Characterization of two melanoma cell lines resistant to BRAF/MEK inhibitors (vemurafenib and cobimetinib).

BACKGROUND: BRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors. METHODS: Cell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array. RESULTS: The resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRß receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion. CONCLUSIONS: In summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies.

Combinations of EGFR and MET inhibitors reduce proliferation and invasiveness of mucosal melanoma cells.

Mucosal melanoma (MM) is a very rare and aggressive type of cancer for which immunotherapy or targeted therapy such as BRAF/MEK inhibitors, used in cutaneous melanoma, usually fail. Due to our earlier experience showing the high effectiveness of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) inhibitors in reducing the activation of the MAPK and PI3K/AKT signalling pathways, we aim to test whether these drugs would also be effective for mucosal melanoma. Cells representing two commercially available mucosal melanoma cell lines (GAK and HMVII) and one cell line obtained from a patient's vaginal melanoma were treated with MET or EGFR inhibitors, or combinations of these agents. The dual-inhibitor treatment strategy resulted in a decrease of cell proliferation, migration and invasion. Moreover, combinations of inhibitors led to reduction of pEGFR/EGFR and pMET/MET ratio and downregulation of PI3K/AKT and MEK/ERK1/2-based signalling pathways. Our findings indicate a potential therapeutic strategy based on EGFR and MET inhibitors in mucosal melanoma, which should be further evaluated in vivo and in clinical experiments. They also suggest that targeting multiple receptor tyrosine kinases may block signalling crosstalk and possibly delay the appearance of resistance to kinase inhibitors in mucosal melanoma cells.

Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche.

BACKGROUND: One of the factors that affect the progression of melanoma is the tumor microenvironment, which consists of cellular elements, extracellular matrix, acidification, and a hypoxic state. Adipocytes are one of the types of cell present in the niche and are localized in the deepest layer of the skin. However, the relationship between fat cells and melanoma remains unclear. METHODS: We assessed the influence of melanoma cells on adipocytes using an indirect coculture system. We estimated the level of cancer-associated adipocyte (CAA) markers through quantitative PCR analysis. The fibroblastic phenotype of CAAs was confirmed by cell staining and western blotting analysis. The lipid content was estimated by lipid detection in CAAs using LipidSpot and by quantitative analysis using Oil Red O. The expression of proteins involved in lipid synthesis, delipidation, and metabolic processes were assessed through quantitative PCR or western blotting analysis. Lactate secretion was established using a Lactate-Glo™ assay. Proteins secreted by CAAs were identified in cytokine and angiogenesis arrays. The proliferation of melanoma cells cocultured with CAAs was assessed using an XTT proliferation assay. Statistical analysis was performed using a one-way ANOVA followed by Tukey's test in GraphPad Prism 7 software. RESULTS: Obtained CAAs were identified by decreased levels of leptin, adiponectin, resistin, and FABP4. Adipocytes cocultured with melanoma presented fibroblastic features, such as a similar proteolytic pattern to that of 3T3L1 fibroblasts and increased levels of vimentin and TGFßRIII. Melanoma cells led to a reduction of lipid content in CAAs, possibly by downregulation of lipid synthesis pathways (lower FADS, SC4MOL, FASN) or enhancement of lipolysis (higher level of phosphorylation of ERK and STAT3). Adipocytes cocultured with melanoma cells secreted higher IL6 and SerpinE1 levels and produced less CCL2, CXCL1, and angiogenic molecules. CAAs also showed metabolic changes comprising the increased secretion of lactate and enhanced production of glucose, lactate, and ion transporters. In addition, changes in adipocytes observed following melanoma coculture resulted in a higher proliferation rate of cancer cells. CONCLUSIONS: Melanoma cells led to decreased lipid content in adipocytes, which might be related to enhanced delipidation or reduction of lipid synthesis. Fibroblast-like CAAs showed metabolic changes that may be the reason for accelerated proliferation of melanoma cells.

Novel hybrid composites based on double-decker silsesquioxanes functionalized by methacrylate derivatives and polyvinyl alcohol as potential materials utilized in biomedical applications.

The use of diverse biomaterials for regenerative medicine is constantly evolving. Therefore, looking for easy-to-scale-up materials in terms of preparation, less complex composition, and featuring structural and chemical stability seems justified. In this work, we report the preparation of double-decker silsesquioxane-based (DDSQ-based) composites, which, according to our best knowledge, have never been used as biomaterials. A family of methacrylate-substituted DDSQs was obtained starting from the previously reported hydroxyalkyl double-decker silsesquioxanes. In the resulting hybrids, methacrylate groups are attached to each other's lateral silicon atoms of DDSQ in trans positions, providing an excellent geometry for forming thin layers. In contrast to pure organic methacrylates, the covalent bonding of methacrylate derivatives to inorganic silsesquioxane core improves mechanics, cell adhesion, and migration properties. Furthermore, to increase the hydrophilicity of the resulting DDSQ-based hybrids, polyvinyl alcohol (PVA) was added. The entire system forms an easy-to-obtain two-component (DDSQ-PVA) composite, which was subjected without any upgrading additives to biological tests later in the research. The resulting biomaterials fulfill the requirements for potential medical applications. Human fibroblasts growing on prepared hybrid composites are characterized by proper spindle-shaped morphology, proliferation, and activation status similar to control conditions (cells cultured on PVA), as well as increased adhesion and migration abilities. The obtained results suggest that the prepared biomaterials may be used in regenerative medicine in the future.

The impact of cellular elements of TME on melanoma biology and its sensitivity to EGFR and MET targeted therapy.

Microenvironment of the melanoma consists of cellular elements like fibroblasts, adipocytes, and keratinocytes as well as extracellular matrix and physicochemical conditions. In our previous research, we have established that melanoma influences strongly above mentioned cells present in the tumor niche and recruits them to support cancer progression. In this work, we evaluated the impact of cancer-associated cells, namely fibroblasts (CAFs), adipocytes (CAAs), and keratinocytes (CAKs) on melanoma proliferation, signaling pathways activation, metabolism as well as the effectiveness of used anti-cancer therapy. Obtained results indicated elevated phosphorylation of STAT3, upregulated GLUT1 and GLUT3 as well as downregulated of MCT-1 expression level in melanoma cells under the influence of all examined cells present in the tumor niche. The proliferation of melanoma cells was increased after co-culture with CAFs and CAKs, while epithelial-mesenchymal transition markers' expression level was raised in the presence of CAFs and CAAs. The level of perilipin 2 and lipid content was elevated in melanoma cells under the influence of CAAs. Moreover, increased expression of CYP1A1, gene encoding drug metabolizing protein, in melanoma cells co-cultured with CAFs and CAKs prompted us to verify the effectiveness of the previously proposed by us anti-melanoma therapy based on combination of EGFR and MET inhibitors. Obtained results indicate that the designed therapy is still efficient, even if the fibroblasts, adipocytes, and keratinocytes, are present in the melanoma vicinity.