Toward a Physiological Approach to Breast Cancer Prevention’ · of preventing breast cancer by treating young nulliparous females with hormones that mimic a full-term pregnancy

Vol. 3, 353-364, June 1994 Cancer Epidemiology, Biomarkers & Prevention 353

Review

Toward a Physiological Approach to Breast Cancer Prevention’

Jose Russo2 and Irma H. Russo

Department of Pathology, Fox Chase Cancer Center,Philadelphia, Pennsylvania 19111

Abstrad

Breast cancer is one of the most frequent malignanciesin women, and its incidence is increasing. No redudionin the mortality rate has resulted from advances in earlydiagnosis and new therapeutic modalities; therefore,new approaches to the understanding of this disease arerequired. The observation that, in an experimentalanimal model, full-term pregnancy prior to exposure toa carcinogenic agent proteds the mammary gland frommalignant transformation led us to study themechanisms underlying this phenomenon. The protediveeffed observed after pregnancy did not depend upongestational or ladational hyperplasia of the mammarygland, but upon strudural changes induced in themammary parenchyma by those processes. Thosechanges are permanent, since the protedive effed ismaintained after pregnancy and consists in the completedifferentiation of terminal end buds to lobules. Theprotedive effed exerted by pregnancy can be mimickedby treating young virgin rats with a single placentalhormone, chorionic gonadotropin. Since the possibilityof preventing breast cancer by treating youngnulliparous females with hormones that mimic a full-term pregnancy that results in complete differentiationof the gland is of pradical interest to the human femalepopulation, we undertook the study of the humanbreast. The breast of postpubertal nulliparous women iscomposed of lobular strudures refleding different stagesof development. Lobules type 1 (lob 1 ) are the mostundifferentiated ones. Lobules type 2 evolve from theprevious ones and have a more complex morphology,being composed of a higher number of dudularstrudures per lobule. They progress to lobules type 3and 4, which are present in the pregnant and ladationalperiods of the mammary gland. In nulliparous womenthe structure most frequently found at all ages is the lob1 , whereas in parous women, lob 3 is the most frequentone. Lob 1 are considered to be the site of origin ofdudal carcinoma in situ, which progresses to invasivecarcinoma. Lob 2 originate lobular carcinoma and lob 3originate adenomas, fibroadenomas, sclerosing adenosis,and apocrine cysts. These observations led us to test ifthe degree of lobular development influences the

Received 1 1/1 9/93 ; revised 1/1 /94; accepted 1/4/94.

1 This research was supported by PHS Grants CA48927, CA38921,

CA06927, CA45�38 awarded by the National Cancer Institute, NIH DHHS,

and by American Cancer Society Grant CN52.2 To whom requests for reprints should be addressed, at Department ofPathology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA

19111.

transformation of human breast epithelial cells exposedin vitro to chemical carcinogens. We found that primarycultures derived from breast tissues composed of lobulestype 1 and 2 express phenotypes of cell transformationwhich were not observed in cells derived from lobulestype 3. These data acquire relevance to the light thatwomen with a history of early pregnancy are at a lowerrisk of developing breast cancer than nulliparouswomen, an effect attributed to differences in the degreeof differentiation of the breast. Therefore, the stimulusof pregnancy furthers the differentiation of lobules type1 to lobules type 3 and 4. It is postulated that theirmore differentiated condition makes them refractory toneoplastic transformation. Overall, the data provide asolid basis for developing physiological means of breastcancer prevention and control.

Current Concepts in Breast Cancer Prevention

Breast cancer is one of the most common neoplasms inwomen, and its incidence is reaching epidemic proportionsin the United States (1 ). Whereas it is clear that a betterunderstanding of the biology of breast cancer has beenaccomplished (2), it is a disease with a complexity that isrooted in multifactorial components which comprise the

endocrinology of the female, interacting with exogenousfactors and lifestyle (3). This complexity has led researchersto place a major emphasis on endogenous and exogenous

hormonal factors, the influence of diet, obesity, nulliparity,

age greater than 30 years for the completion of the firstfull-term pregnancy, early menarche, late menopause, andfamily history as risk factors in breast cancer (3, 4).

Hormones and Breast Cancer

Hormones, especially estrogens, have been linked to breast

cancer (4) and their role has been attributed to their abilityto stimulate cell proliferation, which in turn leads to accu-

mulation of random genetic errors that result in neoplasia(5). On the basis of this concept, chemoprevention of breastcancer has been mostly aimed at reducing the rate of cell

division through administration of antihommones (6). Thechemopreventive agents most widely considered are anties-trogenic compounds such as progestagens, tamoxifen, go-nadotropin-releasing hormone analogues, and aromataseinhibitors (7). From all these agents, tamoxifen is the onethat has been proven to be effective in both primary andadvanced breast cancer. Animal studies have shown thattamoxifen interferes with the phases of tumor initiation and

promotion (8, 9). Tamoxifen has shown to be species, tis-

sue, and cell type specific (10). In the pubertal rat, tamox-ifen is capable of promoting full ductal development in themammary gland (1 1 ). In the mature cycling animal, tamox-ifen acts as an antiestrogen causing atrophy of lobular struc-

tures (1 2). In postmenopausal women, tamoxifen treatmentresults in up-regulation of the proportion of ductal cellsexpressing estrogen receptor (1 3). Whereas the use of ta-

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Other Chemopreventive Agents

3 The abbreviations used are: DMBA, 7, 1 2-dimethylbenz(a)anthracene;TEB, terminal end buds; AB, alveolar buds; TD, terminal ducts; hcG, humanchorionic gonadotropin; DNA-LI, DNA labeling index; HBEC, human breast

epithelial cells; BP, benzo)a)pyrene; NMU, N-methyl-N-nitrosourea;MNNG, methyl-N-nitro-nitrosoguanidine; lob, lobule.

354 Review: Human Papillomaviruses and Cervical Neoplasia

moxifen has been considered beneficial in reducing bloodlipids and bone mineral metabolism, reduction in ischemicdisease and osteoporosis (1 4, 1 5), a real concern abouttamoxifen is that little is known about its effects on normalor premalignant breast tissue (1 6). The fact that endometrial

cancer (1 7), increased incidence of contralateral breast can-cer in premenopausal women previously treated with ta-moxifen (1 8), thromboembolic events (1 9), ocular toxicity-inducing disabling retinopathy, and keratinopathy (20) have

been reported raises concerns about the use of tamoxifen asa general chemopreventive agent. Furthermore, the impactof many years of ovarian stimulation by tamoxifen remains

to be evaluated (21).It has been reported that breast epithelial cells undergo

variations in their rate of proliferation during the differentphases of the menstrual cycle, being relatively low in the

follicular phase, when estrogen level prevails, but increas-ing by a factor of two in the middle to late luteal phaseu nder preva il ing progesterone levels. These observationshave led researchers to conclude that the combination of

both estrogen and progesterone appears to have a greater

stimulatory effect than estrogen on the cell division of themammary epithelium (22-24). This realization led other

investigators to postulate the use of gonadotropin releasinghormone analogues that have the property of inhibitingovulation and decreasing the hormonal levels of premeno-pausal women to those of postmenopause (25).

Among other chemopreventive agents proposed to beused in breast cancer are monoterpenes (26) and retin-oids (27-28). The natural and synthetic vitamin A ana-logues have been shown to inhibit mammary tumors

induced by a variety of carcinogens (27, 28). This effectis manifested by a decreased tumor incidence and mul-

tiplicity and an increased latency of tumor development(27, 28). At difference of the known cytodifferentiatingeffect on acute promyelocytic leukemic cells (29), the

mechanism of action of retinoids on the mammary glandhas not been clearly elucidated. It has been suggestedthat the chemopreventive efficacy of pharmacologicaldoses of metinoids might relate to their observed inhibi-

tion of cell proliferation and the subsequent inhibition ofmorphological development (28). On the basis of obser-vations in animal models, a randomized clinical trial ofretinoid efficacy on breast cancer chemoprevention iscurrently underway (30).

Pregnancy as a Protedive Fador in Breast Cancer

Early full-term pregnancy, before age 30 (years), protectsthe breast from malignancies by reducing in a magnitude offourfold the overall risk; the protection conferred is for life(3, 31-34). Although the intimate mechanisms by which anearly first full-term pregnancy confers protection againstbreast cancer have not been completely elucidated, itsimportance lies in the fact that it represents a physiologicalmechanism which might imprint in the breast special char-acteristics that make the organ refractory to cancer (3�-41).The understanding of this process might have significantimplications in our knowledge of the biology of breastcancer and more importantly the development of strategiesfor breast cancer prevention and cure (36, 37). In our lab-

oratory we have studied the mechanistic basis of breast

Period of HighSusceptibilIty

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20 40 60 80 100 120 140

AGE (in days)

Fig. 1. Histogram representing the number of structures/mm2 (ordinate) ofthe rat mammary gland in relation to age (abscissa). Hatched area, period ofhighest susceptibility of the mammary gland to neoplastic transformation by

chemical carcinogens.

cancer prevention, first by utilizing the rat as an experimen-

tal model and second by studying the developmental pat-tern of the post pubertal breast and how this pattern relatesto the ability of the cells to respond to carcinogens in vitro.

The Rat as an Experimental Model

We have determined that differentiation of the mammarygland prior to exposure to a carcinogenic insult protects this

organ from malignant transformation (35-37, 42). This pos-tulate is based upon the observation that the administrationof the chemical carcinogen DMBA3 to young virgin ratsduring the period in which the mammary gland containsnumerous undifferentiated terminal ductal structures or

TEBs actively cleaving into ABs induces the largest numberof mammary tumors, mainly in thoracic mammary glands.This period has been designated the “susceptibility win-

dow” (Fig. 1 ) (43-47). The high susceptibility of the TEB toneoplastic transformation is due to the high proliferativerate of its epithelium, as determined by the mitotic andDNA labeling indices (41 , 44). These two indices are veryhigh in the TEB and decrease toward the ductal or proximal

portions of the gland, and in more differentiated structures

such as ABs and lobules (41 , 44, 46, 47). TEBs affected bychemical carcinogens enlarge due to epithelial prolifera-tion, progressing to intraductal proliferations (43). Intraduc-

tal proliferations become progressively larger and their con-fluence leads to the formation of microtumors that

histologically are classified as intraductal carcinomas andlater, adenocarcinomas (Fig. 2). With further growth these

tumors develop various patterns, such as cribriform, papil-lary, and occasionally comedocarcinoma. Benign lesions,

such as cysts, hyperplastic alveolar nodules, adenomas, andfibroadenomas originate from more differentiated struc-tures, such as the AB and lobules type 1 and 2 (2, 43,47-49). The histopathological criteria for the diagnosis of

these lesions have been published elsewhere (47).



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age), OV (old virgin; 1 80 days of age) and P (parous; 1 80 days of age).

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Post Pregnancy and

Lactational Regression

Fig. 3. Terminal end buds, alveolar buds, and lobules. Arrows indicate

unidirectional differentiation of terminal end buds into alveolar buds andlobular structures. After weaning neither alveolar buds nor lobules regress toterminal end buds.

Yv ov p

Fig. 5. DNA-LI (ordinate) of TEB, ID, and lob in the mammary gland of

young virgin )YV(, old virgin )OV) and parous (P) rats. DNA-LI was deter-mined by autoradiographic detection of I’Hlthymidine incorporation intomammary epithelial cells. Values represent the mean number of positive

cells per total number of cells counted in each structure ± SD.

Cancer Epidemiology, Biomarkers & Prevention 355

I Dtn.r.ntiatioe Pathway

lop IDC. AdCa

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Fig. 2. TEB evolves towards a differentiation pathway to form AB or lobulesif pregnancy or hCG stimulate them. If a carcinogen hits the target (TEB)

during the susceptibility period, it evolves instead to IDP (intraductal prolif-

eration), IDCa (intraductal carcinoma) and AdCa (adenocarcinoma).

Pregnancy and Lactational

Differentiation

Terminal End Alveolar Buds

Buds

DMBA-induced mammary carcinogenesis is signifi-cantly inhibited in rats which have undergone full-termpregnancy or pregnancy and lactation prior to exposure tothe carcinogen (36, 44, SO) (Fig. 2). The protective effectobserved after pregnancy does not depend upon gestationalor lactational hyperplasia of the mammary gland, but upon

permanent structural changes induced in the mammaryparenchyma by those processes. These changes consist inthe complete differentiation of TEBs to lobules, which show

secretory activity during lactation (51). Those glands thatbetween three and nine weeks after weaning have regressedto a pregestational condition do not appear morphologi-

cally different from the glands of virgin animals, except forthe absence of TEBs, fewer TDs, and more ABs and lobules

(36, 38, 39, 43, 44) (Figs. 3 and 4). Administration of DMBAto parous rats when the glands have regressed to such aresting condition induces the lowest incidence of mammary

adenocarcinomas (52). The mechanism of the refractorinessof parous females vis-a-vis young virgin and old virgin

female rats is due to decrease in cell proliferation (Fig. 5),

decreased carcinogen binding, and increased DNA repaircapability of the mammary epithelium, all of which are theresult ofthe increased differentiation ofthe mammary gland

(40, 41 , 53) (Fig. 6). Whereas other mechanisms for the

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protective effect of pregnancy, such as maternal stimulation

of the immune system with enhancement of the tumomicidaleffect of cytotoxic T-cells, have been suggested (54), theobservation that the levels of cytotoxic T-cells return tonormal values by 42 days postpartum, whereas the protec-

tive effect of pregnancy has been detected by 63 dayspostweaning, indicates that other more permanent mecha-nisms are operational (40, 41, 44, 51).

On the basis of our studies on the effect of pregnancyas a physiological mechanism of prevention, we have beenable to mimic the protective effect exerted by pregnancy bytreating young virgin rats with a single placental hormone,hCG (39, 42, 52, 55-58) (Fig. 2). Since the possibility ofpreventing breast cancer by treating young nulliparous fe-males with hormones that mimic pregnancy but without theneed of a full-term pregnancy, is of practical interest to the

human female population, we designed experiments gearedto answer the following questions: (a) What is the effect ofhCG on mammary carcinogenesis? (b) How comparable isthe effect of pregnancy and hCG treatment on mammary

carcinogenesis? (c) Does hCG exert secondary effects onbody weight and endocrine organs? (d) How does hCG

treatment affect the mammary gland structure? (e) Whateffect has hCG treatment on mammary DNA synthesis? ( 1)

Does hCG affect tumor progression? and lastly (g) How

does hCG exert its effect on the mammary gland?

Effed of hCG Treatment on Mammary Carcinogenesis.The observations that architectural and cell kinetic changes



BREAST STATUS

IAMATURE GLAND

C lobules type I

DMBA Preg.+ hCG + DMBA+DMBA DMBA hCG

SEMI-DIFFERENTIATED

C lobules type I S 2

F FULLY-DIFFERENTIATEDL #{149}Iobulestype3&4

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Fig. 7. Histogram showing tumor and adenocarcinoma incidence in virgin

SD rats treated with DMBA at 55 days of age )DMBA), parous rats (Preg. +

DMBA), and animals that following pregnancy and 21 days of rest receivedDMBA. hCG-treated )hCG + DMBA) group virgin rats received 21 doses ofhCG 100 international units/day, and were allowed to rest for 21 days priorto DMBA instillation. DMBA + hCG, virgin rats that received DMBA at 55

days of age and 21 days later started receiving 100 international units/dailyhCG (or 60 days.

induced in the mammary gland by pregnancy markedly

decrease the incidence of chemically induced mammarycarcinomas led us to postulate that differentiation wasthe underlying mechanism responsible for this protection

(36, 42, 43, 46, 59). This hypothesis was confirmed byinducing similar or even greater protection than that af-

forded by pregnancy through induction of gland differenti-ation with the placental hormone hCG (36, 42, 51 , 55-58).hCG treatment of young virgin rats for 21 days, the length ofpregnancy, reduced mammary carcinoma incidence in adose-dependent manner (36, 57, 59). The number of tumorsas well as of adenocarcinomas per animal was drastically

reduced by hCG treatment to values similar to those ob-served in animals inoculated with carcinogen after preg-

nancy (Fig. 7). hCG, like pregnancy, induced full differen-tiation of the mammary gland, which was morphologicallymanifested as a reduction in the number of TEBs (Fig. 8) due

to a profuse lobular development, depression in DNA syn-

Fig. 8. Physiological events in the life span of a woman and their influence

in breast development.

thesis (Fig. 9), inhibition of the binding of the carcinogen tothe cellular DNA, and increased ability ofthe cells to repair

DNA damage (36, 51, 56, 58, 59).

Prolonged Protedive Effed of hCG Treatment on Mam-mary Carcinogenesis: Comparative Study with Pregnancy.In order for a hormone preventive agent to protect the breastefficiently, it should inhibit tumor initiation at the samelevel or better than pregnancy and the protection has to belong-lasting after the termination of treatment. In order todetermine whether the protective effect of hCG on mam-mary carcinogenesis is temporary and decreases with timeafter the cessation of treatment or is long-lasting like that ofpregnancy, SO-day-old Sprague Dawley mats were dividedinto 3 groups: (a) animals undergoing full term pregnancy;(b) virgin animals receiving 100 international units hCGdaily for 21 days; and (C) age-matched virgin control ani-mals. Each group was subdivided into 2 protocols. For

Protocol 1 , a resting period of 21 days elapsed betweentermination of pregnancy or hCG treatment and the admin-istration of 8 mg DMBA/1 00 gbw; for Protocol 2 the threegroups of animals were treated as described above, but the

resting period was prolonged 63 days after pregnancy or


Structure DNA-LI T� Cal cycle 3H-DMBA Lesions

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Fig. 6. Correlation between type of structure, its

DNA-LI, length ofthe cell cycle in hours (Tc), showingthe relative lengthening of Tc due to lengthening of the

G, phase, growth fraction after 5 days of continuousinfusion of I ‘HJthymidine (GF,) and nuclear uptake ofI3HIDMBA, detected by autoradiography and ex-

pressed as the number of grains/nucleus. The lesions

depicted in the last column indicate the histologicaltype.

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FACTORS

f.Age 1I#{149}Hormonal statusI #{149}Dlet/Wight/Height -�---

I#{149}Anovuletory cycles II#{149}Hereditwy factors

#{149}SocIoeconomIc statusTe itormonal statusI DIet HMENSTRUAL CYCLE

C SocIoeconomic status 4,I #{149}Pregnancy iitwuptonj PREGNANCY I1e Age_____ V I________________I- POST-LACTATONAL IIC Hormonal status WdVOLU�flON I

V Ii#{149}Hormonal status �_SENILE INVOLj [ INVOLUTEDe Age



Involutionby aging

Sexual maturity

Extra hormonal

� �‘ �Fig. 9. Life cycle of the breast in the nulliparous woman. The breast is

composed primarily of lobules type 1 with some progression to type 2 andonly minimal formation of lobules type 3 during sexual maturity, involutingto lob 1 at menopause.


termination of hCG treatment. Age-matched controls for

each subgroup received DMBA only. Tumorigenesis was

evaluated 24 weeks postcarcinogen administration (�1).Pregnancy and hCG treatment followed by the 21 -day rest-

ing period prior to DMBA administration significantly de-pressed both mammary tumorigenesis and carci nogenesis.When the resting period was prolonged to 63 days therewas also a significant depression in both tumor and adeno-

carcinoma incidence. Pregnant and hCG-treated animalsstill developed significantly fewer tumors than their age-matched controls, indicating that the lengthened time after

delivery or termination of hormonal treatment did not ame-liorate the protective effect ofthese two events, at differenceof the observed immunological stimulation of the maternal

system (Si, 54) (Fig. 7).

Effed of hCG Treatment on Body Weight, Estrous Cycle,and Endocrine Organs. It has been shown that humanchorionic gonadotrophin has a DNA sequence differentfrom rat CG; however, this difference seems not to affect thebiological effect of the hormone (60, 61). hCG treatmentmodified the estrous cycle by inducing a prolongation of

diestrus but cycles became regular by 21 days posttreat-ment (57). Treated animals did not exhibit significant dif-ferences in pituitary and adrenal gland weights and nohistological abnormalities were noted in these organs. hCG

treatment induced a significant increase (P < 0.01 ) in ovar-ian weight and it remained at a higher value than in virgincontrols, but the difference at the end ofthe experiment was

not significant. No significant differences in uterine weightwere observed between treated and virgin control animals(57). These data indicate that in the rat model the effect ofhCG on endocrine organs is reversible.

Effed of hCG Treatment on Mammary Gland Strudure.The mammary gland structure of young virgin rats treatedwith a daily injection of 1 00 international units hCG was

evaluated in whole mount preparations by a count underthe stereomicroscope of the number of terminal ductalstructures. In control animals the number of TEBs decreasedas a function of age, whereas in treated animals there was

a sharp diminution between S and 1 S days of treatment;

values reached a plateau thereafter which remained always

lower than values in controls. TDs, which in the mammary

gland ofcontrol animals increased progressively with aging,

were decreased by treatment (P< 0.05). The number of ABsin control animals progressively decreased with aging,

whereas treatment maintained their number constant be-

tween the first and 1 0th day of injection decreasing sharply

between the 1 0th and 21 st days of injection, as the number

of lobules started to rise. After cessation of treatment the

number of ABs exhibited a recovery due to the regression of

newly formed lobular structures, and their number me-

mained significantly higher than in controls up to the end of

the observation period. Lobular structures were almost to-

tally absent from the mammary gland at the beginning of

treatment, and their number remained almost unchanged in

control animals. hCG administration stimulated a burst of

lobular formations starting at the 10th day of treatment,reaching a peak by the 1 5th day. Although their number

diminished progressively thereafter, and even more aftertermination of treatment, it remained slightly higher than incontrol animals (57). The morphological pattern observed

under hCG treatment mimicked quite clearly the effect of

pregnancy (51).

Effed of hCG Treatment on DNA Synthesis. Mammaryglands collected from animals treated with a daily injection

of 1 00 international units hCG at the times indicated abovewere processed for DNA-LI determination. The highest

DNA-LI was observed in the TEBs of control animals.DNA-LI was considerably lower in ductal and lobular struc-

tures present in the same mammary gland (57).The effect of hCG treatment on the rate of DNA syn-

thesis varied as a function of time of treatment and type ofstructure considered. Treatment diminished the DNA-LI in

TEBs, an effect evident by the fifth day of injection, and

more markedly throughout the remainder of the injection

period, remaining significantly lower than in control ani-mals up to the end of the experiment (P < 0.001 ). The

response of TDs, ducts, ABs, and lobules to treatment was

similar among themselves, and different from that of TEB5;

the DNA-LI increased in all of these structures, reaching a

peak of maximal activity by the fifth day of injection, thendecreased by the 1 0th day, and more sharply by the 21 stday of injection. There was a slight recovery after hormonal

withdrawal, but DNA-LI values remained always lower

than those of similar structures in control animals. These

data also indicate that there is a difference in response foreach different compartment of the gland.

Effed of hCG Treatment on Tumor Progression. In order todetermine whether hCG inhibits or promotes tumor growth,

1 00 international units hCG, a dose that induces maximal

protection when given before carcinogen administration

(51 , 57, 59), were utilized forthese studies. For this protocol

virgin rats received, when they reached the age of SO days,

8 mg DMBA/1 00 gbw. Starting 21 days after carcinogen

administration, a time at which intraductal proliferations

are already present, the animals started receiving 100 in-

ternational units of hCG for 60 days (40-46). Carcinogen

treatment induced 1 00% tumor incidence in the animalsthat received DMBA alone, whereas tumor incidence and

the number of tumors per animal were significantly de-creased in animals treated with hCG after DMBA adminis-

tration (Fig. 7). No tumors were detected in animals treated

with hCG alone or with the vehicle (56).




Our results show that hCG inhibits tumor progression,since treatment with this hormone after carcinogen admin-istration produces a significant reduction in the number of

tumors and adenocarcinomas per animal. We concludedthat hCG acted on the initiated foci inhibiting their progres-

sion, not merely delaying the time of tumor appearance,since the latency period was not modified (57). It is postu-

lated that hCG act on the intraductal proliferation or IDP

that is considered to be a preneoplastic lesion in the rat.More studies in this area are required to understand the

effect of hCG in these initiated lesions.

Postulated Mechanism of Adion of hCG. Although up tonow it has been accepted that hCG may influence the

differentiation of the mammary gland through its effect on

the ovary (51 , 55-58, 60-70), what is not known is themechanism whereby hCG significantly decreases overall

tumor incidence and tumor burden. The physiological role

of hCG is its activity on the granulosa cells of the ovary

through a lutropin-choriogonadotropin receptor (62, 65),

although a similar receptor has recently been detected inthe thyroid gland (66). In the ovary hCG increases adenylyl

cyclase activity mediated by intracellular membrane asso-

ciated C proteins which result in cyclic AMP increasesleading to steroid and polypeptide hormone synthesis (65).

It has been reported that hCG acts as an immunosuppres-

sive agent (67), a mitogenic agent, and a local growth factoror an activator of c-myc and c-los oncogene (69, 70), which

in turn are important in the regulation of cell differentiationand proliferation. Exogenous administration of hCG affects

endocrine organs, the secretions of which directly or mdi-rectly affect the development and function of the mammarygland (57). We do not know if the endocrmnological milieu

induced by hCG which results in inhibition of tumor pro-gression exerts this effect through inhibition of cell prolif-eration, induction of cell differentiation, or activation ofprogrammed cell death. Our observation that hCG inducesthe synthesis of inhibin by the mammary epithelium (71)

suggests that this may represent the local regulatory mech-anism through which hCG mediates its effect on the mam-mary gland. This hypothesis is supported by the recently

developed inhibin-deficient transgenic mice, in which thisdeletion results in the development of gonadal tumors, thusdemonstrating that inhibin is a secreted protein with tumorsuppressor activity (72).

Inhibins are growth factors with structural homology to

the transforming growth factor �s and Mullemian inhibitingsubstance, among others (72). These nonsteroidal glyco-protein hormones are produced by the gonads and theplacenta (73-75). They feed back to the anterior pituitarygland to inhibit specifically the production and/or secretionof follicle stimulating hormone (75-80) and in the placentathey regulate the synthesis of hCG (81 ). The granulosa cells

of the ovary in the female have been identified as the site ofinhibin synthesis (82, 83). The major form of this protein isa disulfide-linked heterodimer, consistingofan cr-chain andone of two highly homologous p chains, designated A andB. The a chain is an Mr 18,000 peptide and the �3chains are

Mr 14,000 peptides, which lead to the formation of a M,32,000 ��-13 dimer in most species. Depending on the f3chain (A or B), inhibin will be named inhibin A or inhibin

B (80). Although the highest concentration of inhibin hasbeen found in testes and ovary, it is also expressed inseveral nongonadal tissues, including brain, pituitary, pla-centa, and both human and rat mammary epithelium

(83-86). The finding of inhibin subunit mRNAs in nonre-

productive tissues suggests a role for the inhibin proteinoutside the reproductive axis as an important regulator ofcellular differentiation. The development of gonadal tumorsby inhibin-deficient mice homozygous for the null allele

identifies ct-inhibin as an important negative regulator ofcell proliferation (72). Since the synthesis of inhibin by the

ovary is stimulated by the gonadotropic hormones pregnant

mare serum gonadotropmn and hCG (74, 75, 85, 86), hor-

mones known to have a powerful differentiating effect on

the mammary gland (55-58), our work was designed with

the purpose of determining whether inhibin played a role in

this process. Inhibin was immunocytochemically detectedin the mammary gland of virgin and of pregnant control

animals and hCG-treated virgin rats using known antibodiesagainst both inhibin A or B (87). The mammary gland of

virgin control animals did not show any immunoreactivity

(71 ), whereas under hCG treatment immunocytochemicalreactivity was observed in the mammary gland of pregnant

and hCG-treated animals, the intensity of the reaction in-

creasing progressively for reaching a peak by the 1 5th dayof either pregnancy or treatment (71 , 88). Inhibin or �A

were detected by Northern blot of ovaries and mammary

glands collected from hCG-treated and control animals

(89). Hybridization of ovarian RNA with the a-subunit

probe resulted in a single band of hybridization at about 1.5kilobases. The intensity of the hybridization signal was

increased by hCG treatment. Hybridization with the inhibin

fM-subunit probe resulted in a predominant signal at 6.7kilobases, which also showed an increase after hCG treat-

ment. The total RNA of mammary glands hybridized with

the a and f3 subunits, respectively, showing the same 1 .5-and 6.7-kilobase bands corresponding to the inhibin a and

13A-subunit mRNA present in the ovary (89).

The Human Breast

The understanding of the human breast has been a majorbiological puzzle, mainly due to the fact that the mammarygland seems to be the only organ that is not fully developed

at birth (48, 90). No other organ presents such dramaticchanges in size, shape, and function as does the breastduring growth, puberty, pregnancy, and lactation (48, 90,91 ). It is agreed that the developmental phase of the humanbreast starts as early as the stage of nipple epithelium duringembryonic development, continuing steadily with bodygrowth, and undergoing a spurt of growth with lobule for-

mation at puberty (Fig. 8). Four different lobular structureshave been characterized in the breast of postpubertalwomen, each one representing sequential developmental

stages (48). Lobules type 1 are the most undifferentiatedones; they are also called virginal lobules, because they are

present in the immature female breast before menarche.They are composed of clusters of 6 to 1 1 ductules perlobule. Lobules type 2 evolve from the previous ones andhave a more complex morphology, being composed of a

higher number of ductular structures per lobule. Theyprogress to lobules type 3 which are characterized by hay-ing an average of 80 ductules or alveoli per lobule; they arefrequently seen in the breast of women under hormonalstimulation or during pregnancy. A fourth type of lobule,lobule type 4, has been described as being present duringthe lactational period of the mammary gland, but it is not

found in the breast of nulliparous postpubertal women.



Lobule 2Lobule 2

Lobule 3

Fig. 10. Life cycle ofthe breast in the parous woman. The breast undergoesa complete cycle of development through the formation of lobules type 4

with pregnancy and lactation, which later regress.

Lobule 3

Lobule 4


Lobule type 4 is considered to be the maximal expression of

development and differentiation (48) (Fig. 8).The fact that the breast is the source of the most fre-

quent malignancy in the female population, and the knowl-edge that breast cancer is heavily influenced by the repro-ductive history of the individual, requires a thoroughunderstanding of the developmental pattern of the breast

during the life span of a woman. It is known that womenwith a history of early full-term pregnancy are at a lower

risk of developing breast cancer than nulliparous women(31 , 33, 48, 92-96). The protective effect of pregnancy has

been attributed to differences in the degree of differentiationof the breast (97). In the comparative study of the patho-

genesis of chemically induced mammary carcinomas inexperimental animal models with the pathogenesis of hu-man breast cancer (2), it was concluded that the initiation of

the neoplastic process, which was inversely related to thedegree of differentiation of the mammary gland in the ex-

perimental animal, might occur under similar circum-stances in women (2, 41 , 44-46, 96). The study of the

pathogenesis of human breast cancer indicated that thelobules type 1 are the site of origin of preneoplastic lesionssuch as atypical ductal hyperplasias, which evolve to ductal

carcinoma in situ, progressing to invasive carcinoma. Lob-ules type 2 are postulated to be the origin of atypical lobular

hyperplasia and lobular carcinoma in situ (2), and lobules

type 3 to be the site of origin of secretory adenomas, fibro-

adenomas, sclerosing adenosis, and apocrine cysts (2).These observations indicate that the degree of differentia-tion or lobular development of the mammary gland influ-

ence the type of tumors developed in the human breast (2).Our studies have been aimed at answering the followingquestions: (a) Which is the influence of age and parity onthe development of the human breast? (b) Which are the

main morphological differences between the cancerousand noncancerous breast of nulliparous and parouswomen? and (c) How does lobular differentiation affect thesusceptibility of the breast epithelial cells to neoplastictransformation by chemical carcinogens in vitro?

Influence of Age and Parity on the Development of theHuman Breast

The breast of nullipamous women is predominantly com-

posed of undifferentiated structures, such as terminal ductsand lobules type 1 , although occasional lobules type 2 and

3 are present (95, 96). In parous women on the other hand,the predominant structure is the most differentiated lobuletype 3. At difference of the lobule type 1 , which in thenullipamous woman remains constant throughout the lifespan, the lobule type 3 in parous women peaks during theearly reproductive years, decreasing after the fourth decadeof life. In the breast of nulliparous women, lobules type 2are present in moderate numbers during the early years,sharply decreasing after age 23 (years), whereas the number

of lobules type 1 remains significantly higher. This obser-vation suggests that a certain percentage of lobules type 1might have progressed to lobules type 2, but the number oflobules progressing to type 3 is significantly lower than inthe parous woman (Fig. 9). In the case of parous women, itis interesting to note that a history of parity between the agesof 1 4 to 20 years correlates with a significant increase in thenumber of lobules type 3, which remain present in thebreast as the predominant structure until age 40 (years).

After this age, a decrease in the number of lobule type 3

occurs, probably due to their involution to predominantlylobules type 1 (Fig. 1 0). It has been shown by severalauthors that postmenopausal involution is accompanied by

diminution and atrophy of the parenchymal components(97-100). Among the changes described in the lobular

structures is the increase in intralobular fibrosis and hyalin-ization, which are interpreted as a response to the lack of

hormonal stimulation (99). We have observed that lobulestype 1 are present in the breast of both postmenopausalnulliparous and parous women, although the lobules type 1found in the breast of parous women present higher fre-

quency of hyalinization in the intralobular stroma than thelobules of nullipamous women. During the postmenopausal

years, both parous and nulliparous women have breastswith a preponderance of lobules type 1 . Although ductal

breast cancer originates in lobules type 1 , or terminal ductallobular units (2), the epidemiological observation that nul-liparous women exhibit a higher incidence of breast cancer

than parous women (31 , 33) indicates that lobules type 1 inthese two groups of women might be biologically different

or exhibit different susceptibility to carcinogenesis (101,1 02). The presence of lobules type 1 in the breast of parouswomen has been also interpreted as a failure of the mam-mary parenchyma to respond to the influence of pregnancy

and lactation (23). If this is the case, then parous women

could contain in their breast unstimulated as well as re-gressed lobules type 1 . Therefore, the question that remainsto be answered is whether the lobules type 1 which are

present in the breast of parous women are as sensitive tocarcinogenesis as the lobule found in the breast of nulli-pamous women. Although this question cannot be answered

at the present time, it is possible to speculate that thelobules type 1 in the breast ofparous women are terminallydifferentiated structures that are the consequence of a re-gressive process, as depicted in Fig. 1 0. This hypothesis issupported by the observation ofthe presence of intralobularhyalinization in the lobules type 1 of the parous woman’s

breast, but not in the nulliparous one. In addition, theproliferative activity of the lobule type 1 is lower in thosepresent in the breast of parous women than in those ob-served in the breast of nulliparous women (103, 104).

The understanding of breast development requires a

horizontal study in which all the different phases of growth



U)Li

ciI-0ci

I-U)

lob 1�lob 2EJlob 3

60

40

20

0 HnI�Fig. 1 1. Lobules type 1 , 2, and 3 in the human breast. Lobule type corre-

lated with in vitro determination of DNA-LI, DMBA-DNA binding, expressedas imol/P for mol of DNA, survival efficiency (SE.) and colony efficiency

(CE.).

Ca- Ca+

PAROUS

Fig. 12. Percerit�ige o structures (ordinate), i.e., lob type 1, 2, and 3,composing the breast of nulliparous and par :s women without cancer(Ca-) and with cancer )Ca+).


LOBULEType

DNA-LI DMBA-DNAbIndIng

SE.DMBA

C.EDMBA

1(15.8 � 6.2)6.5 t 2.5 /inol DNA14-15 umol P

53% 84%

2 O.99� 1.2 8-lOumoIP/moI DNA

ND. N.D.

[: �‘ 0 25 � 0.3 final DNA7 umol P -77% -17%

are taken into consideration. For example, the analysis ofbreast structures at a single given point, i.e., ages 49-53years, would lead us to conclude that the breast of bothnulliparous and pamous women appears identical. How-ever, the phenomena occurring in prior years have im-printed permanent changes in breast biology that affect thepotential of the breast for neoplasia but are no longer mom-phologically observable. This horizontal study allowed usto determine that parous women truly underwent lobular

differentiation, whereas nulliparous women seldomreached the lobule type 3 stage (Fig. 9). This may furtherexplain why parous women are more protected againstcarcinogenesis. In parallel studies (see below), it has beenshown that lobules type 1 , 2, and 3 exhibit different cellkinetic characteristics; lobules type 1 and 2 grow faster in

vitro and have a higher DNA labeling index and a shorterdoubling time than lobules type 3 (36, 104). They alsoexhibit different susceptibility to carcinogenesis. Lobuletypes 1 and 2 express malignant phenotypes when treatedwith chemical carcinogens in vitro, changes that are notmanifested by lobule type 3 (101) (Fig. 11). We have alsoshown that during the fourth and fifth decades of life thereis a decrease in the number of both lobules type 2 and type

3. Since ithas been reported that the incidence of atypicallobular hyperplasia decreases significantly with advancing

age, it is possible to postulate that the observed diminutionin lobules type 2 is responsible for the decreased incidenceof this type of lesions (105).

Differences between Cancerous and NoncancerousBreast of Nuiiparous and Parous Women

The analysis of breasts taking into consideration the pres-

ence or absence of cancer in addition to parity, revealedthat the breast of nulliparous women free of cancer and ofnulliparous women with cancer had a similar architecture,both having as the predominant structure the Lob 1 , with alower percentage of Lob 2 and even lower Lob 3. In bothgroups the difference in relative percentage between Lob 1

and Lob 3 was highly significant (Fig. 12).The breast of parous women free of cancer contained

the lowest percentage of Lob 1 , and a slightly higher per-

centage of Lob 2. Lob 3 were the predominant structures

(40.S4%) as opposed to Lob 1 (25.82%) (P< 0.03). Instead

--___

Ca- Ca+

NULLIPAROUS

pamous women with breast cancer had as the predominantstructure the lob 1 , whereas lob 2 and lob 3 were repre-sented in lower percentages (Fig. 1 2). Lob 1 versus lob 3were significantly different. These results indicated that par-ous women who developed the disease contained higher

numbers of lobules type 1.The analysis of these samples allowed us to conclude

that although parity influences the architecture of thebreast, there is a similarity in architecture between the

breast of nulliparous women and of pamous women withbreast cancer. These results confirm our hypothesis that the

degree of breast development is of importance in the sus-ceptibility to carcinogenesis and that parous women who

develop breast cancer might exhibit a defective response tothe differentiating effect of the hormones of pregnancy (96).These findings indicate that the study of breast architecturethrough the quantitation and characterization of specific

lobular types, which are indicators of the greatest level ofdifferentiation achieved by the organ, provides valid end-points for assessing breast differentiation. These parameterswould, in turn, allow researchers to assess the risk of thegland to undergo neoplastic transformation when exposedto given genotoxic agents. In addition, they can be utilizedas intermediate endpoints for assessing the effectiveness ofchemopreventive or hormone-preventive agents.

Lobular Differentiation and the Susceptibility of BreastEpithelial Cells to Transformation by ChemicalCarcinogens in Vitro

Although progress has been made in defining some of the

critical processes contributing to carcinogenesis, the spe-cific biochemical and molecular mechanisms underlyingmany of these complex phenomena remain to be eluci-dated. Recent studies support the hypothesis that the devel-

opment of cancer involves the activation of protooncogenesand the inactivation of genes which function as inhibitors of

carcinogenesis (1 06-1 1 1 ). Whereas understanding of themechanism of action of both oncogenes and tumor sup-

pressor genes is mandatory to mechanistically define theneoplastic process, it is also vitally important to understandthe biological sequence of events that drive a normal HBEC

to neoplastic growth, under the action of known chemicalcarcinogens. A major stumbling block in the understandingof HBEC carcinogenesis is the fact that breast cells, as other

human diploid cells, rarely become immortal, meaning




acquire an infinite life span, and full transformation by

carcinogens alone has not been achieved reproducibly(1 1 2, 1 1 3). A likely explanation is that cells usually senesce

before they can acquire the multiple changes required for

immortalization and transformation. Thus, an important first

step in developing in vitro transformation systems for HBEC

is to understand the mechanism of cell immortalization.

Influence of Degree of Mammary Gland Development andof Cell Proliferation on the Growth Rate of Breast PrimaryCultures. On the basis of our previous observations that the

susceptibility of the mammary gland to neoplastic transfor-mation is related to its degree of development and prolif-

erative activity (2), we studied 1 5 reduction mammoplastiesby determining the degree of breast development, meas-

ured based upon the type and number of lobules present

(48). This study allowed us to classify the mammoplasty

specimens into two types: (a) poorly differentiated breasts,those composed of lobules type 1 and 2; and (b) well

differentiated breasts, those composed almost exclusively of

lobules type 3 (40). Portions of the same breasts were

utilized for measuring the rate of DNA synthesis or DNA-LI

by in vitro incorporation of [3H]thymidine and for studying

the in vitro growth characteristics of organoids obtained by

digestion of the tissue prior to plating. Breasts composed

predominantly of lobules type 1 and 2 had a DNA-LI of1 .03 ± 0.48. Cells derived from these samples attached to

the culture dish immediately with a high number of dou-blings (0.64 ± 0.47). Breast tissue composed almost exclu-sively of lobules type 3, with a DNA-LI of 0.05 ± 0.05, hada significantly lower number of doublings (1 02). These dataindicate that the properties ofthe cell in vivo are maintainedin in vitro conditions and reflect the condition of the host.

The proliferative activity of the breast varies dependingupon the phase of the menstrual cycle; although there isgreat variation between specimens, the geometric means of

cell proliferation has been shown to be consistently lowerin the follicular than in the secretory phase (6, 22, 23).However, no studies have correlated the variations in cellproliferation during the menstrual cycle with the location of

proliferating cells within specific lobular types. Since lob

type 1 (terminal ductal lobular units), the site of origin ofductal carcinomas, has significant differences in cell prolif-eration with lobules type 2 and type 3 (2, 48), the use of cellproliferation as an intermediate endpoint for assessing theeffect of chemopreventive agents requires determining theexact location of the cells in which proliferative activity ismeasured. The added advantage that cell kinetic character-

istics of the gland observed in vivo are also reflected in thebehavior of the cells in vitro validates the usefulness of thein vitro models for testing the response of the mammaryepithelium to hormones or genotoxic agents.

Response of Human Breast Epithelial Cells in Primary Cul-tures to Carcinogen Treatment. Based upon the rationalementioned above, we tested the effect of chemical carcin-

ogens known to induce mammary tumors in rodents (2) onprimary cultures of HBEC obtained from reduction mam-moplasties. Normal breast tissues were digested for obten-tion of lobules type 1 , 2, or 3, which were plated in culturemedium. When the cells formed monolayers, they werepassaged and then treated with the following carcinogens:DMBA, NMU, MNNG, and BP. BP and DMBA require

metabolic activation, whereas NMU and MNNG are directacting carcinogens (2, 1 01 , 1 03). After the second or third

passage posttreatment, the cells were seeded in agar metho-

SUSCEPTIBLE TARGET c�’ �

EA�.Y PRE�4ANCY

�

�LOb2Dlfteieiitletloii Pathway

PathwayExposure to ra�ationEn*onmental Factors

DIet I E S P �s�alance

Nulllparlty

Late pregnancy

Fig. 13. Lob 1 and lob 2 are the susceptible targets for different etiological

events that will lead them to neoplastic transformation. It is postulated thatearly pregnancy or hCG treatment may lead lob 1 and lob 2 towards thedifferentiation pathway.

cel in order to measure anchorage independent growth, aphenotype usually expressed by transformed cells which

correlates with tumorigenicity (1 14). Ten of 20 samplestested expressed an increased survival efficiency in agammethocel when compared with control cells. BP andMNNG were the carcinogens that affected the largest num-

ber of samples, 6 and 5, respectively, whereas DMBA andNMU had the lowest effect, since only 2 samples expressedincreased survival efficiency after treatment. The samplesthat reacted with the carcinogens were derived mainly frombreast tissue containing lobules type 1 or 2, alone or com-bined, and were derived from nulliparous women. Thosecells that did not show response were derived from samplesthat contained lobules type 3 (Fig. 1 1 ) obtained form parouswomen. We concluded that the developmental stage of the

gland in vivo correlates with increased survival efficiency inagar methocel which, in turn, is an indicator of increased

lifespan in vitro and an early transformation phenotype.These results confirm previous observations (1 01 ) and in-dicate that breast cells could be divided into two majorgroups: susceptible and nonsusceptible to carcinogens; thesusceptible samples were affected by at least one carcino-

gen and were those derived from less differentiated breasts,whereas the nonsusceptible samples, which were not af-fected by any of the carcinogens, were derived from breastscontaining preponderance of lobules type 3. The differentresponse of more or less differentiated cells to chemical

carcinogens provides evidence that differentiation plays animportant mole in the induction of cell transformation. This

system represents a useful model for testing intermediateendpoints for evaluating chemopreventive or hormone-

preventive agents.

Conclusions and Future Diredions

Our experimental system has allowed us to determine thatpregnancy induces differentiation of the mammary gland,which results in protection of this organ from chemicallyinduced carcinogenesis. The stimulus of pregnancy can besimulated in virgin animals by treatment with exogenoushormones, mainly the placental hormone hCG. The noveltyof our studies is the evidence that hCG protects themammary gland against carcinogenic initiation and pro-gression, mimicking the physiological process of preg-nancy, without crippling other reproductive or endocrine

functions (Fig. 1 3). The importance of both differentiation



362 Review: Human Papillomaviruse.s and Cervical Neoplasia

and cell proliferation in tumor initiation and progressionvalidates the use of these endpoints for assessing theeffect of hormones on the mammary gland prior to andafter the initiation of the process of carcinogenesis. Col-lectively, our results provide solid bases for developingphysiological means of breast cancer prevention and

control. The fact that the protection conferred by these

processes is maintained even after the termination ofeither pregnancy or hormonal treatment clearly indicates

that the differentiation induced by these processes is apermanent modification of the biological characteristicsof the mammary gland, even though the differentiatedstructures have regressed to seemingly more primitive

conditions (Figs. 3 and 10) (53-57). Conclusions drawnin the experimental system have been validated for theirapplication to the human situation through comparative

studies in these two species (2).

AcknowledgmentsSome of the breast samples utilized in these studies were generously pro-vided by Dr. S. R. Bartow, obtained under contract NCI-RFP No. 1-CB-84231/NOl -CN-23928. The authors acknowledge I. A. Bowman for skillfultyping of the manuscript.

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1994;3:353-364. Cancer Epidemiol Biomarkers Prev J Russo and I H Russo Toward a physiological approach to breast cancer prevention.

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