RESUMEN
Resistance to inactive state-selective RASG12C inhibitors frequently entails accumulation of RASGTP, rendering effective inhibition of active RAS potentially desirable. Here, we evaluated the anti-tumor activity of the RAS(ON) multi-selective tri-complex inhibitor RMC-7977 and dissected mechanisms of response and tolerance in KRASG12C-mutant NSCLC. Broad-spectrum, reversible RASGTP inhibition with or without concurrent covalent targeting of active RASG12C yielded superior and differentiated antitumor activity across diverse co-mutational KRASG12C-mutant NSCLC mouse models of primary or acquired RASG12C(ON) or (OFF) inhibitor resistance. Interrogation of time-resolved single cell transcriptional responses established an in vivo atlas of multi-modal acute and chronic RAS pathway inhibition in the NSCLC ecosystem and uncovered a regenerative mucinous transcriptional program that supports long-term tumor cell persistence. In patients with advanced KRASG12C-mutant NSCLC, the presence of mucinous histological features portended poor response to sotorasib or adagrasib. Our results have potential implications for personalized medicine and the development of rational RAS inhibitor-anchored therapeutic strategies.
RESUMEN
RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).
Asunto(s)
Antineoplásicos , Mutación , Neoplasias , Proteína Oncogénica p21(ras) , Proteínas Proto-Oncogénicas p21(ras) , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Guanosina Trifosfato/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.
Asunto(s)
Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Línea Celular Tumoral , Proteínas Proto-Oncogénicas p21(ras)/genética , Femenino , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Guanosina Trifosfato/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , MasculinoRESUMEN
Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. RMC-7977 is a highly selective inhibitor of the active (GTP-bound) forms of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.
RESUMEN
Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.
Asunto(s)
Adenocarcinoma/enzimología , Neoplasias de la Mama/enzimología , Neoplasias del Colon/enzimología , Melanoma Experimental/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/deficiencia , Neoplasias Cutáneas/enzimología , Macrófagos Asociados a Tumores/enzimología , Inmunidad Adaptativa , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Adenocarcinoma/terapia , Animales , Antígenos de Diferenciación/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Femenino , Humanos , Inmunidad Innata , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Células THP-1 , Carga Tumoral , Microambiente Tumoral , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.
Asunto(s)
Neoplasias de la Mama/inmunología , Inmunosupresores/farmacología , Macrófagos/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Microambiente Tumoral/inmunología , Regulación Alostérica , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
New therapies are required for melanoma. Here, we report that multiple cardiac glycosides, including digitoxin and digoxin, are significantly more toxic to human melanoma cells than normal human cells. This reflects on-target inhibition of the ATP1A1 Na(+)/K(+) pump, which is highly expressed by melanoma. MEK inhibitor and/or BRAF inhibitor additively or synergistically combined with digitoxin to induce cell death, inhibiting growth of patient-derived melanomas in NSG mice and synergistically extending survival. MEK inhibitor and digitoxin do not induce cell death in human melanocytes or haematopoietic cells in NSG mice. In melanoma, MEK inhibitor reduces ERK phosphorylation, while digitoxin disrupts ion gradients, altering plasma membrane and mitochondrial membrane potentials. MEK inhibitor and digitoxin together cause intracellular acidification, mitochondrial calcium dysregulation and ATP depletion in melanoma cells but not in normal cells. The disruption of ion homoeostasis in cancer cells can thus synergize with targeted agents to promote tumour regression in vivo.
Asunto(s)
Inhibidores Enzimáticos/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Digitoxina/farmacología , Digitoxina/uso terapéutico , Sinergismo Farmacológico , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , MAP Quinasa Quinasa 1/metabolismo , Masculino , Melanocitos , Melanoma/mortalidad , Melanoma/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Fosforilación , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Piel/patología , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Merkel cell carcinoma (MCC), a rare but aggressive cutaneous neoplasm with high metastatic potential, has a poor prognosis at late stages of disease with no proven chemotherapeutic regimens. Using an enriched culture medium, we established and characterized 11 MCC cell lines for Bcl-2 family profiling and functional studies. Immunoblot analysis revealed collectively high protein levels of prosurvival Bcl-2 members in cell lines and a panel of MCC tumors. Downregulation of individual Bcl-2 proteins by RNAi promoted death in a subset of MCC cell lines, whereas simultaneous inhibition of multiple family members by using the small-molecule antagonist ABT-263 led to a marked induction of cell death in 10 of 11 lines. ABT-263 induced Bax-dependent apoptosis with rapid cleavage of caspase-3 and PARP, regardless of Bcl-2 family profile or the presence of Merkel cell polyomavirus. Furthermore, ABT-263 treatment led to rapid and sustained growth suppression of MCC xenografts from a representative cell line, accompanied by a striking increase in apoptosis. Our results establish that concurrent inhibition of multiple prosurvival Bcl-2 proteins leads to effective induction of apoptosis, and strongly support the concept that targeting MCC dependence on these molecules may be useful therapeutically by reversing an intrinsic resistance to cell death.
Asunto(s)
Carcinoma de Células de Merkel/patología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Neoplasias Cutáneas/patología , Anciano , Anciano de 80 o más Años , Compuestos de Anilina/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células de Merkel/virología , Línea Celular Tumoral , Supervivencia Celular , Femenino , Humanos , Masculino , Poliomavirus de Células de Merkel/aislamiento & purificación , Ratones , Persona de Mediana Edad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/análisis , Neoplasias Cutáneas/virología , Sulfonamidas/farmacología , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
Studies of human cancer metastasis have been limited by a lack of experimental assays in which cancer cells from patients metastasize in vivo in a way that correlates with clinical outcome. This makes it impossible to study intrinsic differences in the metastatic properties of cancers from different patients. We recently developed an assay in which human melanomas readily engraft in nonobese diabetic/severe combined immunodeficient interleukin-2 receptor-γ chain null (NSG) mice. We show that melanomas from 25 patients exhibited reproducible differences in the rate of spontaneous metastasis after transplantation into NSG mice and that these differences correlated with clinical outcome in the patients. Stage IIIB/C melanomas that formed distant metastases within 22 months in patients also formed tumors that metastasized widely in NSG mice, whereas stage IIIB/C melanomas that did not form distant metastases within 22 to 50 months in patients metastasized more slowly in NSG mice. These differences in the efficiency of metastasis correlated with the presence of circulating melanoma cells in the blood of NSG mice, suggesting that the rate of entry into the blood is one factor that limits the rate of metastasis. The study of NSG mice can therefore yield information about the metastasis of human melanomas in vivo, in this case revealing intrinsic differences among stage III melanomas in their ability to circulate/survive in the blood and to metastasize.
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Subunidad gamma Común de Receptores de Interleucina/deficiencia , Melanoma/patología , Neoplasias Cutáneas/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Progresión de la Enfermedad , Humanos , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Mediciones Luminiscentes , Ratones , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patología , Resultado del TratamientoRESUMEN
It is unclear whether neurogenesis occurs in the adult mammalian enteric nervous system (ENS). Neural crest-derived cells capable of forming multilineage colonies in culture, and neurons and glia upon transplantation into chick embryos, persist throughout adult life in the mammalian ENS. In this study we sought to determine the physiological function of these cells. We discovered that these cells could be identified based on CD49b expression and that they had characteristics of enteric glia, including p75, GFAP, S100B, and SOX10 expression. To test whether new neurons or glia arise in the adult gut under physiological conditions, we marked dividing progenitors with a thymidine analog in rodents under steady-state conditions, or during aging, pregnancy, dietary changes, hyperglycemia, or exercise. We also tested gut injuries including inflammation, irradiation, benzalkonium chloride treatment, partial gut stenosis, and glial ablation. We readily observed neurogenesis in a neurogenic region of the central nervous system, but not reproducibly in the adult ENS. Lineage tracing of glial cells with GFAP-Cre and GFAP-CreERT2 also detected little or no adult ENS neurogenesis. Neurogenesis in the adult gut is therefore very limited under the conditions we studied. In contrast, ENS gliogenesis was readily observed under steady-state conditions and after injury. Adult enteric glia thus have the potential to form neurons and glia in culture but are fated to form mainly glia under physiological conditions and after the injuries we studied.
Asunto(s)
Sistema Nervioso Entérico/citología , Tracto Gastrointestinal/citología , Neurogénesis/fisiología , Neuroglía/fisiología , Animales , Diferenciación Celular/fisiología , Linaje de la Célula , Células Cultivadas , Dieta , Sistema Nervioso Entérico/fisiología , Femenino , Tracto Gastrointestinal/inervación , Tracto Gastrointestinal/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Células Madre Multipotentes/citología , Células Madre Multipotentes/fisiología , Cresta Neural/citología , Neuroglía/citología , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated whether melanoma is hierarchically organized into phenotypically distinct subpopulations of tumorigenic and nontumorigenic cells or whether most melanoma cells retain tumorigenic capacity, irrespective of their phenotype. We found 28% of single melanoma cells obtained directly from patients formed tumors in NOD/SCID IL2Rγ(null) mice. All stage II, III, and IV melanomas obtained directly from patients had common tumorigenic cells. All tumorigenic cells appeared to have unlimited tumorigenic capacity on serial transplantation. We were unable to find any large subpopulation of melanoma cells that lacked tumorigenic potential. None of 22 heterogeneously expressed markers, including CD271 and ABCB5, enriched tumorigenic cells. Some melanomas metastasized in mice, irrespective of whether they arose from CD271(-) or CD271(+) cells. Many markers appeared to be reversibly expressed by tumorigenic melanoma cells.
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Melanoma/patología , Fenotipo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adapaleno , Animales , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Humanos , Melanoma/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Naftalenos/metabolismo , Metástasis de la Neoplasia , Trasplante Heterólogo/patologíaRESUMEN
Melanoma, like most cancers, is a disease that wreaks havoc mostly through its propensity to spread and establish secondary tumors at sites that are anatomically distant from the primary tumor. The consideration of models of cancer progression is therefore important to understand the essence of this disease. Previous work has suggested that melanoma may propagate according to a cancer stem cell (CSC) model in which rare tumorigenic and bulk non-tumorigenic cells are organized into stable hierarchies within tumors. However, recent studies using assays that are more permissive for revealing tumorigenic potential indicate that it will not be possible to cure patients by focusing research and therapy on rare populations of cells within melanoma tumors. Studies of the nature of tumorigenic melanoma cells reveal that these cells may gain a growth, metastasis and/or therapy resistance advantage by acquiring new genetic mutations and by reversible epigenetic mechanisms. In this light, efforts to link the phenotypes, genotypes and epigenotypes of melanoma cells with differences in their in vivo malignant potential provide the greatest hope of advancing the exciting progress finally being made against this disease.
Asunto(s)
Melanoma/patología , Neoplasias Cutáneas/patología , Progresión de la Enfermedad , Humanos , Modelos Biológicos , Células Madre Neoplásicas/patologíaRESUMEN
Trefoil (TFF) peptides are involved in gastrointestinal mucosal restitution. An hypoxia inducible factor 1 (HIF-1)-dependent induction of TFF genes has been reported in gastric epithelial cells. Nitric oxide (NO) is associated with mucosal damage and modulates HIF-1 activity. The aim of the present study was to analyze the role of iNOS-derived NO in HIF-1alpha stabilization and TFF gene expression in damaged gastric mucosa. Aspirin caused gastric injury that peaked 6 h after dosing and returned to normality at 24 h. iNOS mRNA expression occurs in the corpus in parallel with damage. Blockade of iNOS activity did not modify gastric lesions induced by aspirin but delayed mucosal healing. Aspirin induced HIF-1alpha stabilization and TFF2 mRNA up-regulation in the mucosa, but these effects were diminished when iNOS activity was inhibited. Results obtained using a coculture setup showed that iNOS-derived NO from activated macrophages induced HIF-1alpha stabilization, TFF gene expression, and accelerated wound healing in cultured epithelial cells. Finally, transient silencing of endogenous HIF-1alpha in epithelial cells significantly undermined activated macrophage-induced TFF gene expression. Evidence suggests that the iNOS-derived NO associated with NSAID-induced gastric injury is implicated in mucosal restitution via the HIF-1-mediated induction of TFF genes.
Asunto(s)
Mucosa Gástrica/lesiones , Mucosa Gástrica/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , Péptidos/genética , Amidinas/farmacología , Animales , Antiinflamatorios no Esteroideos/toxicidad , Aspirina/toxicidad , Secuencia de Bases , Bencilaminas/farmacología , Línea Celular , Técnicas de Cocultivo , Cartilla de ADN/genética , Inhibidores Enzimáticos/farmacología , Mucosa Gástrica/efectos de los fármacos , Humanos , Activación de Macrófagos , Masculino , Ratones , MicroARNs/genética , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factor Trefoil-2 , Regulación hacia Arriba/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
The identification and characterization of cancer stem cells might lead to more effective treatments for some cancers by focusing therapy on the most malignant cells. To achieve this goal it will be necessary to determine which cancers follow a cancer stem cell model and which do not, to address technical issues related to tumorigenesis assays, and to test the extent to which cancer cell heterogeneity arises from genetic versus epigenetic differences.
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Neoplasias/terapia , Células Madre Neoplásicas/patología , Animales , Células Clonales , Humanos , Neoplasias/genética , Neoplasias/inmunologíaRESUMEN
A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells (0.1-0.0001%) form tumours when transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. However, the extent to which NOD/SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/SCID interleukin-2 receptor gamma chain null (Il2rg(-/-)) mice, can increase the detection of tumorigenic melanoma cells by several orders of magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumours under these more permissive conditions. In single-cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumours. Modifications to xenotransplantation assays can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.
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Recuento de Células , Melanoma/patología , Trasplante de Neoplasias , Trasplante Heterólogo , Animales , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/patologíaRESUMEN
The present study analyses the expression and distribution of neuronal nitric oxide synthase (nNOS) in the brainstem of animals pre-treated with Escherichia coli or Helicobacter pylori LPS, at doses that modulate gastric motor function. Systemic administration of H. pylori LPS prevented in a dose-dependent manner (5, 40 and 100 microg kg(-1), i.v.) the increase in intragastric pressure induced by 2-deoxy-D-glucose (200 mg kg(-1), i.v.) in urethane-anaesthetized rats. Quantitative analysis showed a significant increase in the amount of nNOS mRNA induced by E. coli or H. pylori LPS (2 h later), in a segment of the brainstem containing the dorsal vagal complex (DVC). Immunohistochemical studies showed nNOS presence in the DVC of vehicle-treated rats. Both E. coli (40 microg kg(-1), i.p.) and H. pylori LPS (100 microg kg(-1), i.p.) significantly increased (2 h later) the number of nNOS immunoreactive cells in this area, mainly at the most rostral level. The present study shows that systemic administration of E. coli or H. pylori LPS induces a transcriptional up-regulation of the nNOS in the DVC of the brainstem and suggests a role for NO synthesis in this area in the control of gastric motor function under endotoxemia.
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Tronco Encefálico/enzimología , Endotoxemia/enzimología , Proteínas del Tejido Nervioso/biosíntesis , Óxido Nítrico Sintasa/biosíntesis , Animales , Escherichia coli , Lipopolisacáridos , Masculino , Óxido Nítrico Sintasa de Tipo I , Presión , Ratas , Ratas Sprague-Dawley , Estómago/efectos de los fármacos , Regulación hacia Arriba , Nervio Vago/fisiologíaRESUMEN
We have investigated the mechanisms underlying acute changes in gastric motor function triggered by endotoxemia. In fundal strips from rats pre-treated with endotoxin (40 microg/kg, i.p. 30 min), mechanical activity was analyzed and the source of nitric oxide (NO) was visualized by confocal microscopy of tissue loaded with the fluorescent dye DAF-FM. NOS expression was determined by quantitative RT-PCR and Western blot, and enzyme activity by the citrulline assay. Strips from endotoxin-treated rats were hypo-contractile. This was prevented by pre-incubation with the neurotoxin tetrodotoxin, the gangliar blocker hexamethonium, or non-selective and neuronal-specific NOS inhibitors (L-NOARG and TRIM, respectively). The soluble guanylyl cyclase (sGC) inhibitor ODQ and the inhibitor of small conductance Ca2+-activated K+ channels apamin prevented relaxation induced by endotoxin, nicotine, exogenous NO (DETA-NONOate), and the NO-independent sGC activator BAY 41-2272. NO synthesis was observed in neuronal soma, axons, and nerve endings of the myenteric plexus in the fundus of endotoxin-treated rats and was prevented by L-NAME, tetrodotoxin, and hexamethonium. nNOS and iNOS mRNA and protein contents were unchanged. Our findings demonstrate synthesis of NO in post-ganglionic myenteric neurons during early endotoxemia that mediates gastric hypo-contractility. The effect of NO is mediated via sGC and small conductance Ca2+-activated K+channels.
Asunto(s)
Fibras Autónomas Posganglionares/metabolismo , Endotoxemia/metabolismo , Motilidad Gastrointestinal/fisiología , Óxido Nítrico/biosíntesis , Fosfato de Piridoxal/análogos & derivados , Animales , Apamina/farmacología , Carbacol/farmacología , Dexametasona/farmacología , Endotoxemia/fisiopatología , Fundus Gástrico/fisiopatología , Motilidad Gastrointestinal/efectos de los fármacos , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/fisiología , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Nicotina/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitroarginina/farmacología , Compuestos Nitrosos/farmacología , Fragmentos de Péptidos/farmacología , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Canales de Potasio Calcio-Activados/fisiología , Pirazoles/farmacología , Piridinas/farmacología , Fosfato de Piridoxal/farmacología , Ratas , Suramina/farmacología , Tetrodotoxina/farmacología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
The effects of endotoxin on fecal pellet output and the neural mechanisms involved in this response were investigated in conscious rats. E. coli endotoxin (40 micro g/kg i.p.) significantly increased fecal excretion for 3 h after the injection. Water content in feces was not modified by endotoxin. Ablation of primary afferent neurons by systemic administration of high doses of capsaicin (20+30+50 mg/kg s.c.) to adult rats prevented the stimulatory effect of endotoxin and so did abdominal vagotomy. Adrenoceptor blockade with phentolamine (5 mg/kg i.p.) + propranolol (3 mg/kg i.p.) did not modify pellet output in endotoxin-treated rats while muscarinic receptor blockade with atropine (1 mg/kg i.p.) abolished the stimulatory effect of endotoxin. Finally, the increase in pellet output induced by endotoxin was prevented in animals receiving the substance P receptor antagonist D-Pro2, D-Trp7,9-substance P (2 mg/kg i.p.) or the NO-synthase inhibitor L-NAME (10 mg/kg i.p.). None of the above treatments modified pellet output in saline-treated rats. These observations indicate that endotoxin increases fecal pellet output through a nervous reflex in which capsaicin-sensitive afferent neurons and the release of excitatory (acetylcholine and substance P) and inhibitory (NO) neurotransmitters in the colonic wall are involved.