Heteroactivation Amongst Tam Receptor Tyrosine Kinases And Via Egfr In Human Glioma Cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAGlioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumor, and often features a combination of tumor suppressor gene inactivation and multiple oncogene overactivation. The TAM (Tyro3, Axl, Mer) subfamily of receptor tyrosine kinases (RTKs) are recognized as playing prominent roles in tumorigenesis, including in gliomas, in particular in regulation of cell migration, invasion, survival and chemoresistance. In addition to signaling via homodimeric activation of RTKs, diversity in signaling may be achieved by cross-talk amongst different RTK types, including heterodimerization. In lung and breast cancers, Axl has been shown to modulate acquired resistance to EGFR inhibitors. However, the potential for RTK signaling cross-talk with TAMs in glioma is unknown. Therefore, the purpose of this study is to determine such novel, unconventional activation mechanisms for the TAMs in human GBM cells.Two human GBM-derived cell lines that overexpress TAMs were cultured and studied in parallel: SNB-19 and UP007. Western blot was used to detect protein expression and phosphorylation. Both cell lines showed distinct responses in terms of Axl signaling and its inhibition. Stimulation of SNB-19 cells with the natural TAM ligand Gas6 led to concentration-dependent increases in Axl phosphorylation (threefold) and Akt kinase activation further downstream (twofold) over 60 min. However, Gas6 did not stimulate Axl phosphorylation in UP007 cells but did cause a threefold increase in Akt phosphorylation. Tyro3 RTK was not affected by Gas6 in either cell line. In both cell lines, incubation with the Axl-specific small molecule inhibitor BGB324 led to a blockade of not only basal Axl activity, but also Tyro3 phosphorylation, suggesting that Tyro3 is activated through a heterodimeric interaction with Axl. This interaction was confirmed by coimmunoprecipitation of Axl-Tyro3 complexes from GBM cell lysates.In addition, GBM cells stimulated with the growth factor EGF resulted in phosphorylation of Axl, in addition to EGFR. Furthermore, this effect was abolished by preincubation with the EGF-specific inhibitor gefitinib but not the Axl-specific inhibitor BGB324, further supporting the EGFR-Axl functional interaction. Conversely, the Axl-specific inhibitor had no effect on activation of EGFR by EGF, which was abolished by gefitinib. In addition, stimulation of cells with EGF also stimulated Axl kinase activity.In conclusion, these data show for the first time, heteroactivation to occur of (i) Axl via EGFR and (ii) Tyro3 via Axl, in human GBM cells. The exact mechanism and biological functions occurring through these new receptor associations are currently being investigated. Further studies will reveal the divergence of downstream signalling pathways to emanate from TAMs activated in different ways, and how these regulate major functional processes involved in glioma progression, such as migration and invasion.Citation Format: Mikaella Vouri, Qian An, Geoffrey J. Pilkington, Sassan Hafizi. Heteroactivation amongst TAM receptor tyrosine kinases and via EGFR in human glioma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 119. doi:10.1158/1538-7445.AM2015-119