Germline ERBB3 mutation in familial non-small-cell lung carcinoma: expanding ErbB's role in oncogenesis

Abstract

Lung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable, with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. Germline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and subcellular localization were assessed, and cellular proliferation, pAkt/Akt and pERK levels determined. A novel germline variant in ERBB3 (c.1946 T > G: p.Iso649Arg), coding for receptor tyrosine-protein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80 kDa form (which enhances proliferation) compared with the full-length (180 kDa) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localization pre- and post-epidermal growth factor stimulation; however, epidermal growth factor receptor (EGFR) levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared with wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 min post-stimulation compared with wtErbB3 transfection, demonstrating altered signalling pathway activation. Cumulatively, these results support this mutation as tumorogenic. This is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

Figure 1

Germline ERBB3 mutation segregating with NSCLC in an affected family. (A). Family Pedigree. (B). Sanger sequencing chromatograms of germline DNA, demonstrating heterozygosity for ERBB3 c.1946 T > G variant (arrow) in three affected individuals and wild type in the proband’s unaffected mother.

Figure 2

ErbB3 expression, folding, response to stimulation with EGF, and effect on cellular proliferation in HeLa cells transfected with vector-only, wtErbB3, or mutErbB3. (A). Western blot of lysates from transfected cells using two different commercial anti-ErbB3 antibodies. MutErbB3 is stably expressed and normally folded. A higher ratio of 80 kDa to full length 180 kDa form (arrows) is observed with mutErbB3 compared with wtErbB3. (B). Transfected HeLa cells fixed pre- (0’) and post (30’)-EGF stimulation and immunostained for ErbB3 (red), endogenous EGFR (purple) and nuclei stained using DAPI (blue). Right column shows merged image. Scale bars, 20 μm. Without stimulation EGFR co-localized with mutErbB3 in concentrated puncta, less evident with wtErbB3. After stimulation, both wtErbB3 and mutErbB3 increased in the perinuclear region, co-localizing with EGFR. (C). Proliferation assay of transfected cells quantified and described as fold change relative to vector only (data shown as mean ± standard error of mean). MutErbB3-transfected cells showed increased rates of proliferation.

Figure 3

Analysis of protein expression and downstream signalling pathway activation in HeLa cells transfected with vector-only, wtErbB3 or mutErbB3 constructs, before starvation (control), after 3-h starvation (0 min) followed by EGF stimulation (assessed at 10 min and 30 min). (A). Western blots of ErbB3, EGFR, phospho-Akt (pAkt), total Akt (tAkt), phospho-ERK (pERK) and total ERK (tERK), performed on transfected cell lysates. Following starvation and EGF stimulation, mutErbB3-transfected cells demonstrated decreased EGFR levels compared with wtErbB3. (B). Ratios of pAkt/tAkt (upper graphs), and pERK/tERK (lower graphs) quantified and normalized to β-tubulin. By 30 min, mutErbB3-transfected cells show decreased pAkt/tAkt ratio and increased pERK/tERK ratio compared with wtErbB3-transfected cells. Both blot images and ratio quantification are representative of at least three separate biological replicates.