The scavenging capacity of DMBT1 is impaired by germline deletions.

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20-45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.

Differential expression of apoptotic genes PDIA3 and MAP3K5 distinguishes between low- and high-risk prostate cancer.

BACKGROUND: Despite recent progress in the identification of genetic and molecular alterations in prostate cancer, markers associated with tumor progression are scarce. Therefore precise diagnosis of patients and prognosis of the disease remain difficult. This study investigated novel molecular markers discriminating between low and highly aggressive types of prostate cancer. RESULTS: Using 52 microdissected cell populations of low- and high-risk prostate tumors, we identified via global cDNA microarrays analysis almost 1200 genes being differentially expressed among these groups. These genes were analyzed by statistical, pathway and gene enrichment methods. Twenty selected candidate genes were verified by quantitative real time PCR and immunohistochemistry. In concordance with the mRNA levels, two genes MAP3K5 and PDIA3 exposed differential protein expression. Functional characterization of PDIA3 revealed a pro-apoptotic role of this gene in PC3 prostate cancer cells. CONCLUSIONS: Our analyses provide deeper insights into the molecular changes occurring during prostate cancer progression. The genes MAP3K5 and PDIA3 are associated with malignant stages of prostate cancer and therefore provide novel potential biomarkers.

Down-regulation of HLA Class I and NKG2D ligands through a concerted action of MAPK and DNA methyltransferases in colorectal cancer cells.

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.

Gene expression analysis of endobronchial epithelial lining fluid in the evaluation of indeterminate pulmonary nodules.

OBJECTIVE: Making a definitive preoperative diagnosis in patients with indeterminate pulmonary nodules is still a challenge. Gene expression profiling may be a useful adjunctive diagnostic utility in this regard. We investigated the feasibility of bronchoscopic microsampling to collect endobronchial epithelial lining fluid to obtain RNA as a starting point for gene expression profiling. METHODS: In 15 patients, epithelial lining fluid was collected in triplicate from subsegmental bronchi close to the pulmonary nodules and from contralateral lungs. Diagnosis was confirmed by transbronchial biopsy or surgery (non-small cell lung cancer, n = 11; benign or other lesions, n = 4). Total RNA was isolated from the samples and evaluated concerning quantity and quality. The complementary DNA was generated and analyzed by quantitative real-time polymerase chain reaction for potential lung cancer associated genes like matrix metalloprotinase (MMP9). RESULTS: Total RNA of adequate amount (>0.8 microg) and sufficient quality was obtained in 13 (86%) of the 15 patients. In patients with lung cancer, normalized MMP9 gene expression levels in endobronchial lining fluid samples collected close to the lesions were in median 12 times higher than levels in the matching contralateral samples. MMP9 expression levels were particularly high in endobronchial lining fluid samples collected from patients with squamous cell carcinoma but not elevated in the case of benign lesions. CONCLUSIONS: Our results show that quantitative gene expression analysis of endobronchial lining fluid collected by bronchoscopic microsampling is both feasible and reliable and may therefore be a useful additional diagnostic method in patients with indeterminate pulmonary nodules.

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-kappaB activation and to the pathogenesis of Crohn's 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-kappaB activation.

DMBT1 expression distinguishes anorectal from cutaneous melanoma.

AIMS: Anorectal melanoma (AM) forms a rare but highly malignant subset of mucosal melanoma with an extremely poor prognosis. Although AMs display histological and immunohistochemical features very similar to cutaneous melanoma (CM), no association exists either with exposure to ultraviolet light or with melanocytic naevi. While AMs are clearly distinguished from CM by displaying few BRAF mutations, they are commonly indistinguishable from CM at the level of gene expression. The aim was to carry out expression analyses of classical immunohistochemical markers and of the protein deleted in malignant brain tumours 1 (DMBT1) in cases of primary anorectal malignant melanoma and CM. METHODS AND RESULTS: Expression analyses of classical immunohistochemical markers (S100, HMB45, Melan A and MiTF) and of the protein DMBT1 were carried out in 27 cases of primary anorectal malignant melanoma and 26 cases of CM. All AM cases analysed showed expression of at least three of the classical markers for melanoma. However, immunohistochemistry showed 19 out of 27 AM to be positive for DMBT1, which represented a statistically significant difference (P = 0.0009) compared with CM (six out of 26), which more commonly are negative for DMBT1 expression. CONCLUSION: These results identify DMBT1 as a molecular feature that may allow distinction between AM and CM and support the notion that AM represents an entity molecularly distinct from CM.

Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance.

BACKGROUND: In breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer. RESULTS: First, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab), demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation. CONCLUSION: In this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not have potential for treatment of de novo trastuzumab resistant cells. Instead, c-MYC was identified as a novel potential target protein in breast cancer cells.

Reverse-phase protein arrays for application-orientated cancer research.

A detailed and quantitative analysis of disease-relevant signaling will greatly contribute to our understanding of tumorigenesis and cancer progression, and thus open new strategies for drug discovery. However, throughput and sensitivity of currently established methods available for proteome profiling do not comply with the needs of clinical research such as high sample capacity and low sample consumption. Protein microarrays emerged as a promising alternative to analyze the abundance of proteins and their phosphorylation status on a high-throughput level. Here we summarize recent methodological advancements in the field of reverse-phase protein arrays and demonstrate their potential for clinical research as well as for in vitro applications.

Effects of infiltrating lymphocytes and estrogen receptor on gene expression and prognosis in breast cancer.

The involvement of the immune system for the course of breast cancer, as evidenced by varying degrees of lymphocyte infiltration (LI) into the tumor is still poorly understood. The aim of this study was to evaluate the prognostic value of LI in breast cancer samples using microarray-based screening for LI-associated genes. Starting from the observation that most published ER gene signatures are heavily influenced by the LI effect, we developed and applied a novel approach to dissect molecular signatures. Further, a meta-analysis encompassing 1,044 hybridizations showed that LI alone is not sufficient to highlight breast cancer patients with different prognosis. However, for ER positive patients, high LI was associated with shorter survival times, whereas for ER negative patients, high LI is significantly associated with longer survival. Annotation of LI, in addition to ER status, is important for breast cancer patient prognosis and may have implications for the future treatment of breast cancer.

Molecular cancer phenotype in normal prostate tissue.

BACKGROUND: Insufficient sensitivity and specificity of prostate biopsies for cancer detection. OBJECTIVES: Based on evidence from our microarray analyses, we hypothesized that considerable molecular changes precede morphologically detectable malignant transformation of prostate epithelial tissues. The identification of such changes could lead to novel strategies in the clinical management of prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: Histologically normal, fresh prostate tissue from prostate cancer patients, healthy donors, and cancer suspect patients with continuous negative biopsies were analyzed. MEASUREMENTS: To identify molecular changes between 29 tumor-free prostate tissues from healthy donors and 27 patients with proven prostate cancer, we performed a global microarray screening. Based on this screening as well as literature data, we selected a subset of 29 genes for validation by arrayed real-time reverse transcription-polymerase chain reaction (RT-PCR) using histologically tumor-free biopsy samples from 114 patients representing three prostate cancer risk groups. RESULTS AND LIMITATIONS: We identified five genes (FOS, EGR1, MYC, TFRC, and FOLH1), which displayed significant differential expression between morphologically normal prostate tissues from men of each of the three risk groups. These results were independent from age, prostate-specific antigen (PSA), frequency and timing of previous prostate biopsies, tissue composition, tumor stage, and tumor grade. In univariate logistic regression analyses, the transcript levels of these genes were found to be highly indicative for the presence or absence of cancer in the entire prostate. The study was designed as a proof of principle. The clinical relevance of our results has to be evaluated in a larger clinical setting. CONCLUSIONS: Our results suggest a measurable molecular cancer phenotype in histologically normal prostate tissue indicating the presence of prostate cancer elsewhere in the organ.

Global gene expression analysis reveals specific patterns of cell junctions in non-small cell lung cancer subtypes.

Non-small cell lung cancer (NSCLC) can be classified into the major subtypes adenocarcinoma (AC) and squamous cell carcinoma (SCC). Although explicit molecular, histological and clinical characteristics have been reported for both subtypes, no specific therapy exists so far. However, the characterization of suitable molecular targets holds great promises to develop novel therapies in NSCLC. In the present study, global gene expression profiling of 58 human NSCLC specimens revealed large transcriptomic differences between AC and SCC subtypes: more than 1700 genes were found to be differentially expressed. The assignment of these genes to biological processes pointed to the deregulation of distinct sets of genes coding for cell junctions in both tumor subtypes. We focused on 17 cell adhesion genes and 11 reported marker genes for epithelial-mesenchymal transition (EMT), and investigated their expression in matched tumor-normal specimens by quantitative real-time PCR. The majority of the cell adhesion genes was significantly up-regulated in at least one tumor subtype compared to normal tissue, predominantly desmosomes and gap junctions in SCC, and tight junctions in AC. The higher expression of EMT marker transcripts in tumor specimens suggested a large potential for invasion and migration processes in NSCLC. Our results indicate that AC and SCC in the lung are characterized by the expression of distinct sets of cell adhesion molecules which may represent promising targets for novel specific therapies.

Genetic variation of Aflatoxin B1 aldehyde reductase genes (AFAR) in human tumour cells.

AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furthermore, human AFAR1 catalyses the rate limiting step in the synthesis of the neuromodulator gamma-hydroxybutyrate (GHB) and was found elevated in neurodegenerative diseases such as Alzheimer's and dementia with Lewy bodies (DLB). The human AFAR gene family maps to a genomic region in 1p36 of frequent hemizygous deletions in various human cancers. To investigate, if genetic variation of AFAR1 and AFAR2 exists that may alter protein detoxification capabilities and confer susceptibility to cancer, we have analysed a spectrum of human tumours and tumour cell lines for genetic heterogeneity. From 110 DNA samples, we identified nine different amino acid changes; two were in AFAR1 and seven in AFAR2. In AFAR1, we found genetic variation in the proposed substrate-binding amino acid 113, encoding Ala(113) or Thr(113). An AFAR2 variant had a Glu(55) substituted by Lys(55) at a position that is conserved among many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related.

Contact spotting of protein microarrays coupled with spike-in of normalizer protein permits time-resolved analysis of ERBB receptor signaling.

Protein microarrays allow highly accurate comparison and quantification of numerous biological samples in parallel while requiring only little material. This qualifies protein arrays for systems biology and clinical research where only limited sample material is available, but a precise readout is required. With the introduction of signal normalization steps to monitor the drop size of manually contact-spotted RP protein arrays, the usefulness of normalizer proteins to ensure a high-throughput but inexpensive protein analysis was demonstrated. This approach was applied for the analysis of signaling through ERBB receptor activated kinases in the breast cancer cell line MCF-7. Activation of ERK1/2 and AKT by ERBB1 (EGFR), ERRB2 (HER2/neu), and ERBB3-4 was monitored in a time-resolved manner. Analysis of pathway activation by stimulation with epidermal growth factor and heregulin, or inhibition by blocking with gefitinib or herceptin allowed a characterization of the distinct signaling properties of the different ERBB receptor subtypes.

Estimating large-scale signaling networks through nested effect models with intervention effects from microarray data.

MOTIVATION: Targeted interventions using RNA interference in combination with the measurement of secondary effects with DNA microarrays can be used to computationally reverse engineer features of upstream non-transcriptional signaling cascades based on the nested structure of effects. RESULTS: We extend previous work by Markowetz et al., who proposed a statistical framework to score different network hypotheses. Our extensions go in several directions: we show how prior assumptions on the network structure can be incorporated into the scoring scheme by defining appropriate prior distributions on the network structure as well as on hyperparameters. An approach called module networks is introduced to scale up the original approach, which is limited to around 5 genes, to infer large-scale networks of more than 30 genes. Instead of the data discretization step needed in the original framework, we propose the usage of a beta-uniform mixture distribution on the P-value profile, resulting from differential gene expression calculation, to quantify effects. Extensive simulations on artificial data and application of our module network approach to infer the signaling network between 13 genes in the ER-alpha pathway in human MCF-7 breast cancer cells show that our approach gives sensible results. Using a bootstrapping and a jackknife approach, this reconstruction is found to be statistically stable. AVAILABILITY: The proposed method is available within the Bioconductor R-package nem.

Marked gene transcript level alterations occur early during radical prostatectomy.

OBJECTIVES: Gene expression analyses have become an important approach to understand the biology of cancer. However, transcript level patterns and RNA quality could rapidly change in response to ischemic and mechanical stress. Studies have shown that this occurs both perioperatively and after surgical removal of organs. METHODS: To better understand the relative importance of perioperative and postoperative gene expression changes, we performed quantitative reverse transcription-polymerase chain reactions on the transcripts of 91 cancer-related genes from normal and cancerous prostate tissues from 10 patients at eight different time points during surgical manipulation and after removal of the prostate. RESULTS: The mRNA levels of 8 (EGR1, p21, KRT17, PIM1, S100P, TNFRSF, WFDC2, and TRIM29) of 91 genes changed significantly with time of surgery in normal and tumor tissue. Remarkably, all eight genes were up-regulated, a reaction that was most prominent during the early intraoperative period. Additional changes occurred but were much less prominent during the first postoperative hour. CONCLUSIONS: Our results substantially challenge the utility of immediate postoperative tissue sampling. At least for prostate cancer, the data suggest that preoperative tissue collection by core biopsies is optimal for studying molecular changes in normal and neoplastic prostate tissues.

Infrared-based protein detection arrays for quantitative proteomics.

BACKGROUND: Protein microarrays have the potential to join the field of quantitative proteomics as a standard method. This antibody-based experimental platform allows the highly sensitive and highly specific quantification of selected target proteins and excels with high sample capacity. As a key feature, numerous arrays can be produced in parallel, thus minimizing sample consumption. OBJECTIVE: The recent progress made in the field of reverse phase protein arrays is summarized, with a focus on the introduction of normalization measures, as introduced in the authors' infrared-based protein arrays with quantitative readout approach. CONCLUSION: Tumor biology as well as drug discovery applications will soon benefit from the comprehensive description of signaling pathways and protein microarrays are an appropriate tool to achieve this goal.

DMBT1 confers mucosal protection in vivo and a deletion variant is associated with Crohn's disease.

BACKGROUND & AIMS: Impaired mucosal defense plays an important role in the pathogenesis of Crohn's disease (CD), one of the main subtypes of inflammatory bowel disease (IBD). Deleted in malignant brain tumors 1 (DMBT1) is a secreted scavenger receptor cysteine-rich protein with predominant expression in the intestine and has been proposed to exert possible functions in regenerative processes and pathogen defense. Here, we aimed at analyzing the role of DMBT1 in IBD. METHODS: We studied DMBT1 expression in IBD and normal tissues by quantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and mRNA in situ hybridization. Genetic polymorphisms within DMBT1 were analyzed in an Italian IBD case-control sample. Dmbt1(-/-) mice were generated, characterized, and analyzed for their susceptibility to dextran sulfate sodium-induced colitis. RESULTS: DMBT1 levels correlate with disease activity in inflamed IBD tissues. A highly significant fraction of the patients with IBD displayed up-regulation of DMBT1 specifically in the intestinal epithelial surface cells and Paneth cells. A deletion allele of DMBT1 with a reduced number of scavenger receptor cysteine-rich domain coding exons is associated with an increased risk of CD (P = .00056; odds ratio, 1.75) but not for ulcerative colitis. Dmbt1(-/-) mice display enhanced susceptibility to dextran sulfate sodium-induced colitis and elevated Tnf, Il6, and Nod2 expression levels during inflammation. CONCLUSIONS: DMBT1 may play a role in intestinal mucosal protection and prevention of inflammation. Impaired DMBT1 function may contribute to the pathogenesis of CD.

Large scale statistical inference of signaling pathways from RNAi and microarray data.

BACKGROUND: The advent of RNA interference techniques enables the selective silencing of biologically interesting genes in an efficient way. In combination with DNA microarray technology this enables researchers to gain insights into signaling pathways by observing downstream effects of individual knock-downs on gene expression. These secondary effects can be used to computationally reverse engineer features of the upstream signaling pathway. RESULTS: In this paper we address this challenging problem by extending previous work by Markowetz et al., who proposed a statistical framework to score networks hypotheses in a Bayesian manner. Our extensions go in three directions: First, we introduce a way to omit the data discretization step needed in the original framework via a calculation based on p-values instead. Second, we show how prior assumptions on the network structure can be incorporated into the scoring scheme using regularization techniques. Third and most important, we propose methods to scale up the original approach, which is limited to around 5 genes, to large scale networks. CONCLUSION: Comparisons of these methods on artificial data are conducted. Our proposed module network is employed to infer the signaling network between 13 genes in the ER-alpha pathway in human MCF-7 breast cancer cells. Using a bootstrapping approach this reconstruction can be found with good statistical stability. The code for the module network inference method is available in the latest version of the R-package nem, which can be obtained from the Bioconductor homepage.

Deleted in Malignant Brain Tumors 1 (DMBT1) is present in hyaline membranes and modulates surface tension of surfactant.

BACKGROUND: Deleted in Malignant Brain Tumors 1 (DMBT1) is a secreted scavenger receptor cysteine-rich protein that binds various bacteria and is thought to participate in innate pulmonary host defense. We hypothesized that pulmonary DMBT1 could contribute to respiratory distress syndrome in neonates by modulating surfactant function. METHODS: DMBT1 expression was studied by immunohistochemistry and mRNA in situ hybridization in post-mortem lungs of preterm and full-term neonates with pulmonary hyaline membranes. The effect of human recombinant DMBT1 on the function of bovine and porcine surfactant was measured by a capillary surfactometer. DMBT1-levels in tracheal aspirates of ventilated preterm and term infants were determined by ELISA. RESULTS: Pulmonary DMBT1 was localized in hyaline membranes during respiratory distress syndrome. In vitro addition of human recombinant DMBT1 to the surfactants increased surface tension in a dose-dependent manner. The DMBT1-mediated effect was reverted by the addition of calcium depending on the surfactant preparation. CONCLUSION: Our data showed pulmonary DMBT1 expression in hyaline membranes during respiratory distress syndrome and demonstrated that DMBT1 increases lung surface tension in vitro. This raises the possibility that DMBT1 could antagonize surfactant supplementation in respiratory distress syndrome and could represent a candidate target molecule for therapeutic intervention in neonatal lung disease.