BMP7, a putative regulator of epithelial homeostasis in the human prostate, is a potent inhibitor of prostate cancer bone metastasis in vivo

Abstract

Bone morphogenic protein 7 (BMP7) counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity. Because epithelial-to-mesenchymal transition is involved in cancer, we investigated whether BMP7 plays a role in prostate cancer growth and metastasis. BMP7 expression in laser-microdissected primary human prostate cancer tissue was strongly down-regulated compared with normal prostate luminal epithelium. Furthermore, BMP7 expression in prostate cancer cell lines was inversely related to tumorigenic and metastatic potential in vivo and significantly correlated to E-cadherin/vimentin ratios. Exogenous addition of BMP7 to human prostate cancer cells dose-dependently inhibited transforming growth factor beta-induced activation of nuclear Smad3/4 complexes via ALK5 and induced E-cadherin expression. Moreover, BMP7-induced activation of nuclear Smad1/4/5 signaling transduced via BMP type I receptors was synergistically stimulated in the presence of transforming growth factor beta, a growth factor that is enriched in the bone microenvironment. Daily BMP7 administration to nude mice inhibited the growth of cancer cells in bone. In contrast, no significant growth inhibitory effect of BMP7 was observed in intraprostatic xenografts. Collectively, our observations suggest that BMP7 controls and preserves the epithelial phenotype in the human prostate and underscore a decisive role of the tumor microenvironment in mediating the therapeutic response of BMP7. Thus, BMP7 can still counteract the epithelial-to-mesenchymal transition process in the metastatic tumor, positioning BMP7 as a novel therapeutic molecule for treatment of metastatic bone disease.

Figure 1

BMP7 mRNA expression in noncancerous prostate epithelium (NP) and prostate cancer cells (PC) of radical prostatectomy specimens. a: BMP7 mRNA expression in patient-matched specimens consisting predominantly of NP and PC tissue (n = 16, P = 0.1, paired t-test), b: BMP7 mRNA expression in NP and neoplastic epithelial cells isolated by LCM (PC) (n = 14, P = 0.0019, paired t-test) c: Mean BMP7 mRNA expression values ± SEM in bulk tissue in NP and PC both normalized to glyceraldehyde-3-phosphate dehydrogenase (P = 0.1, paired t-test). d: Mean BMP7 mRNA expression values ± SEM in microdissected NP and neoplastic epithelium (PC), both normalized to glyceraldehyde-3-phosphate dehydrogenase (*P = 0.0019, paired t-test). BMP7 monoclonal antibody immunoreactivity and in clinical specimens of NP (e and f) and PC (g and h). e: Boxed area is magnified in f. BMP7 immunoreactivity was detected in the apical cytoplasm of the luminal cell layer of the normal acinar epithelium and parts of stroma (arrowhead), but weak or no staining of the basal cells was found. g and h: Prostate cancer cells lack BMP7 immunoreactivity, whereas BMP7 was still detectable in parts of the stroma (arrowhead). Scale bars = 100 μm.

Figure 2

Real-time mRNA expression of E-cadherin, vimentin, and BMPs in prostate cancer cell lines with different tumorigenic potential in vitro and in experimentally induced bone metastases. a: E-cadherin/vimentin ratios in prostate cancer cell lines. b: BMP7 mRNA expression in prostate cancer cell lines. c: Correlation between E-cadherin/vimentin ratio and BMP7 mRNA expression in prostate cancer cell lines with different tumorigenic potential (R² = 0.98, P = 0.0015). d: Relative expression levels of different BMP mRNA levels in an experimentally induced bone metastasis from PC-3M-Pro4 cells in nude mice. The mRNA levels (±SEM) were quantified by real-time reverse transcriptase-PCR relative to β-actin endogenous control, except for d, where β2-microglobulin was used for normalization as described previously.

Figure 3

Effects of daily systemic administration of 100 μg/kg rhBMP7 on the growth of human prostate cancer cells in bone marrow (a–d) and bone metastasis (e and f) in vivo after intraosseous transplantation or intracardiac inoculation of human PC-3M-Pro4/luc⁺ cells using whole body bioluminescent reporter imaging. a: BMP7 treatment of PC-3M-Pro4/luc⁺ cells growing in bone marrow of nude mice as monitored by BLI. b: Representative examples of bioluminescent photon emission in a vehicle- and BMP7-treated animal at day 21. c: Representative radiographs at day 21 after intraosseous transplantation of PC-3M-Pro4/luc⁺ cells growing in bone marrow (arrows indicate bone lesions in tibiae of nude mice). d: Immunohistochemical analyses of phosho-Smad1 localization at day 21 after intraosseous transplantation of PC-3M-Pro4/luc⁺ cells growing in bone marrow. The animals were either vehicle-treated (upper left panel) or treated with rhBMP7 (top right) 6 hours before explantation and fixation of the tibiae. The number of phospho-Smad1-positive cells in the BMP7-treated group is increased significantly. e: Effect of BMP7 treatment on the formation and growth of bone metastases by PC-3M-Pro4/luc⁺ in nude mice as monitored by BLI (tumor burden, number of bone metastases, representative examples of BLI at day 19). Relative light units (±SEM) emitted was quantified at least once weekly starting at day 0 after intraosseous implantation or intracardiac inoculation of the prostate cancer cells. n = 8 for each experimental group for intraosseous transplantation experiment (a–d); n = 10 for intracardiac inoculation (e). Values are expressed as means ± SEM. *P < 0.05, **P < 0.001 (analysis of variance).

Figure 4

Effects of daily systemic administration of 100 μg/kg rhBMP7 on intraprostatic growth of human PC-3M-Pro4/luc⁺ cells and formation of loco-regional lymph node metastases nude mice. a: Tumor take of PC-3M-Pro4/luc⁺ cells growing in prostate on treatment with vehicle solution or daily 100 μg/kg rhBMP7 as monitored by BLI. b: The number of affected lymph nodes in nude mice after 21 days as detected by BLI. c: Representative example of bioluminescent photon emission of a mouse with multiple loco-regional lymph node metastases 21 days after intraprostatic inoculation of PC-3M-Pro4/luc⁺ cells. The mouse prostate was removed before bioluminescent reporter imaging to obtain sensitive measurements of affected lymph nodes (b). The presence of micrometastatic deposits in lymph nodes was confirmed by H&E staining. Relative light units emitted was quantified at least once weekly starting at day 0 after intraprostatic implantation of the prostate cancer cells. n = 8 for each experimental group. Values are expressed as means ± SEM (analysis of variance).

Figure 5

The effects of BMP7 on gene promoter luciferase constructs in human PC-3M-Pro4 prostate cancer cells. a: Challenge of the SBE₄ construct, consisting of four SBEs, demonstrates the presence of functional BMP7 and TGFβ receptors (Smad-mediated signaling). *P ≤ 0.01 versus Co. b: BMP7, but not TGFβ, induces BRE₄-luciferase activity, indicating the presence and functionality of BMP receptors. Signaling of BMP to stimulate the expression of BRE₄-luciferase is transduced by BMP type I receptors and mediated by Smad1, Smad4, and Smad5, which form a complex with this reporter construct. Strikingly, signaling of BMP7 to stimulate BRE₄-luciferase is significantly enhanced when coincubated in the presence of TGFβ. ^#P < 0.01 versus Co; *P < 0.01 versus BMP7. c and d: The presence of functionally active TGFβ receptor complexes, particularly ALK5 (TGFβ type 1 receptor), in PC-3M-Pro4 cells was demonstrated by the CAGA-luciferase reporter, whose activity depends on binding of activated Smad3/Smad4 transcription factor complexes. The CAGA boxes confer TGFβ but not BMP stimulation to the promoter reporter constructs. The addition of BMP7 to TGFβ-stimulated PC-3M-Pro4 cells dose-dependently inhibited TGFβ-driven CAGA-luciferase activity. *P < 0.01 versus Co, **P < 0.01 versus TGFβ alone (c); and *P < 0.01 versus TGFβ alone, ^#P < 0.01 versus Co (d).

Figure 6

The effects of BMP7 on E-cadherin promoter luciferase constructs and protein expression of E-cadherin/vimentin in human PC-3M-Pro4 prostate cancer cells. a and b: BMP7 induces a dose-dependent and significant increase in E-cadherin promoter-luciferase construct activity (P < 0.01 at >0.2 μg/ml BMP7). E-Cadherin promoter activity is significantly and synergistically enhanced when BMP7 and TGFβ were coincubated (P < 0.05 versus BMP7 alone). *P ≤ 0.01 versus Co (a); *P < 0.05 versus Co, **P < 0.05 versus other conditions (b). c: E-cadherin and vimentin protein expression in PC-3M-Pro4 cells in the presence or absence of rhBMP7 and/or TGFβ for 48 hours as determined by immunoblotting. Treatment of PC-3M-Pro4 cells with 0.5 μg/ml rhBMP7 in the presence of TGFβ induces protein expression of E-cadherin. Values are expressed as E-cadherin/Vimentin ratio. d: Vimentin monoclonal antibody immunoreactivity of cultured PC-3M-Pro4 cells. In the presence of BMP7, the TGFβ-induced vimentin expression and cellular redistribution was completely abolished. Values are expressed as means ± SD (n = 4, analysis of variance).