Bone morphogenetic protein 7 in dormancy and metastasis of prostate cancer stem-like cells in bone

Abstract

Metastatic disease is the major cause of cancer deaths, and recurrent tumors at distant organs are a critical issue. However, how metastatic tumor cells become dormant and how and why tumors recur in target organs are not well understood. In this study, we demonstrate that BMP7 (bone morphogenetic protein 7) secreted from bone stromal cells induces senescence in prostate cancer stem-like cells (CSCs) by activating p38 mitogen-activated protein kinase and increasing expression of the cell cycle inhibitor, p21, and the metastasis suppressor gene, NDRG1 (N-myc downstream-regulated gene 1). This effect of BMP7 depended on BMPR2 (BMP receptor 2), and BMPR2 expression inversely correlated with recurrence and bone metastasis in prostate cancer patients. Importantly, this BMP7-induced senescence in CSCs was reversible upon withdrawal of BMP7. Furthermore, treatment of mice with BMP7 significantly suppressed the growth of CSCs in bone, whereas the withdrawal of BMP7 restarted growth of these cells. These results suggest that the BMP7-BMPR2-p38-NDRG1 axis plays a critical role in dormancy and recurrence of prostate CSCs in bone and suggest a potential therapeutic utility of BMP7 for recurrent metastatic disease.

Figure 1.

CM of bone stromal cells induces senescence in prostate cancer cells. (A) The prostate cancer cell line PC3 mm was cultured in the presence of CM from human bone stromal cells (HS5), mouse BM stroma (MBMS), mouse embryonic fibroblasts (NIH3T3), or human lung fibroblasts (MRC5) or serum-free medium (−). Expression of p21, p27, and β-tubulin was examined by qRT-PCR (n = 3) and Western blot. (B) The prostate tumor cell lines LNCaP, C4, C4-2, C4-2B, PC3, DU145, and ALVA were cultured in the presence of CM of HS5 or serum-free medium (−), and the expression of p21 was examined by qRT-PCR (n = 3). (C) PC3 mm was cultured in the presence of CM of HS5, MBMS, NIH3T3, or MRC5 or serum-free medium (−), and the activation of p38 (phosphorylated p38, p-p38; total p38, p38) and the expression of NDRG1 and β-tubulin were examined by Western blot. (D) LNCaP, C4, C4-2, C4-2B, PC3, ALVA, and DU145 were cultured in the presence or absence of CM of HS5, and the expression of p-p38, p38, and β-tubulin was examined by Western blot. (E) PC3 mm was cultured in the presence or absence of CM of HS5 or MRC5 for 48 h, and cell viability was measured by the MTT assay (n = 3). (F) PC3 mm was cultured in the presence or absence of HS5-CM for 48 h, and SA–β-gal staining (blue-green) was performed (n = 4). The inset shows representative images of three independent experiments. Bar, 100 µm. (G) PC3 mm was cultured in the presence of CM of HS5, MRC5, or NIH3T3 or serum-free medium (−), and the expression of NDRG1 was measured by qRT-PCR (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed three times independently, and representative data are shown. Results are shown as mean ± SEM.

Figure 2.

BMP7 up-regulates NDRG1 through activation of p38. (A) PC3 mm cells were grown in the presence of HS5-CM with 3 µg/ml BMP inhibitor Noggin (+) or vehicle (−), and the expression of NDRG1, p-p38, p38, and β-tubulin was examined by Western blot and qRT-PCR (n = 3; *, P < 0.05 vs. first bar; #, P < 0.05 vs. second bar). (B) The effect of BMP2, BMP4, BMP5, BMP6, BMP7, and FGF2 on the expression of p-p38, p38, p21, p27, NDRG1, and β-tubulin in PC3 mm and DU145 cells was examined by Western blot. (C) LNCaP, C4, C4-2, C4-2B, and ALVA were cultured with or without 200 ng/ml BMP7, and the expression of p-p38, p38, and β-tubulin was examined by Western blot. (D) The effect of the indicated concentrations of BMP7 on NDRG1 expression in PC3 mm and DU145 was examined by qRT-PCR (n = 3; *, P < 0.05). (E) The effect of the indicated concentrations of BMP7 on the expression of NDRG1 in PC3 mm was examined by NDRG1 reporter assay (n = 3; **, P < 0.01; ***, P < 0.001). (F and G) Western blot for BMP7 in the CM from bone stromal cells (hBMSC, hFOB1.19, and HS5) and prostate cancer cells (PC3 mm, DU145, C4-2B, and ALVA; F) or CM from HS5 that had either scrambled shRNA (scramble) or shRNA for BMP7 (shBMP7; G). (H) PC3 mm cells were cultured with the CM from HS5/scramble or HS5/shBMP7, and the expression of p-p38, NDRG1, p21, and β-tubulin was examined by Western blot. (I and J) PC3 mm cells were treated with or without 200 ng/ml BMP7 in the presence or absence of 10 µM of the p38 inhibitor SB203580 (SB), and the expression of NDRG1 was examined by qRT-PCR (I) and by NDRG1 reporter assay (J; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). All experiments were performed three times independently, and representative data are shown. Results are shown as mean ± SEM.

Figure 3.

BMP7 up-regulates NDRG1 by activating p38 and p21. (A) Effect of 200 ng/ml BMP7 on senescence in PC3 mm was examined by SA–β-gal staining (n = 4). −, control vehicle. The inset shows representative images of three independent experiments. Bar, 100 µm. (B and C) Effect of 200 ng/ml BMP7 on p21 was measured by qRT-PCR and p21 reporter assay in PC3 mm (B and C) and DU145, C4, C4-2, and C4-2B cells (B; n = 3). Control, control vehicle. (D) PC3 mm cells were transiently transfected with dominant-negative Smad4 (DN-Smad4) or empty vector and then cultured with or without BMP7 for 48 h. Expression of p21 was assessed by qRT-PCR (n = 3). (E) PC3 mm cells were treated with or without 200 ng/ml BMP7 and/or 10 µM SB203580 (SB), and the expression of p21 was examined by qRT-PCR (n = 3). (F) PC3 mm cells that had either scrambled shRNA (scramble) or shRNA for p21 (shp21) were treated with or without BMP7, and the expression of p-p38, p38, p21, NDRG1, and β-tubulin was examined by Western blot. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed three times independently, and representative data are shown. Results are shown as mean ± SEM.

Figure 4.

BMP7 induces reversible senescence. (A) Effect of BMP7 on the growth of PC3 mm cells was measured by the MTT assay. Cells were treated with 200 ng/ml BMP7 continuously (green), with BMP7 between day 4 and 11 followed by BMP7 withdrawal (red), or with control vehicle (black; n = 5). (B) PC3 mm cells were cultured with 10 ng/ml EGF or 200 ng/ml BMP7 alone, or EGF with BMP7, or EGF with BMP7 followed by BMP7 withdrawal, and the expression of p-Erk, Erk, p-p38, p38, and β-tubulin was examined by Western blot. (C) Effect of BMP7 on growth arrest was examined by SA–β-gal staining (top) and cell cycle analysis (bottom). PC3 mm cells (left) and C4-2B cells (right) were cultured with (+/+) or without (−/−) BMP7 for 96 h. +/− cells were treated with BMP7 for 48 h followed by withdrawal of BMP7 and further incubation for 48 h (n = 3). (D) Effect of BMP7 on senescence was examined by SA–β-gal staining. DU145, LNCaP, C4, and ALVA were cultured with (+/+) or without (−/−) BMP7 for 96 h. +/− cells were treated with BMP7 for 48 h followed by withdrawal of BMP7 and further incubation for 48 h (n = 3). (C and D) The insets show representative images of three independent experiments. Bars, 50 µm. (E and F) PC3 mm cells that had either scrambled shRNA (scramble) or shRNA for NDRG1 (shNDRG1) were cultured with BMP7 or control vehicle, and the knockdown of NDRG1 was confirmed by qRT-PCR (n = 3; E). These cells were stained for SA–β-gal activity (n = 4; F). (G) Effect of NDRG1 on senescence was examined by SA–β-gal staining (bottom). PC3 mm cells stably expressing Tet-NDRG1 were cultured with (+/+) or without (−/−) tetracycline for 96- or 48-h treatment followed by 48-h withdrawal of tetracycline (+/−; n = 4). The expression of NDRG1 and β-tubulin was examined by Western blot (top). *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experiment in A was performed twice, experiments in B–G were performed three times independently, and representative data are shown. Results are shown as mean ± SEM.

Figure 5.

The expression of BMPR2 receptor is inversely correlated with recurrence and bone metastasis of prostate cancer patients. (A) The relation between recurrence-free survival and the expression (positive or negative) of the indicated BMP receptors was analyzed using an existing cohort data of prostate cancer patients (n = 21; Glinsky et al., 2004). *, P = 0.0485 (Log-rank test). (B, top) Representative images of immunohistochemical staining for BMPR2 on prostate cancer patient samples with or without bone metastasis. Bar, 50 µm. (bottom) The correlation between BMPR2 expression and bone metastasis (met) status was evaluated by Fisher’s exact test.

Figure 6.

BMPR2 mediates BMP7-induced reversible senescence. (A) The expression of BMPR2 and β-tubulin in PC3 mm that had either scrambled shRNA (scramble) or shRNA for BMPR2 (shBMPR2) was examined by Western blot (top) and qRT-PCR (bottom; n = 3). (B) Kaplan-Meier analysis for bone metastasis–free survival of mice after intracardiac injection of PC3 mm cells that carried either scrambled shRNA (scramble; n = 9) or shRNA for BMPR2 (shBMPR2; n = 10) followed by treatment with vehicle or BMP7. Scramble versus Scramble + BMP7: ***, P < 0.0001; shBMPR2 versus shBMPR2 + BMP7: NS; Scramble + BMP7 versus shBMPR2 + BMP7: ***, P = 0.0002 by Log-rank test. (C) PC3 mm/scramble cells and PC3 mm/shBMPR2 cells were treated with or without BMP7, and the expression of p-p38, p38, p21, NDRG1, and β-tubulin was examined by Western blot. (D) The PC3 mm cells stably expressing Tet-shBMPR2 were cultured with (+) or without (−) induction of shBMPR2 in the presence or absence of BMP7, followed by assaying SA–β-gal. −/−, no induction; +/+, continuous induction; +/−, 48-h induction followed by 48-h withdrawal of tetracycline (n = 3). ***, P < 0.001. The expression of BMPR2 and β-tubulin was examined by Western blot (inset). Experiments in A, C, and D were performed three times, the experiment in B was performed twice independently, and representative data are shown. Results are shown as mean ± SEM.

Figure 7.

BMP7 induces reversible senescence in CSCs through activation of p38, p21, and NDRG1. (A) CSCs isolated from PC3 mm were injected subcutaneously into nude mice, and the growth of tumors was monitored by BLI. (B) The CSCs were treated with the HS5-CM or BMP7, and the expression of p-p38, NDRG1, and β-tubulin was examined by Western blot. (C and D) The CSCs were treated with or without BMP7 and/or SB203580 (SB), and the expression of NDRG1 (C) and p21 (D) was examined by qRT-PCR (n = 3). *, P < 0.05; ***, P < 0.001. (E) Effect of BMP7 on the sphere forming ability of CSCs was measured. −/−/−, no treatment throughout; −/+/−, no treatment for 5 d, treated with BMP7 for 4 d, and no treatment thereafter; +/+/−, treatment for 9 d and no treatment thereafter (n = 5). *, P < 0.001 versus −/−/−; #, P < 0.001 versus −/−/−. (F) CSCs were cultured in the presence (+/+), absence (−/−), or withdrawal after treatment (+/−) of BMP7, and SA–β-gal staining was performed (n = 6). ***, P < 0.001. Bar, 10 µm. (G and H) The CSCs were treated with BMP7, and the expression of NDRG1 (G) and p21 (H) was measured by qRT-PCR. −/−, no treatment control; +/+, continuous treatment with BMP7 for 96 h; +/−, 48-h treatment followed by 48-h withdrawal of BMP7 (n = 3). *, P < 0.05; **, P < 0.01. (I) A similar experiment was performed as in E for CSCs from PC3 mm/Tet-NDRG1 with (+) or without (−) induction of NDRG1, followed by assaying sphere formation. −/−/−, no induction throughout; −/+/−, no induction for 5 d, induction for 3 d, and no induction thereafter; +/+/−, induction for 8 d and no treatment thereafter (n = 5). *, P < 0.001 versus −/−/−; #, P < 0.001 versus −/−/−. (J) A similar experiment was performed as in F for CSCs from PC3 mm/Tet-NDRG1. They were cultured with (+/+) or without (−/−) induction or withdrawal after induction of NDRG1 (+/−), and SA–β-gal staining was performed (n = 8). ***, P < 0.001. The expression of NDRG1 and β-tubulin was examined by Western Blot (top). The experiment in A was performed twice, experiments in B–J were performed three times independently, and representative data are shown. Results are shown as mean ± SEM.

Figure 8.

BMP7 suppresses tumor growth, but withdrawal induces tumor recurrence of CSCs in vivo. (A) CSCs isolated from PC3 mm were coinjected into tibiae of nude mice with control (scramble; n = 11) or BMP7–knocked down (shBMP7 #1, n = 14; #2, n = 10) HS5 cells. (left) BLI and x-ray radiography of bone lesions from representative mice in each group 4 wk after inoculation. Osteolytic lesions are indicated by arrowheads. (middle) Normalized BLI signals after 4 wk. *, P < 0.05 by Mann-Whitney test. (right) Representative images of immunohistochemical staining of tumors in the tibiae for p-p38 and NDRG1. (B) CSCs isolated from PC3 mm/shNDRG1 (n = 12) or PC3 mm/scramble (n = 12) were coinjected with HS5 into tibiae of nude mice. (left) BLI and x-ray radiography of representative mice in each group 4 wk after inoculation. Osteolytic lesions are indicated by arrowheads. (middle) BLI after 4 wk. **, P < 0.01 by Mann-Whitney test. (right) H&E staining of the tibiae from representative mice 4 wk after inoculation. The asterisk indicates a tumor. (C) Kaplan-Meier bone metastasis–free survival curve of mice after intracardiac injection with CSCs isolated from PC3 mm (left) or C4-2B (right) followed by treatment with vehicle or BMP7. Control, control vehicle treatment (PC3 mm, n = 9; C4-2B, n = 10); BMP7, continuous treatment with BMP7 (PC3 mm, n = 15; C4-2B, n = 10); BMP7 withdrawal, BMP7 treatment (2 wk for PC3 mm and 3 wk for C4-2B) followed by withdrawal of BMP7 (PC3 mm, n = 9; C4-2B, n = 10). ***, P = 0.0008; and **, P = 0.0012 versus control; and ###, P < 0.0001; and #, P = 0.0289 versus BMP7 by Log-rank test. (D) Effect of BMP7 on senescence was examined by SA–β-gal staining. The bone derivatives of PC3 mm (PC3 mm–BM) were cultured with (+/+) or without (−/−) BMP7 for 96 h. +/− cells were treated with BMP7 for 48 h followed by withdrawal of BMP7 and further incubation for 48 h (n = 3). ***, P < 0.001. (E, top) After injection of CSCs into mouse tibiae, BMP7 was administered daily i.v. Control, control vehicle treatment (n = 13); BMP7, continuous treatment with BMP7 (n = 10); withdrawal, 10-d treatment followed by withdrawal of BMP7 (n = 10). *, P < 0.05 versus control; ###, P < 0.001 versus BMP7 treatment. (bottom) Representative SA–β-gal staining (blue-green) of tumors in the tibiae of mice at the end point. Experiments in A–C and E were performed twice, the experiment in D was performed three times independently, and representative data are shown. Results are shown as mean ± SEM. Bars: (A, D, and E) 50 µm; (B) 200 µm.