RESUMEN
INTRODUCTION: The clinical benefit of the anti-CTLA-4 monoclonal antibody (mAb) ipilimumab has been well established but limited by immune-related adverse events, especially when ipilimumab is used in combination with anti-PD-(L)1 mAb therapy. To overcome these limitations, we have developed XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 mAb. METHODS: XTX101 consists of an anti-human CTLA-4 mAb covalently linked to masking peptides that block the complementarity-determining regions, thereby minimizing the mAb binding to CTLA-4. The masking peptides are designed to be released by proteases that are typically dysregulated within the tumor microenvironment (TME), resulting in activation of XTX101 intratumorally. Mutations within the Fc region of XTX101 were included to enhance affinity for FcγRIII, which is expected to enhance potency through antibody-dependent cellular cytotoxicity. RESULTS: Biophysical, biochemical, and cell-based assays demonstrate that the function of XTX101 depends on proteolytic activation. In human CTLA-4 transgenic mice, XTX101 monotherapy demonstrated significant tumor growth inhibition (TGI) including complete responses, increased intratumoral CD8+T cells, and regulatory T cell depletion within the TME while maintaining minimal pharmacodynamic effects in the periphery. XTX101 in combination with anti-PD-1 mAb treatment resulted in significant TGI and was well tolerated in mice. XTX101 was activated in primary human tumors across a range of tumor types including melanoma, renal cell carcinoma, colon cancer and lung cancer in an ex vivo assay system. CONCLUSIONS: These data demonstrate that XTX101 retains the full potency of an Fc-enhanced CTLA-4 antagonist within the TME while minimizing the activity in non-tumor tissue, supporting the further evaluation of XTX101 in clinical studies.
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Antineoplásicos , Melanoma , Humanos , Ratones , Animales , Antígeno CTLA-4 , Ipilimumab/uso terapéutico , Antineoplásicos/uso terapéutico , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Melanoma/tratamiento farmacológico , Modelos Animales de Enfermedad , Ratones Transgénicos , Péptidos/uso terapéutico , Microambiente TumoralRESUMEN
Deep learning has emerged as the technique of choice for identifying hidden patterns in cell imaging data but is often criticized as "black box." Here, we employ a generative neural network in combination with supervised machine learning to classify patient-derived melanoma xenografts as "efficient" or "inefficient" metastatic, validate predictions regarding melanoma cell lines with unknown metastatic efficiency in mouse xenografts, and use the network to generate in silico cell images that amplify the critical predictive cell properties. These exaggerated images unveiled pseudopodial extensions and increased light scattering as hallmark properties of metastatic cells. We validated this interpretation using live cells spontaneously transitioning between states indicative of low and high metastatic efficiency. This study illustrates how the application of artificial intelligence can support the identification of cellular properties that are predictive of complex phenotypes and integrated cell functions but are too subtle to be identified in the raw imagery by a human expert. A record of this paper's transparent peer review process is included in the supplemental information. VIDEO ABSTRACT.
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Aprendizaje Profundo , Melanoma , Animales , Inteligencia Artificial , Humanos , Ratones , Redes Neurales de la ComputaciónRESUMEN
Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αß and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αß T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αß T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αß and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.
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Antígenos CD/inmunología , Butirofilinas/antagonistas & inhibidores , Butirofilinas/inmunología , Linfocitos Intraepiteliales/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/genética , Butirofilinas/genética , Femenino , Humanos , Inmunoterapia/métodos , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CD137 (4-1BB) is a member of the TNFR superfamily that represents a promising target for cancer immunotherapy. Recent insights into the function of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as critical features, and these observations help explain the limited activity and toxicity seen with clinically tested CD137 agonists. Here, we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 that engages a unique epitope that is shared by human, cynomolgus monkey, and mouse and is associated with a differentiated pharmacology and toxicology profile. In vitro, CTX-471 increased IFN-γ production by human T cells in an Fcγ receptor-dependent (FcγR-dependent) manner, displaying an intermediate level of activity between 2 clinical-stage anti-CD137 antibodies. In mice, CTX-471 exhibited curative monotherapy activity in various syngeneic tumor models and showed a unique ability to cure mice of very large (~500 mm3) tumors compared with validated antibodies against checkpoints and TNFR superfamily members. Extremely high doses of CTX-471 were well tolerated, with no signs of hepatic toxicity. Collectively, these data demonstrate that CTX-471 is a unique CD137 agonist that displays an excellent safety profile and an unprecedented level of monotherapy efficacy against very large tumors.
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Anticuerpos Monoclonales/inmunología , Inmunoterapia/métodos , Neoplasias/terapia , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Mapeo Epitopo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Inmunoterapia/efectos adversos , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/metabolismo , Macaca fascicularis , Ratones , Ratones Desnudos , Neoplasias/inmunología , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Emerging evidence suggests that crosstalk between glioma cells and the brain microenvironment may influence brain tumor growth. To date, known reciprocal interactions among these cells have been limited to the release of paracrine factors. Combining a genetic strategy with longitudinal live imaging, we find that individual gliomas communicate with distinct sets of non-glioma cells, including glial cells, neurons, and vascular cells. Transfer of genetic material is achieved mainly through extracellular vesicles (EVs), although cell fusion also plays a minor role. We further demonstrate that EV-mediated communication leads to the increase of synaptic activity in neurons. Blocking EV release causes a reduction of glioma growth in vivo. Our findings indicate that EV-mediated interaction between glioma cells and non-glioma brain cells alters the tumor microenvironment and contributes to glioma development.
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Neoplasias Encefálicas/patología , Encéfalo/patología , Comunicación Celular , Vesículas Extracelulares/metabolismo , Glioma/patología , Animales , Astrocitos/patología , Encéfalo/fisiopatología , Neoplasias Encefálicas/fisiopatología , Fusión Celular , Línea Celular Tumoral , ADN de Neoplasias/genética , Fenómenos Electrofisiológicos , Vesículas Extracelulares/ultraestructura , Glioma/fisiopatología , Humanos , Ratones Endogámicos C57BL , Ratones Desnudos , Neuronas/patología , Imagen de Lapso de TiempoRESUMEN
We screen ion channels and transporters throughout the genome to identify those required by human melanoma cells but not by normal human melanocytes. We discover that Mucolipin-1 (MCOLN1), which encodes the lysosomal cation channel TRPML1, is preferentially required for the survival and proliferation of melanoma cells. Loss of MCOLN1/TRPML1 function impairs the growth of patient-derived melanomas in culture and in xenografts but does not affect the growth of human melanocytes. TRPML1 expression and macropinocytosis are elevated in melanoma cells relative to melanocytes. TRPML1 is required in melanoma cells to negatively regulate MAPK pathway and mTORC1 signaling. TRPML1-deficient melanoma cells exhibit decreased survival, proliferation, tumor growth, and macropinocytosis, as well as serine depletion and proteotoxic stress. All of these phenotypes are partially or completely rescued by mTORC1 inhibition. Melanoma cells thus increase TRPML1 expression relative to melanocytes to attenuate MAPK and mTORC1 signaling, to sustain macropinocytosis, and to avoid proteotoxic stress.
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Sistema de Señalización de MAP Quinasas , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Melanoma/metabolismo , Proteostasis , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Masculino , Melanoma/genética , Melanoma/patología , Ratones , Fenotipo , Pinocitosis , Canales de Potencial de Receptor Transitorio/genética , Células Tumorales CultivadasRESUMEN
Soft agar anchorage-independent growth assays have been commonly used as an indicator of cellular transformation in cell culture. Protocols listed here are optimized to allow for all steps, including plasmid purification, virus production, transduction, and soft agar colony formation, to be performed in 96-well plates. These modifications decrease hands-on time, increase fidelity of the assay, and make it possible to screen 500-1000 short-hairpin RNAs (shRNA) in "one-shRNA-one-well" format in parallel. These protocols can also be used to conduct functional cDNA or CRISPR screens for modulators of anchorage-independent growth.
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Adhesión Celular , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Silenciador del Gen , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/genética , Agar/química , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
Genomic diversity among melanoma tumors limits durable control with conventional and targeted therapies. Nevertheless, pathologic activation of the ERK1/2 pathway is a linchpin tumorigenic mechanism associated with the majority of primary and recurrent disease. Therefore, we sought to identify therapeutic targets that are selectively required for tumorigenicity in the presence of pathologic ERK1/2 signaling. By integration of multigenome chemical and genetic screens, recurrent architectural variants in melanoma tumor genomes, and patient outcome data, we identified two mechanistic subtypes of BRAFV600 melanoma that inform new cancer cell biology and offer new therapeutic opportunities. Subtype membership defines sensitivity to clinical MEK inhibitors versus TBK1/IKBKε inhibitors. Importantly, subtype membership can be predicted using a robust quantitative five-feature genetic biomarker. This biomarker, and the mechanistic relationships linked to it, can identify a cohort of best responders to clinical MEK inhibitors and identify a cohort of TBK1/IKBKε inhibitor-sensitive disease among nonresponders to current targeted therapy.Significance: This study identified two mechanistic subtypes of melanoma: (1) the best responders to clinical BRAF/MEK inhibitors (25%) and (2) nonresponders due to primary resistance mechanisms (9.9%). We identified robust biomarkers that can detect these subtypes in patient samples and predict clinical outcome. TBK1/IKBKε inhibitors were selectively toxic to drug-resistant melanoma. Cancer Discov; 7(8); 832-51. ©2017 AACR.See related commentary by Jenkins and Barbie, p. 799This article is highlighted in the In This Issue feature, p. 783.
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Biomarcadores de Tumor/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Carcinogénesis/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/clasificación , Melanoma/patología , Ratones , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This is the first prospective study of a combination therapy involving a cardenolide and a MEK inhibitor for metastatic melanoma. Whereas BRAF mutant melanomas can exhibit profound responses to treatment with BRAF and MEK inhibitors, there are fewer options for BRAF wild-type melanomas. In preclinical studies, we discovered that cardenolides synergize with MEK inhibitor to promote the regression of patient-derived xenografts irrespective of BRAF mutation status. We therefore conducted a phase 1B study of digoxin 0.25 mg and trametinib 2 mg given orally once daily in 20 patients with advanced, refractory, BRAF wild-type melanomas. The most common adverse events were rash, diarrhea, nausea, and fatigue. The response rate was 4/20 or 20% with response durations of 2, 4, 6, and 8 months. The disease control rate (including partial responses and stable disease) was 13/20 or 65% of patients, including 5/6 or 83% of patients with NRAS mutant melanomas and 8/14 or 57% of NRAS wild-type melanomas. Patients with stable disease had disease control for 2, 2, 2, 4, 5, 6, 7, 10, and 10 months. Xenografts from four patients recapitulated the treatment responses observed in patients. Based on these pilot results, an expansion arm of digoxin plus MEK inhibitor is warranted for NRAS mutant metastatic melanoma patients who are refractory or intolerant of immunotherapy. KEY POINTS: Digoxin plus trametinib is well tolerated and achieves a high rate of disease control in BRAF wild-type metastatic melanoma patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Digoxina/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Melanoma/patología , Ratones , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Retratamiento , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ETS transcription factors are commonly deregulated in cancer by chromosomal translocation, overexpression or post-translational modification to induce gene expression programs essential in tumorigenicity. Targeted destruction of these proteins may have therapeutic impact. Here we report that Ets-1 destruction is regulated by the deubiquitinating enzyme, Usp9x, and has major impact on the tumorigenic program of metastatic melanoma. Ets-1 deubiquitination blocks its proteasomal destruction and enhances tumorigenicity, which could be reversed by Usp9x knockdown or inhibition. Usp9x and Ets-1 levels are coincidently elevated in melanoma with highest levels detected in metastatic tumours versus normal skin or benign skin lesions. Notably, Ets-1 is induced by BRAF or MEK kinase inhibition, resulting in increased NRAS expression, which could be blocked by inactivation of Usp9x and therapeutic combination of Usp9x and MEK inhibitor fully suppressed melanoma growth. Thus, Usp9x modulates the Ets-1/NRAS regulatory network and may have biologic and therapeutic implications.
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Carcinogénesis/patología , GTP Fosfohidrolasas/genética , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Humanos , Melanoma/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Estabilidad Proteica , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Mutations in the adenomatous polyposis coli (APC) gene are common in colorectal cancer (CRC), and more than 90% of those mutations generate stable truncated gene products. We describe a chemical screen using normal human colonic epithelial cells (HCECs) and a series of oncogenically progressed HCECs containing a truncated APC protein. With this screen, we identified a small molecule, TASIN-1 (truncated APC selective inhibitor-1), that specifically kills cells with APC truncations but spares normal and cancer cells with wild-type APC. TASIN-1 exerts its cytotoxic effects through inhibition of cholesterol biosynthesis. In vivo administration of TASIN-1 inhibits tumor growth of CRC cells with truncated APC but not APC wild-type CRC cells in xenograft models and in a genetically engineered CRC mouse model with minimal toxicity. TASIN-1 represents a potential therapeutic strategy for prevention and intervention in CRC with mutant APC.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias del Colon/genética , Neoplasias Colorrectales/genética , Terapia Molecular Dirigida , Piperidinas/farmacología , Sulfonamidas/farmacología , Animales , Proliferación Celular , Colesterol/química , Neoplasias del Colon/patología , Neoplasias Colorrectales/patología , Femenino , Genes Supresores de Tumor , Células HCT116 , Humanos , Masculino , Ratones , Ratones Desnudos , Mutación , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Transgenes , 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.
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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
The RASSF1A gene is one of the most frequently inactivated genes in over 30 different types of cancers (H. Donninger, M. D. Vos, and G. J. Clark, J. Cell Sci. 120:3163-3172, 2007, http://dx.doi.org/10.1242/jcs.010389). Despite the prevalence of RASSF1A silencing in human cancer, the mechanism by which RASSF1A functions as a tumor suppressor is not well understood. Characterization of the consequences of RASSF1A loss on epithelial cell proliferation revealed that RASSF1A expression suppresses both microRNA 21 (miR-21) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. The mechanism of the former is through restraint of SCF(ßTrCP)-dependent destruction of the repressor element 1 silencing transcription factor (REST) tumor suppressor and consequent inhibition of miR-21 promoter activation. The mechanism of the latter is through physical sequestration of MST2, which results in accumulation of inactivating S259 phosphorylation of RAF1. Whether or not inactivation of these RASSF1A regulatory relationships can unleash enhanced proliferative capacity is dependent upon the coupling of SCF(ßTrCP) and miR-21 to suppression of SKP2 protein translation and stability. Airway epithelial cultures retain this coupling and therefore respond to RASSF1A inactivation by p27-dependent cell cycle arrest. In contrast, colonic crypt-derived epithelial cells have uncoupled SCF(ßTrCP) from SKP2 and respond to RASSF1A inactivation by enhanced proliferation rates. These observations help account for context-specific molecular etiology of oncogenic transformation and suggest intervention strategies for recently developed SKP2 inhibitors.
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Ciclo Celular/genética , Genes Supresores de Tumor , Oncogenes , Transducción de Señal/genética , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Masculino , MicroARNs/metabolismo , Datos de Secuencia Molecular , Proteínas Represoras/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
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 TratamientoAsunto(s)
Neoplasias Colorrectales/genética , Células Epiteliales/patología , Genes Supresores de Tumor , Interferencia de ARN , Proteína de la Poliposis Adenomatosa del Colon/genética , Fosfatidilinositol 3-Quinasa Clase I , Neoplasias Colorrectales/patología , Genes p53 , Humanos , Mutación , Invasividad Neoplásica/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , ARN Interferente Pequeño , Análisis de Secuencia de ADN , Proteínas ras/genéticaRESUMEN
Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for antioxidant and anti-inflammation enzymes that binds to its endogenous inhibitor protein, Kelch-like ECH (erythroid cell-derived protein with CNC homology)-associated protein 1, in the cytoplasm under normal conditions. Various endogenous or environmental oxidative stresses, such as ionizing radiation (IR), can disrupt the Nrf2-Kelch-like ECH-associated protein 1 complex. This allows Nrf2 to translocate from the cytoplasm into the nucleus to induce transcription of heme oxygenase-1 and other cytoprotective enzymes through binding to antioxidant responsive elements. However, how Nrf2 protects cells from IR-induced damage remains unclear. Here, we report that Nrf2 activation by the synthetic triterpenoids, bardoxolone methyl (BARD) and 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide, protects colonic epithelial cells against IR-induced damage, in part, by enhancing signaling of the DNA damage response. Pretreatment with BARD reduced the frequency of both G1 and S/G2 chromosome aberrations and enhanced the disappearance of repairosomes (C-terminal binding protein interacting protein, Rad51, and p53 binding protein-1 foci) after IR. BARD protected cells from IR toxicity in a Nrf2-dependent manner. The p53 binding protein-1 promoter contains three antioxidant responsive elements in which Nrf2 directly binds following BARD treatment. In addition, 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide provided before exposure to a lethal dose of whole-body irradiation protected WT mice from DNA damage and acute gastrointestinal toxicity, which resulted in improved overall survival. These results demonstrate that Nrf2 activation by synthetic triterpenoids is a promising candidate target to protect the gastrointestinal tract against acute IR in vitro and in vivo.
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Colon/efectos de la radiación , Daño del ADN , Mucosa Intestinal/efectos de la radiación , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal , Animales , Línea Celular Transformada , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Radiación IonizanteRESUMEN
Normal human colonic epithelial cells (HCECs) are not immortalized by telomerase alone but also require CDK4. Some human cell types growth-arrest due to stress- or aberrant signaling-induced senescence (stasis). Stasis represents the consequences of growth conditions culture that are inadequate to maintain long-term proliferation. Overexpressed CDK4 titers out p16 and allows cells to ignore the growth arrest signals produced by stasis. To identify factors contributing to the inadequate culture environment, we used a 62,000-member shRNA library to knock down factors cooperating with human telomerase reverse transcriptase (hTERT) in the immortalization of HCECs. Knockdown of Klotho gamma (KLG; also known as KLPH and LCTL) allowed hTERT to immortalize HCECs. KLG is one isoform of the Klotho family of factors that coordinate interaction between different FGF ligands and the FGF receptor. We also found that knockdown of KLG induced another member of the Klotho family, Klotho beta (KLB). Induction of KLB was maintained and could activate ERK1/2 in immortalized cells. Supplementation of the culture medium with the KLB ligand FGF19 had a similar effect on hTERT-expressing HCECs as knockdown of KLG regarding both immortalization and down-regulation of the tumor suppressor Klotho alpha. Together, these data suggest that KLB is an important regulator in the immortalization of HCECs by facilitating FGF19 growth factor signaling.
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Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Colon/citología , Células Epiteliales/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , ARN Interferente Pequeño/fisiología , Western Blotting , Línea Celular , Factores de Crecimiento de Fibroblastos/farmacología , Glucuronidasa/genética , Humanos , Proteínas Klotho , ARN Interferente Pequeño/genética , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Specific information about how telomerase acts in vivo is necessary for understanding telomere dynamics in human tumor cells. Our results imply that, under homeostatic telomere length-maintenance conditions, only one molecule of telomerase acts at each telomere during every cell division and processively adds â¼60 nt to each end. In contrast, multiple molecules of telomerase act at each telomere when telomeres are elongating (nonequilibrium conditions). Telomerase extension is less processive during the first few weeks following the reversal of long-term treatment with the telomerase inhibitor Imetelstat (GRN163L), a time when Cajal bodies fail to deliver telomerase RNA to telomeres. This result implies that processing of telomerase by Cajal bodies may affect its processivity. Overexpressed telomerase is also less processive than the endogenously expressed telomerase. These findings reveal two major distinct extension modes adopted by telomerase in vivo.
Asunto(s)
Homeostasis , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismo , Western Blotting , Línea Celular Tumoral , Cuerpos Enrollados/metabolismo , Fase G1 , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Modelos Genéticos , Oligonucleótidos/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S , Telomerasa/antagonistas & inhibidores , Telomerasa/genéticaRESUMEN
Landmark cancer genome resequencing efforts are leading to the identification of mutated genes in many types of cancer. The extreme diversity of mutations being detected presents significant challenges to subdivide causal from coincidental mutations to elucidate how disrupted regulatory networks drive cancer processes. Given that a common early perturbation in solid tumor initiation is bypass of matrix-dependent proliferation restraints, we sought to functionally interrogate colorectal cancer candidate genes (CAN-genes) to identify driver tumor suppressors. We have employed an isogenic human colonic epithelial cell (HCEC) model to identify suppressors of anchorage-independent growth by conducting a soft agar-based short hairpin RNA (shRNA) screen within the cohort of CAN-genes. Remarkably, depletion of 65 of the 151 CAN-genes tested collaborated with ectopic expression of K-RAS(V12) and/or TP53 knockdown to promote anchorage-independent proliferation of HCECs. In contrast, only 5 of 362 random shRNAs (1.4%) enhanced soft agar growth. We have identified additional members of an extensive gene network specifying matrix-dependent proliferation, by constructing an interaction map of these confirmed progression suppressors with approximately 700 mutated genes that were excluded from CAN-genes, and experimentally verifying soft agar growth enhancement in response to depletion of a subset of these genes. Collectively, this study revealed a profound diversity of nodes within a fundamental tumor suppressor network that are susceptible to perturbation leading to enhanced cell-autonomous anchorage-independent proliferative fitness. Tumor suppressor network fragility as a paradigm within this and other regulatory systems perturbed in cancer could, in large part, account for the heterogeneity of somatic mutations detected in tumors.