RESUMO
Chemotherapy is used as neoadjuvant therapy for all subgroups of breast cancer, including ER-positive, and HER2-negative cases. However, studies have suggested that using aromatase inhibitors combined with CDK4/6-inhibitors might be an appropriate alternative in selected patients. Thus, the NEOLETRIB trial evaluates the response of ER-positive, HER2-negative luminal A/B breast cancer to the combination of letrozole and ribociclib in the neoadjuvant setting. Comprehensive molecular biology procedures, including sequential single-cell RNA-sequencing of tumor biopsies, are performed during 6 months of treatment with extensive biobanking of blood samples, tumor biopsies and gut microbiome specimens. Our findings will hopefully contribute to an improved selection of patients who may benefit from this drug combination and give new insights into the intra-tumoral changes during this treatment.Trial registration number: NCT05163106 (ClinicalTrials.gov).
[Box: see text].
RESUMO
Lung cancer is still a leading cause of death worldwide. In recent years, knowledge has been obtained of the mechanisms modulating ion channel kinetics and thus of cell bioelectric properties, which is promising for oncological biomarkers and targets. The complex interplay of channel expression and its consequences on malignant processes, however, is still insufficiently understood. We here introduce the first approach of an in-silico whole-cell ion current model of a cancer cell, in particular of the A549 human lung adenocarcinoma, including the main functionally expressed ion channels in the plasma membrane as so far known. This hidden Markov-based model represents the electrophysiology behind proliferation of the A549 cell, describing its rhythmic oscillation of the membrane potential able to trigger the transition between cell cycle phases, and it predicts membrane potential changes over the cell cycle provoked by targeted ion channel modulation. This first A549 in-silico cell model opens up a deeper insight and understanding of possible ion channel interactions in tumor development and progression, and is a valuable tool for simulating altered ion channel function in lung cancer electrophysiology.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Canais Iônicos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Modelos Biológicos , Células A549 , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Biologia Computacional , Simulação por Computador , Humanos , Transporte de Íons , Cadeias de Markov , Potenciais da Membrana , Técnicas de Patch-ClampRESUMO
It is intuitively obvious that the ability of a cell to repair DNA damage is saturable, either by limitation of enzymatic activities, the time allotted to achieve their function, or both. However, very little is known regarding the mechanisms that establish such a threshold. Here we demonstrate that the CUL4A ubiquitin ligase restricts the cellular repair capacity by orchestrating the concerted actions of nucleotide excision repair (NER) and the DNA damage-responsive G1/S checkpoint through selective degradation of the DDB2 and XPC DNA damage sensors and the p21/CIP1/WAF1 checkpoint effector. We generated Cul4a conditional knockout mice and observed that skin-specific Cul4a ablation dramatically increased resistance to UV-induced skin carcinogenesis. Our findings reveal that wild-type cells do not operate at their full DNA repair potential, underscore the critical role of CUL4A in establishing the cellular DNA repair threshold, and highlight the potential augmentation of cellular repair proficiency by pharmacological CUL4A inhibition.
Assuntos
Proteínas Culina/metabolismo , Dano ao DNA , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Animais , Células Cultivadas , Proteínas Culina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Genes cdc , Camundongos , Camundongos Knockout , Neoplasias Cutâneas/patologia , Transgenes , Raios UltravioletaRESUMO
Mutation of the TRIM (tripartite motif)-NHL family members brat and mei-P26 perturb the differentiation of transit-amplifying progenitor cells resulting in tumour-like phenotypes. The NHL (named after the NCL1, HT2A and LIN41 repeat) domain is essential for their growth suppressive activity, and they can induce cell-cycle exit in a RING-independent manner. TRIM3 is the only bona fide tumour suppressor in the mammalian TRIM-NHL subfamily and similar to the other members of this family, its ability to inhibit cell proliferation depends on the NHL domain. However, whether the RING domain was required for TRIM3-dependent cell-cycle exit had not been investigated. In the present study, we establish that the RING domain is required for TRIM3-induced growth suppression. Furthermore, we show that this domain is necessary to promote ubiquitination of p21 in a reconstituted in vitro system where UbcH5a is the preferred E2. Thus the ability of TRIM3 to suppress growth is associated with its ability to ubiquitinate proteins.
Assuntos
Proteínas de Transporte/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Camundongos , Mutação , Estrutura Terciária de Proteína , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
p27Kip1, a cyclin-cdk inhibitor, is a tumor suppressor. An overwhelming amount of data correlate p27 abundance to tumor prognosis in humans. Mouse models have supported the importance of decreasing p27 to tumor incidence. Inactivation of most tumor suppressors occurs at the level of gene mutation or silencing, but p27 is regulated posttranscriptionally, and how its level is reduced in cancer is largely unknown. Reports on a series of allelic mice with p27 mutations affecting different posttranscriptional regulatory pathways are emerging and being used to examine which pathways are necessary for p27 turnover associated with tumor development, with surprising results.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Proteínas Quinases Associadas a Fase S/metabolismoRESUMO
PURPOSE: We conducted research on CDK4/6 inhibitors (CDK4/6i) simultaneously in the preclinical and clinical spaces to gain a deeper understanding of how senescence influences tumor growth in humans. PATIENTS AND METHODS: We coordinated a first-in-kind phase II clinical trial of the CDK4/6i abemaciclib for patients with progressive dedifferentiated liposarcoma (DDLS) with cellular studies interrogating the molecular basis of geroconversion. RESULTS: Thirty patients with progressing DDLS enrolled and were treated with 200 mg of abemaciclib twice daily. The median progression-free survival was 33 weeks at the time of the data lock, with 23 of 30 progression-free at 12 weeks (76.7%, two-sided 95% CI, 57.7%-90.1%). No new safety signals were identified. Concurrent preclinical work in liposarcoma cell lines identified ANGPTL4 as a necessary late regulator of geroconversion, the pathway from reversible cell-cycle exit to a stably arrested inflammation-provoking senescent cell. Using this insight, we were able to identify patients in which abemaciclib induced tumor cell senescence. Senescence correlated with increased leukocyte infiltration, primarily CD4-positive cells, within a month of therapy. However, those individuals with both senescence and increased TILs were also more likely to acquire resistance later in therapy. These suggest that combining senolytics with abemaciclib in a subset of patients may improve the duration of response. CONCLUSIONS: Abemaciclib was well tolerated and showed promising activity in DDLS. The discovery of ANGPTL4 as a late regulator of geroconversion helped to define how CDK4/6i-induced cellular senescence modulates the immune tumor microenvironment and contributes to both positive and negative clinical outcomes. See related commentary by Weiss et al., p. 649.
Assuntos
Aminopiridinas , Lipossarcoma , Humanos , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Lipossarcoma/tratamento farmacológico , Lipossarcoma/patologia , Senescência Celular , Quinase 4 Dependente de Ciclina , Microambiente TumoralRESUMO
SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Transformação Celular Neoplásica/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína Fosfatase 2/metabolismo , Substituição de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Humanos , Camundongos , Mutação de Sentido Incorreto , Proteína Fosfatase 2/genética , ProteóliseRESUMO
Phosphorylation of Tyr-88/Tyr-89 in the 3(10) helix of p27 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This modification does not affect the interaction of p27 with cyclin-CDK complexes but does interfere with van der Waals and hydrogen bond contacts between p27 and amino acids in the catalytic cleft of the CDK. Thus, it had been suggested that phosphorylation of this site could switch the tumor-suppressive CDK inhibitory activity to an oncogenic activity. Here, we examined this hypothesis in the RCAS-PDGF-HA/nestin-TvA proneural glioma mouse model, in which p21 facilitates accumulation of nuclear cyclin D1-CDK4 and promotes tumor development. In these tumor cells, approximately one-third of the p21 is phosphorylated at Tyr-76 in the 3(10) helix. Mutation of this residue to glutamate reduced inhibitory activity in vitro. Mutation of this residue to phenylalanine reduced the tumor-promoting activity of p21 in the animal model, whereas glutamate or alanine substitution allowed tumor formation. Consequently, we conclude that tyrosine phosphorylation contributes to the conversion of CDK inhibitors from tumor-suppressive roles to oncogenic roles.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Tirosina/química , Sequência de Aminoácidos , Animais , Proliferação de Células , Neoplasias do Sistema Nervoso Central/metabolismo , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Glioblastoma/metabolismo , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , FosforilaçãoRESUMO
Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C-cyclin D-CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. SIGNIFICANCE: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6-INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275.
Assuntos
Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Proteínas Inibidoras de Quinase Dependente de Ciclina/química , Resistencia a Medicamentos Antineoplásicos , Piperazinas/química , Inibidores de Proteínas Quinases/química , Piridinas/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proteínas Inibidoras de Quinase Dependente de Ciclina/administração & dosagem , Proteínas Inibidoras de Quinase Dependente de Ciclina/uso terapêutico , Feminino , Humanos , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Proteínas Supressoras de Tumor/metabolismoRESUMO
PD-1 blockade (nivolumab) efficacy remains modest for metastatic sarcoma. In this paper, we present an open-label, non-randomized, non-comparative pilot study of bempegaldesleukin, a CD122-preferential interleukin-2 pathway agonist, with nivolumab in refractory sarcoma at Memorial Sloan Kettering/MD Anderson Cancer Centers (NCT03282344). We report on the primary outcome of objective response rate (ORR) and secondary endpoints of toxicity, clinical benefit, progression-free survival, overall survival, and durations of response/treatment. In 84 patients in 9 histotype cohorts, all patients experienced ≥1 adverse event and treatment-related adverse event; 1 death was possibly treatment-related. ORR was highest in angiosarcoma (3/8) and undifferentiated pleomorphic sarcoma (2/10), meeting predefined endpoints. Results of our exploratory investigation of predictive biomarkers show: CD8 + T cell infiltrates and PD-1 expression correlate with improved ORR; upregulation of immune-related pathways correlate with improved efficacy; Hedgehog pathway expression correlate with resistance. Exploration of this combination in selected sarcomas, and of Hedgehog signaling as a predictive biomarker, warrants further study in larger cohorts.
Assuntos
Antineoplásicos Imunológicos , Segunda Neoplasia Primária , Sarcoma , Antineoplásicos Imunológicos/uso terapêutico , Proteínas Hedgehog , Humanos , Interleucina-2/uso terapêutico , Segunda Neoplasia Primária/induzido quimicamente , Nivolumabe/uso terapêutico , Projetos Piloto , Receptor de Morte Celular Programada 1/metabolismo , Sarcoma/tratamento farmacológico , Sarcoma/patologiaRESUMO
How proteins participate in tumorigenesis can be obscured by their multifunctional nature. For example, depending on the cellular context, the cdk inhibitors can affect cell proliferation, cell motility, apoptosis, receptor tyrosine kinase signaling, and transcription. Thus, to determine how a protein contributes to tumorigenesis, we need to evaluate which functions are required in the developing tumor. Here we demonstrate that the RCAS/TvA system, originally developed to introduce oncogenes into somatic cells of mice, can be adapted to allow us to define the contribution that different functional domains make to tumor development. Studying the development of growth-factor-induced oligodendroglioma, we identified a critical role for the Cy elements in p21, and we showed that cyclin D1T286A, which accumulates in the nucleus of p21-deficient cells and binds to cdk4, could bypass the requirement for p21 during tumor development. These genetic results suggest that p21 acts through the cyclin D1-cdk4 complex to support tumor growth, and establish the utility of using a somatic cell modeling system for defining the contribution proteins make to tumor development.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/metabolismo , Oligodendroglioma/metabolismo , Animais , Proteínas Aviárias/metabolismo , Linhagem Celular , Galinhas , Ciclina D , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Ciclinas/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Camundongos , Camundongos Transgênicos , Oligodendroglioma/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Virais/metabolismo , Retroviridae/genéticaRESUMO
Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107(-/-) p27(D51/D51) and p107(-/-) p130(-/-) mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Osteogênese/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Animais , Animais Recém-Nascidos , Peso Corporal , Osso e Ossos/anatomia & histologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Condrócitos/citologia , Condrogênese , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Masculino , Camundongos , Tamanho do Órgão , Fenótipo , Proteína p107 Retinoblastoma-Like/deficiência , Fase S , Análise de SobrevidaRESUMO
MEF is an ETS-related transcription factor with strong transcriptional activating activity that affects hematopoietic stem cell behavior and is required for normal NK cell and NK T-cell development. The MEF (also known as ELF4) gene is repressed by several leukemia-associated fusion transcription factor proteins (PML-retinoic acid receptor alpha and AML1-ETO), but it is also activated by retroviral insertion in several cancer models. We have previously shown that cyclin A-dependent phosphorylation of MEF largely restricts its activity to the G(1) phase of the cell cycle; we now show that MEF is a short-lived protein whose expression level also peaks during late G(1) phase. Mutagenesis studies show that the rapid turnover of MEF in S phase is dependent on the specific phosphorylation of threonine 643 and serine 648 at the C terminus of MEF by cdk2 and on the Skp1/Cul1/F-box (SCF) E3 ubiquitin ligase complex SCF(Skp2), which targets MEF for ubiquitination and proteolysis. Overexpression of MEF drives cells through the G(1)/S transition, thereby promoting cell proliferation. The tight regulation of MEF levels during the cell cycle contributes to its effects on regulating cell cycle entry and cell proliferation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Ligases/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica , Meia-Vida , Humanos , Hidrólise , Leupeptinas/farmacologia , Ligases/genética , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas/genética , Serina/metabolismo , Treonina/metabolismo , Fatores de Transcrição/análise , Fatores de Transcrição/química , Ubiquitina/análiseRESUMO
Senescence is an irreversible form of growth arrest and is generally considered a favorable outcome of cancer therapies, yet little is known about the molecular events that distinguish this state from readily reversible growth arrest (i.e. quiescence). Recently, we discovered that during therapy induced senescence the chromatin remodeling protein α-thalassemia, mental retardation, X-linked (ATRX) represses Harvey rat sarcoma viral oncogene homolog (HRAS), and repression of HRAS is necessary to establish senescence, suggesting how new clinical combinations might be used to achieve durable senescence.
RESUMO
CDK4/6 inhibitors are among a new generation of therapeutics. Building upon the striking success of the combination of CDK4/6 inhibitors and the hormone receptor antagonist letrozole in breast cancer, many other combinations have recently entered clinical trials in multiple diseases. To achieve maximal benefit with CDK4/6 inhibitors it will be critical to understand the cellular mechanisms by which they act. Here we highlight the mechanisms by which CDK4/6 inhibitors can exert their anti-tumor activities beyond simply enforcing cytostatic growth arrest, and discuss how this knowledge may inform new combinations, improve outcomes, and modify dosing schedules in the future.
Assuntos
Antineoplásicos/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Desenho de Fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Humanos , Terapia de Alvo Molecular , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/efeitos adversos , Transdução de SinaisRESUMO
CDK4/6 inhibitors are being used to treat a variety of human malignancies. In well-differentiated and dedifferentiated liposarcoma their clinical promise is associated with their ability to downregulate the MDM2 protein. The downregulation of MDM2 following treatment with CDK4/6 inhibitors also induces many cultured tumor cell lines derived from different types of malignancies to progress from quiescence into senescence. Here we used cultured human cell lines and defined a role for PDLIM7 and CDH18, regulating MDM2 protein in CDK4/6 inhibitor-treated cells. Materials from our previous phase II trials with palbociclib were then used to demonstrate that expression of CDH18 protein was associated with response, measured as both progression-free survival and overall survival. This supports the hypothesis that the biologic transition from quiescence to senescence has clinical relevance for this class of drugs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caderinas/metabolismo , Senescência Celular/fisiologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Linhagem Celular Tumoral , Intervalo Livre de Doença , Humanos , Lipossarcoma/tratamento farmacológico , Lipossarcoma/metabolismo , Piperazinas/farmacologia , Intervalo Livre de Progressão , Piridinas/farmacologiaRESUMO
Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27(kip1) is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G(1) cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCF(skp2) complex.
Assuntos
Quinases relacionadas a CDC2 e CDC28/fisiologia , Proteínas de Ciclo Celular/metabolismo , Ciclina A/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Quinases relacionadas a CDC2 e CDC28/análise , Ciclina A/análise , Ciclina A/genética , Ciclina E/análise , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Citometria de Fluxo , Fase G1 , Células HeLa , Humanos , Mutação , Fase S , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitina/metabolismoAssuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias/terapia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Neoplasias/patologiaRESUMO
Senescence is a state of stable cell cycle exit with important implications for development and disease. Here, we demonstrate that the chromatin remodeling enzyme ATRX is required for therapy-induced senescence. ATRX accumulates in nuclear foci and is required for therapy-induced senescence in multiple types of transformed cells exposed to either DNA damaging agents or CDK4 inhibitors. Mobilization into foci depends on the ability of ATRX to interact with H3K9me3 histone and HP1. Foci form soon after cells exit the cell cycle, before other hallmarks of senescence appear. Eliminating ATRX in senescent cells destabilizes the senescence-associated heterochromatic foci. Additionally, ATRX binds to and suppresses expression from the HRAS locus; repression of HRAS is sufficient to promote the transition of quiescent cells into senescence and preventing repression blocks progression into senescence. Thus ATRX is a critical regulator of therapy-induced senescence and acts in multiple ways to drive cells into this state.Therapy induced senescence (TIS) is a growth suppressive program activated by cytostatic agents in some cancer cells. Here the authors show that the chromatin remodeling enzyme ATRX is a regulator of TIS and drives cells into this state via multiple mechanisms.
Assuntos
Senescência Celular/genética , Proteína Nuclear Ligada ao X/fisiologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/metabolismo , Dano ao DNA , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Inibidores de Proteínas Quinases/efeitos adversos , RNA Mensageiro/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismoRESUMO
IMPORTANCE: More than 90% of well-differentiated or dedifferentiated liposarcomas (WD/DDLS) have CDK4 amplification. The selective CDK4 and CDK6 inhibitor palbociclib inhibits growth and induces senescence in liposarcoma cell lines and xenografts. Our prior phase 2 study demonstrated that treatment with palbociclib (200 mg daily for 14 days every 21 days) resulted in clinical benefit in WD/DDLS but moderate hematologic toxic effects. It is important to understand whether palbociclib at a new dose and schedule-125 mg daily for 21 days every 28 days-results in clinical benefit and manageable toxic effects. OBJECTIVE: To determine the progression-free survival (PFS) at 12 weeks of patients with WD/DDLS treated with palbociclib (PD0332991). DESIGN, SETTING, AND PARTICIPANTS: In this phase 2, nonrandomized, open-label clinical trial conducted at the Memorial Sloan Kettering Cancer Center, 60 patients 18 years and older with advanced WD/DDLS and measurable disease by RECIST 1.1 were enrolled from December 2011 to January 2014 and followed to March 2015. Patients received oral palbociclib at 125 mg daily for 21 days in 28-day cycles. MAIN OUTCOMES AND MEASURES: Primary end point was PFS. Secondary end points included response rate and toxic effects. RESULTS: Overall, 30 patients were enrolled in the initial cohort and 30 more in an expansion cohort. Median (range) age was 61.5 (35-87) years; 31 patients (52%) were male; median (range) Eastern Cooperative Oncology Group score was 0 (0-1). Progression-free survival at 12 weeks was 57.2% (2-sided 95% CI, 42.4%-68.8%), and the median PFS was 17.9 weeks (2-sided 95% CI, 11.9-24.0 weeks). There was 1 complete response. Toxic effects were primarily hematologic and included neutropenia (grade 3, n = 20 [33%]; grade 4, n = 2 [3%]) but no neutropenic fever. CONCLUSIONS AND RELEVANCE: In patients with advanced WD/DDLS, treatment with palbociclib was associated with a favorable PFS and occasional tumor response. This dose and schedule appears active and may have less toxic effects than 200 mg for 14 days. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01209598.