Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.

In addition to mesenchymal cells, endothelial cells may contribute to fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). We investigated whether human intestinal microvascular endothelial cells (HIMEC) undergo EndoMT and contribute to fibrosis in human and experimental inflammatory bowel disease (IBD). HIMEC were exposed to TGF-β1, IL-1β, and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) and evaluated for morphological, phenotypic, and functional changes compatible with EndoMT. Genomic analysis was used to identify transcription factors involved in the transformation process. Evidence of in situ and in vivo EndoMT was sought in inflamed human and murine intestine. The combination of TGF-β1, IL-1β and TNF-α, or activated LPMC supernatants induced morphological and phenotypic changes consistent with EndoMT with a dominant effect by IL-1. These changes persisted after removal of the inducing agents and were accompanied by functional loss of acetylated LDL-uptake and migratory capacity, and acquisition of de novo collagen synthesis capacity. Sp1 appeared to be the main transcriptional regulator of EndoMT. EndoMT was detected in microvessels of inflammatory bowel disease (IBD) mucosa and experimental colonic fibrosis of Tie2-green fluorescent protein (GFP) reporter-expressing mice. In conclusion, chronic inflammation induces transdifferentiation of intestinal mucosal microvascular cells into mesenchymal cells, suggesting that the intestinal microvasculature contributes to IBD-associated fibrosis through the novel process of EndoMT.

Androgen-regulated genes (ARGs) are essential for the development of the prostate. Ironically, ARGs are also responsible for the pathogenesis of prostate cancer. We used oligonucleotide array technology to study the expression profiles of ARGs in LNCaP prostate cancer cells and identified 692 dihydrotestosterone-regulated genes. Representative clusters containing genes with similar expression patterns to prostate-specific antigen and other known ARGs are discussed. Based on functional information, we categorized several candidate targets for prostate cancer therapy and diagnosis. Although many of these candidate targets are known to play an important role in cancer development, several are novel genes to the field of prostate cancer. A cross-comparison study of our results with those that have been previously published from three other array experiments using a similar LNCaP model validated 13 of these candidate targets as androgen-regulated. FKBP51 (FK506-binding immunophilin 51) was found in the same cluster as prostate-specific antigen and its protein expression was increased in LNCaP cells treated with either dihydrotestosterone or synthetic androgen R1881. Results from mining the Gene Logic BioExpress database showed that FKBP51 expression is significantly higher in the prostate cancer group than in the normal and normal adjacent group. Additionally, the androgen-independent prostate tumor xenograft, CWR22R, had higher FKBP51 protein levels than that of the androgen-dependent prostate tumor xenograft, CWR22. A tissue microarray study further revealed that FKBP51 protein expression was higher in prostate cancer specimens than in benign prostate tumor samples. These results suggest the potential value of FKBP51 as a novel diagnostic marker or target for prostate cancer therapy.

Intestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC-released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α, released by injured colonocytes, exacerbated experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α-positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1β. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and activated de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α-mediated IEC-fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. IL-1α may be a target for treating early IBD or preventing the reactivation of IBD.

BackgroundFibrosis of the intestine is a common and poorly understood complication of Crohn’s disease (CD) characterized by excessive deposition of extracellular matrix and accompanied by narrowing and obstruction of the gut lumen. Defining the molecular characteristics of this fibrotic disorder is a vital step in the development of specific prediction, prevention, and treatment strategies. Previous epigenetic studies indicate that alterations in DNA methylation could explain the mechanism by which mesenchymal cells adopt the requisite pro-fibrotic phenotype that promotes fibrosis progression. However, to date, genome-wide analysis of the DNA methylome of any type of human fibrosis is lacking. We employed an unbiased approach using deep sequencing to define the DNA methylome and transcriptome of purified fibrotic human intestinal fibroblasts (HIF) from the colons of patients with fibrostenotic CD.ResultsWhen compared with normal fibroblasts, we found that the majority of differential DNA methylation was within introns and intergenic regions and not associated with CpG islands. Only a low percentage occurred in the promoters and exons of genes. Integration of the DNA methylome and transcriptome identified regions in three genes that inversely correlated with gene expression: wingless-type mouse mammary tumor virus integration site family, member 2B (WNT2B) and two eicosanoid synthesis pathway enzymes (prostacyclin synthase and prostaglandin D2 synthase). These findings were independently validated by RT-PCR and bisulfite sequencing. Network analysis of the data also identified candidate molecular interactions relevant to fibrosis pathology.ConclusionsOur definition of a genome-wide fibrosis-specific DNA methylome provides new gene networks and epigenetic states by which to understand mechanisms of pathological gene expression that lead to fibrosis. Our data also provide a basis for development of new fibrosis-specific therapies, as genes dysregulated in fibrotic Crohn’s disease, following functional validation, can serve as new therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0193-6) contains supplementary material, which is available to authorized users.

Germline heterozygous PTEN mutations cause subsets of Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS); these subsets are characterized by high risks of breast, thyroid, and other cancers and, in one subset, autism spectrum disorder (ASD). Up to 10% of individuals with PTEN MUT CS, CS-like syndrome, or BRRS have germline SDHx (succinate dehydrogenase, mitochondrial complex II) variants, which modify cancer risk. PTEN contributes to metabolic reprogramming; this is a well-established role in a cancer context. Relatedly, SDH sits at the crossroad of the electron transport chain and tricarboxylic acid (TCA) cycle, two central bioenergetic pathways. Intriguingly, PTEN MUT and SDH MUT individuals have reduced SDH catalytic activity, resulting in succinate accumulation; this indicates a common genotype-independent biochemical alteration. Here, we conducted a TCA targeted metabolomics study on 511 individuals with CS, CS-like syndrome, or BRRS with various genotypes (PTEN or SDHx, mutant or wild type [WT]) and phenotypes (cancer or ASD) and a series of 187 population controls. We found consistent TCA cycle metabolite alterations in cases with various genotypes and phenotypes compared to controls, and we found unique correlations of individual metabolites with particular genotype-phenotype combinations. Notably, increased isocitrate (p ¼ 1.2 3 10 À3), but reduced citrate (p ¼ 5.0 3 10 À4), were found to be associated with breast cancer in individuals with PTEN MUT /SDHx WT. Conversely, increased lactate was associated with neurodevelopmental disorders regardless of genotype (p ¼ 9.7 3 10 À3); this finding was replicated in an independent validation series (n ¼ 171) enriched for idiopathic ASD (PTEN WT , p ¼ 5.6 3 10 À4). Importantly, we identified fumarate (p ¼ 1.9 3 10 À2) as a pertinent metabolite, distinguishing individuals who develop ASD from those who develop cancer. Our observations suggest that TCA cycle metabolite alterations are germane to the pathobiology of PTEN-related CS and BRRS, as well as genotype-independent ASD, with implications for potential biomarker and/or therapeutic value.

BACKGROUND & AIMS Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The chromosome-associated protein D3 (dCAP-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, whose products heterodimerize to form an amino acid transporter in HT-29 cells following bacterial infection; levels of SLC7A5–SLC3A2 were increased in tissues from patients with UC, compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5–SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3– deficient cells. CONCLUSIONS CAP-D3 downregulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with active UC; strategies to increase its levels might restore mucosal homeostasis to patients with active UC.

Background Fibrosis of the intestine is currently an irreversible complication of Inflammatory Bowel Disease yet little is understood of the underlying pathogenesis and anti-fibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen - a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (IL1-β, TNF-α and TGF-β) induces Collagen I alpha 2 (COL1A2) mRNA and protein. Methods Chromatin immunoprecipitation assays on pure cultures of human intestinal mucosal endothelial cells undergoing EndoMT were performed with antibodies to specific histone modifications and RNA polymerase II. RT-PCR was used to quantify the levels of Col1A2 and endothelial specific von Willebrand factor (vWF) mRNA. Results We show that cytokines induce selective chromatin modifications (histone 4 hyperacetylation and hypermethylation of histone 3) and phosphorylated RNA polymerase II at the COL1A2 promoter. Hypoacetylated and hypomethylated histone 3 was detected on the repressed vWF gene. Prolonged exposure to cytokines (16 days) retained hyperacetylation of select lysines in H4 on the COL1A2 promoter. Removal of cytokines after 16 days and continued culture for 10 days, showed persistent hyperacetylation at lysine 16 in histone H4. Conclusion This is the first study to show that COL1A2 gene expression is associated with cytokine-induced, temporally ordered and persistent chromatin modifications and suggests that these are important determinants of gene expression in EndoMT and intestinal fibrosis.