| dc.description.abstract | Vimentin, a type III intermediate filament protein, is found in mesenchymal cells along with microfilaments and microtubules. It mainly functions to give structural support and viscoelastic resilience to the cells; in addition, it is also involved in migration, signalling and stress responses. It is normally absent in epithelial cells, however, its expression is induced in cancer cells as they start metastasising, therefore, it is considered a canonical marker of epithelial mesenchymal transition (EMT) and cancer metastasis. In the literature, different supportive roles of vimentin are reported during cancer progression, these include increased cell migration, invasion and proliferation, however, the molecular basis of these functions are still inexplicit. To study the role of vimentin in cancer progression and metastasis, we ectopically expressed full-length vimentin in vimentin-deficient epithelial cell line MCF-7 and compared the structural and functional changes in this cancer cell line before and after vimentin expression. First, we studied the filament assembly of vimentin in MCF-7 by tagging the full-length vimentin with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) at the N- and C-terminus and compared it with the untagged vimentin. Untagged vimentin was able to form normal fully extended intermediate filaments in the cytoplasm. We observed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with β-catenin at cell–cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin in a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by pure untagged vimentin. Our data suggested that placing a tag at the N-terminus may create steric hindrance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Our results show that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into the pre-existing network. The C-terminal tagged vimentin was able to form filaments, however, there were perinuclear bundles of filaments in case of AcGFP tag (VIM-AcGFP) suggesting a possible disruption between the tail and rod domain associations. Second, we investigated genomic and functional implications of ectopic expression of vimentin in MCF-7 cells. Vimentin altered 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. The most downregulated gene was CDH5, an endothelial cadherin. Downregulation of CDH5 using shRNA increased cell migration. Using shRNA mediated knockdown of vimentin in two cell types; MCF-7FV (ectopically expressing vimentin) and MDA-MB-231 (endogenously expressing vimentin), 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. Furthermore, presence of vimentin also altered the lamin expression in MCF-7, which was an unusual finding. Collectively, our data demonstrate, for the first time, that vimentin in cancer cells could change nuclear architecture by affecting lamin expression, which downregulates the genes maintaining cell–cell junctions resulting in increased cell migration. Third, we studied the role of the single cysteine at 328 in vimentin in EMT and cancer progression. We established MCF-7 expressing separately wildtype (WT) and C328S vimentin polypeptides. This genetic transformation induced EMT-like features that include cell proliferation, migration, and invasion and reduced cell adhesion in C328S cells compared to WT. Transcriptomic analysis revealed upregulation of transcription factors that regulate EMT and downregulation of epithelial markers in C328S cells compared to WT. Also, the breast cancer stem cell markers such as CD56, OCT4, PROCR and CD49f were upregulated in C328S-VIM expressing MCF-7 cells. We also observed a stark increase in expression of long non-coding RNA XIST, which is reportedly upregulated in large number of solid tumours. Downregulation of mutant vimentin in C328S cells using shRNA restored cell phenotypes and downregulated XIST expression, thereby establishing a direct link between C328S vimentin and XIST expression which is a novel finding. Furthermore, xenotransplantation studies in nude mice revealed that C328S cells underwent oestrogen-independent tumorigenic transformation. As mutations in vimentin are reported in clinical samples around this region of single cysteine, we propose that C328S vimentin produces signalling that induce EMT-like changes and enhance cancer cell stemness, conversely C328 region safeguards against EMT induced metastasis in breast cancer in a tumour suppressor way. Fourth, we have also studied the response of vimentin to the oxidative stress induced in MCF-7. We have treated MCF-7 cells expressing untagged full-length vimentin with diamide which is a thiol oxidizing agent to induce oxidative stress in these cells. We observed that vimentin filaments extensively reorganize into dotted structures in response to oxidative stresses induced by diamide implying the role of vimentin as a critical stress sensor. In cells where both keratins and vimentin are present, vimentin was the first stress responder. We also showed that vimentin C328 residue is prerequisite for stress sensing role of vimentin. In addition, our results refute the possibility of crosslinking of vimentin cysteine residues as a possible mechanism behind vimentin remodeling in response to diamide. Our data also preclude involvement of p38 and pI3 kinases involvement in this response. In conclusion, our data demonstrate that vimentin supports cancer cell migration and metastasis by downregulating the genes associated with cell-cell junctions via altered lamins expression, making them loosely attached and free to move. This study also reveals the novel role of vimentin C328 region in controlling cancer cell behaviour and highlights the importance of C328 residue in stress sensing role of vimentin. In addition we describe that tagging vimentin with fluorescent or epitope tags for study purposes, affects protein dynamics and filament assembly. | en_US |
