SNU-16 Gastric Cancer Cells Research Articles

Design, synthesis and biological evaluation of a series of novel pyrrolo[2,3-d]pyrimidin/pyrazolo[3,4-d]pyrimidin-4-amine derivatives as FGFRs-dominant multi-target receptor tyrosine kinase inhibitors for the treatment of gastric cancer

Gastric cancer is the second most lethal cancer across the world. With the progress in therapeutic approaches, the 5-year survival rate of early gastric cancer can reach > 95%. However, the prognosis and survival time of advanced gastric cancer is still somber. Therefore, more effective targeted therapies for gastric cancer treatment are urgently needed. FGFR, VEGFR and other receptor tyrosine kinases have recently been suggested as potential targets for gastric cancer treatment. We herein report the discovery of pyrrolo[2,3-d]pyrimidin/pyrazolo[3,4-d]pyrimidin-4-amine derivatives as a new class of FGFRs-dominant multi-target receptor tyrosine kinase inhibitors. SAR assessment identified the most active compounds 8f and 8k, which showed excellent inhibitory activity against a variety of receptor tyrosine kinases. Moreover, 8f and 8k displayed excellent potency in the SNU-16 gastric cancer cell line. Furthermore, 8f and 8k could inhibit FGFR1 phosphorylation and downstream signaling pathways as well as induce cell apoptosis. In vivo, 8f and 8k suppress tumor growth in the SNU-16 xenograft model without inducing obvious toxicity. These findings raise the possibility that compounds 8f and 8k might serve as potential agents for the treatment of gastric cancer.

Abstract 1089: Extrachromosomal DNA (ecDNA)-driven switching of oncogene dependency facilitates resistance to targeted therapy

Abstract Introduction: Tumors with oncogene copy number amplification are aggressive, have poor prognosis and, to date, have been very difficult to treat. Computational analyses in a large pan-cancer study revealed that ecDNA comprises over 50% of highly amplified oncogenes. We sought to determine the underlying mechanisms that render tumors with amplified oncogenes on ecDNA largely refractory to targeted oncogene therapy intervention and the subsequent implications regarding emergent resistance of oncogene amplified tumors to targeted inhibitors. Methods: This study used the SNU16 gastric cancer cell line that contains the common oncogenes, MYC and FGFR2, amplified on ecDNA. We performed a longitudinal assessment of cellular resistance and ecDNA dynamics in response to the FGFR inhibitor, infigratinib (infi), including dosing at EC20, EC50, and EC90. Results: Prior to infi exposure, we characterized broad differences in ecDNA structure and quantified the supporting massively increased genetic heterogeneity at the subcellular level. Strikingly, upon treatment with infi, we observed dose-dependent therapeutic resistance driven by underlying intracellular heterogeneity of oncogenes residing on ecDNA. Suppressing FGFR signaling at the EC90 dose led to a decrease in FGFR2 ecDNA and concomitant exponential increase in EGFR ecDNA, revealing a rapid switch of functional oncogene dependencies and therefore a fitness advantage uniquely enabled by ecDNA. We subsequently exposed the infi-resistant cells (now harboring EGFR amplification) to erlotinib and documented a return from the emergent ecDNA-enabled EGFR dependency back to the basal FGFR2 dependency on ecDNA. In addition to facilitating resistance to targeted therapeutic pressure, this unique oncogene dependency switching mechanism demonstrates that ecDNA-amplified oncogenes constitute cancer driver alterations. We next tested simultaneous upfront inhibition of both FGFR and EGFR with combination targeted therapies. Although initial cytotoxicity was robust, the cell population inevitably became resistant. Resistance to the up-front dual blockade was also driven by ecDNA, with diverse oncogenes including MET and KRAS becoming amplified on ecDNA in a marked heterogeneous pattern within the population. In sum, the cells had a remarkable propensity to amplify a range of oncogenes on ecDNA to enable survival under therapeutic pressure. Conclusions: This data validates previous reports of ecDNA copy number dynamics under therapeutic pressure. The inability to predict which oncogenes would amplify on ecDNA in upfront combinations suggests that an up-front targeted therapy approach, including combination therapy, will not be durably effective for patients with tumors carrying ecDNA and highlights the urgent need to take a new therapeutic approach to disable tumor cells' utilization of ecDNA. Citation Format: Kristen M. Turner, Dean Perusse, Laurence Jadin, Nam-Phuong Nguyen, Christian Hassig, Jason Christiansen. Extrachromosomal DNA (ecDNA)-driven switching of oncogene dependency facilitates resistance to targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1089.

Effects of p-coumaric acid on microRNA expression profiles in SNU-16 human gastric cancer cells.

p-Coumaric acid (p-CA), a phenolic compound abundantly found in edible plants, was found to induce apoptosis in SNU-16 gastric cancer cells. However, the effects of p-CA on the microRNA expression pattern in SNU-16 cells have not been reported. The primary objective of this study was to investigate microRNA expression profiles in p-CA-treated SNU-16 cells. SNU-16 cells were treated with 1.5 mM p-CA for 48 h. High-throughput RNA sequencing was performed. The accuracy and validity of the RNA sequencing results were tested by real-time PCR. Gene Ontology(GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the differentially expressed microRNAs (DEMs). Thirty-two DEMs were identified between p-CA-treated and control cells, 9 of which were upregulated, and 23 downregulated. GO and KEGG pathway analyses of the target genes indicated that these genes were involved in a variety of biological functions and several cancer-related pathways. The miRNA interactive network analysis identified hsa-miR-125a-5p, hsa-miR-30a-5p, and hsa-miR-7-5p as the major nodes. Our findings indicated that p-CA exerted its anticancer effects in SNU-16 gastric cancer cells by modulating the expression of certain microRNAs, providing a better understanding of the anticancer properties of p-CA in gastric cancer cells.

MicroRNA-19a regulates the proliferation, migration and invasion of human gastric cancer cells by targeting CUL5

Gastric cancer is one of the prevalent types of cancers and despite improvements in its treatment, the overall survival is still far from descent. The dearth of efficient biomarkers, chemotherapeutic agents and therapeutic targets form a major hurdle in the treatment of the gastric cancer. Accumulating evidences suggest that MicroRNAs (miRs) may prove important therapeutic targets/agents for the management of cancers including gastric cancer. Herein, we examined the expression of miR-19a by qRT-PCR in gastric cancer and attempted to explore its potential role. It was found that the expression of miR-19a is significantly (p < 0.05) enhanced in the gastric cancer tissues as well as the gastric cancer cell lines. Inhibition of miR-19a in gastric cancer cells suppressed the proliferation migration and invasion of the gastric cancer cells. Bioinformatic analysis revealed CUL5 to be the potential target of miR-19a. Contrary, to the expression of miR-19a, the expression of CUL5 was significantly (p < 0.05) downregulated in all the gastric cancer tissues and cell lines. However, inhibition of miR-19a in SNU-16 gastric cancer cells could cause upsurge of CUL5 expression. Overexpression of CUL-5 was found to exhibit similar effects on the proliferation, migration and invasion of the SNU-16 gastric cancer cells as that of miR-19a suppression. Additionally, overexpression of CUL5 could at least partially abolish the effects of miR-19a suppression on the proliferation, migration and invasion of SNU-16 gastric cancer cells. Finally, overexpression of miR-19a caused inhibition of the xenografted tumors in vivo indicating the potential of miR-19a as therapeutic target for gastric cancer.

Abstract 899: Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells

Abstract Because about 10% of gastric cancers show amplification of FGFR2, inhibition of FGFR2 activation has been regarded as one of therapeutic targets. AZD4547, a selective inhibitor of the FGFR1-3 tyrosine kinases, was developed to inhibit FGFR signaling; however, its efficacy is limited by emergency of the acquired resistance. We tried to clarify a resistance mechanism against the FGFR inhibitor. The cell line resistant against AZD4547 was established using SNU-16 (SNU16R), an FGFR2-amplified gastric cancer cell line, by culturing with increasing concentration of AZD4547. The expression levels of FGFR or phosphorylated FGFR (pFGFR), EphB3 and downstream signaling molecules, proteins involved in epithelial-mesenchymal transition (EMT) and apoptosis, were determined by Western blot. Cell viability was measured with EZ-Cytox kit reagent (DoGen, Seoul, Korea). Relative level of tyrosine phosphorylation of receptor tyrosine kinases (RTKs) was evaluated with Proteome ProfilerTM array by human phosphor-RTK kit (R&amp;D Systems). Apoptosis was determined by Annexin V-FITC apoptosis detection kit (BioBud, Seoul, Korea). Cell cycle assay was performed using Beckman Coulter flow cytometer and WinMDI for determination of cell numbers in G1, S, G2/M phases. Here we show the following: 1) establishment of a gastric cancer cell line (SNU16) resistant to AZD4547 (SNU16R); 2) expression of EphB3 receptor in SNU16R and induction of apoptosis and cell cycle arrest as well as inhibition of cell proliferation and suppressed expression of EMT markers under the treatment EphB3 inhibitor in SNU16R; 3) demonstration of mTOR as a downstream signaling cascade molecule of EphB3 by showing increased expression of phosphorylated (p-) mTOR and its downstream molecules in SNU16R and suppressed expression of p-mTOR by the EphB3 inhibitor. Cell cycle arrest, inhibition of cell proliferation and suppressed expression of p-mTOR under treatment with everolimus were also shown. We concluded that phosphorylation of mTOR through activation of EphB3 receptor is one of the mechanisms that confer resistance to AZD 4547, a selective FGFR inhibitor, in gastric cancer cells. Citation Format: Suk-young Lee, YooJin Na, YoonA Jung, JungLim Kim, Sang Cheul Oh, Dae-Hee Lee. Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 899.

Abstract B037: The establishment and evaluation of anti-ASC amino acid transporter 2 (ASCT2) antibody as a novel therapeutic antibody for gastric cancer

Abstract Introduction: Glutamine is the most abundant amino acid in blood, serving as a major biosynthetic substrate in nucleic acids and proteins. Recent studies have shown that glutamine contributes to cancer cell proliferation, invasion, and metastasis, so targeting the glutamine pathway is expected to be a feasible approach to cancer treatment. In cancer cells, the Na+-dependent neutral amino acid exchanger alanine-serine-cysteine (ASC) transporter 2 (ASCT2) plays a key role in the glutamine uptake. ASCT2 has been shown to be overexpressed in a variety of cancerous tissues, including gastric cancer. In this study, we established a defucosylated humanized anti-human ASCT2 monoclonal antibody, KM8094, that specifically binds to the natural configuration of an extracellular domain of ASCT2. We further evaluated the efficacy of KM8094 as a therapeutic agent for gastric cancer treatment using gastric cancer cell lines. Materials and Methods: Immunohistochemistry was conducted on tumor tissue specimens. The ASCT2 expression on tumor cell-surface was analyzed by flow cytometry using KM8094 and PE-conjugated anti-human IgG monoclonal antibody. The glutamine uptake in human gastric cancer cells (SNU-16) was examined using 14C-labeled glutamine. Cell growth was measured by a CellTiter-Glo Luminescent Cell Viability Assay after 72-h treatment with KM8094 or control IgG. The in vivo anti-tumor activity of KM8094 combined with docetaxel was examined using gastric cancer xenograft models. Results: In the immunohistochemical analysis, we observed the high expression of ASCT2 in human tumor tissues, including gastric cancer. To evaluate the inhibitory activity on the ASCT2 transporter function and tumor cell growth of KM8094, we performed a glutamine uptake assay using 14C-glutamine. As a result, KM8094 inhibited the 14C-glutamine uptake in SNU-16 gastric cancer cells expressing ASCT2. KM8094 also suppressed cell growth of multiple gastric cancer cell lines both in vitro and in vivo. These results suggested that KM8094 has therapeutic potential for gastric cancer by inhibiting ASCT2 transporter activity. Furthermore, in an SNU-16 gastric cancer xenograft model, combination therapy of KM8094 and docetaxel, a therapeutic drug for gastric cancer, showed a more potent antitumor effect than either agent alone. Conclusions: KM8094 may be a potential new therapeutic agent for gastric cancer expressing ASCT2, as it exhibited antitumor growth activities both in vitro and in vivo. Citation Format: Aya Sasakawa, Kenta Hosomi, Toshihiko Ishii, Hiroshi Ando, Masayo Suzuki, Minami Imaizumi, Jian Zou, Noriyuki Kasai, Kazuya Yamano, Kazuyasu Nakamura, Ryuichiro Nakai. The establishment and evaluation of anti-ASC amino acid transporter 2 (ASCT2) antibody as a novel therapeutic antibody for gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B037.

Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26.

Several novel fusion transcripts were identified by next-generation sequencing in gastric cancer; however, the breakpoint junctions have yet to be characterized. The present study characterized a plethora of APIP-FGFR2 genomic breakpoints in the SNU-16 gastric cancer cell line, which harbored homogeneously staining regions (hsrs) and double minute chromosomes. Oligonucleotide microarrays revealed high-level amplifications at chromosomes 8q24.1 (0.8 Mb region), 10q26 (1.1 Mb) and 11p13 (1.1 Mb). These amplicons contained MYC and PVT1 at chromosome 8q24.1, BRWD2, FGFR2 and ATE1 at chromosome 10q26, and 24 genes, including APIP, CD44, RAG1 and RAG2, at chromosome 11p13. Based on these findings, reverse transcription-polymerase chain reaction (PCR) was performed using various candidate gene primers to detect possible fusion transcripts, and several products using primer sets for the APIP and FGFR2 genes were detected. Eventually, three in-frame and two out-of-frame fusion transcripts were detected. Notably, PCR analysis of the entire genomic DNA detected three distinct genomic junctions. The breakpoints were within intron 5 of APIP, which contained three distinct breakpoints, and introns 5, 7 and 9 of FGFR2. Fluorescence in situ hybridization showed several fusion signals within hsrs using two short probes (~10-kb segments of a bacterial artificial chromosome clone) containing exons 2–5 of APIP or exons 11–13 of FGFR2. Although, for any given fusion, a multiplicity of transcripts is thought to be created by alternative splicing of one rearranged allele, the results of the present study suggested that genomic fusions of APIP and FGFR2 are generated in hsrs with a diversity of breakpoints that are then faithfully transcribed.

Epithelial-mesenchymal transition confers resistance to selective FGFR inhibitors in SNU-16 gastric cancer cells.

BackgroundUp to 10 % of primary gastric cancers are characterized by FGFR2 amplification, and fibroblast growth factor receptor (FGFR) inhibitors may represent therapeutic agents for patients with these malignancies. However, long-term benefits of the treatment might be limited owing to the occurrence of drug resistance.MethodsTo investigate the mechanisms of resistance to selective FGFR inhibitors, we established three FGFR2-amplified SNU-16 gastric cancer cell lines resistant to AZD4547, BGJ398, and PD173074, respectively.ResultsThe resistant cell lines (SNU-16R) demonstrated changes characteristic of epithelial-to-mesenchymal transition (EMT). In addition, they displayed loss of expression of FGFR2 and other tyrosine kinase receptors concurrent with activation of downstream signaling proteins and upregulation of the transforming growth factor β (TGF-β) level. However, treatment of parental SNU-16 cells with TGF-β1 did not evoke EMT, and pharmacological inhibition of TGF-β receptor I was not sufficient to reverse EMT changes in the resistant cells. Finally, we showed that the SNU-16R cell lines were sensitive to the human epidermal growth factor receptor 2 inhibitor mubritinib and the heat shock protein 90 inhibitor AUY922.ConclusionIn conclusion, we provide experimental evidence that EMT-mediated resistance might emerge in gastric cancer patients following treatment with FGFR inhibitors, and mubritinib or AUY922 treatment may be an alternative therapeutic strategy for these patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s10120-014-0444-1) contains supplementary material, which is available to authorized users.

Inhibition of histone deacetylase 4 increases cytotoxicity of docetaxel in gastric cancer cells.

New treatment options for gastric cancer are in great demand. Histone deacetylases (HDACs) are exciting therapeutic targets, but only the class I HDACs 1, 2, and 3 have been studied in gastric cancer. We have investigated class IIa HDAC expression and inhibition in gastric cancer cells. We measured the level of 27 (phospho)proteins related to class IIa HDAC expression and function in ten laser-capture microdissection gastric tumor samples compared to patient-matched adjacent normal mucosa. Following, we evaluated class IIa HDAC inhibition by MC1568 in SNU-16 gastric cancer cells alone and in combination with cisplatin or docetaxel. We demonstrate for the first time an increase of HDAC4 in gastric tumor cells. HDAC4 inhibition had a synergistic effect with docetaxel treatment, shifting the cellular response from a cytostatic to a cytotoxic phenotype. This effect was associated with increased levels of cleaved caspases 3 and 9 and increased acetylated histone H3 Lys9/Lys14. These data support in vivo studies investigating the potential clinical use of HDAC4 inhibitors in combination with docetaxel for the treatment of gastric cancer, lowering treatment doses of docetaxel to reduce the burden of adverse side effects on patients.

CEACAM6 promotes tumor migration, invasion, and metastasis in gastric cancer

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) shows increased expression in a wide variety of human cancers, and its over-expression is associated with enhanced migration, invasion, and in vivo metastasis. Here, we reported that CEACAM6 was up-regulated in gastric cancer (GC) cell lines and tumor tissues. Over-expression of CEACAM6 in MKN-45 and SGC-7901 GC cells promoted migration and invasion in vitro and metastasis in athymic mice, whereas migration and invasion of MKN-28 and SNU-16 GC cells were suppressed by knockdown of CEACAM6. We also observed that steroid receptor coactivator (C-SRC) phosphorylation was increased when CEACAM6 was over-expressed in SGC-7901 cells. Taken together, these results suggested that CEACAM6 functions as an oncoprotein in GC and may be an important metastatic biomarker and therapeutic target.

Downregulation of tumor suppressor MBP-1 by microRNA-363 in gastric carcinogenesis

Gastric carcinoma is one of the most common malignancies and the second most lethal cancer worldwide. The mechanisms underlying aggressiveness of gastric cancer still remain obscure. c-Myc promoter binding protein 1 (MBP-1) is a negative regulator of c-myc expression and ubiquitously expressed in normal human tissues. It is produced by alternative translation initiation of α-enolase gene. Both MBP-1 and α-enolase are involved in the control of tumorigenesis including gastric cancer. MicroRNAs (miRNAs) are involved in tumorigenesis and could have diagnostic, prognostic and therapeutic potential. In this study, whether miRNAs modulate tumorigenesis of gastric cancer cells through targeting MBP-1 was evaluated. We found that miR-363 targets 3'-untranslated region of human MBP-1/α-enolase messenger RNA. The exogenous miR-363 promotes growth, viability, progression, epithelial-mesenchymal transition and tumorsphere formation of SC-M1 gastric cancer cells through downregulation of MBP-1, whereas the knockdown of endogenous miR-363 suppresses tumorigenesis and progression of SC-M1 cells via upregulation of MBP-1. The miR-363/MBP-1 axis is also involved in the control of carcinogenesis in KATO III and SNU-16 gastric cancer cells. Furthermore, miR-363 induces the xenografted tumor growth and lung metastasis of SC-M1 cells through MBP-1 in vivo. Taken together, these results suggest that miR-363 plays an important role in the increment of gastric carcinogenesis via targeting MBP-1.

Abstract 4174: miR-17-92 as a therapeutic target in gastric cancer.

Abstract We previously reported that 82 gastric cancer patients overexpress miR-17-5p, compared with healthy volunteers [median tumor/normal ratio, 1.6; false discovery rate&amp;lt;0.001]. Although 13q31.3 amplification has been reported in gastric cancer, the exact frequency of MIR17HG gene amplification in gastric cancer has not been systematically investigated. Using combined CGH and expression array analyses, we identified genomic abnormalities associated with miR-17-5p expression. No copy number alteration of MIR17HG was identified among the 32 study patients. Median or higher levels of miR-17-5p expression in gastric cancer patients were associated with copy number gain and overexpression of known oncogenes such as MYC and FGFR2. Based on mRNA expression microarray, genes differentially expressed between the high miR-17-expressing tumors and healthy volunteers were significantly enriched in MYC target genes (LS P value, 0.007). MYC knockdown in SNU-16 gastric cancer cells significantly decreased miR-17-92 expression. Although transcriptional activation, rather than gene amplification, was identified as the primary mechanism underlying miR-17-92 overexpression in gastric cancer, miR-17-5p inhibition led to suppressed monolayer and anchorage-independent growth and increased G1/S phase ratio in KATO-III cells which harbor FGFR2 gene amplification. Hence, miR-17-92 may serve as a therapeutic target in gastric cancer (supported by the Proteogenomic Research Program through the National Research Foundation of Korea funded by the Korean Ministry of Education, Science and Technology; and by Korean National Cancer Center Grant 121005). Citation Format: Dong M. park, Seok C. Lee, Hark K. Kim. miR-17-92 as a therapeutic target in 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 4174. doi:10.1158/1538-7445.AM2013-4174

Prostaglandin Reductase 2 Modulates ROS-Mediated Cell Death and Tumor Transformation of Gastric Cancer Cells and Is Associated with Higher Mortality in Gastric Cancer Patients

Various prostanoids and peroxisome proliferator-activated receptor γ (PPARγ) ligands play an important role in gastric cancer. Previously, we demonstrated that prostaglandin reductase 2 (PTGR2) catalyzes the reduction of the PPARγ ligand 15-keto-PGE(2) into 13,14-dihydro-15-keto-PGE(2). Here, we present functional data and clinical relevance for the role of PTGR2 in gastric cancer. Using lentiviral technology in AGS and SNU-16 gastric cancer cell lines, we either down-regulated or overexpressed PTGR2. In vitro analysis showed that PTGR2 knockdown resulted in decreased proliferation rate and colony formation, and in vivo xenograft models showed slower growth of tumors. Mechanistically, PTGR2 knockdown induced cell death, altered mitochondrial function, and increased reactive oxygen species production, which led to activation of ERK1/2 and caspase 3, with increased Bcl-2 and suppressed Bax expression. PTGR2 overexpression showed the opposite outcomes. Clinically, immunopathological staining showed strong PTGR2 expression in the gastric tumor portion, relative to nearby nontumor portions, and its expression negatively correlated with survival of patients with intestinal-type gastric cancer. Finally, in contrast to PTGR2-overexpressing cells, PTGR2-knockdown cells were more sensitive to cisplatin and 5-fluorouracil. Taken together, our findings not only provide functional and mechanistic evidence of the involvement of PTGR2 in gastric cancer, but also provide clinical observations affirming the significance of PTGR2 in gastric cancer and suggesting that PTGR2-target based therapy is worth further evaluation.

Tumor suppressor, AT motif binding factor 1 (ATBF1), translocates to the nucleus with runt domain transcription factor 3 (RUNX3) in response to TGF-β signal transduction

Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor, and loss of heterozygosity at ATBF1 locus occurs frequently in gastric cancers. We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer and that ATBF1 enhanced the promoter activity of p21Waf1/Cip1. We also found that ATBF1 moves between cytoplasm and nucleus, but the precise mechanism of translocation is unknown. In this study, we investigated the mechanism of ATBF1 translocation to the nucleus with the runt domain transcription factor 3 (RUNX3) in cooperation with TGF-β signal transduction. Materials and methods: To analyze the expression of ATBF1 and RUNX3 in gastric cancer cells, we performed immunohistochemistry on 98 resected gastric cancer tissue samples and scored the nuclear staining intensity as grade 0 to grade 5. Co-immunoprecipitation (co-IP) of ATBF1 and RUNX3 was performed. Dual luciferase assays were performed by transfecting ATBF1 and RUNX3 with a p21Waf1/Cip1 reporter vector. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3 in response to TGF-β signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-β1 using confocal laser scanning microscopy. Results: Strong immunohistochemical nuclear staining of ATBF1 was observed in 37 (37.8%) of the gastric cancer tissue samples, and RUNX3 nuclear staining was observed in 15 (15.3%). There was a statistically significant correlation between ATBF1 and RUNX3 nuclear localization (rs=0.433, p<0.001). Co-IP revealed a physical association between ATBF1 and RUNX3. ATBF1 and RUNX3 up-regulated p21Waf1/Cip1 promoter activity synergistically. In SNU16 gastric cancer cells, ATBF1 and RUNX3 were cytoplasmic before TGF-β1 stimulation, but after 24h of TGF-β1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus. Conclusion: ATBF1 associates with RUNX3 and translocates to the nucleus in response to TGF-β signal transduction and might function in the nucleus as tumor suppressor and transcriptional regulator.

S1993 Tumor Suppressor At Motif Binding Factor 1 (ATBF1) Negatively Regulates Cell Growth Through the Nuclear Translocation with Runt Domain Transcription Factor 3 (RUNx3) in Cooperation with TGF-β Signal Transduction

Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor (Nat. Genet., 2005) and frequent loss of heterozygosity at ATBF1 locus in gastric cancer was recently reported (Clin Cancer Res., 2007). We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer (Oncogene, 2001) and ATBF1 enhanced the promoter activity of the p21 (Cancer Res., 2003). We also found that ATBF1 translocates between cytoplasm and nucleus (Int. J. Cancer, 2007), but its precise mechanism has not been fully elucidated. In this study, we investigated the mechanism of nuclear translocation of ATBF1 with runt domain transcription factor 3 (RUNX3) in cooperation with TGF-β signal transduction. Materials and Methods: 1. To analyze the expression of ATBF1 in gastric cancer cells, we performed immunohistochemistry of 123 resected gastric cancer tissues. 2. ATBF1 and RUNX3 were over expressed by transfecting myc-tagged ATBF1 expression vector and flag-tagged RUNX3 expression vector. Immunoprecipitation (IP) between ATBF1 and RUNX3 was performed. 3. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3, we examined the subcellular localization of ATBF1 and RUNX3 in SNU-16 gastric cancer cells treated with from 0.2 to 2.0 ng/ml of recombinant TGF-β1 by confocal mirosope. 4. We confirmed ATBF1 and RUNX3 protein expression in cytoplasm and nucleus extracted fraction by western blot. 5. Cell cycle analyses by using FACScan were performed. 6. Dual-luciferase assays were performed by transfecting ATBF1 and RUNX3 with p21 reporter vector. Results: 1. The nuclear localization of ATBF1 was observed in 33 (26.8%) gastric cancers and the cytoplasm localization of ATBF1 was observed in 61 (49.6%) gastric cancers. ATBF1 expression was not observed in 29 (23.6%) gastric cancers. 2. IP revealed that the physical interaction of ATBF1-RUNX3. 3. In SNU-16 gastric cancer cells, ATBF1 and RUNX3 existed in the nucleus before recombinant TGF-β1 stimulation, but after 24 hours of TGF-β1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus by confocal microscopic observation. 4. Western blot also revealed ATBF1 and RUNX3 protein in the nucleus was increased after TGF-β1 stimulation. 5.ATBF1 induced the accumulation in the G0/G1 phase. 6. ATBF1 and RUNX3 synergistically up-regulated p21 promoter activity. Conclusion: ATBF1 associates with RUNX3 and translocates to the nucleus with RUNX3 by TGF-β signal transduction and works as tumor suppressor and transcription regulator in the nucleus.

Gene expression analysis in human gastric cancer cell line treated with trichostatin A and S-adenosyl-L-homocysteine using cDNA microarray.

Trichostatin A (TSA) and S-adenosyl-L-homocysteine (AdoHcy) have been reported to affect histone modifications. To investigate the effects of two drugs that can reportedly affect chromatin remodeling, we analyzed the gene expression profiles of TSA and AdoHcy in a gastric cancer cell line using 14 K cDNA microarray. The significant analysis of microarray (SAM) identified 98 and 43 differentially expressed genes in TSA and AdoHcy treated sets, respectively, and selected genes were functionally classified. In the gastric cancer cell line, genes related to cell communication, cell growth/maintenance, and morphogenesis were highly expressed with TSA, and genes with cell growth/maintenance, metabolism, oxidoreductase activity were upregulated with AdoHcy. Genes downregulated with TSA included those controlling the cell cycle, cell growth/proliferation, DNA binding, and metabolism, whereas genes involved in calcium signaling, cell growth/proliferation, and metabolism were downregulated with AdoHcy. Furthermore, we identified the genes commonly expressed in both drug treatments. Compared to TSA, AdoHcy did not induce apoptosis in the SNU-16 gastric cancer cell line, and RT-PCR was performed for selective genes to confirm the microarray data. This gene expression profile analysis with TSA and AdoHcy should contribute to a greater understanding of the molecular mechanism of chromatin remodeling and cancer, and provide candidate genes for further studies involving the roles of histone modifications in gastric cancer.