Role of ES cell-expressed Ras (ERas) in tumorigenicity of gastric cancer

Abstract

ERas, a unique member of the Ras family, was initially found only in embryonic stem (ES) cells, where it plays a crucial role in the transformation of transplanted ES cells to teratomas. ERas is involved in ES cell survival, and unlike other Ras family members, is constitutively active without any mutations. The aim of this study was to investigate the expression and role of ERas in human gastric cancer. To test whether ERas played a significant role in human cancer cells, we examined its expression and function in gastric cancer. ERas was expressed in gastric cancer cell lines at different levels. Induction of ERas expression activated the phosphatidylinositol 3 kinase (PI3K)/Akt axis and then enhanced anchorage-independent growth and ERas knockdown by siRNA suppressed cell invasion. Immunohistochemical analyses revealed that ERas was expressed in 38.7% (55/142) of human gastric carcinoma tissues, and its expression was significantly associated with metastasis to the liver (P < 0.0001) and lymph nodes (P < 0.05). ERas up-regulated transcription regulatory factors including ZFHX1A, ZFHX1B, and TCF3, which repress E-cadherin. These data suggest that ERas is activated in a significant population of gastric cancer, where it may play a crucial role in gastric cancer cell survival and metastases to liver via down-regulation of E-cadherin.

Figure 1

ERas mRNA and protein expression in nine gastric cancer cell lines and gastric cancer tissues. A: Expression of the ERas gene in gastric cancer cell lines (Lanes 2–10) was examined by RT-PCR. The negative control (Lane 12) indicates no template in the reaction. Primers for GAPDH were used as internal control. B: Expression of ERas protein in nine gastric cancer cell lines was analyzed by Western blots with anti-ERas antibody (Lanes 2–10). C: Expression of ERas protein in four cases of human gastric cancer tissue (Lanes 2–5) and corresponding non-tumor tissue adjacent to gastric cancer (Lanes 6–9) was analyzed by Western blot with anti-ERas antibody. D: Expression of ERas in gastric cancer tissue by immunohistochemical staining. Scale bars: 200 μm (top); 50 μm (bottom).

Figure 2

The role of ERas on signaling pathway, cell growth, and anchorage-independent growth. A: Western blotting of total lysates of control, parental GCIY cells (Lane 1), GCIY cells transfected with empty vector (Lanes 2 and 3), and ERas-overexpressing GCIY clones (GCIY cells transfected with ERas-expressing vector) (Lanes 4 and 5) with anti-phospho-Akt and Akt antibody, or with anti-phospho-MEK1/2 and MEK1/2 antibody. The cell lysates were probed for β-actin to control for equal protein loading. B: Parental GCIY cells, GCIY cells transfected with empty vector, and ERas-overexpressing GCIY clones were plated at 10³ cells per well on 96-well plates. Growth rates were measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium (MTS) assay on subsequent days. This experiment was repeated three times in triplicate. C: Parental GCIY cells, GCIY cells transfected with empty vector, and ERas-overexpressing GCIY clones were plated at 2.5 × 10³ cells per well on 24-well plates in soft agar. After 21 days the colonies were visualized with crystal violet and counted. Columns represent the means of three independent experiments; bars, SD. *P < 0.01 versus control.

Figure 3

Silencing of endogenous ERas expression suppresses invasive activity in gastric cancer cell lines. A: Western blotting of total lysates of GCIY cells and NUGC-4 cells treated with negative control siRNA (Lane 1), GCIY cells and NUGC-4 cells treated with ERas siRNA 1 (Lane 2), or ERas siRNA 2 (Lane 3) with anti-phospho-Akt and Akt antibody. The cell lysates were probed for β-actin to control for equal protein loading. B: GCIY cells and NUGC-4 cells treated with negative control siRNA, ERas siRNA 1, or ERas siRNA 2 were plated at 10³ cells per well on 96-well plates. Growth rates were measured by MTS assay. C: GCIY cells and NUGC-4 cells treated with negative control siRNA, ERas siRNA 1, or ERas siRNA 2 were plated at 5 × 10³ cells per well on upper chamber of membrane coated with Matrigel of 96-well Transwell plates. After 36 hours incubation, invasion of cells through Matrigel-coated membranes were measured. Columns represent the means of three independent experiments; bars, SD. *P < 0.01 versus control.

Figure 4

Immunohistochemical analysis of ERas and E-cadherin protein expression in resected human gastric cancer tissue. Sections of normal adjacent gastric mucosa (top) did not show staining with anti-human ERas antibody but did stain with anti–E-cadherin antibody. ERas protein was expressed in gastric cancer tissue and liver metastasis. E-cadherin was not expressed in gastric cancer tissue and liver metastasis (middle and bottom). Scale bars = 50 μm.

Figure 5

ERas suppresses the expression of E-cadherin. A: Real-time quantitative RT-PCR of E-cadherin mRNA levels in an ERas-overexpressing GCIY clones relative to parental GCIY cells. Columns represent the means of three independent experiments; bars, SD. *P < 0.01 versus control. B: Representative immunofluorescence stainings of cell lines. Nuclei stained blue with 4′,6-diamidino-2-phenylindole (DAPI) (left), ERas stained red with antibody against ERas (middle), and E-cadherin stained green with antibody against E-cadherin (right). C: Western blot analysis of total lysates of parental GCIY cells (Lane 1), GCIY cells transfected with empty vector (Lanes 2 and 3), and ERas-overexpressing GCIY clones (Lanes 4 and 5) with anti–E-cadherin antibody. Western blotting of total lysates of GCIY cells or NUGC-4 cells treated with ERas siRNA 1 or ERas siRNA 2 with anti–E-cadherin antibody. D: mRNA expression profiles of six E-cadherin repressors (SNAI1, SNAI2, ZFHX1A, ZFHX2B, TWIST1, and TCF3) in parental GCIY cells, ERas-overexpressing GCIY clone, and GCIY cells transfected with empty vector were determined by quantitative real-time RT-PCR. Columns represent the means of three independent experiments; bars, SD. *P < 0.001, **P < 0.05 versus control.

Figure 6

ERas promotes metastasis via induction of EMT. A: Cell morphology of GCIY cells transfected with ERas-expressing vector or treated with ERas siRNA. B: Western blot analysis of total lysates of GCIY cells transfected with empty vector and ERas-overexpressing GCIY clones with anti-Fibronectin and Vimentin antibody. C: Western blotting of total lysates of GCIY cells treated with negative control siRNA, ERas siRNA 1, or ERas siRNA 2 with anti-Fibronectin and Vimentin antibody.