Publications by authors named "Pascal Belleau"

24 Publications

  • Page 1 of 1

Intraductal Transplantation Models of Human Pancreatic Ductal Adenocarcinoma Reveal Progressive Transition of Molecular Subtypes.

Cancer Discov 2020 Oct 23;10(10):1566-1589. Epub 2020 Jul 23.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common malignancy, with little improvement in patient outcomes over the past decades. Recently, subtypes of pancreatic cancer with different prognoses have been elaborated; however, the inability to model these subtypes has precluded mechanistic investigation of their origins. Here, we present a xenotransplantation model of PDAC in which neoplasms originate from patient-derived organoids injected directly into murine pancreatic ducts. Our model enables distinction of the two main PDAC subtypes: intraepithelial neoplasms from this model progress in an indolent or invasive manner representing the classical or basal-like subtypes of PDAC, respectively. Parameters that influence PDAC subtype specification in this intraductal model include cell plasticity and hyperactivation of the RAS pathway. Finally, through intratumoral dissection and the direct manipulation of gene dosage, we identify a suite of -regulated secreted and membrane-bound proteins that may represent potential candidates for therapeutic intervention in patients with PDAC. SIGNIFICANCE: Accurate modeling of the molecular subtypes of pancreatic cancer is crucial to facilitate the generation of effective therapies. We report the development of an intraductal organoid transplantation model of pancreatic cancer that models the progressive switching of subtypes, and identify stochastic and RAS-driven mechanisms that determine subtype specification...
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http://dx.doi.org/10.1158/2159-8290.CD-20-0133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664990PMC
October 2020

SOAT1 promotes mevalonate pathway dependency in pancreatic cancer.

J Exp Med 2020 09;217(9)

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.
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http://dx.doi.org/10.1084/jem.20192389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478739PMC
September 2020

Bioactivation of Napabucasin Triggers Reactive Oxygen Species-Mediated Cancer Cell Death.

Clin Cancer Res 2019 12 16;25(23):7162-7174. Epub 2019 Sep 16.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Purpose: Napabucasin (2-acetylfuro-1,4-naphthoquinone or BBI-608) is a small molecule currently being clinically evaluated in various cancer types. It has mostly been recognized for its ability to inhibit STAT3 signaling. However, based on its chemical structure, we hypothesized that napabucasin is a substrate for intracellular oxidoreductases and therefore may exert its anticancer effect through redox cycling, resulting in reactive oxygen species (ROS) production and cell death.

Experimental Design: Binding of napabucasin to NAD(P)H:quinone oxidoreductase-1 (NQO1), and other oxidoreductases, was measured. Pancreatic cancer cell lines were treated with napabucasin, and cell survival, ROS generation, DNA damage, transcriptomic changes, and alterations in STAT3 activation were assayed and . Genetic knockout or pharmacologic inhibition with dicoumarol was used to evaluate the dependency on NQO1.

Results: Napabucasin was found to bind with high affinity to NQO1 and to a lesser degree to cytochrome P450 oxidoreductase (POR). Treatment resulted in marked induction of ROS and DNA damage with an NQO1- and ROS-dependent decrease in STAT3 phosphorylation. Differential cytotoxic effects were observed, where NQO1-expressing cells generating cytotoxic levels of ROS at low napabucasin concentrations were more sensitive. Cells with low or no baseline NQO1 expression also produced ROS in response to napabucasin, albeit to a lesser extent, through the one-electron reductase POR.

Conclusions: Napabucasin is bioactivated by NQO1, and to a lesser degree by POR, resulting in futile redox cycling and ROS generation. The increased ROS levels result in DNA damage and multiple intracellular changes, one of which is a reduction in STAT3 phosphorylation.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891204PMC
December 2019

Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts.

Cancer Discov 2019 08 13;9(8):1102-1123. Epub 2019 Jun 13.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures . Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population "antigen-presenting CAFs" and find that they activate CD4 T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. SIGNIFICANCE: Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II-expressing CAFs with a capacity to present antigens to CD4 T cells, and potentially to modulate the immune response in pancreatic tumors...
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http://dx.doi.org/10.1158/2159-8290.CD-19-0094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727976PMC
August 2019

A type 2 diabetes disease module with a high collective influence for Cdk2 and PTPLAD1 is localized in endosomes.

PLoS One 2018 9;13(10):e0205180. Epub 2018 Oct 9.

Départment of Pediatrics, Faculty of Medicine, Université Laval, Centre de Recherche du CHU de Québec, Québec city, Canada.

Despite the identification of many susceptibility genes our knowledge of the underlying mechanisms responsible for complex disease remains limited. Here, we identified a type 2 diabetes disease module in endosomes, and validate it for functional relevance on selected nodes. Using hepatic Golgi/endosomes fractions, we established a proteome of insulin receptor-containing endosomes that allowed the study of physical protein interaction networks on a type 2 diabetes background. The resulting collated network is formed by 313 nodes and 1147 edges with a topology organized around a few major hubs with Cdk2 displaying the highest collective influence. Overall, 88% of the nodes are associated with the type 2 diabetes genetic risk, including 101 new candidates. The Type 2 diabetes module is enriched with cytoskeleton and luminal acidification-dependent processes that are shared with secretion-related mechanisms. We identified new signaling pathways driven by Cdk2 and PTPLAD1 whose expression affects the association of the insulin receptor with TUBA, TUBB, the actin component ACTB and the endosomal sorting markers Rab5c and Rab11a. Therefore, the interactome of internalized insulin receptors reveals the presence of a type 2 diabetes disease module enriched in new layers of feedback loops required for insulin signaling, clearance and islet biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205180PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177195PMC
March 2019

Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer.

Cancer Discov 2018 09 31;8(9):1112-1129. Epub 2018 May 31.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient-derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection. New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. .
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http://dx.doi.org/10.1158/2159-8290.CD-18-0349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125219PMC
September 2018

Inferring and modeling inheritance of differentially methylated changes across multiple generations.

Nucleic Acids Res 2018 08;46(14):7466

Département de Médecine Moléculaire - Université Laval, Faculté de médecine, Pavillon Ferdinand-Vandry, 1050 avenue de la Médecine, bureau 4633, Québec, QC G1V 0A6, Canada.

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http://dx.doi.org/10.1093/nar/gky477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101620PMC
August 2018

Inferring and modeling inheritance of differentially methylated changes across multiple generations.

Nucleic Acids Res 2018 08;46(14):e85

Département de Médecine Moléculaire - Université Laval, Faculté de médecine, Pavillon Ferdinand-Vandry, 1050 avenue de la Médecine, bureau 4633, Québec, QC G1V 0A6, Canada.

High-throughput methylation sequencing enables genome-wide detection of differentially methylated sites (DMS) or regions (DMR). Increasing evidence suggests that treatment-induced DMS can be transmitted across generations, but the analysis of induced methylation changes across multiple generations is complicated by the lack of sound statistical methods to evaluate significance levels. Due to software design, DMS detection was usually made on each generation separately, thus disregarding stochastic effects expected when a large number of DMS is detected in each generation. Here, we present a novel method based on Monte Carlo sampling, methylInheritance, to evaluate that the number of conserved DMS between several generations is associated to an effect inherited from a treatment and not randomness. Moreover, we developed an inheritance simulation package, methInheritSim, to demonstrate the performance of the methylInheritance method and to evaluate the power of different experimental designs. Finally, we applied methylInheritance to a DNA methylation dataset obtained from early-life persistent organic pollutants (POPs) exposed Sprague-Dawley female rats and their descendants through a paternal transmission. The results show that metylInheritance can efficiently identify treatment-induced inherited methylation changes. Specifically, we identified two intergenerationally conserved DMS at transcription start site (TSS); one of those persisted transgenerationally. Three transgenerationally conserved DMR were found at intra or integenic regions.
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http://dx.doi.org/10.1093/nar/gky362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101575PMC
August 2018

metagene Profiles Analyses Reveal Regulatory Element's Factor-Specific Recruitment Patterns.

PLoS Comput Biol 2016 08 18;12(8):e1004751. Epub 2016 Aug 18.

Centre de Recherche du CHU de Québec - Université Laval, Québec, Québec, Canada.

ChIP-Sequencing (ChIP-Seq) provides a vast amount of information regarding the localization of proteins across the genome. The aggregation of ChIP-Seq enrichment signal in a metagene plot is an approach commonly used to summarize data complexity and to obtain a high level visual representation of the general occupancy pattern of a protein. Here we present the R package metagene, the graphical interface Imetagene and the companion package similaRpeak. Together, they provide a framework to integrate, summarize and compare the ChIP-Seq enrichment signal from complex experimental designs. Those packages identify and quantify similarities or dissimilarities in patterns between large numbers of ChIP-Seq profiles. We used metagene to investigate the differential occupancy of regulatory factors at noncoding regulatory regions (promoters and enhancers) in relation to transcriptional activity in GM12878 B-lymphocytes. The relationships between occupancy patterns and transcriptional activity suggest two different mechanisms of action for transcriptional control: i) a "gradient effect" where the regulatory factor occupancy levels follow transcription and ii) a "threshold effect" where the regulatory factor occupancy levels max out prior to reaching maximal transcription. metagene, Imetagene and similaRpeak are implemented in R under the Artistic license 2.0 and are available on Bioconductor.
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http://dx.doi.org/10.1371/journal.pcbi.1004751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990179PMC
August 2016

Using informative Multinomial-Dirichlet prior in a t-mixture with reversible jump estimation of nucleosome positions for genome-wide profiling.

Stat Appl Genet Mol Biol 2015 Dec;14(6):517-32

Genome-wide mapping of nucleosomes has revealed a great deal about the relationships between chromatin structure and control of gene expression. Recent next generation CHIP-chip and CHIP-Seq technologies have accelerated our understanding of basic principles of chromatin organization. These technologies have taught us that nucleosomes play a crucial role in gene regulation by allowing physical access to transcription factors. Recent methods and experimental advancements allow the determination of nucleosome positions for a given genome area. However, most of these methods estimate the number of nucleosomes either by an EM algorithm using a BIC criterion or an effective heuristic strategy. Here, we introduce a Bayesian method for identifying nucleosome positions. The proposed model is based on a Multinomial-Dirichlet classification and a hierarchical mixture distributions. The number and the positions of nucleosomes are estimated using a reversible jump Markov chain Monte Carlo simulation technique. We compare the performance of our method on simulated data and MNase-Seq data from Saccharomyces cerevisiae against PING and NOrMAL methods.
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http://dx.doi.org/10.1515/sagmb-2014-0098DOI Listing
December 2015

Germline copy number variation in the YTHDC2 gene: does it have a role in finding a novel potential molecular target involved in pancreatic adenocarcinoma susceptibility?

Expert Opin Ther Targets 2014 Aug 16;18(8):841-50. Epub 2014 May 16.

University of Palermo, Department of Surgical, Oncological and Stomatological Sciences, Section of Medical Oncology , Via del Vespro 129, 90127 Palermo , Italy +39 091 6552500 ; +011 39 091 6554529 ;

Objective: The vast majority of pancreatic cancers occurs sporadically. The discovery of frequent variations in germline gene copy number can significantly influence the expression levels of genes that predispose to pancreatic adenocarcinoma. We prospectively investigated whether patients with sporadic pancreatic adenocarcinoma share specific gene copy number variations (CNVs) in their germline DNA.

Patients And Methods: DNA samples were analyzed from peripheral leukocytes from 72 patients with a diagnosis of sporadic pancreatic adenocarcinoma and from 60 controls using Affymetrix 500K array set. Multiplex ligation-dependent probe amplification (MLPA) assay was performed using a set of self-designed MLPA probes specific for seven target sequences.

Results: We identified a CNV-containing DNA region associated with pancreatic cancer risk. This region shows a deletion of 1 allele in 36 of the 72 analyzed patients but in none of the controls. This region is of particular interest since it contains the YTHDC2 gene encoding for a putative DNA/RNA helicase, such protein being frequently involved in cancer susceptibility. Interestingly, 82.6% of Sicilian patients showed germline loss of one allele.

Conclusions: Our results suggest that the YTHDC2 gene could be a potential candidate for pancreatic cancer susceptibility and a useful marker for early detection as well as for the development of possible new therapeutic strategies.
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http://dx.doi.org/10.1517/14728222.2014.920324DOI Listing
August 2014

Analysis of germline gene copy number variants of patients with sporadic pancreatic adenocarcinoma reveals specific variations.

Oncology 2013 9;85(5):306-11. Epub 2013 Nov 9.

Section of Medical Oncology, Department of Surgical, Oncological and Stomatological Sciences,University of Palermo, Palermo, Italy.

Objectives: The rapid fatality of pancreatic cancer is, in large part, the result of diagnosis at an advanced stage in the majority of patients. Identification of individuals at risk of developing pancreatic adenocarcinoma would be useful to improve the prognosis of this disease. There is presently no biological or genetic indicator allowing the detection of patients at risk. Our main goal was to identify copy number variants (CNVs) common to all patients with sporadic pancreatic cancer.

Methods: We analyzed gene CNVs in leukocyte DNA from 31 patients with sporadic pancreatic adenocarcinoma and from 93 matched controls. Genotyping was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix).

Results: We identified 431 single nucleotide polymorphism (SNP) probes with abnormal hybridization signal present in the DNA of all 31 patients. Of these SNP probes, 284 corresponded to 3 or more copies and 147 corresponded to 1 or 0 copies. Several cancer-associated genes were amplified in all patients. Conversely, several genes supposed to oppose cancer development were present as single copy.

Conclusions: These data suggest that a set of 431 CNVs could be associated with the disease. This set could be useful for early diagnosis.
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http://dx.doi.org/10.1159/000354737DOI Listing
January 2014

Analysis of ZNF350/ZBRK1 promoter variants and breast cancer susceptibility in non-BRCA1/2 French Canadian breast cancer families.

J Hum Genet 2013 Feb 15;58(2):59-66. Epub 2012 Nov 15.

Cancer Genomics Laboratory, Oncology and Molecular Endocrinology Research Centre, Centre Hospitalier Universitaire de Québec and Laval University, Québec City, QC, Canada.

ZNF350/ZBRK1 is a transcription factor, which associates with BRCA1 to co-repress GADD45A to regulate DNA damage repair, and the expression of ZNF350 is altered in different human carcinomas. In a previous study, we identified ZNF350 genomic variants potentially involved in breast cancer susceptibility in high-risk non-BRCA1/2 breast cancer individuals, which pointed toward a potential association for variants in the 5'-UTR and promoter regions. Therefore, direct sequencing was undertaken and identified 12 promoter variants, whereas haplotype analyses put in evidence four common haplotypes with a frequency>2%. However, based on their frequency observed in breast cancer and unrelated healthy individuals, these are not statistically associated with breast cancer risk. Luciferase promoter assays in two breast cancer cell lines identified two haplotypes (H11 and H12) stimulating significantly the expression of ZNF350 transcript compared with the common haplotype H8. The high expression of the H11 allele was associated with the variant c.-874A. Using MatInspector and Transcription Element Search softwares, in silico analyses predicted that the variant c.-874A created a binding site for the factors c-Myc and myogenin. This study represents the first characterization step of the ZNF350 promoter. Additional studies in larger cohorts and other populations will be needed to further evaluate whether common and/or rare ZNF350 promoter variants and haplotypes could be associated with a modest risk of breast cancer.
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http://dx.doi.org/10.1038/jhg.2012.127DOI Listing
February 2013

Polymorphic variations in the FANCA gene in high-risk non-BRCA1/2 breast cancer individuals from the French Canadian population.

Mol Oncol 2013 Feb 11;7(1):85-100. Epub 2012 Sep 11.

Cancer Genomics Laboratory, Division of Endocrinology and Genomics of CHUQ Research Centre and Laval University, Québec G1V 4G2, Canada.

The majority of genes associated with breast cancer susceptibility, including BRCA1 and BRCA2 genes, are involved in DNA repair mechanisms. Moreover, among the genes recently associated with an increased susceptibility to breast cancer, four are Fanconi Anemia (FA) genes: FANCD1/BRCA2, FANCJ/BACH1/BRIP1, FANCN/PALB2 and FANCO/RAD51C. FANCA is implicated in DNA repair and has been shown to interact directly with BRCA1. It has been proposed that the formation of FANCA/G (dependent upon the phosphorylation of FANCA) and FANCB/L sub-complexes altogether with FANCM, represent the initial step for DNA repair activation and subsequent formation of other sub-complexes leading to ubiquitination of FANCD2 and FANCI. As only approximately 25% of inherited breast cancers are attributable to BRCA1/2 mutations, FANCA therefore becomes an attractive candidate for breast cancer susceptibility. We thus analyzed FANCA gene in 97 high-risk French Canadian non-BRCA1/2 breast cancer individuals by direct sequencing as well as in 95 healthy control individuals from the same population. Among a total of 85 sequence variants found in either or both series, 28 are coding variants and 19 of them are missense variations leading to amino acid change. Three of the amino acid changes, namely Thr561Met, Cys625Ser and particularly Ser1088Phe, which has been previously reported to be associated with FA, are predicted to be damaging by the SIFT and PolyPhen softwares. cDNA amplification revealed significant expression of 4 alternative splicing events (insertion of an intronic portion of intron 10, and the skipping of exons 11, 30 and 31). In silico analyzes of relevant genomic variants have been performed in order to identify potential variations involved in the expression of these spliced transcripts. Sequence variants in FANCA could therefore be potential spoilers of the Fanconi-BRCA pathway and as a result, they could in turn have an impact in non-BRCA1/2 breast cancer families.
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http://dx.doi.org/10.1016/j.molonc.2012.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528405PMC
February 2013

Specific transcriptional response of four blockers of estrogen receptors on estradiol-modulated genes in the mouse mammary gland.

Breast Cancer Res Treat 2012 Jul 8;134(2):625-47. Epub 2012 Jun 8.

Molecular Endocrinology, Oncology and Human Genomics Research Center, Laval University and Laval University Hospital Research Center, 2705 Laurier Blvd, Quebec, QC, G1V 4G2, Canada.

Novel agents for the endocrine therapy of breast cancer are needed, especially in order to take advantage of the multiple consecutive responses observed in metastatic progressing breast cancer following previous hormone therapy, thus delaying the use of cytotoxic chemotherapy with its frequent poor tolerance and serious side effects. Acolbifene (ACOL) is a novel and unique antiestrogen which represents a unique opportunity to achieve the most potent and specific blockade of estrogen action in the mammary gland and uterus while exerting estrogen-like beneficial effects in other tissues, especially the bones. To better understand the specificity of action of ACOL, we have used Affymetrix GeneChips containing 45,000 probe sets to analyze 34,000 genes to determine the specificity of this compound compared to the pure antiestrogen fulvestrant, as well as to the mixed antagonists/agonists tamoxifen and raloxifene to block the effect of estradiol (E(2)) and to induce effects of their own on the genomic profile in the mouse mammary gland. The genes modulated by E(2) were those identified in two separate experiments and validated by quantitative real-time PCR (qPCR). Three hours after the single subcutaneous injection of E(2) (0.05 μg), the simultaneous administration of ACOL, fulvestrant, tamoxifen, and raloxifene blocked by 98, 61, 43, and 92 % the number of E(2)-upregulated genes, respectively. On the other hand, 70, 10, 25, and 55 % of the genes down-regulated by E(2) were blocked by the same compounds. Of the 128 genes modulated by E(2), 49 are associated with tumorigenesis while 22 are known to be associated with breast cancer. When used alone, ACOL modulated the smallest number of genes also influenced by E(2), namely 4 %, thus possibly explaining potential utilities of this compound in breast cancer prevention and therapy.
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http://dx.doi.org/10.1007/s10549-012-2104-7DOI Listing
July 2012

PITX2 is involved in stress response in cultured human trabecular meshwork cells through regulation of SLC13A3.

Invest Ophthalmol Vis Sci 2011 Sep 29;52(10):7625-33. Epub 2011 Sep 29.

Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.

Purpose: Mutations of the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) and glaucoma. In this study, the authors investigated genes directly regulated by the PITX2 transcription factor to gain insight into the mechanisms underlying these disorders.

Methods: RNA from nonpigmented ciliary epithelium cells transfected with hormone-inducible PITX2 and activated by mifepristone was subjected to microarray analyses. Data were analyzed using dCHIP algorithms to detect significant differences in expression. Genes with significantly altered expression in multiple microarray experiments in the presence of activated PITX2 were subjected to in silico and biochemical analyses to validate them as direct regulatory targets. One target gene was further characterized by studying the effect of its knockdown in a cell model of oxidative stress, and its expression in zebrafish embryos was analyzed by in situ hybridization.

Results: Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) was identified as 1 of 47 potential PITX2 target genes in ocular cells. PITX2 directly regulates SLC13A3 expression, as demonstrated by luciferase reporter and chromatin immunoprecipitation assays. Reduction of PITX2 or SLC13A3 levels by small interfering RNA (siRNA)-mediated knockdown augmented the death of transformed human trabecular meshwork cells exposed to hydrogen peroxide. Zebrafish slc13a3 is expressed in anterior ocular regions in a pattern similar to that of pitx2.

Conclusions: The results indicate that SLC13A3 is a direct downstream target of PITX2 transcriptional regulation and that levels of PITX2 and SLC13A3 modulate responses to oxidative stress in ocular cells.
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http://dx.doi.org/10.1167/iovs.10-6967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183983PMC
September 2011

Fine temporal analysis of DHT transcriptional modulation of the ATM/Gadd45g signaling pathways in the mouse uterus.

Mol Reprod Dev 2009 Mar;76(3):278-88

Oncology and Molecular Endocrinology Research Center, CHUL Research Center, CHUQ, Department of Anatomy and Physiology, Laval University, Québec, Canada.

In rodents, the uterus of a mature female undergoes changes during the uterine cycle, under the control of steroid hormones. 5alpha-Dihydrotestosterone (DHT) is recognized to play an important role in the regulation of androgen action in normal endometrium. Using microarray technology, a screening analysis of genes responding to DHT in the uterus of ovariectomized mice, has allowed us to highlight multiple genes of the ATM/Gadd45g pathway that are modulated following exposure to DHT. Two phases of regulation were identified. In the early phase, the expression of genes involved in the G2/M arrest is rapidly increased, followed by the repression of genes of the G1/S checkpoint, and by the induction of transcriptional regulators. Later, i.e. from 12 to 24 hr, genes involved in G2/M transition, cytoarchitectural and lipid-related genes are stimulated by DHT while immunity-related genes appear to be differentially regulated by the hormone. These results show that a physiological dose of DHT induces the transcription of genes promoting the cell cycle progression in mice. Profile determination of temporal uterine gene expression at the transcriptional level enables us to suggest that the DHT modulation of genes involved in ATM/Gadd45g signaling in an ATM- or p53-independent manner, could play an important role in the cyclical changes of uterine cells in the mouse uterus.
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http://dx.doi.org/10.1002/mrd.20949DOI Listing
March 2009

Prostate-specific genes and their regulation by dihydrotestosterone.

Prostate 2008 Feb;68(3):241-54

Molecular Endocrinology and Oncology Research Center, Laval University Medical Center, Department of Anatomy and Physiology, Laval University, Québec, Canada.

Background: Prostate is a well-known androgen-dependent tissue.

Methods: By sequencing 4,294,186 serial analysis of gene expression (SAGE) tags, we have investigated the transcriptomes of normal mouse prostate, liver, testis, lung, brain, femur, skin, adipose tissue, skeletal muscle, vagina, ovary, mammary gland, and uterus in order to identify the most abundant and tissue-specific transcripts in the prostate, as well as to target the androgen responsive transcripts specifically regulated in the prostate. Small interference RNA (siRNA) in LNCaP cells was applied to validate the roles of prostate-specific/enriched ARGs in the growth of human prostate cancer cells.

Results: The most abundant transcripts were involved in prostatic secretion, energy metabolism and immunity. Previously well-known prostate-specific transcripts, including many transcripts involved in prostatic secretion, polyamine biosynthesis and transport, and immunity were specific/enriched in the prostate. Only 22 transcripts among 114 androgen-regulated genes (ARGs) in the mouse prostate were modulated by dihydrotestosterone (DHT) in two or more tissues. The siRNA results showed that inhibition of HSPA5 and MAT2A gene expression repressed growth of human cancer LNCaP cells.

Conclusions: The current study globally assessed the transcriptome of the prostate and revealed the most abundant and tissue-specific transcripts which are responsible for the unique functions of this organ. These prostate-specific ARGs might be used as targets to develop safe and effective gene-based therapy for the prevention and treatment of prostate cancer.
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http://dx.doi.org/10.1002/pros.20712DOI Listing
February 2008

Gene interactions in depression: pathways out of darkness.

Trends Genet 2007 Nov 25;23(11):547-56. Epub 2007 Oct 25.

Department of Neuroscience, CHUL Research Centre and Laval University, Quebec, QC, Canada.

Mood disorders, including major depressive disorder and bipolar disorder, are influenced by both genetic and environmental factors. Hypotheses about the neurobiology of mood disorders have been postulated and putatively associated genes identified. Recently, the immune-related gene encoding purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7) has been genetically associated with major depressive disorder and bipolar disorder. New candidate genes and emerging gene networks and pathways involved in the aetiology of mood disorders point to a major role for neuronal survival and the adaptive immune systems.
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http://dx.doi.org/10.1016/j.tig.2007.08.011DOI Listing
November 2007

Genetic variants and haplotype analyses of the ZBRK1/ZNF350 gene in high-risk non BRCA1/2 French Canadian breast and ovarian cancer families.

Int J Cancer 2008 Jan;122(1):108-16

Cancer Genomics Laboratory, Oncology and Molecular Endocrinology Research Centre, Centre Hospitalier Universitaire de Québec and Laval University, Quebec City, Canada.

Our current understanding of breast cancer susceptibility involves mutations in the 2 major genes BRCA1 and BRCA2, found in about 25% of high-risk families, as well as few other low penetrance genes such as ATM and CHEK2. Approximately two-thirds of the multiple cases families remain to be explained by mutations in still unknown genes. In a candidate gene approach to identify new genes potentially involved in breast cancer susceptibility, we analyzed genomic variants in the ZBRK1 gene, a co-repressor implicated in BRCA1-mediated repression of GADD45. Direct sequencing of ZBRK1 entire coding region in affected breast cancer individuals from 97 high-risk French Canadian breast/ovarian cancer families and 94 healthy controls led to the identification of 18 genomic variants. Haplotype analyses, using PHASE, COCAPHASE and HaploStats programs, put in evidence 3 specific haplotypes which could potentially modulate breast cancer risk, and among which 2 that are associated with a potential protective effect (p = 0.01135 and p = 0.00268), while another haplotype is over-represented in the case group (p = 0.00143). Further analyses of these haplotypes indicated that a strong component of the observed difference between both groups emerge from the first 5 variants (out of 12 used for haplotype determination). The present study also permitted to determine a set of tagging SNPs that could be useful for subsequent analyses in large scale association studies. Additional studies in large cohorts and other populations will however be needed to further evaluate if common and/or rare ZBRK1 sequence variants and haplotypes could be associated with a modest/intermediate breast cancer risk.
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http://dx.doi.org/10.1002/ijc.23058DOI Listing
January 2008

Temporal analysis of E2 transcriptional induction of PTP and MKP and downregulation of IGF-I pathway key components in the mouse uterus.

Physiol Genomics 2007 Mar;29(1):13-23

Oncology and Molecular Endocrinology Research Center, Centre Hospitalier de l'Université Laval Research Center, Centre Hospitalier Universitaire de Québec, Department of Anatomy and Physiology, Laval University, Quebec, Canada.

17beta-Estradiol (E2) is well known to be associated with uterine cancer, endometriosis, and leiomyomas. Although insulin-like growth factor I (IGF-I) has been identified as a mediator of the uterotrophic effect of E2 in several studies, this mechanism is still not well understood. In the present study, identification of the genes modulated by a physiological dose of E2, in the uterus, has been done in ovariectomized mice using Affymetrix microarrays. The E2-induced genomic profile shows that multiple genes belonging to the IGF-I pathway are affected after exposure to E2. Two phases of regulation could be identified. First, from 0 to 6 h, the expression of genes involved in the cell cycle, growth factors, protein tyrosine phosphatases, and MAPK phosphatases is quickly upregulated by E2, while IGF-I receptor and several genes of the MAPK and phosphatidylinositol 3-kinase pathways are downregulated. Later, i.e., from 6 to 24 h, transporters and peptidases/proteases are stimulated, whereas defense-related genes are differentially regulated by E2. Finally, cytoarchitectural genes are modulated later. The present data show that a physiological dose of E2 induces, within 24 h, a series of transcriptional events that promote the uterotrophic effect. Among these, the E2-mediated activation of the IGF-I pathway seems to play a pivotal role in the uterotrophic effect. Furthermore, the protein tyrosine phosphatases and MAPK phosphatases are likely to modulate the estrogenic uterotrophic action by targeting, at different steps, the IGF-I pathway.
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http://dx.doi.org/10.1152/physiolgenomics.00291.2005DOI Listing
March 2007

Protein diversity is generated within the motin family of proteins by alternative pre-mRNA splicing.

Gene 2005 May 29;350(2):137-48. Epub 2005 Mar 29.

Centre de recherche en rhumatologie et immunologie, Centre de recherche du CHUL, Laval University, Bloc T1-492705, Boul. Laurier, Ste-Foy, Québec, Canada G1V 4G2.

The motin family of proteins is comprised of three polypeptides, angiomotin, angiomotin-like 1, and angiomotin-like 2. Angiomotin is an angiostatin-binding protein that promotes endothelial cell motility and is involved in angiogenesis. The function of human angiomotin-like-1 and angiomotin-like-2, however, remains unknown. In this report, we investigated the potential for molecular diversity within the motin family of proteins through the identification and characterization of alternatively spliced transcripts in endothelial cells, human tissues and a variety of cell lines. We report that the motins display variability at the mRNA level suggesting an intricate regulatory system at the transcriptional and potentially protein level. Some alternative transcripts are expressed in a tissue-specific manner and others give rise to novel protein isoforms. The alternative splicing that occurs within this protein family may have important implications in the regulation of the expression and function of the motins.
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http://dx.doi.org/10.1016/j.gene.2005.02.001DOI Listing
May 2005

Analysis of microsatellite markers and single nucleotide polymorphisms in candidate genes for susceptibility to bipolar affective disorder in the chromosome 12Q24.31 region.

Am J Med Genet B Neuropsychiatr Genet 2005 May;135B(1):50-8

Neuroscience, CHUL Research Center and Laval University, CHUQ Pavillon CHUL, Ste-Foy, Québec, Canada.

Previous results from our genetic analyses using pedigrees from a French Canadian population suggested that the interval delimited by markers D12S86 and D12S378 on chromosome 12 was the most probable genomic region to contain a susceptibility gene for affective disorders. Here we present a more detailed genetic analysis of a 7.7 Mb genomic region located on 12q24.31. This region was saturated with 20 microsatellite markers to refine the candidate region and linkage analysis performed in 41 families from the Saguenay-Lac-St-Jean (SLSJ) region of Quebec. The results of two point parametric analysis using MFLINK supported the presence of a susceptibility locus on chromosome 12q24.31. Association studies with microsatellite markers using a case/control sample from the same population (n = 401) and analyzed with CLUMP revealed significant allelic associations between the bipolar phenotype and markers NBG6 (P = 0.008) and NBG12 (P < 10(-3)). According to these results, we investigated candidate genes in the NBG12 area. We analyzed 32 genes for the presence of polymorphisms in coding sequences and intron/exon junctions and genotyped 22 non-synonymous SNPs in the SLSJ case/control sample. Two uncommon polymorphisms (minor allele frequency < or = 0.03) found in KIAA1595 and FLJ22471 genes, gave P-values below 0.05 with the T1 statistic. Moreover, using haplotype analysis, a nearly significant haplotypic association was observed at the HM74 gene. These results do not give strong support for a role in the susceptibility to bipolar disorder of any of these genes analyzed. However, the significance of rare polymorphisms should be explored by further analyses.
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http://dx.doi.org/10.1002/ajmg.b.30165DOI Listing
May 2005

Tetrahydrogestrinone induces a genomic signature typical of a potent anabolic steroid.

J Endocrinol 2005 Feb;184(2):427-33

Molecular Endocrinology and Oncology Laboratory, Laval University Medical Research Center (CRCHUL) and Laval University, Québec City, G1V 4G2, Canada.

Tetrahydrogestrinone (THG) is a recently identified compound having the greatest impact in the world of sports. In order to obtain a highly accurate and sensitive assessment of the potential anabolic/androgenic activity of THG, we have used microarrays to identify its effect on the expression of practically all the 30,000 genes in the mouse genome and compared it with the effect of dihydrotestosterone (DHT), the most potent natural androgen. Quite remarkably, we found that 671 of the genes modulated by THG in the mouse muscle levator ani are modulated in a similar fashion by DHT, while in the gastrocnemius muscle and prostate, 95 and 939 genes respectively, are modulated in common by the two steroids. On the other hand, THG is more potent than DHT in binding to the androgen receptor, while, under in vivo conditions, THG possesses 20% of the potency of DHT in stimulating prostate, seminal vesicle and levator ani muscle weight in the mouse. The present microarray data provide an extremely precise and unquestionable signature of the androgenic/anabolic activity of THG, an approach which should apply to the analysis of the activity of any anabolic steroid.
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http://dx.doi.org/10.1677/joe.1.05997DOI Listing
February 2005