Estrogen Responsiveness and Control of Normal Human Breast Proliferation

Journal of Mammary Gland Biology and Neoplasia, Vol. 3, No. 1, 1998

Estrogen Responsiveness and Control of Normal HumanBreast Proliferation

Elizabeth Anderson,1,3 Robert B. Clarke,1 and Anthony Howell2

Our understanding of the hormonal control of the proliferation of normal human breast epithe-

lium is still surprisingly meager. However, the results of a number of recent studies have

confirmed that estrogen is the major steroid mitogen for the luminal epithelial cell population

(the usual targets for neoplastic transformation). Estrogen seemingly exerts its effects on cell

division indirectly as there is complete dissociation between the population of luminal epithelial

cells expressing the estrogen receptor (ER)4 and those that proliferate. We suggest that the

ER-negative proliferating cells represent a precursor or stem cell population that differentiates

to ER-containing, nonproliferative cells. In turn, these ER-positive cells act as ` estrogen

sensors’ and transmit positive or negative paracrine growth signals to the precursor cells

depending on the prevailing hormonal environment. As yet there is no direct evidence supporting

this hypothesis but we suggest ways in which it may be obtained. The implication of these

studies is that inhibition of luminal epithelial proliferation with tamoxifen or pure antiestrogens

or by preventing ovarian steroid secretion should be an effective strategy for the prevention

of breast cancer. In addition, we may be able to predict the risk of breast cancer in an individual

by measuring the intrinsic estrogen sensitivity of her breast epithelium. Finally, study of the

paracrine mechanisms of growth control in the normal human breast may provide new, more

specific, therapeutic targets for breast cancer prevention.

KEY WORDS: Human breast; proliferation; estrogen; estrogen receptor.

INTRODUCTION normal breast development, yet we still do not know

exactly how the ovarian steroids exert their effects.In 1984 Jean McManus and Clifford Welsch One reason is that endocrine regulation of breast devel-

wrote, ª The hormonal control of development, growth opment is difficult to study in normal women. Weand differentiation in vivo of the human breast is, cannot obtain normal human tissue at will nor can weunfortunately, still an enigmaº (1). In 1997, our under- manipulate the hormonal environment of the normalstanding of normal human breast growth and develop- breast at will. Consequently, we are reliant on tissuement is still surprisingly incomplete. We accept almost obtained at surgery for the removal of benign lesions oras a fact of life that intact ovaries are required for for breast reduction with attendant reservations about

whether these conditions are really representative of

the normal situation. Because of these constraints,1 Clinical Research Department, Christie Hospital NHS Trust, Wil-important new insights into the effects of ovarian ste-mslow Rd, Manchester M20 4BX.roids on the normal human breast and their underlying2 CRC Department of Medical Oncology, Christie Hospital NHS

Trust, Wilmslow Rd, Manchester M20 4BX. mechanisms are only now emerging.3 To whom correspondence should be addressed. e-mail: The major histological unit of the human breast

[email protected] an.ac.ukis the lobular structure arising from a terminal duct.4 Abbreviations: estrogen receptor (ER); thymidine labeling indexThis structure consists of several small, blind-endingin percent (TLI); epidermal growth factor receptor (EGFR); pro-

gesterone receptor (PR). ductules lined by a continuous layer of luminal epithe-

231083-3021/98/0100-0023$1 5.00/0 q 1998 Plenum Publishing Corporation

24 Anderson, Clarke, and Howell

lial cells surrounded by a second layer of myoepithelial the normal human breast throughout the menstrual

cycle (10±16). In these, and all subsequent studies,cells (Fig. 1). The myoepithelial cells are in direct

contact with the basement membrane and the whole our strategy has been to use normal tissue obtained

either at reduction mammoplasty or at operation forstructure is surrounded by delimiting fibroblasts and

a specialized intra-lobular stroma. The lobule in the the removal of a fibroadenoma (with informed con-sent). We chose to study patients with fibroadenomasnon-pregnant, non-lactating woman is a surprisingly

dynamic structure; several studies have shown that because they were the largest source of relatively

young, pre-menopausal breast tissue and, at the timethere is considerable cyclical variation in the morpho-

logical appearance of the epithelium as well as the of initiating these studies, fibroadenomas were thought

not to be associated with an increased risk of breaststroma and components of the surrounding extracellu-

lar matrix (2±6). Our comments will be restricted to cancer. However, more recently, the relationshipbetween the presence of a fibroadenoma and the riskthe cyclical variation and hormone responsiveness of

the luminal epithelial cells where human breast tumors of breast cancer has become less certain with one group

reporting up to an approximately two-fold increase inappear to arise. Careful histological examination

shows that most human breast tumors are morphologi- relative risk (17). This finding clearly demonstrates

the difficulty in obtaining truly normal human breastcally similar to luminal epithelial cells (7). In addition,

most tumors retain the biochemical characteristics of tissue for the study of hormonal control of function.Although the situation is not ideal, we are continuingluminal cells; for example, they express the same

cytokeratin profile (8), steroid receptors and polymor- to use tissue from fibroadenoma patients in addition

to that from reduction mammoplasties because thesephic epithelial mucin (9). Thus, the purpose of the

present article is to review the cyclical changes in the patients represent the largest group in which we can

study pre-menopausal breast function and becauseluminal epithelial cell population of the normal humanbreast together with the evidence for the estrogenic other benign lesions such as hyperplasia are associated

with a much greater risk of breast cancer. Howevercontrol of these changes that has been generated in invitro and in vivo experimental studies. These studies we have ensured that none of the patients we studied

had a family history of breast cancer although aboutare of more than academic interest; we cannot hope

to develop new, more effective strategies for cancer a quarter were using oral contraceptives at the time

of surgery.prevention if we do not understand how the factorsthat increase breast cancer risk affect the development The proliferative activity of normal tissue taken

at different times of the menstrual cycle was measuredof the normal human mammary epithelium.

by incubation with tritiated thymidine ([3H]-dT) and

subsequent calculation of a thymidine labeling indexCYCLICAL VARIATION IN

from autoradiography of tissue sections (TLI; percentPROLIFERATION AND STEROID RECEPTOR

cells labeled with [3H]-dT). We found considerableEXPRESSION IN NORMAL BREAST

inter-individual variation between the TLIs that couldLUMINAL EPITHELIAL CELLS

not be attributed to assay variability (10); nevertheless,

proliferation was clearly elevated in the luteal phaseWe and several other groups have examined theof the cycle. This result is in agreement with those ofproliferative activity of luminal epithelial cells withinthe other groups who have also found the proliferative

activity of normal human breast epithelial cells to behigher in the last half of the cycle (11±16). Detailed

analysis of other kinetic parameters such as the mitotic

and apoptotic indices showed a similar pattern of

change in that they, also, were higher in the second

half of the cycle compared to the first, although theapoptotic index reached a maximum approximately

three days after the peak of proliferative activity (13).

Cyclical variation in epithelial expression of other pro-

teins related to the proliferative and apoptotic pro-

cesses has also been shown. For example, Sabourin etFig. 1. The arrangement of epithelial cell types in the lobules

of the normal human breast. al. (18) showed that the anti-apoptotic protein bcl-2

Estrogen Responsiveness and Control of Normal Human Breast Proliferation 25

reached peak levels in the middle of the cycle (days when progesterone levels rise in the early luteal phase

(19). Such observations have led several groups to13±17) and fell to a nadir at the time when apoptosis

was greatest. suggest that, unlike the endometrium, the major steroid

mitogen for breast epithelial cells is progesterone eitherThe pattern of change in breast epithelial prolifer-

ative activity throughout the cycle contrasts directly alone or after estradiol priming. Likewise there appearto be important differences in the control of PR expres-with that seen in the endometrium. In this tissue, prolif-

eration is highest in the follicular phase of the cycle sion between the two tissues; the cyclical variation of

PR expression in the endometrium suggests that it isand falls rapidly at the beginning of the luteal phase;

it is almost undetectable in the later stages of the cycle an estrogen-inducible protein (28) whereas the lack of

variation in PR expression in the breast suggests that(19,20). Despite these differences, we assumed that

the cyclical variation in breast epithelial cell prolifera- it might be constitutively expressed. In order to investi-gate the hormonal control of function further, manytion was related in some way to the changes in ovarian

steroid secretion and we wanted to know whether groups have turned to experimental systems where the

steroid exposure of breast epithelial tissue or cells canexpression of the receptors for estrogen and progester-

one (ER and PR) also varied. We found that approxi- be manipulated. Such systems will be reviewed later

but we also hope to show that novel data on the hor-mately 5% of luminal epithelial cells expressed the

ER as determined immunocytochemically on frozen monal control of human breast function can be gleanedfrom relatively simple observations on tissue obtainedsections of the normal tissue used for the proliferative

studies (21, see also 24). The number of cells express- at operation.

ing ER was highest in the follicular phase of the cycle

and was inversely correlated with the TLI. In contrast,

15±20% of epithelial cells expressed the PR and theproportion did not vary during the cycle when mea- NORMAL HUMAN BREAST EPITHELIALsured immunocytochemically on frozen sections of CELLS IN CULTUREnormal breast tissue. A few other groups have exam-

ined steroid receptor expression in normal human

breast tissue throughout the menstrual cycle. The The establishment of human breast epithelial cell

cultures that replicate and retain their original charac-results of these studies are in agreement with those ofour own investigations and show that ER protein is teristics has proved extraordinarily difficult despite the

development of a number of different strategies formore readily detected in tissue removed in the follicu-

lar phase of the cycle compared to the luteal phase epithelial cell isolation and culture. These include sep-

aration of the luminal and myoepithelial cell popula-(22±24). The other groups were also able to show

that PR expression in the normal breast does not vary tions on the basis of specific cell surface antigens (29),

allowing the cells to immortalise spontaneously (30),appreciably during the cycle.Epithelial cell expression of receptors other than immortalization with the SV40 tumor or human papil-

loma viruses (31±34) or mutated tumor suppressorthose for steroids also varies throughout the cycle. For

example, Gompel et al. (25) showed that epidermal genes (35), culture on or in different extracellular

matrix components (36±39) or combinations of thesegrowth factor receptors (EGFR) are more highly

expressed in the luteal phase of the cycle compared approaches (31,37). Studies on epithelial cells cultured

using these techniques have produced much valuablewith the follicular phase. This finding may not besurprising in the light of studies on human breast and interesting data on, for example, the peptide

growth factors that stimulate growth (40±42) and thetumors showing that EGFR and ER expression are

generally inversely related (26). conditions required for branching morphogenesis and

ductule formation (37,39,43). Almost universally theseThe obvious candidates for the control of cyclical

changes in the breast epithelial cell population are methods have failed to produce cultured luminal epi-thelial cells that express steroid receptors and are capa-estradiol and progesterone since they are produced

cyclically by the ovaries (27). Maximal breast prolifer- ble of responding to ovarian steroids. To date, there

are only two reports of the isolation of steroid receptoration coincides with the mid-luteal phase peaks of

estradiol and progesterone secretion whereas prolifera- expressing human mammary epithelial cells. In the

first study, the cultured epithelial cells expressed bothtion of the endometrium, a ª classicalº estrogen target

organ is highest in the follicular phase and declines the ER and the PR and treatment with estradiol not only


increased proliferation but also enhanced expression of NORMAL HUMAN BREAST EPITHELIUMIMPLANTED INTO ATHYMIC NUDEboth types of receptor suggesting that estradiol is theMICEovarian steroid that stimulates luminal epithelial cell

proliferation (44). However, the cells used in this par-Because cultures of primary or immortalizedticular study were obtained at operation for fibrocystic

human breast luminal epithelial cells have proved todisease and may have been derived from the lesionbe poor models for the study of steroid responsiveness,rather than the normal epithelium. In the second study,many researchers, including ourselves, have turned tohuman mammary epithelial cells isolated from reduc-in vivo models of normal human breast tissuetion mammoplasties and immortalised with SV40 wereimplanted into athymic nude mice.found to express a splice variant of the ER (45). This

A system of implanting intact human breast tissueER variant contained a deletion in exon 2 (the DNA-into athymic nude mice was developed in the late 1970sbinding domain) and did not bind to an artificial EREin response to a perceived lack of suitable modelsin gel mobility shift assays. These results imply thatin which the hormonal control of proliferation andthese particular cells were incapable of responding tofunction could be studied (1). In these particular exper-estradiol treatment although this was not shown. Asiments, small pieces of tissue taken from the peripher-far as is known, none of the other studies involvingies of benign breast lesions were implantedisolation of cells from reduction mammoplasties havesubcutaneously into intact female athymic nude mice.produced ER-expressing human luminal epithelialThe mice were subsequently treated with estradiol,cells.progesterone , thyroxine or human placental lactogenThere have been attempts to restore receptoreither singly or in combination and tissue pieces wereexpression in cultures of immortalized human breastretrieved at different time points after the start of treat-epithelial cells by transfection with an ER-expressionment for measurement of proliferative activity by [3H]-vector (46±48). These attempts have successfully pro-dT uptake. This particular study showed, for the firstduced high level ER protein expression but in onlytime, that human breast epithelial cell proliferation

one did subsequent treatment with estradiol stimulatecould be stimulated by estradiol (1). Thyroxine also

proliferation and even then the effect was small (48).stimulated proliferation, human placental lactogen

More often, transfection of ER resulted in the oppositealone was without effect although it enhanced the

effect in that treatment of the transfected cells withactivity of estradiol when given in combination. In

estradiol inhibited proliferation (46,47). One explana-contrast, progesterone did not alter the proliferative

tion for these findings is that the negative effects ofactivity of the breast epithelium either alone or in

estradiol on ER negative cells transfected with ER iscombination with the other agents. At about the same

that the exogenous receptor interferes with ortime, other experiments were being carried out in

` squelches’ the transcriptional factors used to maintainwhich partially enzymatically digested breast tissue

estrogen-independent growth (46). Why over expres-(` organoids’ ) was implanted into intact mammary fat

sion of ER in the MCF-7 cells does not ` squelch’ the pads of athymic nude mice or fat pads that had beentranscriptional factors involved in estrogen-dependent cleared of parenchyma (49±51). In all these reports,growth is not well understood. However, it is becoming the partially digested human tissue re-organized itselfclear that the activity of the ER is highly dependent into primitive glandular structures. Only one groupupon its cellular context and it is possible that the examined the estrogen-responsiveness of such tissueER negative cell lines do not provide the appropriate implants and showed that estradiol did, indeed, stimu-environment in terms of co-factors for correct function late proliferation (50). The other groups concentratedof the exogenous ER (46). At present, it is not known on demonstrating a lactational response in the humanwhy the ER-expressing cells that have been shown to tissue after administration of lactogenic hormones orexist in the luminal epithelial cell population in vivo after mating the xenograft-bearing mice (49,51). Muchdo not survive to grow in vitro. If we could determine later, a response to estrogen treatment was demon-why this happens we might gain valuable new informa- strated in a system where human breast epithelial cells,tion about the steroidal control of proliferation in the dissociated from reduction mammoplasty specimens,normal human breast epithelium as well as an answer were embedded in extracellular matrices (type I colla-to the question of why such a large proportion of breast gen and Matrigel) before being implanted subcutane-

ously into the nude mice (52). Again, the breasttumors contain ER.


epithelial cells were shown to have organized them- The results of our experiments on the effects of

estradiol and progesterone on the proliferation ofselves into primitive branching ductal structures inhuman breast tissue implanted into the athymic nudeboth types of extracellular matrix and there were prolif-mice were described in detail by Laidlaw et al. (53).erative responses to estradiol, cholera toxin and epider-Briefly, when human breast tissue was implanted intomal growth factor (EGF).female, intact, adult mice that received no further treat-These experiments showed that the hormonalment, the TLI of the epithelial cells fell from approxi-environment of normal human breast tissue could, atmately 2% at the time of removal from the patient tolast, be relatively easily manipulated following implan-0.5%, two weeks after implantation. This low level oftation into athymic nude mice. However, very fewproliferation was maintained for at least eight weeksgroups continued to pursue this approach possiblyin the untreated mice as was the structure and integritybecause the experiments were tedious and time-con-of the tissue. Raising the serum estradiol levels suchsuming and because specialist animal facilities werethat they approximated human peak luteal phase levelsrequired. Nevertheless, we decided to revisit the exper-( , 1300 pmol/l) significantly increased the prolifera-iments of McManus and Welsch (1) to further investi-tive activity of the epithelial cells whereas progester-gate the estrogenic response in normal human breastone, also at peak luteal levels, was without effect eithertissue. We decided to implant pieces of intact tissuealone or after priming with luteal phase estradiol levels.into non-ovariectomized adult female mice for severalThe proliferative response to estradiol was dose-depen-reasons, some of them rather pragmatic. Firstly, thedent between median serum estradiol levels of 400 touse of intact pieces of tissue would maintain the archi-1300 pmol/l which were representative of the rangetecture of the normal human breast tissue and the rela-seen during the human menstrual cycle. Raising estra-tionship between the epithelial and stromaldiol levels to those of early pregnancy (a median ofcompartments. Secondly, implantation subcutaneously4400 pmol/l) had no further effect. Thus we confirmedrather than into the mammary fat pad was adoptedthe results of McManus and Welsch (1) and providedbecause it was felt that it would be a less invasivefurther evidence to support the role of estradiol as the

procedure and because the pieces of tissue wouldmajor ovarian steroid mitogen for the normal human

remain easily accessible for serial sampling. Thirdly,breast. Progesterone appeared to be neutral with

implanting intact tissue would be much less time con-respect to its effects on breast epithelial cells in that

suming than using enzymatically disaggregatedit neither stimulated nor inhibited proliferation.

organoids. Finally, we used intact female mice as ovari-We obtained some very interesting results when

ectomy would add another level of complexity to thewe examined steroid receptor expression in the normal

experiments and would be associated with significanthuman breast tissue after estradiol and progesterone

intra- and post-operative mortality. We measured thetreatment. The percentage of normal epithelial cells

endogenous serum estradiol levels in the strain of micethat expressed the ER fell from , 5% when removed

used in our experiments and found them to be lessfrom the patients to , 1% after the two week control

than 100 pmol/l which is equivalent to those found in period and subsequent treatment with estradiol andpost-menopausal women (53). Thus, our first aim was progesterone either alone or in combination had noto confirm the results of McManus and Welsh (1) using further effect. This reduction in the proportion of epi-histologically confirmed normal tissue as opposed to thelial cells expressing the ER may have resulted fromtissue obtained from the periphery of diffuse benign the low levels of circulating estradiol in the untreatedlesions. We also aimed to use calibrated slow-release nude mice ( , 100 pmol/l) or may be related to theestradiol silastic pellets designed to deliver serum lev- immunocytochemical assay used at that time whichels that approximated those in the human menstrual was not very sensitive, especially after it had beencycle instead of the schedule of McManus and Welsch adapted for application to tissue after it had been xeno-(1) which resulted in uncertain amounts of estradiol grafted. The proportion of luminal epithelial cells thatand estrone being administered to the mice. Our third expressed PR was also very small after two weeks inaim was to examine the effects of steroid treatment the untreated mice. However, a 15±20 fold increase inon receptor expression in the breast tissue implants the percentage of cells expressing PR was seen inbecause suitable methods for the immunocytochemical the normal tissue xenografts after 7 and 14 days ofdetection of ER and PR in small tissue samples had, treatment with estradiol at luteal phase levels. Proges-

terone at luteal phase levels was without effect on theby then, been developed.


expression of either of the receptors. These results

suggested that the PR is an estrogen-regulated protein

in normal human breast epithelial cells. This finding

was rather surprising given that studies on breast epi-

thelium throughout the menstrual cycle in vivo showedthat PR expression remained high through the men-

strual cycle. These data had, in turn, been interpreted

to mean that synthesis of the receptor might be consti-

tutive (shown earlier). Accordingly, we carried out

more detailed investigations into the control of PR

expression in normal human breast epitheliumimplanted into the athymic mice with the specific aim

of determining why PR expression did not change

throughout the cycle in women in vivo (54).

PROLIFERATION AND PR EXPRESSIONARE DIFFERENTIALLY SENSITIVE TOESTROGEN

Our first approach to resolving the discrepancy

in PR expression between our experiments on normalhuman breast implanted into athymic mice and the

studies on normal breast throughout the menstrual

cycle was to determine both PR expression and prolif-

erative activity in xenografts removed from mice

treated with different levels of estradiol (54).Fig. 2. Effects of estradiol treatment at differing concentrations onFigure 2A shows the results of the immunocyto-normal human breast tissue implanted into athymic nude mice.chemical detection of PR in sections cut from Carnoy’ s(A) Epithelial cell PR expression. (B) Epithelial cell proliferation

fixed paraffin-embedded specimens of human breastmeasured by the TLI. The columns represent the interquartile ranges

tissue xenografts retrieved from mice after treatment of the measurements whereas the bars indicate the median values.

with follicular or luteal phase levels of estradiol. As P 5 progesterone; Lo 5 follicular phase levels of estradiol; Hi 5luteal phase levels of estradiol; Hi,14 5 14 day treatment withbefore, PR expression was almost undetectable inluteal phase levels of estradiol; * 5 p , 0.001 compared with thebreast tissue removed from untreated mice. However,no treatment value by Mann Whitney U test.

follicular phase levels of estradiol were sufficient to

induce maximal PR expression in the epithelial cells

and the higher luteal phase levels of the steroid had

no further effect. In keeping with the menstrual cycle serum (27) or breast tissue (55) during the cycle are

sufficient to induce maximal PR expression in thedata, the proliferative activity of the breast epithelium

treated with follicular phase levels of estradiol was breast epithelial cells. In contrast, proliferation is onlysignificantly enhanced by much higher serum levelsnot significantly different from that of the control,

untreated tissue (Fig. 2B). But, as expected, treatment of estradiol.

How individual breast epithelial cells might bewith the higher, luteal phase estradiol levels signifi-

cantly increased the proliferative activity of the epithe- differentially sensitive to estradiol is, as yet, unclear.

However, there are data, generated in studies on breastlial cells in the breast tissue implants. These findingsindicated that PR expression was far more sensitive to cancer cells, suggesting that the sensitivity of the ER

may altered by interactions with other intracellularestrogen than proliferation explaining the discrepancy

between the data on PR expression during the men- signaling pathways. In particular, phosphorylation of

the ER by, for example, protein kinase A or mitogen-strual cycle and those obtained from the model of

normal human breast tissue implanted into athymic activated protein kinase has been shown to enhance

DNA binding and transcriptional activity (56). Further-nude mice. Even the lowest levels of estradiol seen in


more, a plethora of steroid receptor co-activators and dent in very few cells suggesting the existence of two

co-repressors have been identified which appear to separate populations of luminal epithelial cells. Anregulate receptor function in a cell- and context-spe- alternative explanation was that PR synthesis had beencific manner (57). It is not difficult to envisage that down-regulated during the S-phase of the cell cyclechanges in the environment of the receptor by, for (the only phase detected by [3H]-dT labeling). Studiesexample, signaling from cell surface receptors or inter- on breast cancer cells have shown that PR proteinactions with co-factors could enhance or reduce the levels vary throughout the cell cycle and are maximalestrogen sensitivity of individual breast epithelial cells. only at the transition into G2 from the S-phase (59).

In order to confirm that PR expression and proliferation

were, indeed, taking place in separate epithelial cells,

we used fluorescent immunocytochemistry to detectTHE RELATIONSHIP BETWEENthe Ki67 proliferation-associated antigen and steroidPROLIFERATION AND STEROIDreceptor expression simultaneously. The Ki67 antigenRECEPTOR EXPRESSION IN LUMINALhas been reported as being present in cell nuclei at allEPITHELIAL CELLSstages of the cell cycle except G0 (60) and should be

co-localized with PR even if, as suggested previously,These findings raised the question of whetherthe receptor has been down-regulated during the S-PR expression and proliferation occurred in the samephase. Fluorescent dual labeling was carried out onepithelial cell which would have to have been sensitiveformalin-fixed sections of normal breast tissue afterto two different concentrations of estradiol or whethermicrowave antigen retrieval and showed that, as forthe two processes took place in separate cell popula-the previous studies, very few epithelial cells were co-tions each of which was sensitive to a different estrogenlabeled with the anti-PR and anti-Ki67 antibodies (Seelevel. An answer to this question was suggested byFig. 3B). Thus our original conclusion that PR expres-studies in which we used dual labeling techniques forsion and proliferation take place in separate epithelialthe simultaneous detection of steroid receptor expres-cell populations was corroborated.sion and proliferation in sections of human breast tis-

Since both the PR expressing and the proliferatingsue (58).luminal epithelial cells were sensitive to estrogen, weIn the first set of experiments we used sectionsassumed that both populations would contain ER.cut from Carnoy’ s fixed breast tissue samples obtainedAccordingly, we examined the co-incidence of ER andat different times of the menstrual cycle. The sectionsPR expression and of ER and Ki67 expression usingwere stained immunocytochemically to reveal the PR-the same methods and samples as detailed earlier. Aexpressing cells and then dipped in photographic emul-number of interesting points were raised by the datasion to determine [3H]-dT uptake. As shown in Fig.

3A, PR expression and [3H]-dT labeling were co-inci- obtained from these studies. The first was that a larger

Fig. 3. The proportion of PR expressing epithelial cells that are also labeled with (A)

[3H]-dT or (B) an antibody against the Ki67 proliferation associated protein. The results

are presented as the percentage of the total number of cells counted in 25 specimens

of normal human breast. At least 1000 cells were assessed per specimen; the total

number of cells counted is indicated in parentheses.


one expressing ER and PR. In addition, the receptor-

expressing cells were distributed relatively evenly

throughout the luminal epithelium. Taken together,

these findings implied that steroid receptor expression

is dissociated from proliferation in the normal humanbreast. They raise some very interesting questions as

to how hormonal control of epithelial growth and func-

tion might be achieved.

A MODEL FOR THE ESTROGENICFig. 4. The proportion of PR expressing cells that were also labeled

CONTROL OF LUMINAL EPITHELIALwith an antibody against the ER. The results are presented as

CELL PROLIFERATIONpercentages of the total number of cells counted (indicated in paren-

theses) across 10 specimens of normal human breast tissue.

Our own data together with those of others sug-

gest a model for the estrogenic control of proliferation

within the human luminal epithelial compartment. Inproportion of luminal cells than expected expressed

the ER compared to our original studies on frozen this model (see Fig. 6), the ER-negative cells are in

close proximity to the ER-positive cells and are stimu-sections cut from samples of the breast tissue that had

been implanted into the athymic nude mice (shown lated to divide by juxtacrine and/or paracrine factorssecreted by the receptor-containing cells in responseearlier). We attributed this to the greater sensitivity of

the immunofluorescent method of detecting the ER to estrogen. The ER-negative cells may represent a

precursor or stem cell population which eventuallywhich included microwave-mediated antigen retrieval

in citrate buffer. Secondly, there was almost complete differentiates to become ER-positive and non-prolifer-

ative. Based on our observations that proliferating cellsco-incidence of ER and PR expression in that 96% of

the cells labeled with the anti-PR antibody also labeled are often immediately adjacent to those expressing thereceptor, we further speculate that the ER-positive cellswith the anti-ER antibody (see Fig. 4). Thirdly, co-

incidence of ER expression and proliferation as mea- might be arranged with their proliferative precursors

into discrete areas of the luminal epithelium or ` prolif-sured by [3H]-dT uptake or by detection of the Ki67

antigen was a very rare event (see Fig. 5). However, erative units’ similar to those described in the epider-

mis (61).almost every proliferating cell was found adjacent to

Fig. 5. The proportion of ER expressing epithelial cells that are also labeled with (A) [3H]-

dT or (B) an antibody against the Ki67 proliferation associated index. The results are

presented as the percentage of the total number of cells counted in (A) 10 specimens or

(B) 25 specimens of normal human breast. At least 1000 cells were assessed per specimen;

the total number of cells counted is indicated in parentheses.


addition, we have shown that estradiol treatment

enhances expression of type 1 insulin-like growth fac-

tor receptor mRNA in normal human tissue implanted

into athymic nude mice (68). These preliminary studies

suggest the potential for paracrine regulation of prolif-eration of the normal human breast epithelium as sug-

gested in this hypothesis.

Recently Cunha and his colleagues (69) carried

out elegant studies in which mammary gland epithe-

lium isolated from ER knockout mice was implanted

into the mammary fat pads of wild type mice and viceversa. The results of these experiments suggest that,

in the mouse as well as in humans, estradiol stimulates

epithelial cell proliferation indirectly via the secretion

of paracrine growth factors. In the case of the mouse,Fig. 6. A model for indirect estrogenic control of normal human

the ` estrogen sensor’ cells appear to be in the stromalbreast epithelial cell proliferation. In this scheme, the cells capable

compartment as wild type epithelial cells could notof proliferation (the stem cell/early transit cell populations) are ER-respond to estrogen treatment when implanted into thenegative and the receptor is only expressed as the cells become more

differentiated. These differentiated cells control the proliferative fat pads of ER-knock out mice. However, in humans,activity of the stem and early transit cells via positive and negative ER expression in the stromal cells surrounding theparacrine signals depending on the prevailing estrogenic

epithelium has never been reported leading to the con-environment.

clusion that the ` estrogen sensing’ cells in our proposedmodel are situated in the epithelial compartment.

Studies on breast cancer cell lines carried out

some time ago provide indirect support for the above CONCLUSIONSmodel of estrogenic control of normal breast epithelial

cell proliferation. Firstly, conditioned media from ER- The studies reviewed in this article all point tothe normal human breast epithelium being an estrogenpositive breast cancer cell lines treated with estradiol

in culture was shown to stimulate the proliferation of target tissue. Unlike the endometrium, however, human

breast epithelial cells do not appear to be very sensitiveER-negative breast cancer cells both in culture and

when injected into athymic nude mice bearing ER- to estrogen. This would seem logical in that a cyclical

estrogenic response in the breast of the same magnitudenegative breast tumor xenografts (62). Furthermore ,

treatment of ER-positive breast cancer cell lines with and speed of that seen in the endometrium would behighly undesirable. An indirect mechanism of control-antiestrogens enhanced the synthesis and secretion of

peptide growth regulators (63). Transforming growth ling proliferation within the breast luminal epithelium

is one means by which estrogen sensitivity could befactors a and b together with insulin-like growth fac-

tor-I were implicated as the mediators of these para- attenuated. During pregnancy, when extensive epithe-

lial proliferation is required, not only are circulatingcrine effects. Secondly, some evidence has been

generated by studies on breast cancer cell lines that estradiol levels much higher than those of the men-strual cycle but there are large numbers of, as yet,ER-negative cells can give rise to ER-positive ones.

For example, small colonies of MCF-7 cells grown in unidentified factors that could enhance the estro-

genic response.soft agar were shown to be ER-negative when sec-

tioned and stained immunocytochemically with anti- For future studies on estradiol control of normal

breast growth and function, we should like to recapitu-ER antibodies whereas large colonies were ER-posi-tive (64). Fractionation of primary tumor cells on the late in culture the full repertoire of luminal epithelial

cell types that we see in vivo. We would not expectbasis of receptor content produced an ER-negative

fraction that gave rise to ER-positive cells in culture to isolate and culture ER-positive cells from normal

breast tissue de novo as the hypothesis presented earlier(65). Expression of growth factors and their cognate

receptors has been demonstrated in both normal human predicts that these cells represent a differentiated, non-

proliferative population. However, given the correctbreast tissue and cultured epithelial cells (66,67). In


culture conditions, we may be able to induce differenti- small replacement doses of estrogens and androgens

to protect the cardiovascular and skeletal systems.ation of the ER-negative precursor cells and use them

as a model in which to study paracrine regulation of Finally, an early first full term pregnancy protects

against breast cancer (70) presumably because a signi-breast epithelial physiology. An alternative strategy

would be to implant isolated ER-negative epithelial ficant proportion of the breast luminal epithelium hasundergone terminal differentiation. A preventativecells embedded in extracellular matrix into athymic

nude mice. Some interesting data have been generated strategy suggested by Russo and colleagues (72) and

tested in rodents is the administration of a differentiat-recently by Kang et al. (45) who showed that splicing

of the ER mRNA to produce the wild type ER protein ing agent (in this case human chorionic gonadotrophin)

at the time when the mammary gland is most suscepti-occurred only when tumor epithelial cells were xeno-

grafted subcutaneously into athymic mice. This sug- ble to carcinogens; i.e., in early reproductive life. Thisstrategy has not been tested using human material orgests that three-dimensional structure and intercellular

interactions are also involved in governing estrogen subjects although the effects of human chorionic

gonadotrophin on proliferation and differentiationresponsiveness. Finally, relatively simple immunocy-

tochemical and/or in situ hybridization surveys of nor- could be tested relatively easily using human tissue

implanted into athymic nude mice.mal human breast tissue under well characterized

estrogenic conditions could still yield interesting data. In conclusion, recent studies on human breastepithelial cells in culture and xenografted into athymicIn addition to providing new insights into the

control of normal breast function, the findings and nude mice have generated new data on the estrogenic

control of proliferation. Future studies of this kindhypothesis presented earlier have important implica-

tions for breast cancer prevention and for predicting may yield new therapeutic targets for breast cancer

prevention or new methods of predicting individualbreast cancer risk. Firstly, we and others, have shownthat there is considerable variation in breast epithelial risk of breast cancer.

cell proliferative activity between individuals but we

do not know, as yet, whether high levels of proliferation

are associated with an increased risk of breast cancer.ACKNOWLEDGMENTS

Current theories on the carcinogenic process suggest

that this may well be the case and it is interesting thatThe authors thank Mr. Ian Laidlaw and Professorthe endocrine breast cancer risk factors such as early

C. Potten for their invaluable contributions to the stud-menarche, late menopause and late first full term preg-ies on normal human breast tissue implanted into nudenancy (70) all serve to increase the number of timesmice. We should also like to acknowledge the involve-the breast epithelium undergoes a cyclical increase inment of Mr. K. Spreckley, Mr. A. Rushton, Dr. S.proliferation. It is also not yet known whether muta-Roberts, Dr. A. Wakeling and Dr. J. Coyne. The studiestions in the BRCA 1 and 2 genes alter the estrogencarried out at the Christie Hospital were supported bysensitivity of the normal breast epithelium or whetherthe Endowment Fund of the Christie Hospital NHSthey exert their effects on breast cancer susceptibilityTrust, Zeneca Pharmaceuticals and the Cancerby some other mechanism. If it could be shown thatResearch Campaign.the estrogen sensitivity of the luminal epithelial cell

population is related to the likelihood of developing

breast cancer, then we can envisage individual riskprediction based on measurement of products of estra-

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Estrogen Responsiveness and Control of Normal Human Breast Proliferation