1.1 Lung cancer
Lung cancer is the most common cancers in the world. Due to lacking of advances in diagnostic and therapeutic techniques, the overall 5-year survival rate of lung cancer remains low, which is less than 18%.1 In Taiwan, lung cancer remains the leading cause of cancer death which recorded the 5-year survival rate of 15.9%, with a median survival of 13.2 months.2 In general, there are two major types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is further categorized into subgroups including adenocarcinomas, squamous cell and large cell carcinomas. NSCLC accounts for approximately 80–85% of lung cancer cases, with squamous cell carcinoma and adenocarcinoma being the most common histological subtypes.3
1.2 Minichromosome maintenance protein 2 (MCM2)
Mcm2-7 is a helicase complex which consists of six different subunits (historically numbered from 2 to 7). MCM2 is also called as DNA replication licensing factor, is one of six members of the minichromosome maintenance (MCM) protein family. Briefly, replication initiation starts with licensing process, which involved the assembly of pre-replicative complexes (pre-RCs) at replication origins. The six subunit origin recognition complex (ORC; subunits Orc1-6), Cdc6 and Cdt1 cooperate to load the hexameric MCM2-7 complex onto double-stranded DNA as an inactive double hexamer.4,5 At the G1/S transition, two kinases, CDK and Dbf4-dependent kinase (DDK), activate the MCM helicase, which involves the recruitment of Cdc45 and the heterotetrameric GINS complex to form the CMG complex.6-8 MCM complex forms the
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part of the pre-replication complex (pre-RC) at origins of DNA replication and acts as of "licensing factor" to initiate DNA replication and to limit replication to one round per cell cycle.9,10
1.3 The MCM2 and cancer
MCM2 have been proposed as an excellent proliferation marker to evaluate the proliferation state in many types of cancer including colon cancer, gastric cancer, breast cancer and prostate cancer.11-14 Previous studies reported that MCM2 is a promising marker in premaglinant lung cells and non-small cell cancer.15,16 MCM genes are associated with lung cancer as evidenced by a study of 178 tumor genomes revealed that 10% of lung squamous cell carcinomas were found to contain amplifications and 12%
of lung squamous cell carcinomas contained point mutations in at least one of the six
MCM genes.
17 In in-vivo animal studies, an experimental reduction of MCM2 expression in transgenic mice caused lymphomas, implied that reductions in MCM2 expression levels are linked to human cancer.18,19 Recent studies revealed the role of MCM2 as a novel therapeutic target of anti-cancer drugs such as TSA in colon cancer cells and lovastatin in NSCLCs, suggested that MCM2 might play an important role in cancer treatment. 20,21 The molecular regulation of MCM2 in lung cancer has not been fully elucidated and the MCM2 offers a novel target for drug development to block uncontrolled cancer proliferation.1.4 Phosphoproteomics
Recently, phosphoproteomic technologies which primarily focusing on post-translation modifications have emerged as useful tools for large-scale study of phosphorylated proteins and contributed to the development of cancer biomarkers and drug targets.
Protein phosphorylation is the most frequent reversible post-translation modifications.
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In eukaryotic proteome, upwards of 30% of the proteins are prone to be phosphorylated at some point during their existence.22 Study of phosphoproteomics is of great importance as protein phosphorylation governs most of the signal transduction processes and regulates a number of cellular processes, including cell cycle, growth, apoptosis, and differentiation. In phosphoproteomic experiments, the problem of low abundant phosphorylated proteins needed to be tackled. To date, there are various methods developed to enrich the low abundant phosphorylated proteins or peptides prior to MS analysis. The common approaches for serine/threonine and tyrosine phosphopeptide enrichment included the immobilized metal affinity chromatography (IMAC), IMAC with methyl esterification, strong cation exchange chromatography and metal oxide chromatography (MOC) using titania, zirconia and alumina.23-32 A more popular phosphopeptides enrichment method using MOC modified with aliphatic hydroxy acids named as Hydroxy Acid-modified Metal Oxide chromatography (HAMMOC) has proven to be successful in large scale phosphoproteomic studies with advantage of reducing non-specific binding of the acidic amino acid residues.33 In this study, we applied mass spectrometry-based phosphoproteomics using HAMMOC phosphopeptides enrichment method to characterize the phosphorylation events of MCM2 in NSCLC. We analyzed and interpreted the phosphoproteome data of NSCLC in response to MCM2 in order to identify the reliable candidate proteins for subsequent experimental studies or downstream analysis, to discover protein-protein interactions and to infer about the regulatory network. Here, we obtained a global view of protein phosphorylation events that occur downstream of MCM2 in lung cancer cells.
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1.5 Aim of the study
1. To acquire the phosphoproteomic profile and characterize the phosphorylation events upon MCM2 overexpression and silencing MCM2.
2. To investigate and validate the biological process regulated by MCM2 in human lung cancer cells.
3. To identify the key phosphoproteins regulated by MCM2 and study its
phospho-regulation function.1.6 Experimental Design
The experimental design of this study is illustrated in Figure 1. We first compared the endogenous MCM2 protein expression level in two lung carcinoma cell lines A549 and H1299. Next, we optimized the transfection condition for MCM2 overexpression and silencing MCM2. For MCM2 overexpression in A549 cells, we transfect cells using 1 ug of MCM2-overexprssing plasmid and harvest cells after 24 hours. For silencing MCM2 in H1299 cells, we transfect cells using siRNA at final concentration of 10 nmol and harvest cells after 48 hours. Protein was extracted from cell lysates, subjected to the phosphoproteomics workflow (as shown in Figure 2) including reduction, alkylation and protein digestion, dimethyl labelling, phosphopeptide enrichment and nanoLC−MS/MS analysis. The raw LC-MS/MS data were analyzed by MaxQuant version 1.5.0.30 using the Andromeda Search engine and searched against the human Swiss-Prot database (reviewed, without isoforms) September 2014 release. We compared the overlap in phosphosites and phosphoproteins from phosphoproteome of MCM2 overxepression in A549 cells versus silencing MCM2 in H1299 cells.
Phosphoproteins that change phosphorylation level significantly are selected by ratio H/L normalized <= 0.67 (1.5-fold reduced) or >= 1.5 (1.5-fold increased). Ratio H/L
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normalized represents peptide ratios provided by the MaxQuant output, which have been normalized for each LC-MS/MS run. Localization of PTM probabilities are required to be at least of 0.75.
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