Prediction of regulatory targets of alternative isoforms of the epidermal growth factor receptor in a glioblastoma cell line

Matthias Kappler,Alexander W. Eckert,Henri Wichmann,David H. Ardell,Dirk Vordermark,Claus Weinholdt,Johanna Kotrba,Ivo Grosse

doi:10.1186/s12859-019-2944-9

Abstract

BackgroundThe epidermal growth factor receptor (EGFR) is a major regulator of proliferation in tumor cells. Elevated expression levels of EGFR are associated with prognosis and clinical outcomes of patients in a variety of tumor types. There are at least four splice variants of the mRNA encoding four protein isoforms of EGFR in humans, named I through IV. EGFR isoform I is the full-length protein, whereas isoforms II-IV are shorter protein isoforms. Nevertheless, all EGFR isoforms bind the epidermal growth factor (EGF). Although EGFR is an essential target of long-established and successful tumor therapeutics, the exact function and biomarker potential of alternative EGFR isoforms II-IV are unclear, motivating more in-depth analyses. Hence, we analyzed transcriptome data from glioblastoma cell line SF767 to predict target genes regulated by EGFR isoforms II-IV, but not by EGFR isoform I nor other receptors such as HER2, HER3, or HER4.ResultsWe analyzed the differential expression of potential target genes in a glioblastoma cell line in two nested RNAi experimental conditions and one negative control, contrasting expression with EGF stimulation against expression without EGF stimulation. In one RNAi experiment, we selectively knocked down EGFR splice variant I, while in the other we knocked down all four EGFR splice variants, so the associated effects of EGFR II-IV knock-down can only be inferred indirectly. For this type of nested experimental design, we developed a two-step bioinformatics approach based on the Bayesian Information Criterion for predicting putative target genes of EGFR isoforms II-IV. Finally, we experimentally validated a set of six putative target genes, and we found that qPCR validations confirmed the predictions in all cases.ConclusionsBy performing RNAi experiments for three poorly investigated EGFR isoforms, we were able to successfully predict 1140 putative target genes specifically regulated by EGFR isoforms II-IV using the developed Bayesian Gene Selection Criterion (BGSC) approach. This approach is easily utilizable for the analysis of data of other nested experimental designs, and we provide an implementation in R that is easily adaptable to similar data or experimental designs together with all raw datasets used in this study in the BGSC repository, https://github.com/GrosseLab/BGSC.

Highlights

The epidermal growth factor receptor (EGFR) is a major regulator of proliferation in tumor cells
The rest of this paper is structured as follows: In Results, we describe the identification of a cell line with an inducible EGFR-signaling pathway, investigate the specificity of small interfering RNAs (siRNAs), introduce the two-step Bayesian Gene Selection Criterion (BGSC) approach for predicting putative target genes regulated by EGF via EGFR isoforms II-IV and not by the full-length EGFR isoform I or other receptors, and describe the quantitative real-time polymerase chain reaction (qPCR) validation experiments
Additional file 1: Figure S.1 shows that the application of siRNAALL and siRNAI reduced the levels of all EGFR splice variants by 70.9% on average and the levels of the full-length EGFR splice variant I by 78.1% on average

Summary

Introduction

The epidermal growth factor receptor (EGFR) is a major regulator of proliferation in tumor cells. All EGFR isoforms bind the epidermal growth factor (EGF). One potential molecular target amplified in 36% of glioblastoma patients is the epidermal growth factor receptor (EGFR), and the expression of EGFR is associated with prognosis in cancer [2]. EGFR is known to affect growth and survival signals and to play a crucial role in the regulation of cell proliferation, differentiation, and migration of various tumor entities [3]. EGFR is well known as a prognostic tumor marker and therapeutic target in different tumor entities. The full-length transmembrane glycoprotein isoform of EGFR consists of three functional domains of which the extracellular domain is capable of binding at least seven different ligands such as EGF, AREG, or TGF-α [4]. Only the fulllength EGFR isoform I translated from EGFR splice variant I is well investigated, but comparatively little is known about the biological significance of the truncated EGFR isoforms II-IV translated from EGFR splice variants II-IV

Results

Discussion

Conclusion