Abstract 3387: Tyrosine kinase inhibitors induce TGF-β2 expression in head and neck squamous cell carcinoma cell lines as a mechanism of acquired resistance.
Authors: Kleczko Emily K. , Heasley Lydia R. , Marshall Marianne E. , Singleton Katherine R. , Heasley Lynn E. 
Published: August 2014

Worldwide, head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer. Because the epidermal growth factor receptor (EGFR) is overexpressed in about 90% of HNSCC tumors, molecularly targeted therapy against EGFR was thought to hold promise for HNSCC treatment. Cetuximab, a monoclonal antibody against EGFR, has been approved to treat HNSCC in the US. Still the 5-year survival rate for HNSCC is only 40-50%, despite the use of cetuximab in the clinic. The modest effect of cetuximab on survival indicates that EGFR inhibition alone will not be effective in treating HNSCC. The efficacy of cetuximab in treating HNSCC may be limited by acquired resistance or the activity of alternative dominant growth factor pathways in HNSCC. Previously, our lab has shown that in addition to EGFR signaling, the fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) participate as an important autocrine signaling pathway in some gefitinib-insensitive HNSCC cell lines, further indicating that inhibiting the EGFR pathway alone will not be effective in treating HNSCC. To identify genes that change in response to EGFR-specific tyrosine kinase inhibitors (TKIs) and to FGFR-specific TKIs that may mediate acquired resistance, we 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. We found that in all three cell lines, TGF-β2 mRNA was upregulated following blockade of the dominant receptor pathway. Both ELISA and qRT-PCR were used to validate this induction of TGF-β2 in these three cell lines. Treatment with a small molecule inhibitor of TGF-β receptor I (TGFβRI) provided an additive reduction of clonogenic growth when combined with EGFR and/or FGFR TKIs. Additionally, silencing TGF-β2 with shRNA in UMSCC25 cells lead to an additive decrease in clonogenic growth in combination with EGFR and/or FGFR TKIs. Furthermore, we observed an induction of NF-κB activity in the cell lines following treatment with EGFR and/or FGFR TKIs. This induction was mitigated by the use of a TGF-β2 neutralizing antibody, suggesting that TGF-β2 is signaling through a non-canonical pathway in our model. In addition to observing a rapid increase in TGF-β2 expression, the cell line UMSCC25 was chronically adapted to increasing concentrations of gefitinib over the course of several months. We observed a sustained increase in TGF-β2 expression in the resistant cells compared to the control cells. This data suggests that TGF-β2 induction may provide a novel mechanism of acquired resistance to TKIs, and supports the hypothesis that combination therapy will be more effective than monotherapy in treating HNSCC.

Citation Format: Emily K. Kleczko, Lydia R. Heasley, Marianne E. Marshall, Katherine R. Singleton, Lynn E. Heasley. Tyrosine kinase inhibitors induce TGF-β2 expression in head and neck squamous cell carcinoma cell lines as a mechanism of acquired resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2013-3387