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. 2012 Jan 1;72(1):88-99.
doi: 10.1158/0008-5472.CAN-10-4621. Epub 2011 Nov 15.

GLI1 inhibition promotes epithelial-to-mesenchymal transition in pancreatic cancer cells

Simon Joost  1 , Luciana L Almada, Verena Rohnalter, Philipp S Holz, Anne M Vrabel, Maite G Fernandez-Barrena, Robert R McWilliams, Michael Krause, Martin E Fernandez-Zapico, Matthias Lauth
Affiliations

GLI1 inhibition promotes epithelial-to-mesenchymal transition in pancreatic cancer cells

Simon Joost et al. Cancer Res. .

Abstract

The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here, we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility, and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC.

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Conflict of interest statement

Conflict of interest: None

Figures

Figure 1
Figure 1. GLI1 regulates CDH1 expression
(A)QPCR analysis of Panc1, Su86.86, AsPC1 and Hs766T cells transiently transfected with control siRNA (siLuc, targeting firefly luciferase) or siRNA targeting GLI1 (siGLI1) or GLI2 (siGLI2). The EMT inducer TGFβ (5 ng/ml for 48 h) was applied as a positive control. (B)Immunoblot detecting of E-Cadherin expression in siGLI-transfected PDAc cell lines. Shown is a representative blot of at least three independent experiments. (C)Immunofluorescence staining of E-Cadherin (green) expression in siRNA-transfected Su86.86 cells. The nuclei are stained blue. The inset shows a magnification. Shown is a representative picture of at least three independent experiments. (D)MiaPaCa2 cells were stably transfected with EGFP (mock) or a GLI1 expression plasmid. QPCR analysis reveals the induction of CDH1 by exogenous GLI1.
Figure 2
Figure 2. CDH1is a GLI1 target gene in PDAC cells
(A)Heatmap of GLI1 and CDH1 expression in 15 PDAC cell lines as measured by qPCR of triplicate samples. The positive correlation is statistically significant. (B)Schematic drawing depicting the predicted GLI binding sites (G) in the human CDH1 promoter. The transcription start site is at +1. (C)Transient transfection of GLI1 into Panc1 cells results in a significant induction of a luciferase reporter construct containing the human CDH1 promoter. (D)Chromatin immunoprecipitation (CHIP) using an antibody against endogenous GLI1 in Panc1 cells. (E)Correlation of CDH1 and GLI1 mRNA copy number in 144 human primary pancreatic cancer samples.
Figure 3
Figure 3. SiGLI1-mediated loss of E-Cadherin expression does not require the induction of known CDH1 repressors
(A)QPCR analysis of SNAI1 (SNAIL) and SNAI2 (SLUG) mRNA expression in siGLI-transfected Panc1 cells. TGFβ serves as a positive control for the induction of SNAI1/2. PAI1 is a target gene of TGFβ. (B)Western Blot showing the lack of SNAIL protein stabilization upon knock-down of GLI1 in Panc1 cells. In contrast SNAIL can readily be detected by treatment of cells with TGFβ. (C)QPCR detection of CDH1, GLI1 and PAI1 gene expression in Panc1 cells exposed to 10 μM of the TGFβ signaling inhibitor SB-431542.
Figure 4
Figure 4. Knock-down of GLI1 expression leads to loss of epithelial markers
(A)Morphological changes of Panc1 (left) and Su86.86 (right) cells after transfection with siGLI1 or exposure to TGFβ. The upper panel shows brightfield images whereas the lower panel depicts fluorescence images after staining the f-actin network with Phalloidin-Texas Red. Nuclei appear in blue. (B)Panc1 cells were transiently transfected with control (scramble) shRNA plasmid (shSCR) or GLI1-specific shRNA (shGLI1). For verification of shRNA efficacy see Nolan-Stevaux et al (18). Cells were cotransfected with GFP-F to label membranes and to better visualize cell morphology. TGFβ was applied as a positive control. (C)QPCR data demonstrating the decrease in epithelial gene (KRT19, EVA1, PTPRM) expression upon knock-down of GLI1 in several cell lines. In contrast, Integrin β1 (ITGB1) is upregulated. (D)Microarray data showing a widespread reduction in Keratin gene expression in siGLI1(Pool of 4 different siRNAs)-transfected compared to control (siLuc)-transfected Panc1 cells. Two independent experiments are shown. (E)Immunostaining of siRNA-transfected Su86.86 cells for Keratin 14 (KRT14) and Keratin 7/8 (KRT7/8).
Figure 5
Figure 5. Adherens junction are disassembled upon loss of GLI1
(A)Immunostaining of E-Cadherin, α-Catenin and β-Catenin expression in Su86.86 cells. Note that the overall levels of α- and β-Catenin decrease in TGFβ-treated and in siGLI1-transfected cells. The remaining β-Catenin is no longer localized to membranes (as in siLuc control cells), but accumulates in the cytoplasm. (B)Destabilization of α- and β-Catenin in siGLI1-transfected Panc1 and Su86.86 cells as verified by western blotting. Shown is a representative blot of at least three independent experiments. (C)WNT luciferase reporter assay in Panc1 cells transfected with control (shSCR) or GLI1-targeting (shGLI1) shRNA.
Figure 6
Figure 6. Low GLI1 levels lead to increased motility of PDAC cells
(A)In vitro scratch assay using Panc1 cells transiently transfected with control (siLuc), CDH1 or GLI1 siRNA. Shown is a representative image of at least three independent experiments performed in triplicate. (B)Quantification of the relative open scratch area (after 24 h) from the wounding experiment depicted in (A). (C)Neutral red assay demonstrating no significant change in cell number within the time frame of the scratch experiment.
Figure 7
Figure 7. Low GLI1 levels synergize with TGFβ and HGF
(A)QPCR detection of CDH1 and GLI1 expression in siLuc- and siGLI1-transfected Panc1 cells exposed to increasing concentrations of TGFβ. (B)Quantification of a scratch assay using siRNA-transfected Panc1 cells treated with low doses of TGFβ or HGF. Note the synergistic effects of moderately reduced GLI1 levels and TGFβ or HGF. (C)Representative image of the in vitro scratch assay shown in (B).
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Comment in

  • GLI1 modulates EMT in pancreatic cancer--letter.
    Inaguma S, Kasai K, Hashimoto M, Ikeda H. Inaguma S, et al. Cancer Res. 2012 Jul 15;72(14):3702-3; author reply 3704-5. doi: 10.1158/0008-5472.CAN-12-0379. Epub 2012 Jul 3. Cancer Res. 2012. PMID: 22761339 No abstract available.

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