Impairment of Assembly of the Vimentin Intermediate Filaments Leads to Suppression of Formation and Maturation of Focal Contacts and Alteration of the Type of Cellular Protrusions.

Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by formation of lamellipodia and/or filopodia, while amoeboid migration is based on bleb formation. Changing of migrational conditions can lead to alteration in the character of cell movement. For example, inhibition of the Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility mode. Ability of the cells to switch from one type of motility to another is called migratory plasticity. Cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryo fibroblasts REF52 with normal VIF organization, fibroblasts with vimentin knockout (REF-/-), and fibroblasts with mutation inhibiting assembly of the full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase in the number of cells with blebs. The fibroblasts with short fragments of vimentin demonstrate the significant increase in number of cells forming blebs both spontaneously and in the presence of CK-666. Disruption of the VIF organization did not lead to the significant changes in the microtubules network or the level of myosin light chain phosphorylation, but caused significant reduction in the focal contact system. The most pronounced and statistically significant decrease in both size and number of focal adhesions were observed in the REF117 cells. We believe that regulation of the membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that organization of VIFs determines the type of cell protrusions, which, in turn, determines the character of cell movement. A novel role of VIFs as a regulator of membrane blebbing, essential for manifestation of the migratory plasticity, is shown.

Plasma Membrane Blebbing Is Controlled by Subcellular Distribution of Vimentin Intermediate Filaments.

The formation of specific cellular protrusions, plasma membrane blebs, underlies the amoeboid mode of cell motility, which is characteristic for free-living amoebae and leukocytes, and can also be adopted by stem and tumor cells to bypass unfavorable migration conditions and thus facilitate their long-distance migration. Not all cells are equally prone to bleb formation. We have previously shown that membrane blebbing can be experimentally induced in a subset of HT1080 fibrosarcoma cells, whereas other cells in the same culture under the same conditions retain non-blebbing mesenchymal morphology. Here we show that this heterogeneity is associated with the distribution of vimentin intermediate filaments (VIFs). Using different approaches to alter the VIF organization, we show that blebbing activity is biased toward cell edges lacking abundant VIFs, whereas the VIF-rich regions of the cell periphery exhibit low blebbing activity. This pattern is observed both in interphase fibroblasts, with and without experimentally induced blebbing, and during mitosis-associated blebbing. Moreover, the downregulation of vimentin expression or displacement of VIFs away from the cell periphery promotes blebbing even in cells resistant to bleb-inducing treatments. Thus, we reveal a new important function of VIFs in cell physiology that involves the regulation of non-apoptotic blebbing essential for amoeboid cell migration and mitosis.

Inactivation of PTEN and ZFHX3 in Mammary Epithelial Cells Alters Patterns of Collective Cell Migration.

Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.

The Role of the Adapter Protein Anks1a in the Regulation of Breast Cancer Cell Motility.

Epithelial-mesenchymal transition (EMT) is a critical step in tumor progression that leads to the acquisition by cancer cells the capacity for migration using the mesenchymal motility mode regulated by the Rac→WAVE→Arp2/3 signaling pathway. Earlier it was shown that proteins interacting with Rac can regulate mesenchymal migration and thus determine the metastatic potential of the cells. The search for new regulators of cell migration is an important theoretical and practical task. The adaptor protein Anks1a is one of the proteins interacting with Rac, whose expression is altered in many types of tumors. The aim of this study was to find whether Anks1a affects the migration of cancer cells and to identify the mechanism underlying this effect. It was suggested that Anks1a can influence cancer cell migration either as a Rac1 effector or by activating human epidermal growth factor receptor 2 (HER2) exchange. We investigated how upregulation and inhibition of Anks1a expression affected migration of breast cancer cells with different HER2 status. Anks1a was shown to interact with the activated form of Rac1. In the MDA-MB-231 cells (triple negative cancer), which lack HER2, Anks1a accumulated at the active cell edge, which is characterized by enrichment with active Rac1, whereas no such accumulation was observed in the HER2-overexpressing SK-BR-3 cells. Downregulation of the ANKS1a expression with esiRNA had almost no effect on the cancer cell motility, except a slight increase in the average migration rate of MDA-MB-231 cells. Among three cell lines tested, overexpression of Anks1a increased the migration rate of HER2-overexpressng SK-BR-3 cells only. We showed that Anks1a is an effector of activated Rac1, but its influence on the cell migration in this capacity was minimal, at least in the studied breast cancer cells. Anks1a affected the motility of breast cancer cells due to its involvement in the EGF receptor exchange.

Transition from mesenchymal to bleb-based motility is predominantly exhibited by CD133-positive subpopulation of fibrosarcoma cells.

BACKGROUND INFORMATION: Metastatic disease is caused by the ability of cancer cells to reach distant organs and form secondary lesions at new locations. Dissemination of cancer cells depends on their migration plasticity - an ability to switch between motility modes driven by distinct molecular machineries. One of such switches is mesenchymal-to-amoeboid transition. Although mesenchymal migration of individual cells requires Arp2/3-dependent actin polymerisation, amoeboid migration is characterised by a high level of actomyosin contractility and often involves the formation of membrane blebs. The acquisition of amoeboid motility by mesenchymal cells is often associated with enhanced metastasis. RESULTS: We studied the ability of mesenchymal HT1080 fibrosarcoma cells to switch to amoeboid motility. We induced the transition from lamellipodium-rich to blebbing phenotype either by down-regulating the Arp2/3 complex, pharmacologically or by RNAi, or by decreasing substrate adhesiveness. Each of these treatments induced blebbing in a subset of fibrosarcoma cells, but not in normal subcutaneous fibroblasts. A significant fraction of HT1080 cells that switched to blebbing behaviour exhibited stem cell-like features, such as expression of the stem cell marker CD133, an increased efflux of Hoechst-33342 and positive staining for Oct4, Sox2 and Nanog. Furthermore, the isolated CD133+ cells demonstrated an increased ability to switch to bleb-rich amoeboid phenotype both under inhibitor's treatment and in 3D collagen gels. CONCLUSIONS: Together, our data show a significant correlation between the increased ability of cells to switch between migration modes and their stem-like features, suggesting that migration plasticity is an additional property of stem-like population of fibrosarcoma cells. This combination of features could facilitate both dissemination of these cells to distant locations, and their establishment self-renewal in a new microenvironment, as required for metastasis formation. SIGNIFICANCE: These data suggest that migration plasticity is a new feature of cancer stem-like cells that can significantly facilitate their dissemination to a secondary location by allowing them to adapt quickly to challenging microenvironments. Moreover, it complements their resistance to apoptosis and self-renewal potential, thus enabling them not only to disseminate efficiently, but also to survive and colonise new niches.

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

Arpin downregulation in breast cancer is associated with poor prognosis.

BACKGROUND: The Arp2/3 complex is required for cell migration and invasion. The Arp2/3 complex and its activators, such as the WAVE complex, are deregulated in diverse cancers. Here we investigate the expression of Arpin, the Arp2/3 inhibitory protein that antagonises the WAVE complex. METHODS: We used qRT-PCR and reverse phase protein arrays in a patient cohort with known clinical parameters and outcome, immunofluorescence in breast biopsy cryosections and breast cancer cell lines. RESULTS: Arpin was downregulated at the mRNA and protein levels in mammary carcinoma cells. Arpin mRNA downregulation was associated with poor metastasis-free survival (MFS) on univariate analysis (P=0.022). High expression of the NCKAP1 gene that encodes a WAVE complex subunit was also associated with poor MFS on univariate analysis (P=0.0037) and was mutually exclusive with Arpin low. Arpin low or NCKAP1 high was an independent prognosis factor on multivariate analysis (P=0.0012) and was strongly associated with poor MFS (P=0.000064). CONCLUSIONS: Loss of the Arp2/3 inhibitory protein Arpin produces a similar poor outcome in breast cancer as high expression of the NCKAP1 subunit of the Arp2/3 activatory WAVE complex.

Clathrin is required for Scar/Wave-mediated lamellipodium formation.

The Scar/Wave complex (SWC) generates lamellipodia through Arp2/3-dependent polymerisation of branched actin networks. In order to identify new SWC regulators, we conducted a screen in Drosophila cells combining proteomics with functional genomics. This screen identified Clathrin heavy chain (CHC) as a protein that binds to the SWC and whose depletion affects lamellipodium formation. This role of CHC in lamellipodium formation can be uncoupled from its role in membrane trafficking by several experimental approaches. Furthermore, CHC is detected in lamellipodia in the absence of the adaptor and accessory proteins of endocytosis. We found that CHC overexpression decreased membrane recruitment of the SWC, resulting in reduced velocity of protrusions and reduced cell migration. By contrast, when CHC was targeted to the membrane by fusion to a myristoylation sequence, we observed an increase in membrane recruitment of the SWC, protrusion velocity and cell migration. Together these data suggest that, in addition to its classical role in membrane trafficking, CHC brings the SWC to the plasma membrane, thereby controlling lamellipodium formation.