Transcriptome analysis reveals vimentin-induced downregulation of cell-cell associations augments cancer cell migration

Abstract

Abstract Background Vimentin is a type III intermediate filament (IF) protein, whose expression correlates with advanced metastatic cancer, reduced patient survival and poor prognosis across many cancers. During EMT-induced metastasis when vimentin begins to express, the epithelial characteristics are lost, and cell motility is augmented. The molecular bases for these changes are not well defined. Methods Ectopic expression of vimentin was carried in MCF-7 using spinfection of retroviruses. shRNA was used to knockdown vimentin in vimentin overexpressing MCF-7 and MDM-MB-231 cells, which express vimentin endogenously. The transcriptome profiling was carried out by RNA-Seq and validated by qPCR. Protein expression was measured by western blotting. Effect of vimentin on MCF-7 was determined by cell proliferation, migration and adhesion assays. Results Vimentin expression elicited a change in cell shape by significantly decreasing major axis, major axis angle and increasing cell migration, with no change in cell proliferation. Vimentin suppresses expression of major keratin genes KRT18, KRT19 and KRT8. 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 downregulation of vimentin in two cell types; MCF-7FV (ectopically expressing vimentin) and MDA-MB-231 (endogenously expressing vimentin), we identified 13 vimentin-responsive protein encoding genes common in both approaches and two long non-coding RNAs, LINC00052 and C15ORF9-AS1. Eight of these gene products CDH5, AXL, PTPRM, TGFBI, CDH10, FOXM1, BCL2 and NES were associated with cell-cell and cell-ECM interactions, E2F1, FOXM1 and CDC45 were in the cell proliferation group and the rest FSD1, BCL2, KIF26A and WISP2 were outside the two groups. Interestingly, downregulation of CDH5 significantly increased MCF-7 cell migration. Furthermore, vimentin expression in MCF-7 reduced nuclear area, altered expression of lamins, which was mostly reversed after its downregulation. Conclusion Collectively, we demonstrate, for the first time, that vimentin expression in cancer cells downregulates genes maintaining cell-cell junctions resulting in increased cell migration. Furthermore, this is the first report linking vimentin expression with LINC00052, which is dysregulated in many cancers.