BACKGROUND & AIMS: We aimed to identify long noncoding RNAs (lncRNAs) that are up-regulated in gastric cancer tissues from patients and study their function in gastric tumor metastasis. METHODS: We collected gastric tumor and nontumor tissues from patients in China and analyzed levels of lncRNAs by microarray analysis, proteins by immunohistochemistry, and RNAs by quantitative reverse-transcription polymerase chain reaction; we compared these with survival times of patients and tumor progression. RNA levels were knocked down or knocked out in BGC-823, SGC-7901, and MKN45 cell lines using small interfering or short hairpin RNAs or clustered regularly interspaced short palindromic repeats (ie, CRISPR)/CRISPR associated protein 9 (ie, Cas9) vectors. Genes were overexpressed from transfected plasmids in HGC-27 cells. Cells were analyzed by Northern blot and immunoblot, polysome profiling assay, and cell invasion assay. Cells were injected into the tail veins or spleens of nude mice or SCID mice; lung and liver tissues were collected, and metastases were counted. lncRNAs were cloned by using rapid amplification of complementary DNA ends. Their interactions with other genes were determined by RNA pulldown and mapping assays. RESULTS: In microarray analyses, we identified 151 lncRNAs expressed at significantly higher levels in gastric tumor vs nontumor tissues. Levels of an lncRNA that we called gastric cancer metastasis associated long noncoding RNA (GMAN) were increased in gastric tumor tissues, compared with nontumor tissues; its up-regulation was associated with tumor metastasis and shorter survival times of patients. The GMAN gene overlaps with the ephrin A1 gene (EFNA1) and was highly expressed in BGC-823 and MKN45 cells. Knockdown of GMAN in these cells did not affect proliferation, colony formation, or adhesion but did reduce their invasive activity in Transwell assays. Ectopic expression of GMAN increased the invasive activity of HGC-27 cells. BGC-823 and MKN45 cells with knockdown of GMAN formed fewer metastases after injection into tail veins of nude mice. Knockdown or knockout of GMAN also reduced levels of ephrin A1 protein in cells. We found that GMAN promoted translation of ephrin A1 messenger RNA into protein by binding to the antisense GMAN RNA (GMAN-AS)-this antisense sequence is also complementary to that of ephrin A1 mRNA. Levels of ephrin A1 protein were also increased in gastric tumors from patients with metastases than in those without metastases. Knockout of ephrin A1 in BGC-823 cells reduced their invasive activity in Transwell assays and ability to form metastases after injection into SCID mice. Ectopic expression of ephrin A1 in BGC823 cells with knockdown or knockout of GMAN restored their invasive activities and ability form metastases in nude or SCID mice. A CRISPR/Cas9-based strategy to disrupt the GMAN gene significantly reduced the numbers of metastases formed from SGC-7901 cells inmice. CONCLUSIONS: We identified an lncRNA, which we call GMAN, that is increased in gastric tumors from patients and associated with survival and formation of metastases. It regulates translation of ephrin A1 mRNA by binding competitively to GMAN-AS. Knockdown or knockout of GMAN or ephrin A1 in gastric cancer cell lines reduces their invasive activity and ability to form metastases after injection into mice. These genes might be targeted to prevent or reduce gastric cancer metastasis.
BACKGROUND & AIMS: microRNAs (miRNAs) are small noncoding RNAs that bind to the 3 0 untranslated regions of mRNAs to promote their degradation or block their translation. Mice with disruption of the trefoil factor 1 gene (Tff1) develop gastric neoplasms. We studied these mice to identify conserved miRNA networks involved in gastric carcinogenesis. METHODS: We performed next-generation miRNA sequencing analysis of normal gastric tissues (based on histology) from patients without evidence of gastric neoplasm (n = 64) and from TFF1-knockout mice (n = 22). We validated our findings using 270 normal gastric tissues (including 61 samples from patients without evidence of neoplastic lesions) and 234 gastric tumor tissues from 3 separate cohorts of patients and from mice. We performed molecular and functional assays using cell lines (MKN28, MKN45, STKM2, and AGS cells), gastric organoids, and mice with xenograft tumors. RESULTS: We identified 117 miRNAs that were significantly deregulated in mouse and human gastric tumor tissues compared with nontumor tissues. We validated changes in levels of 6 miRNAs by quantitative real-time polymerase chain reaction analyses of neoplastic gastric tissues from mice (n = 39) and 3 independent patient cohorts (n = 332 patients total). We found levels of MIR135B5p, MIR196B-5p, and MIR92A-5p to be increased in tumor tissues, whereas levels of MIR143-3p, MIR204-5p, and MIR1333p were decreased in tumor tissues. Levels of MIR143-3p were reduced not only in gastric cancer tissues but also in normal tissues adjacent to tumors in humans and low-grade dysplasia in mice. Transgenic expression of MIR143-3p in gastric cancer cell lines reduced their proliferation and restored their sensitivity to cisplatin. AGS cells with stable transgenic expression of MIR143-3p grew more slowly as xenograft tumors in mice than control AGS cells; tumor growth from AGS cells that expressed MIR143-3p, but not control cells, was sensitive to cisplatin. We identified and validated bromodomain containing 2 (BRD2) as a direct target of MIR143-3p; increased levels of BRD2 in gastric tumors was associated with shorter survival times for patients. CONCLUSIONS: In an analysis of miRNA profiles of gastric tumors from mice and human patients, we identified a conserved signature associated with the early stages of gastric tumorigenesis. Strategies to restore MIR1433p or inhibit BRD2 might be developed for treatment of gastric cancer.
BACKGROUND & AIMS: Wnt signaling contributes to the development of colorectal cancer (CRC). We studied interactions between lysine demethylase 4D (KDM4D or JMJD2D) and b-catenin, a mediator of Wnt signaling, in CRC cell lines and the effects on tumor formation in mice. METHODS: We obtained colorectal tumor specimens and surrounding nontumor colon tissues (controls) from patients undergoing surgery in China; levels of JMJD2D were measured by immunohistochemical or immunoblot analysis. JMJD2D expression was knocked down in CRC (CT26, HCT116, and SW480 cells) using small hairpin RNAs, and cells were analyzed with viability, flow cytometry, colony formation, and transwell migration and invasion assays. Cells were also grown as tumor xenografts in nude mice or injected into tail veins or spleens of mice, and metastases were measured. We performed promoter activity, co-immunoprecipitation, and chromatin immunoprecipitation assays. We also performed studies with Apcmin/thorn and JMJD2D-knockout mice; these mice were crossed, and colorectal tumor formation in offspring (Apcmin/thorn Jmjd2d thorn/thorn and Apcmin/thorn Jmjd2d-/-) was analyzed. JMJD2D-knockout and wild-type (control) mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated CRC; some mice were given the JMJD2D inhibitor 5-chloro-8-hydroxyquinoline (5-c-8HQ) or vehicle to examine the effects of 5-c-8HQ on intestinal tumor formation. RESULTS: Levels of JMJD2D were significantly higher in human colorectal tumors than in control tissues and correlated with levels of proliferating cell nuclear antigen. JMJD2D knockdown reduced CRC cell proliferation, migration, and invasion, as well as growth of xenograft tumors and formation of metastases in mice. JMJD2D was required for expression of b-catenin in CRC cell lines; ectopic expression of JMJD2D increased the promoter activities of genes regulated by b-catenin (MYC, CCND1, MMP2, and MMP9). We found that JMJD2D and b-catenin interacted physically and that JMJD2D demethylated H3K9me3 at promoters of b-catenin target genes. JMJD2D-knockout mice developed fewer colitis-associated colorectal tumors than control mice, and their tumor tissues had lower levels of b-catenin, MYC, cyclin D1, and proliferating cell nuclear antigen than tumors from control mice. Apcmin/thorn Jmjd2d-/-mice developed fewer and smaller colon tumors than Apcmin/thorn mice. Mice given 5-c-8HQ developed smaller and fewer colitisassociated tumors, with lower levels of cell proliferation, than mice given vehicle. Apcmin/thorn mice given 5-c-8HQ also developed fewer tumors in intestines and colons than mice given vehicle. CONCLUSIONS: Levels of the histone demethylase JMJD2D are increased in human colorectal tumors compared with nontumor colon tissues. JMJD2D interacts with b-catenin to activate transcription of its target genes and promote CRC cell proliferation, migration, and invasion, as well as formation of colorectal tumors in mice.
BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. METHODS: We obtained biopsy samples during endoscopy from inflamed and/ or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4DIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin a1 (DEFA1) and supplementation of L-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by highperformance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4DIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4DIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4DIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4DIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.
BACKGROUND & AIMS: NVR 3-778 is a first-in-class hepatitis B virus (HBV) capsid assembly modulator that can inhibit HBV replication. We performed a proof-of-concept study to examine the safety, pharmacokinetics, and antiviral activity of NVR 3-778 in patients with chronic HBV infection. METHODS: We performed a phase 1 study in 73 hepatitis B envelope antigen (HBeAg)-positive patients with chronic HBV infection without cirrhosis. In a 2-part study (part 1 in New Zealand and part 2 in Hong Kong, Singapore, Taiwan, Korea, and the United States), patients were randomly assigned to groups that were given oral NVR 3-778 (100 mg, 200 mg, or 400 mg daily or 600 mg or 1000 mg twice daily) or placebo for 4 weeks. Additional groups received combination treatment with pegylated interferon (pegIFN) and NVR 3-778 (600 mg twice daily) or pegIFN with placebo. RESULTS: Reductions in serum levels of HBV DNA and HBV RNA were observed in patients receiving > 1200 mg/d NVR 3-778. The largest mean reduction in HBV DNA was observed in the group given NVR 3-778 plus pegIFN (1.97 log(10) IU/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.43 log(10) IU/mL and 1.06 log(10) IU/mL, respectively). The mean reduction in HBV RNA was also greatest in the group given NVR 3-778 plus pegIFN (2.09 log(10) copies/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.42 log(10) copies/mL and 0.89 log(10) copies/mL, respectively). There was no significant mean reduction in HBsAg during the 4-week treatment period. There were no discontinuations and no pattern of dose-related adverse effects with NVR 3-778. CONCLUSIONS: In a phase 1 study of HBeAg-positive patients with chronic HBV infection without cirrhosis, NVR 3-778 was well tolerated and demonstrated antiviral activity. The agent reduced serum levels of HBV DNA and HBV RNA, to the greatest extent in combination with pegIFN. The observed reductions in HBV RNA confirmed the novel mechanism of NVR 3-778.
This article has an accompanying continuing medical education activity, also eligible for MOC credit, on page e14. Learning Objective: Upon completion of this CME activity, successful learners will be able to (1) recognize a novel technique for the development of endoscopic treatment, (2) determine easy to detect lymph nodes accurately based on the presence of carbon nanoparticles, and (3) identify how to use a transesophageal endoscopic method for mediastinal lymph node resection.