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Identification and validation of genes with expression patterns inverse to multiple metastasis suppressor genes in breast cancer cell lines.

ABSTRACT: Metastasis suppressor genes (MSGs) have contributed to an understanding of regulatory pathways unique to the lethal metastatic process. When re-expressed in experimental models, MSGs block cancer spread to, and colonization of distant sites without affecting primary tumor formation. Genes have been identified with expression patterns inverse to a single MSG, and found to encode functional, druggable signaling pathways. We now hypothesize that common signaling pathways mediate the effects of multiple MSGs. By gene expression profiling of human MCF7 breast carcinoma cells expressing a scrambled siRNA, or siRNAs to each of 19 validated MSGs (NME1, BRMS1, CD82, CDH1, CDH2, CDH11, CASP8, MAP2K4, MAP2K6, MAP2K7, MAPK14, GSN, ARHGDIB, AKAP12, DRG1, CD44, PEBP1, RRM1, KISS1), we identified genes whose expression was significantly opposite to at least five MSGs. Five genes were selected for further analysis: PDE5A, UGT1A, IL11RA, DNM3 and OAS1. After stable downregulation of each candidate gene in the aggressive human breast cancer cell line MDA-MB-231T, in vitro motility was significantly inhibited. Two stable clones downregulating PDE5A (phosphodiesterase 5A), an enzyme involved in the regulation of cGMP-specific signaling, exhibited no difference in cell proliferation, but reduced motility by 47 and 66 % compared to the empty vector-expressing cells (p = 0.01 and p = 0.005). In an experimental metastasis assay, two shPDE5A-MDA-MB-231T clones produced 47-62 % fewer lung metastases than shRNA-scramble expressing cells (p = 0.045 and p = 0.009 respectively). This study demonstrates that previously unrecognized genes are inversely related to the expression of multiple MSGs, contribute to aspects of metastasis, and may stand as novel therapeutic targets.

SUBMITTER: Marino N

PROVIDER: S-EPMC4948753 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Identification and validation of genes with expression patterns inverse to multiple metastasis suppressor genes in breast cancer cell lines.

Marino Natascia N Collins Joshua W JW Shen Changyu C Caplen Natasha J NJ Merchant Anand S AS Gökmen-Polar Yesim Y Goswami Chirayu P CP Hoshino Takashi T Qian Yongzhen Y Sledge George W GW Steeg Patricia S PS

Clinical & experimental metastasis 20140803 7

Metastasis suppressor genes (MSGs) have contributed to an understanding of regulatory pathways unique to the lethal metastatic process. When re-expressed in experimental models, MSGs block cancer spread to, and colonization of distant sites without affecting primary tumor formation. Genes have been identified with expression patterns inverse to a single MSG, and found to encode functional, druggable signaling pathways. We now hypothesize that common signaling pathways mediate the effects of mult ...[more]

PMID: 25086928

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Project description:NME1 is a well-documented metastasis suppressor gene, with suppressor activity demonstrated across a wide spectrum of human cancers including melanoma and carcinomas of the breast, stomach and thyroid. A primary aim of the current study was to identify profiles of genes whose expression is regulated by NME1 in cell lines of melanoma and thyroid carcinoma origin. Impact of NME1 was determined by forcing its expression transiently in cell lines using a novel Ad5-based adenoviral vector (Ad5-NME1), followed 48 h later by analysis of RNA expression profiles using the U133A microarray chip. Robust NME1 expression was achieved following infection with the Ad5-NME1 adenovirus in the human metastasis-derived cell lines WM1158 (melanoma) and WRO82 (follicular thyroid carcinoma), resulting in wide-ranging effects on gene expression in both settings. A substantial proportion of the NME1-regulated genes identified in the analyses were of clear potential relevance to metastasis, such as matrix metalloproteinase-1 (MMP1), angiopoietin-2 (ANGPT2), SERPINB9 and colony stimulating factor receptor-2B (CSFR2B). Nine genes were identified (false discovery rate <0.1) that were regulated by NME1 in both the WM1158 and WRO82 cell lines, each possessing one or more such metastasis-relevant activities as stress fiber formation and focal adhesion (PPM1E, ZYX, PFN1), chemotaxis (CCR1) epithelial-mesenchymal signaling (WNT6), differentiation and morphogenesis (TBX4, ZFP36L2), and G protein modulation (GPR52 and PFN1). In addition, a number of the NME1-regulated genes were shown to be of prognostic value for distant disease-free survival and overall survival in melanoma and breast cancer. The combined expression of three NME1-regulated genes CSFR2B, MSF4A1 and SERPINB9 provided a strongly synergistic correlation with distant disease-free survival in the basal subtype of breast cancer (p<3.5e(-5), hazard ratio=0.33). Our study demonstrates that analysis of NME1-dependent gene expression is a powerful approach for identifying potential modulators of metastatic potential in multiple cancer types, which in turn may represent useful therapeutic targets. The study also highlights NME1-dependent genes as potential prognostic/diagnostic indices, which are profoundly lacking at present in melanoma.

Project description:DNA methylation is an epigenetic event that involves adding a methyl group to the cytosine (C) site, especially the one that pairs with a guanine (G) site (ie, CG or CpG site), in a human genome. This event plays an important role in both cancerous and normal cell development. Previous studies often assume symmetric methylation on both DNA strands. However, asymmetric methylation, or hemimethylation (methylation that occurs only on 1 DNA strand), does exist and has been reported in several studies. Due to the limitation of previous DNA methylation sequencing technologies, researchers could only study hemimethylation on specific genes, but the overall genomic hemimethylation landscape remains relatively unexplored. With the development of advanced next-generation sequencing techniques, it is now possible to measure methylation levels on both forward and reverse strands at all CpG sites in an entire genome. Analyzing hemimethylation patterns may potentially reveal regions related to undergoing tumor growth. For our research, we first identify hemimethylated CpG sites in breast cancer cell lines using Wilcoxon signed rank tests. We then identify hemimethylation patterns by grouping consecutive hemimethylated CpG sites based on their methylation states, methylation "M" or unmethylation "U." These patterns include regular (or consecutive) hemimethylation clusters (eg, "MMM" on one strand and "UUU" on another strand) and polarity (or reverse) clusters (eg, "MU" on one strand and "UM" on another strand). Our results reveal that most hemimethylation clusters are the polarity type, and hemimethylation does occur across the entire genome with notably higher numbers in the breast cancer cell lines. The lengths or sizes of most hemimethylation clusters are very short, often less than 50 base pairs. After mapping hemimethylation clusters and sites to corresponding genes, we study the functions of these genes and find that several of the highly hemimethylated genes may influence tumor growth or suppression. These genes may also indicate a progressing transition to a new tumor stage.

Dataset Information

Identification and validation of genes with expression patterns inverse to multiple metastasis suppressor genes in breast cancer cell lines.

Publications

Identification and validation of genes with expression patterns inverse to multiple metastasis suppressor genes in breast cancer cell lines.

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