Melanocyte Growth Research Articles

TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

SUMMARYWe screen ion channels and transporters throughout the genome to identify those required by human melanoma cells but not by normal human melanocytes. We discover that Mucolipin-1 (MCOLN1), which encodes the lysosomal cation channel TRPML1, is preferentially required for the survival and proliferation of melanoma cells. Loss of MCOLN1/TRPML1 function impairs the growth of patient-derived melanomas in culture and in xenografts but does not affect the growth of human melanocytes. TRPML1 expression and macropinocytosis are elevated in melanoma cells relative to melanocytes. TRPML1 is required in melanoma cells to negatively regulate MAPK pathway and mTORC1 signaling. TRPML1-deficient melanoma cells exhibit decreased survival, proliferation, tumor growth, and macropinocytosis, as well as serine depletion and proteotoxic stress. All of these phenotypes are partially or completely rescued by mTORC1 inhibition. Melanoma cells thus increase TRPML1 expression relative to melanocytes to attenuate MAPK and mTORC1 signaling, to sustain macropinocytosis, and to avoid proteotoxic stress.

Abstract 1049A: Deregulation of MITF in the context of inactive MC1R leads to melanocyte transformation

Abstract MC1R, the cAMP pathway and the response to solar UV are all elements involved in the growth, differentiation and survival of melanocytes and in malignant melanoma. Here we present a model of melanomagenesis based on the forced expression of MITF-M in the context of MC1R-inactivating variants in a way not dependent on sun exposure. We demonstrate that lentiviral transduction of MITF-M alone leads to consistent transformation of the Hermes4C cell line with inactive MC1R, but not the Hermes3C cell line having a wild type background. Hermes 4c cells transduced with MITF-M are able to form tumors in mice. We mapped MITF binding to chromatin and found that 4C cells displayed enhanced MITF chromatin binding compared to 3C. Expression analysis revealed that the altered binding pattern leads to enhanced transcription of epithelial to mesenchymal transition genes and consequent repression of key melanocyte-specific genes e.g. involved in pigmentation in the background of inactive MC1R. We observed a marked deregulation of AXL and EGFR, which is accompanied by downregulation of PTEN and increased phosphorylation of ERK and PI3K-AKT pathway. In conclusion, we show that MITF-M is a molecular switch in the context of MC1R-inactivating variant which leads to cell reprogramming and melanomagenesis. Citation Format: Tine Norman Alver, Timothy J. Lavelle, Karen-Marie Heintz, Patrik Wernhoff, Vegard Nygaard, Geir F. Øy, Sigurd L. Bøe, Alfonso Urbanucci, Eivind J. Hovig. Deregulation of MITF in the context of inactive MC1R leads to melanocyte transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1049A.

Fission Yeast as a Model System for Studying Cancer Signaling and Drug Discovery: Discovery of ACA-28 as a Novel Inducer of ERK-dependent Apoptosis Reveals a New Cancer Therapy

Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved kinase modules that link extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, and apoptosis. The Ras/Raf/MEK/ERK MAPK pathway is one of the most studied of the mammalian MAPK pathways and has attracted intense research interest because of its critical involvement in the regulation of cell proliferation. The mutational activation of upstream signaling components that constitutively activate ERK MAPKs as seen in various primary tumor samples has validated this pathway for drug discovery. The fission yeast Schizosaccharomyces pombe is an important tool for cancer research. This well-studied model organism has enabled groundbreaking, Nobel Prize-winning discoveries and has provided insights into how both normal and cancerous cells grow and divide. We performed chemical genetic screening using a fission yeast phenotypic assay and demonstrated that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. Importantly, the growth of normal human epidermal melanocytes was less affected by ACA-28. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells oncogenically transformed with HER2/ErbB2 but not in the parental cells. Notably, the ACA-28-induced apoptosis was abrogated when ERK activation was blocked with the specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells as compared with normal human epidermal melanocytes. ACA-28 might serve as a promising seed compound to combat ERK-dependent cancers by stimulating oncogenic signaling.

807 Homocysteine induces apoptosis of melanocytes via PERK-eif2α-CHOP pathway in vitiligo

Hyperhomocysteinemia and folic acid deficiencies have been reported in patients with vitligo, while the mechanism underlying this process is unclear. This study aims to investigate the effect of homocysteine (Hcy) on vitiligo pathogenesis and the underlying mechanisms. Our results demonstrated that Hcy was highly expressed in the serum of patients with vitiligo and associated with the degree of disease progression. In vitro study confirmed that oxidative stress can promote the accumulation of Hcy in melanocytes. Hcy treatment can inhibit melanocyte growth and induce cell apoptosis in a dose-dependent manner. Hcy treatment can upregulate glucose-regulated protein 78(GRP78), activate endoplasmic reticulum stress and induce the expression of C/EBP homologous protein(CHOP). Knockout the IRE1 pathway has no effect on apoptosis, whereas, the level of apoptosis is reduced by knockout the PERK pathway. We also observed activation of pPERK in peripheral melanocytes of vitiligo patient. Folic acid serves as cofactors of homocysteine methyltransferase of methionine from homocysteine. Folic acid supplementation can reduce intracellular Hcy levels, inhibit the activation of the perk-eif2a-CHOP pathway, reduce cell apoptosis rate and promote melanocyte surivival. Our study also showed that homosysteine disrupts melanogenesis via inhibition of MITF TYR TYRP1 and melanA experssion, supplementation of folic acid can restore above molecular expression and restore melanin synthesis. Our data suggest that an elevated Hcy level may be a precipitating factor for vitiligo in predisposed individuals. In addition, Hcy facilitate melanocyte apoptosis through perk-eif2a-CHOP pathway, and decrease melanin synthesis. Importantly, folic acid supplementation can reduce the Hcy toxicity to melanocyte and restore melanin synthesis.

Automated in vivo screen in zebrafish identifies Clotrimazole as targeting a metabolic vulnerability in a melanoma model

Therapeutic approaches for cutaneous melanoma are flourishing, but despite promising results, there is an increasing number of reported primary or secondary resistance to the growing sets of drugs approved for therapy in the clinics. Combinatorial approaches may overcome resistance, as they may tackle specific weaknesses of melanoma cells, not sufficient on their own, but effective in combination with other therapies.The transgenic zebrafish line kita:ras develops melanoma with high frequency. At 3 dpf, transgenic kita:ras larvae show a hyperpigmentation phenotype as earliest evidence of abnormal melanocyte growth. Using this model, we performed a chemical screen based on automated detection of a reduction of melanocyte number caused by any of 1280 FDA or EMA approved drugs of the library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We further tested two compounds for each of the 5 classes, and a farnesyltransferase inhibitor (Lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of Clotrimazole and Lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) in order to investigate the mechanism of action of Clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in Clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the same class, Miconazole. Furthermore, we show that the effects of Clotrimazole are mediated by the inhibition of hexokinase activity, which is lethal to the abnormal metabolic profile of melanoma cells in vitro and in vivo. Thus, our study shows that the zebrafish can provide a phenotype-rich assay for fully automated screening approaches to identify drugs for synthetic lethal treatment in melanoma and suggest further testing of Clotrimazole in combinatorial treatments.

Heterogeneity of cultured melanocyte elongation and proliferation factor in bilateral diffuse uveal melanocytic proliferation

A patient with bilateral diffuse uveal melanocytic proliferation (BDUMP) associated with endometrial cancer was treated with plasmapheresis, but failed therapy with progressive serous retinal detachment. We collected plasma before and after plasmapheresis therapy. Our goal was to determine if the cultured melanocyte elongation and proliferation (CMEP) factor and hepatocyte growth factor (HGF) was present in the IgG enriched fraction and understand why our patient failed plasmapheresis therapy. Melanocytes were cultured for 3–5 days in the presence of control medium, unfractionated pre-plasmapheresis BDUMP medium, IgG enriched or IgG depleted BDUMP medium, or unfractionated post-plasmapheresis BDUMP medium. Subretinal fluid was collected from patients with BDUMP and control retinal detachments and analyzed by electropheresis with immunoblotting. Medium with unfractionated BDUMP plasma stimulated melanocyte growth 1.4–1.5 fold compared to control medium on days 3–5 (p < 0.001 for all). Both IgG enriched and IgG depleted BDUMP medium mildly increased melanocyte growth 1.3 fold (p < 0.05 for enriched, p < 0.01 for depleted) compared to control. In comparison, unfractionated BDUMP medium caused a 1.7-fold increase in melanocyte growth, which was significantly more than the enriched (p < 0.01) and depleted (p < 0.05) fractions. Pre-plasmapheresis and post-plasmapheresis unfractionated BDUMP medium equally stimulated melanocyte growth 1.7-fold (p < 0.05) compared to control. HGF was present in IgG depleted, pre-plasmapheresis, and post-plasmapheresis samples, but absent in the IgG enriched fraction. There was no enrichment of IgG in the subretinal fluid from eyes with BDUMP. In conclusion, CMEP factor is not concentrated in the IgG enriched plasma fraction in our patient who failed plasmapheresis therapy. HGF levels have no correlation with melanocyte growth. Because plasmapheresis preferentially removes immunoglobulins from the plasma, our patient responded poorly to plasmapheresis treatment with worsening retinal detachment.

Quantification of Hypopigmentation Activity In Vitro.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis. In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors.

Coculture with adipose-derived stem cells (ADSCs) can stimulate proliferation and migration of melanocytes. To enhance outcomes of skin disorders caused by melanocyte loss or death, mixed transplantation with ADSCs has been suggested. However, role of cocultured ADSCs in proliferation and migration of melanocytes remains unclear. This study determined the effect of ADSCs on production of growth factors and expression levels of intergrins in primary culture of adult human melanocytes with or without ADSCs and in nude mice grafted with such melanocytes. Higher amounts of growth factors for melanocytes, such as bFGF and SCF were produced and released from ADSCs by coculturing with melanocytes. Relative levels of integrins β1, α5, and α6 as well as adhesion to fibronectin and laminin were increased in melanocytes cocultured with ADSCs. Such increases were inhibited by neutralization of bFGF or SCF. Relative levels of bFGF, SCF and integrins were increased in nude mice skin after grafting with melanocyte+ADSC cocultures. Collectively, these results indicate that ADSCs can stimulate proliferation and migration of melanocytes by increasing expression of integrins in melanocytes through upregulation of production/release of melanocyte growth factors such as bFGF and SCF.

Role of IL‐17A receptor blocking in melanocyte survival: A strategic intervention against vitiligo

Cytokines regulate immune response and inflammation and play an important role in depigmentation process of an autoimmune disease, vitiligo. We sought to determine how inflammatory cytokines influence the progression of vitiligo, and based on that, we develop a logical therapeutic intervention using primary melanocyte culture. Melanocytes were cultured and exposed to IL-17A, IL-1β, IFN-γ and TGF-β for 4days. Melanocytes proliferation, tyrosinase assay and melanin content were measured. Real-Time PCR was used to analyse mRNA expression of genes specific for melanocytes growth and pigmentation. Anti-IL-17A receptor antibody was used to block IL-17A receptors expressed on melanocytes. Protein expression of MITF and TYR was assessed by immunofluorescence and Western blotting. A gradual decline in the melanocyte population, melanin content and tyrosinase activity was observed after different cytokine treatment. The expression of MITF and its downstream genes after blocking with anti-IL-17RA, an increased melanin content, increased expression of TYR, MITF along with its downstream genes, and cell proliferation was observed.

Natural Compounds in the Chemoprevention of Malignant Melanoma.

Malignant melanoma is a very aggressive form of skin cancer, with increasing rates every year, with an etiology that derives from the transformation and uncontrolled growth of melanocytes. There are several treatment options which can be used as unique treatment or combined, depending upon the stage of melanoma including surgical excision, chemotherapy, immunotherapy, targeted therapy. Plant Kingdom displays an unequalled potential for the synthesis of highly diversified chemical structures. Although natural compounds are synthesized in order to help the plant to interact with the environment, a large number of phytochemicals act as drugs within the human or animal body by activating various biochemical pathways. The study aims to review another approach in the management of this highly aggressive form of skin cancer, namely the effect of natural compounds in the chemoprevention of malignant melanoma.

Decreased expression of neuregulin1 in the lesional skin of vitiligo patients.

Paracrine cross-talk exists between the fibroblasts of dermis and epidermal cells through secretions of various growth factors. Melanocytes present at the basement layer of the epidermis and respond to various factors secreted by underlying dermal fibroblasts in the dermis to regulate the function of the skin. Therefore the study was planned to check the expression of fibroblast-derived factor neuregulin1 (NRG1) in vitiligo skin and its effect on melanocytes. For this study, relative gene expression at mRNA level of NRG1 in the vitiligo skin was analyzed by qRT-PCR, and protein analysis was done by immunohistochemistry. Effect of different concentrations of NRG1 was checked on the cultured melanocytes by melanin content assay, proliferation assay, and tyrosinase (TYR) assay. The effect of NRG1 was also checked on the level of melanocyte regulatory genes (MITF, c-KIT, TYR, DCT). Expression of NRG1 was significantly less in lesional dermis of vitiligo patients as compared to nonlesional and healthy control dermis both at mRNA as well as protein level. NRG1 treatment showed significant increase in proliferation, melanin content, TYR level, and gene expression level of melanocyte specific genes. Treatment of NRG1 to the cultured melanocytes increases proliferation and pigmentation. Lower expression of NRG1 in the lesional dermis of vitiligo patients inhibits the melanocyte growth. Therefore this study hypothesized that low expression of NRG1 in lesional skin of vitiligo patients might have a possible role in the melanocyte loss and vitiligo pathogenesis.

Abstract 4503: The paracrine role of p53 in skin

Abstract Exposure to ultraviolet radiation (UVR) in sunlight activates both defensive and repair pathways in the skin. As the major cell type affected by UVR, keratinocytes coordinate critical aspects of the skin response. Molecularly, the transcription factor p53 is induced by UVR in keratinocytes leading to their cell death by apoptosis (peeling). p53 also stimulates the expression of paracrine melanocyte growth factors involved in tanning defined as increase in melanocyte proliferation and melanin synthesis. Here, we investigate the less defined role of p53 in tanning. For this, we use a mouse model that has high p53 levels in keratinocytes and presents with elevated melanocyte number and melanin production. Interestingly, loss of p53 in keratinocytes completely abrogates the hyperpigmentation phenotype. These data demonstrate in a powerful way the paracrine role of p53 expressed in keratinocytes on melanocyte behavior and function. Moreover, it provides an excellent tool to examine molecular and cellular changes that accompany the melanocyte response to sunburn. Sunburn increases melanoma risk in epidemiological studies. To elucidate the role of the p53 response in melanoma, we treated mice with “sunburning” doses of UVB and followed melanocyte proliferation and the expression of keratinocyte-induced melanocyte growth factors. We present our latest findings reinforcing the signs of cross-talk between keratinocytes and melanocytes which support a paracrine role for p53 expressed in keratinocyte on promoting melanocyte proliferation through the release of paracrine factors. Citation Format: Tamara Terzian, Nema Sobhani, Rohan Mylavarapu, Manale El Kharbili, Andrew Parker, Colleen Little, Ethan Krauspe, Neil Box. The paracrine role of p53 in skin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4503.

Immunohistochemical Evaluation of Canine Pituitary Adenomas Obtained by Transsphenoidal Hypophysectomy.

Hypophysectomy specimens from 16 dogs with pituitary adenoma were evaluated with periodic acid-Schiff (PAS), reticulin, and immunohistochemistry for adrenocorticotrophic hormone (ACTH), melanocyte stimulating hormone (MSH), growth hormone (GH), and Ki-67. The reticulin network was obliterated in all adenomas. One adenoma expressed ACTH and GH. Eight corticotroph adenomas were basophilic to chromophobic, and PAS- and ACTH-positive. Seven melanotroph adenomas were distinguished from corticotroph adenomas by expression of MSH. Pituitary-dependent hypercortisolism was diagnosed in 5 of 8 dogs with corticotroph and 4 of 7 with melanotroph adenoma. Pituitary height/brain area (P/B) ratio was elevated in all dogs. Previous canine hypophysectomy studies suggested that melanotroph adenomas were larger and carried a worse prognosis than corticotroph adenomas; however, in this study, corticotroph adenomas in comparison to melanotroph adenomas were larger (median P/B ratio: 1.06 versus 0.76), more proliferative (median Ki-67 index: 9.47% versus 1.99%), and associated with shorter survival (median: 300 versus 793 days). Recommended immunohistochemistry for PAS-positive pituitary adenomas includes ACTH and MSH to distinguish corticotrophs from melanotrophs and Ki-67 for proliferation index.

A preliminary study of growth characteristics of melanocytes co-cultured with keratinocytes in vitro.

To clarify the characteristic growth of melanocytes (MCs) and Keratinocytes (KCs) in vitro and discuss the mechanism of culturing autologous melanocytes in the treatment of vitiligo. Epidermis cells derived from normal skin tissues were isolated and cultured in vitro. Melanocytes in DOPA staining were observed. The expression level of markers in MCs was detected by qRT-PCR and the percentage of MCs and KCs were detected by flow cytometry. Cells derived from normal skin tissues mainly included KCs, MCs, and fibroblasts. There were significant differences between the percentage of KC, MC, fibroblasts (P < 0.05), and the expression of Microphthalmia-associated transcription factor (P < 0.05) and Tyrosinase-related protein-2 (P < 0.05) in the second, 10th, 20th, and 30th day. Significant differences were also found between the average numbers of MC stained by DOPA (P < 0.05) and the average percentage of MCs in the 10th, 20th, and 30th Day (P < 0.05). But there were no significant differences between the average percentage of KCs in the 10th, 20th, and 30th Day (P > 0.05) detected by flow cytometry. The number of MCs co-cultured with KCs in vitro reached the maximum in the 20th Day and this co-cultured model may contribute to the growth of MCs which could be used in the treatment of vitiligo.

Effect of Dickkopf1 on the senescence of melanocytes: in vitro study.

Fibroblasts secrete several growth factors which are important for the regulation of skin pigmentation. Dickkopf1 (DKK1) is also secreted by fibroblasts which inhibit the growth and function of melanocytes. Therefore, the study was designed to check the role of DKK1 in vitiligo pathogenesis. This study confirmed the higher expression of DKK1 in lesional skin of vitiligo patients. In vitro effect of DKK1 on cultured melanocytes revealed decrease in the melanocytes proliferation and pigmentation. In vitro effect of DKK1 was then checked on the melanocytes senescence and found that DKK1 induced senescence in the treated melanocytes. Expression of senescence markers was significantly higher in DKK1 treated melanocytes. This study suggests that higher expression of DKK1 in the dermis induced senescence in melanocytes that may lead to hypopigmentation and play role in vitiligo pathogenesis.

A common intronic variant of PARP1 confers melanoma risk and mediates melanocyte growth via regulation of MITF.

Previous genome-wide association studies have identified a melanoma-associated locus at 1q42.1 that encompasses a ∼100-kb region spanning the PARP1 gene. Expression quantitative trait locus (eQTL) analysis in multiple cell types of the melanocytic lineage consistently demonstrated that the 1q42.1 melanoma risk allele (rs3219090[G]) is correlated with higher PARP1 levels. In silico fine-mapping and functional validation identified a common intronic indel, rs144361550 (-/GGGCCC; r2 = 0.947 with rs3219090), as displaying allele-specific transcriptional activity. A proteomic screen identified RECQL as binding to rs144361550 in an allele-preferential manner. In human primary melanocytes, PARP1 promoted cell proliferation and rescued BRAFV600E-induced senescence phenotypes in a PARylation-independent manner. PARP1 also transformed TERT-immortalized melanocytes expressing BRAFV600E. PARP1-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF, highlighting a new role for PARP1 in melanomagenesis.

Keap1 knockdown in melanocytes induces cell proliferation and survival via HO-1-associated β-catenin signaling

BackgroundNrf2-Keap1 signaling pathway protects cells against photo-oxidative stress. Yet in recent works, its role in melanogenesis together with cell protection functions against oxidative stress has been gaining interest. However, its effect on melanogenesis still has contradictory results from different studies. ObjectiveThe aims of our study were to investigate the effect of Keap1 silencing in melanocyte on melanogenesis and its associated mechanism. MethodsPrimary human epidermal melanocytes and melan-a cell line were used for this experiment. RNA sequencing was done to identify genes involved in melanocyte biology using Keap1 knockdown through siRNA techniques. And melanogenesis and the expression of melanogenesis-associated molecules were evaluated in Keap1 silenced melanocyte to examine the effects of Keap1 on melanogenesis, melanocyte growth, and related pathways. ResultsRNA-sequencing data revealed that Keap1 knockdown in primary human epidermal melanocytes (PHEMs) induced cell survival-related gene expression. Additionally, siRNA-mediated inhibition of Keap1 led to upregulation of MITF and melanogenesis-associated molecules along with Nrf2 activation in PHEMs. HO-1, a major gene that is upregulated in RNA-sequencing using Keap1-silenced PHEMs, protected melanocytes against H2O2-induced cell death and upregulated MITF and β-catenin expression. Further, increased expression of melanogenesis-associated molecules after Keap1 silencing was validated to occur through HO-1-associated β-catenin activation in a Keap1 and HO-1 double knockdown experiment. ConclusionThis work suggests that Keap1 silencing in melanocytes induced melanogenesis and the expression of melanogenesis-associated molecules through HO-1-associated β-catenin activation. Keap1 downregulation in melanocytes is important for cell proliferation and survival.

Identification of ACA‐28, a 1′‐acetoxychavicol acetate analogue compound, as a novel modulator of ERK MAPK signaling, which preferentially kills human melanoma cells

The extracellular signal-regulated kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as melanoma. Melanoma remains incurable despite the use of conventional chemotherapy; consequently, development of new therapeutic agents for melanoma is highly desirable. Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. Notably, the ACA-28-induced apoptosis in melanoma and HER2-transformed cells was abrogated when ERK activation was blocked with a specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells, as compared in NHEM. ACA-28 might serve as a promising seed compound for melanoma treatment.

806 Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes

Ultraviolet (UV)-induced mutations in melanocytes are a well-established cause of melanoma. However, histologically normal “fields” of altered keratinocytes present in sun-exposed epidermis could also drive initiation and progression of melanoma. Supporting this idea, we observed reduced expression of the keratinocyte desmosomal cadherin, desmoglein 1 (Dsg1) (which we previously showed is lost in response to UV) in regions surrounding human in situ and stage I/II melanomas. Loss of Dsg1 was specific as expression of E-cadherin was not significantly altered in early tumors. Dsg1-deficient keratinocytes also exhibited indications of impaired Dsg1-dependent signaling including increased phospho-EGFR and nuclear NFκB. To directly determine whether Dsg1 loss affects melanocyte behavior, we carried out in vitro analyses of Dsg1-silenced keratinocytes. Dsg1 knockdown increased keratinocyte expression of mRNAs encoding multiple pro-inflammatory cytokines, which also occurred in primary human melanocytes co-cultured with these keratinocytes. Growth of melanocytes in media conditioned by Dsg1-deficient keratinocytes resulted in significantly increased average melanocyte dendrite length (104μm vs. 74μm in controls; p=0.002) and dendrite number (3.3 vs. 2.6/cell; p=0.001). Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents exhibited aberrant behavior, including increased numbers and mis-localization to suprabasal layers. Based on these observations, we propose that under homeostatic conditions Dsg1 keeps cytokine signaling in check but that Dsg1 loss due to environmental stress disrupts keratinocyte:melanocyte communication through altered paracrine signaling to promote melanoma initiation and/or progression. We propose Dsg1 loss as a potential biomarker for increased melanoma risk and a therapeutic target for histone deacetylase inhibitors, which we have shown increase Dsg1 expression.

Potential therapeutic targets of epithelial–mesenchymal transition in melanoma

Melanoma is a cutaneous neoplastic growth of melanocytes with great potential to invade and metastasize, especially when not treated early and effectively. Epithelial-mesenchymal transition (EMT) is the process by which melanocytes lose their epithelial characteristics and acquire mesenchymal phenotypes. Mesenchymal protein expression increases the motility, invasiveness, and metastatic potential of melanoma. Many pathways play a role in promotion of mesenchymal protein expression including RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, Wnt/β-catenin, and several others. Downstream effectors of these pathways induce expression of EMT transcription factors including Snail, Slug, Twist, and Zeb that promote repression of epithelial and induction of mesenchymal character. Emerging research has demonstrated that a variety of small molecule inhibitors as well as phytochemicals can influence the progression of EMT and may even reverse the process, inducing re-expression of epithelial markers. Phytochemicals are of particular interest as supplementary treatment options because of their relatively low toxicities and anti-EMT properties. Modulation of EMT signaling pathways using synthetic small molecules and phytochemicals is a potential therapeutic strategy for reducing the aggressive progression of metastatic melanoma. In this review, we discuss the emerging pathways and transcription factor targets that regulate EMT and evaluate potential synthetic small molecules and naturally occurring compounds that may reduce metastatic melanoma progression.