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1
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Chen C, Yang L, Rivandi M, Franken A, Fehm T, Neubauer H. Bioinformatic Identification of a Breast-Specific Transcript Profile. Proteomics Clin Appl 2020; 14:e2000007. [PMID: 32558282 DOI: 10.1002/prca.202000007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2020] [Revised: 04/23/2020] [Indexed: 11/10/2022]
Abstract
PURPOSE To identify a breast-specific transcript profile for the first time, and present an updated bioinformatics strategy for searching tissue-specific transcripts and predicting their significance in cancer. EXPERIMENTAL DESIGN The RNA-seq data of 49 311 transcripts in 88 human tissues from the GTEx, the Illumina Body Map, and the RIKEN FANTOM5 project are integrated to screen breast-specific transcripts. Gene Expression Profiling Interactive Analysis, TGCA, and Kaplan-Meier Plotter are used to examine their expression in cancer tissues and values for prognosis prediction. RESULTS Only 96 transcripts in human genome are breast-specific for women. Among them, ankyrin repeat domain 30A (ANKRD30A) and long intergenic non-protein coding RNA 993 (LINC00993) are further analyzed. The two transcripts are also breast-specific in 33 types of common female cancer and are often dysregulated in breast cancer tissues. Their expression is higher in the luminal breast cancer while significantly downregulated in triple-negative breast cancer. Moreover, the high expression levels of ANKRD30A and LINC0993 in breast cancer tissues indicate a better prognosis of patients with breast cancer. CONCLUSIONS AND CLINICAL RELEVANCE Breast-specific transcripts in human genome are rare and poorly understood currently. The data indicate that these breast-specific biomarkers are promising candidates for screening early cancer, assessing treatment response, monitoring recurrence, identifying metastatic tumor origin, and serving as potential targets for immunotherapy.
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Affiliation(s)
- Chen Chen
- Department of Obstetrics and Gynecology, Heinrich Heine University of Duesseldorf, Duesseldorf, 40225, Germany.,Department of Breast and Thyroid Surgery, The Third Affiliated Hospital of Zunyi Medical University (The First People's Hospital of Zunyi), Zunyi, 563000, China
| | - Liwen Yang
- Department of Obstetrics and Gynecology, Heinrich Heine University of Duesseldorf, Duesseldorf, 40225, Germany
| | - Mahdi Rivandi
- Department of Obstetrics and Gynecology, Heinrich Heine University of Duesseldorf, Duesseldorf, 40225, Germany
| | - André Franken
- Department of Obstetrics and Gynecology, Heinrich Heine University of Duesseldorf, Duesseldorf, 40225, Germany
| | - Tanja Fehm
- Department of Obstetrics and Gynecology, Heinrich Heine University of Duesseldorf, Duesseldorf, 40225, Germany
| | - Hans Neubauer
- Department of Obstetrics and Gynecology, Heinrich Heine University of Duesseldorf, Duesseldorf, 40225, Germany
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2
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Bhattacharjee A, Vishwakarma GK. Time-course data prediction for repeatedly measured gene expression. INT J BIOMATH 2019. [DOI: 10.1142/s1793524519500335] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Variability in time course gene expression data is a natural phenomenon. The intention of this work is to predict the future time point data through observed sample data point. The Bayesian inference is carried to serve the objective. A total of 6 replicates 3 time point’s data of 218 genes expression is adopted to illustrate the method. The estimates are found consistent with HPD interval to predict the future time point gene expression value. This proposed method can be adopted in other gene expression data setup to predict the future time course data.
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Affiliation(s)
- Atanu Bhattacharjee
- Section of Biostatistics, Centre for Cancer Epidemiology, Tata Memorial Centre, Navi Mumbai 410210, India
| | - Gajendra K. Vishwakarma
- Department of Applied Mathematics, Indian Institute of Technology (ISM), Dhanbad-826004, India
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3
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Rangel N, Villegas VE, Rondón-Lagos M. Profiling of gene expression regulated by 17β-estradiol and tamoxifen in estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines. BREAST CANCER-TARGETS AND THERAPY 2017; 9:537-550. [PMID: 29033607 PMCID: PMC5614746 DOI: 10.2147/bctt.s146247] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
One area of great importance in breast cancer (BC) research is the study of gene expression regulated by both estrogenic and antiestrogenic agents. Although many studies have been performed in this area, most of them have only addressed the effects of 17β-estradiol (E2) and tamoxifen (TAM) on MCF7 cells. This study aimed to determine the effect of low doses of E2 and TAM on the expression levels of 84 key genes, which are commonly involved in breast carcinogenesis, in four BC cell lines differentially expressing estrogen receptor (ER) α and HER2 (MCF7, T47D, BT474, and SKBR3). The results allowed us to determine the expression patterns modulated by E2 and TAM in ERα+ and ERα− cell lines, as well as to identify differences in expression patterns. Although the MCF7 cell line is the most frequently used model to determine gene expression profiles in response to E2 and TAM, the changes in gene expression patterns identified in ERα+ and ERα− cell lines could reflect distinctive properties of these cells. Our results could provide important markers to be validated in BC patient samples, and subsequently used for predicting the outcome in ERα+ and ERα− tumors after TAM or hormonal therapy. Considering that BC is a molecularly heterogeneous disease, it is important to understand how well, and which cell lines, best model that diversity.
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Affiliation(s)
- Nelson Rangel
- Department of Medical Sciences, University of Turin, Turin, Italy.,Faculty of Natural Sciences and Mathematics, Universidad del Rosario, Bogotá, Colombia
| | - Victoria E Villegas
- Faculty of Natural Sciences and Mathematics, Universidad del Rosario, Bogotá, Colombia
| | - Milena Rondón-Lagos
- School of Biological Sciences, Universidad Pedagógica y Tecnológica de Colombia, Tunja, Colombia
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Ran D, Daye ZJ. Gene expression variability and the analysis of large-scale RNA-seq studies with the MDSeq. Nucleic Acids Res 2017; 45:e127. [PMID: 28535263 PMCID: PMC5737414 DOI: 10.1093/nar/gkx456] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2016] [Revised: 04/10/2017] [Accepted: 05/19/2017] [Indexed: 12/21/2022] Open
Abstract
Rapidly decreasing cost of next-generation sequencing has led to the recent availability of large-scale RNA-seq data, that empowers the analysis of gene expression variability, in addition to gene expression means. In this paper, we present the MDSeq, based on the coefficient of dispersion, to provide robust and computationally efficient analysis of both gene expression means and variability on RNA-seq counts. The MDSeq utilizes a novel reparametrization of the negative binomial to provide flexible generalized linear models (GLMs) on both the mean and dispersion. We address challenges of analyzing large-scale RNA-seq data via several new developments to provide a comprehensive toolset that models technical excess zeros, identifies outliers efficiently, and evaluates differential expressions at biologically interesting levels. We evaluated performances of the MDSeq using simulated data when the ground truths are known. Results suggest that the MDSeq often outperforms current methods for the analysis of gene expression mean and variability. Moreover, the MDSeq is applied in two real RNA-seq studies, in which we identified functionally relevant genes and gene pathways. Specifically, the analysis of gene expression variability with the MDSeq on the GTEx human brain tissue data has identified pathways associated with common neurodegenerative disorders when gene expression means were conserved.
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Affiliation(s)
- Di Ran
- Mel and Enid Zuckerman College of Public Health, The University of Arizona, Tucson, AZ 85724, USA
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5
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Kosaloglu Z, Bitzer J, Halama N, Huang Z, Zapatka M, Schneeweiss A, Jäger D, Zörnig I. In silico SNP analysis of the breast cancer antigen NY-BR-1. BMC Cancer 2016; 16:901. [PMID: 27863482 PMCID: PMC5116164 DOI: 10.1186/s12885-016-2924-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2016] [Accepted: 11/03/2016] [Indexed: 11/10/2022] Open
Abstract
Background Breast cancer is one of the most common malignancies with increasing incidences every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease. The novel breast cancer associated antigen NY-BR-1 was identified by SEREX analysis and is expressed in the majority (>70%) of breast tumors as well as metastases, in normal breast tissue, in testis and occasionally in prostate tissue. The biological function and regulation of NY-BR-1 is up to date unknown. Methods We performed an in silico analysis on the genetic variations of the NY-BR-1 gene using data available in public SNP databases and the tools SIFT, Polyphen and Provean to find possible functional SNPs. Additionally, we considered the allele frequency of the found damaging SNPs and also analyzed data from an in-house sequencing project of 55 breast cancer samples for recurring SNPs, recorded in dbSNP. Results Over 2800 SNPs are recorded in the dbSNP and NHLBI ESP databases for the NY-BR-1 gene. Of these, 65 (2.07%) are synonymous SNPs, 191 (6.09%) are non-synoymous SNPs, and 2430 (77.48%) are noncoding intronic SNPs. As a result, 69 non-synoymous SNPs were predicted to be damaging by at least two, and 16 SNPs were predicted as damaging by all three of the used tools. The SNPs rs200639888, rs367841401 and rs377750885 were categorized as highly damaging by all three tools. Eight damaging SNPs are located in the ankyrin repeat domain (ANK), a domain known for its frequent involvement in protein-protein interactions. No distinctive features could be observed in the allele frequency of the analyzed SNPs. Conclusion Considering these results we expect to gain more insights into the variations of the NY-BR-1 gene and their possible impact on giving rise to splice variants and therefore influence the function of NY-BR-1 in healthy tissue as well as in breast cancer.
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Affiliation(s)
- Zeynep Kosaloglu
- Clinical Cooperation Unit "Applied Tumor Immunity", National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.,, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany
| | - Julia Bitzer
- Department of Medical Oncology, National Center for Tumor Diseases (NCT) and University Hospital Heidelberg, Heidelberg, Germany.,, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany
| | - Niels Halama
- Department of Medical Oncology, National Center for Tumor Diseases (NCT) and University Hospital Heidelberg, Heidelberg, Germany.,, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany
| | - Zhiqin Huang
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany.,, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany
| | - Marc Zapatka
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany.,, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany
| | - Andreas Schneeweiss
- Department of Obstetrics and Gynecology, National Center for Tumor Diseases (NCT) and University Hospital Heidelberg, Heidelberg, Germany.,, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany
| | - Dirk Jäger
- Clinical Cooperation Unit "Applied Tumor Immunity", National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.,Department of Medical Oncology, National Center for Tumor Diseases (NCT) and University Hospital Heidelberg, Heidelberg, Germany.,, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany
| | - Inka Zörnig
- Department of Medical Oncology, National Center for Tumor Diseases (NCT) and University Hospital Heidelberg, Heidelberg, Germany. .,, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
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6
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Chen C, Li Z, Yang Y, Xiang T, Song W, Liu S. Microarray expression profiling of dysregulated long non-coding RNAs in triple-negative breast cancer. Cancer Biol Ther 2016; 16:856-65. [PMID: 25996380 DOI: 10.1080/15384047.2015.1040957] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Triple-negative breast cancer (TNBC) represents a collection of malignant breast tumors that are often aggressive and have an increased risk of metastasis and relapse. Long non-coding RNAs are generally defined as RNA transcripts measuring 200 nucleotides or longer that do not encode for any protein. During the past decade, increasing evidence has shown that lncRNAs play important roles in oncogenesis and tumor suppression; however, the roles of lncRNAs in TNBC are poorly understood. To address this issue, we used Agilent human lncRNA microarray chips and bioinformatics tools, including Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), to assess lncRNA expression in 3 pairs of TNBC tissues. A dysregulated lncRNA expression profile was identified by microarray and verified by qRT-PCR in 48 pairs of breast cancer subtype tissues. Metastasis is the major cause of cancer-related deaths, including those in TNBC, and the presence of dormant residual disseminated tumor cells (DTC) may be a key factor leading to metastasis. ANKRD30A, a potential target for breast cancer immunotherapy, is currently one of the most used DTC markers. Notably, we found the expression levels of the novel intergenic lncRNA LINC00993 to be associated with the expression levels of ANKRD30A. Furthermore, our qRT-PCR data indicated that the expression of LINC00993 was also associated with the expression of the estrogen receptor. In conclusion, our study identified a set of lncRNAs that were consistently aberrantly expressed in TNBC, and these dysregulated lncRNAs may be involved in the development and/or progression of TNBC.
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Affiliation(s)
- Chen Chen
- a Department of Surgery ; The First Affiliated Hospital of Chongqing Medical University ; Chongqing , China
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ANDERGASSEN ULRICH, KÖLBL ALEXANDRAC, MAHNER SVEN, JESCHKE UDO. Real-time RT-PCR systems for CTC detection from blood samples of breast cancer and gynaecological tumour patients (Review). Oncol Rep 2016; 35:1905-15. [DOI: 10.3892/or.2016.4608] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Accepted: 11/15/2015] [Indexed: 11/06/2022] Open
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Farahmand S, Goliaei S, Ansari-Pour N, Razaghi-Moghadam Z. GTA: a game theoretic approach to identifying cancer subnetwork markers. MOLECULAR BIOSYSTEMS 2016; 12:818-25. [DOI: 10.1039/c5mb00684h] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
The identification of genetic markers (e.g. genes, pathways and subnetworks) for cancer has been one of the most challenging research areas in recent years.
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Affiliation(s)
- S. Farahmand
- Research Laboratory of Computational Biology
- Faculty of New Sciences and Technology
- University of Tehran
- Tehran
- Iran
| | - S. Goliaei
- Research Laboratory of Computational Biology
- Faculty of New Sciences and Technology
- University of Tehran
- Tehran
- Iran
| | - N. Ansari-Pour
- Faculty of New Sciences and Technology
- University of Tehran
- Tehran
- Iran
- School of Biological Sciences
| | - Z. Razaghi-Moghadam
- Faculty of New Sciences and Technology
- University of Tehran
- Tehran
- Iran
- School of Biological Sciences
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9
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Hsu JY, Chang KY, Chen SH, Lee CT, Chang ST, Cheng HC, Chang WC, Chen BK. Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation. Oncotarget 2015; 6:1723-39. [PMID: 25595899 PMCID: PMC4359327 DOI: 10.18632/oncotarget.2783] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2014] [Accepted: 11/19/2014] [Indexed: 12/31/2022] Open
Abstract
Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed by COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E2 (PGE2). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF enhancement of cell migration and invasion, but the addition of PGE2 compensated for this inhibitory effect in COX-2-knockdown cells. COX-2 depletion inhibited EGF-induced matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. The inhibitory effect of COX-2 depletion on MMPs and the fibronectin/Rac1/cdc42 axis were reversed by co-treatment with PGE2. Furthermore, depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results demonstrate that EGF-induced COX-2 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.
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Affiliation(s)
- Jinn-Yuan Hsu
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, ROC
| | - Kwang-Yu Chang
- National Institute of Cancer Research, National Health Research Institutes and Division of Hematology/Oncology, Department of Internal Medicine, National Cheng Kung University, Tainan 701, Taiwan, ROC
| | - Shang-Hung Chen
- Division of Hematology and Oncology, Department of Internal Medicine, Chi-Mei Medical Center, Liouying, Tainan 736, Taiwan, ROC
| | - Chung-Ta Lee
- Department of Pathology, National Cheng Kung University Hospital, Tainan 701, Taiwan, ROC
| | - Sheng-Tsung Chang
- Department of Pathology, Chi-Mei Medical Center, Tainan 710, Taiwan, ROC
| | - Hung-Chi Cheng
- Institute of Biochemistry, National Cheng Kung University, Tainan 701, Taiwan, ROC
| | - Wen-Chang Chang
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, ROC
| | - Ben-Kuen Chen
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, ROC.,Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, ROC.,Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan, ROC
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Differential expression of secretoglobins in normal ovary and in ovarian carcinoma – Overexpression of mammaglobin-1 is linked to tumor progression. Arch Biochem Biophys 2014; 547:27-36. [DOI: 10.1016/j.abb.2014.02.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2013] [Revised: 02/05/2014] [Accepted: 02/21/2014] [Indexed: 11/18/2022]
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Huan J, Wang L, Xing L, Qin X, Feng L, Pan X, Zhu L. Insights into significant pathways and gene interaction networks underlying breast cancer cell line MCF-7 treated with 17β-estradiol (E2). Gene 2013; 533:346-55. [PMID: 23978611 DOI: 10.1016/j.gene.2013.08.027] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2013] [Revised: 07/25/2013] [Accepted: 08/10/2013] [Indexed: 12/31/2022]
Abstract
OBJECTIVE Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. METHODS We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with KEGG pathway enrichment, PPI network construction, module analysis and text mining methods to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. RESULTS Of the genes queried on the Affymetrix Human Genome U133 plus 2.0 microarray, we identified 628 (12h), 852 (24h) and 880 (48 h) differentially expressed genes (DEGs) that showed a robust pattern of regulation by E2. From pathway enrichment analysis, we found out the changes of metabolic pathways of E2 treated samples at each time point. At 12h time point, the changes of metabolic pathways were mainly focused on pathways in cancer, focal adhesion, and chemokine signaling pathway. At 24h time point, the changes were mainly enriched in neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction and calcium signaling pathway. At 48 h time point, the significant pathways were pathways in cancer, regulation of actin cytoskeleton, cell adhesion molecules (CAMs), axon guidance and ErbB signaling pathway. Of interest, our PPI network analysis and module analysis found that E2 treatment induced enhancement of PRSS23 at the three time points and PRSS23 was in the central position of each module. Text mining results showed that the important genes of DEGs have relationship with signal pathways, such as ERbB pathway (AREG), Wnt pathway (NDP), MAPK pathway (NTRK3, TH), IP3 pathway (TRA@) and some transcript factors (TCF4, MAF). CONCLUSIONS Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which E2 operates to achieve its widespread effects on breast cancer cells.
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Affiliation(s)
- Jinliang Huan
- Department of General Surgery, The Eighth People's Hospital of Shanghai, Shanghai 200235, China.
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13
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Zehentner BK, Secrist H, Hayes DC, Zhang X, Ostenson RC, Loop S, Goodman G, Houghton RL, Persing DH. Detection of Circulating Tumor Cells in Peripheral Blood of Breast Cancer Patients During or After Therapy Using a Multigene Real-Time RT-PCR Assay. Mol Diagn Ther 2012; 10:41-7. [PMID: 16646576 DOI: 10.1007/bf03256441] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
AIM To evaluate the utility of a multigene real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay to detect circulating tumor cells in peripheral blood specimens of breast cancer patients during or after treatment. METHOD Using this assay, peripheral blood samples were analyzed for expression levels of mammaglobin and three complementary transcribed breast cancer-specific genes: B305D, gamma-aminobutyrate type A receptor pi subunit (GABA pi; GABRP), and B726P. We examined 172 blood specimens from 82 breast cancer patients during or after therapy for the presence of circulating tumor cells using the multigene real-time RT-PCR assay. RESULTS In 63.4% of the blood samples, a positive signal for mammaglobin and/or three breast cancer-associated gene transcripts was detected. Of breast cancer patients, 75.6% had at least one positive blood sample. Blood specimens from 51 of 53 healthy female volunteers tested negative in the assay whereas two samples had a low expression signal. In addition, three patients were monitored for more than a year during their adjuvant therapy treatment. CONCLUSION This assay could be a valuable tool for monitoring breast cancer patients during and after therapy.
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Okawa T, Kurio Y, Morimoto M, Hayashi T, Nakagawa T, Sasaki N, Okuda M, Mizuno T. Calreticulin expression in neoplastic versus normal dog mammary glands: A cDNA subtraction-based study. Res Vet Sci 2012; 92:80-91. [DOI: 10.1016/j.rvsc.2010.10.028] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2010] [Revised: 10/23/2010] [Accepted: 10/28/2010] [Indexed: 11/25/2022]
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Kim Y, Park T. DNA microarrays to define and search for genes associated with obesity. Biotechnol J 2010; 5:99-112. [PMID: 20024972 DOI: 10.1002/biot.200900228] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
One of the major goals of this review was to identify obesity-specific gene profiles in animal models to help comprehend the pathogenic mechanisms and the prediction of the phenotypic outcomes of obesity and its associated metabolic diseases. The genomic examination of insulin-sensitive tissues, such as the adipose and hepatic tissues, has provided a wealth of information about the changes in gene expression in obesity and its associated metabolic diseases. The overexpression of genes related to inflammation, immune response, adhesion molecules, and lipid metabolism is a major characteristic of white adipose tissue, while the overexpression of the genes related to lipid metabolism, adipocyte differentiation, defense, and stress responses is noticeable in the non-alcoholic fatty liver of obese rodents. The hepatic-gene expression profiles led us to hypothesize that in obese rodents, the livers are supplied with large amounts of free fatty acids under conditions associated with obesity either through increased fatty acid biosynthesis or through decreased fatty acid oxidation, which may lead to increased mitochondrial respiratory activity. The wide list of genes that were identified in previous studies could be a source of potential therapeutic targets because most of these genes are involved in the key mechanisms of obesity development, from adipocyte differentiation to the disturbance of metabolism.
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Affiliation(s)
- Yunjung Kim
- Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University, Seoul, Korea
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Wu N, Matand K, Williams S. A novel mRNA level subtraction method for quick identification of target-orientated uniquely expressed genes between peanut immature pod and leaf. Biol Proced Online 2009; 12:44-55. [PMID: 21406066 PMCID: PMC3055529 DOI: 10.1007/s12575-009-9022-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2009] [Accepted: 11/16/2009] [Indexed: 12/16/2022] Open
Abstract
Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.
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Affiliation(s)
- Ning Wu
- Center for Biotechnology Research and Education, Langston University, P.O. Box 1730, Langston, OK 73050, USA
| | - Kanyand Matand
- Center for Biotechnology Research and Education, Langston University, P.O. Box 1730, Langston, OK 73050, USA
| | - Sonya Williams
- Center for Biotechnology Research and Education, Langston University, P.O. Box 1730, Langston, OK 73050, USA
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17
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GABA receptor expression in benign and malignant thyroid tumors. Pathol Oncol Res 2009; 15:645-50. [PMID: 19381877 DOI: 10.1007/s12253-009-9165-x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2008] [Accepted: 03/24/2009] [Indexed: 12/29/2022]
Abstract
Neurotransmitter systems have recently been shown to be involved in multiple malignancies including breast, colon and prostate cancers. The role of neurotransmitters and neurotrophic factors has not yet been examined in thyroid cancer. To determine the possible involvement of neurotransmitter systems in thyroid carcinogenesis we characterized the patterns of gamma-aminobutyric acid (GABA) receptor expression in normal thyroid and thyroid tumors. We examined the expression patterns of the GABAergic system in 70 human thyroid tumor samples (13 follicular adenomas, 14 follicular carcinomas, 43 papillary carcinomas) and adjacent normal thyroid by immunohistochemistry. GABAergic system mRNA expression in thyroid cancer cell lines derived from primary (FTC133) and metastatic tumors (FTC236 and FTC238) was examined by real time PCR. Overall, GABA receptor expression is increased in tumors compared to normal thyroid tissue. Expression of GABAA receptor beta2 was detected in the vasculature of normal thyroid and thyroid tumors but not in thyroid cancer cells. GABAA alpha2 was detected in metastatic-derived but not in primary-tumor derived cell lines. Expression levels of GABAB R2 and GABA receptor associated protein (GABARAP) are increased in adenomas and thyroid cancer suggesting their role in early stages of thyroid tumorigenesis. This study represents the first demonstration of GABA receptor expression in human thyroid tissue and suggests that the GABAergic system is involved in thyroid carcinogenesis.
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Maemura K, Shiraishi N, Sakagami K, Kawakami K, Inoue T, Murano M, Watanabe M, Otsuki Y. Proliferative effects of gamma-aminobutyric acid on the gastric cancer cell line are associated with extracellular signal-regulated kinase 1/2 activation. J Gastroenterol Hepatol 2009; 24:688-96. [PMID: 19032445 DOI: 10.1111/j.1440-1746.2008.05687.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND AND AIM Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult mammalian brain. However, GABA is found not only in peripheral neuronal tissue, but also in many peripheral non-neuronal tissues, and is thought to have important physiological functions in addition to neurotransmission. We previously reported that GABA participates in chondrocyte proliferation. In the present study, we investigated the effects of GABA on the proliferation of a gastric cancer cell line, KATO III. METHODS Reverse transcription polymerase chain reaction and immunohistochemical analyses were performed to examine the expression of the GABA synthesis enzyme, glutamate decarboxylase (GAD), and that of the GABA(A) and GABA(B) receptor subunits. The production of GABA was confirmed by immunohistochemistry. The proliferative effect of GABA on KATO III cells was analyzed by bromodeoxyuridine incorporation assay, and the activation status of mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase [ERK]-1/2, Jun-N-terminal kinase, and p38) and the expression of cyclin D1 were analyzed by western blotting. RESULTS KATO III cells expressed GAD and GABA. More than five GABA(A) receptor subunits, including the pi subunit, were expressed in KATO III cells; however, GABA(B) receptor subunits were not seen. The addition of GABA to the medium promoted KATO III proliferation, and maximum proliferative effects were observed in the presence of 10 or 1 microM GABA. The addition of 1 microM GABA predominantly activated ERK-1/2 among the three MAP kinases in addition to increasing cyclin D1 expression. CONCLUSION GABA is able to promote KATO III cell proliferation in an autocrine or a paracrine fashion through GABA(A) receptors followed by MAP kinase activation.
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Affiliation(s)
- Kentaro Maemura
- Department of Anatomy and Cell Biology, Division of Basic Medicine I, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan.
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19
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Lancashire LJ, Rees RC, Ball GR. Identification of gene transcript signatures predictive for estrogen receptor and lymph node status using a stepwise forward selection artificial neural network modelling approach. Artif Intell Med 2008; 43:99-111. [PMID: 18420392 DOI: 10.1016/j.artmed.2008.03.001] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2007] [Revised: 02/29/2008] [Accepted: 03/10/2008] [Indexed: 10/22/2022]
Abstract
OBJECTIVE The advent of microarrays has attracted considerable interest from biologists due to the potential for high throughput analysis of hundreds of thousands of gene transcripts. Subsequent analysis of the data may identify specific features which correspond to characteristics of interest within the population, for example, analysis of gene expression profiles in cancer patients to identify molecular signatures corresponding with prognostic outcome. These high throughput technologies have resulted in an unprecedented rate of data generation, often of high complexity, highlighting the need for novel data analysis methodologies that will cope with data of this nature. METHODS Stepwise methods using artificial neural networks (ANNs) have been developed to identify an optimal subset of predictive gene transcripts from highly dimensional microarray data. Here these methods have been applied to a gene microarray dataset to identify and validate gene signatures corresponding with estrogen receptor and lymph node status in breast cancer. RESULTS Many gene transcripts were identified whose expression could differentiate patients to very high accuracies based upon firstly whether they were positive or negative for estrogen receptor, and secondly whether metastasis to the axillary lymph node had occurred. A number of these genes had been previously reported to have a role in cancer. Significantly fewer genes were used compared to other previous studies. The models using the optimal gene subsets were internally validated using an extensive random sample cross-validation procedure and externally validated using a follow up dataset from a different cohort of patients on a newer array chip containing the same and additional probe sets. Here, the models retained high accuracies, emphasising the potential power of this approach in analysing complex systems. These findings show how the proposed method allows for the rapid analysis and subsequent detailed interrogation of gene expression signatures to provide a further understanding of the underlying molecular mechanisms that could be important in determining novel prognostic markers associated with cancer.
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Affiliation(s)
- Lee J Lancashire
- Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.
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20
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O'Brien N, O'Donovan N, Foley D, Hill ADK, McDermott E, O'Higgins N, Duffy MJ. Use of a panel of novel genes for differentiating breast cancer from non-breast tissues. Tumour Biol 2008; 28:312-7. [PMID: 18253069 DOI: 10.1159/000115527] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2007] [Accepted: 09/23/2007] [Indexed: 12/29/2022] Open
Abstract
Existing serum markers for breast cancer such as CA 15-3, BR 27.29 and CEA lack sensitivity and specificity. The aim of this study was to evaluate the value of new putative breast-specific markers for differentiating breast cancer from non-breast tissues. Expression of mammaglobin A (MGA), B726P, small breast epithelial mucin (SBEM) and MUC1 was measured by RT-PCR. MGA mRNA was detected in 86/162 (60%) breast cancers but in only 1/32 (3%) non-breast tissues; B726P was detected in 44/108 (41%) breast cancers but in none of 20 non-breast tissues, while SBEM was present in 52/103 (51%) breast cancers but in only 1/26 non-breast cancer tissues. In contrast to these novel markers, the established breast cancer marker MUC1 was detected in 72/99 (73%) breast cancers and in 22/32 (59%) of non-breast tissues. Combining MGA with B726P separated breast cancer from non-breast tissue with a sensitivity of 71% and a specificity of 95% while combining MGA with SBEM differentiated breast cancer from non-breast tissues with a sensitivity of 76% and a specificity of 89%. Genes such as MGA, B726P and SBEM that are expressed relatively exclusively in breast tissue are potential new markers for breast cancer.
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Affiliation(s)
- Neil O'Brien
- Department of Pathology and Laboratory Medicine, St. Vincent's University Hospital, Dublin, Ireland
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21
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Huang L, Cheng HC, Isom R, Chen CS, Levine RA, Pauli BU. Protein kinase Cepsilon mediates polymeric fibronectin assembly on the surface of blood-borne rat breast cancer cells to promote pulmonary metastasis. J Biol Chem 2008; 283:7616-27. [PMID: 18184652 DOI: 10.1074/jbc.m705839200] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose- and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase Cepsilon (PKCepsilon), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKCepsilon protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKCepsilon and dominant-negative PKCepsilon constructs (e.g. RD-PKCepsilon). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKCepsilon impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis.
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Affiliation(s)
- Lynn Huang
- Cancer Cell Biology Laboratories, Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA
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22
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Gradilone A, Gazzaniga P, Cigna E, Vasaturo F, Vincenzi B, Gandini O, Silvestri I, Ribuffo D, Scarpa S, Scuderi N, Aglianò AM. Fibronectin and laminin expression in sentinel lymph nodes of patients with malignant melanoma. Br J Dermatol 2007; 157:398-401. [PMID: 17573891 DOI: 10.1111/j.1365-2133.2007.07995.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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23
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Seil I, Frei C, Sültmann H, Knauer SK, Engels K, Jäger E, Zatloukal K, Pfreundschuh M, Knuth A, Tseng-Chen Y, Jungbluth AA, Stauber RH, Jäger D. The differentiation antigen NY-BR-1 is a potential target for antibody-based therapies in breast cancer. Int J Cancer 2007; 120:2635-42. [PMID: 17330232 DOI: 10.1002/ijc.22620] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Antibody-based cancer immunotherapy relies on the identification and characterization of target antigens and the development of potent antibodies recognizing the target. Here we report the expression analysis and molecular characterization of the differentiation antigen NY-BR-1, which we previously identified by using the SEREX (serological analysis of recombinant cDNA expression libraries) method. Corroborating methodologies, including mRNA quantitation and immunoblotting show that NY-BR-1 is strongly expressed in >70% of 129 breast tumors. Application of a NY-BR-1 specific antibody demonstrated NY-BR-1 expression in primary and metastastic breast cancers. In contrast, most of the breast cancer cell lines tested, expressed only low NY-BR-1 levels. Importantly, confocal microscopy revealed that ectopically expressed NY-BR-1 localizes to the cytoplasm and the cell membrane. NY-BR-1 localization in breast cancer specimens was also confirmed by immunohistochemistry. Bioinformatic analysis and deletion mutagenesis further show that NY-BR-1 membrane localization is mediated by 2 cis-active membrane targeting domains. Biochemical surface labeling and FACS analysis of live cells further characterize NY-BR-1 as a transmembrane protein, which can be specifically recognized by the anti-NY-BR-1 antibody on the surface of vital cells. The strong expression of NY-BR-1 in breast tumors, its cytoplasmic and membrane localization and accessibility to an ectopically applied antibody now suggest to pursue NY-BR-1 as a potential target for antibody-based therapies in breast cancer patients.
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MESH Headings
- Antibodies, Monoclonal/immunology
- Antibodies, Monoclonal/therapeutic use
- Antigens, Differentiation/chemistry
- Antigens, Differentiation/genetics
- Antigens, Differentiation/immunology
- Antigens, Neoplasm/chemistry
- Antigens, Neoplasm/genetics
- Antigens, Neoplasm/immunology
- Binding Sites
- Breast Neoplasms/drug therapy
- Breast Neoplasms/immunology
- Breast Neoplasms/pathology
- Cell Line, Tumor
- Cell Membrane/metabolism
- Cytoplasm/metabolism
- Female
- Flow Cytometry
- Gene Expression Regulation, Neoplastic
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism
- Humans
- Hydrophobic and Hydrophilic Interactions
- Immunoblotting
- Immunohistochemistry
- Membrane Proteins/chemistry
- Membrane Proteins/genetics
- Membrane Proteins/immunology
- Microscopy, Confocal
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
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Affiliation(s)
- Inka Seil
- Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt, Germany
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24
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Azare J, Leslie K, Al-Ahmadie H, Gerald W, Weinreb PH, Violette SM, Bromberg J. Constitutively activated Stat3 induces tumorigenesis and enhances cell motility of prostate epithelial cells through integrin beta 6. Mol Cell Biol 2007; 27:4444-53. [PMID: 17438134 PMCID: PMC1900039 DOI: 10.1128/mcb.02404-06] [Citation(s) in RCA: 125] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The persistent activation of signal transducer and activator of transcription 3 (Stat3) is a common feature of prostate cancer. However, little is known about the Stat3 targets that may mediate prostate tumorigenesis. The introduction of an activating mutant form of Stat3 (Stat3-C) into immortalized prostate epithelial cells resulted in tumorigenesis. Stat3-C-expressing cells had decreased E-cadherin levels, increased numbers of lamellipodia and stress fibers, and enhanced migratory capacities compared to vector control-expressing cells, with a concomitant increase in the expression of integrin beta6 and its ligand, fibronectin (FN). Exogenously added FN increased cellular migration, with a concomitant loss of E-cadherin expression. The blockade of integrin alphavbeta6 in Stat3-C-expressing cells inhibited migration, increased E-cadherin levels, and reduced colony formation in soft agar. These results demonstrate the sufficiency of constitutively activated Stat3 in mediating prostate tumorigenesis and identify novel Stat3 targets that are involved in promoting cell migration and transformation.
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Affiliation(s)
- Janeen Azare
- Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
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25
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Culleton J, O'Brien N, Ryan BM, Hill ADK, McDermott E, O'Higgins N, Duffy MJ. Lipophilin B: A gene preferentially expressed in breast tissue and upregulated in breast cancer. Int J Cancer 2007; 120:1087-92. [PMID: 17163411 DOI: 10.1002/ijc.22471] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Lipophilin B (LPB), which is also known as BU101, is a secretoglobin which exists in vivo as a complex with the mammary-specific protein, mammaglobin A (MGA). The aim of our study was to investigate the expression of LPB in a panel of breast and nonbreast tissues and compare its expression with that of MGA. Using RT-PCR, LPB mRNA was detected in 16/25 (64%) of normal breast specimens, 23/30 (77%) of fibroadenomas, 102/156 (65%) of primary breast cancers and in 8/36 (22%) nonbreast tissues. Levels of expression of LPB mRNA were significantly higher in breast cancers compared to both normal breast tissues (p = 0.02) and nonbreast tissue (p < 0.001). In the primary breast cancers, expression of LPB mRNA was positively correlated with the estrogen receptor (p = 0.045) but inversely related to both tumor grade (p < 0.001) and proliferation rates (p = 0.0345). Compared to MGA, expression of LPB was more sensitive but less specific for breast cancer. Using Western blotting, LPB migrated with an approximate molecular mass of 7-8 kDa, the expected molecular mass of free LPB. Immunohistochemical analysis of breast cancers showed that LPB expression was predominantly confined to the cytoplasm of tumor cells. We conclude that expression of LPB is preferentially but not exclusively restricted to breast tissue. Since LPB was expressed relatively specifically in breast tissue and was significantly upregulated in breast carcinomas, it is a promising candidate biomarker for breast cancer.
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MESH Headings
- Biomarkers, Tumor/analysis
- Biomarkers, Tumor/genetics
- Blotting, Western
- Breast/chemistry
- Breast/metabolism
- Breast/pathology
- Breast Neoplasms/chemistry
- Breast Neoplasms/genetics
- Breast Neoplasms/pathology
- Carcinoma/chemistry
- Carcinoma/genetics
- Carcinoma/pathology
- Female
- Gene Expression Regulation, Neoplastic
- Humans
- Immunohistochemistry
- Myelin Proteins/analysis
- Myelin Proteins/genetics
- Myelin Proteins/metabolism
- Proteolipids/analysis
- Proteolipids/genetics
- Proteolipids/metabolism
- RNA, Messenger/analysis
- RNA, Messenger/metabolism
- Receptors, Estrogen/analysis
- Receptors, Estrogen/genetics
- Receptors, Estrogen/metabolism
- Secretoglobins
- Tumor Cells, Cultured
- Up-Regulation
- Uteroglobin/analysis
- Uteroglobin/genetics
- Uteroglobin/metabolism
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Affiliation(s)
- Jane Culleton
- Department of Pathology and Laboratory Medicine, St. Vincent's University Hospital, Dublin, Ireland
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26
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Esseghir S, Reis-Filho JS, Kennedy A, James M, O'Hare MJ, Jeffery R, Poulsom R, Isacke CM. Identification of transmembrane proteins as potential prognostic markers and therapeutic targets in breast cancer by a screen for signal sequence encoding transcripts. J Pathol 2007; 210:420-30. [PMID: 17054309 DOI: 10.1002/path.2071] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
This study demonstrates, through a combination of stringent screening methods and thorough validation, that it is possible to identify transmembrane proteins preferentially expressed in primary breast tumour cells. mRNA was extracted from tumour cells isolated from invasive breast cancers and it was then subtracted against normal breast tissue mRNA prior to the generation of a signal sequence-trap library. Screening of the library identified 31 positive clones encoding 12 cell-surface and 12 secreted proteins. The expression of a subset of transmembrane genes was then interrogated using a high-throughput method (tissue microarray) coupled with cutting-edge in situ techniques in a large cohort of patients who had undergone uniform adjuvant chemotherapy. Expression of CD98 heavy chain (CD98HC) and low-level expression of the insulin-like growth factor 2 receptor/mannose-6-phosphate receptor (IGF2R/M6PR) correlated with poor patient prognosis in the whole cohort. Expression of bradykinin receptor B1 (BDKRB1) and testis enhanced gene transcript (TEGT) correlated with good prognosis in woman with oestrogen receptor (ER)-negative breast tumours. These results indicate that this combined approach of isolating primary tumour cells, generating a library to specifically isolate signal-sequence-containing transcripts, and in situ hybridization on tissue microarrays successfully identified novel prognostic markers (BDKRB1, CD98hc, and TEGT) and potential transmembrane therapeutic targets (CD98hc) in breast cancer.
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Affiliation(s)
- S Esseghir
- Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK
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27
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Dolled-Filhart M, Rydén L, Cregger M, Jirström K, Harigopal M, Camp RL, Rimm DL. Classification of Breast Cancer Using Genetic Algorithms and Tissue Microarrays. Clin Cancer Res 2006; 12:6459-68. [PMID: 17085660 DOI: 10.1158/1078-0432.ccr-06-1383] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid-based analysis to predict patient outcome. EXPERIMENTAL DESIGN We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section. RESULTS The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n=223, 5-year survival=95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n=149). CONCLUSIONS With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.
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Affiliation(s)
- Marisa Dolled-Filhart
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520-8023, USA
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28
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Gaggioli C, Robert G, Bertolotto C, Bailet O, Abbe P, Spadafora A, Bahadoran P, Ortonne JP, Baron V, Ballotti R, Tartare-Deckert S. Tumor-derived fibronectin is involved in melanoma cell invasion and regulated by V600E B-Raf signaling pathway. J Invest Dermatol 2006; 127:400-10. [PMID: 16960555 DOI: 10.1038/sj.jid.5700524] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Melanomas are malignant tumors of melanocytes that, if not detected early, are highly aggressive and poorly treatable. Activation of extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase signaling is commonly found in melanomas mainly through oncogenic mutations of B-Raf. We previously reported that activation of ERK/MAP kinase stimulates synthesis of fibronectin by upregulating the transcription factor early growth response-1 (Egr-1). To further analyze the link between ERK/MAP kinase pathway and fibronectin in melanoma, we have studied the regulation and role of fibronectin produced by melanoma cells bearing oncogenic B-Raf mutation. We show that fibronectin is expressed in situ during tumor progression and that high fibronectin and Egr-1 levels are found in cells expressing this mutation. Expression of active mutants of B-Raf induces fibronectin, whereas endogenous fibronectin is inhibited by small interfering RNA (siRNA)-mediated depletion of B-Raf or Egr-1. In contrast, stimulation of ERK pathway is insufficient to promote fibronectin upregulation in normal melanocytes. Finally, we show that suppression of fibronectin by siRNA leads to decreased melanoma cell invasiveness in vitro. These results reveal a tumor-specific regulation of fibronectin by constitutive ERK/MAP kinase signaling and indicate that self-production of fibronectin may play a role in melanoma tumorigenesis, by promoting tumor cell invasion.
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Affiliation(s)
- Cédric Gaggioli
- INSERM, U597, Biologie et Pathologies des Cellules Mélanocytaires, Faculté de Médecine, Nice Cédex 2, France
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29
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Tang SW, Chang WH, Chao YW, Lin CY, Chen HF, Lai YH, Zhan BW, Su YC, Jane SW, Chen YC, Hsu CI, Lin WC, Wang KC, Lai MK, Lin JY. Identification of differentially expressed genes in clear cell renal cell carcinoma by analysis of full-length enriched cDNA library. J Biomed Sci 2006; 13:233-40. [PMID: 16453177 DOI: 10.1007/s11373-005-9059-1] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
Renal cell carcinoma (RCC) is the most common malignancy in adult kidney, and accounts for 3% of malignancies worldwide with increasing incidence. Clear cell RCC (ccRCC) is the major type in RCC. Resection by surgery is the main treatment because the response of ccRCC to traditional therapies is very poor. To identify the tumor-associated genes for better understanding the molecular mechanism of ccRCC, the full-length enriched cDNA libraries of ccRCC and normal kidney tissues were constructed by the oligo-capping method. Nucleotide sequences of the cDNA libraries of ccRCC and normal kidney tissues were sequenced. From the sequence analysis of 19,425 and 12,400 clones of ccRCC and normal kidney tissues, 4356 and 3055 genes were identified, respectively. By comparing the gene-expression patterns of ccRCC and normal tissues, the up- or down-regulated genes were identified. Among these identified genes, the differential expression of annexin A2 and argininosuccinate synthetase genes were further confirmed by quantitative real-time PCR and Western blot analysis.
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Affiliation(s)
- Sai-Wen Tang
- Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
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30
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Wang W, Epler J, Salazar LG, Riddell SR. Recognition of Breast Cancer Cells by CD8+ Cytotoxic T-Cell Clones Specific for NY-BR-1. Cancer Res 2006; 66:6826-33. [PMID: 16818660 DOI: 10.1158/0008-5472.can-05-3529] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Immunotherapy for breast cancer using cytotoxic T cells (CTL) is hindered by the lack of well-characterized breast cancer antigens that are expressed in most breast tumor cells and recognized by CD8+ CTL. A recently described breast tissue differentiation antigen, NY-BR-1, is expressed in >80% breast tumors and elicits a humoral response in a subset of breast cancer patients. To identify potential NY-BR-1 epitopes that are recognized by CTL, CD8+ T cells were stimulated in vitro with autologous dendritic cells pulsed with NY-BR-1 peptides that were predicted to bind to HLA-A2. In multiple normal female donors and breast cancer patients, specific CD8+ CTL responses were detected by enzyme-linked immunospot assay against several NY-BR-1 peptides after two cycles of stimulation. CD8+ CTL clones against three NY-BR-1 epitopes were isolated and recognized peptide-pulsed target cells with high avidity. T-cell clones specific for one of the NY-BR-1 epitopes (p904) also recognized breast tumor cells expressing NY-BR-1, NY-BR-1(-) cells transfected with a cDNA encoding the NY-BR-1 protein, and autologous dendritic cells pulsed with opsonized NY-BR-1+ breast tumor cells. Taken together, these results show that the p904 epitope derived from NY-BR-1 is efficiently processed and presented endogenously and identify NY-BR-1 as a promising target for T-cell-based immunotherapy for breast cancer.
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Affiliation(s)
- Wei Wang
- Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
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31
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Aouacheria A, Navratil V, Barthelaix A, Mouchiroud D, Gautier C. Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues. BMC Genomics 2006; 7:94. [PMID: 16640784 PMCID: PMC1459866 DOI: 10.1186/1471-2164-7-94] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2005] [Accepted: 04/26/2006] [Indexed: 11/24/2022] Open
Abstract
Background Owing to the explosion of information generated by human genomics, analysis of publicly available databases can help identify potential candidate genes relevant to the cancerous phenotype. The aim of this study was to scan for such genes by whole-genome in silico subtraction using Expressed Sequence Tag (EST) data. Methods Genes differentially expressed in normal versus tumor tissues were identified using a computer-based differential display strategy. Bcl-xL, an anti-apoptotic member of the Bcl-2 family, was selected for confirmation by western blot analysis. Results Our genome-wide expression analysis identified a set of genes whose differential expression may be attributed to the genetic alterations associated with tumor formation and malignant growth. We propose complete lists of genes that may serve as targets for projects seeking novel candidates for cancer diagnosis and therapy. Our validation result showed increased protein levels of Bcl-xL in two different liver cancer specimens compared to normal liver. Notably, our EST-based data mining procedure indicated that most of the changes in gene expression observed in cancer cells corresponded to gene inactivation patterns. Chromosomes and chromosomal regions most frequently associated with aberrant expression changes in cancer libraries were also determined. Conclusion Through the description of several candidates (including genes encoding extracellular matrix and ribosomal components, cytoskeletal proteins, apoptotic regulators, and novel tissue-specific biomarkers), our study illustrates the utility of in silico transcriptomics to identify tumor cell signatures, tumor-related genes and chromosomal regions frequently associated with aberrant expression in cancer.
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Affiliation(s)
- Abdel Aouacheria
- Laboratoire de Biométrie et Biologie Evolutive, CNRS UMR 5558, Université Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France
- Current address: Apoptosis and Oncogenesis Laboratory, IBCP, UMR 5086 CNRS-UCBL, IFR 128, Lyon, France
| | - Vincent Navratil
- Laboratoire de Biométrie et Biologie Evolutive, CNRS UMR 5558, Université Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France
| | | | - Dominique Mouchiroud
- Laboratoire de Biométrie et Biologie Evolutive, CNRS UMR 5558, Université Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France
| | - Christian Gautier
- Laboratoire de Biométrie et Biologie Evolutive, CNRS UMR 5558, Université Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France
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Boudreau CR, Yang I, Liau LM. Gliomas: advances in molecular analysis and characterization. ACTA ACUST UNITED AC 2005; 64:286-94; discussion 294. [PMID: 16229087 DOI: 10.1016/j.surneu.2005.03.033] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
BACKGROUND Gliomas represent the most common primary brain tumor. Despite recent advances in diagnostic imaging, neurosurgical technique, radiation therapy, and chemotherapy, significant advances in accurate prognosis and improved survival have not been achieved. Nevertheless, new developments in molecular biology could have potential impact on the clinical management of patients with these brain tumors. This review will describe the technological advances being used to enrich the classification of gliomas, present specific studies that have successfully used the new technologies to identify molecular subtypes of glioblastoma, and discuss the implications of such enhanced classification and molecular characterizations for the prediction of therapeutic response and the design of future brain tumor therapies. RESULTS Innovative techniques using complementary DNA and oligonucleotide microarrays (gene chips), tissue microarrays (tissue chips), and differential immunoabsorption have provided high throughput and potentially comprehensive approaches for the molecular characterization of human gliomas. Alterations of several tumor suppressor genes and oncogenes have already been identified as being critical to glioma transformation and progression. These approaches have led to the subclassification of glioblastoma multiforme into distinct subtypes based on the molecular signatures of the tumors. CONCLUSIONS Classifications of gliomas can now be enhanced with new techniques for comprehensive molecular characterization. Improved and efficient molecular profiling of brain tumors is advancing diagnosis/prognosis and identifying targets for novel and rational therapeutic approaches. Neurosurgeons and neuro-oncologists should be aware of these new developments so they can better advise and treat their patients.
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Affiliation(s)
- Christine R Boudreau
- Division of Neurosurgery, Brain Research Institute, Geffen School of Medicine at UCLA, University of California-Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
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Garib V, Lang K, Niggemann B, Zänker KS, Brandt L, Dittmar T. Propofol-induced calcium signalling and actin reorganization within breast carcinoma cells. Eur J Anaesthesiol 2005; 22:609-15. [PMID: 16119598 DOI: 10.1017/s026502150500102x] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
BACKGROUND AND OBJECTIVE MDA-MB-468 breast carcinoma cells respond to non-volatile anaesthetics such as propofol with an increased migration. Here we investigated the relationship between GABA-A receptor modulators, the mode of calcium oscillation and actin reorganization with regard to breast carcinoma cell migration. METHODS Expression of the GABA-A receptor was determined by Western blot analysis. Calcium-imaging experiments of individual MDA-MB-468 cells as well as visualization of the F-actin distribution were performed by confocal laser scanning microscopy. Cell migration was investigated in a three-dimensional collagen matrix by time-lapse video microscopy. The GABA agonist propofol was used in a final concentration of 6 microg mL(-1). GABA-A receptor antagonist bicuculline (50 micromol) and selective L-type calcium channel blocker verapamil (5 micromol) were used to modulate the propofol effects. RESULTS A functional GABA-A receptor is expressed by MDA-MB-468 cells. Activation with propofol resulted in sustained increased intracellular calcium concentrations concomitant with actin reorganization and induction of migration in MDA-MB-468 cells. These propofol effects were completely blocked by verapamil. Spontaneous migration of MDA-MB-468 cells (64.4 +/- 7.0%) was significantly increased by propofol to 85.0 +/- 5.0%. MDA-MB-468 cells co-treated with propofol and verapamil showed a migratory activity of 63.0 +/- 2.0% indicating that verapamil blocked the propofol effect. Similar results were achieved with the GABA-A receptor inhibitor bicuculline (control: 56.3 +/- 8.5%; propofol: 80.5 +/- 7.1%; propofol + bicuculline: 52.5 +/- 8.6%). CONCLUSION Activation of GABA-A receptor by propofol correlated with an increased migration of MDA-MB-468 breast carcinoma cells, mediated by calcium influx via L-type calcium channels and reorganization of the actin cytoskeleton.
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Affiliation(s)
- V Garib
- University of Witten/Herdecke, Institute of Immunology, Witten, Germany
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Shearstone JR, Wang YE, Clement A, Allaire NE, Yang C, Worley DS, Carulli JP, Perrin S. Application of functional genomic technologies in a mouse model of retinal degeneration. Genomics 2005; 85:309-21. [PMID: 15718098 DOI: 10.1016/j.ygeno.2004.11.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2004] [Accepted: 11/01/2004] [Indexed: 02/03/2023]
Abstract
Generation of tissue-specific, normalized and subtracted cDNA libraries has the potential to characterize the expression of rare transcriptional units not represented on Affymetrix GeneChips. Initial sequence analysis of our murine cDNA clone collections showed that as much as 86, 45, and 30% of clones are not represented on the Affymetrix Mu11k, MG-U74, and MG-430 chip sets, respectively. A detailed study that compared EST sequences of a subtracted library generated from mouse retina to those of MG-430 consensus sequences was undertaken, using UniGene build 124 as the common reference. A set of 1111 nonredundant transcript regions, not represented on the commercial array, was identified. These clusters were used as the primary filter for analyzing a data set produced by assaying samples from the Pde6b(rd1) mouse model of retinal degeneration on a 12,325-feature retinal cDNA microarray. QRT-PCR validated eight unique transcripts identified by microarray. Seven of the transcripts showed retina-specific expression. Full-length cloning strategies were applied to two of the ESTs. The genes discovered by this approach are the full-length mouse homologue of guanylate cyclase 2F (GUCY2F) and a carboxy-truncated splice variant of retinal S-antigen (SAG), known as regulators of the visual phototransduction G-protein-coupled receptor-mediated signaling pathway. These sequences have been assigned GenBank Accession Nos. and , respectively.
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Affiliation(s)
- Jeffrey R Shearstone
- Research Molecular Discovery, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.
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Rebrikov D, Desai S, Kogan YN, Thornton AM, Diatchenko L. Subtractive cloning: new genes for studying inflammatory disorders. ACTA ACUST UNITED AC 2005; 7:17-28. [PMID: 16013213 DOI: 10.1902/annals.2002.7.1.17] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Understanding of the biology of interaction between pathogens and host is the central question in studying inflammatory disorders. Subtractive DNA cloning is one of the most efficient and comprehensive methods available for identifying eukaryotic genes regulated under specific physiological conditions, including inflammation and host response. Here we explore the utility of subtractive DNA cloning and describe suppression subtractive hybridization (SSH), a polymerase chain reaction (PCR)-based DNA subtraction method that has been developed and evolved in our labs over several years. The SSH method possesses a number of advantages as compared to other subtractive cloning techniques, making it one of the most adventitious methods for cloning differentially expressed genes. Besides isolation of differentially expressed eukaryotic mRNAs, subtractive DNA cloning can be used to identify genes that are differentially expressed between diverse bacterial species. These genes can be of great interest, as some may encode strain-specific traits such as drug resistance, or bacterial surface proteins involved in determining the virulence of a particular strain. Other genes may be useful as markers for epidemiological or evolutionary studies. To demonstrate the potential of the SSH technique, we describe here the comprehensive characterization of 2 SSH subtracted libraries constructed in our laboratories. One library was created using eukaryotic cDNA subtraction and is specific for mRNAs up-regulated in CD25 positive cells from mouse lymph nodes as compared to CD25 negative cells. The second subtracted library is specific for a methicillin-resistant Staphylococcus aureus bacterial strain, but not in a methicillin-sensitive strain. The bacterial genomes of these 2 strains have been completely sequenced and this second library provides an excellent reference for testing the ability of SSH to recover all strain-specific gene content. The analysis of these 2 subtracted libraries serves as the basis for a discussion of the strength and limitations of the SSH technique. We will also compare and contrast subtractive DNA cloning to other current technologies used to isolate differentially expressed genes.
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Affiliation(s)
- Denis Rebrikov
- Evrogen JSC; Institute of Bioorganic Chemistry, Moscow, Russia
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O'Brien NA, O'Donovan N, Ryan B, Hill ADK, McDermott E, O'Higgins N, Duffy MJ. Mammaglobin a in breast cancer: existence of multiple molecular forms. Int J Cancer 2005; 114:623-7. [PMID: 15609337 DOI: 10.1002/ijc.20780] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Existing serum-based markers for breast cancer all lack organ specificity. Mammaglobin A (MGA) is a 93 amino acid protein expressed almost exclusively in breast tissue. The aim of our study was to investigate the different forms of MGA protein in fibroadenomas and breast carcinomas. MGA protein was measured by Western blotting in 132 breast cancers, 29 fibroadenomas and 14 nonbreast tissues. MGA protein in breast tissue was found to exist in 2 main forms. These forms migrated with approximate molecular masses of 18 and 25 kDa. Both forms of MGA were detected more frequently in breast carcinomas compared to fibroadenomas. The high molecular weight form of MGA but not the low molecular weight form was found more frequently in hormone receptor-positive than in receptor-negative cancers. Furthermore, an inverse relationship was found between the high molecular weight form of MGA and both tumour grade and proliferation index. No significant correlation was found between the MGA proteins and either tumor size or nodal status. Our results show that MGA protein exists in 2 main forms in breast tissue. As the high molecular weight form correlated positively with hormone receptors and negatively with tumor grade and proliferation rate, its presence is likely to be associated with a favourable prognosis for breast cancer. As expression of MGA is almost breast specific, it is a promising marker for breast cancer. Its most immediate use is likely to be in detecting micrometastases from breast cancer.
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Affiliation(s)
- Neil A O'Brien
- Department of Nuclear Medicine, St. Vincent's University Hospital, Dublin, Ireland
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Gaggioli C, Deckert M, Robert G, Abbe P, Batoz M, Ehrengruber MU, Ortonne JP, Ballotti R, Tartare-Deckert S. HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1. Oncogene 2005; 24:1423-33. [PMID: 15608673 DOI: 10.1038/sj.onc.1208318] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.
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Affiliation(s)
- Cédric Gaggioli
- INSERM Unité 597, Biologie et Pathologies des Cellules Mélanocytaires, 'équipe labellisée la Ligue 2001', IFR 50, 06107 Nice Cédex 2, France
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Kim JM, Sohn HY, Yoon SY, Oh JH, Yang JO, Kim JH, Song KS, Rho SM, Yoo HS, Kim YS, Kim JG, Kim NS. Identification of Gastric Cancer–Related Genes Using a cDNA Microarray Containing Novel Expressed Sequence Tags Expressed in Gastric Cancer Cells. Clin Cancer Res 2005. [DOI: 10.1158/1078-0432.473.11.2] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Abstract
Purpose: Gastric cancer is one of the most frequently diagnosed malignancies in the world, especially in Korea and Japan. To understand the molecular mechanism associated with gastric carcinogenesis, we attempted to identify novel gastric cancer–related genes using a novel 2K cDNA microarray.
Experimental Design: A 2K cDNA microarray was fabricated from 1,995 novel expressed sequence tags (ESTs) showing no hits or a low homology with ESTs in public databases from our 143,452 ESTs collected from gastric cancer cell lines and tissues. An analysis of the gene expression for human gastric cancer cell lines to a normal cell line was done using this cDNA microarray. Data for the different expressed genes were verified using semiquantitative reverse transcription-PCR, Western blotting, and immunohistochemical staining in the gastric cell lines and tissues.
Results: Forty genes were identified as either up-regulated or down-regulated genes in human gastric cancer cells. Among these, genes such as SKB1, NT5C3, ZNF9, p30, CDC20, and FEN1, were confirmed to be up-regulated genes in nine gastric cell lines and in 25 pairs of tissue samples from patients by semiquantitative reverse transcription-PCR. On the other hand, genes such as MT2A and CXX1 were identified as down-regulated genes. In particular, the SKB1, CDC20, and FEN1 genes were overexpressed in ≥68% of tissues and the MT2A gene was down-expressed in 72% of the tissues. Western blotting and immunohistochemical analyses for CDC20 and SKB1 showed overexpression and localization changes of the corresponding protein in human gastric cancer tissues.
Conclusions: Novel genes that are related to human gastric cancer were identified using cDNA microarray developed in our laboratory. In particular, CDC20 and MT2A represent a potential biomarker of human gastric cancer. These newly identified genes should provide a valuable resource for understanding the molecular mechanism associated with tumorigenesis of gastric carcinogenesis and for the discovery of potential diagnostic markers of gastric cancer.
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Affiliation(s)
- Jeong-Min Kim
- 1Genome Research Center, Korea Research Institute of Bioscience and Biotechnology
- 5Department of Microbiology, College of Natural Sciences, Kyungpook National University, Daegu, Korea
| | - Ho-Yong Sohn
- 4Department of Food and Nutrition, Andong National University, Andong, Korea; and
| | - Sun Young Yoon
- 1Genome Research Center, Korea Research Institute of Bioscience and Biotechnology
| | - Jung-Hwa Oh
- 1Genome Research Center, Korea Research Institute of Bioscience and Biotechnology
| | - Jin Ok Yang
- 1Genome Research Center, Korea Research Institute of Bioscience and Biotechnology
| | - Joo Heon Kim
- 2Department of Pathology, Eulji University School of Medicine; and
| | - Kyu Sang Song
- 3Department of Pathology, College of Medicine, Chungnam National University, Daejeon, Korea
| | - Seung-Moo Rho
- 2Department of Pathology, Eulji University School of Medicine; and
| | - Hyan Sook Yoo
- 1Genome Research Center, Korea Research Institute of Bioscience and Biotechnology
| | - Yong Sung Kim
- 1Genome Research Center, Korea Research Institute of Bioscience and Biotechnology
| | - Jong-Guk Kim
- 5Department of Microbiology, College of Natural Sciences, Kyungpook National University, Daegu, Korea
| | - Nam-Soon Kim
- 1Genome Research Center, Korea Research Institute of Bioscience and Biotechnology
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Zehentner BK, Deme A, Toure P, Hawes SE, Brooks L, Feng Q, Hayes D, Zhang X, Persing DH, Critichlow CW, Houghton RL, Kiviat. NB. Mammaglobin as a novel breast cancer biomarker: multigene reverse transcription-PCR assay and sandwich ELISA. Clin Chem 2004; 50:2069-76. [PMID: 15375015 PMCID: PMC1482781 DOI: 10.1373/clinchem.2004.038687] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND The aim of this study was to examine the potential usefulness of a mammaglobin multigene reverse transcription-PCR (RT-PCR) assay and a mammaglobin sandwich ELISA as diagnostic tools in breast cancer. METHODS We studied peripheral blood samples from 147 untreated Senegalese women with biopsy-confirmed breast cancer and gathered patient information regarding demographic, and clinical staging of disease. The samples were tested for mammaglobin and three breast cancer-associated gene transcripts by a multigene real-time RT-PCR assay and for serum mammaglobin protein by a sandwich ELISA assay. RESULTS In 77% of the breast cancer blood samples, a positive signal was obtained in the multigene RT-PCR assay detecting mammaglobin and three complementary transcribed genes. Fifty samples from healthy female donors tested negative. Significant correlations were found between mammaglobin protein in serum, presence of mammaglobin mRNA-expressing cells in blood, stage of disease, and tumor size. Circulating mammaglobin protein was detected in 68% of the breast cancer sera, and was increased in 38% in comparison with a mixed control population. The RT-PCR assay and the ELISA for mammaglobin produced a combined sensitivity of 84% and specificity of 97%. CONCLUSION The ELISA and RT-PCR for mammaglobin and mammaglobin-producing cells could be valuable tools for diagnosis and prognosis of breast cancer.
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Affiliation(s)
- Barbara K. Zehentner
- Corixa Corporation, Seattle, WA
- Corresponding author: Barbara K. Zehentner, Ph.D., Corixa Corporation, 1124 Columbia Street, Seattle, WA 98104, phone 206 753 5932, fax 206 754 5917, e-mail:
| | - Amadou Deme
- University of Dakar, Senegal, West Africa.University of Washington
| | - Papa Toure
- University of Dakar, Senegal, West Africa.University of Washington
| | - Stephen E. Hawes
- Department of Epidemiology, School of Public Health and Community Medicine
| | | | - Qinghua Feng
- Department of Pathology, School of Medicine, Seattle, WA
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Plachot C, Lelièvre SA. DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium. Exp Cell Res 2004; 298:122-32. [PMID: 15242767 DOI: 10.1016/j.yexcr.2004.04.024] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2004] [Revised: 04/14/2004] [Indexed: 10/26/2022]
Abstract
Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation, a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2' deoxycytidine during acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19, a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which mammary epithelial differentiation is coordinated both at the tissue and cellular levels.
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Affiliation(s)
- Cedric Plachot
- Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907-2026, USA
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Jiang Z, Wu CL, Woda BA, Iczkowski KA, Chu PG, Tretiakova MS, Young RH, Weiss LM, Blute RD, Brendler CB, Krausz T, Xu JC, Rock KL, Amin MB, Yang XJ. Alpha-methylacyl-CoA racemase: a multi-institutional study of a new prostate cancer marker. Histopathology 2004; 45:218-25. [PMID: 15330799 DOI: 10.1111/j.1365-2559.2004.01930.x] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
AIM To test whether alpha-methylacyl-CoA racemase (AMACR) is a sensitive and specific marker of prostate cancer. METHODS AND RESULTS The expression levels of AMACR mRNA were measured by real-time polymerase chain reaction. A total of 807 prostatic specimens were further examined by immunohistochemistry specific for AMACR. Quantitative immunostaining analyses were carried out by using the ChromaVision Automated Cellular Imaging System and the Ariol SL-50 Imaging System, respectively. AMACR mRNA levels measured in prostatic adenocarcinoma were 55 times higher than those in benign prostate tissue. Of 454 cases of prostatic adenocarcinoma, 441 were positive for AMACR, while 254 of 277 cases of benign prostate were negative for AMACR. The sensitivity and specificity of AMACR immunodetection of prostatic adenocarcinomas were 97% and 92%, respectively. Both positive and negative predictive values were 95%. By automatic imaging analyses, the AMACR immunostaining intensity and percentage in prostatic adenocarcinomas were also significantly higher than those in benign prostatic tissue (105.9 versus 16.1 for intensity, 45.7% versus 0.02% and 35.03% versus 4.64% for percentage, respectively). CONCLUSIONS We have demonstrated the promising features of AMACR as a biomarker for prostate cancer in this large series and the potential to develop automated quantitative diagnostic tests.
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Affiliation(s)
- Z Jiang
- University of Massachusetts medical School, Worcester, MA, USA
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Han S, Sidell N, Roser-Page S, Roman J. Fibronectin stimulates human lung carcinoma cell growth by inducing cyclooxygenase-2 (COX-2) expression. Int J Cancer 2004; 111:322-31. [PMID: 15221958 DOI: 10.1002/ijc.20281] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Tobacco use is the most important risk factor for the development of lung carcinoma. One characteristic shared by tobacco-related lung diseases is altered lung connective tissue content and composition. In particular, tobacco results in increased expression of fibronectin (FN), a matrix glycoprotein implicated in lung development, injury and repair and in tumor cell invasion. We hypothesized that excessive deposition of FN in lung might promote lung carcinoma cell proliferation. Consistent with this hypothesis, we found that FN stimulated human lung carcinoma cell proliferation and diminished apoptosis in vitro, and that this effect was mediated through the integrin alpha5beta1 and associated with upregulation of cyclooxygenase-2 (COX-2) mRNA and protein expression, and increased prostaglandin E2 (PGE2) biosynthesis. The stimulatory effect of FN on COX-2 was blocked by the specific COX-2 inhibitor NS-398 and by inhibitors of protein kinase C (PKC), Calphostin C, and extracellular signal-regulated kinases (Erks), PD98095. Electrophoretic mobility shift assays revealed that FN increased the nuclear binding activity of cyclic AMP response element binding protein (CREB) and CCAAT/enhancer-binding protein (C/EBP), 2 proteins known to play important roles in the regulation of COX-2 promoter activity. Transient transfection assays with wild-type and mutated constructs of the human COX-2 gene promoter revealed that the stimulatory effect of FN was prevented when either the CRE or the NF-IL6 (C/EBP) sites were mutated. Taken together, the results indicate that FN stimulates human lung carcinoma cell proliferation and diminishes apoptosis by inducing COX-2 gene expression and PGE2 biosynthesis. Activation of PKC and Erk and DNA-protein interactions at CRE and NF-IL6 (C/EBP) sites in the COX-2 gene promoter appear to play key roles in this process. This work demonstrates that signaling through specific matrix-binding beta1 integrins (i.e., alpha5beta1) resulting from exaggerated deposition in lung of the matrix glycoprotein fibronectin might promote lung carcinoma cell growth.
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Affiliation(s)
- Shouwei Han
- Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
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Stolk JA, Jiang Y, Day CH, Klee JI, Zhang X, Dillon DC, Houghton RL, Harlan D, Reed SG, Xu J. P704P, P712P, and P775P: A genomic cluster of prostate-specific genes. Prostate 2004; 60:214-26. [PMID: 15176051 DOI: 10.1002/pros.20012] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
BACKGROUND Discovery of prostate cancer- and tissue-specific genes will lead to an increased understanding of the molecular events associated with the malignant transformation and tumorigenesis of prostate cells. Such understanding will likely result in the development of promising new markers for screening, diagnosis, and prognosis, as well as potential therapeutic approaches for combating this disease. METHODS A PCR-based subtraction method was combined with a high-throughput microarray screening approach to identify prostate tissue- and/or cancer-specific genes. Northern blot and quantitative real-time PCR were used to confirm prostate specificity. Bioinformatics analysis was performed to determine gene localization and to identify the open reading frame of novel genes. RESULTS Three novel cDNA clones, P704P, P712P, and P775P, were identified and characterized to be specific for normal and malignant prostate tissues. Furthermore, P712P mRNA expression was found to be androgen responsive in LNCaP cells. Sequences for all three cDNAs were localized to an 80 kb genomic region on chromosome 22. Attempts to identify full-length transcripts did not reveal any apparent open reading frames, indicating that P704P, P712P, and P775P may belong to a novel class of transcripts with specific patterns of gene expression that do not code for translated proteins. CONCLUSIONS A genomic cluster of prostate-specific genes with no apparent open reading frame has been discovered using a high-throughput approach combining subtraction with microarray. This may represent an important genomic region having possible connections to prostate biology with potential applications in prostate diagnostics and therapy.
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Affiliation(s)
- John A Stolk
- Corixa Corporation, Seattle, Washington 98104, USA.
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Frasor J, Stossi F, Danes JM, Komm B, Lyttle CR, Katzenellenbogen BS. Selective estrogen receptor modulators: discrimination of agonistic versus antagonistic activities by gene expression profiling in breast cancer cells. Cancer Res 2004; 64:1522-33. [PMID: 14973112 DOI: 10.1158/0008-5472.can-03-3326] [Citation(s) in RCA: 275] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Selective estrogen receptor modulators (SERMs) such as tamoxifen are effective in the treatment of many estrogen receptor-positive breast cancers and have also proven to be effective in the prevention of breast cancer in women at high risk for the disease. The comparative abilities of tamoxifen versus raloxifene in breast cancer prevention are currently being compared in the Study of Tamoxifen and Raloxifene trial. To better understand the actions of these compounds in breast cancer, we have examined their effects on the expression of approximately 12,000 genes, using Affymetrix GeneChip microarrays, with quantitative PCR verification in many cases, categorizing their actions as agonist, antagonist, or partial agonist/antagonist. Analysis of gene stimulation and inhibition by the SERMs trans-hydroxytamoxifen (TOT) and raloxifene (Ral) or ICI 182,780 (ICI) and by estradiol (E2) in estrogen receptor-containing MCF-7 human breast cancer cells revealed that (a) TOT was the most E2-like of the three compounds, (b) all three compounds either partially or fully antagonized the action of E2 on most genes, with the order of antagonist activity being ICI > Ral > TOT, (c) TOT and Ral, but not ICI, displayed partial agonist/partial antagonist activity on a number of E2-regulated genes, (d) several stimulatory cell cycle-related genes were down-regulated exclusively by ICI, (e) the estrogen-like activity of Ral nearly always overlapped with that of TOT, indicating that Ral has little unique agonist activity different from that of TOT, and (f) some genes were specifically up-regulated by TOT but not Ral, ICI, or E2. Hence, gene expression profiling can discern fundamental differences among SERMs and provides insight into the distinct biologies of TOT, Ral, and ICI in breast cancer.
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Affiliation(s)
- Jonna Frasor
- Department of Molecular and Integrative Physiology, University of Illinois and College of Medicine, 407 South Goodwin Avenue, Urbana, IL 61801, USA
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Radvanyi L. Discovery and immunologic validation of new antigens for therapeutic cancer vaccines. Int Arch Allergy Immunol 2004; 133:179-97. [PMID: 14764946 DOI: 10.1159/000076625] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Immunotherapy using both active and passive approaches is increasingly being used as a modality to treat human cancer. The last decade has seen a tremendous burst of activity in antigen discovery in cancer, and many new targets have now been identified for both monoclonal antibody therapy and active immunization. In addition, advances have been made in our understanding of the immune response against cancer and how new vaccine vectors, such as poxviruses, alphaviruses and bacterial vectors, can be used to overcome some of the traditional hurdles (e.g. self-tolerance and immune suppression in cancer patients) that have hindered the generation of effective antitumor immune responses. Improvements in genomics technology in the area of DNA microarrays and differential display and subtractive hybridization together with a new wave of mass spectrometry-based proteomics tools, as well as more sensitive assays to validate the immunoreactivity of new antigens, have all accelerated the rate of new antigen discovery in cancer. This rapid progress should initiate a major paradigm shift in how we treat cancer within the next 10 years, where, instead of being a novelty, the combination of targeted T cell-based vaccines and antiangiogenesis therapies will be routinely combined with traditional chemotherapy. The successful combination of these approaches will change the face of cancer from a relatively acute, life-threatening disease to a manageable chronic disorder with long survival times.
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Affiliation(s)
- Laszlo Radvanyi
- Immunology Platform, Aventis Pasteur Canada, Toronto, Ont, Canada.
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Abstract
Estrogens work along with genetic changes to promote the development and growth of breast cancers. Because estrogenic hormones act via the estrogen receptors (ERs), ER-alpha and ER-beta, and the ER is present in more than half of breast tumors, this receptor has been the most widely targeted protein in breast cancer therapy. The presence of the ER in breast tumors predicts improved disease-free survival and response to selective ER modulators (SERMs), such as tamoxifen, or other forms of endocrine therapy. Suppression of ER activity by SERMs has proven to be a great benefit in the treatment of breast cancers and also in the prevention of breast cancer in women at high risk for the disease. The Study of Tamoxifen and Raloxifene trial comparing tamoxifen versus raloxifene effectiveness in breast cancer prevention is currently under way. To understand the balance of beneficial and undesirable effects of SERMs and to optimize their effectiveness, current investigations seek to characterize the genes activated or suppressed by these agents. Elucidation of the gene networks and cell signaling pathways under estrogen and SERM regulation and a clearer definition of the respective roles of ER-alpha and ER-beta and their coregulators in the actions of selective ER ligands, should enable the identification of new gene targets for therapeutic intervention and the development of novel drugs for the optimal treatment and prevention of breast cancer.
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Affiliation(s)
- Benita S Katzenellenbogen
- Department of Molecular and Integrative Physiology, University of Illinois and College of Medicine, Urbana, IL, USA
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Zhang DH, Salto-Tellez M, Chiu LL, Shen L, Koay ESC. Tissue microarray study for classification of breast tumors. Life Sci 2003; 73:3189-99. [PMID: 14561524 DOI: 10.1016/j.lfs.2003.05.006] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Clinical and pathological heterogeneity of breast cancer hinders selection of appropriate treatment for individual cases. Molecular profiling at gene or protein levels may elucidate the biological variance of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters. We studied the immunohistochemical profile of a panel of seven important biomarkers using tumor tissue arrays. The tumor samples were then classified with a monothetic (binary variables) clustering algorithm. Two distinct groups of tumors are characterized by the estrogen receptor (ER) status and tumor grade (p = 0.0026). Four biomarkers, c-erbB2, Cox-2, p53 and VEGF, were significantly overexpressed in tumors with the ER-negative (ER-) phenotype. Eight subsets of tumors were further identified according to the expression status of VEGF, c-erbB2 and p53. The malignant potential of the ER-/VEGF+ subgroup was associated with the strong correlations of Cox-2 and c-erbB2 with VEGF. Our results indicate that this molecular classification system, based on the statistical analysis of immunohistochemical profiling, is a useful approach for tumor grouping. Some of these subgroups have a relative genetic homogeneity that may allow further study of specific genetically-controlled metabolic pathways. This approach may hold great promise in rationalizing the application of different therapeutic strategies for different subgroups of breast tumors.
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Affiliation(s)
- Dao-Hai Zhang
- Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Hospital, Singapore
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Frasor J, Danes JM, Komm B, Chang KCN, Lyttle CR, Katzenellenbogen BS. Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 2003; 144:4562-74. [PMID: 12959972 DOI: 10.1210/en.2003-0567] [Citation(s) in RCA: 589] [Impact Index Per Article: 26.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with quantitative PCR verification in many cases, to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. Of the >12,000 genes queried, over 400 showed a robust pattern of regulation, and, notably, the majority (70%) were down-regulated. We observed a general up-regulation of positive proliferation regulators, including survivin, multiple growth factors, genes involved in cell cycle progression, and regulatory factor-receptor loops, and the down-regulation of transcriptional repressors, such as Mad4 and JunB, and of antiproliferative and proapoptotic genes, including B cell translocation gene-1 and -2, cyclin G2, BCL-2 antagonist/killer 1, BCL 2-interacting killer, caspase 9, and TGFbeta family growth inhibitory factors. These together likely contribute to the stimulation of proliferation and the suppression of apoptosis by E2 in these cells. Of interest, E2 appeared to modulate its own activity through the enhanced expression of genes involved in prostaglandin E production and signaling, which could lead to an increase in aromatase expression and E2 production, as well as the decreased expression of several nuclear receptor coactivators that could impact ER activity. Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which this hormone operates to achieve its widespread effects on breast cancer cells.
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Affiliation(s)
- Jonna Frasor
- Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801, USA
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Waring JF. Overview of microarrays in drug discovery and development. CURRENT PROTOCOLS IN PHARMACOLOGY 2003; Chapter 9:Unit9.6. [PMID: 21956809 DOI: 10.1002/0471141755.ph0906s21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
With the sequencing of the human genome, new tools and technologies have been developed to identify and quantify global gene-expression changes occurring in the cell. One of the main tools being utilized is microarray technology, which allows one to quantitate expression changes of thousands of genes in a single experiment. Microarrays allow researchers to gain an unprecedented understanding of the function and regulation of genes, and are transforming virtually all areas of biological research. In the drug-discovery process, microarrays have the potential to play a role in all stages, from new target discovery through compound profiling and safety assessment. This overview highlights some of these studies and discusses how this technology is transforming the field of drug discovery and development.
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Ding H, Shi GG, Yu X, Yu JP, Huang JA. Modulation of GdCl 3 and Angelica Sinensis polysaccharides on differentially expressed genes in liver of hepatic immunological injury mice by cDNA microarray. World J Gastroenterol 2003; 9:1072-6. [PMID: 12717859 PMCID: PMC4611375 DOI: 10.3748/wjg.v9.i5.1072] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the modulating effect of GdCl3 and Angelica Sinensis polysaccharides (ASP) on differentially expressed genes in liver of hepatic immunological mice by cDNA microarray.
METHODS: Hepatic immunological injury was induced by lipopolysaccharide (LPS ip, 0.2 mg·kg-1) in bacillus calmetteguerin (BCG ip, 1 mg·kg-1) primed mice; A single dose of 20 mg·kg-1 GdCl3 was simultaneously pretreated and 30 mg·kg-1 ASP (ig, qd × 7 d) was administrated when the BCG+LPS was primed. The mice were sacrificed at the end of the 7th day after ip LPS for 6 h and the liver was removed quickly. The PCR products of 512 genes were spotted onto a chemical material-coated glass plate in array. The DNAs were fixed to the glass plate after series of treatments. The total RNAs were isolated from the liver tissue, and were purified to mRNAs by Oligotex. Both mRNAs from the normal liver tissue and the liver tissue from the mice with hepatic immunological injury or that pretreated with GdCl3 or ASP were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for fluorescent signals and showed differences between the two tissues.
RESULTS: Among the 512 target genes, 18 differed in liver tissue of hepatic immunological injury mice, and 6 differed in those pretreated by ASP, 7 differed in those pretreated by GdCl3.
CONCLUSION: cDNA microarray technique is effective in screening the differentially expressed genes between two different kinds of tissue. Further analysis of those obtained genes will be helpful to understand the molecular mechanism of hepatic immunological injury and to study the intervention of drug. Both ASP and GdCl3 can decrease the number of the differentially expressed genes in liver tissue of mice with hepatic immunological injury.
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Affiliation(s)
- Hong Ding
- Medical College, Shantou University, Guangdong Province, China.
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