Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5ß/B6 and ABCB5ß/B9.

ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5ß). Several studies have shown that the ABCB5ß homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5ß/B6 and ABCB5ß/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5ß in melanocytes and melanoma.

A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression.

Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation.

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor-induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

EEF2K silencing inhibits tumour progression through repressing SPP1 and synergises with BET inhibitors in melanoma.

BACKGROUND: Despite the remarkable breakthroughs achieved in the management of metastatic melanoma using immunotherapy and targeted therapies, long-term clinical efficacy is often compromised due to dose-limiting toxicity and innate or acquired resistance. Therefore, it is of vital importance to further explore the molecular mechanisms underlying melanoma progression and identify new targeted therapeutic approaches. METHODS: The function of eukaryotic elongation factor-2 kinase (EEF2K) in melanoma were investigated in vitro and in vivo. RNA-seq and chromatin immunoprecipitation (ChIP) assay were undertaken to explore the mechanisms. The antitumor effect of bromodomain and extra terminal domain (BET) inhibitors combined with cytarabine were assessed in melanoma both in vitro and in vivo. RESULTS: EEF2K silencing markedly attenuated the malignant phenotypes of melanoma cells, including proliferation, migration, invasion and metastasis. In contrast, EEF2K overexpression promoted melanoma cell proliferation, migration and invasion. Mechanistically, we demonstrated that EEF2K upregulates the phosphorylation of STAT3 (p-STAT3) at Tyr705, which binds to the promoter region of SPP1 and enhances its transcription, thus facilitating melanoma progression. Transfection-induced re-expression of SPP1 partly negated the inhibitory effect of EEF2K silencing on melanoma, whereas inhibition of SPP1 or STAT3 significantly abolished the efficacy of EEF2K on melanoma cells. Intriguingly, EEF2K silencing combined with BET inhibitor treatment further inhibited cell proliferation and promoted apoptosis in melanoma. We further screened the US FDA-approved antitumour drug library and identified cytarabine as a potential clinically applicable EEF2K inhibitor that could synergise with BET inhibitors in melanoma treatment. CONCLUSION: EEF2K/p-STAT3/SPP1 may be a novel oncogenic pathway in melanoma progression, which could be a target for novel combination therapy for melanoma.

Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line.

The role of proline dehydrogenase/proline oxidase (PRODH/POX) in the mechanism of antineoplastic activity of metformin (MET) was studied in C32 melanoma cells. PRODH/POX is a mitochondrial enzyme-degrading proline that is implicated in the regulation of cancer cell survival/apoptosis. The enzyme is activated by AMP kinase (AMPK). It has been found that MET induced a significant decrease in cell viability and DNA biosynthesis accompanied by an increase in the expressions of AMPK and PRODH/POX in C32 cells. The mechanism for MET-dependent cytotoxicity on C32 cells was found at the level of PRODH/POX-induced ROS generation and activation of Caspase-3 and Caspase-9 expressions in these cells. The effects were not observed in MET-treated PRODH/POX knock-out C32 cells. Of interest is an MET-dependent increase in the concentration of proline, which is a substrate for PRODH/POX. This phenomenon is due to the MET-dependent inhibition of collagen biosynthesis, which is the main proline-utilizing process. It has been found that the underlying mechanism of anticancer activity of MET involves the activation of AMPK, PRODH/POX, increase in the cytoplasmic concentration of proline, inhibition of collagen biosynthesis, and stimulation of PRODH/POX-dependent ROS generation, which initiate the apoptosis of melanoma cells.

Interaction of Glia Cells with Glioblastoma and Melanoma Cells under the Influence of Phytocannabinoids.

Brain tumor heterogeneity and progression are subject to complex interactions between tumor cells and their microenvironment. Glioblastoma and brain metastasis can contain 30-40% of tumor-associated macrophages, microglia, and astrocytes, affecting migration, proliferation, and apoptosis. Here, we analyzed interactions between glial cells and LN229 glioblastoma or A375 melanoma cells in the context of motility and cell-cell interactions in a 3D model. Furthermore, the effects of phytocannabinoids, cannabidiol (CBD), tetrahydrocannabidiol (THC), or their co-application were analyzed. Co-culture of tumor cells with glial cells had little effect on 3D spheroid formation, while treatment with cannabinoids led to significantly larger spheroids. The addition of astrocytes blocked cannabinoid-induced effects. None of the interventions affected cell death. Furthermore, glial cell-conditioned media led to a significant slowdown in collective, but not single-cell migration speed. Taken together, glial cells in glioblastoma and brain metastasis micromilieu impact the tumor spheroid formation, cell spreading, and motility. Since the size of spheroid remained unaffected in glial cell tumor co-cultures, phytocannabinoids increased the size of spheroids without any effects on migration. This aspect might be of relevance since phytocannabinoids are frequently used in tumor therapy for side effects.

Viscoelastic parameterization of human skin cells characterize material behavior at multiple timescales.

Countless biophysical studies have sought distinct markers in the cellular mechanical response that could be linked to morphogenesis, homeostasis, and disease. Here, an iterative-fitting methodology visualizes the time-dependent viscoelastic behavior of human skin cells under physiologically relevant conditions. Past investigations often involved parameterizing elastic relationships and assuming purely Hertzian contact mechanics, which fails to properly account for the rich temporal information available. We demonstrate the performance superiority of the proposed iterative viscoelastic characterization method over standard open-search approaches. Our viscoelastic measurements revealed that 2D adherent metastatic melanoma cells exhibit reduced elasticity compared to their normal counterparts-melanocytes and fibroblasts, and are significantly less viscous than fibroblasts over timescales spanning three orders of magnitude. The measured loss angle indicates clear differential viscoelastic responses across multiple timescales between the measured cells. This method provides insight into the complex viscoelastic behavior of metastatic melanoma cells relevant to better understanding cancer metastasis and aggression.

Influence of cellular redox environment on aryl hydrocarbon receptor ligands induced melanogenesis.

Many environmental pollutants, natural compounds, as well as endogenous chemicals exert their biological/toxicological effects by reacting with the aryl hydrocarbon receptor (AhR). Previous evidence shed new light on the role of AhR in skin physiology by regulating melanin production. In this study, we investigated the effect of oxidative imbalance induced by AhR ligands on the melanogenesis process in B16 murine melanoma cells. Exposure to 6-formylindolo[3,2-b] carbazole (FICZ) or benzo-α-pyrene (BαP) led to enhanced expression of CTNNB1, MITF, and TYR genes following increased tyrosinase enzyme activity and melanin content in an AhR-dependent manner. Analysis of the presence of reactive oxygen species (ROS) as well as reduced glutathione (GSH) / oxidized glutathione (GSSG) ratio revealed that treatment with AhR ligands is associated with oxidative stress which can be ameliorated with NAC (N-acetyl cysteine) or diphenyleneiodonium chloride (DPI). On the other hand, NAC and DPI enhanced melanogenesis induced by AhR ligands by reducing the level of ROS. We have shown for the first time that a cellular redox status is a critical event during AhR ligand-induced melanogenesis.

Health-related quality of life using the FACT-M questionnaire in patients with malignant melanoma: A systematic review.

BACKGROUND: Since there are no formal definition of health-related quality of life (HRQOL) there may be a lack of coherence and understanding of how to interpret HRQOL-data. The aim of this study is to summarize HRQOL-results that have used the FACT-M questionnaire in patients with melanoma, and specifically to summarize FACT-M between tumor stage. METHODS: This review was conducted in accordance with the PRISMA guidelines. INCLUSION CRITERIA: original studies on cutaneous melanoma between 2005 and 2020, written in English, containing "Functional Assessment of Cancer Therapy Melanoma" OR "Functional Assessment of Cancer Therapy M" OR FACT-M OR FACT/M OR FACTM OR "FACT M" OR FACT-melanoma OR "FACT Melanoma" together with FACT-M numbered data and basic patient characteristics, using the databases Scopus, Web of Science, PubMed and PsycINFO. RESULTS: 16 articles describing 14 patient cohorts published 2008-2020 were included. The majority of the studies did not report subscale scores in accordance with FACT-M guidelines. The results did indicate that FACT-M total scores were inversely correlated with AJCC stage. Subscale analysis demonstrated varying degrees of correlation with AJCC stage. The Melanoma Surgery Subscale score was lowest in stage III patients, probably reflecting more advanced surgical procedures in this group of patients. CONCLUSIONS: Though this review is based on a questionnaire limited to the assessment of melanoma patients, it highlights the universal need for clinical studies to describe their selected HRQOL-questionnaires, its definition of HRQOL and its dimensions, as well as comply with the questionnaire's guidelines when reporting HRQOL-data.

RGS2 Suppresses Melanoma Growth via Inhibiting MAPK and AKT Signaling Pathways.

BACKGROUND/AIM: This study aimed to explore RGS2 as a regulator of melanoma cell growth. MATERIALS AND METHODS: Effect of RGS2 over-expression was analyzed in three melanoma cell lines, and Rgs2 knockdown was performed in zebrafish. RESULTS: RGS2 was differentially expressed among the cell lines. In B16F10 cells, RGS2 over-expression inhibited MAPK and AKT activation, and prevented cell growth. A similar outcome was observed in A375 cells, but the MAPK signals were not suppressed. In A2058 cells, RGS2 repressed AKT activation, but without affecting cell growth. Moreover, MAPK and AKT constitutive activation abolished the RGS2 inhibitory effect on B16F10 cell growth. Rgs2 knockdown caused ectopic melanocyte differentiation, and promoted MAPK and AKT activation in zebrafish embryos. CONCLUSION: RGS2 prevents melanoma cell growth by inhibiting MAPK and AKT, but this effect depends on the overall cell genetic landscape. Further studies are warranted to investigate the anticancer therapeutic potential of RGS2 for melanoma.

Modeling the Ex Post Real Option Value in Metastatic Melanoma Using Real-World Data.

OBJECTIVES: Real option value (ROV) is created when a drug enables a patient to live long enough to benefit from a future innovation. Few studies have quantified ROV in the real world. We aimed to estimate the ex post ROV for ipilimumab in metastatic melanoma using real-world data (RWD). METHODS: We developed a framework for calculating ROV using RWD, accounting for the health gain in the standard therapy arm and the uptake of future innovations. A Markov model was developed to estimate the quality-adjusted life-years (QALYs) gained with ipilimumab compared with chemotherapy for patients with or without subsequent cancer immunotherapy (CIT). A nationwide electronic health record-derived, deidentified database was used to estimate survival and uptake of CIT. RESULTS: The incremental QALYs gained for ipilimumab compared with chemotherapy without subsequent CIT were 1.74. With subsequent CIT, the incremental QALYs compared with chemotherapy increased by 0.92, 0.60, 0.33, 0.18, 0.10, and 0.02 when CIT became available 0, 3, 6, 9, 12, and 24 months after the initiation of first-line treatment, respectively. The results were most sensitive to the survival benefit of ipilimumab, the survival benefit of subsequent CIT, and the uptake of CIT. CONCLUSIONS: This is the first study to estimate ex post ROV using RWD. The ex post ROV was between 1% and 54% of conventional value for patients who received a diagnosis within 2 years before CIT availability. Further studies are needed to understand ROV in other disease areas, particularly those with longer survival times.

Quantitative analysis of tumour spheroid structure.

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

p32 promotes melanoma progression and metastasis by targeting EMT markers, Akt/PKB pathway, and tumor microenvironment.

Melanoma originates from melanin-producing cells called melanocytes. Melanoma poses a great risk because of its rapid ability to spread and invade new organs. Cellular metastasis involves alteration in the gene expression profile and their transformation from epithelial to mesenchymal state. Despite of several advances, metastatic melanoma being a key cause of therapy failure and mortality remains poorly understood. p32 has been found to be involved in various physiological and pathophysiological conditions. However, the role of p32 in melanoma progression and metastasis remains underexplored. Here, we identify the role of p32 in the malignancy of both murine and human melanoma. p32 knockdown leads to reduced cell proliferation, migration, and invasion in murine and human melanoma cells. Furthermore, p32 promotes in vitro tumorigenesis, inducing oncogenes and EMT markers. Mechanistically, we show p32 regulates tumorigenic and metastatic properties through the Akt/PKB signaling pathway in both murine and human melanoma. Furthermore, p32 silencing attenuates melanoma tumor progression and lung metastasis in vivo, modulating the tumor microenvironment by inhibiting the angiogenesis, infiltration of macrophages, and leukocytes in mice. Taken together, our findings identify that p32 drives melanoma progression, metastasis, and regulates the tumor microenvironment. p32 can be a target of a novel therapeutic approach in the regulation of melanoma progression and metastasis.

LncRNA MSC-AS1 motivates the development of melanoma by binding to miR-302a-3p and recruiting IGF2BP2 to elevate LEF1 expression.

Melanoma is considered as the most common malignancy among skin cancers. The roles of many long non-coding RNAs (lncRNAs) have been clearly identified in multiple tumors. Nevertheless, lncRNA MSC antisense RNA 1 (MSC-AS1) has not been deeply investigated melanoma. In the present study, RT-qPCR and western blot analyses were used to measure the expression of RNAs and proteins. Functional and in vivo assays were implemented to detect the function of genes in melanoma. RNA pull-down, RIP and luciferase reporter assays were applied for determining interactions between RNA and protein molecules. It was observed that MSC-AS1 and lymphoid enhancer-binding factor 1 (LEF1) were remarkably up-regulated while microRNA-302a-3p (miR-302a-3p) down-regulated in melanoma cell lines. The silencing of MSC-AS1 hindered cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro and tumor growth in vivo. Furthermore, MSC-AS1 regulated LEF1 expression through sponging miR-302a-3p and recruiting insulin like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Eventually, LEF1 overexpression rescued cell progression impaired by MSC-AS1 knock-down. In summary, our research identified the MSC-AS1/miR-302a-3p/IGF2BP2/LEF1 axis in melanoma development, which indicated that MSC-AS1 is a potential biomarker in the treatment of melanoma.

From Melanocytes to Melanoma Cells: Characterization of the Malignant Transformation by Four Distinctly Different Melanin Fluorescence Spectra (Review).

The melanin fluorescence emitted by pigment cells of the human skin has been a central research topic for decades, because melanin, on the one hand, protects against (solar) radiation in the near-UV range, whereas on the other hand, melanocytes are the starting point for the malignant transformation into melanoma. Until recently, however, melanin fluorescence was not accessible in the context of conventional spectroscopy, because it is ultraweak and is overshadowed by the more intense so-called autofluorescence of endogenous fluorophores. The advent of a new method of laser spectroscopy has made this melanin fluorescence measurable in vivo. A stepwise two-photon absorption with 800 nm photons is used, which more selectively excites melanin (dermatofluoroscopy). Our review summarizes the experimental results on melanin fluorescence of the four types of cutaneous pigment cells from healthy and malignant tissues. Outstanding is the finding that different types of melanocytes (i.e., melanocytes of common nevi, versus dysplastic nevi or versus melanoma cells) show characteristically different fluorescence spectra. The possibilities of using this melanin fluorescence for melanoma diagnosis are shown. Moreover, the uniform fluorescence spectra emitted by different melanoma subtypes are essential. Conclusions are drawn about the molecular processes in the melanosomes that determine fluorescence. Finally, experimental suggestions for further investigations are given.

Slow Transcription of the 99a/let-7c/125b-2 Cluster Results in Differential MiRNA Expression and Promotes Melanoma Phenotypic Plasticity.

Almost half of the human microRNAs (miRNAs) are encoded in clusters. Although transcribed as a single unit, the levels of individual mature miRNAs often differ. The mechanisms underlying differential biogenesis of clustered miRNAs and the resulting physiological implications are mostly unknown. In this study, we report that the melanoma master transcription regulator MITF regulates the differential expression of the 99a/let-7c/125b-2 cluster by altering the distribution of RNA polymerase II along the cluster. We discovered that MITF interacts with TRIM28, a known inhibitor of RNA polymerase II transcription elongation, at the mIR-let-7c region, resulting in the pausing of RNA polymerase II activity and causing an elevation in mIR-let-7c expression; low levels of RNA polymerase II occupation over miR-99a and miR-125b-2 regions decreases their biogenesis. Furthermore, we showed that this differential expression affects the phenotypic state of melanoma cells. RNA-sequencing analysis of proliferative melanoma cells that express miR-99a and miR-125b mimics revealed a transcriptomic shift toward an invasive phenotype. Conversely, expression of a mIR-let-7c mimic in invasive melanoma cells induced a shift to a more proliferative state. We confirmed direct target genes of these miRNAs, including FGFR3, BAP1, Bcl2, TGFBR1, and CDKN1A. Our study demonstrates an MITF-governed biogenesis mechanism that results in differential expression of clustered 99a/let-7c/125b-2 miRNAs that control melanoma progression.

Influence of Tumor Microenvironment and Fibroblast Population Plasticity on Melanoma Growth, Therapy Resistance and Immunoescape.

Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.

Paraneoplastic neuromyelitis optica spectrum disorder associated with malignant melanoma: A case report.

Neuromyelitis optica spectrum disorders (NMOSDs) are autoimmune demyelinating diseases involving the central nervous system, affecting the spinal cord and optic nerves. There are few reports of paraneoplastic NMOSD associated with malignant melanoma. Here, we report a rare case of anti-aquaporin 4 (AQP4) antibody-positive NMOSD associated with malignant melanoma. A 61-year-old Japanese woman was diagnosed with malignant melanoma and lung metastasis four years after a diagnosis of anti-AQP4 antibody-positive NMOSD. When diagnosing and treating patients with NMOSD, physicians should be aware of the development of malignancy for at least several years.

Benzyl and phenethyl isothiocyanates as promising epigenetic drug compounds by modulating histone acetylation and methylation marks in malignant melanoma.

Melanoma is an aggressive skin cancer with increasing incidence rates globally. On the other hand, isothiocyanates are derived from cruciferous vegetables and are known to exert a wide range of anti-cancer activities including, among others, their ability to interact with the epigenome in order to supress cancer progression. The aim of this study was to determine the role of phenethyl and benzyl isothiocyanates in modulating histone acetylation and methylation as a potential epigenetic therapeutic strategy in an in vitro model of malignant melanoma. We report that both isothiocyanates induced cytotoxicity and influenced acetylation and methylation status of specific lysine residues on histones H3 and H4 by modulating the expression of various histone acetyltransferases, deacetylases and methyltransferases in malignant melanoma cells. Our data highlight novel insights on the interaction of isothiocyanates with components of the histone regulatory machinery in order to exert their anti-cancer action in malignant melanoma.