Transcriptome Analysis Reveals Vimentin-Induced Disruption of Cell–Cell Associations Augments Breast Cancer Cell Migration

Abstract

In advanced metastatic cancers with reduced patient survival and poor prognosis, expression of vimentin, a type III intermediate filament protein is frequently observed. Vimentin appears to suppress epithelial characteristics and augments cell migration but the molecular basis for these changes is not well understood. Here, we have ectopically expressed vimentin in MCF-7 and investigated its genomic and functional implications. Vimentin changed the cell shape by decreasing major axis, major axis angle and increased cell migration, without affecting proliferation. Vimentin downregulated major keratin genes KRT8, KRT18 and KRT19. Transcriptome-coupled GO and KEGG analyses revealed that vimentin-affected genes were linked to either cell–cell/cell-ECM or cell cycle/proliferation specific pathways. Using shRNA mediated knockdown of vimentin in two cell types; MCF-7FV (ectopically expressing) and MDA-MB-231 (endogenously expressing), we identified a vimentin-specific signature consisting of 13 protein encoding genes (CDH5, AXL, PTPRM, TGFBI, CDH10, NES, E2F1, FOXM1, CDC45, FSD1, BCL2, KIF26A and WISP2) and two long non-coding RNAs, LINC00052 and C15ORF9-AS1. CDH5, an endothelial cadherin, which mediates cell–cell junctions, was the most downregulated protein encoding gene. Interestingly, downregulation of CDH5 by shRNA significantly increased cell migration confirming our RNA-Seq data. Furthermore, presence of vimentin altered the lamin expression in MCF-7. Collectively, we demonstrate, for the first time, that vimentin in breast cancer cells could change nuclear architecture by affecting lamin expression, which downregulates genes maintaining cell–cell junctions resulting in increased cell migration.

Over-expression of KRT8 is associated with invasion of non-functioning pituitary adenomas

“…KRT8 serves as an upstream regulatory gene in numerous signaling pathways associated with tumorigenesis, exhibiting abnormal expression in various epithelial-origin tumors such as breast cancer, gastric cancer, and pancreatic cancer [15][16][17] .The regulation of KRT8 is mediated by the MEK-ERK signaling pathway 18 . Busch et al have reported that the recombination process of cytokeratin and the metastasis of epithelial tumor cells are in uenced by KRT8 phosphorylation 19 .…”

Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction

“…The mutagenized cDNA from the pBluescript vector is excised out and ligated into an expression vector such as pLPCpuro or pLPChygro in identical restriction sites, usually EcoRI and BamHI [9,10], and used for maxi DNA prep. These constructs can be used for protein expression by western blotting and immunostaining and can be employed for functional analysis.…”