The human breast cancer cell lines MCF-7, BT549, T47-D, MDA-MB-231, and HCC-1806 and the human breast epithelial cell line MCF-10A were obtained from The Cell Bank of Chinese Academy of Sciences and ATCC and American Type Culture Collection (ATCC), and cultured at 37 °C with 5% CO${}_2$. MCF-7 and T47D are estrogen receptor positive human breast cancer cell lines, BT549, MDA-MB-231 and HCC-1806 human triple negative breast cancer cell lines. All experiments were performed using mycoplasma-free cells. All cell lines were authenticated using single nucleotide polymorphisms profiling. One hundred cases of paraffin-embedded diseased tissues and corresponding paired normal tissues were obtained from hospitalized patients with breast carcinoma at the First Affiliated Hospital of Kunming Medical University from September 2014 to July 2021. All paraffin tissue samples were kept by our hospital and were used for immunohistochemical staining, and there was no need to consult the patient. This study was approved by the relevant authoritative organization of the hospital.

TRIzol reagent (15596-026, Invitrogen, Carlsbad, CA, USA) was utilized for the extraction of the aggregate RNA, which was reverse transcribed to cDNA according to the requirements of the RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Scientific, Waltham, MA, USA). PCR amplification was conducted using the SYBR Green Select Master Mix System (4472908, Invitrogen, Carlsbad, CA, USA) on a CFX96TM Real-Time PCR Detection System. The target mRNA expression was normalized to the mRNA expression of GAPDH. The primers were as such follows: MCM6: forward, 5′-TGTCAGTGGTGTTGATGGATATG-3′, reverse 5′-GCTGTCTGTTCCTCATCTCTG-3′; GAPDH: forward, 5′-GGCAACAATATCAGCAACTTC-3′, reverse 5′-GGTCCTTAGGTGTGGTATC‑3′. All qRT‒PCR experiments were conducted no less than three times. MCM6 expression was examined via Western blotting. Briefly, the cells were lysed for 20 min using RIPA buffer (89900, Thermo Fisher Scientific, Waltham, MA, USA), and all cell proteins were extracted after centrifugation. The BCA Protein Assay Kit (23227, Pierce, Waltham, MA, USA) was adopted for the quantification of cell lysate protein. The protein solution was separated using SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, which was treated with evaporated milk (5%) at 4 °C for a night. The PVDF membrane was cultured with anti-MCM6 antibody (13347-2-AP, Proteintech, Rosemont, IL, USA) at 1:1000 at approximately 23 °C and treated with secondary antibody at a 1:10,000 dilution. The internal control used was $\beta$-actin (ZSGB-BIO, Beijing, China, #TA-09).

IHC staining was performed in conformity with the manufacturer’s protocol (Dako REAL EnVision Detection System; Dako, Santa Clara, CA, USA). To elaborate, the tissue segments underwent a baking process at 68 °C for 2 h. The slices were dewaxed with xylene, gradient alcohol and distilled water. Then, the slices were boiled in citric acid buffer (pH 6.0, 0.01 M) for 2 min at high temperature and pressure for antigen repair. The slices were incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity. Subsequently, the slices were cultured with MCM6 antibody (diluted 1:400; 13347-2-AP, Proteintech) at 4 °C overnight and goat anti-mouse IgG-HRP antibody. Diaminobenzidine tetrahydrochloride (DAB) was used to observe the reaction. IHC staining was scored by two independent investigators. The aggregate score was obtained by multiplying the proportion of positive tumor cells [scored as 0 (5%), 1 (6~25%), 2 (26~50%), 3 (51%~75%), 4 (76~100%), respectively] and the staining intensity (scored as 0, 1, 2, and 3 according to severity). Based on the final scores, less than 5% of cells were stained, regardless of intensity, and specimens were categorized as negative; weak, moderate and strong were given marks of 1~4 points, 5~8 points and 9~12 points, respectively. For the following statistical analysis, the negative and weak groups were taken as the low MCM6 expression group, and the moderate and strong groups were considered to be the high MCM6 expression group.

The RNA-seq data and relevant information about the research subjects were acquired from the 2022 version of The Cancer Genome Atlas database (TCGA) (https://www.genome.gov/Funded-Programs-Projects/Cancer-Genome-Atlas) from the official website. The subjects were grouped into two groups according to expression level, namely, the median MCM6 mRNA expression of each sample. The clinicopathological data of the subjects were collected, including age, clinical classification of primary focus, regional lymph node and distant metastasis, represented by TNM collectively, endogenous molecular classification (Luminal A, Luminal B, basal like, HER2 overexpression or normal type) and histological type. The expression level of MCM6 protein in breast cancer patients was obtained in UALCAN database (http://ualcan.path.uab.edu/).

The online websites Kaplan‒Meier Plotter (http://kmplot.com/analysis/) and PrognoScan (http://dna00.bio.kyutech.ac.jp/PrognoScan/) were selected for the prognostic analysis of MCM6 in patients with breast carcinoma. The “rms” R package was used to combine MCM6 expression with clinicopathological features to construct a nomogram risk prediction model for the prediction of OS, and the predictive performance was measured using calibration curves and the C-index [22, 23].

The h.all.v7.5.symbols.gmt [Hallmarks] gene set database was selected. GSEA was deployed to elaborate the functional differences of the gene between the two expression groups, and the MCM6 expression level was considered a label of phenotype. The arrangements of gene sets were arranged out by 1000 times per every analysis. The False Discovery Rate (FDR) q value, the normalized enrichment score (NES) and the nominal p value were established to classify each phenotypic enrichment signaling pathway.

With the RNA-seq data, the MCM6-ICG relations to explore the immune genes related to MCM6 expression were analyzed using the R package, which includes limma, reshape2, ggplot2, ggpubr, and corrplot. The correlation threshold was set to a correlation coefficient $\ge$0.2 and a p value less than 0.05.

Diseased cell proliferation was measured with a sulforhodamine B (SRB) assay. The cells were seeded in a 96-well plate (100 µL/well) and fixed with cold 10% trichloroacetic acid for half an hour at room temperature. The SRB solution (0.4%, Sigma S9012, Wappingers Falls, NY, USA) was treated with deionized water and then added for dyeing for approximately 10 min. Tris solution (10 mM) was incubated at 23 °C for 5 min, and the optical density (OD) of the cells was determined at 490–530 nm wavelengths [24].

The cell samples were collected using trypsin, fixed with 80% ethanol at 4 °C in a dark environment overnight, and then stained in the dark with propidium iodide (0.1 mg/mL, BD Biosciences, Franklin Lake, NJ, USA) and RNase A (1 mg/mL) for 30 minutes. The cell cycle of each group was tested via flow cytometry. The data were analyzed by FlowJo 7.6 (San Carlos, CA, USA) [25].

The collected cells were resuspended in binding buffer, cultured with Annexin V-FITC (BD Biosciences) for 10 minutes in a dark environment at room temperature and subsequently stained with binding buffer and PI (BD Biosciences) in darkness for 30 minutes at 4 °C. Cell measurement was conducted using flow cytometry [25].

The cells were inoculated into 6-well culture plates in complete medium until they were 80% confluence. The cells were scratched with a 200 µL pipette tip. Then the medium was changed to a serum-free medium. The migration process was carefully observed at 0, 24 and 48 h on an inverted microscope (Olympus, Tokyo, Japan). ImageJ (National institutes of Health, Bethesda, MD, USA) was employed to calculate the migration area [25].

The cell suspension was cultured in serum-free medium into the upper layer of the experimental plates coated with Matrigel (BD Biosciences). The serum-containing medium was placed in the lower layer. The cells that invaded the lower layer received methanol treatment and Giemsa staining and were photographed microscopically [26].

The animal experiments won approval from the authoritative organization of the hospital. Six-week-old female BALB/C nude mice were maintained in a pathogen-free environment. MDA-MB-231 cells (1 $\times$ 10${}^6$/point), which were divided into two groups (Normal control group, shMCM6 group), were injected subcutaneously into the mammary fat pads of the mice. The subcutaneous tumor size was observed every 4 days. The tumor size was measured using a caliper, and the formula was as follows: volume = 1/2 (width${}^2$$\times$ length). The mice were sacrificed after 24 days. The diseased tissues were excised and weighed. The tumors were collected and used for additional IHC pathological analysis [25].

For data analysis, SPSS 22.0 software (IBM Corp., Armonk, NY, USA) was adopted. The expression difference between breast tumor and normal breast tissues was analyzed by the Wilcoxon signed-rank test. Univariate and multivariate Cox analyses were selected to analyze the influence of MCM6 expression along with other factors on mouse survival. Logistic regression analysis was performed to determine how MCM6 was related to other clinical factors. One-way analysis of variance was employed to make comparisons between two groups. p 0.05 was deemed to be statistically significant.

We first explored MCM6 expression in a wide range of human malignancies. The mRNA expression of MCM6 increased in various cancers from TCGA database analysis (Fig. 1A,B), and MCM6 expression in breast cancer considerably exceeded that in normal tissues (Fig. 1C,D). The protein expression level of MCM6 in patients with breast cancer outstripped that in normal tissues from the online database UALCAN (http://ualcan.path.uab.edu/) (Fig. 1E). Consistent with the expression pattern from the online database, immunohistochemical staining outcomes indicated higher MCM6 expression in diseased tissues than in adjacent breast tissue. Among 100 cases of breast cancer tissues, 92 cases (92.0%) had overexpression of MCM6, and the rest (8.0%) had low expression of MCM6, while there were 45 cases (45.0%) with overexpression of MCM6 and 55 cases (55.0%) with low expression of MCM6 in adjacent tissues (Fig. 2A–C). In comparison with MCF-10A human breast epithelial cells, MCM6 expression in breast cancer cell lines (MCF-7, BT549, T470, MDA-MB-231, HCC1806) was significantly increased (Fig. 2D,E). These results suggest that MCM6 was overexpressed in patients with breast carcinoma.

Fig. 1.

MCM6 was significantly upregulated in breast cancer based on database analysis. (A,B) MCM6 expression was increased in a variety of tumors according to The Cancer Genome Atlas (TCGA) database analysis. (C) Box plot of TCGA database describes the different expression levels of MCM6 between normal tissues and cancerous tissues. (D) Based on the same number of samples, MCM6 expression in normal and diseased tissues was compared. (E) MCM6 protein expression was higher in normal breast tissue according to the UALCAN database. *p 0.05, **p 0.01, ***p 0.001. ns, not significant; CPTAC, Clinical Proteomic Tumor Analysis Consortium.

Fig. 2.

MCM6 is significantly up-regulated in tissues and cells of breast cancer based on experimental verification. (A–C) Immunohistochemical staining of MCM6 expression in adjacent breast tissue and breast cancer tissues, representative immunohistochemical images and statistical results (n = 100). (D,E) Quantitative Real-Time PCR (qRT‒PCR) and western blotting analyses of MCM6 expression in MCF-10A breast cells and diseased cells, including MCF-7, BT549, T470, MDA-MB-231, and HCC1806 breast cancer cells. Statistics are presented as the means $\pm$ SDs. ***p 0.001.

We further explored how MCM6 expression is correlated with clinicopathologic parameters (age, histological grade, size of tumor, lymph node metastasis, vascular invasion, distant metastasis, and molecular typing). The outcomes demonstrated that MCM6 expression was associated with the size of tumor (p = 0.001) and lymph node metastases (p = 0.012) (Table 1). An analysis of the TCGA database revealed that the expression of MCM6 varied according to age, and there were differences between Asians and Caucasians White (Fig. 3A,B); MCM6 expression in estrogen receptors (ER) and progesterone receptors (PR)-negative breast cancer patients was higher than that in ER- and PR-positive breast cancer patients (Fig. 3C,D); MCM6 expression in HER2-positive subjects was more obvious than in negative breast cancer patients (Fig. 3E); MCM6 expression was elevated in luminal B, basal and HER2 breast cancer (Fig. 3F); MCM6 expression in invasive lobular carcinoma was higher than that in invasive lobular carcinoma tube carcinoma (Fig. 3G); no notable difference in the expression of MCM6 was found in each clinical stage (Fig. 3H); MCM6 expression was different only in the T1 and T2 stages; no considerable difference was shown in MCM6 expression between the N stage and the M stage, which T represents tumor size stage, N represents lymph node metastasis stage, and M represents distant metastasis stage (Fig. 3I–K). Based on the analysis from the PrognoScan online website, breast cancer patients with high MCM6 expression had reduced OS, relapse-free survival (RFS) and distant metastasis-free survival (DMFS) (Fig. 4A–C), which is consistent with the results of the Kaplan-Meier Plot online prognostic analysis (Fig. 4D–F).

Fig. 3.

Expression of MCM6 in different clinicopathological features. (A) MCM6 expression in people under 60 years old and over 60 years old. (B) MCM6 expression in Asian, Black or African American and White individuals. (C–E) Relationship between MCM6 expression and Estrogen Receptors (ER), Progesterone Receptors (PR), and Human Epidermal Growth Factor Receptors 2 (HER2) status. (F) Relationship between MCM6 expression and different cancerous types of molecules. (G) Relationship between MCM6 expression and different histological subtypes of breast cancer. (H) MCM6 expression in different pathologic stages. (I–K) MCM6 expression in the T, N and M phases. Statistics are presented as the means $\pm$ SDs. *p 0.05, **p 0.01, ***p 0.001, ns, not significant.

Fig. 4.

Breast cancer with high MCM6 expression has a poor prognosis. The PrognoScan online website was selected to analyze the prognostic role of MCM6 in breast carcinoma, and (A), (B), and (C) represent Overall Survival (OS), Relapse Free Survival (RFS) and Distant Metastasis Free Survival (DMFS), respectively. The Kaplan Meier Plotter online website was also utilized for the analysis of the prognostic effects of MCM6 in breast cancer, and the results suggested reduced survival time in patients with overexpression of MCM6 in terms of all three indicators mentioned above, and (D), (E), and (F) represent OS, RFS and DMFS, respectively.

In addition, univariate Cox regression analysis revealed that the expression of ER, PR, stage, T stage, M stage, N stage and MCM6 was associated with a low survival rate of patients (Fig. 5A). In multivariate Cox regression, after adjusting for other confounding factors, the expression of age, ER, M, N and MCM6 was an independent indicator (Fig. 5B). Furthermore, various indicators (age, ER, M, N and MCM6 expression) were included in the nomogram prediction model by multivariate Cox regression. The total score was obtained by age, expression of ER, M stage, N stage, MCM6, and prediction of survival probabilities at three years and five years (Fig. 5C). The C-index for the prediction nomogram was 0.817. Calibration curves displayed the predictive power of the predictive model in terms of survival time in patients with breast carcinoma (Fig. 5D,E). These results suggest that overexpression of MCM6 is a dismal prognostic factor for breast cancer.

Fig. 5.

Univariate and multivariable Cox regression analysis and construction of nomograms related to patient prognosis. (A) The prognosis of MCM6 was analyzed by univariate Cox regression. (B) Multivariate Cox regression analysis of the prognosis of MCM6. (C) The nomogram combined with the risk signature and clinicopathological factors. (D,E) Calibration plots for the prediction of prolonged survival of three and five years.

GSEA was performed between subgroups with different levels of MCM6 expression using the whole gene network based on the TCGA dataset with a view to explore the biological pathways via which MCM6 works in diseased tissues. The biological processes involved mainly included DNA repair, different pathways of IL6-JAK-STAT3, MTORC1, PI3K-AKT-MTOR, MYC, etc. (Fig. 6A). KEGG analysis demonstrated that overexpression of MCM6 was mainly involved in the process of mismatch repair, the regulation of ubiquitination proteolysis, ERBB signaling pathway and p53 signaling pathway. In addition, MCM6 is enriched in immune signaling pathways and activates related immune responses, such as B cell and T cell receptor pathways, and cytotoxic effects of natural killer cells (Fig. 6B).

Fig. 6.

Analysis of signaling pathways by using GSEA. (A) GSEA plots showing the NES for HALLMARK, which had close relations to the overexpression of MTM6 in breast carcinoma and involved signaling pathways, including DNA repair, signaling types of IL6 JAK STAT3, MTORC1 and PI3K AKT MTOR, inflammatory and interferon alpha responses, and MYC targets V1. (B) NES for KEGG, which has a close association with the overexpression of MTM6, including B-cell receptor, ERBB, p53, T-cell receptor signaling pathways, mismatch repair, natural killer cell-mediated cytotoxicity, and ubiquitin-mediated proteolysis.

The impact of MCM6 expression on chemosensitivity in patients with breast carcinoma were assessed via the pRRophetic algorithm and the half-maximal inhibitory concentration available in the Genomics of Drug Sensitivity in Cancer (GDSC) database. The results demonstrated that 134 chemotherapeutic drugs were identified to be significantly correlated with MCM6 expression. In the group overexpressing MCM6, we found that there were 14 chemotherapy drugs with significant effects (Fig. 7A–N), especially PD-0332991 (p = 3.7 $\times$ 10${}^{-12}$) and trametinib (p 2.22 $\times$ 10${}^{-16}$). According to these findings, the drug sensitivity of these small molecule compounds is better when MCM6 is highly expressed, but further exploration and experimental verification are needed.

Fig. 7.

MCM6 expression correlates with the chemosensitivity of breast cancer. Box plots show the chemosensitivity of (5Z) –7–oxozeaenol (A), belinostat (B), BEZ235 (C), dabrafenib (D), elesclomol (E), erlotinib (F), lapatinib (G), MK–2206 (H), Nutlin–3a (I), PD-0325901 (J), phenformin (K), sorafenib (L), temsirolimus (M) and trametinib (N) in high- and low-expression of MCM6 in breast cancer. ***p 0.001.

The expression correlation between MCM6 and genes related to immune detection points was analyzed based on the TCGA BRCA database. Our research outcomes indicated a positive correlation between MCM6 and the expression of multiple immune checkpoints and a negative correlation with that of BTNL9 (Fig. 8A). Analysis of the relationship between MCM6 and immunity, including 24 kinds of immune cells, showed a significantly negative correlation with eight immune cells (iDC, CD8 T cell, Th17 cells, eosinophils, mast cells, NK CD56 bright cells, NK cells, pDC) but a significantly positive correlation with fifteen immune cells (cells of Th2, aDC, Th1, Treg, T helper, NK CD56dim, Marcophages, B, Tcm, DC, Tgd, T, Term, Neutrophils, TFH) (Fig. 8B). In addition, MCM6 expression was positively correlated with TMB (R = 0.33, p 2.2 $\times$ 10${}^{-16}$) (Fig. 8C). Finally, we also analyzed the methylation levels of MCM6 in the diseased group and the normal group, and the outcomes revealed that MCM6 had a higher methylation level in breast cancer (Fig. 8D).

Fig. 8.

Correlation analysis of MCM6 and immune checkpoint-related gene expression, immune cells, TMB and methylation. (A) TCGA BRCA database was used in correlational analysis of MCM6 and the expression of immune checkpoint-related genes, and the correlation coefficient threshold was set at 0.2. (B) Correlation analysis of MCM6 expression and immune cell infiltration. (C) Correlation analysis of tumor mutation burden (TMB) with MCM6 expression. (D) TCGA Breast Invasive Carcinoma (BRCA) database was used to analyze the promoter methylation level of MCM6 between normal tissues and diseased tissues.

To make a clarification of the biological role of MCM6 in the progression and prognosis of breast carcinoma, the lentivirus system was used to conduct stable MCM6 knockdown in MDA-MB-231 and MCF-7 cancerous cells (shMCM6), by which the mRNA and protein expression of MCM6 was considerably down-regulated (Fig. 9A,B). MCM6 down-regulation prevents the growth of cancerous cells (Fig. 9C), lead to a higher percentage of apoptotic cells (Fig. 9D). MCM6 silencing caused cancerous cells to reproduce in large numbers in G1 phase and reduced cancerous cells in S phase (Fig. 9E). MCM6 knockdown also reduced the migrating and invasive power of diseased cells (Fig. 9F,G). To test whether MCM6 promoted breast tumor growth in vivo, a subcutaneous transplantation tumor model of BALB/c nude mice was constructed with MDA-MB-231 breast cancer cells (Fig. 10A). The volume and weight of the xenografts of MDA-MB-231 cells with MCM6 knockdown were lighter than those of the control group (Fig. 10B,C). The quantity of ki-67-positive cells in the xenograft tumor with MCM6 knockdown was decreased compared with that in the control group, consistent with MCM6 expression (Fig. 10D). These results suggested that MCM6 knockdown can successfully retrain the growth of cancerous cells both in vivo and in vitro.

Fig. 9.

MCM6 promotes breast cancer cell growth, migration, and invasion and inhibits apoptosis in vitro. (A,B) qRT‒PCR and WB showing MCM6 expression in stably silenced in MDA-MB-231 and MCF-7 cells. (C) CCK8 experiment results show that inhibition of MCM6 significantly inhibits the proliferation of MDA-MB-231 and MCF-7 cells. (D) Inhibition of MCM6 significantly promotes the apoptosis rate of MDA-MB-231 and MCF-7cells. (E) Knockdown MCM6 leads to a large number of G1 stage cancer cells to multiply, and S stage cancer cells to reduce. (F) Wounding healing assay showed that knockdown of MCM6 significantly slowed down the wound healing rate of MDA-MB-231 and MCF-7cells. (G) Invasion chamber experiment confirmed that knockdown of MCM6 significantly inhibited the invasion rate of MDA-MB-231 and MCF-7cells. Statistics are presented as the means $\pm$ SDs. *p 0.05, ***p 0.001; ns, not significant.

Fig. 10.

CKAP4 promotes breast cancer cell growth in vivo. (A) Schematic diagram of the nude mouse xenograft model and all the tumors. (B,C) Knock down MCM6 significantly inhibits the growth of transplanted tumor in nude mice. (D) The immunohistochemical results showed that the expression of MCM6 was significantly decreased in the knockdown group, and the positive rate of KI67 in the knockdown group was significantly lower than that in the control group. Statistics are presented as the means $\pm$ SDs. *p 0.05, ***p 0.001; ****p 0.0001.