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[This corrects the article DOI: 10.18632/oncoscience.395.].
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Resistance to cancer chemotherapy is a major global health burden. Epidermal growth factor receptor (EGFR) is a proven therapeutic target for multiple cancers of epithelial origin. Despite its overexpression in >90% of head and neck squamous cell carcinoma (HNSCC) patients, tyrosine kinase inhibitors such as erlotinib have shown a modest response in clinical trials. Cellular heterogeneity is thought to play an important role in HNSCC therapeutic resistance. Genomic alterations alone cannot explain all resistance mechanisms at play in a heterogeneous system. It is thus important to understand the biochemical mechanisms associated with drug resistance to determine potential strategies to achieve clinical response. We investigated tyrosine kinase signaling networks in erlotinib-resistant cells using quantitative tyrosine phosphoproteomics approach. We observed altered phosphorylation of proteins involved in cell adhesion and motility in erlotinib-resistant cells. Bioinformatics analysis revealed enrichment of pathways related to regulation of the actin cytoskeleton, extracellular matrix (ECM)-receptor interaction, and endothelial migration. Of importance, enrichment of the focal adhesion kinase (PTK2) signaling pathway downstream of EGFR was also observed in erlotinib-resistant cells. To the best of our knowledge, we present the first report of tyrosine phosphoproteome profiling in erlotinib-resistant HNSCC, with an eye to inform new ways to achieve clinical response. Our findings suggest that common signaling networks are at play in driving resistance to EGFR-targeted therapies in HNSCC and other cancers. Most notably, our data suggest that the PTK2 pathway genes may potentially play a significant role in determining clinical response to erlotinib in HNSCC tumors.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Aminoácidos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Resistencia a Antineoplásicos , Clorhidrato de Erlotinib/farmacología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Humanos , Marcaje Isotópico , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , TirosinaRESUMEN
The prognosis of AML is generally poor, with 5-year survival rate of 25%. There has been substantial progress in identification of new therapeutic targets, along with approval of at least three targeted therapies for AML in recent years. Nevertheless, treatment has largely remained unchanged over couple of decades, with ~40% patients not achieving remission. AML is a highly heterogenous disease and there is a need for a preclinical platform to understand the heterogeneity and tumor microenvironment that can guide therapy selection. In this study, we employed an ex vivo tumor explant model to study tumor microenvironment and to select a treatment course for AML patients. Our data reveal dysregulation of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) in a subset of AML patients. Based on this observation, epigenetic modulators azacitidine and panobinostat alone and in combination, were evaluated as treatment regimens in cytarabine refractory tumors. More than 50% of the treated samples showed response to the combination therapy. In order to explore alternate treatment modalities for tumors refractory to these epigenetic modulators, TCGA data analysis was done which revealed increased expression and hypomethylation of IFNGR1/2, suggesting activation of JAK/STAT pathway in AML. This was further interrogated ex vivo, with p-STAT3 expression in patients' samples. Fedratinib, a JAK/STAT inhibitor was evaluated and 78% tumor efficacy response was achieved. Taken together, our data indicate that ex vivo platform derived from patient samples is capable in guiding optimal therapy selection for various classes of drugs including identification of novel targeted therapies.
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Citarabina/uso terapéutico , Epigenómica/métodos , Inmunosupresores/uso terapéutico , Inhibidores de las Cinasas Janus/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Línea Celular Tumoral , Citarabina/farmacología , Humanos , Inmunosupresores/farmacología , Inhibidores de las Cinasas Janus/farmacología , Leucemia Mieloide Aguda/mortalidad , Pronóstico , Análisis de SupervivenciaRESUMEN
Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.
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Antineoplásicos/uso terapéutico , Clorhidrato de Erlotinib/uso terapéutico , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal , Genómica , Humanos , Redes y Vías Metabólicas , Fenotipo , Proteómica , Carcinoma de Células Escamosas de Cabeza y Cuello/enzimología , Secuenciación Completa del GenomaRESUMEN
Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.
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Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in India. Despite improvements in treatment strategy, the survival rates of HNSCC patients remain poor. Thus, it is necessary to identify biomarkers that can be used for early detection of disease. In this study, we employed iTRAQ-based quantitative mass spectrometry analysis to identify dysregulated proteins from a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. We identified 2468 proteins, of which 496 proteins were found to be dysregulated in at least two out of three HNSCC cell lines compared to immortalized normal oral keratinocytes. We detected increased expression of replication protein A1 (RPA1) and heat shock protein family H (Hsp110) member 1 (HSPH1), in HNSCC cell lines compared to control. The differentially expressed proteins were further validated using parallel reaction monitoring (PRM) and western blot analysis in HNSCC cell lines. Immunohistochemistry-based validation using HNSCC tissue microarrays revealed overexpression of RPA1 and HSPH1 in 15.7% and 32.2% of the tested cases, respectively. Our study illustrates quantitative proteomics as a robust approach for identification of potential HNSCC biomarkers. The proteomic data has been submitted to ProteomeXchange Consortium (http://www.proteomecentral.proteomexchange.org) via the PRIDE public data repository accessible using the data identifier - PXD009241.
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Interleukin-33 (IL-33) is a member of the IL-1 family of cytokines that play a central role in the regulation of immune responses. Its release from epithelial and endothelial cells is mediated by pro-inflammatory cytokines, cell damage and by recognition of pathogen-associated molecular patterns (PAMPs). The activity of IL-33 is mediated by binding to the IL-33 receptor complex (IL-33R) and activation of NF-κB signaling via the classical MyD88/IRAK/TRAF6 module. IL-33 also induces the phosphorylation and activation of ERK1/2, JNK, p38 and PI3K/AKT signaling modules resulting in the production and release of pro-inflammatory cytokines. Aberrant signaling by IL-33 has been implicated in the pathogenesis of several acute and chronic inflammatory diseases, including asthma, atopic dermatitis, rheumatoid arthritis and ulcerative colitis among others. Considering the biomedical importance of IL-33, we developed a pathway resource of signaling events mediated by IL-33/IL-33R in this study. Using data mined from the published literature, we describe an integrated pathway reaction map of IL-33/IL-33R consisting of 681 proteins and 765 reactions. These include information pertaining to 19 physical interaction events, 740 enzyme catalysis events, 6 protein translocation events, 4 activation/inhibition events, 9 transcriptional regulators and 2492 gene regulation events. The pathway map is publicly available through NetPath ( http://www.netpath.org /), a resource of human signaling pathways developed previously by our group. This resource will provide a platform to the scientific community in facilitating identification of novel therapeutic targets for diseases associated with dysregulated IL-33 signaling. Database URL: http://www.netpath.org/pathways?path_id=NetPath_120 .
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EGFR-based targeted therapies have shown limited success in smokers. Identification of alternate signaling mechanism(s) leading to TKI resistance in smokers is critically important. We observed increased resistance to erlotinib in H358 NSCLC (non-small cell lung carcinoma) cells chronically exposed to cigarette smoke (H358-S) compared to parental cells. SILAC-based mass-spectrometry approach was used to study altered signaling in H358-S cell line. Importantly, among the top phosphosites in H358-S cells we observed hyperphosphorylation of EGFR (Y1197) and non-receptor tyrosine kinase FAK (Y576/577). Supporting these observations, a transcriptomic-based pathway activation analysis of TCGA NSCLC datasets revealed that FAK and EGFR internalization pathways were significantly upregulated in smoking patients, compared to the never-smokers and were associated with elevated PI3K signaling and lower level of caspase cascade and E-cadherin pathways activation. We show that inhibition of FAK led to decreased cellular proliferation and invasive ability of the smoke-exposed cells, and restored their dependency on EGFR signaling. Our data suggests that activation of focal adhesion pathway significantly contributes to erlotinib resistance, and that FAK is a potential therapeutic target for management of erlotinib resistance in smoke-induced NSCLC.
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BACKGROUND: Dysregulation of miRNAs is associated with the development of non-small cell lung cancer (NSCLC). It is imperative to study the dysregulation of miRNAs by cigarette smoke which will affect their targets, either leading to the overexpression of oncoproteins or downregulation of tumor suppressor proteins. OBJECTIVE AND METHODS: In this study, we carried out miRNA sequencing and SILAC-based proteomic analysis of H358 cells chronically exposed to cigarette smoke condensate. Using bioinformatics analysis, we mapped the dysregulated miRNAs to differentially expressed target proteins identified in our data. Gene ontology-based enrichment and pathway analysis was performed using the deregulated targets to study the role of cigarette smoke-mediated miRNA dysregulation in NSCLC cell line. RESULTS: miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (2 fold, Log Base 2; p-value ≤ 0.05) in H358-Smoke cells. Proteomic analysis of the smoke exposed cells compared to the untreated parental cells resulted in the quantification of 2,610 proteins, of which 690 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway study using Ingenuity Pathway Analysis (IPA) revealed activation of NRF2-mediated oxidative stress response and actin-cytoskeleton signaling, and repression of protein kinase A signaling in H358-Smoke cells. We also identified 5 novel miRNAs in H358-Smoke cells using unassigned reads of small RNA-Seq dataset. CONCLUSION: In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC cell line. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential therapeutic targets and/or molecular markers in NSCLC especially in smokers.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , MicroARNs/genética , Proteoma/metabolismo , Fumar/efectos adversos , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/inducido químicamente , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Biología Computacional , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteómica/métodos , Análisis de Secuencia de ARN/métodos , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Proteomics analysis of chronic cigarette smoke exposure is a rapidly emerging postgenomics research field. While smoking is a major cause of lung cancer, functional studies using proteomics approaches could enrich our mechanistic understanding of the elusive lung cancer global molecular signaling and cigarette smoke relationship. We report in this study on a stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analysis of a human lung mucoepidermoid carcinoma cell line, H292 cells, chronically exposed to cigarette smoke. Using high resolution Orbitrap Velos mass spectrometer, we identified the hyperphosphorylation of 493 sites, which corresponds to 341 proteins and 195 hypophosphorylated sites, mapping to 142 proteins upon smoke exposure (2.0-fold change). We report differential phosphorylation of multiple kinases, including PAK6, EPHA4, LYN, mitogen-activated protein kinase, and phosphatases, including TMEM55B, PTPN14, TIGAR, among others, in response to chronic cigarette smoke exposure. Bioinformatics analysis revealed that the molecules differentially phosphorylated upon chronic exposure of cigarette smoke are associated with PI3K/AKT/mTOR and CDC42-PAK signaling pathways. These signaling networks are involved in multiple cellular processes, including cell polarity, cytoskeletal remodeling, cellular migration, protein synthesis, autophagy, and apoptosis. The present study contributes to emerging proteomics insights on cigarette smoke mediated global signaling in lung cells, which in turn may aid in development of precision medicine therapeutics and postgenomics biomarkers.
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Células Epiteliales/efectos de los fármacos , Nicotiana/efectos adversos , Fosfoproteínas/genética , Proteoma/genética , Mucosa Respiratoria/efectos de los fármacos , Humo/efectos adversos , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Anotación de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Chronic exposure to cigarette smoke markedly increases the risk for lung cancer. Regulation of gene expression at the post-transcriptional level by miRNAs influences a variety of cancer-related interactomes. Yet, relatively little is known on the effects of long-term cigarette smoke exposure on miRNA expression and gene regulation. NCI-H292 (H292) is a cell line sensitive to cigarette smoke with mucoepidermoid characteristics in culture. We report, in this study, original observations on long-term (12 months) cigarette smoke effects in the H292 cell line, using microarray-based miRNA expression profiling, and stable isotopic labeling with amino acids in cell culture-based quantitative proteomic analysis. We identified 112 upregulated and 147 downregulated miRNAs (by twofold) in cigarette smoke-treated H292 cells. The liquid chromatography-tandem mass spectrometry analysis identified 3,959 proteins, of which, 303 proteins were overexpressed and 112 proteins downregulated (by twofold). We observed 39 miRNA target pairs (proven targets) that were differentially expressed in response to chronic cigarette smoke exposure. Gene ontology analysis of the target proteins revealed enrichment of proteins in biological processes driving metabolism, cell communication, and nucleic acid metabolism. Pathway analysis revealed the enrichment of phagosome maturation, antigen presentation pathway, nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, and cholesterol biosynthesis pathways in cigarette smoke-exposed cells. In conclusion, this report makes an important contribution to knowledge on molecular changes in a lung cell line in response to long term cigarette smoke exposure. The findings might inform future strategies for drug target, biomarker and diagnostics innovation in lung cancer, and clinical oncology. These observations also call for further research on the extent to which continuing or stopping cigarette smoking in patients diagnosed with lung cancer translates into molecular and clinical outcomes.
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Fumar Cigarrillos/efectos adversos , Neoplasias Pulmonares/genética , MicroARNs/metabolismo , Biomarcadores/análisis , Carcinoma de Pulmón de Células no Pequeñas , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/diagnósticoRESUMEN
The traditional Indian medicine, Ayurveda, provides insights and practical solutions towards a healthy life style. Rasayana is a branch of Ayurveda known for preserving and promoting health, enhancing the quality of life and delaying the aging process. In the traditional knowledge, the Rasayana herb, Chlorophytum borivilianum (C. borivilanum) is regarded as a general health promoting tonic that delays aging and increases lifespan, cognitive function and physical strength. Aging is a complex and multifactorial physiological phenomenon that manifests itself over a wide range of biological systems, tissues, and functions. Longevity is an obvious marker of physiological aging. Simple model systems such as the single-cell budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode, Caenorhabditis elegans (C. elegans) are widely used to study the aging process and longevity. Here, we show that a polysaccharide fraction obtained from C. borivilianum increases the lifespan of S. cerevisiae and C. elegans, using an automated screening platform (ChronoscreenTM). Chemical analysis of this extract revealed a low molecular weight polysaccharide of 1000 Da, predominantly comprising Glu1â6Glu linkage. This polysaccharide showed significant dose-dependent extension of the median lifespan of S. cerevisiae by up to 41% and of the median lifespan of C. elegans by up to 10%. Taking cue from these results and the traditionally described benefits of Rasayanas on skin rejuvenation, we tested in vitro the polysaccharide for potential skin benefits. In a keratinocyte culture, we observed that this polysaccharide increased cell proliferation significantly, and induced synthesis of hyaluronic acid (HA), a well-known extracellular matrix component. Furthermore, when added to culture medium of human reconstructed epidermis, we observed an enhanced production of epidermal markers, e.g. CD44 and HA that are otherwise diminished in aged skin. Together, these results suggest that in addition to life-span extension of S. cerevisiae and C. elegans, a polysaccharide from the Rasayana herb, C. borivilianum may have beneficial effects on skin aging parameters.
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Asparagaceae , Longevidad/efectos de los fármacos , Medicina Ayurvédica , Extractos Vegetales/farmacología , Polisacáridos/farmacología , Envejecimiento , Animales , Caenorhabditis elegans/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Queratinocitos/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
This corrects the article DOI: 10.1038/srep36132.
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Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.
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Genoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Anotación de Secuencia Molecular , Transcriptoma/genética , Animales , Anopheles/genética , Exones/genética , Perfilación de la Expresión Génica , Proteoma/genética , ProteómicaRESUMEN
Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.
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Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Caspasa 9/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Femenino , Proteína Forkhead Box O3/metabolismo , Harmina/uso terapéutico , Harmina/toxicidad , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Fosfotirosina/análisis , Fosfotirosina/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello , Espectrometría de Masas en Tándem , Análisis de Matrices Tisulares , Trasplante Heterólogo , Proteína bcl-X/metabolismo , Quinasas DyrKRESUMEN
Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated (≥ 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Humo/efectos adversos , Quinasas p21 Activadas/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Receptores ErbB/metabolismo , Silenciador del Gen , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Fosforilación , Proteoma , Pirazoles/farmacología , Pirroles/farmacología , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Productos de Tabaco , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.
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Biomarcadores de Tumor/biosíntesis , Carcinogénesis/genética , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Empalme Alternativo/genética , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Humanos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteómica , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Chewing tobacco is a common practice in certain socio-economic sections of southern Asia, particularly in the Indian subcontinent and has been well associated with head and neck squamous cell carcinoma. The molecular mechanisms of chewing tobacco which leads to malignancy remains unclear. In large majority of studies, short-term exposure to tobacco has been evaluated. From a biological perspective, however, long-term (chronic) exposure to tobacco mimics the pathogenesis of oral cancer more closely. We developed a cell line model to investigate the chronic effects of chewing tobacco. Chronic exposure to tobacco resulted in higher cellular proliferation and invasive ability of the normal oral keratinocytes (OKF6/TERT1). We carried out quantitative proteomic analysis of OKF6/TERT1 cells chronically treated with chewing tobacco compared to the untreated cells. We identified a total of 3,636 proteins among which expression of 408 proteins were found to be significantly altered. Among the overexpressed proteins, stearoyl-CoA desaturase (SCD) was found to be 2.6-fold overexpressed in the tobacco treated cells. Silencing/inhibition of SCD using its specific siRNA or inhibitor led to a decrease in cellular proliferation, invasion and colony forming ability of not only the tobacco treated cells but also in a panel of head and neck cancer cell lines. These findings suggest that chronic exposure to chewing tobacco induced carcinogenesis in non-malignant oral epithelial cells and SCD plays an essential role in this process. The current study provides evidence that SCD can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients who are users of tobacco.
Asunto(s)
Queratinocitos/enzimología , Estearoil-CoA Desaturasa/metabolismo , Uso de Tabaco/metabolismo , Carcinogénesis/metabolismo , Línea Celular , Movimiento Celular , Proliferación Celular , Inducción Enzimática , Humanos , Mucosa Bucal/patología , Neoplasias de la Boca/etiología , Neoplasias de la Boca/patología , Proteoma/metabolismo , Estearoil-CoA Desaturasa/genética , Nicotiana/efectos adversos , Uso de Tabaco/efectos adversos , Uso de Tabaco/patologíaRESUMEN
Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737).