Journal of the National Cancer Institute Advance Access originally published online on April 28, 2009
JNCI Journal of the National Cancer Institute 2009 101(9):663-677; doi:10.1093/jnci/djp063
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© The Author 2009. Published by Oxford University Press.
ARTICLES |
Clusterin, a Haploinsufficient Tumor Suppressor Gene in Neuroblastomas
Affiliations of authors: Molecular Haematology and Cancer Biology Unit, UCL Institute of Child Health, London, UK (OC, DC, IP, GS, SG, NJS, NH, JA, AS); Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell’Esercizio Fisico, Università degli Studi di Parma, Parma, Italy (DC, AEC, SB); National Institute Biostructures and Biosystems (INBB), Rome, Italy (SB); Department of Pathology and Lab Medicine, University of Pennsylvania and the Children's Hospital of Philadelphia, Philadelphia, PA (MD, AT-T); Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, PA (MDH)
Correspondence to: Arturo Sala, PhD, Molecular Haematology and Cancer Biology Unit, UCL Institute of Child Health, 30 Guilford St, London WC1N 1EH, UK (e-mail: a.sala{at}ich.ucl.ac.uk); Saverio Bettuzzi, Dipartimento di medicina Sperimentale, Universita’ degli studi di Parma, via Volturno 39 43100, Parma, Italy (e-mail: saverio.bettuzzi{at}unipr.it).
Background: Clusterin expression in various types of human cancers may be higher or lower than in normal tissue, and clusterin may promote or inhibit apoptosis, cell motility, and inflammation. We investigated the role of clusterin in tumor development in mouse models of neuroblastoma.
Methods: We assessed expression of microRNAs in the miR-17-92 cluster by real-time reverse transcription–polymerase chain reaction in MYCN-transfected SH-SY5Y and SH-EP cells and inhibited expression by transfection with microRNA antisense oligonucleotides. Tumor development was studied in mice (n = 66) that were heterozygous or homozygous for the MYCN transgene and/or for the clusterin gene; these mice were from a cross between MYCN-transgenic mice, which develop neuroblastoma, and clusterin-knockout mice. Tumor growth and metastasis were studied in immunodeficient mice that were injected with human neuroblastoma cells that had enhanced (by clusterin transfection, four mice per group) or reduced (by clusterin short hairpin RNA [shRNA] transfection, eight mice per group) clusterin expression. All statistical tests were two-sided.
Results: Clusterin expression increased when expression of MYCN-induced miR-17-92 microRNA cluster in SH-SY5Y neuroblastoma cells was inhibited by transfection with antisense oligonucleotides compared with scrambled oligonucleotides. Statistically significantly more neuroblastoma-bearing MYCN-transgenic mice were found in groups with zero or one clusterin allele than in those with two clusterin alleles (eg, 12 tumor-bearing mice in the zero-allele group vs three in the two-allele group, n = 22 mice per group; relative risk for neuroblastoma development = 4.85, 95% confidence interval [CI] = 1.69 to 14.00; P = .005). Five weeks after injection, fewer clusterin-overexpressing LA-N-5 human neuroblastoma cells than control cells were found in mouse liver or bone marrow, but statistically significantly more clusterin shRNA-transfected HTLA230 cells (3.27%, with decreased clusterin expression) than control-transfected cells (1.53%) were found in the bone marrow (difference = 1.74%, 95% CI = 0.24% to 3.24%, P = .026).
Conclusions: We report, to our knowledge, the first genetic evidence that clusterin is a tumor and metastasis suppressor gene.
| CONTEXT AND CAVEATS Prior knowledge The role of clusterin in cancer is unclear, and experimental results on its expression and activities are inconsistent across studies. Study design Cultured neuroblastoma cell lines, mouse models of human neuroblastoma, and human patient specimens were used. Expression of clusterin, MYCN, and miR-17-92 microRNAs in neuroblastoma cells was altered by various molecular biology techniques. The impact of such genetic alterations on tumor growth and metastasis was investigated. Contribution Clusterin appears to be a tumor and metastasis suppressor that is negatively regulated by MYCN and miR-17-92 microRNAs. Increased clusterin expression was associated with decreased tumor growth and decreased metastasis to mouse liver or bone marrow, whereas decreased clusterin expression was associated with increased tumor growth and metastasis. Implications More research into the role of clusterin in various cancers is warranted. Limitations Small numbers of mice and of patient specimens were used. Immunocompromised mice were used. The age of patients who contributed neuroblastoma samples was skewed to older ages, and the neuroblastoma stage was skewed to higher stage. From the Editors
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Manuscript received May 7, 2008; revised February 12, 2009; accepted February 27, 2009.
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J Natl Cancer Inst 2009 101: 613.