Original Article Identification of Novel Proteins in Chemoresistant Lung Cancer Cells by Quantitative Proteomics

Int J Clin Exp Pathol 2018;11(3):1101-1111 www.ijcep.com /ISSN:1936-2625/IJCEP0069378 Original Article Identification of novel proteins in chemoresistant lung cancer cells by quantitative proteomics Li Su1, Jiao Liu1, Hongying Zhen2 1Center of Medical and Health Analysis, Peking University, Beijing, China; 2The Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Received November 18, 2017; Accepted December 2, 2017; Epub March 1, 2018; Published March 15, 2018 Abstract: Lung cancer is the leading cause of cancer-related deaths worldwide, with a five-year survival rate of only 18%. Non-small cell lung cancer (NSCLC) in addition to large cell lung cancer, comprise 85%-90% of all lung cancer diagnoses. Chemoresistance of the cancer cells is one of the reasons for the poor survival rate. In this research, we used mitoxantrone-induced resistant (MXR) NCl-H460 cells to find the mechanism of chemoresistance. We found that the MXR-resistant cells had high single-cell clonogenic ability like cancer stem cells. From the quantitative proteomics study, we found that the MXR-resistant cells high upregulated many metabolism and stem cell-related proteins, such as STAT3 and ALDH. The high level expression of histone 3.1 showed the possibility of genetic chang- ing of resistant cells. Using Western blot assays, we confirmed enhancement of EZH2 in MXR-resistant NCl-H460 cells. Therefore, the EZH2-STAT3 pathway has an important role in the MXR-resistant NCI-H460 cancer cells. Both EZH2 and STAT3 can be used as new target proteins for chemotherapy in the treatment of large cell lung cancer. Keywords: Multidrug resistance, mitoxantrone, proteomics, bioinformatics, STAT3, cancer stem cells Introduction of the resistance is not very clear. Cancer stem cells (CSCs) are one of the important reasons. Cancer incidence and mortality have been increasing in China, making cancer the leading Cancer stem cells, also called tumor-initiating cause of death since 2010 and a major public cells, have a high capacity for self-renewal and health problem. Lung cancer is the leading multi-lineage differentiation and are believed to cause of cancer-related deaths worldwide, with be responsible for tumor development, recur- rence and dissemination, as well as the acquisi- a five-year survival rate of only 18% [1]. In China tion of drug resistance [6]. These “stemness” also, lung cancer is the most common and has properties are governed by pathways such as the poorest survival rate [2]. STAT3, NANOG, NOTCH, WNT, and HEDGEHOG, There are two kinds of lung cancer, one is called which are highly dysregulated in CSCs due to genetic and epigenetic changes of cancers [7]. small cell lung cancer (SCLC), which is sensitive The proteins of these pathways are thought of to chemotherapy and has a high survival rate. as the CSCs markers. Different CSCs show dif- The other one is called non-small cell lung can- ferent dysregulation of the pathways. In this cer (NSCLC), which comprises 85%-90% of lung research, we used the non-small cell lung can- cancer diagnoses [3]. Despite drastic treat- cer cell lines, NCI-H460, to find the main dys- ment strategies, including radiotherapy, che- regulated proteins during the mitoxantrone- motherapy, and even immune approaches, the induced chemoresistance by quantitative five-year survival rate for all NSCLC is still < proteomics. We found 60 proteins that were 20% [4, 5]. Chemotherapy is not as useful for dysregulated which might mediate the mixto- large cell lung cancer as it is with small cell lung xantrone-resistance of the NCI-H460 cells, cancer. Multidrug resistance is one of the rea- including upregulation of ALDH1 and the EZH2- sons for its poor survival rate. The mechanism STAT3 pathway, which can be used as the new EZH2-STAT3 pathway and mitoxantrone-resistance in NCl-H460 cells protein targets of large cell lung cancer th- China) and the protein concentration was erapy. determined by BCA assay (Pierce, Thermo Fisher Scientific, MA, USA). Each group was Materials and methods analyzed in triplicate. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- Sample preparation for LC-MS phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), ammonium bicarbonate, sodium deoxycholate, Protein samples (50 μg) from each group were and iodoacetamide were purchased from Sigma processed according to the manufacturer’s (St. Louis, MO, USA). Tris-(2-carboxyethyl) phos- protocol for filter-aided sample preparation phine (TCEP) was acquired from Thermo Sci- (FASP) [10]. In brief, proteins were concentrat- entific (Rockford, Il, USA). Modified sequencing- ed using Vivacon 500 filtration tubes (Cat No. grade trypsin was obtained from Promega (Ma- VNO1HO2, Sartorius Stedim Biotech, UK), dison, WI, USA). All mobile phases and solutions mixed with 100 μL of 8 M urea in 0.1 M Tris/ were prepared with HPLC grade solvents (i.e. HCL (pH 8.5) buffer, and centrifuged at 14,000 water, acetonitrile, methanol, and formic acid) g and 4°C for 15 minutes. This step was per- from Fisher. All other reagents were from com- formed twice, after which 10 μL of 0.05 M TCEP mercial suppliers and were of standard bio- in water was added to the filters, and samples chemical quality. were incubated at 37°C for 1 h. Then, 10 μL of 0.1 M iodoacetamide (IAA) was added to the Cell lines and generation of chemoresistant filters, then the samples were incubated in cancer cells darkness for 30 minutes. Filters were washed twice with 200 μL of 50 mM NH HCO . Finally, Cancer cell line, NCl-H460 was used. Ch- 4 3 1 μg of trypsin in 100 μL of 50 mM NH HCO emotherapeutic drug, mitoxantrone (MXR, 20 4 3 was added to each filter. The protein to enzyme ng/mL) was used, along with a mutant from the ratio was 50:1. Samples were incubated over- previous research [8]. Drug treatments were night at 37°C and released peptides were col- repeated two or three times, which mimicked lected by centrifugation. the clinical regimen that patients with cancers would receive. This strategy ensured that more LC-MS analysis than 95% of cells underwent apoptosis or senescence with the senescent cells eventually LC-MS experiments were performed on a nano- dying, thereby selecting the most resistant flow HPLC system (Easy-nLC 1000, Thermo clones. Fisher Scientific, Waltham, MA, USA) connect- ed to a LTQ-Orbitrap Elite mass spectrometer Single-cell clonogenic assay (Thermo Fisher Scientific), equipped with a To assess self-renewal in vitro, cells were sort- Nanospray Flex Ion Source (Thermo Fisher ed by flow cytometry (FACS Aria II) using the Scientific). 1 μg peptide mixtures (5 μL) were single-cell sorting model, seeded into 6-well separated using a home-made reversed-phase plates (50, 100 or 200 cells per well), and cul- C18 column (150 μm I.D. × 150 mm, 3 μm of tured in a 10% fetal bovine serum (FBS)- particle size) at a flow rate of 300 nL/min. containing RPMI-1640 medium. Cell clones Chromatographic separation was performed were counted after fourteen days when a clone with a 60 min gradient of 2% to 40% acetoni- reached 100-200 cells [8, 9]. trile in 0.1% formic acid. The electrospray volt- age was maintained at 2.2 kV, and the capillary Protein preparation temperature was set at 275°C. The LTQ- Orbitrap was operated in data-dependent After determining the density of the harvested mode to simultaneously measure full scan MS NaCl-H460 (N group) and MXR-chemoresistant spectra (m/z 300-1800) in the Orbitrap with a cells (M group), aliquots containing 107 cells mass resolution of 60,000 at m/z 400. After a were collected and placed into individual tubes full scan survey, the fifteen most abundant ions and washed with PBS. After centrifugation detected in the full MS scan were measured in (1000 rpm, 5 min, room temperature), cells the LTQ-Orbitrap, using collision-induced disso- were lysed in RIPA buffer (Applygen, Beijing, ciation (CID). 1102 Int J Clin Exp Pathol 2018;11(3):1101-1111 EZH2-STAT3 pathway and mitoxantrone-resistance in NCl-H460 cells Western blotting The same protein samples used for the LC-MS analysis were also used for the Western blotting assay. After the addition of the sample loading buffer, protein samples of each group were separated using 10% SDS-PAGE and were subsequently transferred to the PVDF membrane (Millipore). The membrane was incubated in fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline, pH 7.4, containing 5% non- fat dried milk) at room temperature for 1 h and Figure 1. Colony forming efficiency. The MXR-che- then probed with monoclonal mouse anti-STAT3 moresistant cells had higher colony forming effi- antibody (Abcam, Cambridge, UK), mouse anti- ciency compared with the normal NCl-H460 cells EZH2 antibody (BD Bioscience, America), and (P=0.0186). mouse anti-glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) antibody (Zhongshan Gold Bridge Biotechnology Co. Ltd, China) in blocking Protein identification and quantification buffer at 4°C overnight. The membrane was Data analysis was performed with MaxQuant washed three times for 5 minutes each using software (version 1.6.0.16) (http://www.max- PBST (PBS containing 0.1% Tween-20), then quant.org/) [11]. For protein identification, MS/ incubated with the appropriate horseradish MS data were submitted to the Uniprot human peroxidase (HRP)-conjugated secondary anti- protein database using the Andromeda search body at room temperature for 1 h, and washed engine [12] with the following settings: trypsin three more times in PBST buffer. The mem- cleavage; fixed modification of carbamidometh- brane was finally incubated with ECL substrate ylation of cysteine; variable modifications of solution (ECL Kit, Perkinelmer) for 5 minutes, methionine oxidation; a maximum of two according to the manufacturer’s instructions, missed cleavages; false discovery rate of 0.01 and visualized with autoradiographic film.

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