Minichromosome maintenance proteins in lung adenocarcinoma: Clinical significance and therapeutic targets

Lung cancer is the most common cause of cancer‐related death worldwide, accounting for 1.8 million deaths annually. Analysis of The Cancer Genome Atlas data showed that all members of the minichromosome maintenance (MCM) family (hexamers involved in DNA replication: MCM2‐MCM7) were upregulated in lung adenocarcinoma (LUAD) tissues. High expression of MCM4 (P = 0.0032), MCM5 (P = 0.0032), and MCM7 (P = 0.0110) significantly predicted 5‐year survival rates in patients with LUAD. Simurosertib (TAK‐931) significantly suppressed the proliferation of LUAD cells by inhibiting cell division cycle 7‐mediated MCM2 phosphorylation. This finding suggested that MCM2 might be a therapeutic target for LUAD. Moreover, analysis of the epigenetic regulation of MCM2 showed that miR‐139‐3p, miR‐378a‐5p, and miR‐2110 modulated MCM2 expression in LUAD cells. In patients with LUAD, understanding the role of these miRNAs may improve prognoses.

lung cancer has increased owing to the development of effective treatment strategies, including molecularly targeted drugs and immune checkpoint inhibitors [5][6][7].
The prognosis of patients with LUAD is poor (the average 5-year survival rate is below 20%), despite the availability of many treatment strategies [8]. Moreover, many patients with lung cancer are often diagnosed at an advanced stage, and metastasis to other organs occurs frequently [9,10]. Unfortunately, effective treatment regimens for patients with metastatic lung cancer are limited [11]. Therefore, additional studies are needed to identify effective targeted molecular therapies for LUAD.
Minichromosome maintenance (MCM) family proteins are composed of six subunits (MCM2-MCM7), which form a heterohexameric complex, and act as essential players in the initiation and elongation of eukaryotic DNA replication [12]. The MCM complex is also a replication gatekeeper that allows only one DNA replication per cell cycle, and knockdown of these genes induces cell cycle arrest and apoptosis in cancer cells [13]. Aberrant expression of MCM proteins has been reported in several types of cancers, for example, renal cell carcinoma, prostate cancer, and breast cancer [14][15][16].
Because overexpression of MCM proteins promotes the malignant transformation of cancer cells, anticancer drugs targeting MCMs are being studied [17][18][19]. Previous studies have shown that some antibiotics (e.g., heliquinomycin and ciprofloxacin) inhibit the helicase activity of MCMs and suppress the growth of cancer cells [18,20]. Some statins, for example, lovastatin and atorvastatin, which inhibit HMG-CoA reductase to lower blood cholesterol levels, have also been shown to suppress the expression of MCM2 and induce cell cycle arrest and apoptosis [19,21]. Moreover, biologically active compounds isolated from natural products (e.g., breviscapine and cedrol) suppress the expression of MCMs and inhibit cancer cell proliferation [22,23]. MCMs are therapeutic targets for a wide range of cancers, and control of these molecules may improve the prognosis of patients with cancers.
Numerous studies have shown that the noncoding RNAs in the human genome are key players in finetuning gene expression [24,25]. miRNAs are noncoding RNAs that control gene transcription levels by binding to specific sites in target RNAs [26,27]. Notably, a single miRNA can control many RNA transcripts [25]. Therefore, aberrant expression of miRNAs can trigger the malignant transformation of human cells. Several studies have shown that aberrant expression of miR-NAs occurs frequently in cancer cells and that their expression is closely involved in cancer cell progression, metastasis, and drug resistance [28,29]. Previous studies have shown that miR-1296 directly regulates MCM2 expression in prostate cancer cells [30]. Furthermore, miR-214-3p is downregulated in hepatocellular carcinoma cells, and this miRNA controls the expression of both MCM5 and MCM7 [31].
In this study, we investigated the clinical significance of all MCM family members (hexamers involved in DNA replication: MCM2-MCM7) in LUAD cells. Analysis of The Cancer Genome Atlas (TCGA) data showed that all MCM family members (MCM2-MCM7) were upregulated in LUAD tissues. Overexpression of all MCMs in LUAD clinical specimens was confirmed by immunostaining. Notably, high expression of MCM4 (P = 0.0032), MCM5 (P = 0.0032), and MCM7 (P = 0.0110) significantly predicted 5-year survival rates in patients with LUAD. Functional assays showed that MCM2 was a promising therapeutic target in LUAD cells. Simurosertib significantly suppressed the proliferation of LUAD cells by inhibiting MCM2 phosphorylation.
Finally, we investigated the epigenetic regulation of MCM2 and found that miR-139-3p, miR-378a-5p, and miR-2110 controlled MCM2 expression in LUAD cells. Through their functions as DNA helicases, MCM family members were found to be essential for the replication of genomic DNA and were closely involved in the molecular pathogenesis of LUAD. In particular, MCM2 may be a therapeutic target in patients with LUAD, and control of this molecule may improve prognoses in patients with LUAD.

Cell lines and cell culture
Two NSCLC cell lines, A549 and H1299, were used in this study (American Type Culture Collection, Manassas, VA, USA). Both cell lines were grown in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum. Both cell lines were cultured in an incubator at 37°C in an atmosphere containing 5% CO 2 . Cells were cultured as described previously [34]. Confirmation of negativity for Mycoplasma infection in A549 and H1299 cells was carried out using a LookOut Mycoplasma PCR Detection Kit (catalog no.: MP0035; Sigma-Aldrich, MO, USA) and JumpStart Taq DNA Polymerase (catalog no.: D9307; Sigma-Aldrich) according to the manufacturer's protocol.
Transfection with small interfering RNA (siRNA) and miRNA Opti-MEM (Gibco, Carlsbad, CA, USA) and Lipofectamine RNAiMax Transfection Reagent (Invitrogen, Carlsbad, CA, USA) were used to transfect siRNA and miRNA into cell lines. The procedure for siRNA and miRNA transfection was described previously [34][35][36]. The siRNAs and miRNAs used in this study are listed in Table S1.

RNA extraction and reverse transcriptionquantitative PCR
Total RNA from NSCLC cell lines was extracted using Isogen II (NIPPON GENE Co., Ltd., Tokyo, Japan). A NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to check the quantity and quality of total RNA. cDNA was synthesized using a PrimeScript RT Master Mix (catalog no.: RR036A; Takara Bio Inc., Shiga, Japan). TaqMan Real-Time PCR Assays were conducted to analyze gene expression, as described previously [34][35][36]. Reverse transcriptionquantitative PCR (RT-qPCR) was performed using a Ste-pOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The reagents used in this study are listed in Table S1.  Table S1.

Cell proliferation and cell cycle assays
Cell proliferation was evaluated using a Cell Proliferation Kit (XTT based; Biological Industries, Beit-Haemek, Israel). BD Cycletest Plus DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used for the evaluation of the cell cycle distribution on a BD FACSCelesta Flow Cytometer (BD Biosciences). The data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The procedures for cell proliferation and cell cycle analyses were described previously [34][35][36].

Immunohistochemical staining
Tissue microarray slides (catalog no.: LC811a; US Biomax, Inc., Derwood, MD, USA) were used for immunohistochemical staining. Blocking, the primary antibody reaction, secondary antibody reaction, and binding of avidin to the biotin complex were conducted using a VEC-TASTAIN Universal Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's protocol. Dako Real antibody diluent (Agilent, Santa Clara, CA, USA) was used to dilute the primary antibody. The chromogenic reaction was developed using a Dako REAL EnVision Detection System Peroxidase/ DAB+, Rabbit/Mouse (Agilent). The primary antibody used in this study is described in Table S1. The characteristics of patients from whom the tissue samples were derived in this tissue array are described in Table S2. The intensity and area of staining were evaluated based on previous reports [37], and immunohistochemical scores were calculated.

Cytotoxicity assay
For cytotoxicity assays, 100 lL of cell suspension (approximately 2 9 10 3 cellsÁmL À1 ) was seeded in each well of a 96-well plate, and cells were allowed to attach overnight. The next day, culture media were discarded, and the cells were treated with different concentrations of TAK-931 (catalog no.: HY-100888/CS-0020550; MedChemExpress, NJ, USA) ranging from 1 to 1000 nM. The plates were incubated for 5 days, and media containing TAK-931 were replaced every other day. Cells treated with 0.1% DMSO served as a negative control. To investigate cell toxicity, XTT assays were performed using a Cell Proliferation Kit (Biological Industries) according to the manufacturer's protocol. The signals were measured using a microplate reader (Bio-Rad Laboratories, Inc.).

Colony formation assay
For colony formation assays, 1 mL medium containing 300 cells was added to each well of a 12-well plate, and the cells were treated with different concentrations of TAK-931 the next day. TAK-931 was replaced every other day, and the cells were incubated for 10 consecutive days. Cells treated with 0.1% DMSO were used as a control. At the end of the incubation, the colonies were washed with phosphate-buffered saline and subsequently fixed using 4% paraformaldehyde for 15 min at room temperature. After fixation, the colonies were dyed with 0.5% crystal violet (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 25 min. The wells were washed with water, and the plates were air-dried.

The construction of plasmid and dual-luciferase reporter assays
The wild-type sequences containing miRNAs binding sites in the 3 0 -untranslated regions (UTRs) of MCM2 and the deletion-type sequences lacking them were cloned into the -psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). The procedures for transfection and dual-luciferase reporter assays were provided in previous studies [34,35].

Statistical analysis
Statistical analyses were performed using GRAPHPAD PRISM 8 (GraphPad Software, La Jolla, CA, USA) and JMP PRO 14 (SAS Institute Inc., Cary, NC, USA). Differences between the two groups were analyzed using Student's ttests or Mann-Whitney U-tests. Multiple group comparisons were performed using one-way ANOVA and Tukey tests for post hoc analyses. Survival rates were analyzed using Kaplan-Meier survival curves and log-rank tests.

Expression and clinical significance of MCM2-MCM7 in patients with LUAD by TCGA analysis
First, we analyzed the Gene Expression Omnibus (GEO) database (GEO accession no: GSE19188) to identify genes exhibiting oncogenic effects in NSCLC. The upregulated genes in GSE19188 were narrowed down based on the criteria of log 2 (fold change) greater than 1 and P-value less than 0.05; using these parameters, 613 genes were identified. These candidate genes were classified according to the Kyoto Encyclopedia of Genes and Genomes pathways by GeneCodis 4 database (https://genecodis.genyo.es/) (data downloaded on March 17, 2021) [41]. The top 10 annotations based on relative enrichment score were mucin type O-glycan biosynthesis, DNA replication, glycosaminoglycan biosynthesis-heparan sulfate/heparin, RNA degradation, homologous recombination, protein digestion and absorption, Fanconi anemia pathway, cell cycle, ether lipid metabolism, and glycosphingolipid biosynthesis-lacto and neolacto series (Table 1). In our previous reports, we found that cell cycle and DNA replication genes regulated by miRNAs affected lung cancer progression. Twenty-four and eight genes were classified as cell cycle-and DNA replicationrelated genes, respectively (Tables 2 and 3). MCM2, MCM4, MCM6, and MCM7 were common in these two annotations.
MCM2-MCM7 form a hexamer and act as a DNA helicase essential for genomic DNA replication. Notably, MCM2-MCM7 were significantly upregulated in LUAD tissues compared with normal tissues, and the 5-year overall survival rates tended to be poor in patients with high MCM2-MCM7 expression (Fig. 1).
In this study, we focused on MCM2-MCM7.

Overexpression of MCM2-MCM7 in LUAD clinical specimens
We performed immunohistochemistry (IHC) to investigate MCM2-MCM7 protein expression in LUAD. The nuclear expression of MCM2-MCM7 was high in LUAD but low in normal lung tissues. MCM2-MCM7 were significantly overexpressed in LUAD compared with normal lung tissues (Fig. 2). The characteristics of patients are described in Table S2.

Search for miRNAs that negatively regulate MCMs (MCM2-MCM7) expression in LUAD cells
We hypothesized that one of the reasons for the overexpression of MCMs in LUAD cells is that some miR-NAs expression is repressed. We recently created the RNA-sequencing-based miRNA expression signature using LUAD clinical specimens (GEO accession no: GSE230229). Based on this signature, we selected miR-NAs whose expression is repressed in LUAD tissues.

Alteration of mRNA expression of MCMs (MCM2-MCM7) in LUAD patients based on TCGA-LUAD analysis
We investigated the clinical impact of alteration of MCMs (MCM2-MCM7) expression in patients with LUAD using TCGA-LUAD cohort dataset.
Patients with LUAD who had high expression in at least one of the MCM genes (MCM2-MCM7) showed a worse survival outcome compared with patients with no MCM gene alteration (Fig. 5B). Gene set enrichment analysis using TCGA-LUAD dataset revealed that 'Retinoblastoma Gene in Cancer', 'Cell Cycle', 'DNA IR-damage and cellular response via ATR', and 'G1 to S cell cycle control' were identified as enrichment pathways in high expression of MCM genes group (Fig. 5C).

Effects of MCM2-MCM7 knockdown on cell proliferation and the cell cycle
Next, we investigated the effects of MCM2-MCM7 knockdown to evaluate the functions of MCM2-MCM7 in A549 and H1299 cells. First, we confirmed the knockdown efficiencies of MCM2-MCM7 siRNAs using RT-qPCR and western blotting. Transfection with two siRNAs reduced the mRNA and protein expression of MCM2-MCM7 in A549 and H1299 cells (Figs S2 and S3). Next, we conducted cell proliferation assays. Most siRNAs targeting MCM2-MCM7 suppressed cell proliferation (Fig. 6). Lastly, cell cycle assays were performed after transfection with siRNAs targeting MCM2-MCM7. siRNAs targeting MCM2-MCM5 and MCM7 led to G 0 /G 1 arrest, and those targeting MCM6 induced G 2 /M arrest ( Fig. 7; Figs S4 and S5). These results suggested that knockdown of MCM2-MCM7 inhibited cell proliferation and cell cycle progression in A549 and H1299 cells.

Effects of the CDC7 inhibitor TAK-931 on A549 and H1299 cells
Phosphorylation of MCM2 by cell division cycle 7 (CDC7) at the G 1 phase to S phase initiates DNA synthesis [42]. CDC7 is a dumbbell former 4 protein (DBF4)-dependent serine/threonine kinase and plays important roles in DNA replication [43]. CDC7 kinase activity is essential for MCM2-MCM7 hexamerinduced DNA replication. We hypothesized that indirect suppression of MCM2-MCM7 hexamer function caused by CDC7 inhibition may decrease tumor cell progression (Fig. 8A). TAK-931 (simurosertib) was used as a CDC7 inhibitor in this study.
In A549 and H1299 cells, TAK-931 blocked the phosphorylation of MCM2 (Ser40) in a concentrationdependent manner ( Fig. 8B; Fig. S6). Moreover, cell cycle assays showed that cells treated with TAK-931 exhibited late S to late G 2 /M arrest in a concentrationand time-dependent manner ( Fig. 8C; Fig. S7). This result suggested that TAK-931 suppressed the functions of MCM2-MCM7 and that DNA replication was delayed in A549 and H1299 cells.

Discussion
DNA replication is the most critical issue for the normal maintenance of cells, and its regulation is extremely strict. In cancer cells, however, DNA replication proceeds chaotically, which triggers abnormal cell proliferation. Aberrant expression of various genes involved in DNA replication has been reported in cancer cells [44,45].
The hexameric protein complex formed by MCM proteins (MCM2-MCM7) is a key component of the prereplication complex and recruits other DNA replication-associated proteins to form replication forks [42,46]. Aberrant expression of MCM proteins has been reported in several types of cancers, for example, renal cell carcinoma, prostate cancer, and breast cancer, and these events promote genome instability and uncontrolled cell cycle progression [23,47].
Several sets of cohort data in TCGA for LUAD revealed that all MCM family members were significantly upregulated in LUAD tissues. Moreover,  MCM4, MCM5, and MCM7 expression levels may be used as prognostic molecular markers for patients with LUAD. In the future, these molecules may have applications in predicting the efficacy of treatment regimens and in the selection of therapeutic drugs for patients with LUAD. Minichromosome maintenance family members have been reported to be phosphorylated by various kinases and activated in human cells [44,48]. Aberrant activation of the MCM family by excess phosphorylation causes disruption of DNA replication and the cell cycle and is involved in the development and progression of cancer cells [49,50]. MCM7 forms a complex with MCM2, MCM4, and MCM6 and has been shown to regulate the helicase activity of the complex [51]. Aberrant expression of MCM7 has been reported in several types of cancers, and its expression leads to a poor prognosis in these patients [52,53]. MCM7 phosphorylation (Y600) through the epidermal growth factor/Lyn kinase cascade enhances MCM complex assembly and cancer cell proliferation [49]. RACK1 is a highly conserved WD40 repeat scaffold protein that is involved in several cellular processes [50]. Overexpression of RACK1 promotes AKT-dependent phosphorylation of MCM7 and is involved in DNA replication and proliferation in NSCLC cells [50].
CDC7, a serine/threonine kinase, regulates DNA replication, cell cycle, DNA repair, and gene expression by phosphorylating many intracellular target molecules [42,54]. Notably, MCM is a key target of CDC7, and the CDC7/DBF4 complex selectively phosphorylates MCM2 (Ser40 and Ser53) and is involved in regulating the initiation of DNA replication [55,56]. Therefore, CDC7 is a therapeutic target molecule for cancer therapy, and various CDC7 inhibitors have been developed [42]. TAK-931 is a novel CDC7-selective inhibitor that has been shown to exhibit significant antitumor effects both in vitro and in vivo [57]. Our data showed that treatment of LUAD cells with TAK-931 markedly suppressed cell proliferation by targeting MCM2 phosphorylation. The development of therapeutic agents that target the CDC7/MCM2 cascade will contribute to improving the prognosis of patients with LUAD.
Based on the epigenomic perspective, we further investigated the cause of MCM2 upregulation in LUAD cells. Numerous studies have clarified that noncoding RNAs are involved in the regulation of gene expression. In particular, miRNAs play pivotal roles as regulators of gene expression [24,25]. Previous studies have shown that miR-186-3p is downregulated in cervical cancer tissues and cell lines and that the expression of this miRNA directly controls MCM2 expression [58]. In colon cancer, MCM2 expression is regulated by miR-195-5p and miR-497-5p, which are downregulated in cancer tissues and cultured cancer stem cells. Overexpression of MCM2 is involved in the maintenance of stemness [59].
Our current findings showed that miR-139-3p, miR-378a-5p, and miR-2110 were downregulated in LUAD tissues and controlled the expression of MCM2 in LUAD cells. Previous studies have demonstrated that miR-139-3p was downregulated in lung squamous cell carcinoma tissues and that checkpoint kinase 1 (CHEK1) could be targeted by miR-139-3p [60]. Notably, CHEK1 is closely involved in various biological processes, for example, cell cycle arrest, DNA repair, and cell death [61,62].
The Cancer Genome Atlas analysis revealed that aberrant expression of MCM genes (MCM2-MCM7) has a negative impact on prognosis with LUAD patients. In this study, we showed candidate miRNAs that negatively regulate MCM genes. For example, miR-144-3p, miR-2110, miR-30c-2-3p, etc. may regulate multiple MCMs from our data ( Table 4 and Tables S3-S7), and some of them are also downregulated in TCGA-LUAD cohort dataset. Functional analyses of these miRNAs will be a future challenge. Elucidation of the molecular networks regulated by tumor-suppressive miRNAs will reveal the novel oncogenic pathways underlying the malignant transformation of LUAD.

Conclusions
Analysis of TGCA-LUAD datasets and immunostaining revealed that all MCM family members (MCM2-MCM7) were overexpressed in cancer tissues. Notably, high expression of MCM4, MCM5, and MCM7 significantly predicted 5-year survival rates in patients with LUAD. Functional assays showed that knockdown of each MCM gene significantly suppressed the proliferation of LUAD cells. The CDC7-selective inhibitor TAK-931 attenuated the proliferation of LUAD cells by suppressing MCM2 phosphorylation. Moreover, MCM2 expression was controlled by miR-139-3p, miR-378a-5p, and miR-2110. MCM family members have DNA helicase activity that is essential for the replication of genomic DNA and are closely involved in the molecular pathogenesis of LUAD. In particular, regulation of MCM2 phosphorylation may be a promising target when developing novel treatments for this disease.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Fig. S1. Selection of miRNAs regulating MCM3-MCM7 in LUAD cells.    Fig. S6. Full-size images of the western blots shown in Fig. 8. Fig. S7. Effects of CDC7 inhibition on MCM2 phosphorylation and late S to early G 2 /M cell cycle arrest in NSCLC cell lines. Table S1. Reagents used in this study. Table S2. Characteristics of the patients used for IHC.