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SMAD4 haploinsufficiency associates with augmented colonic inflammation in select humans and mice.

ABSTRACT: SMAD4 is a common mediator of the TGF-beta signaling pathway. One of the members of this pathway, TGF-beta 1, has an important role in controlling gut inflammation in relation to the continuous stimulation of the intestinal microbiota. SMAD4 haploinsufficiency in humans has been linked to juvenile polyposis hereditary hemorrhagic telangiectasia syndrome (JP/HHT; OMIM#17505). Hematochezia and colonic mucosal inflammation suggestive of inflammatory bowel diseases (IBD) have been reported in JP/HHT. Stimulated by recent experience with two affected pediatric patients presented here, we explored the potential role of Smad4 haploinsufficiency in a murine model of colonic inflammation. Smad4(+/-) mice were maintained on a mixed C57/129SvEv background. Chronic colitis was induced with repeated administration of dextran sulfate sodium (DSS) in drinking water. The colonic mucosal microbiota was interrogated by massively parallel pyrosequencing of the bacterial 16S rRNA gene. 66.7% of Smad4(+/-) mice were sensitive to DSS colitis compared to 14.3% of wild type (Chi-Square p=0.036). The augmented colitis was associated with microbiota separation in the Smad4(+/-) mice. Enterococcus and Enterococcus faecalis specifically was increased in abundance in the colitis-prone animals. Smad4 haploinsufficiency can associate with increased susceptibility to large bowel inflammation in mammals with variable penetrance in association with the colonic mucosal microbiota. These findings may reveal implications not only towards colonic inflammation in the setting of SMAD4 haploinsufficiency, but for colorectal cancer as well.

SUBMITTER: Szigeti R

PROVIDER: S-EPMC3875295 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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SMAD4 haploinsufficiency associates with augmented colonic inflammation in select humans and mice.

Szigeti Reka R Pangas Stephanie A SA Nagy-Szakal Dorottya D Dowd Scot E SE Shulman Robert J RJ Olive Anthony P AP Popek Edwina J EJ Finegold Milton J MJ Kellermayer Richard R

Annals of clinical and laboratory science 20120101 4

SMAD4 is a common mediator of the TGF-beta signaling pathway. One of the members of this pathway, TGF-beta 1, has an important role in controlling gut inflammation in relation to the continuous stimulation of the intestinal microbiota. SMAD4 haploinsufficiency in humans has been linked to juvenile polyposis hereditary hemorrhagic telangiectasia syndrome (JP/HHT; OMIM#17505). Hematochezia and colonic mucosal inflammation suggestive of inflammatory bowel diseases (IBD) have been reported in JP/HHT ...[more]

PMID: 23090737

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Project description:BACKGROUND:The inactivation of tumor suppressor genes follows Alfred Knudson's 'two-hit' model: both alleles need to be inactivated by independent mutation events to trigger tumor formation. However, in a minority of tumor suppressor genes a single hit is sufficient to initiate tumorigenesis notwithstanding the presence of the wild-type allele, a condition known as haploinsufficiency. The SMAD4 gene is an intracellular mediator of the TGF-beta and BMP signal transduction pathways and a tumor suppressor involved in pancreatic and colorectal tumorigenesis. In Smad4-mutant mouse models, haploinsufficiency characterizes the development of gastrointestinal polyps with initial retention of the wild-type allele and protein expression within the nascent tumors and in their direct microenvironment. Similarly, germline SMAD4 mutations are responsible for a subset of patients affected by juvenile polyposis syndrome, an autosomal dominant intestinal cancer syndrome. To date, the molecular and cellular consequences of SMAD4 haploinsufficiency on TGF-beta and BMP signaling and on genome-wide gene expression have not been investigated. RESULTS:Here we show that, similar to previous observations in Smad4-mutant mouse models, haploinsufficiency characterizes a substantial fraction of the juvenile polyps arising in patients with germline SMAD4 mutations. Also, mouse embryonic and intestinal cells heterozygous for a targeted Smad4 null mutation are characterized by a corresponding 50% reduction of the Smad4 protein levels. Reporter assays revealed that mouse Smad4+/- cells exert intermediate inhibitory effects on both TGF-beta and BMP signaling. Genome-wide expression profiling analysis of Smad4+/- and Smad4-/- cells pinpointed a subset of dosage-dependent transcriptional target genes encompassing, among others, members of the TGF-beta and Wnt signaling pathways. These SMAD4 dosage-dependent transcriptional changes were confirmed and validated in a subset of target genes in intestinal tissues from juvenile polyposis syndrome patients. CONCLUSION:Smad4 haploinsufficiency is sufficient to significantly inhibit both TGF-beta and BMP signal transduction and results in the differential expression of a broad subset of target genes likely to underlie tumor formation both from the mesenchymal and epithelial compartments. The results of our study, performed in normal rather than tumor cells where additional (epi-) genetic alterations may confound the analysis, are relevant for our understanding and elucidation of the initial steps underlying SMAD4-driven intestinal tumorigenesis.

Project description:BackgroundPatients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.MethodsUtilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/- mouse model was used to detect entero-endocrine cell hyperplasia with IHC.ResultsAnalyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/- mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease.ConclusionsHemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

Project description:Background: Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations in a single gene, CDKN1B, with heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear. Methods: Utilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/- mouse model was used to detect entero-endocrine cell hyperplasia with IHC. Results: Analyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/- mouse model. Conclusions: Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

Dataset Information

SMAD4 haploinsufficiency associates with augmented colonic inflammation in select humans and mice.

Publications

SMAD4 haploinsufficiency associates with augmented colonic inflammation in select humans and mice.

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