A Functional and Regulatory Network Associated with PIP Expression in Human Breast Cancer

BACKGROUND: The PIP (prolactin-inducible protein) gene has been shown to be expressed in breast cancers, with contradictory results concerning its implication. As both the physiological role and the molecular pathways in which PIP is involved are poorly understood, we conducted combined gene expression profiling and network analysis studies on selected breast cancer cell lines presenting distinct PIP expression levels and hormonal receptor status, to explore the functional and regulatory network of PIP co-modulated genes. PRINCIPAL FINDINGS: Microarray analysis allowed identification of genes co-modulated with PIP independently of modulations resulting from hormonal treatment or cell line heterogeneity. Relevant clusters of genes that can discriminate between [PIP+] and [PIP-] cells were identified. Functional and regulatory network analyses based on a knowledge database revealed a master network of PIP co-modulated genes, including many interconnecting oncogenes and tumor suppressor genes, half of which were detected as differentially expressed through high-precision measurements. The network identified appears associated with an inhibition of proliferation coupled with an increase of apoptosis and an enhancement of cell adhesion in breast cancer cell lines, and contains many genes with a STAT5 regulatory motif in their promoters. CONCLUSIONS: Our global exploratory approach identified biological pathways modulated along with PIP expression, providing further support for its good prognostic value of disease-free survival in breast cancer. Moreover, our data pointed to the importance of a regulatory subnetwork associated with PIP expression in which STAT5 appears as a potential transcriptional regulator

Deciphering cellular states of innate tumor drug responses

BACKGROUND: The molecular mechanisms underlying innate tumor drug resistance, a major obstacle to successful cancer therapy, remain poorly understood. In colorectal cancer (CRC), molecular studies have focused on drug-selected tumor cell lines or individual candidate genes using samples derived from patients already treated with drugs, so that very little data are available prior to drug treatment. RESULTS: Transcriptional profiles of clinical samples collected from CRC patients prior to their exposure to a combined chemotherapy of folinic acid, 5-fluorouracil and irinotecan were established using microarrays. Vigilant experimental design, power simulations and robust statistics were used to restrain the rates of false negative and false positive hybridizations, allowing successful discrimination between drug resistance and sensitivity states with restricted sampling. A list of 679 genes was established that intrinsically differentiates, for the first time prior to drug exposure, subsequently diagnosed chemo-sensitive and resistant patients. Independent biological validation performed through quantitative PCR confirmed the expression pattern on two additional patients. Careful annotation of interconnected functional networks provided a unique representation of the cellular states underlying drug responses. CONCLUSION: Molecular interaction networks are described that provide a solid foundation on which to anchor working hypotheses about mechanisms underlying in vivo innate tumor drug responses. These broad-spectrum cellular signatures represent a starting point from which by-pass chemotherapy schemes, targeting simultaneously several of the molecular mechanisms involved, may be developed for critical therapeutic intervention in CRC patients. The demonstrated power of this research strategy makes it generally applicable to other physiological and pathological situations

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

publication en ligne. Article dans revue scientifique avec comité de lecture. nationale.National audienceThe human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

ETUDE DE LA GP17, UN MARQUEUR DES SECRETIONS APOCRINES (CONSEQUENCES FONCTIONNELLES DE L'INTERACTION GP17/CD4 SUR LA SENSIBILITE DES LYMPHOCYTES T CD4+ A LA STIMULATION DU RECEPTEUR DE L'ANTIGENE (DOCTORAT : IMMUNOLOGIE))

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Analyse de la région chromosomique 7q34-35 dans le cancer du sein (PIP et ARHGEF5, deux gènes sujets à des altérations)

Le gène PIP est exprimé dans 60% des carcinomes mammaires et localisé en 7q34 dans une région chromosomique souvent touchée par des remaniements dans le cancer du sein. Nous montrons que, dans certains cas de carcinomes primaires du sein, la région 3' du gène PIP est amplifiée et impliquée dans la formation de petites molécules d'ADN extrachromosomique circulaires (spcDNA). La formation de ces molécules est associée à la présence d'une instabilité génétique. L'étude par FISH de la région chromosomique 7q34-q35, dans la lignée de carcinome mammaire T47D qui surexprime PIP, montre que cette région est le siège d'une répétition inversée, située "in loco". Cette altération est caractéristique d'un mécanisme de "cassure-fusion-pont" (CFP), impliqué dans l'amplification d'oncogènes dans différents types de tumeurs. Nous montrons que le site fragile FRA7I borde l'extrémité distale de la duplication et que la chromatide cassée a été cicatrisée par le fragment télomérique 7q. Ceci suggère que le processus de CFP est déclenché par une cassure initiale au niveau de FRA7I et limité à un seul cycle. Cette observation représente un exemple unique des produits initiaux du mécanisme de CFP et de son déclenchement in vivo par activation d'un site fragile. L'oncogène ARHGEF5 étant localisé dans la région dupliquée, nous avons étudié son expression dans le cancer du sein. Il code une protéine de la famille des RhoGEF et est très mal connu. Nous avons découvert des variants d'épissage de ARHGEF5, spécifiques de la glande mammaire tumorale, et caractérisés par l'absence d'un ou plusieurs exons, codant le domaine catalytique DH de la protéine. L'expression cellulaire de la protéine ARHGEF5 sauvage montre qu'elle induit un remodelage du cytosquelette, évocateur d'une activité des Rho GTPases cdc42 et Racl. L'altération de l'activation de ces Rho GTPases par les protéines ARHGEF5 variantes pourrait jouer un rôle important dans la carcinogenèse mammaire.The PIP gene is expressed in 60% of primary and metastatic breast carcinomas and is localized on the long arm of chromosome 7 which is frequently rearranged in breast and others cancers. We characterized PIP gene rearrangements occurring in breast cancer. We demonstrated that the 3' end of the PIP gene is amplified and involved in the formation of extrachromosomal small circular DNA molecules (spcDNA) in some breast cancers. The spcDNA molecules formation has been associated with genome instability. In this frame, we analyzed by FISH the chromosome region 7q34-q35 containing PIP. We demonstrated in the breast carcinoma cells T47D overexpressing PIP, that the gene is duplicated as a part of a large inverted repeat localized at a normal position on 7q. Moreover, we localized FRA7I to 7q35 and we show that FRA7I sets the telomeric boundary of the duplicated region. Altogether, our results strongly suggest that the duplication was generated by one cycle of BFB initiated from a break at FRA7I. Our results highlight for the first time that BFB cycles can be initiated in vivo by events taking place within common fragile site in tumor cells. Because of its localization close to FRA7I, we examined the expression of the ARHGEF5 oncogene in breast cancer. ARHGEF5 encodes a protein of the RhoGEF family. We report the identification of rive novel ARHGEF5 alternative transcripts specifically expressed in breast tumours. These variant transcripts were characterized by the absence of one or several exons, all coding for the catalytic Dbl-homology (DH) domain. Moreover, the expression of recombinant ARHGEF5 protein generates a cytoskeleton reorganization. A Rho GTPase alterated activation mediated by ARHGEF5 variant proteins could be play an important role in breast carcinogenesis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Modifications structurales et transcriptionnelles des gènes PIP et ARHGEF5 dans le cancer du sein (étude du réseau de régulation génique associé à l'expression du gène PIP)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF