Sprouty4 is epigenetically upregulated in human colorectal cancer.

Sprouty4 (SPRY4) has been frequently reported as a tumor suppressor and is therefore downregulated in various cancers. For the first time, we report that SPRY4 is epigenetically upregulated in colorectal cancer (CRC). In this study, we explored DNA methylation and hydroxymethylation levels of SPRY4 in CRC cells and patient samples and correlated these findings with mRNA and protein expression levels. Three loci within the promoter region of SPRY4 were evaluated for 5mC levels in CRC using the combined bisulfite restriction analysis. In addition, hydroxymethylation levels within SPRY4 were measured in CRC patients. Lastly, DNA methylation and mRNA expression data were extracted from CRC patients in multiple high-throughput data repositories like Gene Expression Omnibus and The Cancer Genome Atlas. Combined in vitro and in silico analysis of promoter methylation levels of SPRY4 clearly demonstrates that the distal promoter region undergoes hypomethylation in CRC patients and is associated with increased expression. Moreover, a decrease in gene body hydroxymethylation and an increase in gene body methylation within the coding region of SPRY4 were found in CRC patients and correlated with increased expression. SPRY4 is epigenetically upregulated in CRC by promoter hypomethylation and hypermethylation within the gene body that warrants future investigation of atypical roles of this established tumor suppressor.

SPRY4 gene expression is increased in colorectal cancerThe SPRY4 promoter loses DNA methylation, and the gene body gains DNA methylation in colorectal cancerThe gene body of SPRY4 loses DNA hydroxymethylation.

Aberrant regulation of CXCR4 in cancer via deviant microRNA-targeted interactions.

CXCR4 is involved in many facets of cancer, including being a major player in establishing metastasis. This is in part due to the deregulation of CXCR4, which can be attributed to many genetic and epigenetic mechanisms, including aberrant microRNA-CXCR4 interaction. MicroRNAs (miRNAs) are a type of small non-coding RNA that primarily targets the 3' UTR of mRNA transcripts, which in turn suppresses mRNA and subsequent protein expression. In this review, we reported and characterized the many aberrant miRNA-CXCR4 interactions that occur throughout human cancers. In particular, we reported known target sequences located on the 3' UTR of CXCR4 transcripts that tumour suppressor miRNAs bind and therefore regulate expression by. From these aberrant interactions, we also documented affected downstream genes/pathways and whether a particular tumour suppressor miRNA was reported as a prognostic marker in its respected cancer type. In addition, a limited number of cancer-causing miRNAs coined 'oncomirs' were reported and described in relation to CXCR4 regulation. Moreover, the mechanisms underlying both tumour suppressor and oncomir deregulations concerning CXCR4 expression were also explored. Furthermore, the miR-146a-CXCR4 axis was delineated in oncoviral infected endothelial cells in the context of virus-causing cancers. Lastly, miRNA-driven therapies and CXCR4 antagonist drugs were discussed as potential future treatment options in reported cancers pertaining to deregulated miRNA-CXCR4 interactions.

Hepatocellular Carcinoma: The Role of MicroRNAs.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. HCC is diagnosed in its advanced stage when limited treatment options are available. Substantial morphologic, genetic and epigenetic heterogeneity has been reported in HCC, which poses a challenge for the development of a targeted therapy. In this review, we discuss the role and involvement of several microRNAs (miRs) in the heterogeneity and metastasis of hepatocellular carcinoma with a special emphasis on their possible role as a diagnostic and prognostic tool in the risk prediction, early detection, and treatment of hepatocellular carcinoma.

Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers.

Conventional wisdom is that Sprouty2 (SPRY2), a suppressor of Receptor Tyrosine Kinase (RTK) signaling, functions as a tumor suppressor and is downregulated in many solid tumors. We reported, for the first time, that increased expression of SPRY2 augments cancer phenotype and Epithelial-Mesenchymal-Transition (EMT) in colorectal cancer (CRC). In this report, we assessed epigenetic DNA modifications that regulate SPRY2 expression in CRC. A total of 4 loci within SPRY2 were evaluated for 5mC using Combined Bisulfite Restriction Analysis (COBRA). Previously sequenced 5hmC nano-hmC seal data within SPRY2 promoter and gene body were evaluated in CRC. Combined bioinformatics analyses of SPRY2 CRC transcripts by RNA-seq/microarray and 450K methyl-array data archived in The Cancer Genome Atlas (TCGA) and GEO database were performed. SPRY2 protein in CRC tumors and cells was measured by Western blotting. Increased SPRY2 mRNA was observed across several CRC datasets and increased protein expression was observed among CRC patient samples. For the first time, SPRY2 hypomethylation was identified in adenocarcinomas in the promoter and gene body. We also revealed, for the first time, increases of 5hmC deposition in the promoter region of SPRY2 in CRC. SPRY2 promoter hypomethylation and increased 5hmC may play an influential role in upregulating SPRY2 in CRC.

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies.

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12-CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12-CXCR4/CXCR7 axis as a treatment strategy for CRC.

Natural proteins and polysaccharides in the development of micro/nano delivery systems for the treatment of inflammatory bowel disease.

Treatments for inflammatory bowel disease (IBD) are typically immunosuppressive. Despite a range of treatment options, limited efficacy, systemic toxicities like bone marrow suppression, infections and malignancy are their serious setbacks. There exists an unmet medical need for novel therapeutic agents without safety concerns resulting from chronic, systemic immunosuppression. Of late, several natural agents with better therapeutic potential have been reported. It is very likely that restricting the release of the active molecules to the intestine would further improve their clinical efficacy and safety. To this end, novel polymer-based micro/nano formulations protect the drug from gastric environment and slowly release the drug in the colon. However, cost and side-effects associated to synthetic polymers have led to the development of biocompatible, economic and pharmaceutically well-accepted biomacromolecules in exploring their potential in IBD. Since last few years, biological proteins, polysaccharides and their combinations have shown great efficacy in colitis induced animal models. In this review, micro/nano formulations developed using biomacromolecules like chitosan, zein, pectin, casein, alginate, dextran, glucomannan and hyaluronic acid have been reviewed focusing on their potential in protecting active cargo, avoiding premature release, distal colon targeting along with their impact on reshaping the altered gut microbiota and how it can ameliorate the colitis conditions.

Correction: Stuckel, et al.; Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but Not Decreased Promoter Methylation in Colorectal Cancer. Cancers 2020, 12, 539.

The authors would like to make a correction to their published paper [...].

Natural Product-Based Nanomedicine in Treatment of Inflammatory Bowel Disease.

: Many synthetic drugs and monoclonal antibodies are currently in use to treat Inflammatory Bowel Disease (IBD). However, they all are implicated in causing severe side effects and long-term use results in many complications. Numerous in vitro and in vivo experiments demonstrate that phytochemicals and natural macromolecules from plants and animals reduce IBD-related complications with encouraging results. Additionally, many of them modify enzymatic activity, alleviate oxidative stress, and downregulate pro-inflammatory transcriptional factors and cytokine secretion. Translational significance of natural nanomedicine and strategies to investigate future natural product-based nanomedicine is discussed. Our focus in this review is to summarize the use of phytochemicals and macromolecules encapsulated in nanoparticles for the treatment of IBD and IBD-associated colorectal cancer.

Defects in long-chain 3-hydroxy acyl-CoA dehydrogenase lead to hepatocellular carcinoma: A novel etiology of hepatocellular carcinoma.

The incidence of both nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) have been increasing at an alarming rate. Little is known about NAFLD without cirrhosis as a risk for HCC. Here we report, for the first time, generation of a mouse model with a defect in long-chain 3-hydoxy acyl-CoA dehydrogenase (LCHAD). The LCHAD exon 15 deletion was embryonic lethal to the homozygous mice whereas heterozygous mice (HT) develop significant hepatic steatosis starting at young age (3 months old) and HCC at older age (>13 months old) without any evidence of fibrosis or cirrhosis. None of the wild-type (WT) mice developed steatosis and HCC (n = 39), whereas HT-LCHAD mice (n = 41) showed steatosis and ~20% (8/41) developed liver masses with histological features of HCC. Proteomic analysis of liver tissues from WT-mice and HT-mice with no signs of HCC was conducted. Proteins with significant changes in abundance were identified by mass spectrometry. Abundance of 24 proteins was significantly different (p < 0.01) between WT and HT-LCHAD mice. The proteins found to vary in abundance are associated with different cellular response processes ranging from intermediary metabolism of carbohydrate, protein and lipid to oxidative stress, signal transduction and the process of tumorigenesis. Protein expression pattern of the HT-LCHAD mouse liver indicates predisposition to HCC and suggests that impaired hepatic mitochondrial fatty acid oxidation plays an important role in the development and progression of HCC. To assess the implication of these studies in human disease, we demonstrated significant downregulation of HADHA transcripts in HCC patients.

Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer.

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4's regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

Proteomics approaches for early detection and targeted therapy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related mortality worldwide. HCC incidences have increased worldwide though more prevalent in Asia and Africa. Hepatitis B virus and hepatitis C virus infections are mostly responsible of increased number of HCC cases. Biomarkers can help early detection and improve treatment regimen in patients as advanced stage is chemo-refractive with limited treatment options. Potential of proteomics in finding new biomarkers for early detection has been explored more recently. Future developments in this area rely on how efficiently we manage vast amount of data generated by these techniques and speed up the clinical trials to improve patient care.

Atypical role of sprouty in p21 dependent inhibition of cell proliferation in colorectal cancer.

Sprouty (SPRY) appears to act as a tumor suppressor in cancer, whereas we reported that SPRY2 functions as a putative oncogene in colorectal cancer (CRC) [Oncogene, 2010, 29: 5241-5253]. In general, various studies established inhibition of cell proliferation by SPRY in cancer. The mechanisms by which SPRY regulates cell proliferation in CRC are investigated. We demonstrate, for the first time, suppression of SPRY2 augmented EGF-dependent oncogenic signaling, however, surprisingly decreased cell proliferation in colon cancer cells. Our data suggest that cell cycle inhibitor p21(WAF1/CIP1) transcriptional activity being regulated by SPRY2. Indeed, suppression of SPRY2 significantly increased p21(WAF1/CIP1) mRNA and protein expression as well as p21(WAF1/CIP1) promoter activity. Conversely, overexpressing SPRY2 triggered a decrease in p21(WAF1/CIP1) promoter activity. Concurrent down-regulation of both SPRY1 and SPRY2 also increased p21(WAF1/CIP1) expression in colon cancer cells. Increased nuclear localization of p21(WAF1/CIP1) in SPRY2 downregulated colon cancer cells may explain the inhibition of cell proliferation in colon cancer cells. Underscoring the biological relevance of these findings in SPRY1 and SPRY2 mutant mouse, recombination of floxed SPRY1 and SPRY2 alleles in mouse embryonic fibroblasts (MEFs) resulted in increased expression and nuclear localization of p21(WAF1/CIP1) and decreased cell proliferation. In CRC, the relationship of SPRY with p21 may provide unique strategies for cancer prevention and treatment. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Epigenetic therapy for colorectal cancer.

Aberrations in epigenome that include alterations in DNA methylation, histone acetylation, and miRNA (microRNA) expression may govern the progression of colorectal cancer (CRC). These epigenetic changes affect every phase of tumor development from initiation to metastasis. Since epigenetic alterations can be reversed by DNA demethylating and histone acetylating agents, current status of the implication of epigenetic therapy in CRC is discussed in this article. Interestingly, DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) have shown promising results in controlling cancer progression. The information provided here might be useful in developing personalized medicine approaches.

The renin-angiotensin system mediates EGF receptor-vitamin d receptor cross-talk in colitis-associated colon cancer.

PURPOSE: We previously showed that EGF receptor (EGFR) promotes tumorigenesis in the azoxymethane/dextran sulfate sodium (AOM/DSS) model, whereas vitamin D suppresses tumorigenesis. EGFR-vitamin D receptor (VDR) interactions, however, are incompletely understood. Vitamin D inhibits the renin-angiotensin system (RAS), whereas RAS can activate EGFR. We aimed to elucidate EGFR-VDR cross-talk in colorectal carcinogenesis. EXPERIMENTAL DESIGN: To examine VDR-RAS interactions, we treated Vdr(+/+) and Vdr(-/-) mice with AOM/DSS. Effects of VDR on RAS and EGFR were examined by Western blotting, immunostaining, and real-time PCR. We also examined the effect of vitamin D3 on colonic RAS in Vdr(+/+) mice. EGFR regulation of VDR was examined in hypomorphic Egfr(Waved2) (Wa2) and Egfr(wild-type) mice. Angiotensin II (Ang II)-induced EGFR activation was studied in cell culture. RESULTS: Vdr deletion significantly increased tumorigenesis, activated EGFR and ß-catenin signaling, and increased colonic RAS components, including renin and angiotensin II. Dietary VD3 supplementation suppressed colonic renin. Renin was increased in human colon cancers. In studies in vitro, Ang II activated EGFR and stimulated colon cancer cell proliferation by an EGFR-mediated mechanism. Ang II also activated macrophages and colonic fibroblasts. Compared with tumors from Egfr(Waved2) mice, tumors from Egfr(wild-type) mice showed upregulated Snail1, a suppressor of VDR, and downregulated VDR. CONCLUSIONS: VDR suppresses the colonic RAS cascade, limits EGFR signals, and inhibits colitis-associated tumorigenesis, whereas EGFR increases Snail1 and downregulates VDR in colonic tumors. Taken together, these results uncover a RAS-dependent mechanism mediating EGFR and VDR cross-talk in colon cancer.

Epigenetics of hepatocellular carcinoma: role of microRNA.

Hepatocellular carcinoma (HCC) represents a major form of primary liver cancer in adults. MicroRNAs (miRs), small non-coding single-stranded RNAs of 19-24 nucleotides in length, negatively regulate the expression of many target genes at the post-transcriptional and/or translational levels and play a critical role in the initiation and progression of HCC. In this review we have summarized the information of aberrantly expressed miRs in HCC, their mechanism of action and relationship to cancer. The recent advances in HCC research reveal that miRs regulate expression of various oncogenes and tumor suppressor genes, thereby contributing to the modulation of diverse biological processes including proliferation, apoptosis, epithelial to mesenchymal transition and metastasis. From a clinical viewpoint, polymorphisms within miR-binding sites are associated with the risk of HCC. Polymorphisms in miR related genes have been shown to correlate with survival or treatment outcome in patients. Furthermore, the review focuses on the potential role of miRs as novel biomarkers and their translational applications for diagnosis and therapy in HCC. With further insights into miR deregulation in HCC, it is expected that novel miR-based therapeutics will arise. Also, we orient the readers to other reviews that may provide better understanding of miR research in HCC.

Epigenetics of colon cancer.

Accumulation of genetic and epigenetic alterations transforms normal colonic epithelial cells to adenocarcinoma cells. Genetic alterations include mutations in tumor suppressor genes and oncogenes, whereas epigenetic mechanisms are defined as heritable alterations in gene expression that is independent of changes in the primary DNA sequence. Role of epigenetic mechanisms in development and maintenance of organ- and tissue-specific gene expression is now realized. Disturbances in epigenetic landscape can lead to malignant cellular makeover, and these heritable changes are maintained through various cycles of cell division that renders cells to have discrete identity with similar genetic information. Epigenetic alterations in colorectal cancer (CRC) that transform colonic epithelial cells into adenocarcinoma cells include aberrant DNA methylation, chromatin modifications, and noncoding RNAs, especially microRNA expression. CpG island DNA methylation and aberrant methylation of genes drive the initiation and progression of CRC. Histone modifications impinge on chromatin structure and gene expression and thus play an important role in gene silencing in CRC. DNA hypermethylation also leads to downregulation and inappropriate expression of certain microRNAs that act like tumor suppressor genes. Determining the causes and roles of epigenetic instability in CRC pathogenesis will lead to effective prevention and therapeutic strategies for patients with CRC. Epigenetic drugs that underscore the reversible nature of epigenetic events have led the possibility of epigenetic therapy as a treatment option in CRC.

Aberrant crypt foci in colon cancer epidemiology.

Colonic carcinogenesis is characterized by progressive accumulations of genetic and epigenetic derangements. These molecular events are accompanied by histological changes that progress from mild cryptal architectural abnormalities in small adenomas to eventual invasive cancers. The transition steps from normal colonic epithelium to small adenomas are little understood. In experimental models of colonic carcinogenesis aberrant crypt foci (ACF), collections of abnormal appearing colonic crypts, are the earliest detectable abnormality and precede adenomas. Whether in fact ACF are precursors of colon cancer, however, remains controversial. Recent advances in magnification chromoendoscopy now allow these lesions to be identified in vivo and their natural history ascertained. While increasing lines of evidence suggest that dysplastic ACF harbor a malignant potential, there are few prospective studies to confirm causal relationships and supporting epidemiological studies are scarce. It would be very useful, for example, to clarify the relationship of ACF incidence to established risks for colon cancer, including age, smoking, sedentary lifestyle, and Western diets. In experimental animal models, carcinogens dose-dependently increase ACF, whereas most chemopreventive agents reduce ACF incidence or growth. In humans, however, few agents have been validated to be chemopreventive of colon cancer. It remains unproven, therefore, whether human ACF could be used as reliable surrogate markers of efficacy of chemopreventive agents. If these lesions could be used as reliable biomarkers of colon cancer risk and their reductions as predictors of effective chemopreventive agents, metrics to quantify ACF could greatly facilitate the study of colonic carcinogenesis and chemoprevention.

Ursodeoxycholic acid suppresses Cox-2 expression in colon cancer: roles of Ras, p38, and CCAAT/enhancer-binding protein.

In the azoxymethane (AOM) model of experimental rodent colon cancer, cholic acid and its colonic metabolite deoxycholic acid (DCA) strongly promote tumorigenesis. In contrast, we showed that ursodeoxycholic acid (UDCA), a low abundance bile acid, inhibited AOM tumorigenesis. Dietary UDCA also blocked the development of tumors with activated Ras and suppressed cyclooxygenase-2 (Cox-2) upregulation in AOM tumors. In this study, we compared the effect of dietary supplementation with tumor-promoting cholic acid to chemopreventive UDCA on Cox-2 expression in AOM tumors. Cholic acid enhanced Cox-2 upregulation in AOM tumors, whereas UDCA inhibited this increase and concomitantly decreased CCAAT/enhancer binding protein beta (C/EBPbeta), a transcriptional regulator of Cox-2. In HCA-7 colon cancer cells, DCA activated Ras and increased C/EBPbeta and Cox-2 by a mechanism requiring the mitogen-activated protein kinase p38. UDCA inhibited DCA-induced p38 activation and decreased C/EBPbeta and Cox-2 upregulation. Using transient transfections, UDCA inhibited Cox-2 promoter and C/EBP reporter activation by DCA. Transfection with dominant-negative (17)N-Ras abolished DCA-induced p38 activation and C/EBPbeta and Cox-2 upregulation. Taken together, these studies have identified a transcriptional pathway regulating Cox-2 expression involving Ras, p38, and C/EBPbeta that is inhibited by UDCA. These signal transducers are novel targets of UDCA's chemopreventive actions.

Epidermal growth factor receptor signaling is required for microadenoma formation in the mouse azoxymethane model of colonic carcinogenesis.

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2+/-1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6+/-2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2+/-4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-alpha (6.4+/-1.3-fold) and increased phospho-(active) EGFR (5.9+/-1.1-fold), phospho-(active) ErbB2 (2.3+/-0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3+/-0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P<0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P<0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies.

Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2.

Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.