RESUMEN
The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Células Madre Embrionarias/fisiología , Epigénesis Genética , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Proteína Quinasa CDC2/genética , Diferenciación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , Femenino , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Células MCF-7 , Masculino , Ratones , Ratones Noqueados , Fosforilación , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Melanoma Experimental/patología , Melanoma/patología , Neoplasias Cutáneas/patología , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Femenino , Dosificación de Gen , Humanos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/mortalidad , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/genética , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/genética , Pronóstico , Piel/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/mortalidad , Vimentina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
B-cell receptor (BCR) signaling is essential for the diffuse large B-cell lymphoma (DLBCL) subtype that originates from activated B-cells (ABCs). ABC-DLBCL cells are sensitive to Bruton tyrosine kinase intervention. However, patients with relapsed or refractory ABC-DLBCL had overall response rates from 33% to 37% for Bruton tyrosine kinase inhibitors, suggesting the evaluation of combination-based treatment for improved efficacy. We investigated the efficacy and mechanism of the bromodomain and extraterminal motif (BET) inhibitor AZD5153 combined with the Bruton tyrosine kinase inhibitor acalabrutinib in ABC-DLBCL preclinical models. AZD5153 is a bivalent BET inhibitor that simultaneously engages the 2 bromodomains of BRD4. Adding AZD5153 to acalabrutinib demonstrated combination benefits in ABC-DLBCL cell line and patient-derived xenograft models. Differential expression analyses revealed PAX5 transcriptional activity as a novel downstream effector of this drug combination. PAX5 is a transcription factor for BCR signaling genes and may be critical for perpetually active BCR signaling in ABC-DLBCL. Our analyses further indicated significant alterations in BCR, RELB/alternative NF-κB, and toll-like receptor/interferon signaling. Validation of these results mapped a positive-feedback signaling loop regulated by PAX5. We demonstrated that AZD5153 decreased PAX5 expression, whereas acalabrutinib disruption of BCR signaling inhibited PAX5 activation. Furthermore, several interferon levels were decreased by AZD5153 and acalabrutinib in tumors. Adding interferon-beta1 (IFNß1) to cells treated with acalabrutinib partially rescued PAX5 activation. Our results demonstrate that AZD5153 enhances the efficacy of acalabrutinib through PAX5 and BCR mechanisms that are critical for ABC-DLBCL.
Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Humanos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Interferones , Proteínas de Ciclo Celular , Factor de Transcripción PAX5/genéticaRESUMEN
The cyclin-dependent kinase 5 (CDK5) represents an unusual member of the family of cyclin-dependent kinases, which is activated upon binding to non-cyclin p35 and p39 proteins. The role of CDK5 in the nervous system has been very well established. In addition, there is growing evidence that CDK5 is also active in non-neuronal tissues, where it has been postulated to affect a variety of functions such as the immune response, angiogenesis, myogenesis, melanogenesis and regulation of insulin levels. Moreover, high levels of CDK5 have been observed in different tumour types, and CDK5 was proposed to play various roles in the tumorigenic process. In this review, we discuss these various CDK5 functions in normal physiology and disease, and highlight the therapeutic potential of targeting CDK5.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica , Transducción de Señal , Animales , Humanos , Inmunomodulación , Modelos Biológicos , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/patología , Organogénesis/genética , Organogénesis/inmunologíaRESUMEN
Basal-like triple-negative breast cancers frequently express high levels of c-Myc. This oncoprotein signals to the core cell cycle machinery by impinging on cyclin E. High levels of E-type cyclins (E1 and E2) are often seen in human triple-negative breast tumors. In the current study, we examined the requirement for E-type cyclins in the c-Myc-driven mouse model of breast cancer (MMTV-c-Myc mice). To do so, we crossed cyclin E1- (E1-/-) and E2- (E2-/-) deficient mice with MMTV-c-Myc animals, and observed the resulting cyclin E1-/-/MMTV-c-Myc and cyclin E2-/-/MMTV-c-Myc females for breast cancer incidence. We found that mice lacking cyclins E1 or E2 developed breast cancers like their cyclin Ewild-type counterparts. In contrast, further reduction of the dosage of E-cyclins in cyclin E1-/-E2+/-/MMTV-c-Myc and cyclin E1+/-E2-/-/MMTV-c-Myc animals significantly decreased the incidence of mammary carcinomas, revealing arole for E-cyclins in tumor initiation. We also observed that depletion of E-cyclins in human triple-negative breast cancer cell lines halted cell cycle progression, indicating that E-cyclins are essential for tumor cell proliferation. In contrast, we found that the catalytic partner of E-cyclins, the cyclin-dependent kinase 2 (CDK2), is dispensable for the proliferation of these cells. These results indicate that E-cyclins, but not CDK2, play essential and rate-limiting roles in driving the proliferation of c-Myc overexpressing breast cancer cells.
Asunto(s)
Ciclina E/genética , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias de la Mama Triple Negativas/genética , Animales , Ciclo Celular/genética , División Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Quinasa 2 Dependiente de la Ciclina/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
The mammalian epididymis not only plays a fundamental role in the maturation of spermatozoa, but also provides protection against various stressors. The foremost among these is the threat posed by oxidative stress, which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids, proteins, and nucleic acids. In mice, the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5 (GPX5) as a major luminal scavenger in the proximal caput epididymidal segment. Accordingly, the loss of GPX5-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx5-/- mice. To explore the underlying mechanism, we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged (13 months old) Gpx5-/- mice. This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts, including the downregulation of a subgroup of piRNA pathway genes, in aged Gpx5-/- mice. In agreement with these findings, we also observed the loss of piRNAs, which potentially bind to the P-element-induced wimpy testis (PIWI)-like proteins PIWIL1 and PIWIL2. The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5-/- mice. Importantly, the oxidative stress response genes tend to have more targeting piRNAs, and many of them were among the top increased genes upon the loss of GPX5. Taken together, our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible involvement in the aging and oxidative stress-mediated responses.
Asunto(s)
Epidídimo/metabolismo , Glutatión Peroxidasa/fisiología , ARN Interferente Pequeño/metabolismo , Envejecimiento/metabolismo , Animales , Regulación hacia Abajo , Epidídimo/enzimología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Glutatión Peroxidasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Immunotherapies that target programmed cell death protein 1 (PD-1) and its ligand PD-L1 as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) have shown impressive clinical outcomes for multiple tumours. However, only a subset of patients achieves durable responses, suggesting that the mechanisms of the immune checkpoint pathways are not completely understood. Here, we report that PD-L1 translocates from the plasma membrane into the nucleus through interactions with components of the endocytosis and nucleocytoplasmic transport pathways, regulated by p300-mediated acetylation and HDAC2-dependent deacetylation of PD-L1. Moreover, PD-L1 deficiency leads to compromised expression of multiple immune-response-related genes. Genetically or pharmacologically modulating PD-L1 acetylation blocks its nuclear translocation, reprograms the expression of immune-response-related genes and, as a consequence, enhances the anti-tumour response to PD-1 blockade. Thus, our results reveal an acetylation-dependent regulation of PD-L1 nuclear localization that governs immune-response gene expression, and thereby advocate targeting PD-L1 translocation to enhance the efficacy of PD-1/PD-L1 blockade.
Asunto(s)
Antígeno B7-H1/metabolismo , Núcleo Celular/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Acetilación , Animales , Línea Celular , Línea Celular Tumoral , Proteína p300 Asociada a E1A/metabolismo , Expresión Génica/fisiología , Células HEK293 , Humanos , Inmunoterapia/métodos , Células MCF-7 , Ratones , Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Células RAW 264.7RESUMEN
Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3ß, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3ß phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Reparación del ADN/genética , Fase G1/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular Tumoral , Daño del ADN/genética , Células HEK293 , Células HeLa , Humanos , Mitosis/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
Overexpression of cyclin D1 and its catalytic partner, CDK4, is frequently seen in human cancers. We constructed cyclin D1 and CDK4 protein interaction network in a human breast cancer cell line MCF7, and identified novel CDK4 protein partners. Among CDK4 interactors we observed several proteins functioning in protein folding and in complex assembly. One of the novel partners of CDK4 is FKBP5, which we found to be required to maintain CDK4 levels in cancer cells. An integrative analysis of the extended cyclin D1 cancer interactome and somatic copy number alterations in human cancers identified BAIAPL21 as a potential novel human oncogene. We observed that in several human tumor types BAIAPL21 is expressed at higher levels as compared to normal tissue. Forced overexpression of BAIAPL21 augmented anchorage independent growth, increased colony formation by cancer cells and strongly enhanced the ability of cells to form tumors in vivo. Lastly, we derived an Aggregate Expression Score (AES), which quantifies the expression of all cyclin D1 interactors in a given tumor. We observed that AES has a prognostic value among patients with ER-positive breast cancers. These studies illustrate the utility of analyzing the interactomes of proteins involved in cancer to uncover potential oncogenes, or to allow better cancer prognosis.