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Hedgehog-EGFR cooperation response genes determine the oncogenic phenotype of basal cell carcinoma and tumour-initiating pancreatic cancer cells.

Eberl M, Klingler S, Mangelberger D, Loipetzberger A, Damhofer H, Zoidl K, Schnidar H, Hache H, Bauer HC, Solca F, Hauser-Kronberger C, Ermilov AN, Verhaegen ME, Bichakjian CK, Dlugosz AA, Nietfeld W, Sibilia M, Lehrach H, Wierling C, Aberger F - EMBO Mol Med (2012)

Bottom Line: Interactions between HH/GLI and other oncogenic pathways affect the strength and tumourigenicity of HH/GLI.However, the in vivo relevance of HH-EGFR signal integration and the critical downstream mediators are largely undefined.We describe HH-EGFR cooperation response genes including SOX2, SOX9, JUN, CXCR4 and FGF19 that are synergistically activated by HH-EGFR signal integration and required for in vivo growth of BCC cells and tumour-initiating pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.

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Activation of cooperation response genes requires the specific combination of HH/GLI and EGFR signallingAnalysis of RAS/MEK/ERK signalling and JUN activation in human HaCaT keratinocytes in response to various receptor tyrosine kinase (RTK) ligands. Note that although EGF (10 ng/ml), FGF7 (50 ng/ml), HGF (50 ng/ml) and to a lower extent also VEGF (50 ng/ml) induce ERK1/2 activation (pERK1/2), only EGF treatment is able to stimulate JUN activation (phosphorylated JUN, pJUN).CRG regulation by RTK pathways. qPCR analysis of HH/GLI-EGFR cooperation response gene expression in response to either GLI1 activation, treatment with RTK ligands EGF, VEGF, HGF and FGF7, or a combination of GLI1 and the RTK ligands (see A). PTCH and BCL2 served as reference for direct GLI target genes whose expression is independent of parallel EGFR signalling (Kasper et al, 2006b). As bFGF treatment did not induce activation of MEK/ERK in HaCaT cells (see A), we did not analyse the combination of GLI1/bFGF.CRG and canonical GLI target regulation by GLI-RAS signalling. Expression of JUN, SOX9, FGF19, CXCR4, TGFA, SPP1 and SOX2 (left), and PTCH and HHIP (right) in response to combined GLI1 and oncogenic KRAS (KRAS*). GLI1 transgene levels are unaffected by KRAS* expression. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001, ns: not significant (p > 0.05).
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fig04: Activation of cooperation response genes requires the specific combination of HH/GLI and EGFR signallingAnalysis of RAS/MEK/ERK signalling and JUN activation in human HaCaT keratinocytes in response to various receptor tyrosine kinase (RTK) ligands. Note that although EGF (10 ng/ml), FGF7 (50 ng/ml), HGF (50 ng/ml) and to a lower extent also VEGF (50 ng/ml) induce ERK1/2 activation (pERK1/2), only EGF treatment is able to stimulate JUN activation (phosphorylated JUN, pJUN).CRG regulation by RTK pathways. qPCR analysis of HH/GLI-EGFR cooperation response gene expression in response to either GLI1 activation, treatment with RTK ligands EGF, VEGF, HGF and FGF7, or a combination of GLI1 and the RTK ligands (see A). PTCH and BCL2 served as reference for direct GLI target genes whose expression is independent of parallel EGFR signalling (Kasper et al, 2006b). As bFGF treatment did not induce activation of MEK/ERK in HaCaT cells (see A), we did not analyse the combination of GLI1/bFGF.CRG and canonical GLI target regulation by GLI-RAS signalling. Expression of JUN, SOX9, FGF19, CXCR4, TGFA, SPP1 and SOX2 (left), and PTCH and HHIP (right) in response to combined GLI1 and oncogenic KRAS (KRAS*). GLI1 transgene levels are unaffected by KRAS* expression. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001, ns: not significant (p > 0.05).

Mentions: SMO-independent signal inputs such as RAS-MEK/ERK have been shown to modify the transcriptional activity of GLI proteins (Riobo et al, 2006a; Stecca et al, 2007). We have previously shown that EGFR-dependent activation of RAS-MEK/ERK and JUN/AP1 activation is critical for mediating HH/GLI-EGFR signal cooperation (Schnidar et al, 2009). Since activation of RAS-MEK/ERK is a common event in response to receptor tyrosine kinase (RTK) activation, we addressed whether the activation of HH/GLI-EGFR response genes is specifically mediated by combined GLI and EGFR signalling, or whether other RTK pathways can also cooperate with GLI and induce a similar cooperation response gene profile. Therefore, we tested various RTK ligands including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF7) and basic FGF (bFGF) for their ability to induce RAS/RAF/MEK/ERK and JUN/AP1 activation in human HaCaT keratinocytes. As shown in Fig 4A, EGF, FGF7, HGF and to a lower extent also VEGF stimulated MEK/ERK, though only EGF was able to induce JUN activation (pJUN). Analysis of HH-EGFR cooperation response gene expression in GLI1 expressing keratinocytes simultaneously treated either with EGF, HGF, FGF7, or VEGF revealed that only the combination of GLI1 and EGF led to synergistic induction of JUN, SOX9, FGF19, SPP1, TGFA and SOX2. Only SOX2 expression was also enhanced by GLI1 and FGF7 treatment (Fig 4B). The canonical GLI targets PTCH and BCL2 (Kasper et al, 2006b) were not hyper-activated by any of the combinations, rather we observed an unexplained reduction of PTCH mRNA expression by combined GLI1 and VEGF, HGF or FGF7 treatment (Fig 4B). As expression of oncogenic RAS has been shown to promote the transcriptional and oncogenic activity of GLI (Pasca di Magliano et al, 2006; Stecca et al, 2007), we studied the effect of combined GLI1/RAS activation on the expression of HH-EGFR response genes. As shown in Fig 4C, co-expression of GLI1 and oncogenic KRASG12V (KRAS*) did not lead to increased expression of HH-EGFR cooperation response genes. Rather, KRAS* attenuated the expression of JUN, FGF19, CXCR4, TGFA and SPP1 in the presence of GLI1. By contrast and consistent with previous reports showing enhancement of GLI activity by oncogenic RAS (Stecca et al, 2007), expression of the EGF-independent GLI targets PTCH and HHIP (Kasper et al, 2006b) (data not shown) was significantly increased by GLI1/KRAS*. Thus, the regulation of HH-EGFR cooperation response genes requires the specific combination of HH/GLI and EGFR activation. Whether MEK/ERK-dependent activation of JUN/AP1 is the critical determinant for cooperation of HH/GLI and RTK pathways such as EGFR signalling remains to be addressed.


Hedgehog-EGFR cooperation response genes determine the oncogenic phenotype of basal cell carcinoma and tumour-initiating pancreatic cancer cells.

Eberl M, Klingler S, Mangelberger D, Loipetzberger A, Damhofer H, Zoidl K, Schnidar H, Hache H, Bauer HC, Solca F, Hauser-Kronberger C, Ermilov AN, Verhaegen ME, Bichakjian CK, Dlugosz AA, Nietfeld W, Sibilia M, Lehrach H, Wierling C, Aberger F - EMBO Mol Med (2012)

Activation of cooperation response genes requires the specific combination of HH/GLI and EGFR signallingAnalysis of RAS/MEK/ERK signalling and JUN activation in human HaCaT keratinocytes in response to various receptor tyrosine kinase (RTK) ligands. Note that although EGF (10 ng/ml), FGF7 (50 ng/ml), HGF (50 ng/ml) and to a lower extent also VEGF (50 ng/ml) induce ERK1/2 activation (pERK1/2), only EGF treatment is able to stimulate JUN activation (phosphorylated JUN, pJUN).CRG regulation by RTK pathways. qPCR analysis of HH/GLI-EGFR cooperation response gene expression in response to either GLI1 activation, treatment with RTK ligands EGF, VEGF, HGF and FGF7, or a combination of GLI1 and the RTK ligands (see A). PTCH and BCL2 served as reference for direct GLI target genes whose expression is independent of parallel EGFR signalling (Kasper et al, 2006b). As bFGF treatment did not induce activation of MEK/ERK in HaCaT cells (see A), we did not analyse the combination of GLI1/bFGF.CRG and canonical GLI target regulation by GLI-RAS signalling. Expression of JUN, SOX9, FGF19, CXCR4, TGFA, SPP1 and SOX2 (left), and PTCH and HHIP (right) in response to combined GLI1 and oncogenic KRAS (KRAS*). GLI1 transgene levels are unaffected by KRAS* expression. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001, ns: not significant (p > 0.05).
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fig04: Activation of cooperation response genes requires the specific combination of HH/GLI and EGFR signallingAnalysis of RAS/MEK/ERK signalling and JUN activation in human HaCaT keratinocytes in response to various receptor tyrosine kinase (RTK) ligands. Note that although EGF (10 ng/ml), FGF7 (50 ng/ml), HGF (50 ng/ml) and to a lower extent also VEGF (50 ng/ml) induce ERK1/2 activation (pERK1/2), only EGF treatment is able to stimulate JUN activation (phosphorylated JUN, pJUN).CRG regulation by RTK pathways. qPCR analysis of HH/GLI-EGFR cooperation response gene expression in response to either GLI1 activation, treatment with RTK ligands EGF, VEGF, HGF and FGF7, or a combination of GLI1 and the RTK ligands (see A). PTCH and BCL2 served as reference for direct GLI target genes whose expression is independent of parallel EGFR signalling (Kasper et al, 2006b). As bFGF treatment did not induce activation of MEK/ERK in HaCaT cells (see A), we did not analyse the combination of GLI1/bFGF.CRG and canonical GLI target regulation by GLI-RAS signalling. Expression of JUN, SOX9, FGF19, CXCR4, TGFA, SPP1 and SOX2 (left), and PTCH and HHIP (right) in response to combined GLI1 and oncogenic KRAS (KRAS*). GLI1 transgene levels are unaffected by KRAS* expression. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001, ns: not significant (p > 0.05).
Mentions: SMO-independent signal inputs such as RAS-MEK/ERK have been shown to modify the transcriptional activity of GLI proteins (Riobo et al, 2006a; Stecca et al, 2007). We have previously shown that EGFR-dependent activation of RAS-MEK/ERK and JUN/AP1 activation is critical for mediating HH/GLI-EGFR signal cooperation (Schnidar et al, 2009). Since activation of RAS-MEK/ERK is a common event in response to receptor tyrosine kinase (RTK) activation, we addressed whether the activation of HH/GLI-EGFR response genes is specifically mediated by combined GLI and EGFR signalling, or whether other RTK pathways can also cooperate with GLI and induce a similar cooperation response gene profile. Therefore, we tested various RTK ligands including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF7) and basic FGF (bFGF) for their ability to induce RAS/RAF/MEK/ERK and JUN/AP1 activation in human HaCaT keratinocytes. As shown in Fig 4A, EGF, FGF7, HGF and to a lower extent also VEGF stimulated MEK/ERK, though only EGF was able to induce JUN activation (pJUN). Analysis of HH-EGFR cooperation response gene expression in GLI1 expressing keratinocytes simultaneously treated either with EGF, HGF, FGF7, or VEGF revealed that only the combination of GLI1 and EGF led to synergistic induction of JUN, SOX9, FGF19, SPP1, TGFA and SOX2. Only SOX2 expression was also enhanced by GLI1 and FGF7 treatment (Fig 4B). The canonical GLI targets PTCH and BCL2 (Kasper et al, 2006b) were not hyper-activated by any of the combinations, rather we observed an unexplained reduction of PTCH mRNA expression by combined GLI1 and VEGF, HGF or FGF7 treatment (Fig 4B). As expression of oncogenic RAS has been shown to promote the transcriptional and oncogenic activity of GLI (Pasca di Magliano et al, 2006; Stecca et al, 2007), we studied the effect of combined GLI1/RAS activation on the expression of HH-EGFR response genes. As shown in Fig 4C, co-expression of GLI1 and oncogenic KRASG12V (KRAS*) did not lead to increased expression of HH-EGFR cooperation response genes. Rather, KRAS* attenuated the expression of JUN, FGF19, CXCR4, TGFA and SPP1 in the presence of GLI1. By contrast and consistent with previous reports showing enhancement of GLI activity by oncogenic RAS (Stecca et al, 2007), expression of the EGF-independent GLI targets PTCH and HHIP (Kasper et al, 2006b) (data not shown) was significantly increased by GLI1/KRAS*. Thus, the regulation of HH-EGFR cooperation response genes requires the specific combination of HH/GLI and EGFR activation. Whether MEK/ERK-dependent activation of JUN/AP1 is the critical determinant for cooperation of HH/GLI and RTK pathways such as EGFR signalling remains to be addressed.

Bottom Line: Interactions between HH/GLI and other oncogenic pathways affect the strength and tumourigenicity of HH/GLI.However, the in vivo relevance of HH-EGFR signal integration and the critical downstream mediators are largely undefined.We describe HH-EGFR cooperation response genes including SOX2, SOX9, JUN, CXCR4 and FGF19 that are synergistically activated by HH-EGFR signal integration and required for in vivo growth of BCC cells and tumour-initiating pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.

Show MeSH
Related in: MedlinePlus