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Melanomas from gynecologic sites (MOGS) are rare and have poor survival. MicroRNAs (miRs) regulate gene expression and are dysregulated in cancer. We hypothesized that MOGS would display unique miR and mRNA expression profiles. The miR and mRNA expression profile in RNA from formalin fixed, paraffin embedded vaginal melanomas (relative to vaginal mucosa) and vulvar melanomas (relative to cutaneous melanoma) were measured with the Nanostring Human miRNA assay and Tumor Signaling mRNA assay. Differential patterns of expression were identified for 21 miRs in vaginal and 47 miRs in vulvar melanoma (fold change >2, p<0.01). In vaginal melanoma, miR-145-5p (tumor suppressor targeting TLR4, NRAS) was downregulated and miR-106a-5p, miR-17-5p, miR-20b-5p (members of miR-17-92 cluster) were upregulated. In vulvar melanoma, known tumor suppressors miR-200b-3p and miR-200a-3p were downregulated, and miR-20a-5p and miR-19b-3p, from the miR-17-92 cluster, were upregulated. Pathway analysis showed an enrichment of "proteoglycans in cancer". Among differentially expressed mRNAs, topoisomerase IIα (TOP2A) was upregulated in both MOGS. Gene targets of dysregulated miRs were identified using publicly available databases and Pearson correlations. In vaginal melanoma, suppressor of cytokine signaling 3 (SOCS3) was downregulated, was a validated target of miR-19b-3p and miR-20a-5p and trended toward a significant inverse Pearson correlation with miR-19b-3p (p = 0.093). In vulvar melanoma, cyclin dependent kinase inhibitor 1A (CDKN1A) was downregulated, was the validated target of 22 upregulated miRs, and had a significant inverse Pearson correlation with miR-503-5p, miR-130a-3p, and miR-20a-5p (0.005 < p < 0.026). These findings support microRNAs as mediators of gene expression in MOGS., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 DiVincenzo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ulcerated cutaneous melanoma carries a poor prognosis, and the underlying biology driving its aggressive behavior is largely unexplored. MicroRNAs (miRs) are small, noncoding RNAs that inhibit the expression of specific genes and exhibit dysregulated expression patterns in cancer. We hypothesized that a unique miR profile exists in ulcerated relative to nonulcerated melanoma and that miR expression inversely correlates with target genes of biologic importance. Expression of miRs and mRNAs was assessed in ulcerated and nonulcerated cutaneous melanomas using the NanoString Human miRNA and Tumor Signaling 360 mRNA assays and validated in an independent cohort. Pathway enrichment and functional annotations for differentially expressed miRs and mRNAs were determined using publicly available databases. Pearson correlations were employed to predict potential miR‒mRNA binding pairs. Ulcerated melanoma tissue showed at least 1.5-fold change in relative expression of 24 miRs, including miR-206, miR-1-3p, and miR-4286 (>2.25-fold decrease, P < 0.048) and miR-146a-5p, miR-196b-5p, and miR-363-3p (>2.5-fold increase, P < 0.014). Ulcerated melanomas also had 21 differentially expressed mRNAs relative to nonulcerated tumors (P < 0.01), among which two had an inverse correlation in expression with regulatory miRs (SOCS3 and miR-218-5p and IL7R and miR-376c-5p). This miR expression profile adds to the molecular characterization of the poorly understood histopathologic phenotype of ulcerated melanoma., (Copyright © 2022. Published by Elsevier Inc.)

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis., (© 2022 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Introduction: Myeloid-derived suppressor cells (MDSC) are a subset of immature myeloid cells that inhibit anti-tumor immunity and contribute to immune therapy resistance. MDSC populations were measured in melanoma patients receiving immune checkpoint inhibitors (ICI)., Methods: Patients with melanoma (n=128) provided blood samples at baseline (BL), and before cycles 2 and 3 (BC2, BC3). Peripheral blood mononuclear cells (PBMC) were analyzed for MDSC (CD33 + /CD11b + /HLA- DR lo/- ) and MDSC subsets, monocytic (CD14+, M-MDSC), granulocytic (CD15+, PMN-MDSC), and early (CD14-/CD15-, E-MDSC) via flow cytometry. Statistical analysis employed unpaired and paired t-tests across and within patient cohorts., Results: Levels of MDSC as a percentage of PBMC increased during ICI (BL: 9.2 ± 1.0% to BC3: 23.6 ± 1.9%, p<0.0001), and patients who developed progressive disease (PD) had higher baseline MDSC. In patients who had a complete or partial response (CR, PR), total MDSC levels rose dramatically and plateaued (BL: 6.4 ± 1.4%, BC2: 26.2 ± 4.2%, BC3: 27.5 ± 4.4%; p<0.0001), whereas MDSC rose less sharply in PD patients (BL: 11.7 ± 2.1%, BC2: 18.3 ± 3.1%, BC3: 19.0 ± 3.2%; p=0.1952). Subset analysis showed that within the expanding MDSC population, PMN-MDSC and E-MDSC levels decreased, while the proportion of M-MDSC remained constant during ICI. In PD patients, the proportion of PMN-MDSC (as a percentage of total MDSC) decreased (BL: 25.1 ± 4.7%, BC2: 16.1 ± 5.2%, BC3: 8.6 ± 1.8%; p=0.0105), whereas a heretofore under-characterized CD14+/CD15+ double positive MDSC subpopulation increased significantly (BL: 8.7 ± 1.4% to BC3: 26.9 ± 4.9%; p=0.0425)., Conclusions: MDSC levels initially increased significantly in responders. PMN-MDSC decreased and CD14+CD15+ MDSC increased significantly in PD patients. Changes in MDSC levels may have prognostic value in ICI., 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 © 2021 Sun, Benner, Savardekar, Lapurga, Good, Abood, Nagle, Duggan, Stiff, DiVincenzo, Suarez-Kelly, Campbell, Yu, Wesolowski, Howard, Shah, Kendra and Carson.)

Tumor ulceration is considered one of the most prognostically significant findings in primary cutaneous melanoma, associated with decreased disease-free and overall survival. However, the unique features associated with ulcerated melanoma that contribute to a poor prognosis in affected patients remain poorly defined. microRNAs are small, non-coding RNAs that function to inhibit expression of specific gene targets, therefore altering the functions of cells in which they are expressed. miR-1469 is a novel miR with significantly decreased expression in ulcerated melanoma tissue relative to non-ulcerated tumors. We hypothesized that loss of miR-1469 expression in melanoma contributes to altered tumor cell functions mediating disease progression. Transfection of a miR-1469 mimic resulted in a significant reduction in the migratory and invasive capacity of the CHL1 and MEL39 melanoma cell lines (>58.1% reduction, p < 0.0332), as well as the invasive capacity of the A375 melanoma cell line (>50% reduction, p < 0.0021). Expression of myeloid cell leukemia-1 (MCL1), a miR-1469 target gene, was reduced in the A375 and MEL39 cell lines by immunoblot. No significant differences in viability, resistance to apoptotic stimuli, or proliferation were observed following transfection. These findings together demonstrate how migration and invasion are specific functions through which miR-1469 expression in melanoma cells can contribute to the differences in disease progression associated with tumor ulceration., Competing Interests: The authors have declared no competing interests exist.

The ubiquitin-proteasome signaling pathway is critical for cell cycle regulation and neoplastic growth. Proteasome inhibition can activate apoptotic pathways. Bortezomib, a selective proteasome inhibitor, has anti-melanoma activity. MLN2238 (ixazomib), an oral proteasome inhibitor, has improved pharmacotherapeutic parameters compared to bortezomib. Interferon-alpha (IFN-α), an immune boosting agent, is FDA-approved for treatment of melanoma. In this study in vitro and in vivo evaluation of the antitumor potential of ixazomib and combination treatments with ixazomib and IFN-α were performed. Apoptosis induced by ixazomib was first observed at 12 hours and was maximal at 48 hours with similar levels of cell death compared to bortezomib. IFN-α alone had little effect on cell viability in vitro. However, the combination of ixazomib with IFN-α significantly enhanced ixazomib's ability to induce apoptotic cell death in BRAF V600E mutant and BRAF wild-type human melanoma tumor cells. The combination of ixazomib and IFN-α also enhanced inhibition of cell proliferation in BRAF V600E mutant melanoma tumor cells; however, this was not seen in BRAF wild-type cells. Ixazomib-induced apoptosis was associated with processing of the pro-apoptotic proteins procaspase-3, -7, -8, and -9, and cleavage of poly-ADP-ribose polymerase (PARP). In an in vivo xenograft model of human melanoma, combination treatment with IFN-α-2b and ixazomib demonstrated a significant reduction in tumor volume when compared to vehicle (p = 0.005) and single therapy ixazomib (p = 0.017) and IFN-α-2b (p = 0.036). These pre-clinical results support further evaluation of combination treatment with ixazomib and IFN-α for the treatment of advanced BRAF V600E mutant melanoma.

Purpose: IFN-alpha is administered to melanoma patients and its endogenous production is essential for immune-mediated tumor recognition. We hypothesized that a reduced capacity for signal transducer and activator of transcription (STAT) 1 activation allows melanoma cells to evade the direct actions of IFN-alpha., Experimental Design: Tyr(701)-phosphorylated STAT1 (P-STAT1) was measured by flow cytometry in IFN-alpha-stimulated human melanoma cell lines, melanoma cells derived from patient tumors, and peripheral blood mononuclear cells (PBMC). Expression of other Janus-activated kinase (Jak)-STAT intermediates (STAT1, STAT2, Jak1, tyrosine kinase 2, IFN-alpha receptor, STAT3, and STAT5) was evaluated by flow cytometry, immunoblot, or immunohistochemistry., Results: Significant variability in P-STAT1 was observed in human melanoma cell lines following IFN-alpha treatment (P < 0.05) and IFN-alpha-induced P-STAT1 correlated with the antiproliferative effects of IFN-alpha (P = 0.042). Reduced formation of P-STAT1 was not explained by loss of Jak-STAT proteins or enhanced STAT5 signaling as reported previously. Basal levels of P-STAT3 were inversely correlated with IFN-alpha-induced P-STAT1 in cell lines (P = 0.013). IFN-alpha-induced formation of P-STAT1 was also variable in melanoma cells derived from patient tumors; however, no relationship between P-STAT3 and IFN-alpha-induced P-STAT1 was evident. Because IFN-alpha acts on both tumor and immune cells, we examined the ability of IFN-alpha to induce P-STAT1 in patient-derived melanoma cells and PBMCs. IFN-alpha induced significantly lower levels of P-STAT1 in melanoma cells compared with matched PBMCs (P = 0.046). Melanoma cells and human melanocytes required 10-fold higher IFN-alpha doses to exert P-STAT1 levels comparable with PBMCs., Conclusions: Melanoma cells are variable in their IFN-alpha responsiveness, and cells of the melanocytic lineage exhibit a lower capacity for IFN-alpha-induced Jak-STAT signaling compared with immune cells.