cancer: Current Status, Targeted Therapy and Future Implications.

Biochem Pharmacol (Los Angel) 2016 26;5(2). Epub 2016 Feb 26.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, USA.

The cancer stem cells (CSCs) are biologically distinct subset of rare cancer cells with inherent ability of self-renewal, de-differentiation, and capacity to initiate and maintain malignant tumor growth. Studies have further reported that CSCs prime cancer recurrence and therapy resistance. Therefore, targeting CSCs to inhibit cancer progression has become an attractive anti-cancer therapeutical strategy. Recent technical advances have provided a greater appreciation of the multistep nature of the oncogenesis and also clarified that CSC concept is not universally applicable. Irrespective, the role of the CSCs in gastrointestinal (GI) cancers, responsible for the most cancer-associated death, has been widely accepted and appreciated. However, despite the tremendous progress made in the last decade in developing markers to identify CSCs, and assays to assess tumorigenic function of CSCs, it remains an area of active investigation. In current article, we review findings related to the role and identification of CSCs in GI-cancers and discuss the crucial pathways involved in regulating CSCs populations' development and drug resistance, and use of the tumoroid culture to test novel CSCs-targeted cancer therapies.
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http://dx.doi.org/10.4172/2167-0501.1000202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814166PMC
February 2016

Designing a broad-spectrum integrative approach for cancer prevention and treatment.

Authors:
Keith I Block Charlotte Gyllenhaal Leroy Lowe Amedeo Amedei A R M Ruhul Amin Amr Amin Katia Aquilano Jack Arbiser Alexandra Arreola Alla Arzumanyan S Salman Ashraf Asfar S Azmi Fabian Benencia Dipita Bhakta Alan Bilsland Anupam Bishayee Stacy W Blain Penny B Block Chandra S Boosani Thomas E Carey Amancio Carnero Marianeve Carotenuto Stephanie C Casey Mrinmay Chakrabarti Rupesh Chaturvedi Georgia Zhuo Chen Helen Chen Sophie Chen Yi Charlie Chen Beom K Choi Maria Rosa Ciriolo Helen M Coley Andrew R Collins Marisa Connell Sarah Crawford Colleen S Curran Charlotta Dabrosin Giovanna Damia Santanu Dasgupta Ralph J DeBerardinis William K Decker Punita Dhawan Anna Mae E Diehl Jin-Tang Dong Q Ping Dou Janice E Drew Eyad Elkord Bassel El-Rayes Mark A Feitelson Dean W Felsher Lynnette R Ferguson Carmela Fimognari Gary L Firestone Christian Frezza Hiromasa Fujii Mark M Fuster Daniele Generali Alexandros G Georgakilas Frank Gieseler Michael Gilbertson Michelle F Green Brendan Grue Gunjan Guha Dorota Halicka William G Helferich Petr Heneberg Patricia Hentosh Matthew D Hirschey Lorne J Hofseth Randall F Holcombe Kanya Honoki Hsue-Yin Hsu Gloria S Huang Lasse D Jensen Wen G Jiang Lee W Jones Phillip A Karpowicz W Nicol Keith Sid P Kerkar Gazala N Khan Mahin Khatami Young H Ko Omer Kucuk Rob J Kulathinal Nagi B Kumar Byoung S Kwon Anne Le Michael A Lea Ho-Young Lee Terry Lichtor Liang-Tzung Lin Jason W Locasale Bal L Lokeshwar Valter D Longo Costas A Lyssiotis Karen L MacKenzie Meenakshi Malhotra Maria Marino Maria L Martinez-Chantar Ander Matheu Christopher Maxwell Eoin McDonnell Alan K Meeker Mahya Mehrmohamadi Kapil Mehta Gregory A Michelotti Ramzi M Mohammad Sulma I Mohammed D James Morre Vinayak Muralidhar Irfana Muqbil Michael P Murphy Ganji Purnachandra Nagaraju Rita Nahta Elena Niccolai Somaira Nowsheen Carolina Panis Francesco Pantano Virginia R Parslow Graham Pawelec Peter L Pedersen Brad Poore Deepak Poudyal Satya Prakash Mark Prince Lizzia Raffaghello Jeffrey C Rathmell W Kimryn Rathmell Swapan K Ray Jörg Reichrath Sarallah Rezazadeh Domenico Ribatti Luigi Ricciardiello R Brooks Robey Francis Rodier H P Vasantha Rupasinghe Gian Luigi Russo Elizabeth P Ryan Abbas K Samadi Isidro Sanchez-Garcia Andrew J Sanders Daniele Santini Malancha Sarkar Tetsuro Sasada Neeraj K Saxena Rodney E Shackelford H M C Shantha Kumara Dipali Sharma Dong M Shin David Sidransky Markus David Siegelin Emanuela Signori Neetu Singh Sharanya Sivanand Daniel Sliva Carl Smythe Carmela Spagnuolo Diana M Stafforini John Stagg Pochi R Subbarayan Tabetha Sundin Wamidh H Talib Sarah K Thompson Phuoc T Tran Hendrik Ungefroren Matthew G Vander Heiden Vasundara Venkateswaran Dass S Vinay Panagiotis J Vlachostergios Zongwei Wang Kathryn E Wellen Richard L Whelan Eddy S Yang Huanjie Yang Xujuan Yang Paul Yaswen Clement Yedjou Xin Yin Jiyue Zhu Massimo Zollo

Semin Cancer Biol 2015 Dec;35 Suppl:S276-S304

Centro di Ingegneria Genetica e Biotecnologia Avanzate, Naples, Italy; Department of Molecular Medicine and Medical Biotechnology, Federico II, Via Pansini 5, 80131 Naples, Italy.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered.
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http://dx.doi.org/10.1016/j.semcancer.2015.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819002PMC
December 2015

Claudins and cancer: Fall of the soldiers entrusted to protect the gate and keep the barrier intact.

Semin Cell Dev Biol 2015 Jun 27;42:58-65. Epub 2015 May 27.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, United States. Electronic address:

The role of the tight junctions (TJ) in controlling paracellular traffic of ions and molecules, through the regulation of claudin proteins, is now established. However, it has also become increasingly evident that claudin proteins, as integral components of the TJs, play crucial role in maintaining the cell-cell integrity. In conformity, deregulation of claudin expression and cellular distribution in cancer tissues has been widely documented and correlated with cancer progression and metastasis. However, this correlation is not unidirectional and rather suggests tissue specific regulations. Irrespective, if the widely described correlations between altered claudin expression and cancer initiation/progression could be established, they may serve as important markers for prognostic purposes and potential therapeutic targets. In this review, we summarize data from screening of the cancer tissues, manipulation of claudin expression in cells and animals subjected to cancer models, and how claudins are regulated in cancer. The focus of this article remains analysis of the association between cancer and the claudins and to decipher clinical relevance.
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http://dx.doi.org/10.1016/j.semcdb.2015.05.001DOI Listing
June 2015

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

Mol Cancer 2014 Jul 6;13:167. Epub 2014 Jul 6.

Department of Veterans Affairs, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

Background: The 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.

Methods: APCmin 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.

Results: Claudin-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.

Conclusion: Taken 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.
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http://dx.doi.org/10.1186/1476-4598-13-167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105545PMC
July 2014

Proteogenomic analysis reveals unanticipated adaptations of colorectal tumor cells to deficiencies in DNA mismatch repair.

Cancer Res 2014 Jan 18;74(1):387-97. Epub 2013 Nov 18.

Authors' Affiliations: Departments of Biochemistry, Biomedical Informatics, Surgery, Cancer Biology, and Biostatistics; and Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt University School of Medicine, Nashville, Tennessee.

A growing body of genomic data on human cancers poses the critical question of how genomic variations translate to cancer phenotypes. We used standardized shotgun proteomics and targeted protein quantitation platforms to analyze a panel of 10 colon cancer cell lines differing by mutations in DNA mismatch repair (MMR) genes. In addition, we performed transcriptome sequencing (RNA-seq) to enable detection of protein sequence variants from the proteomic data. Biologic replicate cultures yielded highly consistent proteomic inventories with a cumulative total of 6,513 protein groups with a protein false discovery rate of 3.17% across all cell lines. Networks of coexpressed proteins with differential expression based on MMR status revealed impact on protein folding, turnover and transport, on cellular metabolism and on DNA and RNA synthesis and repair. Analysis of variant amino acid sequences suggested higher stability of proteins affected by naturally occurring germline polymorphisms than of proteins affected by somatic protein sequence changes. The data provide evidence for multisystem adaptation to MMR deficiency with a stress response that targets misfolded proteins for degradation through the ubiquitin-dependent proteasome pathway. Enrichment analysis suggested epithelial-to-mesenchymal transition in RKO cells, as evidenced by increased mobility and invasion properties compared with SW480. The observed proteomic profiles demonstrate previously unknown consequences of altered DNA repair and provide an expanded basis for mechanistic interpretation of MMR phenotypes.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-2488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896054PMC
January 2014

Trichostatin-A modulates claudin-1 mRNA stability through the modulation of Hu antigen R and tristetraprolin in colon cancer cells.

Carcinogenesis 2013 Nov 23;34(11):2610-21. Epub 2013 Jul 23.

Department of Surgery, Vanderbilt University Medical Center, MCN, B-2211, Nashville, TN 37232, USA.

Expression of claudin-1, a tight junction protein, is highly upregulated in colon cancer. We have reported that claudin-1 expression in colon cancer cells is epigenetically regulated as histone deacetylase (HDAC) inhibitors decrease claudin-1 messenger RNA (mRNA) stability and thus expression. In this regard, our data suggested a role of the 3'-untranslated region (UTR) in the regulation of HDAC-dependent regulation of claudin-1 mRNA stability. In the current study, we demonstrate, based on our continued investigation, that the ELAV-like RNA-binding proteins (RBPs), human antigen R (HuR) and tristetraprolin (TTP) associate with the 3'-UTR of claudin-1 mRNA to modulate the latter's stability. Ribonomic and site-directed mutagenesis approaches were used to confirm the binding of HuR and TTP to the 3'-UTR of claudin-1. We further confirmed their roles in the stabilization of claudin-1 mRNA, under conditions of HDAC inhibition. In summary, we report that HuR and TTP are the critical regulators of the posttranscriptional regulation of claudin-1 expression in colon cancer cells. We also demonstrate that inhibition of HDACs by trichostatin treatment decreased the binding of HuR while increasing the binding of TTP to the 3'-UTR of claudin-1. Additionally, we provide data showing transcriptional regulation of claudin-1 expression, through the regulation of transcription factor Sp1. Taken together, we demonstrate epigenetic regulation of claudin-1 expression in colon cancer cells at the transcriptional and posttranscriptional levels.
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http://dx.doi.org/10.1093/carcin/bgt207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3810835PMC
November 2013

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

Gut 2014 Apr 13;63(4):622-34. Epub 2013 Jun 13.

Department of Cancer Biology, Vanderbilt University Medical Center, , Nashville, Tennessee, USA.

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.
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http://dx.doi.org/10.1136/gutjnl-2012-304241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083824PMC
April 2014

Claudin-1 expression confers resistance to anoikis in colon cancer cells in a Src-dependent manner.

Carcinogenesis 2012 Dec 31;33(12):2538-47. Epub 2012 Aug 31.

Department of Surgery, Vanderbilt University Medical Center, Nashville, TN-37232, USA.

Denial of the appropriate cell-matrix interaction in epithelial cells induces apoptosis and is called 'anoikis'. Cancer cells are resistant to anoikis and it is believed that the resistance to anoikis helps promote tumor malignancy especially metastasis. We and others have demonstrated that the expression of tight junction protein claudin-1 is highly upregulated in colorectal cancer (CRC) and helps promote tumor progression and metastasis. However, molecular mechanism/s underlying claudin-1-dependent regulation of CRC progression remains poorly understood. In current study, we have determined that claudin-1 expression modulates anoikis in colon cancer cells to influence colon cancer invasion and thus metastasis. We have further provided data that claudin-1 modulates anoikis in a Src-Akt-Bcl-2-dependent manner. Importantly, claudin-1 physically associates with Src/p-Src in a multiprotein complex that also includes ZO-1, a PDZ-binding tight junction protein. Taken together, our data support the role of claudin-1 in the regulation of CRC progression and suggest that the regulation of anoikis may serve as a key regulatory mechanism in claudin-1-dependent regulation of CRC progression. Our findings are of direct clinical relevance and may open new therapeutic opportunity in colon cancer treatment and/or management.
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http://dx.doi.org/10.1093/carcin/bgs275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510734PMC
December 2012

Caudal homeobox protein Cdx-2 cooperates with Wnt pathway to regulate claudin-1 expression in colon cancer cells.

PLoS One 2012 15;7(6):e37174. Epub 2012 Jun 15.

Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037174PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376107PMC
December 2012

Claudin-1 up-regulates the repressor ZEB-1 to inhibit E-cadherin expression in colon cancer cells.

Gastroenterology 2011 Dec 28;141(6):2140-53. Epub 2011 Aug 28.

Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Background & Aims: Expression of the tight junction protein claudin-1 is dysregulated in colon tumors and associates with their progression. Up-regulation of claudin-1 reduces expression of E-cadherin. We investigated the mechanisms by which claudin-1 regulates E-cadherin expression and its effects in colon cancer cells.

Materials And Methods: We used gene expression analysis, immunoblotting, and reverse transcription polymerase chain reaction to associate expression of the repressor of transcription Zinc Finger E-box binding homeobox-box1 (ZEB-1) with claudin-1. We analyzed SW480 colon cancer cells that overexpressed claudin-1, or SW620 cells in which claudin-1 expression was repressed, to determine the effects on ZEB-1 and E-cadherin expression, invasive activity, and resistance to anoikis. We studied cells that expressed constitutively active or dominant negative forms of factors in the Wnt or phosphotidylinositol-3-kinase signaling pathways and used pharmacologic inhibitors of these pathways to study their role in claudin-1-dependent regulation of ZEB-1. We used microarray analysis to examine gene expression patterns in 260 colorectal tumor and normal colon samples.

Results: Claudin-1 down-regulates E-cadherin expression by up-regulating expression of ZEB-1. Claudin-1 activates Wnt and phosphotidylinositol-3-kinase/Akt signaling. ZEB-1 mediates claudin-1-regulated changes in cell invasion and anoikis. Expression of claudin-1 correlated with that of ZEB-1 in human colon tumor samples. In the progression from normal colonic epithelium to colon adenocarcinoma, levels of E-cadherin decreased, whereas levels of claudin-1 and ZEB-1 increased. Down-regulation of E-cadherin and up-regulation of ZEB-1 in colon tumors were associated with shorter survival times.

Conclusions: Claudin-1 up-regulates the repressor ZEB-1 to reduce expression of E-cadherin in colon cancer cells, increasing their invasive activity and reducing anoikis. This pathway is associated with colorectal cancer progression and patient survival.
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http://dx.doi.org/10.1053/j.gastro.2011.08.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395068PMC
December 2011

Cancer stem cells and colorectal cancer: an overview.

Curr Top Med Chem 2011 ;11(13):1592-8

Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

Normal tissue homeostasis involves a careful balance between the normal cell loss and renewal. Stem and progenitor cells help maintain this precise and fine balance through their ability of self-renewal in a tightly regulated manner. In this regard, the gastrointestinal epithelium is unique in that cell proliferation, differentiation, and apoptosis occur in an orderly fashion along the crypt-villus axis. The colonic crypt is primarily a proliferative compartment, is monoclonal and is maintained by stem cells. The concept of tissue stem cells capable of giving rise to all differentiated cells within a given tissue has led to the concept of a cellular hierarchy in tissues and in tumors including colorectal cancer (CRC). Thus, only a few cells may be necessary and sufficient for tissue repair or tumor regeneration. However, such a proposition also raises questions regarding the precise methods and markers to identify such population and to define the circumstantial evidences and place for the origin and establishment of the early mutant stem-cell population. Thus, it is imperative that we understand what cancer stem cells (CSC) are and their potential association with cancer in a tissue specific manner. In this review, we have summarized the current knowledge of stem cell organization and CSC within the colonic epithelium and discussed the potential role of CSC in the development and/or progression of CRC and as targets for therapeutic interventions.
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http://dx.doi.org/10.2174/156802611796117694DOI Listing
December 2011