Neuregulin Signaling Is a Mechanism of Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma

Abstract

EGFR signaling confers resistance to radiotherapy and is a validated target in head and neck squamous cell carcinoma (HNSCC). The inhibition of EGFR in combination with radiotherapy improves local control and overall survival in these patients; however, therapeutic resistance limits the efficacy of this approach. We therefore sought to identify cellular mechanisms that cause resistance to EGFR inhibition and radiotherapy in HNSCC. Though clonal isolation of carcinoma cells exposed to increasing concentrations of cetuximab, we found that resistant cells upregulate prosurvival ErbB3 and AKT signaling. Using EFM-19 cells and confirmatory analysis of protein levels, we demonstrate that cetuximab resistance is characterized by enhanced neuregulin expression identifying a novel adaptive mechanism of therapeutic resistance. Inhibition of this autocrine loop with CDX-3379 (an ErbB3 specific antibody) was sufficient to block ErbB3/AKT signaling in cetuximab resistant cells. The combination of CDX-3379 and cetuximab reduced proliferation and survival after radiotherapy in several HNSCC cell lines. These in vitro findings were confirmed in xenograft tumor growth experiments including an approach using growth factor-supplemented Matrigel. In vivo, the delivery of EGFR and ErbB3 antibodies significantly reduced tumor growth in cetuximab-resistant FaDu and CAL27 xenografts. In summary, this work demonstrates that autocrine NRG ligand secretion is a mechanism for therapeutic resistance to cetuximab and radiotherapy. This cross-resistance to both therapeutic modalities identifies NRG as an actionable therapeutic target for improving treatment regimens in HNSCC.

Figure 1.. Clonal strategy and screening for cetuximab resistance.

(A.) Overview of the strategy to generate a CR cell line. A431 cells were exposed to increasing concentrations of the antibody over ~14 weeks, then subjected to a selection process to obtain clones. Each shade of grey represents a different clone. (B.) Western blots demonstrating changes in EGFR, ErbB2 and ErbB3 levels and phosphorylation in A431 parental (WT) and cetuximab-resistant (CR) clone cell lines, and (C.) AKT and ERK1/2 phosphorylation and expression. GAPDH was used as a loading control. Data are representative from two experiments.

Figure 2.. Upregulation of neuregulin family ligand expression in A431-CR clones.

(A.) Western blots demonstrating changes in ErbB3, AKT and ERK1/2 phosphorylation and expression in two A431-CR clone cell lines (CR1 and CR2) treated with vehicle, 30 nM CTX, 50 nM CDX-3379, or both for 48 hours. GAPDH was used as a loading control. Representative data from three experiments are shown. (B.) Western blots in EFM-19 cells after the addition ofconditioned media from EFM-19 cell line, A431-WT and two A431-CR clones (CR1 and CR2). Cells were treated with vehicle, 30 nM CTX, 50 nM CDX-3379, or both for 48 hours. GAPDH was used as a loading control. Representative data from three experiments are shown. (C.) Western blots demonstrating changes in neuregulin (NRG) levels and ErbB3 phosphorylation and expression in A431-WT and two A431-CR clone cell lines treated with vehicle, 30 nM CTX, 50 nM CDX-3379, or both for 48 hours. GAPDH was used as a loading control. Representative data from three experiments are shown (D.) Bar graphs representing fold increases in proliferation for A431-WT clone and A431-CR clones after 5 days of drug exposure. Cultures were treated as described in Materials and Methods. The results are mean values ± standard error (SE) for three independent experiments for each cell line. An * indicates a significant difference vs untreated, an ** indicates a significant difference vs CTX, an # indicates a significant difference vs CTX alone, and an ## indicates a significant difference vs CTX + NRG (p ≤ 0.05).

Figure 3.. CDX-3379 blocks the induction of NRG/ErbB3/AKT pathway in HNSCC cell lines.

(A.) Western blots representing levels of NRG expression and AKT phosphorylation and expression in FaDu (F), A431 (A), Detroit562 (D), CAL27 (C), UNC7 (U7) and UNC10 (U10) HNSCC cell lines. GAPDH was used as a loading control. Representative data from two experiments are shown. (B.) Western blots demonstrating changes in NRG levels and ErbB3 and AKT phosphorylation and expression in FaDu, Detroit562 and CAL27 HNSCC cell lines treated with vehicle, 30 nM CTX, 50 nM CDX-3379, or both for 48 hours. GAPDH was used as a loading control. Representative data from two experiments are shown. (C.) Bar graphs representing fold increases in proliferation for FaDu, CAL27 and Detroit562 HNSCC cell lines after 5 days of drug exposure. Cultures were treated as described in Materials and Methods. The results are mean values ± standard error (SE) for three independent experiments for each cell line. An * indicates a significant difference vs untreated, an ** indicates a significant difference vs CTX, an # indicates a significant difference vs CTX alone, and an ## indicates a significant difference vs CTX + NRG (p ≤ 0.05).

Figure 4.. CDX-3379 effectively blocks the neuregulin autocrine survival signaling in FaDu-CR clones.

(A.) Western blots in EFM-19 cells after the addition ofconditioned media from EFM-19 cell line, FaDu-WT and two FaDu-CR clones (CR1 and CR2). Cells were treated with vehicle, 30 nM CTX, 50 nM CDX-3379, or both for 48 hours. GAPDH was used as a loading control. Representative data from three experiments are shown. (B.) Western blots demonstrating changes in NRG levels and in ErbB3 phosphorylation and expression in FaDu-WT and FaDu-CR clone cell lines treated with vehicle, 30 nM CTX, 50 nM CDX-3379, or both for 48 hours. GAPDH was used as a loading control. Representative data from three experiments are shown. (D.) Bar graphs representing fold increases in proliferation for FaDu-CR clone after 5 days of drug exposure. Cultures were treated as described in Materials and Methods. The results are mean values ± standard error (SE) for three independent experiments for each cell line. An * indicates a significant difference vs untreated, an ** indicates a significant difference vs CTX, an # indicates a significant difference vs CTX alone, and an ## indicates a significant difference vs CTX + NRG (p ≤ 0.05).

Figure 5.. Neuregulin enhances colony formation in the presence of cetuximab, CDX-3379 addition reverses this autocrine pathway.

Clonogenic survival of (A. and C.) FaDu-CR clone cell line and (B. and D.) CAL27 cells treated as described in Material and Methods. The results represent data from three independent experiments for each cell line. Data are represented as the mean ± standard error. An * indicates a significant difference vs CTX alone and an ** indicates a significant difference vs CTX + NRG (p ≤ 0.05).

Figure 6.. Therapeutic effects of CDX-3379 in vivo in the presence or absence of cetuximab (CTX), neuregulin (NRG) and/or radiotherapy (RT).

(A.) Mice bearing tumors derived from FaDu-CR cells received vehicle, 2 mg/kg CTX twice a week I.P., 10 mg/kg CDX-3379 twice a week I.P., or both for a week. Values are the means ± SE of eight tumors per group. An * indicates a significant difference (p ≤ 0.05) compared to the vehicle, CTX or CDX-3379 group treatments. (B.) Mice bearing tumors derived from FaDu-CR cells received 5 daily doses of RT of 2 Gy to the tumor using 250-kV orthovoltage unit, RT+2 mg/kg CTX twice a week I.P., RT+10 mg/kg CDX-3379 twice a week I.P., or the triple combination for a week. Values are the means ± SE of eight tumors per group. An * indicates a significant difference (p ≤ 0.05) compared to RT, RT+CTX or RT+CDX-3379 group treatments. (C.) Mice bearing tumors derived from CAL27 cells encapsulated in matrigel in the presence of 1 μg per tumor of NRG received a single I.P. injection of vehicle, 2 mg/kg CTX, 10 mg/kg CDX-3379 or both. Values are the means ± SE of eight tumors per group. An * indicates a significant difference (p ≤ 0.05) compared to vehicle, CTX or CDX-3379 group treatments. (D.) Mice bearing tumors derived from CAL27 cells encapsulated in matrigel in the presence of 1 μg per tumor of NRG received 3 daily fractions of RT of 2 Gy to the tumor using 250-kV orthovoltage unit, RT+ a single I.P. injection 2 mg/kg CTX, RT+ a single I.P. injection 10 mg/kg CDX-3379 or RT+ a single I.P. injection of both. Values are the means ± SE of eight tumors per group. An * indicates a significant difference (p ≤ 0.05) compared to RT, RT+CTX or RT+CDX-3379 group treatments.