TianLing Hu

Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells.

Objective Exposure of the oesophageal mucosa to gastric acid and bile acids leads to the accumulation of reactive oxygen species (ROS), a known risk factor for Barretts oesophagus and progression to oesophageal adenocarcinoma (OAC). This study investigated the functions of glutathione peroxidase 7 (GPX7), frequently silenced in OAC, and its capacity in regulating ROS and its associated oxidative DNA damage. Design Using in-vitro cell models, experiments were performed that included glutathione peroxidase (GPX) activity, Amplex UltraRed, CM-H2DCFDA, Annexin V, 8-oxoguanine, phospho-H2A.X, quantitative real-time PCR and western blot assays. Results Enzymatic assays demonstrated limited GPX activity of the recombinant GPX7 protein. GPX7 exhibited a strong capacity to neutralise hydrogen peroxide (H2O2) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barretts oesophagus cells, BAR-T and CP-A led to resistance to H2O2-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H2O2, intracellular ROS, oxidative DNA damage and double-strand breaks, compared with controls (p<0.01). In addition, these cells demonstrated lower levels of ROS signalling, indicated by reduced phospho-JNK (Thr183/Tyr185) and phospho-p38 (Thr180/Tyr182), and demonstrated lower levels of apoptosis following the exposure to acidic bile acids or H2O2-induced oxidative stress. The knockdown of endogenous GPX7 in immortalised oesophageal squamous epithelial cells (HET1A) confirmed the protective functions of GPX7 against pH4 bile acids by showing an increase in the levels of H2O2, intracellular ROS, oxidative DNA damage, double-strand breaks, apoptosis, and ROS-dependent signalling (p<0.01). Conclusion The dysfunction of GPX7 in oesophageal cells increases the levels of ROS and oxidative DNA damage, which are common risk factors for Barretts oesophagus and OAC.

Silencing of Glutathione Peroxidase 3 through DNA Hypermethylation Is Associated with Lymph Node Metastasis in Gastric Carcinomas

Gastric cancer remains the second leading cause of cancer-related death in the world. H. pylori infection, a major risk factor for gastric cancer, generates high levels of reactive oxygen species (ROS). Glutathione peroxidase 3 (GPX3), a plasma GPX member and a major scavenger of ROS, catalyzes the reduction of hydrogen peroxide and lipid peroxides by reduced glutathione. To study the expression and gene regulation of GPX3, we examined GPX3 gene expression in 9 gastric cancer cell lines, 108 primary gastric cancer samples and 45 normal gastric mucosa adjacent to cancers using quantitative real-time RT-PCR. Downregulation or silencing of GPX3 was detected in 8 of 9 cancer cell lines, 83% (90/108) gastric cancers samples, as compared to non-tumor adjacent normal gastric samples (P<0.0001). Examination of GPX3 promoter demonstrated DNA hypermethylation (≥10% methylation level determined by Bisulfite Pyrosequencing) in 6 of 9 cancer cell lines and 60% of gastric cancer samples (P = 0.007). We also detected a significant loss of DNA copy number of GPX3 in gastric cancers (P<0.001). Treatment of SNU1 and MKN28 cells with 5-Aza-2′ Deoxycytidine restored the GPX3 gene expression with a significant demethylation of GPX3 promoter. The downregulation of GPX3 expression and GPX3 promoter hypermethylation were significantly associated with gastric cancer lymph node metastasis (P = 0.018 and P = 0.029, respectively). We also observed downregulation, DNA copy number losses, and promoter hypermethylation of GPX3 in approximately one-third of tumor-adjacent normal gastric tissue samples, suggesting the presence of a field defect in areas near tumor samples. Reconstitution of GPX3 in AGS cells reduced the capacity of cell migration, as measured by scratch wound healing assay. Taken together, the dysfunction of GPX3 in gastric cancer is mediated by genetic and epigenetic alterations, suggesting impairment of mechanisms that regulate ROS and its possible involvement in gastric tumorigenesis and metastasis.

Location-Specific Epigenetic Regulation of the Metallothionein 3 Gene in Esophageal Adenocarcinomas

Background Metallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs). Methods and Results Using quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from −372 to −306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (−4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from −139 to −49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, −log10(FDR)>3.0]. The DNA methylation levels from −127 to −8 CpG sites showed the strongest correlation with MT3 gene expression (r = −0.4, P<0.0001). Moreover, the DNA hypermethylation from −127 to −8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks. Conclusion In summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

Glutathione peroxidase 7 has potential tumour suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma

Objective To investigate the potential tumour suppressor functions of glutathione peroxidase 7 (GPX7) and examine the interplay between epigenetic and genetic events in regulating its expression in oesophageal adenocarcinomas (OAC). Design In vitro and in vivo cell models were developed to investigate the biological and molecular functions of GPX7 in OAC. Results Reconstitution of GPX7 in OAC cell lines, OE33 and FLO-1, significantly suppressed growth as shown by the growth curve, colony formation and EdU proliferation assays. Meanwhile, GPX7-expressing cells displayed significant impairment in G1/S progression and an increase in cell senescence. Concordant with the above functions, Western blot analysis displayed higher levels of p73, p27, p21 and p16 with a decrease in phosphorylated retinoblastoma protein (RB), indicating its increased tumour suppressor activities. On the contrary, knockdown of GPX7 in HET1A cells (an immortalised normal oesophageal cell line) rendered the cells growth advantage as indicated with a higher EdU rate, lower levels of p73, p27, p21 and p16 and an increase in phosphorylated RB. We confirmed the tumour suppressor function in vivo using GPX7-expressing OE33 cells in a mouse xenograft model. Pyrosequencing of the GPX7 promoter region (−162 to +138) demonstrated location-specific hypermethylation between +13 and +64 in OAC (69%, 54/78). This was significantly associated with the downregulation of GPX7 (p<0.01). Neither mutations in the coding exons of GPX7 nor DNA copy number losses were frequently present in the OAC examined (<5%). Conclusions Our data suggest that GPX7 possesses tumour suppressor functions in OAC and is silenced by location-specific promoter DNA methylation.

Loss of glutathione peroxidase 7 promotes TNF-α-induced NF-κB activation in Barrett’s carcinogenesis

Esophageal adenocarcinoma (EAC) is a classic example of inflammation-associated cancer, which develops through GERD (gastroesophageal reflux disease)-Barretts esophagus (BE)-dysplasia-adenocarcinoma sequence. The incidence of EAC has been rising rapidly in the USA and Western countries during the last few decades. The functions of glutathione peroxidase 7 (GPX7), an antioxidant enzyme frequently silenced during Barretts tumorigenesis, remain largely uncharacterized. In this study, we investigated the potential role of GPX7 in regulating nuclear factor-kappaB (NF-κB) activity in esophageal cells. Western blot analysis, immunofluorescence and luciferase reporter assay data indicated that reconstitution of GPX7 expression in CP-A (non-dysplastic BE cells) and FLO-1 (EAC cells) abrogated tumor necrosis factor-α (TNF-α)-induced NF-κB transcriptional activity (P < 0.01) and nuclear translocation of NF-κB-p65 (P = 0.01). In addition, we detected a marked reduction in phosphorylation levels of components of NF-κB signaling pathway, p-p65 (S536), p-IκB-α (S32) and p-IKKα/β (S176/180), as well as significant suppression in induction of NF-κB target genes [TNF-α, interleukin (IL)-6, IL-8, IL-1β, CXCL-1 and CXCL-2] following treatment with TNF-α in GPX7-expressing FLO-1 cells as compared with control cells. We validated these effects by knockdown of GPX7 expression in HET1A (normal esophageal squamous cells). We found that GPX7-mediated suppression of NF-κB is independent of reactive oxygen species level and GPX7 antioxidant function. Further mechanistic investigations demonstrated that GPX7 promotes protein degradation of TNF-receptor 1 (TNFR1) and TNF receptor-associated factor 2 (TRAF2), suggesting that GPX7 modulates critical upstream regulators of NF-κB. We concluded that the loss of GPX7 expression is a critical step in promoting the TNF-α-induced activation of proinflammatory NF-κB signaling, a major player in GERD-associated Barretts carcinogenesis.

Glutathione Peroxidase 7 Suppresses Bile Salt-Induced Expression of Pro-Inflammatory Cytokines in Barrett's Carcinogenesis.

Esophageal adenocarcinoma (EAC) is the most frequent malignancy in the esophagus in the US and its incidence has been rising rapidly in the past few decades. Chronic gastroesophageal reflux disease (GERD), where the esophageal epithelium is abnormally exposed to acid and bile salts, is a pro-inflammatory condition that is the main risk factor for the development of Barretts esophagus (BE) and its progression to EAC. Glutathione peroxidase 7 (GPX7) is frequently silenced through DNA hypermethylation during Barretts tumorigenesis. In this study, we investigated the role of GPX7 in regulating the bile salts-induced inflammatory signaling in Barretts carcinogenesis. Using quantitative real-time PCR (qRT-PCR), we demonstrated a significant induction in the expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) and chemokines (CXCL-1 and CXCL-2) in esophageal cells after exposure to acidic (pH4) or neutral (pH7) bile salts. Western blot analysis showed that exposure to acidic and neutral bile salts increased p-NF-κB-p65 (S536) protein levels independent of ROS. Reconstitution of GPX7 expression in EAC cells abolished the increase of p-p65 (S536) protein levels and mRNA expression of cytokines and chemokines upon treatment with acidic and neutral bile salts. Examination of human primary EAC tissues by qRT-PCR demonstrated significant overexpression of cytokines (TNF-α, IL-1β and IL-8) in EAC samples, as compared to normal samples, with significant inverse correlation with GPX7 expression level. Taken together, the loss of GPX7 expression promotes bile salt-induced activation of pro-inflammatory cytokines and chemokines; important contributors to GERD-associated Barretts carcinogenesis.

Regulation of Desmocollin3 Expression by Promoter Hypermethylation is Associated with Advanced Esophageal Adenocarcinomas

BACKGROUND: Desmocollin3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and plays an important role in tumor invasion and metastasis. In this study, we investigated the epigenetic mechanism that regulates DSC3 expression in esophageal adenocarcinomas (EACs). METHODS: Expression of DSC3 was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The promoter DNA methylation level of DSC3 was examined using quantitative bisulfite pyrosequencing. RESULTS: The qRT-PCR analysis demonstrated significant down-regulation of the DSC3 mRNA levels in human EAC cell lines and tissue samples (P<.001). In addition, the EAC cell lines and tumor samples have aberrant promoter hypermethylation as compared to normal esophageal samples (P<.001). DSC3 promoter hypermethylation (>10% methylation level) was detected in 97.5% (39/40) of EAC samples whereas none of the normal tissue samples showed hypermethylation (P<.0001). There was a significant inverse correlation between promoter DNA methylation levels and mRNA expression folds for DSC3 (coefficient r=-0.685, P<.0001). Treatment of FLO-1 and SKGT4 EAC cells with 5-Aza-deoxytidine led to a significant reduction in the promoter DNA methylation levels with restoration of the DSC3 expression, suggesting that promoter DNA methylation is a key epigenetic mechanism regulating DSC3 expression. High DSC3 promoter DNA methylation levels were significantly correlated with advanced tumor stage (P<.001) and lymph node metastasis (P<.001). CONCLUSION: Taken together, our results demonstrate that epigenetic silencing of DSC3 is a frequent finding in EAC that is possibly associated with advanced stages.

Integrated molecular analysis reveals complex interactions between genomic and epigenomic alterations in esophageal adenocarcinomas

The incidence of esophageal adenocarcinoma (EAC) is rapidly rising in the United States and Western countries. In this study, we carried out an integrative molecular analysis to identify interactions between genomic and epigenomic alterations in regulating gene expression networks in EAC. We detected significant alterations in DNA copy numbers (CN), gene expression levels, and DNA methylation profiles. The integrative analysis demonstrated that altered expression of 1,755 genes was associated with changes in CN or methylation. We found that expression alterations in 84 genes were associated with changes in both CN and methylation. These data suggest a strong interaction between genetic and epigenetic events to modulate gene expression in EAC. Of note, bioinformatics analysis detected a prominent K-RAS signature and predicted activation of several important transcription factor networks, including β-catenin, MYB, TWIST1, SOX7, GATA3 and GATA6. Notably, we detected hypomethylation and overexpression of several pro-inflammatory genes such as COX2, IL8 and IL23R, suggesting an important role of epigenetic regulation of these genes in the inflammatory cascade associated with EAC. In summary, this integrative analysis demonstrates a complex interaction between genetic and epigenetic mechanisms providing several novel insights for our understanding of molecular events in EAC.

Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer

Gastric cancer (GC) is one of the most common cancers in the world, and remains the third leading cause of cancer-related deaths worldwide. Glutathione peroxidase 7 (GPX7) is a member of GPX family which is downregulated in some cancer types. In this study, we investigated the expression, regulation, and molecular function of GPX7 in gastric cancer using 2D and 3D in vitro models and de-identified human tissue samples. Quantitative real-time RT-PCR, immunofluorescence, Western blot, 3D organotypic cultures, and pyrosequencing assays were used. We detected downregulation of GPX7 in all 7 gastric cancer cell lines that we tested and in approximately half (22/45) of human gastric cancer samples, as compared to histologically normal gastric tissues. Quantitative bisulfite pyrosequencing methylation analysis demonstrated DNA hypermethylation (> 10% methylation level) of GPX7 promoter in all 7 gastric cancer cell lines and in 56% (25/45) of gastric cancer samples, as compared to only 13% (6/45) in normal samples (p < 0.0001). Treatment of AGS and SNU1 cells with 5-Aza-2′-deoxycytidine led to a significant demethylation of GPX7 promoter and restored the expression of GPX7. In vitro assays showed that reconstitution of GPX7 significantly suppressed gastric cancer cell growth in both 2D and 3D organotypic cell culture models. This growth suppression was associated with inhibition of cell proliferation and induction of cell death. We detected significant upregulation of p27 and cleaved PARP and downregulation of Cyclin D1 upon reconstitution of GPX7. Taken together, we conclude that epigenetic silencing of GPX7 could play an important role in gastric tumorigenesis and progression.

Abstract 5482: Constitutive overexpression of nrf2 in esophageal adenocarcinoma protects cancer cells from bile salts-induced DNA damage and favors cancer cell survival

Background: Esophageal adenocarcinoma (EAC) is the major type of malignant cancer of esophagus in the USA. The incidence rate has increased 4-10% per year among men since 1976 in the USA, more rapidly than for any other type of cancer. EAC is known to originate from premalignant Barrett’s esophagus (BE) through BE-dysplasia-EAC process. Normal cells have intact anti-oxidative mechanisms, among which NFE2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular response to various stimuli. Cancer cells have high levels of reactive oxygen species and oxidative stress due to activation of oncogenes, inflammatory microenvironment, and dysfunction of anti-oxidative mechanisms. However, the role of NRF2 in Barrett’s related esophageal carcinogenesis is barely known. Methods and Results: We have found that Nrf2 protein expression is significantly upregulated in EAC cell lines as compared to BE cell lines (CPA, BAR10T) and normal esophageal squamous cell lines. We detected a similar overexpression of NRF2 in primary EAC tissue samples. Using luciferase reporter assay, we demonstrated significant induction of NRF2 transcription activity in EAC cells in response to exposure to acidic bile acids, as compared to controls (P Citation Format: Dunfa Peng, Tianling Hu, Shoumin Zhu, Wael El-Rifai. Constitutive overexpression of nrf2 in esophageal adenocarcinoma protects cancer cells from bile salts-induced DNA damage and favors cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5482. doi:10.1158/1538-7445.AM2017-5482