Abstract P6-05-05: Discistronic reporter screen for internal ribosome entry site (IRES) - mediated translational regulation of truncated p110 ERBB2 isoform

Abstract

Abstract Background: We and others demonstrated that truncated p110 ERBB2 (p110 t-ERBB2, also as 611CTF) is a hyperactive truncated ERBB2 isoform capable of increasing cell migration and invasion in multiple cell types in vitro, and induction of human breast epithelial cell (HMLE) xenograft formation in vivo [Ward, et al., Oncogene (2013) 32, 2463–2474]. p110 t-ERBB2 arises through alternative initiation of translation from methionine 611, however, it is unclear how regulation of its expression may be achieved. mRNA structural elements termed internal ribosome entry sites (IRESs) can initiate translation in a cap-independent manner when canonical cap-dependent translation is severely compromised. By cloning the EGFR 5' untranslated region (UTR) between the Renilla and firefly luciferase open reading frames of pRF, Webb, et al. have reported human EGFR 5' UTR sequence can initiate expression of a downstream open reading frame via an IRES [Oncogenesis (2014) 3, e134]. Therefore, we sought to identify presence of a putative IRES within the 5'UTR ERBB2 mRNA which might mediate alternative ERBB2 protein translation initiation under stress conditions and promote p110 t-ERBB2 biosynthesis. Methods: Discistronic reporter pRF was used as the backbone vector to detect IRES activity. Promoter-less vector pRFΔP were constructed by removing SV40 promoter via restriction digestion. The HER2 mRNA 5'UTR (from both variant 1 and variant 3) and several overlapping sequences from full length ERBB2 (p185 ERBB2) start codon to p110 t-ERBB2 start codon were cloned between the Renilla and firefly luciferase open reading frames of pRF and pRFΔP, then the resultant constructswere transiently transfected into different cell lines(BT474, SK-BR3, MCF-7, HeLa, CHO). Three control constructs pRF (empty vector control), pRF-Tub (negative control, containing βtubulin 5'UTR, which lacks IRES activity) and pRF-myc (positive control, containing the well-characterized c-myc IRES) were parallel-transfected. Luciferase expression was then quantified using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) following manufacturer's instructions. Parallel western blot analysis and qRT-PCR were also conducted. Results: In this report, we demonstrate that in BT474 and SK-BR3 cells, no IRES activity was detected within human ERBB2 5'UTR sequences under non-stressed conditions, or under serum-starvation, hypoxic conditions or thapsgargin-induced endoplasmic reticulum stress -- conditions when global translation was compromised.The construct pRF-+265/+1561 (within ERBB2 mRNA coding sequence, 5' to the p110 t-ERBB2 start codon) displayed a 10-21 fold increase in firefly/Renilla activity when compared with the empty control pRF and negative control pRF-Tub,consistent with the possibility that the region between +265/+1561 may contain a cryptic promoter. Conclusions: These data are inconsistent with the hypothesis that a 5'UTR IRES-mediated mechanism is involved in the translation of p110 t-ERBB2 isoform, and that other mechanisms are operative in alternative translational regulation/biosynthesis of p110 ERBB2 isoform. Citation Format: Zong Y, Li Y, Liu X, Pegram MD. Discistronic reporter screen for internal ribosome entry site (IRES) - mediated translational regulation of truncated p110 ERBB2 isoform [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-05.