RESUMEN
Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization.
Asunto(s)
Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Histona Acetiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Reparación del ADN por Recombinación , ATPasas Asociadas con Actividades Celulares Diversas , Acetilación , Animales , Arginina , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/genética , ADN Helicasas/genética , Inestabilidad Genómica , Células HEK293 , Células HeLa , Histona Acetiltransferasas/genética , Histonas/metabolismo , Humanos , Lisina Acetiltransferasa 5 , Metilación , Ratones , Ratones Transgénicos , Proteína-Arginina N-Metiltransferasas/genética , Interferencia de ARN , Factores de Tiempo , Transfección , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Prolactin (PRL) is a polypeptide hormone/cytokine mainly synthesized by the lactotrophic cells of the adenohypophysis. In addition to the best-known role in mammary gland development and the functional differentiation of its epithelium, PRL is involved in regulation of multiple physiological processes in higher organisms contributing to their homeostasis. PRL has been also associated with pathology, including breast cancer. Therefore, it is relevant to determine the molecular mechanisms by which PRL controls cellular functions. Here, we analyze the role of Src family kinases (SFKs) in the intracellular signaling pathways controlled by PRL in several model systems. The data show that SFKs are essential components in transmitting signals upon PRL receptor stimulation, as they control activation of Jak2/Stat5 and other routes that regulate PRL cellular responses.
Asunto(s)
Prolactina/metabolismo , Familia-src Quinasas/fisiología , Animales , Proliferación Celular/efectos de los fármacos , Activación Enzimática , Humanos , Janus Quinasa 2/metabolismo , Quinasas Janus/fisiología , Prolactina/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/fisiología , Receptores de Prolactina/fisiología , Transducción de SeñalRESUMEN
Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway.
Asunto(s)
Arginina/metabolismo , Proteína Homóloga de MRE11/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Proteínas Represoras/metabolismo , Tioléster Hidrolasas/metabolismo , Ubiquitinación , Arginina/química , Línea Celular , Daño del ADN , Inestabilidad Genómica , Humanos , MetilaciónRESUMEN
The role of Src family kinases (SFKs) in human tumors has been always associated with tyrosine kinase activity and much less attention has been given to the SH2 and SH3 adapter domains. Here, we studied the role of the c-Src-SH2 domain in triple-negative breast cancer (TNBC). To this end, SUM159PT and MDA-MB-231 human cell lines were employed as model systems. These cells conditionally expressed, under tetracycline control (Tet-On system), a c-Src variant with point-inactivating mutation of the SH2 adapter domain (R175L). The expression of this mutant reduced the self-renewal capability of the enriched population of breast cancer stem cells (BCSCs), demonstrating the importance of the SH2 adapter domain of c-Src in the mammary gland carcinogenesis. In addition, the analysis of anchorage-independent growth, proliferation, migration, and invasiveness, all processes associated with tumorigenesis, showed that the SH2 domain of c-Src plays a very relevant role in their regulation. Furthermore, the transfection of two different aptamers directed to SH2-c-Src in both SUM159PT and MDA-MB-231 cells induced inhibition of their proliferation, migration, and invasiveness, strengthening the hypothesis that this domain is highly involved in TNBC tumorigenesis. Therefore, the SH2 domain of c-Src could be a promising therapeutic target and combined treatments with inhibitors of c-Src kinase enzymatic activity may represent a new therapeutic strategy for patients with TNBC, whose prognosis is currently very negative.
RESUMEN
Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment. SIGNIFICANCE: Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses.This article is highlighted in the In This Issue feature, p. 521.
Asunto(s)
Comunicación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/etiología , Glioblastoma/patología , Inmunidad Innata , Biomarcadores de Tumor , Comunicación Celular/genética , Susceptibilidad a Enfermedades , Glioblastoma/metabolismo , Humanos , Inmunidad Innata/genética , Clasificación del Tumor , Estadificación de Neoplasias , Transcriptoma , Microambiente TumoralRESUMEN
Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.
Asunto(s)
Autorrenovación de las Células , Glucosa/metabolismo , Células Madre Neoplásicas/metabolismo , Familia-src Quinasas/metabolismo , Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Células MCF-7 , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Células Madre Neoplásicas/fisiología , Proteoma/genética , Proteoma/metabolismo , Familia-src Quinasas/genéticaRESUMEN
Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens. We find that multiple DUBs may be individually required for hematopoiesis and identify ubiquitin-specific protease 15 (USP15) as essential for HSC maintenance in vitro and in transplantations and Usp15 knockout (KO) mice in vivo. USP15 is highly expressed in human hematopoietic tissues and leukemias. USP15 depletion in murine progenitors and leukemia cells impairs in vitro expansion and increases genotoxic stress. In leukemia cells, USP15 interacts with and stabilizes FUS (fused in sarcoma), a known DNA repair factor, directly linking USP15 to the DNA damage response (DDR). Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSCs and leukemia cells.
Asunto(s)
Enzimas Desubicuitinizantes/fisiología , Células Madre Hematopoyéticas/fisiología , Leucemia/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteasas Ubiquitina-Específicas/fisiología , Animales , Línea Celular , Proliferación Celular , Daño del ADN , Reparación del ADN , Hematopoyesis , Células Madre Hematopoyéticas/enzimología , Humanos , Células K562 , Leucemia/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , UbiquitinaciónRESUMEN
miR205 has a dual activity, as tumor suppressor and as oncogene. Here we analyzed the impact of miR205 ectopic expression in the initial tumorigenic processes of SUM159PT, a triple negative breast cancer cell line with low endogenous levels of miR205. In SUM159PT, miR205 inhibited expression of its targets VEGFA, ErbB3, Zeb1, Fyn and Lyn A/B; it reduced cell proliferation, and Myc/cyclin D1 levels, while increased p27kip1 expression. miR205 abolished anchorage-independent growth, inhibited migration and invasion, Src-kinases/Stat3 axis activation, and levels of secreted MMP9. miR205 also reduced expression of CD44 and TAZ, E2A.E12, Twist, Snail1 and CK5, associated with epithelial-mesenchymal transition (EMT). Importantly, we show that miR205 inhibited SUM159PT cancer-stem cell renewal, expression in mammospheres of CD44 and ALDH1 stem-cell markers, TAZ, and E2A.E12. All these effects of miR205 were reverted by Anti-miR205 co-expression, demonstrating its specificity. Thus, all these results strongly suggest that ectopic expression of miR205 in SUM159PT affected several parameters associated with initial steps of tumorigenesis.
Asunto(s)
Neoplasias de la Mama/patología , MicroARNs/fisiología , Células Madre Neoplásicas/patología , HumanosRESUMEN
Breast cancer progression, treatment resistance, and relapse are thought to originate from a small population of tumor cells, breast cancer stem cells (BCSCs). Identification of factors critical for BCSC function is therefore vital for the development of therapies. Here, we identify the arginine methyltransferase PRMT5 as a key in vitro and in vivo regulator of BCSC proliferation and self-renewal and establish FOXP1, a winged helix/forkhead transcription factor, as a critical effector of PRMT5-induced BCSC function. Mechanistically, PRMT5 recruitment to the FOXP1 promoter facilitates H3R2me2s, SET1 recruitment, H3K4me3, and gene expression. Our findings are clinically significant, as PRMT5 depletion within established tumor xenografts or treatment of patient-derived BCSCs with a pre-clinical PRMT5 inhibitor substantially reduces BCSC numbers. Together, our findings highlight the importance of PRMT5 in BCSC maintenance and suggest that small-molecule inhibitors of PRMT5 or downstream targets could be an effective strategy eliminating this cancer-causing population.
Asunto(s)
Neoplasias de la Mama/genética , Factores de Transcripción Forkhead/genética , Código de Histonas , Células Madre Neoplásicas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/genética , Animales , Neoplasias de la Mama/metabolismo , Proliferación Celular , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/fisiología , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.
Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Familia-src Quinasas/metabolismo , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Proteína 61 Rica en Cisteína/análisis , Proteína 61 Rica en Cisteína/genética , Femenino , Humanos , Proteómica/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/sangre , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Familia-src Quinasas/genéticaRESUMEN
SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.