Faculty Publications

MCF-10F human breast epithelial cells when transformed with 17-beta-estradiol (E2) give rise to highly invasive C5 cells that generate adenocarcinomas in SCID mice. From these tumor, cell lines Such as C5-A6-T6 and C5-A8-T8 have been derived. Variable patterns of chromatin supraorganization have been demonstrated for these cells during the transformation/tumorigenesis progress, when assessing chromatin entropy by image analysis in Feulgenstained preparations. Since epigenetic dysregulation might contribute to the chromatin textural repatterning in transformed MCF-10F cells, the association of the variable chromatin packing states with global DNA methylation was investigated in these cells after their treatment with restriction enzymes followed by Feulgen staining and chromatin entropy evaluation by image analysis. The results indicate that although -C(m)CGG- sequences may affect chromatin supraorganization in some of the analyzed cell types (perhaps due to localized hypermethylation), not all the chromatin condensation patterns in these cells with transformation and/or tumorigenesis are associated with DNA methylation (e.g. E2 cells). Chromatin supraorganization remodeling in C5-A6-T6 and C5-A8-T8 cells may be attained by different mechanisms, with C5-A6-T6 chromatin packing states perhaps being associated with local DNA hypermethylation or other epigenetic factors, and C5-A8-T8 likely being associated with global DNA hypomethylation, as reported in the literature for other cell types. Thus, we assume that a variable epigenetic modulation affecting the higher-order packing states of chromatin in the estrogen-transformed MCF-10F cell model could be evident with the chromatin entropy study by image analysis.

BACKGROUND: Bisphenol A (BPA) is widely used in the manufacture of polycarbonate plastics, including infant formula bottles. OBJECTIVES: Based on the reported endocrine disruptor activity of this polyphenol, we hypothesized that exposure to BPA early in life would elicit developmental changes in the mammary tissue and cause a predisposition for mammary cancer. METHODS: We exposed neonatal/prepubertal rats to BPA via lactation from nursing dams treated orally with 0, 25, and 250 mu g BPA/kg body weight/day. For turmorigenesis studies, female offspring were exposed to 30 mg dimethylbenzanthracene (DMBA)/kg body weight at 50 days of age. RESULTS: The combination of DMBA treatment with lactational exposure to BPA demonstrated a dose-dependent increase in mammary tumor multiplicity and reduced tumor latency compared with controls. In the absence of DMBA treatment, lactational BPA exposure resulted in increased cell proliferation and decreased apoptosis at 50 but not 21 days postpartum (shortly after last BPA treatment). Using Western blot analysis, we determined that steroid receptor coactivators (SRCs) 1-3, Akt, phosphorylated Akt, progesterone receptor A (PR-A), and erbB3 proteins were significantly up-regulated at 50 days of age. CONCLUSIONS: The data presented here provide the first evidence that maternal exposure to BPA during lactation increases mammary carcinogenesis in a DMBA-induced model of rodent mammary cancer. Changes in PR-A, SRC 1-3, erbB3, and Akt activity are consistent with increased cell proliferation and decreased apoptosis playing a role in mammary cancer susceptibility. These alterations provide an explanation of enhanced mammary carcinogenesis after lactational BPA exposure.

Long-term exposure to estrogens influences the development of breast cancer in women, but the precise mechanisms involved are not clearly defined. Our working hypothesis is that estrogen modulates this process by two separate processes. One involves the binding of estradiol to estrogen receptor (ER) alpha with stimulation of cell proliferation. Errors in DNA occurring during replication result in fixed mutations when not repaired. The other process results from the formation of genotoxic metabolites of estradiol, which can bind to DNA, cause depurination, and result in mutations. Our collaborative group, funded by a Department of Defense Center of Excellence grant, has examined this hypothesis using a variety of experimental methods. Estradiol and its catechol-estrogen metabolite 4-OH-estradiol causes mutations in cell culture systems and can transform benign MCF-10F cells, allowing them to cause tumors in SCID mice. We have demonstrated loss of heterozygosity and gains and losses of DNA segments by comparative genomic hybridization methodology. The depurinated estradiol-guanine and -adenine adducts are measurable in MCF-7 breast cancer cells in culture and in mouse mammary tissue. The double transgenic, alpha estrogen receptor knockout/Wnt-1 knockin mouse model allows us to dissect out the separate effects of ER-mediated and ER-independent actions of estradiol. Knock out of the ER alpha delays the onset of breast tumors in this model, demonstrating a role of receptor-mediated actions. Oophorectomy delays the onset of tumors and reduces overall incidence, providing evidence for an ER-independent effect. Taken together, these data suggest that both ER-dependent and genotoxic ER-independent effects of estradiol mediate breast cancer development.

CYP1B1 and CYP19 ( aromatase) have been shown to be expressed in breast tumors. Both enzymes are efficient estrogen hydroxylases, indicating the potential for overlapping substrate and inhibitor specificity. We measured the inhibition properties of aromatase inhibitors (AIs) against CYP1B1-catalyzed hydroxylation of 17 beta-estradiol (E2) to determine whether CYP1B1 affects the disposition of AIs. In addition, we estimated the frequency of coexpression of these enzymes in breast tumor epithelium. Immunohistochemical analyses of CYP19 and CYP1B1 in a panel of 29 cases of invasive ductal carcinoma of the breast showed epithelial cell staining for CYP19 in 76% and for CYP1B1 in 97% of the samples. Statistical analysis showed no significant correlation (0.33) for positive expression of CYP19 and CYP1B1 ( p > 0.07). CYP1B1 inhibition was determined for two steroidal inhibitors: formestane and exemestane and five nonsteroidal inhibitors: aminoglutethimide, fadrozole, anastrozol!

A review. Exptl. studies have confirmed that full-term pregnancy prevents the initiation of chem. induced cancer, a phenomenon mediated by the induction of differentiation of the mammary gland. These observations led to suggestion that activation of this mechanism was the most physiol. approach for breast cancer prevention. A new approach for breast cancer prevention has to be developed capitalizing on the preventive effect of the hormones of pregnancy, which, by inducing differentiation of the breast, imprint a permanent genomic signature that is assocd. with reduced breast cancer risk. The exptl. demonstration that human chorionic gonadotropin, the hormone synthesized by the embryo and the placenta, is the one mediating the preventive effect of pregnancy, made it natural to select this specific hormone for the prevention of breast cancer. [on SciFinder (R)]

Background. Prolonged exposure to estrogens (17b-estradiol)(E2), xenoestrogens, hormone replacement therapy and contraceptives has been recognized as a key etiological factor of human breast cancer. The biol. and carcinogenic effects of E2 and xenoestrogens are mediated via estrogen receptors alpha (ERa) and beta (ERb). Both receptors are localized in the nucleus of E2-targeted cells including human breast cells where they are involved in the regulation of nuclear gene expression. There is increasing evidence indicating that a small fraction of total cellular ERs, particularly ERa, are localized in the membrane of E2-targeted cells where they mediate E2-dependent and/or E2-independent rapid and non-nuclear genomic signal pathways. Results. The present work will present evidence that: (1) there is mitochondrial localization of ERs in human breast cancer cells; (2) there is a functional role of the mitochondrial ERs in the regulation of mitochondrial genes encoding respiratory chain proteins. Conclusions. The potential implications of the mitochondrial ER-mediated pathways in estrogen carcinogenesis, particularly in stimulation of cell proliferation and inhibition of apoptosis, in human breast cancer and the potential nutritional and environmental perspectives of these effects will be addressed. [on SciFinder (R)]

Mechanisms underlying global changes in gene expression during tumour progression are poorly understood. SATB1 is a genome organizer that tethers multiple genomic loci and recruits chromatin- remodelling enzymes to regulate chromatin structure and gene expression. Here we show that SATB1 is expressed by aggressive breast cancer cells and its expression level has high prognostic significance ( P < 0.0001), independent of lymph- node status. RNA- interference- mediated knockdown of SATB1 in highly aggressive ( MDA- MB- 231) cancer cells altered the expression of >1,000 genes, reversing tumorigenesis by restoring breast- like acinar polarity and inhibiting tumour growth and metastasis in vivo. Conversely, ectopic SATB1 expression in non- aggressive ( SKBR3) cells led to gene expression patterns consistent with aggressive- tumour phenotypes, acquiring metastatic activity in vivo. SATB1 delineates specific epigenetic modifications at target gene loci, directly upregulating met!

S100P is expressed in several malignant neoplasms. It was previously demonstrated that S100P is involved in the very early stages of breast carcinogenesis. In the present study we used a retrovirus-mediated transfer of antisenseSIOOP in order to check whether the decrease in expression of this protein could lead to alterations in the cell cycle of epithelial cells of human breast cancer. The T47D breast carcinoma cell line, a human breast epithelial cell that expresses high levels of S100P, was a tool used in this study to investigate the alteration in cell cycle induced by a retrovirus-mediated transfer of antisense-S100P. First we used the real-time PCR technique to quantify the gene expression. The results showed a reduction of 63% of expression within the T47D-S100P-A/S infected population compared with control T47D-LXSN clones. To determine the impact of the SIOOP antisense technique on protein expression in T47D cells, we performed immunofluorescence staining and analyzed the resulting images using a confocal microscope. The images showed much less pronounced antibody marking of the S100P protein in the T47D-SIOOPA/S compared with control cells. To evaluate whether the antisense approach caused any alteration in the cell cycle, we concluded the study with flow cytometric analysis of the cell distribution. Our findings indicated that, in our model, S100P-antisense cells showed a 23 % reduction of cells at the S-phase. Using transduction techniques with an S100P anti sense-retroviral construct we were able to demonstrate a significant reduction in S-phase of the T47D cell cycle. To the best of our knowledge, this is the first time that an antisense approach has been used against S100P mRNA in breast cancer epithelial cells. The results showed here seem to further classify S100P as a protein that might be involved in the cell cycle imbalance observed during breast carcinogenesis.

Breast cancer is a fatal disease whose incidence is gradually increasing in most industrialized countries and in all ethnic groups. Primary prevention is the ultimate goal for the control of this disease. The knowledge that breast cancer risk is reduced by early full-term pregnancy and that additional pregnancies increase the rate of protection has provided novel tools for designing cancer prevention strategies. The protective effect of pregnancy has been experimentally reproduced in virgin rats by treatment with the placental hormone human chorionic gonadotropin (hCG). HCG prevents the initiation and inhibits the progression of chemically induced mammary carcinomas by inducing differentiation of the mammary gland, inhibiting cell proliferation, and increasing apoptosis. It also induces the synthesis of inhibin, a tumor suppressor factor, downregulates the level of expression of the estrogen receptor alpha (ER-alpha) by methylation of CpG islands, imprinting a permanent genomic signature that characterizes the refractory condition of the mammary gland to undergo malignant transformation. The genomic signature induced by hCG is identical to that induced by pregnancy and is specific for this hormone. Comparison of the mammary gland's genomic profile of virgin Sprague-Dawley rats treated daily with hCG for 21 days with that of rats receiving 17beta-estradiol (E2) and progesterone (Pg) (E2 + Pg) revealed that in hCG-treated rats 194 genes were significantly up-modulated (> 2.5 log2-folds) (p < 0.01) and commonly expressed, whereas these genes were not expressed in the E2 + Pg group. The genomic signature induced by hCG and pregnancy included activators or repressors of transcription genes, apoptosis, growth factors, cell division control, DNA repair, tumor suppressor, and cell-surface antigen genes. Our data indicate that hCG, like pregnancy, induces permanent genomic changes that are not reproduced by steroid hormones and in addition regulates gene expression through epigenetic mechanisms that are differentiation-dependent processes, leading us to conclude that hormonally induced differentiation offers enormous promise for the primary prevention of breast cancer.

Both estrogen receptors (ER) alpha (ERalpha) and beta (ERbeta) are localized in the nucleus, plasma membrane, and mitochondria, where they mediate the different physiological effects of estrogens. It has been observed that the relative subcellular localization of ERs is altered in several cancer cells. We have demonstrated that MCF-10F cells, the immortal and non-tumorigenic human breast epithelial cells (HBEC) that are ERalpha-negative and ERbeta-positive, are transformed in vitro by 17beta-estradiol (E(2)), generating highly invasive cells that are tumorigenic in severe combined immunodeficient mice. E(2)-transformed MCF-10F (trMCF) cells exhibit progressive loss of ductulogenesis, invasive (bsMCF) and tumorigenic (caMCF) phenotypes. Immunolocalization of ERbeta by confocal fluorescent microscopy and electron microscopy revealed that ERbeta is predominantly localized in mitochondria of MCF-10F and trMCF cells. Silencing ERbeta expression with ERbeta-specific small interference RNA (siRNA-ERbeta) markedly diminishes both nuclear and mitochondrial ERbeta in MCF-10F cells. The ERbeta shifts from its predominant localization in the mitochondria of MCF-10F and trMCF cells to the nucleus of bsMCF cells, becoming predominantly nuclear in caMCF cells. Furthermore, we demonstrated that the mitochondrial ERbeta in MCF-10F cells is involved in E(2)-induced expression of mitochondrial DNA (mtDNA)-encoded respiratory chain (MRC) proteins. This is the first report of an association of changes in the subcellular localization of ERbeta with various stages of E(2)-induced transformation of HBEC and a functional role of mitochondrial ERbeta in mediating E(2)-induced MRC protein synthesis. Our findings provide a new insight into one of the potential roles of ERbeta in human breast cancer.

Endocrine and reproductive influences significantly affect the lifetime risk of breast cancer. Nulliparity is one of the most firmly established risk factors for breast cancer, whereas early full-term pregnancy and parity confer a significant protection. The breast attains its maximum development during pregnancy and lactation. After menopause the breast regresses in both nulliparous and parous women containing lobular structures designated lobules type 1 (Lob 1). We have postulated that the degree of differentiation acquired through early preanancy changes the 'genomic signature' that differentiates the Lob 1 from the early parous women from that of the nulliparous women by shifting the Stem cell 1 to a Stem cell 2, making this the mechanism of protection conferred by early full-term pregnancy. In order to elucidate the molecular pathways through which pregnancy exerts a protective effect, we have analyzed the genomic profile of Lob 1 present in reduction mammoplasty spe!

Animal and 'in vitro' experiences learned that human chorionic gonadotropin (hCG) is capable to protect from breast cancer. Receptors for hCG/luteinizing hormone (LH) are present on human female and male breast cancer cells. hCG decreases proliferation and invasion of breast cancer MCF-7 cells by inhibiting NF-kappa B, AP-1 activation and other genes. Doxorubicin toxicity is enhanced by conjugation with beta-hCG in MCF-7 cells. All these pieces of evidence suggest that hCG is active in human breast cancer. Direct proof however is missing. We performed a pilot study phase I trial for testing the inhibitory effects or recombinant hCG (rhCG) on primary breast cancer. Twenty-five postmenopausal women with newly diagnosed breast cancers of more than 1.5 cm were biopsied before randomization to receive either 500 mu g rhCG (n=20) or placebo. After 2 weeks, surgery was done and tissues were analysed with regard to morphological, immunohistochemical and biochemical changes in tis!