Extracellular Matrix Protein 1 Research Articles

Long noncoding RNA FALEC inhibits proliferation and metastasis of tongue squamous cell carcinoma by epigenetically silencing ECM1 through EZH2.

Tongue squamous cell carcinoma (TSCC), the most common epithelial cancer identified in the oral cavity, has become one of the most common malignancies across the developing countries. Increasing evidence indicates that long non-coding RNAs (lncRNAs) serve as important regulators in cancer biology. The focally amplified long non-coding RNA in epithelial cancer (FALEC) was found downregulated in the tissues of tongue squamous cell carcinoma (TSCC) and was predicted to present a good prognosis by bioinformatics analysis. Experiments indicated that FALEC knockdown significantly increased the proliferation and migration of TSCC cells both in vitro and in vivo; however, FALEC overexpression repressed these malignant behaviors. RNA pull-down and RNA immunoprecipitation demonstrated that FALEC could recruit enhancer of zeste homolog 2 (EZH2) at the promoter regions of extracellular matrix protein 1 (ECM1), epigenetically repressing ECM1 expression. The data revealed that FALEC acted as a tumor suppressor in TSCC and may aid in developing a novel potential therapeutic strategy against TSCC.

Endoplasmic reticulum-localized ECM1b suppresses tumor growth and regulates MYC and MTORC1 through modulating MTORC2 activation in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease with dismal 5-year survival. Extracellular matrix protein 1 (ECM1) was identified as one of the most downregulated genes by transcriptomic analysis of normal esophageal/ESCC paired tissue samples. ECM1 plays oncogenic roles in cancer development in various cancer types. However, little is known about its role in ESCC. In vivo and in vitro functional assays coupled with analyses on public datasets and detailed molecular and mechanistic analyses were used to study the gene. We demonstrate that as opposed to the previously identified oncogenic role of ECM1a, ECM1b is a novel tumor suppressor in ESCC. ECM1 is significantly downregulated in ESCC and several other squamous cell carcinomas. ECM1b encodes a cellular protein that suppresses MYC protein expression and MTORC1 signaling activity. MTORC2 inactivation leads to suppressed MYC expression and MTORC1 signaling. ECM1b localizes to the endoplasmic reticulum and suppresses MTORC2 activation by inhibiting MTORC2/ribosome association. By regulating MTORC2/MYC/MTORC1 signaling, ECM1b suppresses general protein translation and enhances chemosensitivity. We provide evidence establishing a novel role of ECM1 in cancer that suggests ECM1b as a biomarker for ESCC disease management.

1388-P: Umbilical Cord Blood-Derived Exosomal LRG1 and ECM1 Induce Abnormal Placental Angiogenesis in Women with Gestational Diabetes Mellitus

Background: Gestational diabetes mellitus (GDM) is characterized by a high risk of fetal macrosomia and placenta hypervascularization. There is a need to investigate the underlying mechanism to explain these associate complications. Exosomes had been known participating in the regulation of various angiogenic processes. However, the effects of exosomes from cases of GDM on placental vascular network formation remain unclear. Methods and Results: Capillary branching per placental villi and protein level of CD31 were identified to be increased in GDM. Exosomes derived from umbilical cord blood (UE) enhanced endothelial cell vascularization significantly, especially in GDM pregnancies. The proteome of 10 normal exosomes and 10 GDM exosomes showed different profiles and uncovered two up-regulated proteins in GDM cases, namely leucine-rich alpha-2-glycoprotein-1 (LRG1) and extracellular matrix protein 1 (ECM1). Genetically engineered exosomes that overexpressed LRG1 or ECM1 resulted in hyper vascularization, including proliferation and migration. Conclusions: Our results demonstrate for the first time the expression of LRG1 and ECM1 in normal and GDM UE and their role in regulating pathological placental angiogenesis. These findings support the potential role of LRG1 and ECM1 in monitoring placental pathologies in this increasingly prevalent condition and novel insights into the causes. Disclosure J. Yao: None. T. Duan: None. K. Wang: None. Funding National Natural Science Foundation of China; National Key Research and Development Program of China

Serum Protein Biomarkers of Fibrosis Aid in Risk Stratification of Future Stricturing Complications in Pediatric Crohn's Disease.

Avoiding fibrostenotic complications is of paramount concern in the management of Crohn's disease (CD). We sought to investigate the association of candidate biomarkers of fibrosis collected at diagnosis with the future development of fibrostenotic CD. Using the Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort, a multicenter prospective observational pediatric inception cohort, subjects with an inflammatory phenotype (B1) at diagnosis who later converted to a stricturing phenotype (B2) within 3 years were compared with those who remained B1. Serum collected at diagnosis underwent both parallel reaction monitoring-targeted proteomic analysis and conventional enzyme-linked immunosorbent assay for 10 candidate biomarkers of intestinal fibrosis. Cox proportional hazard regression was used for multivariable analysis of time-dependent outcomes. In 116 subjects 58 subjects with verified B1 phenotype at diagnosis who later converted to B2 disease were compared with 58 subjects who remained B1 over 3 years of follow-up. Extracellular matrix protein 1 (ECM1) levels in the upper quartile (hazard ratio [HR] 3.43, 95% confidence limit [CL] 1.33, 8.42) were associated with future fibrostenotic disease. ASCA IgA (HR 4.99, 95% CL 1.50, 16.68) and CBir levels (HR 5.19, 95% CL 1.83, 14.74) were also associated with future intestinal fibrostenosis, although ECM1 continued to demonstrate independent association with conversion to B2 even with adjustment for serologies in multivariable analysis (HR 5.33, 95% CL 1.29, 22.13). ECM1 and other biomarkers of fibrosis may aid in determining the risk of uncomplicated inflammatory disease converting to B2 stricturing phenotypes in children with CD. Prospective validation studies to verify test performance and optimize clinical utilization are needed before clinical implementation.

Downregulation of Long Non-coding RNA FALEC Inhibits Gastric Cancer Cell Migration and Invasion Through Impairing ECM1 Expression by Exerting Its Enhancer-Like Function.

Long non-coding RNAs (lncRNAs) have been shown to play important roles in many human diseases. However, their functions and mechanisms in tumorigenesis and development remain largely unknown. Here, we demonstrated that focally amplified lncRNA in epithelial cancer (FALEC) was upregulated and significantly correlated with lymph node metastasis, TNM stage in gastric cancer (GC). Further experiments revealed that FALEC knockdown significantly inhibited GC cells migration and invasion in vitro. Mechanistic investigations demonstrated that small interfering RNA-induced silencing of FALEC decreased expression of the nearby gene extracellular matrix protein 1 (ECM1) in cis. Additionally, ECM1 and FALEC expression were positively correlated, and high levels of ECM1 predicted shorter survival time in GC patients. Our results suggest that the downregulation of FALEC significantly inhibited the migration and invasion of GC cells through impairing ECM1 expression by exerting an enhancer-like function. Our work provides valuable information and a novel promising target for developing new therapeutic strategies in GC.

Novel role of extracellular matrix protein 1 (ECM1) in cardiac aging and myocardial infarction.

IntroductionThe prevalence of heart failure increases in the aging population and following myocardial infarction (MI), yet the extracellular matrix (ECM) remodeling underpinning the development of aging- and MI-associated cardiac fibrosis remains poorly understood. A link between inflammation and fibrosis in the heart has long been appreciated, but has mechanistically remained undefined. We investigated the expression of a novel protein, extracellular matrix protein 1 (ECM1) in the aging and infarcted heart.MethodsYoung adult (3-month old) and aging (18-month old) C57BL/6 mice were assessed. Young mice were subjected to left anterior descending artery-ligation to induce MI, or transverse aortic constriction (TAC) surgery to induce pressure-overload cardiomyopathy. Left ventricle (LV) tissue was collected early and late post-MI/TAC. Bone marrow cells (BMCs) were isolated from young healthy mice, and subject to flow cytometry. Human cardiac fibroblast (CFb), myocyte, and coronary artery endothelial & smooth muscle cell lines were cultured; human CFbs were treated with recombinant ECM1. Primary mouse CFbs were cultured and treated with recombinant angiotensin-II or TGF-β1. Immunoblotting, qPCR and mRNA fluorescent in-situ hybridization (mRNA-FISH) were conducted on LV tissue and cells.ResultsECM1 expression was upregulated in the aging LV, and in the infarct zone of the LV early post-MI. No significant differences in ECM1 expression were found late post-MI or at any time-point post-TAC. ECM1 was not expressed in any resident cardiac cells, but ECM1 was highly expressed in BMCs, with high ECM1 expression in granulocytes. Flow cytometry of bone marrow revealed ECM1 expression in large granular leucocytes. mRNA-FISH revealed that ECM1 was indeed expressed by inflammatory cells in the infarct zone at day-3 post-MI. ECM1 stimulation of CFbs induced ERK1/2 and AKT activation and collagen-I expression, suggesting a pro-fibrotic role.ConclusionsECM1 expression is increased in ageing and infarcted hearts but is not expressed by resident cardiac cells. Instead it is expressed by bone marrow-derived granulocytes. ECM1 is sufficient to induce cardiac fibroblast stimulation in vitro. Our findings suggest ECM1 is released from infiltrating inflammatory cells, which leads to cardiac fibroblast stimulation and fibrosis in aging and MI. ECM1 may be a novel intermediary between inflammation and fibrosis.

Extracellular matrix protein 1 (ECM1) is associated with carcinogenesis potential of human bladder cancer.

BackgroundBladder cancer (BCa) is a common urological malignant tumor worldwide, and recurrence and death still remain high. New therapeutic targets are needed to treat patients who are not sensitive to current therapy. Extracellular matrix protein 1 (ECM1) is a key player in multiple epithelial malignancies. However, the knowledge regarding the expression of ECM1 in BCa and the mechanisms by which ECM1 affects BCa tumor progression is unclear.Materials and methodsECM1 expression levels in BCa tissues and cells were detected by quantitative real-time PCR (qRT-PCR), immunohistochemistry and Western blot. ECM1 expression was suppressed by shRNAs. Cell Counting Kit-8 (CCK-8), luminescent cell viability assay and 5-ethynyl-2′-deoxyuridine (EdU) assay were used to detect cell proliferation. Flow cytometry and transwell assay were used to evaluate cell apoptosis and invasion, respectively. All statistical analyses were performed by using the GraphPad Prism 7 software package.ResultsIn this study, the expression of ECM1 in BCa specimens and cell lines was examined and displayed a significant increase compared with noncancerous counterparts, while ECM1-knockdown affected not only cell proliferation and migration, but also cell invasion ability and apoptosis potential, corresponding to the finding that ECM1 overexpression in BCa patients was associated with a poor prognosis. Additionally, after suppression of ECM1, the expression of glucose transporter 1 (GLUT1), lactate dehydrogenase (LDHA) and hypoxia-inducible factor 1α (HIF-1α), genes involved in Warburg effect regulation, were significantly decreased, and the lactate production was also obviously reduced in ECM1-silenced cells.ConclusionOur investigations revealed that the expression of ECM1 was closely associated with tumor cell growth, migration and apoptosis at least in part through regulation of Warburg effect, defining ECM1 as an effective predictor in the carcinogenesis and postoperative recurrence of human BCa.

Lipoid proteinosis and epilepsy: Molecular analysis

Aim: Lipoid proteinosis (LP) is also known as Urbach-Wiethe disease which is a rare autosomal recessive disorder characterized by intracellular accumulation of hyaline material in skin and mucosae. LP has typical neurological, dermatological and radiological findings. The pathogenesis of disease is unknown. In literature several cases have been reported up to date. Mutations in extracellular matrix protein 1 (ECM1) gene have been found to cause the disease. We evaluated the molecular features of a family diagnosed with LP and evaluate the known and novel mutations of LP. Material and Methods: Genomic DNA was extracted from peripheral blood of the patients and family members including clinically normal parents and two siblings of the two affected subjects by using a commercial DNA extraction kit. Polymerase chain reaction was performed for all 10 exons of ECM1 gene by using the primers defined. Results: All of the exons of the ECM1 gene were sequenced and this revealed a 2-bp deletion in exon 7 of the ECM1 gene in both patients and both parents. Patients have the homozygous 2-bp deletions (c735del TG) and the parents and two healthy siblings showed heterozygous 2-bp deletion. Conclusions: Our patients found to be homozygous for the deletion in ECM1 gene. In order to understand the molecular pathology of the disease in detail, further functional analysis of the mutations should be performed.

Extracellular Matrix Protein 1 Gene Mutation in Turkish Patients with Lipoid Proteinosis.

Background:Lipoid proteinosis (LP) is a rare autosomal recessive genodermatosis characterized by mucocutaneous lesions and hoarseness of voice that develop in early childhood. LP is caused by mutation in the extracellular matrix protein 1 (ECM1) gene, which is located on 1q21.2.Aims:This study aimed to present the profile of ECM1 gene mutations and to identify possible novel mutations specific to Turkey.Materials and Methods:The ECM1 gene mutations of 19 LP patients from five families were evaluated using DNA isolated from peripheral blood samples. All ten exons in the ECM1 gene region were amplified by polymerase chain reaction (PCR). The PCR products were analyzed using a DNA sequencing analyzer. The results of DNA sequencing were analyzed with bioinformatics methods.Results:of the 19 LP patients evaluated in our study, we detected defects in exon 6 (c.507delT, 658T>G), exon 9 (157C>T, 727C>T), and exon 10 (c.93_94delGCinsTT) of the ECM1 gene.Conclusions:Our results indicate that defects in exons 6, 9, and 10 of the ECM1 gene were responsible for LP in our country. The identification of these pathogenic mutations is valuable because it facilitates early diagnosis and genetic counseling.

Lichen Sclerosus: An autoimmunopathogenic and genomic enigma with emerging genetic and immune targets.

Lichen sclerosus (LS) is an inflammatory dermatosis with a predilection for anogenital skin. Developing lesions lead to vulvar pain and sexual dysfunction, with a significant loss of structural anatomical architecture, sclerosis, and increased risk of malignancy. Onset may occur at any age in both sexes, but typically affects more females than males, presenting in a bimodal fashion among pre-pubertal children and middle-aged adults. A definitive cure remains elusive as the exact pathogenesis of LS remains unknown. A general review of LS, histologic challenges, along with amounting support for LS as an autoimmune disease with preference for a Th1 immune response against a genetic background is summarized. In addition to the classically referenced ECM1 (extracellular matrix protein 1), a following discussion of other immune and genetic targets more recently implicated as causative or accelerant agents of disease, particularly miR-155, downstream targets of ECM1, galectin-7, p53, and epigenetic modifications to CDKN2A, are addressed from the viewpoint of their involvement in three different, but interconnected aspects of LS pathology. Collectively, these emerging targets serve not only as inherently potential therapeutic targets for treatment, but may also provide further insight into this debilitating and cryptic disease.

Novel compound heterozygous mutations of ECM1 in a Chinese family with lipoid proteinosis

Lipoid proteinosis (LP) is a rare autosomal recessive genodermatosis caused by mutations of the ECM1 gene. The common variations of the ECM1 gene are nonsense and missense mutations, and in rare instance, compound heterozygotes may occur. We describe two siblings of LP from a nonconsanguineous family of China who were detected novel compound heterozygous mutations of c. 157C >T(p. R53X) in exon 3 and c. 857G >A (p. C286Y) in exon 7 of the ECM1 gene. Their mother was a carrier of missense mutation of c. 857G >A in exon 7 of ECM1, their father and one of the old sisters were the carriers of nonsense mutation of c. 157C >T in exon 3, respectively. All the carriers presented normally. The results support the opinion that the mutations of the ECM1 gene for LP are of varieties.

Tumor-derived exosomal proteins as diagnostic biomarkers in non-small cell lung cancer.

Accumulating evidence supports a role for exosomal protein in diagnosis. The purpose of this study was to identify the tumor‐derived exosomal biomarkers in the serum that improve the diagnostic value in Chinese non‐small cell lung cancer (NSCLC) patients. Serum exosomes were isolated from healthy donors (n = 46) and NSCLC patients (n = 125) by ultracentrifugation and were characterized using transmission electron microscopy, qNano, and immunoblotting. Proteomic profiles (by mass spectrometry) revealed multiple differentially expressed proteins in the healthy and NSCLC groups. The exosomal expression levels of alpha‐2‐HS‐glycoprotein (AHSG) and extracellular matrix protein 1 (ECM1) increased significantly in the NSCLC patients compared to the healthy group. Alpha‐2‐HS‐glycoprotein showed diagnostic values with a maximum area under the receiver operating characteristic curve (AUC) as 0.736 for NSCLC vs healthy individuals (P < .0001) and 0.682 for early stage NSCLC vs healthy individuals (P < .01). Extracellular matrix protein 1 showed the diagnostic capacity with AUC values of 0.683 (P < .001) and 0.656 (P < .05) in cancer and early stage NSCLC compared to healthy individuals. When AHSG was combined with ECM1, the AUCs were 0.795 and 0.739 in NSCLC and early stage patients, respectively. Taken together, the combination of AHSG, ECM1, and carcinoembryonic antigen improved the diagnostic potential of NSCLC. The diagnosis values were AUC of 0.938 for NSCLC and 0.911 for early stage NSCLC vs healthy individuals. Our results suggest that novel proteomic signatures found in serum exosomes of NSCLC patients show potential usefulness as diagnostic tools.

Downregulation of Extracellular Matrix and Cell Adhesion Molecules in Cumulus Cells of Infertile Polycystic Ovary Syndrome Women with and without Insulin Resistance.

Objective The extracellular matrix (ECM) of the cumulus oocyte complex (COC) is composed of several molecules that have different roles during follicle development. This study aims to explore gene expression profiles for ECM and cell adhesion molecules in the cumulus cells of polycystic ovary syndrome (PCOS) patients based on their insulin sensitivity following controlled ovarian stimulation (COS). Materials and Methods In this prospective case-control study enrolled 23 women less than 36 years of age who participated in an intracytoplasmic sperm injection (ICSI) program. Patients were subdivided into 3 groups: control (n=8, fertile women with male infertility history), insulin resistant (IR) PCOS (n=7), and insulin sensitive (IS) PCOS (n=8). We compared 84 ECM component and adhesion molecule gene expressions by quantitative real-time polymerase chain reaction array (qPCR-array) among the groups. Results We noted that 21 of the 84 studied genes differentially expressed among the groups, from which 18 of these genes downregulated. Overall, comparison of PCOS cases with controls showed downregulation of extracellular matrix protein 1 (ECM1); catenin (cadherin-associated protein), alpha 1 (CTNNA1); integrin, alpha 5 (ITGA5); laminin, alpha 3 (LAMA3); laminin, beta 1 (LAMB1); fibronectin 1 (FN1); and integrin, alpha 7 (ITGA7). In the IS group, there was upregulation of ADAM metallopeptidase with thrombospondin type 1 motif, 8 (ADAMTS8) and neural cell adhesion molecule 1 (NCAM1) compared with the controls (P<0.05). ConclusionDownregulation of ECM and cell adhesion molecules seem to be related to PCOS. Gene expression profile alterations in cumulus cells from both the IS and IR groups of PCOS patients seems to be involved in the composition and regulation of ECM during the ovulation process. This study highlights the association of ECM gene alteration as a viewpoint for additional understanding of the etiology of PCOS.

Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system.

The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.

Extracellular matrix protein 1 recruits moesin to facilitate invadopodia formation and breast cancer metastasis

Invadopodia are actin-based cortical protrusions of tumour cells, and required for stromal invasion and metastasis. Extracellular matrix protein 1 (ECM1) has long been regarded as a secretory protein, but the mechanism of its precise functions in tumour cells is still obscure. Recently published data suggested a function of ECM1 in remodelling the actin cytoskeleton; however, its role in invadopodia formation remains unknown. Here, we demonstrated for the first time that ECM1 was a membrane protein and was essential for invadopodia formation by breast cancer cells. ECM1 depletion attenuated the ability of tumour cells to matrix attachment, invasion, and spontaneous metastasis to the lungs of mice. Additionally, co-expression of ECM1 and moesin (MSN) was closely related to aggressive breast cancer phenotypes. ECM1 interacted with MSN and recruited it adjacent to the membrane in order to promote MSN membrane translocation and phosphorylation, which facilitated invadopodia formation by breast cancer cells. These results elucidate a novel mechanism underlying the role of ECM1 in breast cancer metastasis and suggest ECM1 as a potential therapeutic target for overcoming tumour dissemination.

Genetic expression profile-based screening of genes and pathways associated with papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer; however, the specific genes and signaling pathways involved in this cancer remain largely unclear. The present study analyzed three profile datasets, GSE6004, GSE29265 and GSE60542, which were comprised of 47 PTC and 41 normal thyroid tissue samples, to identify key genes and pathways associated with PTC. Initially, differentially-expressed genes (DEGs) between PTC and normal thyroid tissue were screened using R 3.4.0 (2017-04-21, R Foundation, Vienna, Austria, http://www.R-project.org/). These DEGs were then clustered by gene ontology functional terms and representative signaling pathways. Additionally, specific key gene nodes were filtered out from a constructed protein-protein interaction (PPI) network. The results identified a total of 423 shared DEGs associated with PTC, including 211 upregulated and 212 downregulated genes. These 423 genes were primarily enriched in glycosaminoglycan binding, sulfur compound binding, heparin binding, enzyme activator activity, peptidase activator activity and hsa04512: Extracellular matrix (ECM)-receptor interaction. A total of 21 central node genes were identified as key genes in the PTC disease process including complement factor D (CFD), Collagen Type I α 1 Chain (COL1A1), Extracellular Matrix Protein 1 (ECM1) and Fibronectin 1 (FN1). These genes are involved in protease binding, G-protein coupled receptor binding, extracellular matrix structural constituent and peptidase regulator activity. To conclude, using bioinformatics analysis, the present study identified candidate DEGs and critical pathways in PTC that may improve the current understanding regarding the underlying mechanisms of PTC. These genes and pathways may be used as potential therapeutic targets of PTC in the future.

Extracellular matrix protein 1 promotes follicular helper T cell differentiation and antibody production

T-follicular helper (TFH) cells are a subset of CD4+ helper T cells that help germinal center (GC) B-cell differentiation and high-affinity antibody production during germinal center reactions. Whether important extracellular molecules control TFH differentiation is not fully understood. Here, we demonstrate that a secreted protein extracellular matrix protein 1 (ECM1) is critical for TFH differentiation and antibody response. A lack of ECM1 inhibited TFH cell development and impaired GC B-cell reactions and antigen-specific antibody production in an antigen-immunized mouse model. ECM1 was induced by IL-6 and IL-21 in TFH cells, promoting TFH differentiation by down-regulating the level of STAT5 phosphorylation and up-regulating Bcl6 expression. Furthermore, injection of recombinant ECM1 protein into mice infected with PR8 influenza virus promoted protective immune responses effectively, by enhancing TFH differentiation and neutralizing antibody production. Collectively, our data identify ECM1 as a soluble protein to promote TFH cell differentiation and antibody production.

PO-134 Cytogenetic and molecular characterisation of new high-grade serous ovarian cancer cell line OVPA8

IntroductionBladder cancer (BC) is the ninth most common cancer worldwide. A quarter of all cases present as muscle invasive BC (MIBC). Treatment for this group has remained largely unchanged for the past 20 years limited mainly to cisplatin based therapy and radical cystectomy. Prognosis for these patients is poor, with 5 year survival <50% and identification of potential new therapeutic approaches is urgently needed. We have explored the possible role of extracellular matrix protein 1 (ECM1) overexpression in MIBC. ECM1 overexpression has been linked to carcinogenesis and progression to a more aggressive phenotype in a number of carcinomas, but its role in BC has not yet been examined. In a previous study, ECM1 was proposed to interact with and stabilise EGFR in a breast cancer cell line leading to increased resistance to ERBB targeting drugs. Despite repeated implication of EGFR in the progression of BC, EGFR targeting agents have so far had little success in the treatment of MIBC in part due to resistance. Improved understanding of the mechanisms of resistance to such agents is vital. The current study examined ECM1 overexpression and the relationship between ECM1 and EGFR in BC.Material and methodsECM1 expression was assessed at the mRNA and protein levels in 47 bladder tumour derived cell lines using qRT-PCR and western blot analysis. ECM1 knockdown cell lines were established using shRNA lentiviral transduction in a panel of cell lines which overexpress the protein. ECM1 knockdown cell lines were analysed for phenotypic effects. Phosphorylation of EGFR and downstream effectors was examined following recombinant ECM1 treatment. Publicly available microarray data was mined to assess the clinical significance of ECM1 expression in patients, relating high expression to tumour stage and overall survival.Results and discussionsECM1 knockdown had an inhibitory effect on wound-healing ability. Evidence suggests ECM1 initiates phosphorylation and thus activation of EGFR and downstream effectors in the pathway, however we have been unable to detect a direct interaction with EGFR as described in breast. Mining of publicly available data revealed a significant association between high ECM1 expression and reduced overall survival.ConclusionECM1 overexpression in MIBC appears to be associated with poorer prognosis in patients. While ECM1 has been shown to influence EGFR signalling, the mechanism of EGFR activation by ECM1 in BC may be different from that previously described in breast cancer.

Identification of novel target genes in human lung tissue involved in chronic obstructive pulmonary disease.

IntroductionAs part of a study aimed at illuminating at least some of the complex molecular events taking place in COPD, we screened tissues by means of transcriptome analyses.Materials and methodsTissues were subjected to transcriptome analysis. Candidate genes were identified and validated by immunohistochemistry. Primary human lung cells were subjected to stimulation with cigarette smoke extract for further validation by real time PCR.ResultsSix candidate genes were selected for further investigations: Aquaporin 3 (AQP3), extracellular matrix protein 1 (ECM1), four and a half LIM domain 1 (FHL1), milk fat globule epidermal growth factor 8 (MFGE8, lactadherin), phosphodiesterase 4D-interacting protein (PDE4DIP), and creatine transporter SLC6A8. All six proteins were allocated to distinct cell types by immunohistochemistry. Upon stimulation with cigarette smoke extract, human type II pneumocytes showed a dose-dependent down-regulation of MFGE8, while ECM1 and FHL1 also tended to be down-regulated. Although present, none of the candidates was regulated by cigarette smoke extract in primary human macrophages.DiscussionMFGE8 turned out to be an interesting new candidate gene in COPD deserving further studies.

PO-131 Over expression of extracellular matrix protein 1 (ECM1) in muscle-invasive bladder cancer

IntroductionThe connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells to directly exchange ions and small molecules. The connexin channels assemble into distinct plasma membrane domains known as gap junctions. Intercellular communication via gap junctions has an important role in regulating cell growth, differentiation and in maintaining tissue homeostasis. The most ubiquitously expressed connexin isoform in human tissues, connexin 43 (Cx43), acts as a tumour suppressor in multiple tissue types and is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the regulation of Cx43 degradation remains poorly understood.Material and methodsThe cervical cancer cell lines HeLa and C33a were used as model systems. Silencing and ectopic overexpression of proteins were performed by siRNA and plasmid transfection, respectively. The Cx43 ubiquitination status was determined using immunoprecipitation and western blotting. Gap junction intercellular communication (GJIC) was measured using the scrape loading dye transfer assay. Cells were imaged using confocal microscopy.Results and discussionsHere, we identify a member of the NEDD4 (neural precursor cell-expressed developmentally downregulated gene 4) family of E3 ubiquitin ligases, termed ITCH, as a novel regulator of Cx43 degradation and GJIC. Depletion of ITCH resulted in increased Cx43 protein levels, gap junction size and GJIC. Ectopic overexpression of ITCH, but not a catalytically inactive ITCH mutant, led to decreased Cx43 protein levels and loss of gap junctions. The data further indicate that ITCH acts in concert with two other members of the NEDD4 family that previously have been shown to regulate Cx43 degradation, termed NEDD4 and SMURF2. Simultaneous depletion of NEDD4, SMURF2, and ITCH by siRNA was found to result in a significantly lower Cx43 ubiquitination level and reduced Cx43 degradation under basal conditions. The triple knock-down of these three E3 ubiquitin ligases was also found to strongly counteract the TPA (12-O-tetradecanoylphorbol 13-acetate)-induced degradation of Cx43.ConclusionThese data identify ITCH as a novel regulator of Cx43 degradation and GJIC. The data also indicate that ITCH acts together with NEDD4 and SMURF2 to mediate the basal and TPA-induced turnover of Cx43.