Role of epigenetic alterations in the pathogenesis of Barrett’s esophagus and esophageal adenocarcinoma

Esophageal adenocarcinoma is rapidly rising in the US and needs newer biomarkers for surveillance and prognostic risk assessment

The risk of esophageal adenocarcinoma (EAC) in patients with Barrett’s esophagus appears to be approximately 30 to 125- fold greater than in the general population with an estimated incidence of 1 in 180-patient years [3,17]. Because of this increased risk, patients with BE usually undergo endoscopic surveillance at regular intervals. However, because the incidence of EAC in Barrett’s esophagus is uncommon, most of these surveillance endoscopies in BE patients do not detect cancer. Therefore, surveillance would benefit from effective markers to stratify patient according to their level of cancer progression risk [3]. So far, dysplasia is the currently accepted marker for cancer risk, with high grade dysplasia being considered more predictive than low-grade dysplasia. Whereas low-grade dysplasia (LGD) has no histologic abnormalities specific for neoplasia given its similarity to those changes occurring in normal tissue in response to injury, by the time high grade dysplasia (HGD) is evident, approximately one third of patients will have already invasive cancer.

In contrast to HGD, the predictive value of LGD for cancer risk assessment is controversial. Therefore, more accurate tissue-based biomarkers capable of predicting the risk of progression to EAC would be of significant use [3]. Moreover, serum biomarkers such as freely circulating plasma DNA abnormalities could be of potential use in predicting level of aggressiveness and behavior of this type of malignancy, as demonstrated in the study performed by Kawakami et al., where high plasma levels of hypermethylated APC DNA were correlated with reduced survival in patients with EAC [34]. At the same time, the study found higher quantity of methylated DNA in esophageal adenocarcinoma than in squamous cell carcinoma, suggesting that quantitative differences in degree of methylation exist between different tumor types.

AKAP12

The A-Kinase anchoring protein 12 (AKAP12) is a multivalent anchoring protein and an important regulator of the beta2-adrenergic receptor complex [39]. The AKAP12 gene maps to chromosome 6q24-25.2 [38], a locus that is frequently deleted in human cancers. It also controls cell signaling, cell adhesion, mitogenesis and differentiation and possesses tumor suppressor activity [39]. In the study by Jin et al. using MSP in 259 esophageal tissues, AKAP12 hypermethylation frequency was not seen in normal esophagus but increased to 38.9% in Barrett’s mucosa derived from patients with Barrett’s alone, 48.3 % in all Barrett’s tissue derived from Barrett’s and EAC, 52.5% in dysplastic tissue, and 52.2% in EAC. In contrast only 2 (7.7%) of 26 esophageal squamous cell carcinoma samples exhibited AKAP12 methylation. AKAP12 methylation also correlated with decreased mRNA levels. Treatment of BIC and OE33 EAC cells with 5-aza-2’-deoxycytidine reduced AKAP12 methylation and increased AKAP12 mRNA expression The above study also found a significant correlation between AKAP12 hypermethylation and BE segment length [39].

APC

The adenomatous polyposis coli (APC) gene maps to 5q21 [40], is a tumor suppressor gene and is involved in cell adhesion through its interaction with beta catenin-cadherin proteins. Furthermore, the APC protein binds to the ends of microtubules that form the spindle apparatus during chromosome segregation, and so APC defects may mediate chromosomal instability during metaphase [19].

APC methylation has been examined in multiple studies [16,26,28,31,41,42]. The rate of methylation of APC promoter has varied from 0 to 25% in normal tissue. Higher levels of promoter methylation have been found in normal appearing mucosa derived from EAC specimens [41]. APC methylation in metaplastic tissue ranges from 50 to 95% and goes up to 61 to 100% in dysplastic tissues. Wang et al. [42] also demonstrated in a nested case control study that APC and p16 hypermethylation is predictive of progression to cancer. In fact, another study Brock et al. [31] demonstrated that hypermethylation of APC promoter was a more powerful predictor of survival and tumor recurrence than age or stage.

CDH13

CDH13 (also known as H-cadherin and T-cadherin), is a member of the cadherin gene superfamily and maps to 16q24 [43], a locus that frequently undergoes deletion in human cancers including esophageal carcinoma [44]. Hypermethylation of the CDH13 promoter was studied by Jin et al. [44] in 259 esophageal biopsy specimens by MSP. CDH13 hypermethylation frequency was 0% in normal esophagus but increased during neoplastic progression, rising to 70% in BE, 77.5% in dysplasia and 76.1% in EAC. In contrast, only 5 (19.2%) of 26 esophageal squamous cell carcinoma samples exhibited CDH13 hypermethylation. Notably, in the above study, CDH13 showed a strong relationship to BE segment length.

DAPK1

Death-associated protein kinase1 (DAPK1) maps to the chr9q34.1 locus [45]. DAPK1 encodes a structurally unique 160-kD calmodulin dependent serine-threonine kinase that is a positive mediator of gamma-interferon induced programmed cell death and therefore is a tumor suppressor [33]. In a study done by Kuester et al. [33] hypermethylation of the DAPK promoter was detected in 20% of normal mucosa, 50% of Barrett’s metaplasia, 53% of dysplasia and 60% of adenocarcinomas and resulted in marked decrease in protein expression. In this study, the loss of this protein was significantly associated with advanced depth of tumor invasion and advanced tumor staging. The severity of the reflux esophagitis also correlated significantly with the DAPK promoter hypermethylation.

GPX and GST

Since one of the most clinical risk factors for development of Barrett’s is reflux, 2 authors studied the effect on glutathione S-transferase superfamily (GST) and glutathione peroxidase (GPX) on the development of EAC [25,46]. Normal cells can handle the oxidative stress through intact antioxidative systems. Glutathione S-transferase superfamily (GST) and glutathione peroxidase (GPX) are major cellular antioxidants. GSTs catalyze the conjugation of the tripeptide glutathione to a wide variety of exogenous and endogenous chemicals with electrophilic functional groups thereby neutralizing their electrophilic sites and rendering the products more water soluble. GPXs catalyze the reduction of H₂O₂, organic hydroperoxide and lipid peroxides by reduced glutathione, thereby protecting cells against oxidative damage [46].

Peng et al. [46] systematically analyzed the promoter regions of 23 genes of the glutathione S-transferase (GST) and glutathione peroxidase (GPX) families. Out of the 14 genes that had CPG islands in their promoter regions, promoter DNA hypermethylation and mRNA downregulation was found for GPX3, GPX7, GSTM2, GSTM3 and GSTM5. They then analyzed 159 primary human samples (37 normal, 11 Barrett’s, 11 dysplasia and 100 adenocarcinoms samples using quantitative bisulphite pyrosequencing. GSTM5 showed higher level of methylation in normal tissues and therefore is not presented in the table. The range of methylation for normal tissues ranged from 0% to 8.1%, for metaplastic tissues from 12.5 to 90%, dysplastic tissues from 37.5% to 87.5% and carcinoma from 15% to 69.1%. A significant inverse correlation between DNA methylation and mRNA expression level was shown for GPX3 (p<0.001), GPX7 (p=0.002), GSTM2 (p<0.001) and GSTM5 (p<0.01). Treatment of esophageal cancer cell lines with 5-aza-2′-deoxycytidine led to reversal of the methylation pattern and re-expression of these genes at the mRNA and protein levels. Immunohistochemical analysis of GPX3, GPX7 and GSTM2 on a tissue microarray that contained 75 BACs with normal squamous esophageal samples demonstrated absent to weak staining in tumors (52% for GPX3, 57% for GPX7 and 45% for GSTM2) and a moderate to strong immunostaining in normal samples.

Lee et al. had earlier studied Glutathione Peroxidase-3 (GPx3) alone by using MSP. They detected GPx3 promoter hypermethylation was detected in 16.7% of normal tissues, 81.1% of Barrett’s metaplasia, 82.2% of dysplasia and 88% of carcinoma samples [25].

MGMT

O-Methylguanine-DNA Methytransferase (MGMT) maps to chromosome 10q26 [49] and codes for a DNA repair enzyme that removes methyl or alkyl groups from guanine after chemical modification and therefore protects cells from G to A mutations. This gene has been studied by multiple authors [16,26,31,41,47,48]. Methylation of normal tissues ranged from 1.8% to 54.8%, metaplastic tissues from 25% to 88.9%, dysplastic tissues from 71.4% to 100% and EAC from 23.4% to 78.7%. Kuester et al. also demonstrated MGMT hypermethylation correlated with down expression of protein and mRNA and with progressed stage of disease [48]. Baumann et al. found that MGMT hypermethylation correlated with MGMT protein expression but not p53 status or with patient outcomes [47].

NELL1

The nel-like1 (NELL1) has been mapped to chromosome 11p15 [50], a locus that frequently shows loss of heterozygosity (LOH) in human cancers including colon cancer and EAC [50]. The NELL1 gene encodes a protein kinase C-binding protein that contains six EGF-like domains and belongs to a new class of signaling molecules controlling cell growth and differentiation. The precise roles of NELL1 in physiology and pathophysiology remain unclear although it has been reported that overexpression of NELL1 promotes apoptosis in osteoblasts and this apoptotic activity may be associated with Fas signaling pathway [51]

NELL1 hypermethylation was examined by methylation specific PCR in 259 human esophageal tissues by Jin et al 2007. NELL1 hypermethylation frequency was 0% in normal esophagus but increased early during neoplastic progression, to 41.7% in BE alone, 46.7 in all tissues with Barrett’s, 52.5% in dysplastic tissues and 47.8% in EAC. Treatment of these cells with 5-Aza-dC led to concomitant increases in mRNA expression and reduction in NELL1 methylation levels. Nell1 hypermethylation showed a strong direct relationship with Barrett’s segment length and hypermethylation of NELL1 was significantly associated with shortened survival in stages I-II EAC patients [36]

REPRIMO/RPRM

Reprimo (RPRM) encodes a cytoplasmic protein that belongs to a family of molecules controlled by p53 and is a candidate tumor suppressor gene involved in regulation of p53 mediated cell cycle arrest at the G2 phase of the cell cycle [52]. The Reprimo gene maps to chromosome 2q23 a locus that commonly shows allelic imbalance in human cancers. In the study done by Hamilton et al., Reprimo methylation was found to 0% in normal tissues, 36% in metaplastic tissues, 63.6% in dysplastic tissues and 62.6% in EAC. In addition, within the set of patients with Barrett’s, those with long-segment BE had significantly more Reprimo promoter methylation than those with short-segment disease [35]. They also treated OE33 cell lines with 5-aza and found reduced methylation and increased expression of gene with increased mRNA levels.

P16/CDKN2A

Inactivation of CDKN2A/P16 tumor suppressor gene is one of the most common genetic abnormalities in human neoplasia [53]. The CDKN2A gene located on 9p21, encodes a cell cycle regulatory protein CDK1 that inhibits cyclin-dependant kinases 4 and 6, preventing the phosphorylation of pRb protein and release of transcription factor E2F. This blocks cell cycle progression from G1 to S phase. Alterations of the CKN2A gene lead to its inactivation, resulting in deregulation of cell proliferation and consequent genomic instability [54] Mutations, loss of heterozygosity and hypermethylation are the most frequent modes of inactivation of the gene. Although homozygous deletions of p16 have been reported in several types of malignancies, it has never been detected in esophageal adenocarcinoma, suggesting hypermethylation of the remaining p16 allele as alternative mechanism of inactivation [16].

A study done by Wong et al. 1977 has provided evidence that p16 may be inactivated in esophageal adenocarcinoma with 9p21 LOH by at least two different mechanisms: mutation or promoter hypermethylation.19 out of 21 patients with Barrett’s esophagus (90%) had LOH and 8 of 21 patients (38%) had p16 promoter hypermethylation suggesting that p16 promoter hypermethylation with 9p21 LOH is a common mechanism for inactivation of the p16 gene in premalignant epithelium and esophageal adenocarcinomas [55].

Multiple authors have studied p16/CDKN2A methylation [16,26,28,31,41,42]. The range of methylation in normal tissues varied from 0% to 24.4%, in metaplastic tissues from 7.4% to 27.5%, in dysplastic tissues from 22.2% to 55.0% and EAC from 39.0 % to 59.5%. Wang et al. 2009 did a nested case control study and found that APC and P16 are significantly more methylated in progressors than non progressors [42].

SFRP

Secreted frizzled related protein (SFRP) genes are a new class of tumor suppressor genes that have been found to be methylated in Barrett’s esophagus and esophageal adenocarcinoma [27]. SFRPs are secreted glycoproteins that work as inhibitive modulators of a putative tumorigenic Wnt pathway [56]. Five SFRP genes have been identified and SFRPs 1, 4 and 5 appeared to be more consistently methylated in Barrett’s esophagus and EAC [27].

In the study performed by Zou et al., the promoter regions of SFRP genes were methylated in 73-93% of esophageal cancers and in 73-89% of Barrett’s epithelia. However SFRP methylation was less common (21-32%) in normal appearing epithelia adjacent to Barrett’s and least common (0-13%) in normal esophageal or gastric epithelia from individuals without Barrett’s. SFRP 2 was the only one that showed high levels of methylation in normal tissues. It was observed that SFRP mRNA and protein expression were absent or markedly decreased in Barrett’s esophagus and cancer but generally high in normal epithelia [27]. Based on the study it was concluded that methylation represents a likely mechanism of SFRP gene silencing in Barrett’s esophagus, EAC and methylated SFRP1, 4 and 5 genes represent potential tumor markers for Barrett’s esophagus and its related neoplasia. Smith et al. [41] and Clement et al. [28] also showed similar findings (see Table 1).

SOCS

SOCS-1 is mapped to chromosome 16p13.13 and SOCS-3 is mapped to 17q25.3 [57]. These genes encode a member of the STAT-induced STAT inhibitors (SSIs), also known as suppressors of cytokine signaling (SOCS). SSI family members are cytokine-inducible negative regulators of cytokine signaling.

Hengge et al. studied 19 Barrett’s adenocarcinomas, 56 Barrett’s intraepithelial neoplasms (29 low grade and 27 high grade), 30 Barrett’s mucosa without neoplasia, 20 samples of normal squamous and gastric epithelium and four cell lines [58]. In normal squamous epithelium and normal gastric mucosa, neither SOCS-3 nor SOCS-1 methylation was observed. In Barrett’s mucosa without intraepithelial neoplasia, SOCS-3 methylation occurred in 4/30 cases (13%) whereas SOCS-1 was unmethylated. A hypermethylated SOCS-3 promoter was found in 14/19 Barrett’s adenocarcinomas (74%) and in 20/29 high and 6/27 low grade intraepithelial neoplasms (69% and 22%, respectively). SOCS-1 promoter hypermethylation occurred in 8/19 adenocarcinomas (42%) and in 6/29 high grade and 1/27 low grade intraepithelial neoplasms (21% and 4%, respectively). They concluded that promoter methylation and subsequent transcript downregulation of SOCS-3 transcripts and, to a much lesser extent, SOCS-1 are involved in the multistep carcinogenesis of Barrett’s adenocarcinoma

SST

Somatostatin whose gene is mapped to 3q28 [59] is produced by the thalamus and also by a variety of endocrine and neuroendocrine tissues. In the GI tract, SST regulates endocrine and exocrine secretion, modulates motor activity, and is the primary inhibitor of gastrin-stimulated gastric acid secretion [60]. SST gene promoter methylation was studied by Jin et al. in 260 esophageal biopsy specimens of different histologies using MSP. Incremental increases in the frequency of SST hypermethylation were observed during progression from NE (9%) to BE (70%), HGD (71.4%) and EAC (71.6%), whereas LGD (63.2%) demonstrated a slightly lower frequency of SST hypermethylation than did BE or HGD [60] SST promoter hypermethylation was significantly more frequent in Long segment Barrett’s esophagus (LSBE), which has a higher malignant potential than does short segment Barrett’s esophagus (SSBE). Thus SST methylation represents a molecular correlate of BE segment length, as well as a potential biomarker of the prediction of BE progression. They concluded that hypermethylation of SST promoter was significantly more frequent in the premalignant lesions, metaplastic BE and BE with high-grade dysplasia, as well as in frank EAC and ESCC, than in normal esophageal epithelium.

TAC1

The tachykinin-1 (TAC) gene has been mapped to chromosome 7q21-22 [61] a locus that frequently undergoes loss of heterozygosity in human cancers including esophageal adenocarcinoma [62]. TAC 1 encodes the neuropeptides substance P, neurokinin A, and neuropeptide K, which act through two types of transmembrane G-protein-coupled receptors, denoted neurokinin-1 and neurokinin-2. The precise involvement of TAC 1 in carcinogenesis is not clear although the neuropeptides like substance P have been shown to have proliferative and antiapoptotic effects.

Methylation of the TAC 1 gene promoter was studied in 258 human endoscopic esophageal specimens and 126 plasma samples by Jin et al. by real-time quantitative methylation-specific PCR from patients or tissues at various stages of neoplastic evolution [63]. The frequency of TAC1 hypermethylation increased dramatically from 7.5% in normal esophagus to 55.6% in BE from patients with Barrett’s metaplasia alone, 57.5% in dysplastic Barrett’s esophagus and 61.2% in EAC [63]. The segment length of BEs with hypermethylated TAC 1 promoters were significantly longer than the segment lengths of BEs with unmethylated promoters and it was noted that the frequency of TAC1 hypermethylation was significantly higher in LSBE than in SSBE. Overall patient survival correlated significantly with TAC1 hypermethylation in patients with squamous cell esophageal tumors but not in EAC patients. In addition, the data in the study suggested that circulating hypermethylated TAC1 DNA is associated with the presence of EAC. They concluded that TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of poor prognosis in SCC of the esophagu [63].

TIMP3

The TIMP-3 gene is located on chromosome 22q12.3, a region in which loss of heterozygosity and silencing of the gene due to CpG island hypermethylation is frequently observed in various cancers [64] TIMP-3 is considered a tumor suppressor gene, because TIMP-3 can inhibit tumor growth, angiogenesis, and invasion and can promote apoptosis [65].

TIMP3 methylation was studied by multiple authors including [16,26,28,31,41]. They reported methylation of TIMP3 in normal tissues from 0% to 19.3%, metaplastic from 54.1 % to 87.5%, dysplastic tissues from 71.4 to 77.8% and EAC from 19.5 % to 86.3%.

WIF1

Many authors have established the crucial role of the Wnt signaling pathway in the pathogenesis of many human cancer [18,28,29]. Overexpression of its signaling components as well as the downregulation of its antagonist (SRFP) have also been confirmed along the EAC carcinogenesis sequence [27-29]

Among other Wnt antagonists, WIF-1 also plays an important role. WIF-1 is mapped to 12q14.3 [66]. Clement et al. found that methyaltion of WIF-1 ranged from 0% in normal tissues to 51.6% in metaplastic tissues and 83.3 in EAC. They also showed that WIF-1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF-1 in cell lines where WIF-1 was epigenetically silenced resulted in growth suppression. Restoration of WIF-1 could sensitize the EAC cells to the chemotherapy drug cisplatin. They therefore concluded that silencing of WIF-1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF-1 might be used as a potential new targeted therapy for the treatment of this malignancy [29].

Aberrantly methylated DNA can be used as prognostic biomarker

Jin et al. conducted a multicenter , retrospective, double blinded trail in 2009 with the 8 genes( p16, RUNX3, HPP1, NELL1, TAC1, SST, AKAP12, and CDH13) [67]. They had studied the genes earlier and had established that all these genes predicted progression risk. Using MSP they studied 145 nonprogressors (NPs) and 50 progressors at 2 and 4 years, they found that the 8-marker panel is more objective, quantifiable and possesses higher predictive sensitivity and specificity than do clinical features, including age. This is an example of how the genes above can be used in combination to predict progression risk and as an early disease marker. Such studies can also be used in the future to predict endoscopic surveillance interval since only about 0.5% of patients with Barrett’s progress to EAC.

Aberrant DNA methylation can be detected in the serum

Ultimately the biomarkers will be more useful if they can be detected in the plasma remote from the site of the tumor. Kawakami et al. published a study showing APC DNA methylation in the plasma could be used as a biomarker [34]. Hypermethylated APC DNA was observed in the plasma of 13 (25%) of 52 adenocarcinoma patients and in two (6.3%) of 32 squamous carcinoma patients. High plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival [34]. In the year 2007 Jin et al. measured TAC1 methylation levels in 126 plasma samples from 35 control subjects, 10 additional patients with Ba, 20 with dysplastic Barrett's esophagus, and 61 with EAC. TAC1 hypermethylation frequencies in plasma samples were significantly higher in EAC patients than in control subjects [63]. Hoffmann et al. used APC and DAPK as biomarkers in plasma. Thirty-six of 59 patients (61.0%) with esophageal cancer had detectable levels of methylated DAPK or APC promoter DNA and preoperative detection was significantly associated with an unfavorable prognosis The combination of both markers significantly increased sensitivity and specificity for discriminating between short (<2.5 years) and long survivors [68]. Postoperative APC detection was significantly different if residual tumor was apparent . Therefore some of the genes studied in the paper could be used as a combination to develop a biomarker that can be used remotely from the site of the tumor and be helpful to a clinician in many ways.