Application of molecular biomarkers in clinical setting might improve diagnostic and management strategy of GC patients. To date, some molecular markers, including human epidermal growth factor receptor-2, vascular endothelial growth factor receptor 2, urothelial cancer associated 1, excision repair cross-complementation group 1, B-cell lymphoma-2, and Ki-67 have been proposed to have diagnostic and prognostic value in the management of GC patients [18, 19]. However, the majority of these markers were not able to accurately reflect prognostic value and therapeutic efficiency in advanced GC. Identification of molecular biomarkers might improve patient therapeutic strategy of GC, hence, we evaluated the CNV and expression of ADAR to reveal its alteration and clinical significance in advanced GC.
We analyzed mRNA expression level of ADAR in GC tumors and adjacent normal tissues. This revealed that ADAR is remarkably overexpressed in GC tumors. In addition, we compared ADAR expression level in GC stage III and IV. Interestingly, the average expression of ADAR mRNA in stage IV was higher than stage III. Besides, overexpression of ADAR was significantly associated with metastasis, stage, histology and size of tumor, which may indicate that ADAR has a pivotal role in stage III to IV progression of GC. According to ADAR function, overexpression of ADAR could leads to hyper-editing [20]. Unbalanced editing process has adverse results, and abnormal RNA editing within the transcriptome detected in many kinds of tumors. RNA editing could leads to recoding of a transcript and contribute to carcinogenesis through reducing the activity of tumor suppressors such as bladder cancer associated protein in hepatocellular carcinoma [21] or enhancing the activity of pro-survival genes such as antizyme inhibitor 1 in cervical cancer [22].
To determine whether ADAR copy number has variation in tumors, QMF-PCR was conducted. We found that ADAR significantly amplifies in advanced GC. Moreover, our results showed a positive correlation between ADAR gene copy number and its expression at the mRNA level. Correlation between ADAR copy number and ADAR expression has also been reported in breast carcinoma, ovarian adenocarcinoma, lung adenocarcinoma and liver carcinoma [22]. ADAR frequently amplifies in human cancers consistent with the elevated expression and editing levels of its substrates [23]. Notably, knockdown of ADAR in lung adenocarcinoma cells with amplified ADAR leads to decreased migration and invasion [12]. Hence, pharmacological targeting of ADAR promise a potential therapeutic application for tumors with ADAR amplification.
Also, we carried out a correlation test between ADAR expression and copy number change in 37 GC cell lines and statistical analysis revealed that ADAR expression was consistently associated with ADAR copy number. The association of gene copy number with gene expression has also been found in other cancer cell lines. Hyman et al. performed a high-resolution analysis in breast cancer cell lines and showed that 44% of the highly amplified genes showing overexpression and 10.5% of the highly overexpressed genes being amplified [24]. These findings further strengthen our hypothesis that ADAR amplification increases its expression.
As previously mentioned, ADAR amplification and overexpression in different cancers have been reported [22, 23]. Yet, the effects of these genomic and transcriptomic changes on clinicopathological features of GC patients have remained largely unknown. We evaluated the association of the ADAR gene CNV and its expression with the clinicopathological features of patients. The mRNA expression levels of ADAR were positively associated with metastasis, stage, histology and size of tumor. Indeed, ADAR amplification was significantly associated with metastasis, stage and size of tumor. Findings of the current study support the previous study reporting the oncogenic potential of ADAR in GC [25]. The association between the tumor aggressiveness and the overexpression of ADAR has also been demonstrated in hepatocellular carcinoma and colorectal cancer [26, 27]. Based on available evidences, the molecular mechanisms of how ADAR promotes GC cell growth and migration might be explained by its role in the regulation of mTOR signaling pathway. Overexpression of ADAR in GC cells increases proliferation and migration but these effects significantly debilitate with rapamycin, the mTOR kinase inhibitor, demonstrating that rapamycin could inhibit the effects of ADAR overexpression on GC cell growth and migration. Together these results suggested that mTOR signaling is important for ADAR mediated GC invasion and metastasis [25].
Currently, prognosis of GC patients is primarily determined using depth of wall invasion, lymph node or distant metastasis status and age; however, these prognostic factors are limited in clinical practice, and may not result in an accurate prognosis [28]. To further confirm the prognostic value of ADAR overexpression in GC patients, Kaplan–Meier analysis and log-rank test were performed. The results showed that the overall survival rate of patients with lower expression of ADAR was better than that of patients with high expression, suggesting its prognostic role. Chen et al. found a correlation between high expression of ADAR and a poor prognosis of cervical cancer [22]. Again, Chan et al. showed a significant correlation between ADAR upregulation and GC patient shorter survival [29].