RESUMO
Melanoma brain metastasis (MBM) represents a frequent complication of cutaneous melanoma. Despite aggressive multi-modality therapy, patients with MBM often have a survival rate of <1 year. Alteration in DNA methylation is a major hallmark of tumor progression and metastasis; however, it remains largely unexplored in MBM. In this study, we generated a comprehensive DNA methylation landscape through the use of genome-wide copy number, DNA methylation and gene expression data integrative analysis of melanoma progression to MBM. A progressive genome-wide demethylation in low CpG density and an increase in methylation level of CpG islands according to melanoma progression were observed. MBM-specific partially methylated domains (PMDs) affecting key brain developmental processes were identified. Differentially methylated CpG sites between MBM and lymph node metastasis (LNM) from patients with good prognosis were identified. Among the most significantly affected genes were the HOX family members. DNA methylation of HOXD9 gene promoter affected transcript and protein expression and was significantly higher in MBM than that in early stages. A MBM-specific PMD was identified in this region. Low methylation level of this region was associated with active HOXD9 expression, open chromatin and histone modifications associated with active transcription. Demethylating agent induced HOXD9 expression in melanoma cell lines. The clinical relevance of this finding was verified in an independent large cohort of melanomas (n = 145). Patients with HOXD9 hypermethylation in LNM had poorer disease-free and overall survival. This epigenome-wide study identified novel methylated genes with functional and clinical implications for MBM patients.
Assuntos
Neoplasias Encefálicas/secundário , Genes Homeobox , Proteínas de Homeodomínio/genética , Metástase Linfática , Melanoma/genética , Proteínas de Neoplasias/genética , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Estudos de Coortes , Ilhas de CpG , Metilação de DNA , Progressão da Doença , Epigênese Genética , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Melanoma/patologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Regiões Promotoras Genéticas , Neoplasias Cutâneas , Taxa de Sobrevida , Resultado do Tratamento , Melanoma Maligno CutâneoRESUMO
In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma. However, despite their growing implication in cancer, little is known about the role of long noncoding RNAs in melanoma progression. We hypothesized that copy number alterations (CNAs) of intergenic nonprotein-coding domains could help identify long intergenic noncoding RNAs (lincRNAs) associated with metastatic cutaneous melanoma. Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 (cancer susceptibility candidate 15) lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines. The locus was actively transcribed in metastatic melanoma cells, and upregulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model. In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC (American Joint Committee on Cancer) stage III lymph node metastasis. Moreover, small interfering RNA (siRNA) knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states. Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes. These findings support a key role for CASC15 in metastatic melanoma.
Assuntos
Cromossomos Humanos Par 6/genética , Regulação Neoplásica da Expressão Gênica/genética , Loci Gênicos/genética , Melanoma/genética , RNA Longo não Codificante/genética , Neoplasias Cutâneas/genética , Animais , Biópsia por Agulha , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Melanócitos/patologia , Melanoma/patologia , Camundongos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell-surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM development. The expression of CD44v6 also enhances the migration of MBM cells by hyaluronic acid and hepatocyte growth factor exposure. Additionally, CD44v6-positive MBM migration is reduced by blocking with a CD44v6-specific monoclonal antibody or knocking down CD44v6 by siRNA. ESRP1 and ESRP2 splicing factors correlate with CD44v6 expression in PRM, and ESRP1 knockdown significantly decreases CD44v6 expression. However, an epigenetic silencing of ESRP1 is observed in metastatic melanoma, specifically in MBM. In advanced melanomas, CD44v6 expression correlates with PTBP1 and U2AF2 splicing factors, and PTBP1 knockdown significantly decreases CD44v6 expression. Overall, these findings open a new avenue for understanding the high affinity of melanoma to progress to MBM, suggesting CD44v6 as a potential MBM-specific factor with theranostic utility for stratifying patients.