HMGA1 Is Induced by Wnt/β-Catenin Pathway and Maintains Cell Proliferation in Gastric Cancer
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090069
ISSN1525-2191
AutoresShin-ichi Akaboshi, Sugiko Watanabe, Yuko Hino, Yoko Sekita, Yang Xi, Kimi Araki, Ken–ichi Yamamura, Masanobu Oshima, Takaaki Ito, Hideo Baba, Mitsuyoshi Nakao,
Tópico(s)Cancer-related molecular mechanisms research
ResumoThe development of stomach cancer is closely associated with chronic inflammation, and the Wnt/β-catenin signaling pathway is activated in most cases of this cancer. High-mobility group A (HMGA) proteins are oncogenic chromatin factors that are primarily expressed not only in undifferentiated tissues but also in various tumors. Here we report that HMGA1 is induced by the Wnt/β-catenin pathway and maintains proliferation of gastric cancer cells. Specific knockdown of HMGA1 resulted in marked reduction of cell growth. The loss of β-catenin or its downstream c-myc decreased HMGA1 expression, whereas Wnt3a treatment increased HMGA1 and c-myc transcripts. Furthermore, Wnt3a-induced expression of HMGA1 was inhibited by c-myc knockdown, suggesting that HMGA1 is a downstream target of the Wnt/β-catenin pathway. Enhanced expression of HMGA1 coexisted with the nuclear accumulation of β-catenin in about 30% of gastric cancer tissues. To visualize the expression of HMGA1 in vivo, transgenic mice expressing endogenous HMGA1 fused to enhanced green fluorescent protein were generated and then crossed with K19-Wnt1/C2mE mice, which develop gastric tumors through activation of both the Wnt and prostaglandin E2 pathways. Expression of HMGA1-enhanced green fluorescent protein was normally detected in the forestomach, along the upper border of the glandular stomach, but its expression was also up-regulated in cancerous glandular stomach. These data suggest that HMGA1 is involved in proliferation and gastric tumor formation via the Wnt/β-catenin pathway. The development of stomach cancer is closely associated with chronic inflammation, and the Wnt/β-catenin signaling pathway is activated in most cases of this cancer. High-mobility group A (HMGA) proteins are oncogenic chromatin factors that are primarily expressed not only in undifferentiated tissues but also in various tumors. Here we report that HMGA1 is induced by the Wnt/β-catenin pathway and maintains proliferation of gastric cancer cells. Specific knockdown of HMGA1 resulted in marked reduction of cell growth. The loss of β-catenin or its downstream c-myc decreased HMGA1 expression, whereas Wnt3a treatment increased HMGA1 and c-myc transcripts. Furthermore, Wnt3a-induced expression of HMGA1 was inhibited by c-myc knockdown, suggesting that HMGA1 is a downstream target of the Wnt/β-catenin pathway. Enhanced expression of HMGA1 coexisted with the nuclear accumulation of β-catenin in about 30% of gastric cancer tissues. To visualize the expression of HMGA1 in vivo, transgenic mice expressing endogenous HMGA1 fused to enhanced green fluorescent protein were generated and then crossed with K19-Wnt1/C2mE mice, which develop gastric tumors through activation of both the Wnt and prostaglandin E2 pathways. Expression of HMGA1-enhanced green fluorescent protein was normally detected in the forestomach, along the upper border of the glandular stomach, but its expression was also up-regulated in cancerous glandular stomach. These data suggest that HMGA1 is involved in proliferation and gastric tumor formation via the Wnt/β-catenin pathway. Gastric cancer is the second leading cause of human cancer deaths worldwide, and it is known to be closely associated with chronic inflammation caused by Helicobacter pylori infection.1Parkin DM Bray F Ferlay J Pisani P Global cancer statistics, 2002.CA Cancer J Clin. 2005; 55: 74-108Crossref PubMed Scopus (17337) Google Scholar, 2Ushijima T Sasako M Focus on gastric cancer.Cancer Cell. 2004; 5: 121-125Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar This disease is an example of human oncogenesis that is etiologically induced by extrinsic or environmental factors. Despite preventive therapies and numerous efforts to identify premalignant lesions, gastric cancer is often diagnosed at the advanced stages.3Kapadia CR Gastric atrophy, metaplasia, and dysplasia: a clinical perspective.J Clin Gastroenterol. 2003; 36: S29-S36Crossref PubMed Scopus (108) Google Scholar, 4Clouston AD Timely topic: premalignant lesions associated with adenocarcinoma of the upper gastrointestinal tract.Pathology. 2001; 33: 271-277Crossref PubMed Scopus (19) Google Scholar It is therefore crucial to understand the molecular basis of gastric tumorigenesis to identify diagnostic and therapeutic targets in this cancer. High-mobility group A proteins (HMGA1 and HMGA2, formerly HMGI/Y and HMGI/C, respectively) are non-histone, architectural chromatin proteins that participate in various cell regulation activities, including cell growth and proliferation.5Sgarra R Rustighi A Tessari MA Di Bernardo J Altamura S Fusco A Manfioletti G Giancotti V Nuclear phosphoproteins HMGA and their relationship with chromatin structure and cancer.FEBS Lett. 2004; 574: 1-8Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 6Fusco A Fedele M Roles of HMGA proteins in cancer.Nat Rev Cancer. 2007; 7: 899-910Crossref PubMed Scopus (575) Google Scholar HMGA1 and HMGA2 are encoded by two distinct genes, and are characterized by the presence of three DNA-binding motifs, named AT hooks, which preferentially bind stretches of AT-rich DNA sequences.7Reeves R Molecular biology of HMGA proteins: hubs of nuclear function.Gene. 2001; 277: 63-81Crossref PubMed Scopus (467) Google ScholarHMGA genes are highly expressed during embryonic development, whereas their expression is down-regulated in differentiated cells in adults,8Zhou X Benson KF Ashar HR Chada K Mutation responsible for the mouse pygmy phenotype in the developmentally regulated factor HMGI-C.Nature. 1995; 376: 771-774Crossref PubMed Scopus (549) Google Scholar, 9Chiappetta G Avantaggiato V Visconti R Fedele M Battista S Trapasso F Merciai BM Fidanza V Giancotti V Santoro M Simeone A Fusco A High level expression of the HMGI (Y) gene during embryonic development.Oncogene. 1996; 13: 2439-2446PubMed Google Scholar though both HMGA1 and HMGA2 can be induced by mitogenic stimuli.7Reeves R Molecular biology of HMGA proteins: hubs of nuclear function.Gene. 2001; 277: 63-81Crossref PubMed Scopus (467) Google Scholar, 10Wood LJ Mukherjee M Dolde CE Xu Y Maher JF Bunton TE Williams JB Resar LM HMG-I/Y, a new c-myc target gene and potential oncogene.Mol Cell Biol. 2000; 20: 5490-5502Crossref PubMed Scopus (168) Google Scholar Notably, HMGA genes are frequently reactivated in many types of human cancer, and the overexpression of HMGA proteins is linked to malignant transformation and progression in human cancers, including gastric cancer.11Abe N Watanabe T Masaki T Mori T Sugiyama M Uchimura H Fujioka Y Chiappetta G Fusco A Atomi Y Pancreatic duct cell carcinomas express high levels of high mobility group I(Y) proteins.Cancer Res. 2000; 60: 3117-3122PubMed Google Scholar, 12Reeves R Edberg DD Li Y Architectural transcription factor HMGI(Y) promotes tumor progression and mesenchymal transition of human epithelial cells.Mol Cell Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar, 13Nam ES Kim DH Cho SJ Chae SW Kim HY Kim SM Han JJ Shin HS Park YE Expression of HMGI(Y) associated with malignant phenotype of human gastric tissue.Histopathology. 2003; 42: 466-471Crossref PubMed Scopus (20) Google Scholar, 14Evans A Lennard TW Davies BR High-mobility group protein 1(Y): metastasis-associated or metastasis-inducing?.J Surg Oncol. 2004; 88: 86-99Crossref PubMed Scopus (50) Google Scholar In addition to the above reports, our recent study determined that HMGA2 maintains epithelial–mesenchymal transition in human pancreatic adenocarcinomas.15Watanabe S Ueda Y Akaboshi S Hino Y Sekita Y Nakao M HMGA2 maintains oncogenic RAS-induced epithelial-mesenchymal transition in human pancreatic cancer cells.Am J Pathol. 2009; 174: 854-868Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar However, the biological roles of the different HMGA proteins in different cancer phenotypes, and the induction mechanism of oncogenic HMGA genes are largely unknown. Among the cancer-related signaling pathways, the canonical Wnt pathway, also known as the Wnt/β-catenin pathway, is involved in gastrointestinal carcinogenesis. Wnt ligands engage their receptor complex, stabilize intracellular levels of β-catenin, and allow the nuclear accumulation of β-catenin, together with the transcription factor lymphoid enhancer-binding factor 1/T cell-specific factor, followed by transcriptional activation of the Wnt/β-catenin target genes such as c-myc and cyclin D.16Peifer M Polakis P Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus.Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1143) Google Scholar In the absence of Wnt, destruction complexes consisting of glycogen synthase kinase-3β, the adenomatous polyposis coli protein, and axin, bind and phosphorylate β-catenin, which is thus targeted for ubiquitination and proteolytic degradation. Constitutive activation of the Wnt/β-catenin pathway can occur due to mutations in the adenomatous polyposis coli, β-catenin, and axin genes during cancer development.17Klaus A Birchmeier W Wnt signalling and its impact on development and cancer.Nat Rev Cancer. 2008; 8: 387-398Crossref PubMed Scopus (1254) Google Scholar, 18Clevers H Wnt/β-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4517) Google Scholar, 19Reya T Clevers H Wnt signalling in stem cells and cancer.Nature. 2005; 434: 843-850Crossref PubMed Scopus (3021) Google Scholar, 20Polakis P Wnt signaling and cancer.Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 21Satoh S Daigo Y Furukawa Y Kato T Miwa N Nishiwaki T Kawasoe T Ishiguro H Fujita M Tokino T Sasaki Y Imaoka S Murata M Shimano T Yamaoka Y Nakamura Y AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1.Nat Genet. 2000; 24: 245-250Crossref PubMed Scopus (837) Google Scholar, 22Liu W Dong X Mai M Seelan RS Taniguchi K Krishnadath KK Halling KC Cunningham JM Boardman LA Qian C Christensen E Schmidt SS Roche PC Smith DI Thibodeau SN Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating β-catenin/TCF signalling.Nat Genet. 2000; 26: 146-147Crossref PubMed Scopus (442) Google Scholar The nuclear localization of β-catenin is a hallmark of gastric cancer tissues.23Clements WM Wang J Sarnaik A Kim OJ MacDonald J Fenoglio-Preiser C Groden J Lowy AM β-Catenin mutation is a frequent cause of Wnt pathway activation in gastric cancer.Cancer Res. 2002; 62: 3503-3506PubMed Google Scholar It has been recently reported that K19-Wnt1/C2mE transgenic mice expressing Wnt1, cyclooxygenase-2 (COX2), and microsomal prostaglandin E synthase-1 in gastric epithelial cells, under the control of the cytokeratin 19 (K19) gene promoter. They develop dysplastic stomach tumors, so providing an animal model of human gastric adenocarcinoma.24Oshima H Matsunaga A Fujimura T Tsukamoto T Taketo MM Oshima M Carcinogenesis in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway.Gastroenterology. 2006; 131: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar Interestingly, the activation of both Wnt and inflammation pathways was required for cancer development, since either altered pathway alone did not lead to tumor formation. Collectively, these observations suggest that the Wnt/β-catenin pathway is involved in gastric tumorigenesis, although the precise mechanisms remain undetermined. During our investigations into chromatin factors, we found that HMGA1 is induced by the Wnt/β-catenin pathway and maintains proliferation of gastric cancer cells. Depletion of HMGA1 resulted in reduced cell proliferation. Wnt3a treatment increased HMGA1, as well as c-myc transcripts, and the Wnt3a-induced expression of HMGA1 was inhibited by c-myc knockdown. Overexpression of HMGA1 was consistently correlated with the nuclear accumulation of β-catenin in human gastric cancer tissues. To visualize the Hmga1 protein in vivo, transgenic mice expressing endogenous Hmga1 fused to enhanced green fluorescent protein (EGFP) were generated and crossed with K19-Wnt1/C2mE mice. Expression of Hmga1-EGFP was normally found in the forestomach, along the upper border of the glandular stomach. In contrast, Hmga1-EGFP was up-regulated in cancerously proliferative glandular stomach. Based on the results of the present study, we discuss the role of HMGA1 in gastric tumor formation via the Wnt/β-catenin pathway. AGS, KATO-III, and Panc1 cells (American Type Culture Collection, Manassas, VA), as well as HEK293 cells (Health Science Research Resources, Osaka, Japan) were used. Two gastric cancer cell lines, HSC39 and HSC57, were a gift from Dr. K. Yanagihara and Dr. T. Ushijima (National Cancer Center Research Institute, Tokyo, Japan). The culture conditions were: RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% (v/v) heat-inactivated fetal bovine serum for AGS, HSC39, HSC57, and KATO-III cells; 1:1 mixture of Dulbecco’s modified Eagle’s minimum essential medium and Ham’s F-12 nutrient medium supplemented with 10% fetal bovine serum for Panc1 cells; and low glucose Dulbecco’s modified Eagle’s minimum essential medium supplemented with 10% fetal bovine serum for HEK293 cells. AGS cells (1 × 105/well) were grown in 6-well plates and treated with 100 μmol/L NS-398 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) or 100 μmol/L indomethacin (Wako Pure Chemical Industries, Ltd.) for 48 hours. Secreted Wnt3a was prepared from culture medium of L9 cells stably expressing Wnt3a, which were a gift from Dr. S. Takada (National Institutes of Natural Sciences, Okazaki, Japan). HEK293 cells were treated with 50% Wnt3a-condition medium for 48 hours. Small interfering (si)RNA duplexes were designed for targeting mRNAs encoding human HMGA1, HMGA2, β-catenin, and c-myc (Japan Bio Services Co., Ltd., Saitama, Japan), and are listed in Table 1. The selected siRNA sequences were submitted to human genome and Expressed Sequence Tags databases to ensure their target specificities. Validated stealth RNA interference against c-myc and its negative control was obtained from Invitrogen (Carsbad, CA). The siRNAs were transfected into the cells using Oligofectamine RNAiMAX (Invitrogen, Carsbad, CA).Table 1Small Interfering RNAs Used in this StudyNamesiRNA sequenceHMGA1 si-S5′-GUGCCAACACCUAAGAGACCUTT-3′HMGA1 si-AS5′-AGGUCUCUUAGGUGUUGGCACTT-3′HMGA1 si-S25′-GCAGGAAAAGGACGGCACUTT-3′HMGA1 si-AS25′-AGUGCCGUCCUUUUCCUGCTT-3′HMGA2 si-S5′-CCGGUGAGCCCUCUCCUAATT-3′HMGA2 si-AS5′-UUAGGAGAGGGCUCACCGGTT-3′c-myc si-S5′-CUAUGACCUCGACUACGACTT-3′c-myc si-AS5′-GUCGUAGUCGAGGUCAUAGTT-3′stealth c-myc-S5′-UUUCAACUGUUCUCGUCGUUUCCGC-3′stealth c-myc-AS5′-GCGGAAACGACGAGAACAGUUGAAA-3′β-catenin si-S5′-CAGUCUUACCUGGACUCUGTT-3′β-catenin si-AS5′-CAGAGUCCAGGUAAGACUGTT-3′GL3 si-S5′-CUUACGCUGAGUACUUCGATT-3′GL3 si-AS5′-UCGAAGUACUCAGCGUAAGTT-3′ Open table in a new tab Cell proliferation was assessed by seeding AGS, HSC57, and KATO-III cells (1 × 105/well) into 6-well plates. The cells were transfected with HMGA1, HMGA2, or control siRNAs (50 pmol) on day 0, using oligofectamine RNAiMAX, according to the manufacturer’s protocols. The number of viable cells was counted using a hemocytometer. Data were obtained from three independent experiments. Two micrograms of the total RNAs were treated with DNase I (Roche Diagnostics, Mannheim, Germany) and reverse-transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). PCR amplification was then performed using specific primers for the indicated transcripts (Tables 2 and 3). For quantification, real-time PCR analysis was performed using Power SYBR Green PCR Master Mix on an ABI Prism 7500 Sequence Detector (Applied Biosystems). PCR amplification was repeated at least three times from more than three independent experiments. The relative fold induction was quantified using the comparative threshold cycle method, and β-actin was used as a normalization control. Primer sets are listed in Table 3.Table 2Oligonucleotides Used for the PCRNamePrimer sequenceHuman HMGA1 S5′-TGAGTCCCGGGACAGCACTGGTAG-3′ HMGA1 AS5′-GCGGCTAAGTGGGATGTTAGCCTTG-3′ HMGA2 S5′-CAGGATGAGCGCACGCGGTGAGGGC-3′ HMGA2 AS5′-CCATTTCCTAGGTCTGCCTCTTGGC-3′ c-myc S5′-TCGTCTCAGAGAAGCTGGCCT-3′ c-myc AS5′-CTTTTCCACAGAAACAACATCG-3′ β-catenin S5′-CAGTTGCTTGTTCGTGCACAT-3′ β-catenin AS5′-CAAGTCCAAGATCAGCAGTCTC-3′ GAPDH S5′-GATGCCCCCATGTTCGT-3′ GAPDH AS5′-CAGGGGTCTTACTCCTTGGA-3′Mouse Hmga1 S5′-ATGAGCGAGTCGGGCTCAAAG-3′ Hmga1 AS5′-TCACTGCTCCTCCTCAGAG-3′ Gapdh S5′-ATCACCATCTTCCAGGAGCGAG-3′ Gapdh AS5′-GTTGTCATGGATGACCTTGGCC-3′ Open table in a new tab Table 3Oligonucleotides Used for the Quantitative Real-time PCRNamePrimer sequenceHuman HMGA1 S5′-TCCAGGAAGGAAACCAAGG-3′ HMGA1 AS5′-AGGACTCCTGCGAGATGC-3′ c-myc S5′-TGCTCCATGAGGAGACACC-3′ c-myc AS5′-CTTTTCCACAGAAACAACATCG-3′ β-catenin S5′-GCTTTCAGTTGAGCTGACCA-3′ β-catenin AS5′-CAAGTCCAAGATCAGCAGTCTC-3′ Cyclin D1 S5′-GAAGATCGTCGCCACCTG-3′ Cyclin D1 AS5′-GACCTCCTCCTCGCACTTCT-3′ YWHAZ S5′-AGACGGAAGGTGCTGAGAAA-3′ YWHAZ AS5′-TCAAGAACTTTTCCAAAAGAGACA-3′ β-actin S5′-CCAACCGCGAGAAGATGA-3′ β-actin AS5′-CCAGAGGCGTACAGGGATAG-3′Mouse Hmga1 S5′-CTCCAGGGAGGAAACCAAG-3′ Hmga1 AS5′-CAGAGGACTCCTGGGAGATG-3′ Wnt1 S5′-ACAGTAGTGGCCGATGGTG-3′ Wnt1 AS5′-CTTGGAATCCGTCAACAGGT-3′ K19 S5′-ATGAGATCATGGCCGAGAAG-3′ K19 AS5′-GGTGTTCAGCTCCTCAATCC-3′ Ki67 S5′-AGGGTAACTCGTGGAACCAA-3′ Ki67 AS5′-TTAACTTCTTGGTGCATACAATGTC-3′ β-actin S5′-CCAACCGTGAAAAGATGACC-3′ β-actin AS5′-CCAGAGGCATACAGGGACAG-3′ Open table in a new tab The human HMGA1 promoter-luciferase construct (a generous gift from Dr. K. Peeters, University of Leuven, Belgium25Cleynen I Huysmans C Sasazuki T Shirasawa S Van de Ven W Peeters K Transcriptional control of the human high mobility group A1 gene: basal and oncogenic Ras-regulated expression.Cancer Res. 2007; 67: 4620-4629Crossref PubMed Scopus (45) Google Scholar) was introduced into HEK293 cells, together with phRL-SV40 (1 ng) (Promega, Madison, WI) using Fugene6 (Roche Diagnosics). Luciferase activities were checked 48 hours after transfection using the dual luciferase reporter assay system (Promega). Firefly luciferase activities were normalized to Renilla luciferase activities. Luciferase activities were determined from more than three independent assays. To generate Hmga-1-EGFP knock-in mice, 2.5- and 4-kb fragments containing the Hmga1 gene were amplified by genomic PCR from mouse bacterial artificial chromosome clone RP23-189L19, derived from C57BL/6J mice. The EGFP gene was fused in-frame to the last open reading frame before the Hmga1 translation stop codon in the 5′ homologous arm. A 2.5-kb 5′ arm of homology (EcoRI to BamHI) including exons 3, 4, and 5 before the stop codon fused EGFP gene, and a 4-kb 3′ arm of homology (XbaI/SpeI to MluI) including exon 5 after the stop codon, were cloned into 5′ and 3′ multiple-cloning sites of the pIRES-neo3 vector (Clontech Laboratories, Inc., Mountain View, CA) that lacked a synthetic intron. After sequence confirmation, the construct was linearized using MluI and introduced into wild-type TT2-KTPU8 F1 mouse embryonic stem (ES) cells by electroporation. The transfected ES cells were then cultured in selection medium containing 0.2 mg/ml G418. Southern blot analysis using a probe 5′ to the BamHI site was performed on G418 resistant colonies to identify the ES cells with correct incorporation of the targeting construct into the genome. The gene targeted ES cells were then aggregated with morulae of ICR mice. The aggregated embryos were transferred to pseudopregnant females and allowed to develop to term. The chimeric mice were bred with C57BL/6 wild-type mice, and the resulting pups were screened for the presence of the heterozygous targeted allele. The genotype of the mice was determined by Southern blot analysis and PCR of genomic DNA isolated from the tail or ear. Heterozygous mice were intercrossed to obtain homozygous mice. Hmga1-EGFP/Hmga1-EGFP mice were also crossed with K19-Wnt1/C2mE mice24Oshima H Matsunaga A Fujimura T Tsukamoto T Taketo MM Oshima M Carcinogenesis in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway.Gastroenterology. 2006; 131: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar or C57BL/6 mice (as a control), to analyze the expression of Hmga1-EGFP in normal tissues and gastric tumors. Mouse tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Histological sections were cut at 3 μm. Human stomach tumor tissue arrays (BioChain Institute, Inc., Hayward, CA) or mouse tissue samples were deparaffinized, and antigens were retrieved by autoclaving at 120°C for 15 minutes for β-catenin and HMGA1, in a buffer solution (0.01 M/L sodium citrate [pH 6.0] for β-catenin, 1 mmol/L EDTA/PBS [pH 9.0] for HMGA1). The slides were then incubated in methanol with 0.3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity. Thereafter, tissue sections were immersed in 0.5% BlockAce (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) in PBS for 30 minutes, covered with primary antibodies, and incubated overnight at 4°C. To detect nuclear β-catenin, mouse monoclonal antibodies for the stabilized (active) form of β-catenin that is dephosphorylated on Ser-37 or Thr-41 (Clone 8E7; Upstate, Charlottesville, VA) were used as the primary antibodies.24Oshima H Matsunaga A Fujimura T Tsukamoto T Taketo MM Oshima M Carcinogenesis in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway.Gastroenterology. 2006; 131: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar. Goat polyclonal HMG-I(Y) antibodies (N-19; Santa Cruz Biotechnology, Inc., CA) were used to detect HMGA1, and rabbit polyclonal GFP antibodies (FL; Santa Cruz Biotechnology, Inc., CA) were used to detect GFP. As the internal positive control, anti-Sp1 antibodies were used (data not shown). Visualization of the immunoreactions was performed using Histofine Simple Stain MAX-PO (Nichirei Bioscience Inc., Tokyo, Japan) and 3,3-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark). The slides were counterstained with hematoxylin and mounted with Malinol (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). Carcinoma cells with moderate or strong nuclear HMGA1 staining were counted as HMGA1-positive, while cells with weak nuclear staining and/or diffuse cytoplasmic staining were counted as negative. Cells with nuclear β-catenin staining were judged as β-catenin-positive, and those with membrane-associated β-catenin or no β-catenin staining were counted as negative. Positive nuclear staining for HMGA1 or β-catenin was exemplified in adenocarcinoma. HMGA1-positive cells and β-catenin-positive cells were quantitatively assessed by counting carcinoma cells (mean, 233; range, 110 to 450) in the same tissue samples. To observe fluorescent images, mouse tissues were fixed in 4% paraformaldehyde for 3 hours, incubated in 20% sucrose overnight, and frozen in Tissue-Tek optimal cutting temperature embedding compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan). Embedded frozen tissues were sectioned at 5 μm. Statistical analyses were performed using JMP 7.0.1 for Windows software (SAS Institute Inc., Cary, NC). Significant differences in real-time PCR quantification were evaluated using two-tailed paired t-tests. The association between HMGA1-positive cells and β-catenin-positive cells was analyzed using Pearson’s correlation coefficient, which varies from a perfect negative correlation (−1) to a perfect positive correlation (+1). Statistical significance was considered at a probability level of 0.05 or less. Several reports have shown that HMGA1 is overexpressed in gastric cancer,13Nam ES Kim DH Cho SJ Chae SW Kim HY Kim SM Han JJ Shin HS Park YE Expression of HMGI(Y) associated with malignant phenotype of human gastric tissue.Histopathology. 2003; 42: 466-471Crossref PubMed Scopus (20) Google Scholar, 26Xiang YY Wang DY Tanaka M Suzuki M Kiyokawa E Igarashi H Naito Y Shen Q Sugimura H Expression of high-mobility group-1 mRNA in human gastrointestinal adenocarcinoma and corresponding non-cancerous mucosa.Int J Cancer. 1997; 74: 1-6Crossref PubMed Scopus (50) Google Scholar, 27Lee S Baek M Yang H Bang YJ Kim WH Ha JH Kim DK Jeoung DI Identification of genes differentially expressed between gastric cancers and normal gastric mucosa with cDNA microarrays.Cancer Lett. 2002; 184: 197-206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar but the precise role of HMGA1 in the malignant phenotype remains undetermined. To examine the expression status of HMGA genes in human gastric cancer cells, we performed reverse transcription (RT)-PCR (Figure 1A). HMGA1 was expressed in all four gastric cancer cell lines (HSC39, HSC57, AGS, and KATO-III), whereas HMGA2 expression was not detected in any of the gastric cancer cells studied. In normal stomach tissue, HMGA1 was expressed at low levels, while HMGA2 was not detected. As a control, both HMGA1 and HMGA2 transcripts were found in HEK293 and Panc1 cells. To test the effect of HMGA1 on cell proliferation, we used siRNAs against HMGA1 or HMGA2 transcripts, whose knockdown effects have been previously demonstrated at both the RNA and protein levels.15Watanabe S Ueda Y Akaboshi S Hino Y Sekita Y Nakao M HMGA2 maintains oncogenic RAS-induced epithelial-mesenchymal transition in human pancreatic cancer cells.Am J Pathol. 2009; 174: 854-868Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar Western blot analysis showed that HMGA1 was expressed and depleted by the specific knockdown in AGS and HSC57 cells (Figure 1B). Quantitative RT-PCR analysis showed that HMGA1 was equally down-regulated by two distinct siRNAs in AGS cells (Figure 1C), and in HSC57 and KATO-III cells (data not shown). Notably, the knockdown of HMGA1 significantly reduced the growth rate of the gastric cancer cells studied, compared with the use of control and HMGA2 siRNAs. Cell death was assessed by fluorescence activated cell sorting analysis and was not increased under knockdown conditions (data not shown). These results indicate that HMGA1 is involved in maintaining the proliferation of the gastric cancer cells. Nuclear localization of β-catenin, a hallmark of Wnt/β-catenin signaling activation, is found in approximately 30% to 50% of gastric cancer tissues and in many kinds of gastric cancer cell lines.23Clements WM Wang J Sarnaik A Kim OJ MacDonald J Fenoglio-Preiser C Groden J Lowy AM β-Catenin mutation is a frequent cause of Wnt pathway activation in gastric cancer.Cancer Res. 2002; 62: 3503-3506PubMed Google Scholar, 28Ikenoue T Ijichi H Kato N Kanai F Masaki T Rengifo W Okamoto M Matsumura M Kawabe T Shiratori Y Omata M Analysis of the β-catenin/T cell factor signaling pathway in 36 gastrointestinal and liver cancer cells.Jpn J Cancer Res. 2002; 93: 1213-1220Crossref PubMed Scopus (50) Google Scholar Our expression studies showed that transcripts for β-catenin and c-myc, known as key factors in the Wnt/β-catenin pathway, were expressed in the gastric cancer cells studied (Figure 1A). To assess the effect of the Wnt/β-catenin pathway on HMGA1 expression, quantitative RT-PCR was performed following the selective knockdown of β-catenin or c-myc(Figure 1C). The depletion of either β-catenin or c-myc significantly reduced the expression of HMGA1 (P < 0.01), as did HMGA1 knockdown. In addition, the loss of β-catenin also decreased c-myc and cyclin D1 transcripts. Since the knockdown of β-catenin or c-myc reduced HMGA1 expression by approximately 50%, we examined whether other factors may mediate the transcriptional up-regulation of HMGA1. The use of COX2 inhibitors, NS-398 and indomethacin, decreased their proliferation but did not affect HMGA1 expression in AGS cells, suggesting that COX2 pathway unlikely influences on the expression of HMGA1 (data not shown). These results suggest that HMGA1 is involved in maintaining the growth activities of the gastric cancer cells, by acting as a downstream target of the Wnt/β-catenin pathway. We used HEK293 cells that have no constitutive Wnt/β-catenin activation but accumulate nuclear β-catenin after treatment with Wnt3a29Hino S Michiue T Asashima M Kikuchi A Casein kinase Iɛ enhances the binding of Dvl-1 to Frat-1 and is essential for Wnt-3a-induced accumulation of β-catenin.J Biol Chem. 2003; 278: 14066-14073Crossref PubMed Scopus (95) Google Scholar, 30Zhu H Mazor M Kawano Y Walker MM Leung HY Armstrong K Waxman J Kypta RM Analysis of Wnt gene expression in prostate cancer: mutual inhibition by WNT11 and the androgen receptor.Cancer Res. 2004; 64: 7918-7926Crossref PubMed Scopus (102) Google Scholar to investigate how the Wnt/β-catenin pathway induced the expression of HMGA1. We used a luciferase reporter assay in HEK293 cells (Figure 2A) to examine the transcriptional role of Wnt/β-catenin in the human HMGA1 gene promoter. An E-box motif in the HMGA1 gene promoter (at position −1353 from the transcriptional start site) has been reported to bind c-myc,10Wood LJ Mukherjee M Dolde CE Xu Y Maher JF Bunton TE Williams JB Resar LM HMG-I/Y, a new c-myc target gene and potential oncogene.Mol Cell Biol. 2000; 20: 5490-5502Crossref PubMed Scopus (168) Google Scholar, 31Pedulla ML Treff NR Resar LM Reeves R Sequence and analysis of the murine Hmgiy (Hmga1) gene locus.Gene. 2001; 271: 51-58Crossref PubMed Scopus (30) Google Scholar and we therefore used a reporter plasmid containing the promoter region (nucleotides −1745 to + 265) upstream of the luciferase gene. Treatment of the cells with Wnt3a increased the HMG
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