miRNA signature associated with outcome of gastric cancer patients following chemotherapy

<p>Abstract</p> <p>Background</p> <p>Identification of patients who likely will or will not benefit from cytotoxic chemotherapy through the use of biomarkers could greatly improve clinical management by better defining appropriate treatment options for patients. microRNAs may be potentially useful biomarkers that help guide individualized therapy for cancer because microRNA expression is dysregulated in cancer. In order to identify miRNA signatures for gastric cancer and for predicting clinical resistance to cisplatin/fluorouracil (CF) chemotherapy, a comprehensive miRNA microarray analysis was performed using endoscopic biopsy samples.</p> <p>Methods</p> <p>Biopsy samples were collected prior to chemotherapy from 90 gastric cancer patients treated with CF and from 34 healthy volunteers. At the time of disease progression, post-treatment samples were additionally collected from 8 clinical responders. miRNA expression was determined using a custom-designed Agilent microarray. In order to identify a miRNA signature for chemotherapy resistance, we correlated miRNA expression levels with the time to progression (TTP) of disease after CF therapy.</p> <p>Results</p> <p>A miRNA signature distinguishing gastric cancer from normal stomach epithelium was identified. 30 miRNAs were significantly inversely correlated with TTP whereas 28 miRNAs were significantly positively correlated with TTP of 82 cancer patients (<it>P</it><0.05). Prominent among the upregulated miRNAs associated with chemosensitivity were miRNAs known to regulate apoptosis, including let-7g, miR-342, miR-16, miR-181, miR-1, and miR-34. When this 58-miRNA predictor was applied to a separate set of pre- and post-treatment tumor samples from the 8 clinical responders, all of the 8 pre-treatment samples were correctly predicted as low-risk, whereas samples from the post-treatment tumors that developed chemoresistance were predicted to be in the high-risk category by the 58 miRNA signature, suggesting that selection for the expression of these miRNAs occurred as chemoresistance arose.</p> <p>Conclusions</p> <p>We have identified 1) a miRNA expression signature that distinguishes gastric cancer from normal stomach epithelium from healthy volunteers, and 2) a chemoreresistance miRNA expression signature that is correlated with TTP after CF therapy. The chemoresistance miRNA expression signature includes several miRNAs previously shown to regulate apoptosis <it>in vitro</it>, and warrants further validation.</p

Distinctions in gastric cancer gene expression signatures derived from laser capture microdissection versus histologic macrodissection

<p>Abstract</p> <p>Background</p> <p>Gastric cancer samples obtained by histologic macrodissection contain a relatively high stromal content that may significantly influence gene expression profiles. Differences between the gene expression signature derived from macrodissected gastric cancer samples and the signature obtained from isolated gastric cancer epithelial cells from the same biopsies using laser-capture microdissection (LCM) were evaluated for their potential experimental biases.</p> <p>Methods</p> <p>RNA was isolated from frozen tissue samples of gastric cancer biopsies from 20 patients using both histologic macrodissection and LCM techniques. RNA from LCM was subject to an additional round of T7 RNA amplification. Expression profiling was performed using Affymetrix HG-U133A arrays. Genes identified in the expression signatures from each tissue processing method were compared to the set of genes contained within chromosomal regions found to harbor copy number aberrations in the tumor samples by array CGH and to proteins previously identified as being overexpressed in gastric cancer.</p> <p>Results</p> <p>Genes shown to have increased copy number in gastric cancer were also found to be overexpressed in samples obtained by macrodissection (LS <it>P </it>value < 10^-5), but not in array data generated using microdissection. A set of 58 previously identified genes overexpressed in gastric cancer was also enriched in the gene signature identified by macrodissection (LS <it>P </it>< 10^-5), but not in the signature identified by microdissection (LS <it>P </it>= 0.013). In contrast, 66 genes previously reported to be underexpressed in gastric cancer were enriched in the gene signature identified by microdissection (LS <it>P </it>< 10^-5), but not in the signature identified by macrodissection (LS <it>P </it>= 0.89).</p> <p>Conclusions</p> <p>The tumor sampling technique biases the microarray results. LCM may be a more sensitive collection and processing method for the identification of potential tumor suppressor gene candidates in gastric cancer using expression profiling.</p

Protein and lipid MALDI profiles classify breast cancers according to the intrinsic subtype

<p>Abstract</p> <p>Background</p> <p>Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of common solid tumors. Using recently developed MALDI matrices for lipid profiling, we evaluated whether direct tissue MALDI MS analysis on proteins and lipids may classify human breast cancer samples according to the intrinsic subtype.</p> <p>Methods</p> <p>Thirty-four pairs of frozen, resected breast cancer and adjacent normal tissue samples were analyzed using histology-directed, MALDI MS analysis. Sinapinic acid and 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument.</p> <p>Results</p> <p>Protein and lipid profiles distinguish cancer from adjacent normal tissue samples with the median prediction accuracy of 94.1%. Luminal, HER2+, and triple-negative tumors demonstrated different protein and lipid profiles, as evidenced by permutation <it>P </it>values less than 0.01 for 0.632+ bootstrap cross-validated misclassification rates with all classifiers tested. Discriminatory proteins and lipids were useful for classifying tumors according to the intrinsic subtype with median prediction accuracies of 80.0-81.3% in random test sets.</p> <p>Conclusions</p> <p>Protein and lipid profiles accurately distinguish tumor from adjacent normal tissue and classify breast cancers according to the intrinsic subtype.</p

DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity

<p>Abstract</p> <p>Background</p> <p>Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.</p> <p>Results</p> <p>Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV<it>-tk</it>) gene in a vector expressing also the <it>neo</it>^Rgene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.</p> <p>Conclusions</p> <p>We demonstrated that all sequences identified by their CTCF binding both <it>in vitro </it>and <it>in vivo </it>had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.</p

Cohesin is required for higher-order chromatin conformation at the imprinted IGF2-H19 locus

Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi-mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF-mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion

The Drosophila homolog of the mammalian imprint regulator, CTCF, maintains the maternal genomic imprint in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>CTCF is a versatile zinc finger DNA-binding protein that functions as a highly conserved epigenetic transcriptional regulator. CTCF is known to act as a chromosomal insulator, bind promoter regions, and facilitate long-range chromatin interactions. In mammals, CTCF is active in the regulatory regions of some genes that exhibit genomic imprinting, acting as insulator on only one parental allele to facilitate parent-specific expression. In <it>Drosophila</it>, CTCF acts as a chromatin insulator and is thought to be actively involved in the global organization of the genome.</p> <p>Results</p> <p>To determine whether CTCF regulates imprinting in <it>Drosophila</it>, we generated <it>CTCF </it>mutant alleles and assayed gene expression from the imprinted <it>Dp(1;f)LJ9 </it>mini-X chromosome in the presence of reduced <it>CTCF </it>expression. We observed disruption of the maternal imprint when <it>CTCF </it>levels were reduced, but no effect was observed on the paternal imprint. The effect was restricted to maintenance of the imprint and was specific for the <it>Dp(1;f)LJ9 </it>mini-X chromosome.</p> <p>Conclusions</p> <p>CTCF in <it>Drosophila </it>functions in maintaining parent-specific expression from an imprinted domain as it does in mammals. We propose that <it>Drosophila </it>CTCF maintains an insulator boundary on the maternal X chromosome, shielding genes from the imprint-induced silencing that occurs on the paternally inherited X chromosome.</p> <p>See commentary: <url>http://www.biomedcentral.com/1741-7007/8/104</url></p

Epigenetic Repression of RARRES1 Is Mediated by Methylation of a Proximal Promoter and a Loss of CTCF Binding

The cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown.We first compared methylation occurring at the sequences (-664~+420) flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (-664~-86), while the downstream element (-85~+420) proximal to the transcriptional start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores.This report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread

Loss of Maternal CTCF Is Associated with Peri-Implantation Lethality of Ctcf Null Embryos

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development

Multiple Organ System Defects and Transcriptional Dysregulation in the Nipbl+/− Mouse, a Model of Cornelia de Lange Syndrome

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75–80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only ∼30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/− mice and in individuals with CdLS