Page 89 - Helicobacter pylori and Gastric Cancer: From Tumor microenvironment to Immunotherapy

presence or absence of the Smoothened inhibitor cyclopamine (Figure 3). We observed strong accumulation of radioactivity in both Hedgehog challenged cells, as well as in cells challenged with Hedgehog in the presence of cyclopamine. We thus concluded that endocytosis following Hedgehog stimulation does not require Smoothened activity and that hence our model system was suitable for investigating at least certain aspects of Smoothened-independent signal transduction. To further characterize these aspects we performed kinome profiling of Smoothened-/- fibroblasts (originally obtained from Drs. James Chen and Philip Beachy and previously described by Varajosalo et al[47], challenged with either a vehicle control or 1 μg/ml Shh for 10 min. The results are summarized in Figure 3 and Supplementary table 1 and reveal that the influence of Smoothened-independent Hedgehog-induced signaling on cellular kinase activity is substantial. Conspicuously lacking, however, is G protein-coupled receptor-associated signal transduction which is obviously in line with the absence of Smoothened-dependent events. In particular activation of cytoskeletal remodeling is seen following addition of Hedgehog, which correlates with a downregulation of activity of the Csk negative regulators of Src activity and may relate to the observed Smoothened-independent effects of Hedgehog on endocytosis described above, especially as kinase enzymatic activity directed against FAK-responsive peptides is co-activated[48, 49]. A prominent effect is increased mTOR activation whereas inflammatory signal transduction was also activated by Hedgehog in Smoothened-/- fibroblasts. Hedgehog in wild type fibroblasts provokes similar effects (see above) and thus these effects of Hedgehog signaling appear thus at least partially to stem from Smoothened-independent signaling. Similarly, activation of Wnt and Notch signaling is also seen and thus this aspect of Hedgehog signaling seems also independent of Smoothened. Interestingly, in the absence of Smoothened, Hedgehog activates rather than inhibits PKA, and it is tempting to speculate that this effect may relate to activating phosphorylation of Smoothened by PKA that has been described in Hedgehog signaling[50]. Control experiments revealed that the effect of Hedgehog on Patched negative fibroblasts was negligible (although generally speaking Patched-negative fibroblasts show more kinase activity as compared to control cells, potentially an effect of unleashed Smoothened signaling), suggesting that a contribution of Patched-independent Hedgehog-dependent signaling is non- existent (Supplementary table 1). In conjunction these results reveal that an unexpectedly large proportion of Hedgehog signal transduction towards the cellular kinome is mediated though non-canonical Patched-depedent Smoothened- independent signaling.
Noncanonical Hedgehog signaling
Noncanonical Hedgehog signalling
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