Tissue transglutaminase-2 promotes gastric cancer progression via the ERK1/2 pathway

Abstract

Gastric cancer (GC) is one of the most common tumors worldwide and involves extensive local tumor invasion, metastasis, and poor prognosis. Understanding mechanisms regulating progression of GC is necessary for developing effective therapeutic strategies. Tissue transglutaminase-2 (TG2), a multifunctional member of the transglutaminase family, has been shown to be critical for tumor initiation and progression. However, how TG2 promotes the progression of GC is unknown. We report that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain- and loss-of-function approaches, we observed that TG2 promoted GC cell proliferation, migration, invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with the ERK1/2 pathway activation and an ERK1/2 inhibitor (U0126) inhibited cell proliferation, migration, and invasion induced by overexpression of TG2. In summary, TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer.

Keywords: ERK1/2; TG2; gastric cancer; tumor progression.

Figure 1. Expression of TG2 in the GC tissues and cell lines

A. TG2 mRNA expression in GC cell lines and a normal gastric epithelial cell line GES-1 was analyzed by qRT-PCR. B. TG2 protein expression in human GC cell lines and a normal gastric epithelial cell line GES-1 was examined by Western blot. C. and D. TG2 mRNA expression in 50 gastric cancer tissues and adjacent non-tumor tissues was analyzed by qRT-PCR. Data are 2^−ΔCt (*P < 0.05). E. Positive ratios of TG2 protein expression in 127 pairs of gastric cancer tissues. F. Expression of TG2 was examined with immunohistochemistry (IHC) in non-tumor gastric tissues, intestinal-type GC tissues, and diffuse-type GC tissues, and classified as strong expression (++), moderate expression (+), weak positive or negative expression (± or −). Original magnification: 200x.

Figure 2. TG2 promotes GC cell proliferation

A. TG2 protein expression in MKN45 and NCI-N87 cells transfected with TG2-shRNA was quantified with Western blot. B. TG2 protein expression in SGC7901 and AGS cells transfected with TG2-constructed plasmid. C. and D. The effect of TG2 knockdown on MKN45 and NCI-N87 cell proliferation was measured by CCK8 assay. E. and F. The effect of TG2 overexpression on SGC7901 and AGS cell proliferation was measured by CCK8 assay. Data are means ± SD of three independent experiments (*P < 0.05).

Figure 3. TG2 enhances migration and invasion of GC cells

A., C., E., and G. Effect of knockdown and overexpression of TG2 on cell migration and invasion was assayed and representative photographs of migrating or invaded cells on membranes with or without Matrigel (magnification, 100×) are shown. B., D., F., and H. Histograms depict migrating and invaded cells. Cells were counted in five randomly selected microscopic fields. Data are means ± SD of three independent experiments, *P < 0.05.

Figure 4. TG2 regulates cell proliferation, migration, and invasion via activation of the ERK1/2 pathway

A. and B. ERK1/2 phosphorylation and TG2 expression in GC cells (MKN45, NCI-N87, SGC7901, AGS, and GES-1) were quantified by Western blot. C. and E. Effects of TG2 on ERK1/2 and phosphorylation were measured. D. and F. Protein ratios of pERK1/2 in MKN45, NCI-N87, SGC-7901, and AGS cells. G. After treatment with U0126 at indicated times, ERK1/2 phosphorylation and TG2 expression in SGC7901/TG2 cells and AGS/TG2 cells were quantified. H. and I. SGC7901/TG2 and AGS/TG2 cell proliferation were assayed in the presence of U0126. J., K., L. and M. Effect of U0126 on cell migration and invasion was assayed and typical photographs (magnification, 100×) and migrating or invaded cells are shown. Data are means ± SD of three independent experiments, *P < 0.05.

Figure 5. Knockdown of TG2 expression inhibits subcutaneous tumor growth and peritoneal and other metastases in nude mice

A. and B. Representative photographs of tumors in nude mice (N = 5 per group) derived from MKN45/TG2-shRNA, MKN45/TG2-NC, NCI-N87/TG2-shRNA, and NCI-N87/TG2-NC cells. C. Tumor volume was monitored weekly after treatment with MKN45/TG2-shRNA, MKN45/TG2-NC, NCI-N87/TG2-shRNA, and NCI-N87/TG2-NC cells in nude mice (*P < 0.05). D. Average tumor weights derived from MKN45/TG2-shRNA, MKN45/TG2-NC, NCI-N87/TG2-shRNA, or NCI-N87/TG2-NC cells (N = 5) (*P < 0.05). E. Expression of TG2, Ki67, and pERK1/2 was quantified by IHC in tumor grafts from MKN45/TG2-shRNA, MKN45/TG2-NC, NCI-N87/TG2-shRNA, or NCI-N87/TG2-NC cells. Original magnification: 200x. F. MKN45/TG2-shRNA, MKN45/TG2-NC, NCI-N87/TG2-shRNA, or NCI-N87/TG2-NC cells were inoculated into nude mice, and the peritoneal nodules (red arrows) were observed after 35 days (N = 5 per group). G. Average peritoneal nodules from nude mice are shown (*P < 0.05). Data are means ± SD of three independent experiments, *P < 0.05.

Figure 6. TG2 overexpression promotes subcutaneous tumor growth and peritoneal spread and metastasis in nude mice

A. Representative photographs of nude mice tumors (N = 5 per group) derived from SGC7901/TG2 cells and SGC7901/Vector. B. Tumor volume was monitored weekly after treatment with SGC7901/TG2 cells or SGC7901/Vector (*P < 0.05). C. Average tumor weight derived from SGC7901/TG2 cells or SGC7901/Vector (N = 5) (*P < 0.05). D. Average peritoneal nodules from nude mice (*P < 0.05). E. SGC7901/TG2 cells or SGC7901/Vector were inoculated into nude mice, and peritoneal nodules (red arrows) were observed after 35 days (N = 5 per group). Data are means ± SD of three independent experiments, *P < 0.05.