Functions and Epigenetic Regulation of Wwox in Bone Metastasis from Breast Carcinoma: Comparison with Primary Tumors

Abstract

Epigenetic mechanisms influence molecular patterns important for the bone-metastatic process, and here we highlight the role of WW-domain containing oxidoreductase (Wwox). The tumor-suppressor Wwox lacks in almost all cancer types; the variable expression in osteosarcomas is related to lung-metastasis formation, and exogenous Wwox destabilizes HIF-1α (subunit of Hypoxia inducible Factor-1, HIF-1) affecting aerobic glycolysis. Our recent studies show critical functions of Wwox present in 1833-osteotropic clone, in the corresponding xenograft model, and in human bone metastasis from breast carcinoma. In hypoxic-bone metastatic cells, Wwox enhances HIF-1α stabilization, phosphorylation, and nuclear translocation. Consistently, in bone-metastasis specimens Wwox localizes in cytosolic/perinuclear area, while TAZ (transcriptional co-activator with PDZ-binding motif) and HIF-1α co-localize in nuclei, playing specific regulatory mechanisms: TAZ is a co-factor of HIF-1, and Wwox regulates HIF-1 activity by controlling HIF-1α. In vitro, DNA methylation affects Wwox-protein synthesis; hypoxia decreases Wwox-protein level; hepatocyte growth factor (HGF) phosphorylates Wwox driving its nuclear shuttle, and counteracting a Twist program important for the epithelial phenotype and metastasis colonization. In agreement, in 1833-xenograft mice under DNA-methyltransferase blockade with decitabine, Wwox increases in nuclei/cytosol counteracting bone metastasis with prolongation of the survival. However, Wwox seems relevant for the autophagic process which sustains metastasis, enhancing more Beclin-1 than p62 protein levels, and p62 accumulates under decitabine consistent with adaptability of metastasis to therapy. In conclusion, Wwox methylation as a bone-metastasis therapeutic target would depend on autophagy conditions, and epigenetic mechanisms regulating Wwox may influence the phenotype of bone metastasis.

Keywords: Wwox; bone metastasis; epigenetic reprogramming; transcription factors.

Figure 1

Structure of WWOX gene and of the mature protein. The nine exons of the gene, and the domains for protein-protein interactions are indicated. WW, WW domain; NLS, nuclear localization sequence; ADH/SDR, short-chain alcohol dehydrogenase/reductase; MTS, mitochondrial targeting sequence; CpG sites, methylation sites; ITCH, E3 ubiquitin ligase; Runx2, Runt-related transcription factor 2; Hyal-2, hyaluronidase-2; GSK-3β, glycogen synthase kinase 3β. P, phospho. For Wwox protein, the amino acid position is indicated by the numbers.

Figure 2

Regulation of HIF-1 (Hypoxia inducible factor-1) activity and glycolysis by exogenous WWOX and WWOX loss. K.O., knockout.

Figure 3

Wwox protein levels in breast carcinoma cell lines, and role of decitabine and Wwox in the expression of autophagy players. All Western blots were performed with 100 µg of total proteins, and the specific signals were examined after immunoblotting and reaction with ECL plus chemiluminescence kit. Vinculin was used for normalization of the densitometric values. (A) Wwox protein levels were evaluated in different cell lines: bone metastatic 1833 cells, derived from invasive MDA-MB231 breast carcinoma cells; MCF-7 non-invasive breast carcinoma cells; (B) the 1833 cells were exposed to decitabine or the vehicle alone for 30 days; WWOX expression vector (e.v.) was transfected for 24 or 48 h [64]. The first lanes correspond to control-untreated cells. After normalization of the densitometric values using vinculin, the data were used for the graphic. The Western blot experiments in (A,B) were repeated three times, and the data show the fold variations versus the first lane. The statistical analysis was performed using ANOVA, with p < 0.05 considered significant. * p < 0.05, ** p < 0.005, *** p < 0.001 versus control; ^Δ p < 0.05, ^ΔΔ p < 0.005 versus vehicle.

Figure 4

Wwox plays various functions in bone metastasis, related to gene expression for mesenchymal epithelial transition and autophagy. EMT, epithelial-mesenchymal transition; MET, mesenchymal-epithelial transition; HGF, hepatocyte growth factor.