Hedgehog signaling antagonist GDC-0449 (Vismodegib) inhibits pancreatic cancer stem cell characteristics: molecular mechanisms

Abstract

Background: Recent evidence from in vitro and in vivo studies has demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in various human cancer stem cells (CSCs). Therefore, the chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. GDC-0449 (Vismodegib), orally administrable molecule belonging to the 2-arylpyridine class, inhibits SHH signaling pathway by blocking the activities of Smoothened. The objectives of this study were to examine the molecular mechanisms by which GDC-0449 regulates human pancreatic CSC characteristics in vitro.

Methodology/principal findings: GDC-0499 inhibited cell viability and induced apoptosis in three pancreatic cancer cell lines and pancreatic CSCs. This inhibitor also suppressed cell viability, Gli-DNA binding and transcriptional activities, and induced apoptosis through caspase-3 activation and PARP cleavage in pancreatic CSCs. GDC-0449-induced apoptosis in CSCs showed increased Fas expression and decreased expression of PDGFRα. Furthermore, Bcl-2 was down-regulated whereas TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was increased following the treatment of CSCs with GDC-0449. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GDC-0449-treated pancreatic CSCs. Thus, activated Gli genes repress DRs and Fas expressions, up-regulate the expressions of Bcl-2 and PDGFRα and facilitate cell survival.

Conclusions/significance: These data suggest that GDC-0499 can be used for the management of pancreatic cancer by targeting pancreatic CSCs.

Figure 1. Expressing components of Sonic Hedgehog (SHH) signaling pathway in human pancreatic cancer cell lines and pancreatic cancer stem cells (CSCs).

Pancreatic cancer cells (AsPC-1, PANC-1 and MIA PaCa-2) and pancreatic CSCs were grown for 48 h. Total RNA was isolated and expression of Shh, Patched-1, Patched-2, Smoothened, Gli-1 and Gli-2 was measured by qRT-PCR. HK-GAPD was used as endogenous normalization control. All assays were performed in triplicate and were calculated on the basis of ΔΔCt method. The n-fold change in mRNAs expression was determined according to the method of 2^-ΔΔCT with GAPDH employed as the endogenous control.

Figure 2. Inhibition of SHH signaling suppressed cell viability in human pancreatic cancer cell lines and pancreatic CSCs by GDC-0449.

Cells were treated with GDC-0449 (0, 1, 5 and 10 µM) for 48 and 72 h. At the end of incubation period, cell viability was measured by XTT in (A) AsPC-1, (B) MIA PaCa-2, (C) PANC-1, and (D) Pancreatic CSCs. Data represent mean ± SD. @ or # significantly different from respective control (P<0.05).

Figure 3. Induction of apoptosis by GDC-0449 in pancreatic cancer cell lines and pancreatic CSCs.

The cells were treated with GDC-0449 (0, 1, 5 and 10 µM) for 48 and 72 h. At the end of incubation period, cells were harvested and apoptosis was measured. Data represent mean ± SD. @ or # significantly different from respective control (P<0.05).

Figure 4. GDC-0449 differentially regulates genes involved in the balance between cell death and cell survival.

Pancreatic CSCs were treated with GDC-0449 (0, 1, 5 and 10 µM) for 48 h. The expression of Fas, DR4/TRAIL-R1, DR5/TRAIL-R2, PARP cleavage, Bcl-2, and Caspase-3 by the Western blot analysis. β-Actin was used as a loading control.

Figure 5. GDC-0449 downregulates the expression of various components of SHH signaling pathway in pancreatic CSCs

. (A), Pancreatic CSCs were treated with GDC-0449 (10 µM) for 36 h. At the end of incubation period, RNA was extracted and the expression of Gli1, Gli2, Patched-1, Patched-2, Smoothened and Shh was measured by qRT-PCR. Data represent the mean ± SD. @ significantly different from respective control (P<0.05). (B), Pancreatic CSCs were treated with and GDC-0449 (0, 1, 5 and 10 µM) for 48 h. Nuclear extracts were prepared and the gelshift experiment was performed. Probe only, control (without GDC-0449), and GDC-0449 treated samples (1, 5 and 10 µM), respectively. The data are representative of three experiments. (C), Gli-dependent luciferase activity is reduced by GDC-0499. Pancreatic CSCs were transduced with lentiviral construct expressing Gli-dependent luciferase reporter, and treated with GDC-0449 (0, 5 and 10 µM) for 48 h. Lysates were prepared, and luciferase activity was measured. Normalized luciferase activity is presented as mean ± SD. @ or # significantly different from respective control (P< 0.05). (D), GDC-0449 inhibits expression of Gli1 and Gli2 in human pancreatic CSCs. The cells were seeded on fibronectin-coated coverslips and treated with GDC-0449 (10 µM) for 48 h. Subsequently, cells were fixed with 4% paraformaldehyde, blocked in 10% normal goat serum and stained with Gli1 and Gli2 primary antibodies (1∶100) for 16 h at 4°C and washed with PBS. Afterwards, cells were incubated with fluorescently labeled secondary antibody (1∶200) along with DAPI (1 mg/ml) for 1 h at room temperature and cells were mounted and visualized under a fluorescent microscope. For better visuality, the color of DAPI was changed from blue to red.

Figure 6. Impact of SHH signaling pathway on the regulation of cell survival and antiproliferative effects of GDC-0449.

(A), Knockout of Gli1 shRNA and Gli2 shRNA in human pancreatic CSCs. Pancreatic CSCs were transduced with lentiviral particles expressing Scrambled, Gli1 shRNA, Gli2 shRNA or Gli1 plus Gli2 shRNA (KO). (B), Pancreatic CSCs were treated with GDC-0449 (0, 1, 5 and 10 µM) for 72 h, and cell viability and apoptosis was measured in scrambled and Gli1 plus Gli2 shRNA CSCs. Data represent mean ± SD, n = 4. @ or # significantly different from respective control (P<0.05). (C), Scrambled and Gli1 plus Gli2 shRNA pancreatic CSCs were treated with GDC-0449 (0 and 10 µM) for 48 h, and lysates were extracted to determine the expression of DR4, DR5, PDGFRα, Fas and Bcl-2 by Western blot analysis. β-Actin was used as the loading control. (D), Inhibition of primary and secondary spheroids by GDC-0449. Pancreatic CSCs (scrambled, and Gli1 + Gli2 shRNA) were seeded in suspension and treated with GDC-0449 (10 µM) for 7 days. At the end of incubation period, spheroids were collected, and dissociated with Accutase (Innovative Cell Technologies, Inc.). For secondary spheroids, cells were reseeded and treated with GDC-0449 (10 µM) for additional 7 days. Cell viability was measured by trypan blue assay. Data represent mean ± SD. @ or # significantly different from respective controls, P<0.05.

Figure 7. Schematic representation of the inhibition of SHH signaling and genes involved in the balance between cellular proliferation and cell death.

Activated Gli1 and Gli2, downstream of SHH-Patched-Smoothened, regulate targets of SHH signaling including Bcl-2, PDGFRα, Fas, and DRs. GDC-0449 (targeting Smoothened) blocks the indirect functions of Gli activators, resulting in cell death.