RESUMEN
CASZ1 is a conserved transcription factor involved in neural development, blood vessel assembly and heart morphogenesis. CASZ1 has been implicated in cancer, either suppressing or promoting tumor development depending on the tissue. However, the impact of CASZ1 on hematological tumors remains unknown. Here, we show that the T-cell oncogenic transcription factor TAL1 is a direct positive regulator of CASZ1, that T-cell acute lymphoblastic leukemia (T-ALL) samples at diagnosis overexpress CASZ1b isoform, and that CASZ1b expression in patient samples correlates with PI3KAKT- mTOR signaling pathway activation. In agreement, overexpression of CASZ1b in both Ba/F3 and T-ALL cells leads to the activation of PI3K signaling pathway, which is required for CASZ1b-mediated transformation of Ba/F3 cells in vitro and malignant expansion in vivo. We further demonstrate that CASZ1b cooperates with activated NOTCH1 to promote T-ALL development in zebrafish, and that CASZ1b protects human T-ALL cells from serum deprivation and treatment with chemotherapeutic drugs. Taken together, our studies indicate that CASZ1b is a TAL1-regulated gene that promotes T-ALL development and resistance to chemotherapy.
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Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.
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Proteínas Serina-Treonina Quinasas/metabolismo , Espermátides/enzimología , Animales , Diferenciación Celular/fisiología , Fertilidad/fisiología , Masculino , Ratones , Ratones Noqueados , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Espermatocitos/citología , Espermatocitos/enzimología , Espermatogénesis/fisiología , Espermatozoides/enzimología , Testículo/enzimologíaRESUMEN
Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.
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Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Línea Celular , Aberraciones Cromosómicas , Activación Enzimática , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Quinasas Janus/metabolismo , Masculino , Persona de Mediana Edad , Fosfohidrolasa PTEN/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of therapeutic approaches for lung diseases. We combine mouse models allowing diphtheria toxin-mediated damage of specific epithelial cell types and parallel GFP-labeling of functionally dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncover cell types, including Krt13+ basal and Krt15+ club cells, detect an intermediate cell state between basal and goblet cells, reveal goblet cells as actively dividing progenitor cells, and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also show that diphtheria toxin-expressing cells can persist in the lung, express specific inflammatory factors, and transcriptionally resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to concise epithelial injury, encompassing proliferation, differentiation, and cell-to-cell interactions.
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Toxina Diftérica , Pulmón , Ratones , Animales , Humanos , Toxina Diftérica/metabolismo , Pulmón/metabolismo , Diferenciación Celular , Perfilación de la Expresión Génica , División CelularRESUMEN
The respiratory system is a vital component of our body, essential for both oxygen uptake and immune defense. Knowledge of cellular composition and function in different parts of the respiratory tract provides the basis for a better understanding of the pathological processes involved in various diseases such as chronic respiratory diseases and cancer. Single-cell RNA sequencing (scRNA-seq) is a proficient approach for the identification and transcriptional characterization of cellular phenotypes. Although the mouse is an essential tool for the study of lung development, regeneration, and disease, a scRNA-seq mouse atlas of the lung in which all epithelial cell types are included and annotated systematically is lacking. Here, we established a single-cell transcriptome landscape of the mouse lower respiratory tract by performing a meta-analysis of seven different studies in which mouse lungs and trachea were analyzed by droplet and/or plate-based scRNA-seq technologies. We provide information on the best markers for each epithelial cell type, propose surface markers for the isolation of viable cells, harmonized the annotation of cell types, and compare the mouse single-cell transcriptomes with human scRNA-seq data of the lung.
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Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Ratones , Humanos , Análisis de Secuencia de ARN , Transcriptoma/genética , Células Epiteliales/metabolismoRESUMEN
Expression of protein kinase CK2 is frequently deregulated in cancer and mounting evidence implicates CK2 in tumorigenesis. Here, we show that CK2 is overexpressed and hyperactivated in chronic lymphocytic leukemia (CLL). Inhibition of CK2 induces apoptosis of CLL cells without significantly affecting normal B and T lymphocytes. Importantly, this effect is not reversed by coculture with OP9 stromal cells, which are otherwise capable of rescuing CLL cells from in vitro spontaneous apoptosis. CLL cell death upon CK2 inhibition is mediated by inactivation of PKC, a PI3K downstream target, and correlates with increased PTEN activity, indicating that CK2 promotes CLL cell survival at least in part via PI3K-dependent signaling. Although CK2 antagonists induce significant apoptosis of CLL cells in all patient samples analyzed, sensitivity to CK2 blockade positively correlates with the percentage of CLL cells in the peripheral blood, ß2 microglobulin serum levels and clinical stage. These data suggest that subsets of patients with aggressive and advanced stage disease may especially benefit from therapeutic strategies targeting CK2 function. Overall, our study indicates that CK2 plays a critical role in CLL cell survival, laying the groundwork for the inclusion of CK2 inhibitors into future therapeutic strategies.
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Quinasa de la Caseína II/metabolismo , Leucemia Linfocítica Crónica de Células B/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Linfocitos B/efectos de los fármacos , Estudios de Casos y Controles , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Activación Enzimática , Femenino , Expresión Génica , Humanos , Técnicas In Vitro , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Fosfohidrolasa PTEN/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , Linfocitos T/efectos de los fármacosRESUMEN
Commitment to the CD4+ or CD8+ T cell lineages is linked to the acquisition of a functional program broadly defined by helper and cytotoxic properties, respectively. The mechanisms underlying these processes in the human thymus remain largely unclear. Moreover, recent thymic emigrants are thought to have some degree of plasticity, which may be important for the shaping of the immune system and adjustment to specific peripheral needs. We show here that IL-4 induces proliferation-independent de novo synthesis of CD8αß in human CD4 single-positive (SP) thymocytes, generating a stable CD8SP population that features a diverse TCRαß repertoire, CD4 expression shut-down and ThPOK downregulation. IL-4 also promotes an innate-like program in both CD4SP and CD8SP thymocytes, characterized by Eomes upregulation in the absence of T-bet, in line with its recognized role in the generation of thymic innate-like CD8+ T cells. The clinical relevance of these findings is further supported by the profile of IL-4 production and IL-4 receptor expression that we identified in the human thymus. Importantly, human cord blood CD4+ T cells preserve the ability to generate Eomes+ CD8+ T cells in the presence of IL-4, with implications in neonatal immunity. Our results support a role for IL-4 in the dynamic regulation of human thymocyte plasticity and identify novel strategies to modulate immune responses.
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Linfocitos T CD4-Positivos , Interleucina-4 , Linfocitos T CD8-positivos , Diferenciación Celular , Sangre Fetal/metabolismo , Humanos , Recién Nacido , Interleucina-4/metabolismoRESUMEN
Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.
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Fosfohidrolasa PTEN/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Humanos , Modelos Biológicos , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Specific inhibition of signaling elements essential for the viability of B-cell chronic lymphocytic leukemia (CLL) cells offers great promise for the design of more efficient therapies. The protein serine/threonine kinase CK2 is frequently upregulated in cancer, and it is overexpressed and hyperactivated in primary CLL cells from untreated patients. We have shown that inhibition of CK2 induces apoptosis of CLL cells, whereas it does not significantly impact normal lymphocytes, demonstrating the selectivity of the CK2 inhibitors toward leukemia cells. Notably, although co-culture with OP9 stromal cells and BCR stimulation both promote leukemia cell survival in vitro, they do not prevent apoptosis of CLL cells treated with CK2 inhibitors. PI3K signaling pathway was previously shown to be essential for CLL cell viability, an observation we confirmed in all patient samples analyzed. Further, we observed that CK2 blockade decreases PTEN phosphorylation, leading to PTEN activation, and that apoptosis of CLL cells upon CK2 inhibition is mediated by PKC inactivation. This suggests that activation of PI3K/PKC signaling pathway is involved in the pro-survival effects of CK2 in CLL cells. Sensitivity to CK2 inhibition does not correlate with expression of ZAP-70 or CD38, or with IGVH mutation status. However, it positively correlates with the percentage of CLL cells in the peripheral blood, ß2 microglobulin levels, and Binet clinical stage. CK2 appears to play an important role in the biology of CLL and constitutes a promising target for the development of leukemia-specific therapies.
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Quinasa de la Caseína II/metabolismo , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/patología , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Separación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/enzimología , Ratones , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
PURPOSE: To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease. EXPERIMENTAL DESIGN: Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698). RESULTS: TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment. CONCLUSIONS: The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.
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Adenocarcinoma del Pulmón/mortalidad , Ácido Aspártico Endopeptidasas/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/mortalidad , Inflamación/patología , Linfocitos Infiltrantes de Tumor/inmunología , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Adulto , Anciano , Anciano de 80 o más Años , Ácido Aspártico Endopeptidasas/genética , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Complejo CD3/inmunología , Complejo CD3/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Bases de Datos Genéticas/estadística & datos numéricos , Femenino , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Proteína A Asociada a Surfactante Pulmonar/genética , Tasa de Supervivencia , Microambiente Tumoral/inmunologíaRESUMEN
Mutations and deletions of the tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) are frequently involved in the development of cancer. However, PTEN is also tightly controlled by various non-genomic mechanisms. This review focuses on those mechanisms, namely on the epigenetic silencing of PTEN, post-transcriptional regulation by non-coding RNAs and post-translational modification. We summarise their involvement in cancer in general, and place some emphasis on leukaemia, where PTEN genetic lesions are relatively uncommon and, strikingly, high levels of PTEN expression frequently associate with PTEN functional inactivation. Overall, it is apparent that rather than looking strictly for PTEN genetic lesions and PTEN expression status, the key to evaluating the real impact of PTEN as a 'quasi-insufficient' tumour suppressor must rely on the complete understanding of PTEN's 'functional dose', incorporating the multiple layers of PTEN regulation in the cell that are ultimately compromised in a given cancer.
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Neoplasias/enzimología , Fosfohidrolasa PTEN/metabolismo , Animales , Línea Celular Tumoral , Epigenómica , Genes Supresores de Tumor , Humanos , Fosfohidrolasa PTEN/biosíntesis , Fosfohidrolasa PTEN/genética , Transducción de SeñalRESUMEN
Chronic lymphocytic leukemia (CLL) remains an incurable malignancy, urging for the identification of new molecular targets for therapeutic intervention. CLL cells rely on overexpression and hyperactivation of the ubiquitous serine/threonine protein kinase CK2 for their viability in vitro. CIGB-300 is a cell-permeable selective CK2 inhibitor peptide undergoing clinical trials for several cancers. Here, we show that CIGB-300 promotes activation of the tumor suppressor PTEN and abrogates PI3K-mediated downstream signaling in CLL cells. In accordance, CIGB-300 decreases the viability and proliferation of CLL cell lines, promotes apoptosis of primary leukemia cells and displays antitumor efficacy in a xenograft mouse model of human CLL. Our studies provide pre-clinical support for the testing and possible inclusion of CK2 inhibitors in the clinical arsenal against CLL.
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Quinasa de la Caseína II/antagonistas & inhibidores , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Péptidos Cíclicos/uso terapéutico , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Fosfohidrolasa PTEN , Péptidos Cíclicos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The importance of microenvironmental factors for driving progression in leukemia has been debated. Previous evidence has pointed to interleukin-7 (IL-7), a fundamental cytokine to normal T-cell development and homeostasis, as an important determinant of the viability and proliferation of T-cell acute lymphoblastic leukemia (T-ALL) cells in vitro. In this study, we report that IL-7 is also a critical determinant of T-ALL progression. T-ALL cell lines and primary T-ALL samples initiated leukemia more slowly when engrafted to immunocompromised Rag2(-/-)IL2rg(-/-) mice lacking IL-7. This effect was not related to reduced engraftment or homing of transplanted cells to the bone marrow. Instead, IL-7 deficiency diminished expansion of leukemia cells in the bone marrow and delayed leukemia-associated death of transplanted mice. Moreover, infiltration of different organs by T-ALL cells, which characterizes patients with advanced disease, was more heterogeneous and generally less efficient in IL-7-deficient mice. Leukemia progression was associated with increased Bcl-2 expression and cell viability, reduced p27(Kip1) expression, and decreased cell-cycle progression. Clinical measurements of IL-7 plasma levels and IL-7 receptor (IL-7R) expression in T-ALL patients versus healthy controls confirmed that IL-7 stimulates human leukemia cells. Our results establish that IL-7 contributes to the progression of human T-cell leukemia, and they offer preclinical validation of the concept that targeting IL-7/IL-7R signaling in the tumor microenvironment could elicit therapeutic effects in T-ALL.
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Interleucina-7/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Animales , Procesos de Crecimiento Celular/fisiología , Supervivencia Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación hacia Abajo , Humanos , Interleucina-7/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-7/biosíntesis , Receptores de Interleucina-7/genética , Trasplante Heterólogo , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.
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Metástasis de la Neoplasia/patología , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/patología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Bevacizumab , Hipoxia de la Célula , Línea Celular Tumoral , Movimiento Celular/efectos de la radiación , Colágeno/metabolismo , Relación Dosis-Respuesta en la Radiación , Combinación de Medicamentos , Células Endoteliales/patología , Células Endoteliales/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Laminina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Ratones , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Microvasos/efectos de la radiación , Neoplasias/genética , Neoplasias/radioterapia , Fosfatidilinositol 3-Quinasas/metabolismo , Proteoglicanos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Dosificación Radioterapéutica , Regeneración/efectos de la radiación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra/embriología , Pez Cebra/fisiologíaRESUMEN
BACKGROUND: The unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vgamma9Vdelta2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its gammadelta T-cell activation and anti-tumor properties. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vgamma9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, CONCLUSIONS/SIGNIFICANCE: The development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic Vgamma9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials.