The receptor tyrosine kinases Tyro3 and Axl are co‐regulated and activate discrete signaling pathways for cancer cell proliferation and invasion respectively

Abstract

The TAM (Tyro3, Axl, Mer) subfamily of receptor tyrosine kinases (RTKs) have been implicated in tumor growth and spread in several different cancers, with Axl in particular being shown to regulate cell invasion as well as resistance to other targeted therapies such as EGFR blockade. However, little is known about Tyro3, which although having a more restricted tissue pattern, is also found overexpressed in several solid cancers. The aim of this study is to elucidate the specific signaling pathways emanating from Tyro3 activation in cancer cells. In addition to signaling via homodimeric activation of RTKs, diversity in signaling may be achieved by cross‐talk amongst different RTKs via e.g. heterodimerization. Therefore, a further aim is to identify such novel, unconventional activation mechanisms for the TAMs in cancer cells.A range of human cancer cell lines that overexpress specific TAM RTKs were cultured for this study. Western blot was used to detect protein expression and phosphorylation. The TAM ligand Gas6 rapidly stimulated Axl phosphorylation within minutes but did not alter Tyro3 phosphorylation. Conversely, the related TAM ligand ProS1 rapidly stimulated Tyro3 phosphorylation but with no effect on Axl. Incubation of cells with the Axl‐selective small molecule inhibitor BGB324 led to a blockade of not only basal Axl activity, but also Tyro3 phosphorylation, suggesting that Tyro3 may be activated through a heterodimeric interaction with Axl. This interaction was confirmed by coimmunoprecipitation of Axl‐Tyro3 complexes from cell lysates, which was further strengthened by Gas6 stimulation. In addition, we found that Tyro3 protein stability was dependent on Axl co‐expression, as Axl siRNA knockdown reduced presence of cellular Tyro3 protein but not its mRNA. This suggests the Axl‐Tyro3 heterodimerization stabilizes Tyro3 presence at the cell surface. Furthermore, siRNA knockdown of Tyro3 in cells revealed that Tyro3 functions as a mediator of signaling for cell survival and cell cycle progression through regulation of cyclin D1 – a role distinct from the known proinvasion role of Axl.Based on these findings, we propose that Tyro3 may be a driver of tumor persistence and chemoresistance through association with Axl and potentially other RTKs. Our current work is further revealing the divergence of downstream signaling pathways to emanate from TAM RTKs activated in different ways, and how these regulate the major cellular behaviors during tumor progression and spread.Support or Funding InformationThis research is funded by the University of Portsmouth and by the Council for At‐Risk Academics (Cara).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.