RESUMEN
Kinase activity is frequently altered in renal cell carcinoma (RCC), and tyrosine kinase inhibitors (TKIs) are part of the standard treatment strategy in patients with metastatic disease. However, there are still no established biomarkers to predict clinical benefits of a specific TKI. Here, we performed protein tyrosine kinase (PTK) profiling using PamChip® technology. The aim of this study was to identify differences in PTK activity between normal and malignant kidney tissue obtained from the same patient, and to investigate the inhibitory effects of TKIs frequently used in the clinics: sunitinib, pazopanib, cabozantinib and tivozanib. Briefly, our results showed that 36 kinase substrates differs (FDR < 0.05) between normal and cancer kidney tissue, where members of the Src family kinases and the phosphoinositide-3-kinase (PI3K) pathway exhibit high activity in renal cancer. Furthermore, ex vivo treatment of clear cell RCC with TKIs revealed that pathways such as Rap1, Ras and PI3K pathways were strongly inhibited, whereas the neurotrophin pathway had increased activity upon TKI addition. In our assay, tivozanib and cabozantinib exhibited greater inhibitory effects on PTK activity compared to sunitinib and pazopanib, implying they might be better suitable as TKIs for selected RCC patients.
Asunto(s)
Carcinoma de Células Renales , Inhibidores de Proteínas Quinasas , Proteínas Tirosina Quinasas , Anilidas , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Humanos , Indazoles , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Factores de Crecimiento Nervioso , Compuestos de Fenilurea , Fosfatidilinositol 3-Quinasas , Fosfatidilinositoles/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/metabolismo , Piridinas , Pirimidinas , Quinolinas , Sulfonamidas , Sunitinib/uso terapéutico , Familia-src QuinasasRESUMEN
Phosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based platforms for measuring reporter peptide signal levels allow for differential phosphorylation analysis between conditions for distinct active kinases. Peptide array technologies like the PamStation12 from PamGene allow for generating high-throughput, multi-dimensional, and complex functional proteomics data. As the adoption rate of such technologies increases, there is an imperative need for software tools that streamline the process of analyzing such data. We present Kinome Random Sampling Analyzer (KRSA), an R package and R Shiny web-application for analyzing kinome array data to help users better understand the patterns of functional proteomics in complex biological systems. KRSA is an All-In-One tool that reads, formats, fits models, analyzes, and visualizes PamStation12 kinome data. While the underlying algorithm has been experimentally validated in previous publications, we demonstrate KRSA workflow on dorsolateral prefrontal cortex (DLPFC) in male (n = 3) and female (n = 3) subjects to identify differential phosphorylation signatures and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar global phosphorylation signals patterns.
Asunto(s)
Corteza Prefontal Dorsolateral/enzimología , Familia de Multigenes , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Programas Informáticos , Algoritmos , Autopsia , Benchmarking , Conjuntos de Datos como Asunto , Corteza Prefontal Dorsolateral/química , Femenino , Expresión Génica , Humanos , Masculino , Fosfoproteínas/clasificación , Fosfoproteínas/genética , Fosforilación , Análisis de Componente Principal , Análisis por Matrices de Proteínas , Proteínas Serina-Treonina Quinasas/clasificación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/clasificación , Proteínas Tirosina Quinasas/genética , Proteómica/métodosRESUMEN
Better understanding of GBM signalling networks in-vivo would help develop more physiologically relevant ex vivo models to support therapeutic discovery. A "functional proteomics" screen was undertaken to measure the specific activity of a set of protein kinases in a two-step cell-free biochemical assay to define dominant kinase activities to identify potentially novel drug targets that may have been overlooked in studies interrogating GBM-derived cell lines. A dominant kinase activity derived from the tumour tissue, but not patient-derived GBM stem-like cell lines, was Bruton tyrosine kinase (BTK). We demonstrate that BTK is expressed in more than one cell type within GBM tissue; SOX2-positive cells, CD163-positive cells, CD68-positive cells, and an unidentified cell population which is SOX2-negative CD163-negative and/or CD68-negative. The data provide a strategy to better mimic GBM tissue ex vivo by reconstituting more physiologically heterogeneous cell co-culture models including BTK-positive/negative cancer and immune cells. These data also have implications for the design and/or interpretation of emerging clinical trials using BTK inhibitors because BTK expression within GBM tissue was linked to longer patient survival.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/metabolismo , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/mortalidad , Glioblastoma/enzimología , Glioblastoma/mortalidad , Proteoma/metabolismo , Transducción de Señal , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Cocultivo/métodos , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/enzimología , Proteómica/métodos , Factores de Transcripción SOXB1/metabolismo , Tasa de SupervivenciaRESUMEN
Kinase drug discovery represents an active area of therapeutic research, with previous pharmaceutical success improving patient outcomes across a wide variety of human diseases. In pancreatic ductal adenocarcinoma (PDAC), innovative pharmaceutical strategies such as kinase targeting have been unable to appreciably increase patient survival. This may be due, in part, to unchecked desmoplastic reactions to pancreatic tumors. Desmoplastic stroma enhances tumor development and progression while simultaneously restricting drug delivery to the tumor cells it protects. Emerging evidence indicates that many of the pathologic fibrotic processes directly or indirectly supporting desmoplasia may be driven by targetable protein tyrosine kinases such as Fyn-related kinase (FRK); B lymphoid kinase (BLK); hemopoietic cell kinase (HCK); ABL proto-oncogene 2 kinase (ABL2); discoidin domain receptor 1 kinase (DDR1); Lck/Yes-related novel kinase (LYN); ephrin receptor A8 kinase (EPHA8); FYN proto-oncogene kinase (FYN); lymphocyte cell-specific kinase (LCK); tec protein kinase (TEC). Herein, we review literature related to these kinases and posit signaling networks, mechanisms, and biochemical relationships by which this group may contribute to PDAC tumor growth and desmoplasia.
Asunto(s)
Adenocarcinoma/genética , Tumor Desmoplásico de Células Pequeñas Redondas/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/patología , Tumor Desmoplásico de Células Pequeñas Redondas/patología , Receptor con Dominio Discoidina 1/genética , Progresión de la Enfermedad , Humanos , Neoplasias Pancreáticas/patología , Proteínas Tirosina Quinasas/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-hck/genética , Transducción de Señal , Familia-src Quinasas/genéticaRESUMEN
Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.
Asunto(s)
Carcinoma Ductal Pancreático/enzimología , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias Pancreáticas/enzimología , Proteínas Tirosina Quinasas/biosíntesis , Carcinoma Ductal Pancreático/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Proteínas Tirosina Quinasas/genética , Proto-Oncogenes MasRESUMEN
Acquired cetuximab resistance is a challenge for oncologists treating advanced head and neck carcinoma (HNC). While intrinsic cetuximab resistance mechanism in colorectal cancer is known, resistance in HNC is unclear. We established two different cetuximab resistant HNC cell lines by culturing epidermal growth factor (EGFR) expressing UM-SCC-1 and UM-SCC-6â¯cell lines in the presence of 5⯵g/ml cetuximab. We then explored potential mechanisms of resistance. We found that the 2â¯cell lines developed resistance by different mechanisms. Specifically, we found that UM-SCC-1 resistant cells (UM-SCC-1R) showed enhanced EGF-induced downstream signals while UM-SCC-6 resistant cells (UM-SCC-6R) demonstrated EGF-independent signaling. Global kinase activity (kinomic) profiling revealed unique signaling differences in the two resistant cell lines. However, both of the resistant lines demonstrated increased phospho-serine 727 and total STAT3 expression compared to the parental lines. STAT3 knockdown promoted increased cytotoxicity both in the presence and absence of cetuximab in the resistant lines suggesting that STAT3 may be a common target in cetuximab resistance.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Cetuximab/farmacología , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Sunitinib is a protein tyrosine kinase (PTK) inhibitor that has immune-modulating properties. In this context, peripheral blood mononuclear cells (PBMC), mainly constituted by lymphocytes, could be a perfect surrogate tissue for identifying and assaying pharmacodynamic biomarkers of sunitinib. In this study, we investigated the changes in lymphocytes count as pharmacodynamic biomarker in metastatic renal cell carcinoma (mRCC) patients under sunitinib therapy. Thereafter, we studied the ex vivo effect of sunitinib and SU12262 (active metabolite) on PBMC from naïve mRCC patients using a high throughput kinomic profiling method. METHODS: The prognostic value of total lymphocytes count between Day 0 and Day 21 (expressed as a ratio D21/D0) was retrospectively investigated in 88 mRCC patients under sunitinib therapy. PTK PamChip® microarrays were used to explore prospectivelythe ex vivo effect of sunitinib and SU12662 on PTK activity in PBMC from 21 naïve mRCC patients. RESULTS: In this retrospective study, D21/D0 lymphocytes ratio (Hazard Ratio, 1.83; CI95%, 1.24-2.71; p=0.0023) was independently associated with PFS. Interestingly, kinomic analysis showed that D21/D0 lymphocytes ratio and Heng prognostic model was statistically associated with the ex vivo sunitinib and SU12662 effect in PBMC. CONCLUSION: The present study highlights that D21/D0 total lymphocytes ratio could be a promising pharmacodynamic biomarker in mRCC patients treated with sunitinib. Additionally, it paves the way to investigate the kinomic profile in PBMC as a prognostic factor in a larger cohort of mRCC patients under sunitinib therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/sangre , Carcinoma de Células Renales/sangre , Indoles/uso terapéutico , Neoplasias Renales/sangre , Leucocitos Mononucleares/efectos de los fármacos , Pirroles/uso terapéutico , Anciano , Carcinoma de Células Renales/tratamiento farmacológico , Supervivencia sin Enfermedad , Femenino , Humanos , Neoplasias Renales/tratamiento farmacológico , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , SunitinibRESUMEN
Alzheimer's disease (AD) is characterized by a long pre-clinical phase (20-30 years), during which significant brain pathology manifests itself. Disease mechanisms associated with pathological hallmarks remain elusive. Most processes associated with AD pathogenesis, such as inflammation, synaptic dysfunction, and hyper-phosphorylation of tau are dependent on protein kinase activity. The objective of this study was to determine the involvement of protein kinases in AD pathogenesis. Protein kinase activity was determined in postmortem hippocampal brain tissue of 60 patients at various stages of AD and 40 non-demented controls (Braak stages 0-VI) using a peptide-based microarray platform. We observed an overall decrease of protein kinase activity that correlated with disease progression. The phosphorylation of 96.7% of the serine/threonine peptides and 37.5% of the tyrosine peptides on the microarray decreased significantly with increased Braak stage (p-value <0.01). Decreased activity was evident at pre-clinical stages of AD pathology (Braak I-II). Increased phosphorylation was not observed for any peptide. STRING analysis in combination with pathway analysis and identification of kinases responsible for peptide phosphorylation showed the interactions between well-known proteins in AD pathology, including the Ephrin-receptor A1 (EphA1), a risk gene for AD, and sarcoma tyrosine kinase (Src), which is involved in memory formation. Additionally, kinases that have not previously been associated with AD were identified, e.g., protein tyrosine kinase 6 (PTK6/BRK), feline sarcoma oncogene kinase (FES), and fyn-associated tyrosine kinase (FRK). The identified protein kinases are new biomarkers and potential drug targets for early (pre-clinical) intervention.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Hipocampo/enzimología , Hipocampo/patología , Proteínas Quinasas/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/diagnóstico , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Ionizing radiation treatment is used in over half of all cancer patients, thus determining the mechanisms of response or resistance is critical for the development of novel treatment approaches. MATERIALS AND METHODS: In this report, we utilize a high-content peptide array platform that performs multiplex kinase assays with real-time kinetic readout to investigate the mechanism of radiation response in vascular endothelial cells. We applied this technology to irradiated human umbilical vein endothelial cells (HUVEC). RESULTS: We identified 49 specific tyrosine phosphopeptides that were differentially affected by irradiation over a time course of 1h. In one example, the Tropomyosin receptor kinase (Trk) family members, TrkA and TrkB, showed transient activation between 2 and 15 min following irradiation. When we targeted TrkA and TrkB using small molecule inhibitors, HUVEC were protected from radiation damage. Conversely, stimulation of TrkA using gambogic amide promoted radiation enhancement. CONCLUSIONS: Thus, we show that our approach not only can identify rapid changes in kinase activity but also identify novel targets such as TrkA. TrkA inhibition resulted in radioprotection that correlated with enhanced repair of radiation-induced damage while TrkA stimulation by gambogic amide produced radiation sensitization.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/efectos de la radiación , Tolerancia a Radiación , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Ciclo Celular/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Reparación del ADN/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Fosforilación , Análisis por Matrices de Proteínas , Proteínas Quinasas/metabolismo , Dosis de Radiación , Tolerancia a Radiación/efectos de los fármacos , Receptor trkA/agonistas , Xantonas/farmacologíaRESUMEN
BACKGROUND: Protein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other. METHODOLOGY: We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles. RESULTS AND CONCLUSION: Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo.