Synergism between Hedgehog-GLI and EGFR Signaling in Hedgehog-Responsive Human Medulloblastoma Cells Induces Downregulation of Canonical Hedgehog-Target Genes and Stabilized Expression of GLI1

Abstract

Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG) was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies.

Targeting class I histone deacetylases by the novel small molecule inhibitor 4SC‐202 blocks oncogenic hedgehog‐GLI signaling and overcomes smoothened inhibitor resistance

“…b and c ) and activation of HH signaling by Smoothened agonist SAG induces HH target genes (e.g. GLI1 and HHIP) at the mRNA and protein level . Notably, 4SC‐202 treatment efficiently repressed SAG‐induced GLI1 (Fig.…”

Mitogen-activated protein kinases and Hedgehog-GLI signaling in cancer: A crosstalk providing therapeutic opportunities?

“…EGF impairs the activation of the target GLI1 upon HH pathway activation with the SMO agonist SAG. Although EGF is able to activate ERK1/2 in these cells, MEK1/2-ERK1/2 activity is not required for inhibitory effect of EGF on GLI [107].…”

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

“…DAOY human medulloblastoma cells have been reported to be responsive to Hh signaling modulation (Gotschel et al , 2013). DAOY cells responded to Smo activation (using SAG; Smo agonist) or inhibition (using cyclopamine; Smo antagonist) by upregulating or downregulating Gli1 mRNA levels, respectively (Fig EV4A).…”