Targeting the HER Family with Pan-HER Effectively Overcomes Resistance to Cetuximab

Abstract

Cetuximab, an antibody against the EGFR, has shown efficacy in treating head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer, and non-small cell lung cancer (NSCLC). Despite the clinical success of cetuximab, many patients do not respond to cetuximab. Furthermore, virtually all patients who do initially respond become refractory, highlighting both intrinsic and acquired resistance to cetuximab as significant clinical problems. To understand mechanistically how cancerous cells acquire resistance, we previously developed models of acquired resistance using the H226 NSCLC and UM-SCC1 HNSCC cell lines. Cetuximab-resistant clones showed a robust upregulation and dependency on the HER family receptors EGFR, HER2, and HER3. Here, we examined pan-HER, a mixture of six antibodies targeting these receptors on cetuximab-resistant clones. In cells exhibiting acquired or intrinsic resistance to cetuximab, pan-HER treatment decreased all three receptors' protein levels and downstream activation of AKT and MAPK. This correlated with decreased cell proliferation in cetuximab-resistant clones. To determine whether pan-HER had a therapeutic benefit in vivo, we established de novo cetuximab-resistant mouse xenografts and treated resistant tumors with pan-HER. This regimen resulted in a superior growth delay of cetuximab-resistant xenografts compared with mice continued on cetuximab. Furthermore, intrinsically cetuximab-resistant HNSCC patient-derived xenograft tumors treated with pan-HER exhibited significant growth delay compared with vehicle/cetuximab controls. These results suggest that targeting multiple HER family receptors simultaneously with pan-HER is a promising treatment strategy for tumors displaying intrinsic or acquired resistance to cetuximab. Mol Cancer Ther; 15(9); 2175-86. ©2016 AACR.

Figure 1. Pan-HER inhibits the proliferation of cetuximab-resistant cells

(A) Pan-HER inhibits the proliferation of cetuximab-resistant clones. Cetuximab-sensitive cells HP and cetuximab-resistant clones (HC1, HC4, and HC8) were plated and treated with vehicle, cetuximab (20 μg/ml) or Pan-HER (0.1, 1, 20, or 100 μg/ml) for 72 hours prior to measuring the proliferation by CCK8 assay. Data points are represented as means ± SEM (n=4) **p≤0.001. (B) Pan-HER down-regulates HER family receptors and decreases downstream AKT and MAPK signalling. Cells were treated with vehicle or Pan-HER (0.1, 1, 20, or 100 μg/ml) for 24 hours. (C) HER family receptors remain down-regulated in cetuximab-resistant clones at 72 hours following Pan-HER treatment. Cells were treated with vehicle or Pan-HER (20 μg/ml) for 24, 48, or 72 hours. Cells were lysed and fractionated on SDS-PAGE, followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control.

Figure 2. Pan-HER effectively down-modulate HER family receptors in cetuximab-resistant clones compared with cetuximab and enhance to cell cycle arrest

(A) Pan-HER, but not cetuximab, treatment leads to decreased expression of HER family receptors and subsequent downstream AKT and MAPK signalling pathways. HP and cetuximab-resistant clones (HC1, HC4, and HC8) were treated with vehicle, cetuximab (20 μg/ml) or Pan-HER (20 μg/ml) for 24 hours. Cells were lysed and fractionated on SDS-PAGE, followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control. (B) Pan-HER induces a G1-phase cell cycle arrest in cetuximab-resistant clones. Cells were treated with 20 μg/ml cetuximab or 20 μg/ml Pan-HER for 24 hours, and cell cycle phase distribution was analyzed as described in the Materials and Methods section. Data points are represented as means ± SEM (n=3) *p<0.05, **p≤0.001.

Figure 3. Pan-HER decreases HER family receptors in acquired cetuximab-resistant HNSCC

(A) Cells were plated and treated with vehicle cetuximab (20 μg/ml) or Pan-HER (20 μg/ml) for 72 hours prior to measuring the proliferation by CCK8 assay. Data points are represented as means ± SEM (n= 4). *p<0.05, **p≤0.001. (B) Pan-HER decreased HER family receptors and inhibits downstream AKT and MAPK signalling. Cells were treated with vehicle, cetuximab (20 μg/ml) or Pan-HER (20 μg/ml) for 24 hours, after which the cell were lysed and fractionated on SDS-PAGE for the indicated proteins. α-Tubulin was used as a loading control.

Figure 4. Pan-HER exhibits greater anti-tumor effects than cetuximab in lung tumors

(A) Pan-HER shows anti-tumor activity and delayed tumor growth of H226 xenografts compared with the cetuximab treatment group. H226 cells were injected into mice and tumors were allowed to grow to 100 mm³. All mice were randomized to treatment or control groups and treated with cetuximab (50 mg/kg), Pan-HER (50 mg/kg), or IgG (50 mg/kg) i.p. twice weekly. (B) Pan-HER-induced HER family receptor down-modulation in vivo. Immunoblot analysis of total HER family receptors in xenograft tumors after IgG, cetuximab or Pan-HER treatment. α-Tubulin was used as a loading control.

Figure 5. Pan-HER treatment delays the growth of tumors with acquired cetuximab-resistance

(A) Growth-inhibitory effects of Pan-HER on cetuximab-resistant tumors in vivo. Acquired cetuximab-resistance tumors were established and evaluated for Pan-HER response as described in Materials and Methods. The black arrows indicate initiation of Pan-HER treatment. The average tumor volume in mice treated with IgG is included in all groups for comparison purposes. Tumor number is indicated for correlation with Figure 5B. (B) Pan-HER-induced HER family receptor down-modulation in vivo. Immunoblot analysis of total HER family receptors in cetuximab-resistant xenograft tumors after cetuximab or Pan-HER treatment. α-Tubulin was used as a loading control. (C) Down-modulation of HER family receptors in cetuximab-resistant tumors corresponds with loss of proliferation. Cetuximab-resistant tumor samples after cetuximab or Pan-HER treatments in vivo were prepared and analyzed for EGFR, HER2, HER3, and proliferation (Ki67) immunohistochemistry.

Figure 6. Pan-HER effectively decreases HER family receptors in intrinsic cetuximab-resistant lung and HNSCC cell lines

(A) Cells were plated and allowed to adhere for 24 hours prior to vehicle, cetuximab (20 μg/mL) or Pan-HER (20 μg/mL) treatment. Cell proliferation assay were described as Materials and Methods. Data points are represented as mean ± SEM (n=6). **p≤0.001. (B) Pan-HER degrades HER family receptors and inhibits downstream AKT and MAPK signalling. Cells were treated with vehicle, cetuximab (20 μg/ml) or Pan-HER (20 μg/ml) for 24 hours. The cells were then lysed and fractionated on SDS-PAGE for the indicated proteins. α-Tubulin was used as a loading control.

Figure 7. Pan-HER can overcome cetuximab-resistant in intrinsically resistant patient-derived xenografts

(A) Mice were injected with HNSCC PDX cetuximab-resistant tumor (UWSCC-1) and tumors were allowed grow to 200 mm³. All mice were randomized to treatment or control groups and treated with either 50 mg/kg of cetuximab, Pan-HER, or vehicle i.p. twice weekly. The black dotted line designates the start of drug treatment (B) Pan-HER-induced HER family receptor down-modulation in PDX HNSCC tumor (UWSCC-1). Immunoblot analysis was performed of total HER family receptors in PDX HNSCC tumors after treatment. α-Tubulin was used as a loading control. (C) Reduction of HER family receptors in PDX HNSCC tumors. Cetuximab-resistant tumor samples after cetuximab or Pan-HER treatment in vivo were prepared and analyzed for EGFR, HER2, and HER3 immunohistochemistry.