Aberrant overexpression of ADAR1 promotes gastric cancer progression by activating mTOR/p70S6K signaling

Abstract

ADAR1, one of adenosine deaminases acting on RNA, modulates RNA transcripts through converting adenosine (A) to inosine (I) by deamination. Emerging evidence has implicated that ADAR1 plays an important role in a few of human cancers, however, its expression and physiological significance in gastric cancer remain undefined. In the present study, we demonstrated that ADAR1 was frequently overexpressed in gastric cancer samples by quantitative real-time PCR analysis. In a gastric cancer tissue microarray, ADAR1 staining was closely correlated with tumor stage (P < 0.001) and N classification (P < 0.001). Functional analysis indicated that ADAR1 overexpression promoted cell proliferation and migration in vitro, whereas ADAR1 knockdown resulted in an opposite phenotypes. Furthermore, ADAR1 knockdown also inhibited tumorigenicity and lung metastasis potential of gastric cancer cells in nude mice models. Mechanistically, ADAR1 expression had a significant effect on phosphorylation level of mTOR, p70S kinase, and S6 ribosomal protein, implying its involvement in the regulation of mTOR signaling pathway. We conclude that ADAR1 contributes to gastric cancer development and progression via activating mTOR/p70S6K/S6 ribosomal protein signaling axis. Our findings suggest that ADAR1 may be a valuable biomarker for GC diagnosis and prognosis and may represent a new novel therapeutic opportunities.

Keywords: ADAR1; gastric cancer; mTOR; metastasis; tumorigenecity.

Figure 1. Expression pattern of ADAR1 in gastric cancer tissues and cell lines

(A) The mRNA expression of ADAR1 was measured in 38 paired gastric cancer samples and adjacent, non-cancerous stomach tissues by quantitative real-time-PCR. Data is shown as –ΔCt, where β-actin was used as internal control. P value was calculated by Student's t test, **P < 0.01. (B) The protein expression of ADAR1 was performed with immunohistochemical staining on a gastric cancer tissue microarray with anti-ADAR1 antibody. (−), representative sections of negative staining; (+), slight positive; (++), moderate positive; (+++), strong positive. Magnification: × 40 (upper) and × 200 (bottom). (C) The positive ratio of ADAR1 staining in TNM stage I, II, III and IV. Weak staining includes (−) and (+); strong staining includes (++) and (+++). *P < 0.01. (D) Kaplan-Meier overall survival plot comparing patients demonstrating high ADAR1 expression (black line; n = 564) and low ADAR1 expression in tumors (red line; n = 115; P < 0.01, Log-rank test). (E) The expression of ADAR1 was evaluated by western blot in five gastric cancer cell lines and two random adjacent non-cancerous stomach tissues.

Figure 2. ADAR1 enhanced GC cell growth and colony formation

(A) The effects of ADAR1 knockdown on cell proliferation in AGS, BGC823 and HGC27 cells. The knockdown efficiency of ADAR1 was determined by western blotting. (B) The effects of ADAR1 knockdown on colony formation capacity of AGS, BGC823 and HGC27 cells. The same amounts of related cells were plated into a 6-well plate. Cell colonies were stained and counted after two weeks. (C) Cell growth was examined by CCK8 method in ADAR1 overexpressed SGC7901 and MGC803 cells. Western blot analysis was used to detect ADAR1 expression in the two cell lines. All the above experiments were repeated at least three times. The data represents mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 3. Knockdown of ADAR1 induced cell death and slowed down cell growth

(A) Cell apoptosis was examined by Annexin-V/PI staining and flow cytometry analysis in two groups of cells stably silencing ADAR1 (AGS/shNC and AGS/shADAR1, SGC7901/shNC and SGC7901/shADAR1). The proportion of cell death was determined by PI-positive staining. Error bars represent mean ± SD for three independent experiments. *p < 0.05, **p < 0.01. (B) EdU incorporation assay was used to measure cell proliferation in AGS/LV-shADAR1, BGC823/LV-shADAR1 cells and their corresponding control cells. Data represented as mean ± SD deviation of three independent experiments. *p < 0.05.

Figure 4. ADAR1 promotes GC cell motility

(A) The migration abilities of MGC803 and SGC7901 cells transfected with ADAR1 overexpression plasmid or empty vector were evaluated by transwell chamber assays, respectively. All experiments were repeated at least three times. Representative fields of migrated cells are shown. Cell numbers were counted in five randomly selected microscopic fields and the data are shown as the mean ± SD. *P < 0.05, **p < 0.01. (B) Migration of AGS, MGC823 and HGC27 cells was evaluated by transwell assay after stably infected with LV-shADAR1, where LV-shNC was used as a control. Cell numbers were counted in five randomly selected microscopic fields and the data is shown as mean ± SD of three independent experiments. *P < 0.05, **p < 0.01. (C) Wound healing experiment was used to analyze the motility ability of BGC823 and AGS cells in which ADAR1 was silenced.

Figure 5. ADAR1 knockdown suppressed the tumorigenicity and metastasis of GC cell in nude mice

(A) The impact of ADAR1 knockdown on tumorigenicity of BGC823 cells in nude mice. Animals were sacrificed and tumor tissues were collected and photographed, tumor weight was measured at the end of the experiment. Values are expressed as mean ± SEM (n = 5). (B) Lung metastasis potential assay for ADAR1 knockdown was examined in SGC7901 cells by tain vein injection. The tumor nodules were observed in lung surface after 40 days receiving 1.5 × 10⁶ SGC7901-shNC or SGC7901-shADAR1 cells. The mean number of tumor nodules from each group (n = 5) and the hematoxylin and eosin (HE) stained lung sections are shown. Magnification: ×200.

Figure 6. Effects of ADAR1 on the phosphorylation level of mTOR signaling in GC cells

(A) Intracellular signaling array was performed in AGS cells before and after silencing ADAR1. The significantly changed protein dots were marked with rectangles. (B) Data quantified by Image Studio Version 3.1 for each protein dots of intracellular signaling array used above. (C) Western blot analysis was used to confirm the phosphorylation level change of mTOR, p70S6 kinase and S6 ribosomal protein in SGC7901 cells (for ADAR1 overexpression) and AGS cells (for ADAR1 knockdown).

Figure 7. Rapamycin partially blocked the effects of ADAR1 overexpression on GC cell growth and migration

(A) The effects of ADAR1 overexpression on SGC7901 and MGC803 cell proliferation with or without rapamycin treatment (10 μM), *p < 0.05. (B) Representative photographs of migratory cells on the transwell membrane (magnification, 200×). The below panel is the average MGC803 cell number of triplicate, *p < 0.05.