RESUMEN
IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.
Asunto(s)
Interleucina-12 , Neoplasias , Humanos , Ratones , Animales , Interleucina-12/metabolismo , Neoplasias/tratamiento farmacológico , Citocinas , Transducción de Señal , Índice Terapéutico , Microambiente TumoralRESUMEN
Antiangiogenesis-based cancer therapies, specifically those targeting the VEGF-A/VEGFR2 pathway, have been approved for subsets of solid tumors. However, these therapies result in an increase in hematologic adverse events. We surmised that both the bone marrow vasculature and VEGF receptor-positive hematopoietic cells could be impacted by VEGF pathway-targeted therapies. We used a mouse model of spontaneous breast cancer to decipher the mechanism by which VEGF pathway inhibition alters hematopoiesis. Tumor-bearing animals, while exhibiting increased angiogenesis at the primary tumor site, showed signs of shrinkage in the sinusoidal bone marrow vasculature accompanied by an increase in the hematopoietic stem cell-containing Lin-cKit(+)Sca1(+) (LKS) progenitor population. Therapeutic intervention by targeting VEGF-A, VEGFR2, and VEGFR3 inhibited tumor growth, consistent with observed alterations in the primary tumor vascular bed. These treatments also displayed systemic effects, including reversal of the tumor-induced shrinkage of sinusoidal vessels and altered population balance of hematopoietic stem cells in the bone marrow, manifested by the restoration of sinusoidal vessel morphology and hematopoietic homeostasis. These data indicate that tumor cells exert an aberrant systemic effect on the bone marrow microenvironment and VEGF-A/VEGFR targeting restores bone marrow function.
Asunto(s)
Médula Ósea/metabolismo , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/terapia , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Femenino , Hematopoyesis , Células Madre Hematopoyéticas/patología , Homeostasis , Inmunohistoquímica , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Receptores de Factores de Crecimiento Endotelial Vascular/inmunología , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
[This corrects the article on p. e55095 in vol. 8.].
RESUMEN
The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S1PRs). Here, we studied the role of S1PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S1PR2-specific antagonist (JTE013), we found that S1PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotoxemia. Experiments with bone marrow chimeras (S1pr2(+/+) â S1pr2(+/+), S1pr2(+/+) â S1pr2(-/-), and S1pr2(-/-) â S1pr2(+/+)) indicate the critical role of S1PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE013 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S1PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S1PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S1PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S1PR2 as a novel therapeutic target for vascular disorders.
Asunto(s)
Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Inflamación/metabolismo , Inflamación/patología , Receptores de Lisoesfingolípidos/metabolismo , Enfermedad Aguda , Animales , Biomarcadores/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiopatología , Permeabilidad Capilar/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Endotoxemia/complicaciones , Endotoxemia/metabolismo , Endotoxemia/patología , Activación Enzimática/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Inmunohistoquímica , Inflamación/complicaciones , Inflamación/fisiopatología , Mediadores de Inflamación/metabolismo , Riñón/metabolismo , Riñón/patología , Ratones , FN-kappa B/metabolismo , Fenotipo , Pirazoles/farmacología , Piridinas/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/genética , Transducción de Señal/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Under steady state conditions, erythropoiesis occurs in the bone marrow. However, in mice, stress or tissue hypoxia results in increased erythropoiesis in the spleen. There is increasing evidence that the hematopoietic microenvironment, including endothelial cells, plays an important role in regulating erythropoiesis. Here, we show that short-term expression of constitutively active Akt in the endothelium of mice results in non-anemic stress erythropoiesis in the spleen. The initiation of this stress response was independent of erythropoietin and BMP4, and was observed in endothelial myrAkt1 mice reconstituted with wild-type bone marrow. Together, these data suggest that endothelial cell hyperactivation is a potentially novel pathway of inducing red cell production under stress.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/metabolismo , Eritropoyesis , Modificación Traduccional de las Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Bazo/citología , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Eritropoyesis/fisiología , Eritropoyetina/metabolismo , Femenino , Expresión Génica , Humanos , Ratones , Embarazo , Bazo/metabolismo , Estrés FisiológicoRESUMEN
Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/metabolismo , Células Endoteliales/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células del Estroma/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Animales , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Ratones , Ratones Transgénicos , Neovascularización Patológica/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Microambiente Tumoral/fisiologíaRESUMEN
RhoB, a member of the Rho subfamily of small GTPases, mediates diverse cellular functions, including cytoskeletal organization, cell transformation and vesicle trafficking. The thymus undergoes progressive decline in its structure and function after puberty. We found that RhoB was expressed in thymic medullary epithelium. To investigate a role of RhoB in the regulation of thymic epithelial organization or thymocyte development, we analyzed the thymi of RhoB-deficient mice. RhoB-deficient mice were found to display earlier thymic atrophy. RhoB deficiency showed significant reductions in thymus weight and cellularity, beginning as early as 5 weeks of age. The enhanced expression of TGF-ß receptor type II (TGFßRII) in thymic medullary epithelium was observed in RhoB-null mice. In addition, the expression of fibronectin, which is shown to be regulated by TGF-ß signaling, was accordingly increased in the mutant thymic medulla. Since there is no age-related change of RhoB expression in the thymus, it is unlikely that RhoB in thymic epithelium directly contributes to age-related thymic involution. Nevertheless, our findings strongly support a physiological role of RhoB in regulation of thymus development and maintenance through the inhibition of TGF-ß signaling in thymic medullary epithelium.
Asunto(s)
Epitelio/metabolismo , Timo/metabolismo , Timo/patología , Proteína de Unión al GTP rhoB/deficiencia , Proteína de Unión al GTP rhoB/metabolismo , Animales , Epitelio/inmunología , Epitelio/patología , Femenino , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Proteína de Unión al GTP rhoB/genéticaRESUMEN
Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34(-)Flt3(-) KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34(-)Flt3(-) KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the modulation of angiocrine factors, with Akt-mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs.
Asunto(s)
Diferenciación Celular , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Comunicación Paracrina , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Crecimiento Transformadores/metabolismo , Animales , Comunicación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Activación Enzimática , Hematopoyesis , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Tumor invasion into blood and/or lymphatic channels is an important component of cancer staging and prognosis. Standard pathological methods do not provide sufficient contrast to discriminate between invasion into each type of vessel and are complicated by tissue retraction artifacts. We evaluated the ability of a triple-stain immunohistochemical method, combining cytokeratin, CD34, and podoplanin stains in a single section, to distinguish blood from lymphatic vascular invasion in oral squamous cell carcinoma and confirmed its results using multispectral analysis. The triple-stain method was significantly more sensitive in detecting invasive events than the standard hematoxylin and eosin staining method and easily discriminated between blood and lymphatic vessel invasion. Invasive events were present in blood and/or lymphatic vessels in the majority of patients with and without presentation of lymph node metastasis, indicating that vessel invasion in this cancer model is common and is not a rate-limiting step for lymph node metastasis.
Asunto(s)
Vasos Sanguíneos/patología , Carcinoma de Células Escamosas/irrigación sanguínea , Carcinoma de Células Escamosas/patología , Vasos Linfáticos/patología , Neoplasias de la Boca/irrigación sanguínea , Neoplasias de la Boca/patología , Antígenos CD34/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Invasividad NeoplásicaRESUMEN
Dendritic cell (DC) function will likely be important for immunological cancer therapies, but our knowledge of the roles of DCs in tumors is limited, in part because most studies were performed before DC subtypes were defined. We studied the distributions of immature epidermal, immature dermal, mature, and plasmacytoid DCs in 63 primary tumors and eight lymph node metastases from oral squamous cell carcinoma patients without evidence of distant metastasis. Immature CD207/Langerin+ and CD209/DC-SIGN+ DCs were present in the primary tumor region, but CD209/DC-SIGN+ cells rarely infiltrated the tumor. Mature DCs were rare, and presence of CD123+ plasmacytoid DCs was associated with poorer outcome. Unexpectedly, migration and maturation of DCs was associated with a worse outcome. Overall, the distribution of DC subtypes indicated that ineffective DC response to tumor is a failure of DC function rather than recruitment, suggesting that a strategy of shifting the balance of secreted factors in the tumor milieu may be more effective in restoring anti-tumor immune function than current methods restoring only one population of DCs to the immunosuppressive tumor region.
Asunto(s)
Carcinoma de Células Escamosas/patología , Células Dendríticas/citología , Neoplasias de la Boca/patología , Anciano , Moléculas de Adhesión Celular/biosíntesis , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-3/biosíntesis , Lectinas Tipo C/biosíntesis , Leucocitos/metabolismo , Ganglios Linfáticos/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias/patología , Plasmacitoma/patología , Receptores de Superficie Celular/biosíntesis , Resultado del TratamientoRESUMEN
Chronic activation of Akt signaling in the endothelium recapitulates the salient features of a tumor vasculature and can be inhibited by rapamycin, an inhibitor of mammalian target of rapamycin. This led to the hypothesis that the antitumor efficacy of rapamycin may be partially dependent on its ability to inhibit endothelial Akt signaling, making rapamycin an antiangiogenic agent and endothelial Akt pathway inhibitor. Dose-response studies with rapamycin showed that primary human endothelial cells and fibroblasts had a bimodal Akt response with effective reductions in phosphorylated Akt (pAkt) achieved at 10 ng/mL. In contrast, rapamycin increased pAkt levels in tumor cell lines. When tumor-bearing mice were treated with rapamycin doses comparable to those used clinically in transplant patients, we observed strong inhibition of mammary tumor growth. To test whether Akt activation in the endothelium was rate-limiting for this antitumor response, we engineered mouse mammary tumor virus-polyoma virus middle T antigen mice with endothelial cell-specific expression of constitutively activated Akt. We observed that the antitumor efficacy of rapamycin was reduced in the presence of elevated endothelial Akt activation. Just as we observed in MCF7 cells in vitro, rapamycin doses that were antiangiogenic resulted in increased pAkt levels in total mouse mammary tumor virus-polyoma virus middle T antigen tumor lysates, suggesting that tumor cells had an opposite Akt response following mammalian target of rapamycin inhibition compared with tumor endothelial cells. Together, these data support the hypothesis that endothelial Akt signaling in the tumor vasculature is an important target of the novel anticancer drug rapamycin.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Células Endoteliales/enzimología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Sirolimus/farmacología , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine, and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible.