Abstract 2474: Smad4 loss induces cetuximab resistance and increased metastatic potential in head and neck squamous cell carcinoma
Authors: Ozawa Hiroyuki , Cheng Haixia , Fertig Elana J. , Howard Jason D. , Markovic Ana , Hughes Robert , Perez Jimena , Quon Harry , Chung Christine H. 
Published: May 2015

Introduction: Cetuximab is a monoclonal antibody that increases survival in head and neck squamous cell carcinoma (HNSCC) patients when used in combination with radiation or chemotherapy. However, this EGFR targeted-agent only has modest activity when administered as a monotherapy and a majority of patients develop therapeutic resistance. Previous work has established the TGF-beta signaling pathway has a critical role in HNSCC development and progression. Modulation of this pathway, and more specifically loss of Smad4 expression, is a common means of developing drug resistance in many cancers. To determine the role of Smad4 loss in HNSCC development, metastasis, and cetuximab resistance, Smad4 was knocked down or reexpressed in HNSCC cell lines and evaluated in vitro and in vivo.

Methods: Smad4 was knocked-down in SCC61 following stable introduction of Smad4-targeting shRNA. Using stable lentiviral transduction, Smad4 was reexpressed in FaDu, a HNSCC cell line which lacks de novo expression of Smad4. Following modulation, changes in cetuximab sensitivity were determined using Matrigel colony formation assays. The effects of Smad4 modulation on various signal transduction pathways were determined by Western blot analysis with total and phospho-specific antibodies. Changes in metastatic potential were evaluated using an orthotopic in vivo model. The subsequent development of lymph node metastases were monitored utilizing luciferin-based imaging. Key proteins associated with metastasis were also assayed using ELISA and protein antibody arrays.

Results: Smad4 loss increased the growth of SCC61 and FaDu and Smad4 status correlated with cetuximab resistance in both cells. Western blot analysis demonstrated Smad4 loss activates JNK and MAPK signaling. Consequently, co-treatment of Smad4 knockdown cells with cetuximab and a JNK inhibitor reversed the previously observed therapeutic resistance. Smad4 knockdown also increased lymph node metastasis in the orthotopic in vivo model. Protein array analysis determined Smad4 knockdown increased the expression of amphiregulin, FGF2, and VEGF, pathways capable of promoting metastasis. Expression of these Smad4-sensitive targets was inhibited with the use of a MAPK inhibitor.

Conclusion: We determined Smad4 loss enhances cetuximab resistance and in vivo metastasis development in HNSCC. Additionally, JNK and MAPK pathway activation play an important role in mediating this process. Our results suggest Smad4 downregulation plays a critical role for prognosis of HNSCC patients, and JNK and MAPK inhibition may contribute to improve therapeutic outcome of those patients.

Citation Format: Hiroyuki Ozawa, Haixia Cheng, Elana J. Fertig, Jason D. Howard, Ana Markovic, Robert Hughes, Jimena Perez, Harry Quon, Christine H. Chung. Smad4 loss induces cetuximab resistance and increased metastatic potential in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2014-2474