Melanoma Tissues 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.

Mitotic activity: A systematic literature review of the assessment methodology and prognostic value in canine tumors.

One of the most relevant prognostic indices for tumors is cellular proliferation, which is most commonly measured by the mitotic activity in routine tumor sections. The goal of this systematic review was to analyze the methods and prognostic relevance of histologically measuring mitotic activity that have been reported for canine tumors in the literature. A total of 137 articles that correlated the mitotic activity in canine tumors with patient outcome were identified through a systematic (PubMed and Scopus) and nonsystematic (Google Scholar) literature search and eligibility screening process. Mitotic activity methods encompassed the mitotic count (MC, number of mitotic figures per tumor area) in 126 studies, presumably the MC (method not specified) in 6 studies, and the mitotic index (MI, number of mitotic figures per number of tumor cells) in 5 studies. A particularly high risk of bias was identified based on the available details of the MC methods and statistical analyses, which often did not quantify the prognostic discriminative ability of the MC and only reported P values. A significant association of the MC with survival was found in 72 of 109 (66%) studies. However, survival was evaluated by at least 3 studies in only 7 tumor types/groups, of which a prognostic relevance is apparent for mast cell tumors of the skin, cutaneous melanoma, and soft tissue tumor of the skin and subcutis. None of the studies using the MI found a prognostic relevance. This review highlights the need for more studies with standardized methods and appropriate analysis of the discriminative ability to prove the prognostic value of the MC and MI in various tumor types. Future studies are needed to evaluate the influence of the performance of individual pathologists on the appropriateness of prognostic thresholds and investigate methods to improve interobserver reproducibility.

Development of Transmission Ambient Pressure Laser Desorption Ionization/Postphotoionization Mass Spectrometry Imaging.

Laser-based high-resolution mass spectrometry imaging at ambient conditions has promising applications in life science. However, the ion yield during laser desorption/ablation is poor. Here, transmission atmospheric pressure laser desorption ionization combined with a compact postphotoionization (t-AP-LDI/PI) assembly with a krypton discharge lamp was developed for the untargeted imaging of various biomolecules. The spatial distributions of numerous lipid classes, fatty acids, neurotransmitters, and amino acids in the subregions of mouse cerebellum tissue were obtained. Compared with single laser ablation, the sensitivities for most analytes were increased by 1 to 3 orders of magnitude by dopant-assisted postphotoionization. After careful optimization, a spatial resolution of 4 μm could be achieved for the metabolites in mouse hippocampus tissue. Finally, the melanoma tissue slices were analyzed using t-AP-LDI/PI MSI, which revealed the metabolic heterogeneity of the melanoma microenvironment and exhibited the phenomenon of abnormal proliferation and invasion trends in tumor cells.

Abstract 876: spatialGE: Empowering researchers to study the tumor microenvironment leveraging spatial transcriptomics

Abstract The use of single-cell RNA-seq (scRNA-seq) to understand the complexity of the tumor microenvironment (TME) has been pivotal in advancing cancer research and treatment strategies. Nevertheless, locating the individual TME components in their spatial contexts is essential to understanding the complexity of the TME, tumorigenesis, metastasis, and drug response. The recent popularity of spatially resolved transcriptomics (SRT) allows researchers to describe the TME and their spatial neighborhoods; however, SRT data analysis can be challenging for non-data scientists. Thus, we have developed spatialGE, a point-and-click web application designed to empower researchers in the analysis of SRT data. Our application offers a suite of functionalities, including the visualization of spatial gene expression activity maps, quantification of the spatial heterogeneity, testing of pathway-level spatial aggregation, tissue domain/niche detection, detection of spatial gradients in expression patterns, and differential expression analysis. Furthermore, spatialGE allows direct comparative analysis of gene expression with the morphology in tissue images, as well as methods for cell/domain phenotyping. Data input has been streamlined and made flexible to receive outputs from multiple SRT platforms. Analyses and results are stored within user-defined projects to facilitate accessibility at any time. We demonstrate the usefulness of spatialGE through its application to leptomeningeal metastatic melanoma (LMM) tissues assayed with the Visium platform. The gene expression tissue domain detection and gradient analysis in spatialGE indicated that SERPINA3 expression is higher in tumor regions closer to stroma. This finding led to cell culture experiments indicating that knocking down SERPINA3 results in the sensitization of tumors to MAPK-targeting therapy. These results highlight the utility of spatialGE for exploring SRT data and the empowerment of cancer researchers to explore the spatial architecture of the TME. Citation Format: Oscar Ospina, Roberto Manjarres-Betancur, Guillermo Gonzalez-Calderon, Alex C. Soupir, Inna Smalley, Kenneth Tsai, Joseph Markowitz, Anders Berglund, Xiaoqing Yu, Brooke L. Fridley. spatialGE: Empowering researchers to study the tumor microenvironment leveraging spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 876.

Abstract 3008: Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma

Abstract Background: Ras oncogene activation is a common and often essential event in cancer development, including melanoma. Ras mutation is present in 20-30% of melanomas and remains an undrugged target. NRAS mutation is the most frequent Ras isoform activated in melanoma, with more than 80% cases of Q61 substitutions, and is closely associated with tumor aggressiveness. Rab25 is a ubiquitously expressed small GTPase protein with a context-dependent role in cancer pathophysiology. Its role remains undiscovered in the case of melanoma. Rab25 deep deletion can be seen in portions of melanoma in databases such as the Cancer Genome Atlas and Cbioportal. Interestingly, Rab25 has a consensus sequence homologous with the binding site of Q61 mutant Ras. We propose that the loss of Rab25 in melanocytes accompanied by Ras oncogenic activation are significant events in melanoma development, and restoration of Rab25 can potentially inhibit constitutively activated N-RAS; Rab25 serves as a tumor suppressor in melanoma genesis. Objectives: To discover the role of Rab25 in melanoma, we studied Rab25 expression via mRNA and protein levels in different melanoma cell lines and screened biopsy-proven melanoma samples through immunohistochemistry in 61 different melanoma sections. Further, to see the tumor suppressive mechanism of Rab25 in vitro, we transduced selected cell lines Rab25. Results: There is an 87% and 62.7% reduction in Rab25 mRNA level in NRASQ61R(SKMEL-2) and NRASQ61L type (WM1366) melanoma cell lines, respectively, also reflected in Rab25 protein level, and statistically significant at p-value <0.005, using student’s T-test. Further, Rab25, prominent in adjacent normal tissues, was significantly lost in biopsy-proven melanoma tissues, with an H-score ranging from 13.28 to 91.77% and 0.00 to-11.24 % for normal skin tissue and melanoma section, respectively, and a negative correlation of -0.350, indicating a promising association of Rab25 loss with the progression of melanoma. Conclusion: There is a loss of Rab25 in NRAS mutant melanoma with cancer progression. We saw a significant increase in Rab25 expression among the transduced sublines of NRAS mutant melanoma cell lines at the mRNA level, which will be further assessed through western blotting, cell proliferation assay, trans well migration assay, soft-agar colony formation assay to confirm NRAS mutant melanoma cell behavior. We will attempt to deliver a liposomally encapsulated six-peptide sequence to therapeutically inhibit the mutant NRAS. Citation Format: Samikshya Kandel, Krishna Rao. Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3008.

Abstract 6173: The development and evaluation of a convolutional neural network for melanoma detection in whole slide images

Abstract The current melanoma staging system is predictive of 74% of the variance in survival, with prognostic biomarkers subject to high levels of inter-observer variation. The application of convolutional neural networks (CNNs) to whole slide images (WSIs), may reveal new insights into tumor morphology and therefore patient prognosis. Melanoma morphology appears to be of greater significance than in other solid tumors, with Breslow thickness remaining the strongest prognostic indicator. Other biomarkers based on tumor morphology have been generated and although some have been found to be prognostically superior to Breslow thickness, none have been integrated into clinical workflows. This may in part be explained by their demands on pathologist time. Therefore, this work outlines the development and evaluation of a CNN for invasive cutaneous melanoma detection in WSIs, which may be used for prognostic biomarker generation. 1,157 WSIs containing cutaneous melanoma from five datasets (three from the University of Leeds, one from the Melanoma Institute Australia, as well as The Cancer Genome Atlas) have been used in the initial development and evaluation of the CNN. A custom-designed 2-class tumor segmentation network with a fully convolutional architecture was trained using annotations. The CNN was evaluated using various methodologies, including comparison at per-pixel and per-tumor levels as compared to manual annotation, as well as variation across 3 scanning platforms (Leica Aperio AT2 (Milton Keynes, UK), Roche Ventana DP600 (Arizona, US) and Hamamatsu NanoZoomer S360 (Hamamatsu City, Japan). The CNN detected and located invasive melanoma tissue of no specific type with an average per-pixel sensitivity and specificity of 97.6% and 99.9% respectively across the 5 test sets. There were no statistical differences between tumor dimensions generated by the CNN as compared to manual annotation. Similarly, there were no statistically significant differences between CNN generated tumor dimensions across three scanning platforms. We have developed and performed initial evaluation of a CNN which appears to accurately detect invasive cutaneous melanoma of no specific type in WSIs for objective evaluation of tumor morphology. Future work should interrogate these data further for its propensity to predict survival outcomes. Citation Format: Emily L. Clarke, Derek Magee, Julia Newton-Bishop, William Merchant, Marlous Hall, Robert Insall, Nigel Maher, Richard Scolyer, Grace Farnworth, Anisah Ali, Sally O'Shea, Darren Treanor. The development and evaluation of a convolutional neural network for melanoma detection in whole slide images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6173.

B7-H3 is associated with the armored-cold phenotype and predicts poor immune checkpoint blockade response in melanoma

Melanoma is the most suitable tumor type for immunotherapy, but not all melanoma patients could respond to immunotherapy. B7 homolog 3 (B7-H3) belongs to the B7 family and is overexpressed in a number of malignant tumors, but the expression pattern of B7-H3 in melanoma has not been well summarized. The expression of B7-H3 was investigated in melanoma and its correlations with features of the tumor microenvironment (TME) by using various public databases, including the Cancer Genome Atlas (TCGA), the GEPIA, and the Human Protein Atlas databases. In addition, the in-house melanoma tissue microarray was applied to validate the results from public databases. Based on the public and in-house cohorts, we found that B7-H3 was overexpressed in melanoma tumor tissues and high B7-H3 expression was related to poor clinical outcome. Moreover, B7-H3 was negatively correlated with levels of tumor-infiltrating lymphocytes (TILs) and positively correlated with collagen infiltration. With clinical translational value, the predictive value of B7-H3 for conventional immunotherapy was detected using the Kaplan-Meier plotter tool, and the results showed that melanoma patients with high B7-H3 expression were insensitive to anti-PD-1 and anti-CTLA-4 immunotherapy. In conclusion, we first investigate the expression of B7-H3 in melanoma and its correlations with the TME features, and indicate B7-H3 as a promising therapeutic target in melanoma patients that are insensitive to conventional immunotherapy.

Tumor-Intrinsic Galectin-3 Suppresses Melanoma Metastasis

Melanoma poses a poor prognosis with high mortality rates upon metastasis. Exploring molecular mechanisms governing melanoma progression paves the way for developing novel approaches to control melanoma metastasis and ultimately enhance patient survival rates. Extracellular galectin-3 (Gal-3) has emerged as a pleiotropic promoter of melanoma metastasis, exerting varying activities depending on its interacting partner. However, whether intracellular Gal-3 promotes melanoma aggressive behavior remains unknown. Here, we explored Gal-3 expression in human melanoma tissues as well as murine melanoma models to examine its causal role in metastatic behavior. We found that Gal-3 expression is downregulated in metastatic melanoma tissues compared to its levels in primary melanomas. Enforced silencing of Gal-3 in melanoma cells promoted migration, invasion, colony formation, in vivo xenograft growth and metastasis, and activated canonical oncogenic signaling pathways. Moreover, loss of Gal-3 in melanoma cells resulted in upregulated expression of the pro-metastatic transcription factor, nuclear factor of activated T cells (NFAT1), and its downstream metastasis-associated proteins, matrix metalloproteinase-3 (MMP-3) and interleukin-8 (IL-8). Overall, our findings implicate melanoma intracellular Gal-3 as a major determinant of its metastatic behavior and reveal a negative regulatory role for Gal-3 on the expression of NFAT1 in melanoma cells.

Tertiary lymphoid structures in cancer: immune mechanisms and clinical implications.

Cancer is a major cause of death globally, and traditional treatments often have limited efficacy and adverse effects. Immunotherapy has shown promise in various malignancies but is less effective in tumors with low immunogenicity or immunosuppressive microenvironment, especially sarcomas. Tertiary lymphoid structures (TLSs) have been associated with a favorable response to immunotherapy and improved survival in cancer patients. However, the immunological mechanisms and clinical significance of TLS in malignant tumors are not fully understood. In this review, we elucidate the composition, neogenesis, and immune characteristics of TLS in tumors, as well as the inflammatory response in cancer development. An in-depth discussion of the unique immune characteristics of TLSs in lung cancer, breast cancer, melanoma, and soft tissue sarcomas will be presented. Additionally, the therapeutic implications of TLS, including its role as a marker of therapeutic response and prognosis, and strategies to promote TLS formation and maturation will be explored. Overall, we aim to provide a comprehensive understanding of the role of TLS in the tumor immune microenvironment and suggest potential interventions for cancer treatment.

Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma

BackgroundIn the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244.MethodsTo directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments.ResultsCD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244−/− bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors.ConclusionOur study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

Multi-omics analysis for ferroptosis-related genes as prognostic factors in cutaneous melanoma.

Melanoma is highly malignant and heterogeneous. It is essential to develop a specific prognostic model for improving the patients' survival and treatment strategies. Recent studies have shown that ferroptosis results from the overproduction of lipid peroxidation and is an iron-dependent form of programmed cell death. Despite this, ferroptosis-related genes (FRGs) and their clinical significances remain unknown in malignant melanoma. This study aims to assess the role of FRGs in melanoma, with the goal of developing a novel prognostic model that provides new insights into personalized treatment and improvement of therapeutic outcomes for melanoma. We systematically characterized the genetic alterations and mRNA expression of 73 FRGs in The Cancer Genome Atlas (TCGA)-skin cutaneous melanoma (SKCM) dataset in this study. The results were validated with real-time RT-PCR and Western blotting. Subsequently, a multi-gene feature model was constructed using the TCGA-SKCM cohort. Melanoma patients were classified into a high-risk group and a low-risk group based on the feature model. As a final step, correlations between ferroptosis-related signatures and immune features, immunotherapy efficacy, or drug response were analyzed. By analyzing melanoma samples from TCGA-SKCM dataset, FRGs exhibited a high frequency of genetic mutations and copy number variations (CNVs), significantly impacting gene expression. Additionally, compared with normal skin tissue, 30 genes with significantly differential expression were identified in melanoma tissues. A prognostic model related to FRGs, constructed using the LASSO Cox regression method, identified 13 FRGs associated with overall survival prognosis in patients and was validated with external datasets. Finally, functional enrichment and immune response analysis further indicated significant differences in immune cell infiltration, mutation burden, and hypoxia status between the high-risk group and the low-risk group, and the model was effective in predicting responses to immunotherapy and drug sensitivity. This study develops a strong ferroptosis-related prognostic signature model which could put forward new insights into target therapy and immunotherapy for patients with melanoma.

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. FFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

Circular RNA circFCHO2(hsa_circ_0002490) promotes the proliferation of melanoma by directly binding to DND1

Circular RNAs (circRNAs) have been documented to play crucial roles in the biology of various cancers. However, their investigation in melanoma is still at an early stage, particularly as a broader mechanism beyond acting as miRNA sponges needs to be explored. We report here that circFCHO2(hsa_circ_0002490), a circRNA encompassing exons 19 and 20 of the FCHO2 gene, exhibited a consistent overexpression in melanoma tissues. Furthermore, elevated circFCHO2 levels demonstrated a positive correlation with the malignant phenotype and poor prognosis among the 158 melanoma patients studied. Besides, we observed that heightened levels of circFCHO2 promoted melanoma cell proliferation, migration, and invasion in vitro, along with contributing to tumor growth in vivo. Furthermore, we found differences in the secondary structure of circFCHO2 compared to most other circular RNA structures. It has fewer miRNA binding sites, while it has more RNA binding protein binding sites. We therefore speculate that circFCHO2 may have a function of interacting with RNA binding proteins. Mechanistically, it was confirmed by fluorescence in situ hybridization (FISH), RNA-pull down, RNA immunoprecipitation (RIP), and western blotting assays that circFCHO2 interacts with dead end protein homolog 1 (DND1) and reverses the inhibition of the PI3K/AKT signaling pathway by binding to DND1. Our findings reveal that circFCHO2 drives melanoma progression by regulating the PI3K/AKT signaling pathway through direct binding to DND1 and may serve as a potential diagnostic biomarker and therapeutic target for the treatment of melanoma.Graphical Headlights: CircFCHO2 is highly expressed in melanoma. High circFCHO2 levels were positively correlated with poor prognosis in 158 melanoma patients. CircFCHO2 is involved in the regulation of the PI3K/AKT signalling pathway by binding to DND1. CircFCHO2 could serve as a potential biomarker and therapeutic target for the management of melanoma.

Immune Infiltrate Profiling of Cutaneous Melanoma with Spatial Transcriptomics

Background and Objective:In 2023, an estimated 97,610 melanomas will be diagnosed in the U.S. Immunotherapy has revolutionized care for melanoma patients and highlighted the prognostic value of characterizing immune infiltrates in melanoma tissue. This project used spatial transcriptomics (ST) to analyze immune infiltrates in cutaneous melanoma specimens.
 Methods:Visium Spatial Gene Expression platform generated datasets from formalin fixed paraffin embedded cutaneous melanoma specimens were analyzed using Loupe Browser software v6.5.0 (10x Genomics Inc.). Unsupervised clustering and manual region selection identified areas enriched in immune cell specific differentially expressed genes (DEGs). The transcript for Collagen alpha-1(XIX) chain (COL19A1) was identified as a DEG co-expressed with B cell marker DEGs. Fluorescence activated cell sorting was used to isolate primary B and T cells from mouse secondary lymphoid organs and human peripheral blood mononuclear cells to measure COL19A1 with quantitative PCR (qPCR).
 Results:Transcripts for CD19 (B cell marker) were concentrated in an immune DEG- rich region adjacent to a melanoma micrometastasis in one specimen. There was overlap between the tertiary lymphoid structure [TLS] markers SELL, CXCR5, CCR7, and CXCL13 with CD19 and/or COL19A1. Though qPCR revealed minimal Col19a1 in mouse B and T cells, COL19A1 was abundant and specific for human B cells.
 Conclusions & Potential Impact:This study demonstrates the feasibility and potential of using ST to characterize immune cell infiltrates in melanoma. Though COL19A1 expression is typically understood to be enriched in muscle and neuronal cells, the studies presented here suggest this transcript could be a novel human (but not mouse) B cell marker. Future studies are needed to determine if collagen XIX (protein) is expressed in B cells and if it is, what the biological functions of this protein are in B cells under physiologic and pathologic (e.g. melanoma) conditions.

Role of PLK1/NUMB/NOTCH in epithelial-mesenchymal transition in human melanoma

Polo-like kinase 1 (PLK1), a serine/threonine kinase, is overexpressed in melanoma and its expression has been associated with poor disease prognosis. PLK1 has been shown to interact with NUMB, a NOTCH antagonist. However, the exact role of PLK1, NUMB, and NOTCH signaling in epithelial-mesenchymal transition (EMT) in melanoma progression is unclear. In this study, Affymetrix microarray analysis was performed to determine differentially expressed genes following shRNA-mediated knockdown of PLK1 in human melanoma cells that showed significant modulations in EMT and metastasis-related genes. Using multiple PLK1-modulated melanoma cell lines, we found that PLK1 is involved in the regulation of cell migration, invasion, and EMT via its kinase activity and NOTCH activation. In vitro kinase assay and mass spectrometry analysis demonstrated a previously unknown PLK1 phosphorylation site (Ser413) on NUMB. Overexpression of non-phosphorylatable (S413A) and phosphomimetic (S413D) mutants of NUMB in melanoma cells implicated the involvement of NUMB-S413 phosphorylation in cell migration and invasion, which was independent of NOTCH activation. To determine the clinical relevance of these findings, immunohistochemistry was performed using melanoma tissue microarray, which indicated a strong positive correlation between PLK1 and N-cadherin, a protein required for successful EMT. These findings were supported by TCGA analysis, where expression of high PLK1 with low NUMB or high NOTCH or N-cadherin showed a significant decrease in survival of melanoma patients. Overall, these results suggest a potential role of PLK1 in EMT, migration, and invasion of melanoma cells. Our findings support the therapeutic targeting of PLK1, NUMB, and NOTCH for melanoma management.

A novel PDPN antagonist peptide CY12-RP2 inhibits melanoma growth via Wnt/β-catenin and modulates the immune cells.

Podoplanin (PDPN) is a highly conserved, mucin-type protein specific to the lymphatic system. Overexpression of PDPN is associated with the progression of various solid tumors, and plays an important roles in the tumor microenvironment by regulating the immune system. However, the role of PDPN-mediated signal activation in the progression of melanoma is still unknown. PDPN expression was first analyzed in 112 human melanoma tissue microarrays and melanoma cell lines. Functional experiments including proliferation, clone formation, migration, and metastasis were utilized to identify the suppressive effects of PDPN. The Ph.D.TM-12 Phage Display Peptide Library was used to obtain a PDPN antagonist peptide, named CY12-RP2. The immunofluorescence, SPR assay, and flow cytometry were used to identify the binding specificity of CY12-RP2 with PDPN in melanoma cells. Functional and mechanistic assays in vivo and in vitro were performed for discriminating the antitumor and immune activation effects of CY12-RP2. PDPN was overexpressed in melanoma tissue and cells, and inhibited melanoma cells proliferation, migration, and metastasis by blocking the EMT and Wnt/β-catenin pathway. PDPN antagonistic peptide, CY12-RP2, could specifically bind with PDPN, suppressing melanoma various functions inducing apoptosis in both melanoma cells and 3D spheroids. CY12-RP2 also enhanced the anti-tumor capacity of PBMC, and inhibited melanoma cells growth both in xenografts and allogeneic mice model. Moreover, CY12-RP2 could inhibit melanoma lung metastasis, and abrogated the immunosuppressive effects of PDPN by increasing the proportion of CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD49b + Granzyme B + NK cells, and CD11b + CD86 + M1-like macrophages and the levels of IL-1β, TNF-α, and IFN-γ. This study has demonstrated the important role of PDPN in the progression of melanoma and formation of immunosuppressive environment, and provided a potential approach of treating melanoma using the novel CY12-RP2 peptide. In melanoma, PDPN is overexpressed in the cancer cells, and promotes melanoma cells growth and metastasis through activating the Wnt/β-catenin pathway. Treatment with the PDPN antagonistic peptide CY12-RP2 could not only inhibit the melanoma growth and metastasis both in vitro and in vivo through Wnt/β-catenin pathway blockade, but also abrogate the immunosuppressive effects of PDPN through modulating immune cells.

LINC00662 promotes melanoma progression by competitively binding miR-107 and activating the β-catenin signaling pathway.

Melanoma is a highly malignant tumor in the body. Long non-coding RNAs (lncRNAs) have been reported to be involved in the development of various tumors. Emerging evidence demonstrates the critical role of lncRNAs in melanoma development. In this study, we aimed to investigate the expression, biological function and regulatory mechanism of LINC00662 in melanomas. First, we found that LINC00662 was up-regulated in melanoma tissues and cell lines. High expression of LINC00662 in melanomas was associated with a poor patient prognosis. Silencing of LINC00662 suppressed the proliferation, migration, and invasion of melanoma cells in vitro and in vivo, while overexpression of LINC00662 promoted melanoma cell proliferation in vitro. Bioinformatics analysis, dual-luciferase assay, and RIP assay confirmed that LINC00662 competitively regulated miR-107. Silencing of LINC00662 upregulated miR-107 expression in a melanoma cell line. Inhibition of miR-107 significantly reversed the inhibitory effect of LINC00662 silencing on cell proliferation and migration. Furthermore, POU3F2 was validated as a downstream target of LINC00662/miR107 and was downregulated when LINC00662 was silenced. Overexpressing POU3F2 attenuated the effect of si-LINC00662 on cellular functions. In addition, the results also showed that the β-catenin pathway was involved in a si-LINC00662-induced function in melanoma. Overall, our results confirmed that LINC00662 promoted melanoma progression by sponging miR107 and inducing POU3F2, highlighting the mechanism of the LINC00662/miR-107/POU3F2 axis in melanoma cell proliferation and invasion.

Combined BET and MEK Inhibition synergistically suppresses melanoma by targeting YAP1.

Rationale: The response rate to the MEK inhibitor trametinib in BRAF-mutated melanoma patients is less than 30%, and drug resistance develops rapidly, but the mechanism is still unclear. Yes1-associated transcriptional regulator (YAP1) is highly expressed in melanoma and may be related to MEK inhibitor resistance. The purpose of this study was to investigate the mechanism of YAP1 in MEK inhibitor resistance in melanoma and to screen YAP1 inhibitors to further determine whether YAP1 inhibition reverses MEK inhibitor resistance. Methods: On the one hand, we analyzed paired melanoma and adjacent tissue samples using RNA-seq and found that the Hippo-YAP1 signaling pathway was the top upregulated pathway. On the other hand, we evaluated the transcriptomes of melanoma samples from patients before and after trametinib treatment and investigated the correlation between YAP1 expression and trametinib resistance. Then, we screened for inhibitors that repress YAP1 expression and investigated the mechanisms. Finally, we investigated the antitumor effect of YAP1 inhibition combined with MEK inhibition both in vitro and in vivo. Results: We found that YAP1 expression levels upon trametinib treatment in melanoma patients were correlated with resistance to trametinib. YAP1 was translocated into the nucleus after trametinib treatment in melanoma cells, which could render resistance to MEK inhibition. Thus, we screened for inhibitors that repress YAP1 expression and identified multiple bromodomain and extra-terminal (BET) inhibitors, including NHWD-870, as hits. BET inhibition repressed YAP1 expression by decreasing BRD4 binding to the YAP1 promoter. Consistently, YAP1 overexpression was sufficient to reverse the proliferation defect caused by BRD4 depletion. In addition, the BET inhibitor NHWD-870 acted synergistically with trametinib to suppress melanoma growth in vitro and in vivo. Conclusions: We identified a new vulnerability for MEK inhibitor-resistant melanomas, which activated Hippo pathway due to elevated YAP1 activity. Inhibition of BRD4 using BET inhibitors suppressed YAP1 expression and led to blunted melanoma growth when combined with treatment with the MEK inhibitor trametinib.

The combination of single-cell and RNA sequencing analysis decodes the melanoma tumor microenvironment and identifies novel T cell-associated signature genes.

Skin cutaneous melanoma (SKCM), a malignant melanocyte-derived skin cancer, potentially leads to fatal outcomes without effective treatment. The variability in immunotherapy responses among melanoma patients is significantly influenced by the intricate immune microenvironment, particularly due to the status of tumor T cells, encompassing their activity, exhaustion levels, and antigen recognition capabilities. This study utilized single-cell RNA sequencing (scRNA-seq) to analyze 34 melanoma samples from two public datasets (GSE215120 and GSE115978). Herein, we extracted 706 marker genes associated with immune checkpoint (ICP) therapy from these T cells, 509 markers of T cells from 11 melanoma tissues, and eventually identified 33 candidate genes. These genes underwent LASSO and COX regression analyses to identify the signature genes. Of the initial 33 candidate genes, we successfully isolated six distinct T cell-associated immunotherapy-related genes (IRTGs). Additionally, the computation of each patient risk score proved beneficial in evaluating the immune cell infiltration level and functions as an independent prognostic factor for melanoma patient survival. The risk score results revealed promising predictive outcomes in determining the response of melanoma patients to immunotherapy. Notably, our study is the first to reveal the potential correlation between signature gene PEB4B and the immune microenvironment in melaoma, which was explored with multiple immunofluorescence (IF) and Immune Infiltration Assessment. In a conclusion, our findings demonstrate the potential utility of a risk score dependent on signature genes as a predictive tool for assessing the prognosis and response to immunotherapeutic interventions in melanoma patients.

Calreticulin regulates the expression of MMP14 and ADAR1 through EIF2AK2 signaling to promote the proliferation and progression of malignant melanoma cells.

It has been demonstrated that calreticulin (CALR) is expressed abnormally in various tumors and is involved in the occurrence and development of tumors. In this study, CALR and EIF2AK2 expression was measured in the clinical specimens of 39 patients with melanoma. Then, we constructed knockdown and overexpression cell models of CALR and EIF2AK2 and used wound healing and Transwell assays to observe cell migration and invasion. Apoptosis, EDU, and ROS assays were used to measure cell apoptosis and proliferation, as well as ROS levels. The effect of CALR on endoplasmic reticulum stress was detected using endoplasmic reticulum fluorescent probes. Western blotting was used to detect protein levels of CALR, EIF2AK2, ADAR1, and MMP14. The results indicated that CALR and EIF2AK2 expression levels were significantly higher in human melanoma tissues than in adjacent non-tumor tissue. In addition, we found a correlation between CALR and the expression of EIF2AK2 and MMP14, and the experimental results indicated that overexpression of CALR significantly upregulated the expression of EIF2AK2, MMP14, and ADAR1, while knockdown of CALR inhibited their expression. Notably, the knockdown of EIF2AK2 in the CALR overexpression group blocked the upregulation of MMP14 and ADAR1 expression by CALR, and the knockdown of both CALR and EIF2AK2 significantly inhibited MMP14 and ADAR1 expression. In conclusion, CALR and EIF2AK2 play a promoting role in melanoma progression, and knockdown of CALR and EIF2AK2 may be an effective anti-tumor target, and its mechanism may be through MMP14, ADAR1 signaling.