Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1

Aberrant transforming growth factor–β (TGF-β) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-β signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-β signaling activity and that stromal cellconditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-β–induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment

TGF-B induced protein IG-H3 is essential for the growth of human liver metastases

Introduction : Transforming growth factor-beta-induced protein ig-h3 (TGFBI) is extracellular matrix component known to be important for cell-collagen interaction. We and others have reported elevated expression of TGFBI in sev- eral human cancers, where its role remains controversial. Aim Current study aims at clarifying the function of TGFBI to date. Methods &Results : CRC-LM and in liver metastases originating from breast, lung and pancreatic tumors. We have next focused on func- tional aspects and have silenced TGFBI expression in SW1222 human colorectal carcinoma cells. The suppression of TGFBI protein led to a marked decrease in cell migration (-70%) and proliferation (-30%) in vitro. To study the effects in vivo we have developed a novel animal model of colorectal carcinoma based on chicken chorioallantoic membrane (CAM) that mimics human CRC-LM. TGFBI silencing resulted in 50% reduction of tumor volume in the CAM tumor model. Notably, the tumors displayed a marked inhibition of vascularization, suggesting an additional anti-angiogenic effect. Indeed, SW1222 cells silenced for TGFBI expression secreted lower levels of VEGFA in vitro. Finally, we have investigated if TGFBI can be used as systemically reachable target for antibody-drug delivery. For this purpose we have The in vivo data demonstrated that TGFBI is an accessible tumor target. Conclusions : Taken together, the present study shows that TGFBI is essential for promoting the development of CRC- LM and therefore represents a promising target for designing novel therapeutic approaches

SPARC regulates transforming growth factor beta induced (TGFBI) extracellular matrix deposition and paclitaxel response in ovarian cancer cells

TGFBI has been shown to sensitize ovarian cancer cells to the cytotoxic effects of paclitaxel via an integrin receptor-mediated mechanism that modulates microtubule stability. Herein, we determine that TGFBI localizes within organized fibrillar structures in mesothelial-derived ECM. We determined that suppression of SPARC expression by shRNA decreased the deposition of TGFBI in mesothelial-derived ECM, without affecting its overall protein expression or secretion. Conversely, overexpression of SPARC increased TGFBI deposition. A SPARC-YFP fusion construct expressed by the Met5a cell line co-localized with TGFBI in the cell-derived ECM. Interestingly, in vitro produced SPARC was capable of precipitating TGFBI from cell lysates dependent on an intact SPARC carboxy-terminus with in vitro binding assays verifying a direct interaction. The last 37 amino acids of SPARC were shown to be required for the TGFBI interaction while expression of a SPARC-YFP construct lacking this region (aa 1–256) did not interact and co-localize with TGFBI in the ECM. Furthermore, ovarian cancer cells have a reduced motility and decreased response to the chemotherapeutic agent paclitaxel when plated on ECM derived from mesothelial cells lacking SPARC compared to control mesothelial-derived ECM. In conclusion, SPARC regulates the fibrillar ECM deposition of TGFBI through a novel interaction, subsequently influencing cancer cell behavior

TGFBI promoter hypermethylation correlating with paclitaxel chemoresistance in ovarian cancer

The purpose of this study is to determine the methylation status of Transforming growth factor-beta-inducible gene-h3 (TGFBI) and its correlation with paclitaxel chemoresistance in ovarian cancer. The methylation status of TGFBI was examined in ovarian cancer and control groups by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). The TGFBI expression and cell viability were compared by Quantitative Real-Time PCR, Western Blotting and MTT assay before and after demethylating agent 5-aza-2'-deoxycytidine (5-aza-dc) treatment in 6 cell lines (SKOV3, SKOV3/TR, SKOV3/DDP, A2780, 2780/TR, OVCAR8). In our results, TGFBI methylation was detected in 29/40 (72.5%) of ovarian cancer and 1/10 (10%) of benign ovarian tumors. No methylation was detected in normal ovarian tissues (P < 0.001). No statistical correlation between RUNX3 methylation and clinicopathological characteristics was observed. A significant correlation between TGFBI methylation and loss of TGFBI mRNA expression was found (P < 0.001). The methylation level of TGFBI was significantly higher in paclitaxel resistant cell lines (SKOV3/TR and 2780/TR) than that in the sensitive pairs (P < 0.001). After 5-aza-dc treatment, the relative expression of TGFBI mRNA and protein increased significantly in SKOV3/TR and A2780/TR cells. However, no statistical differences of relative TGFBI mRNA expression and protein were found in other cells (all P > 0.05), which showed that re-expression of TGFBI could reverse paclitaxel chemoresistance. Our results show that TGFBI is frequently methylated and associated with paclitaxel-resistance in ovarian cancer. TGFBI might be a potential therapeutic target for the enhancement of responses to chemotherapy in ovarian cancer patients

Genotype-Phenotype Correlation for TGFBI Corneal Dystrophies Identifies p.(G623D) as a Novel Cause of Epithelial Basement Membrane Dystrophy.

Purpose: The majority of anterior corneal dystrophies are caused by dominant mutations in TGFBI (transforming growth factor β-induced) collectively known as the epithelial-stromal TGFBI dystrophies. Most cases of epithelial basement membrane dystrophy (EBMD) are thought to result from a degenerative (nongenetic) process; however, a minority of cases are associated with specific TGFBI mutations. We evaluated the spectrum of TGFBI mutations and associated phenotypes in a United Kingdom cohort with typical epithelial-stromal TGFBI dystrophies and an EBMD cohort. Methods: We recruited 68 probands with a clinical diagnosis of epithelial-stromal TGFBI dystrophy and 23 probands with bilateral EBMD. DNA was extracted from peripheral leukocytes, and TGFBI was bi-directly Sanger sequenced. Results: Nine TGFBI mutations were identified. The most common occurred at the mutation hot-spot residues R124 and R555 in 61 probands; these individuals had a genotype-phenotype correlation consistent with prior reports. Four probands with lattice corneal dystrophy carried a mutation in exon 14: p.(A620D), p.(V625D), and p.(H626R). We identified a p.(G623D) mutation in five probands, including two probands from the EBMD cohort. These subjects typically had an onset of severe recurrent corneal epithelial erosion in the fourth decade with mild diffuse or geographic subepithelial corneal opacities and only small anterior stromal lattice structures in older individuals. Symptoms of painful epithelial erosion improved markedly following phototherapeutic keratectomy. Conclusions: There was a strong correlation between genotype and phenotype for the majority of TGFBI mutations. In this cohort, the p.(G623D) mutation caused a greater proportion of TGFBI-associated disease than anticipated, associated with variable phenotypes including individuals diagnosed with EBMD

Progestins Related to Progesterone and Testosterone Elicit Divergent Human Endometrial Transcriptomes and Biofunctions.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use

TGFBI expression is associated with a better response to chemotherapy in NSCLC

<p>Abstract</p> <p>Background</p> <p>Lung cancer is one of the most prevalent neoplasias in developed countries. Advances in patient survival have been limited and the identification of prognostic molecules is needed. Resistance to treatment is strongly related to tumor cell adhesion to the extracellular matrix and alterations in the quantity and nature of molecules constituting the tumor cell niche. Recently, transforming growth factor beta-induced protein (TGFBI), an extracellular matrix adaptor protein, has been reported to be differentially expressed in transformed tissues. Loss of TGFBI expression has been described in several cancers including lung carcinoma, and it has been suggested to act as a tumor suppressor gene.</p> <p>Results</p> <p>To address the importance of TGFBI expression in cancer progression, we determined its expression in NSCLC clinical samples using immunohistochemistry. We identified a strong association between elevated TGFBI expression and the response to chemotherapy. Furthermore, we transiently over-expressed and silenced TGFBI in human NSCLC cell lines. Cells over-expressing TGFBI displayed increased sensitivity to etoposide, paclitaxel, cisplatin and gemcitabine. We observed that TGFBI-mediated induction of apoptosis occurred through its binding to αvβ3 integrin. We also determined that full-length TGFBI did not induce caspase 3/7 activation but its proteolytic fragments that were < 3 kDa in size, were able to activate caspase 3, 7 and 8. This pro-apoptotic effect was blocked by anti-αvβ3 integrin antibodies.</p> <p>Conclusions</p> <p>The results shown here indicate that TGFBI is a predictive factor of the response to chemotherapy, and suggest the use of TGFBI-derived peptides as possible therapeutic adjuvants for the enhancement of responses to chemotherapy.</p

Identification of presumed pathogenic KRT3 and KRT12 gene mutations associated with Meesmann corneal dystrophy.

PurposeTo report potentially pathogenic mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes in two individuals with clinically diagnosed Meesmann corneal dystrophy (MECD).MethodsSlit-lamp examination was performed on the probands and available family members to identify characteristic features of MECD. After informed consent was obtained, saliva samples were obtained as a source of genomic DNA, and screening of KRT3 and KRT12 was performed. Potentially pathogenic variants were screened for in 200 control chromosomes. PolyPhen-2, SIFT, and PANTHER were used to predict the functional impact of identified variants. Short tandem repeat genotyping was performed to confirm paternity.ResultsSlit-lamp examination of the first proband demonstrated bilateral, diffusely distributed, clear epithelial microcysts, consistent with MECD. Screening of KRT3 revealed a heterozygous missense variant in exon 1, c.250C&gt;T (p.(Arg84Trp)), which has a minor allele frequency of 0.0076 and was not identified in 200 control chromosomes. In silico analysis with PolyPhen-2 and PANTHER predicted the variant to be damaging to protein function; however, SIFT analysis predicted tolerance of the variant. The second proband demonstrated bilateral, diffusely distributed epithelial opacities that appeared gray-white on direct illumination and translucent on retroillumination. Neither parent demonstrated corneal opacities. Screening of KRT12 revealed a novel heterozygous insertion/deletion variant in exon 6, c.1288_1293delinsAGCCCT (p.(Arg430_Arg431delinsSerPro)). This variant was not present in either of the proband's parents or in 200 control chromosomes and was predicted to be damaging by PolyPhen-2, PANTHER, and SIFT. Haplotype analysis confirmed paternity of the second proband, indicating that the variant arose de novo.ConclusionsWe present a novel KRT12 mutation, representing the first de novo mutation and the first indel in KRT12 associated with MECD. In addition, we report a variant of uncertain significance in KRT3 in an individual with MECD. Although the potential pathogenicity of this variant is unknown, it is the first variant affecting the head domain of K3 to be reported in an individual with MECD and suggests that disease-causing variants associated with MECD may not be restricted to primary sequence alterations of either the helix-initiation or helix-termination motifs of K3 and K12

Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR

<p>Abstract</p> <p>Background</p> <p>Hypermethylation of the <it>TGFBI </it>promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the <it>TGFBI </it>promoter in human lung and prostate cancer specimens.</p> <p>Methods</p> <p>Methylation-specific primers were designed based on the methylation profiles of the <it>TGFBI </it>promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP.</p> <p>Results</p> <p>Among 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the <it>TGFBI </it>promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the <it>TGFBI </it>promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (<it>p </it>< 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the <it>TGFBI </it>promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (<it>p </it>< 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the <it>TGFBI </it>gene in human lung and prostate tumor cell lines.</p> <p>Conclusion</p> <p>We successfully optimized a MSP method for the precise and efficient screening of <it>TGFBI </it>promoter methylation status. Dense methylation of the <it>TGFBI </it>promoter correlated with the extent of <it>TGFBI </it>gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, <it>TGFBI </it>promoter methylation can be used as a potential prognostic marker for invasiveness and metastasis in prostate and lung cancer patients, respectively.</p

Reproducible probe-level analysis of the Affymetrix Exon 1.0 ST array with R/Bioconductor

The presence of different transcripts of a gene across samples can be analysed by whole-transcriptome microarrays. Reproducing results from published microarray data represents a challenge due to the vast amounts of data and the large variety of pre-processing and filtering steps employed before the actual analysis is carried out. To guarantee a firm basis for methodological development where results with new methods are compared with previous results it is crucial to ensure that all analyses are completely reproducible for other researchers. We here give a detailed workflow on how to perform reproducible analysis of the GeneChip Human Exon 1.0 ST Array at probe and probeset level solely in R/Bioconductor, choosing packages based on their simplicity of use. To exemplify the use of the proposed workflow we analyse differential splicing and differential gene expression in a publicly available dataset using various statistical methods. We believe this study will provide other researchers with an easy way of accessing gene expression data at different annotation levels and with the sufficient details needed for developing their own tools for reproducible analysis of the GeneChip Human Exon 1.0 ST Array