Abstract 3198: Ron tyrosine kinase synergizes with EGFR to confer aggressive phenotype in head and neck cancers
Authors: Keller James C. , Batajoo Shishir K. , Shroyer Kenneth R. , Ischenko Irene , Chandler Christopher S. , Hayman Michael J. , Chan Edward L. 
Published: June 2012

Abstract
Background: Data generated over the past 20 years strongly support a critical role for EGFR in head and neck squamous cell carcinomas (HNSCC). While there have been successes with EGFR inhibitors in the treatment of this cancer, results have not been dramatic and additional molecular targets are needed. We previously showed that the loss of Ron activity negatively impacted squamous cell tumor growth in a murine model. Based on these results, we hypothesized that high Ron expression confers an aggressive phenotype in HNSCC. To test this hypothesis, we analyzed 73 primary HNSCC for Ron and correlated its expression with clinical and pathological features. We also assessed the biological and biochemical response of altering Ron in HNSCC cells.
Methods: Snap frozen primary HNSCC were collected through the Cooperative Human Tissue Network (CHTN). When patients presented to the CHTN institutions for surgery, informed consents to procure tumors were obtained; patients’ medical charts were reviewed to retrieve clinical and pathological data. Frozen tumors were analyzed for EGFR and Ron by immunoprecipitation followed by western blot (IPW) and immunohistochemistry (IHC). Only histologically confirmed, untreated HNSCC of the primary sites were included in the correlation analysis; samples with &lt;50% tumor to stroma ratio were excluded. SCC9 and COS1 cells were used for biological and biochemical analyses. SCC9 cells were infected with a shRNA Ron expressing retroviruses to generate stable clones with Ron knockdown. The migratory ability of these clones was determined by the transwell filter assay. COS1 cells were transiently transfected with a Ron expression vector; endogenous EGFR in the cell lysates, with and without EGFR inhibitor treatment, was analyzed for phosphorylation by IPW.
Results: We evaluated 73 HNSCC for Ron and EGFR expression/phosphorylation by IPW and IHC. We discovered that 55% (40/73) express Ron. Ron expression is limited to the carcinomas. There was no difference in terms of sex, tumor location or differentiation and the presence of perineural or vascular invasion between Ron+ and Ron- tumors. However, patients with Ron+ HNSCC were significantly older. Ron+ tumors were also larger and had higher EGFR expression. In addition, there was a statistically significant interaction between Ron and pEGFR in their association with tumor size and nodal stage (p = 0.05). There was also a highly significant association between detecting Ron and pEGFR in the same tumors (p &lt; 0.001). The knock-down of Ron expression in SCC9 cells significantly blunted their migratory response to Ron ligand and also to EGF. Biochemical analysis also showed that transient overexpression of Ron in COS cells led to transactivation of EGFR. Transactivation of EGFR by Ron partially overcame the inhibitory effect of Tarceva and Erbitux on EGFR signaling.
Conclusion: Ron synergies with EGFR to confer aggressive features 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 3198. doi:10.1158/1538-7445.AM2011-3198