ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization.

BACKGROUND: Melanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet. METHODS: An immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout (ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes. RESULTS: The immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition of ABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin ß3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation. CONCLUSION: Human melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

BACKGROUND: Melanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet. METHODS: An immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout ( ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes. RESULTS: The immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition ofABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin ß3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation. CONCLUSION: Human melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

Isolation of Stably Transfected Melanoma Cell Clones.

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated cell transfection as it can lead to a heterogeneous cell population and variable transfection efficiency, affecting reproducibility. Moreover, they are preferable for large-scale analyses. The generation of stable cell clones is useful for a wide range of applications, such as research on gene functions and recombinant protein production. There are a few methods to obtain a homogenous cell population upon initial transient transfection. Here, we describe the isolation of single cell clones with glass cylinders. Although this method has been known for some time, there are a few crucial steps, and neglecting them may lead to failure. We have successfully used this method to obtain clones stably overexpressing a protein of interest (POI) or with knockout of a gene of interest (GOI). We describe preparation steps such as the optimization of selecting drug concentrations, preparation of glass cylinders, and validation of whether the obtained clones have the desired change in the expression of the GOI by PCR, western blot analysis, immunostaining, or gDNA sequencing (depending on the type of derived clones). We also discuss the phenotypic heterogeneity of well-established cell lines as this might be an issue in obtaining stable cell clones.

SLC35A2 Deficiency Promotes an Epithelial-to-Mesenchymal Transition-like Phenotype in Madin-Darby Canine Kidney Cells.

In mammalian cells, SLC35A2 delivers UDP-galactose for galactosylation reactions that take place predominantly in the Golgi lumen. Mutations in the corresponding gene cause a subtype of a congenital disorder of glycosylation (SLC35A2-CDG). Although more and more patients are diagnosed with SLC35A2-CDG, the link between defective galactosylation and disease symptoms is not fully understood. According to a number of reports, impaired glycosylation may trigger the process of epithelial-to-mesenchymal transition (EMT). We therefore examined whether the loss of SLC35A2 activity would promote EMT in a non-malignant epithelial cell line. For this purpose, we knocked out the SLC35A2 gene in Madin-Darby canine kidney (MDCK) cells. The resulting clones adopted an elongated, spindle-shaped morphology and showed impaired cell-cell adhesion. Using qPCR and western blotting, we revealed down-regulation of E-cadherin in the knockouts, while the fibronectin and vimentin levels were elevated. Moreover, the knockout cells displayed reorganization of vimentin intermediate filaments and altered subcellular distribution of a vimentin-binding protein, formiminotransferase cyclodeaminase (FTCD). Furthermore, depletion of SLC35A2 triggered Golgi compaction. Finally, the SLC35A2 knockouts displayed increased motility and invasiveness. In conclusion, SLC35A2-deficient MDCK cells showed several hallmarks of EMT. Our findings point to a novel role for SLC35A2 as a gatekeeper of the epithelial phenotype.

Gelatin Zymography to Detect Gelatinase Activity in Melanoma Cells.

Melanoma cells, having highly invasive properties, exhibit the formation of invadopodia-structures formed by tumor cells and responsible for the digestion of the surrounding extracellular matrix (ECM). Several metalloproteases (MMPs) are secreted by cells to hydrolyze ECM proteins. They are mainly secreted through structures known as invadopodia. ECM degradation is crucial for tumor cells while forming metastases as the cells heading towards blood vessels must loosen dense tissue. One group of metalloproteases secreted by melanoma cells comprises the gelatinases, i.e., metalloproteases 2 and 9. Gelatinases cleave gelatin (denatured collagen), a few types of collagen (including type IV), and fibronectin, all structural components of ECM. This paper describes a gelatin zymography assay to analyze the gelatinase activity of melanoma cells. This approach is based on analyzing the extent of digestion of a substrate (gelatin) added to a polyacrylamide gel. Several advantages, such as simplicity, sensitivity, low cost, and semiquantitative analysis by densitometry, as well as the detection of both active and inactive forms of MMPs, make this assay valuable and widely used. This protocol describes how to concentrate medium devoid of intact floating cells, cell debris, and apoptotic bodies. Next, it focuses on preparing polyacrylamide gel with gelatin addition, performing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), removing SDS, and staining of the gel to detect gelatin-free bands corresponding to the activity of gelatinases secreted by melanoma cells. Finally, the paper describes how to quantitatively analyze data from this assay. This method is a good alternative for estimating the gelatinase activity of melanoma cells to a fluorescent gelatin degradation assay, western blot, or enzyme-linked immunosorbent assays (ELISAs).

Gelsolin Contributes to the Motility of A375 Melanoma Cells and This Activity Is Mediated by the Fibrous Extracellular Matrix Protein Profile.

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)-an actin-binding protein-levels in melanoma cell lines and GSN's importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin's BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN's influence on parameters associated with cells' ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin's BM.

Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features.

Thymosin ß4 (Tß4) is a small, 44-amino acid polypeptide. It has been implicated in multiple processes, including cell movement, angiogenesis, and stemness. Previously, we reported that melanoma cell lines differ in Tß4 levels. Studies on stable clones with silenced TMSB4X expression showed that Tß4 impacted adhesion and epithelial-mesenchymal transition progression. Here, we show that the cells with silenced TMSB4X expression exhibited altered actin cytoskeleton's organization and subcellular relocalization of two intermediate filament proteins: Nestin and Vimentin. The rearrangement of the cell cytoskeleton resulted in changes in the cells' topology, height, and stiffness defined by Young's modulus. Simultaneously, only for some A375 clones with a lowered Tß4 level, we observed a decreased ability to initiate colony formation in soft agar, tumor formation in vivo, and alterations in Nanog's expression level transcription factor regulating stemness. Thus, we show for the first time that in A375 cells, biomechanical properties are not directly coupled to stemness features, and this cell line is phenotypically heterogeneous.

A Fluorescent Gelatin Degradation Assay to Study Melanoma Breakdown of Extracellular Matrix.

In order to protrude within a dense tissue, tumor cells have to develop the ability to digest the extracellular matrix (ECM). Melanoma cells, similarly to other types of tumor cells, form invadopodia, membranous invaginations rich in filamentous actin and several other proteins including matrix metalloproteinases (MMPs). MMPs degrade ECM structural proteins such as collagens, fibronectin, or laminin. Here we describe an assay that allows the detection of gelatinase activity exhibited by tumor cells under 2D conditions and methods to present obtained data in both a quantitative and a qualitative manner.

The origin of the expressed retrotransposed gene ACTBL2 and its influence on human melanoma cells' motility and focal adhesion formation.

We have recently found that ß-actin-like protein 2 (actbl2) forms complexes with gelsolin in human melanoma cells and can polymerize. Phylogenetic and bioinformatic analyses showed that actbl2 has a common origin with two non-muscle actins, which share a separate history from the muscle actins. The actin groups' divergence started at the beginning of vertebrate evolution, and actbl2 actins are characterized by the largest number of non-conserved amino acid substitutions of all actins. We also discovered that ACTBL2 is expressed at a very low level in several melanoma cell lines, but a small subset of cells exhibited a high ACTBL2 expression. We found that clones with knocked-out ACTBL2 (CR-ACTBL2) or overexpressing actbl2 (OE-ACTBL2) differ from control cells in the invasion, focal adhesion formation, and actin polymerization ratio, as well as in the formation of lamellipodia and stress fibers. Thus, we postulate that actbl2 is the seventh actin isoform and is essential for cell motility.

Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle ß and γ Actin Are Not Equal in Relation to Human Melanoma Cells' Motility and Focal Adhesion Formation.

Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either ß actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of ß and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than ß actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells' FA formation and motility.

Thymosin ß4 Regulates Focal Adhesion Formation in Human Melanoma Cells and Affects Their Migration and Invasion.

Thymosin ß4 (Tß4), a multifunctional 44-amino acid polypeptide and a member of actin-binding proteins (ABPs), plays an important role in developmental processes and wound healing. In recent years an increasing number of data has been published suggesting Tß4's involvement in tumorigenesis. However, Tß4's role in melanoma tumor development still remains to be elucidated. In our study we demonstrate that Tß4 is crucial for melanoma adhesion and invasion. For the purpose of our research we tested melanoma cell lines differing in invasive potential. Moreover, we applied shRNAs to silence TMSB4X (gene encoding Tß4) expression in a cell line with high TMSB4X expression. We found out that Tß4 is not only a component of focal adhesions (FAs) and interacts with several FAs components but also regulates FAs formation. We demonstrate that Tß4 level has an impact on FAs' number and morphology. Moreover, manipulation with TMSB4X expression resulted in changes in cells' motility on non-coated and MatrigelTM (resembling basement membrane composition)-coated surfaces and drastically decreased invasion abilities of the cells. Additionally, a correlation between Tß4 expression level and exhibition of mesenchymal-like [epithelial-mesenchymal transition (EMT)] features was discovered. Cells with lowered TMSB4X expression were less EMT-progressed than control cells. Summarizing, obtained results show that Tß4 by regulating melanoma cells' adhesion has an impact on motility features and EMT. Our study not only contributes to a better understanding of the processes underlying melanoma cells' capacity to create metastases but also highlights Tß4 as a potential target for melanoma management therapy.