Biomedical subjects

Agnes Viale

Publications and source records attributed to Agnes Viale.

At least 19 recordsLinked to original sources

PubMed2006

Oncogenic Kit signaling and therapeutic intervention in a mouse model of gastrointestinal stromal tumor.

Kit receptor-activating mutations are critical in the pathogenesis of gastrointestinal stromal tumors (GIST). We investigated mechanisms of oncogenic Kit signaling and the consequences of therapeutic intervention in a mouse model of human GIST. Treatment of GIST mice with imatinib decreased cell proliferation and increased apoptosis in the tumor. Analysis of tumor tissue from imatinib-treated mice showed diminished phosphatidylinositol 3-kinase (PI3-kinase) and mammalian target of rapamycin (mTOR) signaling suggesting that oncogenic Kit signaling critically contributes to the translational response in GIST. Treatment with RAD001 (everolimus), an mTOR inhibitor, diminished the translational response and cell proliferation in tumor lesions, pointing to mTOR inhibition as a therapeutic approach for imatinib-resistant GIST. Analysis of RNA expression profiles in GIST lesions with and without imatinib treatment showed changes in expression of IFN-inducible genes and cell cycle regulators. These results convincingly show that KitV558Delta/+ mice represent a unique faithful mouse model of human familial GIST, and they demonstrate the utility of these mice for preclinical investigations and to elucidate oncogenic signaling mechanisms by using genetic approaches and targeted pharmacological intervention.

Animals↗

PubMed2006

The ABRF MARG microarray survey 2005: taking the pulse of the microarray field.

Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.

Animals↗

PubMed2005

Genes that mediate breast cancer metastasis to lung.

By means of in vivo selection, transcriptomic analysis, functional verification and clinical validation, here we identify a set of genes that marks and mediates breast cancer metastasis to the lungs. Some of these genes serve dual functions, providing growth advantages both in the primary tumour and in the lung microenvironment. Others contribute to aggressive growth selectively in the lung. Many encode extracellular proteins and are of previously unknown relevance to cancer metastasis.

Breast Neoplasms↗

PubMed2005

Polycomb group protein ezh2 controls actin polymerization and cell signaling.

Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

Actins↗

PubMed2005

Use of laser-capture microdissection for the identification of marker genes for the ventromedial hypothalamic nucleus.

The ventromedial hypothalamic nucleus (VMH) plays an important role in the control of feeding and energy homeostasis. In contrast to other hypothalamic nuclei that are also known to regulate energy balance, there is a paucity of nucleus-specific marker genes for the VMH, limiting the application of molecular approaches for analyzing VMH information processing, function, and circuitry. Here, we report the use of laser-capture microdissection to isolate a set of cDNAs that are enriched in the VMH relative to two adjacent hypothalamic nuclei, the arcuate and dorsomedial hypothalamus. The relative expression levels of nine of the 12 most robustly expressed VMH-enriched genes were confirmed by real-time PCR analysis using separate RNAs from these three nuclei. Three of these VMH-enriched genes were further characterized by in situ hybridization histochemistry, including pituitary adenylate cyclase activating polypeptide, cerebellin 1, and an expressed sequence tag named LBH2. Finally, to test whether some of these genes were coordinately regulated, we monitored their expression in steroidogenic factor 1 (SF-1) knock-out mice. SF-1 is a transcription factor that controls the development of the VMH. The RNA levels for four of these genes were reduced in these knock-out animals, further suggesting that they are direct or indirect targets of this orphan nuclear receptor. The VMH-enriched genes identified here provide a basis for a functional analysis of VMH neuronal subpopulations via the use of bacterial artificial chromosome transgenics and related technologies. These results also demonstrate the utility of laser-capture microdissection coupled with microarray technology to identify nucleus-specific transcriptional networks.

Animals↗

PubMed2005

Gene expression profiling allows distinction between primary and metastatic squamous cell carcinomas in the lung.

Lung neoplasms commonly develop in patients previously treated for head and neck carcinomas. The derivation of these tumors, either as new primary lung cancers or as metastatic head and neck cancers, is difficult to establish based on clinical or histopathologic criteria since both are squamous cell carcinomas and have identical features under light microscopy. However, this distinction has significant treatment and prognostic implications. Gene expression profiling was performed on a panel of 52 sequentially collected patients with either primary lung (n = 21) or primary head and neck (n = 31) carcinomas using the Affymetrix HG_U95Av2 high-density oligonucleotide microarray. Unsupervised hierarchical clustering with Ward linkage and the Pearson correlation metric was performed. To assess robustness, bootstrap resampling was performed with 1,000 iterations. A t test of the normalized values for each gene was used to determine the genes responsible for segregating head and neck from lung primary carcinomas, and those with the most differential expression were used for later analyses. In the absence of a large "test" set of tumors, we used a supervised leave-one-out cross-validation to test how well we could predict the tumor origin. Once a gene expression profile was established, 12 lung lesions taken from patients with previously treated head and neck cancers were similarly analyzed by gene expression profiling to determine their sites of origin. Unsupervised clustering analysis separated the study cohort into two distinct groups which reliably remained segregated with bootstrap resampling. Group 1 consisted of 30 tongue carcinomas. Group 2 consisted of 21 lung cancers and 1 tongue carcinoma. The clustering was not changed even when normal lung or tongue profiles were subtracted from the corresponding carcinomatous lesions, and a leave-one-out cross-validation showed a 98% correct prediction (see Supplementary Data 1). A minimum set of 500 genes required to distinguish these groups was established. Given the ability to segregate these lesions using molecular profiling, we analyzed the lung tumors of undetermined origin. All cases clearly clustered with either lung or tongue tumor subsets, strongly supporting our hypothesis that this technique could elucidate the tissue of origin of metastatic lesions. Although histologically similar, squamous cell carcinomas have distinct gene expression profiles based on their anatomic sites of origin. Accordingly, the application of gene expression profiling may be useful in identifying the derivation of lung nodules and consequently enhances treatment planning.

Algorithms↗

PubMed2005

Identification of cancer-testis genes expressed by melanoma and soft tissue sarcoma using bioinformatics.

Cancer-testis or germ cell antigens (GCAs) are a category of tumor antigens expressed by male germ cells and by cancers of diverse histological origin, but not usually by normal adult somatic tissue. These antigens include products encoded by the MAGE, BAGE, GAGE, SSX, and LAGE/NY-ESO-1 families that encode antigenic peptides recognized by T lymphocytes. In this study, we exploit oligonucleotide technology to identify genes in melanoma and soft tissue sarcoma (STS) that display a cancer-testis/GCA expression profile. We identified 59 such genes, including GCAs we knew to be recognized by T lymphocytes. Among our findings are the expression of PRAME in monophasic synovial sarcoma, PRAME and NY-ESO-1 in myxoid/round cell liposarcoma, and SSX2 and members of the GAGE family in malignant fibrous histiocytoma. Furthermore, the proto-oncogene DBL/MCF2 was identified as encoding a novel candidate GCA expressed by clear cell sarcoma/melanoma of soft parts (MSP). DBL/MCF2 peptides that are bound to HLA-A*0201 were identified and recognized by T lymphocytes. These results show the utility of high-throughput expression analysis in the efficient screening and identification of GCA candidates in cancer, and its application to the discovery of candidate targets for T cell immunity against GCAs expressed by cancer.

Adult↗

PubMed2005

Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays.

A key step in bringing gene expression data into clinical practice is the conduct of large studies to confirm preliminary models. The performance of such confirmatory studies and the transition to clinical practice requires that microarray data from different laboratories are comparable and reproducible. We designed a study to assess the comparability of data from four laboratories that will conduct a larger microarray profiling confirmation project in lung adenocarcinomas. To test the feasibility of combining data across laboratories, frozen tumor tissues, cell line pellets, and purified RNA samples were analyzed at each of the four laboratories. Samples of each type and several subsamples from each tumor and each cell line were blinded before being distributed. The laboratories followed a common protocol for all steps of tissue processing, RNA extraction, and microarray analysis using Affymetrix Human Genome U133A arrays. High within-laboratory and between-laboratory correlations were observed on the purified RNA samples, the cell lines, and the frozen tumor tissues. Intraclass correlation within laboratories was only slightly stronger than between laboratories, and the intraclass correlation tended to be weakest for genes expressed at low levels and showing small variation. Finally, hierarchical cluster analysis revealed that the repeated samples clustered together regardless of the laboratory in which the experiments were done. The findings indicate that under properly controlled conditions it is feasible to perform complete tumor microarray analysis, from tissue processing to hybridization and scanning, at multiple independent laboratories for a single study.

Adenocarcinoma↗

PubMed2005

Distinction of desmoplastic melanoma from non-desmoplastic melanoma by gene expression profiling.

Desmoplastic melanoma (DM) is a variant of melanoma characterized by the presence of amelanotic fusiform melanocytes dispersed in a prominent collagenous stroma. DM behaves differently from conventional non-desmoplastic melanoma (NDM). It has a higher tendency for local recurrence and is less likely to metastasize to regional lymph nodes. In this study, we explored the possibility of distinguishing DM from NDM by gene expression profiling. RNA samples from ten primary cutaneous melanomas of similar depth of invasion were analyzed using the Affymetrix U133A oligonucleotide platform. Four tumors were DM, five were ND, and one tumor showed combined features of desmoplastic and conventional. Hierarchical cluster analysis clearly separated DM from NDM. The expression of a number of melanocyte differentiation genes was decreased in DM compared with NDM, which corresponded to immunohistochemical results. Various genes were upregulated in DM, including neurotrophic factors and genes involved in extracellular matrix production. A novel finding was the high expression of clusterin in DM, which was confirmed by immunohistochemical studies. Our results from gene expression profiling validate the distinction of DM from NDM. They also provide the opportunity to learn more about the biology of DM which had previously not yet been associated with this variant of melanoma.

Biomarkers, Tumor↗

PubMed2004

Evidence for a p27 tumor suppressive function independent of its role regulating cell proliferation in the prostate.

Reduced p27 levels correlate with poor prognosis in a wide spectrum of human tumors and can accelerate tumorigenesis in mouse tissues. To determine whether p27 deficiency can accelerate tumorigenesis in tissues with inactive Rb and p53 pathways, we examined the effect of p27 status on prostate tumorigenesis in mice expressing simian virus 40 large T antigen (LT). In p27-deficient mice expressing LT, tumors progressed from high-grade prostatic intraepithelial neoplasia to poorly differentiated carcinoma at a greatly accelerated rate. p27 deficiency could not collaborate with a mutant of LT that fails to inactivate the Rb pathway alone. Furthermore, p27 deficiency does not increase the proliferation index, reduce the apoptotic index, or affect the expression of E2F-dependent genes in cells expressing LT at any stage of the disease. Expression of LT alone leads to maximal proliferation, but p27 deficiency still increases the amount of cyclin A and cyclin-dependent kinase 2-associated kinase activity in tissues. Interestingly, this model recapitulates an important feature of the human disease, specifically a high frequency of allelic loss of chromosome 16q, which is syntenic to mouse chromosome 8. Loss of heterozygosity may accelerate the inactivation of other tumor suppressors, such as E-cadherin, which are located in this interval. These experiments provide direct physiological and causal evidence that p27 has tumor suppressive functions independent of its role regulating cell proliferation.

Animals↗

PubMed2004

Transformation fingerprint: induced STAT3-C, v-Src and Ha-Ras cause small initial changes but similar established profiles in mRNA.

Induced transformation of mouse fibroblasts was carried out by releasing tetracycline-repressed expression of an oncogenic mutant of STAT3, STAT3-C, or of v-Src or Ha-Ras. At 15 days after derepression of each oncogene, DNA microarrays showed elevation (>3-fold) of a similar group of approximately 25 mRNAs compared to untransformed cells. RT-PCR confirmed a number of these mRNA elevations. RNA samples were then analysed at intervals during the first 24 h after doxycycline removal to determine the time of early changes. Extensive changes were not observed by array analysis, except in v-Src-expressing cells where about 10 mRNAs were elevated threefold or more. However, RT-PCR did uncover changes in each derepressed cell type that included some of the changes observed after the 15-day transformation period. In addition, STAT3-C target genes such as BclXI and cyclin D1, which were not observed on array analysis, were elevated by RT-PCR analysis. We conclude, therefore, that early after oncogene induction, transcriptional changes, including those initiated by STAT3-C, may occur only in scarce mRNA and/or to a limited extent. However, with additional time and probably additional cell division, a new epigenetic state is established that is mirrored by a changed transcriptional profile emblematic of transformation by each of three oncogenes.

Animals↗

PubMed2004

Casein kinase II alpha subunit and C1-inhibitor are independent predictors of outcome in patients with squamous cell carcinoma of the lung.

PURPOSE: Gene expression profiling has been shown to be a valuable tool for prognostication and identification of cancer-associated genes in human malignancies. We aimed to identify potential prognostic marker(s) in non-small cell lung cancers using global gene expression profiles. EXPERIMENTAL DESIGN: Twenty-one previously untreated patients with non-small cell lung cancer were analyzed using the Affymetrix GeneChip high-density oligonucleotide array and comparative genomic hybridization. Identified candidate genes were validated in an independent cohort of 45 patients using quantitative real-time reverse transcription-PCR and Western blot analyses. Follow-up data for these patients was collected and used to assess outcome correlations. RESULTS: Hierarchical clustering analysis yielded three distinct subgroups based on gene expression profiling. Cluster I consisted of 4 patients with adenocarcinoma and 1 with squamous cell carcinoma (squamous cell carcinoma); clusters II and III consisted of 6 and 10 patients with squamous cell carcinoma, respectively. Outcome analysis was performed on the cluster groups containing solely squamous cell carcinoma, revealing significant differences in disease-specific survival rates. Moreover, patients having a combination of advanced Tumor-Node-Metastasis stage and assigned to the poor prognosis cluster group (cluster II) had significantly poorer outcomes. Comparative genomic hybridization analysis showed recurrent chromosomal losses at 1p, 3p, 17, 19, and 22 and gains/amplifications at 3q, 5p, and 8q, which did not vary significantly between the cluster groups. We internally and externally validated a subset of 11 cluster II (poor prognosis)-specific genes having corresponding chromosomal aberrations identified by comparative genomic hybridization as prognostic markers in an independent cohort of patients with lung squamous cell carcinoma identifying CSNK2A1 and C1-Inh as independent predictors of outcome. CONCLUSION: CSNK2A1 and C1-Inh are independent predictors of survival in lung squamous cell carcinoma patients and may be useful as prognostic markers.

Aged↗

PubMed2004

Gene expression in gastrointestinal stromal tumors is distinguished by KIT genotype and anatomic site.

PURPOSE: Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors. EXPERIMENTAL DESIGN: An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction. RESULTS: Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution. CONCLUSIONS: GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.

Cell Line, Tumor↗

PubMed2004

A comparison of cDNA, oligonucleotide, and Affymetrix GeneChip gene expression microarray platforms.

We have conducted a study to compare the variability in measured gene expression levels associated with three types of microarray platforms. Total RNA samples were obtained from liver tissue of four male mice, two each from inbred strains A/J and C57BL/6J. The same four samples were assayed on Affymetrix Mouse Genome Expression Set 430 GeneChips (MOE430A and MOE430B), spotted cDNA microarrays, and spotted oligonucleotide microarrays using eight arrays of each type. Variances associated with measurement error were observed to be comparable across all microarray platforms. The MOE430A GeneChips and cDNA arrays had higher precision across technical replicates than the MOE430B GeneChips and oligonucleotide arrays. The Affymetrix platform showed the greatest range in the magnitude of expression levels followed by the oligonucleotide arrays. We observed good concordance in both estimated expression level and statistical significance of common genes between the Affymetrix MOE430A GeneChip and the oligonucleotide arrays. Despite their apparently high precision, cDNA arrays showed poor concordance with other platforms.

Analysis of Variance↗

PubMed2003

The oncogene Nup98-HOXA9 induces gene transcription in myeloid cells.

The nucleoporin Nup98 gene is frequently rearranged in acute myelogenous leukemia (AML). In most cases this results in fusion of the N terminus of Nup98 to the DNA binding domain of a homeodomain transcription factor. The prototype of these fusions, Nup98-HOXA9, is associated with human AML and induces AML in mouse models. To understand the mechanisms by which Nup98-HOXA9 causes AML, we expressed it in myeloid cells and identified its target genes using high density oligonucleotide microarrays. The analysis was performed in triplicate and was confirmed by quantitative real time PCR. Of the 102 Nup98-HOXA9 target genes identified, 92 were up-regulated, and only 10 were down-regulated, suggesting a transcriptional activation function. A similar analysis of wild-type HOXA9 revealed 13 target genes, 12 of which were up-regulated, and 1 was down-regulated. In contrast, wild-type Nup98 had no effect on gene expression, demonstrating that the HOXA9 DNA binding domain is required for gene regulation. Co-transfection experiments using a luciferase reporter linked to the promoter of one of the Nup98-HOXA9 target genes confirmed up-regulation at the transcriptional level by Nup98-HOXA9 but not by either HOXA9 or Nup98. These data indicate that Nup98-HOXA9 is an aberrant transcription factor whose activity depends on the HOXA9 DNA binding domain but has a stronger and wider transcriptional effect than HOXA9. Several of the genes regulated by Nup98-HOXA9 are associated with increased cell proliferation and survival as well as drug metabolism, providing insights into the pathogenesis and epidemiology of Nup98-HOXA9-induced AML.

Animals↗

PubMed2003

Classification of clear-cell sarcoma as a subtype of melanoma by genomic profiling.

PURPOSE: To develop a genome-based classification scheme for clear-cell sarcoma (CCS), also known as melanoma of soft parts (MSP), which would have implications for diagnosis and treatment. This tumor displays characteristic features of soft tissue sarcoma (STS), including deep soft tissue primary location and a characteristic translocation, t(12;22)(q13;q12), involving EWS and ATF1 genes. CCS/MSP also has typical melanoma features, including immunoreactivity for S100 and HMB45, pigmentation, MITF-M expression, and a propensity for regional lymph node metastases. MATERIALS AND METHODS: RNA samples from 21 cell lines and 60 pathologically confirmed cases of STS, melanoma, and CCS/MSP were examined using the U95A GeneChip (Affymetrix, Santa Clara, CA). Hierarchical cluster analysis, principal component analysis, and support vector machine (SVM) analysis exploited genomic correlations within the data to classify CCS/MSP. RESULTS: Unsupervised analyses demonstrated a clear distinction between STS and melanoma and, furthermore, showed that CCS/MSP cluster with the melanomas as a distinct group. A supervised SVM learning approach further validated this finding and provided a user-independent approach to diagnosis. Genes of interest that discriminate CCS/MSP included those encoding melanocyte differentiation antigens, MITF, SOX10, ERBB3, and FGFR1. CONCLUSION: Gene expression profiles support the classification of CCS/MSP as a distinct genomic subtype of melanoma. Analysis of these gene profiles using the SVM may be an important diagnostic tool. Genomic analysis identified potential targets for the development of therapeutic strategies in the treatment of this disease.

Algorithms↗

PubMed2003

Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes.

In order to determine the global effects of oncogenic Ras and Akt signaling pathways on translational efficiencies, we compared the gene expression profiles of total cellular mRNA and mRNA associated with polysomes. We found that the immediate effect of Ras and Akt signaling blockade on transcription was relatively modest; however, the profile of mRNA associated with polysomes was substantially altered. These observations indicate that the immediate effect of Ras and Akt signaling regulates the recruitment of specific mRNAs to ribosomes to a far greater extent than they regulate the production of mRNAs by transcriptional effects. The mRNAs most affected are those encoding proteins that regulate growth, transcription regulation, cell to cell interactions, and morphology. These data support a model whereby Ras and Akt signaling primarily lead to cellular transformation by altering the transcriptome and producing a radical shift in the composition of mRNAs associated with actively translating polysomes.

Animals↗

PubMed2003

Segmental duplications in euchromatic regions of human chromosome 5: a source of evolutionary instability and transcriptional innovation.

Recent analyses of the structure of pericentromeric and subtelomeric regions have revealed that these particular regions of human chromosomes are often composed of blocks of duplicated genomic segments that have been associated with rapid evolutionary turnover among the genomes of closely related primates. In the present study, we show that euchromatic regions of human chromosome 5-5p14, 5p13, 5q13, 5q15-5q21-also display such an accumulation of segmental duplications. The structure, organization and evolution of those primate-specific sequences were studied in detail by combining in silico and comparative FISH analyses on human, chimpanzee, gorilla, orangutang, macaca, and capuchin chromosomes. Our results lend support to a two-step model of transposition duplication in the euchromatic regions, with a founder insertional event at the time of divergence between Platyrrhini and Catarrhini (25-35 million years ago) and an apparent burst of inter- and intrachromosomal duplications in the Hominidae lineage. Furthermore, phylogenetic analysis suggests that the chronology and, likely, molecular mechanisms, differ regarding the region of primary insertion-euchromatic versus pericentromeric regions. Lastly, we show that as their counterparts located near the heterochromatic region, the euchromatic segmental duplications have consistently reshaped their region of insertion during primate evolution, creating putative mosaic genes, and they are obvious candidates for causing ectopic rearrangements that have contributed to evolutionary/genomic instability.

Animals↗