Figure 4: FAF1 inhibits bone metastasis in transplantable mouse model of metastasis and correlates with good prognosis in breast cancer patients.

(a) Pre-ranked gene set enrichment analysis (GSEA) in FAF1-high versus FAF1-low patients. The consensus signature of the genes correlated with good prognosis (upper panel) or lower invasiveness (lower panel) was enriched in FAF1-high compared with FAF1-low patients. (b) Immunoblot (IB) of control and MCF10A-RAS cells stably expressing FAF1 or depleted of endogenous FAF1 (shFAF1) and treated with TGF-β (5 ng ml⁻¹) and SB431542 (10 μM) as indicated for 48 h. Epithelial-to-mesenchymal transition (EMT) marker proteins were analysed as indicated. (c) Heat map of TGF-β target and EMT-related genes in control or MCF10A-RAS cells stably expressing FAF1 or depleted of FAF1 (#1 and #2) and treated with or without TGF-β (1 ng ml⁻¹) for 8 h. (d) Consensus signature of genes significantly and negatively correlated with breast cancer metastasis are enriched in FAF1-high versus FAF1-low patients, as shown by pre-ranked GSEA. (e) Consensus signature of genes significantly and positively correlated with breast cancer metastasis (left panel), especially bone metastasis (right panel), are enriched in FAF1-low versus FAF1-high patients, as shown by GSEA (right panel). (f) Bioluminescent imaging (BLI) of three representative mice from each group at week 6 following injection with control or MDA-MB-231 cells stably expressing FAF1. Both ventral and dorsal images are shown (upper panel). The BLI signal of every mouse (lower left panel) and the percentage of bone metastasis-free mice (lower right panel) in each experimental group were followed over time. (g) The BLI of three representative mice from each group at week 6 following injection with control or MDA-MB-231 cells stably depleted of FAF1 and pretreated with or without SB431542 (SB, 10 μM) overnight. Both ventral and dorsal images are shown (left panel). The BLI signal of every mouse (right upper panel) and the percentage of bone metastasis-free mice (right lower panel) in each experimental group are shown over time. Data are presented as the mean±s.d.