Abstract 2640: Small molecule compound NCI-8 induces ERK2-dependent mutant-p53 protein degradation
Authors: Zhang Shengliang , Zhou Lanlan , Dicker David T. , EL-Deiry Wafik S. 
Published: August 2015

Abstract
The tumor suppressor p53 is mutated in over 50% of human cancers leading not only to loss of wild-type p53 function, but also to gain-of-function (GOF) mutant p53 which acts as an oncogene. Mutant p53 is expressed at very high levels and can inhibit residual wild-type p53 activity as well as the function of p53 family member proteins such as p73 or p63. Therefore, targeting mutant p53 by either restoring the p53 pathway or depleting its GOF is an attractive strategy for cancer therapy. NCI-8 is a small molecule compound with dual abilities to induce p53 signal pathway and destabilize mutant p53 protein (deplete GOF) in mutant p53 expressing colorectal cancer cells. It remains unclear how NCI-8 regulates mutant p53 protein degradation. ERK2 is a member of the MAP kinase family which plays a critical role in regulating cell growth and differentiation by phosphorylating substrates including wild-type p53. We demonstrate that NCI-8-induces mutant p53 protein degradation via activation of ERK2 signaling. We observed a sustained phosphorylation of ERK2 upon NCI-8 treatment of cancer cells, but not in normal cells. MEK inhibitor U0126 treatment completely blocked NCI-8-mediated phosphorylation of ERK2 in cancer cells. These results, taken together, suggest that NCI-8 activates the ERK2 signaling pathway in cancer cells. There was a correlation between mutant p53 degradation and phosphorylation of ERK2 in cancer cells treated with NCI-8. We further examined the role of ERK2 phosphorylation in NCI-8- mediated mutant p53 protein degradation. Cancer cells were treated with U0126 to block ERK2 phosphorylation or transiently transfected with siRNA to knockdown ERK2. U0126 treatment or knockdown of ERK2 rescued mutant p53 from NCI-8-mediated protein degradation, suggesting that NCI-8-mediated phosphorylation of ERK2 is required for mutant p53 degradation. We further found that U0126 treatment inhibited NCI-8 induction of p21, puma and Noxa expressions in cancer cells. Consistently, transient over-expression of exogenous mutant p53 blocked NCI-8-mediated restoration of p53 pathway signaling in mutant-p53 expressing cancer cells. These results suggest that NCI-8 restores p53 signal pathway via ERK2-dependent mutant p53 protein degradation. Correlated with the U0126-rescue of mutant p53 protein, the percentage of cells with sub-G1 content induced by NCI-8 is decreased in response to U0126 treatment in cancer cells, suggesting that cell death induced by NCI-8 depends on ERK2-mediated mutant p53 degradation. Furthermore, combinational indices showed that U0126 antagonized NCI-8- induced cell death, but no antagonism was found in combinational treatment of NCI-8 and EGFR inhibitors in cancer cells. Our data indicate an important role of NCI-8-phosphorylated ERK2 in regulation of mutant p53 protein degradation and cell death in cancer cells, and provide a rationale for clinical testing of NCI-8 and ERK2 pathway-related factors in cancer therapy.
Citation Format: Shengliang Zhang, Lanlan Zhou, David T. Dicker, Wafik S. EL-Deiry. Small molecule compound NCI-8 induces ERK2-dependent mutant-p53 protein degradation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2640. doi:10.1158/1538-7445.AM2015-2640