Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Jayanthi P Gudikote,Tina Cascone,Alissa Poteete,Piyada Sitthideatphaiboon,Qiuyu Wu,Naoto Morikawa,Fahao Zhang,Shaohua Peng,Pan Tong,Lerong Li,Li Shen,Monique Nilsson,Phillip Jones,Erik P Sulman,Jing Wang,Jean-Christophe Bourdon,Faye M Johnson,John V Heymach

doi:10.1016/j.jbc.2021.101163

Abstract

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.

Highlights

Inactivation of p53 is present in almost every tumor, and p53-reactivation strategies are an important aspect of cancer therapy
To investigate whether nonsense-mediated decay (NMD) inhibition stabilizes the mRNA expression of p53β/γ isoforms and generates p53β/γ proteins lacking the negative regulatory region (Fig. 1, A and B), we used non–small cell lung cancer (NSCLC) cell lines (A549, H1944, and H460) and glioblastoma multiforme (GBM) cell lines (GSC289 and GSC231) bearing WT TP53 and MDM2 overexpression (Fig. S1, A and B)
NMDi increased the expression of p53β/γ transcripts (Fig. 5, E and F) and significantly increased mRNA and protein levels of p53 transcriptional targets (Fig. 5, G–K). These results indicate that, for TP53 mutations resulting in premature translation termination codons (PTCs) in C-terminal exons, NMD inhibition may help restore p53 function by two distinct mechanisms: enabling the synthesis of near-full-length p53 protein by preventing the degradation of the mutant transcript and enhancing the expression of functional p53β/γ and UACC-893, two sets of same samples were run side by side on the same gel; one set was probed with p53 and PUMA, another set was probed with p21 and Vinculin

Summary

Introduction

Inactivation of p53 is present in almost every tumor, and p53-reactivation strategies are an important aspect of cancer therapy. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway.

Results

Conclusion