Overall Survival In Melanoma Patients Research Articles

Impact of metformin on melanoma: a meta-analysis and systematic review

BackgroundThere is evidence of a modest reduction in skin cancer risk among metformin users. However, no studies have further examined the effects of metformin on melanoma survival and safety outcomes. This study aimed to quantitatively summarize any influence of metformin on the overall survival (OS) and immune-related adverse effects (irAEs) in melanoma patients.MethodsSelection criteria: The inclusion criteria were designed based on the PICOS principles. Information sources: PubMed, EMBASE, Cochrane Library, and Web of Science were searched for relevant literature published from the inception of these databases until November 2023 using ‘Melanoma’ and ‘Metformin’ as keywords. Survival outcomes were OS, progression-free survival (PFS), recurrence-free survival (RFS), and mortality; the safety outcome was irAEs. Risk of bias and data Synthesis: The Cochrane tool for assessing the risk of bias in randomized trial 2 (RoB2) and methodological index for non-randomized studies (MINORS) were selected to assess the risk of bias. The Cochrane Q and I2 statistics based on Stata 15.1 SE were used to test the heterogeneity among all studies. Funnel plot, Egger regression, and Begg tests were used to evaluate publication bias. The leave-one-out method was selected as the sensitivity analysis tool.ResultsA total of 12 studies were included, involving 111,036 melanoma patients. The pooled HR for OS was 0.64 (95% CI [0.42, 1.00], p = 0.004, I2 = 73.7%), HR for PFS was 0.89 (95% CI [0.70, 1.12], p = 0.163, I2 = 41.4%), HR for RFS was 0.62 (95% CI [0.26, 1.48], p = 0.085, I2 = 66.3%), and HR for mortality was 0.53 (95% CI [0.46, 0.63], p = 0.775, I2 = 0.0%). There was no significant difference in irAEs incidence (OR = 1.01; 95% CI [0.42, 2.41]; p = 0.642) between metformin and no metformin groups.DiscussionThe improvement in overall survival of melanoma patients with metformin may indirectly result from its diverse biological targets and beneficial effects on multiple systemic diseases. While we could not demonstrate a specific improvement in the survival of melanoma patients, the combined benefits and safety of metformin for patients taking the drug are worthy of recognition.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO/, identifier CRD42024518182.

Latent-Transforming Growth Factor β-Binding Protein 1/Transforming Growth Factor β1 Complex Drives Antitumoral Effects upon ERK5 Targeting in Melanoma

Melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. Available treatments have improved the survival, although long-term benefits are still unsatisfactory. The mitogen-activated protein kinase ERK5 promotes melanoma growth, and ERK5 inhibition determines cellular senescence and the senescence-associated secretory phenotype. Here, latent-transforming growth factor β-binding protein 1 (LTBP1) mRNA was found to be upregulated in A375 and SK-Mel-5 BRAFV600E melanoma cells after ERK5 inhibition. In keeping with a key role of LTBP1 in regulating transforming growth factor β (TGF-β), TGF-β1 protein levels were increased in lysates and conditioned media of ERK5-knock down (KD) cells, and were reduced upon LTBP1 KD. Both LTBP1 and TGF-β1 proteins were increased in melanoma xenografts in mice treated with the ERK5 inhibitor XMD8-92. Moreover, treatment with conditioned media from ERK5-KD melanoma cells reduced cell proliferation and invasiveness, and TGF-β1-neutralizing antibodies impaired these effects. In silico datasets revealed that higher expression levels of both LTBP1 and TGFB1 mRNA are associated with better overall survival of melanoma patients, and that increased LTBP1 or TGF-β1 expression proved a beneficial role in patients treated with anti-PD1 immunotherapy, making unlikely a possible immunosuppressive role of LTBP1/TGF-β1 upon ERK5 inhibition. This study, therefore, identifies additional desirable effects of ERK5 targeting, providing evidence of an ERK5-dependent tumor suppressive role of TGF-β in melanoma.

Abstract 7619: Tissue-derived peripheral biomarkers that reflect activity of T-cells, macrophages, and neutrophils in patients with solid tumors

Abstract Background: A toolbox of tissue-derived peripheral biomarkers that reflect the activity of T-cells, macrophages and neutrophils is a medical need for immuno-oncology drug development allowing non-invasive quantification of immune cell activity before - and during - treatment, both for predictive and pharmacodynamic purposes. By identifying and quantifying specific extracellular matrix fragments with neo-epitopes that are generated by proteases specific for T-cells, macrophages, and neutrophils, respectively, it is possible to develop peripheral biomarkers that reflect the activity of these immune cells. Such biomarkers have previously shown associations with outcome of patients treated with immune checkpoint inhibitors. Here we investigated the distribution of 3 such biomarkers in different cancer indications. Moreover, these data were compared to data from melanoma patients showing associations with response to cancer immunotherapy. Methods: Tissue-derived biomarkers related to activity of T-cells (C4G: Collagen-4 degraded by Granzyme B), Neutrophils (CPA9-HNE: Calprotectin degraded by neutrophil elastase), Macrophages (VICM: citrullinated and MMP-degraded vimentin) were measured by validated ELISA/ECLIA in serum from patients with various solid tumor types (bladder-, breast- colorectal-, head and neck-, kidney-, lung-, ovarian-, pancreatic, stomach-, prostate- cancer, and melanoma, n=220) and compared to age-matched healthy controls (n=33) by ANOVA. The biomarker levels were compared to historical data. Results: Median biomarker levels of CPA9HNE (neutrophil activity/NETosis) and VICM (macrophage activity) were significantly increased in all solid tumor types (p<0.001), respectively and with a ~5-fold inter-patient variation as well. Median C4G levels was only slightly elevated in a few indications (p<0.05) and with a 5-10-fold inter-patient variation across tumor types. All biomarker levels were independent of disease stage. Similar biomarkers levels were seen to those found to be associated with objective response rate and overall survival of melanoma patients treated with cancer immunotherapy. Conclusion: Tissue-derived peripheral biomarkers that reflect the activity of T-cells (C4G), macrophages (VICM) and neutrophils (CPA9HNE) has biomarker potential in solid tumors and may serve as prognostic, predictive and pharmacodynamic biomarkers in clinical trials investigating cancer immunotherapy. Citation Format: Nicholas Willumsen, Neel I. Nissen, Morten A. Karsdal. Tissue-derived peripheral biomarkers that reflect activity of T-cells, macrophages, and neutrophils in patients with solid tumors [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 7619.

ATP1A1 is a promising new target for melanoma treatment and can be inhibited by its physiological ligand bufalin to restore targeted therapy efficacy

Despite advancements in treating metastatic melanoma, many patients exhibit resistance to targeted therapies. Our study focuses on ATP1A1, a sodium pump subunit associated with cancer development. We aimed to assess ATP1A1 prognostic value in melanoma patients and examine the impact of its ligand, bufalin, on melanoma cell lines in vitro and in vivo. High ATP1A1 expression (IHC) correlated with reduced overall survival in melanoma patients. Resistance to BRAF inhibitor was linked to elevated ATP1A1 levels in patient biopsies (IHC, qPCR) and cell lines (Western blot, qPCR). Additionally, high ATP1A1 mRNA expression positively correlated with differentiation/pigmentation markers based on data from The Cancer Genome Atlas (TCGA) databases and Verfaillie proliferative gene signature analysis. Bufalin specifically targeted ATP1A1 in caveolae, (proximity ligation assay) and influenced Src phosphorylation (Western blot), thereby disrupting multiple signaling pathways (phosphokinase array). In vitro, bufalin induced apoptosis in melanoma cell lines by acting on ATP1A1 (siRNA experiments) and, in vivo, significantly impeded melanoma growth using a nude mouse xenograft model with continuous bufalin delivery via an osmotic pump. In conclusion, our study demonstrates that ATP1A1 could serve as a prognostic marker for patient survival and a predictive marker for response to BRAF inhibitor therapy. By targeting ATP1A1, bufalin inhibited cell proliferation, induced apoptosis in vitro, and effectively suppressed tumor development in mice. Thus, our findings strongly support ATP1A1 as a promising therapeutic target, with bufalin as a potential agent to disrupt its tumor-promoting activity.

Successful treatment with carboplatin and paclitaxel in melanoma progression after immune-related adverse events.

The overall survival of melanoma patients has improved using antibodies targeting immune checkpoints (anti-PD-1, anti-CTLA-4 andanti-LAG-3). Systemic chemotherapy was administered in melanoma for many years with limited effectiveness. Here we report a case of a patient who experienced immune-mediated adverse effects from checkpoint blockade therapy and subsequently responded to chemotherapy. The patient presented with melanoma and paraneoplastic digital ischemia. She received a combination of ipilimumab/nivolumab and experienced G3 myocarditis, followed by melanoma progression after a steroid taper. This patient achieved a partial and durable response with platinum and taxane-based chemotherapy. This report suggests the possibility of a subset of patients who experience progression after immune-based side effects where chemotherapy may be effective in the modern age of melanoma treatment.

Dermokine mutations contribute to epithelial-mesenchymal transition and advanced melanoma through ERK/MAPK pathways.

To discover vulnerabilities associated with dermokine (DMKN) as a new trigger of the epithelial-mesenchymal transition (EMT) -driven melanoma, we undertook a genome-wide genetic screening using transgenic. Here, we showed that DMKN expression could be constitutively increased in human malignant melanoma (MM) and that this correlates with poor overall survival in melanoma patients, especially in BRAF-mutated MM samples. Furthermore, in vitro, knockdown of DMKN inhibited the cell proliferation, migration, invasion, and apoptosis of MM cancer cells by the activation of ERK/MAPK signaling pathways and regulator of STAT3 in downstream molecular. By interrogating the in vitro melanoma dataset and characterization of advanced melanoma samples, we found that DMKN downregulated the EMT-like transcriptional program by disrupting EMT cortical actin, increasing the expression of epithelial markers, and decreasing the expression of mesenchymal markers. In addition, whole exome sequencing was presented with p.E69D and p.V91A DMKN mutations as a novel somatic loss of function mutations in those patients. Moreover, our purposeful proof-of-principle modeled the interaction of ERK with p.E69D and p.V91A DMKN mutations in the ERK-MAPK kinas signaling that may be naturally associated with triggering the EMT during melanomagenesis. Altogether, these findings provide preclinical evidence for the role of DMKN in shaping the EMT-like melanoma phenotype and introduced DMKN as a new exceptional responder for personalized MM therapy.

Abstract LB017: Potential role of kinesin family member C1 (KIFC1) in melanoma

Abstract According to the Surveillance, Epidemiology, and End Results (SEER) analyses, based on 2017-2019 data, approximately ~2.1 percent of men and women will be diagnosed with melanoma at some point during their lifetime. Melanoma can be fatal if not surgically removed at an early stage, with the 5-year survival being a dismal 29.8% for a stage IV melanoma. Melanoma mortality is primarily due to the distant organ metastases and its resistance to treatment, even with newly developed immunotherapy and/or targeted therapies (BRAF and MEK inhibitors). Thus, the discovery of novel melanoma targets is crucial as they may be able to provide alternative treatment options or be used alone or in combination with current therapies. KIFC1 (kinesin family member C1; also known as HSET, an ortholog in Drosophila melanogaster), a nonessential kinesin motor protein, has an established role in centrosome clustering which is utilized by cancer cells to evade apoptosis and mitotic arrest. This occurs when replicating cells harbor additional centrosomes, a hallmark of cancer. The requirement of KIFC1 in mitosis of cancer cells harboring abnormal centrosomes, but not that of normal cells, makes it a potential drug target for cancer management. The goal of this study was to determine the role and significance of KIFC1 in melanoma. Differential expression analysis of GTEX and TCGA RNA-seq data identified significantly higher levels of KIFC1 in both primary and metastatic melanoma as compared to normal skin. In addition, higher expression of KIFC1 was found to be associated with poorer overall survival of melanoma patients. Furthermore, a quantitative protein estimation with the automated ProteinSimple Jess capillary western blotting system demonstrated higher KIFC1 expression in several melanoma cell lines when compared to normal melanocytes. In addition, we also found significantly increased expression of KIFC1 mRNA in mouse melanoma tumors obtained from the genetically engineered BrafV600E/PtenNULL mice, as compared to the normal skin. Next, we determined the effect of a small molecule KIFC1 inhibitor, AZ82 on A375, G361 and Hs294T melanoma cells. AZ82 is an ATP-competitive inhibitor that binds the KIFC1-microtubule complex, blocking the release of ADP and negating its role as a motor protein. Employing RealTime-Glo MT cell viability assay as well as the endpoint XTT growth assay, we found that AZ82 treatment resulted in a significant growth inhibition of human melanoma cells at 2-3.5 µM concentrations. Additionally, AZ82 treatments resulted in a significant decrease in clonogenic survival in all three human melanoma cell lines. Collectively, our data suggests a potential role of KIFC1 in melanoma progression, and merits further investigation to determine the roles and functional and therapeutic significance of KIFC1 in melanoma. Citation Format: Davis S. Mau, Mary A. Ndiaye, Gabriella R. Zaemisch, Chandra K. Singh, Jess Vera, Sean R. McIlwain, Nihal Ahmad. Potential role of kinesin family member C1 (KIFC1) in melanoma [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 LB017.

Abstract 3753: miR-567 modulates the progression of melanoma cells and functions of macrophages

Abstract Purpose: Target therapy and immunotherapy are advancements in the treatment of melanoma; unfortunately, a subset of patients does not benefit or produces drug resistance. Therefore, an alternative strategy should be investigated to further improve clinical benefits. Macrophages are the abundant immune cells related to the progression of melanoma and resistance to drugs. In this study, we explored the novel roles of miR-567 in the progression of melanoma and effects of macrophages. Methods: miR-567 expression in nevus and melanoma tissues was analyzed by in situ hybridization. miR-567 was transfected into melanoma cells to investigate its biological effects through functional assays such as proliferation, colony formation, soft agar, and migration assays. In addition, miR-567-related signal transduction pathways in the melanoma cells were studied by western blot and next-generation sequencing analyses. Different phenotypes of macrophages polarized from THP-1 monocyte cells were used as in vitro model to investigate the effects of miR-567 on macrophages. Results: Our results showed that miR-567 expression levels were decreased in melanoma cells compared to melanocyte cells. Consistently, expression of miR-567 was 0.403-fold lower in melanoma tissues (n=118) as compared to nevus tissues (n=40). In addition, the receiver operating characteristic presented that the area under the curve value of miR-567 is 0.9495. Importantly, higher expression of miR-567 enhanced the overall survival of melanoma patients. Overexpression of miR-567 significantly reduced proliferation, survival, anchorage-independent growth, migratory, and invasive abilities of melanoma cells and BRAF-inhibitor resistant cells. Furthermore, our results demonstrated that IGF1R, E2F1, and Cyclin B2 are direct targets of miR-567 and knockdown of these genes attenuated proliferation and survival of melanoma cells. Interestingly, introduction of miR-567 downregulated MAPK/ERK and PI3K/AKT pathways in melanoma cells and regulated multiple pathways related to immune response. Of note, overexpression of miR-567 reduced the promoting melanoma growth induced by M2 or melanoma-associated macrophages, indicating the involvement of miR-567 in regulation of macrophages. Conclusion: miR-567 could be served not only as biomarkers but also as potential molecular target for prevention of melanoma progression. Citation Format: Mai-Huong Thi Nguyen, Chen-Huan Lin, Yu-Chi Huang, Mu-Shiun Tsai, Ming-Hong Chen, Azusa Miyashita, Satoshi Fukushima, Nianhan Ma. miR-567 modulates the progression of melanoma cells and functions of macrophages. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3753.

Abstract 1265: Pan-cancer study of glycosylation-related gene expression: The emerging role of galectin-2 in tumor progression

Abstract Aberrant glycosylation has emerged as an important hallmark of cancer playing a critical role in the modulation of tumor cell aggressiveness, invasiveness, and modulation of the immune and vascular programs. However, the cellular and molecular mechanisms implicated in glycan-driven cancer progression remain poorly understood. In this study, we explored the expression profile of a glycosylation-related gene signature (Affymetrix, GlycoV4) and its association with cancer progression in tumors from different primary sites. First, we analyzed published transcriptomic data from patients’ tumor biopsies to identify pan-cancer molecular signatures associated with glycosylation-related genes including components of the glycosylation machinery and glycan-binding proteins or lectins, to determine unsupervised sample clusters with similar glyco-transcriptome profiles. Next, focusing on a melanoma transcriptomic database (TCGA-SKCM), a supervised machine learning algorithm based on Random Forest was applied to classify each biopsy according to its sample type (metastatic or primary tumor). Notably, we found that using 27 glycogenes out of the 570 included in the signature each biopsy could be classified with 87% accuracy as metastatic or primary tumor. These findings highlight the importance of components of the glycosylation machinery as novel biomarkers of tumor progression. To explore possible mechanisms underlying glycosylation changes, we focused on the study of lectins responsible for deciphering the biological information encoded by the glycome. Particularly, galectins, a family of endogenous lectins, are known to be involved in immunomodulation, inflammation, tumor escape, and metastasis. TCGA-SKCM data was used to study the implications of two galectins expression, galectin-2 (Gal-2) mostly unexplored, and galectin-1 (Gal-1), a known regulator of antitumor immune responses. In this dataset, we found that Gal-1 expression was associated with less survival, whereas higher expression of Gal-2 was linked to increased overall survival in melanoma patients. Furthermore, initial findings revealed that Gal-2 expression was significantly augmented in metastatic biopsies whereas Gal-1 was significantly increased in primary solid tumor biopsies. Finally, to study possible mechanisms underlying these correlations, we used an immunocompetent mouse melanoma model to challenge WT and Gal-2-deficient (Lgals2-/-) mice. Interestingly, tumor volume was significantly increased in Lgals2-/- mice (p<0.005) compared to their WT counterpart. To understand the molecular bases of Gal-2-driven tumor growth changes, we are studying the tumor microenvironment. Given the anti-inflammatory role of Gal-1 and the potential pro-inflammatory capacity of Gal-2, we propose that Gal-1 and Gal-2 may play opposing roles, regulating the outcome of tumor progression. Citation Format: Montana N. Manselle Cocco, Florencia Veigas, Yamil D. Mahmoud, Mora Massaro, Sabrina Gatto, Rosa M. Morales, Alejandro Cagnoni, Gabriel A. Rabinovich. Pan-cancer study of glycosylation-related gene expression: The emerging role of galectin-2 in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1265.

A novel gene prognostic signature lymphocyte cytosolic protein 2 regulates melanoma progression by activating tumor-infiltrating CD8+ T-cells through the interferon regulatory factor 5 signaling pathway

Cutaneous malignant melanoma is the most lethal skin cancer. The advent of immunotherapy has revolutionized the status of clinical therapies of melanoma, which brought new hope to these patients. However, only a small proportion of patients are responders. Therefore, the identification of novel prognostic and immune-related biomarkers is crucial to guide the development of melanoma clinical treatments. Herein, RNA-seq data of the cutaneous melanoma from public database were used for identifying prognostic gene signatures, and we found that lymphocyte cytosolic protein 2 (LCP2) was highly expressed in melanoma patient, which was associated with better prognosis for melanoma. Kyoto Encyclopedia of Genes and Genomes and gene ontology analyses demonstrated that the differentially expressed genes are significantly involved in lysosome, B-cell receptor signaling pathways, Fc epsilon RI signaling pathway, and T-cell receptor signaling pathway, indicating that these signaling pathways play important roles in melanoma. LCP2 expression was positively correlated with CD8+ T-cell and the overall survival of melanoma patients, and this positive correlation was directly confirmed by fluorescence-activated cell sorting experiment. The in vivo experiment showed that LCP2 knockdown significantly promoted the melanoma progression and decreased interferon regulatory factor 5 (IRF5) expression. In conclusion, we identified that LCP2 is a possible prognostic gene signature for progression-free survival of melanoma patients and regulates melanoma progression by activating tumor-infiltrating CD8+ T-cells through the IRF5 signaling pathway, indicating that LCP2 could serve as a prognostic biomarker and therapeutic target in immunotherapy.

Single-cell RNA analysis to identify five cytokines signaling in immune-related genes for melanoma survival prognosis.

Melanoma is one of the deadliest skin cancers. Recently, developed single-cell sequencing has revealed fresh insights into melanoma. Cytokine signaling in the immune system is crucial for tumor development in melanoma. To evaluate melanoma patient diagnosis and treatment, the prediction value of cytokine signaling in immune-related genes (CSIRGs) is needed. In this study, the machine learning method of least absolute selection and shrinkage operator (LASSO) regression was used to establish a CSIRG prognostic signature of melanoma at the single-cell level. We discovered a 5-CSIRG signature that was substantially related to the overall survival of melanoma patients. We also constructed a nomogram that combined CSIRGs and clinical features. Overall survival of melanoma patients can be consistently predicted with good performance as well as accuracy by both the 5-CSIRG signature and nomograms. We compared the melanoma patients in the CSIRG high- and low-risk groups in terms of tumor mutation burden, infiltration of the immune system, and gene enrichment. High CSIRG-risk patients had a lower tumor mutational burden than low CSIRG-risk patients. The CSIRG high-risk patients had a higher infiltration of monocytes. Signaling pathways including oxidative phosphorylation, DNA replication, and aminoacyl tRNA biosynthesis were enriched in the high-risk group. For the first time, we constructed and validated a machine-learning model by single-cell RNA-sequencing datasets that have the potential to be a novel treatment target and might serve as a prognostic biomarker panel for melanoma. The 5-CSIRG signature may assist in predicting melanoma patient prognosis, biological characteristics, and appropriate therapy.

Low Expression of CLEC2B Indicates Poor Prognosis in Melanoma.

Melanoma is a highly malignant skin tumor with a poor prognosis. Identification of novel biomarkers might potentially reveal the underlying mechanisms of melanoma progression. We demonstrated the relationship between pan-cancer CLEC2B expression and melanoma samples in The Cancer Genome Atlas (TCGA) database. Next, the Kaplan-Meier plot and Cox regression analysis determined the prognostic value of CLEC2B in melanoma. Biological pathway enrichment was screened by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), enabling the correlation analysis between the immune infiltration level and CLEC2B expression in melanoma. Our final claim was validated using qPCR, immunohistochemistry, Western blot, cell colony formation assays, ethynyldeoxyuridine (Edu) analysis, and cell Invasion assays. Our study revealed that the high CLEC2B expression correlates with poor overall survival of melanoma patients. Moreover, a low expression of CLEC2B was found in the A375 cell line. In addition, CLEC2B has significant prognostic value in melanoma diagnosis, with an AUC of 0.896. Prognostic analysis showed the low expression of CLEC2B to be independently associated with melanoma patients. Moreover, the expression of CLEC2B was significantly correlated with B cells, eosinophils, macrophages, neutrophils, NK cells, T helper cells, Tregs, Th1 cells, Th17 cells, and Th2 cells. PCR and immunohistochemistry indicated CLEC2B to be significantly downregulated in melanoma. The cell colony formation assay showed CLEC2B knockout increased the proliferation of A375 cells. Our study established low levels of CLEC2B to be poor prognostic markers, enabling immunosuppressive cell infiltration in melanoma.

MicroRNAs affecting the susceptibility of melanoma cells to CD8+ T cell-mediated cytolysis.

The regulatory functions of microRNAs (miRNAs) in anti-tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell-target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8+ T cell-mediated cytotoxicity. Luciferase expressing B16F10 melanoma (B16F10 Luci+ ) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nβ) specific for the melanoma-associated antigen tyrosinase-related protein 2. miRNAs with the most pronounced effects on T cell-mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)-mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA-seq) data from miRNA-overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)-derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients' survival. The miRNA screen resulted in the selection of seven miRNAs enhancing CTL-mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa-miR-320a-3p, mmu-miR-7037-5p and mmu-miR-666-3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA-mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA-seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA-mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined. For the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell-mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities.

Tumor Androgen Receptor Protein Level Is Positively Associated with a Better Overall Survival in Melanoma Patients.

Androgen receptor (AR) is expressed in numerous tissues and serves important biologic functions in skin, prostate, immune, cardiovascular, and neural systems, alongside sexual development. Several studies have associated AR expression and patient survival in various cancers, yet there are limited studies examining the relationship between AR expression and cutaneous melanoma. This study used genomics and proteomics data from The Cancer Proteome Atlas (TCPA) and The Cancer Genome Atlas (TCGA), with 470 cutaneous melanoma patient data points. Cox regression analyses evaluated the association between AR protein level with overall survival and revealed that a higher level of AR protein was positively associated with a better overall survival (OS) (p = 0.003). When stratified by sex, the AR association with OS was only significant for both sexes. The multivariate Cox models with justifications of sex, age of diagnosis, stage of disease, and Breslow depth of the tumor confirmed the AR-OS association in all patients. However, the significance of AR was lost when ulceration was included in the model. When stratified by sex, the multivariate Cox models indicated significant role of AR in OS of female patients but not in males. AR-associated genes were identified and enrichment analysis revealed shared and distinct gene network in male and female patients. Furthermore, AR was found significantly associated with OS in RAS mutant subtypes of melanoma but not in BRAF, NF1, or triple-wild type subtypes of melanoma. Our study may provide insight into the well-known female survival advantage in melanoma patients.

3-Bromopyruvate Suppresses the Malignant Phenotype of Vemurafenib-Resistant Melanoma Cells.

(1) BRAF mutations are associated with high mortality and are a substantial factor in therapeutic decisions. Therapies targeting BRAF-mutated tumors, such as vemurafenib (PLX), have significantly improved the overall survival of melanoma patients. However, patient relapse and low response rates remain challenging, even with contemporary therapeutic alternatives. Highly proliferative tumors often rely on glycolysis to sustain their aggressive phenotype. 3-bromopyruvate (3BP) is a promising glycolysis inhibitor reported to mitigate resistance in tumors. This study aimed to evaluate the potential of 3BP as an antineoplastic agent for PLX-resistant melanoma treatment. (2) The effect of 3BP alone or in combination with PLX on viability, proliferation, colony formation, cell death, migration, invasion, epithelial-mesenchymal marker and metabolic protein expression, extracellular glucose and lactate, and reactive species were evaluated in two PLX-resistant melanoma cell lines. (3) 3BP treatment, which was more effective as monotherapy than combined with PLX, disturbed the metabolic and epithelial-mesenchymal profile of PLX-resistant cells, impairing their proliferation, migration, and invasion and triggering cell death. (4) 3BP monotherapy is a potent metabolic-disrupting agent against PLX-resistant melanomas, supporting the suppression of the malignant phenotype in this type of neoplasia.

Vitamin D deficiency in melanoma patients is associated with worse overall survival: a retrospective cohort study.

Recent interest has emerged in the protective role of vitamin D in melanoma survival and is the subject of multiple studies with heterogeneous results. Here, we present a retrospective cohort study of 264 patients with invasive melanoma from a tertiary university hospital. The aim of the study was to analyze the relationship between vitamin D levels and prognosis of melanoma patients. We found that lower vitamin D levels are independently associated with worse overall survival in melanoma patients in concordance with previous studies on other populations. Vitamin D deficiency could play a survival role in melanoma patients,. Future prospective studies are needed to investigate the effect of vitamin D supplementation on melanoma outcomes.

Association of immune-related adverse events with immune-checkpoint inhibitors and treatment response in melanoma patients.

e21522 Background: Immune-related adverse effects (irAE´s) of immune-checkpoint inhibitors (ICIs) have been linked with a better treatment response in melanoma patients, especially cutaneous toxicities. However, little is known regarding other irAE´s which is important as they can be used as clinical markers of an adequate therapeutical response. Methods: We conducted a retrospective study on patients who were diagnosed with melanoma and received treatment with ICI´s between January 2015 until December 2021, immune related adverse events and their relationship with overall survival in melanoma patients treated with ICIs was the main objective of this study. Results: 53 records of patients with advance melanoma treated with ICIs between january 2016 to december 2021, demographic characteristics were as follow: 64.2% were male, mean age at diagnoses was 60.3 years, 41.5% had smoking history and 15.1% were Jewish. At diagnosis 73.6% of patients had a good functional status (ECOG 0-1). The most common histological subtypes were epithelioid (34%), and nodular (22.6%). Lung metastases was the most common affected site (49.1%), followed by brain 43.4% and non-regional nodes 42.5%. BRAF mutations was determined in 81.1% of the biopsies and 36% of them being V600E mutation. ICI´s was the preferred first line treatment in 83% of cases, median number of administered cycles were 6 (range 1-54 cycles), 60.4% of patients received pembrolizumab, 37.7% nivolumab plus ipilimumab, 20.8% nivolumab monotherapy and 5.7% ipilimumab. Throughout the studied period IrAE´s were reported in 34% of patients with 66.7% of them being grade 1-2 and 33.3% grade 3-4. The most common IrAE’s: vitiligo 38.8%, hypothyroidism22% and 3.8% pneumonitis. Median PFS at 12 months and OS was significantly better in the group of patients with irAE´s: Patients who develop an irAE´s are 7 times more likely to be disease free at 12 months and 4.1 times more likely to have a longer OS regardless of severity and type of toxicity. The impact of developing irAEs is significantly important for PFS (HR: 11.9, CI 95%: 3.28-4.71) as median PFS was not yet reached in this group. Conclusions: Development of irAEs is associated with favorable outcomes to ICIs with patients being 7 times more likely to be 12-month disease free and 4.1 times more likely to have a longer OS. irAEs can be used as clinical markers of an adequate treatment response.[Table: see text]

MUC21 controls melanoma progression via regulating SLITRK5 and hedgehog signaling pathway.

Mucins are heavily glycosylated proteins secreted by various cell types, to protect the epithelial surface of the gastrointestinal tract from damage. Currently, increasing studies provided evidence to suggest that mucins play an essential role in regulating tumor progression. However, the role of mucins and the underpinning mechanism of how mucins drive melanoma progression remains elusive. In this study, we first demonstrated that mucin 21 (MUC21) expression was significantly upregulated in metastatic melanoma tissues, and a higher MUC21 expression resulted in poor overall survival in melanoma patients by The Cancer Genome Atlas database analysis. In vitro, MUC21 overexpression markedly promoted proliferative properties and aggressive behavior of melanoma cell A375 and A875, as assessed by Cell Counting Kit-8 and transwell assay. In mechanism, we proved that MUC21 suppressed expression of SLITRK5, an integral membrane protein, leading to activation of prosurvival hedgehog pathway and sustained melanoma development. More importantly, we found that combination of hedgehog pathway inhibitor cyclopamine and chemotherapy revealed an improved anticancer effect in MUC21 overexpression xenograft model. Altogether, our study described a novel role of MUC21 in regulating tumor progression, which offers a promising target for melanoma diagnosis and therapy.

Genome-Wide Association Study Suggests the Variant rs7551288*A within the DHCR24 Gene Is Associated with Poor Overall Survival in Melanoma Patients.

Simple SummaryThe aim of this work was to investigate prognostic genetic factors in melanoma patients. Phenotypic and disease data as well as biomaterial were collected after informed consent from patients followed up in a Skin Cancer Center of a University clinic. Genome-wide analysis (GWAS) was performed with survival data of 556 melanoma patients and genetic data including more than 300,000 common polymorphisms. The SNP rs7551288 reached suggestive genome-wide significance (p = 2 × 10−6). This intronic variant of the DHCR24 gene is involved in the cholesterol synthesis pathway. Further analyses and a literature review suggest an important role of this locus for the clinical course of disease in melanoma patients.Melanoma incidence rates are high among individuals with fair skin and multiple naevi. Established prognostic factors are tumour specific, and less is known about prognostic host factors. A total of 556 stage I to stage IV melanoma patients from Germany with phenotypic and disease-specific data were analysed; 64 of these patients died of melanoma after a median follow-up time of 8 years. Germline DNA was assessed by the HumanCoreExome BeadChip and data of 356,384 common polymorphisms distributed over all 23 chromosomes were used for a genome-wide analysis. A suggestive genome-wide significant association of the intronic allele rs7551288*A with diminished melanoma-specific survival was detected (p = 2 × 10−6). The frequency of rs7551288*A was 0.43 and was not associated with melanoma risk, hair and eye colour, tanning and total naevus count. Cox regression multivariate analyses revealed a 5.31-fold increased risk of melanoma-specific death for patients with the rs7551288 A/A genotype, independent of tumour thickness, ulceration and stage of disease at diagnoses. The variant rs7551288 belongs to the DHCR24 gene, which encodes Seladin-1, an enzyme involved in the biosynthesis of cholesterol. Further investigations are needed to confirm this genetic variant as a novel prognostic biomarker and to explore whether specific treatment strategies for melanoma patients might be derived from it.

2-methoxyestradiol inhibits melanoma cell growth by activating adaptive immunity

Background: The overall survival of melanoma patients remains poor despite advancements in surgical treatment and targeted therapies. Therefore, there is a need to develop new therapeutic strategies for melanoma. 2-methoxyestradiol (2-ME) is a major metabolite of estrogen that has been shown to have anti-tumor effects against many malignancies. However, the effects and mechanisms of action of 2-ME against melanoma remain unclear. Materials and methods: Melanoma cells (B16) were treated with 2-ME in vitro. Cell proliferation was detected by CCK8 and clone formation, transwell was carried out to measure the migration of B16 cells with or without 2-ME. Flow cytometry was performed to measure the apoptosis and cell cycle. C57BL/6 mice were used for tumor-bearing of B16 cells, tumor volumes were measured once a day, and sacrificed after it was over 2000 mm3, then immunofluorescence was implemented to examine the marker of CD3, CD8 and PD-L1. Results: In our study, we found that 2-ME significantly affected the proliferation, migration, apoptosis, and cell cycle of melanoma in vitro. Our results also showed that 2-ME had strong anti-tumor effects against melanoma in vivo and increased the infiltration of tumor-specific cytotoxic lymphocytes CD8+ T cells in the tumor microenvironment. Besides, PD-L1 expression in tumor cells was significantly higher in the 2-ME-treated group than in the control group, indicating that 2-ME could exhibit stronger anti-tumor effects against melanoma if combined with PD-1 blockade therapy. Conclusion: 2-ME suppresses melanoma in vivo and in vitro and is a promising synergistic enhancer of PD-1 blockade immunotherapy.