Human GC Research Articles

LncRNA PTCSC3 Alleviates the Postoperative Distant Recurrence of Gastric Cancer by Suppression of lncRNA HOXA11-AS.

IntroductionIt is worldwide accepted that lncRNA PTCSC3 is a tumor suppressor in glioma and thyroid cancer, whereas its role in the recurrence of gastric cancer is unknown.Patients and MethodsWe recruited 80 GC patients (46 males and 34 females, 44 to 68 years, 56.3±6.7 years) in our study. Two human GC cell lines AGS and SNU-1 were transfected with PTCSC3 and HOXA11-AS expression vectors. Then, qPCR was used to detect the level of relative mRNA. Both invasion and migration assays were performed to detect the effect of the lncRNA on gastric cancer cell motility.ResultsIn the present study, we showed that PTCSC3 was downregulated in plasma of gastric cancer patients than in plasma of healthy controls. Follow-up study indicated that PTCSC3 was further downregulated in patients with distant-recurrence but not in patients with local recurrence only or non-recurrence. LncRNA HOXA11-AS was upregulated in plasma of gastric cancer cells than in plasma of healthy controls and was inversely correlated with PTCSC3 in plasma of gastric cancer patients. PTCSC3 overexpression mediated the downregulation of HOXA11-AS in gastric cancer cells, while HOXA11-AS overexpression failed to significantly affect PTCSC3. PTCSC3 overexpression led to inhibited, while HOXA11-AS overexpression led to promoted migration and invasion of gastric cancer cells. In addition, HOXA11-AS overexpression reduced the effects of PTCSC3 overexpression.DiscussionTherefore, lncRNA PTCSC3 alleviates in the postoperative distant recurrence of gastric cancer possible by suppression of HOXA11-AS.

LOXL1-AS1 Drives The Progression Of Gastric Cancer Via Regulating miR-142-5p/PIK3CA Axis.

BackgroundGastric cancer (GC) is a deadly disease, and its incidence is especially high in East Asia including China. Recently, some long non-coding RNA (lncRNAs) have been identified as oncogenes or tumor suppressors. This study aimed to determine the function and mechanism of lncRNA LOXL1-AS1 on the progression of GC.MethodsRT-PCR was done to measure the expression levels of LOXL1-AS1 and miR-142-5p in GC tissues. The association between pathological indexes and LOXL1-AS1 expression was also analyzed. Human GC cell lines AGS and BGC823 were used as cell models. CCK-8 and colony formation assays were conducted to assess the effect of LOXL1-AS1 on the proliferation of GC cell lines. Transwell assay was conducted to determine the influence of LOXL1-AS1 on cell migration and invasion. Furthermore, luciferase reporter assay was carried out to confirm the relationship of miR-142-5p with LOXL1-AS1. Additionally, Western blot was done to detect the regulatory function of LOXL1-AS1 on PIK3CA, a target of miR-142-5p. In vivo experiment was also performed to validate the roles and mechanism of LOXL1-AS1 on the growth and metastasis of GC cells.ResultsLOXL1-AS1 expression in GC samples was significantly increased, which was correlated with unfavorable pathological indexes. Highly expressed LOXL1-AS1 was closely linked to shorter overall survival time and post-progression survival time of the patients. LOXL1-AS1 markedly modulated the malignant phenotypes of GC cells. Additionally, overexpressed LOXL1-AS1 notably reduced the expression of miR-142-5p, but enhanced the expression level of PIK3CA. In vivo experiments further validated that knockdown of LOXL1-AS1 inhibited the growth and metastasis of GC cells via regulating miR-142-5p and PIK3CA.ConclusionLOXL1-AS1 was a sponge of tumor suppressor miR-142-5p in GC, enhanced the expression of PIK3CA indirectly and functioned as an oncogenic lncRNA.

AK5, a novel prognosis marker, inhibits apoptosis and promotes autophagy as well as proliferation in human gastric cancer.

To explore the relationship between AK5 and gastric cancer. The in situ levels of AK5 in the GC tissues from 255 patients were detected by immunohistochemistry (IHC). The correlation between AK5 expression and the clinicopathological parameters was analyzed by Pearson correlation, and the prognostic factors were identified by Cox regression analysis. The transcriptome data of 14 human GC cell lines deposited in the CCLE database were analyzed, and two lines were selected for functional studies. AK5 was knocked down in the AZ521 and MKN74 cells using siRNA, and their proliferation and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay and Annexin-V staining, respectively. In addition, the apoptosis and autophagy of the markers were detected by Western blotting. Patients expressing high AK5 levels in the tumor tissues had significantly shorter survival compared to low-expressing group. In addition, AK5 expression was associated with T stage and N stage and was an independent prognostic factor. AK5 knockdown in the AZ521 and MKN74 cells significantly inhibited proliferation and autophagy, and increased apoptosis. AK5 is a potential prognostic marker and therapeutic target for GC.

Clinical significance of CD8+ T cell immunoreceptor with Ig and ITIM domains+ in locally advanced gastric cancer treated with SOX regimen after D2 gastrectomy

ABSTRACTGastric cancer (GC) development and progression is significantly associated with tumour immune escape. T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibits T-cell responses and is associated with human cancers and T cell exhaustion phenotypes, but its role in cancers remains unclear. TIGIT and programmed cell death protein (PD)-1 levels were detected in 441 human GC specimens using histochemistry. We used flow cytometry to evaluate percentage of TIGIT+ constituting CD8+ T cells of 23 patients with GC who underwent D2 gastrectomy and the S-1 plus oxaliplatin (SOX) regimen. We investigated the influence of SOX regimen and TIGIT functional antibody on CD8 tumour-infiltrating lymphocytes (TILs). Results showed that PD-1 and TIGIT were significantly over expressed in GC and predicted poorer outcome, agreeing with bioinformatics analysis. Significantly reduced percentages of CD8+ TIGIT+ cells were observed in patients after D2 gastrectomy (pre- vs post-surgery, 38 ± 8.7% vs. 26.7% ± 5.2%, p < 0.0001). TIGIT expression on CD8+T cells was modulated by chemotherapeutics (pre- and post-chemotherapy, 31.3 ± 9% vs. 25.1 ± 4.5%, respectively, p = 0.0047) and higher TIGIT expression in post-chemotherapy group was associated with relapsed GC (p = 0.036). In vitro experiments revealed increased CD8+ TIL proliferation and interferon (IFN)-γ production following SOX regimen and TIGIT functional antibody treatments. In conclusion, TIGIT contributes to CD8+ TILs immune dysfunction in patients with GC. Combination of anti-TIGIT therapy and chemotherapy could be considered a therapy for GC.

Differential effects, on oncogenic pathway signalling, by derivatives of the HNF4 \u03b1 inhibitor BI6015

BackgroundGastric cancer (GC) is a highly heterogeneous disease with few “targeted” therapeutic options. Previously, we demonstrated involvement of the transcription factor HNF4α in human GC tumours, and the developmental signal mediator, WNT5A, as a prognostic GC biomarker. One previously developed HNF4α antagonist, BI6015, while not advancing beyond preclinical stages, remains useful for studying GC.MethodsHere, we characterised the antineoplastic signalling activity of derivatives of BI6015, including transfer of the nitro group from the para position, relative to a methyl group on its benzene ring, to the ortho- and meta positions. We assessed binding efficacy, through surface plasmon resonance and docking studies, while biologic activity was assessed by antimitogenic efficacy against a panel of GC cell lines, and dysregulated transcriptomes, followed by pathway and subpathway analysis.ResultsThe para derivative of BI6105 was found substantially more growth inhibitory, and effective, in downregulating numerous oncogenic signal pathways, including the embryonic cascade WNT. The ortho and meta derivatives, however, failed to downregulate WNT or other embryonic signalling pathways, unable to suppress GC growth.ConclusionStraightforward strategies, employing bioinformatics analyses, to facilitate the effective design and development of “druggable” transcription factor inhibitors, are useful for targeting specific oncogenic signalling pathways, in GC and other cancers.

Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer

BackgroundGastric cancer (GC) is a common cancer in Asia and currently lacks a targeted therapy approach. Mesothelin (MSLN) has been reported to be expressed in GC tissue and could be targeted by chimeric antigen receptor (CAR) T cells. Mesothelin targeting CAR-T has been reported in mesothelioma, lung cancer, breast cancer, and pancreas cancer. However, the feasibility of using anti-MSLN CAR T cells to treat GC remains to be studied.MethodsWe verified MSLN expression in primary human GC tissues and GC cell lines and then redirected T cells with a CAR containing the MSLN scFv (single-chain variable fragment), CD3ζ, CD28, and DAP10 intracellular signaling domain (M28z10) to target MSLN. We evaluated the function of these CAR T cells in vitro in terms of cytotoxicity, cytokine secretion, and surface phenotype changes when they encountered MSLN+ GC cells. We also established four different xenograft GC mouse models to assess in vivo antitumor activity.ResultsM28z10 T cells exhibited strong cytotoxicity and cytokine-secreting ability against GC cells in vitro. In addition, cell surface phenotyping suggested significant activation of M28z10 T cells upon target cell stimulation. M28z10 T cells induced GC regression in different xenograft mouse models and prolonged the survival of these mice compared with GFP-transduced T cells in the intraperitoneal and pulmonary metastatic GC models. Importantly, peritumoral delivery strategy can lead to improved CAR-T cells infiltration into tumor tissue and significantly suppress the growth of GC in a subcutaneous GC model.ConclusionThese results demonstrate that M28z10 T cells possess strong antitumor activity and represent a promising therapeutic approach to GC.

Upregulation of TMEFF2 is involved in the antiproliferative effects of vitamin C and tyrphostin AG490 on GES-1 and AGS cells.

Transmembrane protein with epidermal growth factor (EGF)-like and two follistatin motifs 2 (TMEFF2) is downregulated in human gastric cancer, and its levels are associated with tumor aggressiveness. Herein, a positive correlation was identified between serum vitamin C levels (µg/ml) and mRNA levels of TMEFF2 in gastric cancer tissue. TMEFF2 silencing promotes cell proliferation in GES-1 normal human gastric epithelial cells and AGS human gastric adenocarcinoma cells. Notably, vitamin C and AG490 exerted antiproliferative effects on the two cell lines. The present study demonstrated that small interfering (si)-RNA-TMEFF2 exerts pro-proliferative effects on GES-1 and AGS cells. The results revealed that vitamin C significantly inhibited the growth of GES-1 and AGS cells by reducing cell viability, decreasing the expression of proliferating cell nuclear antigen (PCNA), and blocking the STAT3 pathway. Moreover, siRNA-TMEFF2-induced enhanced cell viability and PCNA expression were significantly reversed by additional vitamin C treatment; notably, markedly enhanced TMEFF2 expression was observed. Upregulated TMEFF2 expression was observed in association with the antiproliferative effect of AG490. In conclusion, serum vitamin C content (µg/ml) was positively correlated with the mRNA levels of TMEFF2 in gastric cancer tissue. Exploring novel drugs that target TMEFF2 is a potential therapeutic strategy for blocking human GC.

The novel mechanism of anticancer effect on gastric cancer through inducing G0/G1 cell cycle arrest and caspase-dependent apoptosis in vitro and in vivo by methionine enkephalin.

BackgroundGastric cancer (GC) is the second cause of cancer-related deaths. Methionine enkephalin (MENK), an endogenous opioid peptide, has immunological and antitumor activity.PurposeThe aim of this work was to investigate whether MENK could exhibit activity against human GC in vitro and in vivo.Materials and methodsHuman GC cells were treated with MENK. Cell viability, colony formation, cell morphology, cell cycle, and apoptosis were assessed. The effects of MENK on gene expression of OGFr, Bax, BCL-2, caspase-3, PARP, Ki67, cyclin D1, c-myc, survivin were quantifed by qRT-PCR. Western blot was used to analyze the effects of MENK on protein expression of OGFr, Bax, BCL-2, caspase-3, PARP. The anti-tumor activity of MENK in gastic carcinoma was also investigated with animal experiments.ResultsThe results indicate that MENK could significantly inhibit the growth of human GC cells SGC7901 and HGC27 in a concentration- and time-dependent manner, decrease the number of cell colonies, and arrest cell cycle in the G0/G1 phase by causing a decrease in Ki67, cyclin D1, and c-myc mRNA. Furthermore, MENK could induce tumor cell apoptosis associated with the upregulation of Bax, a corresponding downregulation of BCL-2 and survivin, and activation of caspase-3 and PARP. Moreover, MENK upregulated the expression of opioid receptors (OGFr) in SGC7901 and HGC27 cells. The interaction between MENK and OGFr in SGC7901 and HGC27 cells appears to be essential for the antitumor activity of MENK.ConclusionWe conclude that MENK may be a potential drug for the treatment of GC.

Knockdown of TIPE2 increases the proliferation in lipopolysaccharide-stimulated gastric cancer cells

BackgroundGastric cancer (GC) is one of the most common malignant diseases with high morbidity and mortality, especially in Asian countries. During the GC developing progress, TIPE2, a member of TNF-alpha induced protein 8-like (TNFAIP8L) family, may play important roles. However, the molecular mechanisms of TIPE2 contributing to cell proliferation and tumor growth are poorly understood in GC. We performed flow cytometry to detect the cell cycle of TIPE2-knockdown GC cells under lipopolysaccharide (LPS) stimulation.MethodsWe measured TIPE2 expression in tumor samples from 46 human GC patients at mRNA level by Realtime PCR and in 68 pairs of GC tissues at protein level by immunohistochemistry. We established stable TIPE2 knockdown SGC7901 and BGC823 cell lines and performed CCK-8 and EdU proliferation assays under the stimulation of LPS. And then we analyzed AKT, IκBα and ERK phosphorylation levels, as well as cycle related proteins CDK4 and CyclinD3 in the stable TIPE2 knockdown SGC7901 and BGC823 cells.ResultsOur present studies indicated that the expression of TIPE2 was significantly decreased in tumor tissues compared to distant mucosa tissues in human GC patients. TIPE2 inhibited proliferation stimulated by LPS in SGC7901 and BGC823 cells. Silencing of TIPE2 significantly decreased cell G0/G1 phase ratio and increased G2/M phase. TIPE2 knockdown SGC7901 and BGC823 cells declined AKT and IκBα phosphorylation. TIPE2’s action on GC cell cycle was.ConclusionsOur results demonstrated that TIPE2 is a novel tumor suppressor gene that inhibits GC growth may mediated via AKT and IκBα phosphorylated activation. We revealed that TIPE2 may effectively interdict neoplasm development, which has potential clinical application values for GC targeted therapies.

DDIT4 promotes gastric cancer proliferation and tumorigenesis through the p53 and MAPK pathways

BackgroundGastric cancer (GC) is one of the most common malignancies worldwide, particularly in China. DNA damage-inducible transcript 4 (DDIT4) is a mammalian target of rapamycin inhibitor and is induced by various cellular stresses; however, its critical role in GC remains poorly understood. The present study aimed to investigate the potential relationship and the underlying mechanism between DDIT4 and GC development.MethodsWe used western blotting, real-time polymerase chain reaction, and immunohistochemical or immunofluorescence to determine DDIT4 expression in GC cells and tissues. High-content screening, cell counting kit-8 assays, colony formation, and in vivo tumorigenesis assays were performed to evaluate cell proliferation. Flow cytometry was used to investigate cell apoptosis and cell cycle distribution.ResultsDDIT4 was upregulated in GC cells and tissue. Furthermore, downregulating DDIT4 in GC cells inhibited proliferation both in vitro and in vivo and increased 5-fluorouracil-induced apoptosis and cell cycle arrest. In contrast, ectopic expression of DDIT4 in normal gastric epithelial cells promoted proliferation and attenuated chemosensitivity. Further analysis indicated that the mitogen-activated protein kinase and p53 signaling pathways were involved in the suppression of proliferation, and increased chemosensitivity upon DDIT4 downregulation.ConclusionDDIT4 promotes GC proliferation and tumorigenesis, providing new insights into the role of DDIT4 in the tumorigenesis of human GC.

PO-297 Oncogenic activity of SOX1 in gastric cancer

IntroductionGastric cancer (GC) remains one of the leading causes of global cancer mortality due to therapy resistance. The infection with Helicobacter pylori is the major risk factor, particularly in patients carrying H. pylori highly virulent strains. GC contains a sub-population of gastric cancer stem cells (gCSC) with capacity of self-renewal that are critical drivers of tumour initiation, recurrence and therapy resistance. SOX transcription factors play a key role in the regulation and maintenance of stem cells during the embryonic development and are also involved in cancer. SOX1 is a well-established tumour suppressor in several types of cancer and has an oncogenic role in glioblastoma, but its impact in GC remains largely unknown. Therefore, we aimed to elucidate its function in GC and its putative role in the action of H. pylori.Material and methodsFor this, we analysed GC patient samples, we co-cultured human GC cell lines with different H. pylori strains derived from patients of Donostia Hospital and we also performed functional studies of gain and loss of SOX1 function. Also, we performed transcriptomic analysis in SOX1-silenced cells and we carried out computational analysis using the ACRG datasets.Results and discussionsOur results revealed that SOX1 is highly expressed in human GC samples. Moreover, among a subset of SOX genes, SOX1 was the most significantly up-regulated in gCSC derived from GC cell lines and also in cisplatin resistant cells, as well as in response to H. pylori exposure, in a virulence-dependent manner. In GC cells, SOX1-silencing impaired self-renewal capacity in vitro and reduced tumorigenicity and tumour growth in vivo. The up-regulation of SOX1 showed the opposite phenotype, indicating that SOX1 exerts an oncogenic role in GC. Notably, we found that in GC cells SOX1 was required for H. pylori-induced proliferation, acquisition of stem cell-like properties and induction of β-catenin. Furthermore, the transcriptomic analysis revealed a significant alteration of the E2F signalling pathway in SOX1 knockdown cells. Consistently, E2F1 silencing phenocopied SOX1 knockdown. Moreover, the inhibition of SOX1 downregulated E2F1, suggesting that E2F1 could be a downstream effector of SOX1 in GC.ConclusionWe identify for the first time an oncogenic role of SOX1 in GC. Our findings establish that SOX1-E2F-b-catenin is a critical axis for gCSC maintenance and for H. pylori action, postulating that its inhibition could constitute a promising strategy to combat therapy resistance in GC.

Abstract 860: Experimental alpha-radioimmunotherapy for liver metastasis of gastric cancer

Abstract Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Early stage of GC is curable by surgical therapy or endoscopic surgery. However, previous studies reported that 35% of patients is present with evidence of distant metastasis at the time of diagnosis of GC. Metastatic GC is fetal and the patients with metastatic GC has extremely poor prognosis because current therapeutic approaches to metastatic GC are still limited. Therefore, the development of new therapeutic options has been eagerly awaited. Liver metastasis of GC (LMGC) is often observed during GC progression. It has been shown that 4-14% of the patients has LMGC. It is believed that LMGC is occurred by blood-borne metastasis of GC cells via blood stream. A previous study reported that approximately 70% of LMGC was positive for human epidermal growth factor receptor 2 (HER2). Therefore, HER2 may be a reliable therapeutic target for LMGC. Alpha-particle radioimmunotherapy (alpha-RIT) is an emerging new therapy, in which radioisotopes emitting cytotoxic alpha-particle radiation are delivered to target cancer cells to kill the cells by target-specific antibody. Among alpha-particle emitters, astatine-211 (At-211) is a promising emitter because it emits highly cytotoxic alpha-particles during its decay process with a half-life of 7.2 hours. The aim of this study is to evaluate the therapeutic efficacy and toxicity of At-211-labled anti-HER2 antibody trastuzumab ([At-211]trastuzumab) in a preclinical mouse model of HER2-positive LMGC. We established a mouse model of LMGC by injection of luciferase-labelled HER2-positive human metastatic GC cells (NCI-N87) into splenic vein of severe combined immunodeficiency mouse. Biodistribution studies revealed that the maximum tumor uptake of [At-211]trastuzumab was about 12% of injected dose per tissue gram 24 hours after injection. In experimental therapies, a systemic injection of [At-211]trastuzumab (1 MBq) significantly reduced a tumor burden in the liver and prolonged the survival. Transient but recoverable leukocytopenia was observed in mice received 1 MBq of [At-211]trastuzumab at 5-7 days after injection. No body weight loss was found during the observation period. In this study, we provides a preclinical evidence that alpha-RIT using [At-211]trastuzumab may be a new therapeutic option against LMGC. Citation Format: Huizi Keiko Li, Sumitaka Hasegawa. Experimental alpha-radioimmunotherapy for liver metastasis of gastric cancer [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 860.

PO-110 Targeted alpha-therapy for gastric cancer metastasized to liver in mice

IntroductionGastric cancer often metastasizes to liver in its advanced stage. Liver metastasis of gastric cancer (LMGC) is a fetal medical condition and the patients with LMGC have extremely poor prognosis because of the lack of efficient therapeutics. Therefore, the development of new therapeutic options has been eagerly awaited. The aim of this study is to evaluate targeted alpha-particle radiation therapy for LMGC in a preclinical mouse model.Material and methodsAstatine-211 (At-211), an alpha-particle emitter radionuclide, was produced by irradiation of alpha-particles to bismuth-209 using an AVF accelerator at our institute. An anti-HER2 antibody trastuzumab was conjugated with At-211 to produce alpha-emitting antibodies targeting HER2 ([At-211]-trastuzumab). To generate a mouse model of LMGC, we injected luciferase-labelled HER2-positive human metastatic NCI-N87 GC cells into splenic vein of severe combined immunodeficiency mouse. Tissue and tumour distribution of [At-211]-trastuzumab was examined in the LMGC mouse model. Therapeutic efficacy and toxicities of [At-211]-trastuzumab were evaluated in the animal model. All animal experiments conducted in this study were approved by the Animal Care and Use Committee of our institute and were undertaken in compliance with the institutional guidelines regarding animal care and handling.Results and discussionsBiodistribution studies showed that the maximum uptake of [At-211]trastuzumab in the liver metastatic tumours was approximately 12% of injected dose per tissue gram at 24 hours after injection.A systemic injection of [At-211]trastuzumab (1 MBq) significantly reduced a tumour burden in the liver and extended the survival of model mouse. Transient leukocytopenia was observed in mice received 1 MBq of [At-211]trastuzumab at 5–7 days after injection. No body weight loss was so far found in the mice treated with [At-211]trastuzumab.ConclusionOur preclinical study provides the evidence that targeted alpha-therapy using [At-211]-trastuzumab is effective for LMGC.

Luteolin exerts an anticancer effect on gastric cancer cells through multiple signaling pathways and regulating miRNAs.

Accumulating studies confirmed that luteolin, a common dietary flavonoid which is widely distributed in plants and has diverse beneficial biological function, including anti-oxidant, anti-inflammation and anticancer properties. However, the detail mechanisms of luteolin on GC are poorly understood. Here, we investigated the anticancer effect of luteolin in GC cells in vitro and in vivo. Luteolin reduced the cell viability in a time and dose-dependent manner. Luteolin significantly inhibited cell cycle progress, colony formation, proliferation, migration, invasion and promoted apoptosis in vitro and in vivo. Luteolin also regulated these biological effects associated regulators. Mechanically, luteolin treatment regulated Notch1, PI3K, AKT, mTOR, ERK, STAT3 and P38 signaling pathways and modulated a series of miRNAs expression. These findings provide novel insight into the molecular function of luteolin which suggest its potential as a therapeutic agent for human GC.

MiR-638 acts as a tumor suppressor gene in gastric cancer.

Gastric cancer is one of the major causes of cancer mortality. Several microRNAs play a role in the tumor growth and invasion. However, the underlying molecular mechanism remains poorly understood. We detected the miR-638 expression levels in tumor samples and adjacent noncancerous tissues from 68 patients with gastric cancer as well as in the gastric cancer cell line SGC-7901 and SC-M1. The cell cycle was analyzed by flow cytometry, cell proliferation was observed by CCK-8 assay and cell invasion was detected using Transwell assay. MiR-638 was down-regulated in human GC tissues and its expression level was negatively correlated to TNM stage and lymph metastasis. In the cell lines, aberrant expression of miR-638 was related to the cell proliferation, cell cycle and invasion. We also found that SOX2 had a negative correlation with miR-638 in GC tissues, and miR-638 overexpression could decrease SOX2 expression level by directly binding the 3’-UTR of SOX2. in vitro, down-regulating SOX2 by siRNA could counteract the effect of miR-638 inhibitor on GC cells proliferation and invasion. Our results demonstrate that miR-638 may play a pivotal role in the growth and invasion of GC.

MiR-34a, as a suppressor, enhance the susceptibility of gastric cancer cell to luteolin by directly targeting HK1

Luteolin is a flavonoid compound derived from Lonicera japonica Thunb, which has been reported to exert anticancer effects on different types of tumors. miRNAs are a kind of endogenous non-coding small RNAs, which involved in occurrence and development of multi cancer, including miR-34a. However, the relationship between miR-34a and luteolin's susceptibility to cancer cells still remains unclear. In this study, we explored the roles of miR-34a and the effects of luteolin on GC cells as well as the underlying mechanism of miR-34a in mediating the susceptibility of GC cell to luteolin. Retrospectively study revealed that miR-34a expression was downregulated in human primary GC tissues compared with non-tumor tissues and low miR-34a expression was associated with a significantly shorter overall survival and disease-free survival. MiR-34a overexpression could inhibit GC cells and induce G1 phase arrest via p53/p21 and MAPK /ERK pathways. Luteolin decreased viability of GC cells in a dose-dependent manner. Meanwhile, miR-34a was found to be markedly upregulated in GC cells induced by luteolin and decreased miR-34a level was found in the artificial luteolin-resistant GC cells. Upregulation of miR-34a in luteolin-resistant GC cell could enhance the sensibility of GC cells to luteolin. On the other hand, miR-34a inhibitor could partly counter the anticancer effect of luteolin. In a further assay, we also found that targeting miR-34a could mediate the susceptibility of mouse xenografts to luteolin. Subsequent study found that HK1 was a direct target of miR-34a and downregulated HK1 mRNA or protein levels were presented after miRNA-34a overexpression in GC cells. Moreover, HK1 protein levels was decreased after luteolin treatment and partly restored when co-treated with luteolin and miR-34a inhibitor. Downregulation of HK1 in luteolin-resistant GC cell could increase the cell's sensitivity to luteolin. Therefore, our findings firstly suggested that miR-34a could modulate the susceptibility of gastric cancer cell to luteolin via targeting HK1, potentially benefiting GC patients' treatment in the future.

DNA helix: the importance of being AT-rich.

The AT-rich DNA is mostly associated with condensed chromatin, whereas the GC-rich sequence is preferably located in the dispersed chromatin. The AT-rich genes are prone to be tissue-specific (silenced in most tissues), while the GC-rich genes tend to be housekeeping (expressed in many tissues). This paper reports another important property of DNA base composition, which can affect repertoire of genes with high AT content. The GC-rich sequence is more liable to mutation. We found that Spearman correlation between human gene GC content and mutation probability is above 0.9. The change of base composition even in synonymous sites affects mutation probability of nonsynonymous sites and thus of encoded proteins. There is a unique type of housekeeping genes, which are especially unsafe when prone to mutation. Natural selection which usually removes deleterious mutations, in the case of these genes only increases the hazard because it can descend to suborganismal (cellular) level. These are cell cycle-related genes. In accordance with the proposed concept, they have low GC content of synonymous sites (despite them being housekeeping). The gene-centred protein interaction enrichment analysis (PIEA) showed the core clusters of genes whose interactants are modularly enriched in genes with AT-rich synonymous codons. This interconnected network is involved in double-strand break repair, DNA integrity checkpoints and chromosome pairing at mitosis. The damage of these genes results in genome and chromosome instability leading to cancer and other 'error catastrophes'. Reducing the nonsynonymous mutations, the usage of AT-rich synonymous codons can decrease probability of cancer by above 20-fold.

Abstract 5532: Functional role of Friend Leukemia Integration-1 (FLI1) in gastric carcinogenesis

Abstract Gastric adenocarcinoma (GC) is the 5th most common cancer worldwide but is the 3rd leading cause of cancer death. FLI1 (Friend leukemia integration-1) is an ETS family transcription factor that regulates genes involved in proliferation and differentiation. FLI1 is implicated in tumorigenesis, such as in Ewing’s sarcoma where a translocation creates an EWS-FLI1 oncogenic fusion protein. However, few studies have examined the role of FLI1 in carcinomas. In human breast cancer, overexpression of FLI1 led to inhibition of apoptosis, thereby promoting survival and malignant potential. In functional studies in a murine breast cancer model, however, downregulation of FLI1 increased malignant potential. In human GCs, we recently reported that FLI1 expression is inversely correlated with its promoter CpG methylation of the FLI1. To determine if decreased expression occurs in GC epithelial cells or background non-epithelial cells, we analyzed 91 human GC tumors by immunohistochemistry (IHC) for FLI1 and compared them to normal gastric mucosa and intestinal metaplasia (IM) using an IHC composite scoring system accounting for intensity and percentage of epithelial cells expressing FLI1. We found that FLI1 is strongly expressed in normal gastric glandular epithelium and in IM, and that decreased expression was seen in most human GCs (P &amp;lt; 1x10-17 vs normal, P &amp;lt; 1x10-26 vs IM). These findings suggest that FLI1 acts as a tumor suppressor gene in GC. To test this hypothesis, we used an invasion assay and the human GC cell lines NUGC3 and SNU638, which have little to no FLI1 expression, respectively. Cultured GC cells were transduced to overexpress FLI1 or control, along with an eGFP reporter from an IRES (Lv203, Genecopoeia). After selection by puromycin, these GC cells were plated in serum-free media in the upper chamber on a Matrigel coated 8µm pore opaque membrane. Complete media with 10% fetal calf serum was plated in the lower chamber. Images were obtained of the lower membrane with an inverted fluorescent microscope and cellSens imaging software. Overexpression of FLI1 significantly decreased invasion by NUGC3 cells at 24 hours (P = 0.013) but not at 48 hours (P = 0.268) as compared to control. Overexpression of FLI1 significantly decreased invasion by SNU638 cells at both 24 and 48 hours (P = 0.027 and 0.012, respectively) as compared to control. Since NUGC3 cells have low FLI1 expression, we knocked down FLI1 by using a FLI1 shRNA lentiviral system with mCherry from an IRES as a reporter (LvRU6MP, Genecopoeia). Using the same invasion assay, knockdown of FLI1 trended towards a significant increase in invasion as compared to control at 48 hrs (P = 0.14), but not at 24 hrs (P = 0.73). In summary, the combined observations in human GC tissue samples and the functional analyses in GC cells support a tumor suppressor role for FLI1 in human GC and also suggest that FLI1 and/or its target genes may be involved in regulatory mechanisms driving invasive properties of GC. Citation Format: Armando Del Portillo, Elena V. Komissarova, Anne Koehne de Gonzalez, Aqiba Bokhari, Helen Remotti, Jorge Sepulveda, Antonia Sepulveda. Functional role of Friend Leukemia Integration-1 (FLI1) in gastric carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5532. doi:10.1158/1538-7445.AM2017-5532

MiR-215 promotes cell migration and invasion of gastric cancer by targeting Retinoblastoma tumor suppressor gene 1

BackgroundGastric cancer (GC) is one of the most common malignant tumor and has high mortality worldwide. microRNAs (miRNAs) play critical roles in carcinogenesis. Previous studied showed that miR-215 was involved in tumorigenesis and progression. This study was designed to clarify the biological function of miR-215 in GC. MethodsqRT-PCR was used to detect the miR-215 expression in GC tissues and 6 human GC cell lines (AGS, SGC-7901, NCI-N87, GES-1, MKN-45 and BGC-823) as well. Transwell assay was used to investigate the biological function of miR-215 in GC. Luciferase reporter assay was used to confirm its effect on the regulation of the target gene Retinoblastoma tumor suppressor gene 1 (RB1). ResultsmiR-215 was frequently up-regulated in GC tissues compared to adjacent non-tumor tissues and GC cell lines. miR-215 expression level was correlated with the progression of tumor invasion and tumor-node-metastasis (TNM) stage. Over-expression miR-215 in GC cell lines promoted cell migration and invasion. Besides, miR-215 could down-regulate the expression of RB1 in vitro via directly binding to its 3′-untranslated region (UTR), while the expression of RB1 would suppress the miR-215-indueced GC cell migration and invasion. ConclusionsmiR-215 promoted cell migration and invasion of gastric cancer by directly targeting RB1.

MiR-148b inhibits glycolysis in gastric cancer through targeting SLC2A1.

Although the molecular biology of GC has been well characterized, early diagnostic biomarkers and effective therapeutic options in gastric cancer are still under investigation. Here, we found that miR‐148b expression decreased in human gastric cancer tissues compared with matched adjacent nontumor tissues by q‐PCR analysis and in situ hybridization. Further investigation revealed that overexpression of miR‐148b limited glycolysis including glucose consumption, lactate production in gastric cancer cell lines BGC‐823 and MKN45. Bioinformatics prediction uncovered that a dedicated transporters solute carrier family 2 member 1 (SLC2A1), also called GLUT1, was the direct target of miR‐148b. The target effects were further confirmed by luciferase assay and western blot analysis. Besides, a reverse correlation was observed between relative SLC2A1 and miR‐148b expression in human GC tissues compared with matched adjacent nontumor tissues. Subsequently, SLC2A1 suppression by SLC2A1 siRNA or specific inhibitor restricted the reduced effects of glycolysis mediated by miR‐148b while SLC2A1 overexpression abrogated the effect of miR‐148b on glycolysis. Our findings provided new evidence of miR‐148b in GC development through restraining glycolysis, highlighting the role of miR‐148b as a new target for GC treatment.