BACKGROUND-The accumulation of reactive oxygen species and subsequent oxidative DNA damage underlie the development of Barrett's esophagus (BE) and its progression to Barrett's dysplasia (BD) and adenocarcinoma (BAC).

P ancreatic cancer is a devastating disease with a very poor prognosis, with a 5-year survival rate of < 3%, and is the fourth or fifth largest cause of cancer-related death worldwide.(1,2)Because ductal carcinomas frequently emerge in pancreases damaged by chronic pancreatitis, (3) at least a proportion of peripheral pancreatic duct epithelia with an inflammatory background may be at the precancerous stage, even though they may show no remarkable histological findings. Recently, Hruban et al.(4) suggested a new nomenclature and classification system for pancreatic intraepithelial neoplasia (PanIN) as a precancerous lesion, and proposed a model of progression from PanIN to ductal carcinoma.(5) Elucidation of genetic and epigenetic alterations in such precancerous conditions and ductal carcinomas showing various clinicopathological features would contribute to a better understanding of the molecular basis of multistage pancreatic carcinogenesis.DNA methylation plays an important role in transcriptional regulation, chromatin remodeling and genomic stability.(6) Overall DNA hypomethylation and regional DNA hypermethylation are commonly observed in various tumors, including pancreatic cancers. (7,8) Furthermore, accumulating evidence suggests that aberrant DNA methylation is involved even in the early and precancerous stages of human carcinogenesis.(9-15) DNA methyltransferase 1 (DNMT1) is the major human DNMT (16) and increased levels of its mRNA and protein expression have been reported in several human precancerous conditions and cancers. (17)(18)(19)(20)(21)(22) We have reported previously that DNMT1 protein overexpression precedes an increase in the proliferating cell nuclear antigen labeling index in precancerous conditions of the urinary bladder, (21) and is significantly correlated with poorer differentiation of liver (19) and stomach (22) cancers and a poor prognosis in patients with liver cancer.However, to our knowledge, there are no reported data on the expression of DNMT1 at both the mRNA and protein levels in pancreatic cancers. In this study we carried out an immunohistochemical analysis of DNMT1 expression in a large series of precancerous conditions and ductal carcinomas of the pancreas to evaluate its significance in multistage pancreatic carcinogenesis. Materials and MethodsPatients and samples. A total of 48 peripheral pancreatic duct epithelia showing no remarkable histological findings without an inflammatory background (DE), 54 peripheral pancreatic duct epithelia with an inflammatory background (DEI, such ducts were surrounded by infiltrating lymphocytes) and 188 pancreatic intraepithelial neoplasias (PanIN; 50 PanIN IA, 126 PanIN IB and 12 PanIN II) were obtained from surgical specimens resected from 100 patients at the National Cancer Center Hospital, Tokyo, between 1997 and. Invasive ductal carcinomas from this cohort frequently showed histological heterogeneity (e.g. well, moderately or poorly differentiated adenocarcinoma components were simultaneously observed even in tissue sections fr...

Background & Aims Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The Aurora kinase A gene (AURKA) is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis. Methods We used quantitative real-time reverse transcription PCR, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1−/− mice, growth of tumor xenografts, and human tissues. Results We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1−/− mice (r = 0.62; P=.0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of NFκBp65 in human gastric cancer samples and mouse epithelial cells, suppressed NFκB reporter activity, and reduced the expression of NFκB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1−/− mice. AURKA was found to regulate NFκB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucuosa of patients had increased levels of AURKA protein and nuclear NFκB, compared with healthy gastric tissue. Conclusions In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be upregulated during chronic inflammation to promote activation of NFκB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.

Purpose Suppression of P53 transcriptional function mediates poor therapeutic response in cancer patients. AURKA and HDM2 are negative regulators of P53. Herein, we examined the role of AURKA in regulating HDM2 and its subsequent effects on P53 apoptotic function in gastric cancer (GC). Experimental design Primary tumors and in vitro GC cell models with overexpression or knockdown of AURKA were used. The role of AURKA in regulating HDM2 and cell survival coupled with P53 expression and activity were investigated. Results Overexpression of AURKA enhanced HDM2 protein level; conversely, knockdown of endogenous AURKA decreased expression of HDM2 in AGS and SNU-1 cells. Dual co-immunoprecipitaion assay data indicated that AURKA was associated with HDM2 in a protein complex. The in vitro kinase assay using recombinant AURKA and HDM2 proteins followed by co-immunoprecipitaion revealed that AURKA directly interacts and phosphorylates HDM2 protein in vitro. The activation of HDM2 by AURKA led to induction of P53 ubiquitination and attenuation of cisplatin-induced activation of P53 in gastric cancer cells. Inhibition of AURKA using an investigational small molecule specific inhibitor, alisertib, decreased HDM2 protein level and induced P53 transcriptional activity. These effects markedly decreased cell survival in vitro and xenograft tumor growth in vivo. Notably, analysis of immunohistochemistry on tissue microarrays revealed significant overexpression of AURKA and HDM2 in human GC samples (P<0.05). Conclusion Collectively, our novel findings indicate that AURKA promotes tumor growth and cell survival through regulation of HDM2-induced ubiquitination and inhibition of P53.

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 and Early T-cell Precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the hematopoietic stem cell. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.

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.

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-STAT Y705 , 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-STAT3 Y705 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.

Objective We investigated the potential tumor suppressor functions of glutathione peroxidase 7 (GPX7) and examined 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, led to significant growth suppression as demonstrated by 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 RB; indicating its increased tumor suppressor activities. On the contrary, knockdown of GPX7 in HET1A cells (an immortalized normal esophageal 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 tumor 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 OACs (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 present in the OACs examined (<5%). CONCLUSIONS Our data suggest that GPX7 possesses tumor suppressor functions in OAC and is silenced by location-specific promoter DNA methylation.