Interleukin enhancer-binding factor 2 promotes cell proliferation and DNA damage response in metastatic melanoma.

BACKGROUND: 1q21.3 amplification, which is frequently observed in metastatic melanoma, is associated with cancer progression. Interleukin enhancer-binding factor 2 (ILF2) is located in the 1q21.3 amplified region, but its functional role or contribution to tumour aggressiveness in cutaneous melanoma is unknown. METHODS: In silico analyses were performed using the TCGA SKCM dataset with clinical annotations and three melanoma microarray cohorts from the GEO datasets. RNA in situ hybridisation and immunohistochemistry were utilised to validate the gene expression in melanoma tissues. Four stable melanoma cell lines were established for in vitro ILF2 functional characterisation. RESULTS: Our results showed that the ILF2 copy number variation (CNV) is positively correlated with ILF2 mRNA expression (r = 0.68, p < .0001). Additionally, ILF2 expression is significantly increased with melanoma progression (p < .0001), and significantly associated with poor overall survival for metastatic melanoma patients (p = .026). The overexpression of ILF2 (ILF2-OV) promotes proliferation in metastatic melanoma cells, whereas ILF2 knockdown decreases proliferation by blocking the cell cycle. Mechanistically, we demonstrated the interaction between ILF2 and the splicing factor U2AF2, whose knockdown reverses the proliferation effects mediated by ILF2-OV. Stage IIIB-C melanoma patients with high ILF2-U2AF2 expression showed significantly shorter overall survival (p = .024). Enhanced ILF2/U2AF2 expression promotes a more efficient DNA-damage repair by increasing RAD50 and ATM mRNA expression. Paradoxically, metastatic melanoma cells with ILF2-OV were more sensitive to ATM inhibitors. CONCLUSION: Our study uncovered that ILF2 amplification of the 1q21.3 chromosome is associated with melanoma progression and triggers a functional downstream pathway in metastatic melanoma promoting drug resistance.

Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival.

The role of post-translational modifications (PTM) of the key epigenetic factor DNMT1 protein has not been well explored in cutaneous metastatic melanoma progression. The acetylated DNMT1 (ac-DNMT1) protein level was assessed using an anti-acetylated lysine antibody in a clinically annotated melanoma patient tumor specimen cohort. In this study, we showed that surgically resected tumors have significantly higher DNMT1 protein expression in metastatic melanoma (stage III metastasis n = 17, p = 0.0009; stage IV metastasis n = 164, p = 0.003) compared to normal organ tissues (n = 19). Additionally, reduced ac-DNMT1 protein levels were associated with melanoma progression. There was a significant inverse correlation between ac-DNMT1 and DNMT1 protein levels in stage IV metastatic melanoma (r = -0.18, p = 0.02, n = 164). Additionally, ac-DNMT1 protein levels were also significantly positively correlated with TIP60 (r = 0.6, p < 0.0001) and USP7 (r = 0.74, p < 0.0001) protein levels in stage IV metastatic melanoma (n = 164). Protein analysis in metastatic melanoma tumor tissues showed that with high ac-DNMT1 (p = 0.006, n = 59), or concurrent high ac-DNMT1 with low DNMT1 (p = 0.05, n = 27), or high TIP60 (p = 0.007, n = 41), or high USP7 (p = 0.01, n = 48) consistently showed better 4-year melanoma-specific survival (MSS). Multivariate Cox proportional hazard analysis showed that ac-DNMT1 level is a significant independent factor associated with MSS (HR, 0.994; 95% confidential interval (CI), 0.990-0.998; p = 0.002). These results demonstrated that low ac-DNMT1 levels may represent an important regulatory factor in controlling metastatic melanoma progression and a promising factor for stratifying aggressive stage IV metastasis.

AIM1 and LINE-1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome.

Aberrations in the methylation status of noncoding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in long interspersed nucleotide element-1 (LINE-1) and absent in melanoma-1 (AIM1; 6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissue (PEAT; n = 133) and in melanoma patients' serum (n = 56). LINE-1 U-Index (hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma (n = 100) was significantly higher than that of normal skin (n = 14) and nevi (n = 12; P = 0.0004). LINE-1 U-Index level was elevated with increasing American Joint Committee on Cancer (AJCC) stage (P<0.0001). AIM1 promoter hypermethylation was found in higher frequency (P = 0.005) in metastatic melanoma (65%) than in primary melanomas (38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival (DFS) and overall survival (OS) in stage I/II patients (P = 0.017 and 0.027, respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS (P = 0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III (n = 20) and stage IV (n = 36) patients compared with healthy donors (n = 14; P = 0.022). Circulating methylated AIM1 was detected in patients' serum and was predictive of OS in stage IV patients (P = 0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients' tumors and serum.

Predictive utility of circulating methylated DNA in serum of melanoma patients receiving biochemotherapy.

PURPOSE: Currently, no validated blood-based assays accurately predict treatment response or outcome in melanoma patients. We hypothesized that methylation of tumor-related genes detected in serum DNA could predict disease outcome and therapeutic response in patients receiving concurrent biochemotherapy (BC) for metastatic melanoma. PATIENTS AND METHODS: American Joint Committee on Cancer stage IV melanoma patients (N = 50) had blood drawn before administration of BC. Patients (n = 47) were classified as BC responders or nonresponders. Responders (n = 23) demonstrated a complete or partial response following BC; nonresponders (n = 24) demonstrated progressive disease. Hypermethylation of Ras association domain family 1 (RASSF1A), retinoic acid receptor-beta2 (RAR-beta2), and O6-methylguanine DNA methyltransferase (MGMT) genes were assessed by methylation-specific polymerase chain reaction. RESULTS: Circulating methylated RASSF1A was significantly less frequent for responders (three of 23 patients; 13%) than nonresponders (10 of 24 patients; 42%; P = .028). Patients with RASSF1A, RAR-beta2, or at least one serum methylated gene had significantly worse overall survival than patients with no methylated genes (log-rank, P = .013, .021, and .01, respectively). Methylated RASSF1A was the only factor that significantly correlated with overall survival and BC response (risk ratio, 2.38; 95% CI, 1.16 to 4.86; P = .018; odds ratio = 0.21; 95% CI, 0.05 to 0.90; P = .036). CONCLUSION: Detection of circulating methylated DNA in serum can predict response to BC and disease outcome.