BMP-7 inhibits TGF-β-induced invasion of breast cancer cells through inhibition of integrin β(3) expression

Abstract

Background: The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.

Methods: In this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay.

Results: BMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin α(v)β(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β(3) negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β(3) counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion.

Conclusion: Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β(3).

Fig. 1

BMP-7, but not BMP-6, inhibits TGF-β-induced invasion in M-IV, but not in M-II. a–d Spheroids were allowed to invade collagen in the presence of BMP-7 (100 ng/ml), TGF-β (5 ng/ml) or TGF-β & BMP-7. a Pictures of representative spheroids of M-II. b Quantification of invasion of M-II experiments shown in (a). c Pictures of representative spheroids of M-IV. d Quantification of invasion of M-IV experiments shown in (c). e Spheroids of M-IV were allowed to invade collagen in the presence of BMP-6 (100 ng/ml), TGFβ (5 ng/ml) or TGF-β & BMP-6. Pictures of representative spheroids are shown. f Quantification of invasion of experiments shown in (e). The results are expressed as mean ± S.D. of at least three spheroids per condition. One representative out of three independent experiments is shown. Significance *** p < 0.001

Fig. 2

Noggin enhances basal invasion through inhibition of BMP-7. Spheroids of M-IV were allowed to invade collagen in the absence or presence of Noggin (200 ng/ml). a Pictures of representative spheroids. b Quantification of spheroids of experiments shown in (a). c Spheroids of M-IV were allowed to invade into collagen in the presence of control antibody or BMP-7 blocking antibody (2 μg/ml). Representative pictures are shown. d Quantification of spheroids of experiments shown in (c). The results are expressed as mean ± S.D. of at least four spheroids per condition. One representative out of three independent experiments is shown. Significance *** p < 0.001

Fig. 3

BMP-6 and BMP-7 induce BMP-Smad signaling, but do not affect TGF-β Smad signaling. a Cells were treated with the indicated ligands (BMP-6 and BMP-7 at 100 ng/ml, TGF-β at 5 ng/ml) for 15 min, lysed and subjected to SDS-PAGE and Western blotting. Blots were incubated with phospho-Smad2, phospho-Smad1 and Smad1 antibodies. Total ERK was used as a loading control. b Cells were transfected with the BRE-Luc reporter construct, starved and subsequently stimulated for 8 h with BMP-6 or 7 (both at 100 ng/ml). Relative luciferase activity (RLU) is expressed as mean ± S.D. of triplicate cultures. c Cells were transfected with the CAGA₁₂-Luc reporter construct, starved and subsequently stimulated for 8 h with the indicated ligands (BMP-6 and BMP-7 at 100 ng/ml, TGF-β at 5 ng/ml). Relative luciferase activity (RLU) is expressed as mean ± S.D. of triplicate cultures. One representative out of at least three independent experiments is shown

Fig. 4

BMP-7 inhibits integrin α_vβ₃ expression, but does not affect MMP2 expression in M-IV. Spheroids were embedded with BMP-6 (100 ng/ml), BMP-7 (100 ng/ml), TGF-β (5 ng/ml), TGF-β & BMP-6 or TGF-β & BMP-7 for 24 h. MMP2 (a), integrin α_v (b) and integrin β₃ (c) mRNA expression was analyzed by Q-PCR . One representative out of three independent experiments is shown. Significance *** p < 0.001 * p < 0.05

Fig. 5

Integrin α_vβ₃ is necessary for TGF-β-induced invasion. a Spheroids were allowed to invade into collagen in the presence of DMSO or GLPG0187 (80 nM) with or without TGF-β (5 ng/ml) Representative pictures of spheroids are shown. b Quantification of experiment shown in (a). c Cells were transduced with lentivirus encoding shRNA against integrin β₃ and control lentivirus.. Spheroids of these cells were allowed to invade in the absence or presence of TGF-β (5 ng/ml). Representative pictures of spheroids are shown. d Quantification of invasion shown in (c). At least four spheroids per condition. were quantified. One representative out of three independent experiments is shown. Significance ** p < 0.01

Fig. 6

Integrin β₃ overexpression rescues TGF-β-induced invasion from BMP-7 inhibition. Cells were transduced with lentivirus containing an integrin β₃ overexpression construct. Integrin β₃ overexpressing cells or wildtype (WT) cells were allowed to invade in BMP-7 (100 ng/ml), TGF-β (5 ng/ml) or TGF-β & BMP-7. a Representative pictures of spheroids are shown. b Quantification of spheroid experiment shown in (a). The results are expressed as mean ± S.D. of at least four spheroids per condition. One representative out of three independent experiments is shown. Significance ** p < 0.01 NS not significant