Inhibition of human androgen-independent PC-3 and DU-145 prostate cancers by antagonists of bombesin and growth hormone releasing hormone is linked to PKC, MAPK and c-jun intracellular signalling

Bombesin/gastrin-releasing peptide (BN/GRP) antagonists RC-3940-II and RC-3940-Et, and growth hormone-releasing hormone (GHRH) antagonists MZ-J-7-118 and RC-J-29-18 inhibit the growth of human androgen-independent PC-3 and DU-145 prostate cancers in nude mice. Additive inhibitory effects were observed after treatment with both classes of analogs. In the present study, we investigated the effects of these antagonists on intracellular signalling pathways of protein kinase C (PKC), mitogen activated protein kinases (MAPK) and c-fos and c-jun oncogenes that are involved in tumour cell proliferation. In PC-3 tumours, antagonists of BN/GRP and GHRH decreased significantly the expression of PKC isoforms alpha (alpha), eta (eta) and zeta (zeta) and increased that of delta (delta) PKC protein. MAPK was not detectable. In DU-145 tumours, which constitutively express MAPK, all treatments strongly decreased the levels of p42/44 MAPK. Treatment with the antagonists tended to reduce m-RNA for c-jun in both tumour models. In proliferation assays in vitro, inhibitors of PKC and MAPK diminished growth of DU-145 and PC-3 cells. These findings suggest that antagonists of BN/GRP and GHRH inhibit the growth of androgen-independent prostate cancer by affecting intracellular signalling mechanisms of PKC, MAPK and c-jun.     

Bombesin stimulates nuclear factor kappa B activation and expression of proangiogenic factors in prostate cancer cells

The majority of deaths from prostate cancer occur in patients with androgen-insensitive metastatic disease. An important early event in the development of the metastatic phenotype is the induction of genes that promote angiogenesis, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), which are released from tumor cells into their microenvironment. Coincident with progression from prostatic carcinoma in situ to metastatic disease is an increase in the number of tumor cells exhibiting neuroendocrine (NE) differentiation. NE cells express a variety of peptide hormones, including the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), and its cognate receptor, GRP-R. Although there is a strong positive correlation between the degree of NE differentiation and the metastatic potential of prostate cancers, a mechanistic link between increased expression of peptide hormone receptors, such as GRP-R, and proangiogenic gene expression has not been established. Here we report that BBS stimulates nuclear factor kappa B (NF kappa B) activation and proangiogenic gene expression in the androgen-insensitive prostate cancer cells lines, PC-3 and DU-145. In PC-3 cells, BBS stimulation of GRP-R resulted in the up-regulation of IL-8 and VEGF expression through a NF kappa B-dependent pathway. We show that BBS treatment induced inhibitor of NF kappa B degradation, NF kappa B translocation to the cell nucleus, increased NF kappa B binding to its DNA consensus sequence, and increased IL-8 and VEGF mRNA expression and protein secretion. Treatment with the proteasome inhibitor, MG-132, blocked BBS-stimulated NF kappa B DNA binding, and IL-8 and VEGF expression and secretion. Finally, media collected from PC-3 cell cultures, after BBS treatment, stimulated an NF kappa B-dependent migration of human umbilical vascular endothelial cells in vitro. Together, our data demonstrate a role for BBS and GRP-R in the NF kappa B-dependent up-regulation of proangiogenic gene expression, and suggest a possible molecular mechanism linking NE differentiation and the increased metastatic potential of androgen-insensitive prostate cancers.     

Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through a dual mechanism

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.     

Role of stimulatory guanine nucleotide binding protein (GSalpha) in proliferation of PC-3M prostate cancer cells

Previous studies have shown that calcitonin-like immunoreactive substances are secreted by primary prostate cells. Furthermore, exogenously added calcitonin stimulates proliferation of androgen-responsive LnCaP cells. To examine the possible effect of calcitonin on growth of invasive prostate cancer cells, we tested its effects on proliferation of PC-3M cells. Calcitonin stimulated DNA synthesis of PC-3M cells in a dose-dependent fashion, and also stimulated adenylyl cyclase and protein kinase C activities. To further delineate the role of these signaling cascades in proliferation of PC-3M prostate cancer cells, we selectively activated these pathways by transfecting cDNAs expressing constitutively active forms of either Gsalpha (Gsalpha-QL) or Gqalpha (Gqalpha-QL). cDNAs expressing wild-type forms of G-proteins (Gsalpha-WT and Gqalpha-WT) were used as vehicle controls. Gqalpha-QL transfectants exhibited growth inhibition and terminal differentiation. Those expressing Gsalpha-QL exhibited a dramatic increase in growth rate. Gsalpha-QL transfectants displayed an almost 3-fold increase in [3H]-thymidine incorporation and over a 4-fold increase in growth rate when compared with parental PC-3M cells or those expressing wild-type Gsalpha (Gsalpha-WT). The growth-promoting action of Gsalpha-QL could not be mimicked by either 8-bromo cAMP or forskolin. However, nifedipine, a calcium channel antagonist, potently and selectively inhibited DNA synthesis in Gsalpha-QL transfectants. These results suggest that the growth-promoting actions of Gsalpha on PC-3M cells may be mediated by nifedipine-sensitive proliferative events.     

Blockade of transforming growth factor-beta signaling suppresses progression of androgen-independent human prostate cancer in nude mice.

We investigated the role of transforming growth factor-beta (TGF-beta) signaling in the growth and metastasis of PC-3MM2 human prostate cancer cells. Highly metastatic PC-3MM2 human prostate cancer cells were engineered to constitutively overexpress a dominant-negative type II TGF-beta receptor (DNR). Transfection of DNR had minimal direct effects on cell growth and attenuated TGF-beta-induced cell growth inhibition and TGF-beta1 production. There were no discernable differences in tumorigenicity (tumor incidence) among PC-3MM2 variants when the cells were implanted into the prostates of nude mice. Growth rate and metastatic incidence of DNR-engineered PC-3MM2 cells, however, were significantly reduced. Most cells in the control tumors were positively stained by an antibody to proliferation cell nuclear antigen and very few cells were stained by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL). In sharp contrast, tumors formed by PC-3MM2-DNR cells contained fewer proliferation cell nuclear antigen-positive cells and many more TUNEL-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3MM2-DNR tumors. Expression of interleukin-8 (IL-8) in tumors formed by PC-3MM2 cells was significantly reduced as revealed by both Northern blotting and ELISA. Finally, transfection of antisense IL-8 cDNA significantly reduced IL-8 production by PC-3MM2 cells and antisense IL-8-transfected PC-3MM2 cells grew slower in comparison with parental and control vector-transfected cells. Taken together, our data suggest that TGF-beta signaling, by regulating IL-8 expression in tumor cells and hence tumor angiogenesis, is critical for progressive growth of PC-3MM2 cells in the prostate of nude mice.     

PI3K, Erk Signaling in BMP7-Induced Epithelial-Mesenchymal Transition (EMT) of PC-3 Prostate Cancer Cells in 2- and 3-Dimensional Cultures

We reported previously that bone morphogenetic protein 7 (BMP7) could induce epithelial-mesenchymal transition (EMT) in PC-3 prostate cancer cells grown in tissue culture plates. In this study, we examined BMP7-induced morphological and molecular expression changes that are characteristic of EMT using these cells under both two- (2D) and three-dimensional (3D) culture conditions. Filamentous outgrowths from spheroid structures that were formed from PC-3 cells in 3D cultures were strikingly evident when the spheroids were exposed to extracellular BMP7. This morphological change in 3D was accompanied by down-regulation of E-cadherin, which is an essential adhesion molecule for the integrity of epithelial phenotype. Invasiveness of the cancer cells was significantly enhanced with BMP7 treatment along with activation and up-regulation of proteases such as MMP1, MMP13, and urokinase plasminogen activator. Signal transduction of EMT conversion was examined by the use of certain pathway-specific inhibitors. Of the chemical inhibitors tested, inhibitors of PI3 kinase and Erk were found to suppress BMP-induced morphological changes both in 2D and 3D conditions. These results suggest that, besides the Smad signaling pathways, BMP-induced activation of PI3K and Erk contribute to EMT morphologic conversion of the PC-3 prostate cancer cells. Together, the results support the notion that the complexity of EMT may be better evaluated in terms of both spatial and temporal processes in 3D cell culture models that are physiologically more relevant than the cell growth in tissue culture plates.     

Lysophosphatidic acid-regulated mitogenic ERK signaling in androgen-insensitive prostate cancer PC-3 cells

Advanced and recurrent prostate tumors contain elevated levels of activated extracellular signal-regulated kinases 1 and 2 (ERK) in comparison to early-stage or benign specimens, and inhibition of ERK activation attenuates growth factor-dependent proliferation of prostate cells, suggesting a potential regulatory role for ERK in prostate tumorigenesis. Factors responsible for ERK activation in prostate cells are not well defined. Here, we show positive cooperative interaction between the G protein-coupled lysophosphatidic acid (LPA) and tyrosine kinase epidermal growth factor (EGF) receptors in androgen-insensitive prostate cancer PC-3 cells. Pre-treatment of the PC-3 cells with LPA decreases the dose of EGF required to elicit maximal activation of EGFR. Furthermore, treatment with LPA alone induces the rapid (maximal signal within 2 min) tyrosine phosphorylation of EGFR, and subsequent (maximal signal after 5 min) activation of ERK, suggesting that EGFR activation precedes ERK phosphorylation and may constitute a required component for signal relay from the LPA receptor to ERK. Accordingly, we show that inhibition of EGFR kinase activity attenuates the LPA-regulated ERK activation. In addition, we find that the LPA-regulated tyrosine phosphorylation of EGFR and activation of ERK are attenuated by batimastat, a generic inhibitor of matrix metalloproteinases (MMP). However, unlike the situation in fibroblasts, we find that the LPA-induced transactivation of EGFR in PC-3 cells is not mediated by shedding of heparin-binding EGF. Together, our data show that LPA and EGF cooperate to induce mitogenic signaling in prostate cancer cells in an MMP-regulated activation of the ERK pathway.     

Phosphoprotein associated with glycosphingolipid microdomains 1 inhibits the proliferation and invasion of human prostate cancer cells in vitro through suppression of Ras activation

Phosphoprotein associated with glycosphingolipid microdomains 1 (PAG) is an important negative regulator of immune signaling in T lymphocytes. However, newly emerging evidence has indicated that PAG may play important roles in tumor cells. Our previously reported cDNA microarray experiments identified PAG as a gene down-regulated in the high metastatic potential prostate cancer cell line PC-3M-1E8. In this study, we investigated the role of PAG in the proliferation, invasion and metastasis of prostate cancer cells and the underlying mechanisms. We confirmed that the expression of PAG at both the mRNA and protein levels was low in PC-3M-1E8 and DU145 cells compared to low metastatic potential prostate cancer cells PC-3M-2B4. In addition, we demonstrated that the reintroduction of PAG to PC-3M-1E8 and DU145 cells led to reduced proliferation through cell cycle arrest, decreased anchorage-independent growth and reduced invasion ability of tumor cells in vitro. This is the first report demonstrating that PAG inhibits the proliferation and invasion potential of prostate cancer cells via the interaction with RasGAP to recruit RasGAP to the cell membrane, where RasGAP hydrolyzes GTP to GDP, reduces the level of activated Ras, and ultimately suppresses the activation of ERK1/2, cyclin D1 and other effectors of the Ras signaling pathway. Morphologically, we observed that PAG could diminish the formation of pseudopodia on the cell surface and redistribute the intracellular F-actin in PC-3M-1E8 cells, which directly leads to the decreased invasion and metastasis potential of tumor cells. Taken together, these results suggest that PAG acts to inhibit the development and metastasis of prostate cancers and represents a novel therapeutic target for prostate cancer.     

Human bone marrow-derived mesenchymal stem cells produced TGFbeta contributes to progression and metastasis of prostate cancer

Human mesenchymal stem cells (hMSCs) play an important role in the development of human cancers. In the present study, we observed that hMSCs promoted human prostate cancer (PCa) cell PC-3 growth in vivo and in vitro. The conditional medium of hMSCs promoted the proliferation, migration, and invasion of PC-3 cells. The expression of MMP-2 and MMP-9 in PC-3 was upregulated by conditional medium of hMSCs. In addition, blocking tumor transformation factor beta (TGFβ) blunted the pro-oncogenic function of hMSCs. These results suggest that hMSCs may play a pro-oncogenic role in the growth of human prostate caner by producing TGFβ.     

Naftopidil, a selective alpha-1 adrenoceptor antagonist, inhibits growth of human prostate cancer cells by G1 cell cycle arrest

Alpha-1 adrenoceptor antagonists are generally prescribed for benign prostate hyperplasia with lower urinary tract symptoms. Naftopidil, a selective alpha-1 adrenoceptor antagonist, is frequently used in Japan because it has fewer side effects. Here we demonstrate for the first time that naftopidil has growth inhibitory effect in androgen-sensitive and -insensitive human prostate cancer cell lines. The concentrations causing 50% inhibition (IC50) of cancer cell growth were 22.2 +/- 4.0 microM in androgen-sensitive LNCaP cells and 33.2 +/- 1.1 microM in androgen-insensitive PC-3 cells. FACS analysis revealed that cell growth inhibition by naftopidil was due to the arrest of the G1 cell cycle. Expressions of p27(kip1) and p21(cip1) were significantly increased in LNCaP cells treated with naftopidil. In PC-3 cells, naftopidil induced p21(cip1) but not p27(kip1). In vivo, oral administration of naftopidil to nude mice inhibited the growth of PC-3 tumors as compared to vehicle-treated controls. These results suggest that naftopidil may be useful in the chemoprevention of prostate cancer and the intervention of hormone refractory prostate cancer.     

Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells

Calcitonin (CT) and its receptor (CTR) are expressed only in basal epithelium of benign prostate and in whole epithelium of malignant prostates. Also, CT and CTR mRNA levels in prostate cancers increase with an increase in tumor grade. We tested the role of the CT/CTR autocrine axis on the tumorigenicity of prostate cancer cells. We enforced the expression of CTR in CT-positive/CTR-deficient PC-3 cells. In contrast, we knocked down CTR expression in CT/CTR-positive PC-3M cells. The effect of CTR modulation on the oncogenicity was evaluated by the rate of cell proliferation, invasion, colony formation and in vivo growth in nude mice. Up-regulation of CTR in PC-3 cells and its down-regulation in PC-3M cells significantly altered their tumorigenicity. Intratumorally administered CTR RNAi in preexisting PC-3M xenografts markedly attenuated their further growth. This treatment also led to a remarkable decrease in endothelial cell populations in the tumors and increase in apoptotic, PCNA-negative cell populations. Tumors receiving CTR RNAi treatment displayed markedly lower levels of urokinase-type plasminogen activator, phospho-Akt and survivin, suggesting CTR activates uPA-uPAR axis and PI-3-kinase-Akt-survivin pathway. These results suggest an important role for CT-CTR autocrine axis in the progression of localized prostate tumor to a metastatic phenotype, and offer a potential therapeutic option for invasive cancers.     

Repulsive guidance molecules, novel bone morphogenetic protein co-receptors, are key regulators of the growth and aggressiveness of prostate cancer cells

Repulsive guidance molecule (RGM) family members RGMA, RGMB and RGMC are GPI-linked membrane proteins recently identified as co-receptor of bone morphogenetic proteins (BMPs). BMPs are a group of proteins enriched in bone and play important roles in prostate cancer. The current study aimed to investigate roles played by RGMs in prostate cancer. Expression of RGMs was examined in prostate cancer cell lines and prostate cancer tissues using RT-PCR and immunohistochemical staining. Knockdown of each RGM in prostate cancer cells was performed using the respective anti-RGMA, RGMB and RGMC transgenes. A variety of in vitro function tests were employed to analyze the influence on cancer cell functions by RGM knockdown. The implications of RGM knockdown in BMP signalling were also examined using both Western blot and real-time quantitative PCR. Knockdown of RGMA had no effect on cell growth, migration and invasion, but promoted cell-matrix adhesion. Knockdown of RGMB and RGMC increased growth and adhesion, but only RGMB knockdown increased capacities of migration and invasion in PC-3 cells. Further investigations showed an increase in Smad-3 activation and reduced levels of Smad-1 in PC-3 cells by RGMB and RGMC knockdown, and also an up-regulation of ID1, a BMP target gene particularly in exposure to BMP7. RGMs play inhibitory roles in prostate cancer by suppressing cell growth, adhesion, migration and invasion. RGMs can coordinate Smad-dependent signalling of BMPs in prostate cancer cells.     

Growth and differentiation factor-9 promotes adhesive and motile capacity of prostate cancer cells by up-regulating FAK and Paxillin via Smad dependent pathway

The majority of advanced prostate cancers metastasis to the bone. Mediators of bone remodelling, the bone morphogenetic proteins have extensively been implicated in the progression and metastasis of prostate cancer. The present study investigated the function of BMP member GDF-9, in prostate cancer. We overexpressed GDF9 in PC-3 cells using a mammalian expression construct, and knocked-down with the use of ribozyme transgenes. These cells were further used in in vitro adhesion and motility assays, in order to determine the effect of GDF-9 on these properties. Recombinant GDF-9 was generated to treat PC-3 WT cells before further analysing the effect on adhesion. The GDF-9 overexpressing PC-3 cells demonstrated a significantly enhanced adhesive and motile capacity compared to their controls. The opposite effect was seen in the GDF-9 knock-down cells. In addition, treating PC-3 cells with rh-GDF-9 resulted in them becoming more adhesive. Both endogenous and exogenous GDF-9 was demonstrated to up-regulate focal adhesion associated proteins FAK and paxillin which contribute to promoted cell adhesion and motility. With the use of a Smad3 inhibitor, this effect was inhibited suggesting that GDF-9 signals via Smad3 to up-regulate expression of these proteins. This study shows that GDF-9 can promote the motile and adhesive capacity of PC-3 prostate cancer cells by up-regulating expression of FAK and paxillin in a Smad dependent manner, suggesting a pro-tumourigenic role for GDF-9 in prostate cancer.     

Glucose-regulated protein 78 mediates hormone-independent prostate cancer progression and metastasis through maspin and COX-2 expression

Glucose-regulated protein 78 (GRP78) plays an essential role in embryonic development and in the progression and therapeutic resistance of many cancers. However, little is known about the function of GRP78 in hormone-independent prostate cancer. Here, we found that the expression levels of GRP78 were higher in PC-3 cells than in DU-145 cells. When the expression of GRP78 was silenced using a GRP78-specific small interfering RNA in PC-3 cells, the growth rate and adhesive ability were reduced. Cell migration was dramatically decreased in GRP78-depleted cells. Dissection of the involved signal pathways revealed that maspin expression was upregulated after silencing GRP78 expression. The upregulation of maspin and downregulation of COX-2 may cause the decrease in cell proliferation and migration observed after silencing GRP78 expression. Silencing GRP78 expression may suppress the proliferation, adhesion, and migration of prostate cancer cells via maspin and COX-2 regulation.     

Diverse biological effect and Smad signaling of bone morphogenetic protein 7 in prostate tumor cells

We found that bone morphogenetic protein (BMP) 7, a member of the BMP family, was strikingly up-regulated during the development of primary prostatic adenocarcinoma in the conditional Pten deletion mouse model. To determine the relevance of this finding to human prostate cancer, we examined the expression of BMPs and BMP receptors (BMPR) as well as the responsiveness to recombinant human BMP7 in a series of human prostate tumor cell lines. All prostatic cell lines tested expressed variable levels of BMP2, BMP4, and BMP7 and at least two of each type I and II BMPRs. In all cases, BMP7 induced Smad phosphorylation in a dose-dependent manner, with Smad5 activation clearly demonstrable. However, the biological responses to BMP7 were cell type specific. BPH-1, a cell line representing benign prostatic epithelial hyperplasia, was growth arrested at G1. In the bone metastasis-derived PC-3 prostate cancer cells, BMP7 induced epithelial-mesenchymal transdifferentiation with classic changes in morphology, motility, invasiveness, and molecular markers. Finally, BMP7 inhibited serum starvation-induced apoptosis in the LNCaP prostate cancer cell line and more remarkably in its bone metastatic variant C4-2B line. Each of the cell lines influenced by BMP7 was also responsive to BMP2 in a corresponding manner. The antiapoptotic activity of BMP7 in the LNCaP and C4-2B cell lines was not associated with a significant alteration in the levels of the proapoptotic protein Bax or the antiapoptotic proteins Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis. However, in C4-2B cells but not in LNCaP cells, a starvation-induced decrease in the level of survivin was counteracted by BMP7. Taken together, these findings suggest that BMPs are able to modulate the biological behavior of prostate tumor cells in diverse and cell type-specific manner and point to certain mechanisms by which these secreted signaling molecules may contribute to prostate cancer growth and metastasis.     

KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer

Molecular mechanisms of prostate cancer progression are poorly understood. Here, we studied gene amplification of the large conductance calcium-activated potassium channel alpha subunit (KCNMA1), which is located at the chromosomal region 10q22. Fluorescence in situ hybridization (FISH) revealed KCNMA1 amplification in 16% of 119 late-stage human prostate cancers and in the hormone-insensitive prostate cancer cell line PC-3. In contrast, KCNMA1 amplification was absent in 33 benign controls, 32 precursor lesions and in 105 clinically organ-confined prostate cancers. Amplification was associated with mRNA and protein overexpression as well as increased density of BK channel protein and beta-estradiol-insensitive BK currents in PC-3 cells as compared to non-amplified control cell lines. Specific blockade of BK channels by iberiotoxin or RNA(i) significantly inhibited K(+) currents and growth of PC-3 cells. The data demonstrate that 10q22 amplification drives KCNMA1 expression and cell proliferation. Thus, KCNMA1 qualifies as a promising diagnostic and therapeutic target in patients with prostate cancer.