J Cell Physiol. 2017 Jun;232(6):1368-1386.

Identification of a novel human E-cadherin splice variant and assessment of its effects upon EMT-related events

 

Authors

María Laura Matos 1, §; Lara Lapyckyj 1, §; Marina Rosso 1 ; María José Besso 1; María Victoria Mencucci 1; Clara Isabel Marín Briggiler 1; Silvina Giustina 1; Laura Inés Furlong 1; Mónica Hebe Vazquez-Levin 1,*

§ both authors equally contributed to this work

1 Instituto de Biología y Medicina Experimental (IBYME) National Research Council (CONICET)-FIBYME. Vuelta de Obligado 2490. Buenos Aires. 1428DN. Argentina.

 

*Corresponding author 

Mónica Vazquez-Levin, PhD.

Instituto de Biología y Medicina Experimental (IBYME)

CONICET-FIBYME

mhvazl@gmail.com

 

Abstract

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%) of 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 cell acquisition of fibroblastic-like 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 molecular basis of tumor progression and improve cancer diagnosis.

 

Citation:

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

Matos ML, Lapyckyj L, Rosso M, Besso MJ, Mencucci MV, Marín-Briggiler CI, Giustina S, Furlong LI,Vazquez-Levin MH.

J Cell Physiol. 2017 Jun;232(6):1368-1386. doi: 10.1002/jcp.25622. Epub 2016 Dec 29.

 

Cover Image, Volume 232, Number 6, June 2017.

Matos ML, Lapyckyj L, Rosso M, Besso MJ, Mencucci MV, Marín-Briggiler CI, Giustina S, Furlong LI,Vazquez-Levin MH.

J Cell Physiol. 2017 Jun;232(6):i. doi: 10.1002/jcp.25866.

 

Supplement

Identification of a human novel E-cadherin variant mRNA

A novel E-cadherin transcript was identified during the screening of a unidirectional lambda ZAP Express (Stratagene, USA) vector expression library prepared with RNA from human epididymis. Alignment with the reference nucleotide E-cadherin wild type sequence (Ecadwt; NM_004360) led to the identification of a 34 nt-deletion in Exon 14. With the NetGene2 server (http://www.cbs.dtu.dk/services/NetGene2/), the site used to produce the novel E-cadherin mRNA sequence was identified as a putative alternative splicing site (Figure 1.A., 1.B.).

The expression of the protein encoded by the variant E-cadherin (Ecadvar) mRNA was analyzed using a set of studies that combined bioinformatics tools and transfection assays. Firstly, the amino acidic sequence of the protein encoded by the Ecadvar mRNA was deduced using the ExPASy Translate software (http://web.expasy.org/translate/). Contrasting with the 882 amino acids of the wild type form, the novel Ecad protein spans a total of 757 residues (Figure 1.C.). Both sequences showed 100% homology up to the residue in position 721. The transcript 34 nt-deletion results in a frame-shift, giving rise to a new peptide sequence of 36 amino acids and a premature termination codon. A sequence analysis run through the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/) predicted a secretory nature of the novel protein (Figure 1.D.). Moreover, the transient expression of the novel Ecadvar mRNA in COS-7 cells identified a 94 KDa protein isoform in the cellular conditioned medium (CM), confirming the bioinformatics predictions.

 

Effects of Ecadvar expression in a human breast cancer cell model

MCF-7 human breast cancer cells stably transfected with a pcDNA3 expression plasmid containing the Ecadvar mRNA (MCF7Ecadvar) showed dramatic changes in cell phenotype, depicting a fibroblast-like morphology and a decrease in cell-cell contacts (Figure 2.A.). In line with these findings, a significant decrease in Ecadwt transcript levels (Figure 2.B.), as well as in protein expression (Figure 2.C.) and localization (Figure 2.D.), was observed. Also, filamentous actin (F-actin) was organized in non-junctional stress fibers oriented towards the boundary between cells (Figure 2.D.), a typical distribution found in invasive cells. In contrast, control cells (MCF7pcDNA3) showed E-cadherin and F-actin localization mainly in cell borders. Concomitant with the decrease in Ecadwt, an increased expression of transcriptional repressors (Twist, Snail, Zeb1 and Slug), loss of cytokeratin 19 and gain of vimentin, and expression of the mesenchymal N-cadherin mRNA, was found in MCF7Ecadvar cells (Figure 2.B.). All these changes have been reported in the Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. In addition, low levels of transcripts encoding ESRP1 and ESRP2 RNA binding proteins, responsible for maintaining an epithelial cellular phenotype, were observed (Figure 2.B.). Moreover, the expression of Dysadherin, a cell membrane glycoprotein highly expressed in several tumors that depict decreased E-cadherin expression, was increased in MCF7Ecadvar cells (Figure 2.B.). In agreement with these molecular changes, MCF7Ecadvar cells depicted reduced adhesiveness and increased motility, migration and invasion properties, all indicative of a more aggressive cellular behavior.

Next, a set of experiments were done to assess whether changes in cell morphology and E-cadherin expression could be attributed, at least in part, to an autocrine/paracrine effect of the secreted novel Ecadvar protein. Firstly, HEK-293 cells were stably transfected with Ecadvar (HEK293Ecadvar) and pcDNA3 empty vector (HEK293pcDNA3). Once the expression of the novel Ecadvar mRNA was verified, the CM of both cell lines was collected and used to challenge MCF-7 cells during 7 days. Cells exposed to the HEK293Ecadvar CM displayed a fibroblastic-like morphology and showed a significant decrease in Ecadwt transcript levels. No changes were observed in cells incubated with HEK293pcDNA3 CM.

 

Assessment of Ecadvar mRNA expression levels in human cancer cell lines

The expression levels of the Ecadvar mRNA were determined in a panel of 42 established human cancer cell lines of diverse tissue origin (urological: prostate, bladder; gynecological: breast, ovary, endometrium; digestive: gastric, liver, pancreas, colon; other: skin, lung) by applying a primer-specific quantitative Real Time PCR strategy. Detectable levels of the Ecadvar transcript were found in 29 cell lines evaluated (69%). When Ecadvar mRNA levels were calculated relative to the wild type transcript, 23 cell lines depicted a 0.05 to 1.2% expression of the novel form, while the remaining showed levels between 2 and 11.6%. A survey done on the COSMIC and CCLE databases on the available cell lines (n=22) showed absence of mutations around the CDH1 Exon 13-Exon 14 constitutive splicing donor and/or acceptor sites. In silico and experimental approaches revealed that differences in Ecadvar mRNA levels may correspond to the Nonsense-Mediated mRNA Decay, a regulatory mechanism usually triggered by premature termination codons which may be altered during tumorigenesis.

Alterations in the expression and function(s) of E-cadherin are a key event in the deregulation of cell-cell adhesion during tumor progression and metastasis. Despite the vast literature in this field, the underlying mechanisms of E-cadherin changes in cancer are not fully understood. The present study reports, for the first time, the identification and characterization of a novel human Ecad alternative spliced-transcript that acts as a dominant negative modulator of Ecadwt and induces alterations in gene expression, cell morphology and behavior characteristic of the EMT process.

Considering the relevance of E-cadherin and EMT changes in progression and aggressiveness of numerous tumors of epithelial origin, understanding the mechanisms that control Ecadvar mRNA expression will contribute to the management of cancer diagnosis and treatment.

The short video summarizes the main findings of the present report.

 

 

 

 

Figure 1. Identification of a human novel E-cadherin variant mRNA

A. Sequence alignment of the novel transcript (Ecadvar) with the reference Ecadwt mRNA showing a 34-bp deletion in the variant form. B. Graphical representation of the constitutive and the alternative splicing mechanisms involved in the generation of Ecadwt (top) and Ecadvar (bottom) mRNA. C. Amino acidic sequence of Ecadwt (amino acids 1-882) and Ecadvar (amino acids 1-757) proteins (Grey box: signal peptide and propeptide sequences; Light blue box: extracellular domain sequence; Orange box: transmembrane domain sequence; Violet box: intracellular domain sequence; in Ecadvar Dark Green box: novel peptide sequence). D. Prediction of the secretory nature of the novel protein encoded by Ecadvar mRNA; analysis done with the TMHMM server.

 

 

Figure 2. Effects of Ecadvar expression in MCF7 human breast cancer cells

A. Representative phase-contrast images of MCF7pcDNA3 and MCF7Ecadvar cells. B. Immunodetection of E-cadherin protein (120 KDa full length form) after SDS-PAGE and Western immunoblotting analysis in both cell lines. Immunodetection of Tubulin (55 KDa) was used as loading control. C. Fluorescence immunocytochemistry and confocal laser microscopy analysis of E-cadherin and F-actin in both cell lines. D. E-cadherin, Twist, Snail, Zeb1, Slug, cytokeratin 19, vimentin, N-cadherin, ESRP1, ESRP1 and Dysadherin mRNA expression levels in MCF7EpcDNA3 and MCF7Ecadvar cells assessed by Real Time PCR. The mRNA expression fold change is also shown.