Abstract
Background/Aim: Ras-related associated with diabetes (RRAD), a member of the Ras-related GTPase superfamily, is frequently methylated in several human cancers, though its methylation profile remains unclear in esophageal cancer. Materials and Methods: We examined RRAD promoter hypermethylation using real-time quantitative methylation-specific PCR in 229 primary human esophageal tissues of contrasting histological types. Results: RRAD hypermethylation showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) from esophageal adenocarcinoma (EAC) or normal esophagus (NE) (p<0.01 and p<0.01, respectively). RRAD normalized methylation values were significantly higher in ESCC (0.0242) than in NE (0.0057, p<0.05) or EAC (0.0139, p<0.01). RRAD hypermethylation frequency was also significantly higher in ESCC (23.1%) than in NE (0%, p<0.05) or EAC (5.4%, p<0.05). Conclusion: Promoter hypermethylation of RRAD is a frequent, tissue-specific event in ESCC, and is uncommon in EAC. The aberrant methylation of RRAD may be involved in the pathogenesis of a subset of ESCC, but not in EAC.
Esophageal cancer ranks eighth as most common cancer and the sixth as most frequent cause of cancer-related death worldwide (1). There are two principal forms of this malignancy, each possessing distinct pathological characteristics: esophageal squamous cell carcinoma (ESCC), which occurs in high frequencies in many developing countries, especially in Asia, including China (2); and esophageal adenocarcinoma (EAC), which is more prevalent in Western countries. In 2008, there were an estimated 482,000 new cases and 407,000 deaths worldwide related to esophageal cancer (1). Despite therapy, five-year survival rates remain dismal (i.e., 17% 5-year survival) (3). Clearly, novel early detection biomarkers and therapeutic targets are needed.
Emerging evidence has suggested that epigenetic changes play a crucial role in esophageal carcinogenesis (4-8). It is now well-established that DNA methylation correlates with inactivation of tumor suppressor genes in human malignancies including esophageal cancer (4, 5, 8, 9). Ras-related associated with diabetes (RRAD), a member of the Ras-related GTPase superfamily, is originally identified as an up-regulated mRNA in skeletal muscle from individuals with type II diabetes by subtraction cloning (10), and is most highly expressed in lung, skeletal and cardiac muscle in humans (10, 11). RRAD displays a wide spectrum of cellular functions, including glucose uptake in cultured muscle and fat cells (11), cytoskeletal remodeling (12, 13), osteoblast differentiation (14), voltage-gated calcium channel activity (15), cell migration (16-18) and cardiomyocyte apoptosis (19). It has been shown that RRAD is frequently methylated in multiple human malignancies, including malignant mesothelioma, prostate, cervical, lung, breast and nasopharyngeal cancer (20-24); however, the methylation profiles of RRAD remain uncharacterized in human esophageal cancer.
Clinicopathologic characteristics and methylation status of RRAD in human esophageal tissues.
Our goal was to determine the methylation profiles of RRAD in human esophageal cancer (EAC and ESCC), pre-malignant lesions (henceforth referred to as Barrett's esophagus (BE)) or Barrett's esophagus with dysplasia (D) and normal esophageal epithelium (NE) by real-time quantitative methylation-specific PCR (qMSP).
Materials and Methods
Tissue samples. In the current study, 56 NEs, 52 BEs, 39 Ds, 56 EACs, and 26 ESCCs were examined. All patients provided written informed consent under a protocol approved by the Institutional Review Boards at the University of Maryland School of Medicine, the Baltimore Veterans Affairs Medical Center, and the Johns Hopkins University School of Medicine. Biopsies were obtained using a standardized biopsy protocol, as described previously (25). Briefly, at each endoscopy, four-quadrant biopsies were obtained at 2-cm intervals throughout the grossly apparent BE segment (or at 1-cm intervals on follow-up after an endoscopy with LGD). Research tissues were obtained from grossly, apparent normal esophageal mucosa, Barrett's epithelium, or mass lesions in patients manifesting these changes at endoscopic examination, and histology was confirmed using parallel aliquots obtained at endoscopy. Outcome data were derived from a comprehensive database maintained by the institution's cancer registry and patients' medical records at the University of Maryland and Baltimore Veterans Affairs Medical Centers. All biopsy specimens were stored in liquid nitrogen up until DNA extraction. Clinicopathologic characteristics of patients are summarized in Table I.
DNA and RNA extraction. Genomic DNA was extracted from biopsies and cultured cells using a DNeasy Tissue Kit (Qiagen, Valencia, CA) and stored at −80°C before analysis.
Bisulfite treatment and real-time methylation-specific PCR. DNA was treated with bisulfite to convert unmethylated cytosines to uracils prior to qMSP, as described previously (26). Briefly, 1.0 μg genomic DNA was denatured by treatment with 2 mol/L NaOH and modified by 3 mol/L sodium bisulfite. DNA samples were purified using Wizard DNA cleanup resin (Promega, Madison, WI), treated with 3 mol/L NaOH, precipitated with 100% ethanol, and resuspended in 50 μL water. Promoter methylation levels of RRAD were determined by qMSP with the ABI 7900 Sequence Detection (Taqman) System, using primers and probes as described previously (26). The PCR mixture consisted of 12.5 μL Taqman Universal Master Mix without UNG (Applied Biosystems, Foster City, CA), 2.0 μL of probe for both Reprimo and β-actin (2.5 μmol/L), 0.25 μL forward and reverse primer for both Reprimo and β-actin (10 μmol/L), 50 ng bisulfite-treated DNA, and water (up to a total volume of 25 μL). Normalized methylation value (NMV) was defined as follows: NMV=(RRAD-S/RRAD-FM)/(ACTB-S/ACTB-FM), where RRAD-S and RRAD-FM represent RRAD methylation levels in sample and fully methylated DNAs, respectively, while ACTB-S and ACTB-FM correspond to β-Actin in sample and fully methylated DNAs, respectively.
Data analysis and statistics. Receiver-operator characteristic (ROC) curve analysis was performed using NMVs for the 56 EAC, 26 ESCC and 566 NE by Analyse-it© software (v.1.71, Analyse-it Software, Leeds, UK). Using this approach, the areas under the ROC curve (AUROC) identitifed optimal sensitivity and specificity levels (i.e., cut-offs) at which to distinguish normal from malignant esophageal tissues, and corresponding NMV thresholds were calculated for RRAD. The cut-off value determined from this ROC curve was applied to determine the frequency of RRAD methylation in each tissue type included in the present study. For all other tests, Statistica (version 6.1; StatSoft, Inc., Tulsa, OK) was used. Differences with p<0.05 were deemed significant.
Results
All qMSP assays were performed in duplicate and results were reproducible and concordant. RRAD promoter hypermethylation showed highly discriminative ROC curve profiles, clearly distinguishing both ESCC (p<0.01) and EAC (p<0.05) from NE, as well as EAC from ESCC (p<0.0001). ROC curves with AUROCs for RRAD of ESCC vs. NE, EAC vs. NE, and EAC vs. ESCC are shown in Figure 1.
Receiver-operator characteristic (ROC) curve analysis of normalized methylation value (NMV). ROC curve analysis of RRAD NMVs of normal esophagus (NE) vs. esophageal squamous cell carcinoma (ESCC) (A), NE vs. esophageal adenocarcinoma (EAC) (B) and EAC vs. ESCC (C).
The cut-off NMV for RRAD (0.02) was chosen from the ROC curve in order to maximize for sensitivity and specificity. Mean NMVs and RRAD hypermethylation frequencies for each tissue type are shown in Table I. NMVs of RRAD were significantly higher in ESCC than in NE or in EAC (p<0.05 and p<0.05, respectively; Mann-Whitney U-test, Figure 2), but not in EAC, in D and in BE than in NE. Mean NMV is stepwise increased from NE (0.0057), to BE (0.0093), D (0.01) and EAC (0.0139). Frequency of RRAD hypermethylation was increased relative to NE (0%) in BE (7.7%; p>0.05), D (10.5%; p<0.05), EAC (5.4%; p>0.05) and ESCC (23.1%; p<0.05). In addition, frequency of RRAD hypermethylation was significantly higher in ESCC (23.1%) than in EAC (5.4%; p<0.05).
No significant associations were observed between RRAD promoter hypermethylation and patient age, survival, BE segment lengths, tumor stage or lymph node metastasis and smoking or alcohol consumption (data not shown).
Discussion
In the current study, we systematically investigated hypermethylation of the RRAD gene promoter in primary human esophageal lesions of differing histological types and neoplastic stages. Our results demonstrate that RRAD promoter hypermethylation occurs frequently in human ESCC (23.1%), but only rarely in EAC (5.4%). In addition, both RRAD hypermethylation frequency and mean NMV were significantly higher in ESCC compared to EAC (23.1% vs. 5.4%; p<0.05 and 0.0242 vs. 0.0139; p<0.05, respectively). Furthermore, ROC curve analysis clearly distinguished ESCC from EAC (AUROC=0.735, p<0.0001). Taken together, these results imply that hypermethylation of RRAD is common in human ESCCs but uncommon in EACs, and that it is a cell type-specific event (i.e., common in ESCC but rare in EAC). Thus, RRAD hypermethylation appears to be a critical event unique to human ESCC, rather than EAC.
Methylation levels of RRAD in normal esophagus (NE), esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC).
The precise roles of RRAD in physiology and pathophysiology remain incompletely elucidated. Few reports show oncogenic effects of RRAD. Over-expression of RRAD promoted cell growth by accelerating cell-cycle transitions by interacting with the GTPase-activating protein, nm23, and was associated with poor prognosis of breast cancer patients (27). RRAD may promote carcinogenesis, at least in part, by interacting with GCIP and inhibiting GCIP-mediated reductions of Rb phosphorylation and cyclin-D1 expression (28). RRAD was up-regulated in primary cultured invasive phenotype hepatocellular carcinoma cells (29).
Interestingly, it has been suggested that RRAD functions as a tumor suppressor gene in some human malignancies. RRAD methylation was found and correlated with SV40 infection in human malignant mesothelioma (20). Frequency of RRAD methylation was significantly higher in prostate cancer tissues (37%) than in non-malignant prostatic tissues (9%) (21). RRAD was frequently methylated in biopsies of invasive cervical cancer (22). RRAD hypermethylation was a frequent event in lung and breast cancers (42% and 62%, respectively) and correlated with smoking history and poorer prognosis in lung adenocarcinomas (23). Expression of RRAD was significantly lower in invasive non-small cell lung carcinomas than in non-neoplastic bronchiolar epithelium by immunohistochemistry (30). In lung cancer cells, p53 up-regulated both mRNA and protein expression of RRAD, and inhibited cell migration by disrupting actin dynamics via RRAD (as a direct p53 transcriptional target) (30). In nasopharyngeal carcinoma, RRAD was epigenetically inactivated, and ectopic expression of RRAD suppresses tumor cell proliferation and migration in vitro (24). In addition, RRAD was up-regulated by Platelet-Derived Growth Factor and overexpression of RRAD attenuated vascular lesion formation by inhibition of vascular smooth muscle cell migration (18, 31). Taken together, these findings suggest that RRAD functions as a tumor suppressor gene in certain human cancers.
As mentioned above, RRAD was frequently methylated in human malignant mesothelioma, prostate, cervical, lung, breast and nasopharyngeal cancer (20-24), also in ESCC as shown in the current study. These results reveal that RRAD may act as a tumor suppressor, though the mechanistic details by which RRAD suppresses tumorigenesis are presently unclear.
The current study indicates, for the first time, that aberrant methylation of RRAD may be involved in pathogenesis of a subset of ESCC, but not in EAC. In addition, RRAD hypermethylation is uncommon in EAC, appearing to represent a cell type-specific biomarker for ESCC as opposed to EAC. The exact role of the RRAD hypermethylation regarding the carcinogenesis of human esophageal cancers should be addressed in future work.
Acknowledgements
This study supported by NSFC grant 81172282, Natural Science Foundation of SZU grants 201108 and T201202 to Z Jin; NIH grant DK087454 to SJ Meltzer
Footnotes
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↵* These Authors contributed equally to this study.
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Conflicts of Interest
All Authors do not have any conflicts of interest relevant to the manuscript.
- Received August 11, 2013.
- Revision received September 24, 2013.
- Accepted September 25, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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