Stephanie Filleur

Two Functional Epitopes of Pigment Epithelial–Derived Factor Block Angiogenesis and Induce Differentiation in Prostate Cancer

Pigment epithelial-derived factor (PEDF), an angiogenesis inhibitor with neurotrophic properties, balances angiogenesis in the eye and blocks tumor progression. Its neurotrophic function and the ability to block vascular leakage is replicated by the PEDF 44-mer peptide (residues 58-101). We analyzed PEDFs three-dimensional structure and identified a potential receptor-binding surface. Seeking PEDF-based antiangiogenic agents we generated and tested peptides representing the middle and lower regions of this surface. We identified previously unknown antiangiogenic epitopes consisting of the 34-mer (residues 24-57) and a shorter proximal peptide (TGA, residues 16-26) with the critical stretch L19VEEED24 and a fragment within the 44-mer (ERT, residues 78-94), which retained neurotrophic activity. The 34-mer and TGA, but not the 44-mer reproduced PEDF angioinhibitory signals hinged on c-jun-NH2-kinase-dependent nuclear factor of activated T cell deactivation and caused apoptosis. Conversely, the ERT, but not the 34-mer/TGA induced neuronal differentiation. For the 44-mer/ERT, we showed a novel ability to cause neuroendocrine differentiation in prostate cancer cells. PEDF and the peptides bound endothelial and PC-3 prostate cancer cells. Bound peptides were displaced by PEDF, but not by each other, suggesting multiple receptors. PEDF and its active fragments blocked tumor formation when conditionally expressed by PC-3 cells. The 34- and 44-mer used distinct mechanisms: the 34-mer acted on endothelial cells, blocked angiogenesis, and induced apoptosis whereas 44-mer prompted neuroendocrine differentiation in cancer cells. Our results map active regions for the two PEDF functions, signaling via distinct receptors, identify candidate peptides, and provide their mechanism of action for future development of PEDF-based tumor therapies.

Nuclear Factor of Activated T Cells Balances Angiogenesis Activation and Inhibition

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial–derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis. We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export. We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain–like interleukin 1β–converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK.

The Wilms' tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun

Thrombospondinu20091 (TSP1) is known for its significant anti-angiogenic properties. In a previous study, we have shown that transient or stable overexpression of the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1. We now demonstrate that the c-Jun-induced repression of TSP1 does not occur directly and does not require binding of c-Jun to the TSP1 promoter. Instead, repression involves a factor secreted by c-Jun-overexpressing cells. This secreted factor triggers a signal transduction pathway from the membrane to the nucleus, and these signals lead to the binding of the product of the Wilms tumor suppressor gene, WT1, to the −210 region of the TSP1 promoter. This region binds WT1 and SP1, but not EGR1, although its sequence fits the consensus binding site for this transcription factor. WT1 overexpression in transfected cells inhibits endogenous TSP1 gene expression and TSP1 transcription in experiments using TSP1 promoter-reporter constructs. The WT1−KTS isoform is more active in repressing TSP1 transcription than WT1+KTS, while EGR1 is inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not require de novo protein synthesis. The above mechanism for TSP1 repression could apply to other genes, thus coordinating their regulation in the vicinity of a c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a result of its tumor suppressor properties, may also possess oncogenic characteristics in the c-Jun transformation process, and thus repress the anti-angiogenic protein, TSP1.

In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment.

Antiangiogenic thrombospondin-1 (TSP1) induces endothelial cell death via a CD95-mediated cascade. We used this signaling pathway, where CD95/Fas is a rate-limiting intermediate, as a target to optimize the efficacy of TSP1 active peptide, DI-TSP. Like TSP1, DI-TSP upregulated endothelial CD95L in vivo. To modulate CD95 levels, we chose chemotherapy agent doxorubicin (DXR). DXR caused sustained upregulation of CD95 in the activated endothelium at 1/100 of the maximal tolerated dose. DI-TSP and DXR synergistically induced endothelial apoptosis in vitro, and in vivo, in developing murine vessels. Fas decoy, TSP1 receptor antibody and Pifithrin, a p53 inhibitor, severely decreased apoptosis and restored angiogenesis by DXR–DI-TSP combination, evidencing critical roles of CD95 and TSP1. Combined therapy synergistically blocked neovascularization and progression of the bladder and prostate carcinoma. Such informed design of a complex antiangiogenic therapy based on the rate-limiting molecular targets is a novel concept, which may yield new approaches to cancer treatment.

Asymptomatic Bacteriuria: Significance for Different Patient Population

Infections of the urinary tract (UTI) are one of the most common infections for which antibiotics are prescribed. Asymptomatic bacteriuria, or asymptomatic urinary infection, is a common condition (Nicolle, 2003). Based on the microbiological definition, urinary tract infections are characterized by the presence of ≥105 colony-forming units per ml (CFUs/mL) of a single bacterial species or multiple organisms in two consecutive urine specimens, properly collected from a person with symptoms or signs of a UTI (Rubin et al., 1992). However, the presence of a significant quantity of bacteria in the urine of an asymptomatic patient is known as asymptomatic bacteriuria. Quantitative criteria to establish the diagnosis of significant bacteriuria in an asymptomatic person are: 1. at least 105 CFUs/mL of urine in a voided midstream, so called clean-catch specimen; and 2. at least 100 CFUs per mL of urine obtained by bladder catheterization. Despite several decades of research, there is still a considerable amount of controversy about the adequate management of bacteriuria. A common dilemma in clinical medicine is whether or not to treat asymptomatic bacteriuria. It is now recognized that certain patient characteristics can led to the development of symptomatic infections based on an asymptomatic bacteriuria. For several clinical scenarios, the antibiotic treatment of asymptomatic bacteriuria has been shown to improve patient outcomes. Based on clinical trials conducted over the last few decades, better recommendations for the management of bacteriuria in different patient population could be established. The Infectious Diseases Society of America (IDSA) established guidelines for the screening and treatment of asymptomatic bacteriuria (Nicolle et al., 2005). The optimal management depends significantly on specific patient characteristics, co-morbidities, and risk factors. Recent studies could prove that for certain patient populations, screening and treatment is beneficial or may be beneficial. However, for other clinical scenarios, screening and treatment for asymptomatic bacteriuria has not proven to be beneficial and therefore is highly controversial. Importantly, overtreatment of asymptomatic bacteriuria is a quality, safety, and cost issue. Recent studies have revealed a substantial gap between clinical practice, published guidelines, and recommendations (need reference). Treating physicians need to be aware that because of an increasing antimicrobial resistance, it is important to treat patients only if there is clear evidence of potential benefits. Therefore, in this chapter we review the most recent literature and provide up-to-date information for treating physicians on how to diagnose and when to treat asymptomatic bacteriuria.

Photodynamic therapy: a promising alternative in oncology

Photodynamic Therapy (PDT) is a treatment modality that is based on the administration of a photosensitizer and the following application of light in a wavelength range matching the absorption spectrum of the photosensitizer. Ideally the photosensitizer retains in the tumor tissue more than in normal tissue and thus allows targeted destruction of cancerous tissue. The use of PDT is slowly being accepted as a standard treatment for certain types of cancer. This includes mainly treatment strategies with only palliative intentions (obstructive esophageal cancer and advanced lung cancer) while for certain malignant conditions new applications exists that are already intended for cure (e.g. early stage of lung cancer). The main advantage of PDT is that the treatment can be repeated multiple times safely without major side effects. PDT can be safely combined with already established treatment options like surgery, chemotherapy or radiotherapy. A disadvantage of PDT is the only localized effect of the therapy, which usually cannot significantly alter the outcome of a systemic disease. In this paper we review the history of PDT as well as current clinical applications in oncology and future directions.

MP66-04 CABAZITAXEL INHIBITS THE PROLIFERATION OF HUMAN CASTRATION-REFRACTORY PROSTATE CANCER CELLS IN VITRO AND ENHANCES THE ANTI-TUMOR PROPERTIES OF THE ANGIO-INHIBITORY PIGMENT EPITHELIUM-DERIVED FACTOR IN VIVO WITH A GREATER EFFICACY THAN DOCETAXEL

therapeutic response. The goal of this study was to evaluate and genetically characterize exosome derived RNA (exoRNA) isolated from blood of metastatic CRPC patients. METHODS: Whole blood samples from 18 consented clinically annotated mCRPC patients and 1 normal control were collected. Exosomes were isolated with ultracentrifugation and exoRNA extracted. Following library prep, paired-end sequencing was performed using Illumina Hi-Seq 2000. A bioinformatics pipeline was used for data prepossessing including alignment, duplicate removal, normalization and variant calling. Visualization and differential analyses were performed with SNP & Variation Suite v8.x. RESULTS: In exoRNA there is evidence of extensive chromosomal rearrangement resulting in a myriad of gene fusions and isoforms. Through preliminary analyses we identified 39 genes commonly expressed in the exosomes of these mCRPC patients. These include PDPK1, USP9X, MAGI2, HMGA2 and PTGFR all of which have been previously expressed in prostate cancer tissue. A diverse variety of lcnRNA, ncRNA and miRNA were also identified in circulating exosomes. Validation of these alterations and additional analyses evaluating translocations and splice variants, PCR validation, and/or direct tumor based nucleic acid assays is ongoing. CONCLUSIONS: These preliminary analyses of circulating exoRNA have identified gene expression signatures of several prostate cancer associated transcripts. Ultimately, the identification of PCa associated transcripts in plasma derived exosomes provides evidence that exosomes and exosomal cargo may serve as biomarker in CRPC patients. Exosomes and exoRNA may provide otherwise unattainable insight into tumor evolution and disease progression. Additional studies evaluating the clinical relevance and prognostic value of exosomal RNA will be critical for biomarker development.

Effect of cabazitaxel on the growth of human castration-refractory prostate cancer cells in vitro compared to docetaxel and on the anti-tumor properties of the angio-inhibitor PEDF in vivo.

205 Background: Docetaxel/DTX and cabazitaxel/CBZ have shown promise in the treatment of metastatic Castration-Refractory Prostate Cancer/mCPRC however, comparative studies are missing. Toxicities of these drugs are significant, urging the need to modify taxane regimens. Recently, low-dose metronomic/LDM treatments using conventional chemotherapeutic drugs have shown benefits in CPRC in improving the effect of anti-angiogenic agents. Previously, we have demonstrated that LDM-DTX in combination with PEDF curbs significantly CRPC growth, limits metastases formation and prolongs survival in vivo. In this study, we intended to compare the cytotoxic effect of CBZ and DTX on CRPC cells in vitro and CL1 tumors in vivo. Methods: PC3, DU145 cell lines were from ATCC.CL1 cells were obtained from androgen-deprived LNCaP cells. Cell proliferation was assessed by crystal violet staining and cell cycle analyses. In vitro cytotoxicity assays were performed on CL1 cells/RAW264.7 macrophages co-cultures treated with PEDF ...

793 PIGMENT EPITHELIUM-DERIVED FACTOR INHIBITS BONE METASTASES FORMATION, PROLONGS SURVIVAL AND ENHANCES THE IN VIVO ANTITUMOR ACTIVITIES OF LOW-DOSE CHEMOTHERAPY IN CASTRATION-REFRACTORY PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: The development of metronomic/low dose administration of conventional chemotherapeutic drugs has shown great promise in the treatment of castration-refractory prostate cancer (CPRC). Pigment Epithelium-Derived Factor (PEDF) is a natural angio-inhibitor which is down-regulated in prostate cancer. We have previously demonstrated that the over-expression of PEDF in human CRPC PC3 cells decreased tumor growth in vivo. In the present study, we further validated PEDF anti-tumor properties in the highly metastatic CRPC LNCaP-derivative CL1 cells. We also hypothesized that PEDF may enhance the cytotoxicity effects of low dose docetaxel (DTX) and cyclophosphamide (CTX) chemotherapies in vivo. METHODS: PC3 and CL1 cell lines were genetically modified to stably express the fluorescent DsRed Express protein with PEDF. Resulting cells were characterized in vitro for PEDF expression by western blot and, for proliferation by growth curves and clone formation in matrigel. PEDF anti-tumor effects were assessed on established s.c. xenografts in mice treated with DTX (5mg/kg ip every 4 days, 1mg/kg ip daily for 10 days, 0.5mg/kg every other day), CTX (10-20mg/kg in the drinking water) or placebo. Survival studies were performed by injecting CL1-PEDF or -control cells into the left lobe of the dorsal prostate of anesthetized mice. For bone metastases, CL1-PEDF or -control cells were injected into the proximal region of the tibia. RESULTS: We showed that PEDF expression inhibits the proliferation and induces the differentiation of CPRC cells in vitro, and decreases by 85% and 70% the development of s.c. PC3 and CL1 tumors, respectively. In vivo, PEDF expression inhibits the formation of bone metastases and bone destruction, and prolongs significantly (P 0.01; 95% confidence interval) the median survival of CL1 tumorbearing mice (53 0.001 days versus 57 1). Furthermore, we demonstrated that PEDF enhances the cytotoxicity effects of low dose chemotherapy on established s.c. tumors (best Doc dose: 1mg/kg for PC3 and 5mg/kg for CL1; best CTX dose: 10mg/kg for PC3 and CL1) and prolongs significantly the survival of tumor-bearing mice undergoing low dose chemotherapy. CONCLUSIONS: These data reinforce the significance of PEDF as a potent target for the treatment of CRPC. It also emphasizes PEDF as a promising new agent to enhance the anti-tumor efficacy of low dose chemotherapies.

Effect of PEDF on the in vivo antitumor activities of low-dose chemotherapy in CRPC.

173 Background: The development of metronomic/low dose administration of conventional chemotherapeutic drugs has shown great promise in the treatment of castration-refractory prostate cancer (CPRC). Pigment Epithelium-Derived Factor (PEDF) is a natural angio-inhibitor which is down-regulated in prostate cancer. We have previously demonstrated that the over-expression of PEDF in human CRPC PC3 cells decreased tumor growth in vivo. In the present study, we further validated PEDF anti-tumor properties in the highly metastatic CRPC LNCaP-derivative CL1 cells. We also hypothesized that PEDF may enhance the cytotoxicity effects of low dose docetaxel (DTX) and cyclophosphamide (CTX) chemotherapies in vivo.nnnMETHODSnPC3 and CL1 cell lines were genetically modified to stably express the fluorescent DsRed Express protein with PEDF. Resulting cells were characterized in vitro for PEDF expression by western blot and, for proliferation by growth curves and clone formation in matrigel. PEDF anti-tumor effects were assessed on established s.c. xenografts in mice treated with DTX (5mg/kg ip every 4 days, 1mg/kg ip daily for 10 days, 0.5mg/kg every other day), CTX (10-20mg/kg in the drinking water) or placebo. Survival studies were performed by injecting CL1-PEDF or -control cells into the left lobe of the dorsal prostate of anesthetized mice.nnnRESULTSnWe showed that PEDF expression inhibits the proliferation and induces the differentiation of CPRC cells in vitro, and decreases by 85% and 70% the development of s.c. PC3 and CL1 tumors, respectively. In the survival study, PEDF expression prolongs significantly (P=0.01; 95% confidence interval) the median survival of CL1 tumor-bearing mice (53±0.001 days versus 57±1). Furthermore, we demonstrated that PEDF enhances the cytotoxicity effects of low dose chemotherapy on established s.c. tumors (best Doc dose: 1mg/kg for PC3 and 5mg/kg for CL1; best CTX dose: 10mg/kg for PC3 and CL1) and prolongs significantly the survival of tumor-bearing mice undergoing low dose chemotherapy.nnnCONCLUSIONSnThese data reinforce the significance of PEDF as a potent target for the treatment of CRPC. It also emphasizes PEDF as a promising new agent to enhance the anti-tumor efficacy of low dose chemotherapies.