RESUMEN
Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/fisiología , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Quinasa CDC2 , Línea Celular Tumoral , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Modelos Teóricos , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Entry into mitosis is driven by the activity of kinases, which phosphorylate over 7000 proteins on multiple sites. For cells to exit mitosis and segregate their genome correctly, these phosphorylations must be removed in a specific temporal order. This raises a critical and important question: how are specific phosphorylation sites on an individual protein removed? Traditionally, the temporal order of dephosphorylation was attributed to decreasing kinase activity. However, recent evidence in human cells has identified unique patterns of dephosphorylation during mammalian mitotic exit that cannot be fully explained by the loss of kinase activity. This suggests that specificity is determined in part by phosphatases. In this review, we explore how the physicochemical properties of an individual phosphosite and its surrounding amino acids can affect interactions with a phosphatase. These positive and negative interactions in turn help determine the specific pattern of dephosphorylation required for correct mitotic exit.
Asunto(s)
Puntos de Control del Ciclo Celular , Mitosis , Monoéster Fosfórico Hidrolasas/metabolismo , Humanos , Especificidad por SustratoRESUMEN
Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3300 unique proteins during early mitotic exit, providing up to eightfold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (â¼ 10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition.
Asunto(s)
Mitosis , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteómica/métodos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Anafase , Secuencia Conservada , Evolución Molecular , Células HeLa , Humanos , Metafase , Modelos Biológicos , Datos de Secuencia Molecular , Fosfopéptidos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
We present a cohort review of TORS resection for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) and its associated oncological outcomes spanning a 10-year period. A retrospective case series review was performed of patients undergoing primary surgical treatment for HPV-associated OPSCC through the St. Vincent's Head and Neck Cancer service from 2011 to 2022. The primary outcomes were to investigate complete resection of the primary tumour, rates of recurrence, and survival analysis. Secondary outcomes included complications, rates of adjuvant therapy, sites of recurrence and rates of percutaneous endoscopic gastrostomy (PEG). 184 patients underwent TORS-based therapy with neck dissection, and guideline-directed adjuvant therapy for HPV-associated OPSCC. Our median follow-up was 46 months. The positive margin rate on final histopathology analysis was 10.9%. Adjuvant therapy was indicated in 85 patients (46%). The local recurrence rate was 10.9% with the majority (80%) of patients recurring in the first 3 years since treatment. The disease-specific survival at 3 years was 98.6% and at 5 years was 94.4%. The 3-year and 5-year OS for the cohort was 96.7% and 92.5%, respectively. The presence of extranodal extension and positive margins were associated with increased risk of recurrence, whereas adjuvant therapy was found to be a protective factor for both overall recurrence and survival. Major complications occurred in 12 patients (6.5%), resulting in one death. This study has demonstrated that primary surgical therapy for HPV-associated OPSCC is a safe and effective treatment modality with low local recurrence and complication rates, and overall survival benefits.
Asunto(s)
Neoplasias Orofaríngeas , Procedimientos Quirúrgicos Robotizados , Humanos , Procedimientos Quirúrgicos Robotizados/métodos , Estudios Retrospectivos , Neoplasias Orofaríngeas/cirugía , Neoplasias Orofaríngeas/virología , Neoplasias Orofaríngeas/patología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Resultado del Tratamiento , Recurrencia Local de Neoplasia , Australia/epidemiología , Adulto , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/cirugía , Carcinoma de Células Escamosas/cirugía , Carcinoma de Células Escamosas/virología , Carcinoma de Células Escamosas/patología , Márgenes de Escisión , Carcinoma de Células Escamosas de Cabeza y Cuello/cirugía , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Disección del Cuello/métodos , Anciano de 80 o más AñosRESUMEN
Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Neoplasias , Proteínas Serina-Treonina Quinasas , Técnicas de Cultivo de Célula , Humanos , Marcaje Isotópico , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
BACKGROUND: Despite advances in immunotherapy and targeted therapy, platinum-based chemotherapy remains crucial for many patients with advanced non-small cell lung cancer (NSCLC). Resistance to platinum chemotherapy is common, and predictive biomarkers are needed to tailor treatment to patients likely to respond. In vitro evidence implicates the transforming growth factor-ß (TGF-ß) superfamily ligands activin-A and growth differentiation factor 11 (GDF-11) in innate platinum resistance. We performed a validation study to assess their utility as predictive biomarkers of platinum chemotherapy response in advanced NSCLC. PATIENTS AND METHODS: Our study included 123 adult patients with advanced NSCLC without a driver mutation treated with platinum chemotherapy. 98 patients were from a retrospective cohort and 25 from a prospective cohort. We performed immunohistochemistry staining for Activin-A, GDF-11 and TGF-ß on tumour samples for each patient and analysed IHC expression with objective radiological response and overall survival. RESULTS: The overall median survival was 14.8 months. We performed statistical analysis around a cytoplasmic score of 8/18 for Activin-A and GDF-11 based on previously published work, and 110/30 for TGF-ß based on a calculated cutpoint for significance. No survival difference was detected between these groups for Activin-A (p=0.35), GDF-11 (p=0.57) or TGF-ß (p=0.34). There was no association between rates of progressive disease and high Activin-A expression (p=0.43), high GDF-11 expression (p=1.0) or high TGF-ß expression p=0.89). CONCLUSION: Within the confines of our study, Activin-A, GDF-11 and TGF-ß expression was not a predictor of objective radiological response to chemotherapy or overall survival.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Compuestos Organoplatinos , Activinas/metabolismo , Activinas/uso terapéutico , Adulto , Biomarcadores , Proteínas Morfogenéticas Óseas , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Factores de Diferenciación de Crecimiento/uso terapéutico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Compuestos Organoplatinos/uso terapéutico , Platino (Metal)/uso terapéutico , Estudios Prospectivos , Estudios Retrospectivos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/uso terapéutico , Factores de Crecimiento Transformadores/uso terapéuticoRESUMEN
To assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared with those from healthy individuals. We performed single-cell RNA sequencing of a total of 247,520 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease. Expression quantitative trait mapping identified a total of 4,443 significant loci across all cell types, 312 of which are specific to the retinal ganglion cell subpopulations, which ultimately degenerate in POAG. Transcriptome-wide association analysis identified genes at loci previously associated with POAG, and analysis, conditional on disease status, implicated 97 statistically significant retinal ganglion cell-specific expression quantitative trait loci. This work highlights the power of large-scale iPSC studies to uncover context-specific profiles for a genetically complex disease.
RESUMEN
There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.
Asunto(s)
Atrofia Geográfica , Degeneración Macular , Humanos , Degeneración Macular/genética , Proteómica , Epitelio Pigmentado de la Retina , Transcriptoma/genéticaRESUMEN
BACKGROUND: Human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) continues to increase in incidence. Patients are younger, non-smokers and most commonly present with a neck mass often with no other symptoms. This altered presentation compared with non-HPV OPSCC may not be recognized by medical practitioners, leading to delayed diagnosis. METHODS: Patients with histopathological confirmation of OPSCC and known HPV and/or P16 status who presented to our institution between 2012-2017 inclusive were included in the study. Demographic data, tumour characteristics and presenting symptoms were retrospectivxely obtained from both electronic- and paper-based records. Descriptive statistics were used to report demographic data and the two sample t-test and Fisher's exact test were used to compare groups based on HPV status. Time to diagnosis was also reported. RESULTS: A total of 184 patients were included in the study. The majority of patients were male (85.4%) and HPV + (85.3%). The tonsillar complex (53.8%) and tongue base (42.4%) were the most common primary sites. HPV+ patients were less likely to smoke (17.8%) and they commonly presented with a neck mass (39.5% alone or with other symptoms 61.2%). Time to diagnosis in the HPV+ group was longer (15 weeks). CONCLUSION: Our review has highlighted the altered presentation of OPSCC due to the increased incidence of HPV infection. We showed a delayed time to diagnosis in HPV+ OPSCC compared with non-HPV disease. This confirms the importance of focusing our efforts on educating medical practitioners and creating further awareness to facilitate early detection and treatment.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/epidemiología , Femenino , Humanos , Masculino , Neoplasias Orofaríngeas/diagnóstico , Neoplasias Orofaríngeas/epidemiología , Papillomaviridae , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/epidemiologíaRESUMEN
We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Antineoplásicos/farmacología , Carboplatino/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Neoplasias Pulmonares/patología , Adenocarcinoma del Pulmón/metabolismo , Animales , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Recombinasa Rad51 , Análisis de la Célula Individual , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The identification of clinically viable strategies for overcoming resistance to platinum chemotherapy in lung adenocarcinoma has previously been hampered by inappropriately tailored in vitro assays of drug response. Therefore, using a pulse model that closely mimics the in vivo pharmacokinetics of platinum therapy, we profiled cisplatin-induced signalling, DNA-damage and apoptotic responses across a panel of human lung adenocarcinoma cell lines. By coupling this data to real-time, single-cell imaging of cell cycle and apoptosis we provide a fine-grained stratification of response, where a P70S6K-mediated signalling axis promotes resistance on a TP53 wildtype or null background, but not a mutant TP53 background. This finding highlights the value of in vitro models that match the physiological pharmacokinetics of drug exposure. Furthermore, it also demonstrates the importance of a mechanistic understanding of the interplay between somatic mutations and the signalling networks that govern drug response for the implementation of any consistently effective, patient-specific therapy.
Lung adenocarcinoma is the most common type of lung cancer, and it emerges because of a variety of harmful genetic changes, or mutations. Two lung cancer patients or indeed, two different sets of cancerous cells within a patient may therefore carry different damaging mutations. A group of drugs called platinum-based chemotherapies are currently the most effective way to treat lung adenocarcinoma. Yet, only 30% of patients actually respond to the therapy. Many studies conducted in laboratory settings have tried to understand why most cases are resistant to treatment, with limited success. Here, Hastings, Gonzalez-Rajal et al. propose that previous research has been inconclusive because studies done in the laboratory do not reflect how the treatment is actually administered. In patients, platinum-based drugs are cleared from the body within a few hours, but during experiments, the treatment is continually administered to cells growing in a dish. Hastings, Gonzalez-Rajal et al. therefore developed a laboratory method that mimics the way cells are exposed to platinum-based chemotherapy in the body. These experiments showed that the lung adenocarcinoma cells which resisted treatment also carried high levels of a protein known as P70S6K. Pairing platinum-based chemotherapy with a drug that blocks the activity of P70S6K killed these resistant cells. This combination also treated human lung adenocarcinoma tumours growing under the skin of mice. However, it was ineffective on cancerous cells that carry a mutation in a protein called p53, which is often defective in cancers. Overall, this work demonstrates the need to refine how drugs are tested in the laboratory to better reflect real-life conditions. It also underlines the importance of personalizing drug combinations to the genetic background of each tumour, a concept that will be vital to consider in future clinical trials.
Asunto(s)
Adenocarcinoma del Pulmón , Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.
Asunto(s)
Heterogeneidad Genética , Neoplasias Pulmonares/genética , Neoplasias Torácicas/genética , Neoplasias Torácicas/secundario , Secuenciación Completa del Genoma/métodos , Adenocarcinoma del Pulmón/genética , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/clasificación , Carcinoma de Células Escamosas/genética , Variaciones en el Número de Copia de ADN , Femenino , Efecto Fundador , Interacción Gen-Ambiente , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mutación , Carcinoma Pulmonar de Células Pequeñas/genética , Microambiente TumoralRESUMEN
Primary cilia are crucial for signal transduction in a variety of pathways, including hedgehog and Wnt. Disruption of primary cilia formation (ciliogenesis) is linked to numerous developmental disorders (known as ciliopathies) and diseases, including cancer. The ubiquitin-proteasome system (UPS) component UBR5 was previously identified as a putative positive regulator of ciliogenesis in a functional genomics screen. UBR5 is an E3 ubiquitin ligase that is frequently deregulated in tumors, but its biological role in cancer is largely uncharacterized, partly due to a lack of understanding of interacting proteins and pathways. We validated the effect of UBR5 depletion on primary cilia formation using a robust model of ciliogenesis, and identified CSPP1, a centrosomal and ciliary protein required for cilia formation, as a UBR5-interacting protein. We show that UBR5 ubiquitylates CSPP1, and that UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery, suggesting that UBR5-mediated ubiquitylation of CSPP1 or associated centriolar satellite constituents is one underlying requirement for cilia expression. Hence, we have established a key role for UBR5 in ciliogenesis that may have important implications in understanding cancer pathophysiology.
Asunto(s)
Centriolos/metabolismo , Cilios/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Biopsia , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Poliubiquitina/metabolismo , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.
Asunto(s)
Inestabilidad Cromosómica/genética , Factores de Transcripción de Tipo Kruppel/fisiología , Neoplasias/genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Senescencia Celular/genética , Embrión de Mamíferos , Femenino , Genes Supresores de Tumor/fisiología , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/patologíaRESUMEN
MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial-mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Inestabilidad Cromosómica/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Serina-Treonina Quinasas/genética , Citoesqueleto de Actina/genética , Animales , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Daño del ADN/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.
Asunto(s)
Activinas/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Platino (Metal)/uso terapéutico , Células A549 , Animales , Carboplatino/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Folistatina/uso terapéutico , Humanos , Masculino , Ratones , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.
Asunto(s)
Progresión de la Enfermedad , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Paclitaxel Unido a Albúmina/farmacología , Paclitaxel Unido a Albúmina/uso terapéutico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Técnicas Biosensibles , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Matriz Extracelular/metabolismo , Humanos , Hígado/patología , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Quinasas Asociadas a rho/metabolismo , Familia-src Quinasas/metabolismo , GemcitabinaRESUMEN
The presence or absence of a phosphorylation on a substrate at any particular point in time is a functional readout of the balance in activity between the regulatory kinase and the counteracting phosphatase. Understanding how stable or short-lived a phosphorylation site is required for fully appreciating the biological consequences of the phosphorylation. Our current understanding of kinases and their substrates is well established; however, the role phosphatases play is less understood. Therefore, we utilized a phosphatase dependent model of mitotic exit to identify potential substrates that are preferentially dephosphorylated. Using this method, we identified >16,000 phosphosites on >3300 unique proteins, and quantified the temporal phosphorylation changes that occur during early mitotic exit (McCloy et al., 2015 [1]). Furthermore, we annotated the majority of these phosphorylation sites with a high confidence upstream kinase using published, motif and prediction based methods. The results from this study have been deposited into the ProteomeXchange repository with identifier PXD001559. Here we provide additional analysis of this dataset; for each of the major mitotic kinases we identified motifs that correlated strongly with phosphorylation status. These motifs could be used to predict the stability of phosphorylated residues in proteins of interest, and help infer potential functional roles for uncharacterized phosphorylations. In addition, we provide validation at the single cell level that serine residues phosphorylated by Cdk are stable during phosphatase dependent mitotic exit. In summary, this unique dataset contains information on the temporal mitotic stability of thousands of phosphorylation sites regulated by dozens of kinases, and information on the potential preference that phosphatases have at both the protein and individual phosphosite level. The compellation of this data provides an invaluable resource for the wider research community.
RESUMEN
Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G 2 phase onwards. Addition of low doses of RO3306 in G 2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G 2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G 2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.