Genomic signature of parity in the breast of premenopausal women.

BACKGROUND: Full-term pregnancy (FTP) at an early age confers long-term protection against breast cancer. Previously, we reported that a FTP imprints a specific gene expression profile in the breast of postmenopausal women. Herein, we evaluated gene expression changes induced by parity in the breast of premenopausal women. METHODS: Gene expression profiling of normal breast tissue from 30 nulliparous (NP) and 79 parous (P) premenopausal volunteers was performed using Affymetrix microarrays. In addition to a discovery/validation analysis, we conducted an analysis of gene expression differences in P vs. NP women as a function of time since last FTP. Finally, a laser capture microdissection substudy was performed to compare the gene expression profile in the whole breast biopsy with that in the epithelial and stromal tissues. RESULTS: Discovery/validation analysis identified 43 differentially expressed genes in P vs. NP breast. Analysis of expression as a function of time since FTP revealed 286 differentially expressed genes (238 up- and 48 downregulated) comparing all P vs. all NP, and/or P women whose last FTP was less than 5 years before biopsy vs. all NP women. The upregulated genes showed three expression patterns: (1) transient: genes upregulated after FTP but whose expression levels returned to NP levels. These genes were mainly related to immune response, specifically activation of T cells. (2) Long-term changing: genes upregulated following FTP, whose expression levels decreased with increasing time since FTP but did not return to NP levels. These were related to immune response and development. (3) Long-term constant: genes that remained upregulated in parous compared to nulliparous breast, independently of time since FTP. These were mainly involved in development/cell differentiation processes, and also chromatin remodeling. Lastly, we found that the gene expression in whole tissue was a weighted average of the expression in epithelial and stromal tissues. CONCLUSIONS: Genes transiently activated by FTP may have a role in protecting the mammary gland against neoplastically transformed cells through activation of T cells. Furthermore, chromatin remodeling and cell differentiation, represented by the genes that are maintained upregulated long after the FTP, may be responsible for the lasting preventive effect against breast cancer.

Oxidized derivative of docosahexaenoic acid preferentially inhibit cell proliferation in triple negative over luminal breast cancer cells.

Omega-3 polyunsaturated fatty acids (PUFAs) exert an anticancer effect by affecting multiple cellular mechanisms leading to inhibition of proliferation and induction of apoptosis. It is well known that breast cancer comprises distinct molecular subtypes which differ in their responsiveness to therapeutic and preventive agents. We tested the hypothesis that n-3FA may preferentially affect triple-negative breast cancer cells for which no targeted intervention is presently available. The in vitro antiproliferative effects of n-3 PUFA docosahexaenoic acid (DHA) and its metabolite, 4-OH-DHA as well as its putative metabolite 4-OXO-DHA, were tested in five triple-negative human basal breast cell lines at different stages of transformation (MCF-10F, trMCF, bsMCF, MDA-MB-231, and BT-549) and three luminal breast cancer cell lines (MCF-7, T-47D, and SK-BR-3). Cell proliferation was measured with the tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. DHA and its oxidized derivatives significantly inhibited cell proliferation (20-90% reduction) of both basal and luminal breast cancer cell lines. The inhibitory effect was more pronounced on triple-negative basal breast cancer cell lines as compared to luminal breast cancer cell lines after 4-OXO-DHA treatment. Our data provide novel information regarding the preferential antitumor effect of oxidized derivatives of DHA on basal type breast cancer.

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.

Mimicking pregnancy as a strategy for breast cancer prevention.

Pregnancy and its effects on breast cancer risk have been widely investigated; there is consensus among researchers that early pregnancy confers protection against breast cancer later in life, whereas nulliparity and late-age parity have been associated with increased risk of developing breast cancer. The answer to the question of how pregnancy reduces breast cancer risk has been elusive; however, pregnancy, like breast cancer, is a similar hormone-dependent entity under direct control of estrogen, progesterone and, of particular importance, human chorionic gonadotropin (hCG). In this report, we emphasize the main changes, previously described by our laboratory, in morphology and gene expression levels of the mammary gland of Sprague-Dawley rats exposed to known cancer-preventative conditions (pregnancy, hCG and progesterone + estrogen). In addition, we postulate a protective mechanism induced by hCG that could reduce the cell's potential to be transformed by carcinogens.

Defining the genomic signature of the parous breast.

BACKGROUND: It is accepted that a woman's 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. METHODS: In 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. RESULTS: We 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. CONCLUSIONS: This 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.

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 publisher's online edition of Nutrition and Cancer to view the supplemental file.

An in vitro-in vivo model of epithelial mesenchymal transition in triple negative breast cancer.

The loss of epithelial expression markers by neoplastic breast cancer cells in the primary tumor is believed to play a pivotal role during breast cancer metastasis. This phenomenon is the hallmark of the epithelial mesenchymal transition (EMT) process. Gene expression microarrays were performed to investigate key functional elements on an in vitro metastasis model derived from human breast epithelial cells (MCF10F) treated with 17 beta estradiol. We identified groups of SLUG associated genes modulated during EMT.

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.

Molecular basis of pregnancy-induced breast cancer prevention.

Reduction in breast cancer risk is associated with elevated circulating levels of human chorionic gonadotropin (hCG) during the first trimester of gestation. The knowledge that hCG also modifies the genomic signature of breast epithelial cells, from highly susceptible to refractory, to undergo neoplastic transformation, and also exerts both preventive and therapeutic effects on chemically induced mammary cancer, lead us to select this hormone for inducing chromatin remodeling in breast epithelial cells as a surrogate end point of complete differentiation and cancer prevention. We have found that chromatin remodeling is the driving force of the differences between the nulliparous and parous breast. In the parous breast, the epithelial cells have a condensed chromatin and increased reactivity with anti-H3K9me2 [di-methyl histone 3 (H3) (Lysine 9)] and H3K27me3 antibodies. This is accompanied by upregulation of noncoding RNA (ncRNA) elements including X2-inactive specific transcript (XIST) and chromatin remodeling genes, such as chromodomain helicase DNA-binding protein 2 (CHD2) and the chromobox homolog 3 (CBX3), whose products are required for controlling recruitment of protein/protein or DNA/protein interactions Another important gene upregulated in the parous breast epithelial cells is the histone-lysine N-methyltransferase or enhancer of zeste homolog 2 (EZH2), a member of the polycomb group (PcG) forming multimeric protein complexes that maintains the transcriptional repressive state of genes over successive cell generations. The fact that recent studies indicate that ncRNAs recruit PcG complexes to the locus of transcription or to sites located elsewhere in the genome cause us to postulate that the increased chromatin condensation in the parous breast has been initiated by ncRNAs, a postulate supported by the observed upregulation of several ncRNAs that included the XIST. The identification of a specific genomic signature of pregnancy has uncovered a novel tool that will serve as a surrogate biomarker for testing new chemopreventive agents and will significantly advance the field of cancer prevention. The clinical impact of this work is that it validates in an experimental system the genomic signature of prevention identified in the human parous breast and establish the bases for the use of the hormone hCG in the prevention of breast cancer, an approach that has not been fully developed until now.

The role of the basal stem cell of the human breast in normal development and cancer.

MCF-10F, an ERα negative human breast epithelial cell line derived from normal breast tissue, is able to form ductal structures in a tridimensional collagen matrix system. MCF-10F cells that are estrogen transformed (trMCF cells) progressively express phenotypes of in vitro cell transformation, including colony formation in agar methocel and loss of the ductulogenic capacity. Selection of these trMCF cells for invasiveness identified cells (bcMCF) that formed tumors in severe combined immunodeficient mice. The cell lines derived from those tumors (caMCF) were poorly differentiated ER, PR, and ERBB2 negative adenocarcinomas. These characteristics are similar to the human basal cell-like carcinomas. This in vitro-in vivo model demonstrates the importance of the basal cell type as a stem cell that reconstitutes the branching pattern of the breast and that is also target of a carcinogenic insult leading to transformation and cancer.

Pregnancy-induced changes in breast cancer risk.

Breast cancer is the malignant disease most frequently diagnosed in women of all races and nationalities. Since the 1970s the worldwide incidence of this disease has increased 30-40% in postmenopausal women, in whom, paradoxically, the risk of developing breast cancer is significantly reduced by an early first full term pregnancy (FTP) as compared to nulliparous and late parous women. Although the cause of breast cancer is not known, the mechanisms mediating the protection conferred by an early FTP have been identified to reside in the breast itself, and to be modulated by endogenous and environmental exposures that might negatively affect this organ during specific windows in its development that extend from prenatal life until the first pregnancy. Soon after conception the embryo initiates the production of human chorionic gonadotropin (hCG), the glycoprotein hormone that is diagnostic of pregnancy. HCG in conjunction with ovarian steroid hormones primes the hypothalamic neuroendocrine system for maintaining the pregnancy. Higher levels of hCG during the first trimester of pregnancy have been associated with a reduction in maternal breast cancer incidence after age 50. In preclinical studies it has been demonstrated that both FTP and hCG treatment of virgin rats prevent the development of chemically-induced mammary tumors, a phenomenon mediated by the differentiation of the mammary gland epithelial cells prior to carcinogen exposure. Complete differentiation proceeds through complex morphological, physiological and molecular changes that occur during pregnancy and lactation, that ultimately result in increased DNA repair capabilities of the mammary epithelium, activation of genes controlling differentiation and programmed cell death and imprinting in the breast epithelium a specific and permanent genomic signature of pregnancy. This signature is indicative of a reduced breast cancer risk and serves as a molecular biomarker of differentiation for evaluating the potential use of chemopreventive agents.

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.

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.

BACKGROUND: Environmental 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. METHODS: In 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. RESULTS: Prenatal 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. CONCLUSIONS: Our 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.

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.

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.

DNA methylation changes in a human cell model of breast cancer progression.

Epigenetic inactivation of genes by DNA hypermethylation plays an important role in carcinogenesis. An in vitro model of human breast epithelial cell transformation was used to study epigenetic changes induced by estradiol during the neoplastic process. Different stages of tumor initiation and progression are represented in this model being MCF-10F the normal stage; trMCF cells, the transformed stage; bsMCF cells, the invasive stage and, caMCF cells, the tumor stage. Global methylation studies by restriction landmark genomic scanning (RLGS) showed an increased DNA methylation during the in the invasive and tumor stages. Expression studies showed that NRG1 (neuregulin 1), CSS3 (chondroitin sulfate synthase 3) and SNIP (SNAP-25-interacting protein) were downregulated in the invasive and tumor cells. The transformed cells showed low expression of STXBP6 (amysin) compared to the parental cells MCF-10F. The treatment of these cells with the demethylating agent 5-aza-dC alone or in combination with the histone deacetylase inhibitor trichostatin increased the expression of NRG1, STXBP6, CSS3 and SNIP confirming that DNA methylation plays an important role in the regulation of the expression of these genes. The NRG1 exon 1 has a region located between -136 and +79 (considering +1, the translational initiation site) rich in CpG sites that was analyzed by methylation specific PCR (MSP). NRG1 exon 1 showed progressive changes in the methylation pattern associated with the progression of the neoplastic process in this model; NRG1 exon 1 was unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. Studies of human breast tissue samples showed that NRG1 exon 1 was partially methylated in 14 out of 17 (82.4%) invasive carcinomas although it was unmethylated in normal tissues (8 out of 10 normal breast tissue samples). Furthermore, NRG1 exon 1 was partially methylated in 9 out of 14 (64.3%) morphologically normal tissue samples adjacent to invasive carcinomas.

Estrogen induced breast cancer is the result in the disruption of the asymmetric cell division of the stem cell.

There is evidence that in the human breast there is a stem cell population that can give rise to many different cell types and have the unique potential to divide asymmetrically. In this way stem cells maintain the stem cell pool and simultaneously generate committed cells that reconstitute the organ for example for preparing the breast for a new pregnancy after the involution from a previous pregnancy and lactation process. In addition to the in vivo models of mammary morphogenesis there are in vitro systems that are more amenable to study in critically determined conditions the ductulogenic pattern of growth of the breast epithelia. Primary mammary epithelial cells grown in collagen matrix are able to form tree-like structures resembling in vivo ductulogenesis. The human breast epithelial cells MCF-10F formed tubules when grown in type I collagen and we demonstrated that treatment of these cells with 17ß-estradiol (E(2)) induces phonotypical changes indicative of neoplastic transformation. The transformation of MCF-10F by E(2) is associated with impaired ductal morphogenesis by altering the stem cells unique potential to divide asymmetrically inducing formation of solid masses mimicking intraductal carcinoma that progress to invasive and tumorigenic phenotype. In the present work we present evidence for the mechanism of cell asymmetry leading to normal ductulogenesis and how the normal stem cell is transformed to cancer stem cell by altering this process. Furthermore, we demonstrate that the carcinogenic agent, in this case E(2), induces a defect in the asymmetric cell division program of the normal mammary stem cell.

Human chorionic gonadotropin (hCG) prevents the transformed phenotypes induced by 17 beta-estradiol in human breast epithelial cells.

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life-long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF-10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 beta-estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF-10F induced by E2. MCF-10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D-cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF-10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland.

Cancer Behavior: An Optimal Control Approach.

With special attention to cancer, this essay explains how Optimal Control Theory, mainly used in Economics, can be applied to the analysis of biological behaviors, and illustrates the ability of this mathematical branch to describe biological phenomena and biological interrelationships. Two examples are provided to show the capability and versatility of this powerful mathematical approach in the study of biological questions. The first describes a process of organogenesis, and the second the development of tumors.

Breast development, hormones and cancer.

Breast cancer originates in undifferentiated terminal structures of the mammary gland. The terminal ducts of the Lob 1 of the human female breast, which are the sites of origin of ductal carcinomas, are at their peak of cell replication during early adulthood, a period during which the breast is more susceptible to carcinogenesis. The susceptibility of Lob 1 to undergo neoplastic transformation has been confirmed by in vitro studies, which have shown that this structure has the highest proliferative activity, estrogen receptor content and rate of carcinogen binding to the DNA. The higher incidence of breast cancer observed in nulliparous women supports this concept, whereas the protection afforded by early full-term pregnancy in women could be explained by the higher degree of differentiation of the mammary gland at the time in which an etiologic agent or agents act.