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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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HH-EGFR cooperation response genes are expressed in mouse BCC models and required for in vivo BCC growthCRG expression in GLI-induced skin tumours. qPCR analysis of gene expression in tumours of K5cre;Cleg2 mice (BCC) compared to normal skin (NS). Tumours express high levels of the human GLI2 transgene as well as its targets Gli1 and Ptch (green bars). Likewise, Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4 mRNA levels are significantly elevated in tumours compared to normal skin (blue bars). EGFR expression itself is not significantly changed between tumour and normal skin samples.CRG expression in BCC allograft tumours. Activation of the HH-EGFR targets Sox2, Sox9, Spp1, Jun, Tgfa, Cxcr4 and Il1r2 during in vivo tumour growth of mouse BCC cells. Ptch−/− ASZ001 BCC cells were grafted onto nude mice and tumours harvested after 4 weeks (ASZ allograft). Values represent the ratio of mRNA levels in ASZ allograft samples to those in ASZ cells grown in vitro before grafting. Note that data in A. and B. are plotted on a log10 scale.EGFR activation in BCC allograft tumours. Western blot analysis showing activation of EGFR (pEGFR) in ASZ allografts compared to ASZ cells grown in vitro before grafting.EGFR-dependent CRG expression in mouse BCC. qPCR analysis of SmoM2-induced activation of Jun, Sox9, Sox2, Tgfa, Cxcr4 and Spp1 in primary mouse keratinocytes isolated from the skin of transgenic mice with the indicated genotype.Role of CRG in BCC growth. RNAi knockdown of Jun (shJun), Sox2 (shSox2) and Cxcr4 (shCxcr4) reduces in vivo tumour growth of ASZ001 BCC cells in nude mice (n = 6). For RNAi knockdown efficiencies see Supporting Information Fig S10. Cont: grafted ASZ001 BCC cells transduced with non-target control shRNA. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001.
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fig05: HH-EGFR cooperation response genes are expressed in mouse BCC models and required for in vivo BCC growthCRG expression in GLI-induced skin tumours. qPCR analysis of gene expression in tumours of K5cre;Cleg2 mice (BCC) compared to normal skin (NS). Tumours express high levels of the human GLI2 transgene as well as its targets Gli1 and Ptch (green bars). Likewise, Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4 mRNA levels are significantly elevated in tumours compared to normal skin (blue bars). EGFR expression itself is not significantly changed between tumour and normal skin samples.CRG expression in BCC allograft tumours. Activation of the HH-EGFR targets Sox2, Sox9, Spp1, Jun, Tgfa, Cxcr4 and Il1r2 during in vivo tumour growth of mouse BCC cells. Ptch−/− ASZ001 BCC cells were grafted onto nude mice and tumours harvested after 4 weeks (ASZ allograft). Values represent the ratio of mRNA levels in ASZ allograft samples to those in ASZ cells grown in vitro before grafting. Note that data in A. and B. are plotted on a log10 scale.EGFR activation in BCC allograft tumours. Western blot analysis showing activation of EGFR (pEGFR) in ASZ allografts compared to ASZ cells grown in vitro before grafting.EGFR-dependent CRG expression in mouse BCC. qPCR analysis of SmoM2-induced activation of Jun, Sox9, Sox2, Tgfa, Cxcr4 and Spp1 in primary mouse keratinocytes isolated from the skin of transgenic mice with the indicated genotype.Role of CRG in BCC growth. RNAi knockdown of Jun (shJun), Sox2 (shSox2) and Cxcr4 (shCxcr4) reduces in vivo tumour growth of ASZ001 BCC cells in nude mice (n = 6). For RNAi knockdown efficiencies see Supporting Information Fig S10. Cont: grafted ASZ001 BCC cells transduced with non-target control shRNA. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001.

Mentions: Having demonstrated a critical function of EGFR in HH/GLI-driven BCC, we next focused on the possible role of selected HH-EGFR cooperation response genes in mediating the oncogenic effect of HH-EGFR cooperation. Therefore, we first analysed the expression of cooperation response genes (i.e. Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4; note that closely related FGF19 homologues do not exist in mouse) in BCC-like tumours of mice with epidermis-specific expression of a dominant active form of GLI2 (K5cre;Cleg2, Grachtchouk et al, 2011; Pasca di Magliano et al, 2006) as well as in allograft tumours established from Ptch−/− mouse BCC cells (ASZ001). We found elevated mRNA levels of the HH-EGFR targets Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4 in GLI2-induced BCC-like tumours compared to normal skin of control mice (Fig 5A). Likewise, allograft tumours established from grafted Ptch−/− BCC cells expressed elevated mRNA levels of Spp1, Tgfa, Sox9, Jun, Sox2, Cxcr4 and the known HH-EGFR target gene Il1r2 (Kasper et al, 2006b), when compared to levels in Ptch−/− BCC cells cultured in vitro before grafting (Fig 5B). By contrast, levels of Gli1 and the EGF-independent GLI target Bcl2 (Kasper et al, 2006b) did not differ between allografts and in vitro cultured BCC cells. These data suggest activation of EGFR signalling during in vivo tumour growth of ASZ001 BCC cells. Indeed, only allograft tumours from Ptch−/− BCC cells showed high levels of activated EGFR (pEGFR), while in vitro cultured BCC cells did not (Fig 5C). Allograft tumours established from Ptch−/− BCC cells also showed activation of Mek/Erk and Jun, similar to Ptch−/− BCC cells treated with EGF in vitro (Supporting Information Fig S6). To show in vivo regulation of cooperation response genes by HH-EGFR signalling, we analysed the expression of Jun, Sox2, Sox9, Tgfa, Cxcr4 and Spp1 in epidermal cells of tamoxifen-treated K5creER;SmoM2;EGFR+/+ and K5creER;SmoM2;EGFRfl/fl mice (n = 3 for each genotype). As shown in Fig 5D, SmoM2 expression led to enhanced levels of Jun, Sox9, Sox2, Tgfa, Cxcr4 and Spp1 mRNA in K5creER;SmoM2;EGFR+/+(+TAM) mice. Deletion of EGFR resulted in decreased levels of HH-EGFR response genes (K5creER;SmoM2;EGFRfl/fl (+TAM)). Together with the finding that HH-EGFR response genes are expressed in mouse BCC-like tumours and human BCC (Supporting Information Fig S7), these results suggest that combined HH/GLI and EGFR signalling control the activation of cooperation response genes in BCC.


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)

HH-EGFR cooperation response genes are expressed in mouse BCC models and required for in vivo BCC growthCRG expression in GLI-induced skin tumours. qPCR analysis of gene expression in tumours of K5cre;Cleg2 mice (BCC) compared to normal skin (NS). Tumours express high levels of the human GLI2 transgene as well as its targets Gli1 and Ptch (green bars). Likewise, Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4 mRNA levels are significantly elevated in tumours compared to normal skin (blue bars). EGFR expression itself is not significantly changed between tumour and normal skin samples.CRG expression in BCC allograft tumours. Activation of the HH-EGFR targets Sox2, Sox9, Spp1, Jun, Tgfa, Cxcr4 and Il1r2 during in vivo tumour growth of mouse BCC cells. Ptch−/− ASZ001 BCC cells were grafted onto nude mice and tumours harvested after 4 weeks (ASZ allograft). Values represent the ratio of mRNA levels in ASZ allograft samples to those in ASZ cells grown in vitro before grafting. Note that data in A. and B. are plotted on a log10 scale.EGFR activation in BCC allograft tumours. Western blot analysis showing activation of EGFR (pEGFR) in ASZ allografts compared to ASZ cells grown in vitro before grafting.EGFR-dependent CRG expression in mouse BCC. qPCR analysis of SmoM2-induced activation of Jun, Sox9, Sox2, Tgfa, Cxcr4 and Spp1 in primary mouse keratinocytes isolated from the skin of transgenic mice with the indicated genotype.Role of CRG in BCC growth. RNAi knockdown of Jun (shJun), Sox2 (shSox2) and Cxcr4 (shCxcr4) reduces in vivo tumour growth of ASZ001 BCC cells in nude mice (n = 6). For RNAi knockdown efficiencies see Supporting Information Fig S10. Cont: grafted ASZ001 BCC cells transduced with non-target control shRNA. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001.
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fig05: HH-EGFR cooperation response genes are expressed in mouse BCC models and required for in vivo BCC growthCRG expression in GLI-induced skin tumours. qPCR analysis of gene expression in tumours of K5cre;Cleg2 mice (BCC) compared to normal skin (NS). Tumours express high levels of the human GLI2 transgene as well as its targets Gli1 and Ptch (green bars). Likewise, Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4 mRNA levels are significantly elevated in tumours compared to normal skin (blue bars). EGFR expression itself is not significantly changed between tumour and normal skin samples.CRG expression in BCC allograft tumours. Activation of the HH-EGFR targets Sox2, Sox9, Spp1, Jun, Tgfa, Cxcr4 and Il1r2 during in vivo tumour growth of mouse BCC cells. Ptch−/− ASZ001 BCC cells were grafted onto nude mice and tumours harvested after 4 weeks (ASZ allograft). Values represent the ratio of mRNA levels in ASZ allograft samples to those in ASZ cells grown in vitro before grafting. Note that data in A. and B. are plotted on a log10 scale.EGFR activation in BCC allograft tumours. Western blot analysis showing activation of EGFR (pEGFR) in ASZ allografts compared to ASZ cells grown in vitro before grafting.EGFR-dependent CRG expression in mouse BCC. qPCR analysis of SmoM2-induced activation of Jun, Sox9, Sox2, Tgfa, Cxcr4 and Spp1 in primary mouse keratinocytes isolated from the skin of transgenic mice with the indicated genotype.Role of CRG in BCC growth. RNAi knockdown of Jun (shJun), Sox2 (shSox2) and Cxcr4 (shCxcr4) reduces in vivo tumour growth of ASZ001 BCC cells in nude mice (n = 6). For RNAi knockdown efficiencies see Supporting Information Fig S10. Cont: grafted ASZ001 BCC cells transduced with non-target control shRNA. Error bars represent SEM. *p < 0.05, **p < 0.01; ***p < 0.001.
Mentions: Having demonstrated a critical function of EGFR in HH/GLI-driven BCC, we next focused on the possible role of selected HH-EGFR cooperation response genes in mediating the oncogenic effect of HH-EGFR cooperation. Therefore, we first analysed the expression of cooperation response genes (i.e. Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4; note that closely related FGF19 homologues do not exist in mouse) in BCC-like tumours of mice with epidermis-specific expression of a dominant active form of GLI2 (K5cre;Cleg2, Grachtchouk et al, 2011; Pasca di Magliano et al, 2006) as well as in allograft tumours established from Ptch−/− mouse BCC cells (ASZ001). We found elevated mRNA levels of the HH-EGFR targets Sox2, Sox9, Spp1, Jun, Tgfa and Cxcr4 in GLI2-induced BCC-like tumours compared to normal skin of control mice (Fig 5A). Likewise, allograft tumours established from grafted Ptch−/− BCC cells expressed elevated mRNA levels of Spp1, Tgfa, Sox9, Jun, Sox2, Cxcr4 and the known HH-EGFR target gene Il1r2 (Kasper et al, 2006b), when compared to levels in Ptch−/− BCC cells cultured in vitro before grafting (Fig 5B). By contrast, levels of Gli1 and the EGF-independent GLI target Bcl2 (Kasper et al, 2006b) did not differ between allografts and in vitro cultured BCC cells. These data suggest activation of EGFR signalling during in vivo tumour growth of ASZ001 BCC cells. Indeed, only allograft tumours from Ptch−/− BCC cells showed high levels of activated EGFR (pEGFR), while in vitro cultured BCC cells did not (Fig 5C). Allograft tumours established from Ptch−/− BCC cells also showed activation of Mek/Erk and Jun, similar to Ptch−/− BCC cells treated with EGF in vitro (Supporting Information Fig S6). To show in vivo regulation of cooperation response genes by HH-EGFR signalling, we analysed the expression of Jun, Sox2, Sox9, Tgfa, Cxcr4 and Spp1 in epidermal cells of tamoxifen-treated K5creER;SmoM2;EGFR+/+ and K5creER;SmoM2;EGFRfl/fl mice (n = 3 for each genotype). As shown in Fig 5D, SmoM2 expression led to enhanced levels of Jun, Sox9, Sox2, Tgfa, Cxcr4 and Spp1 mRNA in K5creER;SmoM2;EGFR+/+(+TAM) mice. Deletion of EGFR resulted in decreased levels of HH-EGFR response genes (K5creER;SmoM2;EGFRfl/fl (+TAM)). Together with the finding that HH-EGFR response genes are expressed in mouse BCC-like tumours and human BCC (Supporting Information Fig S7), these results suggest that combined HH/GLI and EGFR signalling control the activation of cooperation response genes in BCC.

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