Frequent p16-Independent Inactivation of p14ARF in Human Melanoma

The CDKN2A/INK4A locus at chromosome 9p21, which includes two tumor suppressor genes that share a common second exon, is a critical target of inactivation in cancer biology. p16INK4A (p16) is transcribed from exons 1α, 2, and 3, with exon 3 encoding only four amino acids. p14ARF (ARF) is encoded by an alternative exon 1 (1β) and the shared exon 2. Exon 1β is located approximately 20 kb upstream of p16 exon 1α. The p16 and ARF transcripts are translated in different reading frames; thus the two proteins have no physical homology. Both proteins function as tumor suppressors, acting through different pathways: ARF via the p53 pathway (1, 2) and p16 via the retinoblastoma (RB) pathway (3). Evidence of a role for p16 in human melanoma includes frequent genetic and epigenetic alterations in human melanoma specimens (4–6) and cell lines (7), the presence of germline mutations in this gene in 10%–50% of familial melanoma cases (8–11), and data from murine models in which the specific deletion of exon 1α combined with melanocyte-specific expression of a mutant Ras transgene produced melanomas (12). In vitro studies of cells from a patient with biallelic germline mutations at 9p21 suggested that in humans it is p16, rather than ARF, that is critical in Ras-induced arrest of fibroblasts (13). The role of ARF in susceptibility to melanoma has therefore been questioned. More recent evidence, however, challenges the central role of p16 in melanomagenesis. Members of several melanoma families have been reported to share an exon 1β germline deletion (14–16) or mutation in either the coding region or splice donor site of this exon (17–19), suggesting an important role for the ARF gene in the human disease. In murine melanoma models using either a Ras transgene driven by the melanocyte-specific tyrosinase promoter or the Met ligand hepatocyte growth factor/scatter factor driven by the metallothonein promoter, it has been observed that targeted deletion of exon 1β alone leads to spontaneous melanoma. Moreover, melanomas develop in these mice with a substantially shorter latency period than similarly constructed mice with a deletion of only exon 1α (12, 20). Most recently, ARF has been demonstrated to be a regulator of melanocyte senescence, independent of p53 activity (20). These data suggest a p16-independent role for ARF in melanomagenesis. Somatic ARF inactivation has also been observed in a variety of human tumors (21–23), but no in-depth studies have, to our knowledge, directly assessed the status of the ARF gene in human melanoma tumors. Therefore, we performed a comprehensive analysis of the pattern of genetic and epigenetic alterations to the p16 and ARF tumor suppressor loci in melanoma.