Bone metastatic process of breast cancer involves methylation state affecting E-cadherin expression through TAZ and WWOX nuclear effectors

Abstract

We investigated the involvement of Hippo-related pathways in bone metastasis from breast cancer, by evaluating E-cadherin expression downstream of WWdomain-containing oxidoreductase (Wwox) and transcriptional co-activator with PDZ-binding motif (TAZ). These nuclear effectors functioned in a context-specific fashion on transcriptome, depending on breast-cancer aggressiveness and methylation state. Wwox and E-cadherin were found in human specimens of bone metastasis but not in primary-ductal breast carcinoma, while TAZ showed a characteristic localisation in metastasis nuclei. Wwox and E-cadherin were higher in 1833-metastatic clone with bone avidity than in parental-MDA-MB231 cells, while only metastatic cells presented TAZ. In 1833 cells, a complex interplay of transcriptional signalling controlled E-cadherin transactivation. Wwox and TAZ activated Hypoxia inducible factor-1 (HIF-1) binding to E-cadherin promoter, while Peroxisome proliferator-activated receptor γ (PPARγ) intervened in E-cadherin transactivation favouring and preventing Wwox and TAZ functions, respectively. Methylation impinged on Hippo-related pathways through Wwox and TAZ, modifying metastatic phenotype. The protract exposure to 5-azacytidine (Aza), by affecting methylation state modified the shape of 1833 cells, becoming mesenchymal as that of MDA-MB231 cells and reduced spontaneous-Matrigel invasion. The underlying-molecular mechanisms were diminutions of E-cadherin, Wwox, matrix metalloproteases 2 and 9, HIF-1- and PPARγ-activities, inversely correlated to Snail and nuclear-TAZ accumulations. Exogenous WWOX restored 1833-Aza invasion. Thus, 1833-Aza cells permitted to study the role played by methylation in metastasis plasticity, being E-cadherin loss part of an entire-gene reprogramming. Of note, bone-metastasis formation in 1833-Aza xenograft was partially impaired, prolonging mice survival. In conclusion, the methylation-heritable changes seemed important for cancer progression to establish bone metastasis engraftment/growth, by affecting steps requiring homotipic and/or heterotypic-adhesive properties and matrix degradation.