RESUMEN
Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies.
Asunto(s)
Proteínas de Ciclo Celular/efectos de los fármacos , Proteína Forkhead Box M1/metabolismo , Infecciones por Papillomavirus/tratamiento farmacológico , Proteínas Tirosina Quinasas/efectos de los fármacos , Pirazoles/antagonistas & inhibidores , Pirimidinonas/antagonistas & inhibidores , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Proteína Quinasa CDC2/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Neoplasias de Cabeza y Cuello , Humanos , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Regulación hacia ArribaRESUMEN
The Fbw7 (F-box/WD repeat-containing protein 7) ubiquitin ligase targets multiple oncoproteins for degradation and is commonly mutated in cancers. Like other pleiotropic tumor suppressors, Fbw7's complex biology has impeded our understanding of how Fbw7 mutations promote tumorigenesis and hindered the development of targeted therapies. To address these needs, we employed a transfer learning approach to derive gene-expression signatures from The Cancer Gene Atlas datasets that predict Fbw7 mutational status across tumor types and identified the pathways enriched within these signatures. Genes involved in mitochondrial function were highly enriched in pan-cancer signatures that predict Fbw7 mutations. Studies in isogenic colorectal cancer cell lines that differed in Fbw7 mutational status confirmed that Fbw7 mutations increase mitochondrial gene expression. Surprisingly, Fbw7 mutations shifted cellular metabolism toward oxidative phosphorylation and caused context-specific metabolic vulnerabilities. Our approach revealed unexpected metabolic reprogramming and possible therapeutic targets in Fbw7-mutant cancers and provides a framework to study other complex, oncogenic mutations.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Metaboloma , Mitocondrias/metabolismo , Mutación , Respiración de la Célula , Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica , Humanos , Mitocondrias/patología , Fosforilación Oxidativa , Estrés Oxidativo , Fosforilación , Ubiquitina , UbiquitinaciónRESUMEN
The Fbw7 tumor suppressor targets a broad network of proteins for ubiquitylation. Here we show critical functions for Fbw7 dimerization in regulating the specificity and robustness of degradation. Dimerization enables Fbw7 to target substrates through concerted binding to two suboptimal and independent recognition sites. Accordingly, an endogenous dimerization-deficient Fbw7 mutation stabilizes suboptimal substrates. Dimerization increases Fbw7's robustness by preserving its function in the setting of mutations that disable Fbw7 monomers, thereby buffering against pathogenic mutations. Finally, dimerization regulates Fbw7 stability, and this likely involves Fbw7 trans-autoubiquitylation. Our study reveals novel functions of Fbw7 dimerization and an unanticipated complexity in substrate degradation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/química , Dimerización , Proteínas F-Box/química , Proteína 7 que Contiene Repeticiones F-Box-WD , Células HCT116 , Humanos , Ligasas/química , Ligasas/metabolismo , Mutación , Unión Proteica , Estabilidad Proteica , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
The Mediator complex is an essential transcription regulator that bridges transcription factors with RNA polymerase II. This interaction is controlled by dynamic interactions between Mediator and the CDK8 module, but the mechanisms governing CDK8 module-Mediator association remain poorly understood. We show that Fbw7, a tumor suppressor and ubiquitin ligase, binds to CDK8-Mediator and targets MED13/13L for degradation. MED13/13L physically link the CDK8 module to Mediator, and Fbw7 loss increases CDK8 module-Mediator association. Our work reveals a novel mechanism regulating CDK8 module-Mediator association and suggests an expanded role for Fbw7 in transcriptional control and an unanticipated relationship with the CDK8 oncogene.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Complejo Mediador/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Proteolisis , Proteínas Ligasas SKP Cullina F-box/genética , UbiquitinaciónRESUMEN
Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/metabolismo , Replicación del ADN/fisiología , Fase S/fisiología , Transducción de Señal/fisiología , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Daño del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Campo Pulsado , Citometría de Flujo , Técnicas de Sustitución del Gen , Inestabilidad Genómica/fisiología , Humanos , Microfluídica , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Puntos de Control de la Fase S del Ciclo Celular/fisiología , Factores de Transcripción/metabolismoRESUMEN
c-Myc (hereafter, Myc) is a cancer driver whose abundance is regulated by the SCFFbw7 ubiquitin ligase and proteasomal degradation. Fbw7 binds to a phosphorylated Myc degron centered at threonine 58 (T58), and mutations of Fbw7 or T58 impair Myc degradation in cancers. Here, we identify a second Fbw7 phosphodegron at Myc T244 that is required for Myc ubiquitylation and acts in concert with T58 to engage Fbw7. While Ras-dependent Myc serine 62 phosphorylation (pS62) is thought to stabilize Myc by preventing Fbw7 binding, we find instead that pS62 greatly enhances Fbw7 binding and is an integral part of a high-affinity degron. Crystallographic studies revealed that both degrons bind Fbw7 in their diphosphorylated forms and that the T244 degron is recognized via a unique mode involving Fbw7 arginine 689 (R689), a mutational hotspot in cancers. These insights have important implications for Myc-associated tumorigenesis and therapeutic strategies targeting Myc stability.
RESUMEN
The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, and Notch). Fbw7 is a tumor suppressor and tumors often contain mutations in FBXW7, the gene that encodes Fbw7. The complexity of its substrate network has obscured the mechanisms of Fbw7-associated tumorigenesis, yet this understanding is needed for developing therapies. We used an integrated approach employing RNA-Seq and high-resolution mapping (cleavage under target and release using nuclease) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of misregulated Fbw7 substrates in colorectal cancer (CRC) cells with engineered tumor-associated FBXW7 null or missense mutations. Both Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy: some were common to both Fbw7 mutant cell lines, whereas others were mutation specific. We identified loci where both Jun and Myc were coregulated by Fbw7, suggesting that substrates may have synergistic effects. One coregulated gene was CIITA, the master regulator of MHC Class II gene expression. Fbw7 loss increased MHC Class II expression and Fbw7 mutations were correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunotherapeutic implications for Fbw7-associated cancers. Analogous studies in neural stem cells in which FBXW7 had been acutely deleted closely mirrored the results in CRC cells. Gene set enrichment analyses revealed Fbw7-associated pathways that were conserved across both cell types that may reflect fundamental Fbw7 functions. These analyses provide a framework for understanding normal and neoplastic context-specific Fbw7 functions.
Asunto(s)
Neoplasias Colorrectales , Proteínas F-Box , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorrectales/patología , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , Mutación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Cyclin-dependent kinase 2 (CDK2) controls cell division and is central to oncogenic signaling. We used an "in situ" approach to identify CDK2 substrates within nuclei isolated from cells expressing CDK2 engineered to use adenosine 5'-triphosphate analogs. We identified 117 candidate substrates, ~40% of which are known CDK substrates. Previously unknown candidates were validated to be CDK2 substrates, including LSD1, DOT1L, and Rad54. The identification of many chromatin-associated proteins may have been facilitated by labeling conditions that preserved nuclear architecture and physiologic CDK2 regulation by endogenous cyclins. Candidate substrates include proteins that regulate histone modifications, chromatin, transcription, and RNA/DNA metabolism. Many of these proteins also coexist in multi-protein complexes, including epigenetic regulators, that may provide new links between cell division and other cellular processes mediated by CDK2. In situ phosphorylation thus revealed candidate substrates with a high validation rate and should be readily applicable to other nuclear kinases.
RESUMEN
BACKGROUND: Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. RESULTS: We studied the consequences of deregulated cyclin E expression in primary cells and found that cyclin E initiated a p53-dependent response that prevented excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity was not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E led to increased p53 serine15 phosphorylation that was sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 was rendered nonfunctional, then the excess cyclin E became catalytically active and caused defects in S phase progression, increased ploidy, and genetic instability. CONCLUSIONS: We conclude that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Ciclo Celular , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Fibroblastos/metabolismo , Humanos , Fosforilación , Reacción en Cadena de la Polimerasa , Serina/metabolismo , Proteína p53 Supresora de Tumor/químicaRESUMEN
Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCFFbw7 ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic.
Asunto(s)
Biocatálisis , Ciclina E/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteolisis , Ciclo Celular , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD , Células HCT116 , Células HeLa , Humanos , Fosforilación , Fosfoserina/metabolismo , Estabilidad Proteica , Subunidades de Proteína/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A-cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.
Asunto(s)
Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa del Factor 2 de Elongación/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Mitosis , Datos de Secuencia Molecular , Factor 2 de Elongación Peptídica/química , Factor 2 de Elongación Peptídica/genética , Fosforilación , Mutación Puntual , Serina/química , Serina/genética , Treonina/química , Treonina/metabolismoRESUMEN
Colorectal cancer (CRC) remains a major cause of cancer mortality worldwide. Murine models have yielded critical insights into CRC pathogenesis, but they often fail to recapitulate advanced-disease phenotypes, notably metastasis and chromosomal instability (CIN). New models are thus needed to understand disease progression and to develop therapies. We sought to model advanced CRC by inactivating two tumor suppressors that are mutated in human CRCs, the Fbw7 ubiquitin ligase and p53. Here we report that Fbw7 deletion alters differentiation and proliferation in the gut epithelium and stabilizes oncogenic Fbw7 substrates, such as cyclin E and Myc. However, Fbw7 deletion does not cause tumorigenesis in the gut. In contrast, codeletion of both Fbw7 and p53 causes highly penetrant, aggressive, and metastatic adenocarcinomas, and allografts derived from these tumors form highly malignant adenocarcinomas. In vitro evidence indicates that Fbw7 ablation promotes genetic instability that is suppressed by p53, and we show that most Fbw7â»/â»; p53â»/â» carcinomas exhibit a CIN⺠phenotype. We conclude that Fbw7 and p53 synergistically suppress adenocarcinomas that mimic advanced human CRC with respect to histopathology, metastasis, and CIN. This model thus represents a novel tool for studies of advanced CRC as well as carcinogenesis associated with ubiquitin pathway mutations.
Asunto(s)
Adenocarcinoma , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorrectales , Modelos Animales de Enfermedad , Proteínas F-Box/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , Movimiento Celular/genética , Transformación Celular Neoplásica , Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The SCF(FBW7) ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7alpha isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas F-Box/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Estabilidad de Enzimas , Proteína 7 que Contiene Repeticiones F-Box-WD , Marcación de Gen , Humanos , Isoenzimas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Especificidad por SustratoRESUMEN
E-type cyclins are thought to drive cell-cycle progression by activating cyclin-dependent kinases, primarily CDK2. We previously found that cyclin E-null cells failed to incorporate MCM helicase into DNA prereplication complex during G(0) --> S phase progression. We now report that a kinase-deficient cyclin E mutant can partially restore MCM loading and S phase entry in cyclin E-null cells. We found that cyclin E is loaded onto chromatin during G(0) --> S progression. In the absence of cyclin E, CDT1 is normally loaded onto chromatin, whereas MCM is not, indicating that cyclin E acts between CDT1 and MCM loading. We observed a physical association of cyclin E with CDT1 and with MCMs. We propose that cyclin E facilitates MCM loading in a kinase-independent fashion, through physical interaction with CDT1 and MCM. Our work indicates that-in addition to their function as CDK activators-E cyclins play kinase-independent functions in cell-cycle progression.