Tumor-induced peripheral immunosuppression promotes brain metastasis in patients with non-small cell lung cancer.

INTRODUCTION: Brain metastases are a significant source of morbidity and mortality for patients with lung cancer. Lung cancer can induce local and systemic immunosuppression, promoting tumor growth and dissemination. One mechanism of immunosuppression is tumor-induced expansion of programmed death-ligand 1 (PD-L1) expressing myeloid cells. Here, we investigate peripheral blood immune phenotype in NSCLC patients with or without brain metastasis. METHODS: Peripheral blood was collected from patients with lung metastatic brain tumors and pre-metastatic lung cancer. Immunosuppressive monocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) were quantified through flow cytometry. T cell reactivity was analyzed via ELISpot. Brain metastasis conditioned media was collected from tumor-derived cell cultures and analyzed for cytokines by ELISA. Naïve monocytes were stimulated with brain metastasis conditioned media to evaluate PD-L1 stimulation. RESULTS: Patients with brain metastatic lung carcinoma demonstrated increased peripheral monocyte PD-L1, MDSC abundance, and Treg percentage compared to early stage pre-metastatic patients and healthy controls. Patients with elevated peripheral monocyte PD-L1 had less reactive T cells and worse survival. Brain metastasis conditioned media stimulation increased monocyte PD-L1, and conditioned media IL-6 levels correlated with PD-L1 induction. Treatment with anti-IL-6 or anti-IL-6 receptor antibodies reduced PD-L1 expression. In summary, patients with lung cancer and brain metastases exhibit multiple markers of peripheral immunosuppression. CONCLUSIONS: The frequency of PD-L1+ myeloid cells correlated with the presence of brain metastases. Tumor-derived IL-6 was capable of inducing PD-L1+ myeloid cells in vitro, suggesting that monitoring of immunosuppressive factors in peripheral blood may identify new targets for therapeutic intervention in selected patients.

Lymphopenia predicts response to stereotactic radiosurgery in lung cancer patients with brain metastases.

BACKGROUND: Stereotactic radiosurgery (SRS) can enhance immune activation and improve disease control through stimulation of anti-tumor immunity. However, patients with cancer receiving chemotherapy are often immunosuppressed, which may impact the efficacy of SRS. Here we investigate the relationship between systemic lymphopenia and response to SRS in patients with brain-metastatic lung cancer. METHODS: We reviewed 125 patients with lung cancer brain metastases treated with SRS between January 2014 and May 2017. Complete blood counts from the time of SRS were reviewed, and lymphopenia was defined as absolute lymphocyte count < 1×109 cells/L. Kaplan-Meier survival analysis and cox proportional-hazards models were used to evaluate risks of progression and death. RESULTS: The median age was 65 years (range 43-86), with 54% female patients. Lymphopenia was present in 60 patients. In univariate analysis, lymphopenic patients had significantly shorter PFS (HR = 2.995, p < 0.0001) and OS (HR = 3.928, p < 0.0001). When accounting for age, gender, smoking history, ECOG score, surgery, and tumor histology in a multivariate model, lymphopenia remained significantly predictive of worse PFS (HR = 1.912, p = 0.002) and OS (HR = 2.257, p < 0.001). Patients who received immunotherapy within 3 months of SRS demonstrated significantly shorter PFS (HR = 3.578, p = 0.006) and OS (HR = 6.409, p = 0.001) if lymphopenic. CONCLUSIONS: Brain-metastatic lung cancer patients with lymphopenia treated with SRS had significantly worse PFS and OS. The effect of lymphopenia was even more pronounced in patients receiving immunotherapy. These data demonstrate the significant impact of deficient immunity on disease control and survival. Lymphopenic patients may benefit from interventions to improve immune function prior to SRS for brain metastases.

Systemic and local immunosuppression in patients with high-grade meningiomas.

AIM: Despite current treatments, high-grade meningiomas continue to have a poor prognosis. Immunotherapy targeting immune checkpoints, such as PD-L1, has demonstrated significant success in controlling numerous malignancies. In this study, we investigate the extent of systemic and local immunosuppression in meningiomas to assess the potential benefit of immune checkpoint inhibitors for the treatment of high-grade meningiomas. METHODS: Peripheral blood was collected from patients undergoing resection of meningiomas (WHO grade I, n = 18; grade II, n = 25; grade III, n = 10). Immunosuppressive myeloid cells (CD45+CD11b+PD-L1+), myeloid-derived suppressor cells (MDSCs) (CD11b+CD33+HLA-DRlow), and regulatory T cells (Tregs) (CD3+CD4+CD25+FoxP3+) were quantified through flow cytometry. Tissue sections from the same patients were assessed for PD-L1 expression and T cell infiltration via immunohistochemistry. RESULTS: Patients with grade III meningiomas demonstrated increased peripheral monocyte PD-L1 compared to patients with grade I/II meningiomas and healthy controls. Peripheral MDSC abundance was increased in grades II and III meningioma patients. PD-L1 staining of meningioma tissue demonstrated increased positivity in grade III meningiomas. Intratumoral PD-L1 was not associated with progression-free survival. High-grade meningiomas had increased T-cell infiltration. However, a significant proportion of these T cells were exhausted PD1+ T cells and immunosuppressive Tregs. CONCLUSIONS: Patients with meningiomas exhibit signs of peripheral immunosuppression, including increased PD-L1 on myeloid cells and elevated MDSC abundance proportional to tumor grade. Additionally, the tumors express substantial PD-L1 proportional to tumor grade. These results suggest a role for immune checkpoint inhibitors targeting the PD-L1/PD-1 pathway in combination with standard therapies for the treatment of high-grade meningiomas.

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

PURPOSE: Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential targets for immune modulation. EXPERIMENTAL DESIGN: Conditioned media from patient-derived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate naïve myeloid cells. Myeloid PD-L1 induction was quantified by flow cytometry. Candidate cytokines correlated with PD-L1 induction were evaluated in tumor sections and plasma for relationships with survival and myeloid PD-L1 expression. The role of identified cytokines on immunosuppression and survival was investigated in vivo utilizing immunocompetent C57BL/6 mice bearing syngeneic GL261 and CT-2A tumors. RESULTS: GBM-derived IL6 was identified as a cytokine that is necessary and sufficient for myeloid PD-L1 induction in GBM through a STAT3-dependent mechanism. Inhibition of IL6 signaling in orthotopic murine glioma models was associated with reduced myeloid PD-L1 expression, diminished tumor growth, and increased survival. The therapeutic benefit of anti-IL6 therapy proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. CONCLUSIONS: Our findings suggest that disruption of IL6 signaling in GBM reduces local and systemic myeloid-driven immunosuppression and enhances immune-mediated antitumor responses against GBM.

The immune checkpoint protein PD-L1 induces and maintains regulatory T cells in glioblastoma.

Glioblastoma (GBM) promotes immunosuppression through upregulation of PD-L1 and regulatory T cell (Treg) expansion, but the association of these suppressive factors has not been well elucidated. Here, we investigate a role of PD-L1 in expanding Tregs and the value of targeting the PD-1 receptor to inhibit Treg expansion. Quantitative RNA sequencing data from The Cancer Genome Atlas were evaluated for an association between CD274 and FOXP3 transcript expressions and impact of FOXP3 on clinical outcomes. Peripheral leukocytes from patients with newly diagnosed GBM were profiled for PD-L1+ myeloid expressions and Treg abundance. Healthy lymphocytes were assessed for impact of recombinant PD-L1 on expansion of the inducible Treg (iTreg) population. iTreg function was evaluated by the capacity to suppress effector T cell proliferation. Specificity of responses were confirmed by pharmacologic inhibition of the PD-1 receptor. Increased PD-L1 mRNA expression in GBM corresponded to increased FOXP3 mRNA (p = 0.028). FOXP3 elevation had a negative impact on overall survival (HR = 2.0; p < 0.001). Peripheral PD-L1 positivity was associated with an increased Treg fraction (p = 0.008). Lymphocyte activation with PD-L1 co-stimulation resulted in greater iTreg expansion compared to activation alone (18.3% vs. 6.5%; p < 0.001) and improved preservation of the Treg phenotype. Suppressive capacity on naïve T cell proliferation was sustained. Nivolumab inhibited PD-L1-induced Treg expansion (p < 0.001). These results suggest that PD-L1 may expand and maintain immunosuppressive Tregs, which are associated with decreased survival in glioma patients. Blockade of the PD-L1/PD-1 axis may reduce Treg expansion and further improve T cell function beyond the direct impact on effector cells.

Sonic hedgehog delivery from self-assembled nanofiber hydrogels reduces the fibrotic response in models of erectile dysfunction.

Erectile dysfunction (ED) is a serious medical condition in which current treatments are ineffective in prostatectomy and diabetic patients, due to injury to the cavernous nerve (CN), which causes irreversible remodeling of the penis (decreased smooth muscle and increased collagen), through a largely undefined mechanism. We propose that sonic hedgehog (SHH) and neural innervation, are indispensable regulators of collagen in the penis, with decreased SHH protein being an integral component of the fibrotic response to loss of innervation. We examined collagen abundance and morphology in control (Peyronie's), prostatectomy and diabetic patients, and in rat models of penile development, CN injury, SHH inhibition and under regenerative conditions, utilizing self-assembling peptide amphiphile (PA) nanofiber hydrogels for SHH delivery. Collagen abundance increased in penis of ED patients. In rats, collagen increased with CN injury in a defined time frame independent of injury severity. An inverse relationship between SHH and collagen abundance was identified; SHH inhibition increased and SHH treatment decreased penile collagen. SHH signaling in the pelvic ganglia (PG)/CN is important to maintain CN integrity and when inhibited, downstream collagen induction occurs. Collagen increased throughout penile development and with age, which is important when considering how to treat fibrosis clinically. These studies show that SHH PA treatment reduces collagen under regenerative post-prostatectomy conditions, indicating broad application for ED prevention in prostatectomy, diabetic and aging patients and in other peripheral nerve injuries. The PA nanofiber protein vehicle may be widely applicable as an in vivo delivery tool. STATEMENT OF SIGNIFICANCE: We use self-assembling peptide amphiphiles (PA) as biological delivery vehicles to prevent cavernous nerve (CN) injury induced erectile dysfunction (ED). These versatile hydrogels were molecularly pre-programmed for sonic hedgehog (SHH) protein delivery, either from an injectable solution with fast, in situ assembly into a soft hydrogel, or by highly aligned monodomain nanofiber bundles. We used PAs to examine a novel neuronal component to collagen regulation and the role of SHH in the fibrotic response to CN injury. SHH perturbation in the penis or the CN, selectively impacts collagen, with SHH inhibition increasing and SHH treatment suppressing collagen. These results suggest that SHH treatment by PA has translational potential to suppress collagen induction and remodelling, an irreversible component of ED development.

Therapeutic manipulation of angiogenesis with miR-27b.

BACKGROUND: Multiple studies demonstrated pro-angiogenic effects of microRNA (miR)-27b. Its targets include Notch ligand Dll4, Sprouty (Spry)-2, PPARγ and Semaphorin (SEMA) 6A. miR-27 effects in the heart are context-dependent: although it is necessary for ventricular maturation, targeted overexpression in cardiomyocytes causes hypertrophy and dysfunction during development. Despite significant recent advances, therapeutic potential of miR-27b in cardiovascular disease and its effects in adult heart remain unexplored. Here, we assessed the therapeutic potential of miR-27b mimics and inhibitors in rodent models of ischemic disease and cancer. METHODS: We have used a number of models to demonstrate the effects of miR-27b mimicry and inhibition in vivo, including subcutaneous Matrigel plug assay, mouse models of hind limb ischemia and myocardial infarction and subcutaneous Lewis Lung carcinoma. RESULTS: Using mouse model of myocardial infarction due to the coronary artery ligation, we showed that miR-27b mimic had overall beneficial effects, including increased vascularization, decreased fibrosis and increased ejection fraction. In mouse model of critical limb ischemia, miR-27b mimic also improved tissue re-vascularization and perfusion. In both models, miR-27b mimic clearly decreased macrophage recruitment to the site of hypoxic injury. In contrast, miR-27b increased the recruitment of bone marrow derived cells to the neovasculature, as was shown using mice reconstituted with fluorescence-tagged bone marrow. These effects were due, at least in part, to the decreased expression of Dll4, PPARγ and IL10. In contrast, blocking miR-27b significantly decreased vascularization and reduced growth of subcutaneous tumors and decreased BMDCs recruitment to the tumor vasculature. CONCLUSIONS: Our study demonstrates the utility of manipulating miR-27b levels in the treatment of cardiovascular disease and cancer.

Chemopreventive apigenin controls UVB-induced cutaneous proliferation and angiogenesis through HuR and thrombospondin-1.

Plant flavonoid apigenin prevents and inhibits UVB-induced carcinogenesis in the skin and has strong anti-proliferative and anti-angiogenic properties. Here we identify mechanisms, by which apigenin controls these oncogenic events. We show that apigenin acts, at least in part, via endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1). TSP1 expression by the epidermal keratinocytes is potently inhibited by UVB. It inhibits cutaneous angiogenesis and UVB-induced carcinogenesis. We show that apigenin restores TSP1 in epidermal keratinocytes subjected to UVB and normalizes proliferation and angiogenesis in UVB-exposed skin. Importantly, reconstituting TSP1 anti-angiogenic function in UVB-irradiated skin with a short bioactive peptide mimetic representing exclusively its anti-angiogenic domain reproduced the anti-proliferative and anti-angiogenic effects of apigenin. Cox-2 and HIF-1α are important mediators of angiogenesis. Both apigenin and TSP1 peptide mimetic attenuated their induction by UVB. Finally we identified the molecular mechanism, whereby apigenin did not affect TSP1 mRNA, but increased de novo protein synthesis. Knockdown studies implicated the RNA-binding protein HuR, which controls mRNA stability and translation. Apigenin increased HuR cytoplasmic localization and physical association with TSP1 mRNA causing de novo TSP1 synthesis. HuR cytoplasmic localization was, in turn, dependent on CHK2 kinase. Together, our data provide a new mechanism, by which apigenin controls UVB-induced carcinogenesis.

The many facets of PEDF in drug discovery and disease: a diamond in the rough or split personality disorder?

INTRODUCTION: Pigment epithelium-derived factor (PEDF) was discovered as a neurotrophic factor secreted by retinal pigment epithelial cells. A decade later, it re-emerged as a powerful angiogenesis inhibitor guarding ocular function. Since then, significant advances were made identifying PEDF's mechanisms, targets and biomedical applications. AREAS COVERED: The authors review several methodologies that have generated significant new information about the potential of PEDF as a drug. Furthermore, the authors review and discuss mechanistic and structure-function analyses combined with the functional mapping of active fragments, which have yielded several short bioactive PEDF peptides. Additionally, the authors present functional studies in knockout animals and human correlates that have provided important information about conditions amenable to PEDF-based therapies. EXPERT OPINION: Through its four known receptors, PEDF causes a wide range of cellular events vitally important for the organism, which include survival and differentiation, migration and invasion, lipid metabolism and stem cell maintenance. These processes are deregulated in multiple pathological conditions, including cancer, metabolic and cardiovascular disease. PEDF has been successfully used in countless preclinical models of these conditions and human correlates suggest a wide utility of PEDF-based drugs. The most significant clinical application of PEDF, to date, is its potential therapeutic use for age-related macular degeneration. Moreover, PEDF-based gene therapy has advanced to early stage clinical trials. PEDF active fragments have been mapped and used to design short peptide mimetics conferring distinct functions of PEDF, which may address specific clinical problems and become prototype drugs.

miR-200b inhibits prostate cancer EMT, growth and metastasis.

miRNA regulate gene expression at post-transcriptional level and fine-tune the key biological processes, including cancer progression. Here, we demonstrate the involvement of miR-200 b in the metastatic spread of prostate cancer. We identified miR-200 b as a downstream target of androgen receptor and linked its expression to decreased tumorigenicity and metastatic capacity of the prostate cancer cells. Overexpression of miR-200 b in PC-3 cells significantly inhibited their proliferation and the formation of subcutaneous tumors. Moreover, in an orthotopic model, miR-200 b blocked spontaneous metastasis and angiogenesis by PC-3 cells. This decreased metastatic potential was likely due to the reversal of the epithelial-to-mesenchymal transition, as was evidenced by increased pan-epithelial marker E-cadherin and specific markers of prostate epithelium, cytokeratins 8 and 18. In contrast, mesenchymal markers, fibronectin and vimentin, were significantly downregulated by miR-200 b. Our results suggest an important role for miR-200 b in prostate cancer progression and indicate its potential utility for prostate cancer therapy.

Androgen receptor drives cellular senescence.

The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence in vitro and in vivo. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor.

miR-27b controls venous specification and tip cell fate.

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR-27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets.

Biomimetic high density lipoprotein nanoparticles for nucleic acid delivery.

We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy that combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy, and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery.

Natural angiogenesis inhibitor signals through Erk5 activation of peroxisome proliferator-activated receptor gamma (PPARgamma).

Erk-5, a member of the MAPK superfamily, has a catalytic domain similar to Erk1/2 and a unique C-terminal domain enabling binding with transcription factors. Aberrant vascularization in the Erk5-null mice suggested a link to angiogenesis. Ectopic expression of constitutively active Erk5 blocks endothelial cell morphogenesis and causes HIF1-alpha destabilization/degradation. However the mechanisms by which endogenous Erk5 regulates angiogenesis remain unknown. We show that Erk5 and its activating kinase MEK5 are the upstream mediators of the anti-angiogenic signal by the natural angiogenesis inhibitor, pigment epithelial-derived factor (PEDF). We demonstrate that Erk5 phosphorylation allows activation of PPARgamma transcription factor by displacement of SMRT co-repressor. PPARgamma, in turn is critical for NFkappaB activation, PEDF-dependent apoptosis, and anti-angiogenesis. The dominant negative MEK5 mutant and Erk5 shRNA diminished PEDF-dependent apoptosis, inhibition of the endothelial cell chemotaxis, and angiogenesis. This is the first evidence of Erk5-dependent transduction of signals by endogenous angiogenesis inhibitors.

The genome of Beet cryptic virus 1 shows high homology to certain cryptoviruses present in phylogenetically distant hosts.

UNLABELLED: This study determined the complete nucleotide sequence of Beet cryptic virus 1 (BCV1). As expected by analogy to previously sequenced alphacryptoviruses, dsRNA1 (2008 bp) encodes a 72.5-kDa protein containing sequence motifs characteristic for RNA-dependent RNA polymerases (RdRp). In addition to the full-length dsRNA1, a truncated form was also detected in dsRNA extracts. dsRNA2 (1783 bp) codes for the viral coat protein (CP) as proven by the identity of the predicted CP sequence to peptide sequences of the purified virion protein. The amino acid sequence of BCV1 RdRp as well as the 5'- and 3'-UTRs show 81-85% identity to the corresponding regions of Vicia cryptic virus (VCV), White clover cryptic virus 1 (WCCV1) and Carrot cryptic virus (CaCV). The amino acid sequence identity of the CP is about 55-62%, moreover, a strong conservation of predicted alpha-helical regions was observed. The high degree of similarity of these seed- and pollen-transmitted viruses persisting in phylogenetically distant hosts, together with their high similarity to fungal partitiviruses strongly supports the hypothesis that horizontal transfer by a fungus played a role in the emergence of the present cryptovirus species. The change in the distribution of cryptic viruses may also be due to human influence: While earlier BCV1 occurred frequently in sugar beet cultivars, it is very rare in cultivars currently used in agricultural practice and was detected in only one of the 28 cultivars investigated in our experiments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-009-0432-4) contains supplementary material, which is available to authorized users.

PPARgamma and Agonists against Cancer: Rational Design of Complementation Treatments.

PPARgamma is a member of the ligand-activated nuclear receptor superfamily: its ligands act as insulin sensitizers and some are approved for the treatment of metabolic disorders in humans. PPARgamma has pleiotropic effects on survival and proliferation of multiple cell types, including cancer cells, and is now subject of intensive preclinical cancer research. Studies of the recent decade highlighted PPARgamma role as a potential modulator of angiogenesis in vitro and in vivo. These observations provide an additional facet to the PPARgamma image as potential anticancer drug. Currently PPARgamma is regarded as an important target for the therapies against angiogenesis-dependent pathological states including cancer and vascular complications of diabetes. Some of the studies, however, identify pro-angiogenic and tumor-promoting effects of PPARgamma and its ligands pointing out the need for further studies. Below, we summarize current knowledge of PPARgamma regulatory mechanisms and molecular targets, and discuss ways to maximize the beneficial activity of the PPARgamma agonists.

Pigment epithelium-derived factor and interleukin-6 control prostate neuroendocrine differentiation via feed-forward mechanism.

PURPOSE: PEDF (pigment epithelium-derived factor) promotes the differentiation and survival of neuronal cells, and expands the adult neuronal stem cell niche. In the prostate PEDF is suppressed by androgen with unclear physiological consequences. We report that PEDF induced the neuroendocrine differentiation of prostate cancer cells, which was accompanied by neurite outgrowth and chromogranin A expression. MATERIALS AND METHODS: We performed neuroendocrine differentiation assay, Western blot analysis, immunostaining and reverse transcriptase-polymerase chain reaction in the human prostate cancer cell lines LNCaP, PC-3 and DU145, and the prostate epithelial strain RWPE-1 (ATCC). RESULTS: Ectopic and endogenous PEDF caused neuroendocrine differentiation of prostate cancer cells, as manifested by neurite-like outgrowths and chromogranin A expression. The transdifferentiated cells expressed axonal and dendritic markers, as ascertained by immunoblotting for specific markers. Neuroendocrine cells formed multiple synaptophysin positive protrusions resembling dendritic spines and vesicles containing serotonin, pointing to possible synapse formation. The known transdifferentiating agent interleukin-6 induced PEDF secretion. Moreover, PEDF neutralizing antibodies abolished the transdifferentiation of interleukin-6 treated cells, suggesting an autocrine loop. Neurogenic events were independent of cyclic adenosine monophosphate. Instead, PEDF activated in this order RhoA, nuclear factor kappaB and Stat3. Inhibitors of the Rho, nuclear factor kappaB and STAT pathways abolished differentiation and synapse formation. Additionally, nuclear factor kappaB activation caused interleukin-6 expression. CONCLUSIONS: We discovered that nuclear factor kappaB controls the formation of neuronal communications in the prostate due to PEDF. We defined a feed-forward loop, in which nuclear factor kappaB induction elicits Stat3 activation and pro-differentiating interleukin-6 expression causes the further expansion of neuroendocrine communications. Our findings point to the role of nuclear factor kappaB and PEDF in coordinated prostate development.

Transient potential receptor channel 4 controls thrombospondin-1 secretion and angiogenesis in renal cell carcinoma.

Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel-Lindau tumor suppressor, stabilization of hypoxia inducible factor-1 transcription factor and increased vascular endothelial growth factor. To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells. Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells. However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic. Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported. Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor. In HNK cells, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, a calcium chelator, simulated TSP1 retention, mimicking the RCC phenotype. Further analysis revealed a profound decrease in transient receptor potential canonical ion channel 4 (TRPC4) Ca(2+) channel expression in RCC cells. TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion. Double labeling of the secretory system components revealed TSP1 colocalization with coatomer protein II (COPII) anterograde vesicles in HNK cells. In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfolding. Our study indicates that TRPC4 loss in RCC leads to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP1, thus enabling angiogenic switch during RCC progression.

Androgen receptor targets NFkappaB and TSP1 to suppress prostate tumor growth in vivo.

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NFkappaB, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NFkappaB suppression, since it was restricted to the cells lacking nuclear (active) NFkappaB. Thus we for the first time identified combined decrease of NFkappaB and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (c) 2007 Wiley-Liss, Inc.

Searching for a new putative cryptic virus in Pinus sylvestris L.

Double-stranded RNAs (dsRNAs) were detected in different pine populations in Germany and Hungary. Two dsRNA species of 1.5 and 1.58 kbp, respectively, persisted in the same trees for at least 2 years and their presence was not associated with any symptoms. The dsRNAs were found to sediment in the VLP (virus-like particles) fraction and to be protected by protein(s) against RNase A digestion at low salt. cDNA cloning and sequencing of the smaller segment (dsRNA2) led to the identification of a putative RNA-dependent RNA-polymerase (RdRp) containing the GDD, as well as three other, conserved motifs. Sequence comparison with different RNA viruses and phylogenetic analysis indicates that the putative RdRp from pine shows highest similarity to the homologous proteins of Beet cryptic virus 3 and of a cryptic virus of Pyrus pyrifolia. On the basis of these results we suggest that the 1.5 and 1.58 kbp dsRNAs in P. sylvestris may represent the genomic segments of a new plant cryptic virus, Cryptoviruses have not yet been reported to occur in Gymnosperms.