RESUMEN
Epithelial-mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-ß signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-ß signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-ß canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-ß signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.
Asunto(s)
Transición Epitelial-Mesenquimal , Proteína de la Leucemia Promielocítica/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Movimiento Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Immunoblotting , Estimación de Kaplan-Meier , Carioferinas/genética , Carioferinas/metabolismo , Masculino , Invasividad Neoplásica , Fosforilación , Proteína de la Leucemia Promielocítica/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Exportina 1RESUMEN
The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5+ malignant melanoma-initiating cells (ABCB5+ MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK-STAT (janus kinase-signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK-STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro. Finally, using a stem cell proliferation assay and tumour xenotransplantation assay in non-obese diabetic/severe combined immunodeficiency mice, we show that HAGE promotes MMICs-dependent tumour initiation and tumour growth by preventing the anti-proliferative effects of interferon-α (IFNα). Our results suggest that the helicase HAGE has a key role in the resistance of ABCB5+ MMICs to IFNα treatment and that cancer therapies targeting HAGE may have broad implications for the treatment of malignant melanoma.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , ARN Helicasas DEAD-box/metabolismo , Interferón-alfa/farmacología , Melanoma/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Nucleares/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Animales , Línea Celular Tumoral , Proliferación Celular , ARN Helicasas DEAD-box/genética , Resistencia a Antineoplásicos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Janus Quinasa 1/metabolismo , Melanoma/enzimología , Melanoma/genética , Melanoma/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Proteínas Nucleares/genética , Fosforilación , Proteína de la Leucemia Promielocítica , Interferencia de ARN , ARN Mensajero/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Esferoides Celulares , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , TYK2 Quinasa/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Transfección , Carga Tumoral , Proteínas Supresoras de Tumor/genética , UbiquitinaciónRESUMEN
Interferons (IFNs) are a family of secreted proteins with antiviral, antiproliferative and immunomodulatory activities. The different biological actions of IFN are believed to be mediated by the products of specifically induced cellular genes in the target cells. The promyelocytic leukaemia (PML) protein localizes both in the nucleoplasm and in matrix-associated multi-protein complexes known as nuclear bodies (NBs). PML is essential for the proper formation and the integrity of the NBs. Modification of PML by the Small Ubiquitin MOdifier (SUMO) was shown to be required for its localization in NBs. The number and the intensity of PML NBs increase in response to interferon (IFN). Inactivation of the IFN-induced PML gene by its fusion to retinoic acid receptor alpha alters the normal localization of PML from the punctuate nuclear patterns of NBs to micro-dispersed tiny dots and results in uncontrolled growth in Acute Promyelocytic Leukaemia. The NBs-associated proteins, PML, Sp100, Sp140, Sp110, ISG20 and PA28 are induced by IFN suggesting that nuclear bodies could play a role in IFN response. Although the function of PML NBs is still unclear, some results indicate that they may represent preferential targets for viral infections and that PML could play a role in the mechanism of the antiviral action of IFNs. Viruses, which require the cellular machinery for their replication, have evolved different ways to counteract the action of IFN by inhibiting IFN signalling, by blocking the activities of specific antiviral mediators or by altering PML expression and/or localization on nuclear bodies.
Asunto(s)
Estructuras del Núcleo Celular/metabolismo , Infecciones por Virus ADN/metabolismo , Proteínas de Unión al GTP , Interferones/metabolismo , Proteínas de Neoplasias/metabolismo , Orgánulos/metabolismo , Infecciones por Virus ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Estructuras del Núcleo Celular/efectos de los fármacos , Enzimas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Interferones/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Resistencia a Mixovirus , Proteínas Nucleares/metabolismo , Orgánulos/efectos de los fármacos , Proteína de la Leucemia Promielocítica , Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de TumorRESUMEN
The promyelocytic leukaemia (PML) protein localizes in the nucleus both in the nucleoplasm and in matrix-associated multiprotein complexes known as nuclear bodies (NBs). The number and the intensity of PML NBs increase in response to interferon (IFN). Overexpression of PML affects the replication of vesicular stomatitis virus and influenza virus. However, PML has a less powerful antiviral activity against these viruses than the IFN mediator MxA. Here, we show that overexpression of PML, but not that of Mx1 or MxA, leads to a drastic decrease of a complex retrovirus, the human foamy virus (HFV), gene expression. PML represses HFV transcription by complexing the HFV transactivator, Tas, preventing its direct binding to viral DNA. This physical interaction requires the N-terminal region of Tas and the RING finger of PML, but does not necessitate PML localization in NBs. Finally, we show that IFN treatment inhibits HFV replication in wild-type but not in PML-/- cells. These findings point to a role for PML in transcriptional repression and suggest that PML could play a key role in mediating an IFN-induced antiviral state against a complex retrovirus.