Germline mutations identified in melanoma-prone kindreds can impair the function of the p14ARF tumour suppressor

The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered sequences in human cancer. The dual coding capacity of the INK4a/ARF locus may account for its high disruption rate in human tumours. This locus encodes two distinct proteins, p^16INK4a and p14ARF, which regulate the cell cycle via the pRb and p53 pathways respectively.

Despite the fact current data indicate that tumour-associated INK4a/ARF alterations favour p^16INK4a for inactivation, a significant proportion of these mutations (approximately 20%) alter the amino acid sequence of both p^16INK4a and p14ARF. If these tumour-associated mutations disrupt the function of p14ARF, the regulation of p53 stability could be a critical component in melanoma susceptibility.

Our recent work leaves no doubt that INK4a/ARF mutations, found in tumours and in melanoma susceptible individuals, disrupt p14ARF function. The effect of compromising p14ARF function is being investigated in melanomas. This cancer frequently retains wild type p53 function and yet does not retain the ability to respond to DNA-damaging agents. This indicates that downstream p53 function is compromised possibly via p14ARF inactivation.

p14ARF induces p53-independent growth arrest by modulating the activities of the e4f and e2f transcription factors

Although p53 stabilisation is the best understood function of ARF, recent evidence confirms that p14ARF also functions in the pRb pathway. The growth inhibitory activity of ARF remains active when the p53 pathway is inactivated but can be suppressed by inactivation of both the pRb and p53 pathways, at least in some cell types. Further evidence that the ARF-p53-hdm2 pathway is not strictly linear comes from comprehensive genetic and immunohistochemical analyses that did not find an inverse correlation between p14ARF alterations and p53 mutations in human tumours.

The various functions of p14ARF presumably reflect its multiple binding partners. In addition to hdm2, p14ARF interacts with the pRb-associated activating transcription factors E2F-1, -2, and -3, and we have recently shown that p14ARF interacts with the E1A-regulated transcriptional repressor, E4F. We propose that functional p14ARF, can compensate for deregulated pRb or p53 function. We find that ectopic expression of p14ARF induces a potent G1 arrest in p53+ cell lines and a consistent dose dependent S-phase decrease in the p53- WMM1175 (pRb+) melanoma cell line. The ARF-induced growth suppression is associated with a decrease in E2F-1 and E2F-2 protein levels and is enhanced by the E4F transcriptional repressor. We suggest that the inhibitory affect of p14ARF in a p53-null background occurs via a network of interactions, including the interaction of p14ARF with the E2F transcription factors and the transcriptional repressor E4F. p14ARF-induced degradation of E2F factors and p14ARF-stimulation of E4F transcriptional repression, can compensate for deregulated p53 function, to induce growth arrest or apoptosis. The loss of p16INK4a, which occurs frequently, but at a late stage of melanoma progression, will stimulate proliferation in cells that retain functional pRb, and in this setting will increase the burden of the p14ARF-pathway. The inactivation of p14ARF would drive proliferation in a manner that favours rapid tumour formation.

Reference:
Rizos H, Diefenbach E, Badhwar P, Woodruff S, Becker TM, Rooney RJ, Kefford RF.
Association of p14ARF with the p120^E4F transcriptional repressor enhances cell cycle inhibition. J Biol Chem. 2003; 278:4981-4989.

SUMO protein modification contributes to the function of the p14ARF melanoma tumour suppressor

p14ARF has recently been shown to promote the accumulation of hdm2 and Werners helicase13 conjugated to the small ubiquitin-like protein SUMO-1. Sumoylation is analogous to ubiquitination, and is the process by which the SUMO protein is conjugated to a target protein via an isopeptide bond between the C-terminal glycine in SUMO and an ε-amino group of a lysine residue in the acceptor protein. The effects of this modification are target specific and include control of protein stability, formation of subnuclear structures and regulation of transcription factor activities (reviewed in Melchior, 2000). The diverse functional consequences of sumoylation provide a possible explanation for the versatility of p14ARF downstream effects which include inhibition of the hdm2 ubiquitin ligase activity for p53, stimulation of topoisomerase I, inhibition of E2F-1, co- operation with CARF and p120^E4F to induce arrest, inhibition of HIF-1α activity and degradation of B23.

We have recently shown that p14ARF promotes the sumoylation of its binding partners hdm2, E2F-1, HIF-1α, TBP-1 and p120^E4F. p14ARF, like the Pc2 and PIAS E3 ligases, interacts with the SUMO conjugative enzyme Ubc9 in vitro. Furthermore, the sumoylation activity of p14ARF is differentially altered by melanoma-associated p14ARF mutations. Thus, we have recently suggested the unifying concept that p14ARF exerts its cell cycle and tumour suppressive functions by mediating the sumoylation of target proteins. We are currently investigating the impact of p14ARF-induced sumoylation on function of target proteins.

Figure: p14ARF promotes SUMO-1 conjugation of its binding partners

U20S cells were transfected with His₆-SUMO-1, p14ARF-FLAG5b and a plasmid encoding (A) hdm2 and (B) E2F-1. After 24 hours the cells were lysed in 6M guanidinium-HCl, proteins were purified on nickel agarose and analysed by Western blotting with antibodies to hdm2 (SMP14), E2F-1 (C-20) and FLAG (M2) as indicated. Upper panel: Ectopic protein expression was analysed by direct Western blot using approximately 2% of cells.

References:
Melchior F. SUMO-nonclassical ubiquitin. Annu Rev Cell Dev Biol  2000; 16:591-626.

Rizos H, Woodruff S, Kefford RF. p14ARF interacts with the SUMO-conjugating enzyme Ubc9 and promotes the sumoylation of its binding partners. Cell Cycle 2005; 4:597-607.