Melanoma Gene Research Articles

Identification of a prognostic model using cuproptosis-related genes in uveal melanoma.

The most common intraocular malignancy in adults remains uveal melanoma (UVM), and those with metastatic disease have a poor outlook. Proliferation, angiogenesis, and metastasis of tumor cells can be triggered by cuproptosis, affecting the survival of cancer patients. Nonetheless, cuproptosis-related genes (CRGs) have not been identified in UVM. In this study, we analyzed 10 CRGs in 80 patients with UVM in the Cancer Genome Atlas (TCGA) database regarding the alterations of the genes including copy number variation and methylation. We further constructed a prognostic gene model using these CRGs and built the risk score formula. Univariate and multivariate Cox regression was applied to validate the risk score as an independent prognostic factor. The prognostic model was validated using 63 UVM samples from the GSE22138 cohort, an independent validation data set. Based on the risk scores for 80 patients with UVM from TCGA, we categorized the patients into high- and low-risk groups. Differentially expressed genes (DEGs) between groups were enriched in allograft rejection, hypoxia, glycolysis, TNFα signaling via NF-κB, and interferon-γ responses via Gene set enrichment analysis (GSEA). CD8 T cells and exhausted T cells were notably enriched in the high-risk group. In conclusion, the alteration of CRGs is related to patients with UVM, and the constructed CRG-related model may be helpful to predict the prognosis of such patients.

Pharmacological attenuation of melanoma by tryptanthrin pertains to the suppression of MITF-M through MEK/ERK signaling axis.

Melanoma is the most aggressive among all types of skin cancers. The current strategies against melanoma utilize BRAFV600E, as a focal point for targeted therapy. However, therapy resistance developed in melanoma patients against the conventional anti-melanoma drugs hinders the ultimate benefits of targeted therapies. A major mechanism by which melanoma cells attain therapy resistance is via the activation of microphthalmia-associated transcription factor-M (MITF-M), the key transcription factor and oncogene aiding the survival of melanoma cells. We demonstrate that tryptanthrin (Tpn), an indole quinazoline alkaloid, which we isolated and characterized from Wrightia tinctoria, exhibits remarkable anti-tumor activity towards human melanoma through the down-regulation of MITF-M. Microarray analysis of Tpn-treated melanoma cells followed by a STRING protein association network analysis revealed that differential expression of genes in melanoma converges at MITF-M. Furthermore, in vitro and in vivo studies conducted using melanoma cells with differential MITF-M expression status, endogenously or ectopically, demonstrated that the anti-melanoma activity of Tpn is decisively contingent on its efficacy in down-regulating MITF-M expression. Tpn potentiates the degradation of MITF-M via the modulation of MEK1/2-ERK1/2-MITF-M signaling cascades. Murine models demonstrate the efficacy of Tpn in attenuating the migration and metastasis of melanoma cells, while remaining pharmacologically safe. In addition, Tpn suppresses the expression of mutated BRAFV600E and inhibits Casein Kinase 2α, a pro-survival enzyme that regulates ERK1/2 homeostasis in many tumor types, including melanoma. Together, we point to a promising anti-melanoma drug in Tpn, by virtue of its attributes to impede melanoma invasion and metastasis by attenuating MITF-M.

Four Immune Modulating Genes in Primary Melanoma That Predict Metastatic Potential

IntroductionHistologic characteristics cannot adequately predict which patients are at risk of developing metastatic disease after excision of primary cutaneous melanoma. The aim of this study was to identify immunomodulatory genes in primary tumors associated with development of distant metastases. Materials and methodsThirty-seven patients with primary melanoma underwent surgical excision. RNA was extracted from the primary tumor specimens. cDNA was synthesized and used with Human Gene Expression microarray. Differential expression of 74 immunomodulatory genes was compared between patients who developed distant metastases and those who did not. ResultsSix of 37 patients developed distant metastases during the time of the study. Differential expression of microarray data showed upregulation of four immunomodulatory genes in this group. These four genes—c-CBL, CD276, CXCL1, and CXCL2—were all significantly overexpressed in the metastatic group with differential expression fold change of 1.15 (P = 0.01), 1.16 (P = 0.04), 2.51 (P < 0.001), and 1.68 (P < 0.02), respectively. CXCL1 had particularly high predictive value with an area under the curve of 0.80. Multivariate analysis showed only expression of CXCL1 (P = 0.01) remains predictive of distant metastases in melanoma patients. This result was confirmed using quantitative real-time polymerase chain reaction. ConclusionsCXCL1, CXCL2, c-CBL, and CD276 are immunomodulatory genes present in primary melanoma that are strongly associated with development of metastatic disease. Identification of their presence, particularly CXCL1, in the primary tumor could be used as a predictor of future risk of metastatic disease and thereby to identify patients who might benefit early from immunotherapy.

Differential Gene Expression and Methylation Analysis of Melanoma in TCGA Database to Further Study the Expression Pattern of KYNU in Melanoma

Background: The aim of this study was to analyze and compare melanoma gene expression profiles in TCGA database through the application of different genes to explore the pathogenesis of melanoma. Furthermore, we confirmed the extent of the role of KYNU in melanoma and whether it can be a potential target for the diagnosis and treatment of melanoma. Methods: The gene expression profiles of melanoma samples were downloaded from TCGA database, and matrix files were synthesized to screen differential genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis and GCDA broad institute were used to analyze common gene locus mutations and expression changes in melanoma, as well as methylation. In addition, the expression patterns of KYNU in melanoma were quantified by immunohistochemistry, Western blotting, qRT-PCR, software such as GEO DataSets and the Human Protein Atlas, and meta-analysis of skin diseases. KYNU was overexpressed in keratinocytes (HaCaT and HEKα) and melanoma cells (A375 and H1205-lu). CFDA-SE, Annexin V–PI double staining, and PI single staining were used to investigate the mechanism of KYNU in melanoma and its effects on melanoma proliferation, apoptosis, invasion, and migration. Results: The main signaling pathways involved in melanoma were EGF/EGFR–RAS–BRAF–MEK–ERK–CyclinD1/CDK4, Ras–PI3K–PTEN–PKB/AKT, and p14/p16 (CDKN2A)–MDM2–p53–p21–cyclinD1/CDK4/6–Rb/E2F. Moreover, MITF, KIT, CDH1. NRAS, AKT1, EGFR, TP53, KIT, and CDK4 were elevated in melanoma, whereas PTEN, cAMP, and BCL2 were reduced in melanoma. The copy number of tumor-promoting genes increased, while the copy number of tumor suppressor genes decreased. Changes in the copy number of the above tumor genes enriched in chromosomes were found through SNP gene mutations. The genes whose expression was negatively regulated by DNA methylation in melanoma included KRT18, CDK2, JAK3, BCL2, MITF, MET, CXCL10, EGF, SOX10, SOCS3, and KIT. The mutation rate of KYNU was high according to TCGA database. The KYNU level was decreased in melanoma. Overexpression of KYNU can promote changes in apoptotic BCL-2, metabolic KYN, 3-HAA, invasion and migration MMP9, E-cadherin, and other related proteins in melanoma. Fluorescence staining and flow analysis showed that a slower proliferation rate led to a stronger fluorescence intensity. In melanoma tumor cells with a low expression of KYNU, overexpression of KYNU could promote tumor cell apoptosis. IL-10 induced immunoregulatory changes in melanoma. The expression of MMP9 and AMPK decreased in A375, but the change in BCL-2 was not obvious. The expression of BCL-2 decreased significantly in H1205-lu. A375 showed cell-cycle arrest, indicating that IL-10 could slow down the cell cycle of melanoma. Conclusions: These results provide insights into the pathologic mechanisms of melanoma target genes and KYNU as a biomarker and potential therapeutic factor for melanoma.

Expression Patterns of Necroptosis-Related Genes: Predicting Prognosis and Immunotherapeutic Effects in Cutaneous Melanoma.

Background Increasing evidence has shown a strong correlation between necroptosis and antitumor immunity. However, precise expression patterns of necroptosis-related genes in cutaneous melanoma (CM) have not been clearly elucidated nor have their effects on the immune cell infiltration in the tumor microenvironment. Method We investigated the expression patterns of necroptosis-related genes of individuals with cutaneous melanoma based on 67 necroptotic genes and methodically associated the expression patterns with the comprehensive characterization of tumor immune microenvironment. Using principal component analysis methods, the NRG score was developed to quantify the expression patterns of necroptotic genes in CM patients. Result Three different necroptotic subtypes were determined with marked survival differences, showing distinct characteristics of immune cell infiltration. The high NRG score group with comprehensive immunosuppression was characterized by the worse immunotherapeutic efficacy and the poor prognosis, while the low NRG score group indicated a robust activation of immune function and a better response to immunotherapy, which may be responsible for a better prognosis. Furthermore, the predictive ability of the NRG score on prognosis and immunotherapeutic benefits was further revalidated using the other independent datasets of cutaneous melanoma. The results indicated that patients with low NRG scores exhibited prolonged survival. Surprisingly, all patients with CM with clinical response, including complete response/partial response, belonged to the low NRG score group. Conclusion Our present work revealed the close association between expression patterns of necroptosis-associated genes and tumor immune microenvironment. NRG score can serve as a potential predictor to independently assess patients' prognosis with CM and effectively estimate the response to immunological therapy, thus facilitating the identification of appropriate candidates with CM for immunotherapy and the formulation of individualized therapeutic approaches.

Bioinformatic analysis identifies epidermal development genes that contribute to melanoma progression.

Several diagnostic and prognostic markers for melanoma have been identified in last few years. However, their actual contribution to melanoma progression have not been investigated in detail. This study was aimed to identify genes, biological processes, and signaling pathways implicated in melanoma progression by applying bioinformatics analysis. We identified nine differentially expressed genes (DEGs) (IL36RN, KRT6A, KRT6B, KRT16, S100A7, SPRR1A, SPRR1B, SPRR2B, and KLK7) that were upregulated in primary melanoma compared with metastatic melanoma in all five datasets analyzed. All these genes except IL36RN, both form a protein-protein interaction network and have cellular functions associated with constitutive processes of keratinocytes. Thus, they were generically termed Epidermal Development and Cornification (EDC) genes. The differential expression of these genes in primary and metastatic melanoma was confirmed in the TCGA-SKCM cohort. High expression of the EDC genes correlated with reduced tumor thickness in primary melanoma and shorter survival in metastatic melanoma. Analysis of DEGs from primary melanoma patients displaying high or low expression of all eight EDC revealed that the upregulated genes are enriched in biological process related to cell migration, extracellular matrix organization, invasion, and Epithelial-Mesenchymal Transition. Further analysis of enriched curated oncogenic genesets together with RPPA data of phosphorylated proteins revealed the activation of MEK, ATF2, and EGFR pathways in tumors displaying high expression of EDC genes. Thus, EDC genes may contribute to melanoma progression by promoting the activation of MEK, ATF2, and EGFR pathways together with biological processes associated with tumor aggressiveness.

Correlation of preferentially expressed antigen of melanoma (PRAME) gene expression with clinical characteristics in acute leukemia patients

BackgroundPreferentially expressed antigen of melanoma (PRAME) gene is regularly overexpressed in acute leukemia (AL) and other malignant diseases which are recognized by human leucocyte antigen (HLA-24) located in the human chromosome of 22q11 coded by 509 amino acids. To rule out the PRAME gene expression in AL patients and its correlation with clinical characteristics in the Indian population set up by RT-qPCR. ResultsA total of 42 samples collected, 29 (69.4%) were males, and 13 (30.95%) were females, with a mean and standard deviation for age were 39.07 ± 22.22 years. Of which AML were of 22 (52.38%) cases, ALL were of 14 (33.33%) cases, and 6 (14.2%) cases which included other forms of leukemia. PRAME gene expression was highly expressed in thirty-three 27 (64.28%) AL patients compared to the least expression in healthy individuals. No significant difference between the different forms of AL (p=0.3203) was observed. Cytogenetic analysis of normal karyotype (NK), abnormal karyotype (Ab. K), and culture failure (CF) displayed statistical non-significance (p=0.5801). Among cytogenetic abnormalities obtained, no significant differences between the groups were observed (p=0.8507). Chloride, potassium, and absolute lymphocyte count (ALC) was found to be statistically significant with p=0.0038**, p=0.0358*, and p=0.0216*, respectively, between all other clinical characteristics. There was no correlation between the PRAME gene expression and clinical parameters. ConclusionPRAME gene expression in AL patients was highly expressed, comparable to studies reported globally with significant cytogenetic results. PRAME gene could be used as a potential diagnostic marker for monitoring the malignancies and minimal residual disease in AL.

Analysis of Melanoma Gene Expression Signatures at the Single-Cell Level Uncovers 45-Gene Signature Related to Prognosis.

Since the current melanoma clinicopathological staging system remains restricted to predicting survival outcomes, establishing precise prognostic targets is needed. Here, we used gene expression signature (GES) classification and Cox regression analyses to biologically characterize melanoma cells at the single-cell level and construct a prognosis-related gene signature for melanoma. By analyzing publicly available scRNA-seq data, we identified six distinct GESs (named: “Anti-apoptosis”, “Immune cell interactions”, “Melanogenesis”, “Ribosomal biogenesis”, “Extracellular structure organization”, and “Epithelial-Mesenchymal Transition (EMT)”). We verified these GESs in the bulk RNA-seq data of patients with skin cutaneous melanoma (SKCM) from The Cancer Genome Atlas (TCGA). Four GESs (“Immune cell interactions”, “Melanogenesis”, “Ribosomal biogenesis”, and “Extracellular structure organization”) were significantly correlated with prognosis (p = 1.08 × 10−5, p = 0.042, p = 0.001, and p = 0.031, respectively). We identified a prognostic signature of melanoma composed of 45 genes (MPS_45). MPS_45 was validated in TCGA-SKCM (HR = 1.82, p = 9.08 × 10−6) and three other melanoma datasets (GSE65904: HR = 1.73, p = 0.006; GSE19234: HR = 3.83, p = 0.002; and GSE53118: HR = 1.85, p = 0.037). MPS_45 was independently associated with survival (p = 0.002) and was proved to have a high potential for predicting prognosis in melanoma patients.

Abstract 2358: Impact pathway analysis of FOXC2-correlated genes in melanoma: A meta-analysis approach to understand the tumor-promoting functions of the FOXC2 transcription factor

Abstract Melanoma is a cancer that arises from pigment-producing melanocytes in the skin. Because of the increasing incidence of melanoma and its resistance to many therapies, insights into melanoma progression are needed. FOXC2, a member of the forkhead box family of transcription factors, has been shown to have oncogenic activity in many epithelial cancer types, and we have recently reported a role for FOXC2 in melanoma progression as well. To gain insight into the role of FOXC2 in melanoma progression, we analyzed publicly available RNA-sequencing data from tumor biopsies of melanoma patients, including a cohort of 479 patients catalogued in The Cancer Genome Atlas as well as additional cohorts from other independent studies. We performed Spearman correlation analyses to identify genes positively and negatively co-expressed with FOXC2, and we performed Impact Pathway Analysis and Gene Ontology Analysis to determine which biological pathways and processes are most impacted by FOXC2-associated genes in melanoma. Our data highlight critical pathways impacted by the expression of FOXC2, and they identify specific genes influenced by this transcription factor that provide mechanistic insight into the oncogenic activity of FOXC2 in melanoma and potentially other cancers. Citation Format: James B. Wall, Kristian M. Hargadon. Impact pathway analysis of FOXC2-correlated genes in melanoma: A meta-analysis approach to understand the tumor-promoting functions of the FOXC2 transcription factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2358.

Abstract 3818: Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma

Abstract Introduction: Loss-of-heterozygosity (LOH) events in chromosome 6p, comprising the human leukocyte antigen (HLA) genes, have been reported in about 10% of cutaneous melanoma (compared to 20-40% of squamous cell carcinomas), while copy number gains in this region have been observed in over 50% of melanoma. Recent studies focused in HLA allelic loss have been restricted to DNA-based approaches, and have not been validated orthogonally at the RNA or protein levels. Here, using clinical melanoma biopsies and patient-derived melanoma cell lines, we show that genetic alterations in HLA genes results in imbalanced allele expression, subsequently skewing antigen presentation by melanoma cells. Methods: Whole exome and RNA sequencing (WES, RNAseq) analyses were performed on 760 melanoma biopsies and 60 patient-derived melanoma cell lines. Patient-matched normal WES was used to perform HLA haplotyping across Class I and II HLA genes. Tumor WES was analyzed for copy number alterations in chromosome 6, identifying which alleles were lost or gained. Differential expression of HLA alleles was quantified in tumor RNAseq, correlating the allelic imbalance at the DNA and RNA levels. Melanoma cell lines heterozygous for HLA-A*02, A*03, and A*24 were analyzed by flow cytometry for surface-level HLA protein expression using allele-specific antibodies to quantify allelic densities and compare the imbalance of HLA-A alleles at the DNA, RNA, and protein levels. Results: Across 760 melanoma biopsies, copy number alterations in chromosome 6p were identified in 76% of tumors; 12% had LOH, and 54% had copy number gains that resulted in imbalanced copies of alleles. In paired tumor WES and RNAseq (N=682), genetic imbalance was correlated with imbalanced expression of HLA alleles in the classical Class I HLA genes (Spearman rho=0.64-0.7; p=2.2e-16); this association was strengthened in tumors with high tumor cellularity, and was not associated with the total expression of the HLA genes.These patterns were explored in a 60 patient-derived melanoma cell lines with matched tumor WES and RNAseq, confirming that alleles gained at the genetic level were also expressed at higher levels than alleles that were not gained or lost. In 10 cell lines heterozygous for either HLA-A*02, A*03, or A*24, allelic imbalance at the DNA and RNA level resulted in correlative imbalanced surface presentation of alleles at the protein level. Conclusions: Evaluation of paired tumor WES and RNAseq revealed orthogonal validation of HLA allelic imbalance, and analysis in cell lines suggested that these patterns were likely tumor intrinsic. Experimental validation of these findings at the protein level suggests that antigen presentation density can be modulated by chromosomal gains, and not just allelic loss, in HLA genes. This knowledge is important for the design of cancer vaccines or T cell therapies targeting neoantigens presented by HLA class I complexes. Citation Format: Katie M. Campbell, Justin Saco, Egmidio Medina, Meelad Amouzgar, Shannon M. Pfeiffer, Cynthia R. Gonzalez, Gabriela Steiner, Ameya Champhekar, Cristina Puig Saus, Jesse Zaretsky, Gabriel Abril Rodriguez, Agustin Vega-Crespo, Ignacio Baselga Carretero, Mito Tariveranmoshabad, Anusha Kalbasi, Christine Spencer, Zachary L. Skidmore, Malachi Griffith, Obi L. Griffith, Daniel K. Wells, Antoni Ribas. Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3818.

Abstract 1388: Mutations in transcriptional activators of MHC I antigen presentation, NLRC5 and CIITA, are mutually exclusive and have different effects on melanoma gene transcripts

Abstract Response to many immunotherapies depends on effective recognition and eradication of tumor cells by the immune system. This is mediated by the successful presentation of tumor-specific antigens on major histocompatibility complex class I (MHC I) molecules on the surface of tumor cells. Studies have focused on alterations in MHC I complex proteins (e.g. HLA, B2M) and gene regulatory elements (e.g. NLRC5). However, the impact on tumor response of individual defects in components regulating and composing the MHC I antigen processing and presentation machinery (APM) have not been extensively studied. Using a harmonized dataset of six clinical trials of patients with melanoma treated with ICB, whole-exome sequencing (WES, n= 244) and RNA sequencing (RNAseq, n= 426) data from untreated tumor samples were assessed to identify mutations in genes associated with MHC I APM and their regulation. Somatic alterations were correlated with clinical response to ICB and with gene expression profiles in tumor-matched RNAseq data. Mutations in MHC I and MHC I-related genes were found in 63% of patients (n= 154/244). The most recurrently mutated genes were NCAM1 (11%, n=28), TRIM48 (7%, n=17), and CAMK2B (6.6%, n=16), and the transcriptional regulators NLRC5 (9.4%, n=23) and CIITA (9.4%, n=23). Mutations in NLRC5 and CIITA trended toward being mutually exclusive from one another (Fisher’s exact test, p= 0.157) and mutations were overrepresented in tumors with stable or progressive disease following ICB therapy. In addition, lower expression of NLRC5 and CIITA were associated with stable or progressive disease status. NLRC5-mutant tumors that progressed following ICB therapy trended towards lower expression of transcriptional target HLA-A (Kruskal-Wallis, p=0.27); however, there was no difference in HLA-A expression across NLRC5-wildtype tumors. For class II transactivating factor, CIITA, which can induce MHC I expression, mutational status was not associated with HLA-A expression across all clinical response groups. There are genetic alterations spanning MHC I and MHCI-associated genes that may be responsible for inactivating class I antigen presentation by melanoma cells. The impact of these alterations may result in varied clinical responses to ICB therapies. The landscape of these alterations needs to be further delineated to understand how tumors are directly evading class I-mediated recognition and elimination. Citation Format: Mildred Galvez, Katie M. Campbell, Egmidio Medina, Antoni Ribas. Mutations in transcriptional activators of MHC I antigen presentation, NLRC5 and CIITA, are mutually exclusive and have different effects on melanoma gene transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1388.

Clinical features and prognosis of childhood B-lineage acute lymphoblastic leukemia expressing the PRAME gene

To study the clinical and prognostic significance of the preferentially expressed antigen of melanoma (PRAME) gene in the absence of specific fusion gene expression in children with B-lineage acute lymphoblastic leukemia (B-ALL). A total of 167 children newly diagnosed with B-ALL were enrolled, among whom 70 were positive for the PRAME gene and 97 were negative. None of the children were positive for MLL-r, BCR/ABL, E2A/PBX1, or ETV6/RUNX1. The PRAME positive and negative groups were analyzed in terms of clinical features, prognosis, and related prognostic factors. Compared with the PRAME negative group, the PRAME positive group had a significantly higher proportion of children with the liver extending >6 cm below the costal margin (P<0.05). There was a significant reduction in the PRAME copy number after induction chemotherapy (P<0.05). In the minimal residual disease (MRD) positive group after induction chemotherapy, the PRAME copy number was not correlated with the MRD level (P>0.05). In the MRD negative group, there was also no correlation between them (P>0.05). The PRAME positive group had a significantly higher 4-year event-free survival rate than the PRAME negative group (87.5%±4.6% vs 73.5%±4.6%, P<0.05), while there was no significant difference between the two groups in the 4-year overall survival rate (88.0%±4.4% vs 85.3%±3.8%, P>0.05). The Cox proportional-hazards regression model analysis showed that positive PRAME expression was a protective factor for event-free survival rate in children with B-ALL (P<0.05). Although the PRAME gene cannot be monitored as MRD, overexpression of PRAME suggests a good prognosis in B-ALL.

Genetic risk factors in melanoma etiopathogenesis and the role of genetic counseling: A concise review

Melanoma is a highly aggressive cancer originating from melanocytes. Its etiopathogenesis is strongly related to genetic, epigenetic, and environmental factors. Melanomas encountered in clinical practice are predominantly sporadic, whereas hereditary melanomas account for approximately 10% of the cases. Hereditary melanomas mainly develop due to mutations in the cyclin-dependent kinase 2A (CDKN2A) gene, which encodes two tumor suppressor proteins involved in the cell cycle regulation. CDKN2A, along with CDK4, TERT, and POT1 genes, are high-risk genes for melanoma. Among the genes that carry a moderate risk are MC1R and MITF, whose protein products are involved in melanin synthesis. The environment also contributes to the development of melanoma. Patients at risk of melanoma should be offered genetic counseling to discuss genetic testing options and the importance of skin UV protection, avoidance of sun exposure, and regular preventive dermatological examinations. Although cancer screening cannot prevent the development of the disease, it allows for early diagnosis when the survival rate is the highest.

Comprehensive Gene Expression Analysis to Identify Differences and Similarities between Sex- and Stage-Stratified Melanoma Samples.

Overall lower incidence and better prognosis are observed in female melanoma patients compared to males. As sex and stage differences in the context of melanoma gene expression are understudied, we aim to highlight them through statistical analysis of melanoma gene expression datasets. Data from seven online datasets, including normal skin, commonly acquired nevi, and melanomas, were collected and analyzed. Sex/stage-related differences were assessed using statistical analyses on survival, gene expression, and its variability. Significantly better overall survival in females was observed in stage I, II but not in stage III. Gene expression variability was significantly different between stages and sexes. Specifically, we observed a significantly lower variability in genes expressed in normal skin and nevi in females compared to males, as well as in female stage I, II melanomas. However, in stage III, variability was lower in males. Similarly, class comparison showed that the gene expression differences between sexes are most notable in non-melanoma followed by early-stage-melanoma samples. Sexual dimorphism is an important aspect to consider for a holistic understanding of early-stage melanomas, not only from the tumor characteristics but also from the gene expression points of view.

Integrative Genomic Profiling Uncovers Therapeutic Targets of Acral Melanoma in Asian Populations.

Acral melanoma is the major subtype of melanoma seen in Asian patients with melanoma and is featured by its insidious onset and poor prognosis. The genomic study that elucidates driving mutational events is fundamental to the development of gene-targeted therapy. However, research on genomic profiles of acral melanoma in Asian patients is still sparse. We carried out whole-exome sequencing (WES) on 60 acral melanoma lesions (with 55 primary samples involved), targeted deep sequencing in a validation cohort of 48 cases, RNA sequencing in 37 acral melanoma samples (all from the 60 undergoing WES), and FISH in 233 acral melanoma specimens (54 of the 60 undergoing WES included). All the specimens were derived from Asian populations. BRAF, NRAS, and KIT were discerned as significantly mutated genes (SMG) in acral melanoma. The detected COSMIC signature 3 related to DNA damage repair, along with the high genomic instability score, implied corresponding pathogenesis of acral melanoma. Moreover, the copy number gains of EP300 were associated with the response of acral melanoma to targeted therapy of A485 (a p300 inhibitor) and immune checkpoint blockade treatment. In addition, the temporal order in mutational processes of the samples was reconstructed, and copy-number alterations were identified as early mutational events. Our study provided a detailed view of genomic instability, potential therapeutic targets, and intratumoral heterogeneity of acral melanoma, which might fuel the development of personalized strategies for treating acral melanoma in Asian populations.

Impact of CYLD on chromatin structure and histone methylation in malignant melanoma.

The tumor suppressive role of CYLD lysine 63 deubiquitinase (CYLD) is known in melanoma. To the best of our knowledge, however, the precise mechanism underlying the tumor suppressive function of CYLD has yet to be clarified. In the present study, a novel melanoma mouse model was generated, which revealed accelerated tumor growth in Cyld-knockout (Cyld−/−) compared with Cyld-wild-type (Cyld+/+) mice. To determine the underlying molecular mechanism, mutation analysis of primary tumor-derived cell lines from Cyld+/+ and Cyld−/− mice was performed using RNA sequencing data. Variant calling revealed no common mutations in Cyld−/− compared with Cyld+/+ cells. Thus, the epigenetic processes influencing development and progression of melanoma were investigated. Initial analysis of expression pattern of known hypermethylated genes in melanoma (suppressor of cytokine signalling, methylthioadenosine phosphorylase, cadherin 1) in the presence or absence of 5′-Aza-deoxyctidine treatment revealed that CYLD does not play a key role in DNA methylation. Chromatin accessibility and histone H3 modification assay uncovered a role of CYLD in the formation of chromatin structure. Subsequent inhibitor experiments confirmed the effect of CYLD on H3K9me2 level associated with heterochromatin. Furthermore, enhanced H3K9 dimethylation in Cyld−/− melanoma cells was associated with upregulation of euchromatic histone lysine methyltransferase 2 (EHMT2). Moreover, the specific inhibitor of EHMT2, CM272, resulted in decreased proliferation and relaxation of compact chromatin in Cyld-deficient melanoma cells. These results reveal a novel role of CYLD in histone methylation and chromatin packaging.

Construction and Validation of a Ferroptosis-Related Prognostic Signature for Melanoma Based on Single-Cell RNA Sequencing.

Melanoma, the deadliest type of skin cancer, is on the rise globally. The generally poor prognosis makes melanoma still an enormous public health problem. Ferroptosis is a newly emerging form of iron-dependent regulated cell death, which has been implicated in the development and treatment of several tumors. However, whether there is a connection between ferroptosis-related genes and the prognosis of melanoma patients remains an enigma. In the present study, we identified a ferroptosis-related genes signature to predict the prognosis of melanoma patients by analyzing single-cell RNA-sequencing data from the Gene Expression Omnibus (GEO). Single-cell trajectory analysis was performed to explore malignant differentiation. CellChat was used to investigate intercellular communications in melanoma. Collectively, a novel four-gene signature (CP, MAP1LC3A, transferrin, and TP53) was constructed for prognosis prediction. COX proportional hazards regression analysis showed that the established ferroptosis-associated risk model was an independent prognostic predictor for melanoma patients (HR = 2.3293; 95%CI 1.1528–4.706) (p < 0.018). Patients with low-risk scores had significantly better overall survival (OS) than those with high-risk scores in The Cancer Genome Atlas, GSE59455, and GSE22153 dataset (p = 0.0015, p = 0.031, p = 0.077). Furthermore, the gene expression level of the four genes were verified in multistrain melanoma cell lines and normal human epidermal melanocytes (NHEM). The protein expression level of the four genes in clinical samples were further verified in the Human Protein Atlas (HPA) databases. Taken together, our study identified the prognostic significance of the ferroptosis-related genes in melanoma and developed a novel four-gene prognostic signature, which may shed light on the prognostic assessment and clinical decision making for melanoma patients.

Establishing a Prognostic Model Based on Ulceration and Immune Related Genes in Melanoma Patients and Identification of EIF3B as a Therapeutic Target.

Ulceration and immune status are independent prognostic factors for survival in melanoma patients. Herein univariate Cox regression analysis revealed 53 ulcer-immunity-related DEGs. We performed consensus clustering to divide The Cancer Genome Atlas (TCGA) cohort (n = 467) into three subtypes with different prognosis and biological functions, followed by validation in three merged Gene Expression Omnibus (GEO) cohorts (n = 399). Multiomics approach was used to assess differences among the subtypes. Cluster 3 showed relatively lesser amplification and expression of immune checkpoint genes. Moreover, Cluster 3 lacked immune-related pathways and immune cell infiltration, and had higher proportion of non-responders to immunotherapy. We also constructed a prognostic model based on ulceration and immune related genes in melanoma. EIF3B was a hub gene in the intersection between genes specific to Cluster 3 and those pivotal for melanoma growth (DepMap, https://depmap.org/portal/download/). High EIF3B expression in TCGA and GEO datasets was related to worst prognosis. In vitro models revealed that EIF3B knockdown inhibited melanoma cell migration and invasion, and decreased TGF-β1 level in supernatant compared with si-NC cells. EIF3B expression was negatively correlated with immune-related signaling pathways, immune cell gene signatures, and immune checkpoint gene expression. Moreover, its low expression could predict partial response to anti-PD-1 immunotherapy. To summarize, we established a prognostic model for melanoma and identified the role of EIF3B in melanoma progression and immunotherapy resistance development.

The histone demethylase PHF8 regulates TGFβ signaling and promotes melanoma metastasis.

The contribution of epigenetic dysregulation to metastasis remains understudied. Through a meta-analysis of gene expression datasets followed by a mini-screen, we identified Plant Homeodomain Finger protein 8 (PHF8), a histone demethylase of the Jumonji C protein family, as a previously unidentified prometastatic gene in melanoma. Loss- and gain-of-function approaches demonstrate that PHF8 promotes cell invasion without affecting proliferation in vitro and increases dissemination but not subcutaneous tumor growth in vivo, thus supporting its specific contribution to the acquisition of metastatic potential. PHF8 requires its histone demethylase activity to enhance melanoma cell invasion. Transcriptomic and epigenomic analyses revealed that PHF8 orchestrates a molecular program that directly controls the TGFβ signaling pathway and, as a consequence, melanoma invasion and metastasis. Our findings bring a mechanistic understanding of epigenetic regulation of metastatic fitness in cancer, which may pave the way for improved therapeutic interventions.

Disruption of LTBP4 Inhibition-Induced TGFβ1 Activation Promoted Cell Proliferation and Metastasis in Skin Melanoma by Inhibiting the Activation of the Hippo-YAP1 Signaling Pathway.

Melanoma is a malignant tumor derived from melanocytes, which is the most fatal skin cancer. The present study aimed to explore and elucidate the candidate genes in melanoma and its underlying molecular mechanism. A total of 1,156 differentially expressed genes were obtained from the GSE46517 dataset of Gene Expression Omnibus database using the package “limma” in R. Based on two algorithms (LASSO and SVM-RFE), we obtained three candidate DEGs (LTBP4, CDHR1, and MARCKSL1). Among them, LTBP4 was identified as a diagnostic marker of melanoma (AUC = 0.985). Down-regulation of LTBP4 expression was identified in melanoma tissues and cells, which predicted poor prognosis of patients with melanoma. Cox analysis results discovered that LTBP4 with low expression was an independent prognostic factor for overall survival in patients with melanoma. LTBP4 inhibition reduced cell apoptosis and promoted cell proliferation and metastasis. These changes were correlated with the expression levels of caspase-3, Ki67 and E-cadherin. Further, as indicated by tumor formation study of nude mice, LTBP4 silencing improved the tumorigenic ability of melanoma cells. Knockdown of LTBP4 increased the percentage of active TGFβ1 secreted by melanoma cells. CTGF, Gyr61, and Birc5 expression levels were reduced, YAP1 phosphorylation was inhibited, and YAP1 was translocated from the cytoplasm to the nucleus in melanoma cells treated with TGF-β1. These effects were reversed by LTBP4 overexpression. As evidenced by immunofluorescent staining, Western blotting and luciferase reporter assay, LTBP4 overexpression activated the Hippo signaling pathway, which was characterized by the increased nuclear-cytoplasmic translocation of YAP1 and the enhanced phosphorylation of YAP1, MST1, and MOB1. In addition, the effects of LTBP4 overexpression on inhibiting CTGF, Cyr61 and Birc5 expression, promoting the apoptosis, and inhibiting the metastasis and proliferation of melanoma cells were reversed by the overexpression of YAP1 or MST1. In conclusion, the LTBP4-TGFβ1-Hippo-YAP1 axis is a critical pathway for the progression of skin melanoma.