RESUMO
The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.
Assuntos
Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Estatmina/metabolismoRESUMO
In epithelial ovarian cancer (EOC), response to platinum (PT)-based chemotherapy dictates subsequent treatments and predicts patients' prognosis. Alternative splicing is often deregulated in human cancers and can be altered by chemotherapy. Whether and how changes in alternative splicing regulation could impact on the response of EOC to PT-based chemotherapy is still not clarified. We identified the splicing factor proline and glutamine rich (SFPQ) as a critical mediator of response to PT in an unbiased functional genomic screening in EOC cells and, using a large cohort of primary and recurrent EOC samples, we observed that it is frequently overexpressed in recurrent PT-treated samples and that its overexpression correlates with PT resistance. At mechanistic level, we show that, under PT treatment, SFPQ, in complex with p54nrb, binds and regulates the activity of the splicing factor SRSF2. SFPQ/p54nrb complex decreases SRSF2 binding to caspase-9 RNA, favoring the expression of its alternative spliced antiapoptotic form. As a consequence, SFPQ/p54nrb protects cells from PT-induced death, eventually contributing to chemoresistance. Overall, our work unveils a previously unreported SFPQ/p54nrb/SRSF2 pathway that in EOC cells plays a central role in regulating alternative splicing and PT-induced apoptosis and that could result in the design of new possible ways of intervention to overcome PT resistance.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Cisplatino/farmacologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Fator de Processamento Associado a PTB/fisiologia , Proteínas de Ligação a RNA/fisiologia , Fatores de Processamento de Serina-Arginina/fisiologia , Animais , Antineoplásicos Alquilantes/uso terapêutico , Apoptose , Caspase 8/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Neoplasias Ovarianas/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Recidiva , Spliceossomos/metabolismoRESUMO
Platinum-based chemotherapy is the therapy of choice for epithelial ovarian cancer (EOC). Acquired resistance to platinum (PT) is a frequent event that leads to disease progression and predicts poor prognosis. To understand possible mechanisms underlying acquired PT-resistance, we have recently generated and characterized three PT-resistant isogenic EOC cell lines. Here, we more deeply characterize several PT-resistant clones derived from MDAH-2774 cells. We show that, in these cells, the increased PT resistance was accompanied by the presence of a subpopulation of multinucleated giant cells. This phenotype was likely due to an altered progression through the M phase of the cell cycle and accompanied by the deregulated expression of genes involved in M phase progression known to be target of mutant TP53. Interestingly, we found that PT-resistant MDAH cells acquired in the TP53 gene a novel secondary mutation (i.e., S185G) that accompanied the R273H typical of MDAH cells. The double p53S185G/R273H mutant increases the resistance to PT in a TP53 null EOC cellular model. Overall, we show how the selective pressure of PT is able to induce additional mutation in an already mutant TP53 gene in EOC and how this event could contribute to the acquisition of novel cellular phenotypes.
Assuntos
Carcinoma Epitelial do Ovário/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Supressora de Tumor p53/genética , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Mitose/genética , Mutação , Neoplasias Ovarianas/genética , Ovário/patologia , Platina/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Standard of care for Epithelial Ovarian Cancer (EOC) patients relies on platinum-based therapy. However, acquired resistance to platinum occurs frequently and predicts poor prognosis. To understand the mechanisms underlying acquired platinum-resistance, we have generated and characterized three platinum-resistant isogenic EOC cell lines. Resistant cells showed 3-to 5- folds increase in platinum IC50. Cross-resistance to other chemotherapeutic agents commonly used in the treatment of EOC patients was variable and dependent on the cell line utilized. Gene expression profiling (GEP) of coding and non-coding RNAs failed to identify a common signature that could collectively explain the mechanism of resistance. However, we observed that all resistant cell lines displayed a decreased level of DNA platination and a faster repair of damaged DNA. Furthermore, all platinum resistant cell lines displayed a change in their morphology and a higher ability to grown on mesothelium. Overall, we have established and characterized three new models of platinum-resistant EOC cell lines that could be exploited to further dissect the molecular mechanisms underlying acquired resistance to platinum. Our work also suggests that GEP studies alone, at least when performed under basal culture condition, do not represent the optimal way to identify molecular alterations linked to DNA repair pathway defects.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Fenótipo , Platina/farmacologia , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Epithelial ovarian cancer (EOC) is an infrequent but highly lethal disease, almost invariably treated with platinum-based therapies. Improving the response to platinum represents a great challenge, since it could significantly impact on patient survival. Here, we report that silencing or pharmacological inhibition of CDK6 increases EOC cell sensitivity to platinum. We observed that, upon platinum treatment, CDK6 phosphorylated and stabilized the transcription factor FOXO3, eventually inducing ATR transcription. Blockage of this pathway resulted in EOC cell death, due to altered DNA damage response accompanied by increased apoptosis. These observations were recapitulated in EOC cell lines in vitro, in xenografts in vivo, and in primary tumor cells derived from platinum-treated patients. Consistently, high CDK6 and FOXO3 expression levels in primary EOC predict poor patient survival. Our data suggest that CDK6 represents an actionable target that can be exploited to improve platinum efficacy in EOC patients. As CDK4/6 inhibitors are successfully used in cancer patients, our findings can be immediately transferred to the clinic to improve the outcome of EOC patients.
Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Proteína Forkhead Box O3/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinoma Epitelial do Ovário , Morte Celular , Linhagem Celular Tumoral , Quinase 6 Dependente de Ciclina/genética , Dano ao DNA , Feminino , Proteína Forkhead Box O3/genética , Humanos , Camundongos , Camundongos Nus , Neoplasias Epiteliais e Glandulares/enzimologia , Neoplasias Ovarianas/enzimologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Platina/uso terapêutico , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tumor suppressor protein p27Kip1 plays a pivotal role in the control of cell growth and metastasis formation.Several studies pointed to different roles for p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin.Here, using in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4BV12 transformed cells, p27kip1 acted mainly in a CDK-dependent but stathmin-independent manner.Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice.Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies.
Assuntos
Neoplasias da Mama/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Sarcoma/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Quinases Ciclina-Dependentes/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Humanos , Camundongos , Camundongos Nus , Fosforilação , Sarcoma/genética , Sarcoma/patologia , Estatmina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Estatmina/genética , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proliferação de Células , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Feminino , Fase G1 , Perfilação da Expressão Gênica , Gigantismo/metabolismo , Gigantismo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Hipófise/metabolismo , Hipófise/patologia , Fase S , Estatmina/deficiência , Timo/metabolismo , Timo/patologiaRESUMO
p27(kip1) (p27) is an inhibitor of cyclin/cyclin-dependent kinase complexes, whose nuclear loss indicates a poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/stathmin (stathmin), a microtubule (MT)-destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three-dimensional (3D)matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D environments, and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility, MT network composition, and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D contexts.