Stefan Lyer

Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-α, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-κB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn’s disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-κB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn’s disease.

DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands.

Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial‐binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS‐induced TLR4‐mediated NF‐κB activation and to the pathogenesis of Crohns disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen‐binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1‐mediated bacterial aggregation via interaction with its bacterial‐recognition motif. Competition and ELISA studies identify poly‐sulfated and poly‐phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose–response studies in Dmbt1−/− and Dmbt1+/+ mice utilizing the DSS‐induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern‐recognition molecule for poly‐sulfated and poly‐phosphorylated ligands providing a molecular basis for its broad bacterial‐binding specificity and its inhibitory effects on LPS‐induced TLR4‐mediated NF‐κB activation.

The SRCR/SID region of DMBT1 defines a complex multi-allele system representing the major basis for its variability in cancer.

Deleted in malignant brain tumors 1 (DMBT1) at 10q25.3–q26.1 has been proposed as a candidate tumor‐suppressor gene for brain and epithelial cancer. DMBT1 encodes a multifunctional mucin‐like protein presumably involved in epithelial differentiation and protection. The gene consists of highly homologous and repeating exon and intron sequences. This specifically applies to the region coding for the repetitive scavenger receptor cysteine‐rich (SRCR) domains and SRCR‐interspersed domains (SIDs) that constitutes the major part of the gene. This particular structure may previously have interfered with the delineation of DMBT1 alterations in cancer. Uncovering these, however, is of mechanistic importance. By a combined approach, we conducted a detailed mutational analysis, starting from a panel of 51 tumors, including 46 tumor cell lines and five primary tumors. Alterations in the repetitive region were present in 22/31 (71%) tumors that were investigated in detail. Six tumors showed presumably de novo mutations, among these three with point mutations in combination with a loss of heterozygosity. However, none of the alterations unambiguously would be predicted to lead to an inactivation of DMBT1. We define seven distinct DMBT1 alleles based on variable numbers of tandem repeats (VNTRs). At least 11 tumors exclusively harbored these VNTRs. The data suggest that the SRCR/SID region defines a complex multi‐allele system that has escaped previous analyses and that represents the major basis for the variability of DMBT1 in cancer. DMBT1 thus compares to mucins rather than to conventional tumor suppressors.

Carcinogen inducibility in vivo and down-regulation of DMBT1 during breast carcinogenesis

Deleted in malignant brain tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor for brain and epithelial cancer. Initial studies suggested loss of expression rather than mutation as the predominant mode of DMBT1 inactivation. However, in situ studies in lung cancer demonstrated highly sophisticated changes of DMBT1 expression and localization, pointing to a chronological order of events. Here we report on the investigation of DMBT1 in breast cancer in order to test whether these principles might also be attributable to other tumor types. Comprehensive mutational analyses did not uncover unambiguous inactivating DMBT1 mutations in breast cancer. Expression analyses in the human and mouse mammary glands pointed to the necessity of DMBT1 induction. While age‐dependent and hormonal effects could be ruled out, 9 of 10 mice showed induction of Dmbt1 expression after administration of the carcinogen 7,12‐dimethybenz(α)anthracene prior to the onset of tumorigenesis or other histopathological changes. DMBT1 displayed significant up‐regulation in human tumor–flanking tissues compared to in normal breast tissues (P < 0.05). However, the breast tumor cells displayed a switch from lumenal secretion to secretion to the extracellular matrix and a significant down‐regulation compared to that in matched normal flanking tissues (P < 0.01). We concluded that loss of expression also is the predominant mode of DMBT1 inactivation in breast cancer. The dynamic behavior of DMBT1 in lung carcinoma is fully reflected in breast cancer, which suggests that this behavior might be common to tumor types arising from monolayered epithelia.

Molecular characterisation of non-absorptive and absorptive enterocytes in human small intestine

Background and aims: Perturbation of differentiation of the crypt-villus axis of the human small intestine is associated with several intestinal disorders of clinical importance. At present, differentiation of small intestinal enterocytes in the crypt-villus axis is not well characterised. Subjects and methods: Expression profiling of microdissected enterocytes lining the upper part of crypts or the middle of villi was performed using the Affymetrix X3P arrays and several methods for confirmation. Results: A total of 978 differentially expressed sequences representing 778 unique UniGene IDs were found and categorised into four functional groups. In enterocytes lining the upper part of crypts, cell cycle promoting genes and transcription/translation related genes were predominantly expressed, whereas in enterocytes lining the middle of villi, high expression of cell cycle inhibiting genes, metabolism related genes, and vesicle/transport related genes was found. Conclusion: Two types of enterocytes were dissected at the molecular level, the non-absorptive enterocyte located in the upper part of crypts and the absorptive enterocyte found in the middle of villi. These data improve our knowledge about the physiology of the crypt-villus architecture in human small intestine and provide new insights into pathophysiological phenomena, such as villus atrophy, which is clinically important.

Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine

Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology. Screening of antibodies from a hybridoma library led to the identification of an acyl‐CoA synthetase 5‐specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl‐CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohns disease or coeliac disease, respectively. Acyl‐CoA synthetase 5 mRNA and protein levels were substantially reduced in injured small intestinal mucosa. Moreover, impaired synthesis of the acyl‐CoA synthetase 5 protein was reflected by a decrease in intramucosal enzyme activity. Subtle changes of the acyl‐CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl‐CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine. Copyright

Identification of a DMBT1 polymorphism associated with increased breast cancer risk and decreased promoter activity

According to present estimations, the unfavorable combination of alleles with low penetrance but high prevalence in the population might account for the major part of hereditary breast cancer risk. Deleted in Malignant Brain Tumors 1 (DMBT1) has been proposed as a tumor suppressor for breast cancer and other cancer types. Genomewide mapping in mice further identified Dmbt1 as a potential modulator of breast cancer risk. Here, we report the association of two frequent and linked single‐nucleotide polymorphisms (SNPs) with increased breast cancer risk in women above the age of 60 years: DMBT1 c.–93C>T, rs2981745, located in the DMBT1 promoter; and DMBT1 c.124A>C, p.Thr42Pro, rs11523871(odds ratio [OR]=1.66, 95% confidence interval [CI]=1.21–2.29, P=0.0017; and OR=1.66; 95% CI=1.21–2.28, P=0.0016, respectively), based on 1,195 BRCA1/2 mutation‐negative German breast cancer families and 1,466 unrelated German controls. Promoter studies in breast cancer cells demonstrate that the risk‐increasing DMBT1 –93T allele displays significantly decreased promoter activity compared to the DMBT1 –93C allele, resulting in a loss of promoter activity. The data suggest that DMBT1 polymorphisms in the 5′‐region are associated with increased breast cancer risk. In accordance with previous results, these data link decreased DMBT1 levels to breast cancer risk. Hum Mutat 30:1–7, 2009.

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression.

Small intestinal mucosa expression of putative chaperone fls485.

BackgroundMaturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa.Methodsfls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies.Resultsfls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c.ConclusionsExpression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.