Recurrent Patterns of Dual RB and p53 Pathway Inactivation in Melanoma

Abstract

Two growth inhibitory hurdles that must be overcome by the evolving cancer cell include pathways regulated by RB and p53. In human melanoma cells, inactivation of a single locus, CDKN2A, can lead to abrogation of both RB and p53 functionality through loss of the two CDKN2A cognate transcripts-p16 and p14ARF, respectively. We thus set out to assess how recurrent patterns of genetic injury at three critical human melanoma loci-CDKN2A, TP53, and CDK4-cooperate to disrupt both RB and p53 pathways. Overall, 77.8% of the melanoma cell lines analyzed showed genetic evidence of dual RB and p53 pathway compromise; this percentage is even higher if protein expression loss is considered. Although homozygous deletion of all three critical CDKN2A exons (exons 1 beta, 1 alpha, and 2) represent the most common mechanism, concurrent loss of CDKN2A(Exon1 alpha) and CDKN2A(Exon1 beta) and simultaneous point mutagenesis of CDK4 and TP53 reflect alternative cassettes of dual inactivation. In cell lines with isolated CDKN2A(Exon2) mutations, coincident alterations in TP53 or deletion of CDKN2A(Exon1 beta) suggest that p16 transcript may be preferentially targeted over the p14ARF transcript as additional p53 pathway lesions are recruited. Moreover, predictive modeling of CDKN2A(Exon2) missense mutations further suggests that the amino acid substitutions in this region negatively impact p16 to a greater extent than p14ARF. Taken together, our data point to a clear need in human melanoma cell lines, as in its murine counterpart, to disrupt both RB and p53 pathways and recurrent mechanisms may play into the unique genetic vulnerabilities of this tumor type.

Melanoma: from mutations to medicine

“…With the recognition that p14ARF (an exon 1b-specific transcript of CDKN2A) down-regulates HDM2 (Kamijo et al 1998;Pomerantz et al 1998;Stott et al 1998;Zhang et al 1998), it became clear that functional inactivation of p53 could be achieved by the melanoma cell through CDKN2A/p14ARF loss. In fact, concomitant deletion of the CDKN2A locus does occur in the face of activating CDK4 mutations (Yang et al 2005), suggesting that abrogation of the RB pathway through a CDK4-mediated mechanism is still insufficient and that secondary inactivation of p53 is needed and accomplished through abrogation of p14ARF. If direct TP53 mutagenesis and p14ARF represent two possible mechanisms for p53 pathway injury, one would expect HDM2 amplification as a third potential mechanism; indeed, HDM2 amplification has been described in 3%-5% of human melanomas (Muthusamy et al 2006).…”

Role of the pro-survival molecule Bfl-1 in melanoma

“…With little pRb and p53 present in the cell, the G1 cell cycle checkpoint is lost and uncontrollable cell growth can occur. Studies have determined that dual loss of the pRb and p53 pathways are present in up to 80% of melanoma cell-lines 76 , and that this concomitant dysregulation of both pathways could be important in the transformation of human melanocytes. …”

Rapid asymmetric evolution of a dual-coding tumor suppressor INK4a/ARF locus contradicts its function

“…Overall, in various cancers, 206 nucleotides in the overlap between the two reading frames (Fig. 1) carry combinations of 117 point mutations, of which 40, 15, and 62 affect INK4a, ARF, or both proteins, respectively (15,16). What is the evolutionary benefit of maintaining overlapping reading frames in INK4a/ARF, especially in light of the paucity of dual-coding genes in eukaryotic genomes?…”