PARP inhibitors chemopotentiate and synergize with cisplatin to inhibit bladder cancer cell survival and tumor growth.

BACKGROUND: Management of bladder cancer (BLCA) has not changed significantly in the past few decades, with platinum agent chemotherapy being used in most cases. Chemotherapy reduces tumor recurrence after resection, but debilitating toxicities render a large percentage of patients ineligible. Recently approved immunotherapy can improve outcomes in only a third of metastatic BLCA patients. Therefore, more options for therapy are needed. In this study, we explored the efficacy of PARP inhibitors (PARPi) as single agents or as combinations with platinum therapy. METHODS: We treated BLCA cells with PARPi (olaparib, niraparib, rucaparib, veliparib, or talazoparib) alone or as the combination of cisplatin with PARPi. We then measured their survival, proliferation, apoptosis, as well as their ability to form colonies. BLCA xenografts in male SCID mice were treated similarly, followed by the assessment of their growth, proliferation, and apoptosis. RESULTS: PARPi niraparib and talazoparib were effective in reducing BLCA cell survival as single agents. Combinations of Cisplatin with talazoparib and niraparib effectively reduced the survival of BLCA cells, while veliparib was not effective even at high concentrations. In vivo, the combinations of cisplatin with niraparib, rucaparib, or talazoparib reduced BLCA xenograft growth significantly. CONCLUSIONS: We provide evidence that PARPi can be effective against BLCA as single agents or as combinatorial therapy with cisplatin.

Lin28 induces resistance to anti-androgens via promotion of AR splice variant generation.

BACKGROUND: Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). METHODS: Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. RESULTS: We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. CONCLUSIONS: Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression.

Interleukin-6 induces neuroendocrine differentiation (NED) through suppression of RE-1 silencing transcription factor (REST).

BACKGROUND: Paracrine interleukin-6 (IL-6) can mediate neuroendocrine (NE) features, including the acquisition of a neurite-like phenotype and growth arrest in prostate cancer cells. However, little is known about the mechanisms underlying neuroendocrine differentiation induced by IL-6. METHODS: Immunoblotting was performed to determine the status of RE1-silencing transcription factor (REST) and of neuroendocrine markers such as Neuron-specific Enolase (NSE), chromogranin A and synaptophysin in LNCaP cells treated with IL-6. To further study the impact of REST-mediated repression on neuroendocrine differentiation (NED) in LNCaP cells, either wild-type REST or a dominant-positive form of REST, REST-VP16, in which both repressor domains of REST were replaced with the activation domain of the herpes simplex virus protein VP16, was introduced into LNCaP cells. RESULTS: In this study, we show that REST is suppressed in IL-6-induced neuroendocrine differentiation in LNCaP cells. Overexpression of exogenous REST abrogated IL-6-induced NED in prostate cancer cells. Expression of the recombinant REST-VP16 fusion protein activated REST target genes and other neuronal differentiation genes and produced neuronal physiological properties. In addition, REST protein turnover was accelerated in IL-6 induced NE differentiated LNCaP cells via the ubiquitin-proteasome pathway, accompanied by a decrease in the expression of the deubiquitylase HAUSP, indicating that pathway(s) priming REST degradation may be involved in IL-6 induced NE differentiation. CONCLUSIONS: These results demonstrate that REST functions as a major switch of IL-6 induced neuroendocrine differentiation in LNCaP cells.

Lin28 promotes growth of prostate cancer cells and activates the androgen receptor.

Prostate cancer (CaP) progresses to a castration-resistant state assisted by multifold molecular changes, most of which involve activation of the androgen receptor (AR). Having previously demonstrated the importance of the Lin28/let-7/Myc axis in CaP, we tested the hypothesis that Lin28 is overexpressed in CaP and that it activates AR and promotes growth of CaP cells. We analyzed human clinical CaP samples for the expression of Lin28 by quantitative real-time RT-PCR, Western blot analysis, and IHC. Growth characteristics of CaP cell lines transiently and stably expressing Lin28 were examined. The clonogenic ability of CaP cells expressing Lin28 was determined by colony formation and soft agar assays. Increase in expression of AR and subsequent increase in transcription of AR-target genes were analyzed by quantitative real-time RT-PCR, luciferase assays, and ELISA. LNCaP cells stably expressing Lin28 were injected into nude mice, and tumorigenesis was monitored. We found that Lin28 is overexpressed in clinical CaP compared to benign prostates. Overexpression of Lin28 enhanced, while down-regulation reduced, growth of CaP cells. Lin28 enhanced the ability of CaP cells to form colonies in anchorage-dependent and anchorage-independent conditions. LNCaP cells stably expressing Lin28 exhibited significantly higher tumorigenic ability in vivo. Lin28 induced expression of the AR and its target genes such as PSA and NKX3.1. Collectively, our findings demonstrate a novel role for Lin28 in CaP development and activation of the AR axis.

MicroRNA let-7c suppresses androgen receptor expression and activity via regulation of Myc expression in prostate cancer cells.

Castration-resistant prostate cancer continues to rely on androgen receptor (AR) expression. AR plays a central role in the development of prostate cancer and progression to castration resistance during and after androgen deprivation therapy. Here, we identified miR-let-7c as a key regulator of expression of AR. miR-let-7c suppresses AR expression and activity in human prostate cancer cells by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation of human prostate cancer cells. Down-regulation of Let-7c in prostate cancer specimens is inversely correlated with AR expression, whereas the expression of Lin28 (a repressor of let-7) is correlated positively with AR expression. Our study demonstrates that the miRNA let-7c plays an important role in the regulation of androgen signaling in prostate cancer by down-regulating AR expression. These results suggest that reconstitution of miR-let-7c may aid in targeting enhanced and hypersensitive AR in advanced prostate cancer.

RhoGDIα downregulates androgen receptor signaling in prostate cancer cells.

INTRODUCTION: Treatment of primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state due to aberrant activation of androgen receptor (AR). Understanding the mechanisms leading to the aberrant activation of AR is critical to develop effective therapy. We have previously identified Rho GDP Dissociation Inhibitor alpha (GDIα) as a novel suppressor in prostate cancer. In this study, we examine the effect of GDIα on AR signaling in prostate cancer cells. METHODS: GDIα was transiently or stably transfected into several prostate cancer cell lines including LNCaP, C4-2, CWR22Rv1, and DU145. The regulation of AR expression by GDIα was analyzed by qRT-PCR and Western blot. AR activity was measured by luciferase reporter assays and electrophoretic mobility shift analysis (EMSA). Immunofluorescence assay was performed to study AR nuclear translocation. The interaction between GDIα and AR was examined by co-immunoprecipitation assays. RESULTS: In this study, we have identified GDIα as a negative regulator of AR signaling pathway. Overexpression of GDIα downregulates AR expression at both mRNA and protein levels. Overexpression of GDIα is able to prevent AR nuclear translocation and inhibit transactivation of AR target genes. Co-immunoprecipitation assays showed that GDIα physically interacts with the N-terminal domain of AR. CONCLUSIONS: GDIα suppresses AR signaling through inhibition of AR expression, nuclear translocation, and recruitment to androgen-responsive genes. GDIα regulatory pathway may play a critical role in regulating AR signaling and prostate cancer growth and progression.

Functional p53 determines docetaxel sensitivity in prostate cancer cells.

BACKGROUND: Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer. METHODS: The effects of docetaxel on human DU145, PC3, LNCaP, and C4-2 prostate cancer cells were examined in cell culture, and p53 expression were analyzed by Western blot analysis. The potential role of p53 in docetaxel sensitivity in prostate cancer cells was tested by either p53 silencing using shRNA or p53 overexpression by introducing wild-type p53. RESULTS: We found that DU145 (mutant p53) and PC3 (p53 null) cells were less sensitive than LNCaP and C4-2 cells expressing functional p53 in response to docetaxel. Docetaxel treatment induces considerably higher apoptosis in LNCaP and C4-2 cells than in DU145 and PC3 cells in a dose dependent manner. Docetaxel increases the levels of ser15 phosphorylation of p53 in a dose dependent manner in both LNCaP and C4-2 cells, while has no effect on the levels of ser15 phosphorylation of p53 in DU145 cells. These results suggest that p53 phosphorylation is associated with docetaxel sensitivity in prostate cancer cells. To further confirm whether p53 activation can induce cell sensitivity to docetaxel treatment, we used p53 shRNA to knock down p53 expression in C4-2 cells and determined the cells response to docetaxel treatment. Knockdown of p53 significantly down regulated p53 phosphorylation and blocked docetaxel induced apoptotic cell death compared to the vector control. To further confirm this observation, we established a stable knock out p53 in C4-2 cells. Down regulation of p53 in the stable p53 knock out C4-2 cells significantly inhibited docetaxel induced apoptotic cell death. We also used wild-type (WT) p53 to over express p53 in DU145 cells, and found that expression of WT-p53 in DU145 cells increased their sensitivity to docetaxel. CONCLUSIONS: These results demonstrate that docetaxel induces p53 phosphorylation and that p53 status is a crucial determinant of docetaxel sensitivity in prostate cancer cells.

RhoGDIα suppresses growth and survival of prostate cancer cells.

BACKGROUND: Treatment for primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state. The mechanisms involved in the development and progression of castration-resistant prostate cancer (CRPC) remain unknown. We have previously generated LNCaP-IL6+ cells by treating LNCaP cells chronically with interleukin-6 (IL-6), which have acquired the ability to grow in androgen-deprived conditions. METHODS: We compared the protein expression profile of LNCaP and LNCaP-IL6+ cells using two-dimensional gel electrophoresis. The gels were then silver stained in order to visualize proteins and the differentially expressed spots were identified and characterized by micro sequencing using MALDI-PMF mass spectrometry. RESULTS: In this study, we have identified RhoGDIα (GDIα) as a suppressor of CaP growth. Expression of GDIα was reduced in LNCaP-IL6+ cells and was down-regulated in more aggressive CaP cells compared to LNCaP cells. Over expression of GDIα inhibited the growth of CaP cells and caused LNCaP-IL6+ cells reversal to androgen-sensitive state, while down-regulation of GDIα enhanced growth of androgen-sensitive LNCaP CaP cells in androgen-deprived conditions. In addition, GDIα suppressed the tumorigenic ability of prostate tumor xenografts in vivo. CONCLUSIONS: These results demonstrate that loss of GDIα expression promotes the development and progression of prostate cancer.

Inhibition of Stat3 activation by sanguinarine suppresses prostate cancer cell growth and invasion.

BACKGROUND: Signal transducer and activator of transcription 3 (Stat3) is an oncogenic transcriptional factor that plays a critical role in carcinogenesis and cancer progression and is a potential therapeutic target. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified previously as a novel inhibitor of survivin that selectively kills prostate cancer cells over "normal" prostate epithelial cells. METHODS: DU145, C4-2B, and LNCaP cells were treated with sanguinarine. The phosphorylation status of Stat3 and related proteins were measured with Western blots. Activation of transcription by Stat3 was measured with luciferase reporter assay. The effect of sanguinarine on anchorage-independent growth was examined with soft agar assay, and on cell migration and invasion of DU145 cells were measured with scratch assay and invasion assay, respectively. RESULTS: In this study, we identified sanguinarine as a potent inhibitor of Stat3 activation which was able to suppress prostate cancer growth, migration, and invasion. Sanguinarine inhibits constitutive as well as IL6-induced phosphorylation of Stat3 at both Tyr705 and Ser727 in prostate cancer cells. The inhibition of Stat3 phosphorylation by sanguinarine correlates with reduction of Janus-activated Kinase 2 (Jak2) and Src phosphorylation. Sanguinarine downregulates the expression of Stat3-mediated genes such as c-myc and survivin and inhibits the Stat3 responsive element luciferase reporter activity. Sanguinarine inhibits the anchorage-independent growth of DU145 and LN-S17 cells expressing constitutively activated Stat3. Migration and invasion abilities of DU145 cells were also inhibited by sanguinarine in a manner similar to the dominant negative form of Stat3. CONCLUSIONS: These data demonstrate that sanguinarine is a potent Stat3 inhibitor and it could be developed as a therapeutic agent for prostate cancer with constitutive activation of Stat3.

Mechanisms of persistent activation of the androgen receptor in CRPC: recent advances and future perspectives.

BACKGROUND: The emergence of castration resistance has remained the primary obstacle in prostate cancer therapy for several decades. Mechanisms likely to be involved in castration-resistant progression have been studied extensively, but have failed to yield many meaningful and effective targets. The re-activation of the androgen receptor (AR) in castration-resistant prostate cancer (CRPC) is now recognized as the central event in this process, and therapeutic modalities are being devised to combat it. METHODS: A review of literature was performed to highlight the important factors that play a role in the aberrant activation of the AR in CRPC. RESULTS: Seminal and exciting advances made in the past few years in the discovery of the roles of new intrinsic factors such as intracrine androgens, gene fusions involving the ETS oncogenes, and splice variants of the AR are reviewed. New and emerging hypotheses about the involvement of factors such as cytokines and other signaling pathways are discussed. CONCLUSIONS: This review summarizes the most recent advances in the persistent activation of the androgen receptor signaling pathway and provides a perspective about their significance in CRPC progression.

Serum bilirubin as a predictor of graft outcomes after renal transplant.

Bilirubin is a signaling molecule that alters the immune response and metabolism. While bilirubin has been employed as a marker of renal and cardiovascular health, its role in renal transplant recipients is not known. In this study, we sought to determine the impact of bilirubin (total, direct and indirect) on the estimated glomerular filtration rate (eGFR) after renal transplantation. We conducted a retrospective review of pre- and postoperative bilirubin levels in 457 renal transplant recipients at a single center. Pre- and post-rejection bilirubin levels were also assessed in those patients who experienced a rejection episode. No statistically significant differences were found in bilirubin levels during the pre-transplant to post-rejection period among patients who experienced rejection with kidney allograft survival. No statistically significant associations were observed between baseline bilirubin and post-transplant eGFR in the full patient group or within the gender- or race-stratified groups. Baseline bilirubin was not correlated with time to rejection. Our results suggest that bilirubin may not offer renoprotection in renal transplant recipients.

False Positive Pregnancy Test Before Kidney Transplant: Case Report and Review of Literature.

Pregnancy tests are routinely done before any surgery under general anesthesia including kidney transplantation. Positive test usually leads to more investigations to detect a possible pregnancy or malignancy and the surgery gets canceled or postponed. Because a kidney transplant from a deceased donor is not elective, it usually gets canceled in this scenario. Some groups have reported on normally elevated human chorionic gonadotrophin (hCG) levels in perimenopausal women and in patients with chronic kidney disease. This is thought to be from the pituitary. We present a highly sensitized prospective kidney transplant recipient with a positive pregnancy test with low levels of serum human chorionic gonadotrophin. She underwent additional preoperative testing after which we proceeded with the kidney transplant. Herein, we discuss the management of patients who have an unexpected positive pregnancy test before transplant.

Sperm centriole assessment identifies male factor infertility in couples with unexplained infertility - a pilot study.

Unexplained infertility affects about one-third of infertile couples and is defined as the failure to identify the cause of infertility despite extensive evaluation of the male and female partners. Therefore, there is a need for a multiparametric approach to study sperm function. Recently, we developed a Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) assay to determine sperm centriole quality. Here, we perform a pilot study of sperm from 10 fertile men and 10 men in couples with unexplained infertility, using three centriolar biomarkers measured at three sperm locations from two sperm fractions, representing high and low sperm quality. We found that FRAC can identify men from couples with unexplained infertility as the likely source of infertility. Higher quality fractions from 10 fertile individuals were the reference population. All 180 studied FRAC values in the 10 fertile individuals fell within the reference population range. Eleven of the 180 studied FRAC values in the 10 infertile patients were outliers beyond the 95% confidence intervals (P = 0.0008). Three men with unexplained infertility had outlier FRAC values in their higher quality sperm fraction, while four had outlier FRAC values in their lower quality sperm fraction (3/10 and 4/10, P = 0.060 and P = 0.025, respectively), suggesting that these four individuals are infertile due, in part, to centriolar defects. We propose that a larger scale study should be performed to determine the ability of FRAC to identify male factor infertility and its potential contribution to sperm multiparametric analysis.

The Emerging Role of Poly (ADP-Ribose) Polymerase Inhibitors as Effective Therapeutic Agents in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is the sixth most common cancer in the US. However, no significant changes in management have occurred since the tyrosine kinase era until the recent breakthrough with checkpoint inhibitors. Therefore, the need for more therapeutic options is paramount. Our objective was to determine whether PARP inhibition represents a novel therapeutic option for RCC. We used publicly available COSMIC, GDC Data Portal, and cBioPortal databases to explore mutations in DNA repair genes in RCC tissues from the TCGA cohort. We treated a human normal renal epithelial cell line RPTEC/TERT1 and two human renal cancer cell lines ACHN and CAKI-2 with PARPi niraparib, olaparib, rucaparib, veliparib, and talazoparib. Cell survival, cell proliferation, clonogenic ability, and apoptosis were assessed. RCC xenografts in SCID mice were treated with PARPi to evaluate their efficacy in vivo. Data mining revealed that ~27-32% of RCC tissues contain mutations in homologous recombination genes. Niraparib and talazoparib were the most effective at reducing cell survival, proliferation, and clonogenic ability in vitro. Niraparib, talazoparib, and rucaparib were the most effective in reducing RCC xenograft growth in vivo. Agents such as PARPi that exploit mutations in DNA damage repair genes may be effective therapeutic options for RCC.

Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) Finds Patient Age and Sperm Morphology Are Associated With Centriole Quality.

A large proportion of infertility and miscarriage causes are unknown. One potential cause is a defective sperm centriole, a subcellular structure essential for sperm motility and embryonic development. Yet, the extent to which centriolar maladies contribute to male infertility is unknown due to the lack of a convenient way to assess centriole quality. We developed a robust, location-based, ratiometric assay to overcome this roadblock, the Fluorescence-based Ratiometric Assessment of Centrioles (FRAC). We performed a case series study with semen samples from 33 patients, separated using differential gradient centrifugation into higher-grade (pellet) and lower-grade (interface) sperm fractions. Using a reference population of higher-grade sperm from infertile men with morphologically standard sperm, we found that 79% of higher-grade sperm of infertile men with substandard sperm morphology have suboptimal centrioles (P = 0.0005). Moreover, tubulin labeling of the sperm distal centriole correlates negatively with age (P = 0.004, R = -0.66). These findings suggest that FRAC is a sensitive method and that patient age and sperm morphology are associated with centriole quality.

Microarray analysis reveals potential target genes of NF-kappaB2/p52 in LNCaP prostate cancer cells.

PURPOSE: Our previous studies showed that NF-kappaB2/p52 is involved in the castration-resistant growth of the androgen-sensitive LNCaP prostate cancer cells. The role of NF-kappaB2/p52 in lymphomagenesis has been studied extensively, but its target genes in other cancers remain unknown. In order to identify genes potentially regulated by p52 in prostate cancer cells, we performed a genome-wide microarray analysis of genes differentially up- or down-regulated by the overexpression of p52 by adenoviral-mediated gene delivery in LNCaP cells. EXPERIMENTAL DESIGN: Total RNAs from vector control-infected and Adeno-p52-infected LNCaP cells were used to prepare cDNAs, which were hybridized to the Whole Genome Human 44k Microarray chips (Agilent Technologies). Data analysis was performed using GeneSpring and Ingenuity Pathway Analysis software. Validation of microarray results was performed by real-time quantitative RT-PCR and Western blot analyses. RESULTS: Expression of approximately 130 genes was differentially upregulated by >5-fold, whereas approximately 60 genes were differentially downregulated by >2-fold in p52-expressing LNCaP cells. Pathway analysis revealed that the upregulated genes belong to functional categories like cell growth and proliferation, cellular movement, cell-to-cell signaling and interaction, cancer, cell cycle, etc., whereas the downregulated genes were represented by functional categories like cell movement, antigen presentation, and cell death. Six of the top upregulated genes including annexin A2, PLAU, RND3, Twist2, VEGFC, and CXCL1 were validated by real-time PCR and Western blot analysis. CONCLUSIONS: This study provides a comprehensive analysis of genes potentially regulated by NF-kappaB2/p52 in the LNCaP prostate cancer cell line and provides a rationale for the induction of castration-resistant growth by p52 in LNCaP cells.

Interleukin-4 activates androgen receptor through CBP/p300.

BACKGROUND: Aberrant activation of androgen receptor (AR) plays an important role in the progression of castration resistant prostate cancer. Interleukin-4 (IL-4) enhances AR activation in the absence of androgen and stimulates castration resistant growth of androgen-sensitive prostate cancer cells. However, the mechanism of IL-4 mediated AR activation has not yet been revealed. METHODS: The effect of IL-4 on CBP/p300 expression was examined by Western blot analysis. The effect of IL-4 on the interactions of AR and CBP/p300 was examined by co-immunoprecipitation and ChIP assays. CBP/p300 siRNA was used to knockdown CBP/p300 expression to examine the role of CBP/p300 expression on IL-4 mediated AR activation. RESULTS: We found that IL-4 increases CBP/p300 protein expression and enhances interaction of AR with CBP/p300 proteins through an increase in the recruitment of CBP/p300 protein to the androgen responsive elements in the promoters of androgen responsive genes. Down regulation of CBP/p300 expression using CBP/p300 specific siRNA abolished IL-4 mediated AR activation, suggesting that CBP/p300 is responsible for AR activation induced by IL-4. Furthermore, AR activation can be enhanced by AR acetylation induced by IL-4 in prostate cancer cells. The IL-4 mediated AR acetylation can be blocked by knocking down CBP/p300 expression using CBP/p300 specific siRNA. CONCLUSION: These results suggest that IL-4 activates AR through enhanced expression of CBP/p300 and its histone acetyltransferase activity.

NF-kappaB2/p52 enhances androgen-independent growth of human LNCaP cells via protection from apoptotic cell death and cell cycle arrest induced by androgen-deprivation.

PURPOSE: Androgen-deprivation therapy only causes a temporary regression of prostate cancer, as all tumors will eventually progress to refractory to hormonal therapy after 1-3 years of treatment. The underlying mechanisms of prostate cancer androgen refractory progression are incompletely understood. In this study, we employed in vitro as well as in vivo models to examine the role of NF-kappaB2/p52 in prostate cancer growth and androgen independent progression. EXPERIMENTAL DESIGN: The effects of NF-kappaB2/p52 on cell growth, androgen responsiveness, cell cycle and apoptosis were examined in androgen sensitive LNCaP cells. The effect of NF-kappaB2/p52 on tumor growth was examined in intact and castrated male mice. RESULTS: Overexpression of NF-kappaB2/p52 enhances androgen-sensitive LNCaP human prostate cancer cell growth and clonogenic ability in androgen-deprived condition in vitro. NF-kappaB2/p52 induced androgen-independent growth is through protecting LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. In addition, NF-kappaB2/p52 stimulates Cyclin D1 expression and knock down of Cyclin D1 expression by siRNA abolished NF-kappaB2/p52-induced cell growth in vitro. Adenoviral mediated NF-kappaB2/p52 expression in LNCaP cells enhances tumor growth in intact male nude mice and induces tumor growth in castrated male nude mice, suggesting that overexpression of NF-kappaB2/p52 induces androgen-independent growth of androgen-sensitive LNCaP cells. CONCLUSIONS: Overexpression of NF-kappaB2/p52 protects androgen sensitive LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. NF-kappaB2/p52 activation induces androgen-independent growth in vitro and in vivo.

Transcriptional regulation of human RANK ligand gene expression by E2F1.

Receptor activator of nuclear factor kappa B ligand (RANKL) is a critical osteoclastogenic factor involved in the regulation of bone resorption, immune function, the development of mammary gland and cardiovascular system. To understand the transcriptional regulation of RANKL, we amplified and characterized a 1890bp 5'-flanking sequence of human RANKL gene (-1782bp to +108bp relative to the transcription start site). Using a series of deletion mutations of the 1890bp RANKL promoter, we identified a 72bp region (-172 to -100bp) mediating RANKL basal transcriptional activity. Sequence analysis revealed a putative E2F binding site within this 72bp region in the human RANKL promoter. Overexpression of E2F1 increased RANKL promoter activity, while down-regulation of E2F1 expression by small interfering RNA decreased RANKL promoter activity. RT-PCR and enzyme linked immunosorbent assays (ELISA) further demonstrated that E2F1 induced the expression of RANKL. Electrophoretic gel mobility shift assays (EMSA) and antibody competition assays confirmed that E2F1 proteins bind to the consensus E2F binding site in the RANKL promoter. Mutation of the E2F consensus binding site in the RANKL promoter profoundly reduced the basal promoter activity and abolished the transcriptional modulation of RANKL by E2F1. These results suggest that E2F1 plays an important role in regulating RANKL transcription through binding to the E2F consensus binding site.

Prostate-specific antigen modulates genes involved in bone remodeling and induces osteoblast differentiation of human osteosarcoma cell line SaOS-2.

PURPOSE: The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation. EXPERIMENTAL DESIGN: Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells. RESULTS: SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation. We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-kappaB ligand expression was up-regulated in cells expressing PSA compared with controls. CONCLUSIONS: Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin-receptor activator of NF-kappaB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.