Abstract
Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide, with 600,000 new diagnoses each year. Due to the fact that over 90% of HNSCC tumors overexpress the epidermal growth factor receptor (EGFR), molecularly targeted therapy against EGFR was approved to treat HNSCC. However, the 5-year survival rate for HNSCC patients, largely unchanged over the past several decades, remains at a dismal 40–50%, and the use of cetixumab, an EGFR-specific monoclonal antibody, has only had a marginal effect on overall survival. Likewise, the use of tyrosine kinase inhibitors (TKIs) that target EGFR has not resulted in an increased survival among HNSCC patients. The modest effect of these drugs on overall survival in HNSCC patients may be a result of tumors becoming resistant to treatment in responding patients. Acquired drug resistance has become a significant limitation to the efficacy of targeted therapeutics. Previously, our lab has identified fibroblast growth factor 2 (FGF2) and its receptor (FGFR) as being involved in an autocrine loop in gefitinib-insensitive cell lines, indicating that the FGFR pathway plays an important role in HNSCC as well. However, targeting FGFR with TKIs will almost undoubtedly lead to drug resistance in these FGFR-dependent cell lines, just as it does when targeting EGFR. To identify pathways important in acquired resistance to TKIs, our lab performed an Affymetrix GeneChip screen in which the cell lines UMSCC25, 584-A2, and Ca9-22 were treated for 4 days with 0.3μM AZ8010, a TKI selective for FGFRs, and/or 0.1μM gefitinib, an EGFR-selective TKI. TGF-β2 was consistently upregulated after treatment with TKIs that inhibited the dominant growth pathway in the HNSCC cell lines. Both ELISA and qRT-PCR confirmed this induction of TGF-β2 in these three cell lines. Treatment with a TGF-β2 neutralizing antibody and with small-molecule TGF-β receptor inhibitors provided additive reduction of growth in response to TKIs. Additionally, silencing TGF-β2 with shRNA in the cell line UMSCC25 leads to decreased clonogenic growth in combination with TKIs compared to a non-silencing control (NSC). Moreover, we found that an induction of NF-kB activity occurs in response to treatment with TKIs, and this induction is mitigated by the addition of a TGF-β2 neutralizing antibody. Furthermore, the cell lines UMSCC25 and UMSCC22A were chronically adapted to gefitinib over the course of several months, and we have observed a stable, prolonged increase in TGF-β2 mRNA expression in the resistant lines compared to the DMSO control lines. Taken together, this data suggests that TGF-β2 induction may provide a novel mechanism of acquired resistance to TKIs, and possibly contributes to oncogene switching programs in HNSCC.
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