Hedgehog signaling pathway is a potential therapeutic target for gallbladder cancer

Abstract

Gallbladder cancer (GBC) is a particularly deadly type of cancer with a 5-year survival rate of only 10%. New effective therapeutic strategies are greatly needed. Recently, we have shown that Hedgehog (Hh) signaling is reactivated in various types of cancer and is a potential therapeutic target. However, little is known about the biological significance of Hh signaling in human GBC. In this study, we determined whether Hh signaling could be a therapeutic target in GBC. The Hh transcription factor Gli1 was detected in the nucleus of GBC cells but not in the nucleus of normal gallbladder cells. The expression levels of Sonic Hh (Shh) and Smoothened (Smo) in human GBC specimens (n = 37) were higher than those in normal gallbladder tissue. The addition of exogenous Shh ligand augmented the anchor-dependent and anchor-independent proliferation and invasiveness of GBC cells in vitro. In contrast, inhibiting the effector Smo decreased the anchor-dependent and anchor-independent proliferation. Furthermore, the suppression of Smo decreased GBC cell invasiveness through the inhibition of MMP-2 and MMP-9 expression and inhibited the epithelial-mesenchymal transition. In a xenograft model, tumor volume in Smo siRNA-transfected GBC cells was significantly lower than in control tumors. These results suggest that Hh signaling is elevated in GBC and may be involved in the acquisition of malignant phenotypes, and that Hh signaling may be a potential therapeutic target for GBC.

Keywords: Epithelial-mesenchymal transition; Hedgehog; Smo protein; gallbladder cancer; matrix metalloproteinase.

Figure 1

Expression of Hedgehog (Hh) components in gallbladder cancer is significantly higher than in normal gallbladder tissue. Immunohistochemical staining for Hh components in gallbladder cancer (a) or normal gallbladder (b) specimens. Clockwise (from upper left) are antibodies to Gli1, Smoothened (Smo), and Sonic Hh (Shh). A positive reaction is brown in color. Red arrows indicate Gli1 expression in the nucleus of cancer cells. Original magnification, ×400.

Figure 2

Expression of Hedgehog (Hh) components in gallbladder cancer (GBC) cells is also evident at mRNA and protein levels. (a) Total RNA from GBC cells (GBd15, TGBC2TKB) was reverse transcribed and the cDNA was subjected to conventional RT-PCR for the indicated mRNAs. GAPDH was used as control. (b) Protein extracted from GBC cells (GBd15, TGBC2TKB) was subjected to Western blot analysis for Hh components. Tubulin was used as the loading control. Shh, Sonic Hh; Smo, Smoothened.

Figure 3

Recombinant human Sonic Hedgehog (rhShh) augments the proliferative and invasive phenotypes of gallbladder cancer cells. (a) GBd15 and TGBC2TKB cells were seeded onto 96-well plates at a density of 5000 cells/well and incubated with rhShh (at 0.1, 1.0, or 2.0 μg/mL) for 24, 48, or 72 h as indicated. (b) GBd15 and TGBC2TKB cells were incubated for 16 h in the presence or absence of rhShh (at 0.1 or 1.0 μg/mL). Migrated cells were quantified by bright-field microscopy. Error bars represent standard deviations. *P < 0.05.

Figure 4

Cyclopamine (Cyclo) and Smo siRNA suppress proliferative and invasive phenotypes of gallbladder cancer cells. (a) GBd15 and TGBC2TKB cells were seeded onto 96-well plates and incubated with cyclopamine (at 5, 10, or 20 μM) for 24, 48, or 72 h. (b) GBd15 and TGBC2TKB cells were incubated for 16 h in the presence or absence of cyclopamine (at 5 or 10 μM). Migrated cells were quantified by bright-field microscopy. (c) GBd15 and TGBC2TKB cells were transfected with Smo siRNA (siSmo) and incubated for 24, 48, or 72 h. (d) GBd15 and TGBC2TKB cells were transfected with Smo siRNA and incubated for 16 h. Migrated cells were quantified by bright-field microscopy. Error bars represent standard deviations. *P < 0.05. siCont, control siRNA.

Figure 5

Smoothened (Smo)-regulated invasion is induced through MMP-2 and MMP-9. (a) Quantitative RT-PCR, (b) Western blotting, and (c) gelatin zymography assay using Smo siRNA (siSmo) transfected GBd15 and TGBC2 cells. (d) Cell invasion assay using MMP-2 siRNA (siMMP-2) and MMP-9 siRNA (siMMP-9) transfected GBd15 and TGBC2TKB cells. siCont, control siRNA. *P < 0.05.

Figure 6

Smoothened (Smo)-regulated invasion is mediated through epithelial–mesenchymal transition. (a) Quantitative RT-PCR (b) and Western blotting of E-cadherin and vimentin in Smo-siRNA transfected GBd15 and TGBC2TKB cells. (c) GBd15 and TGBC2TKB cells transfected with control siRNA (siCont) or Smo siRNA (siSmo) were immunostained with E-cadherin antibody (green) and DAPI (blue). Red arrows, E-cadherin accumulated in the membrane of Smo siRNA transfected cells. Original magnification, ×200. (d) GBd15 and TGBC2TKB cells transfected with Smo siRNA for 48 h before the number of spindle-shaped cells were counted using bright-field microscopy. Error bars represent standard deviations. *P < 0.05.

Figure 7

Hedgehog (Hh) signaling enhances gallbladder cancer colony formation in vitro and inhibition of Smoothened (Smo) suppresses tumor growth in vivo. (a, b) GBd15 and TGBC2TKB cells were incubated with recombinant human Sonic Hh (rhShh) (a, at 0.1 or 1.0 μg/mL) or cyclopamine (Cyclo) (b, at 5 or 10 μM) for 14 days before colony formation was assessed. (c) GBd15 and TGBC2TKB cells were transfected with Smo siRNA (siSmo) and incubated for 14 days before colony formation was assessed. (d, e) TGBC2TKB cells were transfected with control siRNA (siCont) (n = 5) or Smo siRNA (n = 5), and then implanted into the flanks of athymic nude mice. The data represent the volume of each tumor (red arrows). Student's t-test was carried out between the tumor volumes of the two groups. (f) Gli1 and Smo expression were confirmed by immunohistochemistry in tumor tissue explants from the nude mice. Original magnification, ×400. Error bars represent standard deviations. *P < 0.05.