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Correlation of WWOX, RUNX2 and VEGFA protein expression in human osteosarcoma

2013-12-15 18:23:38

BMC Medical Genomics; 2013, 6:56; 15 Dec 2013; DOI:10.1186/1755-8794-6-56



Jilong Yang, Linru Zhao, Wei Tian, Zhichao Liao, Hong Zheng, Guowen Wang and Kexin Chen



Abstract



BACKGROUND



To investigate associations between WW domain-containing oxidoreductase (WWOX), runt-related transcription factor 2 (RUNX2) and vascular endothelial growth factor alpha (VEGFA) in human osteosarcoma (OS).



METHODS



Copy number aberrations of WWOX, RUNX2and VEGFA genes were detected by microarray comparative genomic hybridization (aCGH) in 10 fresh OS tissue samples. VEGFA gene alterations were also investigated and validated by fluorescence in situ hybridization (FISH) in 54 formalin-fixed and paraffin-embedded (FFPE) OS samples. Protein expression of WWOX, RUNX2 and VEGFA were examined in 54 FFPE OS samples by immunohistochemistry (IHC).



RESULTS



Analysis of previously published OS aCGH data (GSE9654) and aCGH data from this study (GSE19180) identified significant deletion of WWOX in 30% (6/20) of OS samples, whilst significant increase in both RUNX2 and VEGFA gene copy numbers were detected in 55% (11/20) and 60% (12/20) of OS samples, respectively. FISH demonstrated increased VEGFA gene copy number in 65.9% (31/47) of evaluable samples, in either focal or large fragment forms. Compared with positive expression of WWOX in 38.9% of the OS samples, positive expression of RUNX2 and VEGFA protein was found in 48.1 and 75.9% of samples. Although there was no significant association between gene copy number aberration and protein expression for WWOX and RUNX2, significant positive correlation between increased VEGFA gene copy number and VEGFA protein expression was observed. Although there was no significant reverse association between WWOX and RUNX2 expression, a significantly positive relationship was observed between RUNX2 and VEGFA protein expression.



CONCLUSIONS



Our data show increased RUNX2 and VEGFA gene copy numbers and elevation of their respective proteins in human OS. Positive correlation of RUNX2 and VEGFA suggests that both increased VEGFA gene copy number and RUNX2 overexpression facilitate increased expression of VEGFA.



Background



Osteosarcoma (OS) is the most common, primary, malignant bone tumor within the non-hematopoietic system. OS frequently occurs in the metaphysis of actively growing long bones and is characteristic of short and rapid progression. It has high incidence of pulmonary metastasis and poor prognosis, and mainly affects children and adolescents. Because of a complexity of karyotypes and a highly unstable genome, OS usually exhibits both numerical and structural chromosomal alterations. As a putative tumor suppressor gene, WWOX is located at chromosome 16q23.3-q24.1, spanning common fragile site FRA16D. WWOX is detected as functional loss or frequent attenuation of protein expression in combination with poor prognosis. This often results from abnormal mRNA splicing of WWOX, missing exons, loss of heterozygosity (LOH) and hypermethylation in numerous carcinomas. WWOX might play tumor-suppressor function through interaction with TNF, p53, Bcl-2, ErbB-4 and c-Jun. A recent study indicated that WWOX was physically and functionally associated with RUNX2 and can suppress RUNX2 transactivation by interaction between the first WW domain and RUNX2. In addition, absence of WWOX seems to contribute to increased RUNX2 expression, further affecting bone growth and metabolism, initiating OS tumorigenesis.



The RUNX2 region (6p12-21) is often detected in OS using gene amplification and protein overexpression, suggesting upregulation of this gene and its protein is associated with tumorigenesis, progression, metastasis and unfavorable outcome. Importantly, Kyle and colleagues observed an inverse relationship between WWOX and RUNX2 expression in WWOX-deficient mice and OS cell lines. Additionally, RUNX2 is a critical element for VEGF mRNA transcription and protein expression in tumorigenesis. Aqeilan and colleagues found that ectopic expression of WWOX in MDAMB231 breast cancer reduced expression of RUNX2 and its target genes, including VEGF. However, little is known about the correlation of WWOX, RUNX2 and VEGF in human OS tissues. In this study, we observed WWOX, RUNX2 and VEGFA gene copy number status and protein expression levels using microarray comparative genomic hybridization (aCGH), immunohistochemistry (IHC) staining and fluorescence in situ hybridization (FISH), in order to investigate correlations between these components.



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