Development of the human breast

Maturitas 49 (2004) 2–15

Development of the human breast Jose Russo∗ , Irma H. Russo Breast Cancer Research Laboratory, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA Received 16 December 2003; received in revised form 7 April 2004; accepted 19 April 2004

Abstract The human breast undergoes a complete series of changes from intrauterine life to senescence. These changes can be divided into two distinct phases; the developmental phase and the differentiation phase. The developmental phase includes the early stages of gland morphogenesis, from nipple epithelium to lobule formation. In lobule formation, both processes, development and differentiation, take place almost simultaneously. For example, the progressive transition of lobule type 1 to types 2, 3, and 4 requires active cell proliferation, to acquire the cell mass necessary for the function of milk secretion. This later process implies differentiation of the mammary epithelium. Therefore, the presence of lobule type 4 is the maximal expression of development and differentiation in the adult gland, whereas the presence of lobule type 3 could indicate that the gland has already been developed. It is important to point out that the presence of proteins that are indicative of milk secretion, such as ␣-lactalbumin, casein, or milk fat lobule type membrane protein, also indicates cellular differentiation of breast epithelium. However, only when all the other components of milk, (such as lactose, ␣-lactalbumin, casein and milk fat) are coordinately synthesized within the appropriate structure can full differentiation of the mammary gland be acknowledged. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Breast; Differentiation; Cell proliferation; Phenotypic changes; Genotypic changes; Estrogen; Estrogen receptors

1. Introduction The breast is a bilateral organ that in the female undergoes dramatic changes in size, shape, and function in association with infantile growth, puberty, pregnancy, lactation, and postmenopausal regression [1–4]. The breast is also the source of the most frequently diagnosed malignancy in the female popu∗ Corresponding author. Tel.: +1 215 728 4782; fax: +1 215 728 2180 E-mail address: J [email protected] (J. Russo).

lation [5]. The knowledge that the risk of developing breast cancer is heavily influenced by endocrinological influences and the reproductive history of the host [2–4] requires a thorough understanding of how the endocrinological milieu, especially that created by pregnancy, influences the development of this organ. The development of the human breast is a progressive process initiated during embryonic life. The main spurt of growth occurs with lobule formation at puberty, but the development and differentiation of the breast are completed only by the end of the first full term

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pregnancy [4]. It has long been known that the risk of breast cancer shows an inverse relationship with early parity [1,3,4,6–10]. Case control studies have demonstrated that breast cancer risk increases with the age at which a woman bears her first child; the important factor in this protection seems to be related to the interval of time between menarche and the first pregnancy, since increased risk has been reported when this interval is lengthened over 14 years [10]. Thus, to be protective, however, pregnancy has to occur before age 30—indeed women first becoming pregnant after that age appears to have a risk above that of nulliparous women [10]. Although multiparity appears to confer additional protection, the protective effect remains largely limited to the first birth. The protection conveyed by an early reproductive event persists at all subsequent ages, even until women older than 75 years of age [1,10]. Although the ultimate mechanisms through which an early first full term pregnancy protects the breast from cancer development are not known, a likely explanation has been provided by studies performed in an experimental animal model. Induction of mammary carcinomas with chemical carcinogens in rats has revealed that full term pregnancy inhibits carcinogenic initiation through the induction of differentiation. It can be postulated that gland differentiation activates specific genes such as inhibin [11], mammary derived growth factor inhibitor [12], a Serpin like gene [13] and other genes which function still must be determined [14], that imprint the breast epithelia to subsequent hormonal milieu, and that this is also responsible for the protection that an early full term pregnancy confers to women. There is no explanation for the higher risk to develop malignancies exhibited by nulliparous and late parous women. The fact that experimentally induced rat mammary carcinomas develop only when the carcinogen interacts with the undifferentiated and highly proliferating mammary epithelium of young nulliparous rats [3,4,15–19], suggests that the breast of late parous and of nulliparous women might exhibit some of the undifferentiated and/or cell proliferative characteristics that predispose the tissue to undergo neoplastic transformation. The fact that the lobules 1 are the one that have the highest proliferative index, the higher concentration of estrogen receptors and that the number of blood vessels per lobular structure, clearly indicate that this structure is the natural target of malignancy

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[4,17,20] as it has been demonstrated by their ability to be transformed in vitro by chemical carcinogens [15,16].

2. Prenatal and perinatal development The mammary gland parenchyma arises from a single epithelial ectodermal bud. Most authors agree on the successive stages of development of the mammary gland during the embryonic and fetal stages, there are variations in nomenclature, and the exact time of appearance of each structure varies whether the authors choose to express the age of the embryo based on the estimated time of conception, the last missed menstrual period, or the length of the embryo. Because of difficulties in establishing precisely the day of conception, we consider it more accurate to correlate the phases of mammary gland development with embryonal or fetal length. Mammary gland development can be divided into 10 different stages (Table 1). Colostrum production is found in the last stage or end vesicle stage. In the newborn breast, there are very primitive structures, composed of ducts ending in short ductules lined by one to two layer of epithelial and one of myoepithelial cells. The epithelial cells have an eosinophilic cytoplasm, with typical apocrine secretion. The fine cytoplasmic vacuolization observed in the epithelial ells is due to the presence of lipid droplets, as confirmed by electron microscopy. However, secretory activity does not seem to be confined to the primitive alveolar structures, since the whole ductal system appears dilated, secretion filled, and lined by a secretory-type epithelium. These observations suggest that secretory activity is a generalized response of all the mammary epithelium to maternal hormonal levels. The secretory activity of the newborn gland subsides within 3–4 weeks [4,21].

3. Postnatal development Mammary gland development during childhood does little more than keep pace with the general growth of the body until the approach of puberty. Although the main changes occurring in the mammary gland are initiated at puberty, ulterior development of the gland varies greatly from woman to woman. Mammary gland development can be defined from the external

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Table 1 Stages of prenatal development of the human breast Stage

Mammary gland development

Embryo-fetal stage

1 2 3 4 5 6 7 8 9 10

Ridge stage Milk hill stage Mammary disc stage lobule type stage Cone stage Budding stage Indentation stage Branching stage Canalization stage End-vesicle stage, in which the end vesicles are composed of a monolayer of epithelium and contain colostrums

Less than 5 mm embryo More than 5.5-mm embryo Around 10–11 mm embryo 11.0–25.0-mm embryo 25–30-mm embryo 30–68-mm embryo 68-mm to 10 cm 10 cm fetus 20 and 32 weeks of gestation Newborn

appearance of the breast or by determination of mammary gland area, volume, degree of branching, or degree of structures whose appearance indicates the level of differentiation of the gland, such as lobule type formation. The adolescent period begins with the first signs of sexual change at puberty and terminates with sexual maturity [22,23]. Puberty in the female starts when pubic hair appears. With the approach of puberty, the rudimentary mammae begin to show growth activity both in the glandular tissue and in the surrounding stroma. Glandular increase is due to the growth and division of small bundles of primary and secondary ducts. They grow and divide partly dichotomously (from the Greek word dichotomos, or repeated bifurcation) and partly sympodially (from Greek syn + podion base, involving the formation of an apparent main axis from successive secondary axes), on a dichotomous basis. The ducts grow, divide, and form club-shaped terminal end buds. Terminal end buds give origin to new branches, twigs, and small ductules or alveolar buds (Fig. 1). We have coined the term alveolar bud to identify those structures that are morphologically more developed than the terminal end bud but yet more primitive than the terminal structure of the mature resting organ, which is called acinus. Alveolar buds cluster around a terminal duct, forming the lobule type 1 or virginal lobule (Fig. 1) and each cluster is composed of approximately 11 alveolar buds. Terminal ducts and alveolar buds are lined by a two-layered epithelium. Lobule formation in the female breast occurs within 1–2 years after onset of the first menstrual period. Full differentiation of the mam-

mary gland is a gradual process taking many years, and in some cases, if pregnancy does not supervene, is never attained [4,19]. It is acknowledged that hormonal influences play a significant role in breast development; however, the effect of their fluctuations during the menstrual cycle on parenchymal proliferation has not been definitively

Fig. 1. Diagrammatic representation of the lobular structures of the human breast.

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elucidated. Normal breast epithelium undergoes cyclic variations of DNA synthesis, as determined in normal breast samples cultured in the presence of 3 H-thymidine. Even though cell proliferation and cell death seem balanced to maintain the equilibrium of the resting breast, mammary development induced by ovarian hormones during a menstrual cycle never fully returns to the starting point of the preceding cycle. Accordingly, each ovulatory cycle fosters slightly more mammary development with new budding of structures that continues until about age 35 [4]. The study of normal breast tissue of adult women contains two identifiable types of lobules, in addition to the already described type 1. These are designated lobule types 2 and 3 (Fig. 1). The transition from lobule type 1 to type 2, and of type 2 to type 3, is a gradual process of sprouting of new alveolar buds. In lobule type 2 and type 3, these are now called ductules; they increase in number from approximately 11 in the lobule types 1–47 and 80 in lobule types 2 and 3, respectively (Table 2). The increase in number results in a concomitant increase in size of the lobules and a reduction in size of each individual structure. The alveolar buds composing a lobule type 1, measure an average of 0.232 × 10−2 mm2 , practically twice the size of the ductules composing lobule type 2, whereas the reduction in size in ductules composing lobules type 3 is less dramatic, although still significant. The breast of nulliparous women contains more undifferentiated structures such as terminal ducts and lobules type 1 (Fig. 2), although occasional lobules type 2 and 3 were seen (Fig. 3). In parous women on the other hand, the predominant structure is the most differentiated lobules type 3 (Figs. 4–6). Lobules type 1 remains constant throughout the lifespan of nulliparous women. Lobules type 3 in parous women peaks during the early

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reproductive years, decreasing after the fourth decade of life (Fig. 6). In the breast of nulliparous women, lobules type 2 are present in moderate numbers during the early years, sharply decreasing after age 23, whereas the number of lobules type 1 remains significantly higher (Fig. 2). This observation suggests that a certain percentage of lobules type 1 might have progressed to lobules type 2, but the number of lobules type progressing to type 3 is significantly lower than in parous women. In the case of parous women, it is interesting to note that a history of parity between the ages of 14–20 years correlates with a significant increase in the number of lobules type 3 that remain present as the predominant structure until the age of 40, the time at which a decrease in the number of lobules type 3 occurs, probably due to their involution to predominantly lobules types 2 and 1 (Figs. 4 and 5) [4].

4. Analysis of breast architecture in the four different quadrants of the breast The development of the human breast during the post pubertal years has been measured by quantitation of the various types of lobules which represent different stages of development and differentiation. The next question addressed was to determine if the architecture of the breast was identical in all the quadrants of the breast. For this purpose whole breasts were serially sectioned and prepared for whole mount for reconstructing them in a three-dimensional fashion. The total number of ducts, lobules type 1, type 2, or type 3 were quantitated in each quadrant of the breast of nulliparous and parous women. The patient population consisted of 16 women, of which 6 were nulliparous and ranged in age from 20 to 61 years, and 10 were parous, ranging in age from 20 to 63 years. Upon removal, each breast

Table 2 Characteristics of the lobular structures of the human breast Structure

Lobular areaa (␮m2 )

No. of ductules/lobuleb

No. of cells/cross-sectionc

Lob 1 Lob 2 Lob 3

48 ± 44 60 ± 26 129 ± 49

11.2 ± 6.3 47.0 ± 11.7 81.0 ± 16.6

32.4 ± 14.1 13.1 ± 4.8 11.0 ± 2.0

a Student’s t-tests were done for all possible comparisons. Lobular areas showed significant differences between Lob 1 vs. Lob 3 and Lob 2 vs. Lob 3 (P < 0.005). b The number of ductules per lobule was different (P < 0.01) in all the comparisons. c The number of cells per cross section was significantly different in ductules of Lob 1 vs. Lob 2 and Lob 3 (P < 0.01).

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Fig. 2. Percentage of lobules type 1 (Lob 1) in the four quadrants of the nulliparous breast. UOQ, upper outer quadrant; LOQ, lower outer quadrant; LIQ, lower inner quadrant; UIQ, upper inner quadrant.

sample was fixed in 10% neutral buffered formalin and then divided into four quadrants: UOQ, LOQ, LIQ, and UIQ, taking the nipple and the axillary tail of the breast as reference points. Each breast was serially cut with a meat slicer into 0.2–0.5-cm thick slices and processed as described elsewhere [18]. Serial sectioning of whole breasts yielded a number of slices that ranged from 6 to 26 for each quadrant, depending upon the size of the breast. The total number of lobules type 1, 2, or 3 (Figs. 2–6) present in each slice was counted and the total number of each lobule type was added for obtaining the number for each specific

quadrant and for the gland as a whole; the number of structures per cm2 of tissue examined was obtained as a ratio between number of structures and size of the area examined. The analysis of results revealed that in nulliparous women the breast architecture did not differ significantly, both having as the predominant structure in the breast the Lob 1 with a lower percentage of Lob 2 (Figs. 2 and 3). The breast tissue of the parous women contained the lowest percentage of Lob 1, a slightly higher percentage of Lob 2, and the Lob 3 as the predominant structure (Figs. 4, 5 and 6). Parous women

Fig. 3. Percentage of lobules type 2 (Lob 2) in the four quadrants of the nulliparous breast. UOQ, upper outer quadrant; LOQ, lower outer quadrant; LIQ, lower inner quadrant; UIQ, upper inner quadrant.

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Fig. 4. Percentage of lobules type 1 (Lob 1) in the four quadrants of the parous breast. UOQ, upper outer quadrant; LOQ, lower outer quadrant; LIQ, lower inner quadrant; UIQ, upper inner quadrant.

Fig. 5. Percentage of lobules type 2 (Lob 2) in the four quadrants of the parous breast. UOQ, upper outer quadrant; LOQ, lower outer quadrant; LIQ, lower inner quadrant; UIQ, upper inner quadrant.

contained a greater number of all the structures in the UOQ; however, their relative percentage was not different from the percentages in the other quadrants of the breast.

5. Pregnancy and postlactational changes During pregnancy, the breast attains its maximum development; it occurs in two distinctly dominant phases characteristic of the early and late states of pregnancy. The early stage is characterized by growth

consisting of proliferation of the distal elements of the ductal tree, resulting in the formation of ductules that at this stage, can be called acini, thus developing a lobule type 3 into a lobule type 4. The intensity of budding and degree of lobule formation goes beyond what has been observed in the virginal breast. By the third month of pregnancy, the number of well-formed lobules exceeds the number of primitive budding stages; however, primitive budding stages are still found. In newly formed lobules, the epithelial cells composing each acinus not only increase greatly in number due to active cell division but they also increase in size mainly

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Fig. 6. Percentage of lobules type 3 (Lob 3) in the four quadrants of the parous breast. UOQ, upper outer quadrant; LOQ, lower outer quadrant; LIQ, lower inner quadrant; UIQ, upper inner quadrant.

because of cytoplasm enlargement [4]. In the middle of pregnancy, the lobules are further enlarged and increased in number. They surround the duct from which their central branch proceeds so thickly that the chief duct, the terminal or intra lobular terminal duct can no longer be recognized. The transition between the terminal ducts and the budding acini is gradual, making the histological distinction between the two of them difficult, since both show evidence of early secretory activity. The definitive structure of the ductal tree is essentially settled by the end of the first half of pregnancy; the mammary changes that characterize the second half of pregnancy are chiefly continuation and accentuation of the secretory activity. Further progressive branching continues with less prominent bud formation. At this time, the formation of true secreting units or acini, the differentiated structures, becomes increasingly evident. Proliferation of new acini is reduced to a minimum, and the luminae of those already formed become distended by accumulation of secretory material or colostrum. The epithelium is vacuolated due to the accumulation of lipids. Under the electron microscopy the mammary epithelia shows numerous lipid droplets and proteinaceous material. In the lobule type 4 or lactating the reactivity against milk fat lobule protein is highly expressed. The secretory acinus formed during pregnancy is a terminal outgrowth that marks the end of glandular differentiation. However, just before and during parturition, there is a new wave of mitotic

activity with an increase in the total DNA of the gland. During lactation, the process of growth and differentiation may be observed in the same lobule type, side by side with the process of milk secretion [3,4,11,21]. From mid-pregnancy on, a yellowish fluid containing a high concentration of protein is secreted into the mammary alveoli and may be expelled from the nipple. After postpartum withdrawal of placental lactogen and sex steroids, which appear to prevent the action of prolactin on the mammary epithelium, lactation starts. Colostrum is secreted during the first week postpartum, followed by a 2–3-week period of transitional milk secretion, leading to the secretion of mature milk [4,20,21]. No major morphological changes of the mammary gland are observed during lactation. The mammary lobules are enlarged and the acini have a dilated lumen filled with granular, slightly basophilic material admixed with fat. There is a significant variation in lobule size throughout the gland, suggestive of a variation in lactogenic activity from lobule to lobule. Milk is synthesized and released into the mammary acini and ductal system, although it can be stored for up to 48 h before the rate of milk synthesis and secretion begins to decrease. As long as milk is removed regularly from the mammary gland, the alveolar cells continue to secrete milk [4,20,21]. The accumulation of milk in the ductoacinar lumina and within the cytoplasm of the lactogenic epithelial cells that occurs after weaning has an inhibitory effect

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on further milk synthesis. This effect is followed by a series of involutional changes in the mammary gland consisting of a multifocal asynchronous process of reduction in volume of the secretory epithelial cells and further inhibition of their secretory activity. It is considered that postlactational regression is due to two complementary mechanisms, cell autolysis, with collapse of acinar structures and narrowing of the tubules, and appearance of round cell infiltration and phagocytes in and about the disintegrating lobules, and finally, regeneration of the periductal and peri lobular connective tissue with renewed budding and proliferation in the terminal tubules. Until menopausal involution sets in, the parous organ shows more glandular tissue than if pregnancy or pregnancy and lactation had never occurred [4,20,21].

6. The menopausal breast Menopause supervenes as the consequence of the atresia of more than 99% of the 400,000 follicles that are present in the ovaries of a female fetus at a gestational age of 5 months. Gonadotropin-releasing hormone secretion is also implicated in this phenomenon, indicating that a hypothalamic process is involved in the development of menopause. The most characteristic sign of menopause is amenorrhea, which is the result of the almost complete cessation of ovarian estrogen and progesterone production. The years leading up to the final menstrual period, until menopause sets in generally at around age 51 years, constitute the perimenopause. During this time, many women ovulate irregularly, either because the rise in estrogen during the follicular phase to insufficient for triggering an LH surge, or because the remaining follicles are resistant to the ovulatory stimulus. The increase in human longevity occurring in our society has caused a considerable increment in the number of women that will live one third or more of their lives in the menopausal period, namely without natural estrogen and progesterone. After menopause, the breast undergoes a regressive phenomenon both in nulliparous and parous women. This regression is manifested as an increase in the number of lobule type 1, and a concomitant decline in the number of lobule type 2 and lobule type 3. At the end of the fifth decade of life, the breast of both nulliparous and parous women contains lobule

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type 1 [4,19]. These observations led us to conclude that the understanding of breast development requires a horizontal study in which all the different phases of growth are taken into consideration. For example, the analysis of breast structures at a single given point, i.e., age 50 years, would lead us to conclude that the breast of both nulliparous and parous women appears identical. However, the phenomena occurring in prior years might have imprinted permanent changes in breast biology and affect the potential of the breast for neoplasm but are no longer morphologically observable. Thus, from a quantitative point of view, the regressive phenomenon occurring in the breast at menopause differs in nulliparous and parous women. In the breast of nulliparous women, the predominant structure is the lobule type 1, which comprises 65–80% of the total lobule type components and their relative percentage is independent of age. Second, in frequency is the lobule type 2 that represents 10–35% of the total. The least frequent are the lobule type 3, which represent only 0–5% of the total lobular population. In the breast of premenopausal parous women, on the other hand, the predominant lobular structure is the lobule type 3, which comprises 70–90% of the total lobule component. Only after menopause do they decline in number, and the relative proportion of the three lobule types present approach that observed in nulliparous women. These observations led to conclude that early parous women truly underwent lobule differentiation, which was evident at a younger age, whereas nulliparous women seldom reached the lobule type 3, and never the lobule type 4 stages [19]. Even though during the postmenopausal years in the breast of both parous and nulliparous women the preponderant structure is the Lob 1, only the nulliparous women are at high risk of developing breast cancer, whereas parous women remain protected [19]. Since ductal breast cancer originates in Lob 1 (TDLU) [17], the epidemiological observation that nulliparous women exhibit a higher incidence of breast cancer than parous women [3,4] indicates that Lob 1 in these two groups of women might be biologically different, or exhibit different susceptibility to carcinogenesis [15,24–26]. The presence of Lob 1 in the breasts of parous women has also been interpreted as a failure of the mammary parenchyma to respond to the influences of pregnancy and lactation [19,20]. It is possible to postulate that unresponsive lobules that fail to undergo

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full differentiation under the stimuli of pregnancy and lactation are responsible of cancer development despite the parity history of a woman. If this were the case, then this unresponsive Lob 1 would be as sensitive to carcinogenesis as the lobules found in the breasts of nulliparous women. We have reported the presence of intralobular hyalinization and lower proliferative activity in the Lob 1 of the parous woman’s breast, whereas hyalinization is absent and cell proliferation is higher in the Lob 1 of the nulliparous woman’s breast. We have also shown that during the fourth and fifth decades of life there is a decrease in the number of Lob 2. We postulate that this type of lobule is the site of origin of both lobular hyperplasia and carcinoma in situ [17,19,27]. Since it has been reported that the incidence of atypical lobular hyperplasia decreases significantly with advancing age, it is possible to postulate that the observed diminution in Lob 2 is responsible for the decreased incidence of this type of lesions. In addition to the differences in proliferative activity the three types of lobules exhibit variations in their in vitro growth characteristics. Lob l and Lob 2 grow faster have a higher DNA labeling index, and a shorter doubling time than Lob 3 [16]. They also exhibit different susceptibility to carcinogenesis [13,15]. Cells obtained from Lob l and Lob 2 express in vitro phenotypes indicative of neoplastic transformation when treated with chemical carcinogens, whereas cells obtained from Lob 3 do not manifest those changes [13,15]. Collectively, our data establish a baseline for understanding the evolution of glandular development, and how it is influenced by age and parity. This knowledge is of utmost importance for understanding the role of differentiation in the protection of the mammary gland against carcinogenesis [28–30]. In addition, these data establish well-defined endpoints for studying the response of the mammary gland to hormonal or chemo preventive agents, which could be utilized in modulating the susceptibility of the breast to carcinogenesis.

7. Cell proliferation and hormone receptors in relation to breast structure The proliferative activity is determined by counting the number of cells that react positive to the antibody Ki67. Lobules type 1 has the highest proliferative ac-

tivity compared to type 2 and 3 in the nulliparous and in the parous women (Table 3). The proliferative index in the lobules type 1 of the nulliparous women breasts is two fold higher than the lob. 1 of the parous women (P < 0.02) (Table 3). Lobule 1 has a higher proliferative index than lobule 2, and 3. These differences are not abrogated when the phases of the menstrual cycle are taken into consideration [25]. Parity, in addition of exerting an important influence in the lobular composition of the breast, as described above, profoundly influences the proliferative activity of the mammary epithelium. Lobule 1 and 2 present in the breast of premenopausal nulliparous women exhibit a significantly higher proliferative activity than those lobules found in the breast of parous women. After menopause sets in, the proliferative activity of the mammary epithelium decreases, but although less pronounced, the differences between the nulliparous women and parous women’s cell proliferation in breast structures are maintained. Estrogens and progesterone are known to promote proliferation and differentiation in the normal breast epithelium. Both steroids act intracellularly through a receptor which, when activated by its binding with the hormone, regulates the expression of specific genes [31]. However, the mechanism by which these molecules exert their mitogenic and differentiation effect has not been clearly established [32–42]. One of the accepted mechanisms of action of steroid hormones postulates that the proliferation of cells is the response to direct stimulation, as the result of the interaction of the estradiol bound to the estrogen receptor (ER) with the DNA [32]. Measurements of the levels of ER and progesterone receptor (PgR) in normal breast in the cytosol fraction, using standard biochemical techniques, is inaccurate because of the low cellularity of the tissue. The use of monoclonal antibodies which specifically recognize ER and PgR makes it possible to identify and to quantitate the cells expressing these receptors [42]. Both ER and PgR are present in the nucleus of epithelial cells. The percentage of cells expressing these receptors, however, varies as a function of the degree of lobular development of the breast, and therefore of the type of lobular structure analyzed. Lobule 1 are the structures more consistently containing a higher percentage of E2R and PgR positive cells than lobules 2, 3 and 4 (Table 3), an observation that indicates that a progressive decrease in the percentage of cells exhibiting an immunocytochemically positive reaction for these

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a

Lobules percentage of lobules type 1 (Lob 1), type 2 (Lob 2), type 3 (Lob 3), and type 4 (Lob 4), positive cells percentage of cells positive for K16. Proliferative activity of Lob 1 Nulliparous vs. Lob 1 parous t = 2.44, P ≤ 0.002.

markers occurs as the structures become more differentiated. These data allowed us to conclude that degree of differentiation of the breast is an important determinant in the expression of both ER and PgR, in addition to modulate the proliferative activity of the breast epithelium. Neither age nor parity history affects the percentage of cells reacting for both receptors.

b

2.00 ± 0.75 1.47 ± 0.19 0 0 Nulli-parous Parous 7 25

40.85 ± 10.93 48.76 ± 11.00

81.13 ± 6.90 66.84 ± 7.55

4.97 ± 2.51b 1.56 ± 0.29b

18.87 ± 6.89 23.10 ± 6.37

0.62 ± 0.30 0.85 ± 0.31

00.00 8.03 ± 4.67

0 0.22 ± 0.18

0 0

Percentage of Ki67 positive cells Percentage Percentage of of lobular Ki67 positive structures cells Percentage of lobular structures Percentage of Ki67 positive cells Percentage of lobular structures

Percentage of Ki67 positive cells

Percentage of lobular structures

Percentage of Ki67 positive cells

Ductal Lob 4 Lob 3 Lob 2 Lob 1 Donor’s age Group n

Table 3 Influence of age and parity on the lobular composition and proliferative activity of the breast lobular structures and proliferative activitya

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8. Genomic profile of the lobular structures This study was performed using the RNA obtained from microdissected lobules type 1 and 3 of three nulliparous and three parous premenopausal women. Microarray hybridization showed that 82 genes were differentially detectable. We have clustered these genes according to functional properties (Table 4). With this array, it is clear that lobules type 3 have a gene expression profile significantly different from those of lobules type 1 of the nulliparous breast. Of interest is the The Rho-E gene is amplified 14 folds in the lobules type 3 of the parous breast (Table 4). This gene belongs to a small G protein superfamily consists of the Ras, Rho, Rab, Arf, Sarl, and Ran families. Members of the Rho family of small guanosine triphosphatases have emerged as key regulators of the actin cytoskeleton, and furthermore, through their interaction with multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion [43]. Rho-E is a Rho protein that binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase activating proteins. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras. Replacing all three positions in Rho-E with conventional amino acids completely restores GTPase activity [44,45]. Rho-E may act to inhibit signaling downstream of Rho-A, altering some Rho-A-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation. In vivo, Rho-E is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state [44,45]. This could be an important function considering that may be this genes is maintained activated even after involution of the lobules type 3 to 1 in the postmenopausal state. The other

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Table 4 Gene expression in the Lob 3 of parous women vs. Lob 1 of nulliparous women Gene namea

Fold mean ± S.D.

Intracellular kinase network

CDC7-related kinase c-jun N-terminal kinase 2 (JNK2) Ribosomal protein S6 kinase II Caspases Caspase-4 (CASP4) Protein turnover Ubiquitin-conjugating enzyme E2 Basigin precursor (BSG) Heterogen nuclear ribonucleoprot K Calcium binding proteins Calmodulin 1 Guanine nucleotide-binding protein Metabolism Cytosolic superoxide dismutase 1 (SOD1) Glutathione-S-transferase (GST) homolog l-lactate dehydrogenase M subunit (LDHA) l-lactate dehydrogenase H subunit (HDHB) Replication factors, DNA damage and repair G/T mismatch-specific thymine DNA glycosylase MutL protein homolog, DNA mismatch repair Proliferating cyclic nuclear antigen (PCNA); cyclin Cell cycle and proliferation related G1/S-specific cyclin D1 (CCND1) Cell cycle protein P38-2G4 homolog, HG4-1 Cyclin-dependent kinase inhibitor 1 (CDKN1A) Btg protein precursor Cdc2-related protein kinase PISSLRE PTPCAAX1 nuclear tyrosine phosphatase (PRL-1) DNA binding nuclear proteins DNA-binding protein CPBP Undefined Interferon-induced 56-kDa protein (IFI-56K) TRAM protein BENE Metalloproteinases and protease inhibitors PRSM1 metallopeptidase Matrix metalloproteinase 3 (MMP3) Matrix metalloproteinase 7 (MMP7); matrilysin Metalloproteinase inhibitor 3 precursor Leukocyte elastase inhibitor (LEI) Bikunin; hepatocyte growth factor activator inhibitor 2 Metalloproteinase inhibitor 1 precursor (TIMP1) Growth factors and hormones

Gene namea

Fold mean ± S.D.

Oncogenes and tumor suppressor genes, transcription activators and repressors, intracellular transducers, effectors and modulators 2.4 ± 0.8 4.5 ± 1.2 1.6 ± 0.4 2.3 ± 0.5 −2.0 ± 0.5 −2.0 ± 0.4 3.2 ± 0.8 7.0 ± 1.6 10.5 ± 2.5 1.6 ± 0.3 2.0 ± 0.5 6.5 ± 1.5 3.2 ± 0.8 3.5 ± 0.9 −3 ± 0.8 3 ± 0.8 3.5 ± 1.6 3.0 ± 1.1 −1.5 ± 0.9 −3.6 ± 1.1 −2 ± 0.6 5.0 ± 0.0 2.2 ± 0.4 1.6 ± 0.5 13.0 ± 1.9 4.0 ± 0.8 −2.0 ± 0.7 16.0 ± 1.9 4.0 ± 1.3 3.2 ± 1.0 3.0 ± 0.5 −1.5 ± 0.7 2.9 ± 1.0

c-jun proto-oncogene; transcription factor AP-1 Interferon-inducible protein 9–7 Neurogenic locus notch protein (N) c-myc oncogene c-myc binding protein MM-1 Cyclin-dependent kinase 4 inhibitor, p16Epidermal growth factor receptor (EGFR) Fos-related antigen (FRA1) Active breakpoint cluster region-related protein ETS-related protein ETS domain protein elk-3, NET Purine-rich single-stranded DNA-binding protein Early growth response protein 1 (hEGR1) Neutrophil gelatinase-associated lipocalin precursor Extracellular matrix, cell adhesion, cellular matrix Cadherin 3 (CDH3); placental cadherin precursor Integrin beta 6 precursor (ITGB6) Integrin beta 4 (ITGB4); CD104 antigen Integrin beta 8 precursor (ITGB8) Paxillin Alpha catenin (CTNNA1) Desmoplakin I and II (DSP, DPI and DPII) Polycystin precursor Wnt-8B Vimentin (VIM) Type I cytoskeletal 13 keratin Nm23-H4; nucleoside-diphosphate kinase Type II cytoskeletal 2 epidermal keratin (KRT2E) BIGH3 Fibronectin precursor (FN) Receptors Arylhydrocarbon receptor (AH receptor) Signaling lymphocytic activation molecule Bone morphogenetic protein 4 type II receptor precursor Oncostain M-specific Receptor beta subunit IgG receptor FC large subunit P51 precursor CD59 glycoprotein precursor

Intracellular transducers, effector modulators, symporters and antiporters 14-3-3-Protein sigma, stratifin; epithelial cell marker protein T3 receptor-associating cofactor 1

2.8 ± 0.5 2.0 ± 0.3 3.0 ± 0.7 −1.5 ± 0.4 9.5 ± 1.2 −2.0 ± 0.4 1.6 ± 0.3 5.0 ± 0.9 2.5 ± 1.0 1.6 ± 0.4 3.0 ± 0.8 −1.6 ± 0.4 2.0 ± 0.2 2.0 ± 0.5 −2.0 ± 0.7 4.5 ± 0.9 −1.8 ± 0.5 4.0 ± 1.2 1.6 ± 0.3 2.2 ± 0.6 −2.0 ± 0.2 −3.3 ± 1.8 −2.3 ± 0.9 1.6 ± 0.8 −2.4 ± 1.2 −2.5 ± 0.5 −2.0 ± 0.5 3.0 ± 0.8 5.2 ± 1.6 5.0 ± 1.1 −3.0 ± 0.6 1.8 ± 0.4 1.6 ± 0.5 −3.0 ± 1.0 2.0 ± 0.4

−2.0 ± 0.4 −2.0 ± 0.4

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Table 4 (Continued) Gene namea

Fold mean ± S.D.

Gene namea

Vascular endothelial growth factor precursor (VEGF)

2.6 ± 0.6

Macrophage inhibitory cytokine I (MIVI) Insulin-like Growth factor binding protein 3 precursor (IGF-bind) Interleukin-1 beta precursor (I)

2.1 ± 0.4 9.5 ± 1.8

Growth factor receptor-bound protein 2 (GRB2) isoform Elongation factor 1 alpha (EF1 alpha) ADP/ATP carrier protein

1.5 ± 0.3

ATP synthase coupling factor 6 mitochondrial precursor

Fold mean ± S.D. 2.5 ± 0.7 1.6 ± 1.3 3.0 ± 0.2 1.6 ± 1.7

a Genes classified according to function. Mean fold increase (decrease) of gene expression in lobules type 3 (Lob 3) in three parous women compared with Lob 1 of three nulliparous women. S.D., standard deviation of the mean.

gene that is significantly overexpressed (five-fold) in the lobule type 3 is the protein tyrosine phosphatase (HPTPCAAX1 or PRL-1) (Table 4). This gene encodes a unique nuclear protein–tyrosine phosphatase, that is regulated by a mechanism different from those of other immediate early genes such as c-fos and c-jun [46]. Importantly this gene has been shown to be upregulated in villus but not crypt enterocytes and in confluent differentiated but not undifferentiated proliferating Caco-2 colon carcinoma cells. In other systems is also related to differentiation, development and regeneration [47–50]. Therefore its function in the breast epithelial cells of lobules type 3 may be related to a differentiation role more than a proliferative related process, due that these lobules have a lower proliferative activity than the lobules type 1 (Table 4). Insulin-like growth factor-binding protein-3 (IGFBP-3) is significantly overexpressed in the lobules type 3 over the lobules type 1 of the breast of premenopausal women. This gene is also overexpressed in the parous breast. This gene codes a specific binding protein for the insulin-like growth factors (IGFs). IGFBP-3 modulates the mitogenic and metabolic effects of IGFs [51]. IGFBP-3 forms a ternary complex with IGF-I or IGF-II and an 85 kDa glycoprotein acid-labile subunit ALS. IGFBP-3 may also play more active, IGF-independent, roles in growth regulation of cancer cells [52]. IGFBP-3 protein levels are developmentally regulated and influenced by a number of hormonal stimuli both in vitro and in vivo. p53 may regulate apoptosis in tumor cells via transactivation of the insulin-like growth factor-binding protein 3 (IGFBP3) gene [52–54]. It has been shown that IGBP-3 could be modulated by hCG

and be one important pathway in the differentiation effect of this hormone in the mammary gland. Their expression in the Lob 3 is a new finding that requires further investigation. The final biological significance of these genes and the other genes, described in Table 4, in the process of differentiation of the breast and their expression during postmenopausal involution and how are they regulated by the reproductive history of the woman is no known. There is also not a clear understanding how these or other genes are working in the protection induced by pregnancy. It was of interest the observation that there was no difference between Lob 1 and Lob 3 in the expression cytoskeletal proteins such as cytokeratins 18, 19, and 8, that are overexpressed in tumor cells [55]. Known genes that are overexpressed in breast cancer, such as HER-2/neu [56] and mucin [57,58] were not expressed in any of the lobular structures. Other genes, like fibroblast-like growth factor 1 (FGF-1), insulin growth factor-1 (IGF-1) binding protein-2, and zinca-2-glycoprotein [59], that are generally up or down regulated in the neoplastic process, were not differentially expressed in the Lob 3 vs. Lob 1. In addition, cells derived from the differentiated Lob 3 are resistant to grow in vitro and do not express transformation phenotypes upon carcinogen treatment, as cells from Lob 1 do [13,15,60]. We have confirmed the differential expression of some those genes that were overexposed in the lobules type 3 using semiquantitative RT-PCR. The results with RT-PCR were consistent with the microarray results. The results of the RT-PCR might not quantitatively reflect the fold difference found in by microarray, but they agree with the results. In none of the four genes tested the RT-PCR contradict the microarray results.

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