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

Abstract

Background The 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. Results We 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. Conclusions By 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 . Electronic supplementary material The online version of this article (10.1186/s12859-019-2944-9) contains supplementary material, which is available to authorized users.

Prognostic impact of cytoplasmatic EGFR upregulation in patients with oral squamous cell carcinoma: A pilot study

“…However, at the end of such a strongly induced EGFR-pathway the EGFR protein is no longer detectable, although the pathway is highly active (13). This indicates that very low levels of EGFR protein might have two opposite reasons i) very low expression of EGFR or II) a very high active EGFR-pathway associated with high level of internalization and degradation of the receptor.…”

“…Briefly, the tumor slides were deparaffinized with xylol and transferred into a series of ethanol dilution. The EGFR antibody (D38B1) (Cell Signaling Technology Inc.), tested by us in a previous work (13,16), and routinely established for IHC staining in the Institute of Pathology (DRK Kliniken Berlin Westend), was used. The antibody was diluted according to the manufactory specifications (1:50).…”

“…There is evidence that some cytoEGFR molecules can be translocated into the nucleus of tumor cells (9,29). In addition, different isoforms of EGFR could even have different targets/induce different pathways which could have different biological and therapeutic effects (13,19). Nuclear EGFR functioned as a transcription factor and induced proliferation-associated genes and increase the chemo-and radioresistance of tumor cells (3).…”

“…Another possible reason for the ambiguous prognostic influence of EGFR for OSCC patients could be the occurrence of alternative EGFR isoforms, which could induce different signal transduction pathways compared to full length EGFR (12,13). Therefore, we investigated the level and the localization (membranous/cytoplasmatic) of EGFR protein in OSCC samples and their effects on patient's outcome.…”

Genetic variant rs10251977 (G>A) in EGFR-AS1 modulates the expression of EGFR isoforms A and D

“…EGFR generates a functionally important four splice variants through alternative splicing where 10.5 kb and 5.8 kb belongs to variant 1 class encoding 170 kDa protein called isoform A. This variant will be generated by skipping of two exons 15a and 15b in EGFR , whereas 1.8 kb, 2.4 kb and 3.0 kb are three other isoforms formed from a read-through of a exon–intron boundary and incorporation of alternate exons 15a and 15b, which encodes 60, 80 and 110 kDa proteins known as isoform B, C and D, respectively 10 , 11 .…”