Page 88 - Helicobacter pylori and Gastric Cancer: From Tumor microenvironment to Immunotherapy

for analysis as to specific and divergent phosphorylation following stimulation with Hedgehog. To this end results were collapsed on elective signal transduction categories (see experimental procedures and [41]. The results are shown in Figure 2 and detailed in Supplementary table 1. They show that Hedgehog challenge provokes fast and substantial remodeling of cellular signaling. Particularly notable is the upregulation of mTOR signaling which may fit recent data that indicate that mTORC1-mediated translation is a key component of hedgehog signaling and is a putative target for treating Hedgehog-driven cancers[42]. Other interesting points include an upregulation of g-protein-coupled receptor kinase enzymatic activity, which is line with observation in Drosophila that a G-protein-coupled receptor kinase controls Smoothened activity[43]. Strong regulation of PKA is also observed and may be in line with the notion that this kinase is a regulator of Hedgehog signaling[44], whereas the observation that PKC enzymatic activity is upregulated is conform the canonical mode of action of G-protein coupled receptor like Smoothened. A variety of pro-inflammatory signaling modules is activated as well (including Lyn, Fyn and peptides that are consensus substrates for Bruton’s tyrosine kinase) but as embryonic fibroblasts are not immunologically-relevant cells, the importance of this observation is uncertain. Maybe unsurprisingly (in view of its role of a Wnt antagonist in the epithelium of the intestine[7, 30], we see strong inhibition of Wnt signaling in our model system following Hedgehog application although this observation is at bay with the data of Shin et al. that show that bladder stromal cells respond to Shh with Wnt activation[45] and also to the recent report of Regan et al.[29] describing that Hedgehog can stimulate Wnt signaling in cancer stem cells. In addition, the upregulation of substrate peptides for p21-activated kinase (Pak) activity and related molecules indicates that Hedgehog stimulation stimulates actin reorganization and morphological changes. In conjunction these experiments show that the effect of Hedgehog on the cellular kinome is rapid and profound.
Patched-dependent Smoothened-independent effects on cellular kinase activity
The existence of Patched-dependent Smoothened-independent signal transduction is supported by various observations[46] and appears highly relevant in that it is essential for cancer stem cell survival in colorectal cancer[29]. To test whether such signaling is present in our model system we incubated MEFs with 3H-sucrose (which is membrane impermeable and is only taken up via endocytosis in most cell types) and challenged the cells with either a vehicle control or 1 μg/ml Shh, in the
Chapter 4
Chapter 4
82
86