AMPK Phosphorylates ZDHHC13 to Increase MC1R Activity and Suppress Melanomagenesis.

Inherited genetic variations in the melanocortin-1 receptor (MC1R) responsible for human red hair color (RHC) variants are associated with impaired DNA damage repair and increased melanoma risk. MC1R signaling is critically dependent on palmitoylation, primarily mediated by the protein acyltransferase zinc finger DHHC-type palmitoyltransferase 13 (ZDHHC13). A better understanding of how ZDHHC13 is physiologically activated could help identify approaches to prevent melanomagenesis in redheads. Here, we report that AMP-activated protein kinase (AMPK) phosphorylates ZDHHC13 at S208 to strengthen the interaction between ZDHHC13 and MC1R-RHC, leading to enhanced MC1R palmitoylation in redheads. Consequently, phosphorylation of ZDHHC13 by AMPK increased MC1R-RHC downstream signaling. AMPK activation and MC1R palmitoylation repressed UVB-induced transformation of human melanocytes in vitro and delayed melanomagenesis in vivo in C57BL/6J-MC1R-RHC mice. The importance of AMPK to MC1R signaling was validated in human melanomas where AMPK upregulation correlated with expression of factors downstream from MC1R signaling and with prolonged patient survival. These findings suggest AMPK activation as a promising strategy to reduce melanoma risk, especially for individuals with red hair. SIGNIFICANCE: Phosphorylation of ZDHHC13 by AMPK at S208 promotes MC1R activation and suppresses melanocyte transformation, indicating activation of AMPK as a potential approach to prevent melanoma in people with red hair.

Cajanin Suppresses Melanin Synthesis through Modulating MITF in Human Melanin-Producing Cells.

Despite its classification as a non-life-threatening disease, increased skin pigmentation adversely affects quality of life and leads to loss of self-confidence. Until now, there are no recommended remedies with high efficacy and human safety for hyperpigmentation. This study aimed to investigate anti-melanogenic activity and underlying mechanism of cajanin, an isoflavonoid extracted from Dalbergia parviflora Roxb. (Leguminosae) in human melanin-producing cells. Culture with 50 µM cajanin for 48-72 h significantly suppressed proliferation in human melanoma MNT1 cells assessed via MTT viability assay. Interestingly, cajanin also efficiently diminished melanin content in MNT1 cells with the half maximum inhibitory concentration (IC50) at 77.47 ± 9.28 µM. Instead of direct inactivating enzymatic function of human tyrosinase, down-regulated mRNA and protein expression levels of MITF and downstream melanogenic enzymes, including tyrosinase, TRP-1 and Dct (TRP-2) were observed in MNT1 cells treated with 50 µM cajanin for 24-72 h. Correspondingly, treatment with cajanin modulated the signaling pathway of CREB and ERK which both regulate MITF expression level. Targeted suppression on MITF-related proteins in human melanin-producing cells strengthens the potential development of cajanin as an effective treatment for human hyperpigmented disorders.

The relationship between melanin production and lipofuscin formation in Tyrosinase gene knockout melanocytes using CRISPR/Cas9 system.

Age spots are a significant phenotypic marker of aging formed by lipofuscin. Melanin is another skin pigment molecule responsible for skin aging. The present study aims to investigate the relationship between melanin production and lipofuscin synthesis in normal mouse melanoma cell line B16F1 cells and Tyrosinase (TYR) gene knockout cells. TYR gene KO cells were successfully developed using CRISPR/Cas9 system and confirmed by Sanger DNA sequencing analysis. Furthermore, the melanin production and lipofuscin formation were validated through RT-PCR and Western blot analysis. The expression levels of gene microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosine-related protein-1 (TRP-1), tyrosine-related protein-2 (TRP-2), and antioxidant proteins such as methionine sulfoxide reductase A (MSRA), Catalase and Glutathione reductase (GR) related to melanogenesis was found to be decreased in TYR gene KO cells compared with normal cells. Moreover, lipofuscin formation was increased in TYR gene KO cells compared to normal cells. Therefore, the above findings suggest that melanin production and lipofuscin formation could be linked by the TYR gene in melanocytes.

Antioxidant and anti-aging carbon quantum dots using tannic acid.

Overexpression of collagenase, elastase, and tyrosinase is caused by external factors such as ultraviolet (UV) radiation and stress, resulting in wrinkle formation and freckles through the loss of skin elasticity and skin pigmentation. In this study, we developed novel carbon quantum dots (CQDs) with antioxidant and anti-aging properties using tannic acid as a carbon source through a simple microwave-assisted pyrolysis method. The synthesized tannic acid-derived CQDs (T-CQDs) showed bright blue fluorescence (QY = 28.2 ± 4.0%), exhibiting maximum emission at 430 nm under 350 nm excitation. Even though small amount of the T-CQDs (3µg ml-1) was used, they exhibited excellent free radical scavenging ability (82.8 ± 4.3%). Also, the T-CQDs (10µg ml-1) revealed remarkable inhibitory activity against skin aging-related collagenase (77.6 ± 4.8%), elastase (52.6 ± 1.0%), and tyrosinase (44.2 ± 1.3%), demonstrating their antioxidant and anti-aging effects. Furthermore, their antioxidant and anti-aging properties were superior to those of tannic acid, L-ascorbic acid, and quercetin used as positive controls. Finally, the T-CQDs effectively suppressed UV-induced reactive oxygen species generation by 30% at the cellular levels and showed high cell viability (99.7 ± 0.8%) even at 500µg ml-1. These results demonstrate that the T-CQDs with superior antioxidant, anti-aging properties, and low cytotoxicity can be utilized as novel anti-aging materials in cosmetic and nanomedicine fields.

Telomerase Reverse Transcriptase Protein Expression Is More Frequent in Acral Lentiginous Melanoma Than in Other Types of Cutaneous Melanoma.

CONTEXT.­: Molecularly distinct from cutaneous melanomas arising from sun-exposed sites, acral lentiginous melanomas (ALMs) typically lack ultraviolet-signature mutations, such as telomerase reverse transcriptase (TERT) promoter mutations. Instead, ALMs show a high degree of copy number alterations, often with multiple amplifications of TERT, which are associated with adverse prognosis. The prognostic value of TERT protein expression in acral melanomas, however, is not established. OBJECTIVE.­: To evaluate the frequency and pattern of TERT immunoreactivity and assess the potential utility of TERT expression as a prognostic indicator in ALMs. DESIGN.­: TERT expression by immunohistochemistry was analyzed in a series of 57 acral and nonacral melanocytic lesions, including 24 primary and 6 metastatic ALMs. Clinical outcome in patients with ALMs by TERT expression was assessed. RESULTS.­: TERT expression was more frequent in ALMs than in nonlentiginous acral melanomas and nonacral cutaneous melanomas, and was absent in acral nevi (P = .01). When present, TERT expression in ALMs was cytoplasmic and more intense than TERT expression in other melanocytic lesions (P = .05) with a higher H-score (P = .01). There was a trend toward decreased overall survival in patients with ALMs with TERT immunoreactivity, but it did not reach statistical significance. Furthermore, no correlation was found between TERT expression and disease-specific survival in patients with ALMs. CONCLUSIONS.­: Although TERT protein expression was frequently detected in both primary and metastatic ALMs, TERT immunoreactivity in ALMs did not correlate with survival in our study. Further studies with larger cohorts are needed to elucidate the prognostic value of TERT expression in ALMs.

ERK1/2 Pathway Is Involved in the Enhancement of Fatty Acids from Phaeodactylum tricornutum Extract (PTE) on Hair Follicle Cell Proliferation.

Extractions from Phaeodactylum tricornutum have been widely studied and evaluated to various biological effects. The aim of this study was to investigate the promotional effect of P. tricornutum extract (PTE) on the ERK1/2 signaling pathway involved in hair follicle cell proliferation. In order to illuminate the enhancement of PTE on hair growth by promoting proliferation of hair follicle cells, the activities of human hair follicle outer root sheath cell (HFORSC), human hair follicle germinal matrix cells (HFGMC), and hair epithelial melanocytes (HEM) were observed under PET treatment. Levels of keratins, PKCζ, ERK1/2, and p38 MAPK in hair follicle cells were determined by Western blotting to illustrate the mechanisms of PTE effects on hair growth. Analyzed by GC-MS, the main polyunsaturated fatty acids which were 9.43% of total fatty acids in PTE were linolenic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid. Melanin content and tyrosinase activity in HEM were measured. The results showed that PTE exhibited remarkable enhancement on cell proliferation. Melanin production was inhibited by PTE treatment, while keratin-14, keratin-15, and keratin-17 levels on hair follicle cells were elevated at different concentrations. The promotions of ERK1/2 and p38 MAPK levels indicated that the ERK1/2 signaling pathway is involved in the proliferation of hair follicle cells. These results are the evidence that PTE potentially deserves further study as a new natural candidate for hair care applications.

ß-Galactosylceramidase Promotes Melanoma Growth via Modulation of Ceramide Metabolism.

Disturbance of sphingolipid metabolism may represent a novel therapeutic target in metastatic melanoma, the most lethal form of skin cancer. ß-Galactosylceramidase (GALC) removes ß-galactose from galactosylceramide and other sphingolipids. In this study, we show that downregulation of galcb, a zebrafish ortholog of human GALC, affects melanoblast and melanocyte differentiation in zebrafish embryos, suggesting a possible role for GALC in melanoma. On this basis, the impact of GALC expression in murine B16-F10 and human A2058 melanoma cells was investigated following its silencing or upregulation. Galc knockdown hampered growth, motility, and invasive capacity of B16-F10 cells and their tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos. Galc-silenced cells displayed altered sphingolipid metabolism and increased intracellular levels of ceramide, paralleled by a nonredundant upregulation of Smpd3, which encodes for the ceramide-generating enzyme neutral sphingomyelinase 2. Accordingly, GALC downregulation caused SMPD3 upregulation, increased ceramide levels, and inhibited the tumorigenic activity of human melanoma A2058 cells, whereas GALC upregulation exerted opposite effects. In concordance with information from melanoma database mining, RNAscope analysis demonstrated a progressive increase of GALC expression from common nevi to stage IV human melanoma samples that was paralleled by increases in microphthalmia transcription factor and tyrosinase immunoreactivity inversely related to SMPD3 and ceramide levels. Overall, these findings indicate that GALC may play an oncogenic role in melanoma by modulating the levels of intracellular ceramide, thus providing novel opportunities for melanoma therapy. SIGNIFICANCE: Data from zebrafish embryos, murine and human cell melanoma lines, and patient-derived tumor specimens indicate that ß-galactosylceramidase plays an oncogenic role in melanoma and may serve as a therapeutic target.

Advances in the Design of Genuine Human Tyrosinase Inhibitors for Targeting Melanogenesis and Related Pigmentations.

Human tyrosinase (hsTYR) is the key enzyme ensuring the conversion of l-tyrosine to dopaquinone, thereby initiating melanin synthesis, i.e., melanogenesis. Although the protein has long been familiar, knowledge about its three-dimensional structure and efficient overexpression protocols emerged only recently. Consequently, for decades medicinal chemistry studies aiming at developing skin depigmenting agents relied almost exclusively on biological assays performed using mushroom tyrosinase (abTYR), producing a plethoric literature, often of little useful purpose. Indeed, several recent reports have pointed out spectacular differences in terms of interaction patterns and inhibition values between hsTYR and abTYR, including for widely used standard tyrosinase inhibitors. In this review, we summarize the last developments regarding the potential role of hsTYR in human pathologies, the advances in recombinant expression systems and structural data retrieving, and the pioneer generation of true hsTYR inhibitors. Finally, we present suggestions for the design of future inhibitors of this highly attractive target in pharmacology and dermocosmetics.

Guanine Deaminase in Human Epidermal Keratinocytes Contributes to Skin Pigmentation.

Epidermal keratinocytes are considered as the most important neighboring cells that modify melanogenesis. Our previous study used microarray to show that guanine deaminase (GDA) gene expression is highly increased in melasma lesions. Hence, we investigated the role of GDA in skin pigmentation. We examined GDA expression in post-inflammatory hyperpigmentation (PIH) lesions, diagnosed as Riehl's melanosis. We further investigated the possible role of keratinocyte-derived GDA in melanogenesis by quantitative PCR, immunofluorescence staining, small interfering RNA-based GDA knockdown, and adenovirus-mediated GDA overexpression. We found higher GDA positivity in the hyperpigmentary lesional epidermis than in the perilesional epidermis. Both UVB irradiation and stem cell factor (SCF) plus endothelin-1 (ET-1) were used, which are well-known melanogenic stimuli upregulating GDA expression in both keratinocyte culture alone and keratinocyte and melanocyte coculture. GDA knockdown downregulated melanin content, while GDA overexpression promoted melanogenesis in the coculture. When melanocytes were treated with UVB-exposed keratinocyte-conditioned media, the melanin content was increased. Also, GDA knockdown lowered SCF and ET-1 expression levels in keratinocytes. GDA in epidermal keratinocytes may promote melanogenesis by upregulating SCF and ET-1, suggesting its role in skin hyperpigmentary disorders.

Chronic stress induces fur color change from dark to brown by decreasing follicle melanocytes and tyrosinase activity in female C57BL/6 mice.

Increasing evidence suggests that stress may induce changes in hair color, with the underlying mechanism incompletely understood. In this study, female C57BL/6 mice subjected to electric foot shock combined with restraint stress were used to build chronic stress mouse model. The melanin contents and tyrosinase activity were measured in mouse skin and B16F10 melanoma cells. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor α (TNF-α), interleukin- 1ß (IL-1ß) and interleukin-6 (IL-6) in the mouse skin. The content of nuclear factor κB (NFκB)/p65 subunit in mouse skins was valued by immunofluorescence staining. The results demonstrated that under chronic stress, the fur color turned from dark to brown in C57BL/6 mice due to the decrease of follicle melanocytes and tyrosinase activity in C57BL/6 mouse skin. Simultaneously, inflammatory responses in skins were detected as shown by increased NFκB activity and TNF-α expression in stressed mouse skin. In cultured B16F10 melanoma cells, TNF-α reduced the melanogenesis and tyrosinase activity in a dose-dependent manner. These findings indicate that chronic stress induces fur color change by decreasing follicle melanocytes and tyrosinase activity in female C57BL/6 mice, and TNF-α may play an important role in stress-induced hair color change.

Transcripts 202 and 205 of IL-6 confer resistance to Vemurafenib by reactivating the MAPK pathway in BRAF(V600E) mutant melanoma cells.

BRAF mutations occur in approximately 50% of melanoma patients. The mutated BRAF kinase continuously activates the mitogen-activated protein kinase (MAPK) pathway to promote cell growth and proliferation. Vemurafenib as a specific BRAF inhibitor can significantly prolong progression-free survival in melanoma patients. However, most patients developed resistance to Vemurafenib after 6 months. The mechanism of drug resistance is not yet fully understood. In this study, we found that proteins secreted by drug-resistant cells protect sensitive cells from Vemurafenib. By RNA-seq, we compared differentially expressed genes between resistant and sensitive cells. We demonstrated that drug-resistant cells secrete more IL-6 protein than sensitive cells. For the first time, we found that IL-6 expressed by drug-resistant cells consists of the following transcripts: IL6-201, IL6-202 and IL6-205. We confirmed that it is the IL6-202 and IL6-205 transcripts that confer drug resistance to Vemurafenib by reactivating the MAPK pathway while IL6-201 is not responsible for the resistance in A375 melanoma cells. Neutralizing IL-6 significantly increased the sensitivity of drug-resistant cells to Vemurafenib. Overall, these results reveal a new mechanism of drug resistance in melanoma.

Pathologic Characteristics of Spitz Melanoma With MAP3K8 Fusion or Truncation in a Pediatric Cohort.

Spitz melanoma is a rare variant of melanoma defined by distinct clinical, histologic, and genetic features and affecting patients of all ages. Half of these tumors are driven by fusion of kinase genes including ALK, NTRK1/3, ROS1, RET, MET, or BRAF. We recently reported recurrent fusion or truncation of the potentially targetable serine-threonine kinase gene MAP3K8 in 33% of Spitz melanomas. Here we describe the histologic features of these MAP3K8-rearranged tumors (16 pediatric Spitz melanomas; 1 atypical Spitz tumor), using hematoxylin-eosin slides, p16 immunohistochemistry, and CDKN2A fluorescence in situ hybridization. The lesions consisted of a compound melanocytic proliferation, ranging in thickness from 1.5 to 13.4 mm (median, 3.1 mm), with 8 having a predominant dermal and 3 having a predominant junctional component. The predominant cell type was epithelioid (94%). The epithelioid melanocytes were generally monomorphic and amelanotic, arranged in expansile epithelial aggregates, confluent hypercellular nests, or enlarged syncytial nodules in the dermis. Ulceration was present in 9 of 17 tumors (53%) and deep mitotic figures were seen in 15 of 17 tumors (88%). Complete loss of p16 expression and homozygous CDKN2A deletion were observed in 82% and 70% of tumors, respectively. Recognition of MAP3K8-altered Spitz melanoma may thus be facilitated by these morphologic features, most notably presence of cohesive cellular nodules in the dermis and an epithelioid-cell phenotype.

DNp73-induced degradation of tyrosinase links depigmentation with EMT-driven melanoma progression.

Melanoma is an aggressive cancer with poor prognosis, requiring personalized management of advanced stages and establishment of molecular markers. Melanomas derive from melanocytes, which specifically express tyrosinase, the rate-limiting enzyme of melanin-synthesis. We demonstrate that melanomas with high levels of DNp73, a cancer-specific variant of the p53 family member p73 and driver of melanoma progression show, in contrast to their less-aggressive low-DNp73 counterparts, hypopigmentation in vivo. Mechanistically, reduced melanin-synthesis is mediated by a DNp73-activated IGF1R/PI3K/AKT axis leading to tyrosinase ER-arrest and proteasomal degradation. Tyrosinase loss triggers reactivation of the EMT signaling cascade, a mesenchymal-like cell phenotype and increased invasiveness. DNp73-induced depigmentation, Slug increase and changes in cell motility are recapitulated in neural crest-derived melanophores of Xenopus embryos, underscoring a previously unnoticed physiological role of tyrosinase as EMT inhibitor. This data provides a mechanism of hypopigmentation accompanying cancer progression, which can be exploited in precision diagnosis of patients with melanoma-associated hypopigmentation (MAH), currently seen as a favorable prognostic factor. The DNp73/IGF1R/Slug signature in colorless lesions might aid to clinically discriminate between patients with MAH-associated metastatic disease and those, where MAH is indeed a sign of regression.

Ganoderma lucidum polysaccharide inhibits UVB-induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways.

Ultraviolet (UV)-induced pigmentation is very common in clinical practice, but the current treatments are rarely effective, accompanied by some side effects. Ganoderma lucidum polysaccharide (GLP) is a natural antioxidant with no toxic side effects, which can antagonize UVB-induced fibroblast photo aging. The study aims to explore the role of GLP in inhibiting UVB-induced melanogenesis and its possible mechanism. The expression of melanogenesis genes such as microphthalmia-associated transcription factor (MITF), tyrosine (TYR), tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2), ras-related protein Rab-27A (Rab27A), and Myosin shows an upward trend after exposure of B16F10 and PIG1 cells to UVB irradiation, but GLP can downregulate the expression of genes related to UVB-induced melanogenesis. GLP can inhibit UVB-activated protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signaling pathways. Besides, GLP protects mitochondria from UVB damage and inhibits reactive oxygen species (ROS) production. Also, UVB-induced cyclic adenosine monophosphate (cAMP) can be inhibited. It has been found in the experiments of UVB-induced skin pigmentation in zebrafish that GLP is capable of inhibiting UVB-induced skin pigmentation. Meanwhile, it can greatly relieve erythema reaction in guinea pig skin caused by high-dosage UVB irradiation. In conclusion, this study shows that GLP can inhibit UVB-induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways and is a potential natural safe whitening sunscreen additive.

Poria cocos Wolf extracts represses pigmentation in vitro and in vivo.

In skin, melanocytes determine skin color using melanogenesis, which induces protective mechanism to oxidative stress and UV damage. However, when melanin is excessive produced by the various stimulus, the accumulated melanin induces hyperpigmentation disease such as melasma, freckles, Melanism ware induced. Therefore, it is implicated to finding potential agents for whitening to be used in cosmetic products. In our present study, we show that Poria cocos Wolf extracts decreased melanin synthesis in B16F10. And then this inhibition of melanogenesis was provoked by regulation of tyrosinase activity and tyrosinase and MITF expression. Moreover, Poria cocos Wolf extracts contained cream improved skin tone using increase of bright value. Overall, these results provide evidence to potential agent for whitening to be used in cosmetic products.

Melanocytic Myxoid Spindle Cell Tumor With ALK Rearrangement (MMySTAR): Report of 4 Cases of a Nevus Variant With Potential Diagnostic Challenge.

Melanocytic tumors rarely display extensive dermal myxoid deposits except in the myxoid variant of melanoma. We describe in 4 patients the unusual association of morphologic and genetic features. All cases occurred in males and were located on the limbs or proximal girdle area. Age at diagnosis ranged from 8 to 47 years. Size ranged from 6 to 11 mm. Microscopic analysis showed compound, but mainly dermal melanocytic nevi, all presenting a deep dermal expansion with fascicules of amelanotic spindled cells floating in a myxoid background. Cytologic atypia and mitotic activity were low. The superficial portion was either of spitzoid or nevoid cytology with a limited junctional component. In the initial case, the dermal myxoid component was predominant with rare, barely visible, superficial melanocytic nests. This peculiar morphology was responsible for a delayed diagnostic, which required an extensive panel of antibodies ruling out most, potentially myxoid, soft tissue tumors. We later observed the presence of similar, but more limited, dermal morphologic features in 3 other cases. Immunohistochemistry in the deep myxoid areas was melanA, ALK, SOX10, and MiTF. Molecular studies confirmed the ALK rearrangement by an ALK break-apart fluorescence in situ hybridization technique and by RNA sequencing. The latter identified 4 different 5'-fusion partners. Two gene fusions were undescribed: FBXO28(e2)-ALK(e19) and NPAS2(e2)-ALK(e19), and 2 previously described: TPM3(e7)-ALK(e20) and PPFIBP1(e9)-ALK(e19). No relapse or metastatic evolution was seen during follow-up (3 to 24 mo). We denominated this potentially challenging new variant of compound nevus linked to a kinase fusion: Melanocytic Myxoid Spindle Cell Tumor with ALK Rearrangement.

Finasteride inhibits melanogenesis through regulation of the adenylate cyclase in melanocytes and melanoma cells.

Finasteride is a well-known 5α-reductase inhibitor used for treatment of alopecia and prostate cancer. But the effect of finasteride in regulating melanogenesis is still unclear. In the present study the role of finasteride on melanogenesis was investigated. Finasteride decrease melanin level in melanocyte melan-a cells and B16F10 melanoma cells without inducing cytotoxicity. MC1R (melanocortin 1 receptor) protein expression was also inhibited by finasteride thereby decreasing the expression of adenylate cyclase, MITF (Melanogenesis associated transcription factor), tyrosinases, TRP (tyrosinase-related protein) -1 and -2. Thus our study suggest that finasteride inhibits melanogenesis in melanocyte and melanoma cells by inhibiting MC1R.

Nicotinamide Phosphoribosyltransferase (NAMPT) as a Therapeutic Target in BRAF-Mutated Metastatic Melanoma.

Background: One of the effects of oncogenic signaling is metabolic reprogramming of tumor cells to support anabolic growth, opening the way to therapeutic targeting of metabolic pathways. Methods: We studied NAD biosynthesis in BRAF inhibitor (BRAFi)-resistant (BiR) melanoma cell lines. Data in cell lines were confirmed by immunohistochemistry in biopsies from 17 patients with metastatic melanoma (MM) before and after the acquisition of resistance to BRAFi. Therapeutic potential of NAD biosynthesis inhibitors was determined by invitro monitoring cell growth and death and in mouse xenograft models. Mice (n = 6-10 mice/group) were treated with nicotinamide phosphoribosyltranferase inhibitor (NAMPTi), BRAFi, or their combination, and tumor growth and survival were analyzed. All statistical tests were two-sided. Results: BiR cells had higher NAD levels compared with their BRAFi-sensitive counterparts (P < .001 and P = .001 for M14 and A375, respectively) and with normal melanocytes (P < .001), achieved through transcriptional upregulation of the enzyme NAMPT, which became the master regulator of NAD synthesis. Conversely, treatment with BRAFi or MEK inhibitors decreased NAMPT expression and cellular NAD levels. Robust NAMPT upregulation was documented in tissue biopsies from MM patients after development of resistance to BRAFi (P < .001). Treatment of melanoma cells with NAMPTi depleted NAD and ATP, depolarized mitochondrial membrane, and led to reactive oxygen species production, blocking cells in the G2/M phase and inducing apoptosis. Treatment of BiR xenografts with NAMPTi improved mouse survival (median survival of vehicle-treated mice was 52 days vs 100 days for NAMPTi-treated ones in M14/BiR, while in A375/BiR median survival of vehicle-treated mice was 23.5 days vs 43 days for NAMPTi-treated ones, P < .001). Conclusions: BiR melanoma cells overexpress NAMPT, which acts as a connecting element between BRAF oncogenic signaling and metabolism, becoming an actionable target for this subset of MM patients.

Photodynamic therapy inhibits melanogenesis through paracrine effects by keratinocytes and fibroblasts.

Photodynamic therapy (PDT) is a treatment option for skin cancer and premalignant skin diseases and exhibits rejuvenation effects, including reducing fine wrinkles and whitening, on aged skin. In this study, we investigated the mechanism underlying the whitening effects of PDT on melanocytes (MCs) in vitro and in vivo. Exposure of MCs to PDT in vitro reduced their melanin content and tyrosinase activity without, however, affecting cell survival. Interestingly, melanogenesis was also inhibited by exposing MCs to conditioned media of PDT-treated keratinocytes or dermal fibroblasts. This paracrine effect was likely due to a decreased release of melanocyte-stimulating cytokines such as Kit ligand and hepatocyte growth factor from these cells. Furthermore, we observed that PDT reduced mottled hyperpigmentation of photoaged patient skin in vivo, highlighting the clinical importance of skin whitening by PDT.