Cancer is tightly associated with abnormal uncontrolled proliferation. A new perspective of cancer progression proposes that DNA replication stress is induced by sustained proliferation, subsequently causing two other cancer hallmarks, escape from apoptosis and genomic instability.42 Small molecule inhibitors that block uncontrolled cancer proliferation by targeting leading or lagging strand synthesis have been used in cancer therapy, such as topoisomerases, DNA polymerases, DNA ligase, proliferating cell nuclear antigen (PCNA), ribonucleotide reductase, and telomerase.43-47 Eukaryotic replication factors are emerged as potential chemotherapeutic targets in cancer treatment but to date, little is known about the inhibitors targeting MCM proteins, which serve as a licensing factor in replication initiation. MCM2 is well-known for its good association with cell proliferation but its precise functional role in lung cancer cells has not been reported. Understanding the molecular basis of MCM2 in lung cancer cells enables us to discover alternative target for lung cancer therapy.
ZNF322 (Zinc finger protein 322) was isolated from an early human embryo heart cDNA library which encodes a zinc-finger protein containing 9 tandem repeated C2H2 type zinc fingers. Overexpression of ZNF322 increase transcription activities of SRE and AP-1, suggested the role of ZNF322 as transcriptional activator in MAPK signaling pathways.48 From our previous studies, proteomics analysis of ZNF322A in human lung adenocarcinoma cells A549 using Isobaric tags for relative and absolute quantitation (iTRAQ) identified several candidate proteins that are regulated by ZNF322A.49MCM2 is one of the candidate proteins that are up-regulated by ZNF322A and their regulation relationship was validated by western blot analysis.
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In the present study, we explored the role of MCM2 in lung cancer cells. We first evaluated the endogenous expression of MCM2 in NSCLC cell lines A549 and H1299.
Western blotting demonstrated that H1299 cells showed higher level of endogenous MCM2 protein as compared to A549 cells. Cell viability analysis showed that MCM2 overexpression promotes cell proliferation in A549 cells and silencing MCM2 inhibits cell proliferation in H1299 cells. This is consistent with the essential role of MCM2 in cellular proliferation. Aberrant protein phosphorylation is often correlated with diseases.50,51 To better elucidate the post-translational events and to reveal the regulatory networks behind these effects, we set up a large-scale mass spectrometry-based quantitative phosphoproteomics study of MCM2 using HAMMOC-TiO2 phosphopeptide enrichment strategies. We compared the differential regulation of phosphorylated proteins in the NSCLC cell lines A549 and H1299 cells in response to MCM2 overexpression and silencing MCM2 respectively. Phosphoproteomic analysis using two different cell lines would provide a more comprehensive profiling of MCM2 downstream events. Surprisingly, our results show that nearly all phosphosites are uniquely response to MCM2 overexpression or silencing MCM2, respectively. There is only a small overlap in differentially regulated phosphosites between the two phosphoproteomes, suggesting that MCM2 downstream regulations are cell-type dependent.
Bioinformatic analysis showed that the regulated phosphoproteins from both phosphoproteome are enriched most in biological functions such as RNA splicing, protein folding, regulation of protein complex assembly, regulation of cytoskeleton organization, regulation of actin filament polymerization, macromolecular complex subunit organization, microtubule-based movement, DNA replication and cell cycle.
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RNA has been linked to cancer-related functions in which defects in alternative splicing lead to various human diseases, primarily neurodegenerative diseases and cancer.52 Cancer cells with aberrant splicing profiles expressing isoforms that can promote cell proliferation, survival, migration and invasion, affect metabolism and in general involved in any aspect of tumor progression and maintenance.53 It has been also reported that MCM2 involved in regulation of cell migration and invasion in medulloblastoma.54 Not surprisingly, increased cell migration ability induced by MCM2 overexpression in A549 cells and reduced cell migration ability upon MCM2 knockdown in H1299 cells were observed in our study. Previous studies demonstrated that knockdown of MCM2 led to cell cycle arrest and cell apoptosis in colon cancer cells (HCT116) and NSCLCs (A549 and GLC-82).20,21 Our data from flow cytometric analysis indicated that silencing MCM2 in p53 null cell line H1299 induces cell cycle arrest in G1/S phase may result from a p53-independent pathway.
Phosphorylation of MCM2 occurs at multiple sites which resulted in a conformational change of the complex and activation of the helicase activity is essential for the initiation of DNA replication in human. Previous studies reported that three sites Ser40, Ser53 and Ser108 on MCM2 have been identified as Cdc7/Dbf4 phosphorylation sites in HeLa cells.55 The Ser108 site was found to be the site of phosphorylation by ATR in the presence of DNA damage.56 Furthermore, the Ser27, Ser41 and Ser139 sites have been identified as major Cdc7 phosphorylation sites in vitro and in HeLa cells confirming Ser139 as a Cdc7 phosphorylation site.57 Perturbations of MCM2 protein in cancer cells by overexpression or suppression would probably induce changes in phosphorylation level of MCM2, thereby alter the process of initiation of DNA replication. Our data identified four sites (Ser26, Ser27, Ser108 and Ser 139) on MCM2
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itself to be differentially phospho-regulated and all of these sites have been reported previously. Of these phosphosites, Ser108 is identified only in A549 cells upon MCM2 overexpression while Ser26 is identified only in H1299 cells upon silencing MCM2.
The discrepancies of MCM2 phosphorylation sites between the two phosphoproteomes may due to the differences of cell lines used and the differences of perturbation to cell lines.
High mobility group (HMG) proteins are the most abundant non-histone chromatin associated proteins. The HMG proteins are categorized into three families based on their DNA binding domains: HMGA (containing AT-hooks), HMGB (containing HMG-boxes) and HMGN (containing nucleosomal binding domains).58,59 High mobility group A (HMGA) proteins bind the minor groove of AT-rich DNA sequences through three N-terminal basic domains called “AT-hooks”. The HMGA family consists of four members: HMGA1a, HMGA1b and HMGA1c (which are encoded through alternative splicing by the HMGA1 gene) and HMGA2 (encoded by the HMGA2gene).60,61 Recent study reported that elevated expression of HMGA1 correlates with the malignant status and prognosis of non-small cell lung cancer.62 HMGA1 protein is highly regulated by post-translational modifications, such as acetylation, methylation and phosphorylation.
Nearly all of HMGA1a and HMGA1b proteins are constitutively phosphorylated at the two or three serine occurred predominantly at the residues of the C-terminal peptide 88-106, a region known to be critical for HMGA1-DNA contact.63,64 HMGA1 phosphorylation reduced DNA-binding activity of HMGA1 whereas protein dephosphorylation increases HMGA1-DNA binding affinity, resulted in gene repression or activation.65,66 Constitutive and inducible phosphorylation at serine residues Ser98, Ser101 and Ser102 has been reported to be dependent on PI-3K via a casein kinase 2
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(CK2)-like specificity.64,66 HMGA1, specifically the HMGA1a isoform has proposed as a novel downstream target of the INSR signaling pathway, which may play an important role in the regulation of insulin signaling and action in vivo.67 In our phosphoproteome of MCM2 overexpression, tri-phosphorylated HMGA1 protein (Ser99, Ser102 and Ser103) was detected in A549 cells after 24 hours MCM2 overexpression but only the phosphosite of Ser99 show the significant increase in phosphorylation level. In phosphoproteome of silencing MCM2, a significant decrease of the tri-phosphorylated HMGA1 protein (Ser99, Ser102 and Ser103) was detectable in H1299 cells at 48 hours siMCM2 post-transfection. Interestingly, phosphorylated HMGN1 protein (Non-histone chromosomal protein HMG-14) at phosphosites of Ser86 and Ser89 was detected upon MCM2 overexpression but not in silencing MCM2. The phosphosite of Ser99 on HMGA1 was found to be differentially up-regulated upon MCM2 overexpression and differentially down-regulated upon silencing MCM2, thus is presented as a novel candidate for phosphoproteomics analysis. To further investigate the biological significances of HMGA1 phosphorylation at Ser99, we examined the cell viability of HMGA1 phosphorylation site mutants expressed in A549 cells. Our study showed that mutation of Ser99 to alanine significantly decreased cell viability of A549 cells, suggesting that phosphorylation of Ser99 of HMGA1 may play important roles in cancer proliferation. The function of this phosphorylation site is yet clearly understood.
Future studies are needed to clarify the importance of HMGA1 phosphorylation in lung cancer study which might result in a profound impact on lung cancer treatment.
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