RESUMEN
Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 µg). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 µM, PEDF 336 (100 µM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neovascularización Coroidal/prevención & control , Proteínas del Ojo/uso terapéutico , Factores de Crecimiento Nervioso/uso terapéutico , Oligopéptidos/uso terapéutico , Serpinas/uso terapéutico , Administración Oftálmica , Inhibidores de la Angiogénesis/química , Animales , Apoptosis , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Portadores de Fármacos , Electrorretinografía , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Proteínas del Ojo/química , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/química , Oligopéptidos/química , Soluciones Oftálmicas , Profármacos , Conejos , Ratas , Serpinas/químicaRESUMEN
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.
Asunto(s)
Arterias/embriología , Endotelio Vascular/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , MicroARNs/fisiología , Neovascularización Fisiológica , Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Aorta/citología , Aorta/metabolismo , Arterias/metabolismo , Biomarcadores/metabolismo , Northern Blotting , Western Blotting , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Diferenciación Celular , Movimiento Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Endotelio Vascular/metabolismo , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas , Seudópodos/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
Sucrose nonfermenting 1 (Snf1)-related kinase (SNRK) is a serine/threonine kinase with sequence similarity to AMP-activated protein kinase (AMPK); however, its function is not well characterized. We conducted a gene array to determine which genes are regulated by SNRK. The array demonstrated that SNRK overexpression increased the levels of genes involved in cell proliferation, including calcyclin-binding protein (CacyBP), a member of the ubiquitin ligase complex that targets nonphosphorylated ß-catenin for degradation. We confirmed that SNRK increased CacyBP mRNA and protein, and decreased ß-catenin protein in HCT116 and RKO colon cancer cells. Furthermore, SNRK inhibited colon cancer cell proliferation, and CacyBP down-regulation reversed the SNRK-mediated decrease in proliferation and ß-catenin. SNRK overexpression also decreased ß-catenin nuclear localization and target gene transcription, and ß-catenin down-regulation reversed the effects of SNRK knockdown on proliferation. SNRK transcript levels were reduced in human colon tumors compared to normal tissue by 35.82%, and stable knockdown of SNRK increased colon cancer cell tumorigenicity. Our results demonstrate that SNRK is down-regulated in colon cancer and inhibits colon cancer cell proliferation through CacyBP up-regulation and ß-catenin degradation, resulting in reduced proliferation signaling. These findings reveal a novel function for SNRK in the regulation of colon cancer cell proliferation and ß-catenin signaling.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Neoplasias del Colon/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , beta Catenina/metabolismo , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiología , Transcriptoma , beta Catenina/genéticaRESUMEN
Extracellular factors control the angiogenic switch in endothelial cells (ECs) via competing survival and apoptotic pathways. Previously, we showed that proangiogenic and antiangiogenic factors target the same signaling molecules, which thereby become pivots of angiogenic balance. Here we show that in remodeling endothelium (ECs and EC precursors) natural angiogenic inhibitors enhance nuclear factor-kappaB (NF-kappaB) DNA binding, which is critical for antiangiogenesis, and that blocking the NF-kappaB pathway abolishes multiple antiangiogenic events in vitro and in vivo. NF-kappaB induction by antiangiogenic molecules has a dual effect on transcription. NF-kappaB acts as an activator of proapoptotic FasL and as a repressor of prosurvival cFLIP. On the FasL promoter, NF-kappaB increases the recruitment of HAT p300 and acetylated histones H3 and H4. Conversely, on cFLIP promoter, NF-kappaB increases histone deacetylase 1 (HDAC1), decreases p300 and histone acetylation, and reduces the recruitment of NFAT, a transcription factor critical for cFLIP expression. Finally, we found a biphasic effect, when HDAC inhibitors (HDACi) were used to test the dependence of pigment epithelial-derived factor activity on histone acetylation. The cooperative effect seen at low doses switches to antagonistic as the concentrations increase. Our study defines an interactive transcriptional network underlying angiogenic balance and points to HDACi as tools to manipulate the angiogenic switch.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , FN-kappa B/fisiología , Factores de Transcripción NFATC/fisiología , Neovascularización Fisiológica , Inhibidores de la Angiogénesis/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Proteínas del Ojo/farmacología , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Femenino , Histona Desacetilasa 1/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Factores de Transcripción NFATC/genética , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Factores de Crecimiento Nervioso/farmacología , Regiones Promotoras Genéticas , Serpinas/farmacología , Transducción de Señal , Trombospondina 1/farmacologíaRESUMEN
The formation of a blood supply is critical for tumor growth and metastasis; however, understanding the relationship of cellular transformation to tumor angiogenesis has been limited by the multifactorial nature of both processes. In this issue of Cancer Cell, Watnick and colleagues use a genetically defined tumor model system to determine the link between ras, myc, and angiogenesis and identify Thrombospondin-1 as being the critical regulator of angiogenesis in this system (Watnick et al., 2003).
Asunto(s)
Neovascularización Patológica/fisiopatología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Trombospondina 1/metabolismo , Proteínas ras/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Trombospondina 1/genéticaRESUMEN
Id proteins are helix-loop-helix transcription factors that regulate tumor angiogenesis. In order to identify downstream effectors of Id1 involved in the regulation of angiogenesis, we performed PCR-select subtractive hybridization on wild-type and Id1 knockout mouse embryo fibroblasts (MEFs). Here we demonstrate that thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis, is a target of transcriptional repression by Id1. We also show that Id1-null MEFs secrete an inhibitor of endothelial cell migration, which is completely inactivated by depletion of TSP-1. Furthermore, in vivo studies revealed decreased neovascularization in matrigel assays in Id1-null mice compared to their wild-type littermates. This decrease was completely reversed by a TSP-1 neutralizing antibody. We conclude that TSP-1 is a major target for Id1 effects on angiogenesis.
Asunto(s)
Regulación de la Expresión Génica , Neovascularización Patológica/genética , Proteínas Represoras , Trombospondina 1/genética , Factores de Transcripción/fisiología , Animales , Bovinos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Elementos E-Box , Embrión de Mamíferos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Fibroblastos/fisiología , Humanos , Inmunohistoquímica , Proteína 1 Inhibidora de la Diferenciación , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Trombospondina 1/antagonistas & inhibidores , Trombospondina 1/biosíntesis , Trombospondina 1/farmacología , Factores de Transcripción/deficiencia , Transcripción Genética , Regulación hacia ArribaRESUMEN
Dysregulated Myc signaling is a key oncogenic pathway in glioblastoma multiforme (GBM). Yet, effective therapeutic targeting of Myc continues to be challenging. Here, we demonstrate that exosomes generated from human bone marrow mesenchymal stem cells (MSCs) engineered to encapsulate siRNAs targeting Myc (iExo-Myc) localize to orthotopic GBM tumors in mice. Treatment of late stage GBM tumors with iExo-Myc inhibits proliferation and angiogenesis, suppresses tumor growth, and extends survival. Transcriptional profiling of tumors reveals that the mesenchymal transition and estrogen receptor signaling pathways are impacted by Myc inhibition. Single nuclei RNA sequencing (snRNA-seq) shows that iExo-Myc treatment induces transcriptional repression of multiple growth factor and interleukin signaling pathways, triggering a mesenchymal to proneural transition and shifting the cellular landscape of the tumor. These data confirm that Myc is an effective anti-glioma target and that iExo-Myc offers a feasible, readily translational strategy to inhibit challenging oncogene targets for the treatment of brain tumors.
RESUMEN
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.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Células Endoteliales/metabolismo , Proteínas del Ojo/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Neovascularización Fisiológica/fisiología , Factores de Crecimiento Nervioso/metabolismo , PPAR gamma/metabolismo , Serpinas/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Proteínas del Ojo/genética , Femenino , Humanos , MAP Quinasa Quinasa 5/genética , MAP Quinasa Quinasa 5/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Desnudos , Proteína Quinasa 7 Activada por Mitógenos/genética , Mutación , FN-kappa B/genética , FN-kappa B/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Factores de Crecimiento Nervioso/genética , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , PPAR gamma/genética , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Serpinas/genéticaRESUMEN
Natural inhibitors of angiogenesis are able to block pathological neovascularization without harming the preexisting vasculature. Here we show that two such inhibitors, thrombospondin-1 and pigment epithelium-derived factor, derive specificity for remodeling vessels from their dependence on Fas/Fas ligand (FasL)-mediated apoptosis to block angiogenesis. Both inhibitors upregulated FasL on endothelial cells. Expression of the essential partner of FasL, Fas/CD95 receptor, was low on quiescent endothelial cells and vessels but greatly enhanced by inducers of angiogenesis, thereby specifically sensitizing the stimulated cells to apoptosis by inhibitor-generated FasL. The anti-angiogenic activity of thrombospondin-1 and pigment epithelium-derived factor both in vitro and in vivo was dependent on this dual induction of Fas and FasL and the resulting apoptosis. This example of cooperation between pro- and anti-angiogenic factors in the inhibition of angiogenesis provides one explanation for the ability of inhibitors to select remodeling capillaries for destruction.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Proteínas del Ojo , Neovascularización Fisiológica/efectos de los fármacos , Factores de Crecimiento Nervioso , Proteínas/farmacología , Serpinas/farmacología , Trombospondina 1/farmacología , Receptor fas/fisiología , Animales , Apoptosis/efectos de los fármacos , Caspasa 8 , Caspasa 9 , Caspasas/fisiología , Células Cultivadas , Endotelio Vascular/citología , Proteína Ligando Fas , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/fisiología , RatonesRESUMEN
Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.
RESUMEN
The morphogen Sonic hedgehog (Shh) promotes neovascularization in adults by inducing pro-angiogenic cytokine expression in fibroblasts; however, the direct effects of Shh on endothelial cell (EC) function during angiogenesis are unknown. Our findings indicate that Shh promotes capillary morphogenesis (tube length on Matrigel increased to 271+/-50% of the length in untreated cells, p=0.00003), induces EC migration (modified Boyden chamber assay, 191+/-35% of migration in untreated cells, p=0.00009), and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN) mRNA (real-time RT-PCR), which are essential for Shh-induced angiogenesis both in vitro and in vivo. Shh activity in ECs is mediated by Rho, rather than through the "classic" Shh signaling pathway, which involves the Gli transcription factors. The Rho dependence of Shh-induced EC angiogenic activity was documented both in vitro, with dominant-negative RhoA and Rho kinase (ROCK) constructs, and in vivo, with the ROCK inhibitor Y27632 in the mouse corneal angiogenesis model. Finally, experiments performed in MMP-9- and OPN-knockout mice confirmed the roles of the ROCK downstream targets MMP-9 and OPN in Shh-induced angiogenesis. Collectively, our results identify a "nonclassical" pathway by which Shh directly modulates EC phenotype and angiogenic activity.
Asunto(s)
Aorta/metabolismo , Neovascularización de la Córnea/metabolismo , Endotelio Vascular/metabolismo , Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Neovascularización Fisiológica/fisiología , Quinasas Asociadas a rho/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Apoptosis , Western Blotting , Bovinos , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Neovascularización de la Córnea/patología , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Proteínas Hedgehog/genética , Humanos , Factores de Transcripción de Tipo Kruppel/fisiología , Metaloproteinasa 9 de la Matriz/fisiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Morfogénesis , Proteínas del Tejido Nervioso/fisiología , Osteopontina/fisiología , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína Gli3 con Dedos de Zinc , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
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 1beta-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.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas del Ojo , Péptidos y Proteínas de Señalización Intracelular , Neovascularización Fisiológica , Factores de Crecimiento Nervioso , Proteínas Nucleares , Factores de Transcripción/fisiología , Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/genética , Células Cultivadas , ADN/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Proteína Quinasa 9 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Factores de Transcripción NFATC , Fosforilación , Regiones Promotoras Genéticas , Proteínas/fisiología , Serpinas/fisiología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
PURPOSE: Pigment epithelial-derived factor (PEDF) is a potent angiogenesis inhibitor with multiple other functions, some of which enhance tumor growth. Our previous studies mapped PEDF antiangiogenic and prosurvival activities to distinct epitopes. This study was aimed to determine the minimal fragment of PEDF, which maintains antiangiogenic and antitumor efficacy. EXPERIMENTAL DESIGN: We analyzed antigenicity, hydrophilicity, and charge distribution of the angioinhibitory epitope (the 34-mer) and designed three peptides covering its COOH terminus, P14, P18, and P23. We analyzed their ability to block endothelial cell chemotaxis and induce apoptosis in vitro and their antiangiogenic activity in vivo. The selected peptide was tested for the antitumor activity against mildly aggressive xenografted prostate carcinoma and highly aggressive renal cell carcinoma. To verify that P18 acts in the same manner as PEDF, we used immunohistochemistry to measure PEDF targets, vascular endothelial growth factor receptor 2, and CD95 ligand expression in P18-treated vasculature. RESULTS: P14 and P18 blocked endothelial cell chemotaxis; P18 and P23 induced apoptosis. P18 showed the highest IC50 and blocked angiogenesis in vivo: P23 was inactive and P14 was proangiogenic. P18 increased the production of CD95 ligand and reduced the expression of vascular endothelial growth factor receptor 2 by the endothelial cells in vivo. In tumor studies, P18 was more effective in blocking the angiogenesis and growth of the prostate cancer than parental 34-mer; in the renal cell carcinoma, P18 strongly decreased angiogenesis and halted the progression of established tumors. CONCLUSIONS: P18 is a novel and potent antiangiogenic biotherapeutic agent that has potential to be developed for the treatment of prostate and renal cancer.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Carcinoma de Células Renales/irrigación sanguínea , Proteínas del Ojo/química , Neoplasias Renales/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Factores de Crecimiento Nervioso/química , Fragmentos de Péptidos/farmacología , Neoplasias de la Próstata/irrigación sanguínea , Serpinas/química , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Células Cultivadas , Quimiotaxis , Neovascularización de la Córnea , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Datos de Secuencia Molecular , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Conformación Proteica , Homología de Secuencia de Aminoácido , Venas Umbilicales/citologíaRESUMEN
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.
Asunto(s)
Proteínas del Ojo/fisiología , Interleucina-6/fisiología , Factores de Crecimiento Nervioso/fisiología , Próstata/citología , Serpinas/fisiología , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Humanos , Masculino , Sistemas Neurosecretores/citología , Neoplasias de la Próstata/patologíaRESUMEN
We have previously demonstrated that apigenin promotes the expression of antiangiogenic protein thrombospondin-1 (TSP1) via a mechanism driven by mRNA-binding protein HuR. Here, we generated a novel mouse model with whole-body THBS-1 gene knockout on SKH-1 genetic background, which allows studies of UVB-induced acute skin damage and carcinogenesis and tests TSP1 involvement in apigenin's anticancer effects. Apigenin significantly inhibited UVB-induced carcinogenesis in the wild-type (WT) animals but not in TSP1 KO (TKO) mice, suggesting that TSP1 is a critical component of apigenin's chemopreventive function in UVB-induced skin cancer. Importantly, TKO mice presented with the elevated cutaneous inflammation at baseline, which was manifested by increased inflammatory infiltrates (neutrophils and macrophages) and elevated levels of the two key inflammatory cytokines, IL-6 and IL-12. In agreement, maintaining normal TSP1 expression in the UVB-irradiated skin of WT mice using topical apigenin application caused a marked decrease of circulating inflammatory cytokines. Finally, TKO mice showed an altered population dynamics of the bone marrow myeloid progenitor cells (CD11b+), with dramatic expansion of the population of neutrophil progenitors (Ly6ClowLy6Ghigh) compared to the WT control. Our results indicate that the cutaneous tumor suppressor TSP1 is a critical mediator of the in vivo anticancer effect of apigenin in skin, specifically of its anti-inflammatory action.
Asunto(s)
Apigenina/farmacología , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/efectos de la radiación , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Protectores Solares/farmacología , Rayos Ultravioleta/efectos adversos , Animales , Antiinflamatorios , Línea Celular Tumoral , Quimioprevención , Modelos Animales de Enfermedad , Genotipo , Humanos , Inflamación/etiología , Inflamación/patología , Inflamación/prevención & control , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/prevención & control , Trombospondina 1/genética , Trombospondina 1/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Carcinoma de Células Renales/irrigación sanguínea , Neoplasias Renales/irrigación sanguínea , Neovascularización Patológica/patología , Trombospondina 1/metabolismo , Animales , Western Blotting , Vesículas Cubiertas por Proteínas de Revestimiento/efectos de los fármacos , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Calcio/metabolismo , Calcio/farmacocinética , Carcinoma de Células Renales/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Quelantes/farmacología , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Neoplasias Renales/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Trombospondina 1/genética , TransfecciónRESUMEN
Overexpression of a novel oncogene MCT-1 (multiple copies in a T cell malignancy) causes malignant transformation of murine fibroblasts. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 breast cancer cells negative for endogenous MCT-1 (MCF7-MCT-1). Overexpression of MCT-1 in these cells resulted in a slight elevation of estrogen receptor-alpha, and higher rates of DNA synthesis and growth in response to estradiol compared with the empty vector control (MCF7-EV). The pure antiestrogen fulvestrant inhibited the estradiol-stimulated proliferation of MCF7-MCT-1 cells. The MCF7-MCT-1 clones showed increased invasiveness in the presence of 50% serum compared with the MCF7-EV. In a tumor xenograft model, MCT-1-overexpressing cells showed higher take rates and formed significantly larger tumors than MCF7-EV controls. When we examined angiogenic phenotype and molecular mediators of angiogenesis in MCF7-MCT-1 tumors in vivo, we found greater microvascular density and lower apoptosis in the MCF7-MCT-1 tumors compared with MCF7-EV controls accompanied by a dramatic decline in the levels of angiogenesis inhibitor, thrombospondin-1 (TSP1). In vitro, blocking TSP1 in the medium conditioned by MCT-1-negative cells restored its angiogenic potential to that of the MCF7-MCT-1 cells. Conversely, despite an increase in mRNA encoding vascular endothelial growth factor upon MCT-1 overexpression, vascular endothelial growth factor protein levels have not been notably altered. Taken together, our results suggest that MCT-1 may contribute to the pathogenesis and progression of human breast cancer via at least two routes: promotion of angiogenesis through the decline of TSP1 and inhibition of apoptosis.
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Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Proteínas Oncogénicas/genética , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Estradiol/análogos & derivados , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Fulvestrant , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica/genética , TransfecciónRESUMEN
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.
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Epítopos/fisiología , Proteínas del Ojo/inmunología , Neovascularización Patológica/prevención & control , Factores de Crecimiento Nervioso/inmunología , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/patología , Serpinas/inmunología , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/inmunología , Inhibidores de la Angiogénesis/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Diferenciación Celular/inmunología , Línea Celular Tumoral , Neovascularización de la Córnea , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/fisiología , Células Endoteliales/citología , Células Endoteliales/inmunología , Mapeo Epitopo , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Neovascularización Patológica/inmunología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/fisiología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Neoplasias de la Próstata/inmunología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/fisiologíaRESUMEN
BACKGROUND: Although inflammation and prostate cancer (PCa) have been linked, the molecular interactions between macrophages and PCa cells are poorly explored. Pigment Epithelium-Derived Factor (PEDF) is an anti-angiogenic and anti-tumor factor. We previously showed that PEDF induces macrophages recruitment in vitro, correlates with macrophages density in human prostate, and stimulates macrophages polarization towards the classically activated pathway. Here, we demonstrate that PEDF modulates the interaction between macrophages and PCa cells through a bidirectional signalling leading to tumor cell apoptosis and phagocytosis. METHODS: RAW 264.7 and THP-1 cells, and BMDMs were grown in vitro as mono- or co-cultures with PC3 or CL1 tumor cells. The effects of PEDF and its derived P18 peptide were measured on macrophages differentiation, migration, and superoxide production, and tumor cell apoptosis and phagocytosis. PEDF receptors (ATP5B, PNPLA2, and LRP6) and CD47 mRNA and protein expression were quantified in macrophages and tumor cells by quantitative RT-PCR, western blot, immunofluorescence and flow cytometry. RESULTS: We found that PEDF induced the migration of macrophages towards tumor 3D spheroids and 2D cultures. In co-culture, PEDF increased PCa cells phagocytosis through an indirect apoptosis-dependent mechanism. Moreover, PEDF stimulated the production of superoxide by macrophages. Conditioned media from macrophages exposed to PEDF induced tumor cells apoptosis in contrast to control conditioned media suggesting that ROS may be involved in tumor cells apoptosis. ATP5B and PNPLA2 PEDF receptors on macrophages and CD47 on tumor cells were respectively up- and down-regulated by PEDF. As PEDF, blocking CD47 induced phagocytosis. Inhibiting ATP5B reduced phagocytosis. Inversely, PNPLA2 inhibition blocks differentiation but maintains phagocytosis. CD47-induced phagocytosis was partially reverted by ATP5B inhibition suggesting a complementary action. Similar effects were observed with P18 PEDF-derived peptide. CONCLUSIONS: These data established that modulating the molecular interactions between macrophages and PCa cells using PEDF may be a promising strategy for PCa treatment.
Asunto(s)
Antineoplásicos/farmacología , Proteínas del Ojo/farmacología , Macrófagos/fisiología , Factores de Crecimiento Nervioso/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Serpinas/farmacología , Animales , Antígeno CD47/metabolismo , Línea Celular Tumoral , Movimiento Celular , Técnicas de Cocultivo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lipasa/metabolismo , Macrófagos/efectos de los fármacos , Masculino , Ratones , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Fagocitosis , Compuestos de Fenilurea/farmacología , Células RAW 264.7 , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Superóxidos/metabolismoRESUMEN
Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These "non-metastatic" exosomes stimulate an innate immune response through the expansion of Ly6Clow patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.