RESUMEN
While Epstein-Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B-cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B-cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B-cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Animales , Proteínas de Ciclo Celular , Infecciones por Virus de Epstein-Barr/complicaciones , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Latencia del Virus , Quinasa Tipo Polo 1RESUMEN
Cervical cancer is the fourth most frequently diagnosed and fatal gynecological cancer. 15-61% of all cases metastasize and develop chemoresistance, reducing the 5-year survival of cervical cancer patients to as low as 17%. Therefore, unraveling the mechanisms contributing to metastasis is critical in developing better-targeted therapies against it. Here, we have identified a novel mechanism where nuclear Caspase-8 directly interacts with and inhibits the activity of CDK9, thereby modulating RNAPII-mediated global transcription, including those of cell-migration- and cell-invasion-associated genes. Crucially, low Caspase-8 expression in cervical cancer patients leads to poor prognosis, higher CDK9 phosphorylation at Thr186, and increased RNAPII activity in cervical cancer cell lines and patient biopsies. Caspase-8 knock-out cells were also more resistant to the small-molecule CDK9 inhibitor BAY1251152 in both 2D- and 3D-culture conditions. Combining BAY1251152 with Cisplatin synergistically overcame chemoresistance of Caspase-8-deficient cervical cancer cells. Therefore, Caspase-8 expression could be a marker in chemoresistant cervical tumors, suggesting CDK9 inhibitor treatment for their sensitization to Cisplatin-based chemotherapy.
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ARN Polimerasa II , Neoplasias del Cuello Uterino , Humanos , Femenino , ARN Polimerasa II/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Fosforilación , Caspasa 8/genética , Caspasa 8/metabolismo , Cisplatino/farmacología , Inhibidores de Proteínas Quinasas , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismoRESUMEN
Ovarian cancer (OC) accounts for approximately 4% of cancer deaths in women worldwide and is the deadliest gynecologic malignancy. High-grade serous ovarian cancer (HGSOC) is the most predominant ovarian cancer, in which BRCA1/2 gene mutation ranges from 3 to 27%. PARP inhibitors (PARPi) have shown promising results as a synthetically lethal therapeutic approach for BRCA mutant and recurrent OC in clinical use. However, emerging data indicate that BRCA-deficient cancers may be resistant to PARPi, and the mechanisms of this resistance remain elusive. We found that amplification of KRAS likely underlies PARPi resistance in BRCA2-deficient HGSOC. Our data suggest that PLK1 inhibition restores sensitivity to PARPi in HGSOC with KRAS amplification. The sequential combination of PLK1 inhibitor (PLK1i) and PARPi drastically reduces HGSOC cell survival and increases apoptosis. Furthermore, we were able to show that a sequential combination of PLK1i and PARPi enhanced the cellular apoptotic response to carboplatin-based chemotherapy in KRAS-amplified resistant HGSOC cells and 3D spheroids derived from recurrent ovarian cancer patients. Our results shed new light on the critical role of PLK1 in reversing PARPi resistance in KRAS-amplified HGSOC, and offer a new therapeutic strategy for this class of ovarian cancer patients where only limited options currently exist.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cistadenocarcinoma Seroso , Neoplasias Ováricas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína BRCA1/genética , Carboplatino/uso terapéutico , Cistadenocarcinoma Seroso/tratamiento farmacológico , Femenino , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ftalazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Quinasa Tipo Polo 1RESUMEN
T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Tolerancia Inmunológica/inmunología , Proteínas de la Membrana/metabolismo , Receptores Virales/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos CD/química , Antígenos CD/inmunología , Autoinmunidad/inmunología , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Línea Celular , Neoplasias Colorrectales/inmunología , Modelos Animales de Enfermedad , Femenino , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Inflamación/inmunología , Inflamación/patología , Ligandos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Conformación Proteica , Multimerización de Proteína , Receptores Virales/química , Receptores Virales/inmunologíaRESUMEN
Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. To do this, we used a specific anaphase-promoting complex/cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the antiapoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells lines with CCNE1 amplification that are challenged with clinically relevant dose of paclitaxel. Using time-laps microscopy, we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cell lines and restores death in mitosis. Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient's survival. Thus, our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel-based efficacy in this highly lethal gynecological disease.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Ciclina E/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Ciclina E/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Resistencia a Antineoplásicos , Femenino , Amplificación de Genes , Humanos , Mitosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Clasificación del Tumor , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patologíaRESUMEN
Although essential for T cell function, the identity of the T cell receptor "inside-out" pathway for lymphocyte function-associated antigen 1 (LFA-1) adhesion has proved elusive. Here, we define the "inside-out" pathway mediated by N-terminal SKAP1 (SKAP-55) domain binding to the C-terminal SARAH domain of RapL. TcR induced Rap1-RapL complex formation and LFA-1 binding failed to occur in Skap1(-/-) primary T cells. SKAP1 generated a SKAP1-RapL-Rap1 complex that bound to LFA-1, whereas a RapL mutation (L224A) that abrogated SKAP1 binding without affecting MST1 disrupted component colocalization in vesicles as well as T cell-dendritic cell (DC) conjugation. RapL expression also "slowed" T cell motility in D011.10 transgenic T cells in lymph nodes (LNs), an effect reversed by the L224A mutation with reduced dwell times between T cells and DCs. Overall, our findings define a TCR "inside-out" pathway via N-SKAP1-C-RapL that regulates T cell adhesion, motility, and arrest times with DCs in LNs.
Asunto(s)
Ganglios Linfáticos/inmunología , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Proteínas de Unión al GTP rap1/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Reguladoras de la Apoptosis , Adhesión Celular , Movimiento Celular , Células Cultivadas , Células Dendríticas/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/inmunología , Mutación , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Unión Proteica , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismo , Proteínas de Unión al GTP rap1/inmunologíaRESUMEN
The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, "3Y") as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4+ primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación de Linfocitos/fisiología , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Humanos , Células Jurkat , Ratones , Mutación Missense , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosforilación/fisiología , Dominios Proteicos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , TirosinaRESUMEN
BACKGROUND: Metaphors, mainly proverbs and idiomatic expressions of ordinary life are commonly used as a model for concretism. Previous studies have shown impaired metaphor comprehension in patients with schizophrenia-spectrum disorders compared to either psychiatric or non-psychiatric control subject. The aim of this study was to detect possible quantitative differences in figurative processing between patients with schizophrenia-spectrum disorders and healthy controls. METHODS: In order to analyse possible dissociations of different aspects of figurative speech, a range of metaphor tasks was used to distinguish between recognition of familiar metaphors, paraphrasing the meaning of the latter and generating novel metaphors: we used a standard proverb test for conventional metaphors consisting of a multiple-choice and a paraphrasing task, and the Metaphoric Triads Test for the assessment of novel metaphors. We included 40 patients with schizophrenia-spectrum disorders and 43 healthy control subjects. RESULTS: Our results showed that patients had impaired figurative speech processing regarding novel and conventional metaphors. Associations with cognitive functions were detected. Performance on the paraphrasing task was associated with the severity of negative symptoms. CONCLUSION: We conclude that patients with schizophrenia-spectrum disorders do exhibit impairments in the recognition and paraphrasing of conventional and the generation of novel metaphors and that some cognitive domains as well the extent of negative symptoms might be associated with these deficits.
Asunto(s)
Comprensión , Trastornos del Lenguaje/diagnóstico , Trastornos del Lenguaje/psicología , Metáfora , Trastornos Psicóticos/diagnóstico , Trastornos Psicóticos/psicología , Esquizofrenia/diagnóstico , Psicología del Esquizofrénico , Adulto , Trastornos del Conocimiento/diagnóstico , Trastornos del Conocimiento/psicología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas/estadística & datos numéricos , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Psicometría , Valores de ReferenciaRESUMEN
BACKGROUND: The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. METHODS: To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. RESULTS: Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. CONCLUSIONS: The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.
Asunto(s)
Neoplasias Ováricas , Proteína p53 Supresora de Tumor , Animales , Ratones , Humanos , Femenino , Proteína p53 Supresora de Tumor/genética , ARN Mensajero/genética , Clasificación del Tumor , Línea Celular Tumoral , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Inestabilidad Cromosómica , MamíferosRESUMEN
T-cell activation is a complex process involving a network of kinases and downstream molecular scaffolds or adaptors that integrate surface signals with effector functions. One key immune-specific adaptor is Src kinase-associated phosphoprotein 1 (SKAP1), which is also known as src kinase-associated protein of 55 kDa (SKAP55). This mini-review explains how SKAP1 plays multiple roles in regulating integrin activation, the "stop-signal", and the optimization of the cell cycling of proliferating T cells through interactions with various mediators, including the Polo-like kinase 1 (PLK1). Ongoing research on SKAP1 and its binding partners will likely provide important insights into the regulation of immune function and have implications for the development of new treatments for disease states such as cancer and autoimmunity.
Asunto(s)
Fosfoproteínas , Linfocitos T , Linfocitos T/metabolismo , Fosfoproteínas/metabolismo , Oligonucleótidos , Integrinas/metabolismo , Familia-src Quinasas/metabolismo , Proliferación CelularRESUMEN
Ovarian cancer is one of the most lethal gynecological cancers worldwide, with approximately 70% of cases diagnosed in advanced stages. This late diagnosis results from the absence of early warning symptoms and is associated with an unfavorable prognosis. A standard treatment entails a combination of primary chemotherapy with platinum and taxane agents. Tumor recurrence following first-line chemotherapy with Carboplatin and Paclitaxel is detected in 80% of advanced ovarian cancer patients, with disease relapse occurring within 2 years of initial treatment. Platinum-resistant ovarian cancer is one of the biggest challenges in treating patients. Second-line treatments involve PARP or VEGF inhibitors. Identifying novel biomarkers and resistance mechanisms is critical to overcoming resistance, developing newer treatment strategies, and improving patient survival. In this study, we have determined that low Caspase-8 expression in ovarian cancer patients leads to poor prognosis. High-Grade Serous Ovarian Cancer (HGSOC) cells lacking Caspase-8 expression showed an altered composition of the RNA Polymerase II-containing transcriptional elongation complex leading to increased transcriptional activity. Caspase-8 knockout cells display increased BRD4 expression and CDK9 activity and reduced sensitivities to Carboplatin and Paclitaxel. Based on our work, we are proposing three potential therapeutic approaches to treat advanced ovarian cancer patients who exhibit low Caspase-8 expression and resistance to Carboplatin and/or Paclitaxel-combinations of (1) Carboplatin with small-molecule BRD4 inhibitors; (2) Paclitaxel with small-molecule BRD4 inhibitors, and (3) small-molecule BRD4 and CDK9 inhibitors. In addition, we are also proposing two predictive markers of chemoresistance-BRD4 and pCDK9.
RESUMEN
Salt-inducible kinases 1-3 (SIK1-3) are key regulators of the LKB1-AMPK pathway and play an important role in cellular homeostasis. Dysregulation of any of the three isoforms has been associated with tumorigenesis in liver, breast, and ovarian cancers. We have recently developed the dual pan-SIK/group I p21-activated kinase (PAK) chemical probe MRIA9. However, inhibition of p21-activated kinases has been associated with cardiotoxicity in vivo, which complicates the use of MRIA9 as a tool compound. Here, we present a structure-based approach involving the back-pocket and gatekeeper residues, for narrowing the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one-based inhibitors towards SIK kinases, eliminating PAK activity. Optimization was guided by high-resolution crystal structure analysis and computational methods, resulting in a pan-SIK inhibitor, MR22, which no longer exhibited activity on STE group kinases and displayed excellent selectivity in a representative kinase panel. MR22-dependent SIK inhibition led to centrosome dissociation and subsequent cell-cycle arrest in ovarian cancer cells, as observed with MRIA9, conclusively linking these phenotypic effects to SIK inhibition. Taken together, MR22 represents a valuable tool compound for studying SIK kinase function in cells.
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Proteínas Quinasas Activadas por AMP , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas Activadas por AMP/metabolismo , Hígado/metabolismo , Isoformas de Proteínas , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Although essential for T cell function, the identity of the T cell receptor (TCR) "inside-out" pathway for the activation of lymphocyte function-associated antigen 1 (LFA-1) is unclear. SKAP1 (SKAP-55) is the upstream regulator needed for TCR-induced RapL-Rap1 complex formation and LFA-1 activation. In this paper, we show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway. A SKAP1 PH domain-inactivating mutation (i.e. R131M) markedly impaired RapL translocation to membranes for Rap1 and LFA-1 binding and the up-regulation of LFA-1-intercellular adhesion molecule 1 (ICAM-1) binding. Further, N-terminal myr-tagged SKAP1 for membrane binding facilitated constitutive RapL membrane and Rap1 binding and effectively substituted for PI3K and TCR ligation in the activation of LFA-1 in T cells.
Asunto(s)
Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Células Jurkat , Antígeno-1 Asociado a Función de Linfocito/genética , Proteínas de Unión al GTP Monoméricas/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Receptores de Antígenos de Linfocitos T/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Cervical cancer is one of the most serious health conditions, with nearly 500,000 women developing the disease each year worldwide. At present, the treatment of recurrent cervical cancer remains largely ineffective, and efforts in cancer drug development are currently focused on critical serine/threonine kinases, such as death-associated protein kinase 1 (DAPK1) and polo-like kinase 1 (PLK1). In the current study, we aimed at exploring the cell cycle roles of DAPK1 and PLK1 in cervical cancer cells. To achive this goal, we used multiple methods including western blotting and assays for studying kinase activity, apoptosis, cell cycle, cell proliferation, immunofluorescence and proximity ligation. The present study demonstrated that, in cervical cancer cells, the enzymatic activity of DAPK1 was regulated in a cell cycle-specific manner. NIMA-related kinases, CDKs, PLKs and Aurora kinases regulate the function of centrosomes by orchestrating the separation of chromosomes during cell division. The present study added DAPK1 to this group of protein kinases due to its localization at centrosomes during mitosis. It was shown that DAPK1 was autophosphorylated at Ser308 in the G2 phase and during mitosis. From prophase to metaphase, the colocalization of PLK1 and DAPK1 at centrosomes was observed. Furthermore, the interaction of both these kinases could be demonstrated using proximity ligations assays and immunoprecipitations. DAPK1 was found to be a substrate of PLK1. Topotecan is an effective drug used for the treatment of cervical cancer. Therefore, the current study examined the role of DAPK1 in topotecan-induced cervical cancer cell death, and it was identified that RNA interference-based silencing of DAPK1 decreased the apoptotic effect of topotecan. Thus, these findings suggested that DAPK1 could be a biomarker and a potential target for the response to topotecan during the therapy of patients with cervical cancer.
RESUMEN
Polo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Activación Enzimática/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Técnicas de Cultivo de Célula , Dimerización , Humanos , Transfección , Quinasa Tipo Polo 1RESUMEN
Introduction: After primary platinum-based chemoradiation of locally advanced uterine cervical cancer, a substantial proportion of women present with persistent, recurrent or metastatic disease, indicating an unmet need for biomarker development. Methods: We evaluated the clinical records of 69 cervical cancer patients (Federation of Gynecology and Obstetrics, FIGO Stage > IB3) who were subjected to definitive CRT. Immunohistochemical scoring of caspase-8, cyclin dependent kinase 9 (CDK9) and phosphorylated (phospho-)CDK9 (threonine (Thr) 186) was performed on pretreatment samples and correlated with the histopathological and clinical endpoints, including relapse-free survival (RFS), distant metastasis-free survival (DMFS), cancer-specific survival (CSS) and overall survival (OS). Results: Lower levels of caspase-8 were more prevalent in patients with a higher T-stage (p = 0.002) and a higher FIGO stage (p = 0.003), and were significantly correlated with CDK9 expression (p = 0.018) and inversely with pCDK9 detection (p = 0.014). Increased caspase-8 levels corresponded to improved RFS (p = 0.005), DMFS (p = 0.038) and CSS (p = 0.017) in the univariate analyses. Low CDK9 expression was associated with worse RFS (p = 0.008), CSS (p = 0.015) and OS (p = 0.007), but not DMFS (p = 0.083), and remained a significant prognosticator for RFS (p = 0.003) and CSS (p = 0.009) in the multivariate analyses. Furthermore, low pCDK9 staining was significantly associated with superior RFS (p = 0.004) and DMFS (p = 0.001), and increased CSS (p = 0.022), and remained significant for these endpoints in the multivariate analyses. Conclusion: Increased caspase-8 and CDK9 levels correlate with improved disease-related outcomes in cervical cancer patients treated with CRT, whereas elevated pCDK9 levels predict worse survival in this patient population.
RESUMEN
Inhibitors of mitotic protein kinases are currently being developed as non-neurotoxic alternatives of microtubule-targeting agents (taxanes, vinca alkaloids) which provide a substantial survival benefit for patients afflicted with different types of solid tumors. Among the mitotic kinases, the cyclin-dependent kinases, the Aurora kinases, the kinesin spindle protein and Polo-like kinases (PLKs) have emerged as attractive targets of cancer therapeutics. The functions of mammalian PLK1-5 are traditionally linked to the regulation of the cell cycle and to the stress response. Especially the key role of PLK1 and PLK4 in cellular growth and proliferation, their overexpression in multiple types of human cancer and their druggability, make them appealing targets for cancer therapy. Inhibitors for PLK1 and PLK4 are currently being tested in multiple cancer trials. The clinical success of microtubule-targeting agents is attributed not solely to the induction of a mitotic arrest in cancer cells, but also to non-mitotic effects like targeting intracellular trafficking on microtubules. This raises the question whether new cancer targets like PLK1 and PLK4 regulate critical non-mitotic functions in tumor cells. In this article we summarize the important roles of PLK1-5 for the regulation of non-mitotic signaling. Due to these functions it is conceivable that inhibitors for PLK1 or PLK4 can target interphase cells, which underscores their attractive potential as cancer drug targets. Moreover, we also describe the contribution of the tumor-suppressors PLK2, PLK3 and PLK5 to cancer cell signaling outside of mitosis. These observations highlight the urgent need to develop highly specific ATP-competitive inhibitors for PLK4 and for PLK1 like the 3rd generation PLK-inhibitor Onvansertib to prevent the inhibition of tumor-suppressor PLKs in- and outside of mitosis. The remarkable feature of PLKs to encompass a unique druggable domain, the polo-box-domain (PBD) that can be found only in PLKs offers the opportunity for the development of inhibitors that target PLKs exclusively. Beyond the development of mono-specific ATP-competitive PLK inhibitors, the PBD as drug target will support the design of new drugs that eradicate cancer cells based on the mitotic and non-mitotic function of PLK1 and PLK4.