NOLC1, an Enhancer of Nasopharyngeal Carcinoma Progression, Is Essential for TP53 to Regulate MDM2 Expression
2009; Elsevier BV; Volume: 175; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080931
ISSN1525-2191
AutoresYu-Chyi Hwang, Tung‐Ying Lu, Dah‐Yeou Huang, Yuan‐Sung Kuo, Cheng-Fu Kao, Ning-Hsing Yeh, Han‐Chung Wu, Chin‐Tarng Lin,
Tópico(s)RNA modifications and cancer
ResumoNasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells. Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells. 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The MDM2 gene is a cellular proto-oncogene, that is often amplified in ∼7% of all human cancers.26Oliner JD Kinzler KW Meltzer PS George DL Vogelstein B Amplification of a gene encoding a p53-associated protein in human sarcomas.Nature. 1992; 358: 80-83Crossref PubMed Scopus (1802) Google Scholar Two promoters have been identified in MDM2 gene structure: a constitutive promoter and a TP53-response intronic promoter (P2).27Barak Y Gottlieb E Juven-Gershon T Oren M Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential.Genes Dev. 1994; 8: 1739-1749Crossref PubMed Scopus (287) Google Scholar, 28Zauberman A Flusberg D Haupt Y Barak Y Oren M A functional p53-responsive intronic promoter is contained within the human mdm2 gene.Nucleic Acids Res. 1995; 23: 2584-2592Crossref PubMed Scopus (256) Google Scholar From our previous study, the expression of MDM2 gene can be indirectly enhanced in the EBV-infected NPC cells through enhancement of TP53 activation.29Wu HC Lin YJ Lee JJ Liu YJ Liang ST Peng Y Chiu YW Wu CW Lin CT Functional analysis of EBV in nasopharyngeal carcinoma cells.Lab Invest. 2003; 83: 797-812Crossref PubMed Scopus (23) Google Scholar Using RNA interference in vivo to examine the role of NOLC1 in the pathogenesis of NPC, we found that NOLC1 was crucial for NPC cell growth and that reduction of its expression in transfected xenografts resulted in retardation of tumor growth and apparent apoptosis and necrosis. We subsequently examined several genes related to this function and found that the depletion of NOLC1 resulted in a reduction of the MDM2 expression. Moreover, we found that NOLC1 and TP53 synergistically co-regulated MDM2 expression in NPC cells. Fourteen NPC cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (Gibco BRL, Gaithersburg, MD). They included NPC-TW01, 02, 03, 04, 05, 06, 07, 08, 09, and 10, all established in our laboratory30Lin CT Wong CI Chan WY Tzung KW Ho JK Hsu MM Chuang SM Establishment and characterization of two nasopharyngeal carcinoma cell lines.Lab Invest. 1990; 62: 713-724PubMed Google Scholar, 31Lin CT Chan WY Chen W Huang HM Wu HC Hsu MM Chuang SM Wang CC Characterization of seven newly established nasopharyngeal carcinoma cell lines.Lab Invest. 1993; 68: 716-727PubMed Google Scholar; NPC-CGBM-1, a gift from Dr. S. K. Liao (Chang-Gung University, Taoyuan, Taiwan)32Liao SK Perng YP Shen YC Chung PJ Chang YS Wang CH Chromosomal abnormalities of a new nasopharyngeal carcinoma cell line (NPC-BM1) derived from a bone marrow metastatic lesion.Cancer Genet Cytogenet. 1998; 103: 52-58Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar; and three other lines CNE1, CNE2, and HONE-1 cells, all originating from China.33Chinese Academy of Medical Sciences, Chungshan Medical College Establishment of an epithelial cell line and fusiform cell line from a patient with nasopharyngeal carcinoma (in Chinese).Sci Sin. 1978; 21: 113-118Google Scholar, 34Gu SY Tang WP Zeng Y Zhao ML Zhao EWP Deng WH Li K An epithelial cell line established from poorly differentiated nasopharyngeal carcinoma (in Chinese).Chin J Cancer. 1983; 2: 70-72Google Scholar The NPC-TW06 cell line contains a heterozygous point mutation in the tumor protein 53 (TP53) gene; the TP53 protein is retained in the cytoplasm and lost the transcriptional activity.35Hwang JK Lin CT Co-localization of endogenous and exogenous p53 proteins in nasopharyngeal carcinoma cells.J Histochem Cytochem. 1997; 45: 991-1003Crossref PubMed Scopus (12) Google Scholar TW01, 02, 05, and 08 are keratinizing squamous cell carcinoma lines (World Health Organization Type I), and TW03 is an undifferentiated carcinoma (World Health Organization Type III), also called lymphoepitheliometous carcinoma. The other cell lines were all undifferentiated carcinoma types (World Health Organization Type III). The four NNM cell cultures, NNM-9, NNM-11, NNM-12, and NNM-13, were primary cultured cells from nasal polyps as described previously,17Lee YC Hwang YC Chen KC Lin YS Huang DY Huang TW Kao CY Wu HC Lin CT Huang CY Effect of Epstein-Barr virus infection on global gene expression in nasopharyngeal carcinoma.Funct Integr Genomics. 2007; 7: 79-93Crossref PubMed Scopus (20) Google Scholar and were grown in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum. NPC paraffin blocks were obtained from the archives of the Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan. The use of human specimens in this research was approved by the Institutional Review Board (IRB-926170459) of National Taiwan University Hospital. SSH was used to isolate genes present in the NPC-TW04 or NNM cells. Total RNAs from these cells were isolated using the acid guanidinium thiocyanate-phenol-chloroform method (TRIzol; Invitrogen Life Technologies, Carlsbad, CA). The mRNA from NPC-TW04 cells was used as the "tester" and the mRNA from NNM cells was used as the "driver" for cDNA subtraction. The construction was performed following the SSH procedure using a PCR-select cDNA subtraction kit (Clontech, Palo Alto, CA). Briefly, equal amounts of mRNA from the tester and driver populations were converted to double-stranded cDNA by reverse transcription followed by digestion with RsaI separately. The digested tester cDNA was subdivided into two populations, each ligated with a different adaptor. Ligation efficiency was evaluated using PCR using primers specific to chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and the adaptor sequences. Following ligation, two hybridization steps was performed. For the first hybridization, an excess of driver was added to each tester, which was denatured, and allowed to anneal. The target sequences in the testers became enriched for differentially expressed genes in NPC-TW04 cells (NPC-TW04-NNM cDNA). The same procedure was repeated using mRNA from NNM cells as tester and mRNA from NPC-TW04 cells as a reference for cDNA subtraction. This produced other target sequences that also became enriched for differentially expressed in NNM cells (NNM-NPC-TW04 cDNA). In the second hybridization step, the subtracted target cDNAs were specifically amplified by nested PCR using adaptor-specific primer pairs and labeled with the addition of digoxigenin- or biotin-labeled nucleotides (Roche Molecular Biochemicals, Indianapolis, Ind) to obtain the digoxigenin-NPC-TW04-NNM cDNA and biotin-NNM-NPC-TW04 cDNA, respectively. A nylon microarray membrane containing 9600 cloned expressed sequence tag DNAs was constructed using cDNA clones from the National Taiwan University Hospital Microarray Core Facility for Genomic Medicine, Taiwan. The DNA microarray analysis was based on the colorimetry detection method on human eye recognition. The probes derived from the subtraction experiments were added to the membrane, the substrate was added, and the two colors developed. The image of each DNA dot was digitized by scanning on a high-resolution flat bed scanner (Umax Magic Scan at 3000 dpi). The digitized images were separated into cyan and magenta colors. The most significantly different hybridizing cDNAs were selected for further analysis. Clones containing the chosen expressed sequence tag cDNA were used to screen an NPC cDNA library prepared from the NPC-TW01 cell line in 1998 by Dr. P. Ouyang of the Department of Anatomy, Chang-Gung University, Taiwan, following a standard procedure.36Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989: 11.38-11.98Google Scholar The full-length nucleotide sequences were compared with sequences in the database using nucleotide BLAST from the National Center for Biotechnology Information BLAST website (http://www.ncbi.nlm.nih.gov/BLAST/). RNA was obtained from 14 NPC, three NNM, NS (nonspecific)-short hairpin (sh)RNA-NPC-TW03, shNOLC1-1-NPC-TW03, and shNOLC1-2-NPC-TW03 cell lines, and four xenograft tumors, as described above in the RNA isolation method. Reverse transcription was performed using the SuperScript First-Strand Synthesis Kit (Invitrogen Life Technologies, Carlsbad, CA). Touchdown PCR was performed for each of 29 genes (Table 1) following procedures described in Don et al,37Don RH Cox PT Wainwright BJ Baker K Mattick JS 'Touchdown' PCR to circumvent spurious priming during gene amplification.Nucleic Acids Res. 1991; 19: 4008Crossref PubMed Scopus (2245) Google Scholar with denaturation performed at 95°C for 30 seconds, anneling of 10 cycles at 65–56° C and 20 cycles at 55°C for 30 sec per cycle, and extension at 72°C for 1 minute. The PCR products were analyzed on a 1.2% agarose gel. The endogenous reference gene was ACTB (encoding β-actin). We performed quantitative reverse transcription (QRT-PCR) analysis using the comparative threshold cycle method using an ABI PRISM 7700 Sequence Detector System and SYBR Green PCR Master Mix Kit (Perkin Elmer, Applied Biosystems, Wellesley, MA), according to the manufacturers' instructions and as described in our recent paper.17Lee YC Hwang YC Chen KC Lin YS Huang DY Huang TW Kao CY Wu HC Lin CT Huang CY Effect of Epstein-Barr virus infection on global gene expression in nasopharyngeal carcinoma.Funct Integr Genomics. 2007; 7: 79-93Crossref PubMed Scopus (20) Google Scholar The endogenous reference gene used was GAPDH.Table 1The Primers Used for RT-PCR and QRT-PCRA. For RT-PCRUniGene numberGene symbolOfficial full namePrimersHs.467020BCL-2B-cell CLL/lymphoma 25′-ACTTGTGGCCCAGATAGGCACCCAG-3′5′-CGACTTCGCCGAGATGTCCAGCCAG-3′Hs.631546BAXBCL2-associated X protein5′-GCTCTGAGCAGATCATGAAGACAG-3′5′-CACAAAGATGGTCACGGTCTGC-3′Hs.2490CASP1caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase)5′-TTTGATTGACTCCGTTATTC-3′5′-TCTCTGCCGACTTTTGTTTC-3′Hs.23960CCNB1cyclin B15′-GAAGCTACTGGAAACATG-3′5′-CTACAGCTCGTTGTATGA-3′Hs.153752CDC25Bcell division cycle 25B5′-CACGCCCGTGCAGAATAAGC-3′5′-ATGACTCTCTTGTCCAGGCTACAGG-3′Hs.370771CDKN1Acyclin-dependent kinase inhibitor 1A (p21, Cip1)5′-ATGTCAGAACCGGCTGGGGATG-3′5′-GCAGGCCAAGGCCCCGCAC-3′Hs.644056CSNK2A1casein kinase 2, alpha 15′-ATGACCACCAGTCACGGCTTAC-3′5′-GGTTCAGACACGGTGCTTCTG-3′Hs.488293EGFRepidermal growth factor receptor5′-CATAGACGACACCTTCCTCC-3′5′-GGGTCTAAGAGCTAATGCGG-3′Hs.244139FASFas (TNF receptor superfamily, member 6)5′-TAGCTCCTATATTTTCGGCTT-3′5′-CTCACCAGCAACACCAAGTGC-3′Hs.396530HGFhepatocyte growth factor5′-ACTGGCTCTTTTAGGCACTGACTC-3′5′-TGTTCCCTTGTAGCTGCGTCCTTT-3′Hs.132966METMet proto-oncogene (hepatocyte growth factor receptor)5′-ACTCCCCCTGAAAACCAAAGCC-3′5′-GGCTTACACTTCGGGCACTTAC-3′Hs.567303MDM2Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse)5′-GCAGGGGAGAGTGATACAGAT-3′5′-GATGGCTGAGAATAGTCTTCA-3′Hs.513617MMP2Matrix metallopeptidase 25′-GGGGCCTCTCCTGACATT-3′5′-CATTCCCTGCAAAGAACACA-3′Hs.297413MMP9Matrix metallopeptidase 95′-TGGGCTACGTGACCTATGAC-3′5′-CAAAGGTGAGAAGAGAGGGC-3′Hs.202453MYCv-myc myelocytomatosis viral oncogene homolog5′-GTGGCACCTCTTGAGGACCA-3′5′-TGGTGCTCCATGAGGAGACA-3′Hs.463456NME1Non-metastatic cells 1, protein (NM23A)5′-TGCTGCGAACCACGTGGGT-3′5′-ATGTGGTCTGCCCTCCTGT-3′Hs.645227TGFB1Transforming growth factor, beta 15′-CTCCGAGAAGCGGTACCTGAAC-3′5′-CACTTGCAGTGTGTTATCCCT-3′Hs.522632TIMP-1TIMP metallopeptidase inhibitor 15′-CTGGAAAACTGCAGGATGGA-3′5′-CGCTGAGCTAAGGTCAGGCT-3′Hs.633514TIMP-2TIMP metallopeptidase inhibitor 25′-CTCATTGCAGGAAAGGCCGA-3′5′-TGGGTGGTGCTCAGGGTGTC-3′Hs.591665TIMP-4TIMP metallopeptidase inhibitor 45′-CCAGAGGTCAGGTGGTAA-3′5′-ACAGCCAGAAGCAGTATC-3′Hs.241570TNFTumor necrosis factor (TNF superfamily, member 2)5′-CTTCTGCCTGCTGCACTTTGGA-3′5′-TCCCAAAGTAGACCTGCCCAGA-3′Hs.81791TNFRSF11BTumor necrosis factor receptor superfamily, member 11b (osteoprotegerin)5′-AGGAAATGCAACACACGACAAC-3′5′-AGGAACAGCAAACCTGAAGAATG-3′Hs.156346TOP2Atopoisomerase (DNA) II alpha 170kDa5′-TGGTCAGAAGAGCATATGAT-3′5′-CTCACAATCTGATCAGCTAC-3′Hs.408312TP53Tumor protein p535′-CTATGTCGAAAAGTGTTTCTGTCATC-3′5′-CAGCCAAGTCTGTGACTTGCACGTAC-3′Hs.111779SPARCSecreted protein, acidic, cysteine-rich (osteonectin)5′-ACTGAAGCTTCCCAGCACCATG-3′5′-GAGAGGATCCGGTACTGTGG-3′Hs.585572SOX5SRY (sex determining region Y)-box 55′-CAACCTTGGTGCTGCTGTATCT-3′5′-GTCTTGGGTTTAGCTGATAGGTTCA-3′Hs.73793VEGFVascular endothelial growth factor A5′-CGATCGTTCTGTATCAGTCTTTCC-3′5′-GAAGTGGTGAAGTTCATGGATGTC-3′Hs.523238NOLC1Nucleolar and coiled-body phosphoprotein 15′-AGAAAAGAAAAAGGCGGCAG-3′5′-TCCTCATCAAGACCCTCACC-3′Hs.520640ACTBActin, beta5′-CACTCTTCCAGCCTTCCTTC-3′5′-GCCATGCCAATCTCATCTTG-3′Hs.544577GAPDHGlyceraldehyde-3-phosphate dehydrogenase5′-CGGGAAGCTTGTGATCCATGG-3′5′-GGCAGTGATGGCATGGACTG-3′B. For QRT-PCRGenePrimersARF5′-GAAGCCAAGGAAGAGGAATGAG-3′5′-CAAATATGTTCCCCCCTTCAGA-3′BAX5′-ATGTTTTCTGACGGCAACTTCA-3′5′-CAGTTCCGGCACCTTGGT-3′CASP15′-GCAGGACAACCCAGCTATGC-3′5′-TCTGCCGACTTTTGTTTCCAT-3′MMP95′-AGTTTGCCGGATACAAACTGGTA-3′5′-GAAACACTCCAACAAAAAACAAAGGT-3′NOLC15′-GACTGCATCTTCTCGTTTTTTACAGTATA-3′5′-GATCAGTGATTCTCAACCATGTAGGA-3′MDM25′-CCTTAGCTGACTATTGGAAATGCA-3′5′-CAGGAAGCCAATTCTCACGAA-3′TNF5′-CCTGCCCCAATCCCTTTATT-3′5′-CCCTAAGCCCCCAATTCTCTT-3′TP535′-GGGTTAGTTTACAATCAGCCACATT-3′5′-GGGCCTTGAAGTTAGAGAAAATTCA-3′VEGF5′-TCTACCTCCACCATGCCAAGT-3′5′-CTGCGCTGATAGACATCCATGA-3′β-actin5′-ACGTGGACATCCGCAAAGAC-3′5′-CTCAGGAGGAGCAATGATCTTGAT-3′GAPDH5′-TGGTATCGTGGAAGGACTCA-3′5′-AGTGGGTGTCGCTGTTGAAG-3′TP53 binding site of MDM2 promoter5′-TAGTCTGGGCGGGATTG-3′5′-TGCAGTTTCGGAACGTG-3′Exson 2 of MDM2 promoter5′-TGGCGATTGGAGGGTAGA-3′5′-ACCTGGATCAGCAGAGAA-3′GAPDH promoter5′-TCCAAGCGTGTAAGGGT-3′5′-GAAGGGACTGAGATTGGC-3′Beta-Hemoglobulin promoter5′-ATCTGAGCCAAGTAGAAGACCTTTTC-3′5′-TCTGCCTGGACTAATCTGCAAG-3′ Open table in a new tab Both NPC cell lines and NPC biopsy specimens were subjected to routine immunohistochemical staining using a monoclonal antibody directed against NOLC1,38Pai CY Chen HK Sheu HL Yeh NH Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis.J Cell Sci. 1995; 108: 1911-1920PubMed Google Scholar according to a previously described method.13Lin CT Lin CR Tan GK Chen W Dee AN Chan WY The mechanism of Epstein-Barr virus infection in nasopharyngeal carcinoma cells.Am J Pathol. 1997; 150: 1745-1756PubMed Google Scholar Immunoreactivity, defined as the number of positive tumor cells over total tumor cells, was scored independently by two researchers. The number of NOLC1-positive and negative NPC cells was counted under light microscope at a magnification of ×400, with only the cells displaying brown nucleoli on the section considered NOLC1-positive. For each slide, 7 to 10 microscopic fields were randomly chosen. Positive scores were the categorized into weak staining (only one nucleolus was stained), moderate staining (more than one nucleolus was stained), and strong staining (both nucleus and nucleolus of the tumor cell staining). The average percentage of NOLC1-positive NPC cells was then calculated for each group. Lysates from the cultured cells were subjected to routine Western blotting as described previously.39Wu HC Lin CT Association of heterotrimeric GTP binding regulatory protein (Go) with mitosis.Lab Invest. 1994; 71: 175-181PubMed Google Scholar The antibodies used were monoclonal anti-mouse antibodies against NOLC1,38Pai CY Chen HK Sheu HL Yeh NH Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis.J Cell Sci. 1995; 108: 1911-1920PubMed Google Scholar TP53, MDM2, and α-tubulin, and polyclonal rabbit antibodies against MMP9 and CASP1. Antibodies of TP53, MDM2, MMP9, α-tubulin, and CASP1 were purchased from Lab Vision Co. (Fremont, CA). The results shown are representative of two independent experiments. The shRNA constructs described in Table 2 were purchased from Open Biosystems (Huntsville, AL). When the NPC cultured cells had reached 70% to 80% confluence, the shRNA constructs were transfected into the NPC cells using the Arrest-In Transfection Reagent for RNAi (Open Biosystems). After incubation for 48 hours, the cells were selected with puromycin to establish of two stable lines: shNOLC1-1-NPC-TW03 and shNOLC1-2-NPC-TW03 line. To avoid the individual clonal variation of gene expression, we used mixed clones of all of the candidate cells that were successfully selected after adding the antibiotics. Then we checked the RNA and protein levels to confirm that these selected clones contained shRNA.Table 2The Construct Sequences of shRNAshRNA symbolSequenceNOLC1-1 shRNA5′-TGCTGTTGACAGTGAGCGCGACATCTAAGTCTGCAGTTAATAGTGAAGCCACAGATGTATTAACTGCAGACTTAGATGTCTTGCCTACTGCCTCGGA-3′NOLC1-2 shRNA5′-TGCTGTTGACAGTGAGCGAACAGTTAAAGCTCAGACTAAATAGTGAAGCCACAGATGTATTTAGTCTGAGCTTTAACTGTCTGCCTACTGCCTCGGA-3′NS-shRNA5′-TGCTGTTGACAGTGAGCGAACCACTAAGCTTCTGTCTTAATAGTGAAGCCACAGATGTATTAAGACAGAAGCTTAGTGGTCTGCCT5′-ACTGCCTCGGA-3′ Open table in a new tab To establish an animal model for the functional analysis of NOLC1, 24 six-week-old NOD/severe combined immunodeficient female mice were obtained from the National Taiwan University Hospital Experimental Animal Center. The animals were divided into four
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