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.

Prevalence, outcomes and associated factors of SARS-CoV-2 infection in psoriasis patients of Southwest China: a cross-sectional survey.

In this study we aimed to investigate the prevalence of SARS-CoV-2 infection in psoriasis patients, and outcomes of SARS-CoV-2 infection and associated risk factors. A cross-sectional survey was conducted from February 2023 to March 2023. Information was obtained with online questionnaire about psoriasis patients on demographic characteristics, clinical characteristics, SARS-CoV-2 infection and outcomes, vaccination, and routine protection against COVID-19. Logistic regression analysis was used to explore risk factors with SARS-CoV-2 infection and exacerbation of psoriasis. A total of 613 participants were recruited. 516 (84.2%) were infected, and associated factors were sex, working status, routine protection against COVID-19, COVID-19 vaccination, impaired nail, infection exacerbate psoriasis, and severity of psoriasis. Among the patients infected with SARS-CoV-2, 30 (5.8%) required hospitalization, 122 (23.6%) had psoriasis exacerbation due to SARS-CoV-2 infection, and associated factors were subtype of psoriasis, discontinuation of psoriasis treatment during SARS-CoV-2 infection, response following COVID-19 vaccination, and severity of psoriasis. Booster dose vaccination contributed a low probability of COVID-19 sequelae. COVID-19 vaccine's effectiveness was unsatisfactory, while booster dose vaccination reduced the occurrence of COVID-19 sequelae in psoriasis patients of Southwest China. Patients treated with psoriasis shown to be safe, without a higher incidence of SARS-CoV-2 infection or COVID-19hospitalization compared to untreated patients. Stopping treatment during SARS-CoV-2 infection led to psoriasis exacerbation, so psoriasis treatment could be continued except severe adverse reaction.

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.

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.

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.

A novel strategy for combination of clofarabine and pictilisib is synergistic in gastric cancer.

Gastric cancer (GC) is frequently characterized by resistance to standard chemotherapeutic regimens and poor clinical outcomes. We aimed to identify a novel therapeutic approach using drug sensitivity testing (DST) and our computational SynerySeq pipeline. DST of GC cell lines was performed with a library of 215 Federal Drug Administration (FDA) approved compounds and identified clofarabine as a potential therapeutic agent. RNA-sequencing (RNAseq) of clofarabine treated GC cells was analyzed according to our SynergySeq pipeline and identified pictilisib as a potential synergistic agent. Clonogenic survival and Annexin V assays demonstrated increased cell death with clofarabine and pictilisib combination treatment (P<0.01). The combination induced double strand breaks (DSB) as indicated by phosphorylated H2A histone family member X (γH2AX) immunofluorescence and western blot analysis (P<0.01). Pictilisib treatment inhibited the protein kinase B (AKT) cell survival pathway and promoted a pro-apoptotic phenotype as evidenced by quantitative real time polymerase chain reaction (qRT-PCR) analysis of the B-cell lymphoma 2 (BCL2) protein family members (P<0.01). Patient derived xenograft (PDX) data confirmed that the combination is more effective in abrogating tumor growth with prolonged survival than single-agent treatment (P<0.01). The novel combination of clofarabine and pictilisib in GC promotes DNA damage and inhibits key cell survival pathways to induce cell death beyond single-agent treatment.

NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1.

BACKGROUND: H. pylori infection is the main risk factor for gastric cancer. In this study, we investigated H. pylori-mediated activation of STAT3 and NF-κB in gastric cancer, using in vitro and in vivo models. METHODS: To investigate the activation of NF-κB and STAT3 by H. pylori strains we used in vitro and in vivo mouse models, western blots, immunofluorescence, ChIP Assay, luciferase and quantitative real-time PCR assays. RESULTS: Following infection with H. pylori in vitro, we found an earlier phosphorylation of NF-kB-p65 (S536), followed by STAT3 (Y705). Immunofluorescence, using in vitro and in vivo models, demonstrated nuclear localization of NF-kB and STAT3, following H. pylori infection. NF-kB and STAT3 luciferase reporter assays confirmed earlier activation of NF-kB followed by STAT3. In vitro and in vivo models demonstrated induction of mRNA expression of IL-6 (p < 0.001), VEGF-α (p < 0.05), IL-17 (p < 0.001), and IL-23 (p < 0.001). Using ChIP, we confirmed co-binding of both NF-kB-p65 and STAT3 on the IL6 promoter. The reconstitution of Trefoil Factor 1 (TFF1) suppressed activation of NF-kB with reduction in IL6 levels and STAT3 activity, in response to H. pylori infection. Using pharmacologic (BAY11-7082) and genetic (IκB super repressor (IκBSR)) inhibitors of NF-kB-p65, we confirmed the requirement of NF-kB-p65 for activation of STAT3, as measured by phosphorylation, transcription activity, and nuclear localization of STAT3 in in vitro and in vivo models. CONCLUSION: Our findings suggest the presence of an early autocrine NF-kB-dependent activation of STAT3 in response to H. pylori infection. TFF1 acts as an anti-inflammatory guard against H. pylori-mediated activation of pro-inflammatory networks.

Silencing of miR490-3p by H. pylori activates DARPP-32 and induces resistance to gefitinib.

Infection with Helicobacter pylori (H. pylori) is the main risk factor for gastric carcinogenesis. In this study, we investigated the expression, molecular functions, and downstream effectors of miR490-3p in gastric cancer. We used in vitro and in vivo models to investigate the role of H. pylori in regulating miR490-3p, DARPP-32-dependent functions, and therapeutic resistance. Human and mouse neoplastic gastric lesions demonstrated a negative correlation between DARPP-32 and miR490-3p expression (R = -0.58, P < 0.01). This was also detected following infection with H. pylori (R = -0.66, P < 0.01). Molecular assays confirmed DARPP-32 as a direct target of miR490-3p. CHRM2, the host gene of miR490-3p, was hypermethylated and downregulated in neoplastic gastric tissues (P < 0.05). H. pylori induced methylation and downregulation of CHRM2 and miR490-3p. Functionally, the reconstitution of miR490-3p sensitized cancer cells to gefitinib by inactivating DRAPP-32-dependent AKT and STAT3 pathways. Patients with low miR490-3p or high DARPP-32 expression had decreased overall survival (P < 0.05). Hypermethylation-mediated silencing of CHRM2 and miR490-3p by H. pylori increased DARPP-32 expression. Downregulation of miR490-3p in gastric cancer plays a role in gefitinib response by inducing DARPP-32-mediated activation of PI3K/AKT, STAT3 signaling pathways.

PRDX2 protects against oxidative stress induced by H. pylori and promotes resistance to cisplatin in gastric cancer.

BACKGROUND: Helicobacter pylori (H. pylori) infection is the main risk factor for gastric cancer. The role of antioxidant enzyme peroxiredoxin 2 (PRDX2) in gastric tumorigenesis remains unknown. In vitro (AGS and SNU-1 cell lines) and in vivo mouse models were utilized to investigate the role of PRDX2 in response to H. pylori infection (7.13, J166 or PMSS1 strain). We detected high levels of PRDX2 expression in gastric cancer tissues. Gastric cancer patients with high expression levels of PRDX2 had significantly worse overall and progression-free survival than those with low levels. H. pylori infection induced activation of NF-κB with increased expression of PRDX2, in in vitro and in vivo models. The knockdown of PRDX2 led to an increase in the levels of reactive oxygen species (ROS), oxidative DNA damage, and double-strand DNA breaks, in response to H. pylori infection, as measured by H2DCFDA, 8-oxoguanine, and p-H2AXγ assays. Luciferase reporter and ChIP assays confirmed the presence of a putative binding site of NF-κB-p65 on PRDX2 promoter region. The inhibition of PRDX2 significantly sensitized AGS and SNU-1 cells to cisplatin treatment. Our data suggest that the future development of therapeutic approaches targeting PRDX2 may be useful in the treatment of gastric cancer.

Activation of STAT3 signaling is mediated by TFF1 silencing in gastric neoplasia.

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.

APE1 Upregulates MMP-14 via Redox-Sensitive ARF6-Mediated Recycling to Promote Cell Invasion of Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) is an aggressive malignancy with poor clinical outcome. The incidence of EAC has been rising rapidly in the past three decades. Here, we showed that apurinic/apyrimidinic endonuclease (APE1) is overexpressed in EAC cell lines, and patients' samples of dysplasia and EAC. Downregulation of APE1 or inhibition of its redox function significantly repressed invasion. Overexpression of a redox-defective mutant, C65A, abrogated the proinvasive phenotype of APE1. APE1 regulated invasion via upregulation of matrix metalloproteinase 14 (MMP-14), which subsequently activated MMP-2, leading to degradation of the extracellular matrix in a redox-dependent manner. Downregulation of APE1 or inhibition of its redox function decreased the rate of endocytosis and recycling of MMP-14 protein. APE1 interacted with ARF6, a key regulator of MMP-14 recycling, which maintained ARF6 activity in an APE1-redox-dependent manner, promoting its ability to regulate MMP-14 recycling to the cell surface. In summary, these findings identify a novel redox-sensitive APE1-ARF6-MMP-14 signaling axis that mediates cellular invasion in esophageal carcinogenesis. SIGNIFICANCE: This study demonstrates the association between oxidative stress and the development and metastatic behavior of esophageal adenocarcinoma.

Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells.

Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is frequently overexpressed in early stages of gastric cancers. We utilized in vitro assays, 3D gastric gland organoid cultures, mouse models, and human tissue samples to investigate the biological and molecular impact of DARPP-32 on activation of IGF1R and STAT3 signaling and gastric tumorigenesis. DARPP-32 enhanced phosphorylation of IGF1R (Y1135), a step that was critical for STAT3 phosphorylation at Y705, nuclear localization, and transcription activation. By using proximity ligation and co-immunoprecipitation assays, we found that IGF1R and DARPP-32 co-existed in the same protein complex. Binding of DARPP-32 to IGF1R promoted IGF1R phosphorylation with subsequent activation of downstream SRC and STAT3. Analysis of gastric tissues from the TFF1 knockout (KO) mouse model of gastric neoplasia, demonstrated phosphorylation of STAT3 in the early stages of gastric tumorigenesis. By crossing the TFF1 KO mice with DARPP-32 (DP) knockout (KO) mice, that have normal stomach, we obtained double knockout (TFF1 KO/DP KO). The gastric mucosa from the double KO mice did not show phosphorylation of IGF1R or STAT3. In addition, the TFF1 KO/DP KO mice had a significant delay in developing neoplastic gastric lesions. Analysis of human gastric cancer tissue microarrays, showed high levels of DARPP-32 and positive immunostaining for nuclear STAT3 in cancer tissues, as compared to non-cancer histologically normal tissues. In summary, the DARPP-32-IGF1R signaling axis plays a key role in regulating the STAT3 signaling, a critical step in gastric tumorigenesis.

NRF2 antioxidant response protects against acidic bile salts-induced oxidative stress and DNA damage in esophageal cells.

Gastroesophageal reflux disease (GERD) is the main risk factor for Barrett's tumorigenesis. In this study, we investigated the role of NRF2 in response to exposure to acidic bile salts (ABS), in conditions that mimic GERD, using Barrett's esophagus cell models. We detected an increase in NRF2 protein levels, following exposure to ABS. We found oxidization of cysteines (cysteines with oxidized thiol groups) in KEAP1 protein with a weaker interaction between NRF2 and KEAP1, following ABS exposure. Treatment with bile salts increased nuclear NRF2 levels, enhancing its transcription activity, as measured by an ARE (antioxidant response element) luciferase reporter assay. The mRNA expression levels of NRF2 target genes, HO-1 and GR, were increased in response to ABS exposure. Using genetic overexpression and knockdown of NRF2, we found that NRF2 has a critical role in suppressing ABS-induced ROS levels, oxidative DNA damage, DNA double strand breaks, and apoptosis. Collectively, our results suggest that transient induction of NRF2 in response to ABS plays a pivotal role in protecting esophageal cells by maintaining the levels of oxidative stress and DNA damage below lethal levels under GERD conditions.

Activation of EGFR-DNA-PKcs pathway by IGFBP2 protects esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage.

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.

Helicobacter pylori-induced miR-135b-5p promotes cisplatin resistance in gastric cancer.

Helicobacter pylori infection is a major risk factor for the development of gastric cancer. Aberrant expression of microRNAs is strongly implicated in gastric tumorigenesis; however, their contribution in response to H. pylori infection has not been fully elucidated. In this study, we evaluated the expression of miR-135b-5p and its role in gastric cancer. We describe the overexpression of miR-135b-5p in human gastric cancer tissue samples compared with normal tissue samples. Furthermore, we found that miR-135b-5p is also up-regulated in gastric tumors from the trefoil factor 1-knockout mouse model. Infection with H. pylori induced the expression of miR-135b-5p in the in vitro and in vivo models. miR-135b-5p induction was mediated by NF-κB. Treatment of gastric cancer cells with TNF-α induced miR-135b-5p in a NF-κB-dependent manner. Mechanistically, we found that miR-135b-5p targets Krüppel-like factor 4 (KLF4) and binds to its 3' UTR, leading to reduced KLF4 expression. Functionally, high levels of miR-135b-5p suppress apoptosis and induce cisplatin resistance. Our results uncovered a mechanistic link between H. pylori infection and miR-135b-5p-KLF4, suggesting that targeting miR-135b-5p could be a potential therapeutic approach to circumvent resistance to cisplatin.-Shao, L., Chen, Z., Soutto, M., Zhu, S., Lu, H., Romero-Gallo, J., Peek, R., Zhang, S., El-Rifai, W. Helicobacter pylori-induced miR-135b-5p promotes cisplatin resistance in gastric cancer.

Integrated Analysis of Mouse and Human Gastric Neoplasms Identifies Conserved microRNA Networks in Gastric Carcinogenesis.

BACKGROUND & AIMS: microRNAs (miRNAs) are small noncoding RNAs that bind to the 3' untranslated regions of mRNAs to promote their degradation or block their translation. Mice with disruption of the trefoil factor 1 gene (Tff1) develop gastric neoplasms. We studied these mice to identify conserved miRNA networks involved in gastric carcinogenesis. METHODS: We performed next-generation miRNA sequencing analysis of normal gastric tissues (based on histology) from patients without evidence of gastric neoplasm (n = 64) and from TFF1-knockout mice (n = 22). We validated our findings using 270 normal gastric tissues (including 61 samples from patients without evidence of neoplastic lesions) and 234 gastric tumor tissues from 3 separate cohorts of patients and from mice. We performed molecular and functional assays using cell lines (MKN28, MKN45, STKM2, and AGS cells), gastric organoids, and mice with xenograft tumors. RESULTS: We identified 117 miRNAs that were significantly deregulated in mouse and human gastric tumor tissues compared with nontumor tissues. We validated changes in levels of 6 miRNAs by quantitative real-time polymerase chain reaction analyses of neoplastic gastric tissues from mice (n = 39) and 3 independent patient cohorts (n = 332 patients total). We found levels of MIR135B-5p, MIR196B-5p, and MIR92A-5p to be increased in tumor tissues, whereas levels of MIR143-3p, MIR204-5p, and MIR133-3p were decreased in tumor tissues. Levels of MIR143-3p were reduced not only in gastric cancer tissues but also in normal tissues adjacent to tumors in humans and low-grade dysplasia in mice. Transgenic expression of MIR143-3p in gastric cancer cell lines reduced their proliferation and restored their sensitivity to cisplatin. AGS cells with stable transgenic expression of MIR143-3p grew more slowly as xenograft tumors in mice than control AGS cells; tumor growth from AGS cells that expressed MIR143-3p, but not control cells, was sensitive to cisplatin. We identified and validated bromodomain containing 2 (BRD2) as a direct target of MIR143-3p; increased levels of BRD2 in gastric tumors was associated with shorter survival times for patients. CONCLUSIONS: In an analysis of miRNA profiles of gastric tumors from mice and human patients, we identified a conserved signature associated with the early stages of gastric tumorigenesis. Strategies to restore MIR143-3p or inhibit BRD2 might be developed for treatment of gastric cancer.

Methylation of the HOXA10 Promoter Directs miR-196b-5p-Dependent Cell Proliferation and Invasion of Gastric Cancer Cells.

The cross-talk between epigenetics and miRNA expression plays an important role in human tumorigenesis. Herein, the regulation and role of miR-196b-5p in gastric cancer was investigated. qRT-PCR demonstrated that miR-196b-5p is significantly overexpressed in human gastric cancer tissues (P < 0.01). In addition, it was determined that HOXA10, a homeobox family member and host gene for miR-196b-5p, is overexpressed and positively correlated with miR-196b-5p expression levels (P < 0.001). Quantitative pyrosequencing methylation analysis demonstrated significantly lower levels of DNA methylation at the HOXA10 promoter in gastric cancer, as compared with nonneoplastic gastric mucosa specimens. 5-Aza-2'-deoxycytidine treatment confirmed that demethylation of HOXA10 promoter induces the expression of HOXA10 and miR-196b-5p in gastric cancer cell model systems. Using the Tff1 knockout mouse model of gastric neoplasia, hypomethylation and overexpression of HOXA10 and miR-196b-5p in gastric tumors was observed, as compared with normal gastric mucosa from Tff1 wild-type mice. Mechanistically, reconstitution of TFF1 in human gastric cancer cells led to an increased HOXA10 promoter methylation with reduced expression of HOXA10 and miR-196b-5p. Functionally, miR-196b-5p reconstitution promoted human gastric cancer cell proliferation and invasion in vitro In summary, the current data demonstrate overexpression of miR-196b-5p in gastric cancer and suggest that TFF1 plays an important role in suppressing the expression of miR-196b-5p by mediating DNA methylation of the HOXA10 promoter. Loss of TFF1 expression may promote proliferation and invasion of gastric cancer cells through induction of promoter hypomethylation and expression of the HOXA10/miR-196b-5p axis.Implications: This study indicates that loss of TFF1 promotes the aberrant overexpression of HOXA10 and miR-196b-5p by demethylation of the HOXA10 promoter, which provides a new perspective of TFF1/HOXA10/miR-196b-5p functions in human gastric cancer. Mol Cancer Res; 16(4); 696-706. ©2018 AACR.