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

PLoS One. 2013 Jun 10;8(6):e65403. doi: 10.1371/journal.pone.0065403. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / metabolism
  • Blotting, Western
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Proliferation
  • Cells, Cultured
  • Cerebellar Neoplasms / drug therapy
  • Cerebellar Neoplasms / metabolism*
  • Cerebellar Neoplasms / pathology
  • Cyclohexylamines / pharmacology
  • Dermis / cytology
  • Dermis / drug effects
  • Dermis / metabolism
  • Drug Synergism
  • Enzyme Inhibitors / pharmacology
  • Enzyme-Linked Immunosorbent Assay
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Gene Expression Profiling
  • HEK293 Cells
  • Hedgehog Proteins / agonists
  • Hedgehog Proteins / genetics
  • Hedgehog Proteins / metabolism*
  • Humans
  • Interleukin-8 / genetics
  • Interleukin-8 / metabolism
  • Matrix Metalloproteinase 7 / genetics
  • Matrix Metalloproteinase 7 / metabolism
  • Medulloblastoma / drug therapy
  • Medulloblastoma / metabolism*
  • Medulloblastoma / pathology
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Oligonucleotide Array Sequence Analysis
  • Patched Receptors
  • Patched-1 Receptor
  • Phosphorylation / drug effects
  • Protein Array Analysis
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Thiophenes / pharmacology
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism
  • Veratrum Alkaloids / pharmacology
  • Zinc Finger Protein GLI1

Substances

  • Biomarkers, Tumor
  • Carrier Proteins
  • Cyclohexylamines
  • Enzyme Inhibitors
  • GLI1 protein, human
  • HHIP protein, human
  • Hedgehog Proteins
  • Interleukin-8
  • Membrane Glycoproteins
  • PTCH1 protein, human
  • Patched Receptors
  • Patched-1 Receptor
  • RNA, Messenger
  • Receptors, Cell Surface
  • SAG compound
  • SHH protein, human
  • Thiophenes
  • Transcription Factors
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Veratrum Alkaloids
  • Zinc Finger Protein GLI1
  • EGFR protein, human
  • ErbB Receptors
  • MMP7 protein, human
  • Matrix Metalloproteinase 7
  • cyclopamine

Grant support

Work was funded by the German Federal Ministry of Education and Science (BMBF) and by the Austrian Genome Research Program GEN-AU/Austrian Science Fund FWF as bi-national Medical Systems Biology projects 0315394A/0315394B and P20652, respectively (http://pybios.molgen.mpg.de/MoGLI). Further funding was received from the Medical Systems Biology project 0315396B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.