Identification of a Novel Human E‐Cadherin Splice Variant and Assessment of Its Effects Upon EMT‐Related Events

• Published in: Journal of cellular physiology Vol. 232; no. 6; pp. 1368 - 1386
• Main Authors: Matos, María Laura, Lapyckyj, Lara, Rosso, Marina, Besso, María José, Mencucci, María Victoria, Briggiler, Clara Isabel Marín, Giustina, Silvina, Furlong, Laura Inés, Vazquez‐Levin, Mónica Hebe
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.06.2017
• Subjects: Adhesion; Adult; Alternative splicing; Alternative Splicing - drug effects; Alternative Splicing - genetics; Amino Acid Sequence; Base Sequence; Bioinformatics; Biomarkers, Tumor - genetics; Biomarkers, Tumor - metabolism; Biotechnology; Cadherins - chemistry; Cadherins - genetics; Cadherins - metabolism; Calcium; Cancer; Cell adhesion; Cell adhesion & migration; Cell culture; Cell Line; Cell migration; Cell Movement - drug effects; Cell Movement - genetics; Conditioning; Culture Media, Conditioned - pharmacology; E-cadherin; Epididymis - drug effects; Epididymis - metabolism; Epithelial-Mesenchymal Transition - drug effects; Epithelial-Mesenchymal Transition - genetics; Gene expression; Gene Expression Regulation, Neoplastic - drug effects; Gene Library; Humans; Male; Mesenchyme; Models, Biological; Molecular modelling; mRNA turnover; N-Cadherin; Neoplasm Invasiveness; Nonsense-mediated mRNA decay; Proteins; Repressors; Ribonucleic acid; RNA; RNA Stability - drug effects; RNA Stability - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-binding protein; Signal transduction; Snail protein; Splicing; Transcription factors; Transfection; Tumor cell lines; Vimentin; Wound healing
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.25622

Epithelial Cadherin (E‐cadherin) is involved in calcium‐dependent cell–cell adhesion and signal transduction. The E‐cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E‐cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E‐cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense‐Mediated mRNA Decay (NMD). The novel E‐cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E‐cadherin variant expression relative to the wild type mRNA = 0.05–11.6%). Stable transfection of the novel E‐cadherin variant in MCF‐7 cells (MCF7Ecadvar) resulted in downregulation of wild type E‐cadherin expression (transcript/protein) and EMT‐related changes, among them acquisition of a fibroblastic‐like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N‐cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell–cell adhesion (Hanging‐drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF‐7 cells incubated with culture medium supplemented with conditioned medium from HEK‐293 cells transfected with the E‐cadherin variant mRNA. Further characterization of the novel E‐cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 232: 1368–1386, 2017. © 2016 Wiley Periodicals, Inc. This report describes the identification and partial characterization of a novel human E‐cadherin variant mRNA produced by alternative splicing. Stable transfection of the E‐cadherin variant mRNA in MCF‐7 cells (MCF7Ecadvar cells) resulted in downregulation of wild type E‐cadherin and EMT‐related changes, that is, acquisition of a fibroblast‐like cellular phenotype, increased expression of E‐cadherin transcriptional repressors, vimentin and N‐cadherin, and loss of cytokeratins. MCF7Ecadvar cells depicted dramatic changes in cellular behavior, showing reduced cell–cell adhesion and increased cell migration and invasiveness.