Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma.

OBJECTIVE: Oesophageal adenocarcinoma (EAC) arises in the setting of Barrett's oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic bile salts, the mimicry of reflux, in activation of NOTCH signaling in EAC. DESIGN: This study used public databases, EAC cell line models, L2-IL1ß transgenic mouse model and human EAC tissue samples to identify mechanisms of NOTCH activation under reflux conditions. RESULTS: Analysis of public databases demonstrated significant upregulation of NOTCH signaling components in EAC. In vitro studies demonstrated nuclear accumulation of active NOTCH1 cleaved fragment (NOTCH intracellular domain) and upregulation of NOTCH targets in EAC cells in response to reflux conditions. Additional investigations identified DLL1 as the predominant ligand contributing to NOTCH1 activation under reflux conditions. We discovered a novel crosstalk between APE1 redox function, reflux-induced inflammation and DLL1 upregulation where NF-κB can directly bind to and induce the expression of DLL1. The APE1 redox function was crucial for activation of the APE1-NF-κB-NOTCH axis and promoting cancer cell stem-like properties in response to reflux conditions. Overexpression of APE1 and DLL1 was detected in gastro-oesophageal junctions of the L2-IL1ß transgenic mouse model and human EAC tissue microarrays. DLL1 high levels were associated with poor overall survival in patients with EAC. CONCLUSION: These findings underscore a unique mechanism that links redox balance, inflammation and embryonic development (NOTCH) into a common pro-tumorigenic pathway that is intrinsic to EAC cells.

Multivalent tyrosine kinase inhibition promotes T cell recruitment to immune-desert gastric cancers by restricting epithelial-mesenchymal transition via tumour-intrinsic IFN-γ signalling.

OBJECTIVE: Gastric cancer (GC) ranks fifth in incidence and fourth for mortality worldwide. The response to immune checkpoint blockade (ICB) therapy in GC is heterogeneous due to tumour-intrinsic and acquired immunotherapy resistance. We developed an immunophenotype-based subtyping of human GC based on immune cells infiltration to develop a novel treatment option. DESIGN: A algorithm was developed to reclassify GC into immune inflamed, excluded and desert subtypes. Bioinformatics, human and mouse GC cell lines, syngeneic murine gastric tumour model, and CTLA4 blockade were used to investigate the immunotherapeutic effects by restricting receptor tyrosine kinase (RTK) signalling in immune desert (ICB-resistant) type GC. RESULTS: Our algorithm restratified subtypes of human GC in public databases and showed that immune desert-type and excluded-type tumours are ICB-resistant compared with immune-inflamed GC. Moreover, epithelial-mesenchymal transition (EMT) signalling was highly enriched in immune desert-type GC, and syngeneic murine tumours exhibiting mesenchymal-like, compared with epithelial-like, properties are T cell-excluded and resistant to CTLA4 blockade. Our analysis further identified a panel of RTKs as potential druggable targets in the immune desert-type GC. Dovitinib, an inhibitor of multiple RTKs, strikingly repressed EMT programming in mesenchymal-like immune desert syngeneic GC models. Dovitinib activated the tumour-intrinsic SNAI1/2-IFN-γ signalling axis and impeded the EMT programme, converting immune desert-type tumours to immune inflamed-type tumours, sensitising these mesenchymal-like 'cold' tumours to CTLA4 blockade. CONCLUSION: Our findings identified potential druggable targets relevant to patient groups, especially for refractory immune desert-type/ 'cold' GC. Dovitinib, an RTK inhibitor, sensitised desert-type immune-cold GC to CTLA4 blockade by restricting EMT and recruiting T cells.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

Induction of Fibroblast Growth Factor Receptor 4 by Helicobacter pylori via Signal Transducer and Activator of Transcription 3 With a Feedforward Activation Loop Involving SRC Signaling in Gastric Cancer

BACKGROUND & AIMS: Helicobacter pylori (H pylori) infection is the main risk factor for gastric cancer. The role of fibroblast growth factor receptors (FGRFs) in H pylori-mediated gastric tumorigenesis remains largely unknown. This study investigated the molecular and mechanistic links between H pylori, inflammation, and FGFR4 in gastric cancer. METHODS: Cell lines, human and mouse gastric tissue samples, and gastric organoids models were implemented. Infection with H pylori was performed using in vitro and in vivo models. Western blot, real-time quantitative reverse-transcription polymerase chain reaction, flow cytometry, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and luciferase reporter assays were used for molecular, mechanistic, and functional studies. RESULTS: Analysis of FGFR family members using The Cancer Genome Atlas data, followed by validation, indicated that FGFR4 messenger (m)RNA was the most significantly overexpressed member in human gastric cancer tissue samples (P < .001). We also detected high levels of Fgfr4 mRNA and protein in gastric dysplasia and adenocarcinoma lesions in mouse models. Infection with J166, 7.13, and PMSS1 cytotoxin-associated gene A (CagA)+ H pylori strains induced FGFR4 mRNA and protein expression in in vitro and in vivo models. This was associated with a concordant activation of signal transducer and activator of transcription 3 (STAT3). Analysis of the FGFR4 promoter suggested several putative binding sites for STAT3. Using chromatin immunoprecipitation assay and an FGFR-promoter luciferase reporter containing putative STAT3 binding sites and their mutants, we confirmed a direct functional binding of STAT3 on the FGFR4 promoter. Mechanistically, we also discovered a feedforward activation loop between FGFR4 and STAT3 where the fibroblast growth factor 19­FGFR4 axis played an essential role in activating STAT3 in a SRC proto-oncogene non-receptor tyrosine kinase dependent manner. Functionally, we found that FGFR4 protected against H pylori-induced DNA damage and cell death. CONCLUSIONS: Our findings demonstrated a link between infection, inflammation, and FGFR4 activation, where a feedforward activation loop between FGFR4 and STAT3 is established via SRC proto-oncogene non-receptor tyrosine kinase in response to H pylori infection. Given the relevance of FGFR4 to the etiology and biology of gastric cancer, we propose FGFR4 as a druggable molecular vulnerability that can be tested in patients with gastric cancer.

Helicobacter pylori-induced RASAL2 Through Activation of Nuclear Factor-κB Promotes Gastric Tumorigenesis via ß-catenin Signaling Axis.

BACKGROUND & AIMS: Helicobacter pylori infection is the predominant risk factor for gastric cancer. RAS protein activator like 2 (RASAL2) is considered a double-edged sword in carcinogenesis. Herein, we investigated the role of RASAL2 in response to H pylori infection and gastric tumorigenesis. METHODS: Bioinformatics analyses of local and public databases were applied to analyze RASAL2 expression, signaling pathways, and clinical significance. In vitro cell culture, spheroids, patient-derived organoids, and in vivo mouse models were used. Molecular assays included chromatin immunoprecipitation, co-immunoprecipitation, Western blotting, quantitative polymerase chain reaction, and immunocyto/histochemistry. RESULTS: H pylori infection induced RASAL2 expression via a nuclear factor-κB (NF-κB)-dependent mechanism whereby NF-κB was directly bound to the RASAL2 promoter activating its transcription. By gene silencing and ectopic overexpression, we found that RASAL2 upregulated ß-catenin transcriptional activity. RASAL2 inhibited protein phosphatase 2A activity through direct binding with subsequent activation of the AKT/ß-catenin signaling axis. Functionally, RASAL2 silencing decreased nuclear ß-catenin levels and impaired tumor spheroids and organoids formation. Furthermore, the depletion of RASAL2 impaired tumor growth in gastric tumor xenograft mouse models. Clinicopathological analysis indicated that abnormal overexpression of RASAL2 correlated with poor prognosis and chemoresistance in human gastric tumors. CONCLUSIONS: These studies uncovered a novel signaling axis of NF-κB/RASAL2/ß-catenin, providing a novel link between infection, inflammation and gastric tumorigenesis.

Integrated expression analysis identifies transcription networks in mouse and human gastric neoplasia.

Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. The Tff1 knockout (KO) mouse model develops gastric lesions that include low-grade dysplasia (LGD), high-grade dysplasia (HGD), and adenocarcinomas. In this study, we used Affymetrix microarrays gene expression platforms for analysis of molecular signatures in the mouse stomach [Tff1-KO (LGD) and Tff1 wild-type (normal)] and human gastric cancer tissues and their adjacent normal tissue samples. Combined integrated bioinformatics analysis of mouse and human datasets indicated that 172 genes were consistently deregulated in both human gastric cancer samples and Tff1-KO LGD lesions (P < .05). Using Ingenuity pathway analysis, these genes mapped to important transcription networks that include MYC, STAT3, ß-catenin, RELA, NFATC2, HIF1A, and ETS1 in both human and mouse. Further analysis demonstrated activation of FOXM1 and inhibition of TP53 transcription networks in human gastric cancers but not in Tff1-KO LGD lesions. Using real-time RT-PCR, we validated the deregulated expression of several genes (VCAM1, BGN, CLDN2, COL1A1, COL1A2, COL3A1, EpCAM, IFITM1, MMP9, MMP12, MMP14, PDGFRB, PLAU, and TIMP1) that map to altered transcription networks in both mouse and human gastric neoplasia. Our study demonstrates significant similarities in deregulated transcription networks in human gastric cancer and gastric tumorigenesis in the Tff1-KO mouse model. The data also suggest that activation of MYC, STAT3, RELA, and ß-catenin transcription networks could be an early molecular step in gastric carcinogenesis.

Helicobacter pylori-induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32.

OBJECTIVE: DARPP-32 is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between Helicobacter pylori infection, proinflammatory NF-κB activation and regulation of DARPP-32. DESIGN: The study used in vivo and in vitro experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, H. pylori infection, tissue microarrays and immunohistochemical assays were used. RESULTS: Our results indicated that H. pylori infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. H. pylori infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level in vitro and in vivo. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). Both H. pylori infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by H. pylori infection counteracted H. pylori-induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r2=0.43, p<0.01). CONCLUSIONS: Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following H. pylori infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis.

Gastric tumour-derived ANGPT2 regulation by DARPP-32 promotes angiogenesis.

OBJECTIVE: Overexpression of dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated isoform (t-DARPP) are associated with gastric tumorigenesis. Herein, we investigated the role of DARPP-32 proteins in regulating angiopoietin 2 (ANGPT2) and promoting tumour angiogenesis. DESIGN: Quantitative real-time RT-PCR, immunoblotting, luciferase reporter, immunofluorescence, immunohistochemistry and angiogenesis assays were applied to investigate the regulation of angiogenesis by DARPP-32 proteins. RESULTS: Overexpression of DARPP-32 significantly increased the mRNA and protein levels of ANGPT2 in gastric cancer cells. The overexpression of DARPP-32 T34A mutant or the N-terminal truncated isoform, t-DARPP, led to similar effects ruling out the T34-dependent regulation of protein phosphatase 1 activity in regulating ANGPT2. DARPP-32 proteins induced a secreted form of ANGPT2, which was detectable in the media, functionally active, and able to induce angiogenesis, measured by the human umbilical vein endothelial cells tube formation assay. Antibody blocking of the secreted ANGPT2 abrogated its function. To identify the mechanism by which DARPP-32 regulates ANGPT2, we examined the activities of NF-κB and signal transducer and activator of transcription 3 (STAT3), known regulators of angiogenesis. The results ruled out NF-κB and showed induction of STAT3 phosphorylation, activation and nuclear localisation. Inhibition or knockdown of STAT3 significantly attenuated the induction of ANGPT2 by DARPP-32 proteins. In vivo xenograft models demonstrated that overexpression of DARPP-32 promotes angiogenesis and tumour growth. Analyses of human gastric cancer tissues showed a strong correlation between DARPP-32 and ANGPT2. CONCLUSIONS: Our novel findings establish the role of DARPP-32-STAT3 axis in regulating ANGPT2 in cancer cells to promote angiogenesis and tumorigenesis.

Activation of ß-catenin signalling by TFF1 loss promotes cell proliferation and gastric tumorigenesis.

OBJECTIVE: In this study, we investigated the role of Trefoil factor 1 (TFF1) in regulating cell proliferation and tumour development through ß-catenin signalling using in vivo and in vitro models of gastric tumorigenesis. DESIGN: Tff1-knockout (Tff1-KO) mice, immunohistochemistry, luciferase reporter, qRT-PCR, immunoblot, and phosphatase assays were used to examine the role of TFF1 on ß-catenin signalling pathway. RESULTS: Nuclear localisation of ß-catenin with transcriptional upregulation of its target genes, c-Myc and Ccnd1, was detected in hyperplastic tissue at an early age of 4-6 weeks and maintained during all stages of gastric tumorigenesis in the Tff1-KO mice. The reconstitution of TFF1 or TFF1 conditioned media significantly inhibited the ß-catenin/T-cell factor (TCF) transcription activity in MKN28 gastric cancer cells. In agreement with these results, we detected a reduction in the levels of nuclear ß-catenin with downregulation of c-MYC and CCND1 mRNA. Analysis of signalling molecules upstream of ß-catenin revealed a decrease in phosphorylated glycogen synthase kinase 3ß (p-GSK3ß) (Ser9) and p-AKT (Ser473) protein levels following the reconstitution of TFF1 expression; this was consistent with the increase of p-ß-catenin (Ser33/37/Thr41) and decrease of p-ß-catenin (Ser552). This TFF1-induced reduction in phosphorylation of GSK3ß, and AKT was dependent on protein phosphatase 2A (PP2A) activity. The treatment with okadaic acid or knockdown of PP2A abrogated these effects. Consistent with the mouse data, we observed loss of TFF1 and an increase in nuclear localisation of ß-catenin in stages of human gastric tumorigenesis. CONCLUSIONS: Our data indicate that loss of TFF1 promotes ß-catenin activation and gastric tumorigenesis through regulation of PP2A, a major regulator of AKT-GSK3ß signalling.

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)-Barrett's 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 Barrett's 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/ß (S176/180), as well as significant suppression in induction of NF-κB target genes [TNF-α, interleukin (IL)-6, IL-8, IL-1ß, 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 Barrett's carcinogenesis.

Aurora kinase A promotes inflammation and tumorigenesis in mice and human gastric neoplasia.

BACKGROUND & AIMS: Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The aurora kinase A (AURKA) gene 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 polymerase chain reaction, 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 nuclear factor-κB (NF-κB) p65 in human gastric cancer samples and mouse epithelial cells, suppressed NF-κB reporter activity, and reduced expression of NF-κB 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 NF-κB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucosa of patients had increased levels of AURKA protein and nuclear NF-κB, compared with healthy gastric tissue. CONCLUSIONS: In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be up-regulated during chronic inflammation to promote activation of NF-κB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.

Lmo2 induces hematopoietic stem cell-like features in T-cell progenitor cells prior to leukemia.

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.

Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

Helicobacter pylori (H. pylori) is the leading risk factor for gastric carcinogenesis. Fibroblast growth factor receptor 4 (FGFR4) is a member of transmembrane tyrosine kinase receptors that are activated in cancer. We investigated the role of FGFR4 in regulating the cellular response to H. pylori infection in gastric cancer. High levels of oxidative stress signature and FGFR4 expression were detected in gastric cancer samples. Gene set enrichment analysis (GSEA) demonstrated enrichment of NRF2 signature in samples with high FGFR4 levels. H. pylori infection induced reactive oxygen species (ROS) with a cellular response manifested by an increase in FGFR4 with accumulation and nuclear localization NRF2. Knocking down FGFR4 significantly reduced NRF2 protein and transcription activity levels, leading to higher levels of ROS and DNA damage following H. pylori infection. We confirmed the induction of FGFR4 and NRF2 levels using mouse models following infection with a mouse-adapted H. pyloristrain. Pharmacologic inhibition of FGFR4 using H3B-6527, or its knockdown, remarkably reduced the level of NRF2 with a reduction in the size and number of gastric cancer spheroids. Mechanistically, we detected binding between FGFR4 and P62 proteins, competing with NRF2-KEAP1 interaction, allowing NRF2 to escape KEAP1-dependent degradation with subsequent accumulation and translocation to the nucleus. These findings demonstrate a novel functional role of FGFR4 in cellular homeostasis via regulating the NRF2 levels in response to H. pylori infection in gastric carcinogenesis, calling for testing the therapeutic efficacy of FGFR4 inhibitors in gastric cancer models.

SOX9 is regulated by AURKA in response to Helicobacter pylori infection via EIF4E-mediated cap-dependent translation.

Helicobacter pylori (H. pylori) infection is the main risk factor for gastric cancer. The SRY-Box Transcription Factor 9 (SOX9) serves as a marker of stomach stem cells. We detected strong associations between AURKA and SOX9 expression levels in gastric cancers. Utilizing in vitro and in vivo mouse models, we demonstrated that H. pylori infection induced elevated levels of both AURKA and SOX9 proteins. Notably, the SOX9 protein and transcription activity levels were dependent on AURKA expression. AURKA knockdown led to a reduction in the number and size of gastric gland organoids. Conditional knockout of AURKA in mice resulted in a decrease in SOX9 baseline level in AURKA-knockout gastric glands, accompanied by diminished SOX9 induction following H. pylori infection. We found an AURKA-dependent increase in EIF4E and cap-dependent translation with an AURKA-EIF4E-dependent increase in SOX9 polysomal RNA levels. Immunoprecipitation assays demonstrated binding of AURKA to EIF4E with a decrease in EIF4E ubiquitination. Immunohistochemistry analysis on tissue arrays revealed moderate to strong immunostaining of AURKA and SOX9 with a significant correlation in gastric cancer tissues. These findings elucidate the mechanistic role of AURKA in regulating SOX9 levels via cap-dependent translation in response to H. pylori infection in gastric tumorigenesis.

Targeting SMAD3 Improves Response to Oxaliplatin in Esophageal Adenocarcinoma Models by Impeding DNA Repair.

PURPOSE: TGFß signaling is implicated in the progression of most cancers, including esophageal adenocarcinoma (EAC). Emerging evidence indicates that TGFß signaling is a key factor in the development of resistance toward cancer therapy. EXPERIMENTAL DESIGN: In this study, we developed patient-derived organoids and patient-derived xenograft models of EAC and performed bioinformatics analysis combined with functional genetics to investigate the role of SMAD family member 3 (SMAD3) in EAC resistance to oxaliplatin. RESULTS: Chemotherapy nonresponding patients showed enrichment of SMAD3 gene expression when compared with responders. In a randomized patient-derived xenograft experiment, SMAD3 inhibition in combination with oxaliplatin effectively diminished tumor burden by impeding DNA repair. SMAD3 interacted directly with protein phosphatase 2A (PP2A), a key regulator of the DNA damage repair protein ataxia telangiectasia mutated (ATM). SMAD3 inhibition diminished ATM phosphorylation by enhancing the binding of PP2A to ATM, causing excessive levels of DNA damage. CONCLUSIONS: Our results identify SMAD3 as a promising therapeutic target for future combination strategies for the treatment of patients with EAC.

Gastric adenocarcinoma has a unique microRNA signature not present in esophageal adenocarcinoma.

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.

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 Barrett's 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-H(2)DCFDA, 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 (H(2)O(2)) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barrett's oesophagus cells, BAR-T and CP-A led to resistance to H(2)O(2)-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H(2)O(2), 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 H(2)O(2)-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 H(2)O(2), 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 Barrett's oesophagus and OAC.

Differential Expression of NEK Kinase Family Members in Esophageal Adenocarcinoma and Barrett's Esophagus.

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.

CDK1 bridges NF-κB and ß-catenin signaling in response to H. pylori infection in gastric tumorigenesis.

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-3ß activity through direct binding with subsequent accumulation and activation of ß-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 ß-catenin. The results demonstrate a mechanistic link between infection, inflammation, and gastric tumorigenesis where CDK1 plays a critical role.