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.

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.

Targeted Molecular Therapies in the Treatment of Esophageal Adenocarcinoma, Are We There Yet?

Esophageal adenocarcinoma is one of the leading causes of cancer-related deaths worldwide. The incidence of esophageal adenocarcinoma has increased at an alarming rate in the Western world and long-term survival remains poor. Current treatment approaches involve a combination of surgery, chemotherapy, and radiotherapy. Unfortunately, standard first-line approaches are met with high rates of recurrence and metastasis. More recent investigations into the distinct molecular composition of these tumors have uncovered key genetic and epigenetic alterations involved in tumorigenesis and progression. These discoveries have driven the development of targeted therapeutic agents in esophageal adenocarcinoma. While many agents have been studied, therapeutics targeting the human epidermal growth factor receptor (HER2) and vascular endothelial growth factor (VEGF) pathways have demonstrated improved survival. More recent advances in immunotherapies have also demonstrated survival advantages with monoclonal antibodies targeting the programmed death ligand 1 (PD-L1). In this review we highlight recent advances of targeted therapies, specifically agents targeting receptor tyrosine kinases, small molecule kinase inhibitors, and immune checkpoint inhibitors. While targeted therapeutics and immunotherapies have significantly improved survival, the benefits are limited to patients whose tumors express biomarkers such as PD-L1 and HER2. Survival remains poor for the remainder of patients with esophageal adenocarcinoma, underscoring the critical need for development of novel treatment strategies.

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.