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.     

A UHPLC-Mass Spectrometry View of Human Melanocytic Cells Uncovers Potential Lipid Biomarkers of Melanoma

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the “malignancy lipid signature” features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma.     

New Therapeutic Approaches for Conjunctival Melanoma—What We Know So Far and Where Therapy Is Potentially Heading: Focus on Lymphatic Vessels and Dendritic Cells

Conjunctival melanoma (CM) accounts for 5% of all ocular melanomas and arises from malignantly transformed melanocytes in the conjunctival epithelium. Current therapies using surgical excision in combination with chemo- or cryotherapy still have high rates for recurrences and metastatic disease. Lately, novel signal transduction-targeted and immune checkpoint inhibitors like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein-1 (PD-1) receptor inhibitors, BRAF- or MEK-inhibitors for systemic treatment of melanoma have improved the outcome even for unresectable cutaneous melanoma, improving patient survival dramatically. The use of these therapies is now also recommended for CM; however, the immunological background of CM is barely known, underlining the need for research to better understand the immunological basics when treating CM patients with immunomodulatory therapies. Immune checkpoint inhibitors activate tumor defense by interrupting inhibitory interactions between tumor cells and T lymphocytes at the so-called checkpoints. The tumor cells exploit these inhibitory targets on T-cells that are usually used by dendritic cells (DCs). DCs are antigen-presenting cells at the forefront of immune response induction. They contribute to immune tolerance and immune defense but in the case of tumor development, immune tolerance is often prevalent. Enhancing the immune response via DCs, interfering with the lymphatic pathways during immune cell migration and tumor development and specifically targeting tumor cells is a major therapeutic opportunity for many tumor entities including CM. This review summarizes the current knowledge on the function of lymphatic vessels in tumor growth and immune cell transport and continues to compare DC subsets in CM with related melanomas, such as cutaneous melanoma and mucosal melanoma.     

Anti-Melanoma Capability of Contactless Cold Atmospheric Plasma Treatment

In this study, we demonstrated that the widely used cold atmospheric plasma (CAP) jet could significantly inhibit the growth of melanoma cells using a contactless treatment method, The flow rate of helium gas was a key operational parameter to modulate electromagnetic (EM) effect on melanoma cells. Metal sheets with different sizes could be used as a strategy to control the strength of EM effect. More attractive, the EM effect from CAP could penetrate glass/polystyrene barriers as thick as 7 mm. All these discoveries presented the profound non-invasive nature of a physically based CAP treatment, which provided a solid foundation for CAP-based cutaneous/subcutaneous tumor therapy.     

The Interplay between HGF/c-met Axis and Nox4 in BRAF Mutated Melanoma

Background: Melanoma is the leading cause of death due to cutaneous malignancy and its incidence is on the rise. Several signaling pathways, including receptor tyrosine kinases, have a role in the development and progression of melanocytic lesions and malignant melanoma. Among those, the hepatocyte growth factor (HGF)/c-met axis is emerging as a critical player because it can play a role in drug resistance. Indeed, 50% of melanoma patients present BRAF mutations, however, all responders develop resistance to the inhibitors typically within one year of treatment. Interestingly, BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells, therefore, the aim of this study was to investigate a possible interplay between HGF/c-met and ROS sources, such as NADPH oxidases (Nox). Methods: The expression of c-met and Nox were quantified in 60 patients with primary cutaneous melanoma. In vitro experiments on melanoma primary cells and the cell line were performed to dissect the underpinned molecular mechanism. Results: The outcome of interest was the correlation between the high positivity for both Nox4 and c-met and metastasis occurring at least 1 year later than melanoma diagnosis in BRAF mutated patients, in contrast to nonmutated. In vitro experiments demonstrated that the axis HGF/c-met/Nox4/ROS triggers the epithelial-mesenchymal transition. Conclusions: The observed correlation suggests an interplay between c-met and Nox4 in promoting the onset of metastasis. This study suggests that Nox4 inhibitors could be associated to the current therapy used to treat melanoma patients with BRAF mutations.     

The Role of Calcium Signaling in Melanoma

Calcium signaling plays important roles in physiological and pathological conditions, including cutaneous melanoma, the most lethal type of skin cancer. Intracellular calcium concentration ([Ca²⁺]i), cell membrane calcium channels, calcium related proteins (S100 family, E-cadherin, and calpain), and Wnt/Ca²⁺ pathways are related to melanogenesis and melanoma tumorigenesis and progression. Calcium signaling influences the melanoma microenvironment, including immune cells, extracellular matrix (ECM), the vascular network, and chemical and physical surroundings. Other ionic channels, such as sodium and potassium channels, are engaged in calcium-mediated pathways in melanoma. Calcium signaling serves as a promising pharmacological target in melanoma treatment, and its dysregulation might serve as a marker for melanoma prediction. We documented calcium-dependent endoplasmic reticulum (ER) stress and mitochondria dysfunction, by targeting calcium channels and influencing [Ca²⁺]i and calcium homeostasis, and attenuated drug resistance in melanoma management.     

Disruption of Redox Homeostasis by Alterations in Nitric Oxide Synthase Activity and Tetrahydrobiopterin along with Melanoma Progression

Cutaneous melanoma emerges from the malignant transformation of melanocytes and is the most aggressive type of skin cancer. The progression can occur in different stages: radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Reactive oxygen species contribute to all phases of melanomagenesis through the modulation of oncogenic signaling pathways. Tetrahydrobiopterin (BH4) is an important cofactor for NOS coupling, and an uncoupled enzyme is a source of superoxide anion (O₂^•−) rather than nitric oxide (NO), altering the redox homeostasis and contributing to melanoma progression. In the present work, we showed that the BH4 amount varies between different cell lines corresponding to distinct stages of melanoma progression; however, they all presented higher O₂^•− levels and lower NO levels compared to melanocytes. Our results showed increased NOS expression in melanoma cells, contributing to NOS uncoupling. BH4 supplementation of RGP cells, and the DAHP treatment of metastatic melanoma cells reduced cell growth. Finally, Western blot analysis indicated that both treatments act on the PI3K/AKT and MAPK pathways of these melanoma cells in different ways. Disruption of cellular redox homeostasis by the altered BH4 concentration can be explored as a therapeutic strategy according to the stage of melanoma.     

Ketoprofen Combined with UVA Irradiation Exerts Higher Selectivity in the Mode of Action against Melanotic Melanoma Cells than against Normal Human Melanocytes

Malignant melanoma is responsible for the majority of skin cancer-related deaths. The methods of cancer treatment include surgical removal, chemotherapy, immunotherapy, and targeted therapy. However, neither of these methods gives satisfactory results. Therefore, the development of new anticancer therapeutic strategies is very important and may extend the life span of people suffering from melanoma. The aim of this study was to examine the effect of ketoprofen (KTP) and UVA radiation (UVAR) therapy on cell proliferation, apoptosis, and cell cycle distribution in both melanotic melanoma cells (COLO829) and human melanocytes (HEMn-DP) in relation to its supportive effect in the treatment of melanoma. The therapy combining the use of pre-incubation with KTP and UVAR causes a significant increase in the anti-proliferative properties of ketoprofen towards melanoma cells and the co-exposure of melanotic melanoma cells induced apoptosis shown as the mitochondrial membrane breakdown, cell-cycle deregulation, and DNA fragmentation. Moreover, co-treatment led to GSH depletion showing its pro-apoptotic effect dependent on ROS overproduction. The treatment did not show a significant effect on normal cells—melanocytes—which indicates its high selectivity. The results suggest a possible benefit from the use of the ketoprofen and ultraviolet A irradiation as a new concept of melanotic melanoma therapy.     

Molecular Profiling and Novel Therapeutic Strategies for Mucosal Melanoma: A Comprehensive Review

Mucosal melanoma is a rare and aggressive subtype of melanoma. Unlike its cutaneous counterpart, mucosal melanoma has only gained limited benefit from novel treatment approaches due to the lack of actionable driver mutations and poor response to immunotherapy. Over the last years, whole-genome and exome sequencing techniques have led to increased knowledge on the molecular landscape of mucosal melanoma. Molecular studies have underlined noteworthy findings with potential therapeutic implications, including the presence of KIT mutations, which are potential targets of tyrosine kinase inhibitors currently in use in the clinic (imatinib), but also SF3B1 mutation, CDK4 amplifications, and CDKN2A gene deletions, which are presently under investigation in clinical trials. Recent results from a pooled analysis of patients with mucosal melanoma treated with immunotherapy have suggested that the combination of immune checkpoint inhibitors might improve survival outcomes in this subset of patients, as compared with single-agent immunotherapy. However, these results are not confirmed across different studies, and combo-immunotherapy correlates with a higher rate of adverse events. In this review, we describe the clinical, biological, and genetic features of mucosal melanoma. We also provide an update on the results of approved systemic treatment in this setting and overview the therapeutic strategies currently under investigation in clinical trials.     

Normal Skin Cells Increase Aggressiveness of Cutaneous Melanoma by Promoting Epithelial-to-Mesenchymal Transition via Nodal and Wnt Activity

Melanoma is a lethal form of skin cancer triggered by genetic and environmental factors. Excision of early-stage, poorly aggressive melanoma often leads to a successful outcome; however, left undiagnosed these lesions can progress to metastatic disease. This research investigates whether the exposure of poorly aggressive melanoma to certain normal skin cells can explain how non-metastatic melanoma becomes more aggressive while still confined to the skin. To this end, we used a serial co-culture approach to sequentially expose cells from two different, poorly aggressive human melanoma cell lines against normal cells of the skin beginning with normal melanocytes, then epidermal keratinocytes, and finally dermal fibroblasts. Protein extraction of melanoma cells occurred at each step of the co-culture sequence for western blot (WB) analysis. In addition, morphological and functional changes were assessed to detect differences between the serially co-cultured melanoma cells and non-co-cultured cells. Results show that the co-cultured melanoma cells assumed a more mesenchymal morphology and displayed a significant increase in proliferation and invasiveness compared to control or reference cells. WB analysis of protein from the co-cultured melanoma cells showed increased expression of Snail and decreased levels of E-cadherin suggesting that epithelial-to-mesenchymal transition (EMT) is occurring in these co-cultured cells. Additional WB analysis showed increased levels of Nodal protein and signaling and signs of increased Wnt activity in the co-cultured melanoma cells compared to reference cells. These data suggest that interaction between poorly aggressive melanoma cells with normal cells of the skin may regulate the transition from localized, poorly aggressive melanoma to invasive, metastatic disease via Nodal and/or Wnt induced EMT.     

Melanocytes Affect Nodal Expression and Signaling in Melanoma Cells: A Lesson from Pediatric Large Congenital Melanocytic Nevi

Expression of Nodal, a Transforming Growth Factor-beta (TGF-β) related growth factor, is associated with aggressive melanoma. Nodal expression in adult dysplastic nevi may predict the development of aggressive melanoma in some patients. A subset of pediatric patients diagnosed with giant or large congenital melanocytic nevi (LCMN) has shown increased risk for development of melanoma. Here, we investigate whether Nodal expression can help identify the rare cases of LCMN that develop melanoma and shed light on why the majority of these patients do not. Immunohistochemistry (IHC) staining results show varying degree of Nodal expression in pediatric dysplastic nevi and LCMN. Moreover, median scores from Nodal IHC expression analysis were not significantly different between these two groups. Additionally, none of the LCMN patients in this study developed melanoma, regardless of Nodal IHC levels. Co-culture experiments revealed reduced tumor growth and lower levels of Nodal and its signaling molecules P-SMAD2 and P-ERK1/2 when melanoma cells were grown in vivo or in vitro with normal melanocytes. The same was observed in melanoma cells cultured with melanocyte conditioned media containing pigmented melanocyte derived melanosomes (MDM). Since MDM contain molecules capable of inactivating radical oxygen species, to investigate potential anti-oxidant effect of MDM on Nodal expression and signaling in melanoma, melanoma cells were treated with either N-acetyl-l-cysteine (NAC), a component of the anti-oxidant glutathione or synthetic melanin, which in addition to providing pigmentation can also exert free radical scavenging activity. Melanoma cells treated with NAC or synthetic melanin showed reduced levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated melanoma cells. Thus, the potential role for Nodal in melanoma development in LCMN is less evident than in adult dysplastic nevi possibly due to melanocyte cross-talk in LCMN capable of offsetting or delaying the pro-melanoma effects of Nodal via anti-oxidant effects of MDM.     

Recent Advances in Phenolic Metabolites and Skin Cancer

Skin cancer represents any tumor development from the cutaneous structures within the epidermis, dermis or subcutaneous tissue, and is considered to be the most prevalent type of cancer. Compared to other types of cancer, skin cancer is proven to have a positive growth rate of prevalence and mortality. There are available various treatments, including chemotherapy, immunotherapy, radiotherapy and targeted therapy, but because of the multidrug resistance development, a low success has been registered. By this, the importance of studying naturally occurring compounds that are both safe and effective in the chemoprevention of skin cancer is emphasized. This review focuses on melanoma because it is the deadliest form of skin cancer, with a significantly increasing incidence in the last decades. As chemopreventive agents, we present polyphenols and their antioxidant activity, anti-inflammatory effect, their ability to balance the cell cycle and to induce apoptosis and their various other effects on skin melanoma. Besides chemoprevention, studies suggest that polyphenols can have treating abilities in some conditions. The limitations of using polyphenols are also pointed out, which are related to their poor bioavailability and stability, but as the technology is well developed, it is possible to augment the efficacy of polyphenols in the case of melanoma.     

Melanoma Brain Metastasis: Mechanisms,Models, and Medicine

The development of brain metastases in patients with advanced stage melanoma is common, but the molecular mechanisms responsible for their development are poorly understood. Melanoma brain metastases cause significant morbidity and mortality and confer a poor prognosis; traditional therapies including whole brain radiation, stereotactic radiotherapy, or chemotherapy yield only modest increases in overall survival (OS) for these patients. While recently approved therapies have significantly improved OS in melanoma patients, only a small number of studies have investigated their efficacy in patients with brain metastases. Preliminary data suggest that some responses have been observed in intracranial lesions, which has sparked new clinical trials designed to evaluate the efficacy in melanoma patients with brain metastases. Simultaneously, recent advances in our understanding of the mechanisms of melanoma cell dissemination to the brain have revealed novel and potentially therapeutic targets. In this review, we provide an overview of newly discovered mechanisms of melanoma spread to the brain, discuss preclinical models that are being used to further our understanding of this deadly disease and provide an update of the current clinical trials for melanoma patients with brain metastases.     

Additive Role of Immune System Infiltration and Angiogenesis in Uveal Melanoma Progression

Uveal melanoma (UM) is a malignant tumor that arises in the melanocytes of the uveal tract. It is the most frequent eye cancer, and despite new therapeutic approaches, prognosis is still poor, with up to 50% of patients developing metastasis with no efficient treatment options available. In contrast to cutaneous melanoma, UM is considered an “immune-cold” tumor due to the low mutational burden and the unique immunosuppressive microenvironment. To gain insight into the role of the UM microenvironment in regard to prognosis and metastatic progression, we have performed a pool analysis characterizing the UM microenvironment by using a bioinformatic approach. A variety of scores based on gene expression measuring stromal infiltration were calculated and used to assess association with prognosis. As a result, the highest immune and stromal scores were associated with poor prognosis. Specifically, stromal cells (fibroblasts and endothelial cells), T cells CD8+, natural killer (NK) cells, and macrophages M1 and M2 infiltration were associated with poor prognosis. Contrary to other tumors, lymphocytic infiltration is related to poor prognosis. Only B cells were associated with more favorable prognosis. UM samples scoring high in both angiogenesis (Angio) and antigen presentation (AP) pathways showed a poor prognosis suggesting an additive role of both functions. Almost all these tumors exhibited a chromosome 3 monosomy. Finally, an enrichment analysis showed that tumors classified as high Angio-high AP also activated metabolic pathways such as glycolysis or PI3K-AKT-MTOR. In summary, our pool analysis identified a cluster of samples with angiogenic and inflammatory phenotypes exhibiting poor prognosis and metabolic activation. Our analysis showed robust results replicated in a pool analysis merging different datasets from different analytic platforms.     

Targeting Genome Stability in Melanoma—A New Approach to an Old Field

Despite recent groundbreaking advances in the treatment of cutaneous melanoma, it remains one of the most treatment-resistant malignancies. Due to resistance to conventional chemotherapy, the therapeutic focus has shifted away from aiming at melanoma genome stability in favor of molecularly targeted therapies. Inhibitors of the RAS/RAF/MEK/ERK (MAPK) pathway significantly slow disease progression. However, long-term clinical benefit is rare due to rapid development of drug resistance. In contrast, immune checkpoint inhibitors provide exceptionally durable responses, but only in a limited number of patients. It has been increasingly recognized that melanoma cells rely on efficient DNA repair for survival upon drug treatment, and that genome instability increases the efficacy of both MAPK inhibitors and immunotherapy. In this review, we discuss recent developments in the field of melanoma research which indicate that targeting genome stability of melanoma cells may serve as a powerful strategy to maximize the efficacy of currently available therapeutics.     

Higher Mutation Burden in High Proliferation Compartments of Heterogeneous Melanoma Tumors

Melanoma tumors are the most heterogeneous of all tumor types. Tumor heterogeneity results in difficulties in diagnosis and is a frequent cause of failure in treatment. Novel techniques enable accurate examination of the tumor cells, considering their heterogeneity. The study aimed to determine the somatic variations among high and low proliferating compartments of melanoma tumors. In this study, 12 archival formalin-fixed paraffin-embedded samples of previously untreated primary cutaneous melanoma were stained with Ki-67 antibody. High and low proliferating compartments from four melanoma tumors were dissected using laser-capture microdissection. DNA was isolated and analyzed quantitatively and qualitatively. Libraries for amplicon-based next-generation sequencing (NGS) were prepared using NEBNext Direct Cancer HotSpot Panel. NGS detected 206 variants in 42 genes in melanoma samples. Most of them were located within exons (135, 66%) and were predominantly non-synonymous single nucleotide variants (99, 73.3%). The analysis showed significant differences in mutational profiles between high and low proliferation compartments of melanoma tumors. Moreover, a significantly higher percentage of variants were detected only in high proliferation compartments (39%) compared to low proliferation regions (16%, p < 0.05). Our results suggest a significant functional role of genetic heterogeneity in melanoma.     

Deciphering the Functional Role of RIPK4 in Melanoma

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1β level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4^high) or independent (RIPK4^low) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).