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The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.
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Proteoma/metabolismo , Proteómica , Adulto , Células Cultivadas , Bases de Datos de Proteínas , Feto/metabolismo , Análisis de Fourier , Perfilación de la Expresión Génica , Genoma Humano/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Internet , Espectrometría de Masas , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Especificidad de Órganos , Biosíntesis de Proteínas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteoma/análisis , Proteoma/química , Proteoma/genética , Seudogenes/genética , ARN no Traducido/genética , Reproducibilidad de los Resultados , Regiones no Traducidas/genéticaRESUMEN
Chronic exposure to arsenic is associated with dermatological and nondermatological disorders. Consumption of arsenic-contaminated drinking water results in accumulation of arsenic in liver, spleen, kidneys, lungs, and gastrointestinal tract. Although arsenic is cleared from these sites, a substantial amount of residual arsenic is left in keratin-rich tissues including skin. Epidemiological studies suggest the association of skin cancer upon arsenic exposure, however, the mechanism of arsenic-induced carcinogenesis is not completely understood. We developed a cell line based model to understand the molecular mechanisms involved in arsenic-mediated toxicity and carcinogenicity. Human skin keratinocyte cell line, HaCaT, was chronically exposed to 100 nM sodium arsenite over a period of 6 months. We observed an increase in basal ROS levels in arsenic-exposed cells. SILAC-based quantitative proteomics approach resulted in identification of 2111 proteins of which 42 proteins were found to be overexpressed and 54 downregulated (twofold) upon chronic arsenic exposure. Our analysis revealed arsenic-induced overexpression of aldo-keto reductase family 1 member C2 (AKR1C2), aldo-keto reductase family 1 member C3 (AKR1C3), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) among others. We observed downregulation of several members of the plakin family including periplakin (PPL), envoplakin (EVPL), and involucrin (IVL) that are essential for terminal differentiation of keratinocytes. MRM and Western blot analysis confirmed differential expression of several candidate proteins. Our study provides insights into molecular alterations upon chronic arsenic exposure on skin.
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Aminoácidos/metabolismo , Arsénico/toxicidad , Marcaje Isotópico/métodos , Queratinocitos/metabolismo , Proteómica/métodos , Piel/citología , Secuencia de Aminoácidos , Western Blotting , Línea Celular , Biología Computacional , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Queratinocitos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteoma/química , Proteoma/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Curcumin, derived from the rhizome Curcuma longa, is a natural anti-cancer agent and has been shown to inhibit proliferation and survival of tumor cells. Although the anti-cancer effects of curcumin are well established, detailed understanding of the signaling pathways altered by curcumin is still lacking. In this study, we carried out SILAC-based quantitative proteomic analysis of a HNSCC cell line (CAL 27) to investigate tyrosine signaling in response to curcumin. RESULTS: Using high resolution Orbitrap Fusion Tribrid Fourier transform mass spectrometer, we identified 627 phosphotyrosine sites mapping to 359 proteins. We observed alterations in the level of phosphorylation of 304 sites corresponding to 197 proteins upon curcumin treatment. We report here for the first time, curcumin-induced alterations in the phosphorylation of several kinases including TNK2, FRK, AXL, MAPK12 and phosphatases such as PTPN6, PTPRK, and INPPL1 among others. Pathway analysis revealed that the proteins differentially phosphorylated in response to curcumin are known to be involved in focal adhesion kinase signaling and actin cytoskeleton reorganization. CONCLUSIONS: The study indicates that curcumin may regulate cellular processes such as proliferation and migration through perturbation of the focal adhesion kinase pathway. This is the first quantitative phosphoproteomics-based study demonstrating the signaling events that are altered in response to curcumin. Considering the importance of curcumin as an anti-cancer agent, this study will significantly improve the current knowledge of curcumin-mediated signaling in cancer.
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Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and â¼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.
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Genoma/genética , Proteoma/análisis , Proteoma/genética , Transcriptoma/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Espectrometría de Masas , Anotación de Secuencia Molecular , Proteómica , Análisis de Secuencia de ARNRESUMEN
Plasma Proteome Database (PPD; http://www.plasmaproteomedatabase.org/) was initially described in the year 2005 as a part of Human Proteome Organization's (HUPO's) pilot initiative on Human Plasma Proteome Project. Since then, improvements in proteomic technologies and increased throughput have led to identification of a large number of novel plasma proteins. To keep up with this increase in data, we have significantly enriched the proteomic information in PPD. This database currently contains information on 10,546 proteins detected in serum/plasma of which 3784 have been reported in two or more studies. The latest version of the database also incorporates mass spectrometry-derived data including experimentally verified proteotypic peptides used for multiple reaction monitoring assays. Other novel features include published plasma/serum concentrations for 1278 proteins along with a separate category of plasma-derived extracellular vesicle proteins. As plasma proteins have become a major thrust in the field of biomarkers, we have enabled a batch-based query designated Plasma Proteome Explorer, which will permit the users in screening a list of proteins or peptides against known plasma proteins to assess novelty of their data set. We believe that PPD will facilitate both clinical and basic research by serving as a comprehensive reference of plasma proteins in humans and accelerate biomarker discovery and translation efforts.
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Proteínas Sanguíneas/análisis , Bases de Datos de Proteínas , Proteoma/análisis , Humanos , Internet , Proteómica , Vesículas Secretoras/químicaRESUMEN
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).
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Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Cisplatino/farmacología , Resistencia a Antineoplásicos , Fluorouracilo/farmacología , Proteína HMGB2/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Interferencia de ARN , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular , Línea Celular Tumoral , Proteína HMGB2/análisis , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Proteómica , ARN Interferente Pequeño/genética , Carcinoma de Células Escamosas de Cabeza y Cuello , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. METHODS: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. RESULTS: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. CONCLUSIONS: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.
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Biomarcadores de Tumor/biosíntesis , Neoplasias de la Vesícula Biliar/genética , Oxidorreductasas Intramoleculares/biosíntesis , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Pronóstico , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Detección Precoz del Cáncer , Neoplasias de la Vesícula Biliar/diagnóstico , Neoplasias de la Vesícula Biliar/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Macrófagos/metabolismo , Macrófagos/patología , Proteínas de Neoplasias/biosíntesis , ProteómicaRESUMEN
Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.
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Bile serves diverse functions from metabolism to transport. In addition to acids and salts, bile is composed of proteins secreted or shed by the hepatobiliary system. Although there have been previous efforts to catalog biliary proteins, an in-depth analysis of the bile proteome has not yet been reported. We carried out fractionation of non-cancerous bile samples using a multipronged approach (SDS-PAGE, SCX and OFFGEL) followed by MS analysis on an LTQ-Orbitrap Velos mass spectrometer using high resolution at both MS and MS/MS levels. We identified 2552 proteins - the largest number of proteins reported in human bile till date. To our knowledge, there are no previous studies employing high-resolution MS reporting a more detailed catalog of any body fluid proteome in a single study. We propose that extensive fractionation coupled to high-resolution MS can be used as a standard methodology for in-depth characterization of any body fluid. This catalog should serve as a baseline for the future studies aimed at discovering biomarkers from bile in gallbladder, hepatic, and biliary cancers.
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Bilis/química , Proteoma/análisis , Proteómica/métodos , Adulto , Biomarcadores/análisis , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Humanos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana EdadRESUMEN
The study of the human urinary proteome has the potential to offer significant insights into normal physiology as well as disease pathology. The information obtained from such studies could be applied to the diagnosis of various diseases. The high sensitivity, resolution, and mass accuracy of the latest generation of mass spectrometers provides an opportunity to accurately catalog the proteins present in human urine, including those present at low levels. To this end, we carried out a comprehensive analysis of human urinary proteome from healthy individuals using high-resolution Fourier transform mass spectrometry. Importantly, we used the Orbitrap for detecting ions in both MS (resolution 60 000) and MS/MS (resolution 15 000) modes. To increase the depth of our analysis, we characterized both unfractionated as well as lectin-enriched proteins in our experiments. In all, we identified 1,823 proteins with less than 1% false discovery rate, of which 671 proteins have not previously been reported as constituents of human urine. This data set should serve as a comprehensive reference list for future studies aimed at identification and characterization of urinary biomarkers for various diseases.
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Proteoma/química , Adulto , Anciano , Secuencia de Aminoácidos , Biomarcadores/orina , Cromatografía de Afinidad , Femenino , Glicoproteínas/orina , Humanos , Lectinas/química , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Fragmentos de Péptidos , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
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
[This corrects the article DOI: 10.18632/oncoscience.395.].
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Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.
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BACKGROUND: Skin acts as a protective barrier against direct contact with pollutants but inhalation and systemic exposure have indirect effect on keratinocytes. Exposure to diesel exhaust has been linked to increased oxidative stress. OBJECTIVE: To investigate global proteomic alterations in diesel particulate extract (DPE)/ its vapor exposed skin keratinocytes. METHODS: We employed Tandem Mass Tag (TMT)-based proteomics to study effect of DPE/ DPE vapor on primary skin keratinocytes. RESULTS: We observed an increased expression of oxidative stress response protein NRF2, upon chronic exposure of primary keratinocytes to DPE/ its vapor which includes volatile components such as polycyclic aromatic hydrocarbons (PAHs). Mass spectrometry-based quantitative proteomics led to identification 4490 proteins of which 201 and 374 proteins were significantly dysregulated (≥1.5 fold, p ≤ 0.05) in each condition, respectively. Proteins involved in cellular processes such as cornification (cornifin A), wound healing (antileukoproteinase) and differentiation (suprabasin) were significantly downregulated in primary keratinocytes exposed to DPE/ DPE vapor. These results were corroborated in 3D skin models chronically exposed to DPE/ DPE vapor. Bioinformatics analyses indicate that DPE and its vapor affect distinct molecular processes in skin keratinocytes. Components of mitochondrial oxidative phosphorylation machinery were seen to be exclusively overexpressed upon chronic DPE vapor exposure. In addition, treatment with an antioxidant like vitamin E partially restores expression of proteins altered upon exposure to DPE/ DPE vapor. CONCLUSIONS: Our study highlights distinct adverse effects of chronic exposure to DPE/ DPE vapor on skin keratinocytes and the potential role of vitamin E in alleviating adverse effects of environmental pollution.
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Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we propose that chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.
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Células Epiteliales/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Extractos Vegetales/farmacología , Tabaco sin Humo/efectos adversos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/patología , Neoplasias Esofágicas/inducido químicamente , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Humanos , Células Madre Neoplásicas/patología , FenotipoRESUMEN
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
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers with high mortality rate. Cigarette smoke and chewing tobacco are well known risk factors associated with ESCC. However, molecular mechanisms associated with development of ESCC among smokers and chewers are poorly understood. MicroRNAs play an important role in regulating physiological and disease processes including esophageal cancer. OBJECTIVE AND METHODS: In this study, we developed an in vitro model by treating non-neoplastic Het- 1A esophageal cell line with cigarette smoke and chewing tobacco. We carried out miRNA sequencing on Illumina HiSeq 2500 platform and compared miRNA expression pattern across cigarette smoke and chewing tobacco treated Het-1A cells with untreated cells. RESULTS: We identified and quantified 433 miRNAs in both smoke exposed and chewing tobacco treated cells, of which 13 miRNAs showed significantly altered expression in cigarette smoke exposed cells while 25 miRNAs showed significantly altered expression in chewing tobacco treated cells. In addition, we predicted novel miRNAs from these data-sets. We evaluated miRNAs that showed selective or context dependent expression pattern in cigarette smoke exposed or chewing tobacco treated cells. CONCLUSION: In this study, we have comprehensively mapped miRNA expression pattern in response to cigarette smoke and chewing tobacco in Het-1A cells. We identified miRNAs that show altered expression in these cell models.
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Transformación Celular Neoplásica/genética , Esófago/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/genética , Fumar/efectos adversos , Tabaco sin Humo/efectos adversos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/patología , Células Cultivadas , Esófago/metabolismo , Esófago/patología , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia de ARN/métodosRESUMEN
BACKGROUND: Skin acts as a protective barrier against direct contact with pollutants but inhalation and systemic exposure have indirect effect on keratinocytes. Exposure to diesel exhaust has been linked to increased oxidative stress. OBJECTIVE: To investigate global proteomic alterations in diesel particulate extract (DPE)/its vapor exposed skin keratinocytes. METHODS: We employed Tandem Mass Tag (TMT)-based proteomics to study effect of DPE/DPE vapor on primary skin keratinocytes. RESULTS: We observed an increased expression of oxidative stress response protein NRF2, upon chronic exposure of primary keratinocytes to DPE/its vapor which includes volatile components such as polycyclic aromatic hydrocarbons (PAHs). Mass spectrometry-based quantitative proteomics led to identification 4490 proteins of which 201 and 374 proteins were significantly dysregulated (≥1.5 fold, p≤0.05) in each condition, respectively. Proteins involved in cellular processes such as cornification (cornifin A), wound healing (antileukoproteinase) and differentiation (suprabasin) were significantly downregulated in primary keratinocytes exposed to DPE/DPE vapor. These results were corroborated in 3D skin models chronically exposed to DPE/DPE vapor. Bioinformatics analyses indicate that DPE and its vapor affect distinct molecular processes in skin keratinocytes. Components of mitochondrial oxidative phosphorylation machinery were seen to be exclusively overexpressed upon chronic DPE vapor exposure. In addition, treatment with an antioxidant like vitamin E partially restores expression of proteins altered upon exposure to DPE/DPE vapor. CONCLUSIONS: Our study highlights distinct adverse effects of chronic exposure to DPE/DPE vapor on skin keratinocytes and the potential role of vitamin E in alleviating adverse effects of environmental pollution.
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Queratinocitos/efectos de los fármacos , Material Particulado/toxicidad , Proteoma/efectos de los fármacos , Piel/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Antioxidantes/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Humanos , Queratinocitos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Cultivo Primario de Células , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal/efectos de los fármacos , Piel/metabolismo , Espectrometría de Masas en Tándem , Factores de Tiempo , Vitamina E/farmacologíaRESUMEN
Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco.
Asunto(s)
Regulación de la Expresión Génica , Queratinocitos/metabolismo , MicroARNs/genética , Mucosa Bucal/citología , Fumar , Tabaco sin Humo , Biomarcadores , Biología Computacional/métodos , Exposición a Riesgos Ambientales/efectos adversos , Humanos , Queratinocitos/patología , FenotipoRESUMEN
Smoking is the leading cause of preventable death worldwide. Though cigarette smoke is an established cause of head and neck cancer (including oral cancer), molecular alterations associated with chronic cigarette smoke exposure are poorly studied. To understand the signaling alterations induced by chronic exposure to cigarette smoke, we developed a cell line model by exposing normal oral keratinocytes to cigarette smoke for a period of 12 months. Chronic exposure to cigarette smoke resulted in increased cellular proliferation and invasive ability of oral keratinocytes. Proteomic and phosphoproteomic analyses showed dysregulation of several proteins involved in cellular movement and cytoskeletal reorganization in smoke exposed cells. We observed overexpression and hyperphosphorylation of protein kinase N2 (PKN2) in smoke exposed cells as well as in a panel of head and neck cancer cell lines established from smokers. Silencing of PKN2 resulted in decreased colony formation, invasion and migration in both smoke exposed cells and head and neck cancer cell lines. Our results indicate that PKN2 plays an important role in oncogenic transformation of oral keratinocytes in response to cigarette smoke. The current study provides evidence that PKN2 can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients with a history of smoking.