The plasticizer butyl benzyl phthalate induces genomic changes in rat mammary gland after neonatal/prepubertal exposure.

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.

Methodological approach to study the genomic profile of the human breast.

One of the key end-points for understanding the molecular basis of the breast in its normal and cancer status is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. The objective of this study was to determine the optimal RNA isolation and amplification to perform genomic expression analysis using the microarray technique from normal breast paraffin-embedded tissue samples using laser capture microdissection (LCM). We isolated epithelial and interlobular stroma cells from normal breast tissue and the total RNA was amplified using a PCR methodology developed by us, and in parallel the same starting material was used for amplification using the linear methodology. After two rounds of RNA amplification, we checked the quality of each amplified RNA and carried out the hybridization with cDNA glass-microarrays employing 15,000 genes for each replicate. In conclusion, we have successfully demonstrated that our PCR methodology is accurate and precise and give us a higher yield of amplified RNA from small number of cells obtained from LCM compared with the typical linear amplification methodology.

The breast of parous women without cancer has a different genomic profile compared to those with cancer.

Our studies are aimed at determining whether pregnancy induces a specific genomic signature in the postmenopausal breast that is responsible for the protective effect elicited by this physiological process. For this purpose we designed a study to compare the gene expression profiles in normal breast tissue from parous postmenopausal women with (case) and without (control) breast cancer. We have used breast samples from 18 parous controls and 41 parous cases. The epithelium and the interlobular stroma were dissected using laser capture microdissection and the RNA of each compartment and each sample was isolated, amplified using PCR methodology, and hybridized to cDNA glass-microarrays containing 40,000 genes, placing the human reference RNA in the green channel (Cy3) and the breast tissue samples in the red channel (Cy5). The normalization and statistical analysis of the expression data were carried out by using the LIMMA software package for the R programming environment which provides functions to summarize the results using the linear model perform hypothesis tests and adjust the p-values for multiple testing. We were able to identify 126 genes that were upregulated and 103 that were downregulated in the parous control group. There were only 56 genes differentially expressed in the interlobular stroma in the parous control group in relation to the other group of women under study. The gene categories that were overrepresented in the breast epithelium of the parous control breast are related to apoptosis, DNA repair, response to exogenous agents and transcription regulation. In the present study we demonstrate that full-term pregnancy imprints a specific genomic signature in the breast epithelium of postmenopausal parous control women that is significantly different from women who have developed cancer. This genomic signature induced by pregnancy could help to predict in which women parity is protective.

The genomic signature of breast cancer prevention.

Early pregnancy imprints in the breast permanent genomic changes or a signature that reduces the susceptibility of this organ to cancer. 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 Lob.1. The Lob 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 undergoing neoplastic transformation, these cell are called Stem cells 1, whereas Lob 1 structures found in the breast of early 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. The degree of differentiation acquired through early pregnancy has changed 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 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. The data obtained thus far is supporting the concept that the lifetime protective effect of an early pregnancy against breast cancer is due to the complete differentiation of the mammary gland, which results in the replacement of the Stem cell 1 that is a component of the nulliparous breast epithelium with a new stem cell, called Stem cell 2, which is characterized by a specific genomic signature. The pattern of gene expression of the stem cell 2 could potentially be used as useful intermediate end points for evaluating the degree of mammary gland differentiation and for evaluating preventive agents such as human chorionic gonadotropin.

Identification of cripto-1 as a novel serologic marker for breast and colon cancer.

PURPOSE: Human Cripto-1 (CR-1), a cell membrane glycosylphosphatidylinositol-anchored glycoprotein that can also be cleaved from the membrane, is expressed at high levels in several different types of human tumors. We evaluated whether CR-1 is present in the plasma of patients with breast and colon cancer, and if it can represent a new biomarker for these malignancies. EXPERIMENTAL DESIGN: We determined CR-1 plasma levels using a sandwich-type ELISA in 21 healthy volunteers, 54 patients with breast cancer, 33 patients with colon carcinoma, and 21 patients with benign breast lesions. Immunohistochemical analysis was also used to assess CR-1 expression in cancerous tissues. RESULTS: Very low levels of CR-1 (mean+/-SD) were detected in the plasma of healthy volunteers (0.32+/-0.19 ng/mL). A statistically significant increase in the levels of plasma CR-1 was found in patients with colon carcinoma (4.68+/-3.5 ng/mL) and in patients with breast carcinoma (2.97+/-1.48 ng/mL; P<0.001). Although moderate levels of plasma CR-1 were found in women with benign lesions of the breast (1.7+/-0.99 ng/mL), these levels were significantly lower than in patients with breast cancer (P<0.001). Finally, immunohistochemical analysis and real-time reverse transcription-PCR confirmed strong positivity for CR-1 in colon and/or breast tumor tissues. CONCLUSION: This study suggests that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas.

The concept of stem cell in the mammary gland and its implication in morphogenesis, cancer and prevention.

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 early 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 specific signature, collectively, the data presently available provides evidence 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.

Genomic signature induced by pregnancy in the human breast.

We have postulated that the lifetime protective effect of an early pregnancy against breast cancer is due to the complete differentiation of the mammary gland characterized by a specific genomic signature imprinted by the physiological process of pregnancy. For demonstrating this hypothesis we compared the genomic profile of the epithelium and the stroma of normal breast tissues from reduction mammoplasties performed in postmenopausal parous and nulliparous women. The epithelium and the stroma were separately dissected using laser capture microdissection (LCM) and the RNA of each compartment and each sample was isolated, amplified using PCR methodology, and hybridized to cDNA glass-microarrays containing 40,000 human cDNA features. The separation of the epithelial compartment from the interlobular stroma of Lob 1 using LCM allowed us to determine that the epithelial component contained 4,828 genes that were equally expressed in both nulliparous and parous women. There were 73 known genes that included immune-modulation-, DNA repair-, programmed cell death-, chromatin remodeling- and transcription-related genes, whereas in the breast of nulliparous women there were 20 different known genes that were upregulated. Our data provide evidence that breast tissues of postmenopausal parous women express in both the epithelial and the stromal compartments numerous genes that differ significantly from those present in breast tissues of post-menopausal nulliparous women, which could be important contributors to the genomic signature induced by an early full term pregnancy.

Molecular basis of pregnancy-induced breast cancer protection.

We have postulated that the lifetime protective effect of an early pregnancy against breast cancer is due to the complete differentiation of the mammary gland characterized by a specific genomic signature imprinted by the physiological process of pregnancy. In the present work, we show evidence that the breast tissue of postmenopausal parous women has had a shifting of stem cell 1 to stem cell 2 with a genomic signature different from similar structures derived from postmenopausal nulliparous women that have stem cell 1. Those genes that are significantly different are grouped in major categories on the basis of their putative functional significance. Among them are those gene transcripts related to immune surveillance, DNA repair, transcription, chromatin structure/activators/co-activators, growth factor and signal transduction pathway, transport and cell trafficking, cell proliferation, differentiation, cell adhesion, protein synthesis and cell metabolism. From these data, it was concluded that during pregnancy there are significant genomic changes that reflect profound alterations in the basic physiology of the mammary gland that explain the protective effect against carcinogenesis. The implication of this knowledge is that when the genomic signature of protection or refractoriness to carcinogenesis is acquired by the shifting of stem cell 1 to stem cell 2, the hormonal milieu induced by pregnancy or pregnancy-like conditions is no longer required. This is a novel concept that challenges the current knowledge that a chemopreventive agent needs to be given for a long period to suppress a metabolic pathway or abrogate the function of an organ.

Breast differentiation and its implication in cancer prevention.

Sporadic breast cancer is a fatal disease most frequently diagnosed in American women from all ethnic groups, suggesting that primary prevention should be the ultimate goal for breast cancer control. We have developed a novel paradigm for breast cancer prevention arising from the well-established knowledge that an early first full-term pregnancy protects the breast against neoplastic transformation, as well as from our studies of the biological principle underlying this protection. We have shown experimentally that the first pregnancy induces the expression of a specific genomic signature in the breast that results from the completion of a cycle in this organ's differentiation driven by the reproductive process. This signature, in turn, is a biomarker associated with a possible overall lifetime decrease in breast cancer risk. We have shown in an experimental model that a short treatment with human chorionic gonadotropin, a placental hormone secreted during pregnancy, induces the same genomic signature that occurs in pregnancy, inhibiting not only the initiation but also the progression of mammary carcinomas, and stopping the development of early lesions such as intraductal proliferations and carcinoma in situ. These observations indicate that human chorionic gonadotropin given for a very short period, only until this genomic signature is acquired, has significant potential as a chemopreventive agent, protecting the normal cell from becoming malignant. This is a novel concept which challenges the current knowledge that a chemopreventive agent needs to be given for a long period of time to suppress a metabolic pathway or abrogate the function of an organ.

The protective role of pregnancy in breast cancer.

Epidemiological, clinical, and experimental data indicate that the risk of developing breast cancer is strongly dependent on the ovary and on endocrine conditions modulated by ovarian function, such as early menarche, late menopause, and parity. Women who gave birth to a child when they were younger than 24 years of age exhibit a decrease in their lifetime risk of developing breast cancer, and additional pregnancies increase the protection. The breast tissue of normally cycling women contains three identifiable types of lobules, the undifferentiated Lobules type 1 (Lob 1) and the more developed Lobules type 2 and Lobules type 3. The breast attains its maximum development during pregnancy and lactation (Lobules type 4). After menopause the breast regresses in both nulliparous and parous women containing only Lob 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 Lob 1 in these two groups of women might be biologically different, or might exhibit different susceptibility to carcinogenesis. Based on these observations it was postulated that Lob 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 are therefore susceptible to undergo neoplastic transformation. These epithelial cells are called Stem cells 1, whereas Lob 1 structures found in the breast of early parous postmenopausal women free of mammary pathology, on the contrary, 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 Lob 1 of the early parous women from that of the nulliparous women by shifting the Stem cells 1 to Stem cells 2 that are 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 cells 1) has, in the past decade, reached 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 further work needs to be carried out in order to better understand the role of the Stem cells 2 and their interaction with the genes that confer them a specific signature, collectively the data presently available provide evidence that pregnancy, through the process of cell differentiation, shifts Stem cells 1 to Stem cells 2 - cells that exhibit a specific genomic signature that could be responsible for the refractoriness of the mammary gland to carcinogenesis.

Serological levels of mutated p53 protein are highly detected at early stages in breast cancer patients.

The aim of this study was to compare the sensitivity of the serological level of anti-p53 antibodies in breast cancer patients and to correlate its expression level with patient age, histological stage and grade of tumor differentiation. Total p53 protein expression (mutant and wild-type) was also determined in the breast cancer tissues using immunohistochemistry (IHC). The serological levels of mutant p53 expression were found to be age-dependent, reaching the highest level at 50 years of age. Faint or low detection was observed in patients ≤30 years of age. Anti-p53-antibodies were detected in patients ≤40 and ≥61 years of age. The serological levels of mutant p53 protein were highly detected in all stages of breast cancer, including the early stages. However, anti-p53 antibodies reached a high level of detection only in stage III breast carcinomas. No expression was found in patients with benign breast disease. The detection of p53 mutations was dependent on the grade of tumor differentiation, achieving the highest level in the poorly differentiated breast carcinomas. Results from IHC were highly correlated with serological p53 mutational analysis. Our findings indicate that mutant p53 in serum is a promising novel parameter for the evaluation of cellular biology and the prognosis of breast cancer from its early stages using blood samples. Anti-p53 antibodies were demonstrated to be less sensitive in this study. It is also possible to use the expression of mutant p53 protein as a molecular marker to differentiate benign breast disease from breast carcinoma prior to surgery.