RESUMEN
Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer survival but are associated with treatment-limiting hypertension, often attributed to endothelial cell (EC) dysfunction. Using phosphoproteomic profiling of VEGFRi-treated ECs, drugs were screened for mitigators of VEGFRi-induced EC dysfunction and validated in primary aortic ECs, mice, and canine cancer patients. VEGFRi treatment significantly raised systolic blood pressure (SBP) and increased markers of endothelial and renal dysfunction in mice and canine cancer patients. α-Adrenergic-antagonists were identified as drugs that most oppose the VEGFRi proteomic signature. Doxazosin, one such α-antagonist, prevented EC dysfunction in murine, canine, and human aortic ECs. In mice with sorafenib-induced-hypertension, doxazosin mitigated EC dysfunction but not hypertension or glomerular endotheliosis, while lisinopril mitigated hypertension and glomerular endotheliosis without impacting EC function. Hence, reversing EC dysfunction was insufficient to mitigate VEGFRi-induced-hypertension in this mouse model. Canine cancer patients with VEGFRi-induced-hypertension were randomized to doxazosin or lisinopril and both agents significantly decreased SBP. The canine clinical trial supports safety and efficacy of doxazosin and lisinopril as antihypertensives for VEGFRi-induced-hypertension and the potential of trials in canines with spontaneous cancer to accelerate translation. The overall findings demonstrate the utility of phosphoproteomics to identify EC-protective agents to mitigate cardio-oncology side effects.
Asunto(s)
Doxazosina , Células Endoteliales , Hipertensión , Receptores de Factores de Crecimiento Endotelial Vascular , Animales , Perros , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Hipertensión/fisiopatología , Doxazosina/farmacología , Doxazosina/uso terapéutico , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteómica/métodos , Presión Sanguínea/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Lisinopril/farmacología , Lisinopril/uso terapéutico , Masculino , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Antihipertensivos/farmacología , Antihipertensivos/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Antagonistas de Receptores Adrenérgicos alfa 1/farmacología , Antagonistas de Receptores Adrenérgicos alfa 1/uso terapéutico , Sorafenib/farmacología , Sorafenib/uso terapéuticoRESUMEN
Epidermal growth factor receptor (EGFR) is commonly over-expressed in metastatic breast cancer yet metastatic breast cancer is generally resistant to anti-EGFR therapies, and the mechanism for resistance to EGFR inhibitors in this setting is not fully understood. Hormonally up-regulated neu-associated kinase (HUNK) kinase is up-regulated in aggressive breast cancers and is thought to play a role in breast cancer metastasis. However, no studies have been conducted to examine a relationship between EGFR and HUNK in breast cancer metastasis. We performed a kinase substrate screen and identified that EGFR is phosphorylated by HUNK. Our studies show that HUNK phosphorylates EGFR at T654, enhancing receptor stability and downstream signaling. We found that increased phosphorylation of T654 EGFR correlates with increased epithelial to mesenchymal, migration and invasion, and metastasis. In addition, we found that HUNK expression correlates with overall survival and distant metastasis free survival. This study shows that HUNK directly phosphorylates EGFR at T654 to promote metastasis and is the first study to show that the phosphorylation of this site in EGFR regulates metastasis.
Asunto(s)
Neoplasias de la Mama/patología , Receptores ErbB/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Transformación Celular Neoplásica , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación , Transducción de SeñalRESUMEN
BACKGROUND: Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination of the Beclin-1 protein complex. Rubicon is a Beclin-1 associated protein that suppresses autophagy by impairing the activity of the class III PI3K, Vps34. However, very little is known about the molecular mechanisms that regulate Rubicon function. METHODS: In this study, co-immunoprecipitation and kinase assays were used to investigate the ability of Hormonally Upregulated Neu-associated Kinase (HUNK) to bind to and phosphorylate Rubicon. LC3B was monitored by immunofluorescence and immunoblotting to determine whether phosphorylation of Rubicon by HUNK controls the autophagy suppressive function of Rubicon. RESULTS: Findings from this study identify Rubicon as a novel substrate of HUNK and show that phosphorylation of Rubicon inhibits its function, promoting autophagy.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia/fisiología , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Fagosomas/metabolismoRESUMEN
BACKGROUND: Epigenetic alterations comprise key regulatory events that dynamically alter gene expression and their deregulation is commonly linked to the pathogenesis of various diseases, including cancer. Unlike DNA mutations, epigenetic alterations involve modifications to proteins and nucleic acids that regulate chromatin structure without affecting the underlying DNA sequence, altering the accessibility of the transcriptional machinery to the DNA, thus modulating gene expression. In cancer cells, this often involves the silencing of tumor suppressor genes or the increased expression of genes involved in oncogenesis. Advances in laboratory medicine have made it possible to map critical epigenetic events, including histone modifications and DNA methylation, on a genome-wide scale. Like the identification of genetic mutations, mapping of changes to the epigenetic landscape has increased our understanding of cancer progression. However, in contrast to irreversible genetic mutations, epigenetic modifications are flexible and dynamic, thereby making them promising therapeutic targets. Ongoing studies are evaluating the use of epigenetic drugs in chemotherapy sensitization and immune system modulation. With the preclinical success of drugs that modify epigenetics, along with the FDA approval of epigenetic drugs including the DNA methyltransferase 1 (DNMT1) inhibitor 5-azacitidine and the histone deacetylase (HDAC) inhibitor vorinostat, there has been a rise in the number of drugs that target epigenetic modulators over recent years. CONCLUSION: We provide an overview of epigenetic modulations, particularly those involved in cancer, and discuss the recent advances in drug development that target these chromatin-modifying events, primarily focusing on novel strategies to regulate the epigenome.
Asunto(s)
Antineoplásicos/uso terapéutico , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Metilación de ADN/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias/genéticaRESUMEN
HUNK is a protein kinase that is implicated in HER2-positive (HER2+) breast cancer progression and resistance to HER2 inhibitors. Though prior studies suggest there is therapeutic potential for targeting HUNK in HER2+ breast cancer, pharmacological agents that target HUNK are yet to be identified. A recent study showed that the broad-spectrum kinase inhibitor staurosporine binds to the HUNK catalytic domain, but the effect of staurosporine on HUNK enzymatic activity was not tested. We now show that staurosporine inhibits the kinase activity of a full length HUNK protein. Our findings further suggest that inhibiting HUNK with staurosporine has a strong effect on suppressing cell viability of HER2/neu mammary and breast cancer cells, which express high levels of HUNK protein and are dependent on HUNK for survival. Significantly, we use in vitro and in vivo methods to show that staurosporine synergizes with the HER2 inhibitor lapatinib to restore sensitivity toward HER2 inhibition in a HER2 inhibitor resistant breast cancer model. Collectively, these studies indicate that pharmacological inhibition of HUNK kinase activity has therapeutic potential for HER2+ breast cancers, including HER2+ breast cancers that have developed drug resistance.
RESUMEN
The extracellular-regulated kinases ERK1 and ERK2 are evolutionarily conserved, ubiquitous serine-threonine kinases that are involved in regulating cellular signaling in both normal and pathological conditions. Their expression is critical for development and their hyperactivation is a major factor in cancer development and progression. Since their discovery as one of the major signaling mediators activated by mitogens and Ras mutation, we have learned much about their regulation, including their activation, binding partners and substrates. In this review I will discuss some of what has been discovered about the members of the Ras to ERK pathway, including regulation of their activation by growth factors and cell adhesion pathways. Looking downstream of ERK activation I will also highlight some of the many ERK substrates that have been discovered, including those involved in feedback regulation, cell migration and cell cycle progression through the control of transcription, pre-mRNA splicing and protein synthesis.
Asunto(s)
Antineoplásicos/uso terapéutico , Adhesión Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Proteínas ras/metabolismo , Animales , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. We evaluated the role of ERK1/2 on the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene, a master regulator of mitochondrial function. The potent and specific MEK1/2 inhibitor trametinib rapidly blocked ERK1/2 phosphorylation, decreased cytosolic and nuclear FOXO3a/1 phosphorylation, and increased PGC-1α gene expression and its downstream mitochondrial biogenesis (MB) targets under physiological conditions in the kidney cortex and in primary renal cell cultures. The epidermal growth factor receptor (EGFR) inhibitor erlotinib blocked ERK1/2 phosphorylation and increased PGC-1α gene expression similar to treatment with trametinib, linking EGFR activation and FOXO3a/1 inactivation to the down-regulation of PGC-1α and MB through ERK1/2. Pretreatment with trametinib blocked early ERK1/2 phosphorylation following ischemia/reperfusion kidney injury and attenuated the down-regulation of PGC-1α and downstream target genes. These results demonstrate that ERK1/2 rapidly regulates mitochondrial function through a novel pathway, EGFR/ERK1/2/FOXO3a/1/PGC-1α, under physiological and pathological conditions. As such, ERK1/2 down-regulates mitochondrial function directly by phosphorylation of upstream regulators of PGC-1α and subsequently decreasing MB.
Asunto(s)
Lesión Renal Aguda/patología , Regulación de la Expresión Génica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Daño por Reperfusión/complicaciones , Daño por Reperfusión/fisiopatología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/metabolismo , Animales , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Riñón/citología , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , PPAR gamma/genética , PPAR gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fosforilación , Conejos , Transducción de SeñalRESUMEN
The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by small interfering RNA (siRNA) induced apoptosis in A2780ip2, OVCAR5 and ES-2 ovarian cancer cells, correlating with loss of the prosurvival protein myeloid cell leukemia sequence 1 (Mcl-1) through a glycogen synthase kinase 3 beta-independent mechanism. SiRNA to EDD or Mcl-1 induced comparable levels of apoptosis in A2780ip2 and ES-2 cells. Stable overexpression of Mcl-1 protected cells from apoptosis following EDD knockdown, accompanied by a loss of endogenous, but not exogenous, Mcl-1 protein, suggesting that EDD regulated Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 messenger RNA and EDD transfection enhanced murine Mcl-1 promoter-driven luciferase expression 5-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and showed that these cells were 4- to 21-fold more sensitive to cisplatin. Moreover, transient EDD overexpression in COS-7 cells was sufficient to promote cisplatin resistance 2.4-fold, dependent upon its E3 ligase activity. In vivo, mouse intraperitoneal ES-2 and A2780ip2 xenograft experiments showed that mice treated with EDD siRNA by nanoliposomal delivery [1,2-dioleoyl-sn-glycero-3-phophatidylcholine (DOPC)] and cisplatin had significantly less tumor burden than those treated with control siRNA/DOPC alone (ES-2, 77.9% reduction, P = 0.004; A2780ip2, 75.9% reduction, P = 0.042) or control siRNA/DOPC with cisplatin in ES-2 (64.4% reduction, P = 0.035), with a trend in A2780ip2 (60.3% reduction, P = 0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest that EDD is a therapeutic target for ovarian cancer.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma Epitelial de Ovario , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cisplatino/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Proteolisis , Transcripción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RGS10 regulates ovarian cancer cell growth and survival, and RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance. However, the mechanisms governing RGS10 expression in ovarian cancer are poorly understood. Here we report RGS10 suppression in primary ovarian cancer and CAOV-3 ovarian cancer cells compared to immortalized ovarian surface epithelial (IOSE) cells, and in A2780-AD chemoresistant cells compared to parental A2780 cells. RGS10-1 and RGS10-2 transcripts are expressed in ovarian cancer cells, but only RGS10-1 is suppressed in A2780-AD and CAOV-3 cells, and the RGS10-1 promoter is uniquely enriched in CpG dinucleotides. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increased RGS10 expression, suggesting potential regulation by DNA methylation. Bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. DNA methylation in CAOV-3 and IOSE cells was similar to A2780 cells. More marked differences were observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associated with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Similarly, acetylated histone levels at the RGS10-1 promoter were markedly lower in CAOV-3 cells compared to IOSE cells, and HDAC1 binding was doubled in CAOV-3 cells. Finally, we show that pharmacological inhibition of DNMT or HDAC enzymes in chemoresistant A2780-AD cells increases RGS10 expression and enhances cisplatin toxicity. These data suggest that histone de-acetylation and DNA methylation correlate with RGS10 suppression and chemoresistance in ovarian cancer. Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics.
Asunto(s)
Metilación de ADN/genética , Histonas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Proteínas RGS/genética , Acetilación , Línea Celular Tumoral , Femenino , HumanosRESUMEN
Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.
Asunto(s)
Empalme Alternativo , Movimiento Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Sitios de Empalme de ARN , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Cortactina/metabolismo , Exones , Fibronectinas/metabolismo , Humanos , Mutación , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Factores de Empalme de ARN , ARN Mensajero/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Phosphorylation of epidermal growth factor receptor (EGFR) on tyrosine 845 by c-Src has been shown to be important for cell proliferation and migration in several model systems. This cross talk between EGFR and Src family kinases (SFKs) is one mechanism for resistance to EGFR inhibitors both in cell models and in the clinic. Here, we show that phosphorylation of tyrosine 845 on EGFR is required for proliferation and transformation using several cell models of breast cancer. Overexpression of EGFR-Y845F or treating cells with the SFK inhibitor dasatinib abrogated tyrosine 845 phosphorylation, yet had little to no effect on other EGFR phosphorylation sites or EGFR kinase activity. Abrogation of Y845 phosphorylation inhibited cell proliferation and transformation, even though extracellular signal-regulated kinase (ERK) and Akt remained active under these conditions. Importantly, cotransfection of mitogen-activated protein kinase (MAPK) kinase 3 and p38 MAPK restored cell proliferation in the absence of EGFR tyrosine 845 phosphorylation. Taken together, these data demonstrate a novel role for p38 MAPK signaling downstream of EGFR tyrosine 845 phosphorylation in the regulation of breast cancer cell proliferation and transformation and implicate SFK inhibitors as a potential therapeutic mechanism for overcoming EGFR tyrosine kinase inhibitor resistance in breast cancer.
RESUMEN
Sphingosine kinase 1 (SK1) is an important enzyme involved in the production of the bioactive lipid sphingosine 1-phosphate (S1P). SK1 is overexpressed in many forms of cancer, however, the contribution of SK1 to cancer progression is still unclear. One of the best characterized mutations found in several forms of human cancer is an activating point mutation in the Ras oncogene, which disrupts its GTPase activity and leads to stimulation of the MEK/ERK pathway. Because SK1 activity and subcellular localization have been shown to be regulated by ERK, we wished to investigate the effect of oncogenic Ras, a potent activator of the Raf/MEK/ERK pathway, on the activity of SK1 and sphingolipid metabolism. Using HEK293T cells transiently transfected with the K-RasG12V oncogene and both wild type and Sphk1(-/-) mouse embryonic fibroblasts stably infected with retroviral K-RasG12V, we found that K-RasG12V increases the production of S1P and decreases the production of ceramide in a SK1-dependent manner. In addition, we found that expression of the K-RasG12V oncogene leads to plasma membrane localization of SK1 and a reduction in cytosolic levels of SK1. This effect is likely mediated by the Raf/MEK/ERK pathway as constitutively active B-Raf or MEK1 are able to activate SK1, but constitutively active Akt1 is not. We believe this research has important implications for how sphingolipids may be contributing to oncogenic transformation and provide some of the first evidence for oncogenes inducing specific changes in sphingolipid metabolism through SK1 regulation.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Esfingolípidos/química , Proteínas ras/metabolismo , Animales , Membrana Celular/metabolismo , Transformación Celular Neoplásica , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Fosforilación , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Fracciones Subcelulares/metabolismo , Proteínas ras/fisiologíaRESUMEN
The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38α also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H(2)O(2), UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular , Línea Celular , Proliferación Celular , Cartilla de ADN/genética , Fibronectinas/genética , Fibronectinas/metabolismo , Células HeLa , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Fosforilación , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , Factores de Empalme de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Receptor fas/genéticaRESUMEN
The acquisition of resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell's response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype.
Asunto(s)
Empalme Alternativo , Resistencia a Antineoplásicos/genética , Neoplasias/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
UBR5 (ubiquitin protein ligase E3 component n-recognin 5)/EDD (E3 ligase identified by differential display) is an E3 ubiquitin ligase that is a potential biomarker for poor prognosis for recurrent, platinum-resistant ovarian cancer. UBR5 has a role in the DNA damage response and many such proteins are regulated by phosphorylation. UBR5 is a 309 kDa nuclear phosphoprotein that we previously identified as a substrate of the MAP kinase ERK2. With its 477 potential phosphorylation sites, little is known about UBR5 phosphorylation and how it may regulate protein function. Currently, thirty-four sites of phosphorylation on UBR5 have been reported in the literature, mostly identified by large scale proteomics studies of tissues or of cells after various treatments; however, no studies have specifically targeted the identification of UBR5 phosphorylation sites. In this study, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain a total sequence coverage of 64.3% from combining tryptic and GluC digests on UBR5 isolated from transfected COS-1 cells. We identified 24 sites of phosphorylation, 18 of which are novel sites. This data enhances our knowledge of UBR5 phosphorylation and provides a framework for the study of how phosphorylation affects UBR5 function.
Asunto(s)
Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Cromatografía Liquida , Fosforilación , Espectrometría de Masas en Tándem , Ubiquitina-Proteína Ligasas/químicaRESUMEN
The Mitogen-Activated Protein Kinase (MAPK) family of signaling molecules regulates a number of cellular processes through the direct phosphorylation and regulation of a plethora of cellular proteins. Identifying the direct substrates of the MAPK pathway proteins is important for determining how the effects of MAPK activation have such profound effects on cell biology. In this chapter, we describe one method for specific labeling and identification of direct MAPK substrates. A single or double point mutation is generated within the ATP binding domain at a particular residue(s) termed the "gatekeeper" that comes into close contact with the N6 position of ATP. Most kinases contain an amino acid larger than alanine at this position. Mutation of the residue(s) to glycine or alanine generates a "pocket" that allows the mutant kinase to bind and uniquely utilize an analog of ATP that contains a chemical substituent at the N6 position. When radiolabeled analog is added to the mutant kinase and a complex mixture of cellular proteins, the only proteins that become radiolabeled are direct substrates of the mutant kinase. To label biologically relevant substrates, we take advantage of the direct binding of MAPKs to their substrates. An epitope tagged mutant kinase is expressed in cells and immunoprecipitated with associated substrates, which are then radiolabeled in an in vitro kinase reaction using (gamma-(32)P) ATP analog. Larger, unlabeled kinase reactions are run in parallel and used to identify the substrates by mass spectrometry.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ingeniería de Proteínas , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Colodión/química , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoprecipitación , Espectrometría de Masas , Membranas Artificiales , Proteínas Quinasas Activadas por Mitógenos/química , Datos de Secuencia Molecular , Mutación , Radioisótopos de Fósforo/metabolismo , Fosforilación , Coloración y Etiquetado , TransfecciónRESUMEN
A thiazolidine-2,4-dione derivative, 3-(2-aminoethyl)-5-(3-phenyl-propylidene)-thiazolidine-2,4-dione (2), was identified as a dual inhibitor of the Raf/MEK/ extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades. The discovered compound inhibited cell proliferation, induced early apoptosis, and arrested cells in G(0)/G(1) phase in human leukemia U937 cells. These results indicate its potential as a new lead compound to develop novel dual signaling pathway inhibitors and anticancer agents.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Tiazolidinas/química , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Tiazolidinas/síntesis química , Tiazolidinas/farmacologíaRESUMEN
A series of analogs of 3-(2-amino-ethyl)-5-(4-ethoxy-benzylidene)-thiazolidine-2,4-dione, a putative substrate-specific ERK1/2 inhibitor, were synthesized and biologically characterized in human leukemia U937 cells to define its pharmacophore. It was discovered that shift of ethoxy substitution from the 4- to the 2-position on the phenyl ring significantly improved functional activities of inhibiting cell proliferation and inducing apoptosis. This may provide access to a new lead for developing ERK1/2 substrate-specific inhibitors.