Contrasting activity of Hedgehog and Wnt pathways according to gastric cancer cell differentiation: relevance of crosstalk mechanisms

Abstract

Gastric cancer displays different biological behaviors according to histological differentiation. The different biological behavior might involve the activation of distinct signaling pathways necessary for the growth and survival of cancer cells in gastric cancer. We investigated the differentiation-related signal interaction between Hedgehog and Wnt pathways in gastric cancer cells. Differentiation of gastric cancer cells was induced by sodium butyrate. The sonic Hedgehog (SHH) signal expressions were increased during cellular differentiation. In contrast, the expression of Wnt signaling was decreased during differentiation. Ectopic expression of glioma-associated oncogene-1 (GLI1) increased the level of secreted frizzled related protein-1 (SFRP1) transcript, whereas inhibition of GLI1 reduced the level of SFRP1 transcript. ChIP assay showed that GLI1 induced the transcriptional regulation of SFRP1 gene expression. Ectopic expression of GLI1 decreased the nuclear beta-catenin staining, but the inhibition of GLI1 induced the reversal of nuclear beta-catenin overexpression. Ectopic expression of beta-catenin also decreased the expression of GLI1 in the butyrate treated cancer cells. SHH and GLI1 immunoexpression was greater in well differentiated gastric cancer tissues compared to poorly differentiated tissues, and nuclear beta-catenin immunoexpression was lower in well differentiated compared to poorly differentiated tissues. The SHH and Wnt pathways are differentially involved according to gastric cancer cell differentiation.

Figure 1

Immunohistochemical staining for glioma‐associated oncogene‐1 (GLI1; a,b), and β‐catenin (c,d) in well differentiated (WD; a,c) and poorly differentiated (PD; b,d) gastric carcinoma tissues. Nuclear GLI1 staining revealed stronger expression in WD than PD tissues. In contrast, the expression of nuclear β‐catenin was stronger in PD than WD tissues. Magnification, ×400.

Figure 2

Increased alkaline phosphatase (ALP) activity by differentiation. (a) Subconfluent AGS and MKN‐45 gastric cancer cells were treated with sodium butyrate (0–3 μM) for 48 h. ALP activity was increased in AGS and MKN‐45 cells. Error bars were calculated from three experiments. (b) Subconfluent AGS cells were treated with all‐trans retinoic acid (0–3 μM) for 48 h. ALP activity was increased after 1 μM retinoid acid treatment. Error bars were calculated from three experiments.

Figure 3

Overexpression of carcinoembryonic antigen (CEA) and Brahma (Brm) by sodium butyrate treatment (NaBU) in AGS (a,c) and MKN‐45 (b,d) gastric cancer cells. RT‐PCR (left column) and Western blot (right column) were carried out with β‐actin as internal standard.

Figure 4

Transepithelial electrical resistance (TER) during gastric cancer cell differentiation. TER values are presented as relative to the values at control. TER was increased in AGS and MKN‐45 cells. Error bars were calculated from three experiments.

Figure 5

Overexpression of sonic Hedgehog (SHH; a,b), glioma‐associated oncogene‐1 (GLI1; c) and Patched‐1 (PTC1; d) by sodium butyrate (NaBU) treatment for 48 h. RT‐PCR (left column) and Western blot (right column) for SHH were carried out in AGS (a) and MKN‐45 (b) gastric cancer cells. Western blot for GLI1 and PTC1 was done in AGS (left column) and MKN‐45 (right column) cells.

Figure 6

Decreased nuclear β‐catenin expression by sodium butyrate (NaBU) treatment in AGS (a,b) and MKN‐45 (c,d) gastric cancer cells. Western blot for β‐catenin was carried out after sequential extraction of cytosolic and nuclear proteins from cells in non‐ionic detergent. Lamin B1 (a nuclear protein) and β‐actin were used as internal standards. Protein expression levels of nuclear β‐catenin were analyzed by densitometry analysis (b,d). Densitometry data presented in graphical form are “fold change” compared with control after normalization with respective loading control (Lamin B1). Similar results were obtained in three independent experiments. Statistical significance was determined by one‐way ANOVA with multiple comparison test. *P < 0.05, statistically significant difference compared with control/PBS. †P < 0.05, statistically significant difference from nuclear β‐catenin of NaBU previous dose‐treated group.

Figure 7

Regulation of secreted frizzled related protein‐1 (SFRP1) transcript by glioma‐associated oncogene‐1 (GLI1) in gastric cancer cells. (a,b) Overexpression of SFRP1 by sodium butyrate (NaBU) treatment for 48 h in AGS (a) and MKN‐45 (b) cells. RT‐PCR (left column) and Western blot (right column) analyses for SFRP1 were carried out with β‐actin as the internal standard. (c) Increased GLI1 expression by GLI1 vector or 2 μM NaBU increased the expression of SFRP1 in RT‐PCR (left column) and Western blot (right column) analyses. The maximal expression of SFRP1 was found when GLI1 was overexpressed by vector and NaBU. (d) ChIP analysis of SFRP1 promoter region in AGS cells. Overexpressed GLI1 by NaBU or GLI1 vector was directly binding to the SFRP1 promoter region. Mouse IgG was used as the negative control and anti‐RNA polymerase beads were used as the positive control. (e) Decreased expression of SFRP1 by siRNA against GLI1. The increased expression of SFRP1 by Hedgehog signal activation after NaBU treatment was significantly decreased by siRNA against GLI1 in RT‐PCR (left column) and Western blot (right column) analyses.

Figure 8

The inverse correlation between Hedgehog and Wnt signal pathways in gastric cancer cells. (a) Overexpressed glioma‐associated oncogene‐1 (GLI1) suppressed nuclear β‐catenin in AGS cells. Western blot for β‐catenin was carried out after sequential extraction of cytosolic and nuclear proteins from cells in non‐ionic detergent. Lamin B1 (a nuclear protein) and β‐actin were used as internal standards. Increased GLI1 expression by GLI1 vector or sodium butyrate (NaBU) decreased nuclear β‐catenin proteins. The most decreased nuclear β‐catenin proteins were found when GLI1 was overexpressed by vector and NaBU. (b) AGS cells were transfected with Topflash (OT) or Fopflash (OF), a control, and the indicated amount of a GLI1 vector. Transfection with GLI1 vector decreased TCF transcription factor activity compared with control (bars, SD; *P < 0.001 vs control). (c) Decreased nuclear β‐catenin after NaBU treatment was recovered by siRNA against GLI1 in AGS cells. (d) Increased expression of GLI1 by NaBU treatment was decreased after β‐catenin overexpression by the vector in MKN‐45 cells in RT‐PCR (left column) and Western blot (right column) analyses.