Person:

Fletcher, Jonathan

The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

Background: Improved mesothelioma patient survival will require development of novel and more effective pharmacological interventions. TP53 genomic mutations are uncommon in mesothelioma, and recent data indicate that p53 remains functional, and therefore is a potential therapeutic target in these cancers. In addition, the tumour suppressor NF2 is inactivated by genomic mechanisms in more than 80% of mesothelioma, causing upregulation of FAK activity. Because FAK is a negative regulator of p53, NF2 regulation of FAK–p53–MDM2 signalling loops were evaluated. Methods: Interactions of FAK–p53 or NF2–FAK were evaluated by phosphotyrosine-p53 immunoaffinity purification and tandem mass spectrometry, and p53, FAK, and NF2 immunoprecipitations. Activation and/or expression of FAK, p53, and NF2 were also evaluated in mesotheliomas. Effects of combination MDM2 and FAK inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, expression of cell cycle checkpoints, and cell cycle alterations. Results: We observed constitutive activation of FAK, a known negative regulator of p53, in each of 10 mesothelioma cell lines and each of nine mesothelioma surgical specimens, and FAK was associated with p53 in five of five mesothelioma cell lines. In four mesotheliomas with wild-type p53, FAK silencing by RNAi induced expression and phosphorylation of p53. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared with four mesothelioma cell lines with nonmutant p53. Additive effects were obtained through a coordinated reactivation of p53, by FAK knockdown/inhibition and MDM2 inhibition, as demonstrated by immunoblots, cell viability, and cell-cycle analyses, showing increased p53 expression, apoptosis, anti-proliferative effects, and cell-cycle arrest, as compared with either intervention alone. Our results also indicate that NF2 regulates the interaction of FAK–p53 and MDM2–p53. Conclusions: These findings highlight novel therapeutic opportunities in mesothelioma.