Minichromosome Maintenance Complex Component Research Articles

Expression of SASP, DNA Damage Response, and Cell Proliferation Factors in Early Gastric Neoplastic Lesions: Correlations and Clinical Significance.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated senescence-associated secretory phenotype (SASP) pathway has recently been identified in the suppression and promotion of cancers. However, its practical role in carcinogenesis remains to be comprehensively elucidated. Here, we describe an investigation analysing SASP activity and its correlations with DNA damage response (DDR), genomic mutations, and cell proliferation in gastric carcinogenesis among 30 cases with available endoscopic submucosal dissection (ESD) specimens of early neoplastic lesions (including low-grade dysplasia [LGD], high-grade dysplasia [HGD], and intramucosal carcinoma). The positive cells of senescence-associated β-galactosidase staining and cGAS, STING, interferon-regulatory factor 3 (IRF3), and signal transducer and activator of transcription 6 (STAT6) expression levels using immunostaining were elevated in HGD and in cancers. Similarly, increased expression of the Fanconi anemia group D2 (FANCD2) protein, tumour suppressor p53 binding protein 1 (TP53BP1), and replication protein A (RPA2) (i.e., primary DDR factors) was detected in HGD and in cancers; these increased expression levels were closely correlated with high expression of Ki67 and minichromosome maintenance complex component 7 (MCM7) proteins. Moreover, genomic mutations in TP53 gene were detected in 56.67% of the evaluated cases (17/30) using next-generation sequencing, and positive staining was verified in HGD and in cancers. Statistical analysis revealed that cell proliferation closely correlated with the expression of DDR factors, of which TP53BP1 was positively associated with SASP factors and IRF3 was positively correlated with cell proliferation. In addition, an analysis evaluating clinical features demonstrated that STAT6-positive cases showed a longer progression-free survival time than STAT6-negative cases. Our evaluation, conducted using a limited number of specimens, suggests SASP may be prevalent in early gastric neoplastic lesions and could be activated by accelerated cell proliferation-induced DDR. The clinical significance of SASP still needs to be determined.

Palbociclib regulates the expression of dihydrofolate reductase and the cell cycle to inhibit t (11;14) multiple myeloma.

BackgroundMultiple myeloma (MM) with t (11;14) has a unique biology. New combinations of novel agents are needed to improve the prognosis of patients with t (11;14) MM. As a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), palbociclib (PAL) has been used to treat various malignancies, including breast cancer, ovarian cancer, and so on. However, the effects of PAL on MM remain unclear, and thus, further investigations are warranted.MethodsTwo t (11;14) MM cell lines, U266 and KMS27, were used in this study. The cell viability and cell cycle were detected to evaluate the anti-myeloma effect of the combination of pralatrexate (PTX) and methotrexate (MTX) with PAL. Three-dimensional (3D) spheroid and mouse models were built to verify the effect of the combination treatment. Western blot was used to detect the expressions of Minichromosome Maintenance Complex Component 2 (MCM2), Minichromosome Maintenance Complex Component 3 (MCM3), and dihydrofolate reductase (DHFR).ResultsIt was observed that PAL had obvious anticancer effects on U266 and KMS27 cells, which had synergistic effects together with PTX and MTX. Importantly, it was found that PAL could promote cell cycle genes including MCM2 and MCM3, and increase the number of MM cells in the G1 phase. Moreover, PAL reduced the expression level of DHFR and exerted its anticancer effects on MM cells by targeting DHFR. It was also determined that PAL exerted anticancer effects on MM in the spheroid and mouse models.ConclusionsPAL exerted obvious anticancer effects on t (11;14) MM cells, which had synergistic effects together with PTX and MTX. PAL could promote cell cycle genes and the G1 phase of MM cells. Moreover, PAL reduced the expression level of DHFR and exerted its anticancer effects on t (11;14) MM cells by targeting DHFR.

P723: LOSS OF THE NK MOTIF AND ANKYRIN REPEAT DOMAIN 1 (KANK1) LEADS TO LYMPHOID COMPARTMENT DYSREGUATION IN MICE

Background: We identified a novel germline loss of heterozygosity (LOH) mutation encompassing the NK Motif And AnKyrin Repeat Domain 1 (KANK1) gene in a young Myelodysplastic syndrome (MDS) patient and his healthy father, with no additional MDS-related mutations or chromosomal abnormalities. Clinical investigation suggested development of MDS following severe infection, likely triggered by an autoimmune mechanism. KANK1 protein plays a role in the control of cytoskeleton formation by regulating actin polymerization. A t(5;9) translocation resulting in a fusion of the platelet-derived growth factor receptor beta gene (PDGFRB) and KANK1 was detected in a patient with myeloid neoplasm (MN) characterized with severe thrombocythemia. The protein is thought to have a tumour suppressor function, as its expression is downregulated or missing in several tumor tissues, while over-expressing the protein was reported to inhibit the proliferation of tumor cells in solid cancer models. In addition, defects in cytoskeletal proteins are reported to contribute to immunological dysfunction and disruption of normal immune processes. Aims: To investigate the role of KANK1 in the regulation of normal hematopoiesis and the development of myeloid disorders. Methods: We generated a new genetically modified KANK1 (Kank1-/-) knockout mouse model and analysed the phenotypic and developmental changes in hematopoiesis in young and aged mice. Results: We confirmed the loss of Kank1 mRNA and protein expression by q-PCR and targeted protein mass-spectrometry respectively. The mice are viable and fertile, and reproduced according to mendelian ratio. Over the two years observation period, Kank1-/- mice did not exhibit any signs of disease, and their peripheral blood parameters remained within the normal range. Flow cytometry analysis of bone marrow (BM) cells from 17-20 weeks kank1-/- old mice showed minor increase in CD3+ T-cell population, while the expression of the double positive B220/CD19 B-cells population was reduced in Kank1-/- compared to Kank1+/+. Comprehensive flow cytometry analysis of spleen and thymus cells from young (n=8 each, 6-7 weeks old) and aged (n=8, 68-71 weeks old) Kank1-/- mice showed further dysregulation of the lymphoid compartment when compared to Kank1+/+. In young mice, a slight decrease of Treg cells is noticed in the spleen, coupled with a slight decrease of activated CD4 in the peripheral blood. In the spleen of aged Kank1-/- mice, the proportions of macrophages and Treg cells were decreased, while the central memory CD4+ T-cells were increased. Total protein analysis from BM cells of Kank1-/- (n=3) compared to Kank1+/+ mice (n=3) showed a significant reduction (FC >0.5, adj.p. <0.05) in proteins associated with cytoskeleton formation such as Myosin1 (MYH1), Tropomysin-1 (TDM1) and Annexin (ANXA2), and extracellular matrix components such as Collagena(CO1A1) and Matrix metalloproteinase9 (MMP9). We also detected a significant increase in expression of the DNA licensing factor minichromosome maintenance complex component 7 (MCM7), and the mitochondrial membrane associated transporter ATP binding Cassette Subfamily B Member 10 (ABCBA). Summary/Conclusion: Dysregulation of the immune system has been widely recognized to play a role in the development of MDS. Based on our results, we propose a role for KANK1 in regulating the development of immune cells in the mouse through it invovlement in cytoskeleton regulation. In vivo infectious challenge experiments using our model will futher elucidate the importance of KANK1 in the development of MDS.

The Minichromosome Maintenance Complex Component 2 (MjMCM2) of Meloidogyne javanica is a potential effector regulating the cell cycle in nematode-induced galls

Root-knot nematodes Meloidogyne spp. induce enlarged multinucleate feeding cells—galls—in host plant roots. Although core cell-cycle components in galls follow a conserved track, they can also be usurped and manipulated by nematodes. We identified a candidate effector in Meloidogyne javanica that is directly involved in cell-cycle manipulation—Minichromosome Maintenance Complex Component 2 (MCM2), part of MCM complex licensing factor involved in DNA replication. MjMCM2, which is induced by plant oxilipin 9-HOT, was expressed in nematode esophageal glands, upregulated during parasitic stages, and was localized to plant cell nucleus and plasma membrane. Infected tomato hairy roots overexpressing MjMCM2 showed significantly more galls and egg-mass-producing females than wild-type roots, and feeding cells showed more nuclei. Phylogenetic analysis suggested seven homologues of MjMCM2 with unknown association to parasitism. Sequence mining revealed two RxLR-like motifs followed by SEED domains in all Meloidogyne spp. MCM2 protein sequences. The unique second RxLR-like motif was absent in other Tylenchida species. Molecular homology modeling of MjMCM2 suggested that second RxLR2-like domain is positioned on a surface loop structure, supporting its function in polar interactions. Our findings reveal a first candidate cell-cycle gene effector in M. javanica—MjMCM2—that is likely secreted into plant host to mimic function of endogenous MCM2.

Screening and expression verification of key genes in cutaneous squamous cell carcinoma.

BackgroundCutaneous squamous cell carcinoma (cSCC), a common skin malignancy, often occurs at exposed sites, and patients’ appearance after surgical resection can be affected. This study sought to screen the key genes of cSCC via a bioinformatics analysis and explore the clinical significance and possible potential mechanisms of these genes in cSCC.MethodsWe screened differentially expressed genes (DEGs) between cSCC and normal skin tissues from the Gene Expression Omnibus database, performed functional enrichment and protein interaction network analyses, and used Cytoscape software to identify the key genes. The expression of the genes was proved by immunohistochemistry.ResultsA total of 164 DEGs were screened, and the functional enrichment analysis showed that the DEGs were significantly enriched in deoxyribonucleic acid replication and the cell-cycle pathway. By constructing a protein-to-protein interaction network, kinesin family member 11 (KIF11), aurora kinase A (AURKA), minichromosome maintenance complex component 2 (MCM2), minichromosome maintenance 10 replication initiation factor (MCM10), and denticleless E3 ubiquitin protein ligase homolog (DTL) were identified as 5 key genes with the highest connectivity. The expression of KIF11, AURKA, and MCM2 were investigated by immunohistochemistry. Compared to the normal skin tissues, the positive rates of the KIF11 and MCM2 proteins in the cSCC tissues were 70.0% and 90.0%, respectively, and the difference was statistically significant (P<0.05). The positive rates of AURKA protein expression in the cSCC and normal skin tissues were 13.9% and 0%, respectively, but the difference was not statistically significant. There was no correlation between the above-mentioned 3 key genes.ConclusionsKIF11 and MCM2 were highly expressed in cSCC, and may be involved in tumorigenesis, and represent novel targets for the clinical diagnosis and treatment of cSCC.

The High Expression of Minichromosome Maintenance Complex Component 5 Is an Adverse Prognostic Factor in Lung Adenocarcinoma.

Background Minichromosome maintenance (MCM) genes are crucial for genomic DNA replication and are important biomarkers in tumor biology. In this study, we aimed to identify the diagnostic, therapeutic, and prognostic value of the MCM2–10 genes in patients with lung cancer. Methods We examined the expression levels, gene networks, and protein networks of lung cancer using data from the ONCOMINE, GeneMANIA, and STRING databases. We conducted a functional enrichment analysis of MCM2–10 using the clusterProfiler package using TCGA data. The correlation between the MCM2–10 expression and lung cancer prognosis was evaluated using Cox regression analysis. The influence of clinical variables on overall survival (OS) was evaluated using univariate and multivariate analyses. The TIMER database was used to evaluate the correlation between tumor infiltrating levels and lung cancer. Kaplan–Meier Plotter pan-cancer RNA sequencing was used to estimate the correlation between the MCM5 expression and OS in different immune cell subgroups in patients with lung adenocarcinoma (LUAD). Finally, the 1-, 3-, and 5-year predictions of LUAD were performed using nomogram and calibration analysis. Results The expression of MCM2, 3, 4, 5, 6, 7, 8, and 10 in lung cancer was higher than that for normal samples. The MCM5 expression was associated with poor OS in patients with LUAD, and prognosis was related to TNM stage, smoking status, and pathological stage. The MCM5 expression is correlated with immune invasion in LUAD and may affect prognosis due to immune infiltration. Conclusion MCM5 may serve as a molecular biomarker for LUAD prognosis.

Incorporation of a histone mutant with H3K56 site substitution perturbs the replication machinery in mouse embryonic stem cells.

Sense mutations in several conserved modifiable sites of histone H3 have been found to be strongly correlated with multiple tissue-specific clinical cancers. These clinical site mutants acquire a distinctively new epigenetic role and mediate cancer evolution. In this study, we mimicked histone H3 at the 56th lysine (H3K56) mutant incorporation in mouse embryonic stem cells (mESCs) by lentivirus-mediated ectopic expression and analyzed the effects on replication and epigenetic regulation. The data show that two types of H3K56 mutants, namely H3 lysine 56-to-methionine (H3K56M) and H3 lysine 56-to-alanine (H3K56A), promote replication by recruiting more minichromosome maintenance complex component 3 and checkpoint kinase 1 onto chromatin compared with wild-type histone H3 and other site substitution mutants. Under this condition, the frequency of genomic copy number gain in H3K56M and H3K56A cells globally increases, especially in the Mycl1 region, a known molecular marker frequently occurring in multiple malignant cancers. Additionally, we found the disruption of H3K56 acetylation distribution in the copy-gain regions, which indicates a probable epigenetic mechanism of H3K56M and H3K56A. We then identified that H3K56M and H3K56A can trigger a potential adaptation to transcription; genes involved in the mitogen-activated protein kinase pathway are partially upregulated, whereas genes associated with intrinsic apoptotic function show obvious downregulation. The final outcome of ectopic H3K56M and H3K56A incorporation in mESCs is an enhanced ability to form carcinomas. This work indicates that H3K56 site conservation and proper modification play important roles in harmonizing the function of the replication machinery in mESCs.

Combined screening analysis of aberrantly methylated-differentially expressed genes and pathways in hepatocellular carcinoma.

Methylation plays an important role in hepatocellular carcinoma (HCC) by altering the expression of key genes. The aim of this study was to screen the aberrantly methylated-differentially expressed genes (DEGs) in HCC and elucidate their underlying molecular mechanism. Gene expression microarrays (GSE101685) and gene methylation microarrays (GSE44909) were selected. DEGs and differentially methylated genes (DMGs) were screened. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the Database for Annotation, Visualization, and Integrated discovery (DAVID). The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyze the functional protein-protein interaction (PPI) network. Molecular Complex Detection (MCODE) analysis was performed using the Cytoscape software. Hub genes were verified in The Cancer Genome Atlas (TCGA) database. A total of 80 hypomethylation-high expression genes (Hypo-HGs) were identified. Pathway enrichment analysis showed DNA replication, cell cycle, viral carcinogenesis, and the spliceosome. The top 5 hub genes were minichromosome maintenance complex component 3 (MCM3), checkpoint kinase 1 (CHEK1), kinesin family member 11 (KIF11), PDZ binding kinase (PBK), and Rac GTPase activating protein 1 (RACGAP1). In addition, 189 hypermethylation-low expression genes (Hyper-LGs) were identified. Pathway enrichment analysis indicated enrichment in metabolic pathways, drug metabolism-other enzymes, and chemical carcinogenesis. The top 5 hub genes were leukocyte immunoglobulin like receptor B2 (LILRB2), formyl peptide receptor 1 (FPR1), S100 calcium binding protein A9 (S100A9), S100 calcium binding protein A8 (S100A8), and myeloid cell nuclear differentiation antigen (MNDA). The methylation status and mRNA expression of MCM3, CHEK1, KIF11, PBK, and S100A9 were consistent in the TCGA database and significantly correlated with the prognosis of patients. Combined screening of aberrantly methylated-DEGs based on bioinformatic analysis may provide new clues for elucidating the epigenetic mechanism in HCC. Hub genes, including MCM3, CHEK1, KIF11, PBK, and S100A9, may serve as biomarkers for the precise diagnosis of HCC.

Bioinformatics approaches identified dasatinib and bortezomib inhibit the activity of MCM7 protein as a potential treatment against human cancer

Minichromosome Maintenance Complex Component 7 (MCM7) is a key component of the DNA replication licensing factor and hexamer MCM (MCM2–7) complex that regulates the DNA replication process. The MCM7 protein is associated with tumor cell proliferation that plays an important role in different human cancer progression. As the protein is highly expressed during the cancer development process, therefore, inhibition of the protein can be utilized as a treatment option for different human cancer. However, the study aimed to identify potential small molecular drug candidates against the MCM7 protein that can utilize treatment options for human cancer. Initially, the compounds identified from protein-drugs network analysis have been retrieved from NetworkAnalyst v3.0 server and screened through molecular docking, MM-GBSA, DFT, pharmacokinetics, toxicity, and molecular dynamics (MD) simulation approach. Two compounds namely Dasatinib (CID_3062316) and Bortezomib (CID_387447) have been identified throughout the screening process, which have the highest negative binding affinity (Kcal/mol) and binding free energy (Kcal/mol). The pharmacokinetics and toxicity analysis identified drug-like properties and no toxicity properties of the compounds, where 500 ns MD simulation confirmed structural stability of the two compounds to the targeted proteins. Therefore, we can conclude that the compounds dasatinib and bortezomib can inhibit the activity of the MCM7 and can be developed as a treatment option against human cancer.

MCM6 is a critical transcriptional target of YAP to promote gastric tumorigenesis and serves as a therapeutic target.

Rationale: Hyperactivation of Hippo-Yes-associated protein (YAP) signaling pathway governs tumorigenesis of gastric cancer (GC). Here we reveal that minichromosome maintenance complex component 6 (MCM6) is a critical transcriptional target of YAP in GC. We aim to investigate the function, mechanism of action, and clinical implication of MCM6 in GC.Methods: The downstream targets of YAP were screened by RNA sequencing (RNA-seq) and microarray, and further validated by chromatin immunoprecipitation PCR and luciferase reporter assays. The clinical implication of MCM6 was assessed in multiple GC cohorts. Biological function of MCM6 was evaluated in vitro, in patient-derived organoids, and in vivo. RNA-seq was performed to unravel downstream signaling of MCM6. Potential MCM6 inhibitor was identified and the effect of MCM6 inhibition on GC growth was evaluated.Results: Integrative RNA sequencing and microarray analyses revealed MCM6 as a potential YAP downstream target in GC. The YAP-TEAD complex bound to the promoter of MCM6 to induce its transcription. Increased MCM6 expression was commonly observed in human GC tissues and predicted poor patients survival. MCM6 knockdown suppressed proliferation and migration of GC cells and patient-derived organoids, and attenuated xenograft growth and peritoneal metastasis in mice. Mechanistically, MCM6 activated PI3K/Akt/GSK3β signaling to support YAP-potentiated gastric tumorigenicity and metastasis. Furthermore, MCM6 deficiency sensitized GC cells to chemo- or radiotherapy by causing DNA breaks and blocking ATR/Chk1-mediated DNA damage response (DDR), leading to exacerbated cell death and tumor regression. As there are no available MCM6 inhibitors, we performed high-throughput virtual screening and identified purpureaside C as a novel MCM6 inhibitor. Purpureaside C not only suppressed GC growth but also synergized with 5-fluorouracil to induce cell death.Conclusions: Hyperactivated YAP in GC induces MCM6 transcription via binding to its promoter. YAP-MCM6 axis facilitates GC progression by inducing PI3K/Akt signaling. Targeting MCM6 suppresses GC growth and sensitizes GC cells to genotoxic agents by modulating ATR/Chk1-dependent DDR, providing a promising strategy for GC treatment.

Immunohistochemical analysis of proliferating cell nuclear antigen and minichromosome maintenance complex component 7 in benign and malignant salivary gland tumors.

Background:Proliferation markers have been used to determine the behavior and prognosis of benign and malignant tumors; this study was aimed to compare the immunohistochemical (IHC) expression of proliferating cell nuclear antigen (PCNA) and novel marker minichromosome maintenance complex component 7 (MCM7) in common salivary gland tumors including pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (AdCC), to find a possible significant correlation between benign and malignant tumors.Materials and Methods:In this cross-sectional study, a total of 90 cases, including 30 PAs, 30 MECs, and 30 AdCCs, were collected. The IHC expressions of PCNA and MCM7 were evaluated. Their expressions were compared with each other and between benign and malignant tumors. Statistical analysis was performed by Chi-square and Tukey's test. P value was considered 0.05.Results:Out of 30 cases of PA, 28 cases (93.3%) were PCNA positive and 28 cases (93.3%) were MCM7 positive. In the AdCC cases, 29 cases (96.6%) were PCNA positive and 29 cases (96.6%) were MCM7 positive. In the MEC cases, all cases (100%) were PCNA positive and 23 cases (76.6%) were MCM7 positive. The labeling index (LI) of MCM7 and PCNA was evaluated, and this index was lower in MCM7 LI than PCNA in all tumors. The MCM7 and PCNA expression showed a significant difference in PA and MEC (P < 0.001).Conclusion:PCNA expression was higher than MCM7 expression in salivary gland tumors. However, more studies are needed to evaluate the malignant activity of these tumors with group of markers such as MCM family members.

Reaction of the Liver upon Long-Term Treatment of Fluoxetine and Atorvastatin Compared with Alcohol in a Mouse Model.

Background Alcoholism is known to cause liver toxicity and is extensively researched. On the other hand, stress, depression, and obesity are interrelated conditions with alcoholism, and their medications would affect the liver itself. In this study, we investigated the effects of the drugs fluoxetine and atorvastatin on the liver and compared with those of alcohol in a mouse model. Methods Comparisons of animals treated with the three drugs were carried out: serum aspartate transaminase (AST), alanine transaminase (ALT), and albumin were measured; liver tumor necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta-1) levels were evaluated; proliferative cells were detected via immunohistochemistry (IHC) targeting on proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2); for apoptosis, IHC targeting on activated caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were employed; and histopathology was also documented in all groups. Results For ALT, AST, albumin, and liver TNF alpha, only the ethanol group surged to significantly higher levels. For TGF beta-1, both ethanol and atorvastatin groups reached a significantly higher level. PCNA and MCM2 showed increased proliferation in the livers of all three groups, with the ethanol group having the highest number of positive cells followed by atorvastatin and then the fluoxetine group. As for cell death, both ethanol and fluoxetine groups showed significantly more apoptosis than control in TUNEL and activated caspase-3, while in the atorvastatin group, activated caspase-3 positive cells increased significantly, but the increase in TUNEL-positive cells did not reach statistical significance.

MCM3 is a novel proliferation marker associated with longer survival for patients with tubo-ovarian high-grade serous carcinoma.

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36-0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

Dissecting GATA1 Protein Interactions in Normal and Malignant Human Erythroblasts

Acute erythroid leukemia (AEL) is characterized by uncontrolled accumulation of transformed erythroblasts. Previous analysis of murine and human AEL revealed aberrant regulation of the master regulator GATA1, which controls terminal erythroid differentiation in multi-protein complexes acting as activators or repressors of gene expression. Although most malignant erythroblasts constitutively express abundant GATA1 protein, terminal erythroid differentiation is impaired. Notably, overexpression of GATA1 significantly induced partial or complete terminal erythroid differentiation of the human AEL cell line K562 or immortalised HUDEP2 human erythroblasts, respectively. These observations led us to hypothesize that blocked terminal erythroid differentiation in AEL might be the consequence of titratable dose-dependent aberrant GATA1 protein interactions. We comparatively analysed nuclear extracts from three human AEL cell lines (F36P, K562, KMOE2) and primary cells from an AEL patient. In addition, we analysed HUDEP2 and primary human erythroblasts (hEBST) from healthy donors that retain the potential for complete in vitro terminal erythroid differentiation. We quantified protein expression using a tandem mass tag (TMT) based approach (n=3/cell type) and we compared putative GATA1 interactions by immunoprecipitation (IP) followed by liquid chromatography mass spectrometry (MS) (n=3/cell type). Quantitative proteomics identified 6774 commonly expressed proteins in AEL and “normal” erythroblasts with a high reproducibility (mean coefficients of variation <10%) for all six different cell types. Unsupervised hierarchical clustering displayed a clear separation of the AEL cells from the “normal erythroblasts” (hEBST, HUDEP2). 386 proteins were higher expressed in the AEL group (logFC>=2; q<0.05), whereas 623 were more abundant in normal erythroblasts (logFC>=2; q<0.05). IP-MS analysis of nuclear lysates from the AEL cell lines, the AEL primary sample, HUDEP2 and hEBST resulted in a matrix containing 1616 proteins from which 126 proteins seem to significantly differentially interact with GATA1. 54 proteins were more enriched in the AEL group, whereas 72 proteins were more enriched in “normal” erythroblasts (q<0.5). Principal component analysis (PCA) showed for all cell lines a similar clustering pattern, accounting for 24% and 32% of the variance. Pulled-down proteins in hEBST and HUDEP2 clustered together and were closer to F36P and KMOE, than LAM49 and K562. Notably, we found significant enrichment (validated by immunoblotting) of the SKI protooncogene in AEL cells (logFC=1.82; q=0.013), a finding which not only confirmed previous findings in murine AEL models (MEL cells, erythroblasts from Nsd1 -/- mice) but also speaks for the functionality of our approach. Similarly, the LRPPRC leucine-rich PPR-motif-containing protein overexpressed in several cancers, as well the lactate dehydrogenases A and B (LDHA, LDHB) were significantly enriched in malignant erythroblasts (logFC>2; q<0.05). Furthermore, the ZEB2 zinc finger E-box-binding homeobox 2 protein, was significantly enriched in AEL cells (logFC=2.02; q=0.005). In contrast, the hematopoietic master transcription factor Runt-related transcription factor 1 (RUNX1) (logFC=2.48; q=0.0018) as well as DNA binding protein Ikaros (IKZF1) (logFC=1.71; q=0.13) were significantly enriched (validated by immunoblotting) in HUDEP2 and hEBST. Moreover, the MCM6 DNA binding mini-chromosome maintenance complex component 6 critical for proper DNA replication was enriched in normal erythroblasts (logFC=1.99; q=0.0001). Interestingly, one of the most strongly enriched (and validated by immunoblotting) proteins in normal erythroblasts was the nuclear pore complex protein NUP155 (logFC=6.1; q=0.0000001). Integration of the quantitative proteomics and the IP-MS analysis identified 118 proteins differentially expressed and differentially pulled-down by GATA1-IP, of which 49 were enriched in malignant and 69 proteins in normal erythroblasts (q<0.5). This shows that we reproducibly identified proteins that are differentially associated with GATA1 which are also differentially expressed in AEL cells versus normal erythroblasts. A targeted CRISPR/Cas9 screen is under way to identify GATA1-interacting proteins responsible for impaired erythroid differentiation of AEL cells. DisclosuresValent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

P8‐10: Minichromosome maintenance complex component (MCM) genes in lung adenocarcinoma: MCM‐family acts as a potential therapeutic target

P8‐10: Minichromosome maintenance complex component (MCM) genes in lung adenocarcinoma: MCM‐family acts as a potential therapeutic target

Discovering of Genomic Variations Associated to Growth Traits by GWAS in Braunvieh Cattle.

A genome-wide association study (GWAS) was performed to elucidate genetic architecture of growth traits in Braunvieh cattle. Methods: The study included 300 genotyped animals by the GeneSeek® Genomic Profiler Bovine LDv.4 panel; after quality control, 22,734 SNP and 276 animals were maintained in the analysis. The examined phenotypic data considered birth (BW), weaning (WW), and yearling weights. The association analysis was performed using the principal components method via the egscore function of the GenABEL version 1.8-0 package in the R environment. The marker rs133262280 located in BTA 22 was associated with BW, and two SNPs were associated with WW, rs43668789 (BTA 11) and rs136155567 (BTA 27). New QTL associated with these liveweight traits and four positional and functional candidate genes potentially involved in variations of the analyzed traits were identified. The most important genes in these genomic regions were MCM2 (minichromosome maintenance complex component 2), TPRA1 (transmembrane protein adipocyte associated 1), GALM (galactose mutarotase), and NRG1 (neuregulin 1), related to embryonic cleavage, bone and tissue growth, cell adhesion, and organic development. This study is the first to present a GWAS conducted in Braunvieh cattle in Mexico providing evidence for genetic architecture of assessed growth traits. Further specific analysis of found associated genes and regions will clarify its contribution to the genetic basis of growth-related traits.

Pharmacoinformatics-based phytochemical screening for anticancer impacts of yellow sweet clover, Melilotus officinalis (Linn.) Pall

To date, much attention has been paid to phytochemicals because of their diverse pharmacological effects on a variety of diseases such as cancer. In this regard, computer-aided drug design, as a cost- and time-effective approach, is primarily applied to investigate the drug candidates before their further costly in vitro and in vivo experimental evaluations. Accordingly, different signaling pathways and proteins can be targeted using such strategies. As a key protein for the initiation of eukaryotic DNA replication, mini-chromosome maintenance complex component 7 (MCM7) overexpression is related to the initiation and progression of aggressive malignancies. The current study was conducted to identify new potential natural compounds from the yellow sweet clover, Melilotus officinalis (Linn.) Pall, by examining the potential of 40 isolated phytochemicals against MCM7 protein. A structure-based pharmacophore model to the protein active site cavity was generated and followed by virtual screening and molecular docking. Overall, four compounds were selected for further evaluation based on their binding affinities. Our analyses revealed that two novel compounds, namely rosmarinic acid (PubChem CID:5281792) and melilotigenin (PubChem CID:14059499) might be druggable and offer safe usage in human. The stability of these two protein-ligand complex structures was confirmed through molecular dynamics simulation. The findings of this study reveal the potential of these two phytochemicals to serve as anticancer agents, while further pharmacological experiments are required to confirm their effectiveness against human cancers.

Immunohistochemical evaluation of D2-40, Galectin-3, Maspin and MCM7 expression in palate squamous cell carcinomas.

Squamous cell carcinoma (SCC) is the most frequent cancer in oral cavity and its prognosis has exhibited little improvement in the last decades. Although much less common palate SCCs manifests a higher local aggression invading very quickly the adjacent muscles and jawbones, thus being able frequently to lead to dysfunctions in chewing, swallowing, and speech. To elucidate what underlies such local aggression, we investigated the immunohistochemical expression in palate SCCs of Podoplanin (D2-40), Galectin-3 (Gal-3), mammary serine protease inhibitor (Maspin) and minichromosome maintenance complex component 7 (MCM7), markers that are known to be involved in tumor invasiveness. We found a progressive increase in reactivity for D2-40 and MCM7 from the normal epithelium toward dysplastic epithelium and respectively to SCC, which suggests the intervention of these markers in the early stages of squamous cell carcinogenesis in the palate. The highest D2-40, Gal-3 and MCM7 reactivity was observed in basaloid and in poorly differentiated (G3) palate SCCs, while for Maspin the well-differentiated (G1) palate SCCs were the most reactive. The first three markers mentioned above were most intensely expressed at the invasion front, while the Maspin reactivity was low or absent at this level. Statistically, we found significant stratification on localization, grading, muscle invasion, and survival for all investigated markers, but with very high direct correlations between D2-40, Gal-3, and MCM7 immunoreactive score (IRS) values, while between the Maspin and each of the previous markers there were very high inverse correlations. Overall, all these investigate markers proved to be responsible for the local invasiveness and regional lymph node metastasis, thus allowing a prognostic and therapeutic stratification of patients with palate SCCs.

Computational Screening of Natural Compounds for Identification of Potential Anti-Cancer Agents Targeting MCM7 Protein.

Minichromosome maintenance complex component 7 (MCM7) is involved in replicative licensing and the synthesis of DNA, and its overexpression is a fascinating biomarker for various cancer types. There is currently no effective agent that can prevent the development of cancer caused by the MCM7 protein. However, on the molecular level, inhibiting MCM7 lowers cancer-related cellular growth. With this purpose, this study screened 452 biogenic compounds extracted from the UEFS Natural Products dataset against MCM protein by using the in silico art of technique. The hit compounds UEFS99, UEFS137, and UEFS428 showed good binding with the MCM7 protein with binding energy values of −9.95, −8.92, and −8.71 kcal/mol, which was comparatively higher than that of the control compound ciprofloxacin (−6.50). The hit (UEFS99) with the minimum binding energy was picked for molecular dynamics (MD) simulation investigation, and it demonstrated stability at 30 ns. Computational prediction of physicochemical property evaluation revealed that these hits are non-toxic and have good drug-likeness features. It is suggested that hit compounds UEFS99, UEFS137, and UEFS428 pave the way for further bench work validation in novel inhibitor development against MCM7 to fight the cancers.

MCM5 Aggravates the HDAC1-Mediated Malignant Progression of Lung Cancer.

BackgroundHistone deacetylase 1 (HDAC1) is essential in the malignant progression of tumors. However, there is no obvious relationship between the expression of HDAC1 and the survival of lung cancer patients. Herein, we explored the involvement of minichromosome maintenance complex component 5 (MCM5) and HDAC1 interaction in the epithelial-to-mesenchymal transition (EMT)-dependent malignant progression of lung cancer.MethodsWe analyzed the expression of MCM5 and HDAC1 in The Cancer Genome Atlas database and clinical samples, as well as their impact on patient survival. Cell and animal experiments were performed to verify the promotion of EMT in lung cancer cells mediated by MCM5 and HDAC1.ResultsWe found that lung adenocarcinoma patients with high expression of MCM5 and HDAC1 had poor survival time. Overexpression of MCM5 and HDAC1 in A549 and H1975 cells can promote proliferation and invasion in vitro and tumor growth and metastasis in vivo. Moreover, astragaloside IV can block the interaction between HDAC1 and MCM5, which can then inhibit the malignant progression of lung cancer in vivo and in vitro.ConclusionThe interaction between MCM5 and HDAC1 aggravated the EMT-dependent malignant progression of lung cancer. Astragaloside IV can block the interaction between MCM5 and HDAC1 to inhibit the progression of lung cancer.