p63 signalling in BRAFi resistance melanoma.
Ankit Patel1, Catherine Harwood1, Daniele Bergamaschi1
1Blizard Institute, Centre for Cell Biology and Cutaneous Research, Queen Mary University of London, UK.

In the past decade, major advances have been made in the understanding of melanoma. New predisposition genes have been reported and key somatic events, such as BRAF mutation, directly translated into therapeutic management. The combination of BRAF inhibitors and MEK inhibitors might improve progression-free survival further and, possibly, increase overall survival. Targeted drugs induce responses in most patients, but nearly all of them relapse because of pre-existing or acquired resistance. Thus, the long-term prognosis of metastatic melanoma remains poor. In the past decades several mechanisms/pathways have been linked to the acquired resistance in these patients, including acquired mutations in N-RAS and MEK1, increased expression of MAP3K, Cot1, cyclin D1 and several growth factor receptors.
P63, a member of the p53/p73 family, was found to be responsible for inhibiting the apoptosis induced by p53 upon exposure to the genotoxic drugs in melanoma.
Here we showed significant correlation between p63 expression and mutational status of BRAF/NRAS in melanoma clinical samples. We also observed that BRAFi resistant cells, constitutively exposed to vemurafenib drug, have increased expression of several p63 isoforms at mRNA as well as protein levels. Furthermore, p63 protein stability is controlled by the FBXW7 (E3 ubiquitin ligase) in BRAFi resistant melanoma cells. FBXW7 is a potent tumor suppressor which ubiquitinates many oncoproteins including cyclin E1, c-myc and Notch. We also observed significant decrease in FBXW7 and upregulation of MDM2 in the nucleus of these cells, where MDM2 interacts with FBXW7 and regulates its protein stability. FBXW7 is either mutated (~8%) or inactivated and MDM2 is often upregulated in melanoma clinical samples. In vemurafenib resistant cells, MDM2 increase in nucleus degrades FBXW7, which stabilizes p63 protein and leads to the survival of melanoma cells. Also, upon nutlin-3 treatment on melanoma cells, FBXW7 expression increases and P63 protein level is abrogated in a dose dependent manner. Therefore, based on the initial results we hypothesized that, in melanoma and BRAFi resistant cells FBXW7 regulates the stability of P63 protein via ubiquitin proteasomal degradation pathway. Hence the combination of nutlin-3 with vemurafenib could be a very effective treatment for tackling melanoma compared to the single agent treatment.

p63 signalling in BRAF inhibitor resistant melanoma