RB loss contributes to aggressive tumor phenotypes in MYC-driven triple negative breast cancer

Triple negative breast cancer (TNBC) is characterized by multiple genetic events occurring in concert to drive pathogenic features of the disease. Here we interrogated the coordinate impact of p53, RB, and MYC in a genetic model of TNBC, in parallel with the analysis of clinical specimens. Primary mouse mammary epithelial cells (mMEC) with defined genetic features were used to delineate the combined action of RB and/or p53 in the genesis of TNBC. In this context, the deletion of either RB or p53 alone and in combination increased the proliferation of mMEC; however, the cells did not have the capacity to invade in matrigel. Gene expression profiling revealed that loss of each tumor suppressor has effects related to proliferation, but RB loss in particular leads to alterations in gene expression associated with the epithelial-to-mesenchymal transition. The overexpression of MYC in combination with p53 loss or combined RB/p53 loss drove rapid cell growth. While the effects of MYC overexpression had a dominant impact on gene expression, loss of RB further enhanced the deregulation of a gene expression signature associated with invasion. Specific RB loss lead to enhanced invasion in boyden chambers assays and gave rise to tumors with minimal epithelial characteristics relative to RB-proficient models. Therapeutic screening revealed that RB-deficient cells were particularly resistant to agents targeting PI3K and MEK pathway. Consistent with the aggressive behavior of the preclinical models of MYC overexpression and RB loss, human TNBC tumors that express high levels of MYC and are devoid of RB have a particularly poor outcome. Together these results underscore the potency of tumor suppressor pathways in specifying the biology of breast cancer. Further, they demonstrate that MYC overexpression in concert with RB can promote a particularly aggressive form of TNBC.     

COPD 差异表达基因的生物信息学分析及在LUSC样本中的表达

为确定慢性阻塞性肺病(COPD)的分子标记物及COPD与肺鳞状细胞癌(LUSC)共存的差异表达基因,探寻COPD合并肺癌的预测因子,发现新的治疗靶点。本研究采用生物信息学方法,从GEO数据库中筛选3套基因芯片数据集,挖掘COPD患者小气道上皮细胞(SAEC)的差异表达基因(DEG)以及潜在的生物标记物,并通过基因本体(GO)、京都基因与基因组百科全书(KEGG)富集分析预测DEGs的功能及参与的代谢途径。继而对DEGs构建PPI网络,使用Cytoscape软件筛选子模块和Hub基因,并将Hub基因通过TCGA数据库分析其在LUSC中的差异表达情况及差异基因间的相关性。结果共获得52个上调基因和24个下调基因,代谢通路主要集中在细胞色素P450对外源物质的代谢、化学致癌、花生四烯酸代谢及甲状腺激素合成四条途径上,通过Cytoscape软件从PPI网络中筛选得到2个功能模块和10个Hub基因,进一步验证发现其中5个基因在TCGA数据库中的LUSC样本中同样差异表达。由此推测SPP1、ALDH3A1、SPRR3、KRT6A和SPRR1B 可能为COPD 分子标记物及COPD与LUSC共存的DEGs,从而为研究COPD和LUSC的发病机制及二者潜在关系奠定良好的基础。     

Comprehensive gene expression analysis reveals multiple signal pathways associated with prostate cancer

Prostate cancer (PC) depends on androgenic signaling for growth and survival. To data, the exact molecular mechanism of hormone controlling proliferation and tumorigenesis in the PC remains unclear. Therefore, in this study, we explored the differentially expressed genes (DEGs) and identified featured genes related to hormone stimulus from PC. Two sets of gene expression data, including PC and normal control sample, were downloaded from Gene Expression Omnibus (GEO) database. The t-test was used to identify DEGs between PC and controls. Gene ontology (GO) functional annotation was applied to analyze the function of DEGs and screen hormone-related DEGs. Then these hormone-related DEGs were further analyzed in constructed cancer network and Human Protein Reference Database to screen important signaling pathways they participated in. A total of 912 DEGs were obtained which included 326 up-regulated genes and 586 down-regulated genes. GO functional enrichment analysis identified 50 hormone-related DEGs associated with PC. After pathway and PPI network analysis, we found these hormone-related DEGs participated in several important signaling pathways including TGF-β (TGFB2, TGFB3 and TGFBR2), MAPK (TGFB2, TGFB3 and TGFBR2), insulin (PIK3R3, SHC1 and EIF4EBP1), and p53 signaling pathways (CCND2 and CDKN1A). In addition, a total of five hormone-related DEGs (SHC1, CAV1, RXRA, CDKN1A and SRF) were located in the center of PPI network and 12 hormone-related DEGs formed six protein modules. These important signal pathways and hormone-related DEGs may provide potential therapeutic targets for PC.     

应用GSEA和WGCNA方法分析细菌性败血症宿主关键差异基因及意义

【目的】采用生物信息学方法分析公共数据库来源的细菌性败血症患者全血转录组学表达谱,探讨细菌败血症相关的宿主关键差异基因及意义。【方法】基于GEO数据库中GSE80496和GSE72829全血转录组基因数据集,采用GEO2R、基因集富集分析(GSEA)联用加权基因共表达网络分析(WGCNA)筛选细菌性败血症患者相比健康人群显著改变的差异基因,通过R软件对交集基因进行GO功能分析和KEGG富集分析。同时,通过String 11.0和Cytoscape分析枢纽基因,验证枢纽基因在数据集GSE72809(Health组52例,Definedsepsis组52例)全血标本中的表达情况,并探讨婴儿性别、月(胎)龄、出生体重、是否接触抗生素等因素与靶基因表达谱间的关系。【结果】分析GSE80496和GSE72829数据集分别筛选得到932个基因和319个基因,联合WGCNA枢纽模块交集得到与细菌性败血症发病相关的10个枢纽基因(MMP9、ITGAM、CSTD、GAPDH、PGLYRP1、FOLR3、OSCAR、TLR5、IL1RN和TIMP1);GSEA分析获得关键通路(氨基酸糖类-核糖代谢、PPAR信号通路、聚糖生物合成通路、自噬调控通路、补体、凝血因子级联反应、尼古丁和烟酰胺代谢、不饱和脂肪酸生物合成和阿尔兹海默症通路)及生物学过程(类固醇激素分泌、腺苷酸环化酶的激活、细胞外基质降解和金属离子运输)。【结论】本项研究通过GEO2R、GSEA联用WGCNA分析,筛选出与细菌性败血症发病相关的2个枢纽模块、10个枢纽基因以及一些关键信号通路和生物学过程,可为后续深入研究细菌性败血症致病机制奠定理论依据。     

Extensive alteration in the expression profiles of TGFB pathway signaling components and TP53 is observed along the gastric dysplasia-carcinoma sequence

Aims: The expression patterns of TGFB signaling proteins, such as TGFB1/2, TGFBR1(ALK5), TGFBR2, SMAD1/2/3, SMAD2/3, SMAD4, SMAD7, and of downstream targets of TGFB signaling, CDKN1A (p21CIP1), CDKN1B (p27KIP1), MYC, CDC25A, TP53, and RELA (p65NF-kB) were investigated in gastric carcinomas and other gastric lesions. Methods and results: A total of 112 gastric carcinomas, 37 dysplasias, 54 intestinal metaplasias, 29 chronic atrophic gastritis and 54 normal gastric epithelium were analyzed by tissue microarray-based immunohistochemical analysis. Extensive changes in expression profiles of these proteins were observed. Three types of expression patterns were observed along the normal epithelium-atrophic gastritis-dysplasia-carcinoma sequence. (1) Expression of TGFB1/2, TGFBR1, MYC, and TP53 continually increased along this sequence. (2) Expression of SMAD4, CDKN1A, SMAD1/2/3, SMAD2/3, and CDKN1B was enhanced in dysplasia but decreased in carcinoma. (3) Expression of TGFBR2, SMAD7, RELA, and CDC25A was enhanced in dysplasia and the enhanced level was maintained in carcinoma. In addition, we also evaluated the clinical significance of the expression of TGFB signaling proteins in gastric carcinoma. TGFB and MYC were positively correlated with advanced stages, whereas SMAD1/2/3 and SMAD4 were strongly associated with earlier stages. Conclusions: The extensive change in expression of TGFB signaling components is implicated during tumorigenesis of gastric neoplasias.     

Systematic analysis on multiple Gene Expression Omnibus data sets reveals fierce immune response in hepatitis B virus‐related acute liver failure

Acute liver failure (ALF) caused by hepatitis B virus (HBV) is common type of liver failure in the world, with high morbidity and mortality rates. However, the prevalence, genetic background and factors determining the development of HBV‐related ALF are rarely studied. In this study, we examined three Gene Expression Omnibus (GEO) data sets by bioinformatics analysis to identify differentially expressed genes (DEGs), key biological processes and pathways. Immune infiltration analysis showed high immune cells infiltration in HBV‐related ALF tissue. We then confirmed natural killer cells and macrophages infiltration in clinical samples by immunohistochemistry assay, implying these cells play a significant role in HBV‐ALF. We found 1277 genes were co‐up‐regulated and that 1082 genes were co‐down‐regulated in the 3 data sets. Inflammation‐related pathways were enriched in the co‐up‐regulated genes and synthetic metabolic pathways were enriched in the co‐down‐regulated genes. WGCNA also revealed a key module enriching in immune inflammation response and identified 10 hub genes, differentially expressed in an independent data set. In conclusion, we identified fierce immune inflammatory response to elucidate the immune‐driven mechanism of HBV‐ALF and 10 hub genes based on gene expression profiles.     

Identification of hub genes and pathways in adrenocortical carcinoma by integrated bioinformatic analysis

Adrenocortical carcinoma (ACC), a rare malignant neoplasm originating from adrenal cortical cells, has high malignancy and few treatments. Therefore, it is necessary to explore the molecular mechanism of tumorigenesis, screen and verify potential biomarkers, which will provide new clues for the treatment and diagnosis of ACC. In this paper, three gene expression profiles (GSE10927, GSE12368 and GSE90713) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were obtained using the Limma package. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched by DAVID. Protein‐protein interaction (PPI) network was evaluated by STRING database, and PPI network was constructed by Cytoscape. Finally, GEPIA was used to validate hub genes’ expression. Compared with normal adrenal tissues, 74 up‐regulated DEGs and 126 down‐regulated DEGs were found in ACC samples; GO analysis showed that up‐regulated DEGs were enriched in organelle fission, nuclear division, spindle, et al, while down‐regulated DEGs were enriched in angiogenesis, proteinaceous extracellular matrix and growth factor activity; KEGG pathway analysis showed that up‐regulated DEGs were significantly enriched in cell cycle, cellular senescence and progesterone‐mediated oocyte maturation; Nine hub genes (CCNB1, CDK1, TOP2A, CCNA2, CDKN3, MAD2L1, RACGAP1, BUB1 and CCNB2) were identified by PPI network; ACC patients with high expression of 9 hub genes were all associated with worse overall survival (OS). These hub genes and pathways might be involved in the tumorigenesis, which will offer the opportunities to develop the new therapeutic targets of ACC.     

Cell Cycle Regulation Targets of MYCN Identified by Gene Expression Microarrays

Background: We have previously shown that MYCN knockdown causes a G1 arrest in MYCN amplified (MNA), p53 wild type (wt) and p53 mutant MNA neuroblastoma cell lines, with increases in p21WAF1 and hypo RB in p53 wt cell lines. 1 Hypothesis: MYCN acts by inhibiting p21WAF1, and also by p21 independent mechanisms to override the G1 checkpoint in exponentially growing cells. Methods: Genes potentially regulated by MYCN were identified using gene expression microarrays in p53 wt MNA IMR-32 and p53 mutant MNA SKNBE(2c) neuroblastoma cell lines treated with MYCN or scrambled siRNA. Results were validated using qRT-PCR and confirmed using the regulatable MYCN expression system (SHEP Tet21N). Results: MYCN knockdown altered the expression of several cell cycle related genes. SKP2 was down regulated in both cell lines, and up regulated in MYCN+ Tet21N cells. Expression of the WNT antagonist DKK3 increased in both cell lines and decreased in MYCN+ Tet21N cells. Expression of CDKN1C (p57cip2) and TP53INP1 also increased after MYCN knockdown. Conclusions: MYCN may override the G1 checkpoint through down-regulation of SKP2 and TP53INP1 resulting in reduced p21WAF1 expression in p53 wt cell lines, and in addition may act through the WNT signalling pathway in a p53 independent manner.     

Restoration of mutant TP53 to normal TP53 function by glycerol as a chemical chaperone

We have previously reported that heat stress induces expression of wild-type TP53 (formerly known as p53) activated factor 1 (CDKN1A, formerly known as WAF1) only when TP53 protein is wild-type using cells of a human glioblastoma cell line (A-172) and cells of its transformant (A-172/mp53/ 143) with a mutant TP53 (point mutation at codon 143 from Val to Ala) vector. Transfection of A-172 cells with the mutant TP53 vector abolished the heat-induced expression of CDKN1A, demonstrating the dominant negative nature of this TP53 mutant over the endogenous wild-type TP53. This kind of dominant negative TP53 mutant occurs frequently in various types of cancer. Overcoming this dominance or restoring the normal functions to these TP53 mutants is a new strategy for TP53-targeted cancer therapies. We examined whether glycerol can act as a chemical chaperone to correct the mutant TP53 conformation. No CDKN1A expression was induced after heating or treatment with glycerol at concentrations of 0.6 and 1.2 M in these transformants. In contrast, A-172/mp53/ 143 cells showed CDKN1A expression when they were heated in the presence of glycerol at 0.6 or 1.2 M, which was similar to the response of the parental and neo vector-transfected control cells. To test the generality of the effects of glycerol on mutant TP53, we used human osteosarcoma Saos-2 cells (lacking TP53) transfected with mutant TP53 and cells of two other human glioblastoma cell lines carrying mutant TP53. These cells showed similar CDKN1A expression when heated in the presence of glycerol at 0.6 or 1.2 M. These results suggest that glycerol is effective in restoring several TP53 mutants to normal TP53 function, leading to normal CDKN1A expression after heat stress. This observation provides a novel tool for correction of mutant TP53 conformation and may be applicable for TP53-targeted cancer therapy.     

Exploring the mechanism of ellagic acid against gastric cancer based on bioinformatics analysis and network pharmacology

Ellagic acid (EA) is a natural polyphenolic compound. Recent studies have shown that EA has potential anticancer properties against gastric cancer (GC). This study aims to reveal the potential targets and mechanisms of EA against GC. This study adopted methods of bioinformatics analysis and network pharmacology, including the weighted gene co-expression network analysis (WGCNA), construction of protein–protein interaction (PPI) network, receiver operating characteristic (ROC) and Kaplan–Meier (KM) survival curve analysis, Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, molecular docking and molecular dynamics simulations (MDS). A total of 540 EA targets were obtained. Through WGCNA, we obtained a total of 2914 GC clinical module genes, combined with the disease database for screening, a total of 606 GC-related targets and 79 intersection targets of EA and GC were obtained by constructing Venn diagram. PPI network was constructed to identify 14 core candidate targets; TP53, JUN, CASP3, HSP90AA1, VEGFA, HRAS, CDH1, MAPK3, CDKN1A, SRC, CYCS, BCL2L1 and CDK4 were identified as the key targets of EA regulation of GC by ROC and KM curve analysis. The enrichment analysis of GO and KEGG pathways of key targets was performed, and they were mainly enriched in p53 signalling pathway, PI3K-Akt signalling pathway. The results of molecular docking and MDS showed that EA could effectively bind to 13 key targets to form stable protein–ligand complexes. This study revealed the key targets and molecular mechanisms of EA against GC and provided a theoretical basis for further study of the pharmacological mechanism of EA against GC.     

TP53INP1 inhibits hypoxia‐induced vasculogenic mimicry formation via the ROS/snail signalling axis in breast cancer

Tumour protein p53‐inducible nuclear protein 1 (TP53INP1) is a tumour suppressor associated with malignant tumour metastasis. Vasculogenic mimicry (VM) is a new tumour vascular supply pattern that significantly influences tumour metastasis and contributes to a poor prognosis. However, the molecular mechanism of the relationship between TP53INP1 and breast cancer VM formation is unknown. Here, we explored the underlying mechanism by which TP53INP1 regulates VM formation in vitro and in vivo. High TP53INP1 expression was not only negatively correlated with a poor prognosis but also had a negative relationship with VE‐cadherin, HIF‐1α and Snail expression. TP53INP1 overexpression inhibited breast cancer invasion, migration, epithelial‐mesenchymal transition (EMT) and VM formation; conversely, TP53INP1 down‐regulation promoted these processes in vitro by functional experiments and Western blot analysis. We established a hypoxia model induced by CoCl₂ and assessed the effects of TP53INP1 on hypoxia‐induced EMT and VM formation. In addition, we confirmed that a reactive oxygen species (ROS)‐mediated signalling pathway participated in TP53INP1‐mediated VM formation. Together, our results show that TP53INP1 inhibits hypoxia‐induced EMT and VM formation via the ROS/GSK‐3β/Snail pathway in breast cancer, which offers new insights into breast cancer clinical therapy.     

Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.     

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.     

Identification of gene expression profiles and key genes in subchondral bone of osteoarthritis using weighted gene coexpression network analysis

Osteoarthritis (OA) significantly influences the quality life of people around the world. It is urgent to find an effective way to understand the genetic etiology of OA. We used weighted gene coexpression network analysis (WGCNA) to explore the key genes involved in the subchondral bone pathological process of OA. Fifty gene expression profiles of GSE51588 were downloaded from the Gene Expression Omnibus database. The OA‐associated genes and gene ontologies were acquired from JuniorDoc. Weighted gene coexpression network analysis was used to find disease‐related networks based on 21756 gene expression correlation coefficients, hub‐genes with the highest connectivity in each module were selected, and the correlation between module eigengene and clinical traits was calculated. The genes in the traits‐related gene coexpression modules were subject to functional annotation and pathway enrichment analysis using ClusterProfiler. A total of 73 gene modules were identified, of which, 12 modules were found with high connectivity with clinical traits. Five modules were found with enriched OA‐associated genes. Moreover, 310 OA‐associated genes were found, and 34 of them were among hub‐genes in each module. Consequently, enrichment results indicated some key metabolic pathways, such as extracellular matrix (ECM)‐receptor interaction (hsa04512), focal adhesion (hsa04510), the phosphatidylinositol 3'‐kinase (PI3K)‐Akt signaling pathway (PI3K‐AKT) (hsa04151), transforming growth factor beta pathway, and Wnt pathway. We intended to identify some core genes, collagen (COL)6A3, COL6A1, ITGA11, BAMBI, and HCK, which could influence downstream signaling pathways once they were activated. In this study, we identified important genes within key coexpression modules, which associate with a pathological process of subchondral bone in OA. Functional analysis results could provide important information to understand the mechanism of OA.