interleukin-8 and cyclopamine

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.

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

The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted β-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.

Topics: Apoptosis; beta Catenin; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Butyrates; Cell Survival; Chemokine CCL2; Colonic Neoplasms; Cytokines; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Hedgehog Proteins; HT29 Cells; Humans; Interleukin-8; Kruppel-Like Transcription Factors; Oncogene Proteins; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Signal Transduction; Trans-Activators; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2