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Background Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. Objective To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. Methods We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. Results Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. Conclusions Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate ‘metabolic switch’ characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.

Adenoviruses harboring the herpes simplex virus thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme targeting human telomerase reverse transcriptase (hTERT-TR) show marked and specific antitumor activity. In addition to inducing tumor cell death by direct cytotoxicity, it is becoming clear that HSVtk also induces antitumor immunity. Programmed death ligand 1 (PD-L1) expressed on tumor cell surfaces mediates tumor-induced immunoresistance by inhibiting PD1-expressing tumor-infiltrating T cells. Here, we explored whether a soluble form of PD1 (sPD1-Ig), which blocks PD-L1, could synergize with TERT-TR-regulated HSVtk to enhance the adenoviral therapeutic efficacy by boosting antitumor immunity. Tumor antigen released by HSVtk-transduced tumors successfully primed tumor antigen-specific CD8 T cells via dendritic cells (DC). Regression of murine tumors was markedly enhanced when sPD1-Ig was incorporated into the adenovirus as compared with a single-module adenovirus expressing only HSVtk. This effect was abolished by CD8 T-cell depletion. Consistent with this, following adoptive transfer of tumor antigen-specific CD8 T cells into tumor-bearing Rag1(-/-) mice, dual-module adenovirus significantly enhanced CD8 T cell-mediated tumor rejection. In addition, secondary tumor challenge at a distal site was completely suppressed in mice treated with a dual-module adenovirus. These results suggest that a dual-targeting strategy to elicit both tumor antigen priming and tumor-induced immunoresistance enhances CD8 T cell-mediated antitumor immunity.

This study attempts to combine two findings toward developing a rational strategy for improved therapy for glioma. One of the findings, made in this pre-clinical study, is that an hTERT-targeting ribozyme-controlled HSVtk gene (hTERT.Rz.HSVtk) exerts anti-tumor effects. The second observation is that the over-expression of a small noncoding RNA, miR-145, causes down-regulation of metastasis-related genes, such as PLAUR, SPOCK3, ADAM22, SLC7A5 and FASCN1. While blocking in vivo tumor growth only slightly, over-expression of miR-145 significantly inhibits both the migration and invasion of U87MG/U373MG glioma cells. We hypothesized that a simultaneous adenoviral-mediated over-expression of miR-145 might enhance the anti-tumor effects of hTERT.Rz.HSVtk and that a combination therapy with miR-145 and the HSVtk gene would be an effective approach for treating glioma. We tested this by developing adenoviral vectors that over-express miR-145 under the CMV promoter and employing them in combination with hTERT.Rz.HSVtk expression, both in vitro and in vivo in animal studies. We found that the adenovirus Ad5CMV.Rz.HSVtk.miR145 harboring an HSVtk expression cassette plus miR-145 produced prolonged survival benefits compared to administration of Ad5CMV.Rz.HSVtk or Ad5CMV.miR-145 alone. This study demonstrates that combination therapy using the hTERT.Rz.HSVtk gene together with miR-145 over-expression produces enhanced anti-tumor effects compared to that resulting from hTERT.Rz.HSVtk gene therapy alone.

Purpose: Our previous studies suggested that human telomerase reverse transcriptase (hTERT) RNA-targeting trans-splicing ribozyme could be a useful tool for cancer gene therapy. Here, we investigated whether adenoviruses harboring this ribozyme can be systemically delivered to mice, and whether they selectively mark tumors expressing hTERT and sensitize them to ganciclovir treatments. Experimental Design: We constructed adenoviral vectors containing modified hTERT-targeting trans-splicing ribozyme with downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by a cytomegalovirus promoter. The tumor-specific trans-splicing reaction and the tumor-killing effect of adenoviruses harboring ribozyme were investigated both in vitro and in vivo using mice with intrahepatic colon cancer metastasis via systemic administration. The safety of systemic administration of the viruses was also evaluated. Results: We showed that Ad-Ribo-LacZ, when injected i.v., performs a highly specific trans-splicing reaction on hTERT mRNA and that it selectively marks tumors expressing hTERT in mice. More importantly, i.v. injection of Ad-Ribo-HSVtk plus ganciclovir significantly reduced tumor burden, with minimal liver toxicity, in mice with metastatic liver cancer, compared with the untreated group (P = 0.0009). Moreover, animals receiving Ad-Ribo-HSVtk showed improved survival compared with controls (P < 0.0001). Conclusions: This study shows that systemically delivered adenovirus harboring trans-splicing ribozyme can recognize cancer-specific transcripts and reprogram them to combat the cancer cells. Use of trans-splicing ribozymes seems to be a potentially useful gene therapy for cancer.

// Seung-Hyun Hong 1,* , Sung-Ho Goh 2,* , Sang Jin Lee 3 , Jung-Ah Hwang 1 , Jieun Lee 1 , Il-Ju Choi 4 , Hyehyun Seo 3 , Jong-Hoon Park 6 , Hiromu Suzuki 7 , Eiichiro Yamamoto 7 , In-Hoo Kim 5 , Jin Sook Jeong 8 , Mi Ha Ju 8 , Dong-Hee Lee 9 , and Yeon-Su Lee 1 1 Cancer Genomics Branch, Research Institute, National Cancer Center, Republic of Korea, 2 New Experimental Therapeutics Branch, Research Institute, National Cancer Center, Republic of Korea, 3 Genitourinary Cancer Branch, Research Institute, National Cancer Center, Republic of Korea, 4 Center for Gastric Cancer, Research Institute, National Cancer Center, Republic of Korea, 5 Molecular Imaging and Therapy Branch, Research Institute, National Cancer Center, Republic of Korea, 6 Department of Biological Science, Sookmyung Women’s University, Seoul, Republic of Korea, 7 Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan, 8 Department of Pathology, Dong-A University College of Medicine, Busan, Republic of Korea, 9 Department of Life Science, Ewha Woman’s University, Seoul, Republic of Korea * These two authors contributed equally to this work. Correspondence: Yeon-Su Lee, email: // Dong-Hee Lee, email: // Keywords : gastric cancer, adenylate cyclase, tumorigenesis, cAMP/PKA/CREB pathway, promoter methylation Received : August 23, 2013 Accepted : September 28, 2013 Published : September 30, 2013 Abstract Adenylate cyclase 3 ( ADCY3 ) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3′,5′-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren’s intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells; conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 ( MMP2 ) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatment with a protein kinase A (PKA) inhibitor decreased MMP2 and MMP9 expression levels in ADCY3 -overexpressing cells. Knockdown of ADCY3 expression by stable shRNA in human gastric cancer cells suppressed tumor growth in a tumor xenograft model. Thus, ADCY3 overexpression may exert its tumor-promoting effects via the cAMP/PKA/CREB pathway. Additionally, bisulfite sequencing of the ADCY3 promoter region revealed that gene expression was reduced by hypermethylation of CpG sites, and increased by 5-Aza-2′-deoxycytidine (5-Aza-dC)-induced demethylation. Our study is the first to report an association of ADCY3 with gastric cancer as well as its tumorigenic potentials. In addition, we demonstrate that the expression of ADCY3 is regulated through an epigenetic mechanism. Further study on the mechanism of ADCY3 in tumorigenesis will provide the basis as a new molecular target of gastric cancer.

c-MYC is a promising target for cancer therapy but its use is restricted by unwanted, devastating side effects. We explored whether intravesical instillation of the c-MYC inhibitor KSI-3716 could suppress tumor growth in murine orthotopic bladder xenografts.The small molecule KSI-3716, which blocks c-MYC/MAX binding to target gene promoters, was used as an intravesical chemotherapy agent. KSI-3716 action was assessed by electrophoretic mobility shift assay, chromatin immunoprecipitation, transcription reporter assay and quantitative reverse transcriptase-polymerase chain reaction. Inhibition of cell proliferation and its mechanism was monitored by cell cytotoxicity assay, EdU incorporation assay and flow cytometry. The in vivo efficacy of KSI-3716 was examined by noninvasive luminescence imaging and histological analysis after intravesical instillation of KSI-3716 in murine orthotopic bladder xenografts.KSI-3716 blocked c-MYC/MAX from forming a complex with target gene promoters. c-MYC mediated transcriptional activity was inhibited by KSI-3716 at concentrations as low as 1 μM. The expression of c-MYC target genes, such as cyclin D2, CDK4 and hTERT, was markedly decreased. KSI-3716 exerted cytotoxic effects on bladder cancer cells by inducing cell cycle arrest and apoptosis. Intravesical instillation of KSI-3716 at a dose of 5 mg/kg significantly suppressed tumor growth with minimal systemic toxicity.The c-MYC inhibitor KSI-3716 could be developed as an effective intravesical chemotherapy agent for bladder cancer.

To the editors We found an error in our published article, Vol. 29, No. 3: 343-350, 2014. An error in published journal: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012R1A1A1002559). This study was also supported by the Biomedical Science Scholarship Grants, Department of Medicine, Chung-Ang University in 2013. Corrected acknowledgement of the above article. This study was supported by the Biomedical Science, Department of Medicine Research Scholarship Grants, Chung-Ang University in 2013.

Stomach cancer is the most prevalent cancer in Eastern Asia(1). Given the diverse route to oncognesis, it is highly attractive to develop targeted therapy based on each patient's biology(2). Several molecular targeting agents that show survival advantage in other cancer types are being under clinical investigation in gastric cancer (GC) (3-6). These agents focus on a few known oncogenic genes, yet its true efficacy of their biologically targeted therapies in gastric cancer is still unknown. To identify high-quality therapeutic targets, we utilized PATHOME (pathway and genome information), a computational method to identify therapeutic target through sub-pathway employing simple statistic-based approach (unpublished). PATHOME design had two stages: 1) discovery stage aimed at selecting target signaling pathway, and 2) validation stage aimed at identifying small number of reproducible genes from sub-pathways by using independent dataset to provide a novel theragnostic strategy in GC patients. Four sets of GEO database datasets were used: GSE13861 (84 Korean GC), GSE15081 (56 Japanese GC) for the discovery stage, and GSE36968 (25 Korean GC), and GSE27342 (160 Chinese GC) were added for validation. Using Vogelstein et al. as the gold standard, DAVID and GSEA vs. PATHOME were applied for comparison of potency in prediction of signaling pathways(7). Previous study showed that the energy sensing mechanism by AMPKα is involved in Asian GC(8). Metformin is a drug used to treat diabetes, which affects AMPKα in the cell. We treated GC cell line with metformin and saw the expression level of proteins that were selected by PATHOME to test its predictability of biological context. 13 up-regulated genes were identified using 3 datasets (GSE 13861, GSE 15081 and GSE36968), and 5 (GENE1, GENE2, GENE3, GENE4 and GENE5) from all 4 datasets using PATHOME. Transcription factor binding site analysis showed that two of the 5 genes contained GENE6 binding sites. Implications of AMPKα as a potential therapeutic target has been published(9). Metformin is shown to increase PRKAA2 level, which encodes AMPKα, also inhibits HNF4α(8). To see if metformin has any effect on the genes with HNF4α binding site, we observed the expression level of GENE4 and related proteins. Metformin treated GC cell lines (NCI-N87 and AGS) result in decreased viability compared to untreated cells. On Western-blot analysis, metformin treated cells show a decrease of GENE4, GENE6, but not GENE7 or GENE8. Utilizing PATHOME, which showed greater consistency compared to DAVID and GSEA in terms of cancer-related pathways, we showed Wnt pathway to have a potential impact on GC in East Asia. Therefore, our approach has simplified insight of multiplexed signals apply in molecular targeted therapies. We report a cross-talk between AMPKα and Wnt pathway in gastric cancer, which is known to be involved in other cancer types. This finding will lead to other potential drug targets for GC. Citation Format: Hae Ryung Chang, Seungyoon Nam, Hae Rim Jung, ChangHyuk Kwon, Hye-Hyun Seo, Frances S. Sung, Hee Seo Park, Taesung Park, Yon Hui Kim. Crosstalk between Wnt and AMPK pathway in Asian gastric cancer by metformin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5540. doi:10.1158/1538-7445.AM2013-5540

The LKB1/AMPK signaling pathway has been well elucidated and recent evidence suggests its involvement in cancer cell biology, demonstrating that the reinforcement of the tumor suppressive functions of LKB1/AMPK is a valuable therapeutic strategy for cancers. Interest in metformin as a novel anticancer agent for breast cancer and other solid tumors continue to grow, currently being investigated in several cancer types in both neoadjuvant and metastatic settings. The biological effect of metformin on cancer cells is driven by its ability to activate AMPK through upstream kinase LKB1, tumor suppressor gene in epithelial tissues. Metformin increases intracellular AMP level, which allosterically activates AMPK. We have previously identified the AMPKα as a modulator in gastric cancer (GC) and through experimental evidence. We show the impact of LKB1/AMPK modulation of HNF4α, a dramatic suppression of cancer cell growth. GC samples were collected and sequenced on SOLiD v 3.0 for both WT-seq and small RNA-seq. Computational analysis showed that 356 out of 18,890 genes were identified as GC related differentially expressed genes in the five-group comparison (normal, tumor stage I, II, III or IV). 28 genes were identified as stage-specific differentially expressed genes, and 13 out of the 28 genes were within the network between HNF4α and HNF1α. In order to test the anti-proliferation activity of metformin in GC cell lines associated with activation of PRKAA1, PRKAA2 and LKB1, and by HNF4α suppression, 4 GC cell lines (NCI-N87, AGS, HS 746T and MKN 45) were treated with metformin. Both PRKAA1/2 showed increased gene expression level when the cells were treated with 10mM of metformin. As for STK11 (LKB1 gene) and HNF4A gene expression level, LKB1 increased and HNF4A decreased with metformin treatment on all four cell lines. Metformin treated NCI-N87 and AGS show that it is involved in cell cycle arrest. Western-blot analysis shows, decreased protein expression of Cyclin A/B and D1 on metformin treated. Lastly, NCI-N87 xenograft study show metformin treated suppression of tumor progression compared to non-treated mouse. During the 28 day treatment of metformin, PRKAA1 and PRKAA2 expression level increased compared to the untreated with. Consistent with in vitro assay, LKB1 level was elevated in the metformin treated tumor compared to the non-treated, and HNF4α level decreased over time in the metformin treated tumor. Study shows that AMPK is a strong therapeutic tumor suppressor target and that metformin is a potential drug for Asian early gastric cancer patient. In our research in progress, we observe potential relationships between the Wnt pathway and AMPKα in light of WNT druggability with metformin. In conclusion LKB1/AMPK by HNF4α inhibition suggests metformin could be a candidate for gastric cancer treatment, probably in combination with conventional chemotherapy and/or as a maintenance therapy. Citation Format: Hae Rim Jung, Hae Ryung Chang, Hye-Hyun Seo, Robert Lemos, Hee Seo Park, Han Liang, Garth Powis, Yon Hui Kim. Metformin increases AMPKα activity by inhibition of AMPKα and cell cycle proliferation in Asian gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5534. doi:10.1158/1538-7445.AM2013-5534