Promotes Prostate Cancer Growth Research Articles

Activation of Androgen Receptor, Lipogenesis, and Oxidative Stress Converged by SREBP-1 Is Responsible for Regulating Growth and Progression of Prostate Cancer Cells

We previously reported that sterol regulatory element-binding protein-1 (SREBP-1) is involved in the transcriptional regulation of androgen receptor (AR) and formation of fatty acid through altered expression of fatty acid synthase (FASN). In this article, we provide a new finding that SREBP-1 induced oxidative stress in prostate cancer cells through increased production of reactive oxygen species (ROS) and expression of NADPH oxidase 5 (Nox5). We have shown that (i) expression of SREBP-1 protein is positively associated with the clinical Gleason grades in human prostate cancer; (ii) genetic overexpression or knockdown of SREBP-1 in prostate cancer cells resulted in corresponding increased or decreased AR, FASN and Nox5 expression, fatty acid and lipid droplet accumulation, and ROS generation; and (iii) SREBP-1 induces and promotes the growth, migration, invasion, and castration-resistant progression of prostate cancer cells in vitro and in vivo. Our data show a novel molecular mechanism by which SREBP-1 promotes prostate cancer growth and progression through alterations in the concerted intracellular metabolic and signaling networks involving AR, lipogenesis, and ROS in prostate cancer cells.

Ligand-dependent Corepressor Acts as a Novel Androgen Receptor Corepressor, Inhibits Prostate Cancer Growth, and Is Functionally Inactivated by the Src Protein Kinase

The activated androgen receptor (AR) promotes prostate cancer (PCa) growth. AR antagonists repress the AR by recruitment of corepressors. Not much is known about the inactivation of AR by corepressors in the presence of agonists (androgens). Here we show that the corepressor LCoR acts as an androgen-dependent corepressor that represses human PCa growth in vivo. In line with this, progressive decrease of ligand-dependent corepressor expression was observed in the PCa TRAMP mouse model with increasing age. LCoR interacts with AR and is recruited to chromatin in an androgen-induced manner. Unexpectedly, the LXXLL motif of LCoR is dispensable for interaction with the AR. Rather, the data indicate that LCoR interacts with the AR DNA binding domain on DNA. Interestingly, the interaction of LCoR with AR is inhibited by signaling pathways that are associated with androgen-independent PCa. Here we also show that the Src kinase inactivates the corepressive function of LCoR. Interfering with endogenous Src function by a dominant negative Src mutant, the growth inhibitory activity of LCoR is enhanced in vivo in a xenograft mouse model system. Thus, our studies indicate a role of LCoR as an AR corepressor and a tumor suppressor. Further, the decreased expression or inactivation of LCoR is as an important step toward PCa carcinogenesis in vivo.

Potential Prostate Cancer Drug Target: Bioactivation of Androstanediol by Conversion to Dihydrotestosterone

High-affinity binding of dihydrotestosterone (DHT) to the androgen receptor (AR) initiates androgen-dependent gene activation, required for normal male sex development in utero, and contributes to prostate cancer development and progression in men. Under normal physiologic conditions, DHT is synthesized predominantly by 5α-reduction of testosterone, the major circulating androgen produced by the testis. During androgen deprivation therapy, intratumoral androgen production is sufficient for AR activation and prostate cancer growth, even though circulating testicular androgen levels are low. Recent studies indicate that the metabolism of 5α-androstane-3α, 17β-diol by 17β-hydroxysteroid dehydrogenase 6 in benign prostate and prostate cancer cells is a major biosynthetic pathway for intratumoral synthesis of DHT, which binds AR and initiates transactivation to promote prostate cancer growth during androgen deprivation therapy. Drugs that target the so-called backdoor pathway of DHT synthesis provide an opportunity to enhance clinical response to luteinizing-hormone-releasing hormone (LHRH) agonists or antagonists, AR antagonists, and inhibitors of 5α-reductase enzymes (finasteride or dutasteride), and other steroid metabolism enzyme inhibitors (ketoconazole or the recently available abiraterone acetate).

The IL-8–Regulated Chemokine Receptor CXCR7 Stimulates EGFR Signaling to Promote Prostate Cancer Growth

The proinflammatory chemokine receptor CXCR7 that binds the ligands CXCL11 and CXCL12 (SDF-1a) is elevated in a variety of human cancers, but its functions are not understood as it does not elicit classical chemokine receptor signaling. Here we report that the procancerous cytokine IL-8 (interleukin-8) upregulates CXCR7 expression along with ligand-independent functions of CXCR7 that promote the growth and proliferation of human prostate cancer cells (CaP cells). In cell culture, ectopic expression or addition of IL-8 selectively increased expression of CXCR7 at the level of mRNA and protein production. Conversely, suppressing IL-8 signaling abolished the ability of IL-8 to upregulate CXCR7. RNAi-mediated knockdown of CXCR7 in CaP cells caused multiple antitumor effects, including decreased cell proliferation, cell-cycle arrest in G(1) phase, and decreased expression of proteins involved in G(1) to S phase progression. In contrast, addition of the CXCR7 ligand SDF-1a and CXCL11 to CaP cells did not affect cell proliferation. Over expression of CXCR7 in normal prostate cells increased their proliferation in a manner associated with increased levels of phospho-EGFR (epidermal growth factor receptor; pY1110) and phospho-ERK1/2. Notably, coimmunoprecipitation studies established a physical association of CXCR7 with EGFR, linking CXCR7-mediated cell proliferation to EGFR activation. Consistent with these findings, CXCR7-depleted CaP tumors grew more slowly than control tumors, expressing decreased tumor-associated expression of VEGF, cyclin D1, and p-EGFR. Together, these results reveal a novel mechanism of ligand-independent growth promotion by CXCR7 and its coregulation by the proinflammatory factor IL-8 in prostate cancer.

Vitamin D deficiency promotes prostate cancer growth in bone

Vitamin D is considered as an important determinant of bone turnover as well as cancer growth. Using a murine model of bone metastasis, we investigated the effect of vitamin D deficiency on prostate cancer cell growth in bone. Three-week-old male nude mice were fed either normal chow (control) or a diet deficient in vitamin D. The latter diet resulted in severe hypovitaminosis D within 6 weeks. At this point of time, 5 × 10(4) cells of the prostate cancer cell line, PC-3, were injected either into the bone marrow (tibia) or subcutaneously into soft tissues. Osteoprotegerin (OPG) was co-administered in subgroups of mice to suppress bone remodeling. Osteolytic lesions were monitored by serial X-ray, while soft tissue tumor growth was measured by caliper. All tissues were analyzed by micro-CT and histology at endpoint. Bone turnover was significantly accelerated in vitamin D deficient compared to vitamin D sufficient mice from week 6 onwards. Intra-tibially implanted PC-3 cells resulted in mixed osteolytic and osteosclerotic lesion. At endpoint, osteolytic and osteosclerotic lesion areas, total tumor area, and tumor mitotic activity were all significantly increased in vitamin D deficient mice compared to controls. Regardless of diet, OPG reduced bone turnover, total tumor, and osteosclerotic area as well as tumor mitotic activity, while promoting cell apoptosis. In contrast, vitamin D deficiency did not alter tumor growth in soft tissues. Vitamin D deficiency stimulates prostate cancer growth in bone through modulating the bone microenvironment.

Human prostate cancer xenografts in lit/lit mice exhibit reduced growth and androgen‐independent progression

The growth hormone/insulin-like growth factor I (GH/IGF-I) axis has been linked to prostate cancer (PCa) risk. Although previous studies indicate that human breast cancers and a murine PCa model develop more slowly in murine hosts homozygous for a missense mutation in the GH-releasing hormone receptor (lit/lit) whose "little" dwarfed phenotype is caused by suppressed GH and IGF-I production, the role of these two hormones remains controversial. To assess how the GH/IGF-I axis influences androgen-responsive, castration-resistant (CR), and androgen-independent (AI) growth of human PCa, we compared xenograft growth of the androgen-responsive human PCa cells, LNCaP, and AI human PCa cells, PC3, in intact and castrate Nod/SCID lit/lit and lit/+ mice, and in vitro growth of these cell lines in lit/lit and lit/+ serum-containing media supplemented with GH or IGF-I. Tumor growth and PSA accumulation rates were suppressed in LNCaP tumor-bearing lit/lit mice pre- and post-castration. Growth of PC3 xenografts in lit/lit mice was also suppressed. In vitro proliferation of LNCaP and PC3 cells cultured in media containing lit/lit mouse serum was decreased as compared to growth in media containing lit/+ serum. Suppressed growth in lit/lit serum could be restored by the addition of IGF-I, and to a lesser extent, GH. Differences in growth correlated with differences in steady-state AKT and ERK1/2 activation. This study demonstrates that circulating GH and IGF-I can promote androgen-responsive growth, CR progression, and AI expansion of PTEN-deficient human PCa cell xenografts and indicates that IGF-I can promote PCa growth in a suppressed GH environment.

Chronic systemic hypoxia promotes LNCaP prostate cancer growth in vivo

Solid tumors contain underperfused regions where hypoxia-inducible factor-1alpha (HIF-1alpha) over-expression induces hypoxia adaptation and cell proliferation. We test the hypothesis that systemic hypoxia promotes prostate cancer growth in vivo and examine HIF-1alpha centrality in this effect. Male athymic mice were xenografted with 3 x 10(6) LNCaP cells per each flank and exposed for 28 days to either chronic hypoxia (CH, 10% O(2)) or CH with reoxygenation (CHReox, 3 times/week for 1 hr), with normoxia as control (n = 17, 9, and 20, respectively). At the end of the observation, mice were euthanized and tumors harvested for analyses. The successful xenografts grew faster in CH and CHReox than in normoxia (first-order rate constants 0.15 +/- 0.01, 0.18 +/- 0.03, and 0.09 +/- 0.01 day(-1), P < 0.05, n = 18, 15, and 25, respectively). Furthermore, the tumor masses at the end were 4.09 +/- 0.58, 3.42 +/- 0.55, and 1.86 +/- 0.25 mg/g bw (P < 0.05), respectively. HIF-1alpha, assayed by Western blot and immunofluorescence, was slightly increased in CH with respect to normoxia, but markedly over-expressed (5-10 times) in CHReox (P < 0.001). The tumor hemoglobin content, higher in CH and CHReox than in normoxia, reflected the higher blood hemoglobin concentration, not neovascularization, as supported by similar expression levels of vascular endothelial growth factor (VEGF) in the three groups. By contrast, protein kinase B (Akt) was more phosphorylated in both hypoxic groups than in normoxia (P < 0.01). In vivo systemic hypoxia promotes prostate cancer growth regardless of HIF-1alpha expression level and neovascularization, suggesting an important role for hypoxia-dependent pathways that do not involve HIF-1alpha, as the phosphatidyl inositol-3-phosphate signaling cascade.

Abstract 1418: Stromal cells of prostate, not the bone marrow, facilitate prostate cancer osteoblastic tumor growth in bone

Abstract Stromal cells promote prostate cancer initiation and progression. Metastasized prostate cancers form osteoblastic bone lesions. The prostate cancer associated stroma expresses alpha-smooth muscle actin (alpha-SMA). Additionally, 69% of human prostate cancer tissues lost stromal TGFbeta type II receptor (Tgfbr2) expression. Conditional stromal knockout of the Tgfbr2 mouse model developed prostate adenocarcinoma. In parallel to the primary site, we observed human prostate cancer associated stroma in bone metastasis gained the expression of androgen receptor and alpha-SMA, which were negative in the normal bone marrow. Tgfbr2 expression was also lost in prostate cancer associated stroma in bone marrow, compared to its high expression in the normal bone marrow. This led us to question whether the stromal cells from the primary site or the bone marrow contribute the prostate cancer osteoblastic bone lesion development. In vitro, the conditioned media from Tgfbr2-flox or Tgfbr2-KO mouse prostate stromal cells (PSC) or bone marrow stromal cells (BMSC) were incubated on the C42B prostate cancer epithelia. Expressions of PTHrP, RANKL osteolytic factors and ET1, RUNX2, BMP2 osteoblastic factors by C42B cells were analyzed by qRT-PCR. The Tgfbr2-flox PSC conditioned media significantly increased the C42B cell expression of all the osteolytic and osteoblastic factors examined compared to C42B cells without conditioned media. However, the Tgfbr2-KO PSC conditioned media decreased the expression of PTHrP and RANKL, while further increased the expression of osteoblastic factors, ET1, RUNX2 and BMP2 by C42B cells by at least 2-fold. Conditioned media of Tgfbr2-flox and Tgfbr2-KO BMSC increased the expression of C42B cells on osteolytic factors (10-fold PTHrP and 2-fold RANKL), but neither has significant effect on expression of osteoblastic factors. Importantly, Tgfbr2-KO PSC and BMSC conditioned media increased C42B proliferation similarly by 2-fold over that of respective Tgfbr2-Tgfbr2-flox conditioned media. We found that the reduced PTHrP and RANKL expression was associated with respective promoter methylation, when C42B cells were incubated with Tgfbr2-KO PSC, but not BMSC. Epigenetic silencing of such osteolytic genes, would suggest for the first time, that the prostate stroma could influence osteoblastic tumor at a distant site. So, GFP labeled C42B cells, following pre-incubation with PSC or BMSC conditioned media, were injected into the tibia of SCID mice. X-rays of the bone lesions and fluorescence imaging of GFP-expressing tumor growth were measured. The data suggested, that the stromal cells from primary prostates, but not from the second metastasis site of bone marrow, promotes prostate cancer growth in the bone and osteoblastic bone lesion development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1418.

An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB.

Metastasis is responsible for the majority of prostate cancer-related deaths; however, little is known about the molecular mechanisms that underlie this process. Here we identify an oncogene-tumor suppressor cascade that promotes prostate cancer growth and metastasis by coordinately activating the small GTPase Ras and nuclear factor-kappaB (NF-kappaB). Specifically, we show that loss of the Ras GTPase-activating protein (RasGAP) gene DAB2IP induces metastatic prostate cancer in an orthotopic mouse tumor model. Notably, DAB2IP functions as a signaling scaffold that coordinately regulates Ras and NF-kappaB through distinct domains to promote tumor growth and metastasis, respectively. DAB2IP is suppressed in human prostate cancer, where its expression inversely correlates with tumor grade and predicts prognosis. Moreover, we report that epigenetic silencing of DAB2IP is a key mechanism by which the polycomb-group protein histone-lysine N-methyltransferase EZH2 activates Ras and NF-kappaB and triggers metastasis. These studies define the mechanism by which two major pathways can be simultaneously activated in metastatic prostate cancer and establish EZH2 as a driver of metastasis.

Expression of the Bcl-2 Protein BAD Promotes Prostate Cancer Growth

BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be “addicted” to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.

Amyloid Precursor Protein Is a Primary Androgen Target Gene That Promotes Prostate Cancer Growth

Androgen receptor (AR) is a critical transcription factor that regulates various target genes and contributes to the pathophysiology of prostate cancer hormone dependently. Here, we identify amyloid precursor protein (APP) as a primary androgen target through chromatin immunoprecipitation (ChIP) combined with genome tiling array analysis (ChIP-chip). ChIP-treated DNA were obtained from prostate cancer LNCaP cells with R1881 or vehicle treatment using AR or acetylated histone H3 antibodies. Ligand-dependent AR binding was further enriched by PCR subtraction. Using chromosome 21/22 arrays, we identified APP as one of the androgen-regulated genes with adjacent functional AR binding sites. APP expression is androgen-inducible in LNCaP cells and APP immunoreactivity was correlated with poor prognosis in patients with prostate cancer. Gain-of-function and loss-of-function studies revealed that APP promotes the tumor growth of prostate cancer. The present study reveals a novel APP-mediated pathway responsible for the androgen-dependent growth of prostate cancer. Our findings will indicate that APP could be a potential molecular target for the diagnosis and treatment of prostate cancer.

Stromal Anti-Apoptotic Androgen Receptor Target Gene c-FLIP in Prostate Cancer

The tumor microenvironment significantly influences prostate cancer progression. Androgen receptor exerts its effect through downstream target genes to regulate prostate cancer cell proliferation. The c-FLIP gene was recently shown to be an androgen receptor target gene. c-FLIP is an inactive homologue of caspase-8 and, thus, it inhibits the death receptor mediated apoptosis pathway. c-FLIP over expression was shown to accelerate the progression of prostate cancer cells to androgen independence. We evaluated the role of c-FLIP expression in stromal cells in prostate cancer development. We examined c-FLIP expression in 53 androgen dependent and 21 androgen independent prostate cancer stromal cells by immunohistochemical analysis. The effects of c-FLIP over expression in stromal cells on the growth and invasion of LNCaP and PC3 prostate cancer cells were determined in indirect coculture systems. At the androgen dependent stage the stromal c-FLIP level was increased in prostate cancer tissue. The expression level of stromal c-FLIP was associated with tumor differentiation. However, stromal c-FLIP expression was not increased in androgen independent human prostate cancer. c-FLIP over expression in stromal cells stimulated the growth and invasion of prostate cancer, including LNCaP and PC3 cells in vitro. These results indicate the over expression of stromal c-FLIP and its function for promoting prostate cancer growth and invasion.

Activation of MCP-1/CCR2 axis promotes prostate cancer growth in bone

Prostate cancer (PCa) frequently metastasizes to bone resulting in a mixture of osteolytic and osteoblastic lesions. We have previously reported that monocyte chemotactic protein-1 (MCP-1) is chemotactic for PCa cells, and its receptor, CCR2 expression, correlates with pathological stages. However, the role of MCP-1/CCR2 axis on PCa progression in bone remains unclear. We first evaluated the serum levels of MCP-1 in patients with bone metastases or localized PCa by enzyme-linked immunosorbent assay. We found that MCP-1 levels were elevated in patients with bone metastases compared to localized PCa. We further determined the effects of knockdown CCR2 or MCP-1 on PCa cell invasion and the tumor cell-induced osteoclast activity in vitro, respectively. PCa C4-2B and PC3 cells were transfected stably with either CCR2 short hairpin RNA (shRNA) or a scrambled RNA. CCR2 knockdown significantly diminished the MCP-1-induced PCa cell invasion. In addition, the MCP-1 production was knocked down by MCP-1 shRNA in C4-2B and PC3 cells. Conditioned media (CM) was collected and determined for the CM-induced osteoclast formation in vitro. MCP-1 knockdown significantly decreased the PCa CM-induced osteoclast formation. Finally, MCP-1 knockdown PC3 cells were implanted into the tibia of SCID mice for 4 weeks. Tumor volume was determined by histopathology and bone histomorphometry. MCP-1 knockdown diminished PC3 tumor growth in bone. We concluded that activation of MCP-1/CCR2 axis promotes PCa growth in bone. This study suggests that MCP-1 may be a target for PCa progression.

The Estradiol-Dihydrotestosterone model of prostate cancer

BackgroundThe exact relationship between hormonal activity and prostate cancer(PCa) has not yet been clearly defined. One of the key hormones associated with PCa is testosterone(T). However, both in vitro and in vivo studies have shown that under some conditions T is capable of either promoting PCa growth or death. This article proposes a theory which resolves this apparent paradox.ModelThe Estradiol-Dihydrotestosterone(E-D) model introduced in this paper proposes that 17β-estradiol(E2) is essential for initiating the growth of PCa cells through the formation of telomeres. It also proposes that T is responsible for increasing the expression of proteins which cause apoptosis, or programmed cell death, and that 5α-dihydrotestosterone(DHT) is essential for preventing this. In addition, it is known that some T is converted to both E2 and DHT, which means that depending on the conditions, T is capable of either promoting the growth of or the killing of PCa.

Oxidative 3α-hydroxysteroid dehydrogenase activity of human type 10 17β-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17β-hydroxysteroid dehydrogenase (17β-HSD10) catalyzes the oxidation of 5α-androstane-3α,17β-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17β-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17β-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17β-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17β-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3α-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17β-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.

Transforming growth factor-beta 1 overproduction in prostate cancer: effects on growth in vivo and in vitro.

We found previously that transforming growth factor-beta 1 (TGF beta 1) mRNA levels are markedly elevated in rat prostate cancer (Dunning R3327 sublines) compared to levels in normal prostate. Our goal was to determine whether elevated expression of TGF beta 1 is biologically relevant to prostate cancer growth in vivo. We chose as our model the R3327-MATLyLu prostate cancer epithelial cell line, which produces metastatic anaplastic tumors when reinoculated in vivo. Our approach was to stably transfect MATLyLu cells with an expression vector that codes for latent TGF beta 1 and to isolate subclones of cells that over-expressed TGF beta 1 mRNA. We also isolated a subclone of MATLyLu cells transfected with a control vector lacking the TGF beta 1 cDNA insert. We then studied the growth of these cells in vivo and in vitro. Twenty days after sc inoculation of 10(6) cells in vivo, TGF beta 1-overproducing MATLyLu tumors were 50% larger, markedly less necrotic, and produced more extensive metastatic disease (lung metastases in 73% of all lobes and lymph node metastases in 88% of animals) compared to control MATLyLu tumors (lung metastases, 21%; lymph node metastases, 7%). Thus, TGF beta 1 produced in vivo is biologically active and can promote prostate cancer growth, viability, and aggressiveness, perhaps via effects on the host and/or on the tumor cells themselves. When followed in vitro, TGF beta 1-overproducing cells became growth inhibited, but this effect was transient as cells subsequently resumed proliferating. Growth inhibition was due to TGF beta, because it could be prevented by TGF beta-neutralizing antibody. Therefore, prostate cancer cells can activate and respond to secreted latent TGF beta 1, and although the cells are transiently inhibited in vitro, there is no net inhibition of growth. The ability of the cells to respond to endogenously produced TGF beta 1 suggests that TGF beta 1 overexpression enhances tumor growth in vivo at least in part via an effect of TGF beta 1 on the tumor cells themselves.