Duane T. Smoot

SILAC-based quantitative proteomic analysis of gastric cancer secretome

Gastric cancer is a commonly occurring cancer in Asia and one of the leading causes of cancer deaths. However, there is no reliable blood‐based screening test for this cancer. Identifying proteins secreted from tumor cells could lead to the discovery of clinically useful biomarkers for early detection of gastric cancer.

GKN1–miR-185–DNMT1 Axis Suppresses Gastric Carcinogenesis through Regulation of Epigenetic Alteration and Cell Cycle

Purpose: Gastrokine 1 (GKN1) functions to protect the gastric antral mucosa and promotes healing by facilitating restoration and proliferation after injury. GKN1 is downregulated in Helicobacter pylori–infected gastric epithelial cells and loss of GKN1 expression is closely associated with gastric carcinogenesis, but underlying mechanisms of the tumor-suppressing effects of GKN1 remain largely unknown. Experimental Design: AGS, MKN1, MKN28 gastric cancer cells and HFE-145 immortalized non-neoplastic gastric mucosal cells were transfected with GKN1 or shGKN1. We conducted molecular and functional studies of GKN1 and miR-185 and investigated the mechanisms of alteration. We also analyzed epigenetic alterations in 80 gastric cancer tissues. Results: Restoration of GKN1 protein suppressed gastric cancer cell growth by inducing endogenous miR-185 that directly targets epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. In addition, ectopic expression of GKN1 upregulated Tip60 and downregulated HDAC1 in an miR-185–independent manner, thereby inducing cell-cycle arrest by regulating cell-cycle proteins in gastric cancer cells. Notably, GKN1 expression was inversely correlated with DNMT1 and EZH2 expression in a subset of 80 gastric cancer tissues and various gastric cancer cell lines. Interestingly, it was found that GKN1 exerted a synergistic anti-cancerous effect with 5-fluorouracil on tumor cell growth, which suggests a possible therapeutic intervention method for gastric cancer. Conclusion: Our results show that GKN1 has an miR-185–dependent and -independent mechanism for chromatic and DNA epigenetic modification, thereby regulating the cell cycle. Thus, the loss of GKN1 function contributes to malignant transformation and proliferation of gastric epithelial cells in gastric carcinogenesis. Clin Cancer Res; 19(17); 4599–610. ©2013 AACR.

Gastrokine 1 inhibits the carcinogenic potentials of Helicobacter pylori CagA

Helicobacter pylori CagA directly injected by the bacterium into epithelial cells via a type IV secretion system, leads to cellular changes such as morphology, apoptosis, proliferation and cell motility, and stimulates gastric carcinogenesis. We investigated the effects of cytotoxin-associated gene A (CagA) and gastrokine 1 (GKN1) on cell proliferation, apoptosis, reactive oxygen species (ROS) production, epithelial-mesenchymal transition (EMT) and cell migration in CagA- or GKN1-transfected gastric epithelial cells and mucosal tissues from humans and mice infected with H.pylori. On the molecular level, H.pylori CagA induced increased cell proliferation, ROS production, antiapoptotic activity, cell migration and invasion. Moreover, CagA induced activation of NF-κB and PI3K/Akt signaling pathways and EMT-related proteins. In addition, H.pylori CagA reduced GKN1 gene copy number and expression in gastric cells and mucosal tissues of humans and mice. However, GKN1 overexpression successfully suppressed the carcinogenic effects of CagA through binding to CagA. These results suggest that GKN1 might be a target to inhibit the effects from H.pylori CagA.

PD‐L1 expression is mainly regulated by interferon gamma associated with JAK‐STAT pathway in gastric cancer

Despite multidisciplinary treatment for patients with advanced gastric cancer, their prognosis remains poor. Therefore, the development of novel therapeutic strategies is urgently needed, and immunotherapy utilizing anti‐programmed death 1/‐programmed death ligand‐1 mAb is an attractive approach. However, as there is limited information on how programmed death ligand‐1 is upregulated on tumor cells within the tumor microenvironment, we examined the mechanism of programmed death ligand‐1 regulation with a particular focus on interferon gamma in an in vitro setting and in clinical samples. Our in vitro findings showed that interferon gamma upregulated programmed death ligand‐1 expression on solid tumor cells through the JAK‐signal transducer and activator of transcription pathway, and impaired the cytotoxicity of tumor antigen‐specific CTL against tumor cells. Following treatment of cells with anti‐programmed death ligand‐1 mAb after interferon gamma‐pre‐treatment, the reduced anti‐tumor CTL activity by interferon gamma reached a higher level than the non‐treatment control targets. In contrast, programmed death ligand‐1 expression on tumor cells also significantly correlated with epithelial‐mesenchymal transition phenotype in a panel of solid tumor cells. In clinical gastric cancer samples, tumor membrane programmed death ligand‐1 expression significantly positively correlated with the presence of CD8‐positive T cells in the stroma and interferon gamma expression in the tumor. The results suggest that gastric cancer patients with high CD8‐positive T‐cell infiltration may be more responsive to anti‐programmed death 1/‐programmed death ligand‐1 mAb therapy.

Epigenomic Promoter Alterations Amplify Gene Isoform and Immunogenic Diversity in Gastric Adenocarcinoma

Promoter elements play important roles in isoform and cell type-specific expression. We surveyed the epigenomic promoter landscape of gastric adenocarcinoma, analyzing 110 chromatin profiles (H3K4me3, H3K4me1, H3K27ac) of primary gastric cancers, gastric cancer lines, and nonmalignant gastric tissues. We identified nearly 2,000 promoter alterations (somatic promoters), many deregulated in various epithelial malignancies and mapping frequently to alternative promoters within the same gene, generating potential pro-oncogenic isoforms (RASA3). Somatic promoter-associated N-terminal peptides displaying relative depletion in tumors exhibited high-affinity MHC binding predictions and elicited potent T-cell responses in vitro, suggesting a mechanism for reducing tumor antigenicity. In multiple patient cohorts, gastric cancers with high somatic promoter usage also displayed reduced T-cell cytolytic marker expression. Somatic promoters are enriched in PRC2 occupancy, display sensitivity to EZH2 therapeutic inhibition, and are associated with novel cancer-associated transcripts. By generating tumor-specific isoforms and decreasing tumor antigenicity, epigenomic promoter alterations may thus drive intrinsic tumorigenesis and also allow nascent cancers to evade host immunity.Significance: We apply epigenomic profiling to demarcate the promoter landscape of gastric cancer. Many tumor-specific promoters activate different promoters in the same gene, some generating pro-oncogenic isoforms. Tumor-specific promoters also reduce tumor antigenicity by causing relative depletion of immunogenic peptides, contributing to cancer immunoediting and allowing tumors to evade host immune attack. Cancer Discov; 7(6); 630-51. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 539.

Reduced lysosomal clearance of autophagosomes promotes survival and colonization of Helicobacter pylori

Evasion of autophagy is key for intracellular survival of bacteria in host cells, but its involvement in persistent infection by Helicobacter pylori, a bacterium identified to invade gastric epithelial cells, remains obscure. The aim of this study was to functionally characterize the role of autophagy in H. pylori infection. Autophagy was assayed in H. pylori‐infected human gastric epithelium and the functional role of autophagy was determined via genetic or pharmacological ablation of autophagy in mouse and cell line models of H. pylori infection. Here, we showed that H. pylori inhibited lysosomal function and thereby promoted the accumulation of autophagosomes in gastric epithelial cells. Importantly, inhibiting autophagosome formation by pharmacological inhibitors or genetic ablation of BECN1 or ATG5 reduced H. pylori intracellular survival, whereas inhibition of lysosomal functions exerted an opposite effect. Further experiments demonstrated that H. pylori inhibited lysosomal acidification and the retrograde trafficking of mannose‐6‐phosphate receptors, both of which are known to positively regulate lysosomal function. We conclude that H. pylori subverts autophagy into a pro‐survival mechanism through inhibition of lysosomal clearance of autophagosomes. Disruption of autophagosome formation offers a novel strategy to reduce H. pylori colonization in human stomachs. Copyright

Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation

MicroRNA (miR) sponges containing miR binding sequences constitute a potentially powerful molecular therapeutic strategy. Recently, naturally occurring circular RNAs (circRNAs) were shown to function as efficient miR sponges in cancer cells. We hypothesized that synthetic circRNA sponges could achieve therapeutic loss-of-function targeted against specific miRs. Linear RNA molecules containing miR-21 binding sites were transcribed in vitro; after dephosphorylation and phosphorylation, circularization was achieved using 5′-3′ end-ligation by T4 RNA ligase 1. circRNA stability was assessed using RNase R and fetal bovine serum. Competitive inhibition of miR-21 activity by a synthetic circRNA sponge was assessed using luciferase reporter, cell proliferation, and cell apoptosis assays in three gastric cancer cell lines. circRNA effects on downstream proteins were also delineated by Tandem Mass Tag (TMT) labeling (data available via ProteomeXchange identifier PRIDE: PXD008584), followed by western blotting. We conclude that artificial circRNA sponges resistant to nuclease digestion can be synthesized using simple enzymatic ligation steps. These sponges inhibit cancer cell proliferation and suppress the activity of miR-21 on downstream protein targets, including the cancer protein DAXX. In summary, synthetic circRNA sponges represent a simple, effective, convenient strategy for achieving targeted loss of miR function in vitro, with potential future therapeutic application in human patients.

YAP/TAZ initiates gastric tumorigenesis via upregulation of MYC

YAP and TAZ play oncogenic roles in various organs, but the role of YAP/TAZ in gastric cancer remains unclear. Here, we show that YAP/TAZ activation initiates gastric tumorigenesis in vivo and verify its significance in human gastric cancer. In mice, YAP/TAZ activation in the pyloric stem cell led to step-wise tumorigenesis. RNA sequencing identified MYC as a decisive target of YAP, which controls MYC at transcriptional and posttranscriptional levels. These mechanisms tightly regulated MYC in homeostatic conditions, but YAP activation altered this balance by impeding miRNA processing, causing a shift towards MYC upregulation. Pharmacologic inhibition of MYC suppressed YAP-dependent phenotypes in vitro and in vivo, verifying its functional role as a key mediator. Human gastric cancer samples also displayed a significant correlation between YAP and MYC. We reanalyzed human transcriptome data to verify enrichment of YAP signatures in a subpopulation of gastric cancers and found that our model closely reflected the molecular pattern of patients with high YAP activity. Overall, these results provide genetic evidence of YAP/TAZ as oncogenic initiators and drivers for gastric tumors with MYC as the key downstream mediator. These findings are also evident in human gastric cancer, emphasizing the significance of YAP/TAZ signaling in gastric carcinogenesis.Significance: YAP/TAZ activation initiates gastric carcinogenesis with MYC as the key downstream mediator. Cancer Res; 78(12); 3306-20. ©2018 AACR.

Clinicians’ perspectives on and interest in participating in a clinical data research network across the Southeastern United States

BackgroundPartnerships between clinicians and researchers could increase the generalizability of research findings and increase uptake of research results across populations. Yet engaging clinicians in research is challenging. Clinical Data Research Networks (CDRNs) provide access to a broad array of clinical data, patients, clinicians and health systems by building on existing health records (EHRs) to facilitate multi-site community engaged research (CEnR).MethodsA mixed-methods sequential explanatory design was employed. Sixty semi-structured interviews with clinicians from various disciplines and healthcare settings were conducted using five open-ended questions. Inductive content analysis was used to identify emerging themes in the data.ResultsWe identified the following emerging themes: 1) Research with relevance and benefits to clinics and provider’s patient population; 2) Difficulties of engaging in research with existing patient care demands; 3) Clear and continuous two-way communication about research, coordinated with provider and clinic needs; 4) Tailored compensation approaches meet provider preferences; 5) Increasing clinician awareness about Clinical Data Research Networks (CDRNs).ConclusionOur interview study provides insight into community clinician perspectives on Clinical Data Research Networks, indicating motivations and challenges to research involvement including consequences of time spent on research participation, barriers to expanding research and meaningful involvement in research governance. Findings can be used to guide the development of strategies to better engage providers in research in clinical settings, which could ultimately improve patient outcomes.