Multi-omics pan-cancer analyses identify MCM4 as a promising prognostic and diagnostic biomarker.

Minichromosome Maintenance Complex Component 4 (MCM4) is a vital component of the mini-chromosome maintenance complex family, crucial for initiating the replication of eukaryotic genomes. Recently, there has been a growing interest in investigating the significance of MCM4 in different types of cancer. Despite the existing research on this topic, a comprehensive analysis of MCM4 across various cancer types has been lacking. This study aims to bridge this knowledge gap by presenting a thorough pan-cancer analysis of MCM4, shedding light on its functional implications and potential clinical applications. The study utilized multi-omics samples from various databases. Bioinformatic tools were employed to explore the expression profiles, genetic alterations, phosphorylation states, immune cell infiltration patterns, immune subtypes, functional enrichment, disease prognosis, as well as the diagnostic potential of MCM4 and its responsiveness to drugs in a range of cancers. Our research demonstrates that MCM4 is closely associated with the oncogenesis, prognosis and diagnosis of various tumors and proposes that MCM4 may function as a potential biomarker in pan-cancer, providing a deeper understanding of its potential role in cancer development and treatment.

EZH2 regulates pancreatic cancer cells through E2F1, GLI1, CDK3, and Mcm4.

Pancreatic cancer (PC) is one of the most common malignant tumors in digestive tract. To explore the role of epigenetic factor EZH2 in the malignant proliferation of PC, so as to provide effective medical help in PC. Sixty paraffin sections of PC were collected and the expression of EZH2 in PC tissues was detected by immunohistochemical assay. Three normal pancreas tissue samples were used as controls. The regulation of EZH2 gene on proliferation and migration of normal pancreatic cell and PC cell were determined by MTS, colony forming, Ki-67 antibody, scratch and Transwell assays. Through differential gene annotation and differential gene signaling pathway analysis, differentially expressed genes related to cell proliferation were selected and verified by RT-qPCR. EZH2 is mainly expressed in the nuclei of pancreatic tumor cells, but not in normal pancreatic cells. The results of cell function experiments showed that EZH2 overexpression could enhance the proliferation and migration ability of PC cell BXPC-3. Cell proliferation ability increased by 38% compared to the control group. EZH2 knockdown resulted in reduced proliferation and migration ability of cells. Compared with control, proliferation ability of cells reduced by 16%-40%. The results of bioinformatics analysis of transcriptome data and RT-qPCR demonstrated that EZH2 could regulate the expression of E2F1, GLI1, CDK3 and Mcm4 in normal and PC cells. The results revealed that EZH2 might regulate the proliferation of normal pancreatic cell and PC cell through E2F1, GLI1, CDK3 and Mcm4.

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

Rice (Oryza sativa) TIR1 and 5'adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in Schizosaccharomyces pombe.

The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing Schizosaccharomyces pombe AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for S. pombe that uses the TIR1 from Oryza sativa (OsTIR1) not fused to Skp1. Furthermore, we demonstrate that degradation efficiency can be improved by pairing an OsTIR1 auxin-binding site mutant, OsTIR1F74A, with an auxin analogue, 5'adamantyl-IAA (AID2). We provide evidence for the enhanced functionality of the OsTIR1 AID and AID2 systems by application to the essential DNA replication factor Mcm4 and to a non-essential recombination protein, Rad52. Unlike AID, no detectable auxin-independent depletion of AID-tagged proteins was observed using AID2.

MCM4 in human hepatocellular carcinoma: a potent prognostic factor associated with cell proliferation.

Hepatocellular carcinoma (HCC) remains a major public health problem. MCM4, a constitutive member of the minichromosomal maintenance protein family, has been reported to play a vital role in cancer malignancy behavior. However, the function of MCM4 in HCC remains largely unknown. The present study explored the specific role of MCM4 in HCC. The data from public datasets including TCGA and GTEx showed that MCM4 was overexpressed in HCC and significantly associated with poor prognosis. Immunohistochemistry results from 102 HCC patients suggested that high-level expression of MCM4 was correlated with tumor size. Then a series of in vivo and in vitro experiments were performed to investigate the function of MCM4 in HCC tumor cells. MCM4 silencing suppressed the cell proliferation and sphere formation of hepatoma cells. Moreover, silencing MCM4 significantly decreased the growth of tumors in a xenograft tumor model. In conclusion, the results of the present study indicated that MCM4 was a potential prognostic predictor associated with poor outcomes of HCC patients and even a therapeutic target for HCC.

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle.

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.

Effect of the natural compound trans­resveratrol on human MCM4 gene transcription.

trans­Resveratrol (Rsv) is a natural compound contained in red wine and grape skins that has various beneficial effects for organisms such as lengthening of their life span. Rsv induces expression of the human TP53 and HELB genes, which are involved in the regulation of DNA maintenance. In the present study, a luciferase expression vector containing 309 bp of the 5' upstream end of the human MCM4 gene was transfected into HeLa S3 cells. A luciferase assay revealed that Rsv treatment increased the minichromosome maintenance 4 (MCM4) gene promoter activity by GC­box and GGAA (TTCC) motifs. Electro phoretic mobility shift assay revealed that the specific binding factor (complex) contains PU.1 (SPI1). Quantitative reverse transcription­polymerase chain reaction analysis indicated that MCM4 gene expression was transiently induced by Rsv. Moreover, western blotting revealed that the SP1/PU.1 ratio markedly increased after Rsv treatment, indicating that a balance or profile of these transcription factors may control Rsv­inducible initiation of transcription. These observations indicated that the beneficial effects of Rsv can be attributed to induction of the chromosomal DNA maintenance factor encoding gene expression.

Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant.

Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress.

DNA unwinding mechanism of a eukaryotic replicative CMG helicase.

High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.

Antitumor Activity of Ohmyungsamycin A through the Regulation of the Skp2-p27 Axis and MCM4 in Human Colorectal Cancer Cells.

Ohmyungsamycin A (1), a novel cyclic peptide discovered from a marine Streptomyces sp., was previously reported with antibacterial and anticancer activities. However, the antitumor activities and the underlying molecular mechanisms of 1 remain to be elucidated. Compound 1 inhibited the proliferation and tumor growth of HCT116 human colorectal cancer cells based on both in vitro cell cultures and an in vivo animal model. A cDNA microarray analysis revealed that 1 downregulated genes involved in cell cycle checkpoint control. Compound 1 also induced G0/G1 cell cycle arrest that was mediated by the regulation of S-phase kinase-associated protein 2 (Skp2)-p27 axis and minichromosome maintenance protein 4 (MCM4). Furthermore, a longer exposure of 1 exhibited an accumulation of a sub-G1 phase cell population, which is characteristic of apoptotic cells. The induction of apoptosis by 1 was also associated with the modulation of caspase family proteins. Compound 1 effectively suppressed tumor growth in a xenograft mouse model subcutaneously implanted with HCT116 cells. In addition, analysis of tumors revealed that 1 upregulated the expression of the CDK inhibitor p27 but downregulated the expression of Skp2 and MCM4. These findings demonstrate the involvement of 1 in cell cycle regulation and the induction of apoptosis in human colorectal cancer cells.

Minichromosome Maintenance Complex (MCM) Genes Profiling and MCM2 Protein Expression in Cervical Cancer Development.

OBJECTIVE: Minichromosome maintenance complex (MCM) proteins are essential for the process of DNA replication and cell division. This study aimed to evaluate MCM genes expression profiles and MCM2 protein in HPV-associated cervical carcinogenesis. METHODOLOGY: MCM2, 4, 5 and 7 genes expression profiles were evaluated in three cervical tissue samples each of normal cervix, human papillomavirus (HPV)-infected low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC), using Human Transcriptome Array 2.0 and validated by nCounter® PanCancer Pathway NanoString Array. Immunohistochemical expression of MCM2 protein was semi-quantitatively assessed by histoscore in tissue microarrays containing 9 cases of normal cervix, 10 LSIL, 10 HSIL and 42 cases of SCC. RESULTS: MCM2, 4, 5 and 7 genes expressions were upregulated with increasing fold change during the progression from LSIL to HSIL and the highest in SCC. MCM2 gene had the highest fold change in SCC compared to normal cervix. Immunohistochemically, MCM2 protein was localised in the nuclei of basal cells of normal cervical epithelium and dysplastic-neoplastic cells of CIN and SCC. There was a significant difference in MCM2 protein expression between the histological groups (P = 0.039), and histoscore was the highest in HSIL compared to normal cervix (P = 0.010). CONCLUSION: The upregulation of MCM genes expressions in cervical carcinogenesis reaffirms MCM as a proliferative marker in DNA replication pathway, whereby proliferation of dysplastic and cancer cells become increasingly dysregulated and uncontrolled. A strong expression of MCM2 protein in HSIL may aid as a concatenated screening tool in detecting pre-cancerous cervical lesions.

Involvement of Dual Strands of miR-143 (miR-143-5p and miR-143-3p) and Their Target Oncogenes in the Molecular Pathogenesis of Lung Adenocarcinoma.

Our analyses of tumor-suppressive microRNAs (miRNAs) and their target oncogenes have identified novel molecular networks in lung adenocarcinoma (LUAD). Moreover, our recent studies revealed that some passenger strands of miRNAs contribute to cancer cell malignant transformation. Downregulation of both strands of the miR-143 duplex was observed in LUAD clinical specimens. Ectopic expression of these miRNAs suppressed malignant phenotypes in cancer cells, suggesting that these miRNAs have tumor-suppressive activities in LUAD cells. Here, we evaluated miR-143-5p molecular networks in LUAD using genome-wide gene expression and miRNA database analyses. Twenty-two genes were identified as potential miR-143-5p-controlled genes in LUAD cells. Interestingly, the expression of 11 genes (MCM4, RAD51, FAM111B, CLGN, KRT80, GPC1, MTL5, NETO2, FANCA, MTFR1, and TTLL12) was a prognostic factor for the patients with LUAD. Furthermore, knockdown assays using siRNAs showed that downregulation of MCM4 suppressed cell growth, migration, and invasion in LUAD cells. Aberrant expression of MCM4 was confirmed in the clinical specimens of LUAD. Thus, we showed that miR-143-5p and its target genes were involved in the molecular pathogenesis of LUAD. Identification of tumor-suppressive miRNAs and their target oncogenes may be an effective strategy for elucidation of the molecular oncogenic networks of this disease.

MCM2, MCM4, and MCM6 in Breast Cancer: Clinical Utility in Diagnosis and Prognosis.

Breast cancer is a heterogeneous disease comprising the estrogen receptor (ER)-positive luminal subtype which is subdivided into luminal A and luminal B and ER-negative breast cancer which includes the triple-negative subtype. This study has four aims: 1) to examine whether Minichromosome Maintenance (MCM)2, MCM4, and MCM6 can be used as markers to differentiate between luminal A and luminal B subtypes; 2) to study whether MCM2, MCM4, and MCM6 are highly expressed in triple-negative breast cancer, as there is an urgent need to search for surrogate markers in this aggressive subtype, for drug development purposes; 3) to compare the prognostic values of these markers in predicting relapse-free survival; and 4) to compare the three approaches used for scoring the protein expression of these markers by immunohistochemistry (IHC). MCM2, MCM4, MCM6, and MKI67 mRNA expression was first studied using in silico analysis of available breast cancer datasets. We next used IHC to evaluate their protein expression on tissue microarrays using three scoring methods. MCM2, MCM4, and MCM6 can help in distinction between luminal A and luminal B whose therapeutic management and clinical outcomes are different. MCM2, MCM4, MCM6, and Ki-67 are highly expressed in breast cancer of high histological grades that comprise clinically aggressive tumors such as luminal B, HER2-positive, and triple-negative subtypes. Low transcript expression of these markers is associated with increased probability of relapse-free survival. A positive relationship exists among the three scoring methods of each of the four markers. An independent validation cohort is needed to confirm their clinical utility.

Function of the amino-terminal region of human MCM4 in helicase activity.

The amino-terminal region of eukaryotic MCM4 is characteristic of the presence of a number of phosphorylation sites for CDK and DDK, suggesting that the region plays regulatory roles in the MCM2-7 helicase function. However, the roles are not fully understood. We analyzed the role of the amino-terminal region of human MCM4 by using MCM4/6/7 helicase as a model for MCM2-7 helicase. First we found that deletion of 35 amino acids at the amino-terminal end resulted in inhibition of DNA helicase activity of the MCM4/6/7 complex. Conversion of arginine at amino acid no. 10 and 11 to alanine had similar effect to the deletion mutant of Δ1-35, suggesting that these arginine play a role in the DNA helicase activity. The data suggest that expression of these mutant MCM4 in HeLa cells perturbed the progression of the S phase. Substitution of six CDK phosphorylation sites (3, 7, 19, 32, 54 and 110) in the amino-terminal region by phospho-mimetic glutamic acids affected the hexamer formation of the MCM4/6/7 complex. MCM4 phosphorylation by CDK may play a role in DNA replication licensing system, and the present results suggest that the phosphorylation interferes MCM function by lowering stability of MCM complex.

Effect of daidzein and equol on DNA replication in MCF-7 cells.

It has been reported that daidzein and equol stimulate DNA replication and proliferation of MCF-7 cells. However, their molecular mechanisms of action are still unclear. We examined the effects of daidzein and equol on DNA replication of MCF-7 cells, focusing on MCM2-7 proteins, which function as the replicative helicase. In the presence of either 1 µM of daidzein or equol, the number of cells in S-phase, which was determined by detecting bromodeoxyuridine incorporated into replicated DNA, almost doubled. The total amounts of MCM7 protein and chromatin-bound MCM7 protein increased in the presence of daidzein. The data suggest that phytoestrogens facilitate cell cycle progression in G1-phase by increasing the level of MCM proteins. In the presence of phytoestrogens, phosphorylation of Rb and levels of MCM2, 3 and 7 mRNA increased, suggesting that stimulation of MCM2-7 transcription is involved in the cell cycle progression. Under the same conditions, double-stranded DNA breakage in logarithmically growing MCF-7 cells, which was detected using anti-γ-H2AX antibodies, did not increase in the presence of equol.

ATR kinase inhibition induces unscheduled origin firing through a Cdc7-dependent association between GINS and And-1.

ATR kinase activity slows replication forks and prevents origin firing in damaged cells. Here we describe proteomic analyses that identified mechanisms through which ATR kinase inhibitors induce unscheduled origin firing in undamaged cells. ATR-Chk1 inhibitor-induced origin firing is mediated by Cdc7 kinase through previously undescribed phosphorylations on GINS that induce an association between GINS and And-1. ATR-Chk1 inhibitor-induced origin firing is blocked by prior exposure to DNA damaging agents showing that the prevention of origin firing does not require ongoing ATR activity. In contrast, ATR-Chk1 inhibitor-induced origins generate additional replication forks that are targeted by subsequent exposure to DNA damaging agents. Thus, the sequence of administration of an ATR kinase inhibitor and a DNA damaging agent impacts the DNA damage induced by the combination. Our experiments identify competing ATR and Cdc7 kinase-dependent mechanisms at replication origins in human cells.

Bortezomib-induced miRNAs direct epigenetic silencing of locus genes and trigger apoptosis in leukemia.

MicroRNAs (miRNAs) have been suggested to repress transcription via binding the 3'-untranslated regions of mRNAs. However, the involvement and details of miRNA-mediated epigenetic regulation, particularly in targeting genomic DNA and mediating epigenetic regulation, remain largely uninvestigated. In the present study, transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, a clinical and highly selective drug for leukemia treatment, and contributed to bortezomib-induced cell death. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 could target CpG islands in the 5'-flanking region of the CEBPD gene. We previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for CCAAT/enhancer binding protein delta (CEBPD) gene inactivation; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. The miRNA/Ago2/YY1/PcG group protein/DNMT complex linked the inactivation of CEBPD and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Moreover, we revealed that miRNA binding is necessary for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. The present study successfully characterized the interactions of the miRNA/Ago2/YY1/PcG group protein/DNMT complex and provided new insights for miRNA-mediated epigenetic regulation in bortezomib-induced leukemic cell arrest and cell death.

Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity.

MCM7, a subunit of mini-chromosome maintenance proteins (MCM) complex, plays an important role in initiating DNA replication during the G1 phase and extending DNA strands during the S phase. Here, we demonstrated that MCM7 is not only sustained but maintains association with chromatin during M phase. Remarkably, MCM7 siRNA can accelerate mitotic exit. MCM7 depletion leads to CDK1 inactivation and promotes subsequent cohesin/RAD21 cleavage, which eventually leads to sister chromatin segregation. Moreover, MCM7 is co-localized with tubulin in the mitotic cells and MCM7 depletion results in aberrant mitosis. Our results indicate that MCM7 may exert certain functions on spindle formation to prevent cytokinesis during early mitosis by regulating CDK1 activity.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex.

An MCM4 mutation detected in cancer cells affects MCM4/6/7 complex formation.

An MCM4 mutation detected in human cancer cells from endometrium was characterized. The mutation of G486D is located within MCM-box and the glycine at 486 in human MCM4 is conserved in Saccharomyces cerevisiae MCM4 and Sulfolobus solfataricus MCM. This MCM4 mutation affected human MCM4/6/7 complex formation, since the complex containing the mutant MCM4 protein is unstable and the mutant MCM4 protein is tend to be degraded. It is likely that the MCM4 mutation affects the interaction with MCM7 to destabilize the MCM4/6/7 complex. Cells with abnormal nuclear morphology were detected when the mutant MCM4 was expressed in HeLa cells, suggesting that DNA replication was perturbed in the presence of the mutant MCM4. Role of the conserved amino acid in MCM4 function is discussed.