The TAM subfamily (Tyro3, Axl, MerTK) of receptor tyrosine kinases are implicated in several cancers, where they have been shown to support primary tumorigenesis as well as secondary resistance to cancer therapies. Relatively little is known about the oncogenic role of Tyro3, including its ligand selectivity and signalling in cancer cells. Tyro3 showed widespread protein and mRNA expression in a variety of human cancer cell lines. In SCC-25 head and neck cancer cells expressing both Tyro3 and Axl, Western blotting showed that both natural TAM ligands ProS1 and Gas6 rapidly stimulated Tyro3 and Erk kinase phosphorylation, with ProS1 eliciting a greater effect. In contrast, Gas6 was the sole stimulator of Axl and Akt kinase phosphorylation. In MGH-U3 bladder cancer cells, which express Tyro3 alone, ProS1 was again the stronger stimulator of Tyro3 and Erk stimulation but additionally stimulated Akt phosphorylation. Conditioned medium from ProS1-secreting 786-0 kidney cancer cells replicated the kinase activation effects of recombinant ProS1 in SCC-25 cells, with specificity confirmed by ProS1 ligand traps and warfarin. In addition, ProS1 protected cancer cells from acute apoptosis induced by staurosporine, as well as additionally, long-term serum starvation-induced apoptosis in MGH-U3 cells (Tyro3 only), which reflects its additional coupling to Akt signalling in these cells. In conclusion, we have shown that ProS1 is a tumour-derived functional ligand for Tyro3 that supports cancer cell survival. Furthermore, the ProS1-Tyro3 interaction is primarily coupled to Erk signalling although it displays signalling diversity dependent upon its representative expression as a TAM receptor in tumour cells.

The TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and protein S (ProS1), are implicated in tumorigenesis and chemoresistance in various cancers. The β-galactoside binding protein galectin-3 (Gal-3), which is also implicated in oncogenesis, has previously been shown to be a ligand for MerTK. However, the selectivity of Gal-3 for the other TAM receptors, and its TAM-mediated signalling and functional properties in cancer cells, remain to be explored. The present study was aimed at determining these, including through direct comparison of Gal-3 with the two canonical TAM ligands. Exogenous Gal-3 rapidly stimulated Tyro3 receptor phosphorylation to the same extent as the Tyro3 ligand ProS1, but not Axl, in the cultured human cancer cell lines SCC-25 (express both Tyro3 and Axl) and MGH-U3 (express Tyro3 only). Gal-3 also activated intracellular Erk and Akt kinases in both cell lines and furthermore protected cells from acute apoptosis induced by staurosporine but not from serum-starvation induced apoptosis. In addition, Gal-3 significantly stimulated cancer cell migration rate in the presence of the Axl blocker BGB324. Therefore, these results have shown Gal-3 to be a novel agonist for Tyro3 RTK, activating a Tyro3-Erk signalling axis, as well as Akt signalling, in cancer cells that promotes cell survival, cell cycle progression and cell migration. These data therefore reveal a novel mechanism of Tyro3 RTK activation through the action of Gal-3 that contrasts with those of the known TAM ligands Gas6 and ProS1.

Tyro3 is a member of the TAM subfamily of receptor tyrosine kinases alongside Axl and MerTK, which are activated by homologous ligands Gas6 and protein S. The TAMs activate signalling pathways that mediate diverse functions including cell survival, proliferation, phagocytosis and immune regulation, and defects in TAM-dependent processes are associated with the development of human autoimmune diseases and numerous cancers. In this study, we have focused on the signalling and functional roles of Tyro3, about which much remains unknown. For this purpose, we used cultured human cancer cell lines with different levels of TAM expression to reveal the relative significance of Tyro3 amongst the TAMs. Knockdown of Tyro3 expression by siRNA in MGH-U3 cells, which express Tyro3 as sole TAM, caused a reduction in cell viability, which could not be rescued by TAM ligand, demonstrating the dependence of these cells solely on Tyro3. In contrast, the reduced viability of SCC-25 cells upon Tyro3 knockdown could be rescued by Gas6 as these cells express both Tyro3 and Axl and hence Axl expression was preserved. An increase in the fraction of Tyro3 knockdown cells in the early apoptotic phase was observed in four different cell lines each with a different TAM expression profile, revealing a broad anti-apoptotic function of Tyro3. Furthermore, in the Tyro3-dependent cells, Tyro3 depletion caused a significant increase in cells in the G0/G1 phase of the cell cycle concomitant with decreases in the G2/M and S phases. In addition, a cancer pathway gene discovery array revealed distinct sets of genes that were altered in expression in cancer cells upon Tyro3 knockdown. Together, these results have elucidated further a role of Tyro3 in promoting multiple tumour-supporting pathways in human cancer cells, which differs in extent depending on the presence of other TAMs in the same cells.

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.

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