Fig 2: A model of EZH2’s interaction with TGFß signaling in enhancing breast cancer bone metastasis.High EZH2-expressing cells have strong ability to invade and metastasize from the primary tumor to the bone. In bone-metastatic tumor cells, EZH2 functions as a transcription co-factor to increase ITGB1 transcription. Integrin ß1 activates FAK and induces phosphorylation at Y397 of FAK, which phosphorylates TGFßRI at Y182. pY182-TGFßRI increases binding with TGFßRII, thereby activating pS465/467-Smad2, PTHLH expression, and the vicious cycle of breast cancer bone metastasis.

Fig 3: Treatment with a clinically applicable FAK inhibitor blocks EZH2-induced breast cancer bone metastasis.a Representative bioluminescence images of and quantification of the fold change in BLI intensity in the right leg region in three subgroups of mice (n = 5 in each group) intratibially injected with MDA-MB-231 cells and given treatment with a vehicle, GSK126 (100 mg/kg/day, i.p. injection), or the FAKi VS-6063 (50 mg/kg, twice a day, oral gavage) beginning on day 18 of injection. The BLI signal was normalized according to the signal on the first day after injection. Data are presented as means ± S.D. t-test (two-sided). b, c Representative pictures of IHC staining and quantification of pS465/467-Smad2 and PTHLP expression in the bone metastasis samples obtained from the three subgroups of mice in a. Data are presented as means ± S.E.M. t-test (two-sided). n = 4 bone metastasis slices in vehicle subgroup; n = 5 bone metastasis slices in GSK126 subgroup; n = 3 bone metastasis slices in FAKi subgroup. Scale bars, 50 µm. d The Pearson r correlation for EZH2 RNA expression and bone metastasis-free survival years (bmfs Yr) in breast cancer patients (GSE2603 dataset). e The Pearson r correlation for EZH2 and PTHLH RNA mRNA expression in bone metastasis samples obtained from breast cancer patients (GSE14020 dataset). All P-values are indicated in the figures.

Fig 4: EZH2 regulates the vicious cycle of breast cancer bone metastasis.a Model of triple co-culture of breast cancer cells with preosteoclasts and osetoblasts. Murine RAW 264.7 preosteoclasts were seeded into the wells of six-well plates. GFP-labeled breast cancer cells and MC3T3 osteoblasts were seeded into Millicell Hanging Cell Culture Inserts (Millipore) in the six-well plates and treated with TGFß (5 ng/mL). b Quantification of MDA-MB-231, 231.sgCtrl., 231.KO#1, and 231.KO#2 cells after co-culture with osteoclasts and MC3T3 osteoblasts treated with TGFß (5 ng/mL) for 6 days. Data are presented as means ± S.E.M. t-test (two-sided). Three biologically independent experiments. c Representative staining images and quantification of mature TRAP+ osteoclasts after culture with MC3T3 osteoblasts and the indicated cancer cells, and treatment of them with TGFß (5 ng/mL) for 6 days. The arrows indicate multinuclear mature TRAP+ osteoclasts. Scale bars, 200 µm. Data are presented as means ± S.E.M. t-test (two-sided). Ten random vision fields examined over three biologically independent experiments. d Quantification of 231.KO#1.pLenti. cells treated with vehicle, 231.KO#1.EZH2 cells treated with vehicle, 231.KO#1.EZH2 cells treated with 2 µM GSK126, 231.KO#1.H689A cells treated with vehicle, after co-culture with osteoclasts and MC3T3 osteoblasts treated with TGFß (5 ng/mL) for 6 days. Data are presented as means ± S.E.M. t-test (two-sided). Three biologically independent experiments. e Representative staining images and quantification of mature TRAP+ osteoclasts after co-culture with MC3T3 osteoblasts and the indicated cancer cells and treatment of them with TGFß (5 ng/mL) for 6 days. The arrows indicate multinuclear mature TRAP+ osteoclasts. Scale bars, 200 µm. Data are presented as means ± S.E.M. t-test, (two-sided). Three biologically independent experiments. f Representative bioluminescence image (BLI), X-ray images and quantification of BLI signals of bone-metastatic lesions in the two subgroups of mice (n = 5 in each group) intratibially injected with 231.KO#1.EZH2 or 231.KO#1.H689A cells 5 weeks post-injection. Data are presented as means ± S.E.M. t-test (two-sided). The arrows indicate osteolytic bone lesions in X-ray images. g qRT-PCR analysis of PTHLH mRNA expression in the indicated cells treated with a vehicle or TGFß (5 ng/mL, 2 h). Data are presented as means ± S.E.M. t-test (two-sided). N.S. not significant. Three biologically independent experiments. h qRT-PCR analysis of Pthlh mRNA expression in the indicated cells treated with a vehicle or TGFß (5 ng/mL, 2 h). Data are presented as means ± S.E.M. t-test (two-sided). Three biologically independent experiments. All P values are indicated in the figures. The TGFß used in all experiments in this study is TGFß1.