The HER2 inhibitor lapatinib potentiates doxorubicin-induced cardiotoxicity through iNOS signaling

Abstract

Rationale: Lapatinib (LAP) is a crucial alternative to trastuzumab upon the onset of drug resistance during treatment of metastatic human epidermal growth factor receptor 2-positive breast cancer. Like trastuzumab, LAP is commonly used alongside anthracyclines as a combination therapy, due to enhanced anti-cancer efficacy. However, this is notably associated with cardiotoxicity so it is imperative to understand the mechanisms driving this cardiotoxicity and develop cardioprotective strategies. To this end, here we utilize human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), which exhibit several characteristics representative of in vivo cardiomyocytes that make them breakthrough models to study drug toxicity. Methods: We investigated LAP- and doxorubicin (DOX)-induced toxicity in hPSC-CMs and evaluated the involvement of inducible nitric oxide (NO) synthase (iNOS). The significance of iNOS-mediated cardiotoxicity was furthermore evaluated in animal studies. Results: LAP synergistically increased DOX toxicity in hPSC-CMs in a dose- and time-dependent manner. At concentrations that were otherwise non-apoptotic when administered separately, LAP significantly potentiated DOX-induced hPSC-CM apoptosis. This was accompanied by increased iNOS expression and pronounced production of NO. iNOS inhibition significantly reduced hPSC-CM sensitivity to LAP and DOX co-treatment (LAP-plus-DOX), leading to reduced apoptosis. Consistent with our observations in vitro, delivery of an iNOS inhibitor in mice treated with LAP-plus-DOX attenuated myocardial apoptosis and systolic dysfunction. Moreover, inhibition of iNOS did not compromise the anti-cancer potency of LAP-plus-DOX in a murine breast cancer xenograft model. Conclusions: Our findings suggest that iNOS inhibition is a promising cardioprotective strategy to accompany HER2-inhibitor/anthracycline combination therapies. Furthermore, these results support the promise of hPSC-CMs as a platform for investigating cardiotoxicity and developing cardioprotectants as a whole.

Keywords: HER2 inhibitor; cardiotoxicity; doxorubicin; iNOS; iPSC.

Figure 1

Human iPSC-derived cardiomyocytes are capable of modeling the synergistic cardiotoxicity of lapatinib (LAP)-plus-doxorubicin (DOX) combination treatment. (A) Representative phase contrast (left) and sarcomeric protein immunostaining (right) images of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ACTN1: actinin alpha 1. Scale bars: 100 μm and 20 μm. (B-C) Flow cytometry analysis of cardiac troponin I (cTnI) and human epidermal growth factor receptor-2 (HER2) expression in hiPSC-CMs. (D) Effect of DOX treatment (after 24 h) on viability of hiPSC-CM and RUES2-CM using a TetraZ-based dye assay. (E) Quantification of cell viability in a representative hiPSC-CM line treated with LAP-plus-DOX, LAP-plus-trastuzumab, or LAP-plus-afatinib at different pharmacological plasma level concentrations. (F) Effects of LAP (2.5 μM) on DOX (1 μM)-induced hiPSC-CM, RUES2-CM, HUVEC, and HEK293 toxicity at different time-points based on quantification of cell viability. (G) Representative images of early (annexin V⁺) and late (annexin V⁺7-AAD⁺) apoptotic hiPSC-CMs 24 h after treatment with LAP-plus-DOX using image-in-flow cytometry. (H) Pro-apoptotic effects of LAP-plus-DOX on hPSC-CMs based on flow cytometry analysis of annexin V⁺ and 7-AAD⁺ cells. All data are presented as mean ± SD (n≥3). *P < 0.05 vs. control; † P < 0.05 vs. DOX.

Figure 2

N⁶-(1-iminoethyl)-L-lysine (L-NIL)-mediated inhibition of inducible nitric oxide synthase (iNOS) in hPSC-derived cardiomyocytes decreases LAP-plus-DOX-induced toxicity. (A) Cytoplasmic iNOS protein expression as determined in hiPSC-CMs treated with vehicle (Control), LAP alone (2.5 μM), DOX alone (1.0 μM), or both LAP-plus-DOX. Cells were labeled with anti-iNOS, or an isotype-matched antibody. Percentage of iNOS-expressing cells are displayed as representative flow cytometry histogram plots (left) and quantitative analysis (right). (B) iNOS activity, as a function of total nitric oxide product formation (nitrate + nitrite), was measured using a colorimetric assay. (C) Proportion of viable, apoptotic and necrotic cells, based on flow cytometry analysis of 7-AAD and Annexin V co-labeling. (D) Quantification of fold changes in apoptosis upon L-NIL co-administration. (E) Quantification of L-NIL-mediated iNOS inhibition based on total nitric oxide product formation. (F) Mitochondrial function of hiPSC-CMs cultured in each experimental group. Oxygen consumption rate (OCR) of hiPSC-CMs demonstrating basal OCR and spare OCR respiratory capacity measured after consecutive addition of oligomycin, FCCP, and antimycin. (G) Flow cytometry analysis of HER2 expression in hiPSC-CMs. (H) Effects of L-NIL on LAP-plus-DOX-induced HER2 activity, PI3K/Akt activation, IκBα phosphorylation, and nuclear translocation of NF-κB p65 subunit. All data are presented as mean ± SD (n≥3). *P < 0.05 vs. control; † P < 0.05 vs. DOX; ‡ by P < 0.05 vs. LAP + DOX.

Figure 3

L-NIL-mediated iNOS inhibition attenuates myocardial apoptosis and systolic dysfunction in mice treated with LAP-plus-DOX. (A) Representative flow cytometry scatter plots and (B) statistical analysis of annexin V⁺ and 7-AAD⁺ cells from digested hearts of mice treated with different dose-regimens of LAP-plus-DOX. (C) Caspase-3 activity assay performed on ventricular extracts from hearts of mice treated with different drug-regimens for 0 to 7 days as indicated. (D) TUNEL immunostaining of left-ventricular sections at 7 days after drug administration. Percentages of TUNEL-expressing cTnI⁺ cells (white arrows) are displayed as representative images and cumulative data. TUNEL, red; cTnI, green; DAPI, blue. Scale bar: 10 μm. (E) Echocardiographic measurements of mice treated with different drug-regimens taken 7 days after commencement of treatment. (F) Hematoxylin and eosin-staining (left) and immunohistochemical localization of iNOS expression (right) in myocardial sections. Scale bar: 50 μm. (G) Western blots showing iNOS expression and (H) NO levels in the myocardium. In Figures (D-H): Control = vehicle; LAP = lapatinib 10 mg/kg; DOX = doxorubicin 5 mg/kg; Combo = LAP 10 mg/kg + DOX 5 mg/kg; L-NIL = vehicle + L-NIL 10 mg/kg; Combo + L-NIL = LAP 10 mg/kg + DOX 5 mg/kg + L-NIL 10 mg/kg. LVEF: left-ventricular ejection fraction; FS: fractional shortening; EDV: end-diastolic volume; ESV: end-systolic volume. All data are presented as mean ± SD (n≥4). *P < 0.05 vs. control; † P < 0.05 vs. DOX; ‡ by P < 0.05 vs. Combo.

Figure 4

L-NIL-mediated iNOS inhibition maintains anti-cancer potency of LAP-plus-DOX combined treatment in a murine SK-BR-3 breast cancer xenograft model. (A) Tumor progression displayed as representative serial, non-invasive In Vivo Imaging System images. (B) Time-course chart showing tumor size as determined by bioluminescent signal intensity in the region of interest, expressed in photons per second. Control = vehicle; Low = low dose of LAP (5 mg/kg) + DOX (5 mg/kg); High = high dose of LAP (10 mg/kg) + DOX (10 mg/kg); L-NIL = high dose of LAP + DOX + L-NIL (10 mg/kg). In (A), red arrows indicate the time points of drug administration. All data are presented as mean ± SD (n≥4). *P < 0.05 vs. Ctl; † P < 0.05 vs. Low; ‡ P < 0.05 vs. High.

Figure 5

L-NIL-mediated iNOS inhibition alleviates LAP-plus-DOX-induced cardiotoxicity in a murine SK-BR-3 breast cancer xenograft model. (A) Immunoblots indicating iNOS and (B) NO levels in the myocardium. Cardiotoxicity was evaluated by (C) animal survival rate, (D) body weight and heart weight, (E) echocardiography, (F) catheter-derived hemodynamic measurements, (G) TUNEL-expressing cTnI⁺ cells (white arrows), (H) collagen I expression, (I) plasma levels of cTnI, and (J) gene expression of stress markers. Scale bars indicate 10 μm in (G) and 50 μm in (H), respectively. Control = vehicle; Low = low dose of LAP (5 mg/kg) + DOX (5 mg/kg); High = high dose of LAP (10 mg/kg) + DOX (10 mg/kg); L-NIL = high dose of LAP + DOX + L-NIL (10 mg/kg). LVEF: left-ventricular ejection fraction; LVFS: left ventricular fractional shortening; LVEDV: left ventricular end-diastolic volume; LVESV: left ventricular end-systolic volume; LVESP: left ventricular end-systolic pressure; LVEDP: left ventricular end-diastolic pressure; ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide. All data are presented as mean ± SD (n≥4). *P < 0.05 vs. Ctl; † P < 0.05 vs. Low; ‡ P < 0.05 vs. High.