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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]

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]

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]

Here, we designed, synthesized and characterized three new cyclometalated Ru(II) complexes, [Ru(phen) 2 (1-(4-Ph-Ph)-IQ)]+ (phen = 1,10-phenanthroline, IQ = isoquinoline, RuIQ9), [Ru(phen) 2 (1-(4-Ph-Ph)-7-OCH 3 -IQ)]+ (RuIQ10), and [Ru(phen) 2 (1-(4-Ph-Ph)-6,7-(OCH 3) 2 -IQ)]+ (RuIQ11). The cytotoxicity experiments conducted on both 2D and 3D multicellular tumor spheroids (MCTSs) indicated that complexes RuIQ9–11 exhibited notably higher cytotoxicity against A549 and A549/DDP cells when compared to the ligands and precursor compounds as well as clinical cisplatin. Moreover, the Ru(II) complexes displayed low toxicity when tested on normal HBE cells in vitro and exposed to zebrafish embryos in vivo. In addition, complexes RuIQ9–11 could inhibit A549 and A549/DDP cell migration and proliferation by causing cell cycle arrest, mitochondrial dysfunction, and elevating ROS levels to induce apoptosis in these cells. Mechanistic studies revealed that RuIQ9–11 could suppress the expression of Nrf2 and its downstream antioxidant protein HO-1 by inhibiting Nrf2 gene transcription in drug-resistant A549/DDP cells. Simultaneously, they inhibited the expression of efflux proteins MRP1 and p-gp in drug-resistant cells, ensuring the accumulation of the complexes within the cells. This led to an increase in intracellular ROS levels in drug-resistant cells, ultimately causing damage and cell death, thus overcoming cisplatin resistance. More importantly, RuIQ11 could effectively inhibit the migration and proliferation of drug-resistant cells within zebrafish, addressing the issue of cisplatin resistance. Accordingly, the prepared Ru(II) complexes possess significant potential for development as highly effective and low-toxicity lung cancer therapeutic agents to overcome cisplatin resistance. Three novel cyclometalated Ru(II) complexes RuIQ9–11 could suppress the expression of Nrf2 and its downstream antioxidant protein HO-1 by inhibiting Nrf2 gene transcription in A549/DDP cells, ultimately causing cell death. RuIQ9–11 also exerted a prominent inhibitory effect on 3D MCTSs of A549 and A549/DPP cells. RuIQ11 could effectively inhibit the migration and proliferation of A549/DPP cells within zebrafish. [Display omitted] • Three new cyclometalated Ru(II) complexes were synthesized and characterized. • RuIQ9–11 exhibited relative low toxicity on both normal HBE cells in vitro and zebrafish embryos in vivo. • RuIQ9–11 could suppress the expression of Nrf2 and its downstream antioxidant protein HO-1, ultimately causing cell death. • RuIQ9–11 exerted a prominent inhibitory effect on 3D multicellular tumor spheroid model. • RuIQ11 could effectively inhibit the migration and proliferation of drug-resistant cells within zebrafish. [ABSTRACT FROM AUTHOR]

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]