Faculty Publications

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.

Breast cancer risk has traditionally been linked to nulliparity or late first full-term pregnancy, whereas young age at first childbirth, multiparity, and breast-feeding are associated with a reduced risk. Early pregnancy confers protection by inducing breast differentiation, which imprints a specific and permanent genomic signature in experimental rodent models. For testing whether the same phenomenon was detectable in the atrophic breast of postmenopausal parous women, we designed a case-control study for the analysis of the gene expression profile of RNA extracted from epithelial cells microdissected from normal breast tissues obtained from 18 parous and 7 nulliparous women free of breast pathology (controls), and 41 parous and 8 nulliparous women with history of breast cancer (cases). RNA was hybridized to cDNA glass microarrays containing 40,000 genes; arrays were scanned and the images were analyzed using ImaGene software version 4.2. Normalization and statistical a!

BACKGROUND: Phthalate esters like n-butyl benzyl phthalate (BBP) are widely used plasticizers. BBP has shown endocrine-disrupting properties, thus having a potential effect on hormone-sensitive tissues. The aim of this study is to determine the effect of neonatal/prepubertal exposure (post-natal days 2-20) to BBP on maturation parameters and on the morphology, proliferative index and genomic signature of the rat mammary gland at different ages of development (21, 35, 50 and 100 days). RESULTS: Here we show that exposure to BBP increased the uterine weight/body weight ratio at 21 days and decreased the body weight at time of vaginal opening. BBP did not induce significant changes on the morphology of the mammary gland, but increased proliferative index in terminal end buds at 35 days and in lobules 1 at several ages. Moreover, BBP had an effect on the genomic profile of the mammary gland mainly at the end of the exposure (21 days), becoming less prominent thereafter. By this age a significant number of genes related to proliferation and differentiation, communication and signal transduction were up-regulated in the glands of the exposed animals. CONCLUSION: These results suggest that BBP has an effect in the gene expression profile of the mammary gland.

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.

We have developed a new approach for breast cancer prevention, capitalizing in the preventive effect of early first full-term pregnancy, hormonally induced differentiation and our ability to identify specific genomic signatures that allow us to predict risk reduction. Early pregnancy imprints in the breast permanent genomic changes or a 'signature' that reduces the susceptibility of this organ to cancer. At cellular level, what we have achieved is the shifting of the Stem Cell 1 population, highly susceptible to cancer, to a population of Stem Cell 2 that is refractory to carcinogenesis. In a case-control study, we have compared the gene expression profile in normal breast tissue from nulliparous and parous postmenopausal women with (case) and without (control) breast cancer. We have determined that early first full-term pregnancy induces a specific genomic signature in the postmenopausal breast that is the biomarker for the Stem cell 2. The Stem cell 2 contains specific genes controlling transcription, RNA processing, immune response, apoptosis and DNA repair. We have further detected in the plasma, using an ELISA assay, the proteins coded by the gene signature. We are developing clinical trials to demonstrate the proof of the principle that r-hCG can induce in the human breast a genomic signature of the Stem cell 2. This is a concept that challenges the currently available chemopreventive agents that need to be given for extended periods for maintaining the suppression of a specific metabolic pathway or the abrogation of the function of an organ.

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!

An elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17 beta-estradiol (E-2), 4-hydroxy-estradiol (4-OHE2) or 2-hydroxyestradiol (2-OHE2) induces phenotypical changes indicative of neoplastic transformation. MCF-10F cells treated with E-2, 4-OHE2 or 2-OHE2, formed colonies in agar methocel and lost their ductulogenic capacity in collagen, expressing phenotypes similar to those induced by the carcinogen benzo[a]pyrene. To investigate whether the transformation phenotypes were associated with genomic changes, cells treated with E-2, 4-OHE2 or 2-OHE2 at different doses were analyzed using microsatellite markers. Since microsatellite instability (MSI) and loss of heterozygosity (LOH) in chromosomes 13 and 17 have been reported in human breast carcinomas, we tested these parameters in MCF-10F cells treated wi! th E-2, 2-OHE2, or 4-OHE2 alone or in combination with the antiestrogen ICI182780. MCF-10F cells treated with E2 or 4-OHE2, either alone or in combination with ICI182780, exhibited LOH in the region 13q12.3 with the marker D13S893 located at similar to 0.8 cM telomeric to BRCA2. Cells treated with E-2 or 4-OHE2 at doses of 0.007 and 70 nM and 2-OHE2 only at a higher dose (3.6 mu M) showed a complete loss of 1 allele with D13S893. For chromosome 17, differences were found using the marker TP53-Dint located in exon 4 of p53. Cells treated with E-2 or 4-OHE2 at doses of 0.007 nM and 70 nM and 2-OHE2 only at a higher dose (3.6 mu M) exhibited a 5 bp deletion in p53 exon 4. Our results show that E-2 and its catechol estrogen metabolites are mutagenic in human breast epithelial cells. ICI182780 did not prevent these mutations, indicating that the carcinogenic effect of E-2 is mainly through its reactive metabolites 4-OHE2 and 2-OHE2, with 4-OHE2 and E-2 being mutagenic at lower d! oses than 2-OHE2. (c) 2005 Wiley-Liss, Inc.

The breast attains its maximum development during pregnancy and lactation. After menopause the breast regresses in both nulliparous and parous women containing lobular structures that have been designated lobules type 1. Despite the similarity in the lobular composition of the breast at menopause, the fact that nulliparous women are at higher risk of developing breast cancer than parous women, indicates that lobules type 1 in these two groups of women might be biologically different, or exhibit different susceptibility to carcinogenesis. Based on these observations it was postulated that the lobule type 1 found in the breast of nulliparous women and of parous women with breast cancer never went through the process of differentiation, retaining a high concentration of epithelial cells that are targets for carcinogens and therefore susceptible to undergo neoplastic transformation, these cell are called Stem cells 1, whereas lobules type 1 structures found in the breast of earl y parous postmenopausal women free of mammary pathology, on the other hand, are composed of an epithelial cell population that is refractory to transformation called Stem cells 2. It was further postulated that the degree of differentiation acquired through early pregnancy has changed the " genomic signature" that differentiates the lobule type 1 from the early parous women from that of the nulliparous women by shifting the Stem cell 1 to a Stem cell 2 that is refractory to carcinogenesis, making this the postulated mechanism of protection conferred by early full term pregnancy. The identification of a putative breast stem cell ( Stem cell 1) has reached in the last decade a significant impulse and several markers also reported for other tissues have been found in the mammary epithelial cells of both rodents and humans. Although still more work needs to be done in order to better understand the role of the Stem cell 2 and its interaction with the genes that confer it a spec ific signature, collectively, the data presently available provides ev idence that pregnancy, through the process of cell differentiation, shifts the Stem cell 1 to Stem cell 2, cells that exhibit a specific genomic signature that could be responsible for the refractoriness of the mammary gland to carcinogenesis.

Breast cancer is a malignancy whose dependence on estrogen exposure has long been recognized even though the mechanisms whereby estrogens cause cancer are not clearly understood. This work was performed to determine whether 17beta-estradiol (E2), the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells and whether nonreceptor mechanisms are involved in the initiation of breast cancer. For this purpose, the effect of four 24 h alternate periods of 70 nM E2 treatment of the estrogen receptor alpha (ER-alpha) negative MCF-10F cell line on the in vitro expression of neoplastic transformation was evaluated. E2 treatment induced the expression of anchorage-independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11-13. Only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER-alpha and progesterone receptor-negative adenocarcinomas that expressed keratins, EMA, and E-cadherin. Tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of MCF-10F cells in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.

Breast cancer is the most frequent malignancy in women. It is well recognized that tumorigenesis is a multistep process resulting from the accumulation of sequential genetic alterations. In breast cancers LOH has been described on one or both arms of multiple chromosomes. Comparative genomic hybridization (CGH) analysis was performed to identify chromosomal imbalances in the breast epithelial cells (HBEC). We have used a human in vitro-in vivo system in which the environmental carcinogen benz(a)pyrene (BP) and the c-Ha-ras oncogene were utilized for inducing in vitro transformation of HBEC. Immortal MCF-10F cells were treated with BP which resulted in the transformed cell line BP-1 that was further enhanced by transfection with the c-Ha-ras to generate the cell line BP-1-Tras. This cell line is tumorigenic when injected in severe combined immunodeficient (SCID) mice, generating the tumor cell line BP-1-Tras T J#4. Our comparative genomic hybridization analysis indicates that the most overrepresented segment after cell transformation and in the BP-1, BP-1-Tras and in the tumor cell line were 1p (80%), 5q21-ter (80%), 8q24.1 (90%) and Xq27-28 (60%). DNA sequence amplification at 10p14-15 was observed in BP-1-Tras T J#4 cells. Allelic losses of chromosome 4, 8p11-21 and 15q11-12, occur after cell transformation and are maintained consistently during tumorigenesis.

The elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory has demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17beta-estradiol (E-2) or its metabolites 4-hydroxy-estradiol (4-OH-E-2) and 2-hydroxy-estradiol (2-OH-E-2) induces phenotypical changes indicative of neoplastic transformation. The MCF-10F cells treated with E-2,E- 2-OH-E-2 and 4-OH-E-2 form colonies in agar methocel and lost their ductulogenic capacity in collagen matrix, expressing phenotypes similar to those induced by the carcinogen benz(a)pyrene (BP). To investigate whether these phenotypic changes were associated with genomic changes, MCF-10F cells treated with cither E-2, 2-OH-E-2, or 4-OH-E2 at different doses (0.007 nM, 70 nM and 3.6 muM) were analyzed using a combination of standard G-banding and comparative genomic hybridization (CGH). Whereas no aneuploidy was observed in any of the t ransformed cells, the CGH revealed instead that only cells treated with 4-OH-E-2 at the highest concentration (3.6 muM) exhibited DNA gains at 8q24, 9q34 and 20q13 and losses at 13q21.

Questions of reproducibility and efficacy of histologic malignancy grading relative to alternative proliferation index measurements for outcome prediction remain unanswered. Microsections of specimens from the Cooperative Breast Cancer Tissue Resource (CBCTR) were evaluated by seven pathologists for reproducibility of grade and classification. Nuclear figure classification was assessed using photographs. Grade was assigned by the Bloom - Richardson method, Nottingham modification. Proliferation index was evaluated prospectively by deoxyribose nucleic acid precursor uptake with thymidine (autoradiographic) or bromodeoxyuridine ( immunohistochemical) labeling index using fresh tissue from 631 node-negative breast cancer patients accessioned at St Luke's Hospital. A modified Nottingham - Bloom - Richardson grade was derived from histopathologic data. Median post-treatment observation was 6.4 years. Agreement on classification of nuclear figures (N = 43) was less than good by ka ppa statistic (kappa = 0.38). Grade was moderately reproducible in four trials ( N = 10,10,19, 10) with CBCTR specimens (kappa = 0.50 - 0.59). Of components of Bloom - Richardson grade, agreement was least for nuclear pleomorphism (kappa = 0.37 - 0.50), best for tubularity (kappa = 0.57 - 0.83), and intermediate for mitotic count ( kappa = 0.45 - 0.64). Bloom - Richardson grade was a univariate predictor of prognosis in node-negative St Luke's patients, and was improved when mitotic count was replaced by labeling index ( low, mid, or high). When labeling index was added to a multivariate model containing tumor size and vessel invasion, grade was no longer a significant predictor of tumor-specific relapse-free or overall survival. Mitotic index predicted best when intervals were lowered to 0 - 2, 3 - 10, and >10 mitotic figures per ten 0.18 mm(2) high-power fields. We conclude that Nottingham - Bloom - Richardson grades remain only modestly reproducible. Prognostically usefu l components of grade are mitotic index and tubularity. The Nottingham - Bloom Richardson system can be improved by lowering cutoffs for mitotic index and by counting 20 - 30 rather than 10 high-power fields. Measurement of proliferation index by immunohistochemically detectable markers will probably give superior prognostic results in comparison to grade.

Prolonged exposure to 17beta-estradiol (E2) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERalpha and ERbeta. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na+, K+-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and act ivity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na+, K+-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na+, K+-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.