Abstract 1631: FGF2 activation of FGFR1 in head and neck squamous cell carcinoma is associated with more invasive disease and can be attenuated by FGFR inhibition
Authors: English Isabel A. , Martinez Jacqueline , El Rassi Edward , Schmidt Mark , Langer Ellen , Bornstein Sophia , Gleysteen John , Wong Melissa , Druker Brian , Traer Elie 
Published: July 2016

Abstract
Introduction. Head and neck squamous cell carcinomas (HNSCCs) account for nearly 600,000 deaths worldwide annually and have limited treatment options. Approximately 20% of HNSCCs harbor amplifications of fibroblast growth factor receptor 1 (FGFR1) on chromosome 8p, however FGFR1 amplification by itself does not predict clinical response to FGFR inhibitors. We hypothesized that FGF2, or basic FGF, ligand expression is a better marker of FGFR activation and predictor of response to FGFR inhibitors.
Results. A tissue micro array (TMA) of HNSCC patient biopsies was stained and quantitated for FGF2 expression by Aperio ImageScope software. FGF2 was significantly increased in recurrent tissue samples (p = 0.04). We examined a number of immortalized HNSCC cell lines and found that overexpression of both FGF2 and FGFR1 predicted response to the selective FGFR inhibitor PD173074. FGFR inhibition did not cause apoptosis, but rather induced a G0/G1 arrest and growth inhibition. FGFR inhibition also induced a change in cell morphology, with a significant increase in cell size and adherence. The expression of epithelial-to-mesenchymal transition (EMT) proteins was examined and FGF2-FGFR1 activation was associated with a more mesenchymal phenotype. Accordingly, FGFR inhibition reversed invasiveness as measured using the Incucyte WoundMaker scratch assay, suggesting that HNSCCs with FGF2-FGFR1 activation have more metastatic potential. Invasiveness of these cells in vivo was confirmed using orthotopic injection into the buccal pad of NSG mice. Once primary tumors reached 0.8 cm in size, mice were sacrificed and buccal mucosa, lung, liver, and neck tissue were examined post-mortem. All of the injected animals developed local invasion, and distant metastases in the lungs. 5/7 mice also had metastases in the liver and this model is being used to test the ability of FGFR inhibition to prevent metastasis.
The mechanism of autocrine FGF2-FGFR1 activation was further explored and FGF2 was found to be secreted in association with extracellular vesicles (ECVs). Interestingly, inhibition of FGFR reduced secretion of ECVs and FGF2, providing a novel approach to target autocrine and paracrine FGFR1 activation within the tumor. We further tested a number of small molecule inhibitors in combination with PD173074 to look for synergistic combinations of kinase inhibitors and found significant synergy between EGFR and FGFR inhibitors suggesting this combination may be most effective in patients with HNSCC.
Conclusions. Increased FGF2 in HNSCC patient samples is correlated with recurrent disease. FGF2-FGFR1 activation increases invasiveness through activation of EMT genes both in vitro, and in an orthotopic model. Inhibition of FGF2-FGFR1 reversed the invasive phenotype in vitro and may be an effective therapeutic strategy to reduce metastases in HNSCC patients.
Citation Format: Isabel A. English, Jacqueline Martinez, Edward El Rassi, Mark Schmidt, Ellen Langer, Sophia Bornstein, John Gleysteen, Melissa Wong, Brian Druker, Elie Traer. FGF2 activation of FGFR1 in head and neck squamous cell carcinoma is associated with more invasive disease and can be attenuated by FGFR inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1631.