Background
RNA editing refers to the post-transcriptional epigenetic modification of RNA sequences through insertion, deletion, or nucleotide conversion, resulting in an increased diversity of the transcriptomic repertoire. Adenosine-to-inosine (A-to-I) RNA editing is primarily mediated by adenosine deaminase acting on RNA (ADAR) enzymes, and is the predominant form of RNA editing in humans [
1]. It involves the conversion of adenosine to inosine, which acts in a similar manner to guanosine in the general cellular machinery [
2]. However, RNA editing in the coding region of mRNA molecules change the amino acid sequence of the encoded protein, with subsequent negative impact on the functionality of the corresponding protein [
3]. Although genome-wide A-to-I editing was initially thought to be a rare event which was limited to coding exons, advances in next-generation sequencing technologies and bioinformatic tools have now allowed revealed identification of hundreds of thousands of RNA editing sites throughout the human transcriptome. Not surprisingly, RNA editing is gaining attention in the field of cancer research, and emerging evidence suggests that A-to-I RNA editing permits transcript localization and degradation via protein recoding, alternative splicing, and microRNA regulation; thus facilitating cancer evolution and pathogenesis [
4,
5]. Furthermore, the selective distribution of RNA editing loci and their biological roles indicate the potential for clinically relevant diagnostic and prognostic tools capable of accurately assessing aberrant RNA editing involved in cancer progression and therapeutic resistance.
Chen and colleagues performed the first systematic and comprehensive analysis using a sequencing-based approach [
6], and demonstrated that
AZIN1 RNA was specifically enhanced in HCC tissues, and significantly correlated with disease progression in HCC patients.
AZIN1 belongs to the antizyme inhibitor family, and plays a role in maintaining polyamine homeostasis which are important for various cellular functions, including cell growth [
7,
8]. This hypothesis was supported by the findings that neutralization of a key inhibitor of the polymerase synthesis pathway through
AZIN1 RNA editing permitted unimpeded tumor growth and proliferation [
6]. Recently, our group also revealed that
AZIN1 confers a gain-of-function phenotype frequently through A-to-I conversions via ADAR1, which can promote ornithine decarboxylase (ODC) and polyamines accumulation—conditions that are associated with aggressive tumors [
6,
9].
The incidence of gastric cancer (GC) in developed countries has fallen significantly, however, this malignancy remains the fourth most common cancer and the second leading cause of cancer-related deaths worldwide [
10]. Approximately, one third of GC patients are first diagnosed at late stages with a locally-advanced or metastatic disease. This highlights the need for identification and development of robust biomarkers that can allow early detection, as well as predict postoperative tumor recurrence, to improve the overall morbidity and mortality associated with gastric neoplasia.
Currently, several well-known antigens, including carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9) and cancer antigen 72-4 (CA72-4), or serological biopsy using Pepsinogen I and II have been investigated in the context of GC [
11,
12]. Although various targets have been suggested to serve as potential biomarkers in patients with GC, biomarkers with adequate sensitivity and specificity for implementation in GC screening and risk stratification remain unavailable, but represent an active area of research.
Work from our group and others have previously identified several epigenetic alterations that could serve as biomarkers for diagnosis, prognosis, and metastasis prediction in patients with various gastrointestinal cancers [
13‐
16]. More recently, we have also revealed the role of altered RNA editing levels and its functional consequence in colorectal cancer [
17]. In the current study, we for the first time, investigate the RNA editing status of the antizyme inhibitor 1 gene (
AZIN1) and the expression pattern of its regulatory protein, ADAR1, in the primary tumor tissues and matched normal mucosa from patients with gastric cancer, with an emphasis to gain insights into the clinical significance of these events in this malignancy.
Discussion
Emerging evidence indicates that epigenetic alterations encompassing RNA editing may play a central role in post-transcriptional gene regulation, and might control the expression of various cancer-related genes. However, the role of RNA-editing and the expression patterns of its regulatory enzymes, as well as their clinical significance in patients with gastric cancer (GC) remains unclear. Our study provides first evidence supporting a potential clinical impact of dysregulated RNA editing and its regulatory enzymes in GC. First, we observed that AZIN1 RNA editing levels were significantly higher in GC tissues compared to matched normal mucosa. Second, we noted that the expression of AZIN1-regulatory enzyme, ADAR1, was also up-regulated in GC tissues, and significantly correlated with increased RNA editing in this malignancy. Third, increased AZIN1 RNA editing and ADAR1 over-expression in GC tissues significantly correlated with key clinico-pathological factors for disease progression, including advanced tumor depth, the presence of lymph node and distant metastases, and higher TNM stages in GC patients. Fourth, both AZIN1 RNA hyper-editing and elevated ADAR1 expression significantly correlated with poor overall survival (OS), with significant associations between AZIN1 editing and poor disease free survival (DFS). Fifth, multivariate Cox regression analysis revealed that increased AZIN1 editing emerged as an independent prognostic factor for both OS and DFS in GC patients. Finally, our data revealed that higher levels of AZIN1 RNA editing were an independent risk factor for lymph node metastasis (LNM) in patients with GC, highlighting its biomarker potential in the identification of high-risk patients that may experience tumor recurrence post-surgical treatments.
Cancer cells acquire specific characteristics, including escape of cell cycle checkpoint controls, continuous proliferation, invasion, and metastasis. Over the past decades, data suggest that alterations in DNA sequences of the key tumor suppressors and oncogenes may influence the progression of tumorigenesis through functional control of the proteins encoded by these genes [
22]. Although these genetic factors are undeniably important, recent years have witnessed an increased emphasis on the contributions of epigenetic alterations mediating cancer pathogenesis, as well as their role as disease biomarkers in various cancer types. Well-studied epigenetic alterations to date include aberrant DNA methylation, and dysregulated expression of noncoding RNAs (e.g. microRNAs, long non-coding RNAs), and various histone modifications. In this context, RNA editing has recently emerged as one of the most recent discoveries, and the most common type of RNA editing in humans is A-to-I conversion catalyzed by ADAR enzymes [
1,
23,
24]. RNA editing is consequential, since it directly impacts protein coding sequences, through aberrant transcript splicing, instability, and dysregulated expression levels [
25], vindicating the paradigm that site-specific dysregulation of RNA editing may play a pivotal role in cancer development [
26‐
29]. Chen and colleagues performed the first systematic and comprehensive analysis using a sequencing-based approach [
6], and demonstrated that
AZIN1 RNA was specifically enhanced in HCC tissues, and that significantly correlated with disease progression in HCC patients. This group recently expanded their research to other cancers within the upper gastrointestinal tract, and successfully demonstrated that reciprocal changes in ADAR1 and ADAR2 coordinated cancer pathogenesis via hypo-editing of the podocalyxin-like protein 1 gene (
PODXL) [
30]. However, the clinical significance of
AZIN1 RNA editing and expression pattern of ADAR1 in GC patients remains unclear; which was the very basis of undertaking the present study. A key finding of our current study was our observation for the increased RNA editing levels of
AZIN1, as well as dysregulated expression of its regulatory enzyme, ADAR1, in GC tissues vis-à-vis normal gastric mucosa. To date, overexpression of ADAR1 and increased
AZIN1 RNA editing in cancer tissues has been demonstrated in various other types of cancers, including HCC, non-small-cell lung [
31], esophageal [
9], and colorectal cancer [
17]. In line with these evidences, our study for the first time revealed that such dysregulated pattern in GC tissues, and suggested that altered ADAR1 expression and
AZIN1 RNA editing is an important phenomenon in this malignancy as well. Furthermore, enhanced
AZIN1 RNA editing and over-expression of ADAR1, significantly correlated with poor OS and
AZIN1 RNA editing also significantly correlated with poor DFS. Hyper-edited levels of
AZIN1 RNA were an independent prognostic factor for both OS and DFS in GC patients. Taken together, our data for the first time, suggest that measurement of
AZIN1 RNA editing status could be a promising prognostic biomarker for tumor recurrence and survival in patients with GC.
Our study also demonstrated an intimate correlation between
AZIN1 RNA editing levels and lymph node metastasis in GC patients. The presence of regional LNM mainly affects disease recurrence and prognosis in GC patients following curative resection. While recent neoadjuvant therapies may offer treatment options for GC patients with LNM [
32,
33], and endoscopic techniques for primary tumor resection, (e.g. endoscopic resection and laparoscopic-assisted gastrectomy) may be used to treat early GC patients without LNM [
34], neither of these approaches are robust at identification of high-risk GC patients. Therefore, an accurate risk assessment of which patients may be truly LNM-positive might reduce overly invasive surgeries and improve the prognosis in GC patients. Interestingly, the current study clearly demonstrated that hyper-editing status of
AZIN1 RNA in primary tumor tissues significantly correlated with the presence of LNM, while logistic regression analysis identified it to be an independent risk factor for identifying LNM in GC patients. These findings suggest that the assessment of
AZIN1 RNA editing levels in pre-surgical biopsy specimens might potentially help in the identification of high-risk GC patients with lymph node metastasis.
We would like to acknowledge several potential limitations of our study. First, we focused on AZIN1 as a most representative RNA editing site in this study, and emerging evidence suggests that there may be potentially other oncogenic RNA editing sites in other genes, which remain an area of active investigation. Therefore, further studies including a broader, unbiased comprehensive analysis may potentially identify additional RNA editing-based markers to assess the risk for oncological outcomes with a higher sensitivity and specificity in GC patients in future. Second, although we have successfully demonstrated our novel findings using a relative-large cohort GC specimens, our cohort was still somewhat smaller, retrospective in nature, and lacked an external independent validation cohort GC specimens. In addition, the clinical materials analyzed in this study were solely from Asian patients, and analytical method for AZIN1 RNA editing level maybe influenced by several cofounding factors. To overcome these hurdles, larger prospective, multi-institutional studies using same analytical method may be needed to further clarify the prognostic potential of AZIN1 RNA editing and ADAR1 expression for GC patients.
Authors’ contributions
Study concept and design (YO, YT, KS, AG); provision of samples (TS, TI, HY, HF, SY, JH, MO, TA, MK); acquisition of data (YO, TS, TI, HY, HF, SY, JH, MO, TA); analysis and interpretation of data (YO, YT, KS); statistical analysis (YO, YT); drafting of the manuscript (YO, YT, MK, AG).