RESUMEN
BACKGROUND: N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) RNA editing are two of the most abundant RNA modification events affecting adenosines in mammals. Both these RNA modifications determine mRNA fate and play a pivotal role in tumor development and progression. RESULTS: Here, we show that METTL3, upregulated in glioblastoma, methylates ADAR1 mRNA and increases its protein level leading to a pro-tumorigenic mechanism connecting METTL3, YTHDF1, and ADAR1. We show that ADAR1 plays a cancer-promoting role independently of its deaminase activity by binding CDK2 mRNA, underlining the importance of ADARs as essential RNA-binding proteins for cell homeostasis as well as cancer progression. Additionally, we show that ADAR1 knockdown is sufficient to strongly inhibit glioblastoma growth in vivo. CONCLUSIONS: Hence, our findings underscore METTL3/ADAR1 axis as a novel crucial pathway in cancer progression that connects m6A and A-to-I editing post-transcriptional events.
Asunto(s)
Adenosina Desaminasa/genética , Carcinogénesis/genética , Glioblastoma/genética , Metiltransferasas/genética , Proteínas de Unión al ARN/genética , Adenosina/metabolismo , Adulto , Animales , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Glioblastoma/patología , Humanos , Masculino , Mutagénesis , Isoformas de Proteínas , ARN Mensajero/metabolismoRESUMEN
Expression of type 5 phosphodiesterase (PDE5), a cGMP-specific hydrolytic enzyme, is frequently altered in human cancer, but its specific role in tumorigenesis remains controversial. Herein, by analyzing a cohort of 69 patients affected by glioblastoma multiforme (GBM) who underwent chemo- and radiotherapy after surgical resection of the tumor, we found that PDE5 was strongly expressed in cancer cells in about 50% of the patients. Retrospective analysis indicated that high PDE5 expression in GBM cells significantly correlated with longer overall survival of patients. Furthermore, silencing of endogenous PDE5 by short hairpin lentiviral transduction (sh-PDE5) in the T98G GBM cell line induced activation of an invasive phenotype. Similarly, pharmacological inhibition of PDE5 activity strongly enhanced cell motility and invasiveness in T98G cells. This invasive phenotype was accompanied by increased secretion of metallo-proteinase 2 (MMP-2) and activation of protein kinase G (PKG). Moreover, PDE5 silencing markedly enhanced DNA damage repair and cell survival following irradiation. The enhanced radio-resistance of sh-PDE5 GBM cells was mediated by an increase of poly(ADP-ribosyl)ation (PARylation) of cellular proteins and could be counteracted by poly(ADP-ribose) polymerase (PARP) inhibitors. Conversely, PDE5 overexpression in PDE5-negative U87G cells significantly reduced MMP-2 secretion, inhibited their invasive potential and interfered with DNA damage repair and cell survival following irradiation. These studies identify PDE5 as a favorable prognostic marker for GBM, which negatively affects cell invasiveness and survival to ionizing radiation. Moreover, our work highlights the therapeutic potential of targeting PKG and/or PARP activity in this currently incurable subset of brain cancers.
Asunto(s)
Neoplasias Encefálicas/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Femenino , Glioblastoma/enzimología , Glioblastoma/terapia , Humanos , Estimación de Kaplan-Meier , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica , Poli(ADP-Ribosa) Polimerasas/metabolismo , Interferencia de ARN , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resultado del TratamientoRESUMEN
Epigenetic alterations, including dysregulated DNA methylation and histone modifications, govern the progression of colorectal cancer (CRC). Cancer cells exploit epigenetic regulation to control cellular pathways, including apoptotic and metastatic signals. Since aberrations in epigenome can be pharmacologically reversed by DNA methyltransferase and histone deacetylase inhibitors, epigenetics in combination with standard agents are currently envisaged as a new therapeutic frontier in cancer, expected to overcome drug resistance associated with current treatments. In this study, we challenged this idea and demonstrated that the combination of azacitidine and romidepsin with IFN-α owns a high therapeutic potential, targeting the most aggressive cellular components of CRC, such as metastatic cells and cancer stem cells (CSCs), via tight control of key survival and death pathways. Moreover, the antitumor efficacy of this novel pharmacological approach is associated with induction of signals of immunogenic cell death. Of note, a previously undisclosed key role of IFN-α in inducing both antiproliferative and pro-apoptotic effects on CSCs of CRC was also found. Overall, these findings open a new frontier on the suitability of IFN-α in association with epigenetics as a novel and promising therapeutic approach for CRC management.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Colorrectales/patología , Interferón-alfa/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Azacitidina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Depsipéptidos/farmacología , Epigénesis Genética/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: Murine neural stem cells (NSCs) were homografted onto the injured spinal cord (SC) to assess their potential to improve motor behavior, to differentiate as neurons, and to establish synapse-like contacts with the descending axonal paths of the host. In addition, we investigated whether transduced NSCs over-expressing vascular endothelial growth factor might exert any angiogenetic effect in the injured SC. METHODS: NSCs derived from mouse embryos were transduced to express either green fluorescent protein or vascular endothelial growth factor. The cells were engrafted in mice where an extended dorsal funiculotomy had been performed at the T8-T9 level. At intervals from 4 to 12 weeks after grafting, motor behavior was assessed using an open field locomotor scale and footprint analysis. At the same time points, the SC was studied by conventional histology, immunohistochemistry, and fluorescence microscopy. The interactions between the grafted NSCs and descending axonal paths were investigated using anterogradely transported fluorescent axonal tracers. RESULTS: By the 12-week time point, mice engrafted with NSCs significantly improved both their locomotor score on open field test and their base of support on footprint analysis. Histological studies showed that green fluorescent protein-positive NSCs survived as long as 12 weeks after grafting, migrated from the grafting site with a tropism toward the lesion, and either remained undifferentiated or differentiated into the astrocytic phenotype without neuronal or oligodendrocytic differentiation. Interestingly, the NSC-derived astrocytes expressed vimentin, suggesting that these cells differentiated as immature astrocytes. The tips of severed descending axonal paths came adjacent to grafted NSCs without forming synapse-like structures. When genetically engineered to over-express vascular endothelial growth factor, the grafted NSCs significantly increased vessel density in the injured area. CONCLUSION: In the traumatically injured mice SC, NSC grafting improves motor recovery. Although differentiation of engrafted NSCs is restricted exclusively toward the astrocytic phenotype, the NSC-derived astrocytes show features that are typical of the early phase after SC injury when the glial scar is still permissive to regenerating axons. The immature phenotype of the NSC-derived astrocytes suggests that these cells might support neurite outgrowth by the host neurons. Thus, modifying the glial scar with NSCs might enhance axonal regeneration in the injured area. The use of genetically engineered NSCs that express trophic factors appears to be an attractive tool in SC transplantation research.