Minichromosome Maintenance Protein 7 is a potential therapeutic target in human cancer and a novel prognostic marker of non-small cell lung cancer

Cancer cell lines used in this study were as follows: lung adenocarcinoma (ADC) NCI-H1781, NCI-H1373, LC319, A549 and PC-14; lung squamous cell carcinoma (SCC) SK-MES-1, NCI-H2170, NCI-H520, NCI-H1703 and RERF-LC-AI; lung large cell carcinoma (LCC) LX1; small cell lung cancer (SCLC) SBC-3, SBC-5, DMS273 and DMS114; bladder cancer SW780, RT4 and 5637; liver cancer HepG2, SNU475 and Huh7; colorectal cancer SW480, LoVo and HCT116. Human small airway epithelial cells (SAECs), normal human lung fibroblasts (IMR-90, WI-39 and HFL-1) and normal human colon fibroblasts (CCD-18Co) were used as normal control cells. All cell lines were grown in monolayers in appropriate media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma). All cell lines were maintained at 37°C in humid air with 5% CO₂ (except for SW780), or without CO₂ (SW780). Cells were transfected with FuGENE6™ (Roche Applied Science, Penzberg, Germany) according to the manufacturer's protocols. Primary non-SCLC (NSCLC) tissue samples as well as their corresponding normal tissues adjacent to resection margins, from patients having no anticancer treatment before tumor resection, had been obtained earlier with informed consent [17‐19]. All tumors were staged on the basis of the pathologic tumor-node-metastasis classification of the International Union Against Cancer. A total of nine frozen primary lung cancer tissues for RNA extraction were obtained as published earlier [18, 19]. Formalin-fixed primary lung tumors and adjacent normal lung tissue samples used for immunostaining on tissue microarrays had been obtained from 331 patients undergoing curative surgery at Saitama Cancer Center (Saitama, Japan) [20, 21]. To be eligible for this study, tumor samples were selected from patients who fulfilled all of the following criteria: (a) patients suffered primary NSCLC with histologically confirmed stage (only pT1 to pT4, pN0 to pN2, and pM0); (b) patients underwent curative surgery, but did not receive any preoperative treatment; (c) among them, NSCLC patients with positive lymph node metastasis (pN1-pN3) were treated with platinum-based adjuvant chemotherapies after surgical resection, whereas patients with pN0 did not receive adjuvant chemotherapies; and (d) patients whose clinical follow-up data were available. This study and the use of all clinical materials mentioned were approved by individual institutional ethics committees.

Bladder tissue samples and RNA preparation were described previously [22‐24]. Briefly, 136 surgical specimens of primary urothelial carcinoma were collected, either at cystectomy or transurethral resection of bladder tumor (TURBT), and snap frozen in liquid nitrogen. Twenty-three specimens of normal bladder urothelial tissue were collected from areas of macroscopically normal bladder urothelium in patients with no evidence of malignancy. Approximately 10,000 cells were microdissected from both stromal and epithelial/tumor compartments in each tissue. RNA was extracted using an RNeasy Micro Kit (QIAGEN, Crawley, UK). Areas of cancer or stroma containing significant inflammatory areas of tumor or stroma containing significant inflammatory cell infiltration were avoided to prevent contamination [24]. Use of tissues for this study was approved by Cambridgeshire Local Research Ethics Committee (Ref 03/018).

We established a genome-wide cDNA microarray with 36,864 cDNAs selected from UniGene database of the National Center for Biotechnology Information (NCBI). This microarray system was constructed essentially as described previously [18, 25, 26]. Briefly, the cDNAs were amplified by RT-PCR using poly (A)⁺ RNAs isolated from various human organs as templates; the lengths of the amplicons ranged from 200 to 1,100 bp, without any repetitive or poly (A) sequences. Many types of tumor and corresponding non-neoplastic tissues were prepared in 8-μm, as described previously [25]. A total of 30,000 - 40,000 cancer or noncancerous cells were collected selectively using the EZ cut system (SL Microtest GmbH, Jena, Germany) according to the manufacturer's protocol. Extraction of total RNA, T7-based amplification, and labeling of probes were performed as described previously [25]. A measure of 2.5-μg aliquots of twice-amplified RNA (aRNA) from each cancerous and noncancerous tissue was then labeled, respectively, with Cy3-dCTP or Cy5-dCTP.

As described previously, we prepared 136 bladder cancer and 23 normal bladder tissues in Addenbrooke's Hospital, Cambridge [27]. Specific primers for human GAPDH (housekeeping gene) and MCM7 were designed (primer sequences in Additional file 1). PCR reactions were performed using the LightCycler^® 480 System (Roche Applied Science) following the manufacture's protocol.

siRNA oligonucleotide duplexes were purchased from Sigma-Aldrich (St. Louis, MO, USA) for targeting the human MCM7 transcripts. siEGFP and siNegative control (siNC), which consists of three different oligonucleotide duplexes, were used as control siRNAs. The siRNA sequences are described in Additional file 2. siRNA duplexes (100 nM final concentration) were transfected into lung and bladder cancer cell lines with Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) for 72 h, and cell growth was examined using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) [28].

Whole cell lysates were prepared from the cells with RIPA-like buffer, and total protein (10 μg) was transferred to nitrocellulose membrane. The membrane was probed with anti-MCM7 antibody (141.2, Santa Cruz Biotechnology, Santa Cruz, CA, USA). ACTB (I-19, Santa Cruz Biotechnology) was used to ensure equal loading and transfer of proteins. Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (GE Healthcare, Little Chalfont, UK) and visualizing with Enhanced Chemiluminescence (GE Healthcare).

BrdU labeling and immunocytochemistry were performed according to previously reported protocols [23, 27, 29]. A549, SBC5 and SW780 cells were incubated with appropriate media containing 2 μM BrdU (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min, and fixed and permeabilized with 100% methanol for 5 min at room temperature. The cells were washed with PBS, and then blocked by 3% BSA for 1 h at 37°C. Then, the cells were incubated with an anti-MCM7 antibody in 3% BSA overnight at 4°C. After incubation with 1^st antibody, the cells were reacted with Alexa Fluor 594-conjugated goat anti-mouse IgG for 1 h at 37°C in the blocking solution. They were then re-fixed, treated with 4 M HCl for 30 min at room temperature and incubated with FITC-conjugated anti-BrdU (BD Biosciences), diluted 1:300, for 1 h at room temperature, followed by observation with confocal microscopy (Leica Microsystems, Wetzlar, Germany).

Immunohistochemical analysis was performed using a specific mouse-MCM7 antibody as described previously [27, 30]. For clinical lung cancer tissues and liver tissue microarray (70 cases), ENVISION+ kit/horseradish peroxidase (Dako, Glostrup, Denmark) was applied. VECTASTAIN^® ABC KIT (Vector Laboratories, Burlingame, CA, USA) was used for bladder tissue microarray and normal tissue slides. Tumor tissue microarrays were constructed with 331 primary NSCLCs which had been obtained by a single institutional group (see above) with an identical protocol to collect, fix, and preserve the tissues after resection [31‐33]. Considering the histologic heterogeneity of individual tumors, a tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide. Three, four or five tissue cores (diameter, 0.6 mm; depth, 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments, Sun Prairie, WI, USA). A core of normal tissue was punched from each case, and 5-μm sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semiquantitatively assessed MCM7 positivity without prior knowledge of clinicopathologic data. Because the intensity of staining within each tumor tissue core was mostly homogeneous, the intensity of MCM7 staining was semiquantitatively evaluated using the following criteria: negative (no appreciable staining in tumor cells) and positive (brown staining appreciable in more than 30% of the nucleus of tumor cells). Cases were accepted as positive only if all reviewers independently defined them as such.

The Kruskal-Wallis test was used to examine the difference between several independent subgroups. Student's t-test or Mann-Whitney's U-test was used to analyze the difference between two independent subgroups. Survival curves were calculated from the date of surgery to the time of death related to NSCLC or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for MCM7 expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were done with the Cox proportional hazard regression model to determine associations between clinicopathological variables and cancer-related mortality. First, we analyzed associations between death and possible prognostic factors including age, gender, histology, pT classification and pN classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced strong MCM7 expression into the model, along with any and all variables that satisfied an entry level of P < 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.

We previously reported that PRMT6, a type I arginine methytransferase, is involved in human carcinogenesis [27]. To clarify detailed functions of PRMT6 in cancer, we performed IP-MS analysis and identified MCM7 as a binding partner (data not shown). The interaction was confirmed by co-immunoprecipitation and immunocytochemistry (Additional file 3). Intriguingly, quantitative real-time PCR showed that expression levels of MCM7 in 9 lung cancer tissues (6 NSCLC cases and 3 SCLC cases) were significantly higher than those in 11 normal tissues containing lung, brain, colon, esophagus, eye, liver, rectum, stomach, bladder and kidney (Figure 1A). Therefore, we hypothesized that MCM7 can also be involved in human carcinogenesis. To validate these results, we conducted immunohistochemical analysis on tissue microarray containing tissue sections from 331 NSCLC patients, who had undergone surgical resection. Immunohistochemistry using an MCM7-specific antibody showed nuclear localization in cancer tissues, but nothing was detected in normal lung tissues (Figure 1B). Importantly, specific MCM7 signals were hardly detected in normal brain, heart, lung, liver, pancreas, stomach, testis, kidney and bladder tissues (Figure 1C), indicating that MCM7 may be specifically overexpressed in cancer tissues. Of 331 cases, MCM7 stained positively in 196 cases (61.1%) and negatively in 135 cases (38.9%; Table 1). Subsequently, we analyzed the association of MCM7 expression with clinical outcomes, and found that expression of MCM7 in NSCLC patients was significantly associated with male gender (P < 0.0001, Fisher's exact test; Table 1), non-adenocarcinoma (ADC) histology (P < 0.0001), presence of lymph node metastasis (pN1-2; P < 0.0001) and tumor-specific 5-year survival after the resection of primary tumors (P = 0.0055 by log-rank test; Figure 1D). We then applied univariate analysis to evaluate associations between patient prognosis and several factors, including MCM7 expression, age, gender, histologic type (non-ADC versus ADC), pT stage (tumor size, T1 versus T2 + T3) and pN stage (node status, N0 versus N1+N2). All those parameters were significantly associated with poor prognosis (Table 2). However, multivariate analysis revealed that MCM7 status did not show the statistical significance as an independent prognostic factor for surgically treated NSCLC patients enrolled in this study, whereas age, pT and pN stages did so (Table 2). This result might be due to the preponderance of MCM7 expression up to the pN stage.

In addition to lung tissues, we examined expression levels of MCM7 in bladder tissues. Quantitative real-time PCR analysis using 23 normal bladder tissues, 124 bladder transitional cell carcinomas (TCCs) and 12 upper urinary tract transitional cell carcinomas (UUT-TCCs) showed elevated mRNA levels of MCM7 in bladder and especially UUT-TCCs compared with normal bladder tissues (Figure 2A). Although there were significant differences between normal bladder and bladder cancers of all pT stages, no significant differences were observed between each pT stage nor tumor grade (Figure 2B; data not shown). Subsequently, immunohistochemical analysis using a bladder tissue microarray revealed that MCM7 was up-regulated in bladder cancer tissues at the protein level (21/29, 72.4%; Figure 2C and Additional file 4). In our microarray expression analysis, we confirmed elevated MCM7 expression in various types of cancers including lung, bladder and liver (Table 3), and tissue microarray immunohistochemical analysis showed up-regulation of MCM7 in liver cancer tissues at the protein level (25/70, 35.7%; Additional files 5 and 6).

In order to examine whether elevated expression of MCM7 plays a critical role in the proliferation of cancer cells, we prepared siRNA oligonucleotide duplexes to specifically suppress the expression of MCM7 (siMCM7#1, #2), and transfected each of them into cancer cells. Firstly, we examined the expression profile of MCM7 in various types of normal and cancer cell lines. Quantitative real-time PCR and western blot analyses showed that MCM7 expression levels in cancer cell lines were significantly higher than those in normal human cell lines at the RNA and protein levels (Additional file 7). Knockdown of MCM7 in A549, SBC5 and SW780 cells was confirmed by western blot and immunocytochemical analyses as shown in Figure 3A. Immunocytochemical analysis showed a decreased proportion of cells incorporating BrdU after treatment with MCM7 siRNAs and this abrogation of cancer cell growth was confirmed by the cell viability assay using cell counting kit 8 (Figure 3A). After confirming that the normal HFL-1 and CCD-18Co cell lines show almost the same growth rate as the cancer cell lines (Additional file 8), we examined effects of these siRNAs on the normal cells (Figure 3B). The cell viability analysis revealed that MCM7 siRNAs had only slight effects on these normal cell lines relative to cancer cell lines, implying that the marked suppression of cancer cell growth by treatment with these specific siRNAs was not dependent on off-target effects. In addition, we demonstrated the effects of heliquinomycin, an inhibitor of MCM4/6/7-dependent DNA helicase activity [34], on the growth of cancer cells. Importantly, it effectively suppressed the growth of cancer cells compared with HFL-1 cells and this effect was in a dose dependent manner (Figure 3C). In particular, after treatment with 4.0 μM of heliquinomycin, no growth-suppressive effects were observed in HFL-1 cells even though significant growth suppression was observed in cancer cells. These data indicate that MCM7 plays an important role in the proliferation of cancer cells, and inactivation of this gene should be a promising therapeutic target in various types of cancer.