Abstract 2118: Nonsense-mediated decay regulates the expression of tumor suppressor transcripts and cancer pathways in non-small cell lung cancer cell lines

Abstract

Abstract Background: Nonsense-mediated decay (NMD) is an RNA surveillance pathway that selectively degrades mRNAs bearing premature translation termination codons (PTCs). In addition, NMD also regulates the expression of a subset of physiological mRNAs containing upstream open reading frames (uORFs), spliceable introns in the 3′ untranslated region (3′-UTR) and those that undergo aberrant alternative splicing. Cancer cells produce many mRNAs harboring PTCs because of high frequency of mutations either in the coding region or in the splice sites that result in aberrant splicing. Thus, cancer cells may depend on NMD for survival. Inhibiting NMD provides an attractive cancer therapeutic strategy as it rescues mRNAs bearing NMD features, some of which may produce functional proteins. Therefore, it is important to identify NMD targets and their NMD-inducing features in cancer cells. In the current study, by using NMD inhibition approach, we identified mRNA transcripts and pathways that are regulated by NMD in non-small cell lung cancer (NSCLC) cell lines. Methods: To identify NMD targets, UPF1, a key component of NMD pathway was depleted by RNAi in NSCLC cell lines. mRNAs regulated by NMD were identified by micro array and RNAseq approaches and selected NMD targets were confirmed by real-time PCR. Reverse phase protein array (RPPA) was performed to identify cancer pathways affected by NMD inhibition. Migration, proliferation and clonogenic assays were performed to assess the effect of NMD inhibition on cancer phenotype. Results: Our microarray and RNAseq analysis results indicated that in NSCLC cell lines, NMD regulates the expression of a number of physiological transcripts derived from non-mutant genes. These include transcripts involved in integrated stress response, apoptotic pathways and tumor suppressor activity. We showed that tumor suppressors DMTF1, and p53 undergo NMD-susceptible alternative splicing in NSCLC cell lines and that NMD-regulated p53 isoforms are partially functional. Our results also demonstrated that mRNA transcripts derived from TSC1 and GADD45A, tumor suppressors that negatively regulate mTOR pathway, are also upregulated upon NMD inhibition in NSCLC cell lines. Concomitantly, RPPA analysis revealed that NMD inhibition resulted in downregulation of components involved in mTOR pathway. NMD inhibition also resulted in reduced cell viability and migration. Conclusions: NMD regulates the expression of physiological transcripts involved in integrated stress response, apoptosis and tumor suppressor activity and impacts cancer phenotype in NSCLC cell line models. Our findings suggest that NMD could serve as an attractive therapeutic target. Citation Format: Jayanthi P. Gudikote, Hao Zhao, Jing Wang, Youhong Fan, Lixia Diao, Lauren A. Byers, Uma Giri, Monique Nilsson, John V. Heymach. Nonsense-mediated decay regulates the expression of tumor suppressor transcripts and cancer pathways in non-small cell lung cancer cell lines. [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 2118. doi:10.1158/1538-7445.AM2015-2118