An integrative model of prostate cancer interaction with the bone microenvironment.

Despite advanced efforts in early diagnosis, aggressive surgical treatment, and use of targeted chemotherapies, the prognosis for many cancers is still dismal. This emphasizes the necessity to develop new strategies for understanding tumor growth and metastasis. Here we use a systems approach that combines mathematical modeling and numerical simulation to develop a predictive computational model for prostate cancer and its subversion of the bone microenvironment. This model simulates metastatic prostate cancer evolution, progressing from normal bone and hormone levels to quantifiable diseased states. The simulations clearly demonstrate phenomena similar to those found clinically in prostate cancer patients. In addition, the major prediction of this model is the existence of low and high osteogenic states that are markedly different from one another. The existence and potential realization of these steady states appear to be mediated by the Wnt signaling pathway and by the effects of PSA on TGF-ß, which encourages the bone microenvironment to evolve. The model is used to explore several potential therapeutic strategies, with some potential drug targets showing more promise than others: in particular, completely blocking Wnt and greatly increasing DKK-1 had significant positive effects, while blocking RANKL did not improve the outcome.

Fibulin-3 promotes muscle-invasive bladder cancer.

Urothelial carcinoma is the most common type of bladder cancer and can be categorized as either non-muscle-invasive (Ta-T1) or muscle-invasive (⩾T2). The majority of bladder cancers are non-muscle-invasive at presentation; however, the recurrence rate for these tumors is high and a subset can progress to T2. In this study, we aimed to identify genes differentially expressed between T1 vs T2 bladder cancer to identify key regulators of bladder cancer progression and/or invasion. We performed RNA-Seq on T1 and T2 bladder cancer tissues and used publicly available bladder cancer profiling studies to prioritize differentially expressed genes for validation and functional assessment. This integrative approach nominated an extracellular matrix glycoprotein, fibulin-3 (FBLN3, also known as EFEMP1), as being highly expressed in T2 vs T1 bladder cancer and aggressive vs indolent disease. We confirmed the overexpression of fibulin-3 in ⩾T2 vs non-muscle-invasive bladder cancer (NMIBC) by quantitative reverse transcriptase-PCR. Consistent with these findings, fibulin-3 expression level correlated with the invasive ability of several bladder cancer cell lines and modulation of fibulin-3 expression directly affected invasion. Fibulin-3 knockdown in bladder cancer cells decreased the incidence of MIBCs in a murine orthotopic bladder cancer model and decreased the expression of insulin-like growth factor-binding protein-5 (IGFBP5). Restoring IGFBP5 in these cells rescued their invasive and migratory potential. These results indicate that fibulin-3 serves as a pro-invasive factor in bladder cancer, which may be mediated through modulation of IGFBP5 expression. This also suggests fibulin-3 and IGFBP5 may have potential as biomarkers of aggressive bladder cancer or therapeutic targets.

Role of Runx2 phosphorylation in prostate cancer and association with metastatic disease.

The osteogenic transcription factor, Runx2, is abnormally expressed in prostate cancer (PCa) and associated with metastatic disease. During bone development, Runx2 is activated by signals known to be hyperactive in PCa including the RAS/MAP kinase pathway, which phosphorylates Runx2 on multiple serine residues including S301 and S319 (equivalent to S294 and S312 in human Runx2). This study examines the role of these phosphorylation sites in PCa. Runx2 was preferentially expressed in more invasive PCa cell lines (PC3>C4-2B>LNCaP). Furthermore, analysis using a P-S319-Runx2-specific antibody revealed that the ratio of P-S319-Runx2/total Runx2 as well as P-ERK/total ERK was highest in PC3 followed by C4-2B and LNCaP cells. These results were confirmed by immunofluorescence confocal microscopy, which showed a higher percentage of PC3 cells staining positive for P-S319-Runx2 relative to C4-2B and LNCaP cells. Phosphorylated Runx2 had an exclusively nuclear localization. When expressed in prostate cell lines, wild-type Runx2 increased metastasis-associated gene expression, in vitro migratory and invasive activity as well as in vivo growth of tumor cell xenografts. In contrast, S301A/S319A phosphorylation site mutations greatly attenuated these Runx2 responses. Analysis of tissue microarrays from 129 patients revealed strong nuclear staining with the P-S319-Runx2 antibody in primary PCas and metastases. P-S319-Runx2 staining was positively correlated with Gleason score and occurrence of lymph node metastases while little or no Runx2 phosphorylation was seen in normal prostate, benign prostate hyperplasia or prostatitis indicating that Runx2 S319 phosphorylation is closely associated with PCa induction and progression towards an aggressive phenotype. These studies establish the importance of Runx2 phosphorylation in prostate tumor growth and highlight its value as a potential diagnostic marker and therapeutic target.

Effects of zoledronic acid on bone fusion in osteoporotic patients after lumbar fusion.

UNLABELLED: Treatment with zoledronic acid in osteoporotic patients with spinal fusion shortens the duration of time to fusion, improves the fusion rate, prevents the subsequent adjacent vertebral compression fractures, improves the clinical outcomes, and prevents immobilization-induced bone loss in the hip. INTRODUCTION: The objective of the study was to explore the effects of zoledronic acid on the healing process in osteoporotic patients following spinal fusion in a randomized, placebo-controlled, and triple-blinded study. METHODS: Seventy-nine osteoporotic patients with single-level degenerative spondylolisthesis were randomly assigned to receive either zoledronic acid infusion (zoledronic acid group) or saline infusion (controls) after spinal fusion. Functional radiography and CT scans were used to evaluate fusion status. Bone formation was graded into three categories: Grade A (bridging bone bonding with adjacent vertebral bodies), Grade B (bridging bone bonding with either superior or inferior vertebral body), or Grade C (incomplete bony bridging). A solid fusion was defined as less than 5° of angular motion with Grade A or B bone formation. Adjacent vertebral compression fractures (VCF) were assessed on MRI at 12 months after surgery. Serum level of carboxy terminal cross-linked telopeptide of type I collagen (ß-CTX) and amino-terminal propeptide of type I procollagen (PINP) was measured. Bone mineral density (BMD) was measured by DXA. Oswestry Disability Index (ODI) was used to assess the clinical outcomes. RESULTS: Grade A or B bridging bone was more frequently observed in zoledronic acid group at 3, 6, and 9 months post-operation compared to the control group (p < 0.05). At 12 -months post-operation, bridging bone and solid fusion were not significantly different between groups. No patients in zoledronic acid group showed aVCF, whereas six patients (17 %) in the control group did (p < 0.05). Both ß-CTX and PINP were suppressed in zoledronic acid group. BMD at the femoral neck decreased rapidly and did not return to the preoperative level in the controls at 3 (-1.4 %), 6 (-2.5 %), and 12 (-0.8 %) months after surgery. Zoledronic acid prevented this immobilization-induced bone loss and increased BMD. ODI showed the improved clinical outcomes compared with controls at 9 and 12 months post-surgery. CONCLUSION: Treatment with zoledronic acid in osteoporotic patients with spinal fusion shortens the time to fusion, improves the fusion rate, prevents subsequent aVCFs, and improves clinical outcomes.

IL-6 secreted by cancer-associated fibroblasts induces tamoxifen resistance in luminal breast cancer.

Cancer-associated fibroblasts (CAFs) have been implicated in the development of resistance to anticancer drugs; however, the role and mechanism underlying CAFs in luminal breast cancer (BrCA) tamoxifen resistance are unclear. We found that stromal fibroblasts isolated from the central or peripheral area of BrCA have similar CAF phenotype and activity. In vitro and in vivo experiments showed that CAFs derived from clinical-luminal BrCAs induce tamoxifen resistance through decreasing estrogen receptor-α (ER-α) level when cultured with luminal BrCA cell lines MCF7 and T47D. CAFs promoted tamoxifen resistance through interleukin-6 (IL-6) secretion, which activates Janus kinase/signal transducers and activators of transcription (JAK/STAT3) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways in tumor cells, followed by induction of epithelial-mesenchymal transition and upregulation of E3 ubiquitin ligase anaphase-promoting complex 10 activity, which targeted ER-α degradation through the ubiquitin-proteasome pathway. Inhibition of proteasome activity, IL-6 activity or either the JAK/STAT3 or PI3K/AKT pathways markedly reduced CAF-induced tamoxifen resistance. In xenograft experiments of CAFs mixed with MCF7 cells, CAF-specific IL-6 knockdown inhibited tumorigenesis and restored tamoxifen sensitivity. These findings indicate that CAFs mediate tamoxifen resistance through IL-6-induced degradation of ER-α in luminal BrCAs.Oncogene advance online publication, 9 June 2014; doi:10.1038/onc.2014.158.

Parathyroid hormone-related protein inhibits DKK1 expression through c-Jun-mediated inhibition of ß-catenin activation of the DKK1 promoter in prostate cancer.

Prostate cancer (PCa)bone metastases are unique in that majority of them induce excessive mineralized bone matrix, through undefined mechanisms, as opposed to most other cancers that induce bone resorption. Parathyroid hormone-related protein (PTHrP) is produced by PCa cells and intermittent PTHrP exposure has bone anabolic effects, suggesting that PTHrP could contribute to the excess bone mineralization. Wnts are bone-productive factors produced by PCa cells, and the Wnt inhibitor Dickkopfs-1 (DKK1) has been shown to promote PCa progression. These findings, in conjunction with the observation that PTHrP expression increases and DKK1 expression decreases as PCa progresses, led to the hypothesis that PTHrP could be a negative regulator of DKK1 expression in PCa cells and, hence, allow the osteoblastic activity of Wnts to be realized. To test this, we first demonstrated that PTHrP downregulated DKK1 mRNA and protein expression. We then found through multiple mutated DKK1 promoter assays that PTHrP, through c-Jun activation, downregulated the DKK1 promoter through a transcription factor (TCF) response element site. Furthermore, chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that PTHrP mediated this effect through inducing c-Jun to bind to a transcriptional activator complex consisting of ß-catenin, which binds the most proximal DKK1 promoter, the TCF response element. Together, these results demonstrate a novel signaling linkage between PTHrP and Wnt signaling pathways that results in downregulation of a Wnt inhibitor allowing for Wnt activity that could contribute the osteoblastic nature of PCa.

Understanding and targeting osteoclastic activity in prostate cancer bone metastases.

Bone metastasis is a debilitating side effect of advanced prostatic carcinoma impacting nearly all of the men developing this disease. Even though a majority of these lesions are considered osteoblastic, it is believed that there is an underlying osteolytic component. Lytic processes are governed primarily by osteoclasts, the primary bone resorptive cell. Osteolysis has been implicated in tumor cell seeding and nourishment of tumor growth via development of pro-tumorigenic changes in the microenvironment. Herein, we provide a current view of the processes involved in regulating osteolysis in the presence of prostate cancer bone metastases. Several factors have been implicated in the division, differentiation, and activation of osteoclasts, including, but not limited to, interleukin-6, receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and parathyroid hormone-related protein (PTHrP). Effector molecules in bone resorption play a significant role, such as matrix metalloproteinases (MMPs), cathepsins, and acid secretion. The primary method for treating skeletal events associated with prostate cancer bone metastases has been bisphosphonates. However, a new therapeutic, denosumab, a monoclonal antibody that inhibits RANKL in a mechanism similar to that attributed to the endogenous mediator OPG, has received approval for treatment of skeletally associated metastases. Additional novel targets are continuously being developed for bone metastases. In this review, we describe the processes involved in osteolysis of the prostate cancer bone microenvironment, and introduce therapeutics that may play a role in inhibiting tumor growth leading to increased survival and quality of life.

Prostate cancer stem cell biology.

The cancer stem cell (CSC) model provides insights into pathophysiology of cancers and their therapeutic response. The CSC model has been both controversial, yet provides a foundation to explore cancer biology. In this review, we provide an overview of CSC concepts, biology and potential therapeutic avenues. We then focus on prostate CSC including 1) their purported origin as either basal-derived or luminal-derived cells; 2) markers used for prostate CSC identification; 3) alterations of signaling pathways in prostate CSCs; 4) involvement of prostate CSCs in metastasis of PCa; and 5) microRNA-mediated regulation of prostate CSCs. Although definitive evidence for the identification and characterization of prostate CSCs still remains unclear, future directions pursuing therapeutic targets of CSCs may provide novel insights for the treatment of PCa.

RANKL inhibition is an effective adjuvant for docetaxel in a prostate cancer bone metastases model.

BACKGROUND: Docetaxel induces an anti-tumor response in men with advanced prostate cancer (PCa); however, the side effects associated with docetaxel treatment can be severe, resulting in discontinuation of therapy. Thus, identification of an effective adjuvant therapy to allow lower doses of docetaxel is needed. Advanced PCa is typically accompanied by skeletal metastasis. Receptor activator of NFkB ligand (RANKL) is a key pro-osteoclastic factor. Targeting RANKL decreases establishment and progression of PCa growth in bone in murine models. METHODS: The efficacy of inhibiting RANKL, using a recombinant soluble RANK extracellular domain fused with the immunoglobulin Fc domain (RANK-Fc), was tested as an adjuvant therapy with docetaxel for PCa bone metastasis in a murine intra-tibial model. RESULT: The combination of RANK-Fc and docetaxel reduced tumor burden in bone greater than either treatment alone. CONCLUSION: The combination of docetaxel with a RANKL-inhibiting agent merits further investigation for treatment of advance PCa.

Snail is a repressor of RKIP transcription in metastatic prostate cancer cells.

Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. Our results therefore suggest that RKIP is a novel component of the Snail transcriptional regulatory network important for the progression and metastasis of cancer.

Comparison of Fc-osteoprotegerin and zoledronic acid activities suggests that zoledronic acid inhibits prostate cancer in bone by indirect mechanisms.

Zoledronic acid (ZA) has been shown to inhibit prostate tumor growth in vitro and have beneficial effects in patients with advanced prostate cancer (CaP). The aim of this study was to determine whether ZA exhibits direct anti-tumor effects on CaP cells in vivo. To distinguish the effects of inhibition of osteolysis and direct anti-tumor activity of ZA in vivo, we compared the results of treatment with ZA and osteoprotegerin (Fc-OPG), which inhibits osteolysis, but without significant direct anti-tumor effects. In vitro Fc-OPG had no significant effects on C4-2 proliferation, whereas ZA decreased proliferation. However, both agents decreased tumor growth in bone. Moreover, both increased bone volume and prevented the overall decreases in BMD associated with growth of C4-2 cells in bone. Our study provides novel and significant observations that the in vivo effects of ZA are consistent with indirect effects mediated by osteoclasts.

The effect of osteoprotegerin administration on the intra-tibial growth of the osteoblastic LuCaP 23.1 prostate cancer xenograft.

Osteoprotegerin (OPG) plays a central role in controlling bone resorption. Exogenous administration of OPG has been shown to be effective in preventing osteolysis and limiting the growth of osteolytic metastasis. The objective of this study was to investigate the effects of OPG on osteoblastic prostate cancer (CaP) metastases in an animal model. LuCaP 23.1 cells were injected intra-tibially and Fc-OPG (6.0 mg/kg) was administered subcutaneously three times a week starting either 24 hours prior to cell injection (prevention regimen) or at 4 weeks post-injection (treatment regimen). Changes in bone mineral density at the tumor site were determined by dual x-ray absorptiometry. Tumor growth was monitored by evaluating serum prostate specific antigen (PSA). Fc-OPG did not inhibit establishment of osteoblastic bone lesions of LuCaP 23.1, but it decreased growth of the tumor cells, as determined by decreases in serum PSA levels of 73.0 +/- 44.3% (P < 0.001) and 78.3 +/- 25.3% (P < 0.001) under the treatment and prevention regimens, respectively, compared to the untreated tumor-bearing animals. Administration of Fc-OPG decreased the proliferative index by 35.0% (P = 0.1838) in the treatment group, and 75.2% (P = 0.0358) in the prevention group. The results of this study suggest a potential role for OPG in the treatment of established osteoblastic CaP bone metastases.

Transgenic models of metabolic bone disease: impact of estrogen receptor deficiency on skeletal metabolism.

Estrogen has protective effects on the skeleton via its inhibition of bone resorption. Mechanisms for these effects and the selectivity to the estrogen receptor alpha (ER alpha) or ER beta are unclear. The purpose of our study was to determine the impact of the ER alpha on skeletal metabolism using murine models with targeted disruption of the ER alpha and beta. Mice generated by homologous recombination and Cre/loxP technology yielding a deletion of the ER alpha exon 3 were evaluated and also crossed with mice with a disruption of the exon 3 of the ER beta to result in double ER alpha and ER beta knockout mice. Skeletal analysis of long bone length and width, radiographs, dual X-ray absorptiometry, bone histomorphometry, micro computerized tomography, biomechanical analysis, serum biochemistry, and osteoblast differentiation were evaluated. Male ER alpha knockout mice had the most dramatic phenotype consisting of reduced bone mineral density (BMD), and bone mineral content (BMC) of femurs at 10 and 16 weeks and 8-9 months of age. Female ER alpha knockout mice also had reduced density of long bones but to a lesser degree than male mice. The reduction of trabecular and cortical bone in male ER alpha knockout mice was statistically significant. Male double ER alpha and ER beta knockouts had similar reductions in bone density versus the single ER alpha knockout mice suggesting that the ER alpha is more protective than the ER beta in bone. In vitro analysis revealed no differences in osteoblast differentiation or mineralized nodule formation among cells from ER alpha genotypes. These data suggest that estrogens are important in skeletal metabolism in males; the ER alpha plays an important role in estrogen protective effects; osteoblast differentiation is not altered with loss of the ER alpha; and compensatory mechanisms are present in the absence of the ER alpha and/or another receptor for estrogen exists that mediates further effects of estrogen on the skeleton.

The comparative biology of skeletal metastasis.

Bone metastasis, a very common sequelae of cancer, is often associated with great morbidity. Understanding the biology of bone metastases may lead to therapeutic interventions to target the metastases. In addition to replacing bone marrow elements, the presence of tumour cells in bone modulates the normal bone remodelling process. Some tumours result in primarily osteolytic bone lesions, whereas others are associated with osteoblastic bone lesions. In either case, the resulting changes in the bone structure result in weakened bone that induces pain and is predisposed to fracture. The mechanisms through which cancer cells modulate bone remodelling are not clearly defined, but ongoing research using a variety of animal models will hopefully provide clues to prevent or slow the progress of bone metastases.

Hydrogen peroxide activates NFkappaB and the interleukin-6 promoter through NFkappaB-inducing kinase.

Aging is associated not only with oxidant stress, but also with increased interleukin-6 (IL-6) levels. To determine if oxidative stress could contribute to the age-associated increase IL-6 expression, we exposed LNCaP prostate carcinoma cells and HeLa cervical carcinoma cells to H2O2 as an oxidant challenge. We found that H2O2 induced IL-6 expression through activation of the IL-6 promoter. Furthermore, H2O2-induced activation of the promoter was mediated through nuclear factor-kappaB (NFkappaB) secondary to H2O2-induced phosphorylation and degradation of IkappaBalpha. NFkappaB-inducing kinase (NIK) is upstream of the IkappaB kinase complex that induces IkappaBalpha degradation. Accordingly, we explored if H2O2 induces IL-6 expression through NIK. In addition to H2O2 inducing NIK autophosphorylation, transfection of LNCaP cells with a dominant negative NIK diminished H2O2-mediated NFkappaB and IL-6 promoter activity. Taken together, these results demonstrate that H2O2 induces the IL-6 promoter by activating NFkappaB through NIK. These data provide a candidate mechanism through which oxidant challenge induces IL-6 gene expression with age.

Anti-interleukin-6 monoclonal antibody induces regression of human prostate cancer xenografts in nude mice.

BACKGROUND: Despite clinical associations and in vitro data suggesting that autocrine interleukin-6 (IL-6) production contributes to prostate cancer progression or chemotherapy resistance, there have been no reports that explore the role of IL-6 on prostate tumors in vivo. In the present study, we investigated the effect of IL-6 inhibition on the growth of human prostate cancer xenografts in nude mice. METHODS: To determine if autocrine IL-6 production contributes to prostate cancer growth and chemotherapy resistance in vivo, xenografts of a human prostate cancer cell line that produces IL-6 (PC-3) were established in nude mice. The mice were randomly divided into four treatment groups: (1) saline (vehicle control) + murine IgG (isotype control); (2) etoposide + murine IgG; (3) saline + anti-IL-6 monoclonal antibody; and (4) etoposide + anti-IL-6 monoclonal antibody. Tumors were measured twice weekly during a 4-week treatment period. At the conclusion of the study, all mice were sacrificed, and in addition to final volume, tumors were evaluated for the degree of apoptosis by TUNEL analysis. RESULTS: Anti-IL-6 Ab (with saline or etoposide) induced tumor apoptosis and regression ( approximately 60% compared to initial tumor size). Etoposide alone did not induce tumor regression or apoptosis in this animal model, and there was no synergy between anti-IL-6 Ab and etoposide. CONCLUSIONS: These studies suggest that IL-6 contributes to prostate cancer growth in vivo, and that targeting IL-6 may contribute to prostate cancer therapy.

Humoral hypercalcemia of malignancy: severe combined immunodeficient/beige mouse model of adult T-cell lymphoma independent of human T-cell lymphotropic virus type-1 tax expression.

The majority of patients with adult T-cell leukemia/lymphoma (ATL) resulting from human T-cell lymphotropic virus type-1 (HTLV-1) infection develop humoral hypercalcemia of malignancy (HHM). We used an animal model using severe combined immunodeficient (SCID)/beige mice to study the pathogenesis of HHM. SCID/beige mice were inoculated intraperitoneally with a human ATL line (RV-ATL) and were euthanized 20 to 32 days after inoculation. SCID/beige mice with engrafted RV-ATL cells developed lymphoma in the mesentery, liver, thymus, lungs, and spleen. The lymphomas stained positively for human CD45RO surface receptor and normal mouse lymphocytes stained negatively confirming the human origin of the tumors. The ATL cells were immunohistochemically positive for parathyroid hormone-related protein (PTHrP). In addition, PTHrP mRNA was highly expressed in lymphomas when compared to MT-2 cells (HTLV-1-positive cell line). Mice with lymphoma developed severe hypercalcemia. Plasma PTHrP concentrations were markedly increased in mice with hypercalcemia, and correlated with the increase in plasma calcium concentrations. Bone densitometry and histomorphometry in lymphoma-bearing mice revealed significant bone loss because of a marked increase in osteoclastic bone resorption. RV-ATL cells contained 1.5 HTLV-1 proviral copies of the tax gene as determined by quantitative real-time polymerase chain reaction (PCR). However, tax expression was not detected by Western blot or reverse transcriptase (RT)-PCR in RV-ATL cells, which suggests that factors other than Tax are modulators of PTHrP gene expression. The SCID/beige mouse model mimics HHM as it occurs in ATL patients, and will be useful to investigate the regulation of PTHrP expression by ATL cells in vivo.

Interleukin-6 induces androgen responsiveness in prostate cancer cells through up-regulation of androgen receptor expression.

Interleukin-6 (IL-6) induces prostate cancer (CaP) cell proliferation in vitro. Several lines of evidence suggest that IL-6 may promote CaP progression through induction of an androgen response. In this work, we explored whether IL-6 induces androgen responsiveness through modulation of androgen receptor (AR) expression. We found that in the absence of androgen, IL-6 increased prostate-specific antigen (PSA) mRNA levels and activated several androgen-responsive promoters, but not the non-androgen responsive promoters in LNCaP cells. Bicalutamide, an antiandrogen, abolished the IL-6 effect and IL-6 could not activate the PSA and murine mammary tumor virus reporters in AR-negative DU-145 and PC3 cells. These data indicate the IL-6 induces an androgen response in CaP cells through the AR. Pretreatment of LNCaP cells with SB202190, PD98059, or tyrphostin AG879 [p38 mitogen-activated protein kinase (MAPK), MAP/extracellular signal-regulated protein kinase kinase 1/2, and ErbB2 MAPK inhibitors, respectively) but not wortmannin (PI3-kinase inhibitor) blocked IL-6-mediated induction of the PSA promoter, which demonstrates that IL-6 activity is dependent on a MAPK pathway. Finally, IL-6 activated the AR gene promoter, resulting in increased AR mRNA and protein levels in LNCaP cells. These results demonstrate that IL-6 induces AR expression and are the first report of cytokine-mediated induction of the AR promoter. Taken together, our results suggest that IL-6 induces AR activity through both increasing AR gene expression and activating the AR in the absence of androgen in CaP cells. These results provide a mechanism through which IL-6 may contribute to the development of androgen-independent CaP.