Abstract A65: Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform
Authors: Zong Yu , Li Yulin , Liu Xiaofei , Pegram Mark Daniel 
Published: August 2018

Background: We and others demonstrated that truncated p110 ERBB2 (p110 t-ERBB2, also known 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(19):2463-74). p110 t-ERBB2 arises through alternative initiation of translation from methionine 611, though the mechanism to account for such cap-independent p110 t-ERBB2 translation is not well studied. 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. It has been previously demonstrated that the human EGFR 5’ untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an IRES (Webb TE, et al., Oncogenesis 2015;4:e134). Therefore, we sought to identify presence of a putative IRES within the 5′ UTR that might mediate alternative ERBB2 protein translation initiation under stress conditions and promote p110 t-ERBB2 biosynthesis.

Methods: The ERBB2 mRNA 5 ′ UTR from 2 published human ERBB2 transcripts, and several overlapping sequences from full-length ERBB2 start codon to p110 t-ERBB2 start codon were cloned between the Renilla and firefly luciferase open reading frames of pRF and promoter-less vector pRFΔP (SV40 promoter removed by restriction digestion), then the resultant constructs were 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 β-tubulin 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 nonstressed 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 t-ERBB2 isoform.

Citation Format: Yu Zong, Yulin Li, Xiaofei Liu, Mark Daniel Pegram. Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A65.