Increasing evidence indicates that the tumor microenvironment is a critical factor supporting cancer progression, chemoresistance and metastasis. Recently, cancer-associated fibroblasts (CAFs) have been recognized as a crucial tumor stromal component promoting cancer growth and invasiveness via modulation of the extracellular matrix (ECM) structure, tumor metabolism and immune reprogramming. One of the main sources of CAFs are endothelial cells undergoing the endothelial-mesenchymal transition (EndMT). EndMT is mainly promoted by the Transforming Growth Factor-β (TGF-β) family secreted by tumor cells, though the role of particular members in EndMT regulation remains poorly understood. Our findings demonstrate that TGF-β2 induces mesenchymal transdifferentiation of human microvascular endothelial cells (HMEC-1 cells) to CAF-like cells in association with elongated cell morphology, modulation of stress fiber organization, higher α-SMA protein levels and activation of RhoA and Rac-1 pathways. Such regulation is similar to that observed in cells maintained using conditioned medium from invasive colorectal cancer cell line culture. Furthermore, TGF-β2 stimulation resulted in myocardin-related transcription factor (MRTF) activation and upregulation. Our results demonstrate for the first time that such interaction is sufficient for integrin-linked kinase (ILK) overexpression. ILK upregulation also enhanced MRTF activation via RhoA and Rac-1-MMP9 via inside-out integrin activation. Herein, we propose a new ILK-MMP9-MRTF axis that appears to be critical for EndMT differentiation of endothelial to CAF-like cells. Thus, it might be an attractive target for cancer treatment.

Endothelial–mesenchymal transition (EndMT) is a crucial phenomenon in regulating the development of diseases, including cancer metastasis and fibrotic disorders. The primary regulators of disease development are zinc-finger transcription factors belonging to the Snail family. In this study, we characterized the myocardin-related transcription factor (MRTF)-dependent mechanisms of a human snail promoter regulation in TGF-β-stimulated human endothelial cells. Although in silico analysis revealed that the snail promoter’s regulatory fragment contains one GCCG and two SP1 motifs that could be occupied by MRTFs, the genetic study confirmed that MRTF binds only to SP1 sites to promote snail expression. The more accurate studies revealed that MRTF-A binds to both SP1 elements, whereas MRTF-B to only one (SP1near). Although we found that each MRTF alone is capable of inducing snail expression, the direct cooperation of these proteins is required to reinforce snail expression and promote the late stages of EndMT within 48 hours. Furthermore, genetic and biochemical analysis revealed that MRTF-B alone could induce the late stage of EndMT. However, it requires a prolonged time. Therefore, we concluded that MRTFs might cause EndMT in a fast- and slow-dependent manner. Based on MRTF-dependent Snail upregulation, we recognized that TGF-β1, as an MRTF-B regulator, is involved in slow EndMT induction, whereas TGF-β2, which altered both MRTF-A and MRTF-B expression, promotes a fast EndMT process.

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Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.