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Simple Summary: In the present study, we analyzed gene expression of NEK family members (NEK1-NEK11) in esophageal adenocarcinoma (EAC) and its precancerous condition, Barrett's esophagus (BE), from the TCGA database and eight GEO datasets using a bioinformatics approach. We analyzed genomic alterations of NEKs in EAC samples using cBioPortal For Cancer Genomics and explored the clinical significance of NEKs expression on patients' survival using the Kaplan–Meier Plotter. We validated the findings using qRT-PCR, Western blotting, immunohistochemistry, and immunofluorescence in cell lines from esophagus and primary EAC tissue samples. Our data indicate that upregulation of several NEKs, such as NEK2, NEK3, and NEK7, may be important in EAC. The incidence of esophageal adenocarcinoma (EAC) has risen rapidly during the past four decades, making it the most common type of esophageal cancer in the USA and Western countries. The NEK (Never in mitosis A (NIMA) related kinase) gene family is a group of serine/threonine kinases with 11 members. Aberrant expression of NEKs has been recently found in a variety of human cancers and plays important roles in tumorigenesis, progression, and drug-resistance. However, the expression of the NEKs in EAC and its precancerous condition (Barrett's esophagus, BE) has not been investigated. In the present study, we first analyzed the TCGA and 9 GEO databases (a total of 10 databases in which 8 contain EAC and 6 contain BE) using bioinformatic approaches for NEKs expression in EAC and BE. We identified that several NEK members, such as NEK2 (7/8), NEK3 (6/8), and NEK6 (6/8), were significantly upregulated in EAC as compared to normal esophagus samples. Alternatively, NEK1 was downregulated in EAC as compared to the normal esophagus. On the contrary, genomic alterations of these NEKs are not frequent in EAC. We validated the above findings using qRT-PCR and the protein expression of NEKs in EAC cell lines using Western blotting and in primary EAC tissues using immunohistochemistry and immunofluorescence. Our data suggest that frequent upregulation of NEK2, NEK3, and NEK7 may be important in EAC. [ABSTRACT FROM AUTHOR]

Esophageal adenocarcinoma (EAC), the predominant type of esophageal cancer in the United States, develops through Barrett's esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease, where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator. We detected high NRF2 protein levels in the EAC cell lines and primary tissues. Knockdown of NRF2 significantly enhanced acidic bile salt-induced oxidative stress, DNA damage, and inhibited EAC cell growth. Brusatol, an NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated the NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with cisplatin (CDDP) induced significant lipid peroxidation and ferroptosis, as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. The combination of Brusatol and CDDP significantly inhibited EAC tumor xenograft growth in vivo and confirmed the in vitro data showing ferroptosis as an important mechanism in the tumors treated with Brusatol or Brusatol and CDDP combination. Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC. [ABSTRACT FROM AUTHOR]

Background: Esophageal adenocarcinoma (EAC) is characterized by poor prognosis and low survival rate. Chronic gastroesophageal reflux disease (GERD) is the main risk factor for the development of Barrett's esophagus (BE), a preneoplastic metaplastic condition, and its progression to EAC. Yes-associated protein 1 (YAP1) activation mediates stem-like properties under cellular stress. The role of acidic bile salts (ABS) in promoting YAP1 activation under reflux conditions remains unexplored. Methods: A combination of EAC cell lines, transgenic mice, and patient-derived xenografts were utilized in this study. mRNA expression and protein levels of APE1 and YAP1 were evaluated by qRT-PCR, western blot, and immunohistochemistry. YAP1 activation was confirmed by immunofluorescence staining and luciferase transcriptional activity reporter assay. The functional role and mechanism of regulation of YAP1 by APE1 was determined by sphere formation assay, siRNA mediated knockdown, redox-specific inhibition, and co-immunoprecipitation assays. Results: We showed that YAP1 signaling is activated in BE and EAC cells following exposure to ABS, the mimicry of reflux conditions in patients with GERD. This induction was consistent with APE1 upregulation in response to ABS. YAP1 activation was confirmed by its nuclear accumulation with corresponding up-regulation of YAP1 target genes. APE1 silencing inhibited YAP1 protein induction and reduced its nuclear expression and transcriptional activity, following ABS treatment. Further investigation revealed that APE1-redox-specific inhibition (E3330) or APE1 redox-deficient mutant (C65A) abrogated ABS-mediated YAP1 activation, indicating an APE1 redox-dependent mechanism. APE1 silencing or E3330 treatment reduced YAP1 protein levels and diminished the number and size of EAC spheroids. Mechanistically, we demonstrated that APE1 regulated YAP1 stability through interaction with β-TrCP ubiquitinase, whereas APE1-redox-specific inhibition induced YAP1 poly-ubiquitination promoting its degradation. Conclusion: Our findings established a novel function of APE1 in EAC progression elucidating druggable molecular vulnerabilities via targeting APE1 or YAP1 for the treatment of EAC. [ABSTRACT FROM AUTHOR]

Simple Summary: Esophageal cancer is the 6th most common cause of cancer-related deaths in 2018 worldwide, with a 5-year survival rate of around 20%. This study reported that esophageal adenocarcinoma cancer cells take advantage of unfolded protein response to survival. Our data using both in vitro cancer cell models and in vivo mice models discovered, for the first time, that Aurora kinase A hijacks pro-survival unfolded protein response in esophageal adenocarcinoma, promoting the survival of cancer cells under reflux-mediated stress conditions. Unfolded protein response (UPR) protects malignant cells from endoplasmic reticulum stress-induced apoptosis. We report that Aurora kinase A (AURKA) promotes cancer cell survival by activating UPR in esophageal adenocarcinoma (EAC). A strong positive correlation between AURKA and binding immunoglobulin protein (BIP) mRNA expression levels was found in EACs. The in vitro assays indicated that AURKA promoted IRE1α protein phosphorylation, activating prosurvival UPR in FLO-1 and OE33 cells. The use of acidic bile salts to mimic reflux conditions in patients induced high AURKA and IRE1α levels. This induction was abrogated by AURKA knockdown in EAC cells. AURKA and p-IRE1α protein colocalization was observed in neoplastic gastroesophageal lesions of the L2-IL1b mouse model of Barrett's esophageal neoplasia. The combined treatment using AURKA inhibitor and tunicamycin synergistically induced cancer cell death. The use of alisertib for AURKA inhibition in the EAC xenograft model led to a decrease in IRE1α phosphorylation with a significant reduction in tumor growth. These results indicate that AURKA activates UPR, promoting cancer cell survival during ER stress in EAC. Targeting AURKA can significantly reverse prosurvival UPR signaling mechanisms and decrease cancer cell survival, providing a promising approach for the treatment of EAC patients. [ABSTRACT FROM AUTHOR]

Infection with Helicobacter pylori (H. pylori) is the main risk factor for gastric cancer, a leading cause of cancer-related death worldwide. The oncogenic functions of cyclin-dependent kinase 1 (CDK1) are not fully understood in gastric tumorigenesis. Using public datasets, quantitative real-time PCR, western blot, and immunohistochemical (IHC) analyses, we detect high levels of CDK1 in human and mouse gastric tumors. H. pylori infection induces activation of nuclear factor κB (NF-κB) with a significant increase in CDK1 in in vitro and in vivo models (p < 0.01). We confirm active NF-κB binding sites on the CDK1 promoter sequence. CDK1 phosphorylates and inhibits GSK-3β activity through direct binding with subsequent accumulation and activation of β-catenin. CDK1 silencing or pharmacologic inhibition reverses these effects and impairs tumor organoids and spheroid formation. IHC analysis demonstrates a positive correlation between CDK1 and β-catenin. The results demonstrate a mechanistic link between infection, inflammation, and gastric tumorigenesis where CDK1 plays a critical role. [Display omitted] • Infection with H. pylori is the main risk factor for gastric tumorigenesis • H. pylori infection induces CDK1 expression via an NF-κB-dependent transcription • CDK1 promotes activation of β-catenin signaling and cellular expansion Zhu et al. show induction of CDK1 in response to H. pylori infection via NF-κB-dependent transcription regulation. CDK1-mediated activation of β-catenin signaling, promoting expansion of cancer cells. The results demonstrate a mechanistic link between infection, inflammation, and gastric tumorigenesis where CDK1 plays a critical role. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes (MDS) are a group of hematopoietic malignancies characterized by ineffective hematopoiesis. Recently, we identified MDS-associated microRNAs (miRNAs) that are down-regulated in MDS. This study examines possible explanations for that observed down-regulation of miRNA expression in MDS. Since genomic losses are insufficient to explain the down-regulation of all our MDS-associated miRNAs, we explored other avenues. We demonstrate that these miRNAs are predominantly intragenic, and that, in many cases, they and their host genes are expressed in a similar pattern during myeloid maturation, suggesting their co-regulation. This co-regulation is further supported by the down-regulation of several of the host genes in MDS and increased methylation of the shared promoters of several miRNAs and their respective host genes. These studies identify a role of hypermethylation of miRNA promoters in the down-regulation of MDS-associated miRNAs, unifying research on miRNAs in MDS and epigenetic regulation in MDS into a common pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND MicroRNAs (miRNAs) play critical roles in tumor development and progression. The finding that a single miRNA can regulate hundreds of genes places miRNAs at critical hubs of signaling pathways. For the current study, the authors investigated the miRNA expression profile of gastric adenocarcinomas and compared it with esophageal adenocarcinomas to better identify a unique miRNA signature of gastric adenocarcinoma. METHODS miRNA expression profiles were obtained using 2 different proprietary microarray platforms on primary gastric adenocarcinoma tissue samples. The cross comparison of results identified 17 up-regulated miRNAs and 12 down-regulated miRNAs that overlapped in both platforms. Quantitative real-time polymerase chain reaction was performed for independent validation of a representative set of 8 miRNAs in gastric and esophageal adenocarcinomas compared with normal gastric mucosa or esophageal mucosa, respectively. RESULTS The deregulation of miR-146b-5p, miR-375, miR-148a, miR-31, and miR-451 was associated significantly with gastric adenocarcinomas. Conversely, deregulation of miR-21 (up-regulation) and miR-133b (down-regulation) was detectable in both gastric and esophageal adenocarcinomas. It was noteworthy that miR-200a was significantly down-regulated in gastric adenocarcinoma samples ( P = .04) but was up-regulated in esophageal adenocarcinoma samples ( P = .001). In addition, the expression level of miR-146b-5p displayed a strong correlation with the tumor stage of gastric cancer. CONCLUSIONS Gastric adenocarcinoma displayed a unique miRNA signature that distinguished it from esophageal adenocarcinoma. This specific signature may reflect differences in the etiology and/or molecular signaling in these 2 closely related cancers. The current findings suggest important miRNA candidates that can be investigated for their biological functions and for their possible diagnostic, prognostic, and therapeutic role in gastric adenocarcinoma. Cancer 2013;119:1985-1993. © 2013 American Cancer Society. [ABSTRACT FROM AUTHOR]

LIM domain only 2 ( Lmo2) is frequently deregulated in sporadic and gene therapy-induced acute T-cell lymphoblastic leukemia (T-ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2 expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2 confers clonal growth advantage in T-cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2-Lmo2 transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2 expression. Most impressively, Lmo2 transgenic T-cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in hematopoietic stem cells (HSCs) and early T-cell precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the HSC. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL. S TEM C ELLS 2013;31:882-894 [ABSTRACT FROM AUTHOR]

Obesity is associated with increased markers of oxidative stress. We examined whether oxidative stress is reduced within the first week after Roux-en-Y gastric bypass (RYGB) surgery and could be related to changes in adipose tissue depots. The reactive oxygen species (ROS) marker 8-iso-prostaglandin F2α (8-iso-PGF2α) and activity of antioxidant glutathione peroxidases (GPX) in plasma were compared before and ~1 week after RYGB. The effects of RYGB on subcutaneous adipose tissue and interstitial fluid 8-iso-PGF2α levels and subcutaneous adipose tissue expression of GPX-3 were also assessed. Levels of 8-iso-PGF2α in subcutaneous and visceral adipose tissue were determined. Plasma 8-iso-PGF2α levels decreased (122 ± 75 to 56 ± 15 pg/ml, P = 0.001) and GPX activity increased (84 ± 18 to 108 ± 25 nmol/min/ml, P = 0.003) in the first week post-RYGB. RYGB also resulted in reductions of 8-iso-PGF2α in subcutaneous adipose tissue (1,742 ± 931 to 1,132 ± 420 pg/g fat, P = 0.046) and interstitial fluid (348 ± 118 to 221 ± 83 pg/ml, P = 0.046) that were comparable to plasma (26-33%, P = 0.74). Adipose GPX-3 expression was increased (6.7 ± 4.7-fold, P = 0.004) in the first postoperative week. The improvements in oxidative stress occurred with minimal weight loss (2.4 ± 3.4%, P = 0.031) and elevations in plasma interleukin-6 (18.0 ± 46.8 to 28.0 ± 58.9 pg/ml, P = 0.004). Subcutaneous and visceral adipose tissues express comparable 8-iso-PGF2α levels (1,204 ± 470 and 1,331 ± 264 pg/g fat, respectively; P = 0.34). These data suggest that RYGB affects adipose tissue leading to the restoration of adipose redox balance within the first postoperative week and that plasma 8-iso-PGF2α is primarily derived from subcutaneous adipose tissue. [ABSTRACT FROM AUTHOR]

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 2372 to 2306 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 (24 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from 2139 to 249 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, 2log10(FDR).3.0]. The DNA methylation levels from 2127 to 28 CpG sites showed the strongest correlation with MT3 gene expression (r =20.4, P<0.0001). Moreover, the DNA hypermethylation from 2127 to 28 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. [ABSTRACT FROM AUTHOR]

Background: Somatostatin (SST), a primary inhibitor of gastrin-stimulated gastric acid secretion, has potent antitumor and anti-secretory activity in several human cancers.Aims: This study was performed to investigate the SST gene expression levels and possible epigenetic mechanisms that regulate expression of SST in gastric adenocarcinomas.Methods: Quantitative real-time (RT)-PCR and quantitative bisulfite pyrosequencing were used to study primary gastric cancer tissue samples and cell lines.Results: Quantitative RT-PCR analysis revealed down-regulation of the SST transcript in 93% of gastric carcinoma samples (30/32), compared with 21 normal samples (P<0.001). Because of the presence of a large CpG island in the SST promoter, we next examined its promoter DNA methylation levels by use of quantitative bisulfite pyrosequencing. The results revealed a significant increase in SST promoter DNA methylation in tumor samples compared with normal samples (P<0.05). Promoter DNA hypermethylation and silencing of SST was also detected in seven gastric cancer cell lines that we tested. To confirm the role of promoter DNA methylation as an epigenetic mechanism regulating SST expression, AGS gastric cancer cells were treated with 5-Aza-dc. This treatment led to reduction of promoter DNA methylation levels of SST accompanied by restoration of its mRNA expression.Conclusions: Our results indicate that promoter DNA methylation levels play a critical role in regulating SST expression in gastric cancer. This finding provides a foundation for further studies on the role of SST in gastric carcinogenesis and its potential as a biomarker for gastric cancers. [ABSTRACT FROM AUTHOR]

The article focuses on a study which examined the epigenetic and genetic mechanisms that regulate checkpoint with forkhead-associated domain and RING-finger domain (CHFR) expression in esophageal adenocarcinoma (EACs). Study authors analyzed the promoter DNA hypermethylation of CHFR using bisulfite pyrosequencing technology. They found that epigenetic and genetic mechanisms are involved in CHFR silencing in patients with EACs.

Simple Summary: Esophageal adenocarcinoma has become a major clinical challenge in the western world due to its rapid increasing incidence and poor overall prognosis. Understanding the molecular events of its tumorigenesis is the key to better diagnosis and development of better therapeutic strategies. In the current study we aimed to identify epigenetic alteration targets in esophageal adenocarcinoma. We focused on a candidate gene, NDRG4 (N-myc downregulated gene 4). We found that NDRG4 was frequent downregulated in esophageal adenocarcinoma through DNA hypermethylation of its promoter region. Re-expression of NRDG4 in cancer cells significantly suppressed tumor growth via inhibition of cell proliferation. These results will improve our understanding on how dysfunction of NDRG4 contributes to esophageal adenocarcinoma. DNA hypermethylation of NDRG4 may be a useful biomarker in clinical monitoring of esophageal adenocarcinoma patients. The incidence of esophageal adenocarcinoma (EAC) has been rising dramatically in the past few decades in the United States and Western world. The N-myc downregulated gene 4 (NDRG4) belongs to the human NDRG family. In this study, we aimed to identify the expression levels, regulation, and functions of NDRG4 in EAC. Using an integrative epigenetic approach, we identified genes showing significant downregulation in EAC and displaying upregulation after 5-Aza-deoxycitidine. Among these genes, likely to be regulated by DNA methylation, NDRG4 was among the top 10 candidate genes. Analyses of TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) data sets and EAC tissue samples demonstrated that NDRG4 was significantly downregulated in EAC (p < 0.05). Using Pyrosequencing technology for quantification of DNA methylation, we detected that NDRG4 promoter methylation level was significantly higher in EAC tissue samples, as compared to normal esophagus samples (p < 0.01). A strong inverse correlation between NDRG4 methylation and its gene expression levels (r = −0.4, p < 0.01) was observed. Treatment with 5-Aza restored the NDRG4 expression, confirming that hypermethylation is a driving force for NDRG4 silencing in EAC. Pathway and gene set enrichment analyses of TCGA data suggested that NDRG4 is strongly associated with genes related to cell cycle regulation. Western blotting analysis showed significant downregulation of Cyclin D1, CDK4 and CDK6 in EAC cells after overexpression of NDRG4. Functionally, we found that the reconstitution of NDRG4 resulted in a significant reduction in tumor cell growth in two-dimensional (2D) and three-dimensional (3D) organotypic culture models and inhibited tumor cell proliferation as indicated by the EdU (5-ethynyl-2′-deoxyuridine) proliferation assay. [ABSTRACT FROM AUTHOR]

Aurora kinase A (AURKA) is frequently overexpressed in several cancers. miRNA sequencing and bioinformatics analysis indicated significant downregulation of miR-4715-3p. We found that miR-4715-3p has putative binding sites on the 3UTR region of AURKA. Upper gastrointestinal adenocarcinoma (UGC) tissue samples and cell models demonstrated significant overexpression of AURKA with downregulation of miR-4715-3p. Luciferase reporter assays confirmed binding of miR-4715-3p on the 3UTR region of AURKA. miR-4715-3p mediated a reduction in AURKA levels leading to G2/M delay, chromosomal polyploidy, and cell death. We also detected a remarkable decrease in GPX4, an inhibitor of ferroptosis, with an increase in cleaved PARP and caspase-3. Inhibition of AURKA using siRNA produced similar results, suggesting a possible link between AURKA and GPX4. Analysis of UGC samples and cell models demonstrated increased methylation levels of several CpG nucleotides upstream of miR-4715-3p. 5-Aza-2′-deoxycytidine induced demethylation of several CpG nucleotides, restoring miR-4715-3p expression, leading to downregulation of AURKA. In conclusion, our data identified a novel epigenetic mechanism mediating silencing of miR-4715-3p and induction of AURKA in UGCs. Inhibition of AURKA or reconstitution of miR-4715-3p inhibited GPX4 and induced cell death, suggesting a link between AURKA and ferroptosis. [ABSTRACT FROM AUTHOR]

TFF1, a secreted protein, plays an essential role in keeping the integrity of gastric mucosa and its barrier function. Loss of TFF1 expression in the TFF1-knockout (KO) mouse leads to a pro-inflammatory phenotype with a cascade of gastric lesions that include low-grade dysplasia, high-grade dysplasia, and adenocarcinomas. In this study, we demonstrate nuclear localization of p-STATY705, with significant overexpression of several STAT3 target genes in gastric glands from the TFF1-KO mice. We also show frequent loss of TFF1 with nuclear localization of STAT3 in human gastric cancers. The reconstitution of TFF1 protein in human gastric cancer cells and 3D gastric glands organoids from TFF1-KO mice abrogates IL6-induced nuclear p-STAT3Y705 expression. Reconstitution of TFF1 inhibits IL6-induced STAT3 transcription activity, suppressing expression of its target genes. TFF1 blocks IL6Rα-GP130 complex formation through interfering with binding of IL6 to its receptor IL6Rα. These findings demonstrate a functional role of TFF1 in suppressing gastric tumorigenesis by impeding the IL6-STAT3 pro-inflammatory signaling axis. Trefoil factor 1 (TFF1) is a protein secreted by the gastric mucosa that protects against gastric tumourigenesis. Here, the authors show that TFF1 inhibits the oncogenic inflammatory response and IL-6-mediated STAT3 activation by interfering with the binding of IL6 to its receptor IL6Rα. [ABSTRACT FROM AUTHOR]

Background: The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks. Methods: Real-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage. Results: Our results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett's cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure. Conclusions: IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]