Identification of biomarkers associated with acquired resistance to cetuximab in head and neck tumor cells

The epidermal growth factor receptor (EGFR) is a protein leads to the activation of intracellular pathways that act directly on cell function, changes and survival. Cetuximab (CTX) is an anti-EGFR monoclonal antibody approved for the treatment of patients with head and neck squamous cell carcinoma (HNC) and metastatic colorectal cancer (mCRC). The response of patients with mCRC to cetuximab has already been associated with wild-type KRAS, NRAS and BRAF. However, for patients with HNSCC so far, no biomarker predictive of response to CTX has been validated, being a selector challenge that may be the beneficiary of this agent. Our study suggests that the development of cetuximab resistance in HNSCC is a complex mechanism that involves an increased number of genetic aberrations and protein dysregulation. The cetuximab acquired-resistant model was successfully established and demonstrated that the overexpression of the RhoA-mTOR-PIK3-AKT pathway and stem/mesenchymal phenotype are potentially novel biomarkers founded in cetuximab-resistance.

Authors and Affiliations:

Izabela N. F. Gomes ^1,† , Renato J. da Silva-Oliveira ^1,2,†, Luciane Sussuchi da Silva ¹, Olga Martinho ³, Adriane F. Evangelista ¹, André van Helvoort Lengert ¹, Letícia Ferro Leal ^1,2, Viviane Aline Oliveira Silva ¹, Stéphanie Piancenti dos Santos ⁴, Flávia Caroline Nascimento ³, André Lopes Carvalho ¹ and Rui Manuel Reis ^1,3,4,5

¹ Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos 14784-400, Brazil;

² Barretos School of Medicine Dr. Paulo Prata—FACISB, Barretos 14785-002, Brazil

³ Life and Health Sciences Research Institute (ICVS), Medical School, University of Minho, 4710-057 Braga, Portugal;

⁴ Laboratory of Molecular Diagnosis, Barretos Cancer Hospital, Barretos 14784-400, Brazil;

⁵ 3ICVS/3B’s-PT Government Associate Laboratory, 4710-057 Braga, Portugal

^† These authors contributed equally to this work.

Abstract:

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed byWestern blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.

Journal: Cells

Link: https://doi.org/10.3390/cells11010154