Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model.

Bcl-2 has emerged as a critical regulator of apoptosis in a variety of cell systems and is up-regulated during progression to androgen independence in prostate cancer cells. The objectives of this study were to characterize changes in Bcl-2 after androgen withdrawal and during progression to androgen independence in the human prostate LNCaP tumor model and determine whether adjuvant use of antisense Bcl-2 oligodeoxynucleotides (ODNs) with androgen ablation delays progression to androgen independence. Bcl-2 expression in LNCaP cells is down-regulated to undetectable levels by androgen in vitro and up-regulated after castration in vivo. Antisense Bcl-2 ODN treatment reduced LNCaP cell Bcl-2 messenger RNA and protein levels by >90% in a sequence-specific and dose-dependent manner at concentrations >50 nM. Bcl-2 mRNA levels returned to pretreatment levels by 48 h after discontinuing treatment. Athymic male mice bearing SQ LNCaP tumors were castrated and injected i.p. with 12.5 mg/kg/day with two-base mismatch ODN control, reverse polarity ODN control, or antisense Bcl-2 ODN. Tumor volume in control mice gradually increased 5-fold (range, 3-6) by 12 weeks after castration compared to a 10-50% decrease in precastrate tumor volume in mice treated with antisense Bcl-2 ODN. Changes in serum PSA paralleled changes in tumor volume, increasing 4-fold faster above nadir in controls than in mice treated with antisense Bcl-2 ODN. After decreasing 70% by 1 week after castration, PSA increased 1.6-fold above precastrate levels by 11 weeks in controls while staying 30% below precastrate levels in antisense-treated mice. In a second group of experiments, LNCaP tumor growth and serum PSA levels were 90% lower (P<0.01) in mice treated with antisense Bcl-2 ODN compared with mismatch or reverse polarity ODN controls. These results support the hypothesis that Bcl-2 helps mediate progression to androgen independence and is an appropriate target for antisense therapy.     

Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.     

NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.     

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.     

Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor

We have shown recently that a 15-mer phosphorothioate oligodeoxynucleotide (ODNas750/15) that hybridizes to the (CAG)n polyglutamine region of mRNA encoding human androgen receptor (AR) inhibits the expression of AR in LNCaP prostate cancer cells in vitro. This AR downregulation was accompanied by significant cell growth inhibition and reduced PSA secretion. In the present study we investigated the effects of this antisense AR ODN on prostate tumor growth in vivo using a mouse xenograft model. Via subcutaneously implanted diffusion pumps, either ODNas750/15 or a scrambled control sequence ODNsr750/15 was continuously administered into LNCaP tumor-bearing male nude mice for 7 weeks. Compared with untreated control animals, treatment with ODNas750/15 resulted in significant tumor growth inhibition. Retardation of tumor growth was also significant in castrated mice, whereas the scrambled control ODN did not exert any effects. No side effects such as loss of body weight were observed at any time of treatment. ODN treatment was well tolerated and, in contrast to castration, did not induce shrinkage of mouse prostates. Both AR expression in the tumor and PSA levels in mouse serum correlated with tumor size. However, we failed to demonstrate a correlation between tumor retardation and Ki-67 antigen expression and the number of apoptotic cells, respectively. Testing of antisense-treated LNCaP cells revealed that expression levels of other proteins that contain shorter polyglutamine sequence stretches such as HDAC2, TFIID, and c-jun were not affected. The present study demonstrates that downregulation of AR with antisense ODNas750/15 causes prostate tumor growth inhibition. These results further point out the important role of the AR in prostate tumors and support further testing of AR downregulation for treatment of prostate cancer.     

Interleukin-4 enhances prostate-specific antigen expression by activation of the androgen receptor and Akt pathway

Androgen receptor (AR) plays an important role in the development and progression of prostate cancer upon the action of androgen through the binding of the androgen-responsive elements (AREs) on the target genes. Abnormal activation of the AR by nonandrogen has been implicated in the progression of androgen-independent prostate cancer. The levels of interleukin-4 (IL-4) are significantly elevated in sera of patients with hormone refractory prostate cancer. The potential role of IL-4 on the activation of AR was investigated in prostate cancer cells. IL-4 enhances AR-mediated prostate-specific antigen (PSA) expression and ARE-containing gene activity through activation of the AR in the androgen ablation condition in human prostate cancer cells. The AR can also be sensitized by IL-4 and activated by significantly lower levels of androgen (10 pM of R1881) in prostate cancer cells. IL-4 enhances nuclear translocation of AR and increases binding of the AR to the ARE in LNCaP prostate cancer cells. Blocking of the Akt pathway by an Akt-specific inhibitor LY294002 abrogates IL-4-induced PSA expression and AR signaling. These results demonstrate that IL-4 enhances PSA expression through activation of the AR and Akt signaling pathways in LNCaP prostate cancer cells. Understanding IL-4-induced signaling leading to abnormal activation of AR will provide insights into the molecular mechanisms of androgen-independent progression of prostate cancer cells.     

Castration-induced increases in insulin-like growth factor-binding protein 2 promotes proliferation of androgen-independent human prostate LNCaP tumors

Activation of alternative growth factor pathways after androgen withdrawal is one mechanism mediating androgen-independent (AI) progression in advanced prostate cancer. Insulin-like growth factor (IGF) I activation is modulated by a family of IGF binding proteins (IGFBPs). Although IGFBP-2 is one of the most commonly overexpressed genes in hormone refractory prostate cancer, the functional significance of changes in IGF-I signaling during AI progression remains poorly defined. In this article, we characterize changes in IGFBP-2 in the LNCaP tumor model after androgen withdrawal and evaluate its functional significance in AI progression using gain-of-function and loss-of-function analyses. IGFBP-2 mRNA and protein levels increase 2-3-fold after androgen withdrawal in LNCaP cells in vitro in LNCaP tumors during AI progression in vivo. Increased IGFBP-2 levels after castration were also identified using a human prostate tissue microarray of untreated and posthormone therapy-treated prostatectomy specimens. LNCaP cell transfectants that stably overexpressed IGFBP-2 progressed more rapidly after castration than control tumors. Antisense oligonucleotides (ASOs) targeting the translation initiation site of IGFBP-2 reduced IGFBP-2 mRNA and protein expression by >70% in a dose-dependent and sequence-specific manner. ASO-induced decreases in IGFBP-2-reduced LNCaP cell growth rates and increased apoptosis 3-fold. LNCaP tumor growth and serum prostate-specific antigen levels in mice treated with castration plus adjuvant IGFBP-2 ASOs were significantly reduced compared with mismatch control oligonucleotides. Increased IGFBP-2 levels after androgen ablation may represent an adaptive response that helps potentiate IGF-I-mediated survival and mitogenesis and promote androgen-independent tumor growth. Inhibiting IGFBP-2 expression using ASO technology may offer a treatment strategy to delay AI progression.     

Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G(1) phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.     

Down-Regulation of prostate-specific antigen expression by ligands for peroxisome proliferator-activated receptor gamma in human prostate cancer

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Recent studies found that ligand-activated PPARgamma regulated differentiation and clonal growth of several types of cancer cells, including prostate cancer, suggesting that PPARgamma could be a tumor suppressor. Troglitazone was a widely used antidiabetic drug that activates PPARgamma. Recently, we reported that this agent had antiprostate cancer effects in vitro and in vivo. In this study, we administered troglitazone for over 1.5 years to an individual with occult recurrent prostate cancer. Using the prostate-specific antigen (PSA) levels as a surrogate marker of the disease, the oral administration of troglitazone (600-800 mg/day) reduced the increase velocity of PSA levels, suggesting clinical efficacy of troglitazone in prostate cancer. PSA promoter/ enhancer reporter assays showed that the PPARgamma ligands troglitazone (10(-5) M), pioglitazone (10(-5) M), or 15-deoxy-delta12,14-prostaglandin J2 (10(-5) M) down-regulated androgen-stimulated reporter gene activity in LNCaP cells, a prostate cancer cell line. The PSA promoter contains androgen receptor response elements (AREs). Reporter gene studies showed that troglitazone inhibited androgen activation of the AREs in the PSA regulatory region. Consistent with inhibition of gene expression, 2 days of incubation of LNCaP with troglitazone dramatically suppressed PSA protein expression without suppressing AR expression, suggesting that troglitazone inhibited ARE activation by a mechanism other than down-regulation of expression of the AR. Taken together, ligands of PPARgamma may be a useful therapeutic approach for the treatment of prostate cancer and may be acting, in part, by inhibiting transactivation of androgen-responsive genes.     

The nuclear factor-kappaB pathway controls the progression of prostate cancer to androgen-independent growth

Typically, the initial response of a prostate cancer patient to androgen ablation therapy is regression of the disease. However, the tumor will progress to an "androgen-independent" stage that results in renewed growth and spread of the cancer. Both nuclear factor-kappaB (NF-kappaB) expression and neuroendocrine differentiation predict poor prognosis, but their precise contribution to prostate cancer progression is unknown. This report shows that secretory proteins from neuroendocrine cells will activate the NF-kappaB pathway in LNCaP cells, resulting in increased levels of active androgen receptor (AR). By blocking NF-kappaB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-kappaB signaling in vivo by the absence of the IkappaBalpha inhibitor prevents regression of the prostate after castration by sustaining high levels of nuclear AR and maintaining differentiated function and continued proliferation of the epithelium. Furthermore, the NF-kappaB pathway was activated in the ARR(2)PB-myc-PAI (Hi-myc) mouse prostate by cross-breeding into a IkappaBalpha(+/-) haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-kappaB is sufficient to maintain androgen-independent growth of prostate and prostate cancer by regulating AR action. Thus, the NF-kappaB pathway may be a potential target for therapy against androgen-independent prostate cancer.     

Castration-induced up-regulation of insulin-like growth factor binding protein-5 potentiates insulin-like growth factor-I activity and accelerates progression to androgen independence in prostate cancer models

Although insulin-like growth factor binding protein-5 (IGFBP-5) has been shown to be implicated in prostate cancer progression, the functional role of IGFBP-5 in progression to androgen-independence remains largely undefined. Here, we demonstrate substantial up-regulation of IGFBP-5 during castration-induced regression and androgen-independent (AI) progression in the mouse androgen-dependent (AD) Shionogi tumor model. To analyze the functional significance of these changes in IGFBP-5, human AD LNCaP prostate cancer cells were stably transfected with IGFBP-5 gene, and IGFBP-5-overexpressing LNCaP tumors progressed significantly faster to androgen independence after castration compared with controls. Antisense mouse IGFBP-5 oligodeoxynucleotides (ODNs) were then designed that reduced IGFBP-5 expression in Shionogi tumor cells in vitro in a dose-dependent and sequence-specific manner. Growth of Shionogi tumor cells was inhibited by antisense IGFBP-5 ODN treatment in a time- and dose-dependent manner, which could be reversed by exogenous IGF-I. However, antisense IGFBP-5 ODN treatment had no additive inhibitory effect on Shionogi tumor cell growth when IGF-I activity was neutralized by anti-IGF-I antibody. Antisense IGFBP-5 ODN treatment resulted in decreased mitogen-activated protein kinase activity and number of cells in the S + G2-M phases of the cell cycle that directly correlated with reduced proliferation rate of Shionogi tumor cells. Systemic administration of antisense IGFBP-5 ODN in mice bearing Shionogi tumors after castration significantly delayed time to progression to androgen independence and inhibited growth of AI recurrent tumors. These findings suggest that up-regulation of IGFBP-5 after castration serves to enhance IGF bioactivity and raise the possibility that the response of prostate cancer to androgen withdrawal can be enhanced by strategies, such as antisense IGFBP-5 ODN therapy, that target IGF signal transduction.     

Stromal TGF-β signaling induces AR activation in prostate cancer

AR signaling is essential for the growth and survival of prostate cancer (PCa), including most of the lethal castration-resistant PCa (CRPC). We previously reported that TGF-β signaling in prostate stroma promotes prostate tumor angiogenesis and growth. By using a PCa/stroma co-culture model, here we show that stromal TGF-β signaling induces comprehensive morphology changes of PCa LNCaP cells. Furthermore, it induces AR activation in LNCaP cells in the absence of significant levels of androgen, as evidenced by induction of several AR target genes including PSA, TMPRSS2, and KLK4. SD-208, a TGF-β receptor 1 specific inhibitor, blocks this TGF-β induced biology. Importantly, stromal TGF-β signaling together with DHT induce robust activation of AR. MDV3100 effectively blocks DHT-induced, but not stromal TGF-β signaling induced AR activation in LNCaP cells, indicating that stromal TGF-β signaling induces both ligand-dependent and ligand-independent AR activation in PCa. TGF-β induces the expression of several growth factors and cytokines in prostate stromal cells, including IL-6, and BMP-6. Interestingly, BMP-6 and IL-6 together induces robust AR activation in these co-cultures, and neutralizing antibodies against BMP-6 and IL-6 attenuate this action. Altogether, our study strongly suggests tumor stromal microenvironment induced AR activation as a direct mechanism of CRPC.