title: The genetic basis for inactivation of Wnt pathway in human osteosarcoma.
abstract: BACKGROUND
  Osteosarcoma is a highly genetically unstable tumor with poor prognosis. We performed microarray-based comparative genomic hybridization (aCGH), transcriptome sequencing (RNA-seq), and pathway analysis to gain a systemic view of the pathway alterations of osteosarcoma.
  
  
  METHODS
  aCGH experiments were carried out on 10 fresh osteosarcoma samples. The output data (Gene Expression Omnibus Series accession number GSE19180) were pooled with published aCGH raw data (GSE9654) to determine recurrent copy number changes. These were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify altered pathways in osteosarcoma. Transcriptome sequencing of six osteosarcomas was performed to detect the expression profile of Wnt signaling pathway genes. Protein expression of WNT1, β-catenin, c-myc, and cyclin D1 in the Wnt pathway was detected by immunohistochemistry (IHC) in an independent group of 46 osteosarcoma samples.
  
  
  RESULTS
  KEGG pathway analysis identified frequent deletions of Wnt and other Wnt signaling pathway genes. At the mRNA level, transcriptome sequencing found reduced levels of mRNA expression of Wnt signaling pathway transcripts. While WNT1 protein expression was detected by IHC in 69.6% (32/46) of the osteosarcomas, no β-catenin protein was detected in the nucleus. β-catenin protein expression was, however, detected in the membrane and cytoplasm of 69.6% (32/46) of the osteosarcomas. c-myc protein expression was detected in only 47.8% (22/46) and cyclin D1 protein expression in 52.2% (24/46) of osteosarcoma samples. Kaplan-Meier survival analysis showed that WNT1-negative patients had a trend towards longer disease free survival than WNT1-positive patients. Interestingly, in WNT1-negative patients, those who were also cyclin D1-negative had significantly longer disease free survival than cyclin D1-positive patients. However, there was no significant association between any of the investigated proteins and overall survival of human osteosarcoma patients.
  
  
  CONCLUSIONS
  Frequent deletions of Wnt and other Wnt signaling pathway genes suggest that the Wnt signaling pathway is genetically inactivated in human osteosarcoma.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Du
authors.affiliation: Department of Bone and Soft Tissue Tumors, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Tianjin 30060, China. yangjilong@tjmuch.com.
authors.forename: Jilong
authors.initials: J
authors.lastname: Yang
authors.forename: Da
authors.initials: D
authors.lastname: Yang
authors.forename: Wei
authors.initials: W
authors.lastname: Tian
authors.forename: Ze
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D000293:Adolescent; D000328:Adult; D001859:Bone Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism* / Q000401:mortality; D002648:Child; D005260:Female; D005784:Gene Amplification; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D012516:Osteosarcoma / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism* / Q000401:mortality; D060449:Wnt Signaling Pathway; D055815:Young Adult