Melanocortin-1 Receptor Expression Research Articles

Melanocortin-1 Receptor Expression as a Marker of Progression in Melanoma.

Melanocortin-1 receptor (MC1R) plays a critical role in human pigmentation and DNA repair mechanisms. MC1R-targeting agents are being investigated in clinical trials in patients with melanoma, yet large studies investigating the rate and degree of MC1R expression in primary and metastatic human melanoma tissue are lacking. Using tissue microarrays containing three large cohorts of 225 cases of benign nevi, 189 with primary melanoma, and 271 with metastatic melanoma, we applied quantitative immunofluorescence and immunohistochemistry to comprehensively study MC1R protein expression. We show a stepwise elevation of MC1R expression in different stages of melanoma progression (nevi, primary, metastasis). Higher MC1R expression was seen in deeper (>1 mm) primary lesions and ulcerated lesions and was associated with shorter survival in primary and metastatic tumors. On multivariable analysis, Breslow thickness, male sex, and chronic sun exposure were independent predictors of worse overall survival in the primary melanoma cohort. Our data suggest that MC1R might be a valuable drug target in aggressive melanoma. Additional studies are warranted to determine its functional significance in melanoma progression and its utility as a predictive biomarker in patients receiving MC1R-directed therapies.

Activation of melanocortin-1 receptor signaling in melanoma cells impairs T cell infiltration to dampen antitumor immunity

Inhibition of T cell infiltration dampens antitumor immunity and causes resistance to immune checkpoint blockade (ICB) therapy. By in vivo CRISPR screening in B16F10 melanoma in female mice, here we report that loss of melanocortin-1 receptor (MC1R) in melanoma cells activates antitumor T cell response and overcomes resistance to ICB. Depletion of MC1R from another melanocytic melanoma model HCmel1274 also enhances ICB efficacy. By activating the GNAS-PKA axis, MC1R inhibits interferon-gamma induced CXCL9/10/11 transcription, thus impairing T cell infiltration into the tumor microenvironment. In human melanomas, high MC1R expression correlates with reduced CXCL9/10/11 expression, impaired T cell infiltration, and poor patient prognosis. Whereas MC1R activation is restricted to melanoma, GNAS activation by hotspot mutations is observed across diverse cancer types and is associated with reduced CXCL9/10/11 expression. Our study implicates MC1R as a melanoma immunotherapy target and suggests GNAS-PKA signaling as a pan-cancer oncogenic pathway inhibiting antitumor T cell response.

Melanocortin 1 Receptor (MC1R): Pharmacological and Therapeutic Aspects.

Melanocortins play crucial roles in regulating the stress response, inflammation, and skin pigmentation. In this review, we focus on the melanocortin 1 receptor (MC1R), a G protein-coupled receptor primarily known for regulating skin pigmentation and exhibiting anti-inflammatory effects. First, we provide an overview of the structure, signaling pathways, and related diseases of MC1R. Next, we discuss the potential therapeutic use of synthetic peptides and small molecule modulators of MC1R, highlighting the development of various drugs that enhance stability through amino acid sequence modifications and small molecule drugs to overcome limitations associated with peptide characteristics. Notably, MC1R-targeted drugs have applications beyond skin pigmentation-related diseases, which predominantly affect MC1R in melanocytes. These drugs can also be useful in treating inflammatory diseases with MC1R expression present in various cells. Our review underscores the potential of MC1R-targeted drugs to treat a wide range of diseases and encourages further research in this area.

EGCG, GCG, TFDG, or TSA Inhibiting Melanin Synthesis by Downregulating MC1R Expression

Without affecting cell viability, epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), theaflavine-3,3′-digallate (TFDG), or theasinensin A (TSA) have been found to effectively reduce intracellular melanin content and tyrosinase (TYR) activity. However, studies on the anti-melanogenic mechanism of the above samples remain weak, and the activities of these samples in regulating melanogenesis at the molecular level lack comparison. Using B16F10 cells with the α-melanocyte-stimulating hormone (α-MSH) stimulation and without the α-MSH stimulation as models, the effects of EGCG, GCG, TFDG, or TSA on cell phenotypes and expression of key targets related to melanogenesis were studied. The results showed that α-MSH always promoted melanogenesis with or without adding the four samples. Meanwhile, the anti-melanogenic activities of the four samples were not affected by whether the α-MSH was added in the medium or not and the added time of the α-MSH. On this basis, the 100 µg/mL EGCG, GCG, TFDG, or TSA did not affect the TYR catalytic activity but inhibited melanin formation partly through downregulating the melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), and the TYR family. The downregulation abilities of catechins on the TYR family and MITF expression were stronger than those of dimers at both the transcription and translation levels, while the ability of dimers to downregulate the MC1R expression was stronger than that of catechins at both the transcription and translation levels to some extent. The results of molecular docking showed that these four samples could stably bind to MC1R protein. Taken together, this study offered molecular mechanisms for the anti-melanogenic activity of the EGCG, GCG, TFDG, and TSA, as potential effective components against the UV-induced tanning reactions, and a key target (MC1R) was identified.

Silver Carp (Hypophthalmichthys molitrix) Scale Collagen Peptides-1 (SCPs1) Inhibit Melanogenesis through Downregulation of the cAMP-CREB Signaling Pathway.

The mechanism of silver carp scale collagen peptides (SCPs1) on melanogenesis and its mechanism of action were examined in mouse melanoma cells (B16). The cell viability and effects of SCPs1 on intracellular tyrosinase (TYR) activity and melanin, reactive oxygen species (ROS), glutathione (GSH) and cyclic adenosine monophosphate (cAMP) content were examined. The regulatory mechanism of SCPs1 on the cAMP response element-binding protein (CREB) signaling pathway was analyzed. The cell viability of the SCPs1 group was >80% (0.01-1 mg/mL) and the inhibitory rate of SCPs1 on B16 cell melanin increased in a dose-dependent manner. The highest inhibitory rate of SCPs1 on melanin content reaching 80.24%. SCPs1 significantly increased the GSH content and decreased the tyrosinase activity, as well as the content of ROS and cAMP. Western blot analysis showed that SCPs1 significantly inhibited melanocortin-1 receptor (MC1R) expression and CREB phosphorylation in the cAMP-CREB signaling pathway, leading to downregulation of microphthalmia-associated transcription factor (MITF) and the expression of TYR, TYR-related protein-1 (TRP-1) and TRP-2. SCPs1 also inhibited the expression of MC1R, MITF, TYR, TRP-1 and TRP-2 at the transcriptional level. Taken together, SCPs1 inhibited melanin synthesis through the downregulation of the cAMP-CREB signaling pathway. Fish-derived collagen peptides could potentially be applied in skin whitening products.

Abstract CT104: MC1R imaging and histology in the targeted imaging of melanoma for alpha-particle radiotherapy (TIMAR1) trial

Abstract Purpose: Melanocortin 1 receptor (MC1R) has been shown in animal models to be a target for effective radionuclide therapy. This first-in-human clinical trial studied the safety and biodistribution of two novel MC1R-targeted imaging tracers designed to support the development of MC1R-targeted alpha particle therapy through image-based identification of MC1R and radiation dosimetry. Imaging was compared to MC1R expression by immunohistochemistry (IHC) of subjects’ melanoma tumors. Methods: Subjects with stage IV melanoma were randomized to receive either [203Pb]VMT01 SPECT/CT imaging followed by [68Ga]VMT02 PET/CT imaging approximately 1 month later, or vice versa in a cross-over design during their normal melanoma treatment course. Patient selection included a positive [18F]FDG PET/CT within 30 days of imaging tracer injection. 555 - 925 mBq of [203Pb]VMT01 was injected IV with imaging performed at 1, 4, and 24 hours. 74 - 277 mBq [68Ga]VMT02 was injected IV and imaging performed dynamically up to 1 hour, and at 2 and 3 hours. Due to an interruption in drug supply, only 3 subjects received [203Pb]VMT01 imaging. Key FDG-avid melanoma lesions were marked and presented to three experienced Radiologists serving as blinded reviewers. The blinded reviewers compared [18F]FDG PET/CT to experimental imaging and scored scans and key lesions on multiple metrics. Imaging positivity was defined as tumor uptake and retention of tracer in ≥ 1 melanoma tumors above background liver activity. Melanoma biopsy tissue was obtained and MC1R IHC performed as available. Results: 7 subjects were enrolled; 6 were imaged and 5 had available pathology. 3 of 6 imaged subjects had MC1R positive tumors via experimental imaging tracers. [68Ga]VMT02 PET/CT at 3 hours led to the best tumor to background ratio, and [203Pb]VMT01 SPECT/CT showed tumor retention at 24 hours. 4 of 5 subjects had positive MC1R staining by IHC. Of the 3 subjects with negative imaging, 2 had positive staining by IHC. Of the 3 subjects with positive imaging, 2 had available biopsies and all stained positive for MC1R by IHC. Conclusion: A subgroup of advanced melanoma patients were identified to express MC1R using new MC1R-targeted imaging agents. Full concordance between imaging and IHC of positive scans, but not with negative scans, suggests that IHC is a more sensitive biomarker detection method. Additional testing is needed to establish frequency of positive MC1R imaging in stage IV melanoma and its correlation to histological findings and treatment outcomes. Preferential tumor retention provides proof-of-concept that the MC1R-targeting therapy trial for [212Pb]VMT01 alpha-emitting therapy will deliver localized radiation to high MC1R expressing tumors as screened by [203Pb]VMT01 or [68Ga]VMT02. Imaging quality is sufficient for subject selection and dosimetry but may exclude subjects with MC1R receptor expression only detectable by IHC. Citation Format: Brenna M. Marks, Matthew Block, Geoffrey B. Johnson, Carrie B. Hruska, Mukesh K. Pandey, Andrew Paulsen, Michael K. Schultz, Michael A. McDonald, Edwin A. Sagastume, Frances L. Johnson. MC1R imaging and histology in the targeted imaging of melanoma for alpha-particle radiotherapy (TIMAR1) trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT104.

Atraric Acid Ameliorates Hyperpigmentation through the Downregulation of the PKA/CREB/MITF Signaling Pathway.

Atraric acid (AA) is derived from lichens and is widely used in perfumes for its desirable scent. It has been reported as having anti-inflammatory and antioxidant activity. Hyperpigmentation is the underlying cause of a variety of dermatological diseases that have a significant impact on patients' quality of life and are frequently difficult to treat. This study aimed to explore the inhibitory effects of AA on hyperpigmentation in vitro and in vivo and its potential molecular mechanisms. The cytological results revealed that at a dose of 250 μM, AA may reduce melanin content and tyrosinase levels without causing cytotoxicity. Furthermore, the expression of melanocortin-1 receptor (MC1R), phosphorylated protein kinase A (pPKA) and phosphorylated cAMP response element binding protein (pCREB) were downregulated in AA-administrated cells. In vivo, histological analysis showed that AA could inhibit melanin production and tyrosinase activity, and 3% AA had the best activity, with almost no side effects. Furthermore, the results of Western blot analysis and RT-PCR suggested that AA may suppress the mRNA transcription of microphthalmia-associated transcription factor (MITF) protein and tyrosine protease by decreasing the expression of MC1R, consequently decreasing the phosphorylation of PKA and CREB. Finally, the MC1R inhibitor MSG606 verified the hypothesis that AA suppresses melanin formation by downregulating the PKA/CREB/MITF signaling pathway. Taken together, our study offers valuable information for the development of AA as a possible ingredient in skin-lightening cosmeceuticals and hyperpigmentation inhibitors.

Dersimelagon, a novel oral melanocortin 1 receptor agonist, demonstrates disease-modifying effects in preclinical models of systemic sclerosis

BackgroundActivation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc).MethodsThe effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-β (TGF-β)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed.ResultsProphylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-β-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117.ConclusionsMT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress.

Expression of Melanocortin-1 Receptor and Serum Melanin in Canities at Young Male students

BACKGROUND: Molecular genetics suggest that hair pigmentation is related to the melanocortin-1 receptor gene. It is a G protein-coupled receptor, which is activated by a number of peptides to stimulate melanogenesis. Melanocortin-1 receptor gene mutation tends to produce functional variability in premature hair graying (PHG) or canities. AIM: Therefore, the objective of this study was to determine melanocortin-1 receptor expression and asses serum melanin in college students canities at Universitas Sumatera Utara. METHODS: This study was a cross-sectional design. We recruited 80 subjects equally divided into normal (control) and premature hair graying groups. The sample included males, aged 25 years who had gray hair and had no history of pigmentary disorders of the skin. Expression of melanocortin-1 receptor was detected with conventional PCR and serum melanin was measured with Elisa using Elisa kit melanin for human. RESULTS: The results showed that the Mean ± SD graying age was 20.28 ± 1.99 years with an age range of 19–24 years and the average age in the control group was 21.25 ± 2.02 years with an age range of 18–24 years. Melanocortin-1 receptor gene expression was shown in the control and PHG groups. Serum melanin levels were decreased significantly (p = 0.0001) in the gray-haired group was 9.27 ± 1.62 μg/dl and the control group was 10.72 ± 1.78 μg/dl. CONCLUSION: Melanocortin-1 receptor gene plays a role in hair graying at young age and there serum melanin levels were low significantly.

Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson\u2019s disease

BackgroundEpidemiological studies suggest a link between the melanoma-related pigmentation gene melanocortin 1 receptor (MC1R) and risk of Parkinson’s disease (PD). We previously showed that MC1R signaling can facilitate nigrostriatal dopaminergic neuron survival. The present study investigates the neuroprotective potential of MC1R against neurotoxicity induced by alpha-synuclein (αSyn), a key player in PD genetics and pathogenesis.MethodsNigral dopaminergic neuron toxicity induced by local overexpression of aSyn was assessed in mice that have an inactivating mutation of MC1R, overexpress its wild-type transgene, or were treated with MC1R agonists. The role of nuclear factor erythroid 2-related factor 2 (Nrf2) in MC1R-mediated protection against αSyn was characterized in vitro. Furthermore, MC1R expression was determined in human postmortem midbrain from patients with PD and unaffected subjects.ResultsTargeted expression of αSyn in the nigrostriatal pathway induced exacerbated synuclein pathologies in MC1R mutant mice, which were accompanied by neuroinflammation and altered Nrf2 responses, and reversed by the human MC1R transgene. Two MC1R agonists were neuroprotective against αSyn-induced dopaminergic neurotoxicity. In vitro experiments showed that Nrf2 was a necessary mediator of MC1R effects. Lastly, MC1R was present in dopaminergic neurons in the human substantia nigra and appeared to be reduced at the tissue level in PD patients.ConclusionOur study supports an interaction between MC1R and αSyn that can be mediated by neuronal MC1R possibly through Nrf2. It provides evidence for MC1R as a therapeutic target and a rationale for development of MC1R-activating strategies for PD.

BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model.

Neuronal apoptosis induced by oxidative stress plays an important role in the pathogenesis and progression of hypoxic-ischemic encephalopathy (HIE). Previous studies reported that activation of melanocortin-1 receptor (MC1R) exerts antioxidative stress, antiapoptotic, and neuroprotective effects in various neurological diseases. However, whether MC1R activation can attenuate oxidative stress and neuronal apoptosis after hypoxic-ischemic- (HI-) induced brain injury remains unknown. Herein, we have investigated the role of MC1R activation with BMS-470539 in attenuating oxidative stress and neuronal apoptosis induced by HI and the underlying mechanisms. 159 ten-day-old unsexed Sprague-Dawley rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. The novel-selective MC1R agonist BMS-470539 was administered intranasally at 1 h after HI induction. MC1R CRISPR KO plasmid and Nurr1 CRISPR KO plasmid were administered intracerebroventricularly at 48 h before HI induction. Percent brain infarct area, short-term neurobehavioral tests, Western blot, immunofluorescence staining, Fluoro-Jade C staining, and MitoSox Staining were performed. We found that the expression of MC1R and Nurr1 increased, peaking at 48 h post-HI. MC1R and Nurr1 were expressed on neurons at 48 h post-HI. BMS-470539 administration significantly attenuated short-term neurological deficits and infarct area, accompanied by a reduction in cleaved caspase-3-positive neurons at 48 h post-HI. Moreover, BMS-470539 administration significantly upregulated the expression of MC1R, cAMP, p-PKA, Nurr1, HO-1, and Bcl-2. However, it downregulated the expression of 4-HNE and Bax, as well as reduced FJC-positive cells, MitoSox-positive cells, and 8-OHdG-positive cells at 48 h post-HI. MC1R CRISPR and Nurr1 CRISPR abolished the antioxidative stress, antiapoptotic, and neuroprotective effects of BMS-470539. In conclusion, our findings demonstrated that BMS-470539 administration attenuated oxidative stress and neuronal apoptosis and improved neurological deficits in a neonatal HI rat model, partially via the MC1R/cAMP/PKA/Nurr1 signaling pathway. Early administration of BMS-470539 may be a novel therapeutic strategy for infants with HIE.

MC1R Is a Prognostic Marker and Its Expression Is Correlated with MSI in Colorectal Cancer.

Melanocortin 1 receptor (MC1R) is thought to be a marker of poor prognosis and a potential target for the treatment of melanoma. Studies have found that MC1R promotes several tumor behaviors, including cell proliferation and differentiation, pigment formation, and genome damage repair. Some single-nucleotide polymorphisms (SNPs) of MC1R are involved in the occurrence and development of melanoma. A few studies have reported a relationship between MC1R and colorectal cancer (CRC). In this research, our objective was to examine MC1R expression and MC1R SNPs and investigate their correlation with the clinicopathological features of human CRC tissues. We evaluated MC1R mRNA expression by performing bioinformatic analyses on human CRC expression datasets. We used Western blotting and RT-qPCR to compare MC1R expression in CRC tissues with that in normal tissues, and MC1R SNPs in CRC tissues were detected by PCR-direct sequencing (DS). The expression of MC1R was significantly decreased in CRC tissues compared with normal tissue, and its expression was negatively associated with P53 expression, MLH1 expression, and PMS2 expression, and high MC1R expression was significantly associated with microsatellite instability (MSI). MC1R SNPs were also associated with the clinicopathological characteristics of CRC; for example, the rs2228479 locus genotype was correlated with Ki67 status, and the rs885479 locus genotype was correlated with age and T stage. In conclusion, MC1R plays a crucial role in the progression of CRC and may be a marker of poor prognosis in CRC.

Factors affecting gene expression associated with the skin color of black-bone chicken in Thailand

The objective of this study was to investigate the effect of breed, sex, and age on the gene expression level of melanocortin 1 receptor (MC1R), DOPA chrome tautomerase (DCT), tyrosinase-related protein 1 (TYRP1), tyrosinase (TYR), and agouti signaling protein (ASIP) genes in Thai commercial chicken lines. All chicken have received Newscastle vaccination, and no antibiotics or any drugs were used in this study. Four chicken breeds including Black-Chinese, KU-Phuparn, Sri Mok, and Pradu Hang Dam were used in this study. These breeds can be classified by their skin color into 3 group including black (Black Chinese and KU-Phuparn), light black (Sri Mok), and yellowish white (Pradu Hang Dam). One hundred chickens per breed were used in this study. Breast skin tissue was randomly collected from 8 chickens (4 males, 4 females) per breed at 4, 8, 12, and 16 wk of age. The mRNA expression was analyzed using qRT-PCR and the gene expression level was calculated as 2−ΔΔCT. From the results, breed significantly (P < 0.01) affected the expression level for the 5 genes evaluated. Birds with the black skin color had greater TYRP1 and TYR gene expression when compared to chickens with light black and yellowish-white skin color, respectively. Whereas, chickens with yellowish-white skin color had greater ASIP gene expression when compared to chickens having the other skin colors. Sex significantly affected DCT, TYRP1, and TYR gene expression where the gene expression in males was greater when compared to females (P < 0.05). Age affected all gene expression levels (P < 0.01). At 4 wk of age, MC1R, DCT, TYRP1, and TYR gene expression was the highest and decreased as bird age increased (P < 0.05); however, ASIP gene expression was greatest at 8 wk of age. After 8 wk of age all gene expression for the genes evaluated in this study decreased as age increased. In addition, an interaction between breed and sex (P < 0.05) impacted DCT and ASIP gene expression. The results from this study showed that all genes evaluated can be used as candidate markers to further improve the blackness of the chicken's skin because the most desired skin color is black in the Thai black-bone chicken population.

Abstract 1171: Assessing Melanocortin 1 receptor as a target for metastatic melanoma drug delivery

Abstract In as little as six weeks, melanoma can turn life-threatening. Not only is melanoma a fast growing skin cancer, but its annual incidence rate is nearly 100,000 in the United States. Disease identified early can be cured by surgery, but metastatic melanoma is lethal. Despite the striking progress in treating metastatic melanoma using MAPK inhibitors and immune checkpoint inhibitors, improvements in objective response rate (&amp;lt;50%) and 5-yr survival (&amp;lt;30%) are critically needed. Melanocortin 1 receptor (MC1R), a G protein-coupled receptor, has been investigated as a potential target for drug delivery (e.g., peptide receptor radionuclide therapies, peptide-drug conjugates, etc.) to metastatic melanoma cells, as it is overexpressed in melanomas but largely absent in normal tissues. This study aims to further investigate the potential of MC1R as a target for drug delivery, by determining its expression in melanoma cancer cell lines, human melanoma biopsies, as well as normal tissue specimen. Bmax and MC1Rs per cell in five human melanoma cell lines (TXM13, TXM40, Sk-Mel-28, A375, and A2058) were calculated from saturation binding assays using synthetic MC1R ligand [125I]Nle-D-Phe-α-MSH (125I-NDP-α-MSH). An immunohistochemistry (IHC) staining protocol was developed using paraffin-embedded xenografts of TXM13, TXM40, Sk-Mel-28, A375, and A2058 human melanoma cells. IHC staining of MC1R was performed using human melanoma biopsy samples of stage III and IV metastatic melanoma obtained from University of Iowa Tissue Procurement Core Facility. MC1R-IHC was also performed on Caucasian North American normal tissue specimens including skin, muscle, pituitary gland, adrenal gland, cerebellum, and testis obtained from Amsbio. 125I-NDP-α-MSH binding assay results revealed positive staining in all melanoma cell lines, with the density of 2,762 receptors/cell in TXM13, 461 receptors/cell in Sk-Mel-28, 343 receptors/cell in TXM40, 164 receptors/cell in A375, and 93 receptors/cell in A2058. The IHC staining also demonstrated positive immunoreactivity against MC1R in stage III and IV human metastatic melanoma biopsy samples. Heterogenous expression of MC1R was found within the biopsy samples and among tumor biopsy samples, and the staining intensity fell within the range of the melanoma cancer cell line xenografts. Conversely, negative IHC staining of MC1R was found in normal tissue specimen, indicating that the expression of MC1R in normal tissues is largely absent. This study establishes an MC1R-IHC staining protocol optimized for clinical applications, and our data demonstrates high expression of MC1R in human melanoma biopsy samples as well as in melanoma cancer cell lines. The expression of MC1R in normal tissue specimens is largely absent. These results indicate that MC1R-targeted therapies, such as peptide receptor radionuclide therapies, are a promising alternative to current therapies for metastatic melanoma. Citation Format: Brenna Marks, Mengshi Li, Edwin Sagastume, Michael Schultz, Frances Johnson. Assessing Melanocortin 1 receptor as a target for metastatic melanoma drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1171.

Specific mutations in the genes of MC1R and TYR have an important influence on the determination of pheomelanin pigmentation in Korean native chickens.

Objective:The TYR (Tyrosinase) and MC1R (Melanocortin 1 receptor) genes are recognized as important genes involved in plumage pigmentation in Korean native chickens. Specifically, most color patterns in chicken result from differential expression of the TYR gene. In this study, the co-segregation of the pigmentation and sequence of the TYR and MC1R genes was investigated through intercrosses between red (R1q1), red with black and black plumage color types of native Korean chickens.Materials and Methods:Using DNA, RNA, and tissue by plumage color of each Korean native chickens, the role of major genes in pigmentation of pheomelanin was evaluated. Reverse transcription polymerase chain reaction, sequencing, western blot, and immunohistochemical were performed to determine the effect of TYR and MC1R genes on plumage pigmentation in Korean native chickens.Results:The KCO line (Korean chicken Ogol: Black-line) with an EEC _ genotype exhibited black feathers, whereas red and red mixed with black chicken with EeC genotype exhibited white feathers. There were notable differences between the base sequences of MC1R and TYR in three Korean chicken breeds, with the highest variation in TYR. Perhaps this is the key characteristics of Korean chicken. Further, we analyzed the expression patterns of MC1R and TYR genes in each type of Korea native chicken and observed that TYR expression was high in feather follicle (R1q2) of KCO tissue. However, native red (Korean chicken red) and native red with black (Korean chicken red dark) chickens have increased TYR expression in the tissue. However, the expression of MC1R was much different from that of TYR.Conclusion:In this study, our results suggest that the differences in position and TYR expression levels exert more influence on plumage pigmentation in native Korean chicken breeds than changes in MC1R expression levels.

Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma.

Melanocortin 1 receptor (MC1R) is under investigation as a target for drug delivery for metastatic melanoma therapy and imaging. The purpose of this study was to determine the potential of using BRAF inhibitors (BRAFi) and histone deacetylase inhibitors (HDACi) to enhance the delivery of MC1R-targeted radiolabeled peptide ([212Pb]DOTA-MC1L) by pharmacologically upregulating the MC1R expression in metastatic melanoma cells and tumors. MC1R expression was analyzed in de-identified melanoma biopsies by immunohistochemical staining. Upregulation of MC1R expression was determined in BRAFV600E cells (A2058) and BRAF wild-type melanoma cells (MEWO) by quantitative real-time polymerase chain reaction, flow cytometry, and receptor-ligand binding assays. The role of microphthalmia-associated transcription factor (MITF) in the upregulation of MC1R was also examined in A2058 and MEWO cells. The effectiveness of [212Pb]DOTA-MC1L α-particle radiotherapy in combination with BRAFi and/or HDACi was determined in athymic nu/nu mice bearing A2058 and MEWO human melanoma xenografts. High expression of MC1R was observed in situ in clinical melanoma biopsies. BRAFi and HDACi significantly increased the MC1R expression (up to 10-fold in mRNA and 4-fold in protein levels) via MITF-dependent pathways, and this increase led to enhanced ligand binding on the cell surface. Inhibition of MITF expression antagonized the upregulation of MC1R in both BRAFV600E and BRAFWT cells. Combining [212Pb]DOTA-MC1L with BRAFi and/or HDACi improved the tumor response by increasing the delivery of 212Pb α-particle emissions to melanoma tumors via augmented MC1R expression. These data suggest that FDA-approved HDACi and BRAFi could improve the effectiveness of MC1R-targeted therapies by enhancing drug delivery via upregulated MC1R.

Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.

MC1R CpG island regulates MC1R expression and is methylated in a subset of melanoma tumours.

Melanocortin 1 receptor (MC1R) is a G protein-coupled receptor expressed in melanocytes where it plays an important role in skin pigmentation and in the UV response, and has implications in melanoma development. Here we show that methylation of a CpG island (CGI) within the MC1R gene can control expression of MC1R in melanoma. This CGI overlaps with a potential enhancer region, and is unmethylated in normal melanocytes but highly methylated in other skin cells, suggesting a melanocyte specific function. Analysis showed that MC1R was the only gene significantly differentially expressed by methylation of this region. Within several data sets, this region is methylated in a subset of melanoma tumours (55%-74% of tumours) and results in reduced MC1R expression and significantly longer overall survival.

Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse cholesterol transport.

A quantitative analysis of multilineage-differentiating stress-enduring (Muse) cells in human adipose tissue and efficacy of melanocytes induction

We have shown previously that multilineage-differentiating stress-enduring (Muse) cells in neonatal fibroblasts can differentiate into functional melanocytes. In this study, we quantitate Muse cells in adipose-mesenchymal stem cells (adipose-MSCs) of human subcutaneous tissue obtained from 11 subjects of various ages, and measured efficacy of melanocytes induction from Adipose-MSC-derived Muse cells (hASC-Muse cells). There was a statistically significant negative correlation between the age of donors and the numbers of adipose-MSCs recovered per g fat as well as the percentage of SSEA3+ cells in the adipose-MSC populations, but isolated hASC-Muse cells showed pluripotency and growth curves equally regardless the age of donors. Adipose-Muse cells sequentially expressed melanocyte-related genes including KIT, MITF, TYRP1 PMEL, DCT, melanocortin 1 receptor (MC1R), and TYR at a comparable level to melanocytes during 6-week culture. Parallel with MC1R expression, adipose-Muse cells increased melanin content by α-MSH stimulation. By quantitating the cell numbers recovered at each step, we found that 10g of adipose tissue could produce at least 2.5×106 melanocytes after 6 weeks of culture. These studies suggest that induction of melanocytes from adipose-Muse is a novel approach to obtain sufficient numbers of melanocytes for clinical application and in vitro study of melanocyte differentiation.