Chronic pharmacologic inhibition of EGFR leads to cardiac dysfunction in C57BL/6J mice

Abstract

Molecule-targeted therapies like those against the epidermal growth factor receptor (EGFR) are becoming widely used in the oncology clinic. With improvements in treatment efficacy, many cancers are being treated as chronic diseases, with patients having prolonged exposure to several therapies that were previously only given acutely. The consequence of chronic suppression of EGFR activity may lead to unexpected toxicities like altered cardiac physiology, a common organ site for adverse drug effects. To explore this possibility, we treated C57BL/6J (B6) mice with two EGFR small molecule tyrosine kinase inhibitors (TKIs), irreversible EKB-569 and reversible AG-1478, orally for 3 months. In B6 female mice, chronic exposure to both TKIs depressed body weight gain and caused significant changes in left ventricular (LV) wall thickness and cardiac function. No significant differences were observed in heart weight or cardiomyocyte size but histological analysis revealed an increase in fibrosis and in the numbers of TUNEL-positive cells in the hearts from treated female mice. Consistent with histological results, LV apoptotic gene expression was altered, with significant downregulation of the anti-apoptotic gene Bcl2l1. Although there were no significant differences in any of these endpoints in treated male mice, suggesting sex may influence susceptibility to TKI mediated toxicity, the LVs of treated male mice had significant upregulation of Egf, Erbb2 and Nppb over controls. Taken together, these data suggest that chronic dietary exposure to TKIs may result in pathological and physiological changes in the heart.

Fig. 1

Immunoblot analysis of liver lysates. Wild-type B6 male mice were exposed to the EGFR small molecule inhibitor AG-1478 in AIN 93G diet (AG-1478) or AIN 93G diet alone (control) for three months (n=2). (A) Western blots and (B) quantification of EGFR activation.

Fig. 2

Effects of EGFR inhibitors on weight gain in B6 mice. Female mice were treated with EKB-569 (A) or AG-1478 (B) or male mice with (C) AG-1478. Left plots, body weight; right plots, percent change in body weight from start of diet. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3

Pathological changes in hearts from B6 mice chronically exposed to EGFR inhibitors. Representative images depicting LV wall thinning (A) interstitial fibrosis (B) and increased numbers of apoptotic cardiac cells as detected by TUNEL staining (C) in the hearts of EKB-569 and AG-1478 exposed female mice. Arrows point to fibrosis (B) or TUNEL positive nuclei (C). Insets in (C) are higher magnification of TUNEL positive cells. D) Quantification of TUNEL-positive cardiac cells in female and male mice chronically exposed to EGFR inhibitors compared to controls. E) Relative fold changes in gene expression of proapoptotic and anti-apoptotic genes in the LV of B6 female and male mice chronically exposed to AG-1478 compared to controls. Bcl2l1= B-cell leukemia/lymphoma 2, Bax= Bcl2-associated X protein and Bad= Bcl2-associated death promoter. *p < 0.05.

Fig. 4

Pathological changes in the aortic valves of B6 mice chronically exposed to EGFR inhibitors. Calcification was detected in the aortic valve leaflets of male and female mice exposed to EGFR inhibitors by von Kossa stain (A). Aortic valve leaflets were thickened in EGFR inhibitor-exposed B6 mice of both sexes (female, B and male, C) compared to controls fed normal chow (NC) or AIN-93G chow (Control). Arrows in A indicate positive staining (black) for calcium deposition. Ao = aortic outflow tract, AV= aortic valve, LV=left ventricle. *p < 0.05. Bar = 100 μm.

Fig. 5

Relative fold changes in gene expression in the LV of B6 male mice chronically exposed to AG-1478 compared to controls. Nppa = nautriuretic peptide precursor A; Nppb = natriuretic peptide precursor B; Egf = epidermal growth factor; Erbb2 = erythroblastic leukemia viral oncogene homolog 2 neuro/glioblastoma derived oncogene homolog (avian); Dtr = Diptheria toxin receptor/heparin-binding epidermal growth factor. *p < 0.05, **p < 0.01.