Journal: EMBO Molecular Medicine

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

doi: 10.1002/emmm.201100201

Figure Lengend Snippet: CRG regulation by GLI1 and EGFR signalling. qPCR analysis of CRG mRNA expression in response to GLI1 expression, EGFR signalling (EGF) or a combination of both stimuli (GLI1/EGF) after 4.5, 9 and 18 h of single or combined activation (three biological replicates measured in duplicate, values are fold change compared to untreated controls). Location of GLI binding sites in CRG promoters. Genomic locus of CRG with in silico predicted GLI binding site clusters located in their 5′ upstream regulatory regions. Numbers indicate the position of binding sites relative to the transcriptional start site (TSS). Direct transcriptional regulation of CRG by GLI. Luciferase reporter assays showing activation of CRG promoters in response to GLI1 expression. For FGF19, CXCR4, TGFA and SOX9 selected GLI binding sites were mutated (FGF19mut, CXCR4mut, TGFAmut, SOX9mut). The mutated GLI binding sites are indicated by asterisks in B. Error bars represent SEM. * p < 0.05, ** p < 0.01.

Article Snippet: For lentiviral RNAi knockdown experiments, the following shRNA constructs (Sigma-Aldrich mission TRC library) were used: shEGFR (TRCN0000055220), shRNA GLI1 (TRCN0000020486), shRNA SMOH (TRCN0000014363), shRNA human and mouse SOX2 (TRCN0000010753), shRNA SOX9 (TRCN0000020386), shRNA FGF19 (TRCN0000040258), shRNA mouse Cxcr4 (TRCN0000028750), shRNA JUN (TRCN0000039590), shRNA mouse Jun (TRCN0000055207), control scrambled shRNA (SHC002) and shRNA against human CXCR4 (clone 12272, Addgene; Orimo et al, ).

Techniques: Expressing, Activation Assay, Binding Assay, In Silico, Luciferase

Journal: EMBO Molecular Medicine

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

doi: 10.1002/emmm.201100201

Figure Lengend Snippet: Analysis 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, ). 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).

Article Snippet: For lentiviral RNAi knockdown experiments, the following shRNA constructs (Sigma-Aldrich mission TRC library) were used: shEGFR (TRCN0000055220), shRNA GLI1 (TRCN0000020486), shRNA SMOH (TRCN0000014363), shRNA human and mouse SOX2 (TRCN0000010753), shRNA SOX9 (TRCN0000020386), shRNA FGF19 (TRCN0000040258), shRNA mouse Cxcr4 (TRCN0000028750), shRNA JUN (TRCN0000039590), shRNA mouse Jun (TRCN0000055207), control scrambled shRNA (SHC002) and shRNA against human CXCR4 (clone 12272, Addgene; Orimo et al, ).

Techniques: Activation Assay, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

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

doi: 10.1002/emmm.201100201

Figure Lengend Snippet: CRG 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 . Cont: grafted ASZ001 BCC cells transduced with non-target control shRNA. Error bars represent SEM. * p < 0.05, ** p < 0.01; *** p < 0.001.

Article Snippet: For lentiviral RNAi knockdown experiments, the following shRNA constructs (Sigma-Aldrich mission TRC library) were used: shEGFR (TRCN0000055220), shRNA GLI1 (TRCN0000020486), shRNA SMOH (TRCN0000014363), shRNA human and mouse SOX2 (TRCN0000010753), shRNA SOX9 (TRCN0000020386), shRNA FGF19 (TRCN0000040258), shRNA mouse Cxcr4 (TRCN0000028750), shRNA JUN (TRCN0000039590), shRNA mouse Jun (TRCN0000055207), control scrambled shRNA (SHC002) and shRNA against human CXCR4 (clone 12272, Addgene; Orimo et al, ).

Techniques: Expressing, Gene Expression, Activation Assay, In Vivo, In Vitro, Western Blot, Isolation, Transgenic Assay, Knockdown, Transduction, Control, shRNA

Journal: EMBO Molecular Medicine

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

doi: 10.1002/emmm.201100201

Figure Lengend Snippet: Sphere growth of rare pancreatic cancer cells. If grown in 3D culture, human pancreatic cancer cells (here: L3.6sl) form macrospheres (MS) at low frequency (0.8–1.2%). If single macrospheres are isolated from 3D cultures (arrows in left image) and grown for serial passages in 2D, the cells will again form macrospheres at a constant low frequency if re-seeded in 3D cultures (right image, also see and Supporting Information). Tumour-initiation by sphere-forming pancreatic cancer cells. Limiting dilution xenograft experiments showing that as few as 100 pancreatic cancer cells (here L3.6sl) derived from single isolated macrospheres are sufficient to initiate tumour growth in vivo ( n = 4/4). By contrast, at least 10 4 pancreatic cancer cells grown in 2D culture need to be grafted to establish tumours in vivo ( n = 4 for each dilution experiment), showing that macrospheres isolated from 3D cultures of pancreatic cancer cells are highly enriched for tumour-initiating cells. CRG expression in tumour-initiating pancreatic cancer cells. qPCR analysis ( n = 4) showing elevated mRNA levels of stemness genes (CD133, Nanog, OCT4, red bars), HH pathway genes (SHH, DHH, IHH, GLI1) (green bars) and HH-EGFR cooperation response genes (JUN, SOX9, FGF19, TGFA, SPP1, CXCR4, blue bars) in tumour-initiating macrosphere cells compared to the corresponding cancer cells grown in 2D cultures. Graphs show results for three human pancreatic cancer cells lines (L3.6sl, Panc-1 and L3.6pl). EGFR signalling in pancreatic cancer cells. Western blot analysis of pancreatic cancer cells (L3.6sl, Panc-1 and L3.6pl) showing activation of EGFR/ERK/JUN signalling in response to EGF treatment. Pharmacological inhibition of EGFR by Erlotinib (Erlo) prevented activation of the EGFR/MEK/JUN cascade. Regulation of CRG expression by GLI-EGFR signalling. Induction of HH-EGFR cooperation response genes (JUN, SOX9, FGF19, CXCR4, TGFA, SPP1) by combined GLI1/EGFR activation in L3.6sl pancreatic cancer cells. Unlike HH-EGFR response genes, transcriptional activation of the canonical GLI target PTCH is independent of EGF treatment. Error bars represent SEM. * p < 0.05, ** p < 0.01, ns: not significant ( p > 0.05).

Article Snippet: For lentiviral RNAi knockdown experiments, the following shRNA constructs (Sigma-Aldrich mission TRC library) were used: shEGFR (TRCN0000055220), shRNA GLI1 (TRCN0000020486), shRNA SMOH (TRCN0000014363), shRNA human and mouse SOX2 (TRCN0000010753), shRNA SOX9 (TRCN0000020386), shRNA FGF19 (TRCN0000040258), shRNA mouse Cxcr4 (TRCN0000028750), shRNA JUN (TRCN0000039590), shRNA mouse Jun (TRCN0000055207), control scrambled shRNA (SHC002) and shRNA against human CXCR4 (clone 12272, Addgene; Orimo et al, ).

Techniques: Isolation, Derivative Assay, In Vivo, Expressing, Western Blot, Activation Assay, Inhibition

Journal: EMBO Molecular Medicine

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

doi: 10.1002/emmm.201100201

Figure Lengend Snippet: GLI1-dependent sphere growth of pancreatic cancer cells. RNAi mediated inhibition of GLI1 expression reduces macrosphere growth in 3D cultures of L3.6sl, Panc-1 and L3.6pl cells. Combined inhibition of GLI and EGFR/JUN signalling. Panc-1 cells were treated with erlotinib (erlo) or GANT61 (GANT) at the concentrations indicated and/or stably transduced with lentiviral shRNA against EGFR (shEGFR), GLI1 (shGLI1) or JUN (shJUN). Note that the combination of shEGFR/GANT, shGLI1/erlo and shJUN/GANT nearly completely abolishes the formation of tumour-initiating macrospheres. CRG function in tumour-initiating pancreatic cancer cells. RNAi-mediated inhibition of the GLI-EGFR targets CXCR4, FGF19, SOX9 and SOX2 in pancreatic cancer cells (data shown for L3.6sl cells) significantly reduces the growth of tumour-initiating macrosphere cells in 3D cultures. Quantification of 3D cultures shown in C. For RNAi knockdown efficiencies see Supporting Information . Data represent the mean values of 4 independent experiments. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For lentiviral RNAi knockdown experiments, the following shRNA constructs (Sigma-Aldrich mission TRC library) were used: shEGFR (TRCN0000055220), shRNA GLI1 (TRCN0000020486), shRNA SMOH (TRCN0000014363), shRNA human and mouse SOX2 (TRCN0000010753), shRNA SOX9 (TRCN0000020386), shRNA FGF19 (TRCN0000040258), shRNA mouse Cxcr4 (TRCN0000028750), shRNA JUN (TRCN0000039590), shRNA mouse Jun (TRCN0000055207), control scrambled shRNA (SHC002) and shRNA against human CXCR4 (clone 12272, Addgene; Orimo et al, ).

Techniques: Inhibition, Expressing, Stable Transfection, Transduction, shRNA, Knockdown

Journal: EMBO Molecular Medicine

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

doi: 10.1002/emmm.201100201

Figure Lengend Snippet: A-F. Effect of RNAi knockdown of GLI1, JUN, SOX9, FGF19, CXCR4 and SMOH on in vivo growth of pancreatic cancer cells. 1 × 10 6 pancreatic cancer cells (L3.6sl) either transduced lentivirally with non-target control shRNA (cont) or with shRNA specific for the respective target were grafted contralaterally onto the lower flank of nude mice ( n = 8 for each group). Similar to RNAi-mediated GLI1 inhibition ( A ), shRNA against the HH/GLI-EGFR targets JUN ( B ), SOX9 ( C ), FGF19 ( D ) and CXCR4 ( E ) interfere with in vivo tumour growth. By contrast, RNAi against SMOH ( F ) does not significantly affect in vivo growth of pancreatic cancer cells. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant ( p > 0.05).

Article Snippet: For lentiviral RNAi knockdown experiments, the following shRNA constructs (Sigma-Aldrich mission TRC library) were used: shEGFR (TRCN0000055220), shRNA GLI1 (TRCN0000020486), shRNA SMOH (TRCN0000014363), shRNA human and mouse SOX2 (TRCN0000010753), shRNA SOX9 (TRCN0000020386), shRNA FGF19 (TRCN0000040258), shRNA mouse Cxcr4 (TRCN0000028750), shRNA JUN (TRCN0000039590), shRNA mouse Jun (TRCN0000055207), control scrambled shRNA (SHC002) and shRNA against human CXCR4 (clone 12272, Addgene; Orimo et al, ).

Techniques: Knockdown, In Vivo, Control, shRNA, Inhibition

Journal: EMBO Molecular Medicine

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

doi: 10.1002/emmm.201100201

Figure Lengend Snippet: CRG expression in reciprocal RNAi knockdown studies. Pancreatic cancer cells were lentivirally transduced with non-target control shRNA (shcont) or with shRNA against JUN (shJUN), SOX9 (shSOX9), FGF19 (shFGF19) or CXCR4 (shCXCR4). 72 h post transduction, transduced cells were analysed by qPCR for the expression of the HH/GLI-EGFR cooperation response genes JUN, SOX9, FGF19, CXCR4 and SPP1, as well as for expression of the canonical GLI target PTCH. Measurement of the mRNA of the respective shRNA target served as control for successful RNAi knockdown (e.g. JUN mRNA level in shJUN cells, SOX9 mRNA level in shSOX9 cells, etc). Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant ( p > 0.05) Model of HH/GLI-EGFR signal integration and activation of CRG expression in HH/GLI-associated tumourigenesis. Arrows illustrate positive regulatory interactions of gene expression between selected cooperation response genes. Width of arrows is proportional to the regulatory effect shown in A.

Article Snippet: For lentiviral RNAi knockdown experiments, the following shRNA constructs (Sigma-Aldrich mission TRC library) were used: shEGFR (TRCN0000055220), shRNA GLI1 (TRCN0000020486), shRNA SMOH (TRCN0000014363), shRNA human and mouse SOX2 (TRCN0000010753), shRNA SOX9 (TRCN0000020386), shRNA FGF19 (TRCN0000040258), shRNA mouse Cxcr4 (TRCN0000028750), shRNA JUN (TRCN0000039590), shRNA mouse Jun (TRCN0000055207), control scrambled shRNA (SHC002) and shRNA against human CXCR4 (clone 12272, Addgene; Orimo et al, ).

Techniques: Expressing, Knockdown, Transduction, Control, shRNA, Activation Assay, Gene Expression