mRNA Expression Datasets Research Articles

Identifying Potential Prognostic Markers for Muscle-Invasive Bladder Urothelial Carcinoma by Weighted Gene Co-Expression Network Analysis.

Muscle-invasive bladder urothelial carcinoma (MIBC) is characterized as a genetic heterogeneous cancer with a high percentage of recurrence and worse prognosis. Identify the prognostic potentials of novel genes for muscle-invasive urothelial bladder cancer could at least provide important information for early detection and clinical treatment. Weighted gene co-expression network analysis (WGCNA) algorithm, a powerful systems biology approach, was utilized to extract co-expressed gene networks from mRNA expression dataset to construct transcriptional modules in MIBC samples, which was associated with demographic and clinical traits of MIBC patients. The potential prognostic markers of MIBC were screened out in the discovery dataset and verified in an independent external validation dataset. A total of 8 co-expression modules were detected through the WGCNA algorithm in the discovery datasets based on 401 MIBC samples. One transcriptional module enriched in cell development was observed to be correlated with the MIBC prognosis in the discovery datasets (HR = 1.48, 95%CI = 1.04-2.11) and independently verified in an external dataset (HR = 3.59, 95%CI = 1.09-11.79). High expression of hub genes including discoidin domain receptor tyrosine kinase 2 (DDR2), PDZ and LIM domain 3 (PDLIM3), zinc finger protein 521 (ZNF521), methionine sulfoxide reductase B3 (MSRB3) were significantly associated with the unfavorable survival of MIBC patients. We identified and validated four novel potential biomarkers associated with prognosis of MIBC patients by constructing genes co-expression networks. The discovery of these genetic markers may provide a new target for the development of MIBC chemotherapeutic drugs.

Platform-independent approach for cancer detection from gene expression profiles of peripheral blood cells.

Peripheral blood gene expression intensity-based methods for distinguishing healthy individuals from cancer patients are limited by sensitivity to batch effects and data normalization and variability between expression profiling assays. To improve the robustness and precision of blood gene expression-based tumour detection, it is necessary to perform molecular diagnostic tests using a more stable approach. Taking breast cancer as an example, we propose a machine learning-based framework that distinguishes breast cancer patients from healthy subjects by pairwise rank transformation of gene expression intensity in each sample. We showed the diagnostic potential of the method by performing RNA-seq for 37 peripheral blood samples from breast cancer patients and by collecting RNA-seq data from healthy donors in Genotype-Tissue Expression project and microarray mRNA expression datasets in Gene Expression Omnibus. The framework was insensitive to experimental batch effects and data normalization, and it can be simultaneously applied to new sample prediction.

Hypoxia-Regulated miRNAs in Human Mesenchymal Stem Cells: Exploring the Regulatory Effects in Ischemic Disorders.

Human mesenchymal/stromal stem cells (hMSC) are the most promising cell source for adult cell therapies in regenerative medicine. Many clinical trials have reported the use of autologous transplantation of hMSCs in several disorders, but with limited results. To exert their potential, hMSCs could exhibit efficient homing and migration toward lesion sites among other effects, but the underlying process is not clear enough. To further increase the knowledge, we studied the co-regulation between hypoxia-regulated genes and miRNAs. To this end, we investigated the miRNA expression profile of healthy hMSCs in low oxygen/nutrient conditions to mimic ischemia and compared with cells of patients suffering from critical limb ischemia (CLI). miRNAs are small, highly conserved, non-coding RNAs, skilled in the control of the target’s expression level in a fine-tuned way. After analyzing the miRNOme in CLI-derived hMSC cells and healthy controls, and intersecting the results with the mRNA expression dataset under hypoxic conditions, we identified two miRNAs potentially relevant to the disease: miR-29b as a pathological marker of the disease and miR-638 as a therapeutic target. This study yielded a deeper understanding of stem cell biology and ischemic disorders, opening new potential treatments in the future.

Seeking for Correlative Genes and Signaling Pathways With Bone Metastasis From Breast Cancer by Integrated Analysis.

Background: Bone metastasis frequently occurs in advanced breast cancer patients, and it is one of major causes of breast cancer associated mortality. The aim of the current study is to identify potential genes and related signaling pathways in the pathophysiology of breast cancer bone metastasis.Methods: Three mRNA expression datasets for breast cancer bone metastasis were obtained from Gene Expression Omnibus (GEO) dataset. The differentially expressed genes (DEGs) were obtained. Functional analyses, protein-protein interaction (PPI) network, and transcription factors (TFs)-target genes network was constructed. Real-time PCR using clinical specimens was conducted to justify the results from integrated analysis.Results: A 749 DEGs were obtained. Osteoclast differentiation and rheumatoid arthritis were two significantly enriched signaling pathways for DEGs in the bone metastasis of breast cancer. SMAD7 (degree = 10), TGFBR2 (degree = 9), VIM (degree = 8), FOS (degree = 8), PDGFRB (degree = 7), COL5A1 (degree = 6), ARRB2 (degree = 6), and ITGAV (degree = 6) were high degree genes in the PPI network. ETS1 (degree = 12), SPI1 (degree = 12), FOS (degree = 10), FLI1 (degree = 5), KLF4 (degree = 4), JUNB (degree = 4), NR3C1 (degree = 4) were high degree genes in the TFs-target genes network. Validated by QRT-PCR, the expression levels of IBSP, MMP9, MMP13, TNFAIP6, CD200, DHRS3, ASS1, RIPK4, VIM, and PROM1 were roughly consistent with our integrated analysis. Except PROM1, the other genes had a diagnose value for breast cancer bone metastasis.Conclusions: The identified DEGs and signaling pathways may make contribution for understanding the pathological mechanism of bone metastasis from breast cancer.

Regulatory network reconstruction of five essential microRNAs for survival analysis in breast cancer by integrating miRNA and mRNA expression datasets

Although many of the genetic loci associated with breast cancer risk have been reported, there is a lack of systematic analysis of regulatory networks composed of different miRNAs and mRNAs on survival analysis in breast cancer. To reconstruct the microRNAs-genes regulatory network in breast cancer, we employed the expression data from The Cancer Genome Atlas (TCGA) related to five essential miRNAs including miR-21, miR-22, miR-210, miR-221, and miR-222, and their associated functional genomics data from the GEO database. Then, we performed an integration analysis to identify the essential target factors and interactions for the next survival analysis in breast cancer. Based on the results of our integrated analysis, we have identified significant common regulatory signatures including differentially expressed genes, enriched pathways, and transcriptional regulation such as interferon regulatory factors (IRFs) and signal transducer and activator of transcription 1 (STAT1). Finally, a reconstructed regulatory network of five miRNAs and 34 target factors was established and then applied to survival analysis in breast cancer. When we used expression data for individual miRNAs, only miR-21 and miR-22 were significantly associated with a survival change. However, we identified 45 significant miRNA-gene pairs that predict overall survival in breast cancer out of 170 one-on-one interactions in our reconstructed network covering all of five miRNAs, and several essential factors such as PSMB9, HLA-C, RARRES3, UBE2L6, and NMI. In our study, we reconstructed regulatory network of five essential microRNAs for survival analysis in breast cancer by integrating miRNA and mRNA expression datasets. These results may provide new insights into regulatory network-based precision medicine for breast cancer.

Prioritizing Crohn's disease genes by integrating association signals with gene expression implicates monocyte subsets.

Genome-wide association studies have identified ~170 loci associated with Crohn's disease (CD) and defining which genes drive these association signals is a major challenge. The primary aim of this study was to define which CD locus genes are most likely to be disease related. We developed a gene prioritization regression model (GPRM) by integrating complementary mRNA expression datasets, including bulk RNA-Seq from the terminal ileum of 302 newly diagnosed, untreated CD patients and controls, and in stimulated monocytes. Transcriptome-wide association and co-expression network analyses were performed on the ileal RNA-Seq datasets, identifying 40 genome-wide significant genes. Co-expression network analysis identified a single gene module, which was substantially enriched for CD locus genes and most highly expressed in monocytes. By including expression-based and epigenetic information, we refined likely CD genes to 2.5 prioritized genes per locus from an average of 7.8 total genes. We validated our model structure using cross-validation and our prioritization results by protein-association network analyses, which demonstrated significantly higher CD gene interactions for prioritized compared with non-prioritized genes. Although individual datasets cannot convey all of the information relevant to a disease, combining data from multiple relevant expression-based datasets improves prediction of disease genes and helps to further understanding of disease pathogenesis.

Combined Therapy Sensitivity Index Based on a 13-Gene Signature Predicts Prognosis for IDH Wild-type and MGMT Promoter Unmethylated Glioblastoma Patients.

Glioblastoma (GBM) is one of the lethal tumors with poor prognosis. However, prognostic prediction approaches need to be further explored. Therefore, we developed an evaluation system that could be used for prognostic prediction of GBM patients. Published mRNA expression datasets from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Chinese Glioma Genome Atlas (CGGA) were analyzed. Quantitative Realtime-PCR of signature genes and molecular aberrations of 178 Xiangya GBM patients were used for confirmation. Gene set enrichment analysis (GSEA) was performed for functional annotation. As a result, we established a 13-gene signature which named Combined Therapy Sensitivity Index (CTSI). Based on a cutoff point, we divided patients into high-risk group and low-risk group. Based on Kaplan-Meier analysis and multivariate Cox regression analysis, we found that patients in the high-risk group had a shorter overall survival time than patients in the low-risk group (p<0.001 in TCGA and CGGA datasets, p=0.047 in GSE4271 dataset, p=0.008 in Xiangya GBM cohort, HR: 1.65-3.42). By comparing the status of IDH mutation, TERT promoter mutation (TERTp-mut) and MGMT promoter methylation, CTSI was predictable in IDH wild-type (IDH-wt)/MGMT promoter unmethylated (MGMTp-unmeth) patients (p=0.037 in IDH-wt/TERTp-mut/MGMTp-unmeth subgroup, HR: 1.98; p=0.032 in IDH-wt/TERTp-wt/MGMTp-unmeth subgroup, HR: 2.09). Based on GESA, the Gene Ontology (GO) gene sets were enriched differently between CTSI high-risk and low-risk groups. Our results showed CTSI risk score can predict the prognosis of IDH-wt/MGMTp-unmeth GBM patients. Based on CTSI, combined with the status of IDH mutation, TERT promoter mutation and MGMT promoter methylation, a stepwise prognosis evaluation system which can provide precise prognosis prediction for GBM patients was established.

Overexpression of the ASPM gene is associated with aggressiveness and poor outcome in bladder cancer.

Abnormal spindle-like microcephaly-associated (ASPM) protein is essential for mitotic spindle function during cell replication. The present study aimed to evaluate the hypothesis that ASPM serves a critical role in cancer invasiveness and may act as a prognostic biomarker in bladder cancer. In total, 6 independent worldwide bladder cancer microarray mRNA expression datasets (n=1,355) with clinical and follow-up annotations were collected from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Reverse transcription-quantitative polymerase chain reaction analysis revealed that ASPM mRNA expression was higher in bladder cancer tissue compared with adjacent normal bladder mucosae in 10 paired human tissue samples (P=0.004). ASPM overexpression in human bladder cancer samples was consistent with the mRNA expression datasets from GEO and TCGA. Bioinformatics analysis indicated that ASPM mRNA expression was significantly associated with grade and tumor node metastasis (TNM) stage in bladder cancer, based on pooled GEO and TCGA datasets (P<0.05). Stratification analysis indicated that the clinical significance of ASPM was particularly pronounced in low-grade or papillary subtypes of bladder cancer. Individual Cox and pooled Kaplan-Meier analyses suggested that ASPM expression was significantly directly correlated with poor overall (OS) and progression-free survival (PFS) in bladder cancer. Multivariate and stratification analyses demonstrated that the prognostic significance of ASPM was evident in low-grade or papillary bladder cancers, yet not in high-grade or non-papillary subgroups. Increased expression of ASPM was associated with poor OS in muscle-invasive bladder cancer and with poor PFS in non-muscle-invasive bladder cancer (P<0.05). Bioinformatics analysis identified the top 11 ASPM-related genes on STRING-DB.org. The expression of the majority of these genes was associated with poor outcomes of bladder cancer with statistical significance. Gene set enrichment analysis indicated that the high expression of ASPM could enrich gene signatures involved in mitosis, differentiation and metastasis in bladder cancer. Further analysis of TCGA datasets indicated that increased ASPM expression was significantly associated with higher Gleason score, T stage, N stage and poor clinical outcome in prostate cancer. It was also significantly associated with late TNM stage and poor PFS in renal cell carcinoma. In summary, ASPM may serve as a novel prognostic biomarker for low-grade or papillary bladder cancer.

Screening of key candidate genes and pathways for osteocytes involved in the differential response to different types of mechanical stimulation using a bioinformatics analysis.

This study aimed to predict the key genes and pathways that are activated when different types of mechanical loading are applied to osteocytes. mRNA expression datasets (series number of GSE62128 and GSE42874) were obtained from Gene Expression Omnibus database (GEO). High gravity-treated osteocytic MLO-Y4 cell-line samples from GSE62128 (Set1), and fluid flow-treated MLO-Y4 samples from GSE42874 (Set2) were employed. After identifying the differentially expressed genes (DEGs), functional enrichment was performed. The common DEGs between Set1 and Set2 were considered as key DEGs, then a protein-protein interaction (PPI) network was constructed using the minimal nodes from all of the DEGs in Set1 and Set2, which linked most of the key DEGs. Several open source software programs were employed to process and analyze the original data. The bioinformatic results and the biological meaning were validated by in vitro experiments. High gravity and fluid flow induced opposite expression trends in the key DEGs. The hypoxia-related biological process and signaling pathway were the common functional enrichment terms among the DEGs from Set1, Set2 and the PPI network. The expression of almost all the key DEGs (Pdk1, Ccng2, Eno2, Egln1, Higd1a, Slc5a3 and Mxi1) were mechano-sensitive. Eno2 was identified as the hub gene in the PPI network. Eno2 knockdown results in expression changes of some other key DEGs (Pdk1, Mxi1 and Higd1a). Our findings indicated that the hypoxia response might have an important role in the differential responses of osteocytes to the different types of mechanical force.

CAV3.1 knockdown suppresses cell proliferation, migration and invasion of prostate cancer cells by inhibiting AKT.

BackgroundAberrant expression of CAV3.1, one of T-type Ca2+ channels, is reported to exert important functions in pathological processes, including carcinogenesis. However, its expression pattern and function in prostate cancer (PCa) remains unclear.Materials and methodsThe expression pattern of CAV3.1 was analyzed in multiple ways, including online analysis in Oncomine database, experimental analyses in cell lines, and collected clinical specimens using immunohistochemistry, quantitative reverse transcription polymerase chain reaction, and Western blot. Then, CAV3.1 was downregulated in PCa cells to explore its functions.ResultsUpregulated CAV3.1 in PCa tissues and cells was confirmed by analyzing mRNA expression datasets from Oncomine and quantitative reverse transcription polymerase chain reaction detection, respectively. Accordingly, significantly higher CAV3.1 protein level in PCa tissues specimens than that in benign prostatic hyperplasia tissues was indicated by immunohistochemical staining. In addition, CAV3.1 upregulation was positively associated with metastasis. Depletion of CAV3.1 impaired the proliferation, migration, and invasion ability of PCa cells demonstrating by cell functional experiments, such as CCK-8, cell cycle distribution, plate clone formation, scratch wound healing, and transwell invasion assays. Mechanistically, due to constrained Akt activity, CAV3.1 knockdown resulted in decreased level of CCND1, N-cadherin, and Vimentin, and increased level of E-cadherin whose expressions could be reversed by ectopic Akt expression. Similarly, ectopic Akt expression also rescued the inhibitory effects of CAV3.1 knockdown on cell functions like proliferation and migration in PCa cells.ConclusionUpregulated CAV3.1 is positively associated with the development of PCa. CAV3.1 knockdown can inhibit PCa cell proliferation, migration, and invasion by suppressing AKT activity.

Abstract B36: FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer

Abstract Purpose: FOXM1 is located at 12p13.33, which shows copy number gains in the majority of high-grade serous ovarian cancer (HGSC) cases. The 12p13.33 amplicon contains 33 genes, some of which may cooperate with FOXM1 to exert oncogenic function. Although FOXM1 overexpression is associated with genomic instability in cancer, whether FOXM1 contributes directly to this phenotype is unclear. We hypothesized that FOXM1 induces DNA replication stress (DRS), resulting in ATR-CHK1 activation. Intriguingly, we noted that RHNO1, a component of the 9-1-1 complex required for ATR-CHK1 activation, is contained within the 12p13.33 amplicon and is arranged in a head-to-head orientation with FOXM1. Here, we characterized the FOXM1/RHNO1 bidirectional promoter, FOXM1/RHNO1 expression, and determined the role of FOXM1/RHNO1 in DRS, the DRS response, and clonogenic growth of HGSC cells. Methods: We used reporter assays to characterize the FOXM1/RHNO1 bidirectional promoter and FOXM1 transcriptional activity. We analyzed copy number, mRNA, and protein expression datasets in TCGA, CCLE, and GTEX. We measured FOXM1/RHNO1 expression in HGSC cell lines and tumors. We overexpressed FOXM1 in human immortalized fallopian tube epithelial (FTE) cells to determine its contribution to DRS and DNA damage. We knocked down RHNO1 in HGSC cells to determine its contribution to the DRS response and clonogenic growth. We performed Western blot analyses to measure markers of DRS and DNA damage. We characterized FOXM1-induced DNA damage at single-cell resolution using Comet assays and flow cytometry measurement of γ-H2AX. Results: FOXM1/RHNO1 showed copy number gains or amplifications in 58% of TCGA HGSC tumors, and FOXM1/RHNO1 mRNA expression significantly correlated in TCGA HGSC tumors, CCLE HGSC lines, and GTEX normal tissues. The FOXM1/RHNO1 bidirectional promoter showed balanced activity in each direction in FTE and HGSC cells, and bidirectional promoter activity correlated with endogenous mRNA expression. TCGA HGSC data indicated that FOXM1 expression associates with markers of DNA replication, global copy number alterations, and CHK1-Ser345-P, a canonical marker of DRS. In agreement, FOXM1 overexpression in FTE cells induced phosphorylation of ATR-Thr1989, CHK1-Ser317/345, H2AX-Ser139, and the proportion of DNA in Comet tails. In contrast, a DNA-binding domain mutant of FOXM1 did not induce DRS or DNA damage. RHNO1 knockdown attenuated CHK1-Ser345-P in response to DRS and RHNO1 shRNA knockdown or CRISPR-Cas9 knockout severely reduced HGSC cell clonogenic growth. Importantly, the effects of RHNO1 disruption were rescued by WT RHNO1 expression, but not by a RHNO1 mutant defective for interaction with the 9-1-1 complex. Conclusion: Our data reveal that FOXM1/RHNO1 share a bidirectional promoter and that this may account for their frequent coexpression in HGSC. FOXM1 induces DRS, and this is dependent on its ability to bind DNA. Conversely, RHNO1 contributes to ATR-CHK1 signaling and promotes the clonogenic growth of HGSC cells. We hypothesize that balanced FOXM1/RHNO1 expression promotes HGSC development and progression. Ongoing studies are characterizing the impact of FOXM1/RHNO1 on DRS and genomic instability in model systems. Finally, our data suggest that the ATR-CHK1 pathway is a synthetic lethal vulnerability in HGSC tumors that overexpress FOXM1. Citation Format: Carter J. Barger, Linda Chee, Connor Branick, Kunle Odunsi, Ronny Drapkin, Adam R. Karpf. FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B36.

Increased HSF1 expression predicts shorter disease-specific survival of prostate cancer patients following radical prostatectomy.

Prostate cancer is a highly heterogeneous disease and the clinical outcome is varying. While current prognostic tools are regarded insufficient, there is a critical need for markers that would aid prognostication and patient risk-stratification. Heat shock transcription factor 1 (HSF1) is crucial for cellular homeostasis, but also a driver of oncogenesis. The clinical relevance of HSF1 in prostate cancer is, however, unknown. Here, we identified HSF1 as a potential biomarker in mRNA expression datasets on prostate cancer. Clinical validation was performed on tissue microarrays from independent cohorts: one constructed from radical prostatectomies from 478 patients with long term follow-up, and another comprising of regionally advanced to distant metastatic samples. Associations with clinical variables and disease outcomes were investigated. Increased nuclear HSF1 expression correlated with disease advancement and aggressiveness and was, independently from established clinicopathological variables, predictive of both early initiation of secondary therapy and poor disease-specific survival. In a joint model with the clinical Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score, nuclear HSF1 remained a predictive factor of shortened disease-specific survival. The results suggest that nuclear HSF1 expression could serve as a novel prognostic marker for patient risk-stratification on disease progression and survival after radical prostatectomy.

Abstract 3321: Dysregulation of m6A RNA methylation regulators in colorectal cancer: Clinical implication as prognostic biomarkers and potential therapeutic targets

Abstract Purpose Among more than 100 known post-transcriptional modifications, N6-Methyladenosine (m6A) represents the most prevalent internal modification in mammalian mRNAs. This epigenomic modification is orchestrated by a distinct group of enzymes and cofactors, categorized as writers, erasers and readers. Writers consist of a multicomponent complex consisting of at least three proteins, namely methyltransferase-like 3 (METTL3), METTL14, and Wilms' tumor 1-associating protein (WTAP1). These m6A marks are erased by two RNA demethylases, fat mass and obesity-associated protein (FTO) and alkylated DNA repair protein alkB homolog 5 (ALKBH5). Lastly, YTHDF1/2/3 family genes constitute the ‘readers' that preferentially bind to RNA harboring m6A modification. Accumulating evidence suggests that m6a RNA methylation plays a crucial role in cancer progression. Herein, we for the first time elucidate the role of m6a regulators in colorectal cancer (CRC), in order to gain insights into their functional role and potential clinical significance in this malignancy. Experimental design Two large independent publicly available genome-wide mRNA expression datasets (N=509, TCGA and N=566, CIT) were used to study the expression of m6a RNA methylation regulators and their association with prognosis and gene expression based Consensus Molecular Subtypes (CMS) in CRC. Furthermore, we studied the tumorigenic properties of the RNA demethylase, FTO, by knockdown and overexpression strategies in CRC cells. Results The risk-score derived from a seven gene mRNA expression classifier consisting of METTL3, METTL14, WTAP, YTHDF1, YTHDF2, FTO and ALKBH5 was associated with poor disease-free survival in the training (TCGA) and validation (CIT) cohorts, with corresponding hazard ratios of 1.90 (95% CI: 1.24-2.92, p&amp;lt;0.002) and 1.86 (95% CI: 1.25-2.78, p&amp;lt;0.001) respectively. Furthermore, a high-m6a risk-score was significantly associated with a mesenchymal, CMS4 subtype, with FTO being the most overexpressed gene in CMS4 subtype of CRCs. Functionally, knockdown of the FTO gene in CMS4 cell lines (MDST8 and NCIH716) dramatically inhibited cellular proliferation, invasion and colony formation abilities. In contrast, overexpression of FTO in RKO cells resulted in enhanced proliferation, invasion and colony formation. Conclusions In summary, we for the first time report the dysregulation of m6a RNA methylation regulators in CRC, wherein we discovered their significant association with poor prognosis and a CMS4 CRC subtype, as well as elucidated the functional role of FTO in CRC cell lines. Besides the potential of utilizing this seven gene m6a regulator gene expression classifier to predict prognosis, our data suggest that targeting FTO might represent an attractive strategy for therapeutic intervention in patients with a mesenchymal CRC subtype with poor prognosis. Citation Format: Raju Kandimalla, Feng Gao, Chun Ruan, Michael Hsieh, Xin Wang, Ajay Goel. Dysregulation of m6A RNA methylation regulators in colorectal cancer: Clinical implication as prognostic biomarkers and potential therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3321.

Screening therapeutic targets of ribavirin in hepatocellular carcinoma.

The objective of the present study was to screen the key genes of ribavirin in hepatocellular carcinoma (HCC) and provide novel therapeutic targets for HCC treatment. The mRNA expression datasets of GSE23031 and GSE74656, as well as the microRNA (miRNA) expression dataset of GSE22058 were downloaded from the Gene Expressed Omnibus database. In the GSE23031 dataset, there were three HCC cell lines treated with PBS and three HCC cell lines treated with ribavirin. In the GSE74656 dataset, five HCC tissues and five carcinoma adjacent tissues were selected. In the GSE22058 dataset, 96 HCC tissues and 96 carcinoma adjacent tissues were selected. The differentially expressed genes (DEGs) and differentially expressed miRNAs were identified via the limma package of R. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed with the Database for Annotation, Visualization and Integrated Discovery. The target mRNAs of DEMs were obtained with TargetScan. A total of 559 DEGs (designated DEG-Ribavirin) were identified in HCC cells treated with ribavirin compared with PBS and 632 DEGs (designated DEG-Tumor) were identified in HCC tissues compared with carcinoma adjacent tissues. A total of 220 differentially expressed miRNAs were identified in HCC tissues compared with carcinoma adjacent tissues. In addition, 121 GO terms and three KEGG pathways of DEG-Ribavirin were obtained, and 383 GO terms and 25 KEGG pathways of DEG-Tumor were obtained. A total of five key miRNA-mRNA regulated pairs were identified, namely miR-183→CCNB1, miR-96→DEPDC1, miR-96→NTN4, miR-183→NTN4 and miR-145→NTN4. The present study indicated that certain miRNAs (including miR-96, miR-145 and miR-183) and mRNAs (including NAT2, FBXO5, CCNB1, DEPDC1 and NTN4) may be associated with the effects of ribavirin on HCC. Furthermore, they may provide novel therapeutic targets for HCC treatment.

Bioinformatic screening for key miRNAs and genes associated with myocardial infarction.

Despite significant advances in understanding of the causes of and treatment of myocardial infarction (MI) in recent years, morbidity and mortality is still high. The aim of this study was to identify miRNA and genes potentially associated with MI. mRNA and miRNA expression datasets were downloaded from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/). Interactions between miRNA and the expression and function of target genes were analyzed, and a protein–protein interaction network was constructed. The diagnostic value of identified miRNA and genes was assessed. Quantitative RT‐PCR was applied to validate the results of the bioinformatics analysis. MiR‐27a, miR‐31*, miR‐1291, miR‐139‐5p, miR‐204, miR‐375, and target genes including CX3CR1,HSPA6, and TPM3 had potential diagnostic value. The genes TFEB,IRS2,GRB2,FASLG,LIMS1,CX3CR1,HSPA6,TPM3,LAT2,CEBPD,AQP9, and MAPKAPK2 were associated with recovery from MI. In conclusion, the identified miRNA and genes might be associated with the pathology of MI.

Abstract P1-10-09: EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity

Abstract Background: Effective treatment options for triple-negative inflammatory breast cancer (TN-IBC), the most aggressive form of breast cancer, are currently lacking. We previously reported that mediators of inflammation promote the growth of TN-IBC xenografts. Eicosapentaenoic acid (EPA), an omega-3 fatty acid (fish oil) with anti-inflammatory properties, is an emerging FDA-approved therapeutic with a favorable toxicology profile. Here we aimed to develop a novel approach to enhance EPA efficacy against TN-IBC by identifying a kinase inhibitor that synergizes with EPA's antitumor activity. Methods and Results: Using a high-throughput siRNA screen in the TN-IBC cell line SUM149PT, we identified inhibition of ephrin type-A receptor 2 (EPHA2), an oncogenic receptor tyrosine kinase, as a target that sensitizes TN-IBC cells to EPA therapy. To determine the clinical relevance of EPHA2, we investigated a meta-analysis of breast cancer mRNA expression data sets and found that high EPHA2 tumor expression, compared with low expressing, correlated significantly with poor overall survival in TN-IBC patients (P = 0.01), while not with other subtypes. Similar findings were observed in vitro, were EPHA2 protein and mRNA overexpression occurred predominantly in the TN subtypes among 49 and 51 breast cancer cell lines (63% and 47%, respectively), highlighting EPHA2 translational potential. Functional expression studies using proliferation and apoptosis assays in vitro, and xenografts in vivo, were performed in two EPHA2-expressing TN-IBC cell lines, SUM149PT and BCX010, to validate EPHA2 as a synergistic combinational target with EPA. EPHA2 gene silencing in combination with EPA significantly reduced cell growth, and enhanced apoptosis, compared with untreated and monotherapy in vitro (P &amp;lt; 0.05), and in vivo (P &amp;lt; 0.001). To translate our findings to the clinic, we validated dasatinib, an FDA-approved small molecule inhibitor of EPHA2, in combination to EPA to significantly enhance apoptosis of TN-IBC cells in vitro (P &amp;lt; 0.05) and in vivo (P &amp;lt; 0.05), compared with untreated and monotherapies. Using membrane fluidity assessment and cholesterol quantification we determined that apoptosis induction after combination therapy was due to increased membrane rigidity and cholesterol concentrations in the plasma membrane of TN-IBC cells (P &amp;lt; 0.05, compared with monotherapies). Finally, we discovered by western blot and gain/loss-of-expression studies that combination therapy inhibited the cholesterol efflux protein ATP-binding cassette sub-family A member 1 (ABCA1), which plays a significant role mediating increased cellular cholesterol (P &amp;lt; 0.05), cell membrane rigidity (P &amp;lt; 0.05), and induction of apoptosis (P &amp;lt; 0.05) in TN-IBC after EPA and EPHA2-targeting combination therapy. Conclusions: This is the first study demonstrating that EPA can enhance conventional targeted therapy against breast cancer. Our study provides molecular and preclinical evidence to support the development of an EPA/EPHA2-inhibition–based phase I clinical trial for patients with EPHA2-positive TN-IBC; our study further suggests the use of EPHA2 and ABCA1 protein expression as biomarkers for patient selection and therapeutic response. Citation Format: Torres-Adorno AM, Vitrac H, Qi Y, Tan L, Levental KR, Fan Y-Y, Yang P, Chapkin RS, Eckhardt BL, Ueno NT. EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-09.

Long Noncoding RNA NEAT1, Regulated by the EGFR Pathway, Contributes to Glioblastoma Progression Through the WNT/β-Catenin Pathway by Scaffolding EZH2

Purpose: Long noncoding RNAs have been implicated in gliomagenesis, but their mechanisms of action are mainly undocumented. Through public glioma mRNA expression data sets, we found that NEAT1 was a potential oncogene. We systematically analyzed the clinical significance and mechanism of NEAT1 in glioblastoma.Experimental Design: Initially, we evaluated whether NEAT1 expression levels could be regulated by EGFR pathway activity. We subsequently evaluated the effect of NEAT1 on the WNT/β-catenin pathway and its target binding gene. The animal model supported the experimental findings.Results: We found that NEAT1 levels were regulated by EGFR pathway activity, which was mediated by STAT3 and NFκB (p65) downstream of the EGFR pathway. Moreover, we found that NEAT1 was critical for glioma cell growth and invasion by increasing β-catenin nuclear transport and downregulating ICAT, GSK3B, and Axin2. Taken together, we found that NEAT1 could bind to EZH2 and mediate the trimethylation of H3K27 in their promoters. NEAT1 depletion also inhibited GBM cell growth and invasion in the intracranial animal model.Conclusions: The EGFR/NEAT1/EZH2/β-catenin axis serves as a critical effector of tumorigenesis and progression, suggesting new therapeutic directions in glioblastoma. Clin Cancer Res; 24(3); 684-95. ©2017 AACR.

A Stack-based Ensemble Framework for Detecting Cancer MicroRNA Biomarkers

MicroRNA (miRNA) plays vital roles in biological processes like RNA splicing and regulation of gene expression. Studies have revealed that there might be possible links between oncogenesis and expression profiles of some miRNAs, due to their differential expression between normal and tumor tissues. However, the automatic classification of miRNAs into different categories by considering the similarity of their expression values has rarely been addressed. This article proposes a solution framework for solving some real-life classification problems related to cancer, miRNA, and mRNA expression datasets. In the first stage, a multiobjective optimization based framework, non-dominated sorting genetic algorithm II, is proposed to automatically determine the appropriate classifier type, along with its suitable parameter and feature combinations, pertinent for classifying a given dataset. In the second page, a stack-based ensemble technique is employed to get a single combinatorial solution from the set of solutions obtained in the first stage. The performance of the proposed two-stage approach is evaluated on several cancer and RNA expression profile datasets. Compared to several state-of-the-art approaches for classifying different datasets, our method shows supremacy in the accuracy of classification.

Identification of key miRNA-gene pairs in chronic lymphocytic leukemia through integrated analysis of mRNA and miRNA microarray.

The aim of the present study was to explore the miRNA-Gene regulatory mechanism in chronic lymphocytic leukemia (CLL), and identify new targets for the therapy of CLL. The miRNA expression dataset GSE62137 and mRNA expression dataset GSE22529 were downloaded from National Center of Biotechnology Information Gene Expression Omnibus database. In CLL samples compared with normal B cell samples, differentially expressed miRNAs (DEMs) were identified via the GEO2R instrument of GEO and differentially expressed genes (DEGs) were obtained via the limma package of R. Functional enrichment analysis of the DEGs was performed via the Database for Annotation, Visualization and Integrated Discovery. The targets of the DEMs were identified based on the miRNAWalk platform. The overlaps between the DEGs and the targets of the DEMs were selected, and the miRNA-Gene regulatory network was constructed based on the overlaps and the corresponding DEMs. A total of 63 DEMs and 504 DEGs were identified in CLL samples compared with normal B cell samples. Eleven enriched functional clusters of the DEGs were obtained. 405 miRNA-Gene regulatory pairs were identified. The miRNA-Gene regulatory pairs contained 351 target genes of the DEMs, including 9 overlaps with the DEGs. A miRNA-Gene regulatory network was constructed. Bioinformatics methods could help us develop a better understanding of the molecular mechanism of CLL. MiRNAs may play a critical role in regulating the process of CLL. They may affect CLL by regulating the processes of immunoreactivity and protein degradation. Genes such as Neurogenic Locus Notch Homolog Protein 2, PR/SET domain 4 and A-kinase anchoring protein 12 may be their regulating targets in CLL.

Abstract 1235: EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. We have previously reported that mediators of inflammation, such as COX-2, promote the growth of Triple-Negative receptor (TN) IBC xenografts; therefore, inflammation in TN-IBC has a unique opportunity as a therapeutic strategy. Eicosapentaenoic acid (EPA), a non-toxic omega-3 fatty acid with anti-inflammatory properties, has partially reduced tumor growth in pre-clinical models of TN-IBC. Therefore, our goal is to develop a novel non-toxic approach that enhances EPA efficacy against TN-IBC in combination with targeted therapy. Methods and Results: Using a high-throughput, siRNA screen (939 genes) in the TN-IBC cell line SUM149PT, we identified Ephrin type-A receptor 2 (EPHA2), an oncogenic cell-surface receptor tyrosine kinase, as a target that modulates the sensitivity of TN-IBC cells to EPA treatment. To determine the clinical relevance of EPHA2, we interrogated a meta-analysis of breast cancer mRNA expression data sets, and found that high EPHA2 tumor expression was significantly correlated with poor overall survival in TN-IBC patients, compared to low EPHA2 expressing tumors (P = 0.01). We observed no significant correlations to other breast cancer subtypes. Similar findings were observed in vitro were EPHA2 expression predominantly occurred in the TN-IBC subtypes (19 of 30) among 49 breast cancer cell lines. Gain/loss-of-expression studies were performed to functionally validate EPHA2 as a synergistic combinational target with EPA in two EPHA2-expressing TN-IBC models, SUM149PT and BCX010, using proliferation and apoptosis assays in vitro and established tumor xenografts in vivo. EPHA2 gene silencing significantly reduced cell growth and induced apoptosis in combination with EPA when compared with untreated control and monotherapy in vitro (P &amp;lt; 0.05) and in vivo (P &amp;lt; 0.001), while vector-induced EPHA2 expression reversed cell growth reduction and apoptosis induction following combination treatment with EPA in vitro (P &amp;lt; 0.05). To translate our findings to the clinic, we validated that dasatinib, a small molecule inhibitor of EPHA2, in combination with EPA significantly enhanced cell death of SUM149PT and BCX010 cells in vitro when compared to non-treated and monotherapy (P &amp;lt; 0.05). Finally, using membrane fluidity assessment and reverse-phase protein array (300 antibodies), we determined that combination treatment efficacy depended on EPA/EPHA2 inhibition-mediated increase in cell membrane rigidity (P &amp;lt; 0.001, compared to monotherapy), which subsequently inhibited receptor tyrosine kinase signaling activity, potentially resulting in induction of apoptosis. Conclusions: Our preclinical findings provide a rationale for the development of a phase 1 clinical trial investigating combination EPA and EPHA2-inhibitors in patients with EPHA2-positive TN-IBC. Citation Format: Angie M. Torres-Adorno, Heidi Vitrac, Yuan Qi, Yiwen Yang, Peiying Yang, Bedrich L. Eckhardt, Naoto T. Ueno. EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2017-1235