RESUMEN
Every organ in the body requires blood vessels for efficient delivery of oxygen and nutrients, but independent vascular beds are highly specialized to meet the individual needs of specific organs. The vasculature of the brain is tightly sealed, with blood-brain barrier (BBB) properties developing coincident with neural vascularization. G protein-coupled receptor 124 (GPR124) (tumor endothelial marker 5, TEM5), an orphan member of the adhesion family of G protein-coupled receptors, was previously identified on the basis of its overexpression in tumor vasculature. Here, we show that global deletion or endothelial-specific deletion of GPR124 in mice results in embryonic lethality associated with abnormal angiogenesis of the forebrain and spinal cord. Expression of GPR124 was found to be required for invasion and migration of blood vessels into neuroepithelium, establishment of BBB properties, and expansion of the cerebral cortex. Thus, GPR124 is an important regulator of neurovasculature development and a potential drug target for cerebrovascular diseases.
Asunto(s)
Barrera Hematoencefálica/embriología , Sistema Nervioso Central/irrigación sanguínea , Sistema Nervioso Central/embriología , Embrión de Mamíferos/irrigación sanguínea , Receptores Acoplados a Proteínas G/fisiología , Animales , Barrera Hematoencefálica/metabolismo , Western Blotting , Cartilla de ADN/genética , Embrión de Mamíferos/metabolismo , Citometría de Flujo , Técnicas Histológicas , Hibridación in Situ , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.
Asunto(s)
Inmunoconjugados , Neoplasias , Humanos , Ratones , Animales , Inmunoconjugados/farmacología , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Anticuerpos Monoclonales Humanizados , Factores de Transcripción , Neoplasias/tratamiento farmacológico , Antígenos B7RESUMEN
The G(1)/S-phase transition is a well-toned switch in the mammalian cell cycle. Cdk2, Cdk4, and the rate-limiting tumor suppressor retinoblastoma protein (Rb) have been studied in separate animal models, but interactions between the kinases and Rb in vivo have yet to be investigated. To further dissect the regulation of the G(1) to S-phase progression, we generated Cdk2(-/-)Cdk4(-/-)Rb(-/-) (TKO) mutant mice. TKO mice died at midgestation with major defects in the circulatory systems and displayed combined phenotypes of Rb(-/-) and Cdk2(-/-)Cdk4(-/-) mutants. However, TKO mouse embryonic fibroblasts were not only resistant to senescence and became immortal but displayed enhanced S-phase entry and proliferation rates similar to wild type. These effects were more remarkable in hypoxic compared with normoxic conditions. Interestingly, depletion of the pocket proteins by HPV-E7 or p107/p130 shRNA in the absence of Cdk2/Cdk4 elicited a mechanism for the G(1)/S regulation with increased levels of p27(Kip1) binding to Cdk1/cyclin E complexes. Our work indicates that the G(1)/S transition can be controlled in different ways depending on the situation, resembling a regulatory network.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/deficiencia , Quinasa 4 Dependiente de la Ciclina/deficiencia , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/citología , Interfase/genética , Proteína de Retinoblastoma/deficiencia , Animales , Proliferación Celular , Células Cultivadas , Senescencia Celular/genética , Fase G1 , Hipoxia , Ratones , Ratones Noqueados , Complejos Multiproteicos/fisiología , Fase SRESUMEN
Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma. Tumor-associated stromal cells processed collagen into glutamine, which was then released and internalized by cancer cells. Under chronic nutrient starvation, a condition driven by the high metabolic demand of tumors, cancer cells exploited glutamine to survive, an effect that could be reversed by blocking collagen uptake with TEM8 neutralizing antibodies. These studies reveal that cancer cells exploit collagen-consuming stromal cells for survival, exposing an important vulnerability across solid tumors with implications for developing improved anticancer therapy.
Asunto(s)
Inmunoconjugados , Neoplasias , Humanos , Ratones , Animales , Supervivencia Celular , Glutamina , Colágeno/metabolismo , Proteínas de Microfilamentos , Receptores de Superficie CelularRESUMEN
Mouse knockouts of Cdk2 and Cdk4 have demonstrated that, individually, these genes are not essential for viability. To investigate whether there is functional redundancy, we have generated double knockout (DKO) mice. Cdk2-/- Cdk4-/- DKOs die during embryogenesis around E15 as a result of heart defects. We observed a gradual decrease of Retinoblastoma protein (Rb) phosphorylation and reduced expression of E2F-target genes, like Cdc2 and cyclin A2, during embryogenesis and in embryonic fibroblasts (MEFs). DKO MEFs are characterized by a decreased proliferation rate, impaired S phase entry, and premature senescence. HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes, senescence, and proliferation in DKO MEFs. In contrast, loss of p27 did not rescue Cdk2-/- Cdk4-/- phenotypes. Our results demonstrate that Cdk2 and Cdk4 cooperate to phosphorylate Rb in vivo and to couple the G1/S phase transition to mitosis via E2F-dependent regulation of gene expression.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/deficiencia , Quinasa 4 Dependiente de la Ciclina/deficiencia , Embrión de Mamíferos/anomalías , Proteína de Retinoblastoma/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/genética , Factores de Transcripción E2F/antagonistas & inhibidores , Fibroblastos/citología , Silenciador del Gen , Hematopoyesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus , Fenotipo , Fosforilación , Proteína de Retinoblastoma/antagonistas & inhibidores , Proteína de Retinoblastoma/químicaRESUMEN
Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8+ tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.
Asunto(s)
Inmunoconjugados/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Receptores de Superficie Celular/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/deficiencia , Biomarcadores de Tumor/genética , Brentuximab Vedotina , Línea Celular Tumoral , Femenino , Humanos , Inmunoconjugados/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Proteínas de Microfilamentos , Neoplasias/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genética , Células del Estroma/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.
Asunto(s)
Antígenos B7/genética , Antígenos B7/metabolismo , Inmunoconjugados/farmacología , Neoplasias/irrigación sanguínea , Animales , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Antígenos B7/inmunología , Benzodiazepinas/farmacología , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Línea Celular Tumoral , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Humanos , Inmunoconjugados/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Neoplasias/patología , Neoplasias/terapia , Oligopéptidos/farmacología , Pirroles/farmacología , ConejosRESUMEN
G protein-coupled receptor 124 (GPR124) is an orphan receptor in the adhesion family of GPCRs, and previous global or endothelial-specific disruption of Gpr124 in mice led to defective CNS angiogenesis and blood-brain barriergenesis. Similar developmental defects were observed following dual deletion of Wnt7a/Wnt7b or deletion of ß-catenin in endothelial cells, suggesting a possible relationship between GPR124 and canonical WNT signaling. Here, we show using in vitro reporter assays, mutation analysis, and genetic interaction studies in vivo that GPR124 functions as a WNT7A/WNT7B-specific costimulator of ß-catenin signaling in brain endothelium. WNT7-stimulated ß-catenin signaling was dependent upon GPR124's intracellular PDZ binding motif and a set of leucine-rich repeats in its extracellular domain. This study reveals a vital role for GPR124 in potentiation of WNT7-induced canonical ß-catenin signaling with important implications for understanding and manipulating CNS-specific angiogenesis and blood-brain barrier-genesis.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Secuencias de Aminoácidos , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Dominios PDZ , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/deficienciaRESUMEN
Antiangiogenic agents that block vascular endothelial growth factor (VEGF) signaling are important components of current cancer treatment modalities but are limited by alternative ill-defined angiogenesis mechanisms that allow persistent tumor vascularization in the face of continued VEGF pathway blockade. We identified prostaglandin E2 (PGE2) as a soluble tumor-derived angiogenic factor associated with VEGF-independent angiogenesis. PGE2 production in preclinical breast and colon cancer models was tightly controlled by cyclooxygenase-2 (COX-2) expression, and COX-2 inhibition augmented VEGF pathway blockade to suppress angiogenesis and tumor growth, prevent metastasis, and increase overall survival. These results demonstrate the importance of the COX-2/PGE2 pathway in mediating resistance to VEGF pathway blockade and could aid in the rapid development of more efficacious anticancer therapies.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Neoplasias Mamarias Experimentales/prevención & control , Neoplasias Mamarias Experimentales/secundario , Ensayos Antitumor por Modelo de Xenoinjerto , Inhibidores de la Angiogénesis/farmacología , Animales , Axitinib , Carcinogénesis/patología , Línea Celular Tumoral , Células Clonales , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/metabolismo , Femenino , Humanos , Imidazoles/farmacología , Indazoles/farmacología , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratones , Terapia Neoadyuvante , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.
Asunto(s)
Pulmón/efectos de los fármacos , Surfactantes Pulmonares/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Pulmón/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Current antiangiogenic agents used to treat cancer only partially inhibit neovascularization and cause normal tissue toxicities, fueling the need to identify therapeutic agents that are more selective for pathological angiogenesis. Tumor endothelial marker 8 (TEM8), also known as anthrax toxin receptor 1 (ANTXR1), is a highly conserved cell-surface protein overexpressed on tumor-infiltrating vasculature. Here we show that genetic disruption of Tem8 results in impaired growth of human tumor xenografts of diverse origin including melanoma, breast, colon, and lung cancer. Furthermore, antibodies developed against the TEM8 extracellular domain blocked anthrax intoxication, inhibited tumor-induced angiogenesis, displayed broad antitumor activity, and augmented the activity of clinically approved anticancer agents without added toxicity. Thus, TEM8 targeting may allow selective inhibition of pathological angiogenesis.
Asunto(s)
Proteínas de Neoplasias/fisiología , Neoplasias/irrigación sanguínea , Neovascularización Patológica , Receptores de Superficie Celular/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoglobulina G/farmacología , Inmunoglobulina G/toxicidad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Melanoma/genética , Melanoma/patología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Neovascularización Patológica/genética , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/inmunología , Trasplante Heterólogo , Cicatrización de Heridas/genéticaRESUMEN
The loss of p53 induces spontaneous tumors in mice, and p53 mutations are found in approximately 50% of human tumors. These tumors are generally caused by a number of events, including genomic instability, checkpoint defects, mitotic defects, deregulation of transcriptional targets, impaired apoptosis, and G(1) deregulation or a combination of these effects. In order to determine the role of proteins involved in G(1) control in tumorigenesis, we focused on Cdk2 and Cdk4, two cyclin-dependent kinases that in association with cyclin E and cyclin D promote the G(1)/S phase transition. We analyzed the consequence of loss of Cdk2 in p53-null animals by generating Cdk2(-/-) p53(-/-) mice. These mice are viable and developed spontaneous tumors, predominantly lymphoblastic lymphomas, similar to p53(-/-) mice. In contrast, the genotypes Cdk4(-/-) p53(-/-) were mostly lethal, with few exceptions, and Cdk2(-/-) Cdk4(-/-) p53(-/-) mice die during embryogenesis at embryonic day 13.5. To study the oncogenic potential, we generated mouse embryonic fibroblasts (MEFs) and found that p53(-/-), Cdk2(-/-) p53(-/-), Cdk4(-/-) p53(-/-), and Cdk2(-/-) Cdk4(-/-) p53(-/-) MEFs grew at similar rates without entering senescence. Ras-transformed MEFs of these genotypes were able to form colonies in vitro and induce tumors in nude mice. Our results suggest that tumorigenicity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates considering the use of broad-spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , Transformación Celular Neoplásica , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ciclinas/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Trasplante de Neoplasias , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Tumor endothelial marker 8 (TEM8) was initially identified as a gene overexpressed in the vasculature of human tumors and was subsequently identified as an anthrax toxin receptor. To assess the functional role of TEM8, we disrupted the TEM8 gene in mice by targeted homologous recombination. TEM8(-/-) mice were viable and reached adulthood without defects in physiologic angiogenesis. However, histopathologic analysis revealed an excess of extracellular matrix in several tissues, including the ovaries, uterus, skin, and periodontal ligament of the incisors, the latter resulting in dental dysplasia. When challenged with B16 melanoma, tumor growth was delayed in TEM8(-/-) mice, whereas the growth of other tumors, such as Lewis lung carcinoma, was unaltered. These studies show that host-derived TEM8 promotes the growth of certain tumors and suggest that TEM8 antagonists may have utility in the development of new anticancer therapies.
Asunto(s)
Melanoma Experimental/patología , Receptores de Péptidos/fisiología , Animales , Biomarcadores de Tumor , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Femenino , Masculino , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Neovascularización Patológica/patología , Receptores de Superficie Celular , Receptores de Péptidos/biosíntesis , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genéticaRESUMEN
Cdk1 was proposed to compensate for the loss of Cdk2. Here we present evidence that this is possible due to premature translocation of Cdk1 from the cytoplasm to the nucleus in the absence of Cdk2. We also investigated the consequence of loss of Cdk2 on the maintenance of the G1/S DNA damage checkpoint. Cdk2(-/-) mouse embryonic fibroblasts in vitro as well as regenerating liver cells after partial hepatectomy (PH) in Cdk2(-/-) mice, arrest promptly at the G1/S checkpoint in response to gamma-irradiation due to activation of p53 and p21 inhibiting Cdk1. Furthermore re-entry into S phase after irradiation was delayed in Cdk2(-/-) cells due to prolonged and impaired DNA repair activity. In addition, Cdk2(-/-) mice were more sensitive to lethal irradiation compared to wild-type and displayed delayed resumption of DNA replication in regenerating liver cells. Our results suggest that the G1/S DNA damage checkpoint is intact in the absence of Cdk2, but Cdk2 is important for proper repair of the damaged DNA.