Proteomics Profiling of Madin-Darby Canine Kidney Plasma Membranes Reveals Wnt-5a Involvement during Oncogenic H-Ras/TGF-β-mediated Epithelial-Mesenchymal Transition

Abstract

Epithelial-mesenchymal transition (EMT) describes a process whereby polarized epithelial cells with restricted migration transform into elongated spindle-shaped mesenchymal cells with enhanced motility and invasiveness. Although there are some molecular markers for this process, including the down-regulation of E-cadherin, our understanding of plasma membrane (PM) and associated proteins involved in EMT is limited. To specifically explore molecular alterations occurring at the PM, we used the cationic colloidal silica isolation technique to purify PM fractions from epithelial Madin-Darby canine kidney cells during Ras/TGF-␤-mediated EMT. Proteins in the isolated membrane fractions were separated by one-dimensional SDS-PAGE and subjected to nano-LC-MS/MS-based protein identification. In this study, the first membrane protein analysis of an EMT model, we identified 805 proteins and determined their differential expression using labelfree spectral counting. These data reveal that MadinDarby canine kidney cells switch from cadherin-mediated to integrin-mediated adhesion following Ras/TGF-␤-mediated EMT. Thus, during the EMT process, E-cadherin, claudin 4, desmoplakin, desmoglein-2, and junctional adhesion molecule A were down-regulated, whereas integrins ␣6␤1, ␣3␤1, ␣2␤1, ␣5␤1, ␣V␤1, and ␣V␤3 along with their extracellular ligands collagens I and V and fibronectin had increased expression levels. Conspicuously, Wnt-5a expression was elevated in cells undergoing EMT, and transient Wnt-5a siRNA silencing attenuated both cell migration and invasion in these cells. Furthermore, Wnt-5a expression suppressed canonical Wnt signaling induced by Wnt-3a. Wnt-5a may act through the planar cell polarity pathway of the non-canonical Wnt signaling pathway as several of the components and modulators (Wnt-5a, -5b, frizzled 6, collagen triple helix repeat-containing protein 1, tyrosine-protein kinase 7, RhoA, Rac, and JNK) were found to be up-regulated during Ras/TGF-

Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes

“…As exosomes are of endosomal origin they typically contain proteins involved in multivesicular body (MVB) biogenesis, membrane transport and fusion (e.g., RabGTPases) [66], tetraspanins and their associated proteins that are functionally organised into tetraspanin-enriched microdomains -also referred to as the tetraspanin web [67], and proteins that may be involved in downstream exosome recognition and internalisation in a recipient cell [68]. Proteome profiling using mass spectrometry and label-free spectral counting [63] was employed to determine enrichment of these protein classes in the three purification methods studied.…”

Critical role of Wnt5a-Ror2 signaling in motility and invasiveness of carcinoma cells following Snail-mediated epithelial-mesenchymal transition

“…2003). More recently, it has been shown that expression of Wnt5a and collagen triple helix repeat‐containing protein 1 (Cthrc1) is highly up‐regulated during oncogenic H‐Ras/TGF‐β‐induced EMT of MDCK cells (Chen et al. 2010).…”

“…These results are consistent with previous report showing that expression of Wnt5a is up-regulated during Snail-mediated EMT of A431 cells . More recently, it has been shown that expres- sion of Wnt5a and collagen triple helix repeat-containing protein 1 (Cthrc1) is highly up-regulated during oncogenic H-Ras ⁄ TGF-b-induced EMT of MDCK cells (Chen et al 2010). Cthrc1 can associate with multiple Wnt and Frizzled proteins as well as Ror2 and enhance the complex formation of Wnt-Frizzled-Ror2, thereby activating planar cell polarity (PCP) pathway selectively (Yamamoto et al 2008).…”

Convergence of Wnt, growth factor, and heterotrimeric G protein signals on the guanine nucleotide exchange factor Daple

“…The β-catenin–independent Wnt pathway behaves as a double-edged sword; it suppresses tumorigenesis in normal epithelium and in early tumors, but also serves as a critical driver of epithelial-mesenchymal transition [EMT] and cancer invasion (17–27). We recently defined a novel paradigm in Wnt signaling in which Frizzled receptors (FZDs) activate the G proteins and trigger non-canonical Wnt signaling via Daple (CCDC88C), which is a Dishevelled (Dvl)-binding protein (28).…”