RESUMO
The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway.
Assuntos
Via de Sinalização Hippo , Proteínas Serina-Treonina Quinases , Fatores de Transcrição , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Fatores de Transcrição/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Camundongos , Linhagem Celular Tumoral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Sinalização YAP/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição de Domínio TEA , Proteínas ras/metabolismo , Feminino , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Although KRASG12C inhibitors show clinical activity in patients with KRAS G12C mutated non-small cell lung cancer (NSCLC) and other solid tumor malignancies, response is limited by multiple mechanisms of resistance. The KRASG12C inhibitor JDQ443 shows enhanced preclinical antitumor activity combined with the SHP2 inhibitor TNO155, and the combination is currently under clinical evaluation. To identify rational combination strategies that could help overcome or prevent some types of resistance, we evaluated the duration of tumor responses to JDQ443 ± TNO155, alone or combined with the PI3Kα inhibitor alpelisib and/or the cyclin-dependent kinase 4/6 inhibitor ribociclib, in xenograft models derived from a KRASG12C-mutant NSCLC line and investigated the genetic mechanisms associated with loss of response to combined KRASG12C/SHP2 inhibition. Tumor regression by single-agent JDQ443 at clinically relevant doses lasted on average 2 weeks and was increasingly extended by the double, triple, or quadruple combinations. Growth resumption was accompanied by progressively increased KRAS G12C amplification. Functional genome-wide CRISPR screening in KRASG12C-dependent NSCLC lines with distinct mutational profiles to identify adaptive mechanisms of resistance revealed sensitizing and rescuing genetic interactions with KRASG12C/SHP2 coinhibition; FGFR1 loss was the strongest sensitizer, and PTEN loss the strongest rescuer. Consistently, the antiproliferative activity of KRASG12C/SHP2 inhibition was strongly enhanced by PI3K inhibitors. Overall, KRAS G12C amplification and alterations of the MAPK/PI3K pathway were predominant mechanisms of resistance to combined KRASG12C/SHP2 inhibitors in preclinical settings. The biological nodes identified by CRISPR screening might provide additional starting points for effective combination treatments. SIGNIFICANCE: Identification of resistance mechanisms to KRASG12C/SHP2 coinhibition highlights the need for additional combination therapies for lung cancer beyond on-pathway combinations and offers the basis for development of more effective combination approaches. See related commentary by Johnson and Haigis, p. 4005.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fosfatidilinositol 3-Quinases/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas Proto-Oncogênicas p21(ras)/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Detecção Precoce de Câncer , Inibidores Enzimáticos/uso terapêutico , Mutação , Linhagem Celular TumoralRESUMO
Aberrant chromosomal architecture, ranging from small insertions or deletions to large chromosomal alterations, is one of the most common characteristics of cancer genomes. Chromosomal instability (CIN) underpins much of the intratumoural heterogeneity observed in cancers and drives phenotypic adaptation during tumour evolution. Thus, an urgent need exists to increase our efforts to target CIN as if it were a molecular entity. Indeed, CIN accelerates the development of anticancer drug resistance, often leading to treatment failure and disease recurrence, which limit the effectiveness of most current therapies. Identifying novel strategies to modulate CIN and to exploit the fitness cost associated with aneuploidy in cancer is, therefore, of paramount importance for the successful treatment of cancer. Modern sequencing and analytical methods greatly facilitate the identification and cataloguing of somatic copy-number alterations and offer new possibilities to better exploit the dynamic process of CIN. In this Review, we describe the principles governing CIN propagation in cancer and how CIN might influence sensitivity to immune-checkpoint inhibition, and survey the vulnerabilities associated with CIN that offer potential therapeutic opportunities.
Assuntos
Instabilidade Cromossômica/genética , Variações do Número de Cópias de DNA/genética , Heterogeneidade Genética , Neoplasias/genética , Aneuploidia , Genoma Humano/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca H1047R hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA H1047R mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Centrossomo/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Amplificação de Genes , Genoma , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Classe I de Fosfatidilinositol 3-Quinases/genética , Estudos de Coortes , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Oncogenes , Fosfatidilinositol 3-Quinases/genéticaRESUMO
BACKGROUND: The focus of tumour-specific antigen analyses has been on single nucleotide variants (SNVs), with the contribution of small insertions and deletions (indels) less well characterised. We investigated whether the frameshift nature of indel mutations, which create novel open reading frames and a large quantity of mutagenic peptides highly distinct from self, might contribute to the immunogenic phenotype. METHODS: We analysed whole-exome sequencing data from 5777 solid tumours, spanning 19 cancer types from The Cancer Genome Atlas. We compared the proportion and number of indels across the cohort, with a subset of results replicated in two independent datasets. We assessed in-silico tumour-specific neoantigen predictions by mutation type with pan-cancer analysis, together with RNAseq profiling in renal clear cell carcinoma cases (n=392), to compare immune gene expression across patient subgroups. Associations between indel burden and treatment response were assessed across four checkpoint inhibitor datasets. FINDINGS: We observed renal cell carcinomas to have the highest proportion (0·12) and number of indel mutations across the pan-cancer cohort (p<2·2â×â10-16), more than double the median proportion of indel mutations in all other cancer types examined. Analysis of tumour-specific neoantigens showed that enrichment of indel mutations for high-affinity binders was three times that of non-synonymous SNV mutations. Furthermore, neoantigens derived from indel mutations were nine times enriched for mutant specific binding, as compared with non-synonymous SNV derived neoantigens. Immune gene expression analysis in the renal clear cell carcinoma cohort showed that the presence of mutant-specific neoantigens was associated with upregulation of antigen presentation genes, which correlated (r=0·78) with T-cell activation as measured by CD8-positive expression. Finally, analysis of checkpoint inhibitor response data revealed frameshift indel count to be significantly associated with checkpoint inhibitor response across three separate melanoma cohorts (p=4·7â×â10-4). INTERPRETATION: Renal cell carcinomas have the highest pan-cancer proportion and number of indel mutations. Evidence suggests indels are a highly immunogenic mutational class, which can trigger an increased abundance of neoantigens and greater mutant-binding specificity. FUNDING: Cancer Research UK, UK National Institute for Health Research (NIHR) at the Royal Marsden Hospital National Health Service Foundation Trust, Institute of Cancer Research and University College London Hospitals Biomedical Research Centres, the UK Medical Research Council, the Rosetrees Trust, Novo Nordisk Foundation, the Prostate Cancer Foundation, the Breast Cancer Research Foundation, the European Research Council.
Assuntos
Antígenos de Neoplasias/genética , DNA de Neoplasias/análise , Mutação da Fase de Leitura , Mutação INDEL , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T CD8-Positivos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Análise Mutacional de DNA , Bases de Dados Genéticas , Exoma , Genes cdc , Genômica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Ativação Linfocitária/genética , Melanoma/genética , Melanoma/imunologia , Fenótipo , Regulação para CimaRESUMO
Chromosomal aberrations during cell division represent one of the first recognized features of human cancer cells, and modern detection methods have revealed the pervasiveness of aneuploidy in cancer. The ongoing karyotypic changes brought about by chromosomal instability (CIN) contribute to tumor heterogeneity, drug resistance, and treatment failure. Whole-chromosome and segmental aneuploidies resulting from CIN have been proposed to allow "macroevolutionary" leaps that may contribute to profound phenotypic change. In this review, we will outline evidence indicating that aneuploidy and CIN contribute to cancer evolution.
Assuntos
Aneuploidia , Instabilidade Cromossômica/genética , Neoplasias/genética , Animais , Resistência a Medicamentos/genética , Humanos , Camundongos , Mitose/genéticaRESUMO
Chromosomal instability (CIN) contributes to cancer evolution, intratumor heterogeneity, and drug resistance. CIN is driven by chromosome segregation errors and a tolerance phenotype that permits the propagation of aneuploid genomes. Through genomic analysis of colorectal cancers and cell lines, we find frequent loss of heterozygosity and mutations in BCL9L in aneuploid tumors. BCL9L deficiency promoted tolerance of chromosome missegregation events, propagation of aneuploidy, and genetic heterogeneity in xenograft models likely through modulation of Wnt signaling. We find that BCL9L dysfunction contributes to aneuploidy tolerance in both TP53-WT and mutant cells by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID. Efforts to exploit aneuploidy tolerance mechanisms and the BCL9L/caspase-2/BID axis may limit cancer diversity and evolution.
Assuntos
Aneuploidia , Caspase 2/fisiologia , Neoplasias Colorretais/genética , Cisteína Endopeptidases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Caspase 2/análise , Segregação de Cromossomos , Cisteína Endopeptidases/análise , Proteínas de Ligação a DNA/genética , Células HCT116 , Humanos , Camundongos , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Intercellular heterogeneity, exacerbated by chromosomal instability (CIN), fosters tumor heterogeneity and drug resistance. However, extreme CIN correlates with improved cancer outcome, suggesting that karyotypic diversity required to adapt to selection pressures might be balanced in tumors against the risk of excessive instability. Here, we used a functional genomics screen, genome editing, and pharmacologic approaches to identify CIN-survival factors in diploid cells. We find partial anaphase-promoting complex/cyclosome (APC/C) dysfunction lengthens mitosis, suppresses pharmacologically induced chromosome segregation errors, and reduces naturally occurring lagging chromosomes in cancer cell lines or following tetraploidization. APC/C impairment caused adaptation to MPS1 inhibitors, revealing a likely resistance mechanism to therapies targeting the spindle assembly checkpoint. Finally, CRISPR-mediated introduction of cancer somatic mutations in the APC/C subunit cancer driver gene CDC27 reduces chromosome segregation errors, whereas reversal of an APC/C subunit nonsense mutation increases CIN. Subtle variations in mitotic duration, determined by APC/C activity, influence the extent of CIN, allowing cancer cells to dynamically optimize fitness during tumor evolution. SIGNIFICANCE: We report a mechanism whereby cancers balance the evolutionary advantages associated with CIN against the fitness costs caused by excessive genome instability, providing insight into the consequence of CDC27 APC/C subunit driver mutations in cancer. Lengthening of mitosis through APC/C modulation may be a common mechanism of resistance to cancer therapeutics that increase chromosome segregation errors. Cancer Discov; 7(2); 218-33. ©2017 AACR.See related commentary by Burkard and Weaver, p. 134This article is highlighted in the In This Issue feature, p. 115.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Instabilidade Cromossômica , Edição de Genes/métodos , Genômica/métodos , Neoplasias/genética , Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Células HCT116 , Células HT29 , Humanos , Mitose , Neoplasias/metabolismoRESUMO
Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/C(Cdc20)) [2]. Upon satisfaction of both pathways, the APC/C(Cdc20) elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/C(Cdc20) is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/C(Cdc20) couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit.
Assuntos
Anáfase/fisiologia , Proteína Quinase CDC2/fisiologia , Cinetocoros/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Cromátides/fisiologia , Ciclina B/fisiologia , Ciclina B1/fisiologia , Células HeLa , Humanos , Não Disjunção Genética/fisiologia , Separase/fisiologiaRESUMO
Development and homeostasis require repeated symmetric cell divisions, which in turn depend on a centered mitotic spindle. In this issue, Kiyomitsu and Cheeseman uncover two mechanisms that ensure correct spindle positioning in anaphase: cortical dynein for pulling the spindle to the cell center and asymmetric membrane elongation that adjusts the position of the cell center to the cleavage plane.
Assuntos
Anáfase , Membrana Celular/metabolismo , Dineínas/metabolismo , Fuso Acromático/metabolismo , Animais , HumanosAssuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Instabilidade Genômica , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Proteínas Nucleares/genética , Prognóstico , Proteínas Repressoras/genética , Fatores de TranscriçãoRESUMO
Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2(+) subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome.
Assuntos
Ciclo Celular , Instabilidade Cromossômica/fisiologia , Proteínas de Homeodomínio/fisiologia , Mitose/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular , Replicação do DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Fatores de TranscriçãoRESUMO
The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).
Assuntos
Proteína Quinase CDC2/metabolismo , Núcleo Celular/metabolismo , Ciclina B/metabolismo , Fase G1/fisiologia , Proteínas de Homeodomínio/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Proteína Quinase CDC2/genética , Linhagem Celular , Núcleo Celular/genética , Ciclina B/genética , DNA/genética , DNA/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Proteínas Repressoras/genéticaRESUMO
In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.
Assuntos
Caderinas/genética , Movimento Celular , Regulação para Baixo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Bovinos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Imunoprecipitação da Cromatina , Cães , Regulação da Expressão Gênica , Genes Reporter , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Humanos , Junções Intercelulares/metabolismo , Camundongos , Proteínas Nucleares/genética , Ocludina , Regiões Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Caramujos/genética , Fatores de Transcrição , Transcrição Gênica , Ativação TranscricionalRESUMO
Cux (Cut homeobox) genes are present in all metazoans. Early reports described many phenotypes caused by cut mutations in Drosophila melanogaster. In vertebrates, CUX1 was originally characterized as the CCAAT-displacement protein (CDP). Another line of investigation revealed the presence of CUX1 within a multi-protein complex called the histone nuclear factor D (HiNF-D). Recent studies led to the identification of several CUX1 isoforms with distinct DNA binding and transcriptional properties. While the CCAAT-displacement activity was implicated in the transcriptional repression of several genes, some CUX1 isoforms were found to participate in the transcriptional activation of some genes. The expression and activity of CUX1 was shown to be regulated through the cell cycle and to be a target of TGF-beta signaling. Mechanisms of regulation include alternative transcription initiation, proteolytic processing, phosphorylation and acetylation. Cell-based assays have established a role for CUX1 in the control of cell cycle progression, cell motility and invasion. In the mouse, gene inactivation as well as over-expression in transgenic mice has revealed phenotypes in multiple organs and cell types. While some phenotypes could be explained by the presumed functions of CUX1 in the affected cells, other phenotypes invoked non-cell-autonomous effects that suggest regulatory functions with an impact on cell-cell interactions. The implication of CUX1 in cancer was suggested first from its over-expression in primary tumors and cancer cell lines and was later confirmed in mouse models.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Genes Homeobox , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais , Mutação , Neoplasias/etiologia , Neoplasias/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).
Assuntos
Replicação do DNA/genética , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Ativação Transcricional , Animais , Linhagem Celular , Cromatina/isolamento & purificação , Cromatografia de Afinidade , Genes cdc , Genômica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Fase S/genética , Análise de Sequência de DNA , Fatores de TranscriçãoRESUMO
It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non-cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus.
Assuntos
Catepsinas/genética , Núcleo Celular/enzimologia , Transformação Celular Neoplásica , Cisteína Endopeptidases/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células 3T3 , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Catepsina L , Divisão Celular , Linhagem Celular Tumoral , Precursores Enzimáticos/genética , Feminino , Humanos , CamundongosRESUMO
The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.
Assuntos
Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Testes de Carcinogenicidade , Proliferação de Células , Transformação Celular Neoplásica , Células Cultivadas , Centrifugação , Ciclina A/genética , Ciclina A/metabolismo , Ciclina A2 , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Fibroblastos/citologia , Genes ras , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Mutantes , Camundongos Nus , Proteínas Nucleares/genética , Isoformas de Proteínas , Proteínas Repressoras/genética , Timidina/metabolismo , Fatores de Transcrição/genéticaRESUMO
The subclass of cysteine proteases termed lysosomal cathepsins has long been thought to be primarily involved in end-stage protein breakdown within lysosomal compartments. Furthermore, few specific protein substrates for these proteases have been identified. We show here that cathepsin L functions in the regulation of cell cycle progression through proteolytic processing of the CDP/Cux transcription factor. CDP/Cux processing in situ was increased following ectopic expression of cathepsin L but was reduced in Cat L(-/-) cells. Furthermore, catalytically active cathepsin L was localized to the nucleus during the G1-S transition as detected by immunofluorescence imaging and labeling using activity-based probes. Trafficking of cathepsin L to the nucleus is accomplished through a mechanism involving translation initiation at downstream AUG sites and the synthesis of proteases that are devoid of a signal peptide. Overall, these results uncover an as yet unsuspected role for cysteine proteases in the control of cell cycle progression.