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Recent progress in the treatment of advanced melanoma has led to unprecedented improvements in overall survival and, as these new melanoma treatments have been developed and deployed in the clinic, much has been learned about the natural history of the disease. Now is the time to apply that knowledge toward the design and clinical evaluation of new chemoprevention agents. Melanoma chemoprevention has the potential to reduce dramatically both the morbidity and the high costs associated with treating patients who have metastatic disease. In this work, scientific and clinical melanoma experts from the national Melanoma Prevention Working Group, composed of National Cancer Trials Network investigators, discuss research aimed at discovering and developing (or repurposing) drugs and natural products for the prevention of melanoma and propose an updated pipeline for translating the most promising agents into the clinic. The mechanism of action, preclinical data, epidemiological evidence, and results from available clinical trials are discussed for each class of compounds. Selected keratinocyte carcinoma chemoprevention studies also are considered, and a rationale for their inclusion is presented. These data are summarized in a table that lists the type and level of evidence available for each class of agents. Also included in the discussion is an assessment of additional research necessary and the likelihood that a given compound may be a suitable candidate for a phase 3 clinical trial within the next 5 years., (© 2018 American Cancer Society.)

Beginning in the last decade of the twentieth century, the fields of pigment cell research and melanoma have witnessed major breakthroughs in the understanding of the role of melanocortins in human pigmentation and the DNA damage response of human melanocytes to solar ultraviolet radiation (UV). This began with the cloning of the melanocortin 1 receptor (MC1R) gene from human melanocytes and the demonstration that the encoded receptor is functional. Subsequently, population studies found that the MC1R gene is highly polymorphic, and that some of its variants are associated with red hair phenotype, fair skin and poor tanning ability. Using human melanocytes cultured from donors with different MC1R genotypes revealed that the alleles associated with red hair color encode for a non-functional receptor. Epidemiological studies linked the MC1R red hair color variants to increased melanoma risk. Investigating the impact of different MC1R variants on the response of human melanocytes to UV led to the important discovery that the MC1R signaling activates antioxidant, DNA repair and survival pathways, in addition to stimulation of eumelanin synthesis. These effects of MC1R were absent in melanocytes expressing 2 MC1R red hair color variants that result in loss of function of the receptor. The importance of the MC1R in reducing UV-induced genotoxicity in melanocytes led us to design small peptide analogs of the physiological MC1R agonist α-melanocortin (α-melanocyte stimulating hormone; α-MSH) for the goal of utilizing them for melanoma chemoprevention.

Exposure to solar ultraviolet radiation (UV) is the main etiological factor for skin cancer, including melanoma. Cutaneous pigmentation, particularly eumelanin, afforded by melanocytes is the main photoprotective mechanism, as it prevents UV-induced DNA damage in the epidermis. Therefore, maintaining genomic stability of melanocytes is crucial for prevention of melanoma, as well as keratinocyte-derived basal and squamous cell carcinoma. A critical independent factor for preventing melanoma is DNA repair capacity. The response of melanocytes to UV is mediated mainly by a network of paracrine factors that not only activate melanogenesis, but also DNA repair, anti-oxidant, and survival pathways that are pivotal for maintenance of genomic stability and prevention of malignant transformation or apoptosis. However, little is known about the stress response of melanocytes to UV and the regulation of DNA repair pathways in melanocytes. Unraveling these mechanisms might lead to strategies to prevent melanoma, as well as non-melanoma skin cancer.

Melanocortins, particularly α-Melanocortin (α-Melanocyte stimulating hormone, α-MSH), were first identified as the physiological regulators of pigmentation in many vertebrate species. Their role in regulating human pigmentation was unequivocally demonstrated in the 1990s, with the cloning of the human melanocortin 1 receptor (MC1R) gene from human melanocytes and the demonstration that functional MC1R is expressed by these cells. α-Melanocyte stimulating hormone is a tridecapeptide, with the core sequence His(6)-Phe(7)-Arg(8)-Trp(9) shared with β- and γ-MSH and identified as essential for receptor activation and stimulation of pigmentation. The small size of α-MSH makes it an attractive molecule for drug design. There has been longstanding interest in the development of melanocortin analogs that target the MC1R expressed on normal melanocytes and melanoma cells. The aim has been to develop MC1R agonists that stimulate melanogenesis and confer photoprotection to human melanocytes and thus prevent skin cancer formation. Recent findings that the physiological α-MSH not only stimulates melanogenesis, but also reduces the extent of DNA damage caused by exposure to solar ultraviolet radiation have further rejuvenated the interest in developing synthetic MC1R agonists for skin cancer prevention. α-Melanocortin analogs have also been developed for imaging of melanoma tumors, localization of residual metastasis and specific delivery of radionuclides to eradicate melanoma tumors, sparing normal tissues. The main challenge is to develop specific MC1R agonists that will target melanocytes for skin cancer prevention, or for localization and treatment of metastatic melanoma.

Melanoma is the most devastating form of skin cancer. The steady increase in the incidence of melanoma, its resistance to chemotherapy, together with its high potential to metastasize, have emphasized the importance of its prevention. It is becoming clear that solar ultraviolet radiation is a main culprit in the etiology of melanoma, the same as in basal and squamous cell carcinomas. It is commonly accepted that skin pigmentation and melanin content are principal determinants of the susceptibility to melanoma and other sun-induced skin cancers. Although this is generally true, however, prediction of melanoma risk based solely on pigmentary phenotype is not always precise and fails to identify high-risk individuals with dark skin color. Other important risk factors need to be considered and better defined, particularly DNA repair capacity. Emerging studies have revealed the role of melanoma susceptibility genes in regulating DNA repair, and indicated that melanoma patients have a lower DNA repair capacity than the general population. As the response of human melanocytes to ultraviolet radiation is modulated by an array of paracrine factors, we have focused our investigation on the role of melanocortins and the melanocortin 1 receptor, as well as endothelin-1, in this response. We have discovered novel roles for melanocortins and endothelin-1 as survival factors that rescue human melanocytes from ultraviolet radiation-induced apoptosis, and importantly enhance repair of DNA photoproducts and reduce the release of hydrogen peroxide that can cause oxidative stress. Our findings, together with epidemiological data showing that loss-of-function mutations in the melanocortin-1 receptor gene increase the risk of melanoma, substantiate the role of DNA repair in melanoma genesis, and suggest that responsiveness to melanocortins and endothelin-1 is important for melanoma prevention.

Cellular senescence, the irreversible proliferative arrest seen in somatic cells after a limited number of divisions, is considered a crucial barrier to cancer, but direct evidence for this in vivo was lacking until recently. The best-known form of human cell senescence is attributed to telomere shortening and a DNA-damage response through p53 and p21. There is also a more rapid form of senescence, dependent on the p16-retinoblastoma pathway. p16 (CDKN2A) is a known melanoma susceptibility gene. Here, we use retrovirally mediated gene transfer to confirm that the normal form of senescence in cultured human melanocytes involves p16, since disruption of the p16/retinoblastoma pathway is required as well as telomerase activation for immortalisation. Expression (immunostaining) patterns of senescence mediators and markers in melanocytic lesions provide strong evidence that cell senescence occurs in benign melanocytic naevi (moles) in vivo and does not involve p53 or p21 upregulation, although p16 is widely expressed. In comparison, dysplastic naevi and early (radial growth-phase, RGP) melanomas show less p16 and some p53 and p21 immunostaining. All RGP melanomas expressed p21, suggesting areas of p53-mediated senescence, while most areas of advanced (vertical growth-phase) melanomas lacked both p16 and p21, implying escape from both forms of senescence (immortalisation). Moreover, nuclear p16 but not p21 expression can be induced in human melanocytes by oncogenic BRAF, as found in around 80% of naevi. We conclude that cell senescence can form a barrier to melanoma development. This also provides a potential explanation of why p16 is a melanoma suppressor gene.

Melanoma is the deadliest form of skin cancer, with no cure for advanced disease. We propose a strategy for melanoma prevention based on using analogs of alpha-melanocyte stimulating hormone (alpha-MSH) that function as melanocortin 1 receptor (MC1R) agonists. Treatment of human melanocytes with alpha-MSH results in stimulation of eumelanin synthesis, reduction of apoptosis that is attributable to reduced hydrogen peroxide generation and enhanced repair of DNA photoproducts. These effects should contribute to genomic stability of human melanocytes, thus preventing their malignant transformation to melanoma. Based on these findings, we synthesized and tested the effects of 3 tetrapeptide alpha-MSH analogs, Ac-His-D-Phe-Arg-Trp-NH2, n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2, on cultured human melanocytes. The latter two analogs were more potent than the former, or alpha-MSH, in stimulating the activity of tyrosinase, thus melanogenesis, reducing apoptosis and release of hydrogen peroxide and enhancing repair of DNA photoproducts in melanocytes exposed to UV radiation (UVR). The above analogs are MC1R agonists, as their effects were abrogated by an analog of agouti signaling protein, the physiological MC1R antagonist, and were absent in melanocytes expressing loss-of-function MC1R. Analogs, such as 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2 with prolonged and reversible effects, can potentially be developed into topical agents to prevent skin photocarcinogenesis, particularly melanoma.

Prostaglandins (PGs) E1 and E2 stimulate tyrosinase activity and suppress the proliferation of Cloudman S91 melanoma cells by altering their progression through the cell cycle. Prostaglandin E1 and PGE2 have prolonged or residual effects on melanoma cells. Cells treated for 5 or 24 hours with 10 micrograms/ml PGE1 or cells treated for 8 or 24 hours with 10 micrograms/ml PGE2 demonstrated decreased proliferation and increased tyrosinase activity for 48 hours after removal of the PGs. The effects of PGs on the cell cycle were investigated by determining total DNA content in cells stained with propidium iodide (PI) and analyzed by a fluorescence activated cell sorter (FACS). Prostaglandin E1 blocked cells in G2 phase after 5 hours of treatment, corresponding to when inhibition of proliferation was first evident. Similarly, after 9 hours of treatment with PGE2, more cells were in late S, early G2 phase and less in G1 than their control counterparts. Also, melanoma cells were pulse-labeled with 5-bromo-2'-deoxyuridine (BrdUrd) prior to or at the end of PG treatment and then stained with a fluoresceinated monoclonal antibody to BrdUrd, and with PI. This allows one to observe how BrdUrd-labeled S-phase cells cycle with time. Both PGE1 and PGE2 inhibit proliferation by blocking cells in G2 phase of the cell cycle. The PG-induced block in G2 may be required by melanoma cells to synthesize mRNA and proteins that are essential for stimulation of tyrosinase activity. Ultrastructurally, only a subpopulation of the cells treated with PGE1 or PGE2 contained more mature melanosomes than control cells.

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin), [Nle4,D-Phe7]-alpha-MSH and related fragment analogues, Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 and Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, were studied for their ability to stimulate tyrosinase activity in Cloudman S91 mouse melanoma cells in tissue culture. All of the melanotropins stimulated tyrosinase activity in a dose-dependent manner. [Nle4,D-Phe7]-alpha-MSH was about 100 times more active than alpha-MSH as determined from the minimal effective dose (MED) required to activate the enzyme above control (basal) levels. The MED of this analogue to significantly stimulate tyrosinase activity at 24, 48 and 72 h of incubation was 10(-11) M whereas the MED of alpha-MSH was 10(-9) M at each of these times. The maximum tyrosinase activity achieved from the time of initial incubation in the presence of [Nle4,D-Phe7]-alpha-MSH was approximately 3-, 5- and 6-fold greater than control levels at 24, 48 and 72 h, respectively. The 2 [Nle4,D-Phe7]-substituted fragment analogues were at least as active as the tridecapeptide analogue and therefore at least 100-fold more active than alpha-MSH in stimulating enzyme activity. These [Nle4,D-Phe7]-substituted analogues were more active in the melanoma tyrosinase assay than in the melanoma adenylate cyclase assay or other normal melanocyte (frog and lizard skin) bioassays.

The effects of prostaglandins (PGs) on the Cloudman S91 melanoma CCL 53.1 cell line indicate that melanogenesis and proliferation are regulated by separate mechanisms that are not necessarily cyclic AMP (cAMP) dependent. These cells responded to PGE1 and PGE2 in a dose-dependent manner, by an increase of tyrosinase activity and by inhibition of proliferation. PGA1 and PGD2 inhibited cellular proliferation and tyrosinase activity, while PGF2 alpha had no effect after 24 h of treatment. PGE1, but not PGE2 or PGD2, increased cellular cAMP levels after 30 min of treatment. Treatment with 10 micrograms/ml PGE1 inhibited cellular proliferation after 4 h and enhanced tyrosinase activity after 12 h. Tyrosinase stimulation by PGE1 required de novo transcription and translation. Actinomycin D, cycloheximide, and the tyrosinase inhibitor phenylthiocarbamide blocked tyrosinase activation but did not alter the inhibitory effect of PGE1 on proliferation. Dibutyryl cAMP and 3-isobutyl-1-methylxanthine augmented tyrosinase activation by PGE1 without enhancing the inhibitory action of PGE1 on cell growth. Neither blockage nor enhancement of the PGE1 effect on tyrosinase altered the PGE1-induced retardation of proliferation. These results are in marked contrast to the traditional concept that elevation of cAMP levels in melanoma cells necessarily results in stimulation of melanogenesis and inhibition of proliferation. The data presented propose independent and possibly alternative pathways for the regulation of these two cellular events.

It is known that many immunologic responses to IL-1 are antagonized by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH). This led us to investigate the possible reciprocal effects of IL-1 and the functionally related epidermal cytokines, epidermal cell-derived thymocyte activating factor (ETAF) and IL-6, on the melanogenic effect of alpha-MSH on murine Cloudman melanoma cells. When these cells were treated with ETAF in combination with alpha-MSH or its potent analog [Nle4,D-Phe7]-alpha-MSH, the melanotropin induced increase in tyrosinase activity, and thus melanin synthesis, was abrogated. This inhibitory effect of ETAF was not mediated by competitive binding to the melanotropin receptor, because ETAF also blocked the melanogenic response of melanoma cells to isobutyl methylxanthine (IBMX) and to PGE1 and PGE2. ETAF had no effect on cellular proliferation. Inhibition of the stimulated tyrosinase activity by ETAF was not due to diminished cAMP synthesis or increased cAMP degradation. Cells treated concomitantly with ETAF and alpha-MSH, IBMX, or PGE1 had the same cAMP levels as cells treated with alpha-MSH, IBMX, or PGE1 alone. In contrast to ETAF, human rIL-1 alpha or IL-1 beta alone or in combination did not have an inhibitory effect on melanogenesis. IL-6 significantly inhibited the basal level of tyrosinase and partially abrogated the alpha-MSH-induced tyrosinase activity. IL-6 also stimulated cellular proliferation when added alone or in combination with alpha-MSH. Granulocyte-macrophage colony stimulating factor (GM-CSF) did not alter either the tyrosinase activity or cellular replication at the concentrations tested. IL-1 alpha, GM-CSF, and IL-6 or IL-1 alpha and GM-CSF added together did not significantly affect the MSH-induced tyrosinase activity. These results ascribe a new potential function for ETAF and IL-6 as modulators of the melanogenic response of pigment cells.

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.

alpha-Melanotropin (alpha-melanocyte stimulating hormone, alpha-MSH) stimulates tyrosinase activity in Cloudman S91 murine melanoma cells. Three [Nle4, D-Phe7]-substituted alpha-melanotropin analogues, [Nle4, D-Phe7]-alpha-MSH, Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2, and Ac-[Nle4, D-Phe7]-alpha-MSH4-10-NH2, are at least 100-fold more effective than alpha-MSH in stimulating melanoma tyrosinase, the rate-limiting enzyme in melanin biosynthesis. These [Nle4, D-Phe7]-substituted melanotropin analogues induce tyrosinase activity in melanoma cells with shorter contact times than required by the native hormone, alpha-MSH. [Nle4, D-Phe7]-substituted melanotropins also induce a prolonged (residual) stimulation of melanoma tyrosinase. Following incubation of melanoma cells in the presence of [Nle4, D-Phe7]-alpha-MSH for 24 h, tyrosinase activity is maintained for up to 6 days in the absence of the melanotropin. The shorter 4-10 and 4-11 fragment analogues also exhibit residual melanotropic activity. The prolonged stimulation of tyrosinase in the absence of the analogues is maintained even though melanoma cells continue to divide about every 24 h. These results suggest that melanoma cells possess spare melanotropin receptors and that [Nle4, D-Phe7]-substituted analogues bind almost irreversibly to these receptors or to some other component of the adenylate cyclase enzyme complex responsible for enhancing tyrosinase activity and melanin production.

alpha-Melanocyte-stimulating hormone (MSH) is known to stimulate melanogenesis in murine melanoma, particularly in Cloudman S-91 melanoma cells. The effects of MSH and insulin on the proliferation of S91 murine melanoma cells have aroused controversy; in various reports, both hormones have been reported to either stimulate or inhibit murine melanoma growth. In our studies both MSH and insulin stimulated the colony-forming ability and the proliferative capacity of S-91 murine melanoma cells grown in soft agar with either serum-supplemented or serum-less medium. Unless insulin and/or MSH were present, Cloudman S-91 melanoma cells failed to clone in soft agar. The insulin effect was greater than that of MSH, and was more pronounced in serum-less than in serum-supplemented medium. The concurrent treatment of S91 melanoma cells with both MSH and insulin resulted in a greater increase in the total number of colonies formed than caused by treatment with either hormone alone. The combined MSH-insulin stimulation of anchorage-independent growth was specific, since the effect could not be mimicked by epidermal growth factor (EGF), gonadotropin-releasing hormone (GRH), luteinizing hormone (LH), nerve growth factor (NGF) or platelet-derived growth factor (PDGF). Therefore, MSH and insulin may be specific growth factors for murine melanoma cells.

The superpotent and ultraprolonged melanotropic properties of an alpha-melanotropin analog, [Nle4, D-Phe7]-alpha-MSH, were investigated in a Cloudman S91 (CCL 53.1) melanoma cell line. [Nle4, D-Phe7]-alpha-MSH is 100-fold more effective than the native hormone, alpha-melanocyte stimulating hormone (alpha-MSH), in stimulating melanoma cell tyrosinase activity, as determined from their minimum effective doses (10(-11)M and 10(-9)M, respectively). [Nle4, D-Phe7]-alpha-MSH also exhibits a more sustained effect than alpha-MSH on tyrosinase after removal of the melanotropins from the incubation medium. When cells were exposed to alpha-MSH (10(-7)M) for 24 h, residual activity after removal of the hormone was minimally significant. In contrast, under the same experimental conditions [Nle4, D-Phe7]-alpha-MSH treatment induced tyrosinase activity 2-3 fold above basal level, and maintained remarkable stimulatory effects up to 72 h following melanotropin removal. When the exposure time to melanotropins was reduced to 4 h, alpha-MSH failed to elicit significant tyrosinase activity, whereas [Nle4, D-Phe7]-alpha-MSH stimulated significant tyrosinase activity during the first 24 h subsequent to melanotropin removal. Interestingly, this stimulation by the analog increased at 48 h, reached a maximum at 72 h following removal of the melanotropin analog, and remained significantly stimulated for 6 consecutive days in the absence of the analog.

Melanotropins induce melanogenesis in mouse Cloudman S91 melanoma cells by stimulating the activity of tyrosinase. In monolayer culture, alpha-melanocyte-stimulating hormone and the superpotent analogue 4-norleucine, 7-D-phenylalanine-alpha-melanocyte, which had prolonged effects on tyrosinase activity, did not inhibit the proliferation of melanoma cells even at concentrations that elicited maximal tyrosinase stimulation. In soft agar the melanotropins stimulated the formation of melanized colonies and increased the cloning and proliferative potentials of melanoma cells. Both melanotropins increased the number of small (42-104 microns in diameter) colonies at initial plating densities ranging from 625 to 7,500 cells/dish. The number of larger (greater than 104 microns in diameter) colonies was also increased except at densities 5,000 cells or more/dish, wherein the proliferative capacity was inhibited; yet the cloning efficiency was still increased. Therefore, in bilayer soft agar cultures, melanotropins stimulate the growth of the clonogenic S91 melanoma cell population under conditions that allow for optimal expression of the cloning and proliferative potentials of these cells.

Cell density is a factor that affects the capacity of Cloudman S91 melanoma cells to respond to melanotropins in monolayer culture. Continuous exposure of melanoma cells to alpha-melanotropin or its potent analog [Nle4, D-Phe7]-alpha-MSH, resulted in maximal stimulation of tyrosinase after 2 d of treatment, but the magnitude of stimulation decreased thereafter despite the continued presence of the melanotropins. However, when melanoma cells continually exposed to melanotropins were subcultured to an initial low cell density and maintained in contact with alpha-MSH or [Nle4, D-Phe7]-alpha-MSH (long-term culture), tyrosinase activity was rapidly restored and greatly enhanced. Also, when cells were seeded at initial densities ranging from 0.2 to 3.2 X 10(6) cells/flask, and exposed for 24 h to 10(-7) M alpha-MSH, only the cultures seeded at low densities (0.2 and 0.4 X 10(6) cells/flask) exhibited maximal tyrosinase activity during the 24 h exposure to the melanotropins. Therefore, tyrosinase activity was primarily affected by cell density rather than by the duration of time the cells were in culture or by continuous exposure to melanotropin. Other flasks of various cell densities were treated with 10(-7) M alpha-MSH or [Nle4, D-Phe7]-alpha-MSH for 24 h, followed by removal of the melanotropins from the culture medium. The magnitude and duration of the residual stimulation of melanoma tyrosinase activity by melanotropins were also found to be dependent on the initial cell density. These results reveal that there is a limited range of optimal cell densities at which melanoma cells can respond to melanotropins and express increased tyrosinase activity.