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Background: As a novel immune checkpoint, CD73 has been reported to play prominent roles in several malignancies. However, the significance of CD73 in melanoma remains ambiguous. This study sought to reveal the impact of CD73 on the tumor microenvironment (TME) and patients' prognosis, and to investigate whether CD73 could be a therapeutic target in Chinese melanomas, which were dominated by acral and mucosal subtypes., Methods: Two independent Chinese cohorts of 194 patients with melanoma were enrolled. CD73 and PD-L1 expression as well as CD8 + and CD56 + cell infiltrations were evaluated by immunohistochemistry in 194 resected melanoma samples. Clinical outcomes of patients were assessed utilizing the Kaplan-Meier plotter and Cox proportional hazard analysis. RNA-seq data was obtained from TCGA database. Gene set functional annotations were performed based on GO, KEGG and GSEA analysis. CIBERSORT, ssGSEA and TIMER were used to explore the association between CD73 and immune infiltration. These findings were validated by establishing tumor xenograft model, and functions of tumor-infiltrating immune cells were examined by flow cytometry and immunofluorescence., Results: High CD73 expression showed poorer clinical outcomes and was identified as an independent prognostic indicator for survival in two cohorts. Expression of CD73 was more prevalent than PD-L1 in Chinese melanoma cohorts (54.6% vs 23.2%). Co-expression of both immune checkpoints was infrequent (12.9%) in melanoma, and 54.4% of PD-L1 negative cases showed elevated expression of CD73. CD73 high tumors showed a microenvironment with fewer CD8 + T cells and CD56 + NK cells infiltration, which displayed a dysfunctional phenotype. With the treatment of CD73 inhibitor APCP, the amount of CD8 + T cells and CD56 + NK cells infiltrated in tumors was elevated and the immunosuppressive effect of CD73 was eliminated., Conclusions: High CD73 expression was associated with an inhibitory TME and adverse clinical outcomes of melanoma. In comparison to PD-L1, CD73 was more prevalent and possessed more definite prognostic significance. Therefore, it may serve as a prognostic indicator and immunotherapeutic target next to PD-L1 in melanoma for Chinese population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gao, Wang, Song, Ren, Yang, Li, Shen, Li, Ding, Yang, Zhou, Wei and Gu.)

Macrophages are the key regulator of T-cell responses depending on their activation state. C-C motif chemokine receptor-like 2 (CCRL2), a nonsignaling atypical receptor originally cloned from LPS-activated macrophages, has recently been shown to regulate immune responses under several inflammatory conditions. However, whether CCRL2 influences macrophage function and regulates tumor immunity remains unknown. Here, we found that tumoral CCRL2 expression is a predictive indicator of robust antitumor T-cell responses in human cancers. CCRL2 is selectively expressed in tumor-associated macrophages (TAM) with immunostimulatory phenotype in humans and mice. Conditioned media from tumor cells could induce CCRL2 expression in macrophages primarily via TLR4, which is negated by immunosuppressive factors. Ccrl2 -/- mice exhibit accelerated melanoma growth and impaired antitumor immunity characterized by significant reductions in immunostimulatory macrophages and T-cell responses in tumor. Depletion of CD8 + T cells or macrophages eliminates the difference in tumor growth between WT and Ccrl2 -/- mice. Moreover, CCRL2 deficiency impairs immunogenic activation of macrophages, resulting in attenuated antitumor T-cell responses and aggravated tumor growth in a coinjection tumor model. Mechanically, CCRL2 interacts with TLR4 on the cell surface to retain membrane TLR4 expression and further enhance its downstream Myd88-NF-κB inflammatory signaling in macrophages. Similarly, Tlr4 -/- mice exhibit reduced CCRL2 expression in TAM and accelerated melanoma growth. Collectively, our study reveals a functional role of CCRL2 in activating immunostimulatory macrophages, thereby potentiating antitumor T-cell response and tumor rejection, and suggests CCLR2 as a potential biomarker candidate and therapeutic target for cancer immunotherapy., Competing Interests: The authors declare no competing interest.

Melanoma is an aggressive form of skin cancer characterized by rapid invasion and metastasis. CD147 is known to be functioning in cell invasion. In this study, we showed that CD147 was translocated from the cell membrane to the mitochondria in advanced melanoma. Melanoma patients with CD147 localized to the mitochondria confer a worse prognosis. The mitochondrial CD147 levels are correlated with the invasion potential of various melanoma cell lines as well as mitochondrial energy metabolism. Depletion of CD147 decreased the activity of mitochondrial complex V. STRING analysis for protein-protein interaction networks (PPIN) in CD147-depleted melanoma cells showed that mitochondrial proteins HSP60 and ATP5B, a subunit of mitochondrial complex V, were node proteins. HSP60 upregulation was correlated with a worse prognosis of melanoma patients. Co-immunoprecipitation (Co-IP) assay indicates that CD147 interacts with HSP60. These data suggested that mitochondrial CD147 may prompt HSP60 to activate ATP5B, thereby promoting the mitochondrial aerobic oxidation and the invasive abilities of melanoma cells. Correlation analysis of the data acquired from patients was helpful to draw a 5-year survival curve for patients who screened positive and negative for mitochondrial CD147. This study unravels the function of CD147 in tumor invasion and highlights it as a potential tumor therapeutic target., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Apigenin, an aromatic compound, exhibits antioxidant, anti‑inflammatory and anti‑viral effects. The present study aimed to investigate the effects of apigenin on cell proliferation and apoptosis of human melanoma cells A375P and A375SM. Therefore, melanoma cells were treated with apigenin to determine its anti‑proliferative and survival effects, using wound healing and MTT assays. The results revealed that melanoma cell viability was decreased in a dose‑dependent manner. Furthermore, chromatin condensation, indicating apoptosis, was significantly increased in a dose‑dependent manner, as demonstrated by DAPI staining. In addition, increased apoptosis rate following treatment with apigenin was confirmed by Annexin V‑propidium iodide staining. The changes in the expression levels of apoptosis‑related proteins in A375P and A375SM melanoma cells were subsequently detected using western blot analysis. The results demonstrated that the protein expression levels of Bcl‑2 were decreased, whereas those of Bax, cleaved poly ADP‑ribose polymerase, cleaved caspase‑9 and p53 were upregulated in a dose‑dependent manner in apigenin‑treated cells compared with those noted in untreated cells. In addition, in apigenin‑treated A375P cells, phosphorylated (p)‑p38 was upregulated and p‑extracellular signal‑regulated kinase (ERK), p‑c‑Jun N‑terminal kinase (JNK) and p‑protein kinase B (Akt) were downregulated. However, in A375SM cells, apigenin treatment increased p‑ERK and p‑JNK and decreased p‑p38 and p‑Akt protein expression levels. Subsequently, the inhibitory effect of apigenin on tumor growth was investigated in vivo. Tumor volume was significantly reduced in the 25 and 50 mg/kg apigenin‑treated groups compared with the control group. Additionally, a TUNEL assay was performed to detect apoptotic cells. Immunohistochemical staining also revealed elevated p‑ERK expression in the apigenin‑treated group compared with the control group. Overall, the findings of the present study indicated that apigenin attenuated the growth of A375SM melanoma cells by inducing apoptosis via regulating the Akt and mitogen‑activated protein kinase signaling pathways.

Melanoma is the most dangerous type of skin cancer because of its high invasion and metastasis ability. Bromodomain-containing protein 4 (BRD4), an acetylation-recognizing reader, mediates the proliferation, metastasis, and invasion of melanoma, and is thus a potential therapeutic target. Mounting evidence suggests that inhibition of single bromodomain of BRD4 would improve specificity and reduce cytotoxicity to non-tumor tissues or cells. In this study, a hierarchical virtual screening campaign was performed against BRD4 BD2 from a chemical database including over 90,000 natural/natural-like compounds. Using various biochemical assays, the 7-methoxycoumarin derivative N13 was identified as a potent inhibitor of BRD4 BD2. Compared with the well-known BRD4 inhibitor JQ1, N13 exhibited higher potency against BRD4 BD2 and much higher specificity for BRD4 BD2 over BRD4 BD1. Additionally, N13 inhibited the proliferation of two kinds of BRD4-overexpressing melanoma cell lines. Mechanistically, N13 impaired the protein-protein interaction (PPI) between BRD4 BD2 and its acetylated ligand proteins (Twist1 K73/K76Ac and FOXO3a K242/245Ac), leading to reducing levels of Wnt5A and CDK6 expression, inducing cell senescence of melanoma cancer cells, and ultimately weakening the adhesion, metastasis, and invasion ability of melanoma cancer cells. To our knowledge, N13 is the first 7-methoxybicoumarin-based BRD4 BD2 inhibitor described to date and may function as a new scaffold for developing more specific and potent therapeutic agents against BRD4 BD2. In addition, our research highlights the druggability of BRD4 BD2 as a target for BRD4-overexpressing melanoma and provides a potential mechanism for the anti-melanoma activity of BRD4 BD2 inhibitor., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Several literature has shown that salinomycin (Sal) is able to kill various types of cancer cells through different signaling pathways. However, its effect on melanoma has seldom been reported. We examined the anti-cancer efficacy of Sal in melanoma cell lines, and found six of eight cell lines were sensitive to Sal. Given the fact that the roles of Sal are diverse in different cancer types, we were eager to figure out the mechanism involved in the current study. We noticed the most sensitive line, SK-Mel-19, showed a typical morphological change after Sal treatment. The autophagy inhibitor, 3-MA, could effectively suppress Sal-induced cell death. It could also facilitate the increase of autophagic markers and reduce the turnover of autophagosomes, which resulted in an aberrant autophagic flux. On the other hand, Sal could stimulate endoplasmic reticulum stress and cause an accumulation of dysfunctional mitochondria. We also discovered a potential correlation between LC3B mRNA level and its sensitivity to Sal in 43 clinical melanoma samples. Overall, our results indicated that Sal could have multiple effect on melanoma cells and induce autophagic cell death in certain kinds of cells, which provided a new insight into the chemotherapy for melanoma.

Human melanoma is a highly malignant tumor originating from cutaneous melanocytes. The noncoding RNA microRNA (miR)-21-5p has been reported to be expressed at high levels in malignant melanocytic skin tissues, but its potential functional role in melanoma remains poorly understood. Here, we explored the cellular effects of miR-21-5p on melanoma in vitro and the underlying mechanisms. Quantitative real-time PCR was used to show that miR-21-5p is significantly up-regulated in clinical samples from patients with melanoma as compared with adjacent noncancerous tissues. Overexpression of miR-21-5p significantly enhanced, whereas knockdown attenuated, cell proliferation and G1/S transition in melanoma cell lines (A375 and M14). Luciferase reporter assays were used to show that the cyclin-dependent kinase inhibitor 2C (CDKN2C) is a downstream target of miR-21-5p. Furthermore, miR-21-5p mimics resulted in a decrease in CDKN2C expression, and CDKN2C expression was observed to be inversely correlated with miR-21-5p expression in melanoma tissues. Rescue experiments were performed to show that overexpression of CDKN2C partially reversed the effects of miR-21-5p up-regulation on A375 cells. Consistently, knockdown of CDKN2C abolished the effects of miR-21-5p down-regulation on A375 cells. Overall, our studies demonstrate that miR-21-5p can promote the growth of melanoma cells by targeting CDKN2C, which may induce G0/G1 phase arrest of melanoma cells., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The present study aimed to investigate the effects of lncRNA FENDRR on the migration and invasion of malignant melanoma (MM) cells. The expression levels of FENDRR in MM tissues and MM cell lines were detected using qRT-PCR, followed by construction of FENDRR-knocked down and overexpressed stable cells. Then the effects of FENDRR on cell proliferation, migration and invasion were detected using MTT assay and Transwell assay. The protein expression levels of matrix metallopeptidase 2 (MMP2), MMP9, and related factors in JNK/c-Jun pathway were detected using Western blot. FENDRR was down-regulated in MM tissues and cell lines. Besides, its expression levels in different MM cells were diverse. Knockdown of FENDRR facilitated MM cells proliferation, migration and invasion in A375 cells, while overexpressing FENDRR had reverse results. In addition, MMPs and JNK/c-Jun pathway involved in the FENDRR-mediated regulation of MM cell proliferation, migration and invasion. Our results demonstrated that FENDRR mediated the metastasis phenotype of MM cells by inhibiting the expressions of MMP2 and MMP9 and antagonizing the JNK/c-Jun pathway., (© 2020 The Author(s).)

Toripalimab, a recombinant, humanized programmed death receptor-1 (PD-1) monoclonal antibody that binds to PD-1 and prevents binding of PD-1 with programmed death ligands 1 (PD-L1) and 2 (PD-L2), is being developed by Shanghai Junshi Bioscience Co., Ltd in China for the treatment of various cancers. In December 2018, based on positive efficacy results of a phase 2 trial and safety data from several clinical studies, toripalimab received conditional approval in China for the treatment of unresectable or metastatic melanoma that has failed previous systemic therapy. This article summarizes the milestones in the development of toripalimab leading to this first global approval for the treatment of unresectable or metastatic melanoma.

Melanoma is a malignant tumor that begins in the melanocyte and has the highest mortality rate among all cutaneous tumors. Chinese propolis (CP) has been shown to have a potent antitumor effect against various cancers. In this study, we uncovered the combined effects of antiproliferation and anti-inflammation of CP on suppressing the progression of human melanoma cell line A375. We evaluated the alterations of protein expression after CP treatment by Western blot. After CP treatment, A375 cells underwent intrinsic apoptosis and cell cycle arrest. Furthermore, we found that CP suppressed inflammation in A375 cells. NLRP1 (NLR family pyrin domain containing 1), confirmed as a proinflammatory protein in melanoma progression, was downregulated significantly by CP, as were the NLRP1-related caspase activation and recruitment domains (CARD) proteins, including caspase-1 and caspase-4. Additionally, decreasing mRNA levels of IL-1α , IL-1β , and IL-18 further proved the negative regulation of CP on the melanoma inflammatory environment. We also discovered that CP induced autophagy in A375 cells. Interestingly, inhibiting autophagy in CP-treated cells diminished its antitumor effect, suggesting that the autophagy was attributed to CP-induced apoptosis. Collectively, CP is a promising candidate for drug development for melanoma therapy.

Conjunctival melanoma (CM) is associated with metastases formation, can be fatal, and occurs in all different races. While cell lines are essential for experimental research, all available CM cell lines are derived from Caucasian patients. Furthermore, they are not derived from metastases. We aimed to establish a new CM cell line from a parotid metastasis in a Han Chinese patient and to depict its characteristics. The novel cell line, CM-AS16, was obtained from a surgical parotid sample and determined as a unique one with short tandem repeat (STR) analysis. It has been successively sub-cultured in vitro for more than 100 passages and exhibits rapid proliferation and migration. Chromosome analysis shows abundant chromosome aberrations, while whole exome sequencing (WES) reveals a typical NRAS mutation (Q61R). In vivo tumor growth was successfully established in a NOD/SCID mice model, and the immunophenotypes, such as HMB45, Melan A, S100, SOX10 and Ki67, manifested similar between the original tumor and the xenograft by immunohistochemistry. A MEK inhibitor binimetinib prominently suppressed in vitro cell growth by inhibiting ERK1/2 phosphorylation. In addition, monoclonal cells were used to demonstrate the drug sensitivity of different cells. In conclusion, the first cell line, CM-AS16, that is derived from a CM in a Han Chinese patient has highly malignant characteristics and a typical NRAS mutation. It may be used as a tool for further exploration of the molecular mechanisms of CM., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Malignant melanoma (MM) is a highly malignant melanocytic tumor, it occurs mostly in the skin, the mucous membrane close to the skin, but also in the tunicae rhagoides and the pia mater. The Uyghur is the largest ethnic group living in the Xinjiang Uyghur Autonomous Region of China, accounting for 46% of the total population of 20 million. Large-scale studies on MMs in Asian countries are limited. This study aimed to investigate BRAF mRNA expression and mutations in Chinese Uyghur patients with MMs and to identify the clinical features associated with these parameters.Formalin-fixed, paraffin wax-embedded tumor sections from 60 MMs were analyzed for BRAF expression using reverse transcription polymerase chain reaction (RT-PCR). Exons 11 and 15 of BRAF were analyzed for the presence of mutations using PCR and DNA sequencing. Sixty MMs were followed by mobile phone for survival analysis.BRAF mRNA expression was higher in MMs than in pigmented moles and normal skin tissues. Fourteen of 60 MMs had BRAF mutations. The frequency of BRAF mutations was significantly higher in patients younger than 60 years (10/28, 4/32, P = .02). A significant difference was observed in the frequency of BRAF mutations among specimens of mucosal, acral, chronic sun-induced damage (CSD), and non-CSD MMs (2/10, 3/19, 8/25, 1/6, P = .002). No significant association was found among BRAF mutations, sex, ulceration, or lymph node metastasis. MMs lymph node metastasis (hazard ratio 2.54 [95% confidence interval 1.062 - 6.066], P = .01) affected survival.This study indicated that BRAF mutations and expression might serve as independent adverse prognostic factors in melanoma., (Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Background: The KIT gene plays an important role in the pathogenesis of malignant melanoma (MM). In recent years, activating mutations in KIT have been recognized as oncogenic. A number of therapies have been established, which provide significant clinical benefits for patients with MM with KIT mutations. Thus, detection of KIT mutations can have profound therapeutic implications., Aim: To investigate KIT gene expression in MMs in Chinese Uyghur and Han patients with mutations in KIT, and to identify the clinical features associated with KIT mutations and c-KIT expression., Methods: In total, 105 MMs (56 from Uyghur and 49 from Han patients) were selected from patients in the Uyghur Autonomous region. Formalin-fixed, paraffin wax-embedded tumour sections were analysed for c-KIT expression using immunohistochemistry. Exons 11 and 13 of KIT were analysed for the presence of mutations using PCR amplification and DNA sequencing., Results: Of the 105 MMs, 13 (10 Han and 3 Uyghur) were found to have mutations in KIT. Thus, the frequency of KIT mutations in Han patients was significantly higher than that in Uyghur patients (P = 0.02). We detected c-KIT expression in 71.4% and 42.9% of the tumour tissue samples collected from the Uyghur and Han patients, respectively., Conclusion: In the Xinjiang Uyghur Autonomous Region in China, chronic sun-induced damage MM is the most prevalent MM among Chinese Uyghur patients, whereas acral and mucosal MMs are the most prevalent in Uyghur patients. Mutations in the KIT gene do not correlate with c-KIT expression., (© 2015 British Association of Dermatologists.)