Development Of Human Melanoma Research Articles

From simplicity to complexity in current melanoma models.

Despite the recent impressive clinical success of immunotherapy against melanoma, development of primary and adaptive resistance against immune checkpoint inhibitors remains a major issue in a large number of treated patients. This highlights the need for melanoma models that replicate the tumor's intricate dynamics in the tumor microenvironment (TME) and associated immune suppression to study possible resistance mechanisms in order to improve current and test novel therapeutics. While two-dimensional melanoma cell cultures have been widely used to perform functional genomics screens in a high-throughput fashion, they are not suitable to answer more complex scientific questions. Melanoma models have also been established in a variety of experimental (humanized) animals. However, due to differences in physiology, such models do not fully represent human melanoma development. Therefore, fully human three-dimensional in vitro models mimicking melanoma cell interactions with the TME are being developed to address this need for more physiologically relevant models. Such models include melanoma organoids, spheroids, and reconstructed human melanoma-in-skin cultures. Still, while major advances have been made to complement and replace animals, these in vitro systems have yet to fully recapitulate human tumor complexity. Lastly, technical advancements have been made in the organ-on-chip field to replicate functions and microstructures of in vivo human tissues and organs. This review summarizes advancements made in understanding and treating melanoma and specifically aims to discuss the progress made towards developing melanoma models, their applications, limitations, and the advances still needed to further facilitate the development of therapeutics.

Tumor suppressive microRNA-485-5p targets PRRX1 in human skin melanoma cells, regulating epithelial-mesenchymal transitionand apoptosis.

Melanoma is one of the most aggressive skin cancers. Existing evidence has reported the aberrant expression of microRNAs (miRNAs) in melanoma, but their putative targets and underlying downstream effects remain to be further understood. Herein, we explored the suppressive role of miR-485-5p in melanoma progression. Initial bioinformatics analyses showed that the PRRX1 gene was differentially expressed in melanoma, while miR-485-5p was predicted to be a potential regulatory miRNA binding to PRRX1 mRNA. We confirmed that PRRX1 was upregulated, while miR-485-5p was downregulated in human melanoma samples compared with adjacent normal skin tissues. We then showed that PRRX1 was a target gene of miR-485-5p by dual-luciferase reporter gene assay. Moreover, a reduction in the expression of PRRX1 and downregulation of important proteins of the transforming growth factor-beta (TGFβ) signaling pathway was observed after miR-485-5p overexpression. Furthermore, miR-485-5p overexpression or PRRX1 knockdown suppressed epithelial-mesenchymal transition, cell viability, migration, and invasion, and promoted cell apoptosis in melanoma cells. Our study demonstrates the tumor-suppressive functions of miR-485-5p in the development of human melanoma, providing a potential target for therapy.

Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human melanoma development.

Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2-), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2-, for example by the absence of its cofactor tetrahydrobiopterin (BH4). Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and together with decreased BH4:BH2 ratio are contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2- levels, leading to NOS coupling, which in turn reduced cell viability, cell proliferation and the ability of melanoma cells to form melanoma spheroids. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.

Chromatin remodellers Brg1 and Bptf are required for normal gene expression and progression of oncogenic Braf-driven mouse melanoma.

Somatic oncogenic mutation of BRAF coupled with inactivation of PTEN constitute a frequent combination of genomic alterations driving the development of human melanoma. Mice genetically engineered to conditionally express oncogenic BrafV600E and inactivate Pten in melanocytes following tamoxifen treatment rapidly develop melanoma. While early-stage melanomas comprised melanin-pigmented Mitf and Dct-expressing cells, expression of these and other melanocyte identity genes was lost in later stage tumours that showed histological and molecular characteristics of de-differentiated neural crest type cells. Melanocyte identity genes displayed loss of active chromatin marks and RNA polymerase II and gain of heterochromatin marks, indicating epigenetic reprogramming during tumour progression. Nevertheless, late-stage tumour cells grown in culture re-expressed Mitf, and melanocyte markers and Mitf together with Sox10 coregulated a large number of genes essential for their growth. In this melanoma model, somatic inactivation that the catalytic Brg1 (Smarca4) subunit of the SWI/SNF complex and the scaffolding Bptf subunit of the NuRF complexdelayed tumour formation and deregulated large and overlapping gene expression programs essential for normal tumour cell growth. Moreover, we show that Brg1 and Bptf coregulated many genes together with Mitf and Sox10. Together these transcription factors and chromatin remodelling complexes orchestrate essential gene expression programs in mouse melanoma cells.

Induction of Paracrine Signaling in Metastatic Melanoma Cells by PPARγ Agonist Rosiglitazone Activates Stromal Cells and Enhances Tumor Growth.

In addition to improving insulin sensitivity in type 2 diabetes, the thiazolidinedione family of compounds and the pharmacologic activation of their best-characterized target PPARγ have been proposed as a therapeutic option for cancer treatment. In this study, we reveal a new mode of action for the thiazolidinedione rosiglitazone that can contribute to tumorigenesis. Rosiglitazone activated a tumorigenic paracrine communication program in a subset of human melanoma cells that involves the secretion of cytokines, chemokines, and angiogenic factors. This complex blend of paracrine signals activated nonmalignant fibroblasts, endothelial cells, and macrophages in a tumor-friendly way. In agreement with these data, rosiglitazone promoted human melanoma development in xenografts, and tumors exposed to rosiglitazone exhibited enhanced angiogenesis and inflammation. Together, these findings establish an important tumorigenic action of rosiglitazone in a subset of melanoma cells. Although studies conducted on cohorts of diabetic patients report overall benefits of thiazolidinediones in cancer prevention, our data suggest that exposure of established tumors to rosiglitazone may be deleterious.Significance: These findings uncover a novel mechanism by which the thiazolidinedione compound rosiglitazone contributes to tumorigenesis, thus highlighting a potential risk associated with its use in patients with established tumors. Cancer Res; 78(22); 6447-61. ©2018 AACR.

RORα and RORγ expression inversely correlates with human melanoma progression.

The retinoic acid-related orphan receptors (RORs) regulate several physiological and pathological processes, including immune functions, development and cancer. To study the potential role of RORs in melanoma progression, we analysed RORα and RORγ expression in nevi and primary melanomas and non-lesional skin and metastases in relation to melanoma clinico-pathomorphological features. The expression of RORα and RORγ was lower in melanomas than in nevi and decreased during melanoma progression, with lowest levels found in primary melanomas at stages III and IV and in melanoma metastases. Their expression correlated with pathomorphological pTNM parameters being low in aggressive tumors and being high in tumors showing histological markers of good prognosis. Higher nuclear levels of RORα and RORγ and of cytoplasmic RORγ correlated with significantly longer overall and disease free survival time. Highly pigmented melanomas showed significantly lower level of nuclear RORs. This study shows that human melanoma development and aggressiveness is associated with decreased expression of RORα and RORγ, suggesting that RORs could be important in melanoma progression and host responses against the tumor. Furthermore, it suggests that RORα and RORγ might constitute a novel druggable target in anti-melanoma management using tumor suppressor gene therapy restoring their normal functions.

MiR-769 promoted cell proliferation in human melanoma by suppressing GSK3B expression.

MicroRNAs (miRNAs) are short, non-coding RNAs with post-transcriptional regulatory function, playing crucial roles in cancer development and progression of human melanoma. Previous studies have indicated that miR-769 was implicated in diverse biological processes. However, the underlying mechanism of miR-769 in human melanoma has not been intensively investigated. In this present study, we aimed to investigate the role of miR-769 and its target genes in human melanoma. We found that miR-769 expression was strongly increased in human melanoma cells and clinical tissues compared with their corresponding controls. Overexpression of miR-769 promoted cell proliferation in human melanoma cell line A375, whereas miR-769-in reverses the function. Glycogen synthase kinase-3 Beta (GSK3B), a potential target gene of miR-769, and was validated by luciferase assay. Further studies revealed that miR-769 regulated cell proliferation of human melanoma by directly suppressing GSK3B expression and the knockdown of GSK3B expression reversed the effect of miR-769-in on human melanoma cell proliferation. In summary, our data demonstrated that miR-769 might act as a tumor promoter by targeting GSK3B during development of human melanoma.

Cross-species models of human melanoma.

Although transformation of melanocytes to melanoma is rare, the rapid growth, systemic spread, as well as the chemoresistance of melanoma present significant challenges for patient care. Here we review animal models of melanoma, including murine, canine, equine, and zebrafish models, and detail the immense contribution these models have made to our knowledge of human melanoma development, and to melanocyte biology. We also highlight the opportunities for cross‐species comparative genomic studies of melanoma to identify the key molecular events that drive this complex disease. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Down-regulation of deacetylase HDAC6 inhibits the melanoma cell line A375.S2 growth through ROS-dependent mitochondrial pathway.

Previous studies have shown that histone deacetylase 6 (HDAC6) plays critical roles in many cellular processes related to cancer. However, its biological roles in the development of melanoma remain unexplored. Our aim was to investigate whether HDAC6 has a biological role in human melanoma development and to understand its underlying mechanism. In the present study, HDAC6 expression was up-regulated in melanoma tissues and cell lines. Knockdown of HDAC6 significantly inhibited the proliferation and colony formation ability of A375.S2 cells, promoted cell arrest at G0/G1 phase and apoptosis. Additionally, western blotting assay showed that HDAC6 silencing suppressed Bcl-2 level and enhanced Bax level, then activated caspase-9 and caspase-3, and further activated the release of cytochrome c from mitochondria to cytoplasm, finally induced cell apoptosis involving the mitochondrial pathway. Knockdown of HDAC6 triggered a significant generation of ROS and disruption of mitochondrial membrane potential (MMP). Furthermore, ROS inhibitor, NAC reduced HDAC6 siRNA-induced ROS production, and blocked HDAC6 siRNA-induced loss of MMP and apoptosis. NAC also significantly blocked HDAC6 siRNA-induced mtDNA copy number decrease and mitochondrial biogenesis and degradation imbalance. In conclusion, the results showed that knockdown of HDAC6 induced apoptosis in human melanoma A375.S2 cells through a ROS-dependent mitochondrial pathway.

MicroRNA-328 inhibits proliferation of human melanoma cells by targeting TGFβ2.

Some microRNAs (miRNAs) have been shown to act as oncogenes or tumor suppressor genes in human melanomas. miR-328 is upregulated in blood cells of melanoma patients compared to in healthy controls. This suggests a role for miR-328 in melanoma that warrants investigation. In this study, we demonstrated miR-328 levels to be dramatically decreased in human melanoma cell lines. Moreover, forced expression of miR-328 inhibited proliferation and induced G1-phase arrest of the SK-MEL-1 melanoma cell line. We identified TGFβ2 as a direct target gene for miR-328 using a fluorescent reporter assay and western blotting. Levels of TGFβ2 were dramatically increased in human melanoma cell lines and were inversely correlated with the miR-328 expression level. Our findings provide new insights into the mechanisms of human melanoma development, indicating that miR-328 has therapeutic potential for this disease.

155 - Redox Rebalance Mediated by Nrf2 and Nox1 after UV Radiation in Human Melanoma Development

155 - Redox Rebalance Mediated by Nrf2 and Nox1 after UV Radiation in Human Melanoma Development

Caveolin-1 is a risk factor for postsurgery metastasis in preclinical melanoma models

Melanomas are highly lethal skin tumours that are frequently treated by surgical resection. However, the efficacy of such procedures is often limited by tumour recurrence and metastasis. Caveolin-1 (CAV1) has been attributed roles as a tumour suppressor, although in late-stage tumours, its presence is associated with enhanced metastasis. The expression of this protein in human melanoma development and particularly how the presence of CAV1 affects metastasis after surgery has not been defined. CAV1 expression in human melanocytes and melanomas increases with disease progression and is highest in metastatic melanomas. The effect of increased CAV1 expression can then be evaluated using B16F10 murine melanoma cells injected into syngenic immunocompetent C57BL/6 mice or human A375 melanoma cells injected into immunodeficient B6Rag1−/− mice. Augmented CAV1 expression suppresses tumour formation upon a subcutaneous injection, but enhances lung metastasis of cells injected into the tail vein in both models. A procedure was initially developed using B16F10 melanoma cells in C57BL/6 mice to mimic better the situation in patients undergoing surgery. Subcutaneous tumours of a defined size were removed surgically and local tumour recurrence and lung metastasis were evaluated after another 14 days. In this postsurgery setting, CAV1 presence in B16F10 melanomas favoured metastasis to the lung, although tumour suppression at the initial site was still evident. Similar results were obtained when evaluating A375 cells in B6Rag1−/− mice. These results implicate CAV1 expression in melanomas as a marker of poor prognosis for patients undergoing surgery as CAV1 expression promotes experimental lung metastasis in two different preclinical models.

Abstract 1647: Role of Sirt1 histone deacetylase in melanoma: A target for melanoma management via its inhibition

Abstract Melanoma is one of the most aggressive skin cancers that rapidly metastasizes and is often lethal, if not diagnosed early. The available preventive strategies and treatment options have not been able to effectively manage melanoma. Therefore, novel mechanisms and target-based approaches are needed to combat this deadly neoplasm. A detailed understanding of genetic controls of cellular proliferation and cell division may provide the basis for the rational design of specific targets and therapeutic strategies for melanoma management. This study was designed to test the hypothesis that Sirt1 is differentially expressed in melanoma and could be used as a target for designing novel approaches for the management of this neoplasm. Sirt1 is the most well studied member of the Sirtuin (Sir2) family enzymes, which are nicotinamide adenine dinucleotide-dependent class III histone deacetylases, and have been shown to deacetylate lysine residues on various proteins. Sirt1 has been shown to be involved in a number of cellular processes including gene silencing, DNA repair, recombination, and stress response. Sirt1 also regulates the tumor suppressor p53 and FoxO transcription factors to favor cell survival. To test and support our hypothesis, we determined the expression of Sirt1 in a variety of melanoma cell lines (A375, Hs294T and G361) and normal human epidermal keratinocytes (NHEKs) and normal human melanocytes (NHEMs). We found that compared to normal NHEK and NHEM, Sirt1 was significantly overexpressed at protein (immunoblot analysis) as well as mRNA levels (Real-Time PCR analysis) in all melanoma cell lines tested. In order to determine the functional significance of Sirt1 in human melanoma cells, we determined the effect of Sirt1 knockdown on melanoma cells employing two distinct approaches viz. i) chemical knockdown of Sirt1 enzyme via small molecule inhibitors, and ii) genetic knockdown of Sirt1 gene via short hairpin RNA (shRNA)-mediated RNA interference. We found that shRNA-mediated knockdown as well as small-molecule-mediated activity inhibition resulted in a significant decrease in the viability and growth of melanoma cells. In addition, Sirt1 inhibition was also found to result in a significant decrease in clonogenic survival of melanoma cells. Taken together, our study suggested that i) Sirt1 overexpression could be a contributing factor in human melanoma development and/or progression, and ii) Sirt1 could serve as a potential target towards developing novel strategies for the management of this lethal skin cancer. However, future studies in appropriate in vitro and in vivo systems are needed to substantiate our findings and to define the mechanism(s) by which Sirt1 imparts a growth advantage to melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1647. doi:10.1158/1538-7445.AM2011-1647

A Mediator of Rho-dependent Invasion Moonlights as a Methionine Salvage Enzyme

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named “mediator of RhoA-dependent invasion (MRDI),” that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

Id1 delays senescence of primary human melanocytes

The Id family of helix-loop-helix transcription factors is upregulated in a variety of human malignancies and has been implicated in promoting tumorigenesis through effects on cell growth, differentiation, and tumor angiogenesis. While expression of Id proteins has been associated with tumorigenesis, the precise mechanistic relationship between Id expression and carcinogenesis has not been clearly delineated. We have previously shown that Id1 delays cellular senescence in primary mammalian cells through inhibition of the cell cycle regulatory protein and familial melanoma gene, p16/INK4a. We have also demonstrated that Id1 expression is upregulated in early stage primary human melanomas and may be an important marker for early malignancy. In order to further define the role of Id1 in human melanoma development, we have evaluated the function of Id1 in primary human melanocytes. Here we show that constitutive expression of Id1 in primary human melanocytes leads to delayed cellular senescence and decreased expression of the familial melanoma gene, p16/INK4a. Although melanocytes constitutively expressing Id1 are shown to possess extended lifespans, this is not associated with an appreciable change in cell growth or telomere length. We conclude that Id1 delays cellular senescence in primary human melanocytes through inhibition of p16/INK4a expression and suggest that Id1 may contribute to the malignant conversion of primary human melanocytes through extension of cellular lifespan.

Metabotropic Glutamate Receptor 1 and Glutamate Signaling in Human Melanoma

Recently, several laboratories have started to investigate the involvement of glutamate signaling in cancer. In previous studies, we reported on a transgenic mouse model that develops melanoma spontaneously. Subsequent studies in these mice identified that the aberrant expression of metabotropic glutamate receptor 1 (GRM1) in melanocytes played a critical role in the onset of melanoma. Confirmation of the etiologic role of GRM1 in melanoma development was shown in a second transgenic line with GRM1 expression under the regulation of a melanocyte-specific dopachrome tautomerase promoter. Ectopic expression of GRM1 was also detected in a subset of human melanoma cell lines and biopsies, suggesting that aberrant expression of GRM1 in melanocytes may contribute to the development of human melanoma. GRM1, a seven-transmembrane domain G protein-coupled receptor, is normally expressed and functional in neuronal cells, and its ligand, glutamate, is the major excitatory neurotransmitter. Human melanoma cells are shown here to release elevated levels of glutamate, implying a possible autocrine loop. Treatment of GRM1-expressing human melanoma cells with a GRM1 antagonist (LY367385 or BAY36-7620) or a glutamate release inhibitor (riluzole) leads to a suppression of cell proliferation as well as a decrease in levels of extracellular glutamate. Treatment of human melanoma cell xenografts with riluzole for 18 days via p.o. gavage or i.v. injection leads to inhibition of tumor growth by 50% in comparison with controls. These data suggest the importance of glutamate signaling in human melanoma and imply that the suppression of glutamate signaling may be a new target for melanoma therapy.

American Society of Preventive Oncology Distinguished Career Achievement Lecture 2006—Enjoy the Journey: The Long and Winding Road of Chemoprevention Agent Development

My career in chemoprevention derived from an attempt to find a better treatment for cutaneous malignant melanoma ([1][1]). This led me to the exploration of retinoids as therapeutic modalities in a number of malignant conditions with the identification of activity in several epithelial malignancies

Molecular biology of human melanoma development and progression

In the United States, Australia, Northern Europe, and Canada, malignant melanoma is increasing at a faster rate than any other cancer, with the exception of lung cancer in women. Major advances have been made in the molecular biology and immunology of melanoma. These advances in basic science have led to more rational approaches to specifically targeting melanoma cells, with promising results in the clinic. An increased understanding of how melanoma spreads has led to more selective, less invasive surgical procedures that do not compromise patient health. Combinations of chemotherapy and immunotherapy are now available for patients with advanced melanoma that affect both the length and quality of the patients' lives. This review of the molecular biology of melanoma development and progression discusses the disease's etiology, molecular genetics, cell-surface antigens, experimental models, biological markers, and new forms of treatment. As we continue to learn more about malignant melanoma, we will be able to devise more specific and effective treatments that will give patients with this potentially deadly disease longer and more productive lives.

Progressive loss of sensitivity to endothelium‐derived growth inhibitors expressed by human melanoma cells during disease progression

Tumor progression is frequently associated with changes in responsiveness of tumor cells to paracrine growth factors. A potential major source of such paracrine factors in solid tumors are endothelial cells since this type of cell can constitute a sizeable fraction of the cellular composition of solid tumors. As an initial step to examining the possible effects of endothelial cell-associated growth factors on tumor cell growth, a panel of human melanoma cell lines representative of different stages of tumor progression was employed for studies utilizing endothelial cell-derived growth modulators. Macrovascular or microvascular human endothelial cells from umbilical vein or from skin, respectively, inhibited melanoma cell growth in direct coculture experiments. The potency of this inhibitory effect diminished as a function of melanoma progression. Conditioned media from endothelial cell cultures mimicked the effect of the cell coculture experiments, suggesting the involvement of soluble growth factor(s). Approximately 50-75% of the conditioned media inhibitory effect was abrogated by addition of the neutralizing antibody to interleukin-6 (IL-6). Gel filtration chromatography revealed the presence of additional inhibitors in endothelial cell conditioned medium. Two peaks of activity were detected with apparent molecular weights of approximately 100-150 Kd and 20-30 Kd, the latter containing IL-6 activity. Whereas early-stage radial growth phase (RGP) primary tumor-derived melanoma cells were sensitive to at least three different endothelial products of high or low molecular weight (including IL-6), melanoma cells from more advanced metastatic lesions were resistant to the latter activities, and retained only partial sensitivity to the high molecular weight inhibitor. More advanced vertical growth phase (VGP) primary melanoma cell lines expressed intermediate inhibition-sensitive phenotypes. Thus human melanoma development appears to be associated with progressive loss of sensitivity to the growth inhibitory effects of IL-6 and other factors produced by endothelial cells. This is likely to be a result of a selection process when tumor cells are confronted with adjacent vasculature during the progress of tumor angiogenesis.

Lack of allelic deletion and point mutation as mechanisms of p53 activation in human malignant melanoma.

To investigate the role of the p53 tumor-suppressor gene in the development of human melanoma, loss of heterozygosity (LOH) of p53 was studied in 46 cases of melanoma by a polymerase-chain-reaction/restriction-fragment-length polymorphism (PCR/RFLP) analysis, and p53 mutations were assessed in 51 cases of melanoma by a polymerase-chain-reaction/single-strand-conformation polymorphism (PCR/SSCP) analysis. Frozen tumors and paraffin samples were used in the study. We were not able to detect any allelic loss in 12BstUI informative cases or any single mutation in exons 5 to 8 of the p53 gene. Our results, together with other findings at the DNA level, suggest that the p53 gene appears not to be commonly involved in the development of melanoma, at least by its most frequent mechanisms of deletion of one allele and/or mutation in the other.