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

Frank Götschel,Fritz Aberger,Christian Bender,Christoph Wierling,Hendrik Hache,Reinhard Schwartz-Albiez,Elena Rüngeler,Wolfgang Gruber,Wilfried Nietfeld,Myriam Friedel,Annemarie Frischauf,Hans Lehrach,Markus Eberl,Ulrike Korf,Daniela Berg,Johanna Sonntag

doi:10.1371/journal.pone.0065403

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.

Highlights

During the last decade, it has become obvious that progression and severity of malignant diseases is often not caused by a single genetic aberration or deregulation of a single signaling pathway, but requires the cooperation of oncogenic-signaling pathways in cancer cells
Responsiveness to activation of Shhsignaling was concluded with the following observations (i) accumulation of GLI1 protein and concurrent inhibition of GLI3 repressor formation after stimulation with SMO agonist (SAG) (Figure 1 A), (ii) processing of GLI3 to its repressor form GLI3R and abrogation of GLI1 and GLI2 protein expression after inhibition of SAG-stimulated Daoy cells with cyclopamine (Figure 1 A), (iii) induction of HH target gene expression (GLI1, HHIP, PTCH) in response to Sonic Hedgehogconditioned medium (Shh-N) treatment (Figure 1 B)
Employing a qPCR screen, we identified a high-level expression of the HH-regulated receptor SMO in Daoy cells, and chose to establish a protocol for direct stimulation with SMO agonist (SAG), and of PTCH, a negative regulator of SMO, with Shh-N conditioned medium

Summary

Introduction

It has become obvious that progression and severity of malignant diseases is often not caused by a single genetic aberration or deregulation of a single signaling pathway, but requires the cooperation of oncogenic-signaling pathways in cancer cells. The first insights into HH/ GLI and EGF crosstalk in cancer was provided by Kasper et al and Schnidar, et al, who pointed out that co-activation of both pathways results in the induction of a specific gene expression pattern, which induces malignant transformation of human keratinocytes [4] [6]. The hypothesis that both pathways merge at the level of transcriptional regulation was supported by other studies, which showed that several different genes possess binding sites for GLI and EGF-regulated transcription factors, such as c-JUN/AP-1 [4] [6] [10]. The interaction of HH/GLI with EGF-induced signaling has been described in a number of tumor types such as skin, prostate, and pancreas [15] [16] [7] [17], while other kinases such as PKC [18] and mTOR/S6K [19] positively regulate GLI activity

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