RESUMEN
BACKGROUND & AIMS: The leukocyte composition of tumors is heterogeneous, as is the involvement of each leukocyte subset in promoting or restraining tumorigenesis. This heterogeneity reflects the tissue of origin, tumor stage, and the functional state of leukocyte activation, but its biological roots remain poorly understood. Since tumorigenesis is driven by various genetic events, we assessed the role of driver genes in shaping the profiles and the roles of leukocytes in tumorigenesis. METHODS: Mouse liver tumors were induced by hepatic overexpression of either MYC or the combination of myristoylated AKT and NRAS(V12) oncogenes via hydrodynamic transfection. A comparative, flow cytometry- and histology-based immunophenotyping of liver-infiltrating leukocytes was performed at various stages of liver tumorigenesis. The roles of the most abundant leukocyte subsets in tumorigenesis were addressed by immunodepletion. The contribution of liver injury was assessed by comparing the injury-inducing hydrodynamic transfection model to a model in which MYC is an inducible transgene. RESULTS: Myristoylated AKT and NRAS(V12) promoted a marked recruitment of CD11b(+)Ly6G(hi)Ly6C(int) neutrophils and CD11b(+)Ly6G(-)Ly6C(hi) monocytes to the liver, but their immunodepletion did not alter tumorigenesis. In contrast, despite minimal invasion by monocytes/neutrophils during MYC-driven tumorigenesis, immunodepletion of these cells reduced MYC tumor burden and extended survival. MYC-driven tumor initiation was augmented specifically by Ly6C+ monocytes and their ability to promote liver injury. CONCLUSIONS: Our results demonstrate that leukocyte profiles do not necessarily predict their involvement in tumorigenesis, the functional role of leukocytes can be shaped by oncogenes, and that monocyte-dependent tissue injury selectively cooperates with MYC during tumorigenesis.
Asunto(s)
Genes myc/fisiología , Neoplasias Hepáticas Experimentales/etiología , Monocitos/fisiología , Animales , Antígenos Ly/análisis , Femenino , Genes ras , Ratones , Infiltración Neutrófila , Proteínas Proto-Oncogénicas c-akt/genética , Receptores de Quimiocina/análisisRESUMEN
Tumor cells have a dysregulated cell cycle that may render their proliferation especially sensitive to the inhibition of cyclin-dependent kinases (CDKs), important regulators of cell cycle progression. We examined the effects of CDK1 inhibition in the context of different oncogenic signals. Cells transformed with MYC, but not cells transformed by a panel of other activated oncogenes, rapidly underwent apoptosis when treated with small-molecule CDK1 inhibitors. The inhibitor of apoptosis protein BIRC5 (survivin), a known CDK1 target, is required for the survival of cells overexpressing MYC. Inhibition of CDK1 rapidly downregulates survivin expression and induces MYC-dependent apoptosis. CDK1 inhibitor treatment of MYC-dependent mouse lymphoma and hepatoblastoma tumors decreased tumor growth and prolonged their survival. As there are no effective small-molecule inhibitors that selectively target the MYC pathway, we propose that CDK1 inhibition might therefore be useful in the treatment of human malignancies that overexpress MYC.
Asunto(s)
Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Antineoplásicos/farmacología , Proteína Quinasa CDC2/metabolismo , Muerte Celular , Línea Celular Tumoral , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , Linfoma/tratamiento farmacológico , Linfoma/genética , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Ratas , Survivin , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
UNLABELLED: At least some cancer stem cells (CSCs) display intrinsic drug resistance that may thwart eradication of a malignancy by chemotherapy. We explored the genesis of such resistance by studying mouse models of liver cancer driven by either MYC or the combination of oncogenic forms of activation of v-akt murine thymoma viral oncogene homolog (AKT) and NRAS. A common manifestation of chemoresistance in CSCs is efflux of the DNA-binding dye Hoechst 33342. We found that only the MYC-driven tumors contained a subset of cells that efflux Hoechst 33342. This "side population" (SP) was enriched for CSCs when compared to non-SP tumor cells and exhibited markers of hepatic progenitor cells. The SP cells could differentiate into non-SP tumor cells, with coordinate loss of chemoresistance, progenitor markers, and the enrichment for CSCs. In contrast, non-SP cells did not give rise to SP cells. Exclusion of Hoechst 33342 is mediated by ATP binding cassette drug transporter proteins that also contribute to chemoresistance in cancer. We found that the multidrug resistance gene 1 (MDR1) transporter was responsible for the efflux of Hoechst from SP cells in our MYC-driven model. Accordingly, SP cells and their tumor-initiating subset were more resistant than non-SP cells to chemotherapeutics that are effluxed by MDR1. CONCLUSION: The oncogenotype of a tumor can promote a specific mechanism of chemoresistance that can contribute to the survival of hepatic CSCs. Under circumstances that promote differentiation of CSCs into more mature tumor cells, the chemoresistance can be quickly lost. Elucidation of the mechanisms that govern chemoresistance in these mouse models may illuminate the genesis of chemoresistance in human liver cancer.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Diferenciación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Células Madre Neoplásicas/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Western Blotting , Diferenciación Celular/genética , Línea Celular Tumoral , Supervivencia Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Citometría de Flujo , Humanos , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacología , Inmunohistoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Células Madre Neoplásicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Trasplante HomólogoRESUMEN
The Myc protein and proteins that participate in mitosis represent attractive targets for cancer therapy. However, their potential is presently compromised by the threat of side effects and by a lack of pharmacological inhibitors of Myc. Here we report that a circumscribed exposure to the aurora kinase inhibitor, VX-680, selectively kills cells that overexpress Myc. This synthetic lethal interaction is attributable to inhibition of aurora-B kinase, with consequent disabling of the chromosomal passenger protein complex (CPPC) and ensuing DNA replication in the absence of cell division; executed by sequential apoptosis and autophagy; not reliant on the tumor suppressor protein p53; and effective against mouse models for B-cell and T-cell lymphomas initiated by transgenes of MYC. Our findings cast light on how inhibitors of aurora-B kinase may kill tumor cells, implicate Myc in the induction of a lethal form of autophagy, indicate that expression of Myc be a useful biomarker for sensitivity of tumor cells to inhibition of the CPPC, dramatize the virtue of bimodal killing by a single therapeutic agent, and suggest a therapeutic strategy for killing tumor cells that overexpress Myc while sparing normal cells.
Asunto(s)
Linfoma/tratamiento farmacológico , Piperazinas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Apoptosis/efectos de los fármacos , Aurora Quinasa B , Aurora Quinasas , Autofagia/efectos de los fármacos , Citocinesis , Replicación del ADN , Modelos Animales de Enfermedad , Humanos , Linfoma/genética , Linfoma/metabolismo , Ratones , Microscopía Electrónica , Trasplante de Neoplasias , Piperazinas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , RatasRESUMEN
We have generated a novel transgenic mouse to direct inducible and reversible transgene expression in the melanocytic compartment. The Dopachrome tautomerase (Dct) control sequences we used are active early in the development of melanocytes and so this system was designed to enable the manipulation of transgene expression during development in utero and in the melanocyte stem cells as well as mature melanocytes. We observed inducible lacZ and GFP reporter transgene activity specifically in melanocytes and melanocyte stem cells in mouse skin. This mouse model will be a useful tool for the pigment cell community to investigate the contribution of candidate genes to normal melanocyte and/or melanoma development in vivo. Deregulated expression of the proto-oncogene MYC has been observed in melanoma, however whether MYC is involved in tumorigenesis in pigment cells has yet to be directly investigated in vivo. We have used our system to over-express MYC in the melanocytic compartment and show for the first time that increased MYC expression can indeed promote melanocytic tumor formation.
Asunto(s)
Regulación de la Expresión Génica , Oxidorreductasas Intramoleculares/genética , Melanocitos/metabolismo , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Línea Celular Tumoral , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Operón Lac , Masculino , Melanocitos/citología , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Células 3T3 NIH , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , Células Madre/metabolismo , Tetraciclina/metabolismo , Tetraciclina/farmacología , TransgenesRESUMEN
A variety of circumstantial evidence from humans has implicated the B cell antigen receptor (BCR) in the genesis of B cell lymphomas. We generated mouse models designed to test this possibility directly, and we found that both the constitutive and antigen-stimulated state of a clonal BCR affected the rate and outcome of lymphomagenesis initiated by the proto-oncogene MYC. The tumors that arose in the presence of constitutive BCR differed from those initiated by MYC alone and resembled chronic B cell lymphocytic leukemia/lymphoma (B-CLL), whereas those that arose in response to antigen stimulation resembled large B-cell lymphomas, particularly Burkitt lymphoma (BL). We linked the genesis of the BL-like tumors to antigen stimulus in three ways. First, in reconstruction experiments, stimulation of B cells by an autoantigen in the presence of overexpressed MYC gave rise to BL-like tumors that were, in turn, dependent on both MYC and the antigen for survival and proliferation. Second, genetic disruption of the pathway that mediates signaling from the BCR promptly killed cells of the BL-like tumors as well as the tumors resembling B-CLL. And third, growth of the murine BL could be inhibited by any of three distinctive immunosuppressants, in accord with the dependence of the tumors on antigen-induced signaling. Together, our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, and sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen. The mouse models described here should be useful in exploring further the pathogenesis of lymphomas, and in preclinical testing of new therapeutics.
Asunto(s)
Genes myc , Linfoma de Células B/etiología , Receptores de Antígenos de Linfocitos B/fisiología , Animales , División Celular/efectos de los fármacos , Linaje de la Célula , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Proto-Oncogenes Mas , TransgenesRESUMEN
Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/prevención & control , Proteínas de Neoplasias/inmunología , Proteínas de Fusión Oncogénica/inmunología , Vacunas de ADN/inmunología , Animales , Antineoplásicos/farmacología , Vacunas contra el Cáncer/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/efectos de los fármacos , Proteínas de Fusión Oncogénica/genética , Tretinoina/farmacología , Vacunas de ADN/farmacologíaRESUMEN
Recent evidence demonstrates that senescence acts as a barrier to tumorigenesis in response to oncogene activation. Using a mouse model of breast cancer, we tested the importance of the senescence response in solid cancer and identified genetic pathways regulating this response. Mammary expression of activated Ras led to the formation of senescent cellular foci in a majority of mice. Deletion of the p19(ARF), p53, or p21(WAF1) tumor suppressors but not p16(INK4a) prevented senescence and permitted tumorigenesis. Id1 has been implicated in the control of senescence in vitro, and elevated expression of Id1 is found in a number of solid cancers, so we tested whether overexpression of Id1 regulates senescence in vivo. Although overexpression of Id1 in the mammary epithelium was not sufficient for tumorigenesis, mice with expression of both Id1 and activated Ras developed metastatic cancer. These tumors expressed high levels of p19(Arf), p53, and p21(Waf1), demonstrating that Id1 acts to make cells refractory to p21(Waf1)-dependent cell cycle arrest. Inactivation of the conditional Id1 allele in established tumors led to widespread senescence within 10 days, tumor growth arrest, and tumor regression in 40% of mice. Mice in which Id1 expression was inactivated also exhibited greatly reduced pulmonary metastatic load. These data demonstrate that established tumors remain sensitive to senescence and that Id1 may be a valuable target for therapy.
Asunto(s)
Senescencia Celular , Proteína 1 Inhibidora de la Diferenciación/fisiología , Neoplasias Mamarias Experimentales/etiología , Neoplasias Mamarias Experimentales/patología , Proteínas ras/fisiología , Animales , Trasplante de Células , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Células Epiteliales , Femenino , Humanos , Glándulas Mamarias Animales , Ratones , Metástasis de la Neoplasia , Proteína p53 Supresora de TumorRESUMEN
Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.
Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Diferenciación Celular , Genes myc/genética , Animales , Apoptosis , Conductos Biliares/citología , Conductos Biliares/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Mediciones Luminiscentes , Ratones , Ratones SCID , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
BACKGROUND: We have used a mouse model based on overexpression of c-Myc in B cells genetically engineered to be self-reactive to test the hypothesis that farnesyl transferase inhibitors (FTIs) can effectively treat mature B cell lymphomas. FTIs are undergoing clinical trials to treat both lymphoid and non-lymphoid malignancies and we wished to obtain evidence to support the inclusion of B cell lymphomas in future trials. RESULTS: We report that two FTIs, L-744,832 and SCH66336, blocked the growth of mature B cell lymphoma cells in vitro and in vivo. The FTI treatment affected the proliferation and survival of the transformed B cells to a greater extent than naïve B cells stimulated with antigen. In syngeneic mice transplanted with the transgenic lymphoma cells, L-744,832 treatment prevented the growth of the tumor cells and the morbidity associated with the resulting lymphoma progression. Tumors that arose from transplantation of the lymphoma cells regressed with as little as three days of treatment with L-744,832 or SCH66336. Treatment of these established lymphomas with L-744,832 for seven days led to long-term remission of the disease in approximately 25% of animals. CONCLUSION: FTI treatment can block the proliferation and survival of self-reactive transformed B cells that overexpress Myc. In mice transplanted with mature B cell lymphomas, we found that FTI treatment led to regression of disease. FTIs warrant further consideration as therapeutic agents for mature B cell lymphomas and other lymphoid tumors.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Linfoma de Células B/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Farnesiltransferasa/metabolismo , Citometría de Flujo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/patología , Linfoma de Células B/genética , Linfoma de Células B/patología , Metionina/análogos & derivados , Metionina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Piperidinas/farmacología , Piridinas/farmacología , Inducción de RemisiónRESUMEN
Pak2 is a serine/threonine kinase that participates in the cellular response to stress. Among the potential substrates for Pak2 is the protein Myc, encoded by the proto-oncogene MYC. Here we demonstrate that Pak2 phosphorylates Myc at three sites (T358, S373, and T400) and affects Myc functions both in vitro and in vivo. Phosphorylation at all three residues reduces the binding of Myc to DNA, either by blocking the requisite dimerization with Max (through phosphorylation at S373 and T400) or by interfering directly with binding to DNA (through phosphorylation at T358). Phosphorylation by Pak2 inhibits the ability of Myc to activate transcription, to sustain cellular proliferation, to transform NIH 3T3 cells in culture, and to elicit apoptosis on serum withdrawal. These results indicate that Pak2 is a negative regulator of Myc, suggest that inhibition of Myc plays a role in the cellular response to stress, and raise the possibility that Pak2 may be the product of a tumor suppressor gene.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción , Animales , Apoptosis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Ciclo Celular , Línea Celular , Transformación Celular Neoplásica , ADN/antagonistas & inhibidores , ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/química , Activación Transcripcional , Proteínas Supresoras de Tumor/metabolismo , Quinasas p21 ActivadasRESUMEN
Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Células Madre Neoplásicas/fisiología , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Carcinoma Ductal Pancreático/patología , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Humanos , Ratones , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Pancreáticas/patología , GemcitabinaRESUMEN
Genomic analysis of human hepatocellular carcinoma (HCC) is potentially confounded by the differentiation state of the hepatic cell-of-origin. Here we integrated genomic analysis of mouse HCC (with defined cell-of-origin) along with normal development. We found a major shift in expression of Wnt and RXR-α pathway genes (up and down, respectively) coincident with the transition from hepatoblasts to hepatocytes. A combined Wnt and RXR-α gene signature categorized HCCs into two subtypes (high Wnt, low RXR-α and low Wnt, high RXR-α), which matched cell-of-origin in mouse models and the differentiation state of human HCC. Suppression of RXR-α levels in hepatocytes increased Wnt signaling and enhanced tumorigenicity, whereas ligand activation of RXR-α achieved the opposite. These results corroborate that there are two main HCC subtypes that correspond to the degree of hepatocyte differentation and that RXR-α, in part via Wnt signaling, plays a key functional role in the hepatocyte-like subtype and potentially could serve as a selective therapeutic target.
Asunto(s)
Carcinogénesis , Carcinoma Hepatocelular/patología , Hepatocitos/patología , Neoplasias Hepáticas/patología , Receptor alfa X Retinoide/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Femenino , Genómica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Unión Proteica , Receptor alfa X Retinoide/genética , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have varying silencing capacities, several different siRNAS typically must be screened to obtain a region that will effectively silence the gene of interest. However, RNA interference with synthetic siRNA is inefficient and cost-intensive, especially for large, functional genomic studies. Here, we describe the use of E. coli endoribonuclease III to cleave double-stranded RNA (dsRNA) into esiRNA (endoribonuclease-prepared siRNA) that can target multiple sites within an mRNA. EsiRNA mediates effective RNA interference with no apparent nonspecific effects in cultured mammalian cells. Since the whole gene can be used at once, screening for an active siRNA for an individual gene is eliminated. Because of its simplicity and potency, this approach is useful for large-scale analysis of mammalian gene function.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/biosíntesis , Ribonucleasa III/metabolismo , Secuencia de Bases , Cartilla de ADN , Escherichia coli/enzimología , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , ARN Bicatenario/síntesis química , ARN Bicatenario/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa III/genéticaRESUMEN
Sox9 has gained increasing importance both functionally and as a prognostic factor in cancer. We demonstrate a functional role for Sox9 in inducing a mesenchymal phenotype in lung ADC. We show that Sox9 mRNA and protein are overexpressed in lung ADC, particularly those with KRAS mutations. Sox9 expression correlated with the Notch target gene Hes1, and numerous other Notch pathway components. We observed that Sox9 is a potent inducer of lung cancer cell motility and invasion, and a negative regulator of E-cadherin, a key protein that is lost during epithelial-mesenchymal transition (EMT). Moreover, we show that Notch1 signaling directly regulates Sox9 expression through a SOX9 promoter binding site, independently of the TGF-ß pathway, and that Sox9 participates in Notch-1 induced cell motility, cell invasion, and loss of E-cadherin expression. Together, the results identify a new functional role for a Notch1-Sox9 signaling axis in lung ADC that may explain the correlation of Sox9 with tumor progression, higher tumor grade, and poor lung cancer survival. In addition to Notch and TGF-ß, Sox9 also acts downstream of NF-κB, BMP, EGFR, and Wnt/ß-catenin signaling. Thus, Sox9 could potentially act as a hub to mediate cross-talk among key oncogenic pathways in lung ADC. Targeting Sox9 expression or transcriptional activity could potentially reduce resistance to targeted therapy for lung ADC caused by pathway redundancy.
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Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Receptor Notch1/metabolismo , Factor de Transcripción SOX9/metabolismo , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Animales , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal , Humanos , Neoplasias Pulmonares/genética , Ratones , Datos de Secuencia Molecular , Receptor Notch1/biosíntesis , Receptor Notch1/genética , Factor de Transcripción SOX9/biosíntesis , Factor de Transcripción SOX9/genética , Transducción de SeñalAsunto(s)
Virus del Sarcoma de Rous/genética , Sarcoma Aviar/historia , Integración Viral/genética , Animales , Transformación Celular Neoplásica , ADN Viral/biosíntesis , ADN Viral/aislamiento & purificación , Patos , Historia del Siglo XX , Historia del Siglo XXI , Infecciones por Retroviridae/genética , Sarcoma Aviar/genética , Infecciones Tumorales por Virus/genética , Replicación Viral/genéticaRESUMEN
Self-assembled nanodiamond-lipid hybrid particles (NDLPs) harness the potent interaction between the nanodiamond (ND)-surface and small molecules, while providing a mechanism for cell-targeted imaging and therapy of triple negative breast cancers. Epidermal growth factor receptor-targeted NDLPs are highly biocompatible particles that provide cell-specific imaging, promote tumor retention of ND-complexes, prevent epirubicin toxicities and mediate regression of triple negative breast cancers.
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Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Tolerancia a Medicamentos , Lípidos/química , Nanodiamantes/química , Animales , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptores ErbB/metabolismo , Femenino , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nucleostemin (NS) is a nucleolar GTP-binding protein that was first identified in neural stem cells, the functions of which remain poorly understood. Here, we report that NS is required for mouse embryogenesis to reach blastulation, maintenance of embryonic stem cell (ESC) self-renewal, and mammary epithelial cell (MEC) reprogramming to induced pluripotent stem (iPS) cells. Ectopic NS also cooperates with OCT4 and SOX2 to reprogram MECs and mouse embryonic fibroblasts to iPS cells. NS promotes ESC self-renewal by sustaining rapid transit through the G1 phase of the cell cycle. Depletion of NS in ESCs retards transit through G1 and induces gene expression changes and morphological differentiation through a mechanism that involves the MEK/ERK protein kinases and that is active only during a protracted G1. Suppression of cell cycle inhibitors mitigates these effects. Our results implicate NS in the maintenance of ESC self-renewal, demonstrate the importance of rapid transit through G1 for this process, and expand the known classes of reprogramming factors.
Asunto(s)
Proteínas Portadoras/genética , Reprogramación Celular/fisiología , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Proteínas Nucleares/genética , Animales , Proteínas Portadoras/metabolismo , Ciclo Celular , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Fase G1/fisiología , Proteínas de Unión al GTP , Regulación del Desarrollo de la Expresión Génica , Genotipo , Células Madre Pluripotentes Inducidas/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARNRESUMEN
Overexpression of MYC transforms cells in culture, elicits malignant tumours in experimental animals and is found in many human tumours. We now report the paradoxical finding that this powerful oncogene can also act as a suppressor of cell motility, invasiveness and metastasis. Overexpression of MYC stimulated proliferation of breast cancer cells both in culture and in vivo as expected, but inhibited motility and invasiveness in culture, and lung and liver metastases in xenografted tumours. We show further that MYC represses transcription of both subunits of αvß3 integrin, and that exogenous expression of ß3 integrin in human breast cancer cells that do not express this integrin rescues invasiveness and migration when MYC is downregulated. These data uncover an unexpected function of MYC, provide an explanation for the hitherto puzzling literature on the relationship between MYC and metastasis, and reveal a variable that could influence the development of therapies that target MYC.
Asunto(s)
Integrina alfa5/metabolismo , Integrina beta3/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Silenciador del Gen , Humanos , Integrina alfa5/genética , Integrina beta3/genética , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/patología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
The altered metabolism of tumors has been considered a target for anticancer therapy. However, the relationship between distinct tumor-initiating lesions and anomalies of tumor metabolism in vivo has not been addressed. We report that MYC-induced mouse liver tumors significantly increase both glucose and glutamine catabolism, whereas MET-induced liver tumors use glucose to produce glutamine. Increased glutamine catabolism in MYC-induced liver tumors is associated with decreased levels of glutamine synthetase (Glul) and the switch from Gls2 to Gls1 glutaminase. In contrast to liver tumors, MYC-induced lung tumors display increased expression of both Glul and Gls1 and accumulate glutamine. We also show that inhibition of Gls1 kills cells that overexpress MYC and catabolize glutamine. Our results suggest that the metabolic profiles of tumors are likely to depend on both the genotype and tissue of origin and have implications regarding the design of therapies targeting tumor metabolism.