Staurosporine-induced Growth Inhibition of Glioma Cells is Accompanied by Altered Expression of Cyclins, CDKs and CDK Inhibitors

Staurosporine was found to bring about complete growth inhibition of human glioma cell lines. U87 MG cells were arrested in S phase while U373 MG cells in G2/M phase on staurosporine treatment. Consistent with this observation, no change in G1 phase regulators viz., Cyclin D1, D3 and CDK4 was seen on staurosporine treatment. The levels of CDK2, CDC2, Cyclin A and Cyclin B proteins decreased, while the levels of CDK inhibitors viz., p21 and p27 were found to increase on staurosporine treatment. The mRNA levels of CDK2 and CDC2 genes were also found to decrease on staurosporine treatment. Thus apart from staurosporine’s known direct inhibitory effect on CDK2 and CDC2 activities, staurosporine was found to down-regulate activities of these two kinases by modulating the expression of the kinases themselves as well that of their activating partners (Cyclins) and their inhibitors.     

Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.

Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.     

SARS-CoV-2病毒感染潜在关键分子生物标志物及免疫浸润特征分析

应用生物信息学方法筛选新型冠状病毒肺炎（corona virus disease 2019,COVID-19）感染的潜在关键分子生物标志物并分析其免疫浸润特征。从GEO数据库下载GSE152418数据集,其中COVID-19患者17例,健康对照17例。用加权基因共表达网络分析（weighted gene co-expression network analysis,WGCNA）方法筛选出COVID-19最相关的模块基因。与差异基因取交集得到共同基因,进行功能及信号通路富集分析,构建蛋白互作网络筛选关键基因,构建关键基因的miRNATF-mRNA调控网络,用CIBERSORT算法预测样本免疫细胞浸润特征。差异分析得到2 049个差异基因。WGCNA分析7个模块中“土耳其蓝色”模块与COVID-19相关性最高（r=0.91,P<0.001）。模块中基因显著性和模块隶属度呈显著正相关（r=0.96,P<0.001）。得到共同基因766个,主要参与有丝分裂、微管结合、阳离子通道活性及卵母细胞减数分裂、细胞衰老等。蛋白互作网络筛选到前10位关键基因分别为CDK1、BUB1、CCNA2...     

Detecting ALK,ROS1 and RET Fusion Genes in Cell Block Samples

Whether Cell block (CB) samples are applicable to detect anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and ret proto-oncogene (RET) fusion genes in lung adenocarcinoma is still unknown. In this study, 108 cytological samples that contained lung adenocarcinoma cells were collected, and made into CB. The CB samples all contained at least 30% lung adenocarcinoma cells. In these patients, 48 harbored EGFR mutation. Among the 50 EGFR wild type patients who detected fusion genes, 14 carried EML4-ALK fusion (28%), 2 had TPM3-ROS1 fusion (4%), and 3 harbored KIF5B-RET fusion (6%). No double fusions were found in one sample. Patients with fusion genes were younger than those without fusion genes (p = 0.032), but no significant difference was found in sex and smoking status (p > 0.05). In the thirty-five patients who received first-line chemotherapy, patients with fusion gene positive had disease control rate (DCR) (72.7% VS 50%, p > 0.05) and objective response rate (ORR) (9.1% VS 4.2%, p > 0.05) compared with those having fusion gene negative. The median progression free survival (mPFS) were 4.0 and 2.7 months in patients harbored fusion mutations and wild type, respectively (p > 0.05). We conclude that CB samples could be used to detect ALK, ROS1 and RET fusions in NSCLC. The frequency distribution of three fusion genes is higher in lung adenocarcinoma with wild-type EGFR, compared with unselected NSCLC patient population. Patients with fusion genes positive are younger than those with fusion gene negative, but they had no significantly different PFS in first-line chemotherapy.     

Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.

A new human cyclin, named cyclin E, was isolated by complementation of a triple cln deletion in S. cerevisiae. Cyclin E showed genetic interactions with the CDC28 gene, suggesting that it functioned at START by interacting with the CDC28 protein. Two human genes were identified that could interact with cyclin E to perform START in yeast containing a cdc28 mutation. One was CDC2-HS, and the second was the human homolog of Xenopus CDK2. Cyclin E produced in E. coli bound and activated the CDC2 protein in extracts from human G1 cells, and antibodies against cyclin E immunoprecipitated a histone H1 kinase from HeLa cells. The interactions between cyclin E and CDC2, or CDK2, may be important at the G1 to S transition in human cells.     

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.     

Evaluation of the TRIP13 level in breast cancer and insights into potential molecular pathways

TRIP13 is a member of the large superfamily of the AAA + ATPase proteins and is associated with a variety of activities. Emerging evidence has shown that TRIP13 may serve as an oncogene. However, the function of TRIP13 in breast cancer (BC) has not yet been elucidated. Here, a variety of bioinformatic tools and laboratory experiments were combined to analyse the expression patterns, prognostic value and functional network of TRIP13 in BC. Multiple databases and immunohistochemistry (IHC) indicated a higher TRIP13 expression in BC tissue compared with normal tissue. TRIP13 was highly expressed in lung metastatic lesions compared with primary tumours in a 4T1 cell implantation BALB/c mouse model of BC. Kaplan–Meier plots also revealed that high TRIP13 expression correlated with poor survival in patients with BC. Furthermore, gene set enrichment analysis revealed that TRIP13 was primarily enriched in the signalling pathway of PI3K‐AKT‐mTOR. Suppressing TRIP13 could inhibit the expression of related genes, as well as the proliferation and migration of BC cell. Finally, 10 hub genes with a high score of connectivity were filtered from the protein–protein interaction (PPI) network, including MAD2L1, CDC20, CDC5L, CDK1, CCNA2, BUB1B, RAD51, SPO11, KIF11 and AURKB. Thus, TRIP13 may be a promising prognostic biomarker and an effective therapeutic target for BC.     

人肝细胞癌预后不良相关基因的生物信息学分析及其临床意义

目的:筛选肝细胞癌(HCC)预后不良相关基因,并探讨其临床意义。方法:在基因表达综合数据库(GEO)中获取符合分析条件的肝细胞癌全基因组表达谱数据并分析得到差异表达基因(DEGs),再运用生物学信息注释及可视化数据库 (DAVID) 和蛋白相互作用数据库 (String) 分别进行功能富集分析和蛋白质互作用网络的构建。利用癌症基因组图谱数据库(TCGA)和Cox比例风险回归模型对相关差异基因进行预后分析。结果:找到一个符合条件的人类HCC数据库 (GSE84402),共筛选出1141个差异表达基因(DEGs),其中上调基因720个,下调基因421个。基因功能富集分析和蛋白质互作用分析结果显示CDK1、CDC6、CCNA2、CHEK1、CENPE 、PIK3R1、RACGAP1、BIRC5、KIF11和CYP2B6为HCC预后的关键基因。TCGA数据库和Cox回归模型分析显示CDC6、PIK3R1、RACGAP1和KIF11的表达升高,CENPE的表达降低与HCC预后不良密切相关。结论:CDC6、CENPE、PIK3R1、RACGAP1和KIF11可能和HCC的预后不良相关,可作为未来HCC预后研究的参考标志物。     

Middle Infrared Radiation Induces G2/M Cell Cycle Arrest in A549 Lung Cancer Cells

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3–5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G₂/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G₂/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.     

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8-3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.     

Identification of a panel of mitotic spindle-related genes as a signature predicting survival in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is one of the most malignant tumor types worldwide. Our objective was to identify a genetic signature that could predict the prognosis of patients with LUAD. We extracted gene data sets from The Cancer Genome Atlas and obtained differentially expressed genes that were highly expressed at every stage. These genes were analyzed using gene set enrichment analysis to obtain four biological processes associated with LUAD. Subsequently, Cox univariate and multivariate analyses were performed to generate four optimized models (G2M checkpoint, E2F targets, mitotic spindle, and glycolysis). We identified a mitotic spindle-related signature (KIF15, BUB1, CCNB2, CDK1, KIF4A, DLGAP5, ECT2, and ANLN), which could be an independent prognostic indicator, to predict the prognosis of patients with LUAD. This new discovery should offer opportunities to explore the pathogenesis of LUAD and prove clinically useful in predicting LUAD patient prognosis.     

Identifying breast cancer subtypes associated modules and biomarkers by integrated bioinformatics analysis

Breast cancer is the most common form of cancer afflicting women worldwide. Patients with breast cancer of different molecular classifications need varied treatments. Since it is known that the development of breast cancer involves multiple genes and functions, identification of functional gene modules (clusters of the functionally related genes) is indispensable as opposed to isolated genes, in order to investigate their relationship derived from the gene co-expression analysis. In total, 6315 differentially expressed genes (DEGs) were recognized and subjected to the co-expression analysis. Seven modules were screened out. The blue and turquoise modules have been selected from the module trait association analysis since the genes in these two modules are significantly correlated with the breast cancer subtypes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment show that the blue module genes engaged in cell cycle, DNA replication, p53 signaling pathway, and pathway in cancer. According to the connectivity analysis and survival analysis, 8 out of 96 hub genes were filtered and have shown the highest expression in basal-like breast cancer. Furthermore, the hub genes were validated by the external datasets and quantitative real-time PCR (qRT-PCR). In summary, hub genes of Cyclin E1 (CCNE1), Centromere Protein N (CENPN), Checkpoint kinase 1 (CHEK1), Polo-like kinase 1 (PLK1), DNA replication and sister chromatid cohesion 1 (DSCC1), Family with sequence similarity 64, member A (FAM64A), Ubiquitin Conjugating Enzyme E2 C (UBE2C) and Ubiquitin Conjugating Enzyme E2 T (UBE2T) may serve as the prognostic markers for different subtypes of breast cancer.     

Identification of KIF4A and its effect on the progression of lung adenocarcinoma based on the bioinformatics analysis

Background: Lung adenocarcinoma (LUAD) is the most frequent histological type of lung cancer, and its incidence has displayed an upward trend in recent years. Nevertheless, little is known regarding effective biomarkers for LUAD.Methods: The robust rank aggregation method was used to mine differentially expressed genes (DEGs) from the gene expression omnibus (GEO) datasets. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to extract hub genes from the protein–protein interaction (PPI) network. The expression of the hub genes was validated using expression profiles from TCGA and Oncomine databases and was verified by real-time quantitative PCR (qRT-PCR). The module and survival analyses of the hub genes were determined using Cytoscape and Kaplan–Meier curves. The function of KIF4A as a hub gene was investigated in LUAD cell lines.Results: The PPI analysis identified seven DEGs including BIRC5, DLGAP5, CENPF, KIF4A, TOP2A, AURKA, and CCNA2, which were significantly upregulated in Oncomine and TCGA LUAD datasets, and were verified by qRT-PCR in our clinical samples. We determined the overall and disease-free survival analysis of the seven hub genes using GEPIA. We further found that CENPF, DLGAP5, and KIF4A expressions were positively correlated with clinical stage. In LUAD cell lines, proliferation and migration were inhibited and apoptosis was promoted by knocking down KIF4A expression.Conclusion: We have identified new DEGs and functional pathways involved in LUAD. KIF4A, as a hub gene, promoted the progression of LUAD and might represent a potential therapeutic target for molecular cancer therapy.     

MicroRNA‐206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D

Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa‐miR‐206 was down‐regulated in melanoma (?75.4‐fold, P = 1.7 × 10^?4). MiR‐206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR‐206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3′UTR reporter gene assays revealed that miR‐206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa‐miR‐206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa‐miR‐206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR‐206 targets.     

Design,synthesis and molecular docking of new fused 1H-pyrroles,pyrrolo[3,2-d]pyrimidines and pyrrolo[3,2-e][1, 4]diazepine derivatives as potent EGFR/CDK2 inhibitors

A new series of 1H-pyrrole (6a–c, 8a–c), pyrrolo[3,2-d]pyrimidines (9a–c) and pyrrolo[3,2-e][1, 4]diazepines (11a–c) were designed and synthesised. These compounds were designed to have the essential pharmacophoric features of EGFR Inhibitors, they have shown anticancer activities against HCT116, MCF-7 and Hep3B cancer cells with IC₅₀ values ranging from 0.009 to 2.195 µM. IC₅₀ value of doxorubicin is 0.008 µM, compounds 9a and 9c showed IC₅₀ values of 0.011 and 0.009 µM respectively against HCT-116 cells. Compound 8b exerted broad-spectrum activity against all tested cell lines with an IC₅₀ value less than 0.05 µM. Compound 8b was evaluated against a panel of kinases. This compound potently inhibited CDK2/Cyclin A1, DYRK3 and GSK3 alpha kinases with 10–23% compared to imatinib (1–10%). It has also arrested the cell cycle of MCF-7 cells at the S phase. Its antiproliferative activity was further augmented by molecular docking into the active sites of EGFR and CDK2 cyclin A1.     

Selective Loss of CDC2 and CDK2 Induction by Tumor Necrosis Factor-α in Senescent Human Diploid Fibroblasts

Normal human diploid fibroblasts undergo a finite number of doublings in culture. This process of senescence is accompanied by a loss in the ability to respond to proliferative stimuli and is therefore distinct from the quiescent state induced by nutrient deprivation. We have studied changes in gene expression induced in these cells following exposure to the cytokine, tumor necrosis factor-α (TNF). We observed that TNF induced CDC2 and CDK2 expression in early-passage quiescent WI-38 fibroblasts. However, as cells approached senescence, their ability to induce CDC2 and CDK2, as well as stimulate DNA synthesis in response to TNF, progressively declined, with minimal to absent induction in senescent cells. This occurred despite the TNF-dependent induction of such proliferation-independent genes as manganese superoxide dismutase and interleukin-6 in senescent and quiescent cells. Serum was similarly unable to induce CDC2 or CDK2 expression in senescent cells. These results demonstrate that senescent cells are selectively deficient in TNF-mediated induction of CDC2 and CDK2, genes crucial to DNA synthesis and mitosis.