Abstract
BackgroundEpidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer. The degree of response to EGFR inhibitors measured by tumor shrinkage varies widely among HNSCC patients, and the biological mechanisms that underlie therapeutic heterogeneity amongst HNSCC patients remain ill-defined.MethodsEGFR-dependent human and murine HNSCC cell lines were treated with the EGFR/ERBB inhibitors, gefitinib and AZD8931, and submitted to RNAseq, GSEA, and qRT-PCR. Conditioned media was analyzed by ELISA and Luminex assays. Murine HNSCC tumors were stained for T cell markers by immunofluorescence. Primary HSNCC patient specimens treated with single agent cetuximab were stained with Vectra multispectral immunofluorescence.ResultsThe transcriptional reprogramming response to EGFR/ERBB-specific TKIs was measured in a panel of EGFR-dependent human HNSCC cell lines and interferon (IFN) α and γ responses identified as top-ranked TKI-induced pathways. Despite similar drug sensitivity, responses among 7 cell lines varied quantitatively and qualitatively, especially regarding the induced chemokine and cytokine profiles. Of note, the anti-tumorigenic chemokine, CXCL10, and the pro-tumorigenic factor, IL6, exhibited wide-ranging and non-overlapping induction. Similarly, AZD8931 exerted potent growth inhibition, IFNα/IFNγ pathway activation, and CXCL10 induction in murine B4B8 HNSCC cells. AZD8931 treatment of immune-competent mice bearing orthotopic B4B8 tumors increased CD8 + T cell content and the therapeutic response was abrogated in nu/nu mice relative to BALB/c mice. Finally, Vectra 3.0 analysis of HNSCC patient tumors prior to and after 3–4 weeks of single agent cetuximab treatment revealed increased CD8 + T cell content in specimens from patients exhibiting a therapeutic response relative to non-responders.ConclusionsThe findings reveal heterogeneous, tumor cell-intrinsic, EGFR/ERBB inhibitor-induced IFN pathway activation in HNSCC and suggest that individual tumor responses to oncogene-targeted agents are a sum of direct growth inhibitory effects and variably-induced participation of host immune cells.
Highlights
Epidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer
We recently defined EGFR-dependent human HNSCC cell lines based on sensitivity to EGFR-specific Tyrosine kinase inhibitor (TKI) where the mean IC50 values for gefitinib and AZD8931 across the panel are 20.4 ± 3.1 and 4.1 ± 1.9, respectively [7,8,9, 43]
Gene set enrichment analysis (GSEA) revealed enrichment of the IFNα and IFNγ Hallmark pathways and negative enrichment of proliferation-associated pathways including E2F targets, G2M checkpoint and MYC targets (Fig. 1a, b). Inhibited expression of these proliferation-associated gene sets is consistent with the strong growth inhibition observed in these cell lines in response to EGFR-specific TKIs [9]
Summary
Epidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer. Treatment of head and neck squamous cell carcinoma (HNSCC) requires combined modalities of surgery, radiation and cytotoxic chemotherapy as well as targeted anti-epidermal growth factor receptor (EGFR) agents and most recently, immunotherapies that inhibit the PD1-PD-L1 axis. Despite only modest activity as single agents [3, 4], monoclonal antibodies targeting EGFR such as cetuximab and pan-EGFR/ERBB inhibitors continue to attract significant clinical attention in HNSCC [2, 4,5,6]. The mechanisms accounting for the broad range of clinical response to EGFR-targeted inhibitors in HNSCC patients remain largely unknown and this knowledge gap represents a significant hurdle to their deployment as precision oncology agents
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