Clusterin, a haploinsufficient tumor suppressor gene in neuroblastomas.
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.
Chayka
Olesya
O
Molecular Haematology and Cancer Biology Unit, UCL Institute of Child Health, 30 Guilford St, London WC1N 1EH, UK.
Corvetta
Daisy
D
Dews
Michael
M
Caccamo
Alessandro E
AE
Piotrowska
Izabela
I
Santilli
Giorgia
G
Gibson
Sian
S
Sebire
Neil J
NJ
Himoudi
Nourredine
N
Hogarty
Michael D
MD
Anderson
John
J
Bettuzzi
Saverio
S
Thomas-Tikhonenko
Andrei
A
Sala
Arturo
A
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D051152:Clusterin / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006239:Haplotypes; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000378:metabolism; D000071447:N-Myc Proto-Oncogene Protein; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009367:Neoplasm Staging; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009570:Nitriles / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D014162:Transfection; D014183:Transplantation, Heterologous
C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D051152:Clusterin; C459801:MYCN protein, human; D035683:MicroRNAs; D000071447:N-Myc Proto-Oncogene Protein; D016328:NF-kappa B; D009570:Nitriles; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D034741:RNA, Small Interfering; D013450:Sulfones; D008156:Luciferases