Inactivation of the WNT5A Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2.

Himani Vaidya,Karen S Katula,Candie Rumph

doi:10.1371/journal.pone.0151392

Himani Vaidya, Karen S Katula + Show 1 more

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https://doi.org/10.1371/journal.pone.0151392

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Abstract

WNT5A is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered WNT5A expression is associated with various cancers, although in most studies the focus has been on only one of the known WNT5A isoforms. In this study, we analyzed expression from two of the major WNT5A promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1βare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1β was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 μM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the WNT5A promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the WNT5A alternative promoters A and B is a characteristic of osteosarcomas.

Highlights

WNT5A is a secreted glycoprotein that binds Frizzled (Fz) and Receptor tyrosine kinase-like orphan receptor 1/2 (Ror 1/2) receptors, leading to either inhibition or activation of Wnt signaling pathways, depending on the receptor-context of the cell [1, 2]
WNT5A promoter B activity is reduced in osteosarcoma cell lines and primary tumor tissue in comparison to normal osteoblasts
The levels of WNT5A promoters A and B transcripts were quantified in RNA from normal osteoblasts, osteosarcoma cell lines U2OS and SaOS-2, and osteosarcoma tumor tissue from doi:10.1371/journal.pone.0151392.g002

Summary

Introduction

WNT5A is a secreted glycoprotein that binds Frizzled (Fz) and Receptor tyrosine kinase-like orphan receptor 1/2 (Ror 1/2) receptors, leading to either inhibition or activation of Wnt signaling pathways, depending on the receptor-context of the cell [1, 2]. WNT5A is involved in regulating cell movement and differentiation, of mesenchymal stem cells such as chrondrocytes, adipocytes, and osteoblasts, through activation of the non canonical Wnt RhoA/Rac or calcium (Ca2+) signaling pathways [2]. WNT5A is misregulated in a wide range of cancer types, displaying both increased and decreased expression. Breast and prostate cancers are more variable showing both increased [19, 20, 21, 22] and decreased WNT5A expression [22, 23, 24]. Of particular importance to this study is the finding that the WNT5A gene is silenced by epigenetic mechanisms, primarily DNA methylation and histone modifications, in cancers in which the gene is not expressed [14, 15, 16, 17, 18, 25, 26]

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