Reduced expression of RhoE promotes ROCK-dependent invasion in transformed human fibroblasts
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: giovanna chiorino
PROVIDER: E-GEOD-15742 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Cen3tel cells, obtained by telomerase immortalization of human fibroblasts, gradually underwent neoplastic transformation and became metastatic in immunocompromised mice. Neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal). Tumorigenic cells acquired a clear-cut membrane localization of adhesion molecules, a reorganization of the actin cytoskeleton, increased cell motility and invasiveness. In a 3-dimensional environment, tumorigenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from a mesenchymal to an amoeboid ROCK-dependent movement. Accordingly, cell invasion decreased upon treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro28-2653. The increased invasiveness of tumorigenic cen3tel cells was associated with a reduced expression of RhoE, a cellular inhibitor of ROCK. Ectopic RhoE expression decreased cen3tel invasion capability. These results point to RhoE and ROCK as regulators of invasiveness of mesenchymal tumor cells and indicate ROCK as a possible therapeutic target.
2011-04-20 | GSE15742 | GEO
Project description:Despite malignant cutaneous melanoma is relatively rare compared to other skin cancers, it is still responsible for 80% of all skin cancer-related deaths. To identify molecular signatures of melanoma progression, excisional biopsies from 18 common melanocytic nevi (CMN), 8 primary radial growth phase melanomas (RGPM), 15 primary vertical growth phase melanomas (VGPM) and 5 melanoma metastases (MTS) were profiled using whole genome oligo-microarrays. Differentially expressed genes for each progression step were identified, and validation of selected transcripts by qRT-PCR was performed on an independent cohort of fixed samples. The comparison between CMN and RGPM showed an enrichment of Gene Ontology (GO) terms related to inter and intra-cellular junctions, whereas the transition from RGPM to VGPM was characterized by the deregulation of WNT3, MAPK and AKT pathways. In this step, enrichment analysis underlined the alteration of biological processes linked to apoptosis. Upregulation of genes involved into DNA double-strand breaks repair and downregulation of cellular adhesion genes were observed in MTS respect to VGPM. Futhermore, the gene expression profiles of 11 dysplastic nevi (DN) were compared to all the others. Some genes controlling proliferation were found more expressed than in CMN. Overall, DN displayed an heterogeneous behaviour, with relevant oncogenes, such as MYC and BCL6, less expressed than in RGPM, and a modulation pattern similar to VGPM for a subset of gene families, such as mismatch repair. This suggests that DN is not an intermediate step within melanoma progression, but a separate entity with an independent risk of progression to melanoma Experiment Overall Design: In order to identify genes implicated during the course of melanoma development and progression, a total of 57 excisional biopsies from common melanocytic nevi (n = 18), dysplastic nevi (n = 11), primary radial growth phase malignant melanomas (n = 8), primary vertical growth phase malignant melanomas (n = 15) and melanoma metastases (n = 5) were analyzed. Total RNA was isolated by using RNeasy Mini Kit (Qiagen, Dusseldorf, Germany). TotRNA quality was checked by means of RNA 6000 pico chip assays (Agilent Technologies, Palo Alto, CA) run on the Agilent 2100 bioanalyzer. RNA isolated from each fresh tissue and from the human universal reference (BD™ Human Universal Reference Total RNA, Clontech, Palo Alto, CA) was amplified by means of the Amino Allyl MessageAmp II aRNA Kit (Ambion) to obtain amino allyl antisense RNA (aaRNA) following the method developed by Eberwine and coworkers. Two rounds of amplification were performed to obtain the necessary quantity of aaRNA for labeling. Briefly: mRNA was reverse transcribed in cDNA single strand; after the second strand synthesis (in the second round of amplification), cDNA was in vitro transcribed in aaRNA including amino allyl modified nucleotides (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy3 or Cy5 dyes (Amersham Biosciences, Buckinghamshire UK) able to react with the modified RNA. mRNA quality was checked by means of RNA 6000 nano chip assays (Agilent Technologies). At least 5 ug of mRNA for each sample were labeled and purified with columns. Equal amounts (0.75 ug) of labeled specimens from sample and reference were put together, fragmented and hybridized to oligonucleotide glass arrays representing 41K human unique genes and transcripts (Human Whole Genome Oligo Microarray Array, Agilent Technologies). All steps were performed using the In Situ Hybridization kit-plus (Agilent Technologies) and following the 60-mer oligo microarray processing protocol (Agilent Technologies). Then, slides were washed with the SSPE wash procedure and scanned with the dual-laser microarray scanner Agilent G2505B. For each sample, a dye-swap replicate was performed. To visually inspect the relationships among samples, unsupervised clustering analysis was applied to the ratios between each DN, RGPM , VGPM or MTS sample and the average profile of all the CMN included into the study, just selecting transcripts with p-value less than 0.01 in at least half of the samples considered. Transcripts modulated in each progression step were then selected by applying a cut-off of 2 for the B value yielded by the limma procedure applied on original ratios (sample over common reference). Initially, DN were included into either the CMN class or the RGPM one, according to the degree of their cyto-architectural atypia However, the presence of such lesions among either CMN or RGPM made the distinction between these two classes and between RGPM and VGPM much less clear. Therefore, to prevent the increasing of heterogeneity, DN were excluded and this allowed the detection of many more differentially expressed transcripts between CMN and RGPM and, especially, between RGPM and VGPM. The great difference found between metastases and the rest of the samples was consistent with unsupervised results. Enrichment of Gene Ontology categories (in particular, biological processes) and gene networks or cellular pathways was performed on each list by using DAVID functional annotation tool and applying a cut-off of 0.01 on the enrichment score obtained. For each progression step, some genes previously described as differentially expressed were found. Furthermore, other novel gene transcripts, never reported as involved in melanoma progression, were identified. The expression deregulation of some of these novel genes was then confirmed on an independent cohort of samples by RT-qPCR.
2009-10-10 | E-GEOD-12391 | biostudies-arrayexpress
Project description:Deregulated expression of ETS transcription factors with oncogenic and tumor suppressor function occurs frequently in prostate cancer leading to profound alterations of the cancer transcriptome. By integrating genomic and functional studies we identified key targets of the aberrantly expressed ETS factors, ERG and ESE3. Altered expression of ETS factors led to the induction of the polycomb group protein EZH2 and silencing of the tumor suppressor Nkx3.1. Nkx3.1 was controlled by ERG and ESE3 both directly via binding to ETS binding sites in the gene promoter and indirectly via EZH2-induced histone H3K27 methylation. This may represent a general mechanism linking aberrantly expressed ETS with deregulation of epigenetic pathways and global reprogramming of the prostate epithelial cell transcriptome in prostate tumorigenesis. Keywords: prostate cancer, gene expression profiling, ETS genes In this study we show that deregulated expression of ETS factors with opposite functions is highly frequent in prostate cancer. Our study uncovers a previously unrecognized link between aberrant expression of ETS factors, deregulation of epigenetic pathways and silencing of tumor suppressor genes in prostate cancer and shows that partially distinct transcriptional programs are associated with different ETS gene expression patterns. The presence of distinct prostate cancer subgroups with different biological features may have important clinical implications and suggests that assessment of ETS expression levels might be useful to distinguish tumors with different clinical outcome.
2010-06-09 | E-GEOD-14206 | biostudies-arrayexpress
Project description:We analyzed mutations in Epidermal Growth Factor Receptor (EGFR) Tyrosine kinase (TK) domain, EGFR expression and gene profiling in prostate carcinoma (PC) in order to find out molecular prognostic markers and supply a proof for EGFR targeted therapies. 100 glyofixx-fixed, paraffin-embedded PC specimens were recovered after radical prostatectomy from locally advanced PC patients. Exons from 18 to 21 of EGFR TK domain were amplified and sequenced. For the entire cohort, EGFR protein evaluation by immunohistochemistry was performed. Gene expression profile was analyzed on 51 out of 100 samples by whole genome microarray. Statistical tests were performed in order to detect any significant association between EGFR iperexpression and prognosis. None out of 100 specimens presented mutations in exon 18; 2 point mutations were identified in exon 19, 5 in exon 20 and 6 in exon 21. In addiction, 58 out of 100 patients had the same silent mutation, at codon 787 in exon 20. EGFR iper-expression was found in 36% of specimens and was significantly associated with biochemical relapse. Gene profiling analysis on mutated samples selected 29 modulated genes differentially expressed between mutated EGFR+ and mutated EGFR- samples; 4 down-regulated genes, EAF2, ABCC4, KLK3 and ANXA3 and one up-regulated gene, FOXC1, are involved in prostate cancer progression. Our findings suggest that a subgroup of PC patients could potentially benefit of EGFR targeted therapies. The EGFR protein evaluation could contribute to identify PC relapsers. Keywords: EGFR expression, TK mutations, target therapy, microarray data. In this work we studied mutation status and expression of Epidermal Growth Factor Receptor (EGFR) in a case series of 100 primary prostate cancer tissue specimens. Results indicate that 13% and 36% of PC patients presents EGFR tyrosine kinase domain mutations and iperexpression respectively, suggesting that this receptor could be a therapeutic target in progressive prostate cancer. The analysis of correlation between EGFR protein expression, mutations, clinical parameters and outcome allow us to identify EGFR as significantly associated with biochemical relapse and high Gleason score. Gene expression profiling in 51 of PC tissues led to the identification of a gene list which separated EGFR mutated patients according to EGFR protein expression. These results could give more information on clinical outcome and possible development of new targeted therapies.
2011-06-30 | E-GEOD-16120 | biostudies-arrayexpress
Project description:Gene expression profiles of 402-91 FUS-CHOP type I cells have been compared to 402-91/ET cells made resistant to Trabectedin Experiments were performed on purified RNA from independent batches of 402-91 and 402-91/ET cells without drug treatment.
2010-07-13 | E-GEOD-20350 | biostudies-arrayexpress
Project description:The oncogenic mutation BRAFV600E is a common event in nevi and melanomas, which are aggressive skin tumors characterized by MAPK signaling pathway activation. It has been observed that mutated benign melanocytic lesions can remain unchanged for decades, but also proliferate or undergo oncogene-induced senescence. The purpose of this study was to investigate the presence of a gene expression signature in BRAFV600E mutated nevi compared to wild-type ones, all derived from sun exposed sites. Microdissected tissues from excisional biopsies of acquired nevi were analyzed to detect the presence of BRAF and NRAS mutations and to profile whole genome expression by means of oligonucleotide microarrays. BRAFV600E mutation was evidenced in 64% of nevi, while no NRAS mutations were detected. Functional analysis of genes differentially expressed between wild-type and mutated lesions pointed out the role of oxidative stress in causing the oncogenic BRAF mutation and that the direct consequence of constitutive activation of BRAF-MEK-ERK pathway, in a benign contest, is the activation of apoptosis, autophagy and senescence. Our data also indicate that in mutated lesions, p53 plays a crucial role in the maintenance of the benign status. In our study, in order to understand the role of BRAF mutation in acquired nevi, we performed gene expression profiling analysis on fourteen lesions derived from intermittently exposed sites, using whole genome oligonucleotide microarrays. Expression data were coupled with sequencing results on BRAF and NRAS genes and class comparison algorithms were applied to detect sequences differentially expressed between BRAFV600E and BRAFwt-NRASwt lesions. Some of the obtained results were further validated by Real-Time RT-PCR on an independent cohort of samples. Mutation analysis evidenced BRAF mutations in 64% of the common acquired nevi. All the mutations consisted in the substitution V600E (T1799A). No alteration was found on exon 11 of the BRAF gene nor on exons 1 and 2 of the NRAS gene, as expected since no lesion came from chronically exposed sites. Transcriptome analysis was carried out using oligo-microarrays and differential expression was tested applying the Linear Models package LIMMA available at www.bioconductor.org that uses Bayesian statistics to compute the probability of a gene being differentially expressed. Only 25 transcripts had B value greater then 2. We then performed a one-way ANOVA, dividing samples into three classes according to their BRAF mutational status (wt, ++, +++) as assessed by pyrosequencing. We found 2882 known transcripts by selecting those with ANOVA p-value<0.01 and increasing or decreasing expression trends along the three classes. Functional analysis was performed using the MetaCoreTM software package to identify overrepresented functional processes. BRAF signature, acquired nevi, gene expression profiling "++ and +++" refer to the percentage of alleles carrying the mutation in the *BRAF*V600E lesions. This has been assessed by pyrosequencing analysis (Venesio et al, 2008. PMID= 18408659).
2016-06-30 | E-GEOD-23976 | biostudies-arrayexpress
Project description:Transcription Profile of human 46BR.1G1 fibroblasts (European Collection of Cell Cultures # CB2577) compared with the 46BR.1G1 derivative cell line 7A3 expressing wild type LigI (Soza et al., Mol Cell Biol , 2009, 29: 2032-2041) One-condition experiment 46BR.1G1 vs. 7A3 cells with dye-swap.
2015-07-07 | E-GEOD-56317 | biostudies-arrayexpress
Project description:Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little prospect for cure. Despite some advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, understanding the biological mechanisms underpinning EOC progression is critical for its treatment and to ameliorate patients survival. Recently, we reported that CD157 is expressed in EOC and controls tumor cell migration and invasion. Using stable overexpression and knockdown in OVCAR-3 and OV-90 ovarian cancer cell lines, we demonstrated that CD157 overexpression promotes morphological and functional changes, characterized by downregulation of epithelial marker and upregulation of mesenchymal ones. These are mediated at the transcriptional level by altering the expression of Snail and Zeb1 transcriptional repressors. The effects of CD157 overexpression on ovarian cancer phenotype translate into increased tumor cell motility and mesotelial invasion, while its knockdown significantly reduces the migratory potential, implying a direct correlation between CD157 expression levels and EOC aggressiveness. The analysis of the transcriptomic profiling highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing EOC cells. The overall picture deduced from the analysis of these modulated transcripts indicated that high CD157 expression results in strengthening of a number of biological functions that favour tumor progression (including cell differentiation, cell motility and migration), and weakening of selected biological processes that hinder the tumor progression (such as apoptosis, cell death and response to stress). Collectively, these data support a causal role of CD157 in the control of ovarian cancer progression motivating the existence of a direct correlation between the expression levels of CD157 and the adverse clinical outcome in EOC patients, and suggest that CD157 may represent a valuable therapeutic target. Gene expression analysisi of control cell lines (OVCAR-3/mock and OV-90/mock) and testing cell lines (OVCAR-3/CD157 and OV-90/CD157), with two replicates, with dye swap, performed for each sample.
2012-12-31 | E-GEOD-36364 | biostudies-arrayexpress
Project description:Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is therefore urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the non-receptor tyrosine kinase Src, observing promising antitumor effects of its small molecule inhibitor Saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from BTC patients. Upon Saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28 and TGBC1-TKB. The effect of Saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose Saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G0/G1 phase, and inhibited cell migration. At high concentrations (median dose from 2.26 to 6.99 µM), Saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, Saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. We here provide a demonstration that the targeted inhibition of Src kinase by Saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of Saracatinib-based clinical applications. EGI-1 cell line treated with Saracatinib at the dose of 10 µM vs EGI-1 cell line untreated; EGI-1 xenograft treated with Saracatinib at the dose of 25 mg/Kg/die vs EGI-1 xenograft untreated Transcriptional alteration mediated by Saracatinib in vitro and in vivo
2012-08-02 | E-GEOD-36622 | biostudies-arrayexpress
Project description:Sexual differentiation in vertebrates is initiated during embryogenesis and leads to the development of individuals into phenotypic males or females. The molecular pathways showing sexual dimorphic patterns have been principally investigated in the gonads. In other organs, much less is known about the gender-associated pattern of gene expression. The brain plays a coordinating role on sexual function in both genders including regulation of reproductive development and maturation through the synthesis of neuropeptide hormones and the regulation of sexual behavior in both males and females. In this study, we aimed at identifying molecular pathways regulated in a gender specific manner in breeding zebrafish in order to generate a greater understanding of the gender specific physiology of the brain. To do so, we measured the gene expression profiles of individual brains of 6 males and 5 females using a 17k oligonucleotide microarray, and interrogated the dataset for genes showing a gender-specific gene expression profile. 42 genes were found to be differentially expressed between males and females from which 18 genes were over-expressed in males and 24 genes were over-expressed in females. Sexually mature fish (6 males and 6 females) were kept in a 15L tank and allowed to breed naturally. After a 20-day period, fish were sacrificed humanly and the brain was extracted, fixed in RNAlater (Ambion) and stored at -20oC until required for microarray analysis. RNA was extracted using the TRI reagent method followed by a LiCl precipitation (Sigma) according to the manufacturer's instructions. Total RNA was amplified and labeled using the Amino Allyl MessageAmp aRNA amplification kit (Ambion). A reference design was used for the array hybridizations. Array analysis was performed according to established protocols (described in Santos et al., 2007) and the data was extracted and queried for differences in gene expression profiles between genders.
2010-05-27 | E-GEOD-6853 | biostudies-arrayexpress