Geun Taek Lee

Effect of bone morphogenetic protein-6 on macrophages.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)‐β superfamily which regulates bone formation, haematopoiesis and development. While TGF‐β is known to be a negative regulator of the immune system, the effect of BMPs on the immune system is largely unknown. Herein, the effect of BMP‐6 on the innate immune system was investigated using the murine macrophage cell line RAW 264·7. BMP‐6 altered cellular morphology, inhibited cellular proliferation, increased the fraction of subG1 phase cells, and decreased the fraction of cells in the S and G2M phases, without changing the percentage of apoptotic cells. In addition, BMP‐6 induced expression of pro‐inflammatory inducible nitric oxide synthase (iNOS) and the cytokine tumour necrosis factor (TNF)‐α. Reverse transcription–polymerase chain reaction (RT‐PCR) analysis demonstrated the expression of all three known type II BMP receptors [BMP‐RII, activin (Act)‐RIIA and Act‐RIIB] and two of the three known type I receptors [activin receptor‐like kinase 2 (ALK2) and ALK3]. Over‐expression as well as knock‐down studies using short hairpin RNA (shRNA) demonstrated that BMP‐RII, ALK2 and ALK3 are the functional BMP‐6 receptors in macrophages. Finally, the effect of BMP‐6 was confirmed in murine peritoneal macrophages and the THP‐1 human monocyte cell line. Taken together, these results demonstrate that BMP‐6 regulates the proliferation and gene expression profile of macrophages.

BMP-6 in Renal Cell Carcinoma Promotes Tumor Proliferation through IL-10–Dependent M2 Polarization of Tumor-Associated Macrophages

Dysregulated bone morphogenetic proteins (BMP) may contribute to the development and progression of renal cell carcinoma (RCC). Herein, we report that BMP-6 promotes the growth of RCC by interleukin (IL)-10-mediated M2 polarization of tumor-associated macrophages (TAM). BMP-6-mediated IL-10 expression in macrophages required Smad5 and STAT3. In human RCC specimens, the three-marker signature BMP-6/IL-10/CD68 was associated with a poor prognosis. Furthermore, patients with elevated IL-10 serum levels had worse outcome after surgery. Together, our results suggest that BMP-6/macrophage/IL-10 regulates M2 polarization of TAMs in RCC.

Effect of IN-1130, a Small Molecule Inhibitor of Transforming Growth Factor-β Type I Receptor/Activin Receptor-Like Kinase-5, on Prostate Cancer Cells

PURPOSE Transforming growth factor-beta is a potent immune suppressor that is over expressed by most malignant cells to evade the host immune response. Thus, a potential anticancer therapeutic strategy is the inhibition of transforming growth factor-beta signaling. MATERIALS AND METHODS We investigated the specificity and the antitumor effect of IN-1130, a novel small molecule inhibitor of the transforming growth factor-beta type I receptor ALK-5. RESULTS IN-1130 inhibited transforming growth factor-beta induced cell death and gene transcriptional activity in a concentration dependent manner in the human hepatoma cell line HepG2. Simultaneously immunoblot analysis demonstrated that IN-1130 inhibited the Smad2 phosphorylation induced by transforming growth factor-beta. To determine the specificity of IN-1130 for transforming growth factor-beta signaling the effect on active and bone morphogenic protein signaling was subsequently investigated. Results demonstrated that IN-1130 did not inhibit bone morphogenic protein signaling. However, active signaling was blocked by IN-1130 in a concentration dependent manner. Furthermore, immunoblot analysis for phospho-Smad2 following transfection with constitutively active ALK-1 to 7 demonstrated that IN-1130 inhibited ALK-4 (active receptor type IB), 5 (TbetaRI) and 7 (nodal type I receptor). To investigate the antitumor effect of IN-1130 WT mice were injected subcutaneously with the murine prostate cancer cell line Tramp C2. Seven days later IN-1130 was administered intraperitoneally daily for 30 days. Results demonstrated a dramatic decrease in tumor volume in association with an enhanced immune response in the treatment group. CONCLUSIONS Taken together these results demonstrate that IN-1130 is a relatively nontoxic inhibitor of ALK-4/5/7 that may potentially treat prostate cancer.

Prostate cancer bone metastases acquire resistance to androgen deprivation via WNT5A-mediated BMP-6 induction

Background:Androgen ablation is the first-line therapy for patients with metastatic prostate cancer (CaP). However, castration resistance will eventually emerge. In the present study, we have investigated the role of bone morphogenetic protein-6 (BMP-6) in the development of castration-resistant prostate cancer (CRPC) in the context of bone metastases.Methods:We initially investigated the clinical course of 158 men with advanced CaP who were treated with primary androgen deprivation therapy. To elucidate the underlying mechanism of CRPC in the context of bone metastases, we examined the impact of bone stromal cells on CaP in the absence of androgens using a co-culture model.Results:In the 158 patients, we found that the median time to prostate-specific antigen progression was significantly shorter when bone metastases were present (14 months (95% CI, 10.2–17.8 months) vs 57 months (95% CI, 19.4–94.6 months)). These results suggest that bone–tumour interactions may accelerate castration resistance. Consistent with this hypothesis, in vitro co-cultures demonstrated that CaP cells proliferated under an androgen-depleted condition when incubated with bone stromal cells. Mechanistically, gene expression analysis using quantitative polymerase chain reaction arrays showed a dramatic induction of BMP-6 by CaP cell lines in the presence of bone stromal cells. Further studies revealed that WNT5A derived from bone stromal cells induced the expression of BMP-6 by CaP cells; BMP-6 in turn stimulated cellular proliferation of CaP cells in an androgen-deprived media via a physical interaction between Smad5 and β-catenin. Intracellularly, WNT5A increased BMP-6 expression via protein kinase C/NF-κB pathway in CaP cell lines.Conclusions:These observations suggest that bone–CaP interaction leads to castration resistance via WNT5A/BMP-6 loop.

Bone morphogenetic protein-6 induces the expression of inducible nitric oxide synthase in macrophages

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor‐β (TGF‐β) superfamily. In the present study, we investigated the effect of BMPs on the production of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line, RAW 264.7, and in mouse peritoneal macrophages. Among the BMPs, only BMP‐6 induced iNOS expression in a time‐dependent and dose‐dependent manner in both cell types. Induction of iNOS was inhibited by both cycloheximide and actinomycin D, indicating that the induction of iNOS expression by BMP‐6 requires new protein synthesis. Mechanistic studies revealed that the BMP‐6‐induced iNOS expression requires both Smads and nuclear factor‐kappa B (NF‐κB) signalling pathways. Furthermore, induction of interleukin‐1β (IL‐1β) was necessary for iNOS induction by BMP‐6. These observations suggest that BMP‐6 stimulates macrophages to produce iNOS through IL‐1β via Smad and NF‐κB signalling pathways and that BMP‐6 may be an important regulator of macrophages.

Induction of Interleukin-6 Expression by Bone Morphogenetic Protein-6 in Macrophages Requires Both SMAD and p38 Signaling Pathways

Unlike the prototype transforming growth factor-β (TGF-β), bone morphogenetic protein-6 (BMP-6) activates macrophages. Here, we report that BMP-6 induces the expression of IL-6 in macrophages. Using overexpression and knockdown experiments, we demonstrate that BMP receptor type II and activin-like kinase-2 are necessary for IL-6 induction by BMP-6. At the intracellular level, both Smad and p38 signaling pathways are required for the induction of IL-6. The cross-talk between the two pathways occurs at the level of transcription factor GATA4 and Smad 1/4. These results, taken together, demonstrate a novel BMP-6 signaling mechanism in which both the Smad and non-Smad pathways directly interact to activate the transcription of a target gene.

Clinical Significance of Wnt/β-Catenin Signalling and Androgen Receptor Expression in Prostate Cancer.

Purpose To investigate the relationships among the Wnt/β-catenin pathway, androgen receptor (AR), and clinicopathological factors in hormone-naïve prostate cancer. Materials and Methods This study was conducted with132 cases of hormone-naïve prostate cancer treated by prostatectomy and prostate needle biopsy. An immunohistochemical study using antibodies against β-catenin, matrix metalloproteinase-7 (MMP-7), and the AR was performed. For the in vitro study, PC-3, LNCaP, 22Rv1, and DU145 cell lines were used. Results The clinical or pathological stage ware a localized cancer in 36 patients (27.3%), locally advanced cancer in 31 (23.5%), and metastatic cancer in 65 (49.2%). We detected increased β-catenin, AR, and MMP-7 expression with a high Gleason grade, disease progression, and increasing serum prostate-specific antigen (PSA) levels (p<0.01). In Spearmans rank correlations, the expression of cytoplasmic β-catenin, MMP-7, and the AR were found to be significantly positively correlated. In addition, the expression of β-catenin, MMP-7, and the AR were significantly correlated with clinicopathological variables indicative of a poor prognosis. Forty-nine patients with primary androgen deprivation had short response durations from hormone therapy to PSA progression with elevated MMP-7 expression on the Kaplan-Meier curve (p=0.0036). Conclusions These data show that an activated Wnt/β-catenin pathway and AR expression in prostate cancer are correlated with metastasis and aggressiveness. In addition, the expression of MMP-7 protein, a target of the Wnt/β-catenin pathway, is associated with PSA progression in prostate cancer patients undergoing primary hormone therapy.

Macrophages induce neuroendocrine differentiation of prostate cancer cells via BMP6-IL6 Loop.

Frequently associated with hormone refractory prostate cancer are neuroendocrine cells. Because these cells do not express androgen receptors and are castration‐resistant, further understanding the mechanism of neuroendocrine differentiation (NED) of prostate cancer cells may yield novel intervention methods in hormone refractory prostate cancer. In this regard, the present study investigated the effect of macrophages on prostate cancer NED.

Effect of Dominant Negative Transforming Growth Factor-β Receptor Type II on Cytotoxic Activity of RAW 264.7, a Murine Macrophage Cell Line

Transforming growth factor-beta (TGF-beta) is a potent suppressor of the immune system. In the present study, we investigated the effect of TGF-beta resistance on a murine macrophage cell line, RAW 264.7, by overexpressing a dominant negative TGF-beta receptor type II (TbetaRIIDN) construct. As expected, TbetaRIIDN-expressing RAW cells, designated as RAW-TbetaRIIDN, were resistant to TGF-beta signaling. When these cells were cocultured with the murine renal cell carcinoma cell line, Renca, a dramatic increase in apoptosis of Renca cells was observed. Simultaneously, elevated levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in association with IFN-gamma were detected in RAW-TbetaRIIDN cells. When the effects of TNF-alpha and iNOS were neutralized through the use of neutralizing antibody and N(G)-methyl-L-arginine, respectively, the enhanced cytotoxicity of TbetaRIIDN-RAW cells was partially reversed. Taken together, these results show that TGF-beta-resistant RAW 264.7 murine macrophage cells have increased cytotoxic activity that is in part mediated by iNOS and TNF-alpha.

Bone morphogenetic protein-6 induces castration resistance in prostate cancer cells through tumor infiltrating macrophages

Bone morphogenetic protein (BMP) is a pleiotropic growth factor that has been implicated in inflammation and prostate cancer (CaP) progression. We investigated the potential role of BMP‐6 in the context of macrophages and castration‐resistant prostate cancer. When the androgen‐responsive murine (Tramp‐C1 and PTENCaP8) and human (LNCaP) CaP cell lines were cocultured with macrophages in the presence of dihydrotestosterone, BMP‐6 increased androgen‐responsive promoter activity and cell count significantly. Subsequent studies revealed that BMP‐6 increased the expression level of androgen receptor (AR) mRNA and protein in CaP cell lines only in the presence of macrophages. Simultaneously, the AR antagonists bicalutamide and MDV3100 partially or completely blocked BMP‐6‐induced macrophage‐mediated androgen hypersensitivity in CaP cells. Abolishing interleukin‐6 signaling with neutralizing antibody in CaP/macrophage cocultures inhibited the BMP‐6‐mediated AR upregulation in CaP cells. Using Tramp‐C1 and PTENCaP8 cells with a tetracycline‐inducible expression of BMP‐6, the induction of BMP‐6 in vivo resulted in a significant resistance to castration. However, this resistance was blocked after the removal of macrophages with clodronate liposomes. Taken together, these results show that BMP‐6 induces castration resistance by increasing the expression of AR through macrophage‐derived interleukin‐6.