Higher Frequency of Hypermethylation of p16INK4A Compared to p14ARF among Cutaneous Melanoma Patients from Southern Italy

Background: Epigenetic deregulation due to p14ARF and p16INK4A promoter hypermethylation has been previously reported in many cancers, including cutaneous melanoma (CM). Nevertheless, discriminating the involvement of these genes in CM still remains an open question. Methods: p14ARF and p16INK4A were analyzed in 60 CM formalin-fixed paraffin-embedded tissue sections and in G-361 and GR-M cutaneous melanoma cell lines by methylation-specific PCR and sequencing. Gene expression was evaluated by qReal-time PCR. Cell lines were treated with demethylating agent 5-aza-2’-deoxycytidine. Results: p16INK4A gene promoter methylation was found in 36 of 60 (60%) melanoma tissues, 12 of which were heterozygous and the others homozygous. Conversely, p14ARF was found methylated in heterozygous status in 19/60 (31.67%) cases. Hypermethylation of both genes showed low frequency (10%). G-361 and GR-M cell lines were identified as homozygous methylated in p16INK4A and unmethylated in p14ARF. Loss and decrease gene expression was observed in homozygous and heterozygous status, respectively. All cells exhibited demethylation and induction of gene expression after 5-aza-dC treatment. Conclusions: Here we showed a higher frequency of methylated CpGs in p16INK4A compared to p14ARF in cutaneous melanoma, unlike other studies that have reported the opposite. As this is the first detailed investigation of the frequency of p16INK4A and p14ARF methylation in CM in Southern Italy, it is likely that the well-known high occurrence of melanoma in this region may be associated to aberrant methylation of p16INK4A. To note, CM tissue analysis facilitates a heterogeneity of results not otherwise demonstrable in cell lines.