Bone morphogenetic protein 7 is expressed in prostate cancer metastases and its effects on prostate tumor cells depend on cell phenotype and the tumor microenvironment

Abstract

Bone morphogenetic protein (BMP) signaling is important in prostate development and prostate cancer (PCa) progression. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in PCa. In our studies, we have focused on BMP-7 because it is involved in prostate morphogenesis, and its expression is regulated by androgens. The objective of our study was to determine BMP-7 expression in PCa metastases and investigate the effects of BMP-7 on PCa cells. Our results show that BMP-7 is expressed in metastatic PCa and its levels are increased in castration-resistant PCa versus androgen-dependent PCa, whereas the expression of BMP-7 is decreased in primary PCa versus normal prostate. Our in vitro results show that BMP-7 inhibits proliferation of androgen-sensitive LNCaP cells, stimulates androgen receptor signaling, increases the expression of differentiation-associated genes, and decreases the levels of some wingless-regulated transcripts. Interestingly, these effects were not detected in C4-2 castration-resistant PCa cells. In vivo expression of BMP-7 in castration-resistant C4-2 cells did not alter proliferation when these cells were grown subcutaneously, but their growth was inhibited in the bone environment. In summary, our results show that BMP-7 is expressed at the highest level in advanced castration-resistant PCa cells and that the inhibitory effects of BMP-7 are dependent on the differentiation status of PCa cells and the tumor microenvironment. Further studies are needed to identify changes in BMP-7 signaling that lead to the loss of its control of proliferation during PCa progression.

Figure 1

BMP-7 is expressed in PCa cell lines, xenografts, and metastases. (A) Expression of BMP-7 mRNA in PCa cell lines. (B) Expression of BMP-7 mRNA in four AD and corresponding CR LuCaP xenografts. Results are plotted as mean ± SEM. *CR significant from AD (P < .05; n = 2–6). (C) Representative samples of BMP-7 expression in NP (a and f), PCa (b and g), and bone (c and h), lymph (d and i), and lung (e and j) PCa metastases. Original magnification, x200.

Figure 2

Effect of BMP-7 on cell proliferation and AR activity in LNCaP and C4-2 cells in vitro. (A) Effect of BMP-7 (500 ng) on proliferation in LNCaP, C4-2, C4-2B, and PC-3 cells. (B) Effect of increasing doses of BMP-7 on proliferation in LNCaP and C4-2 cells. (C) Effect of silencing AR on proliferation in LNCaP cells in the presence or absence of BMP-7 (500 ng). (D) Expression of AR mRNA in four AD and corresponding CR LuCaP xenografts. (E) Altered expression of phosphorylated Smad1 (P-Smad1) in LNCaP and C4-2 cells in the presence or absence of BMP-7 (500 ng) for 3 days. Histone H4 (H4) was blotted as a control. (F) Western analysis of nuclear, cytosolic, and total AR (with GAPDH as control for total AR expression) in LNCaP and C4-2 cells with or without BMP-7 (500 ng) for 3 days.

Figure 3

BMP-7 alters AR localization and transcriptional activity in LNCaP cells. (A) Immunohistochemical analysis of the AR in LNCaP and C4-2 cells cultured in charcoal-stripped serum with or without BMP-7 (500 ng) for 2 days. (B) Immunohistochemical analysis of the AR in LNCaP and C4-2 cells cultured in charcoal-stripped serum with or without DHT (10 nM) for 2 days. (C) Effect of BMP-7 on AR activation in LNCaP and C4-2 cells. (D) Altered mRNA expression of markers of epithelial differentiation and Wnt-regulated genes in LNCaP and C4-2 cells by BMP-7 (500 ng) for 3 days. Results are plotted as mean ± SEM. *Significant from control.

Figure 4

BMP-7 alters AR interactions with β-cat in LNCaP cells. (A) Luciferase assay to determine the effect of Smad1 and β-cat on ARE binding and promoter activation in LNCaP cells. (B) Expression of β-cat and AR in LNCaP and C4-2 cells with or without BMP-7 (500 ng) for 3 days. GAPDH was blotted as a control (the same blot as in Figure 2F). (C) Immunoprecipitation of AR/β-cat complexes in the nucleus of LNCaP and C4-2 cells with or without BMP-7 (500 ng) for 3 days. Nuclear β-cat was blotted as a control. (D) Expression of E-cadherin in LNCaP and C4-2 cells with or without BMP-7 (500 ng) for 3 days. GAPDH was blotted as a control. (E) Immunohistochemical analysis of β-cat in LNCaP and C4-2 cells cultured in charcoal-stripped serum with or without BMP-7 (500 ng) for 2 days.

Figure 5

There is a reciprocal relation between AR and Wnt promoter activity in PCa epithelial cells. (A) Luciferase assays to determine the effect of DHT on AR (ARE) and Wnt signaling (TOPflash) in LNCaP cells. (B) Luciferase assays to determine if the AR inhibits TOP-flash activity in PC-3 cells and if increasing doses of β-cat plasmid DNA alter AR inhibition of TOPflash activity in PC-3 cells. (C) Luciferase assays to determine if AR inhibition (siRNA) influences TOPflash activity in LNCaP cells in the presence or absence of β-cat and if AR inhibition (AR siRNA) increases TOPflash activity in LNCaP cells with increasing doses of β-cat plasmid DNA (1 µg plasmid DNA). Results are plotted as mean ± SEM.

Figure 6

Characterization of BMP-7 overexpressing C4-2 cells. (A) Expression of BMP-7 mRNA in BMP-7-C4-2 cells. (B) Western analysis for BMP-7 of medium (10x concentrated) from BMP-7-C4-2 and pcDNA-C4-2 cells. (C) Cell count to determine cell growth of pcDNA-C4-2 and BMP-7-C4-2 cells in vitro. (D) PSA secretion in pcDNA-C4-2 and BMP-7-C4-2 cells in vitro. (E) Alkaline phosphatase activity in osteoblasts treated with conditioned medium from pcDNA-C4-2 cells, BMP-7-C4-2 cells, or BMP-7 alone (500 ng). Results are plotted as mean ± SEM. *Significant from pcDNA-C4-2.

Figure 7

Growth response of BMP-7 overexpressing subcutaneous and intratibial tumors in vivo. (A) Subcutaneous TuVs of pcDNA-C4-2 and BMP-7-C4-2 tumors in SCID mice (n = 5 per group). (B) Serum PSA in animals with subcutaneous pcDNA-C4-2 and BMP-7-C4-2 tumors in SCID mice (n = 5 per group). (C) Serum PSA in animals with intratibial pcDNA-C4-2 and BMP-7-C4-2 tumors in SCID mice (n = 10 per group). (D) BMD of control and tibiae with tumor of BMP-7-C4-2 and pcDNA-C4-2 tumors in SCID mice (n = 10 per group). (E) Control (pcDNA-C4-2) and BMP-7-C4-2 intratibial tumors. Results are plotted as mean ± SEM. *Significance, P < .05.