Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Identification of novel tissue-specific transcription arising from E-cadherin/CDH1 intron2: a novel protein isoform increases gastric cancer cell invasion and angiogenesis.

ABSTRACT: E-cadherin, a protein encoded by the CDH1 gene is the dominant epithelial cell adhesion molecule playing a crucial role in epithelial tissue polarity and structural integrity. The progression of 90% or more carcinomas is believed to be mediated by disruption of normal E-cadherin expression, subcellular localization or function. Despite the strong correlation between E-cadherin loss and malignancy the mechanism through how this occurs is not known in most sporadic and hereditary epithelial carcinomas. Previous works have shown the importance of CDH1 intron 2 sequences for proper gene and protein expression supporting the possibility of these being cis-modulators of E-cadherin expression/function. but when co-expressed it led to reduced cell-cell adhesiveness, increased invasion and angiogenesis. By expression array analysis, IFITM1 and IFI27 levels were found to be increased upon CDH1a overexpression. Importantly, CDH1a was found to be de novo expressed in gastric cancer cell lines when compared to normal stomach. Results: In this sense we have searched for additional CDH1 transcripts arising from intron 2. From the four found, one (CDH1a) was demonstrated to be tissue specific and to be translated in vivo. When overexpressed in an E-cadherin negative context CDH1a replaced the canonic protein functions Conclusions: We have demonstrated, for the first time, transcripts arising from CDH1 intron 2. One of these, CDH1a, was found to modulate E-cadherin function representing a novel mechanism underlying invasion and angiogenesis in epithelial cancers and opening the way for novel therapeutic approaches. Total RNA obtained from human gastric tubular adenocarcinoma Mkn28 cell line. Three independent replicas of each conditions(i.e pLenti transduced cell lines overexpressing CDH1a , pLenti with empty vector and pLenti transduced cell lines overexpressing wild type CDH1) has been employed.

ORGANISM(S): Homo sapiens

SUBMITTER: Danilo Licastro

PROVIDER: E-GEOD-32540 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric cancer cell invasion and angiogenesis.

Pinheiro Hugo H Carvalho Joana J Oliveira Patrícia P Ferreira Daniel D Pinto Marta Teixeira MT Osório Hugo H Licastro Danilo D Bordeira-Carriço Renata R Jordan Peter P Lazarevic Dejan D Sanges Remo R Stupka Elia E Huntsman David D Seruca Raquel R Oliveira Carla C

Human molecular genetics 20120629 19

Disruption of E-cadherin (CDH1 gene) expression, subcellular localization or function arises during initiation and progression of almost 90% of all epithelial carcinomas. Nevertheless, the mechanisms through which this occurs are largely unknown. Previous studies showed the importance of CDH1 intron 2 sequences for proper gene and protein expression, supporting these as E-cadherin cis-modulators. Through RACE and RT-PCR, we searched for transcription events arising from CDH1 intron 2 and discove ...[more]

PMID: 22752307

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Project description:Background & Aims: E-cadherin expression disruption is commonly observed in epithelial cancers and metastasis. Such event is also recognized as a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression and function of pivotal cancer-related genes, we characterized and dissected a pathway initiated by loss of microRNA-101 that causes E-cadherin dysfunction through upregulation of EZH2 expression in GC. Methods: Microarrays were used to profile the expression of microRNAs in human GC. Array-CGH revealed DNA copy number changes that were validated by genomic quantitative PCR and Snapshot. Expression levels of microRNAs, mRNA and protein were determined by quantitative-real time PCR and western-blot/co-immunofluorescence. CDH1 inactivating mechanisms were analyzed. Gain and loss of function experiments were done in KatoIII cells. E-cadherin functionality was assessed by immunofluorescence and flow cytometry. Results: MiR-101 expression was significantly decreased in tumors in comparison with normal gastric mucosas (P<.0001). In 65% of the analyzed GC cases, miR-101 downregulation was caused by deletions and/or microdeletions at miR-101-2 locus. EZH2 overexpression and consequent loss/aberrant E-cadherin expression was found in concomitance with miR-101 downregulation in 41% of the analyzed GC cases. This occurred preferentially in cases retaining allele(s) untargeted by classical CDH1 inactivating mechanisms. MiR-101 gain of function experiments or direct inhibition of EZH2, led to a strong depletion of endogenous EZH2 protein and consequent rescue of functional E-cadherin at the cell membrane, mimicking results obtained with clinical GC samples. Conclusions: Deletions and/or microdeletions at miR-101-2 locus underlying mature miR-101 downregulation and consequent EZH2 overexpression represented a novel cascade of genetic events leading to E-cadherin disruption, preferentially affecting the intestinal-type of GC. In the present study, 37 primary GCs and a pool of 10 normal gastric mucosas were used to acquire the expression of 703 known human mature miRNAs deposited in the Sanger miRBase Sequence Database, Release 10.0 (http://microrna.sanger.ac.uk), as well as 393 novel human miRNAs discovered through deep sequencing and validated by qRT-PCR and array profiling (NCodeM-bM-^DM-" Human miRNA microarray probe set V3 from Invitrogen). The 10 normal gastric mucosa RNA samples were pooled to obtain the normal gastric reference sample, which was labelled and hybridised four times to check arrays' reproducibility.

Project description:Background: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT). Methods: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4bp deletion in CDH1 exon 11. Results: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed. Conclusions: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling. Examination of the impact of E-cadherin (CDH1) loss in an isogenic pair of breast cell lines.

Dataset Information

Identification of novel tissue-specific transcription arising from E-cadherin/CDH1 intron2: a novel protein isoform increases gastric cancer cell invasion and angiogenesis.

Publications

Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric cancer cell invasion and angiogenesis.

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