Julia Santucci-Pereira

Characterization of a Genomic Signature of Pregnancy Identified in the Breast

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer. Cancer Prev Res; 4(9); 1457–64. ©2011 AACR.

Pregnancy-induced chromatin remodeling in the breast of postmenopausal women.

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation‐associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full‐term pregnancy.

Defining the genomic signature of the parous breast

BackgroundIt is accepted that a womans lifetime risk of developing breast cancer after menopause is reduced by early full term pregnancy and multiparity. This phenomenon is thought to be associated with the development and differentiation of the breast during pregnancy.MethodsIn order to understand the underlying molecular mechanisms of pregnancy induced breast cancer protection, we profiled and compared the transcriptomes of normal breast tissue biopsies from 71 parous (P) and 42 nulliparous (NP) healthy postmenopausal women using Affymetrix Human Genome U133 Plus 2.0 arrays. To validate the results, we performed real time PCR and immunohistochemistry.ResultsWe identified 305 differentially expressed probesets (208 distinct genes). Of these, 267 probesets were up- and 38 down-regulated in parous breast samples; bioinformatics analysis using gene ontology enrichment revealed that up-regulated genes in the parous breast represented biological processes involving differentiation and development, anchoring of epithelial cells to the basement membrane, hemidesmosome and cell-substrate junction assembly, mRNA and RNA metabolic processes and RNA splicing machinery. The down-regulated genes represented biological processes that comprised cell proliferation, regulation of IGF-like growth factor receptor signaling, somatic stem cell maintenance, muscle cell differentiation and apoptosis.ConclusionsThis study suggests that the differentiation of the breast imprints a genomic signature that is centered in the mRNA processing reactome. These findings indicate that pregnancy may induce a safeguard mechanism at post-transcriptional level that maintains the fidelity of the transcriptional process.

In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats

BackgroundEnvironmental estrogens are exogenous estrogen-mimicking compounds that can interfere with endogenous endocrine systems. Several of these endocrine disruptors have been shown to alter normal development and influence tumorigenesis in experimental models. N-butyl benzyl phthalate (BBP), a widely used plasticizer, is a well-known endocrine disruptor. The aim of this study was to elucidate the effect of prenatal exposure to BBP on the morphology, proliferative index, and genomic signature of the rat mammary gland at different ages.MethodsIn utero exposure was performed by gavage of pregnant Sprague Dawley CD rats with 120mg or 500mg BBP/kg/day from day 10 post-conception to delivery. Female litters were euthanized at 21, 35, 50 and 100 days. The morphology and proliferative index of the mammary gland were studied from whole mount preparations and BrdU incorporation, respectively. Gene expression profile was assessed by microarrays. Several genes found differentially expressed and related to different functional categories were further validated by real time RT-PCR.ResultsPrenatal exposure of BBP induced delayed vaginal opening and changes in the post-natal mammary gland long after the end of the treatment, mainly by 35 days of age. Exposure to the high dose resulted in modifications in architecture and proliferative index of the mammary gland, mostly affecting the undifferentiated terminal end buds. Moreover, the expression profiles of this gland in the exposed rats were modified in a dose-dependent fashion. Analysis of functional categories showed that modified genes were related to immune function, cell signaling, proliferation and differentiation, or metabolism.ConclusionsOur data suggest that in utero exposure to BBP induced a delayed pubertal onset and modified morphology of the mammary gland. These alterations were accompanied by modifications in gene expression previously associated with an increased susceptibility to carcinogenesis.

Molecular Pathways Involved in Pregnancy-Induced Prevention Against Breast Cancer

Pregnancy produces a protective effect against breast cancer in women who had their first full term pregnancy (FTP) in their middle twenties. The later in life the first delivery occurs, the higher the risk of breast cancer development. Also, transiently during the postpartum period, the risk of developing breast cancer increases. This transient increased risk is taken over by a long-lasting protective period. The genomic profile of parous women has shown pregnancy induces a long-lasting “genomic signature” that explains the preventive effect on breast cancer. This signature reveals that chromatin remodeling is the driver of the differentiation process conferred by FTP. The chromatin remodeling process may be the ultimate step mediating the protection of the breast against developing breast cancer in post-menopausal years.

The Genomic Signature of Breast Cancer Prevention

The breast of parous postmenopausal women exhibits a specific signature that has been induced by a full term pregnancy. This signature is centered in chromatin remodeling and the epigenetic changes induced by methylation of specific genes which are important regulatory pathways induced by pregnancy. Through the analysis of the genes found to be differentially methylated between women of varying parity, multiple positions at which beta-catenin production and use is inhibited were recognized. The biological importance of the pathways identified in this specific population cannot be sufficiently emphasized because they could represent a safeguard mechanism mediating the protection of the breast conferred by full term pregnancy.

Combination of Antiestrogens and Omega-3 Fatty Acids for Breast Cancer Prevention

The molecular and biological heterogeneity of human breast cancer emphasizes the importance of a multitargeted approach for effective chemoprevention. Targeting the estrogen receptor pathway alone with the antiestrogens, Tamoxifen and Raloxifene reduces the incidence of estrogen receptor positive tumors but is ineffective against the development of hormone independent cancers. Our preclinical data indicate that the administration of omega-3 fatty acids potentiates the antitumor effects of Tamoxifen by inhibiting multiple proliferative and antiapoptotic pathways, several of which interact with estrogen receptor signaling. The complementarity in the mechanism of antitumor action of Tamoxifen and omega-3 fatty acids is well supported by our signaling, genomic, and proteomic studies. Furthermore, administration of omega-3 fatty acids allows the use of lower and, hence, likely less toxic doses of Tamoxifen. If these findings are supported in the clinical setting, the combination of omega-3 fatty acids and anteistrogens may emerge as a promising, effective, and safe chemopreventive strategy to be tested in a large multi-institutional trial using breast cancer incidence as the primary endpoint.

Gene expression profile induced by pregnancy in the breast of premenopausal women

We previously reported that having completed a full term pregnancy (FTP) confers specific gene expression patterns in the breast of healthy postmenopausal women [Belitskaya-Levy, I. et al. 2011, Peri, S. et al. 2012 and Russo, J. 2012]. In the present work, we report on gene expression differences in the breast of parous versus nulliparous healthy premenopausal women. Using Affymetrix Human Genome U133 Plus 2.0 microarrays, we analyzed the gene expression profile of breast tissue from 30 nulliparous (NP) and 79 parous (P) premenopausal volunteers between the ages of 30 and 47 years who were free of breast pathology. Because of the known short-term increase in breast cancer risk preceding the long-term protective effect of FTP, we also examined gene expression differences in P vs. NP women as a function of time since last FTP. Through multiple regression analysis, controlling for confounders, we found 416 probesets differentially expressed (fold-change ≥ 1.2 and false discovery rate Citation Format: Julia Santucci-Pereira, Anne Zeleniuch-Jacquotte, Yelena Afanasyeva, Hua Zhong, Eric A. Ross, Michael Slifker, Suraj Peri, Ricardo Lopez de Cicco, Yubo Zhai, Irma H. Russo, Theresa Nguyen, Fathima Sheriff, Alan A. Arslan, Pal Bordas, Per Lenner, Janet Ahman, Anna-Stina L. Eriksson, Robert Johansson, Goran Hallmans, Paolo Toniolo, Jose Russo. Gene expression profile induced by pregnancy in the breast of premenopausal women. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2360. doi:10.1158/1538-7445.AM2014-2360

Fish Oil Alters Tamoxifen-Modulated Expression of mRNAs That Encode Genes Related to Differentiation, Proliferation, Metastasis, and Immune Response in Rat Mammary Tumors

We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publishers online edition of Nutrition and Cancer to view the supplemental file.

Human chorionic gonadotropin and a 15 amino acid hCG fragment of the hormone induce downregulation of the cytokine IL-8 receptor in normal breast epithelial cells.

Abstract Cytokine receptors are associated with tumor cell growth by increasing proliferation, metastasis and regulating self-renewal of cancer stem cells (SCs). There is a strong association between cytokine IL-8 receptor (CXCR1) over-expression and cells displaying SC characteristics. Human chorionic gonadotropin (hCG) causes differentiation, inhibition of cell proliferation and increased apoptosis of the breast epithelium. hCG receptor (LHCGR) expression in breast tumors and in breast cancer cell lines is undetectable or low. In this study, our objective was to assess and compare the effects of hCG and a 15 amino acid hCG fragment of the hormone on mRNA expression of CXCR1 and LHCGR on normal breast epithelial cells (MCF-10F) by real time RT-PCR after treatment with hCG or a hCG fragment for 15 days. Cell proliferation was also measured. hCG and the hCG fragment decreased cell proliferation in both groups. The compounds upregulated LHCGR expression and downregulated CXCR1 expression. It is possible to postulate that an increase of LHCGR mRNA seems to respond to the decrease of CXCR1 expression. These genes probably act synergistically to reduce the amount of cancer SCs in the mammary gland. Thereby, the use of hCG or the hCG fragment as a therapeutic or preventive tool should be considered.