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Heat shock protein 90-α (Hsp90α) is an intracellular molecular chaperone. However, it can also be secreted with the underlying regulatory mechanism remaining far from clear. Here we show that the secreted Hsp90α is a C-terminal truncated form and its secretion is regulated by the C-terminal EEVD motif via interacting with proteins containing tetratricopeptide repeat domains. We also demonstrate that secretion of Hsp90α is determined by the phosphorylation status at residue Thr-90, regulated by protein kinase A and protein phosphatase 5. We further demonstrate that the secretion of Hsp90α is a prerequisite for its proinvasiveness function and blocking the secreted Hsp90α results in significant inhibition of tumor metastasis. Meanwhile, the level of plasma Hsp90α is positively correlated with tumor malignancy in clinical cancer patients. In sum, our results reveal the regulatory mechanism of Hsp90α secretion, and its function in tumor invasiveness, indicating it can be a promising diagnostic marker for tumor malignancy in clinical application.

Abnormalities of the intestinal microbiota are implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC), two spectra of inflammatory bowel disease (IBD). However, the high complexity and low inter-individual overlap of intestinal microbial composition are formidable barriers to identifying microbial taxa representing this dysbiosis. These difficulties might be overcome by an ecologic analytic strategy to identify modules of interacting bacteria (rather than individual bacteria) as quantitative reproducible features of microbial composition in normal and IBD mucosa. We sequenced 16S ribosomal RNA genes from 179 endoscopic lavage samples from different intestinal regions in 64 subjects (32 controls, 16 CD and 16 UC patients in clinical remission). CD and UC patients showed a reduction in phylogenetic diversity and shifts in microbial composition, comparable to previous studies using conventional mucosal biopsies. Analysis of weighted co-occurrence network revealed 5 microbial modules. These modules were unprecedented, as they were detectable in all individuals, and their composition and abundance was recapitulated in an independent, biopsy-based mucosal dataset 2 modules were associated with healthy, CD, or UC disease states. Imputed metagenome analysis indicated that these modules displayed distinct metabolic functionality, specifically the enrichment of oxidative response and glycan metabolism pathways relevant to host-pathogen interaction in the disease-associated modules. The highly preserved microbial modules accurately classified IBD status of individual patients during disease quiescence, suggesting that microbial dysbiosis in IBD may be an underlying disorder independent of disease activity. Microbial modules thus provide an integrative view of microbial ecology relevant to IBD.

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 β1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.

Crohn's disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype. Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry. CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella. Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn's disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium, compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn's disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls. Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn's disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.

Fucosyltransferase 2 ( FUT2 ) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene ( sese ), have increased susceptibility to Crohn’s disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe , 18 Sese and 9 sese ). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2 −/− genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.

Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1). ALDH expression significantly promotes tumor initiation and correlates with the levels of EMP2 expression in both patient samples and tumor cell lines. As therapy against cancer stem cells in endometrial cancer is lacking, the ability of anti-EMP2 IgG1 therapy to reduce primary and secondary tumor formation using xenograft HEC1A models was determined. Anti-EMP2 IgG1 reduced the expression and activity of ALDH and correspondingly reduced both primary and secondary tumor load. Our results collectively suggest that anti-EMP2 therapy may be a novel method of reducing endometrial cancer stem cells.

Background Crohn’s disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype. Methods Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry. Results CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella . Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn’s disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium , compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn’s disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls. Conclusions Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn’s disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.

Dysbiosis has been demonstrated in Crohn's disease (CD) patients using feces, colonic biopsies, and ileal biopsies. The mucosa-associated microbiome has been reported to be more predictive of disease than the fecal microbiome. However, biopsies as a sampling method for microbiome studies are limited by the difficulty of characterizing bacterial proteins and metabolites, which are masked by the preponderance of human material. We have previously demonstrated that lavage sampling of the mucosal-luminal interface (MLI) - representing the surface loose colonic mucus and adherent microbes after bowel preparation - yields suitable material for bacterial proteomics and metabolomics. We undertook a study to characterize the MLI microbiome of CD patients relative to healthy controls and its relationship to disease phenotype, location, and genetics.MethodsEighty-eight CD patients and 110 healthy controls undergoing colonoscopy were recruited at the Cedars-Sinai Medical Center. All CD patients had mucosal healing by endoscopic appearance. The mucosal surface of the cecum and sigmoid colon were rinsed with sterile saline during colonoscopy. DNA was extracted from pelleted microbes by bead beating and the V4 region of 16S ribosomal RNA was sequenced by HiSeq to a mean depth of 575,708 sequences/sample. Alpha and beta diversity analysis were performed in QIIME. Microbial abundances were fitted to negative binomial models for the cecum and sigmoid colon data. Diagnosis (CD versus healthy), disease behavior, disease location, sex, obesity, and CD genetic risk score (an aggregate measure calculated from IBD-associated polymorphisms genotyped by Immunochip) were covariates in these models. Random forests classifiers were used to predict CD status.ResultsCD patients had lower microbial diversity and altered composition compared to controls in both the cecal and sigmoid MLI microbiome. High-depth sequencing revealed 759 differential operational taxonomic units (OTUs) in the sigmoid and 687 in the cecum between CD and controls, predominantly representing enrichment of members of the Proteobacteria phylum and depletion of Firmicutes. Random forests classifiers based on microbial composition had high accuracy for CD diagnosis, with area under the curve of 0.91 in the sigmoid colon and 0.93 in the cecum. B3 (penetrating) and B2 (stricturing) phenotypes were associated with 329 and 321 differential OTUs, respectively, in the cecum relative to B1. Location had a comparatively small effect, with 31 differential OTUs in the cecum between L1 (ileal) and L3 (ileocolonic) and 55 between L2 (colonic) and L3. Healthy controls in the highest quartile of CD genetic risk were found to have 229 differential features in a pooled analysis of cecum and sigmoid, with 88 showing concordant changes in CD versus healthy including depletion of Faecalibacterium prausnitzii.ConclusionsCD had a distinctive MLI microbiome that differentiated patients by disease behavior and location. We further demonstrated that genetic risk factors for CD influenced microbial composition in healthy controls, supporting recent findings from studies of IBD-associated single nucleotide polymorphisms in genes such as FUT2 and NOD2 that have indicated that genetic variation can garden the microbiome. These results support MLI sampling as a strategy for studies investigating the mucosal microbiome of IBD patients.

Inflammatory bowel disease (IBD) is a set of chronic, relapsing inflammatory diseases of the intestine. The two major subtypes of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Although the pathogenesis of IBD remains largely unknown, Crohn's disease is considered to result from the interaction of environmental factors, including intestinal microbiota, with host immune mechanisms in genetically susceptible individuals. Recent advances in sequencing technologies have allowed us to characterize the IBD associated dysbiosis in unprecedented depth. However, phylogenetic profiling can only provide limited information on the functional implication of these alterations. To address this analytical challenge, we developed the novel mucosal lavage sampling approach, which enabled the profiling of multi'omic molecular features including microbiome, metaproteome and metabolome. Combined with host genomic information, these tools can provide us with unprecedented understanding of the dynamics of host-microbial interaction, and help us to investigate the pathogenesis of inflammatory bowel diseases. Another analytical challenge to identify microbial taxa consistently representing IBD associated dysbiosis is the high complexity and low inter-individual overlap of intestinal microbial composition. This difficulty can be overcome by an ecologic analytic strategy to identify modules of interacting bacteria (rather than individual bacteria) as quantitative reproducible features of microbial composition in normal and IBD mucosa. We developed the strategy to analyze microbial composition using microbial co-occurrence network approach. This strategy uncovered 5 reproducible functional microbial communities (FMCs) detectable in the mucosa of all individuals. The quantitative levels of two FMCs were significantly associated with IBD states. Imputed metagenome analysis indicated the functional importance of the disease associated modules reflected by the enrichment of virulent and pathogenic pathways. Thus, these modules appear to define novel microbial communities within the intestinal microbial ecology, some of which are commonly and stably modified by the IBD disease state, and may be of particular relevance for microbial pathogenesis and intervention. Using this experimental and bioinformatic framework, we investigated the microbial gardening effect of FUT2 gene and its link to Crohn's disease. Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease. In healthy individuals, imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism, and glycan biosynthesis and metabolism related pathways; and, the depletion of amino acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2-/- genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. In an extended cohort containing both healthy and CD individuals, the phylogenetic composition of intestinal mucosal microbiota was affected by an interaction of Crohn's disease status and FUT2 genotype. Decreased abundances of Firmicutes were associated with both CD and FUT2 risk allele. At metagenomic level, a distinct signature of amino acid metabolism deficiency was identified in CD and non-secretor microbiome. Such changes were also reflected at metabolomic level in the proximal gut region. Taken together, FUT2 gene increased the risk of Crohn's disease by changing the microbial composition and function to a disease-like state. The CD associated perturbations of metagenome and metabolome were driven by the FUT2 risk allele. The same experimental and bioinformatic approach can also be applied to study the composition and functional changes of mucosal associated microbiota in other chronic inflammatory disease, namely HIV-1 infection. In the rectal mucosa, microbial composition and imputed function in HIV-positive individuals not receiving cART was significantly different from HIV-negative individuals. Genera including Roseburia, Coprococcus, Ruminococcus, Eubacterium, Alistipes and Lachnospira were depleted in HIV-infected subjects not receiving cART, while Fusobacteria, Anaerococcus, Peptostreptococcus , and Porphyromonas were significantly enriched. HIV-positive subjects receiving cART exhibited similar depletion and enrichment for these genera, but were of intermediate magnitude and did not achieve statistical significance. Imputed metagenomic functions, including amino acid metabolism, vitamin biosynthesis, and siderophore biosynthesis differed significantly between healthy controls and HIV-infected subjects not receiving cART. In the cervicovaginal mucosa, significant differences in alpha and beta diversity were observed between HIV-negative and HIV-positive women, with the latter enriched of organisms associated with bacterial vaginosis and depleted of Lactobacilli. These ecologic changes occurred concomitantly with significant metagenomic and immunologic differences. Such functional pathways may represent novel interventional targets for HIV therapy if normalizing the microbial composition or functional activity of the microbiota proves therapeutically useful.

Heat shock protein 90-α (Hsp90α) is an intracellular molecular chaperone. However, it can also be secreted with the underlying regulatory mechanism remaining far from clear. Here we show that the secreted Hsp90α is a C-terminal truncated form and its secretion is regulated by the C-terminal EEVD motif via interacting with proteins containing tetratricopeptide repeat domains. We also demonstrate that secretion of Hsp90α is determined by the phosphorylation status at residue Thr-90, regulated by protein kinase A and protein phosphatase 5. We further demonstrate that the secretion of Hsp90α is a prerequisite for its proinvasiveness function and blocking the secreted Hsp90a results in significant inhibition of tumor metastasis. Meanwhile, the level of plasma Hsp90α is positively correlated with tumor malignancy in clinical cancer patients. In sum, our results reveal the regulatory mechanism of Hsp90α secretion, and its function in tumor invasiveness, indicating it can be a promising diagnostic marker for tumor malignancy in clinical application. [PUBLICATION ABSTRACT]