Multi-Omics Analysis of MCM2 as a Promising Biomarker in Pan-Cancer

Abstract

Minichromosome maintenance 2 (MCM2) is a member of the minichromosomal maintenance family of proteins that mainly regulates DNA replication and the cell cycle and is involved in regulating cancer cell proliferation in various cancers. Previous studies have reported that MCM2 plays a pivotal role in cell proliferation and cancer development. However, few articles have systematically reported the pathogenic roles of MCM2 across cancers. Therefore, the present pan-cancer study was conducted. Various computational tools were used to investigate the MCM2 expression level, genetic mutation rate, and regulating mechanism, immune infiltration, tumor diagnosis and prognosis, therapeutic response and drug sensitivity of various cancers. The expression and function of MCM2 were examined by Western blotting and CCK-8 assays. MCM2 was significantly upregulated in almost all cancers and cancer subtypes in The Cancer Genome Atlas and was closely associated with tumor mutation burden, tumor stage, and immune therapy response. Upregulation of MCM2 expression may be correlated with a high level of alterations rate. MCM2 expression was associated with the infiltration of various immune cells and molecules and markedly associated with a poor prognosis. Western blotting and CCK-8 assays revealed that MCM2 expression was significantly upregulated in melanoma cell lines. Our results also suggested that MCM2 promotes cell proliferation in vitro by activating cell proliferation pathways such as the Akt signaling pathways. This study explored the oncogenic role of MCM2 across cancers, provided data on the underlying mechanisms of these cancers for further research and demonstrated that MCM2 may be a promising target for cancer immunotherapy.

Keywords: MCM2; comprehensive bioinformatics; immune therapy response; prognostic value; skin cutaneous melanoma.

FIGURE 1

The workflow of the study.

FIGURE 2

The expression of MCM2 in human cancers. (A) mRNA expression of MCM2 across cancers from TCGA in adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain lower‐grade glioma (LGG), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), mesothelioma (MESO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thyroid carcinoma (THCA), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS), uveal melanoma (UVM). (B) The protein expression of MCM2 in cancers from the HPA database (IHC).

FIGURE 3

MCM2 variants, localization and single-cell variations. (A) MCM2 expression in normal cell lines from the HPA database. (B) Subcellular distribution of MCM2 within the endoplasmic reticulum (ER), nucleus and microtubules of the U-2 cells lines, according to the HPA database. (C) Plots of single-cell RNA-sequencing data from the FUCCI U-2 cell line showing the correlation between MCM2 RNA expression and cell cycle progression.

FIGURE 4

Correlations between MCM2 expression and (A) molecular subtypes and (B) immune subtypes across TCGA cancers. CIN, chromosomal instability; GS, genomically stable; POLE, Polymerase ε; EBV, Epstein-Barr virus.

FIGURE 5

MCM2 gene alterations in various cancers. (A) MCM2 gene mutation type analysis in various cancers by cBioPortal. (B) 3D protein structure of MCM2. Colored part means the binding region, while grey means the other part of MCM2. (C) The subtypes and distributions of MCM2 somatic mutations. X-axis, amino acids site; Y-axis, number of MCM2 mutations; green dot, missense mutations; grey dot, truncating mutations. (D) The correlation between mutation status and patient prognosis for all TCGA cancers, including overall survival, progression-free survival, disease-specific survival and disease-free survival, analyzed using the cBioPortal tool. Red square indicated the MCM2 alteration group, Blue square indicated non-alteration group. (E) TMB and MSIsensor scores and MSI MANTIS scores in MCM2 nonmutant cancer and different subtypes of MCM2 mutant cancer (including truncating, missense and multiple). Each dot represents one patient. The black line represents the median and the interquartile ranges. X axes indicated different mutation type of MCM2. Y axes indicated scores of cases. (F) The mutant frequencies of MSH6, MSH2, MLH1 and PMS2 in the MCM2 mutant and nonmutant groups. (G) Bar plot showing the frequencies of the ABTB1, PODXL2, MGLL, C3ORF22, CHCHD6, KBTBD12, SLC41A3, TXNRD3, RUVBL1 and SNX4 alterations co-occurring with MCM2 alterations. (H) Waterfall plot showing the cooccurrence patterns of MCM2 alterations with the genetic alterations of ABTB1, PODXL2, MGLL, C3ORF22, CHCHD6, KBTBD12, SLC41A3, TXNRD3, RUVBL1 and SNX4. Color indicated the different mutation type of the 11 genes in cases.

FIGURE 6

Boxplots showing differential MCM2 phosphorylation levels (beta values) between tumors and adjacent normal tissues across TCGA database.

FIGURE 7

Functional enrichment and co-expression network of MCM2 at the gene and protein levels. (A) PPI network. The related proteins of MCM2. The color depth revealed the correlation between MCM2 and other proteins, which means that the darker the color, the closer the relationship. (B) GGI network. (C) KEGG and GO analyses of 50 targeted binding proteins of MCM2 in patients with cancers.

FIGURE 8

The prognostic value of MCM2 in various cancers. (A) The correlation between transcription levels of MCM2 and cancer stage in patients. (B) AUCs of MCM2 in predicting the prognosis of patients with various types of cancer.

FIGURE 9

The clinical value of MCM2 in various cancers. (A) OS of patients with different expression levels of MCM2 in pan-cancer from the Kaplan–Meier Plotter database. (B) The relationship between MCM2 expression level and sensitivity to different drugs. The colors indicated the correlation between MCM2 expression and drug sensitivity. Red indicated positive relationship, while purple negative. The size of the spots indicated the significance of the correlation. The bigger the size, the more significant correlation.

FIGURE 10

MCM2 expression and immune infiltration in various TCGA cancers. (A) Correlations between MCM2 expression and cancer purity and infiltrating levels of B cells, CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils and dendritic cells. (B) Correlations between MCM2 expression and CD274, CTLA4 and PDCD1. (C) Bar plot showing the biomarker relevance of MCM2 compared to that of standardized cancer immune evasion biomarkers in immune checkpoint blockade (ICB) subcohorts. The AUC was applied to evaluate the predictive performance of the test biomarkers on the ICB response status. (D) Comparison between MCM2 and other published biomarkers based on their predictive power of response outcome and OS. Color indicated the different immune biomarkers.

FIGURE 11

MCM2 in SKCM. (A) The top 50 genes with positive co-expression of MCM2 in the TCGA database of SKCM according to the heatmap. (B) The top 10 genes with a strong positive correlation with MCM2 in SKCM. (C) GSEA showing that MCM2 expression was associated with 10 pathways in TCGA SKCM samples.

FIGURE 12

Survival nomogram and calibration curves. Prediction of 1-year and/or 3-year OS in SKCM patients.

FIGURE 13

MCM2 is upregulated and promotes cell proliferation in SKCM in vitro. (A) The relative expression of MCM2 in SKCM cell lines (compared with that of actin) examined via Western blotting. (B) MCM2 was successfully downregulated in the A375 and SK28 cell lines in protein and transcriptional level by siRNA. (C) The MCM2 knockdown significantly suppressed cell proliferation by CCK8 assay. (D) The side-effect of MCM2 knockdown to other MCM proteins. (E) The MCM2 knockdown significantly suppressed the phosphorylation of Akt.