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Keywords:

  • endoglin;
  • hypermethylation;
  • esophageal adenocarcinoma;
  • esophageal squamous cell carcinoma;
  • biomarker

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

BACKGROUND

Endoglin (ENG) is a 180-kilodalton transmembrane glycoprotein that functions as a component of the transforming growth factor-β receptor complex. Recently, ENG promoter hypermethylation was reported in several human cancers.

METHODS

The authors examined ENG promoter hypermethylation using real-time, quantitative, methylation-specific polymerase chain reaction in 260 human esophageal tissues.

RESULTS

ENG hypermethylation demonstrated highly discriminative receiver operating characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < .01). It is interesting to note that ENG normalized methylation values were significantly higher in ESCC compared with normal tissue (P < .01) or EAC (P < .01). The ENG hypermethylation frequency was 46.2% in ESCC and 11.9% in normal esophageal tissue, but increased early and sequentially during EAC-associated neoplastic progression to 13.3% in Barrett metaplasia (BE), 25% in dysplastic BE, and 26.9% in frank EAC. ENG hypermethylation was significantly higher in normal esophageal tissue from patients with ESCC (mean, 0.0186) than in normal tissue from patients with EAC (mean, 0.0117; P < .05). Treatment of KYSE220 ESCC cells with the demethylating agent 5-aza-2′-deoxycytidine was found to reverse ENG methylation and reactivate ENG mRNA expression.

CONCLUSIONS

Promoter hypermethylation of ENG appears to be a frequent, tissue-specific event in human ESCC and exhibits a field defect with promising biomarker potential for the early detection of ESCC. In addition, ENG hypermethylation occurs in a subset of human EAC, and early during BE-associated esophageal neoplastic progression. Cancer 2013;119:3604–3609. © 2013 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Esophageal cancer ranks as the eighth most common cancer worldwide, with 482,000 new cases diagnosed in 2008, and is the sixth most common cause of cancer death, with 407,000 deaths.[1] This malignancy exists in 2 principal forms, each possessing distinct pathological characteristics: esophageal squamous cell carcinoma (ESCC), which occurs at high frequencies in many developing countries, especially Asia (and including China)[2]; and esophageal adenocarcinoma (EAC), which is more prevalent in Western countries. These aggressive malignancies commonly present as locally advanced disease with a very poor prognosis (ie, with a 5-year survival rate of approximately 17%),[3] although significant advances have occurred in treatment. To improve outcome, it will be vitally important to discover early events that can serve as detection biomarkers or targets for chemoprevention and therapy.

Endoglin/CD105 (ENG) is a 180-kilodalton transmembrane glycoprotein that functions as a component of the transforming growth factor-β receptor complex.[4, 5] ENG is expressed predominantly in proliferating vascular endothelial cells, in which it plays a critical role in vascular remodeling and angiogenesis.[6-10] Germline mutations in the ENG gene can lead to an autosomal dominant vascular dysplasia, hereditary hemorrhagic telangiectasia type 1 syndrome.[7, 11] Its critical role in angiogenesis has prompted investigators to evaluate the role of ENG in cancer progression and metastasis. Intratumor microvessel density as assessed by ENG staining strongly correlates with prognosis in patients with different types of cancer.[9, 10, 12, 13] Although ENG hypermethylation has been reported in human lung, colorectal, and breast cancers,[14-16] to the best of our knowledge only 1 study to date has evaluated ENG hypermethylation in 2 patients with ESCC and 16 ESCC cell lines.[17] Therefore, to further investigate ENG hypermethylation in human esophageal carcinogenesis, we investigated whether and at which neoplastic stage promoter hypermethylation of ENG is involved in human esophageal carcinogenesis, using real-time, quantitative, methylation-specific polymerase chain reaction (PCR) (qMSP) analyses of 260 endoscopic esophageal biopsy specimens of differing histologies. We also evaluated the effect of the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC) on reactivation of epigenetically silenced ENG in esophageal cancer cells. The results of the current study established that promoter hypermethylation of ENG is a common event in patients with ESCC but not in those with EAC and occurs early during Barrett-associated esophageal neoplastic progression.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Tissue Samples

The 260 specimens examined in the current study were composed of 67 normal esophageal specimens (including 19 obtained from patients with non-Barrett/nonesophageal cancer [NE], 20 from patients with ESCC [NEcS], and 28 from patients with EAC [NEcA]), 60 nondysplastic Barrett metaplasia (BE) specimens, 40 dysplastic Barrett (D) specimens, 67 EAC specimens, and 26 ESCC specimens. All patients provided written informed consent under a protocol approved by the Institutional Review Boards at the University of Maryland and Baltimore Veterans Affairs Medical Centers, at which all esophagogastroduodenoscopies were performed. Biopsies were performed using a standardized biopsy protocol, as previously described.[18] Research tissues were obtained from macroscopically apparent Barrett epithelium or from mass lesions in patients demonstrating these changes at the time of endoscopic examination, and histology was confirmed using parallel aliquots taken from identical locations at endoscopy. All biopsy specimens were stored in liquid nitrogen before DNA extraction.

Cell Lines

The KYSE220 ESCC cell line was obtained from collaborators at Toyama University and cultured in 47.5% RPMI-1640 and 47.5% F-12 supplemented with 5% fetal bovine serum.

DNA and RNA Extraction

Genomic DNA and total RNA were extracted from biopsies and cultured cells using DNeasy Tissue Kits (Qiagen, Valencia, Calif) and TRIzol reagent (Invitrogen, Carlsbad, Calif), respectively. DNAs and RNAs were stored at −80°C before analysis.

Bisulfite Treatment and Real-Time qMSP

DNA was treated with bisulfite to convert unmethylated cytosines to uracils before qMSP, as described previously.[15] Promoter methylation levels of ENG were determined by real-time qMSP with the ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif) using primers and probes as described previously.[15] The normalized methylation value (NMV) was defined as follows: NMV = (ENG-S/ENG-FM)/(ACTB-S/ACTB-FM), in which ENG-S and ENG-FM represent ENG methylation levels in sample and fully methylated DNAs, respectively, whereas ACTB-S and ACTB-FM correspond to β-actin in sample and fully methylated DNAs, respectively.

Real-Time Quantitative Reverse Transcriptase-PCR

To determine ENG mRNA levels, 1-step, real-time, quantitative reverse transcriptase (RT)-PCR was performed using a Qiagen QuantiTect Probe RT-PCR Kit (Qiagen, Hilden, Germany) and the ABI 7700 Sequence Detection System (Applied Biosystems). β-Actin was used for the normalization of data. Primers and probes for ENG and β-actin were the same as previously reported.[15] A standard curve was generated using serial dilutions of qPCR Reference Total RNA (Clontech Laboratories Inc, Mountain View, Calif). The normalized mRNA value (NRV) was calculated according to the following formula for the relative expression of target mRNA: NRV = (ENG-S/ENG-C)/(ACTB-S/ACTB-C), in which ENG-S and ENG-C represent levels of mRNA expression for ENG in sample and control mRNAs, respectively, whereas ACTB-S and ACTB-C correspond to amplified ACTB levels in sample and control mRNAs, respectively.

5-Aza-dC Treatment of Esophageal Cancer Cell Lines

To determine whether ENG inactivation was due to promoter hypermethylation in patients with esophageal cancer, KYSE220 cells were subjected to treatment with 5-Aza-dC (Sigma, St. Louis, Mo), as previously described.[19, 20] Briefly, 1 × 105 cells/mL were seeded onto a 100-mm dish and grown for 24 hours. Then, 1 μL of 5mM 5-Aza-dC/mL of cells was added every 24 hours for 4 days. DNAs and RNAs were harvested on day 4.

Informed consent was obtained from the subject(s) and/or guardian(s)

Data Analysis and Statistical Analysis

Receiver operating characteristic (ROC) curve analysis was performed using NMVs for the 67 EAC, 26 ESCC, and 67 normal esophagus specimens by Analyze-it software (Version 1.71; Analyze-it Software, Leeds, UK). With this approach, the area under the ROC curve identified optimal sensitivity and specificity levels at which to distinguish normal from malignant esophageal tissues, yielding corresponding NMV thresholds defining the methylation status of ENG. The threshold NMV value determined from this ROC curve was applied to determine the status of ENG methylation in all tissue types included in the current study. For all other statistical tests, Statistica software (version 6.1; StatSoft, Inc, Tulsa, Okla) was used. Differences with a P value < .05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

ENG Promoter Hypermethylation in Esophageal Tissues

Promoter hypermethylation of ENG was analyzed in 67 normal esophagus (including 19 NE, 20 NEcS, and 28 NEcA) specimens, 60 BE specimens, 40 D specimens, 67 EAC specimens, and 26 ESCC specimens. ENG promoter hypermethylation demonstrated highly discriminative ROC curve profiles and areas under the ROC curve, clearly distinguishing ESCC from both normal esophagus and EAC (P < .01 and P < .01, respectively) (Figs. 1A and 1B), as well as NEcS from NEcA (P < .01) (Fig. 1C), but not EAC from normal esophagus (data not shown).

image

Figure 1. Receiver operating characteristic (ROC) curve analysis of the normalized methylation value (NMV) is shown. ROC curve analysis of (A) endoglin (ENG) NMVs of normal esophagus (N) versus esophageal squamous cell carcinoma (ESCC), (B) ESCC versus esophageal adenocarcinoma (EAC), and (C) N specimens from patients with ESCC (NEcS) versus N specimens from patients with EAC (NEcA) is shown. The area under the ROC curve (AUROC) conveys this biomarker's accuracy in distinguishing EAC from N and from ESCC in terms of its sensitivity and specificity.

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The cutoff NMV for ENG (0.02) was identified from ROC curves (ESCC vs normal esophagus) as maximizing both sensitivity and specificity. The mean NMV and frequency of ENG hypermethylation for each tissue type are shown in Table 1. The NMVs of ENG were significantly higher in ESCC specimens than in normal esophagus or EAC specimens (P < .01, Mann-Whitney U test) (Table 1). Moreover, the NMV of ENG was significantly higher in NEcS specimens (mean, 0.0186) compared with NEcA specimens (mean, 0.0115) (P < .05, Mann-Whitney U test) (Table 1). Similarly, the frequency of ENG hypermethylation was significantly higher in ESCC specimens than in normal esophagus (46.2% vs 11.9%; P < .001), and was sequentially increased in BE (13.3%), D (25%), and EAC (26.9%) specimens versus normal esophagus (11.9%) (P > .05, P > .05, and P < .05, respectively, chi-square for independence test). The ENG hypermethylation frequency was higher in ESCC specimens than in EAC specimens, although these differences did not achieve statistical significance (46.2% vs 26.9%; P = .074). There was no significant difference noted for ENG hypermethylation comparing either NEcS versus NE or NEcA versus NE. In the current study, the mean NMV in NEcS specimens was not found to be significantly higher than in NE. This finding could have resulted from differences in sample sizes between these 2 groups.

Table 1. Methylation Status of ENG in Human Esophageal Tissues
 No. of SamplesMean Age, YearsNMVMethylation Status (Cutoff, 0.02)
HistologyMeanPaFrequencyUMMPb
  1. Abbreviations: EAC, esophageal adenocarcinoma; ENG, endoglin; ESCC, esophageal squamous cell carcinoma; M, methylated; NE, normal esophagus from non-Barrett/cancer patients; NEcA, NE from patients with EAC; NEcS, NE from patients with ESCC; NMV: normalized methylation value; UM, unmethylated.

  2. a

    P value was determined using the Mann-Whitney U test.

  3. b

    P value was determined using the Chi-square for independence test.

  4. c

    Comparisons made with NEcS.

  5. d

    Comparisons made with normal esophagus.

  6. e

    Comparisons made with ESCC.

Normal esophagus6764.40.0137 11.9%598 
NE1964.10.0117 5.3%181 
NEcA2866.90.0115<.05c10.7%253>.05c
NEcS2061.30.0186 20.0%164 
Barrett metaplasia6063.70.0123>.05d13.3%528>.05d
Dysplasia in Barrett esophagus4065.30.0141>.05d25.0%3010>.05d
EAC6765.10.0238>.05d/<.01e26.9%3218<.05d/>.05e
ESCC2662.50.0450<.01d46.2%1412<.001d

No significant associations were observed between ENG promoter hypermethylation and patient age, survival, Barrett segment length, EAC tumor stage, lymph node metastasis, or smoking or alcohol consumption (data not shown).

ENG Methylation and mRNA Levels in KYSE220 Cells After Treatment With 5-Aza-dC

KYSE220 cells were subjected to demethylation by 5-Aza-dC. After treatment with 5-Aza-dC, the NMV of ENG was diminished and ENG mRNA levels were found to be increased (Fig. 2).

image

Figure 2. Endoglin (ENG) methylation and mRNA expression in KYSE220 cells after treatment with 5-aza-2′-deoxycytidine (5-Aza-dC) is shown. After treatment with 5-Aza-dC, the normalized methylation value (NMV) of ENG was found to be diminished, whereas the normalized mRNA value (NRV) was increased.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

In the current study, we systematically investigated hypermethylation of the ENG gene promoter in primary human esophageal lesions of contrasting histological types. The results of the current study demonstrate that ENG promoter hypermethylation occurs frequently in human ESCC (46.2%), but only in a smaller subset of EAC specimens (26.9%). However, ENG hypermethylation occurs early and increases sequentially during esophageal adenocarcinogenesis, from 11.9% in normal esophagus (and 10.7% in NEcA) to 13.3% in BE, 25% in D, and 26.9% in EAC. It is interesting to note that methylation levels of ENG were significantly higher in NEcS specimens compared with NEcA specimens, suggesting that ENG exhibits a field defect with potential biomarker value for ESCC lurking nearby, even when analyzing nonneoplastic esophageal mucosa. In addition, ENG was hypermethylated more frequently in ESCC specimens than in EAC specimens. Taken together, these findings suggest that hypermethylation of ENG is a common event in ESCC, occurs early in some patients during the development of EAC, increases in frequency during Barrett-associated esophageal adenocarcinogenesis, and is a cell type-specific event (ie, more common in ESCC than in EAC). Further evidence supporting this tissue specificity was provided by ROC curves, which clearly distinguished ESCC from EAC but not EAC from normal esophagus. Further support for tissue specificity was evident from our finding that the mean ENG NMVs were significantly higher in ESCC specimens than in EAC specimens. Thus, ENG hypermethylation appears to constitute a critical field-defect event in human ESCC.

Despite extensive knowledge regarding intratumor microvessel density assessed by ENG staining as a prognostic factor in different types of cancer,[9, 10, 12, 13] to the best of our knowledge only limited data are available concerning ENG hypermethylation in tumor cells.[14-17] ENG was found to be significantly downregulated in non-small cell lung cancer using the Affymetrix GeneChip assay (Affymetrix, Santa Clara, Calif), and its promoter was aberrantly methylated in 5 of 7 lung cancer cell lines (71%), 11 of 16 primary lung tumors (69%), and 4 of 5 normal lung tissues (80%) based on combined bisulfite restriction analysis.[14] Based on qMSP assays, ENG was found to be methylated in 3 of 34 colon cancer specimens, but not in normal colonic mucosae.[15] In a large cohort of invasive breast cancers, lack of ENG expression in the tumor cell compartment correlated with ENG gene methylation and poor clinical outcome, and its expression in breast tumor cells suppressed invasion and metastasis.[16] ENG was previously shown to be methylated in 2 patients with ESCC and 16 ESCC cell lines by non-qMSP, and its expression suppressed invasion in ESCC cells.[17] To our knowledge, the current study is the first to quantitatively measure the methylation of ENG in a large cohort of human esophageal cancers.

It has been reported that hypermethylation of gene promoters in histologically normal tissue can be related to the initiation of carcinomas.[21-26] For example, methylation of the MLH1 promoter was observed in small foci of normal colonic epithelial cells from patients with colon cancer and was associated with silencing of this gene, but was not observed in sections of normal colon from healthy volunteers, suggesting that tumors with gene silencing due to epigenetic alteration may evolve from rare clones of methylated cells in normal epithelia.[24] Nonneoplastic epithelia taken from patients with ESCC was found to be significantly more methylated than control esophageal epithelia from healthy volunteers in a panel of 14 promoter loci.[25] Data from our group also previously indicated that AKAP12 hypermethylation was significantly higher in NEcA specimens compared with NE or NEcS specimens.[26] Similarly, in the current study, ENG hypermethylation was found to be significantly higher in NEcS specimens than in NEcA specimens. Thus, our highly sensitive real-time qMSP approach allowed us to demonstrate that nonneoplastic esophageal epithelia from patients with ESCC already exhibit low but abnormal levels of ENG promoter methylation. Therefore, it can be hypothesized that increased ENG methylation in normal esophageal cells extends their lifespan enough to put them at higher risk of future malignant evolution. Furthermore, mean ENG NMVs were found to be significantly higher in ESCC specimens than in EAC specimens. These results also further suggest that hypermethylation of ENG is an early and unique event, constituting a potentially powerful biomarker for the early detection of ESCC.

5-Aza-dC and its derivatives have demonstrated effectiveness as therapeutic anticancer drugs.[27, 28] In agreement with previous findings,[16, 17] the results of the current study found that methylation of ENG in ESCC cancer cell lines was associated with silenced or reduced expression of ENG mRNA. Treatment with 5-Aza-dC reactivated mRNA expression and reversed ENG hypermethylation in these cells. Restoration of ENG mRNA expression by treatment with a demethylating agent indicates that DNA hypermethylation was responsible for the silencing of ENG. These findings also suggest the possibility that epigenetic therapies may be useful in at least a subset of these patients. In addition, the known involvement of ENG in angiogenesis[6-10, 12] suggests the possibility that antiangiogenesis therapy may be directed toward a subset of these patients, such as those whose tumors lack ENG methylation. Further studies are needed to address this possibility.

The results of the current study demonstrate that hypermethylation of the ENG promoter, leading to gene silencing, is a common event in human ESCC. In addition, they indicate that ENG hypermethylation occurs early among a subset of patients with Barrett-associated esophageal adenocarcinogenesis. Further large-scale, prospective, longitudinal validation studies of this biomarker as a potential predictive biomarker of ESCC should be stimulated by these data.

FUNDING SUPPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Supported by National Natural Science Foundation of China grant 81172282, Natural Science Foundation of Shenzhen University grants 201108 and T201202 to Dr. Jin, and a National Institutes of Health grant (DK087454) to Dr. Meltzer.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Dr. Meltzer has received a grant from the National Institutes of Health and the American Cancer Society and is an American Cancer Society Clinical Research Professor.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES