Investigating the impact of HER4 mutations on HER2 signaling

Background: HER2 is amplified in ~20% of breast cancers (BCs) and is a member of the HER/ErbB receptor tyrosine kinase family that also includes EGFR, HER2 and HER3. HER4 is mutated in 7% of HER2+ BCs but there are limited data on the impact of HER4 point mutations (MUTs) on HER2 signaling. Previous work has identified EGFR, p38 MAPK, Src and STAT3 as targets impacted by HER4 overexpression. HEK293 cells express wild type (WT) EGFR and negligible levels of HER2, HER3 and HER4. Using plasmid-based overexpression in HEK293 cells, this study examines the co-expression of WT HER2 with WT HER4 or HER4 point MUTs (2 extracellular domain (ECD) and 6 intracellular domain (ICD)). Methods: Eight HER4 MUTs identified from the COSMIC database were investigated. Four were predicted to be deleterious/damaging and four neutral/tolerated (Provean/SIFT). MUT1 (p.E202D) and MUT2 (p.P224R) are in the ECD; MUT3-MUT8 (p.T1036I, p.Y1242C, p.H1245N, p.S1246R, p.L1247M, p.H1255N) are in the ICD. Mutant primers (Sigma Aldrich) were used for site-directed mutagenesis (QuikChange Lightning) with pRK5 HER4 WT used as a template to generate eight validated HER4 MUT plasmids (SequiServe). HEK293 cells were transfected (Fugene) with either empty vector, GFP control, WT HER2 plasmid, WT HER4, WT HER2/WT HER4, or WT HER2 combined with each HER4 MUT. Transfection was confirmed by Western blot. Protein lysates were analyzed in triplicate by reverse phase protein array (RPPA) to quantify 50 total and 34 phospho-proteins (84 targets). Significant changes (p < 0.05) were determined by Student's t-test. Results: Total EGFR levels were unchanged in response to transfection with WT HER2 or WT HER4, however EGFR phosphorylation at Y992 and Y1068 increased significantly (p<0.05) for WT HER2 and WT HER4 individually or in combination. Unlike WT HER4, WT HER2 did not increase phosphorylated levels of p38 MAPK, Src or STAT3. When co-transfected with WT HER2, changes induced by WT HER4 were maintained for Src (Y416) (p=0.02) and STAT3 (Y705) (p<0.01) but not p38 MAPK (T180/Y182) (p=0.11). In the presence of WT HER2, HER4 MUT6 increased phosphorylated HER2 (Y1248) levels (p=0.01), and MUTs 2, 6, 7 and 8 significantly increased levels of phosphorylated EGFR (Y1173). In addition, HER4 MUT6 reduced Src (Y527) phosphorylation (p=0.01). Conclusions: Our data reports HER4-activated intracellular signaling pathways distinct from HER2, and HER4 MUTs that can alter EGFR and HER2 activity. These results provide preliminary evidence for the phenotypic impact of the HER4 MUTs examined on HER2 signaling.