Global proteomic profiling identifies etoposide chemoresistance markers in non-small cell lung carcinoma.

UNLABELLED Chemoresistance is one of the leading health concerns in cancer treatment. Understanding the mechanism of chemoresistance is the best way to improve the survival of the patient. Etoposide and its analogues are widely used as antitumor drugs in lung cancer but many etoposide resistant lung cancer cases has been identified in recent years. The present study aims to explore the cellular response of lung cancer cell lines to etoposide and finding the potential chemoresistant marker proteins. Multiple proteomic platforms like 2-DE, DIGE and iTRAQ have been used to study the global proteome profile of NCI-H460 and etoposide resistant NCI-H460R cell lines. Our study revealed that etoposide treatment leads to alteration of 83 proteins in NCI-H460R cell lines. The functional analysis highlighted the role of the differential expressed proteins in cellular signaling, apoptosis, and cytoskeleton reorganization. Our study has identified several new proteins like RHOC, DLG5, UGDH, TMOD3 in addition to known chemoresistance associated proteins. In silico prediction of the important selected candidates are further validated at protein and mRNA level. Further, functional studies of newly identified candidate genes RHOC and DLG5 revealed that chemotherapeutic resistance is associated with their elevated level and may serve as novel targets for therapeutic intervention. BIOLOGICAL SIGNIFICANCE Etoposide and its analogues have been used for lung cancer treatment for a while and it was reported that many non small cell lung carcinoma patients are resistant to etoposide. Although etoposide show drug resistance, the exact mechanism was not well understood. The present study focused on the global proteome analysis of NCI-H460 and NCI-H460R cell lines using multiple proteomic platforms to understand the potential chemoresistant markers for etoposide. Our multi-proteomic analysis has showed differential expression of 83 proteins involved in oxidative phosphorylation, metabolic, protein folding, cytoskeleton associated protein along with apoptotic pathway has been identified. In addition, quite a few interesting proteins such as RHOC, DLG5, HSP90, citrate synthase, UDP-glucose-6-dehydrogenase, Tropomodulin-3 are involved in chemoresistance has been observed. Overall, this is the first comprehensive proteomic study on etoposide resistant cell line NCI-H460 to explore the mechanism of chemoresistance in lung cancer.

[1]  Y. Hannun,et al.  Hyaluronan Constitutively Regulates Activation of COX-2-mediated Cell Survival Activity in Intestinal Epithelial and Colon Carcinoma Cells* , 2008, Journal of Biological Chemistry.

[2]  Y. Hozumi,et al.  RhoC Upregulation Is Correlated with Reduced E-cadherin in Human Breast Cancer Specimens After Chemotherapy and in Human Breast Cancer MCF-7 Cells , 2014, Hormones and Cancer.

[3]  D. Rigal,et al.  Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells. , 2010, International journal of oncology.

[4]  Lorna Wilkinson-White,et al.  A dye-binding assay for measurement of the binding of Cu(II) to proteins. , 2008, Journal of inorganic biochemistry.

[5]  W. Russell.  The Response of the Central Nervous System of the Rat to Methylcholanthrene. II. The Effect of a Diet Deficient in Thiamine and Riboflavin on the Induction of Tumors Derived from Nervous Tissue , 1945 .

[6]  G. Szakács,et al.  Multidrug Resistance Mediated by MDR-ABC Transporters , 2009 .

[7]  Michael R. Green,et al.  F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage , 2009, Nature.

[8]  S. Ohno,et al.  Role of Lgl/Dlg/Scribble in the regulation of epithelial junction, polarity and growth. , 2008, Frontiers in bioscience : a journal and virtual library.

[9]  C. Peters,et al.  Potential role for Cathepsin D in p53-dependent tumor suppression and chemosensitivity , 1998, Oncogene.

[10]  K. Hande,et al.  Etoposide: four decades of development of a topoisomerase II inhibitor. , 1998, European journal of cancer.

[11]  K. Kavanagh,et al.  UDP-glucose dehydrogenase: structure and function of a potential drug target. , 2010, Biochemical Society transactions.

[12]  Amalia Bosia,et al.  RhoA Silencing Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells , 2008, Molecular Cancer Research.

[13]  U. Gündüz,et al.  Etoposide resistance in MCF-7 breast cancer cell line is marked by multiple mechanisms. , 2014, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[14]  Eric S. Lander,et al.  Genomic analysis of metastasis reveals an essential role for RhoC , 2000, Nature.

[15]  F. Zhou,et al.  Induction of Paclitaxel Resistance by ERα Mediated Prohibitin Mitochondrial-Nuclear Shuttling , 2013, PloS one.

[16]  S. Abhang,et al.  Quantitative tissue proteomic investigation of invasive ductal carcinoma of breast with luminal B HER2 positive and HER2 enriched subtypes towards potential diagnostic and therapeutic biomarkers. , 2016, Journal of proteomics.

[17]  K. Cowan,et al.  Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance. , 1994, Cancer research.

[18]  B. Stewart,et al.  World Cancer Report , 2003 .

[19]  Dong Liu,et al.  Prohibitin silencing reverses stabilization of mitochondrial integrity and chemoresistance in ovarian cancer cells by increasing their sensitivity to apoptosis , 2008, International journal of cancer.

[20]  G. Sathe,et al.  A comprehensive proteomic analysis of totarol induced alterations in Bacillus subtilis by multipronged quantitative proteomics. , 2015, Journal of proteomics.

[21]  S. Shangary,et al.  Involvement of cathepsin D in chemotherapy-induced cytochrome c release, caspase activation, and cell death , 2005, Molecular Cancer Therapeutics.

[22]  G. Mukherjee,et al.  Notch1 regulates the functional contribution of RhoC to cervical carcinoma progression , 2009, British Journal of Cancer.

[23]  M. Santra,et al.  Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture , 2013, BioMed research international.

[24]  Sanjeeva Srivastava,et al.  A Simple Protein Extraction Method for Proteomic Analysis of Diverse Biological Specimens , 2014 .

[25]  M. Simpson,et al.  Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice. , 2002, The American journal of pathology.

[26]  M. Dean,et al.  Multidrug Efflux Pumps and Cancer Stem Cells: Insights Into Multidrug Resistance and Therapeutic Development , 2011, Clinical pharmacology and therapeutics.

[27]  N. Osheroff,et al.  Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. , 1998, Biochimica et biophysica acta.

[28]  W. Mckeehan,et al.  Sequence analysis of LRPPRC and its SEC1 domain interaction partners suggests roles in cytoskeletal organization, vesicular trafficking, nucleocytosolic shuttling, and chromosome activity. , 2002, Genomics.

[29]  Amy S. Lee,et al.  ER chaperones in mammalian development and human diseases , 2007, FEBS letters.

[30]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[31]  Ting Liu,et al.  Citrate Synthase Expression Affects Tumor Phenotype and Drug Resistance in Human Ovarian Carcinoma , 2014, PloS one.

[32]  S. Lindquist,et al.  HSP90 at the hub of protein homeostasis: emerging mechanistic insights , 2010, Nature Reviews Molecular Cell Biology.

[33]  Wen-feng Gou,et al.  The role of RhoC in ovarian epithelial carcinoma: a marker for carcinogenesis, progression, prognosis, and target therapy. , 2013, Gynecologic oncology.

[34]  S. Srinivasula,et al.  Negative regulation of cytochrome c‐mediated oligomerization of Apaf‐1 and activation of procaspase‐9 by heat shock protein 90 , 2000, The EMBO journal.

[35]  Velia M. Fowler,et al.  Tmod3 regulates polarized epithelial cell morphology , 2007, Journal of Cell Science.

[36]  S. Lindquist,et al.  HSP90 and the chaperoning of cancer , 2005, Nature Reviews Cancer.

[37]  Resistance mechanisms and their regulation in lung cancer. , 1996, Critical reviews in oncogenesis.

[38]  K. Jaskiewicz,et al.  Enhanced Citrate Synthase Activity in Human Pancreatic Cancer , 2005, Pancreas.

[39]  M. Mizuguchi,et al.  A novel SOS1 mutation in Costello/CFC syndrome affects signaling in both RAS and PI3K pathways , 2013, Journal of receptor and signal transduction research.