Combination of anti-HER3 antibody MM-121/SAR256212 and cetuximab inhibits tumor growth in preclinical models of head and neck squamous cell carcinoma

Abstract

The EGFR monoclonal antibody cetuximab is the only approved targeted agent for treating head and neck squamous cell carcinoma (HNSCC). Yet resistance to cetuximab has hindered its activity in this disease. Intrinsic or compensatory HER3 signaling may contribute to cetuximab resistance. To investigate the therapeutic benefit of combining MM-121/SAR256212, an anti-HER3 monoclonal antibody, with cetuximab in HNSCC, we initially screened 12 HNSCC cell lines for total and phosphorylated levels of the four HER receptors. We also investigated the combination of MM-121 with cetuximab in preclinical models of HNSCC. Our results revealed that HER3 is widely expressed and activated in HNSCC cell lines. MM-121 strongly inhibited phosphorylation of HER3 and AKT. When combined with cetuximab, MM-121 exerted a more potent antitumor activity through simultaneously inhibiting the activation of HER3 and EGFR and consequently the downstream PI3K/AKT and ERK pathways in vitro. Both high and low doses of MM-121 in combination with cetuximab significantly suppressed tumor growth in xenograft models and inhibited activations of HER3, EGFR, AKT, and ERK in vivo. Our work is the first report on this new combination in HNSCC and supports the concept that HER3 inhibition may play an important role in future therapy of HNSCC. Our results open the door for further mechanistic studies to better understand the role of HER3 in resistance to EGFR inhibitors in HNSCC.

Fig. 1

A, total and activated HER3 expression in HNSCC cell lines. Western blot was used to determine the expression of HER3 and pHER3 expression in 12 HNSCC cell lines. B, MM-121 alone inhibits HER3 and AKT activation in vitro. HNSCC cell lines SCC47 and Tu212 were treated with escalating doses of MM-121 as indicated for 12 and 24 hours. Cell lysates were immunoblotted to detect pHER3 (Tyr1289), pAKT (Ser473), total HER3 and AKT. Experiments were repeated three times.

Fig. 2. The cetuximab and MM-121 (CM) combination simultaneously inhibits PI3K/AKT and ERK signaling pathways

SCC47 and Tu212 cells were treated with 125μg/mL MM-121, 62μg/mL cetuximab and the combination for 48 and 72 hours. Cell lysates were collected and immunoblotted as indicated.

Fig. 3. The CM combination inhibits HNSCC cell growth in vitro

A, for colony formation assay, SCC47 and Tu212 cells at 200/well were treated with 20μg/mL MM-121, 2μg/mL cetuximab or the combination for 9 days. The medium was changed every three days before cells were stained with 0.2% crystal violet. Cell numbers ≥50 were considered as a colony. ImageJ software was used for colony counting. B, cells (2.5 × 10⁵/well) were cultured for overnight and treated with MM-121 125μg/ml, cetuximab 62μg/ml or the combination for 72 hours and then trypsinized and counted. Error bars are mean ± SE from 3 independent experiments. (* indicates p<0.05 vs. ctrl, ** indicates p<0.05 vs. all other groups).

Fig. 4. The CM combination induces cell cycle arrest and apoptosis in vitro

A, for cell cycle analysis, cells (2.5 × 10⁵/well) were treated with 125μg/mL MM-121, 62μg/mL cetuximab and the combination for 72 hours and then analyzed by flow cytometric analysis. The percentage of cells in the G0-G1, S, and G2-M phases of the cell cycle are shown. B, cell lysate in (A) was collected and immunoblotted for cell cycle related cyclinD1 and cyclinE. C, for apoptosis assay, cells were treated for 72 and 96 hours with the same doses of drugs as cell cycle assay and then analyzed by flow cytometry. D, cell lysates from the same treatment as apoptosis assay were collected and immunoblotted for caspase3 and PARP. All data are expressed as mean ± SE from 3 independent experiments. (*indicates p<0.05, ** indicates p<0.01 vs. ctrl).

Fig. 5. The CM combination inhibits HNSCC xenograft tumor growth in vivo

Mice bearing subcutaneous SCC47 and Tu212 tumors of approximately 100 mm³ were treated by intraperitoneal injection (i.p.) twice per week for 4 weeks with: PBS (phosphate-buffered saline) control, cetuximab (6.25μg/dose), MM-121 at low dose (300μg/dose, MM-121.LD), MM-121 at high dose (600μg/dose, MM-121.HD), the combination with LD MM-121 (Comb.LD), and the combination with HD MM-121 (Comb.HD). A, tumor volumes were measured three times a week. B, tumors were harvested and weighed 26 (Tu212) and 29 (SCC47) days after the first treatment. C, major organs were harvested for toxicity evaluation by H&E (Magnification: 100×). Error bars are mean ± SE of 7 mice from each group. (** indicates p<0.01 v.s. Ctrl, ^† indicates p<0.05 v.s. cetuximab)

Fig. 6. Effect of the CM combination on HER3 and EGFR signaling and tumor proliferation, apoptosis and angiogenesis in vivo

A, the CM combination inhibited HER3 and EGFR signaling in vivo. Fresh tumor tissues were collected and stored at -80°C. Three representative tissue lysates from each group were prepared for Western blot analysis. ImageJ software was used for Western blot quantification. All data are expressed as mean ± SD from 3 tissue samples. B, the CM combination significantly increased necrosis, inhibited proliferation and angiogenesis and induced apoptosis in vivo. Data show the representative tumors with hematoxylin and eosin (H&E) staining, Ki67, TUNEL and CD34 staining (Magnification: 200×). Tissue slides were observed by at least two independent personnel. The percentage of necrotic areas, percentage of positive Ki67 and TUNEL staining and CD34 positive signals were counted from five randomly selected areas in each slide at 100× magnification. For TUNEL assay, green fluorescence indicates positive cells. Error bars are mean ± SE of 7 mice from each group. (* indicates p < 0.05 and ** indicates p < 0.01 v.s. Ctrl).