Abstract
BackgroundOsteosarcoma 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.MethodsaCGH 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.ResultsKEGG 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.ConclusionsFrequent deletions of Wnt and other Wnt signaling pathway genes suggest that the Wnt signaling pathway is genetically inactivated in human osteosarcoma.
Highlights
Osteosarcoma is a highly genetically unstable tumor with poor prognosis
Several signaling pathways are genetically altered in osteosarcoma As presented in our previous reports, high-density genomewide array comparative genomic hybridization (aCGH) profiling (GSE19180) of 10 osteosarcoma tissues identified several major regions with significant genetic alterations
This pattern of copy number alterations was strikingly similar to that from an independent aCGH dataset (GSE9654) of osteosarcoma samples obtained from Canadian patients [2,3,11]
Summary
Osteosarcoma is a highly genetically unstable tumor with poor prognosis. The survival of patients with osteosarcoma has not improved significantly in recent years and the prognosis of patients. Most previous studies have suggested that active Wnt signaling contributes to osteosarcoma development, evidenced by cytoplasmic and/or membranous β-catenin staining or detection of Wnt pathway components [6,7]. Cai and colleagues recently reported that the Wnt pathway is inactivated in osteosarcomas [8]. These contradictory findings provoke debate and stimulate further research into the role of Wnt signaling in osteosarcoma [5]
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