Cannabidiol alters mitochondrial bioenergetics via VDAC1 and triggers cell death in hormone-refractory prostate cancer.

In spite of the huge advancements in both diagnosis and interventions, hormone refractory prostate cancer (HRPC) remains a major hurdle in prostate cancer (PCa). Metabolic reprogramming plays a key role in PCa oncogenesis and resistance. However, the dynamics between metabolism and oncogenesis are not fully understood. Here, we demonstrate that two multi-target natural products, cannabidiol (CBD) and cannabigerol (CBG), suppress HRPC development in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model by reprogramming metabolic and oncogenic signaling. Mechanistically, CBD increases glycolytic capacity and inhibits oxidative phosphorylation in enzalutamide-resistant HRPC cells. This action of CBD originates from its effect on metabolic plasticity via modulation of VDAC1 and hexokinase II (HKII) coupling on the outer mitochondrial membrane, which leads to strong shifts of mitochondrial functions and oncogenic signaling pathways. The effect of CBG on enzalutamide-resistant HRPC cells was less pronounced than CBD and only partially attributable to its action on mitochondria. However, when optimally combined, these two cannabinoids exhibited strong anti-tumor effects in TRAMP mice, even when these had become refractory to enzalutamide, thus pointing to their therapeutical potential against PCa.

ß-Galactosylceramidase Deficiency Causes Upregulation of Long Pentraxin-3 in the Central Nervous System of Krabbe Patients and Twitcher Mice.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a neurodegenerative sphingolipidosis caused by genetic deficiency of lysosomal ß-galactosylceramidase (GALC), characterized by neuroinflammation and demyelination of the central (CNS) and peripheral nervous system. The acute phase protein long pentraxin-3 (PTX3) is a soluble pattern recognition receptor and a regulator of innate immunity. Growing evidence points to the involvement of PTX3 in neurodegeneration. However, the expression and role of PTX3 in the neurodegenerative/neuroinflammatory processes that characterize GLD remain unexplored. Here, immunohistochemical analysis of brain samples from Krabbe patients showed that macrophages and globoid cells are intensely immunoreactive for PTX3. Accordingly, Ptx3 expression increases throughout the course of the disease in the cerebrum, cerebellum, and spinal cord of GALC-deficient twitcher (Galctwi/twi) mice, an authentic animal model of GLD. This was paralleled by the upregulation of proinflammatory genes and M1-polarized macrophage/microglia markers and of the levels of PTX3 protein in CNS and plasma of twitcher animals. Crossing of Galctwi/twi mice with transgenic PTX3 overexpressing animals (hPTX3 mice) demonstrated that constitutive PTX3 overexpression reduced the severity of clinical signs and the upregulation of proinflammatory genes in the spinal cord of P35 hPTX3/Galctwi/twi mice when compared to Galctwi/twi littermates, leading to a limited increase of their life span. However, this occurred in the absence of a significant impact on the histopathological findings and on the accumulation of the neurotoxic metabolite psychosine when evaluated at this late time point of the disease. In conclusion, our results provide the first evidence that PTX3 is produced in the CNS of GALC-deficient Krabbe patients and twitcher mice. PTX3 may exert a protective role by reducing the neuroinflammatory response that occurs in the spinal cord of GALC-deficient animals.

D-Peptide analogues of Boc-Phe-Leu-Phe-Leu-Phe-COOH induce neovascularization via endothelial N-formyl peptide receptor 3.

N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune cells in response to pathogen-associated molecular patterns. Three FPRs have been identified in humans (FPR1-FPR3), characterized by different ligand properties, biological function and cellular distribution. Recent findings from our laboratory have shown that the peptide BOC-FLFLF (L-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor by binding to various angiogenic growth factors, including vascular endothelial growth factor-A165 (VEGF). Here we show that the all-D-enantiomer of L-BOC2 (D-BOC2) is devoid of any VEGF antagonist activity. At variance, D-BOC2, as well as the D-FLFLF and succinimidyl (Succ)-D-FLFLF (D-Succ-F3) D-peptide variants, is endowed with a pro-angiogenic potential. In particular, the D-peptide D-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebrafish embryos and adult mice. This activity is related to the capacity of D-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the effects exerted by D-Succ-F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in HUVECs. Finally, D-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion, our data demonstrate that the angiogenic activity of D-Succ-F3 is due to the engagement and activation of FPR3 expressed by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.

ß-Galactosylceramidase Deficiency Causes Bone Marrow Vascular Defects in an Animal Model of Krabbe Disease.

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase ß-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31-/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.

Inflammation and N-formyl peptide receptors mediate the angiogenic activity of human vitreous humour in proliferative diabetic retinopathy.

AIMS/HYPOTHESIS: Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. METHODS: Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. RESULTS: PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45+ cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. CONCLUSIONS/INTERPRETATION: This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR therapy.

ß3 Integrin Promotes Long-Lasting Activation and Polarization of Vascular Endothelial Growth Factor Receptor 2 by Immobilized Ligand.

OBJECTIVE: During neovessel formation, angiogenic growth factors associate with the extracellular matrix. These immobilized factors represent a persistent stimulus for the otherwise quiescent endothelial cells (ECs), driving directional EC migration and proliferation and leading to new blood vessel growth. Vascular endothelial growth factor receptor 2 (VEGFR2) is the main mediator of angiogenesis. Although VEGFR2 signaling has been deeply characterized, little is known about its subcellular localization during neovessel formation. Aim of this study was the characterization and molecular determinants of activated VEGFR2 localization in ECs during neovessel formation in response to matrix-immobilized ligand. APPROACH AND RESULTS: Here we demonstrate that ECs stimulated by extracellular matrix-associated gremlin, a noncanonical VEGFR2 ligand, are polarized and relocate the receptor in close contact with the angiogenic factor-enriched matrix both in vitro and in vivo. GM1 (monosialotetrahexosylganglioside)-positive planar lipid rafts, ß3 integrin receptors, and the intracellular signaling transducers focal adhesion kinase and RhoA (Ras homolog gene family, member A) cooperate to promote VEGFR2 long-term polarization and activation. CONCLUSIONS: A ligand anchored to the extracellular matrix induces VEGFR2 polarization in ECs. Long-lasting VEGFR2 relocation is closely dependent on lipid raft integrity and activation of ß3 integrin pathway. The study of the endothelial responses to immobilized growth factors may offer insights into the angiogenic process in physiological and pathological conditions, including cancer, and for a better engineering of synthetic tissue scaffolds to blend with the host vasculature.

Brain angioarchitecture and intussusceptive microvascular growth in a murine model of Krabbe disease.

Defects of the angiogenic process occur in the brain of twitcher mouse, an authentic model of human Krabbe disease caused by genetic deficiency of lysosomal ß-galactosylceramidase (GALC), leading to lethal neurological dysfunctions and accumulation of neurotoxic psychosine in the central nervous system. Here, quantitative computational analysis was used to explore the alterations of brain angioarchitecture in twitcher mice. To this aim, customized ImageJ routines were used to assess calibers, amounts, lengths and spatial dispersion of CD31(+) vessels in 3D volumes from the postnatal frontal cortex of twitcher animals. The results showed a decrease in CD31 immunoreactivity in twitcher brain with a marked reduction in total vessel lengths coupled with increased vessel fragmentation. No significant changes were instead observed for the spatial dispersion of brain vessels throughout volumes or in vascular calibers. Notably, no CD31(+) vessel changes were detected in twitcher kidneys in which psychosine accumulates at very low levels, thus confirming the specificity of the effect. Microvascular corrosion casting followed by scanning electron microscopy morphometry confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice with reduction in microvascular density, vascular branch remodeling and intussusceptive angiogenesis. Intussusceptive microvascular growth, confirmed by histological analysis, was paralleled by alterations of the expression of intussusception-related genes in twitcher brain. Our data support the hypothesis that a marked decrease in vascular development concurs to the onset of neuropathological lesions in twitcher brain and suggest that neuroinflammation-driven intussusceptive responses may represent an attempt to compensate impaired sprouting angiogenesis.

Long pentraxin-3 as an epithelial-stromal fibroblast growth factor-targeting inhibitor in prostate cancer.

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer.

Inhibition of angiogenesis by ß-galactosylceramidase deficiency in globoid cell leukodystrophy.

Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme ß-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-ß-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine ß-galactosylceramidase complementary DNA. Finally, RNA interference-mediated ß-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that ß-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.

Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR.

The subcutaneous Matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic molecules. However, quantification of the angiogenic response in the plug remains a problematic task. Here we report a simple, rapid, unbiased and reverse transcription-quantitative PCR (RT-qPCR) method to investigate the angiogenic process occurring in the Matrigel plug in response to fibroblast growth factor-2 (FGF2). To this purpose, a fixed amount of human cells were added to harvested plugs at the end of the in vivo experimentation as an external cell tracer. Then, mRNA levels of the pan-endothelial cell markers murine CD31 and vascular endothelial-cadherin were measured by species-specific RT-qPCR analysis of the total RNA and data were normalized for human GAPDH or ß-actin mRNA levels. RT-qPCR was used also to measure the levels of expression in the plug of various angiogenesis/inflammation-related genes. The procedure allows the simultaneous, quantitative evaluation of the newly-formed endothelium and of non-endothelial/inflammatory components of the cellular infiltrate in the Matrigel implant, as well as the expression of genes involved in the modulation of the angiogenesis process. Also, the method consents the quantitative assessment of the effect of local or systemic administration of anti-angiogenic compounds on the neovascular response triggered by FGF2.

Long pentraxin 3/tumor necrosis factor-stimulated gene-6 interaction: a biological rheostat for fibroblast growth factor 2-mediated angiogenesis.

OBJECTIVE: Angiogenesis is regulated by the balance between pro- and antiangiogenic factors and by extracellular matrix protein interactions. Fibroblast growth factor 2 (FGF2) is a major proangiogenic inducer inhibited by the interaction with the soluble pattern recognition receptor long pentraxin 3 (PTX3). PTX3 is locally coexpressed with its ligand tumor necrosis factor-stimulated gene-6 (TSG-6), a secreted glycoprotein that cooperates with PTX3 in extracellular matrix assembly. Here, we characterized the effect of TSG-6 on PTX3/FGF2 interaction and FGF2-mediated angiogenesis. METHODS AND RESULTS: Solid phase binding and surface plasmon resonance assays show that TSG-6 and FGF2 bind the PTX3 N-terminal domain with similar affinity. Accordingly, TSG-6 prevents FGF2/PTX3 interaction and suppresses the inhibition exerted by PTX3 on heparan sulfate proteoglycan/FGF2/FGF receptor complex formation and on FGF2-dependent angiogenesis in vitro and in vivo. Also, endogenous PTX3 exerts an inhibitory effect on vascularization induced by FGF2 in a murine subcutaneous Matrigel plug assay, the inhibition being abolished in Ptx3-null mice or by TSG-6 treatment in wild-type animals. CONCLUSION: TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of FGF2/PTX3 interaction. This may provide a novel mechanism to control angiogenesis in those pathological settings characterized by the coexpression of TSG-6 and PTX3, in which the relative levels of these proteins may fine-tune the angiogenic activity of FGF2.

Iron supplementation enhances RSL3-induced ferroptosis to treat naïve and prevent castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death in the male population commonly treated with androgen deprivation therapy that often relapses as androgen-independent and aggressive castration-resistant prostate cancer (CRPC). Ferroptosis is a recently described form of cell death that requires abundant cytosolic labile iron to promote membrane lipid peroxidation and which can be induced by agents that inhibit the glutathione peroxidase-4 activity such as RSL3. Exploiting in vitro and in vivo human and murine PCa models and the multistage transgenic TRAMP model of PCa we show that RSL3 induces ferroptosis in PCa cells and demonstrate for the first time that iron supplementation significantly increases the effect of RSL3 triggering lipid peroxidation, enhanced intracellular stress and leading to cancer cell death. Moreover, the combination with the second generation anti-androgen drug enzalutamide potentiates the effect of the RSL3 + iron combination leading to superior inhibition of PCa and preventing the onset of CRPC in the TRAMP mouse model. These data open new perspectives in the use of pro-ferroptotic approaches alone or in combination with enzalutamide for the treatment of PCa.

FGFR blockade by pemigatinib treats naïve and castration resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of cancer mortality in the male population commonly treated with androgen deprivation therapy (ADT) and relapsing as aggressive and androgen-independent castration-resistant prostate cancer (CRPC). In PCa the FGF/FGFR family of growth factors and receptors represents a relevant mediator of cancer growth, tumor-stroma interaction, and a driver of resistance and relapse to ADT. In the present work, we validate the therapeutic efficacy the FDA-approved FGFR inhibitor pemigatinib, in an integrated platform consisting of human and murine PCa cells, and the transgenic multistage TRAMP model of PCa that recapitulates both androgen-dependent and CRPC settings. Our results show for the first time that pemigatinib causes intracellular stress and cell death in PCa cells and prevents tumor growth in vivo and in the multistage model. In addition, the combination of pemigatinib with enzalutamide resulted in long-lasting tumor inhibition and prevention of CRPC relapse in TRAMP mice. These data are confirmed by the implementation of a stochastic mathematical model and in silico simulation. Pemigatinib represents a promising FDA-approved FGFR inhibitor for the treatment of PCa and CRPC alone and in combination with enzalutamide.

Vitreous from idiopathic epiretinal membrane patients induces glial-to-mesenchymal transition in Müller cells.

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Macroglial Müller cells appear to play a pivotal role in the pathogenesis of iERM where they may undergo glial-to-mesenchymal transition (GMT), a transdifferentiation process characterized by the downregulation of Müller cell markers, paralleled by the upregulation of pro-fibrotic myofibroblast markers. Previous observations from our laboratory allowed the molecular identification of two major clusters of iERM patients (named iERM-A and iERM-B), iERM-A patients being characterized by less severe clinical features and a more "quiescent" iERM gene expression profile when compared to iERM-B patients. In the present work, Müller MIO-M1 cells were exposed to vitreous samples obtained before membrane peeling from the same cohort of iERM-A and iERM-B patients. The results demonstrate that iERM vitreous induces proliferation, migration, and GMT in MIO-M1 cells, a phenotype consistent with Müller cell behavior during iERM progression. However, even though the vitreous samples obtained from iERM-A patients were able to induce a complete GMT in MIO-M1 cells, iERM-B samples caused only a partial GMT, characterized by the downregulation of Müller cell markers in the absence of upregulation of pro-fibrotic myofibroblast markers. Together, the results indicate that a relationship may exist among the ability of iERM vitreous to modulate GMT in Müller cells, the molecular profile of the corresponding iERMs, and the clinical features of iERM patients.

An Orthotopic Model of Uveal Melanoma in Zebrafish Embryo: A Novel Platform for Drug Evaluation.

Uveal melanoma is a highly metastatic tumor, representing the most common primary intraocular malignancy in adults. Tumor cell xenografts in zebrafish embryos may provide the opportunity to study in vivo different aspects of the neoplastic disease and its response to therapy. Here, we established an orthotopic model of uveal melanoma in zebrafish by injecting highly metastatic murine B16-BL6 and B16-LS9 melanoma cells, human A375M melanoma cells, and human 92.1 uveal melanoma cells into the eye of zebrafish embryos in the proximity of the developing choroidal vasculature. Immunohistochemical and immunofluorescence analyses showed that melanoma cells proliferate during the first four days after injection and move towards the eye surface. Moreover, bioluminescence analysis of luciferase-expressing human 92.1 uveal melanoma cells allowed the quantitative assessment of the antitumor activity exerted by the canonical chemotherapeutic drugs paclitaxel, panobinostat, and everolimus after their injection into the grafted eye. Altogether, our data demonstrate that the zebrafish embryo eye is a permissive environment for the growth of invasive cutaneous and uveal melanoma cells. In addition, we have established a new luciferase-based in vivo orthotopic model that allows the quantification of human uveal melanoma cells engrafted in the zebrafish embryo eye, and which may represent a suitable tool for the screening of novel drug candidates for uveal melanoma therapy.

Impact of VEGF-dependent tumour micro-environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice.

Fibronectin (FN) is an extracellular matrix cell-adhesive glycoprotein. The alternative spliced isoform EDB-FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro-environments on EDB-FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet-FGF2 cells overexpressing fibroblast growth factor-2 (FGF2) driven by the tetracycline-responsive promoter were further transfected with a VEGF antisense cDNA, generating AS-VEGF/Tet-FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast-growing, highly vascularized Tet-FGF2 tumours. Down-regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS-VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro-environment. Quantitative RT-PCR analysis using human EDB-FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up-regulation of EDB-FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki-1 cells. However, in vivo down-regulation of VEGF expression, as occurs in AS-VEGF/Tet-FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline-treated Tet-FGF2 tumour-bearing animals, causes significant inhibition of EDB-FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT-PCR analysis. Accordingly, treatment of Tet-FGF2 tumour-bearing animals with the neutralizing anti-murine VEGF receptor-2 antibody DC101, or of Caki-1 tumour-bearing animals with the anti-VEGF antibody bevacizumab, inhibited EDB-FN expression in tumour grafts. EDB-FN down-regulation was paralleled by a decrease in vascularity, thus confirming EDB-FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro-environment, and in particular the VEGF/VEGFR-2 system, plays a key role in modulating EDB-FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB-FN in combination with anti-angiogenic and/or cytotoxic drugs.

Natural killer cell impairment in ovarian clear cell carcinoma.

In the present study, we report the analysis of NK cells derived from patients suffering from a rare ovarian cancer histotype of clear cell carcinoma (OCCC) resistant to conventional chemotherapies. We analyzed the phenotype of NK cells derived from peripheral blood (PB) and peritoneal fluid (PF) and evaluated cytotoxic interactions between NK cells and autologous tumor cells (ATC) derived from patients. We provided evidence of impaired degranulation capacity of NK cells derived from patients' PF in the presence of ATC. Analyzing tumor cell ligands recognized by NK cell receptors, we found that ATC are characterized by an HLA class I+ phenotype (although the level of HLA-I expression varies among all patients) and by a heterogeneous expression of ligands for activating NK receptors (from normal to decreased expression of some markers). Furthermore, we observed a down-regulation of crucial NK cell activating receptors, primarily DNAX Accessory Molecule-1 (DNAM-1), on tumor-associated NK cells. Based on these results, we propose that this severe lysis defect may be due to both negative interactions between HLA-I-specific inhibitory NK cell receptors/HLA-I molecules and to defective interactions between activating NK receptors and cognate ligands. In conclusion, for the first time, the phenotypic and functional properties of tumor-associated NK cells and their ATC derived from PF of patients with advanced stage of OCCC were characterized. Taken together results indicate altered interactions between NK cells and ATC and shed light on the aggressive mechanisms of this cancer histotype. Further studies on this rare tumor will be helpful to improve and define more effective therapies.

Gene expression analysis identifies two distinct molecular clusters of idiopatic epiretinal membranes.

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Aim of this study was to provide a molecular characterization of iERMs by gene expression analysis. To this purpose, 56 iERMs obtained by pars plana vitrectomy were analyzed for the expression levels of genes encoding biomarkers of the cellular and molecular events occurring in iERMs. RT-qPCR analysis showed significant differences in the levels of cell population, extracellular matrix and cytokine/growth factor biomarkers among the iERMs investigated. Hierarchical clustering of RT-qPCR data identified two distinct iERM clusters, Cluster B samples representing transcriptionally "activated" iERMs when compared to transcriptionally "quiescent" Cluster A specimens. Further, Cluster B could be subdivided in two subgroups, Cluster B1 iERMs, characterized by a marked glial cell activation, and Cluster B2 samples characterized by a more pro-fibrotic phenotype. Preoperative decimal best-corrected visual acuity and post-surgery inner segment/outer grading values were higher in Cluster A patients, that showed a prevalence of fovea-attached type iERMs with near-normal inner retina, than in Cluster B patients, that presented more severe clinical and spectral domain optical coherence tomography (SD-OCT) features. In conclusion, this molecular characterization has identified two major clusters of iERM specimens with distinct transcriptional activities that reflect different clinical and SD-OCT features of iERM patients. This retrospective work paves the way to prospective whole-genome transcriptomic studies to allow a molecular classification of iERMs and for the identification of molecular signature(s) of prognostic and therapeutic significance.

ß-Galactosylceramidase Promotes Melanoma Growth via Modulation of Ceramide Metabolism.

Disturbance of sphingolipid metabolism may represent a novel therapeutic target in metastatic melanoma, the most lethal form of skin cancer. ß-Galactosylceramidase (GALC) removes ß-galactose from galactosylceramide and other sphingolipids. In this study, we show that downregulation of galcb, a zebrafish ortholog of human GALC, affects melanoblast and melanocyte differentiation in zebrafish embryos, suggesting a possible role for GALC in melanoma. On this basis, the impact of GALC expression in murine B16-F10 and human A2058 melanoma cells was investigated following its silencing or upregulation. Galc knockdown hampered growth, motility, and invasive capacity of B16-F10 cells and their tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos. Galc-silenced cells displayed altered sphingolipid metabolism and increased intracellular levels of ceramide, paralleled by a nonredundant upregulation of Smpd3, which encodes for the ceramide-generating enzyme neutral sphingomyelinase 2. Accordingly, GALC downregulation caused SMPD3 upregulation, increased ceramide levels, and inhibited the tumorigenic activity of human melanoma A2058 cells, whereas GALC upregulation exerted opposite effects. In concordance with information from melanoma database mining, RNAscope analysis demonstrated a progressive increase of GALC expression from common nevi to stage IV human melanoma samples that was paralleled by increases in microphthalmia transcription factor and tyrosinase immunoreactivity inversely related to SMPD3 and ceramide levels. Overall, these findings indicate that GALC may play an oncogenic role in melanoma by modulating the levels of intracellular ceramide, thus providing novel opportunities for melanoma therapy. SIGNIFICANCE: Data from zebrafish embryos, murine and human cell melanoma lines, and patient-derived tumor specimens indicate that ß-galactosylceramidase plays an oncogenic role in melanoma and may serve as a therapeutic target.

Modeling Acquired Resistance to the Second-Generation Androgen Receptor Antagonist Enzalutamide in the TRAMP Model of Prostate Cancer.

Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this "TRAMP-based platform" to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC.Significance: Merging mathematical modeling with experimental data, this study presents the "TRAMP-based platform" as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide.