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. 2009 Jan 1;23(1):24-36.
doi: 10.1101/gad.1753809.

GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation

Olivier Nolan-Stevaux  1 , Janet Lau, Morgan L Truitt, Gerald C Chu, Matthias Hebrok, Martin E Fernández-Zapico, Douglas Hanahan
Affiliations

GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation

Olivier Nolan-Stevaux et al. Genes Dev. .

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. We investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. We report that multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh-Ptch-Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, we find that Gli transcription is decoupled from upstream Shh-Ptch-Smo signaling and is regulated by TGF-beta and KRAS, and we show that Gli1 is required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells.

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Figures

Figure 1.
Figure 1.
Hedgehog pathway components are deregulated in PDAC lesions. (A) Expression of Shh, Ptch1, Smo, and Gli1 mRNA in total RNA extracts from ∼12-wk-old LSL-Kras/+; p53F/+ control pancreata (P) (N = 3) or p48-Cre/+; LSL-Kras/+; p53F/+ tumors (T) (N = 3). Levels of mRNAs expressed as a percentage of the mGus control mRNA. Asterisk indicates a P-value <0.01. (B,C) Immuno-histochemical detection of Shh (50×). (B) No Shh detected in normal pancreas. (C) Shh expression detected inside ductal cells in PanIN lesions of 9-wk-old PDAC mice. The panels are representative of multiple fields of pancreatic sections from two control and two PDAC mice.
Figure 2.
Figure 2.
Smoothened is depleted in ductal cells of PDAC SmoF/F mice. (A) Recombination of the Smo locus in PDAC cell lines. PCR amplification of the Smo locus (Smo) or the p48-Cre transgene (p48). The Smo genotyping procedure amplifies the nonrecombined conditional SmoF allele (upper band, F) and the wild-type (wt) Smo allele (lower band, wild type). The upper PCR band is lost upon Cre recombination of the Smo conditional locus. The input genomic DNA used in each PCR reaction is indicated: (Tail) genomic DNA from mouse tail, 100 ng; (Cells) genomic DNA from PDAC-derived tumor cell lines, 10 ng; (+/+) Smo+/+; (F/+) SmoF/+; (F/F) SmoF/F. (B) Depletion of the Smo mRNA in recombined cell lines. Expression of Smo mRNA in total RNA extracts from Smo+/+ (W), SmoF/+ (H), or SmoF/F (F) PDAC cell lines. Levels of mRNAs expressed as a percentage of the m-Gus control mRNA. Total RNA extracts from two cells lines of each genotype were assayed in triplicate. (C) In vivo recombination of the Smo locus. Genomic DNA from ductal structures or stromal areas isolated by laser-capture microdissection (LCM) from PDAC tumors was subjected to the same PCR amplification as in A. Ducts or stromal areas from two PDAC tumors of each genotype were pooled and subjected to PCR amplification. The input genomic DNA used in each PCR reaction is indicated: (Tail) DNA from mouse tail; (Ducts) LCM-captured ducts from two PDAC tumors; (Stroma) LCM-captured stromal-rich area of two PDAC tumors; (+/+) Smo+/+; (F/+) SmoF/+; (F/F) SmoF/F. (D–G) In vivo depletion of the Smo protein. Immunofluorescent detection of Smo (green) and Muc-1 (red) (630×). The Smo protein is detected in a subset of mucin-negative ducts inside PDAC SmoF/+ PanIN-like lesions (D), but not in mucin-positive ducts (white arrows) as well as in mucin-negative PDAC SmoF/+ adenocarcinoma lesions (E). Smo is undetectable in PDAC SmoF/F PanIN-like lesions (F) orin PDAC SmoF/F adenocarcinoma (G). Granular cytoplasmic Smo staining of an individual PDAC SmoF/+ cell (2520×) (D, insert) absent in individual PDAC SmoF/F cells (F, insert). The panels are representative of multiple fields of pancreatic sections from three PDAC SmoF/+ mice and three PDAC SmoF/F mice.
Figure 3.
Figure 3.
Ductal and acinar pancreatic development is normal in the absence of Smo function. (A) Quantitative RT–PCR comparison of Smo mRNA in total RNA extracts from dissected pancreatic buds of 12.5-d Pdx-Cre; Smo+/+ (Smo+) embryos and Pdx-Cre; SmoF/Null (Smo−) embryos. (B,C) Anti-Muc1 and anti-α-amylase staining of pancreatic sections from Pdx-Cre; Smo+/+ mice (B) and Pdx-Cre; SmoF/Null mice (C). (D) H&E staining of pancreatic sections from Pdx-Cre; Smo+/+ mice and Pdx-Cre; SmoF/Null mice. Arrows indicate pancreatic ducts. The panels are representative of multiple fields of pancreatic sections from two Pdx-Cre; Smo+/+ and two Pdx-Cre; SmoF/Null mice.
Figure 4.
Figure 4.
Genetic depletion of Smo in the pancreatic epithelium does not affect PDAC tumorigenesis. (A–D) H&E analysis of the histopathology of PDAC SmoF/+ (A,C) and PDAC SmoF/F (B,D) lesions reveals no overt difference in the presentation of PanIN-like lesions (A,B) or PDAC (C,D). (E) No significant difference in the pancreatic weight of PDAC SmoF/+ mice (F/+; N = 10) and PDAC SmoF/F mice (F/F; N = 11) could be detected [(**) P = 0.378)], but a significant difference is observed between the pancreatic weight of tumor-bearing mice and control non-tumor-bearing mice [N = 13; (*) P < 0.01]. (F) The mean survival of a cohort of PDAC SmoF/+ mice (green line; N = 31) was significantly greater [17 d; (*) P < 0.05] than that of a PDAC SmoF/F mice (red line; N = 31). The panels are representative of multiple fields of pancreatic sections from eight PDAC SmoF/+ mice and eight PDAC SmoF/F mice. (G) In vitro recombination of the Smo locus in the PDAC 4.2 R cell line derived from the nonrecombined PDAC 4.2 NR cell line. PCR amplification of the Smo locus (Smo) or the Betacellulin control locus (Btc). Smo genotyping: unrecombioned SmoF allele (upper band, F) and wild-type Smo allele (lower band, wild type). The upper PCR band is lost upon Cre recombination of the Smo conditional locus. The input genomic DNA is indicated: (Tail) genomic DNA from mouse tail; (4.2 NR) genomic DNA from nonrecombined PDAC cell line; (4.2 R) genomic DNA from in vitro recombined PDAC cell line. (H) Average pancreatic tumor weight from nude mice orthotopically injected with 4.2 NR (N = 8, Smo+) or 4.2 R cells (N = 8, Smo−). No significant difference was detected. (I,J) H&E staining of 4.2 NR (Smo+) and 4.2 R(Smo−) xenograft tumor sections. The panels are representative of multiple fields of pancreatic sections from four 4.2 NR and four 4.2 R pancreatic tumors.
Figure 5.
Figure 5.
Ptch1 and Gli1 expression are maintained in vivo in Smo-depleted neoplastic ductal cells. (A,B) Laser microdissection of pancreatic ducts before (A) and after (B) capture on H&E-stained frozen tumor sections (50×). (C) Expression of Smo, Ptch1, Shh, and Gli1 mRNA in total RNA extracts from pools of laser capture microdissected pancreatic ducts from two PDAC Smo+/+ (W), two PDAC SmoF/+ (H), and two PDAC SmoF/F (F) tumors. Levels of mRNAs expressed as a percentage of the m-Gus control mRNA. (D) Relative expression (% mGus) of Hedgehog/Gli signaling components following stimulation with increasing concentrations of recombinant Shh (0–50–150–500 ng/mL) in primary pancreatic fibroblasts (F) or in PDAC cell line 3.3 (P). Asterisk indicates a P value <0.01 (C) or <0.001 (D).
Figure 6.
Figure 6.
TGF-β and activated Kras signaling impact Gli and Ptch1 expression in a Smo-independent manner. (A) Expression of Smo, Ptch1, E-Cad, Gli1, and Gli3 mRNA in total RNA extracts from wild-type Smo (4.2 NR) or Smo mutant (4.2 R) PDAC cell lines 48 h after stimulation with 5 ng/mL recombinant TGF-β1. Levels of mRNAs expressed as a percentage of the mGus control mRNA. (B) Expression of Kras, Gli1, and Ptch1 mRNA in total RNA extracts from wild-type Smo (4.2 NR) or Smo mutant (4.2 R) PDAC cell 48 h after transfection with control siRNA pools or siRNA pools targeting Kras or Gli1. Levels of mRNAs expressed as a percentage of the mGus control mRNA. (C) Absorbance at 540 nm of two PDAC cell lines (4.2 NR and 3.3) incubated with MTT (see the Materials and Methods) 72 h after transfection with control siRNA pools or siRNA pools targeting Kras or Gli1 and after 24 h of serum starvation. (D) Relative change in Activated Caspase 3 immuno-fluorescent staining of mouse PDAC 3.3 cells 60 h after transfection with control siRNA pools (Ctrl) or siRNA pools targeting Kras or Gli1, following 12 h of serum starvation. Staining was evaluated in five fields each containing at least 500 cells for each condition. Act-Casp-3-positive cells expressed as a percentage of DAPI nuclei; the percentage in Ctrl-treated cells was set at 100%. Asterisk indicates a P-value <0.01 (A), <0.05 (B,C), or <0.005 (D).
Figure 7.
Figure 7.
GLI1 is required for survival and maintenance of the transformed phenotype of human PDAC cancer cells. (A) Relative changes in chromatin condensation/margination and nuclear fragmentation (proxy for apoptosis) in L3.6, PANC1, MiaPaCa2, and BxPC3 pancreatic cancer cells 48 h after transfection with shGLI1 or scramble control shRNA and subsequent treatment with 25 mM cycloheximide. More than 300 cells in four high-power fields were counted; apoptotic cells are expressed as a percentage of total cells. (B) Hoescht 33342 staining of L3.6 PDAC cells transfected with scramble shRNA, shGLI1, or shGLI1 alongside an shGLI1-resistant GLI1 cDNA construct (rGLI1). Arrows indicate nuclear morphological patterns indicative of apoptosis (blue, Hoechst 33342). (C) Relative colony formation (neoplastic anchorage-independent growth) of L3.6, PANC1, MiaPaca2, and BxPC3 pancreatic cancer cells assayed following transfection with shGLI1 or scramble shRNA (control) plasmids. (D) Relative colony formation of BxPC3 pancreatic cancer cells (wild-type KRAS) transfected with oncogenic KRAS and scramble control shRNA, shGli1, or shGli1 alongside an shGLI1-resistant GLI1 cDNA rescue construct (rGli1). (E) Relative changes in Luciferase activity in L3.6, PANC1, MiaPaCa2, and BxPC3 pancreatic cancer cells transfected with a Gli-Luciferase reporter and with shKRAS or scramble control shRNA. (F) Relative changes in Luciferase activity in BXPC3 (wild-type KRAS) pancreatic cancer cells transfected with a GLI-Luciferase reporter with or without KRAS or GLI1 expression constructs. (*) P-value <0.01; (**) P-value <0.05.
Figure 8.
Figure 8.
A new conceptualization of functionally significant hedgehog signaling and GLI transcription in pancreatic ductal carcinogenesis. (A) The conventional model of Hh/Gli signaling in PDAC. Earlier studies suggested that autocrine signaling by the SHH ligand was functionally important in PDAC cells (green arrow). Possible in vivo signaling to the stroma was an untested hypothesis (dotted black arrow), and hedgehog signaling in cancer cells and stromal cells such as cancer-associated fibroblasts was thought to follow canonical hedgehog signaling, whereby SHH binds to PTCH and induces SMO signaling resulting in the nuclear translocation of active GLI transcription factors and transcriptional activation of GLI target genes such as GLI1 and PTCH1. (B) A refined model of Hh/Gli signaling in PDAC. Our study demonstrates that PDAC tumor cells (1) require GLI1 for transformation and survival, (2) do not transduce functionally significant SHH ligand signals through the SMO coreceptor (crossed-out gray arrow), and (3) continue to express GLI transcriptional targets independently of SMO via noncanonical regulation of GLI target genes mediated in part by KRAS (orange arrow) and TGFβ (purple arrow). In parallel to noncanonical GLI transcription in PDAC cancer cells, SHH produced by cancer cells signals in a paracrine manner (green arrow) to the surrounding mesenchyme and may play a key paracrine function in PDAC pathogenesis (Yauch et al. 2008). In response to SHH, fibroblasts secrete signaling molecules that may stimulate tumor cell growth (Yauch et al. 2008) (dotted black arrow).
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