Abstract 731: Nuclear factor-κB activation and cetuximab-resistance in HNSCC cell lines

Abstract

Abstract Background: Cetuximab, a monoclonal antibody targeting EGFR, has been shown to increase survival in patients with head and neck squamous cell carcinoma (HNSCC). However, only selective patients gain therapeutic benefit. To determine a mechanism of primary resistance to cetuximab, we characterized a primary keratinocyte cell line and 34 HNSCC cell lines for somatic mutations and gene expression alterations. Experimental design: Thirty-five cell lines were assessed by 1) multiplex SNaPshot genotyping to detect 38 somatic mutations in the EGFR and its related pathways, 2) DNA sequencing of the tyrosine kinase domains of MET and FGFR3 to detect mutations, 3) qRT-PCR to quantify EGFR, MET and FGFR1 gene copy number (CN), 4) microarray analyses for gene expression profiles, and 5) matrigel colony formation assays to determine sensitivity to cetuximab. Gene mutations and CN changes were examined for potential association with cetuximab sensitivity. Differentially expressed genes between the cetuximab-resistant and -sensitive cell lines were assessed by GeneSpring and Ingenuity Pathways Analysis (IPA). To confirm the expression array data, seven representative cell lines were treated with BAY-11-7082, an IKB kinase inhibitor, to inhibit NF-kB activity. Results: Three of 35 cell lines had mutations in PIK3CA (E542K, E545Q and H1047R). The cell line with E542K mutation was resistant to cetuximab while the cell lines with E545Q and H1047R were sensitive to cetuximab. No mutations were found in MET or FGFR3. CN gains were detected in EGFR and FGFR1 (20 cell lines with EGFR CN 4-10, 10 with EGFR CN >10, and 9 with FGFR1 CN 4-10), but not in MET. Interestingly, many of the cell lines with EGFR CN gains also had FGFR1 CN gains (Spearman rank test, r=0.48, p=0.0032). However, there was no correlation with the CN gains and cetuximab sensitivity. IPA showed genes involved in epithelial-to-mesenchymal transition (EMT) and NF-kB activation to be associated with cetuximab resistance. Cetuximab-resistant cell lines had higher expression of vimentin and lower expression of E-cadherin. BAY-11-7082 inhibited cellular proliferation of four cetuximab-resistant cell lines, but not three sensitive cell lines. Conclusion: Mutations commonly seen in non-HNSCC cancers are rare, but EGFR and FGFR1 CN gains are common in HNSCC cell lines. The EMT phenotype and NF-kB activation may have an important role in cetuximab resistance and in novel therapeutic development in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 731. doi:10.1158/1538-7445.AM2011-731