Hormonal aspects of breast cancer: Growth factors, drugs and stromal interactions

Critical Reviews in Oncology/Hematology, 1992; 121-23 0 1992 Elsevier Science Publishers B.V. All rights reserved 1040-8428/92/$5.00

ONCHEM 00015

Hormonal aspects of breast cancer Growth factors, drugs and stromal interactions

Robert Clarke, Robert B. Dickson and Marc E. Lippman Lombardi Cancer Research Center, Georgetown University Medical Center, Washington DC, U.S.A.

(Accepted 2 August 199 1)

Contents

1.

II.

III.

Introduction................................................................... 2

Steroid hormones and their role in hormonal carcinogenesis 2

Steroid hormones and growth factors in breast cancer 4 A. Steroidhormonereceptors ................................................... 4 B. Autocrinegrowthstimulation ................................................. 4 C. Steroid regulation of growth factor and growth factor receptor expression. .......... 5 D. Further comments on tumor growth factor interactions in breast cancer ............ 7

IV. Paracrine-stromal growth factor interactions. 7 A. B. C.

D.

E.

Stromal populations of lymphoreticular origin in breast neoplasia. ................. 8 Stromal populations of mesenchymal origin in normal breast. ..................... 9 The role of stromal cells of mesenchymal origin in mediating the estrogenic responsivity ofnormalbreasttissue ....................................................... 9 The role of stromal populations of mesenchymal origin in the control of breast tumor growth.. .................................................................. IO Further comments on tumor-stromal interactions in breast cancer ................. 12

V. Hormonal modulation of cytotoxic chemotherapy .................................. 13 A. Hormonal effects on cell cycle modulation and response to cytotoxic drugs .......... 13 B. Effects on drug resistance and MDR-I ......................................... 14

VI. Conclusions ................................................................... 14

References .............................................................................. 15

Robert Clarke received his M.S. and Ph.D. degrees from Queen’s Uni- versity of Belfast, U.K. He did his Postdoctoral at Queen’s University of Belfast, and Medicine Branch N.C.I., N.I.H., U.S.A. Dr. Clarke is presently Director of the Lombardi Cancer Research Center Animal Core Facility, and Assistant Professor, Department of Physiology and Biophysics, Georgetown University. Robert R. Dickson received his M.Phil. and Ph.D. degrees from Yale University, New Haven, CT, and his Postdoctoral fellow from Laboratoy of Molecular Biology, N.C.I., N.I.H., Bethesda, MD. Dr. Dickson is presently Associate Professor of Anatomy and Cell Biology, Georgetown University, and he is a member of the Breast Cancer Working Group (N.C.I. Organ

Systems Program) and the Vincent T. Lombardi Cancer Center Exec- utive Committee, Georgetown University. Marc E. Lippman received his B.A. from Cornell University, Ithaca, NY and his M.D. degree from Yale Medical School, New Haven, CT. Dr. Lippman is presently Director of the Vincent T. Lombardi Cancer Center, and Professor of Medicine and Pharmacology, Georgetown University School of Medi- cine, Washington, DC.

Correspondence: Robert Clarke, Room S128A, Lombardi Cancer Re- search Center, Georgetown University Medical Center, 3800 Reser- voir Road, Washington DC 20007, U.S.A.

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I. Introduction

Breast cancer is the second most common form of cancer among women in western society. Approximate- ly one in ten of all women in the U.S.A. living to age 80 years will develop breast cancer. Furthermore, the incidence of breast cancer appears to be inexorably increasing [13], with an annual worldwide incidence of over one million predicted by the turn of this century [2]. This high incidence of metastatic breast cancer is a major problem, particularly since the probability of further survival beyond 5 years is low for patients with detectable metastatic disease. Unfortunately, there is little evidence that significant progress has been made over the past 20 years in improving overall survival. Thus, a clearer understanding of the biology of breast tumors and the mechanisms responsible for their progression are essential for the development of novel therapeutic strategies.

The development of normal mammary tissue is the result of complex interactions between a number of hormones and growth factors including the steroids, insulin and various pituitary factors. Steroids can influence cellular function by both genomic and genome-independent mechanisms. Genomic mediated effects of steroids, including induction of mitogenesis, are generally not detected less than 30 min following hormone treatment and occur following significant perturbations in the level of expression of a number genes [3]. Non-genomic mediated events that occur following exposure to steroids are generally observed at pharmacological rather than physiological concentrations [3]. The genomic effects 178-estradiol (E2), progesterone and hydrocortisone appear to be essential for the induction of growth and differentiation in normal mammary tissue. For example, estrogens increase nipple differentiation and induce proliferation of the surrounding mesenchymal stroma in the developing mouse fetus [4]. E2 and progesterone are also important in alveolar formation, ductal branching [5,6] and for full lobulo-alveolar development [7]. Pri- mary breast carcinomas appear to arise from the epithelial rather than myoepithelial cells of the glandular epithelium, presenting as either intraductal or intralobular lesions. Invasive tumors spread radially by infiltrating through tissue spaces and can invade both lymphatic and blood vessels. There is a high incidence of distant metastases, particularly in soft tissues, lungs, liver, bones and adrenals. The curative potential of current therapies is restricted by the disseminated nature of the disease and the progression of the overwhelming majority of tumors to a phenotype characterized by resistance to both cytotoxic drugs and hormonal therapies. Whilst progesterone (probably in conjunction with E2) is more

closely associated with control of normal breast tissue [8], E2 is the hormone most closely associated with the control of neoplastic breast tissue. Of the estrogen receptor (ER) positive human breast cancer cell lines most widely available, all exhibit a marked mitogenic response to E2 in vitro [9]. Pharmacological concentrations of antiestrogens inhibit the proliferation in vitro of human breast cancer cells expressing ER as a result of their interactions with this protein [lO,l 11. It is unlikely that this simply represents a competitive inhibition of E2 binding, since these agents are effective even in the apparently total absence of E2. Thus, the antiestrogen/ ER complex may have intrinsic inhibitory properties [10,12,13]. At the higher pharmacological or supraphar- macological concentrations, E2 and tamoxifen are cytotoxic to both ER-positive and ER-negative cells [ 14,151. We have recently demonstrated that this relatively non- specific toxicity is closely associated with alterations in the gross membrane fluidity of the target cells [15]. These observations may reflect the ability of high-dose estrogens and antiestrogens to inhibit some apparently ER-negative breast tumors [ 16,171.

In this review we will address various hormonal aspects of breast cancer. We will begin with a discussion of the role of steroids in hormonal carcinogenesis. This will be followed by an examination of the role of steroids and growth factors in breast tumorigenesis and progression, with particular emphasis on the autocrine hypothesis. The role of stromalepithelial interactions in mediating the effects of steroids in breast tumor growth and progression will be addressed from the perspective of potential paracrine interactions and how these may be addressed in vitro. The final hormonal as- pect to be discussed will be an analysis of the effects of hormonal agents on systemic cytotoxic treatments for metastatic breast cancer.

II. Steroid hormones and their role in hormonal

carcinogenesis

Steroid hormones have been widely implicated in the process of carcinogenesis, however, their precise role remains to be established. Epidemiological evidence in women indicates that the length of exposure of the mammary glands to estrogenic stimuli and to other die- tary, genetic and environmental factors is directly pro- portional to breast cancer risk. Prolonged exposure to endogenous estrogens may occur as a result of early menarche, late menopause and late age at first full-term pregnancy. Obesity also appears to increase the risk of developing breast cancer, perhaps as a result of increasing peripheral aromatization of circulating androgens to estrogen [ 18,193. Risk is decreased with early menopause

(natural or artificial) and with childbearing but increased by first-trimester abortion [20]. Oral contraceptive use does not appear to be a major risk factor but this is currently controversial [21]. Estrogenic stimulation is also implicated as an etiologic agent in vaginal adenocarcinoma, endometrial cancer, ovarian and liver tumors [22-241.

Prolactin has received some attention as a potential etiologic agent. Whilst apparently not a primary mitogen in human breast tissue, prolactin can increase the mitogenic effects of estrogen [25]. In marked contrast, chemically induced mammary tumors in rodents are to- tally prolactin dependent, at least during the early stages of tumor progression [26,27]. Since breast epithelial mi- toses peak in the luteal phase of the menstrual cycle, during which time progesterone levels are at their high- est and estrogen at their lowest, some investigators have suggested that progesterone plays a pivotal role in mitosis [28]. Whilst the role of prolactin and progesterone require further study, one reasonable hypothesis of hormonal carcinogenesis in breast cancer invokes the ‘total cumulative exposure of breast tissue to bioavailable estrogens and the associated cumulative mitotic activity’ as the important etiological factors [20].

Although E2 and progesterone are clearly associated with increased breast proliferation, estrogens may also damage cells directly. This hypothesis has received strong support from the observations concerning the carcinogenic effects of the synthetic estrogen diethylstilbestrol (DES). DES is oxidized by a peroxidase-mediated reaction. Short-lived expoxide and semiquinone in- termediates are produced which appear to be carcinogenic. Whilst genotoxic, no clear evidence of DES-DNA adduct has been reported. Thus, these species may be unstable, or alternative mechanisms may be responsible for mediating the genotoxic effects of DES [29]. For example, DES and other estrogens may disrupt mitosis by altering spindle formation or function [30].

Carcinogenic reactive species may be generated by the direct metabolism of estrogens. E2 and ethinylestradiol may be hydroxylated to their reactive 2 OH and 16 OH metabolites, both DNA and proteins (e.g., ER) being potential targets [3&32]. Hydroxylation of 16a-estradiol increases in breast cancer patients and in women with a high risk of hereditary breast cancer [33,34]. The carcinogenicity of estrogens may involve both mitogenic and metabolic activation components. In Syrian ham- sters, E2 but not 2-fluoro-estradiol can induce renal cancer. These compounds are equally mitogenic in renal tissues but only E2 is metabolized to a reactive 2 OH intermediate. Thus, at least in some tissues the mitogenic and carcinogenic properties of estrogens can be separated [35].

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In rats, hormonal stimulation of the mammary gland occurs for the 6 months following onset of estrus (days 35 to 42). Administration by gavage of rats during this period with the chemical carcinogen 7,12_dimethylbenz- (a)anthracene (DMBA) induces intraductal proliferation in terminal end buds which can develop into intraductal carcinoma. If these terminal end buds are al- lowed to undergo differentiation into alveolar buds, they are no longer susceptible to DMBA-induced carcinogenesis. The rapid growth and differentiation of terminal end buds that occurs during pregnancy is associated with an inhibition of susceptibility to the carcinogenic effects of DMBA.

Chemically induced mammary tumors in rodents have proved very useful for investigating the critical role of activation of the c-H-ras oncogene. Up to 70% of chemically induced rodent mammary tumors express an activated ras oncogene [36,37]. Placing the activated rus oncogene under the control of a mammary lactation- specific whey acidic protein promoter, results in trans- genie animals developing mammary tumors after pregnancy [38]. Similar studies have also implicated the c- myc oncogene in mammary tumorigenesis [39]. There is some evidence indicating that expression of c-myc in human breast cancer cells is E2-regulated [40]. Whilst expression of the c-myc and activated ras oncogenes appear to be important in rodent mammary tumorigenesis, their precise roles in the human disease remain to be clarified [4143].

The ability of both insulin and EGF to increase expression of the c-&B2 protein in breast cancer cells [44], implicate this oncogene in the mediation of some hormone/growth factor effects on tumor initiation and progression [44]. Paradoxically, E2 inhibits c-e&B2 expression in MCF-7 cells [45]. Expression of c-e&B2 may be more closely related to alterations in the differentiation state rather than mitogenesis per se. Overexpression of this oncogene provides important prognostic information, but this may be limited to rather small groups of patients [46-48]. However, the precise function of c- erbB2 oncogene and its role in mammary tumorigenesis requires further study.

It is clear that estrogens can act both directly and indirectly on normal mammary tissue to induce events that are closely associated with a carcinogenic potential. The ability of E2 to stimulate the proliferation of some ER-positive breast tumor cells both in vivo [49] and in vitro [50] clearly implicates estrogens as regulators of tumor growth, subsequent to carcinogenesis. Thus, it is possible that estrogens function as carcinogens, tumor promoters and as regulators of tumor growth.

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III. Steroid hormones and growth factors in breast cancer

III-A. Steroid hormone receptors

The presence of specific, high affinity, low capacity

binding sites appears to be obligatory for cells to respond to hormonal stimulation. The presence of ER in normal mature human breast tissues has been reported [51-531. The stromal cells are clearly ER negative, whereas some of the epithelial cells are ER positive [51,52]. The ER-positive epithelial cells occur as single cells and are more prevalent in the lobules than the in- terlobular ducts [52]. It has been estimated that only 6-7s of the epithelial cells in normal breast are ER positive [52,53] and the levels of ER expression may alter with the phases of the menstrual cycle [51]. Up to 29% of normal breast epithelial cells may be PGR positive [53]. The myoepithelial cells appear to be exclusively ER and PGR negative [53].

The majority of human neoplasms are believed to originate from the clonoal expansion of single cells, the subsequent heterogeneity resulting from the emerging dominance of phenotypically diverse clonal populations which arise from within the original population [54,55]. The generation of these phenotypically different populations may reflect either the inherent genetic instability of the tumor cells and/or epigenetic phenomena operating in conjunction with the selective pressures provided by the host response to the tumors [56]. The presence of ER in breast tumors may indicate that these tumors arise from within the ER-positive epithelial cell populations of normal breast tissue [57]. Approximately two-thirds of all breast tumor express detectable levels of ER protein [58]. The majority of ER-positive tumors (> 90%) are associated with adjacent non-neoplastic tissue which is also ER positive [59]. Further indirect evidence that E2 responsivity is closely associated with early events in carcinogenesis is provided by the observations that breast cancer occurs almost exclusively after puberty [60], young age at first pregnancy produces a protective effect [61] and the absence of ovarian function reduces breast cancer risk to a level equivalent to that reported in men [60]. Factors which can increase exposure to estrogens such as obesity [ 181, early age at commencement of menstruation, and late age at onset of menopause [61,62], have been implicated in an increased risk of developing breast cancer. It is difficult to reconcile these observations with the concept of a majority of breast tumors arising from ER-negative tissues. It is unlikely that these hormonal effects are directly mediated through the normal stromal populations, since these cells appear to be exclusively ER negative in mature human breast

tissue. [52]. The incidence of ER-positive breast tumors increased 131% over the period from 19741985 [l].

By the time a tumor mass is detected there is already marked heterogeneity of hormone receptor expression [63]. The ER-negative populations may arise as a result of dedifferentiation of the ER-positive populations. This hypothesis gains some support from the observation that ER-negative breast tumor cells are frequently less well differentiated than their ER-positive counterparts [64]. Poorly differentiated tumors (histological grade III) are more likely to be ER negative when compared with well differentiated tumors (grade I and grade II) [65-67]. However, we cannot exclude the possibility that ER expression is occasionally acquired during neoplastic transformation [68] or progression. Some predomin- ately ER-negative tumors may arise from within the normal ER-negative subpopulations [69]. The incidence of ER-negative breast tumors increased 22-27% over the period from 19741985 [l].

The point at which cells lose both their dependence upon E2 and their ability to express ER and acquire resistance to endocrine manipulation are critical stages in the progression of the disease. Endocrine therapy would tend to further select for these cells and may represent one mechanism by which endocrine resistance develops in patients.

The serum estrogen levels in nude mice are equivalent to that observed in postmenopausal women [70,71]. Uti- lizing an in vivo selection in nude mice, we have recently isolated cells which express a hormone-independent phenotype from cell populations initially dependent upon E2 for growth in ovariectomized nude mice [49,72]. Whilst not requiring estrogen supplementation for growth, these cells retain ER expression [49] and the ability to respond to both E2 and antiestrogens [13]. Thus, the growth pattern of these tumors appears to be analogous to that of ER-positive tumors in postmenopausal women. Analyses of the phenotypes of these hormone-independent but responsive sublines strongly suggests that loss of ER-expression tends to occur later rather than earlier in the progression of the disease [57]. A similar observation has been reported for the loss of hormone dependence by androgen dependent mouse mammary cell lines [73].

III-B. Autocrine growth stimulation

There are a number of restriction sites within the cell cycle which influence the proliferation of non-transformed fibroblastic cells. Platelet derived growth factor (PDGF) and basic fibroblast growth factor are required for early Gi transit and epidermal growth factor (EGF) is required for mid Gi transit. Transition through the re-

mainder of G, and subsequent commitment to DNA synthesis requires insulin-like growth factor I (IGF-I) [74,75]. Whilst there is no clear evidence to support analogous restriction sites in the cell cycle of epithelial cells, insulin and EGF/transforming growth factor- alpha (TGF-a) are additive in stimulating the proliferation of MCF-7 cells [76,77], COMMA-D mouse mammary epithelial cells [78], and both normal rat and human mammary epithelial cells [79,80].

De Larco and Todaro [81] initially suggested that some tumor cells may produce the factors they require for continued proliferation. These factors could subsequently function in an autostimulatory or ‘autocrine’ manner. Thus, cells would secrete ligands which then bind to their receptors on the surface of the same cell from which they were secreted. Internal autocrine stimulation may also result from ligand-receptor interactions which occur intracellularly, perhaps at the endo- plasmic reticulum-Golgi complexes or within secretory vesicles [82]. We have previously adapted the autocrine hypothesis to explain the control of growth in hormone- responsive breast cancer cells. Thus, we have predicted that EZdependent breast cancer cells might be expected to secrete increased levels of mitogenic growth factors, and lower levels of inhibitory growth factors, in response to E2 [83]. Antiestrogens might increase the production of inhibitory factors, whilst decreasing the production of mitogens. Hormone-independent cells might constitutively express high levels of mitogenic ligands in the absence of hormone and would not respond to hormonal stimulation.

III-C. Steroid regulation of growth factor andgrowth

factor receptor expression

There is considerable circumstantial evidence in support of an adaptation of the autocrine hypothesis to hormone-responsive tumors. EGF and it’s naturally oc- curring homologue TGF-a have both been implicated in the control of normal breast development and in main- taining the malignant phenotype. Both ligands bind to the EGF receptor protein (EGF-R). TGF-a secretion is E2 regulated in most hormone-dependent human breast cancer cell lines [84]. EGF-stimulated cell proliferation in MCF-7 cells growing in the absence of estrogen is inhibited by antiestrogens [12] and EGF/TGF-cr can partially reverse the growth inhibitory effects of antiestrogens [85]. These observations suggest that TGF-a may be involved in mediating the ability of E2 to induce a mitogenic response in these cells. In contrast, TGF-a is constitutively expressed in many hormone-independent cells [84,86.87]. Infusion of EGF can increase the ability of EZdependent MCF-7 human breast cancer cells to

form small transient tumors independent of E2 in ovariectomized nude mice [88].

We have attempted to determine the relevance of these observations by transfecting hormone-dependent MCF-7 cells with a plasmid expression vector directing the constitutive expression of TGF-c(. Transfectants can secrete sufficient TGF-cr to down regulate EGF-R but retain wild-type responses to E2 as determined by induction of PGR and mitogenesis in vitro. When inoculated into ovariectomized athymic nude mice, TGF-a transfected cells fail to form tumors without E2 supplementation [89]. Thus, TGF-a expression alone is not sufficient to fully mediate the effects of E2 in MCF-7 cells growing either in vitro or in vivo.

In addition to the ligand, the receptor for EGF/TGF- a is also hormone regulated. Both E2 and progestins increase EGF-R expression in hormone-responsive tissues [9&92]. Hormone-independent breast cancer cell lines express high levels of EGF-R relative to hormone-dependent cells [93,94]. Primary breast tumors which have either low ER content or have lost the ability to express ER frequently possess high levels of EGF-R [80,95-971. Since E2 increases the levels of both secreted ligand and receptor, perhaps elevated numbers of EGF cell surfaces receptors and increased ligand production are required for mediation of the full effects of E2 through the TGF- CI autocrine loop. NIH 3T3 cells expressing low levels of EGF-R are not transformed when transfected with the TGF-a cDNA but exhibit the fully transformed phenotype when co-transfected with both EGF-R and ligand [98]. Co-transfection of breast cancer cells with both EGF-R and TGF-a cDNAS are currently in progress

[991. IGF-I is a 70 amino acid polypeptide and IGF-II a

67 amino acid polypeptide, both proteins sharing structural and functional homologies with insulin. The IGFs and their receptors have been implicated in the growth of a number of human tumors. Overexpression of the type I IGF receptor can induce neoplastic transformation in NIH 3T3 cells [loo]. These receptors are widely expressed in benign breast disease [IO11 and in human breast cancer cells [84,102]. There is a direct correlation between expression of ER, PGR and the type I IGF receptor [80,103]. More recently, Bonneterre et al. [ 1041 have observed that expression of the type I IGF receptor indicates a good prognosis. The growth of hormone-independent MDA-MB-231 human breast cancer cells both in vivo and in vitro is at least partly inhibited by an antibody which blocks ligand binding to the IGF-I receptor [105,106]. This antibody also inhibits the growth of a number of human breast cancer cell lines in vitro [106]. In contrast, the growth of hormone-depend-

b

ent MCF-7 cells is inhibited in vitro but not in vivo [105,107].

Elevated levels of either IGF-I m-RNA or IGF-I-like proteins have been reported in liposarcoma [108], medullary thyroid [ 1091, leiomyoma and leiomyosarcoma cells [l lo]. Loss of a requirement for an exogenous source of IGF-I has been implicated in the development of human thyroid tumors [ 1111. IGF-I increases the rate of proliferation of some breast cancer cells [92,112,113] and can induce the transient formation of hormone-independent MCF-7 tumors in ovariectomized athymic nude mice [88]. Some breast cancer cell lines produce an EZregulated IGF-like material [77], but this is not authentic IGF-I since RNase protection analyses fail to detect IGF-I mRNA in these cells [ 1141.

Authentic IGF-II mRNA or protein has been observed in breast cancer [86], rhabdomyocarcoma [ 1151, colon carcinoma [108], adrenal pheochromocytoma [ 1161, medullary thyroid [ 1091, leiomyoma and leiomyosarcoma cells [l lo] and in Wilm’s tumors [ 1171. IGF-II mRNA is expressed by T47D cells and both IGF-I and IGF-II stimulate the growth of MCF-7 and T47D breast cancer cells in vitro [118]. The activation of the inhibitory growth factor TGF-P requires binding to the type II IGF receptor [119]. Thus, IGF-II could be indirectly mitogenic by down regulating its own receptor and thereby reducing the levels of active TGF-8. The proportion of human breast cancer cell lines and breast tumor cells that express authentic IGF-I or IGF-II mRNA appears to be low [114,120]. In contrast, significant IGF-I and IGF-II mRNA expression is observed in the stromal components of a number of breast tumors, implying a potential paracrine role for IGFs [ 1141.

The function of the IGFs in breast cancer is complicated by the presence of IGF binding proteins. These proteins have a high affinity for both IGF-I and IGF-II and generally inhibit IGF function. Breast cancer cell lines secrete significant levels of these IGF binding proteins [121-1231, and their addition to cell culture media has been clearly demonstrated to inhibit the mitogenic effects of IGFs in human breast cancer cells [124]. IGF binding proteins can also inhibit the growth of some colon cancer cells [125]. There appears to be a significant difference in the pattern of binding protein secretion in ER-positive compared with ER-negative tumors [ 1261. Breast cancer cell lines appear to secrete at least two forms of binding proteins, species with approximate molecular weights of 49 000, 43 000, 34 000, 29 000 and 24 000 having been reported [84]. Whilst most investigators describe the inhibitory effects of these proteins, one binding protein (26000 molecular weight) has been described as increasing the mitogenic effects of IGF-I in human fibroblastic cells [127]. Whether similar effects

can occur in human breast cancer cells remains to be established. Since breast cancer cells secrete multiple IGF binding proteins [126] and addition of these can inhibit mitogenesis [124], it seems likely that the cumulative effect of IGF binding protein secretion is to partly antago- nize the mitogenic effects of IGFs in breast cancer cell growth. Thus, the precise role of IGFs in directly contri- buting to control of human breast cancer cell growth by an autocrine stimulation remains unclear and requires further study.

Mouse mammary epithelial cells maintained contin- ually in vitro respond mitogenically to basic fibroblast growth factor (bFGF) which can be substituted for both EGF and IGF-I [82]. bFGF is mitogenic in the S 115 androgen-dependent mouse mammary carcinoma cell line [ 1281. Exogenous bFGF increases the expression of two E2-regulated mRNAs, pS2 and cathepsin D [129]. There is co-amplification of coding sequences for both the int- 2 and another FGF-related oncogene hst in approx. 17% of human breast cancers. However, overexpression of the respective mRNA species is only apparent for the hst

oncogene [ 1301. Many of the FGFs are not secreted but may be retained within the cell and released on cell lysis. Incorporation of bFGFs into the extracellular matrix can occur and may be subsequently released by tumor- derived heparan sulfate degrading enzymes [ 13 11. Some human breast cancer cell lines secrete other basic fibroblast growth factor-like activities [I 321. The frequency of distribution of FGFs and their receptors in human breast tumors, and their relevance to the control of breast cancer cell growth, remain to be elucidated.

Transforming growth factor /3 (TGF-p) is structurally unrelated to TGF-a. In contrast to TGF-cr, TGF-j? inhibits epithelial cell growth but stimulates the proliferation of fibroblasts [ 1331. Proliferation of normal human mammary epithelial cells in vitro is inhibited by TGF-j? [ 1341, however, expression of the milk fat globule antigen is stimulated [135]. TGF-j? is secreted by a number of breast cancer cell lines [136], and it can inhibit the proliferation of these cells growing in vitro [137]. This secretion is reduced by E2 but increased by antiestrogens, implicating an increased production of activated

TGF-P in the mechanism of action of antiestrogens [ 1361. However, increased secretion of TGF-j3 alone is not responsible for the action of antiestrogens, since some ER-positive cells respond to antiestrogens but not to exogenous TGF-P [138]. We have recently reviewed elsewhere the role of growth factors in mechanism of action of antiestrogens and the development of resistance [ 1391. The inhibitory potential of TGF-/3 is complicated by the observation that tumor cells frequently secrete TGF-8 in an inactive form. However, this does not appear to be the case for the TGF-P secreted by MCF-7

cells in response to antiestrogens [136]. Conversion of inactive TGF-/3 to its active form requires binding to the type II IGF receptor [ 191. The ability of TGF-P to inhibit some steroid biosynthetic processes [ 1401 may contribute indirectly to the inhibition of E2-dependent breast cancer growth. These data implicate an additional level of control of steroid-induced proliferation which results from altered secretion of potential growth inhibitory factors.

Other hormones can exert an inhibitory effect on breast tumor cells. Somatostatin and some of its analogues inhibit the growth of mammary tumors in vivo [ 141,142] and breast cancer cells growing in vitro [ 143,144]. These analogs also reduce plasma concentrations of both IGF-I [145,146] and EGF [147]. Thus, inhibition of tumor growth may be the result of a combination of indirect endocrine and direct antitumor effects. LH-RH analogues directly inhibit MCF-7 cells in vitro [102]. Some LHRH analogues inhibit the EZ-induced stimulation of proliferation in CG-5 cells, a sub- line of the MCF-7 human breast cancer cell line [148]. However, it is more likely that the major mechanism of action of this class of compound in vivo occurs through an ability to inhibit gonadotrophin release and the consequent reduction in secreted gonadal steroids [ 1491.

Breast cancer cells express a variety of receptors to other growth factors and hormones. For example, functional receptors for glucagon have been reported on a number of breast cancer cell lines [ 1501. The function of glucagon receptors in breast cancer is not known, but they may be involved in modulating insulin responsivity. Some human breast carcinomas express gonadotropin-releasing hormone receptors [ 1511, vasopressin and oxytocin receptors [152]. T47D cells express receptors for vasoactive intestinal polypeptide [153]. An understanding of the relevance of these observations clearly requires further study.

III-D. Further comments on tumor growth factor interactions in breast cancer

The evidence in support of the autocrine/paracine hy- potheses remains largely circumstantial. We and other investigators have attempted to obtain additional supporting data by transfecting hormonally regulated genes of interest into hormone-dependent cells. This approach can provide useful information on what overexpression of a particular growth factor or receptor can or cannot do through an autocrine stimulation in experimental systems. However, the results of transfection experiments can be misleading, and conclusive proof that the phenotypic changes induced by genetically introduced cDNAs are biologically relevant, and that they occur

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during the normal progression of the disease in patients, is more difficult to obtain. For example, transfection of EGF and EGF-R [98] or the type I IGF receptor [IOO] can induce neoplastic transformation in some cell types. However, many breast tissues also express high levels of these proteins [80,154,155] but show no evidence of a malignant phenotype. Expression of these autocrine loops may be essential for normal epithelial cell function [I 54,155]. It is possible that some of the phenotypic changes that occur during carcinogenesis or malignant progression can be obtained by the altered expression of a number of apparently unrelated genes. In vitro studies indicate that cells may become hormone independent by expressing mutated ER [ 156,157], by acquiring the constitutive expression of previously hormone-regulated genes [57], or by overexpressing oncogenes [158]. The relevance of these observations to the clinical disease has yet to be clearly demonstrated.

Evidence that anti-receptor or anti-ligand antibodies can inhibit tumor growth in nude mice suggests potentially important therapeutic approaches [ 105,106], but interpretation of the data within the context of autocrine/paracrine mechanisms is problematic due to possible endocrine effects. Nevertheless, there is an ever increasing amount of indirect evidence which strongly points towards a critical role for autocrine/paracrine growth factor stimulation in the control of breast cancer cell growth and progression. These observations also indicate the possibility that therapeutic interventions that interfere with these interactions may provide new antitumor therapies.

IV. Paracrine-stromal growth factor interactions

There are essentially two different stromal responses to neoplasia. The most widely studied host response is the infiltration of lymphoreticular cells into solid tumors, perhaps reflecting its prognostic significance and its potential to indicate the degree of host immune competence. For example, the length of survival of breast cancer patients with metastatic disease correlates with pretreatment levels of immunocompetence as determined by parameters of cell-mediated immunity (CMI) [159]. The second important stromal response to neoplasia results in the presence of cells of mesenchymal origin in tumors. The majority of infiltrating breast carcinomas are characterized by a marked desmoplastic response [160]. The cells which comprise this response have been extensively studied and their structural characteristics widely reported. Resting and active fibroblastic cells are occasionally observed in addition to myofibroblasts [161]. The interactions among the different stromal populations and the adjacent tumor cells,

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and their ability to influence the progression of the disease, are poorly understood. In the following sections we will discuss these potential interactions and the role of hormones in mediating or influencing these events.

IV-A. Stromalpopulations of lymphoreticular origin in breast neoplasia

There is considerable evidence implicating CM1 in the control of breast cancer growth and metastasis. The degree of CMI, as determined by the skin window proce- dure, indicates a strong correlation between increased metastatic potential and reduced CM1 [162]. The presence of primary tumors over 5 cm and an increase in the number of axillary lymph node metastases are associated with reduced CM1 [163]. Patients with stage IV breast cancer exhibit a significant increase in the number of monocytes and their mononuclear cells exhibit a reduced response to phytohemagglutinin [164]. There is a significant reduction in natural killer cell (NK) activity in stage III and stage IV breast cancer patients [165- 1671. Patients with lower lymphocyte and/or eosinophil counts have a higher risk of recurrence than patients with normal counts [ 1681.

NK cells make up approx. 1-2.5s of peripheral lymphocytes and have been widely demonstrated to possess both antitumor [ 1691 and anti-metastatic activities [ 169- 1721. The poor metastatic potential of most human xenografts growing in nude mice has been partly attributed to the elevated NK activity observed in these rodents [ 169,17 l-l 731. However, some tumors appear to be in- herently resistant to NK cell-mediated cytolysis [I 74 1761, perhaps reflecting an ability to suppress NK activity [177]. Low NK activity is associated with familial breast cancer [ 1781.

Macrophages are widely observed to infiltrate solid tumors [165,179,180] and can kill tumor cells by both phagocytic and non-phagocytic processes [180]. Non- phagocytic cytolysis may include the release of lysoso- ma1 enzymes by exocytosis. Macrophages may recog- nize some tumors on the basis of their abnormal growth [181] or by surface modifications [180] and can produce both antigen-specific and non-specific cytolysis. The tumoricidal properties of macrophages are acquired following activation by contact with either the target cell and/or secreted products or by soluble lymphokines, e.g., interferon-y [174]. The biology of macrophage-induced cytotoxicity is independent of the sensitivity of the target cells to lymphocyte or NK mediated cytolysis [175]. Tumor cells do not appear to acquire resistance to the cytotoxic effects of macrophages, in marked contrast to their ability to develop resistance to NK-mediated cytolysis [174176]. The limiting factor in macro-

phage control of neoplasia appears to be effecter/tumor cell ratio [ 1741. However, the sera from some cancer patients possesses macrophage inhibitory activity [ 1821 and macrophage infiltration can be associated with tumor progression rather than inhibition, implying that some macrophages secrete factors mitogenic for tumor cells [183].

The stroma of axillary lymph nodes containing metastases possess a higher degree of lymphoreticular infiltration than the areas of neoplasia. The predominant tumor infiltrating lymphoreticular cells are monocytes/ macrophages (Mono l+). Lower numbers of T- lymphocytes (Leu- 1 + ) and CD4+ (Leu-3a +) and CD8 + (Leu-2a + ) lymphocytes are observed [ 1841. NK cell activity is generally low or absent [ 184,185] but lym- phokine activated killer cell (LAK) activity has been demonstrated [ 1851.

Markers of the degree of malignant progression such as ER and PGR expression [ 186,187] also correlate with apparent perturbations in immunosurveillance. There is an inverse correlation between the degree of inflamma- tion and both ER and PGR levels [188]. There are significantly fewer T cells and the subgroup of Leu-3+ helper-inducer cells in ER-positive tumors [ 1651. Higher NK activity is observed in ER-negative tumors [189,190]. These observations strongly suggest that the process of malignant progression in breast cancer is accompanied, not only by increased metastatic potential and loss of both hormone dependence and ER expression, but also by an altered responsivity to effecters of CMI. For example, the increased lymphoreticular infiltration observed in ER-negative tumors may indicate that these tumors are either more resistant to CM1 and/ or secrete high levels of chemoattractants for CM1 effec- tor cells. Fulton et al. [191] have suggested that interactions between macrophages and tumor cells may be responsible for progression to a metastatic phenotype in some mouse mammary tumors.

A potential immuno-modulatory role for endocrine therapies in breast cancer has been recently indicated. Screpanti et al. [ 1921 reported that E2 significantly stimulates NK activity athymic nude mice over the first 30 days, with detectable suppression of NK activity being observed after at least 4 weeks. A 4-6-week delay in E2- suppression of NK activity is consistently observed [ 192-1951. The E2 dependence of some human breast cancer cells growing in nude mice could reflect an E2- induced inhibition of NK activity rather than true E2- dependence. However, NK suppression occurs predominantly in the presence of pharmacological but not physiological concentrations of estrogens [193,195]. If NK activity is primarily responsible for modulating human breast tumor growth in nude mice, the growth curves of

9

the appropriate human breast cancer cells would be expected to be biphasic but this is clearly not the case [71,196,197]. More importantly, MCF-7 cells have been shown to fully retain E2 dependency for growth in mice which are deficient in NK cells [198]. The MCF-7ADR cell line is hormone independent and unresponsive and fails to express ER. These cells do not respond to E2 supplementation when grown in athymic nude mice [199]. Screpanti et al. have recently furthered their studies on E2 and NK activity and now report that E2 increases the sensitivity of MCF-7 cells to the cytotoxic effects of NK cells [200]. Thus, any effects of EZreduced NK activity in vivo would be either partly or fully com- pensated by an increased sensitivity in the MCF-7 cells to remaining NK actitivy.

Tamoxifen increases NK activity in nude mice and this could contribute to its antitumor effects [201,202]. However, the significance of this observation is unclear, since responses to tamoxifen are almost exclusively observed in ER-positive tumors. Athymic mice possess normal levels of macrophage activity [203] and other natural cytotoxic cells have been implicated in tumor re- jection by nude mice [204,205]. A number of immunoglobulin subtypes, e.g., IgM have been detected in sera from nude mice [206]. Pharmacological concentrations of E2 produce an increase in IgM secretion [207]. Al- though of limited value as single agents, we and others have clearly demonstrated the ability of interferons to potentiate the effects of antiestrogens in ER-positive human breast cancer cells growing in vitro [209-2121.

IV-B. Stromal populations of mesenchymal origin in

normal breast

The composition of the mesenchymal tissues is critical for normal human breast development. Transplantation of mammary epithelial cells to the stromal cells of other tissues fails to support normal mammary development [213]. The appearance and function of the stromal tissues present in the normal breast can vary significantly depending upon their location. The stroma of the nipple/areola area is composed of dense collagen with substantial numbers of smooth muscle cells [214]. A layer of subcutaneous adipose tissue lies between the skin and the two morphologically distinct stromal compartments which surround the functional tissues of the mature human breast.

The stroma which is in closest association with the secretory epithelium surrounds the terminal duct lobular units. This intralobular stroma is loose, cellular, and highly vascular, containing fine collagen fibers and reti- culin [2 14,2 151. The mucoid nature of this stromal tissue is most apparent on alcian blue staining [214]. The in-

tralobular stromal cell populations may be closely associated with the functional capabilities of the adjacent epithelial structures, since epithelial-mesenchymal interactions are required for many biological functions in rodent mammary tissue [2 16-2 181. The extralobular stroma is a considerably more dense fibrous tissue and less cellular than the intralobular stroma. It is thought that the main function of the extralobular stroma is structural [2 151.

The cell populations present in these morphologically distinct stromal structures have not been fully identified. Unfortunately, the potentially vague term fibroblast has been widely used to describe the stromal cell populations present in normal breast tissue. It is now clear that there is considerable phenotypic heterogeneity within fibroblastic populations, even within the same tissues [219,220]. This heterogeneity may reflect distinct differentiation pathways from a hypothetical pluripotent fibroblastic cell. Schor and Schor [220] have suggested a ‘clonal modulation model of connective tissue function’ to explain this phenotypic heterogeneity. Thus, the functional activity of fibroblastic subpopulations may reflect the expansion or inhibition of preexisting subpopulations and/or the migration/accumulation of specific fibroblastic subpopulations in response to chemotactic factors [220]. The high number of fibrocytic nuclei present in the mature normal intralobular stroma [214] suggests that the predominant cell type may be the fibrocyte (mature fibroblast). These cells appear structurally similar to the fibroblastic cells present in extralobular stroma [221], but may be functionally distinct as a result of their close association with the ductal epithelial and myoepithelial cells. The development of myofibroblas- tomas, a rare benign breast tumor [222], and extensive reports of the presence of myofibroblasts in infiltrating breast carcinomas [ 160,161,223-2271, suggest the presence of a small or perhaps transient population of myofibroblasts in normal breast. There are also occasional smooth muscle cells and other cell types associated with the vascular and lymphatic structures of the normal breast. Occasional lymphocytes, histiocytes, plasma cells and mast cells may be observed in normal intralobular stroma [215].

IV-C. The role of stromal cells of mesenchymal origin in mediating the estrogenic responsivit)r qf normal breast

tissue

There is a considerable amount of evidence implicating the stromal tissues of mesenchymal origin in mediating the estrogenic signals in rodent mammary gland development. In the rodent, the stromal cells of the developing breast express ER, whereas the epithelial

10

and fat pad mesenchymal cells are apparently ER negative [64]. E2 may influence the growth and differentiation of normal rodent breast tissue by an indirect interaction mediated through stromal tissues. E2 only induces epithelial proliferation in vitro in the presence of mammary stromal cells [216,217]. Direct contact between the stromal and epithelial cells is required for induction of the full mitogenic responses in the epithelial component [216]. Similarly, an E2 induction of PGR synthesis in isolated mouse mammary epithelial cells occurs only in the presence of fibroblastic cells [217]. The effects on PGR appear to be more closely related to stromal matrix production, since coating dishes with type I collagen is also effective [228]. Mouse mammary epithelial cells growing on an artificial reconstituted basement membrane in vitro can express some differentiated functions [229]. In contrast, the mitogenic effects of E2 on isolated mouse mammary epithelial cells require metabolically active fibroblasts [228]. The role of stromal tissue in mediating estrogenic signals in the developing normal human breast is poorly understood.

IV-D. The role of stromalpopulations of mesenchymal origin in the control of breast tumor growth

The major mesenchymal component of malignant breast tissue generally results from the proliferation or migration of normal myofibroblasts, perhaps reflecting the reactive nature of the desmoplastic response [160]. Myofibroblasts are the major cell type thought to be responsible for tissue remodelling in the process of wound repair and are found in high numbers in granulation wound tissues [224]. The contractile nature of these cells has led to the suggestion that they are responsible for the tissue retraction often characteristic of primary breast tumors [224]. This retraction may be the result of TGF-/3 secretion by the tumor cells, since TGF-/I has been implicated in the contraction of the desmoplastic stroma of gastric carcinoma [230]. The frequency with which myofibroblasts are observed in breast tumors may indicate that the desmoplastic response is primarily a wound healing response, perhaps reflecting the at- tempts of normal stromal cells to reestablish the critical mesenchymal/parenchymal organization disrupted during the processes of neoplasia.

A paracrine interaction between tumor and stromal cells could result from the ability of tumors to stimulate proliferation of stromal fibroblastic cells. PDGF is a potent mitogen for fibroblastic cells [23 1] but not for breast tumor cells, although, it is secreted by these tumor cells [232]. The mitogenic effects of tumor-derived PDGF may be mediated through stimulation of IGF production, since PDGF stimulates IGF-I production in

human fibroblastic cells [231]. IGF-I is a potent mitogen for fibroblastic cells [23 l] and its effects appear to be mediated specifically through the type I IGF receptor in myoblasts [223] and breast cancer cells [113]. PDGF-induced mitogenesis may also function synergistically with induced IGF secretion to stimulated stromal fibroblastic proliferation. The secretion of tumor-derived PDGF and the induction of stromal IGF may be partly responsible for the marked expansion of myofibroblast populations characteristic of the desmoplastic response to breast tumors. PDGF may also contribute to the process of metastasis. Following stimulation with PDGF, some fibroblastic cells in vitro secrete elevated levels of collagenolytic activity which could enhance the ability of tumor cells to invade through the desmoplastic matrix [234].

In MCF-7 cells, E2 induces a small increase in platelet derived growth factor (PDGF) mRNA production and a larger increase in secreted PDGF biological activity [232]. Whilst these tumor cells do not express PDGF receptors [232], PDGF can stimulate IGF-I production in human fibroblastic cells [231]. Thus, an additional paracrine stimulation of breast cancer cells could occur through the effects of tumor-derived E2-stimulated growth factors inducing stromal cells to secrete IGFs. Stromal-derived IGFs could then stimulate the adjacent tumor cells. Recently, it has been reported that it is the stromal component in breast tumors which is the major source of authentic IGF-I and IGF-II mRNA synthesis [114].

There are a number of potential caveats which could limit the extent of stromal-derived paracrine IGF-stimulation of adjacent tumor cells. Sufficient IGF activity must be produced to saturate both the IGF receptors on the stromal cells themselves (autocrine stimulation) and to evade inactivation by either inhibitory tumor-derived IGF-binding proteins [ 121-1231 or IGF degrading enzymes [235]. IGF-II is a potent inhibitor of aromatase activity [236], therefore, any mitogenic effects of stromal-derived IGF-II could be offset by a reduction in local E2 production by breast tumor cells and adjacent breast adipose tissue. E2 down regulates the type II IGF receptor [237] and could compromise functions mediated through IGF-II interactions with this receptor. Whilst some MCF-7 cells respond mitogenically to IGFs [238,239] others have found these growth factors to produce only small effects on MCF-7 cell proliferation [240]. Some tumors lose their responsivity to IGFs [241,242], while others appear to be inhibited by excess IGF-II [243]. Significantly, there is considerable varia- bility in the ability of some tumor cells to synthesize functional IGF protein from IGF mRNAs [244]. Whether these IGF mRNAs are actively involved in

II

protein synthesis remains to be demonstrated. Thus, the precise role of authentic IGFs in the paracrine stimulation of breast cancer growth remains unclear.

Whilst various complex paracrine-paracrine tumor- stroma interactions may be appealing, the more simple autocrine explanation may suffice. Many breast tumor cells respond mitogenically to stimulation with IGFs. Thus, tumor-derived IGFs may be autocrine mitogens for these cells. For the stroma, the IGFs are essential for regulating the exit of fibroblastic cells from the Gi phase of the cell cycle [245] and may be critical for the mainte- nance of the myofibroblast morphology. For example, both IGF-I and IGF-II stimulate the differentiation of myoblasts by inducing expression of the myogenin gene [246]. This mechanism could also contribute to the differentiation of myofibroblasts. IGFs also stimulate fibroblastic cells to produce large amounts of collagen [247] and collagen deposition is characteristic of the desmoplastic response [160]. The IGFs (and myofibroblasts) are also strongly implicated in tissue repair and remodelling [248,249]. Thus, the production of IGFs by the stromal tissues in infiltrating breast carcinomas may be providing essential autocrine factors for the mainte- nance of the fibroblastic cells responsible for desmoplasia.

Both IGF-I and IGF-II appear to function primarily through the type I IGF-receptor [233,250] and the ultimate source (tumor or stromal) or nature of the IGF (IGF-I, IGF-II or IGF-like) may be irrelevant to both the malignant epithelial and associated mesenchymal cells. The secretion of large amounts of potentially inhibitory IGF-binding proteins [126] may be an attempt by the tumor cells to inhibit the autocrine function of IGFs in the stromal cells and thereby reduce the desmoplastic response. Of potential relevance is the observation that FGFs inhibit myoblast differentiation by inhibiting IGF-II expression [230,25 11. An analogous interaction could result from the secretion of FGF-like activities by breast tumor cells. Thus, FGFs may reduce the degree of IGF-stimulated collagen deposition by altering fibroblastic differentiation and thereby increasing the accessibility of tumor cells to FGF-induced neovascularization.

A recent report by Colletta et al. [252] indicates that antiestrogens may induce TGF-8 secretion in ER-negative human fibroblastic cells. The ability of stromal cells to secrete inhibitory growth factors in response to the appropriate stimuli indicates a potentially important paracrine inhibition effect. It may be possible in the future to design other agents which inhibit tumor cells by caus- ing stromal cells to secrete potent inhibitors of epithelial cell growth.

The effect on tumor growth and metastasis of the

marked mesenchymal desmoplasia associated with infiltrating breast cancers has been investigated. The majority of studies tend to indicate that direct contact between tumor and stromal cells results in inhibition of either tumor growth or metastasis. For example, co-culture experiments indicate that normal mammary cells (mixture of epithelial and fibroblastic cells) can inhibit the proliferation of mouse mammary tumor ceils in vitro 12531. Normal breast fibroblastic cells alone inhibit MCF-7 cell proliferation [254]. Weakly metastatic cells in rat mammary carcinomas form significant intercellular communications with fibroblastic cells [255]. Junctional complexes, desmosomes and gap junctions, are not observed between highly metastatic cells and stromal cells [256]. The extent of matrix deposition by stromal cells has a significant effect upon the metastatic potential of mammary tumors. The degree of desmoplasia as indicated by fibronectin associated with desmoplasia correlates strongly with prognosis. Thus, tumors which exhibit significant levels of fibronectin immunoreactivity (generally indicating a strong desmoplastic response) are poorly metastatic. Highly metastatic breast tumors appear devoid of fibronectin immunoreactivity [257].

Myofibroblasts comprise a significant proportion of the stromal response to mammary and other tumors, In- hibiton of matrix deposition by stromal myofibroblasts. without inhibition of proliferative responses, can significantly increase the ability of tumors to invade locally and metastasize to distant sites [258]. Furthermore, myofibroblasts can secrete significant metalloproteinase inhibitory activity which is associated with organ-specific resistance to metastasis [259]. The ability of stromal cells to inhibit the proteolytic activity secreted by tumor cells may prevent their ability to invade local tissues. Some secreted enzymes are mitogenic for breast cancer cells [260], implying a possible inhibitory role for stroma-derived enzyme inhibitors, The desmoplastic collagen matrix may be important for elements of the lymphoreticular response to breast tumors. The collagen components of extracellular matrix are essential for lymphocyte migration, recognition/activation and differentiation [261]. Thus, inhibition of collagen deposition may also reduce the immune response to neoplasia.

Not all tumor-mesenchymal interactions are potentially inhibitory (for a recent review see Ref. 262). Transformed fibroblasts [263] and glutaraldehyde-killed fibroblasts [264] both increase tumor-take when co-inoculated with tumor cells into nude mice. The latter observation clearly implicates the cell matrix, rather than secreted growth factors, as mediating the increased tumorigenicity. Horgan et al. [264] have observed that live breast fibroblasts increase the growth rate of MCF-7 tumors. This may reflect the ability of these cells to in-

I2

crease 17/Sestradiol dehydrogenase activity in the tumor cells, thereby increasing local E2 production from es- trone [254]. Perturbations in immunogenicity, perhaps as a result of altered sensitivity to CMI, has also been implicated in the increased tumorigenicity of heterogenous tumors [265]. The increased tumor growth rate may also reflect potential paracrine interactions. Condi- tioned media from some [254,266], but not all fibroblastic cell populations [263] are mitogenic for tumor cells growing in vitro.

Clearly, the most important beneficial tumor-stromal interactions result from the processes associated with neovascularization. Unfortunately, a detailed descrip- tion of this phenomenon is beyond the scope of this arti- cle. In the absence of appropriate access to vascular tissues, the growth of most solid tumors is significantly inhibited. From a clinical perspective, more important interactions include the increased potential for disse- mination to distant sites and the reduction in access to tumor tissues by cytotoxic drugs. The importance of this tumor-stromal interaction is most evident in studies into the well documented ‘tumor bed effect’ (for a recent review see Ref. 267). Inhibition of neovascularization by irradiation of stromal tissues produces the equivalent of feeder layers (reproductively dead but metabolically active cells). Whilst these cells would be expected to con- tinue to secrete biologically active growth factors, they are significantly less able to support tumor growth than normal tissues capable of supporting neovascularization [267].

IV-E. Further comments on tumor-stromal interactions in breast cancer

The stromal responses to tumors most probably represent a fluid and dynamic equilibrium which reflect the changing biological properties of the tumor cell populations and their interactions with adjacent stromal cells. For example, myofibroblasts are only rarely detected in significant numbers in either benign breast disease or carcinoma in situ, but are frequently the major mesenchymal cell type in invasive carcinomas [223-2261. This strongly suggests that the phenotypic progression from non-invasive to invasive carcinoma includes perturbations in the signals responsible for either the local migration or expansion of myofibroblast populations. The loss of normal epithelial basement membrane structures, which accompanies invasion, could provide one such signal, since only then would stromal fibroblastic cells come into close contact with tumor cells’ extracellular matri- ces. The intact basement membrane may also provide a physical barrier for the diffusion of some tumor cell-de-

rived secreted factors, access to soluble factors being greatly enhanced on loss of basement membranes.

The expansion of some fibroblastic cell populations may also be altered by the degree of lymphoreticular infiltration. The differentiation of some fibroblastic populations can be markedly perturbed by contact with either macrophages or their secreted products. Thus, the immunogenic properties of tumor cell populations may indirectly influence the properties of the mesenchymal components of tumor-associated stroma by altering the relative proportion of the various cell types comprising the lymphoreticular infihration. An understanding of the factors which influence the dynamics of host stromal responses and their implications for tumor progression may provide an insight into new targets for the development of novel therapeutic strategies.

Co-culture experiments in vitro provide one means to study the clearly important mesenchyme-epithelial cell interactions. However, an inappropriate choice of control cell populations could lead to comparisons being made between normal myofibroblasts, the mesenchymal cells most frequently isolated in vitro from malignant breast tissues [224,268], and other normal fibroblastic cells. Apparent differences in the expression of specific genes in tumor stromal cells compared with normal stromal cells, may reflect the presence of normal reactive cells (myofibroblasts), rather than specific tumor-fibroblast paracrine interactions or novel cell types. For co-culture experiments to provide biologically relevant comparisons, the ultrastructural and biological properties of both the normal control and tumor-derived fibroblastic and epithelial cell populations should be con- firmed [269]. This is particularly important since there may be as many as four subtype of myofibroblastic cells [270]. It should also be recognized that the behavior of fibroblastic cells from tumors may alter significantly once removed to an in vitro environment. For example, fibroblastic cells can produce both extracellular matrix and enzymes capable of degrading this matrix. In desmoplastic tumors these cells clearly produce more matrix than they degrade. As a potential artifact, the equilibrium is shifted significantly towards degradation once these cells are cultured in vitro [262]. Collagen synthesis and deposition by fibroblastic cells also changes sub- stantially with time in vitro [271].

Many investigators have speculated that the process of malignant progression reflects the continuing generation of variants with specific growth advantages. Clear- ly, cells capable of degrading extracellular matrix more rapidly than it is replaced would have a significantly increased metastatic potential. A major result of the marked desmoplasia associated with breast tumors may be to facilitate the ultimate selection of highly invasive/

13

metastatic variants by providing the most appropriate of selective pressures. However, the precise role of desmoplasia in influencing either initiation, promotion or progression remains unclear.

A substantial proportion of breast tumors are detected in postmenopausal women. Many tumors exhibit a slow proliferative capacity and have failed to colonize local lymph nodes at the time of detection. The slow progression of these tumors may be the result of stromal responses, both desmoplastic and immunologic, retard- ing tumor growth and metastasis. However, it is clear that significant numbers of breast tumors are capable of negating these effects and successfully metastasizing to distant sites. The ability of endocrine agents to perturb immunologic functions, including NK activity [192,201,272] and immunoglobulin secretion [207], may have important implications for the interactions among the immune response, tumor cells, and hormonal therapies in breast cancer patients. These interactions may provide the opportunity for the development of endocrine therapies which, in addition to direct antitumor cell activity, may exhibit additional immunologic antitumor activities.

It is clear from the reports cited in the preceding sections that host-stromal interactions are highly complex and probably dynamic in nature. Tumor stroma may provide both inhibitory and stimulatory effects on adjacent tumor cells and these effects may occur at different times during the evolution of the tumor. Much has yet to be learned about the nature of these interactions, both how and when they operate, and their role in tumor initiation, promotion and progression. Neverthe- less, a full understanding of these interactions may provide critical insights into important targets for antineoplastic therapeutic strategies.

V. Hormonal modulation of cytotoxic chemotherapy

The marked heterogeneity of breast tumors is a major restriction of the curative potential of single modality treatments. Even within tumors which express high levels of ER, there are areas which do not possess these receptors [63,273]. The selective pressure applied by endocrine manipulation would be expected to remove the sensitive populations (ER positive) and could facilitate the emergence of tumors comprised of predominantly endocrine-resistant cells (ER negative) [55]. A combination of cytotoxic chemotherapy with hormonal manipulation could result in the cytotoxic drugs killing the ER- negative subpopulations and the hormonal agents eli- minating the hormone-dependent cells [274-2761. This approach has the further advantage that DNA synthesis occurs predominantly in the ER-negative subpopula-

tions of human breast tumors [277] which should be more sensitive to cell cycle-specific cytotoxic drugs. Many breast tumors exhibit a low proliferative rate and small growth fraction [278]. These tumors may be resistant to cell cycle-specific agents by virtue of their kinetic properties. Subsequent regrowth of these ‘kinetically resistant’ tumor subpopulations may be a significant cause of treatment failure. Consequently, chemoendo- crine regimens have been designed to utilize the mitogenic properties of hormones to recruit non-cycling but hormone-dependent cells back into the cell cycle (estrogenic recruitment). An alternative approach has been to produce synchronous cell populations by blocking cells in a particular phase of the cell cycle (e.g., a Go blockade by antiestrogens) followed by release using a hormonal mitogen. e.g., E2 (hormonal synchronization).

V-A. Hormonal eflects on cell c_ycle modulation and response to cytotoxic drugs

E2 recruitment of hormone-responsive breast cancer cells utilizes the ability of E2 to increase the proportion of cells in S phase which reflects an increase in the number of cells drawn into the cell cycle [279]. This increases the percentage of cells sensitive to the inhibitory effects of cell cycle-specific cytotoxic agents. For example, we have previously demonstrated that E2 poten- tiates the cytotoxic effects of methotrexate in ER-positive MCF-7 human breast cancer cells growing in vitro [280]. Treatment with estradiol also increases the cytotoxic effects of adriamycin [281-2831, cytosine arabino- side [279], melphalan [284] and cyclophosphamide [285] in ER-positive human breast cancer cells growing in vitro. A similar cell cycle recruitment appears to be responsible for the ability of androgens to increase the cytotoxicity of adriamycin and melphalan in the androgen-responsive Shionogi AR SC1 15 cell line [286,287]. Insulin increases the cytotoxicity of methotrexate in MCF-7 cells [288], probably reflecting the ability of insulin to increase the percentage of cells in S phase [289,290].

It has been suggested that a combined phase specific- inhibition of hormone-dependent cells by tamoxifen, followed by E2 rescue would produce a greater perturbation in cell cycle profile than estrogenic recruitment alone [291]. Yang and Samaan [292] have reported improved synchronization by treatment with low concentrations of thymidine following blockade and rescue with tamoxifen and estradiol. Cell populations synchro- nized in this manner are at least 50-fold more sensitive to the inhibitory effects of 5-fluorouracil. Unfortunate- ly, data derived from in vivo studies on the cell cycle ef-

14

fects of tamoxifen have failed to confirm the in vitro observations which indicated that tamoxifen induces a blockade in Gt [293]. This may partly explain the con- flicting results obtained in clinical trials investigating this hormonal synchronization approach. Response rates to combined chemohormonal regimens have been poorer than would have been predicted on theoretical grounds. This may reflect suboptimal scheduling of cytotoxic drugs and hormones, adverse interactions between modalities, or inadequate synchronization of tumor cell populations. While not all tumors will respond to cell cycle modifying regimens, few investigators have attempted to confirm adequate synchronization or recruitment by concurrent estimations of either thymidine labelling index, primer dependent U-DNA polymerase index, or cell cycle distribution on fine nee- dle aspirates. However, when these measurements have been performed, improved response rates are observed almost exclusively in those tumors which exhibited significant estrogenic recruitment [2942961.

Not all chemohormonal interactions are beneficial. Pharmacological concentrations of estrogen can lower the intracellular steady-state levels of methotrexate and reduce its cytotoxicity in MDA-MB-436 human breast cancer cells [297]. Tamoxifen increases melphalan efflux and inhibits influx in MCF-7 cells. The resultant reduction in steady-state intracellular concentrations of the drug are probably responsible for the concurrent reduction in cytotoxicity [298]. The mechanism by which steroids and triphenylethylenes inhibit drug uptake is not known. However, we have recently demonstrated that pharmacological concentrations of both estradiol and tamoxifen reduce the membrane fluidity of MCF-7 and MDA-MB-436 human breast cancer cells, implicating altered membrane structure as the potential mechanism

11%

V-B. Efects on drug resistance and MDR-I

The development of a multidrug resistant phenotype is frequently accompanied by the expression of a 170 kDa glycoprotein (MDR-I) The precise contribution of MDR-1 to the multidrug resistance phenotype in human breast cancer is unclear. Low levels of MDR-1 expression have been detected in normal breast 12211. The larg- est study performed to date failed to detect significant MDR-I expression in 248 breast tumors [299]. In marked contrast, a considerable number of smaller studies have clearly demonstrated MDR-1 expression in human breast tumors [221,228,229,30&302]. Expres- sion correlates with either previous cytotoxic chemotherapy [264] and/or failure on previous treatment [300] and in vitro resistance to the appropriate drugs

[229,301]. The explanation for this apparent disparity has yet to be established but may involve differences in the techniques used for analysis of MDR-1 expression. Adequate detection may be compromised by the low level of MDR-I expression frequently observed in breast tumors. This level is generally much lower than that observed in the other solid tumors which can acquire drug resistance by this mechanism [221]. The level of expression required to induce a clinically resistant tumor has not been established. However, the narrow therapeutic window associated with these drugs implies that a low level of expression may be sufficient at least in some tissues. The detection of higher levels in tumors from breast cancer patients that have received prior cytotoxic chemotherapy [264,300] supports the concept that MDR-1 expression may be induced by exposure to the appropriate cytotoxic agents [303]. Acquisition of a multidrug resistance phenotype in vitro by selection in the presence of adriamycin leads to overexpression of the MDR-I gene in MCF-7 cells [199].

A number of hormonal agents have been demonstrated to at least partly reverse multidrug resistance resulting from overexpression of the MDR-1 gene. Tamoxi- fen, 4-hydroxytamoxifen and the close structural analogue toremifene can reverse MDR-I-induced drug resistance in some tumor cells [304306]. Progesterone also reverses the effects of MDR-I expression [307]. The mechanism through which these agents reverse is unclear. Yang et al. [307] have shown that progesterone competes with vinblastine for binding to the MDR-I protein. This results in enhanced accumulation of vinblastine and a consequent increase in sensitivity to the drug. Cells expressing the MDR-1 gene exhibit similar kinetics of [3H]tamoxifen uptake suggesting that tamoxifen is not a substrate for this membrane pump (Clarke et al., unpublished data). Tamoxifen can also inhibit 45Ca transport in PC12 neurosecretory cells [308] and membrane fluidity in both the MCF-7 and MDA-MB- 436 breast cancer cell lines [15]. Thus, tamoxifen may function by altered membrane fluidity directly inhibiting MDR-1 function. Reduced fluidity of the MDR-1 glycoprotein environment may inhibit its function, perhaps by altering conformation or decreasing mobility in the cell membrane [15]. Many membrane proteins are either chemically or structurally dependent on their lipid environment in order to maintain their functionality [309]. Alternatively, tamoxifen may alter the ability of ATP to bind to the appropriate sites on the glycoprotein.

VI. Conclusions

Steroid and polypeptide hormones play multi-faceted roles in the control of breast cancer growth and progres-

sion. Whilst largely cirumstantial, there is an impressive amount of literature implicating growth factors as indirect (autocrine/paracrine) mediators of steroid hormone function. Since treatment with steroids modulates the expression of a number of genes closely associated with the control of proliferation, it seems unlikely that any one gene alone is responsible for mediating these effects. Thus, the concerted action of many growth factors, acting by both autocrine and paracrine pathways, may be required to induce the full range of biological responses observed in human breast tissue. It is also possible, particularly in heterogenous neoplastic tissue, that some clones within the same tumor will utilize different com- binations of growth factors to achieve a similar hormone-dependent or -independent phenotype. The presence of the IGFs, EGF, TGF-ol, TGF$ and some FGFs in normal serum may also indicate potential endocrine routes of tumor cell growth control.

The significance of these observations implicating growth factors in neoplastic growth will partly depend upon how good growth factors and/or their receptors will prove to be as targets for therapeutic intervention. Initial studies have described the ability of anti-EGF-R [310] and anti-type I IGF receptor antibodies [106] to inhibit breast cancer ceils in culture. These growth factors/ receptors are widespread in normal tissues, consequently, the potential for side effects induced by some anti- growth factor therapies may be substantial [311]. The nature of these side effects, and the degree to which they could limit therapeutic activity, will become apparent once the appropriate in vivo studies have been complet- ed. The overexpression of certain growth factors/receptors may also provide the opportunity to target drugs using synthetic ligands or antibodies. Recent reports have described the ability of a TGF-cr Pseudomonus toxin conjugate to inhibit tumor growth in nude mice [312,313].

Tumor-stromal interactions provide a novel therapeutic target. For example, drugs which could increase proliferation and matrix deposition by stromal myofibroblasts might significantly retard metastatic potential. Increasing the general immunocompetence of the host, or infusion of mediators of CMI, could both directly inhibit the tumor cells and also alter fibroblastic cell differentiation or stromal matrix deposition.

The ability of some hormonal agents to reverse MDR- l-induced drug resistance has significant therapeutic potential. For example, concurrent or sequential administration of the appropriate drugs and antiestrogens may prevent or reduce the effects of MDR-1 expression. Drug-hormone conjugates may provide an alternative approach, provided the conjugates retain the ability to both inhibit MDR-I function and induce cytotoxicity.

During the past ten years, a considerable amount of new and exciting data have been generated relating to the possible role of growth factors and hormones in the control of breast cancer growth and progression. The application of this new knowledge to the development of better treatment will be one of the major challenges of the next decade.

References

Glass AG, Hoover RN. Rising incidence of breast cancer: relation-

ship to stage and receptor status. J Nat1 Cancer Inst 82: 693, 1990.

Miller AB, Bulbrook RD. UICC multidisciplinary project on

breast cancer: the epidemiology, aetiology and prevention of

breast cancer. Int J Cancer 37: 173, 1986.

Duval D, Duranr S, Homo-Delarche F. Non-genomic effects of

steroids. Interactions of steroid molecules with membrane struc-

tures and functions. Biochim Biophys Acta 737: 409, 1983.

Raynaud, A. Observations sur les modifications provoquees par

les hormones oestrogenes, du mode de developpement des mame-

lons des foetus de souris. C R Acad Sci (Paris) 240: 674, 1989.

Bronson, FH, Dagg CP, Snell GD. Biology of the laboratory

mouse. Reproduction 187: 1975.

6 Murr, SM, Bradford GE, Geschwind II. Plasma leuteinizing hor-

mone, follicle stimulating hormone and prolactin during pregnan-

cy in the mouse. Endocrinology 94: 112, 1974.

7 Warner MR. Effect of perinatal oestrogen on the pretreatment re-

quired for mouse lobular formation in vitro. J Endocrinol 77: 1.

8

9

10

11

12

13

14

15

16

17

1978.

Wang S, Counterman LJ. Haslam SZ. Progesterone action in

normal mouse mammary gland. Endocrinology 127: 2183, 1990.

Engel LW, Young NA. Human breast carcinoma ceils in contin-

uous culture: a review. Cancer Res 38: 4327. 1978.

Lippman ME, Bolan G, Huff, K. Interactions of antiestrogens

with human breast cancer in long-term tissue culture. Cancer Treat

Rep 60: 1421. 1976.

Aitken SC. Lippman ME. Hormonal regulation of net DNA syn-

thesis in MCF-7 human breast cancer cells in tissue culture. Cancer

Res 42: 1727. 1982.

Vignon F. Bouton MM, Rochefort H. Antiestrogens inhibit the

mitogenic effect of growth factors on breast cancer cells in the total

absence of estrogens. Biochem Biophys Res Commun 146: 1502.

1987.

Clarke R, Briinner N, Thompson EW et al. The inter-relationships

between ovarian-independent growth. antiestrogen resistance and

invasiveness in the malignant progression of human breast cancer.

J Endocrinol 122: 331. 1989.

Bronzert DA, Triche TJ, Gleason P, Lippman ME. Isolation and

characterization of an estrogen-inhibited variant derived from the

MCF-7 breast cancer cell line. Cancer Res 44: 3942, 1984.

Clarke R. van den Berg HW. Murphy RF. Tamoxifen and 17/J-

estradiol reduce the membrane fluidity of human breast cancer

cells. J Nat1 Cancer Inst 82: 1702, 1990.

Magdelenat H. Pouillart P. Steroid hormone receptors in breast

cancer. In: Sheridan PJ, Blum K, Trachtenberg MC. eds. Steroid

Receptors and Disease: Cancer Autoimmune, Bone and Circulato-

ry Disorders. New York: Marcel Dekker, 1988: 435.

Chouvet C. Vicard E, Frappart L. Falette N, Lefebvre MF. Saez

S. Growth inhibitory effect of 4-hydroxy-tamoxifen on the BT 20

mammary cell line. J. Steroid Biochem 3 I: 655, 1988.

16

18 Ingram D, Nottage E, Ng S, Sparrow L, Roberts A, Willcox D.

Obesity and breast cancer. Cancer 64: 1049, 1989.

19 Tartter PI, Papatestas AE, Ioannovich J, Mulvihill MN. Choles-

terol and obesity as prognostic factors in breast cancer. Cancer 47:

2222, 1981.

20 Henderson BE, Ross, R, Bernstein L. Estrogens as a cause of

human cancer. Cancer Res. 48: 246, 1988.

21 Cancer and steroid hormone study of the Centers for Disease Con-

trol and the National Institute of Child Health and Human Devel-

opment. Oral contraceptive use and the risk of breast cancer. N

Engl J Med 315: 405, 1986.

22 Herbst AL, Ulfelder H, Poskanzer DC. Adenocarcinoma of the

vagina: association of maternal stilbestrol therapy with tumor ap-

pearance in young women. N Engl J Med 284: 878, 1971.

23 Jick H, Walker AM, Rothman KJ. The epidemic of endometrial

cancer: a commentary. Am J Public Health 70: 264, 1980.

24 Vana J, Murphy GP, Arnoff BL, Baker HW. Survey of primary

liver tumors and oral contraceptive use. J Toxicol Environ Health

45: 1934, 1979.

25 Muldoon TG. Interplay between estradiol and prolactin in the

regulation of steroid hormone receptor levels, nature and function-

ality in normal mammary tissue. Endocrinology 109: 1339, 198 1.

26 Manni A, Rainieri J, Arafah BM, Finegan HM, Pearson OH. Role

of estrogen and prolactin in the growth and receptor levels of N-

nitrosomethylurea-induced rat mammary tumors. Cancer Res 42:

3492, 1982.

27 Briand P. Hormone-dependent mammary tumors in mice and rats

as a model for human breast cancer. Anticancer Res 3: 273, 1983.

28 Ferguson DJP, Anderson TJ. Morphological evaluation of cell

turnover in relation to the menstrual cycle in the nesting human

breast. Br J Cancer 44: 177, 1981.

29 Metzler M. Biochemical toxicology of diethylstilbestrol. In: Re-

views in Biochemical Toxicology. Hodgson E, Bend JR, Philips

RM, eds. New York: Elsevier, 1984; 191.

30 Tsutsui T, Maizumi H, McLachlan, JA, Barrett, JC. Aneuploidy

induction and cell transformation by diethylstilbestrol: a possible

chromosomal mechanism in carcinogenesis. Cancer Res 43: 3814,

1983.

31 Fishman J. Aromatic hydroxylation of estrogens. Annu Rev Phy-

siol45: 61, 1983.

32 Metzler M. Metabolic activation of xenobiotic stilbene estrogens.

Fed Proc 46: 1855, 1987.

33 Bradlow HL, Hershcopf RE, Fishman J. Oestradiol 16a-hydroxy-

lase: a risk marker for breast cancer. Cancer Surv 5: 573, 1986.

34 Schneider J, Kinne D, Fracchia A, et al. Abnormal oxidative me-

tabolism in women with breast cancer. Proc. Nat1 Acad Sci USA

79: 3047, 1982.

35 Liehr JG, Stance1 GM, Chorich LP, Bousfield GR, Ulubelen AA.

Hormonal carcinogenesis: separation of estrogenicity from car-

cinogenicity. Chem-Biol Interact 59: 173, 1986.

36 Sukumar S, Notario V, Martin-Zanca D, Barbacid M. Induction

of mammary carcinomas in rats by nitroso-methylurea involves

malignant of Ha-rus-I locus by single point mutations. Nature

306: 658, 1983.

37 Zhang R, Haag JD, Gould MN. Reduction in the frequency of ac-

tivated ras oncogenes in rat mammary carcinomas with increasing

N-methyl-N-nitrosourea doses or increasing prolacting levels.

Cancer Res 50: 4286, 1990.

38 Andrea AC, Schoenberger, CA, Groner B, Hennighausen L, Le

Maur M, Gerlinger P. Ha-ras oncogene expression directed by a

milk protein gene promoter: tissue specificity, hormonal regulation

and tumor induction in transgenic mice. Proc Nat1 Acad Sci USA

84: 1299, 1987.

39 Stewart TA, Pattengale, PK. Leder, P. Spontaneous mammary

adenocarcinoma in transgenic mice that carry and express MTV/ my fusion genes. Cell 38: 627, 1984.

40 Santos, GF, Scott, GK, Lee WMF, Liu E, Benz C. Estrogen-in-

duced post-transcriptional modulation of c-myc proto-oncogene

expression in human breast cancer cells. J Biol Chem 263: 9565,

1988.

41 Rochlitz CF. Scott GK, Dodson JM, et al. Incidence of activating

r-as oncogene mutations associated with primary and metastatic

human breast cancer. Cancer Res 49: 357, 1989.

42 Bos JL, ras oncogenes in human cancer: a review. Cancer Res 49:

4682, 1989.

43 Locker AP, Dowle CS, Ellis IO et al. C-myc oncogene product ex-

pression and prognosis in operable breast cancer. Br J Cancer 60:

669. 1989.

44 Lin YJ, LI S, Clinton GM. Insulin and epidermal growth factor

stimulate phosphorylation of ~185 HER-2 in the breast carcinoma

cell line, BT474. Mol Cell Endocrinol69: 111, 1990.

45 Dati C, Antoniotti, S, Taverna D, Perroteau I. De Bortoli M. Inhi-

bition of c-erbB-2 oncogene expression by estrogens in human

breast cancer cells. Oncogene 5: 1001, 1990.

46 Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/ neu proto-oncogene in human breast and ovarian cancer. Science

244: 707, 1989.

47 Paik S, Hazan R, Fisher ER, et al. Pathologic findings from the

National Surgical Adjuvant Breast and Bowel Project: prognostic

significance of erbB-2 protein overexpression in primary breast

cancer. J Clin Oncol8: 103, 1990.

48 Clark GM, McGuire WL. Follow-up of HER-2/neu amplification

in primary breast cancer. Cancer Res 5 1: 944, 199 I. 49 Clarke R, Brtinner N, Katzenellenbogen BS et al. Progression

from hormone dependent to hormone independent growth in

MCF-7 human breast cancer cells. Proc Nat1 Acad Sci USA 86:

3649, 1989.

50 Lippman ME, Bolan G, Huff K. The effects of estrogens and anti-

estrogens on hormone responsive human breast cancer ceils in long

term tissue culture. Cancer Res 36: 4595, 1976.

51 Balakrishnan A, Yang J, Beattie CW, Gupta TKD, Nandi S. Es-

trogen receptor in dissociated and cultured human breast fibro-

adenoma epithehal cells. Br J Cancer 34: 233, 1987.

52 Petersen OW, Hoyer PE, van Deurs B. Frequency and distribution

of estrogen receptor-positive cells in normal, nonlactating human

breast tissue. Cancer Res 47: 5748, 1987.

53 Jacquemier JD, Hassoun J, Torrente M, Martin P-M. Distribution

of estrogen and progesterone receptors in healthy tissue adjacent

to breast lesions at various stages - immunohistochemical study of

107 cases. Breast Cancer Res Treat 15: 109, 1990.

54 Nowell PC. The clonal evolution of tumor cell populations.

Science 194: 23, 1976.

55 Isaacs JT. Clonal heterogeneity in relation to response. In: Stoll

BA, ed. Endocrine Management of Cancer, Vol. 1, Basel: Karger,

1988; 125.

56 Nowell PC. Mechanisms of tumor progression. Cancer Res 46:

2203, 1986.

57 Clarke R, Dickson RB, Briinner N. The process of malignant pro-

gression in human breast cancer. Ann Oncol 1: 401, 1990.

58 McGuire WL, Carbone PP, Sears ME, Escher, GC. Estrogen re-

ceptors in human breast cancer: an overview. In: McGuire WL,

Carbone PP, Vollmer EP, eds. Estrogen Receptors in Human

Breast Cancer. New York: Raven Press, 1975; 1.

59 Netto GJ, Cheek JH, Zachariah NY, et al. Steroid receptors in be-

nign mastectomy tissue. Am J Clin Path01 94: 14, 1990.

60, Akhtar M, Robinson C, Ali MA, Godwin JT. Secretory carcinoma

of the breast. Light and electron microscopic study of three cases

and review of the literature. Cancer 51: 2245, 1983.

17

61 Thomas DB. Do hormones cause breast cancer? Cancer 53: 595,

1984.

62 Pike MC, Krailo MD, Henderson BE, Casagrande JT, Hoe1 DG.

Hormonal risk factors, breast tissue age and the age incidence of

breast cancer. Nature 303: 767, 1983.

63 Van Netten JP, Armstrong JB, Carlyle SS, et al. Estrogen receptor

distribution in the peripheral, intermediate and central regions of

breast cancers. Eur J Cancer Clin Oncol24: 1885, 1988.

64 Narbaitz K, Stumpf WE, Sar M. Estrogen receptors in mammary

gland primordia of fetal mouse. Anat Embryo1 158: 161, 198.

65 Blanc0 G, Alavaikko. M, Ojala A, et al. Estrogen and progeste-

rone receptors in breast cancer: relationships to tumor histopatho-

logy and survival of patients. Anticancer Res 4: 383, 1984.

66 Singh L. Wilson AJ, Baum M, et al. The relationship between

histological grade, oestrogen receptor status, events and survival

at 8 years in the NATO (Nolvadex) trial. Br J Cancer 57: 612.

1988.

67 Henry JA, Nicoison JA, Farndon JR, Westley BR, May F. Meas-

urement of oestrogen mRNA levels in human breast tumours. Br

J Cancer 58; 600, 1988.

68 Ozzello L. Epithelial-stromal junction of normal and dysplastic

mammary glands. Cancer 25: 586, 1970.

69 Briinner N. Clarke R. Lippman ME, Dickson RB. Models for

studying the progression from hormone-dependent to hormone-in-

dependent growth in human breast cancer. In: Lippman ME,

Dickson RB, eds. Growth regulation of cancer II. New York: Alan

R Liss, 1990; 115.

70 Briinner N. Svenstrup B, Spang-Thompsen M, Bennet P, Nielsen,

A, Nielsen JJ. Serum steroid levels in intact and endocrine ablated

Balb\c nude mice and their intact litter mates. J Steroid Biochem

25: 429. 1986.

71 Seibert K. Shafie. SM, Triche TJ, et al. Clonal variation of MCF-7

breast cancer cells in vitro and in athymic nude mice. Cancer Res

43: 2223. 1983.

72 Clarke R. Lippman ME, Dickson RB, Spang-Thompsen M, Briin-

ner N. In viva/in vitro selection of hormone independent cells from

the hormone dependent MCF-7 human breast cell line. In: Wu B.

Zheng J, eds. Immune-deficient Animals in Experimental Medi-

cine. Basel: Karger, 1989; 190.

73 Darbre PD, King RJB. Progression to steroid insensitivity can

occur irrespective of the presence of functional steroid receptors.

Cell 51: 521. 1987.

74 Leof EB. Lyons RM, Cunningham MR. O’Sullivan D. Mid-Cl ar-

rest and epidermal growth factor independence of ras-transfected

mouse cells. Cancer Res 49: 2356, 1989.

75 Campisi J, Pardee AB. Post-transcriptional control of the onset of

DNA synthesis by an insulin-like growth factor. Mol Cell Biol 4:

1807, 1984.

76 Barnes D, Sato G. Growth of a human mammary tumor cell line

in a serum free medium. Nature 281: 388, 1978.

77 Huff KK. Knabbe C. Lindsey R, et al. Multihormonal regulation

of insulin-like growth factor-I-related protein in MCF-7 human

breast cancer cells. Mol Endocrinol2: 200, 1988.

78 Riss TL, Sirbasku DA. Growth and continuous passage of

COMMA-D mouse mammary epithelial cells in hormonally de-

fined serum-free medium. Cancer Res 47: 3776, 1987.

79 Ethier SP. Cundiff KC. Importance of extended growth potential

and growth factor independence on in vivo neoplastic potential of

primary rat mammary carcinoma cells. Cancer Res 47: 5316, 1987.

80 Pekonen F. Partanen S. Makinen T, Rutanen E-M. Receptors for

epidermal growth factor and insulin-like growth factor and their

relation to steroid receptors in human breast cancer. Cancer Res

48: 1343, 1988.

81 De Larco JE, Todaro GJ. Growth factors from murine sarcoma

virus-transformed cells. Proc Nat1 Acad Sci USA 75: 4001, 1978.

82 Browder TM, Dunbar CE, Nienhuis AW. Private and public auto-

crine loops in neoplastic cells. Cancer Cells 1: 9, 1989.

83 Lippman ME, Dickson RB, Kasid A. et al. Autocrine and para-

crine growth regulation of human breast cancer. J Steroid Biochem

24: 147, 1986.

84 Bates SE, Davidson NHE, Valverius EM. et al. Expression of

transforming growth factor-cc and its mRNA in human breast can-

cer: its regulation by estrogen and its possible functional signifi-

cance. Mol Endocrinol2: 543, 1988.

85 Koga M, Sutherland RL. Epidermal growth factor partially re-

verses the inhibitory effects of antiestrogens on T47D human

breast cancer cell growth. Biochem Biophys Res Commun 146:

738. 1987.

86 Peres R, Betsholtz C, Westermark B, Heldin C-H. Frequent ex-

pression of growth factors for mesenchymal cells in human

mammary carcinoma cell lines. Cancer Res 47: 3425, 1987.

87 Perroteau I, Salomon D, DeBertoli M, et al. Immunological detec-

tion and quantitation of alpha transforming growth factors in

human breast carcinoma cells. Breast Cancer Res Treat 7: 201.

1986.

88 Dickson RB. McManaway ME. Lippman ME. Estrogen-induced

factors of breast cancer cells partially replace estrogen to promote

tumor growth. Science 232: 1540. 1987.

89 Clarke R. Briinner N, Katz D, et al. The effects of a constitutive

production of TGF-a on the growth of MCF-7 human breast

cancer cells in vitro and in vivo. Mel Endocrinol 3: 372. 1989.

90 Mukku VR, Stance1 GM. Regulation of epidermal growth factor

receptor by estrogen. J Biol Chem 260: 9820. 1985.

91 Lingham RB, Stance1 GM. Loose-Mitchel DS. Estrogen regula-

tion of epidermal growth factor messenger ribonucleic acid. Mol

Endocrinol2: 230. 1988.

92 Leake. RE, George WD, Godfrey, D. Rinaldi. F. Regulation of

epidermal growth factor receptor synthesis in breast cancer cells.

Br J Cancer 58: 521. 1988.

93 Davidson NE, Gelmann EP. Lippman ME, Dickson RB. Epidermal

growth factor receptor gene expression in estrogen receptor-posi-

tive and -negative human breast cancer cell lines. Mol Endocrinol

1: 216, 1987.

94 Fizpatrick SL. Brightwell J. Wittliff JL, Barrows GH, Schultz GS.

Epidermal growth factor binding by breast tumor biopsies and re-

lationship to estrogen receptor and progestin receptor levels. Can-

cer Res 44: 3448, 1984.

95 Cattoretti G, Andreola S, Clemente C. D’Amato L. Rilke F. Vi-

mentin and p53 expression in epidermal growth factor receptor-

positive oestrogen receptor-negative breast carcinomas. Br J Can-

cer 57: 353. 1988.

96 Sainsbury JRC, Malcolm A, Appelton D. Farndon JR, Harris AL.

Presence of epidermal growth factor receptors as an indicator of

poor prognosis in patients with breast cancer. J Clin Path01 38:

1225. 1985.

97 Perez R. Pascual M, Macias A. Lage A. Epidermal growth factor

receptors in human breast cancer. Breast Cancer Res Treat 4: 189.

1984.

98 Di Marco E. Pierce JA, Fleming TP et al. Autocrine interaction

between TGF-a and the EGF-receptor: quantitative requirements

for induction of the malignant phenotype. Oncogene 4: 83 I, 1989.

99 Cheville A. Blair 0, Clarke R, Gelmann E, Kern FG. Regulation

of epidermal growth factor receptor (EGFR) expression in MCF-7

cells EGFR transfectants. 4th Annual Meeting on Oncogenes.

1989 (Abstr).

18

100 Kaleko ML, Rutter WJ, Miller AD. Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation. Mol Cell Biol 10: 464, 1990.

101 Peyrat JP, Bonneterre J, Laurent JC, et al. Presence and characterization of insulin-like growth factor I receptors in human benign breast disease. Em J Cancer Clin Oncol. 19: 1425, 1988.

102 Foekens JA, Henkelman MS, Fukkink JF, Blankenstein MA, Klijn JGM. Combined effects of buserelin, estradiol and tamoxifen on the growth of MCF-7 human breast cancer cells in vitro. Biochem Biophys Res Commun 140: 550, 1986.

103 Peyrat JP, Bonneterre J, Beuscart R, Djiane J, Demaille A. Insu- lin-like growth factor I receptors in human breast cancer and their relation to estradiol and progesterone receptors. Cancer Res 48: 6429, 1988.

104 Bonneterre J, Peyrat, JP, Beuscart R, Demaille A. Prognostic significance of insulin-like growth factor I receptors in human breast cancer. Cancer Res 50: 693 1, 1990.

105 Rohlik QT, Adams D, Ku11 FC, Jacobs S. An antibody to the receptor for insulin-like growth factor-I inhibits the growth of MCF-7 cells in tissue culture. Biochem Biophys Res Commun 149: 276, 1987.

106 Arteaga CL, Osborne CK. Growth inhibition of human breast cancer cells in vitro with an antibody against the Type I somatomedin receptor. Cancer Res 49: 6237, 1989.

107 Arteaga CL, Kitten L, Coronado E, Jacobs S, Ku11 F, Osborne CK. Blockade of the type-1 somatomedin receptor inhibits growth of human breast cancer cells in athymic mice. J Clin In- vest 84: 1418, 1989.

108 Tricoli JV, Rail LB, Karakousis CP, et al. Enhanced levels of insulin-like growth factor mRNA in human colon carcinomas and liposarcomas. Cancer Res 46: 6169, 1986.

109 Hoppener JWL, Sttenbergh PH, Slebos RJC, et al. Expression of insulin-like growth factor-I and II genes in rat medullary thyroid carcinoma. FEBS Lett 215: 122, 1987.

110 Hoppener JWL, Mosselman S, Roholl PJM, et al. Expression of insulin-like growth factor-I and II genes in human smooth muscle tumors. EMBO J 7: 1379, 1988.

111 Williams G, Howell A, Jones M. The relationship of body weight to response to endocrine therapy, steroid hormone receptors and survival of patients with advanced cancer of the breast. Br J Can- cer 58: 631, 1989.

112 Maya1 Y, Shiu RPC, Bhaumick B, Bala M. Receptor binding and growth-promoting activity of insulin-like growth factors in human breast cancer cells (T-47D) in culture. Cancer Res 44: 5486, 1984.

113 Furlanetto RW, DiCarlo JN. Somatomedin C receptors and growth effects in human breast cancer cells maintained in long term tissue culture. Cancer Res 44: 2122, 1984.

114 Yee D, Paik S, Lebovic GS, et al. Analysis of insulin-like growth factor-I gene expression in malignancy: evidence for a paracrine role in human breast cancer. Mol Endocrinol3: 509, 1989.

115 Scott J, Cowell J, Robertson ME, et al. Insulin-lie growth factor- II expression in Wilm’s tumors and embryonic tissue. Nature 3 17: 260, 1985.

116 Haselbacher GK, Irminger JC, Zapj J, Ziegler WH, Humbel RE. Insulin-like growth factor-II in human adrenal pheochromocyto- mas and Wilm’s tumours: expression at the mRNA and protein levels. Proc Nat1 Acad Sci USA 84: 1104, 1987.

117 Reeve AE, Eccles MR, Wilkins RJ, Bell GI, Millow LJ. Expres- sion of insulin-like growth factor-II transcripts in Wilm’s tumours. Nature 317: 258, 1985.

118 Cullen KJ, Yee D, Paik S, et al. Insulin-like growth factor-II expression and activity in human breast cancer. Proc Am Assoc Cancer Res 29: 238, 1988 (Abstr.).

119 Dennis PA, Ritkin DB. Cellular activation of latent transforming growth factor B requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc Nat1 Acad Sci USA 88: 580, 1991.

120 Travers MT, Barrett-Lee PJ, Berger U, et al. Growth factor expression in normal, benign and malignant breast tissue. Br Med J 296: 1621, 1988.

121 Yee D, Favoni R, Lupu R, et al. The insulin-like growth factor binding protein BP-25 is expressed by human breast cancer cells. Biochem Biophys Res Commun 158: 38, 1989.

122 Kennedy, DG, van den Berg HW, Clarke R, Murphy RF. The effect of the rate of cell proliferation on the synthesis of methotrexate poly-r-glutamates in two human breast cancer cell lines. Biochem Pharmacol34: 3087, 1985.

123 Meyers JS, Rao BR, Stevens SC, White WL. Low incidence of estrogen receptor in breast carcinomas with rapid rates of cellular proliferation. Cancer 40: 2290, 1977.

124 van der Burg B, Isbrucker L, van Selm-Miltenburb AJP, de Laat SW, van Zoelen EJJ. Role of estrogen-induced insulin-like growth factors in the proliferation of human breast cancer cells. Cancer Res 50: 7770, 1990.

125 Culouscou J-M, Shoyab M. Purification of a colon cancer cell growth inhibitor and its identification as an insulin-like growth factor binding protein. Cancer Res 51: 2813, 1991.

126 Clemmons DR, Camacho-Hubner C, Coronado E, Osborne CKJ. Insulin-like growth factor binding protein secretion by breast carcinoma cell lines: correlation with estrogen receptor status. Endocrinology 127: 2679, 1990.

127 Elgin RG, Busby WH, Clemmons DR. An insulin-like growth binding protein enhances the biologic response to IGF-I. Proc Nat1 Acad Sci USA 84: 3254, 1987.

128 Tanaka A. Matsumoto A, Nishizawa Y, et al. Growth stimulation by androgens, glucocorticoids or fibroblast growth factors and the blocking of the stimulated growth by antibody against basic fibroblast growth factor in protein-free culture of Shionogi carcinoma 115 cells. J Steroid Biochem Mol Bio137: 23, 1990.

129 Cavailles V, Garcia M, Rochefort H. Regulation of cathepsin D and pS2 gene expression by growth factors in MCF-7 human breast cancer cells. Mol Endocrinol3: 522, 1989.

130 Theillet C, Le Roy X, De Lapeyriere 0, et al. Amplification of FGF-related genes in human tumors: possible involvement of HST in breast carcinomas. Oncogene 4: 9 15, 1989.

131 Vlodavsky I, Folkman J, Sullivan R, et al. Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Nat1 Acad Sci USA 84: 2292, 1987.

132 Swain S, Dickson RB, Lippman ME. Anchorage independent epithelial colony stimulating activity in human breast cancer cell lines. Proc Am Assoc Cancer Res 27: 213, 1986 (Abstr.).

133 Massague J. The TGF beta family of growth and differentiation factors. Cell 49: 437, 1987.

134 Valverius EM, Walker-Jones D, Bates SE, et al. Production of and responsiveness to transforming growth factor B in normal and oncogene-transformed human mammary epithelial cells. Cancer Res 49: 6269, 1989.

135 Walker-Jones D. Valverius EM, Stampfer MR, Lippman ME, Dickson RB. Stimulation of epithelial membrane antigen expression by transforming growth factor /? in normal and oncogene- transformed human mammary epithelial cells. Cancer Res 49: 6407, 1989.

136 Knabbe C, Lippman ME, Wakefield LM, Flanders KC, Derynck R, Dickson RB. Evidence that transforming growth factor-p is a hormonally regulated negative growth factor in human breast cancer cells. Cell 48: 417, 1987.

19

137 Zugmaier GC. Knabbe C, Deschauer B, Lippman ME, Dickson

RB. Response of estrogen receptor negative and estrogen recep-

tor positive human breast cancer cell lines to TGF-8-I and TGF-

P-2. Proc Am Assoc Cancer Res 29: 238, 1988 (Abstr.).

138 Murphy LC, Dotzlaw H. Regulation of transforming growth fac-

tor-r and transforming growth factor-p messenger ribonucleic

acid abundance in T-47D, human breast cancer cells. Mol En-

docrinol3: 611, 1989.

139 Clarke R, Lippman ME. Antiestrogen resistance: mechanisms

and reversal. In: Teicher BA. ed. Drug Resistance in Oncology.

New York: Marcel Dekker. 1991, in press.

140 Rainey WE, Naville D, Saez JM, et al. Transforming growth fac-

tor-8 inhibits steroid l’la-hydroxylase cytochrome P-450 expres-

sion in ovine adrenocortical cells. Endocrinology 127: 1910, 1990.

141 Vuk-Pavlovic S, Bozikof V. Pavelic K. Somatostatin reduced

proliferation of murine aplastic carcinoma conditioned to dia-

betes. Int J Cancer 29: 638, 1982.

142 Defeudis FV, Moreau JP. Studies on somatostatin analogues

might lead to new therapies for certain types of cancer. Trends

Pharmacol Sci 7: 384, 1986.

143 Nelson J, Cremin M, Murphy RF. Synthesis of somatostatin by

breast cancer cells and their inhibition by exogenous somatosta-

tin and sandostatin. Br J Cancer 59: 739, 1989.

144 Setyono-Han B, Henkelman MS, Foekens JA, Klijn JGM. Direct

inhibitory effects of somatostatin (analogs) on the growth of

human breast cancer cells. Cancer Res 47: 1566, 1987.

145 Lamberts SWJ, Uitterlinden P, Verschoor L, van Dongen KJ,

DelPozo E. Long-term treatment of acromegaly with the soma-

tostatin analogue SMS 201-995. N Engl J Med 313: 1576, 1985.

146 Pollak M, Costantino J. Polychronakos C, et al. Effect of tamoxi-

fen on serum insulinlike growth factor I levels in stage I breast

cancer patients. J Nat1 Cancer Inst 82: 1693, 1990.

147 Ghirlanda G. Uccioli L, Perri F. et al. Epidermal growth factor,

somatostatin and psoriasis. Lancet i: 65. 1983.

148 Scambia G, Panici PB. Baiocchi G, et al. Growth inhibitory effect

of LH-RH analogs on human breast cancer cells. Anticancer Res

8: 187, 1988.

149 Furr BJA. Nicholson RI. Use of analogs of LH-RH for treatment

of breast cancer. J. Reprod Ferti164: 529, 1982.

150 Cremin M, Clarke R, Nelson J, Murphy RF. The response of

human breast cancer cells to glucagon. Biochem Sot Trans 15:

241. 1987.

151 Eidne. KA. Flanagan CA, Millar RP. Gonadotropin-releasing

hormone binding sites in human breast carcinoma. Science 229:

989. 1985.

152 Taylor AH. Ang VTY, Jenkins JS, Silverlight JJ, Coombes RC,

Luqmani YA. Interaction of vasopressin and oxytocin with

human breast carcinoma cells. Cancer Res 50: 7882, 1990.

153 Gespach C, Bawab W. de Cremoucx P, Calvo F. Pharmacology.

molecular identification and functional characteristics of vasoac-

tive intestinal polypeptide in human breast cancer cells. Cancer

Res 48: 5079, 1988.

154 Valverius EM. Bates SE. Stampfer MR, et al. Transforming

growth factor x production and EGF receptor expression in nor-

mal and oncogene transformed human mammary epithelial cells.

Mel Endocrinol 3: 203, 1989.

155 Bates SE, Valverius EM, Ennis BW, et al. Expression of the

transforming growth factor a/epidermal growth factor receptor

pathway in normal breast epithelial cells. Endocrinology 126:

596, 1990.

156 Raam S, Robert N, Pappas CA, Tamura H. Defective estrogen

receptors in human mammary cancers: their significance in defin-

ing hormone dependence. J Nat1 Cancer Inst 80: 756-1988.

157 Graham ML, Krett NL, Miller LA, et al. T47Dco cells. geneti-

cally unstable and containing estrogen receptor mutations, are a

model for the progression of breast cancers to hormone resist-

ance. Cancer Res 50: 6208, 1990.

158 Kasid A, Lippman ME, Papageorge AG, Lowy DR, Gelmann

EP. Transfection of V-r-a? DNA into MCF-7 human breast can-

cer cells bypasses dependence on estrogen for tumorigenicity.

Science 228: 725, 1985.

159 Adler A, Stein JA, Ben-Efraim S. Immunocompetence, immuno-

suppression, and human breast cancer. Cancer 45: 2074, 1980.

160 Barsky SH, Rao CN. Grotendorst GR. Liotta LA. Increased

content of type V collagen in desmoplasia of human breast carci-

noma. Am J Path01 108: 276, 1982.

161 Tamimi SO, Ahmed A. Stromal changes in invasive breast carci-

noma: an ultrastructural study. J Pathol 153: 163. 1987.

162 Black MM, Zachrau RE, Hankey BF, Wesley M. Skin window

reactivity to autologous breast cancer. An index of prognostically

significant cell-mediated immunity. Cancer 62: 72. 1988.

163 Humphrey LJ, Singla 0, Volenec FJ. Immunologic responsive-

ness of the breast cancer patient. Cancer 46: 893, 1980.

164 Ludwig CU. Hartmann D, Landmann R. et al. Unaltered im-

munocompetence in patients with non-disseminated breast cancer

at the time of diagnosis. Cancer 55: 1673. 1985.

165 An T, Sood U. Pietruk T, Cummings G. In situ quantification

of inflammatory mononuclear cells in ductal infiltrating breast

carcinoma. Am J Path01 128: 52, 1987.

166 Contreras 00, Stoliar A. Immunologic changes in human breast

cancer. Eur J Gynecol Oncol9: 502, 1988.

167 Akimoto M. Ishii H, Nakajima Y, Iwasaki H. Assessment of host

immune response in breast cancer patients. Cancer Detect Prev

9: 311. 1986.

168 Ownby HE. Roi L, Isenberg RR. Brennan MJ. Peripheral

lymphocyte and eosinophil counts as indicators of prognosis in

primary breast cancer. Cancer 52: 126. 1983.

169 Wheelock EF, Robinson MK. Endogenous control of the neo-

plastic process. Lab Invest 48: 120, 1983.

170 Talmadge JE, Meyers KM, Prieur DJ, Starkey JR. Role of NK

cells in tumour growth and metastasis in beige mice. Nature 284:

622. 1980.

171 Gorelik E, Wihrout RH, Okumura K. Habu S, Herberman RB.

Role of NK cells in the control of metastatic spread and growth

of tumor cells in mice. Int J Cancer 30: 107. 1982

172 Richie JP. Abrogation of hematogenous metastases in a murine

model by natural killer cells. Surgery 96: 133. 1984.

173 Vose BM, Moore M. Suppressor cell activity of lymphocytes in-

filtrating human lung and breast tumors. Int J Cancer 24: 579.

1979.

174 Fidler IJ. Eradication of cancer metastasis by tumoricidal macro-

phages. Adv Exp Med Bio1233: 415. 1988.

175 Fidler IJ. Schroit AJ. Recognition and destruction of neoplastic

cells by activated macrophages: descrimination of altered self.

Biochim Biophys Acta 948: 151, 1988.

176 Whitworth PM, Pak CC, Esgro J. Kleinerman ES, Fidler IJ.

Macrophages and cancer. Cancer Metastasis Rev 8: 3 19. 1990.

177 Mantovani A, Allavena P. Sessa C, Bolis G. Mangioni C. Natural

killer activity of lymphoid cells isolated from human ascitic

ovarian tumors. Int J Cancer 25: 573. 1980.

178 Strayer DR. Carter WA, Brodsky 1. Familial occurrence of breast

cancer is associated with reduced natural killer cytotoxicity.

Breast Cancer Res Treat 7: 187. 1986.

179 Kelly, PM, Davison RS, Bliss E. McGee JO. Macrophages in

human breast disease: aquantitative immunohistochemical study.

Br J Cancer 57: 174. 1988.

20

180 Key ME, Hoyer L, Bucana C, Hanna MG. Mechanisms of

macrophage-mediated tumor cytolysis. Adv Exp Med Biol 146:

265, 1982.

181 Hibbs JB, Lambert LH, Remington JS. Control of carcinogene-

sis: a possible role for the activated macrophage. Science 177:

9980, 1972.

182 Meredino RA, Arena A, Liberto MC, Iannello D. Influence of

sera from patients affected by neoplasia on some human macro-

phage functions. Cancer Detect Prev 12: 73, 1988.

183 Acero R, Polentarutti N, Bottazzi B. et al. Effect of hydrocorti-

sone on the macrophage content, growth and metastasis of trans-

planted murine tumors. Int J Cancer 33: 95, 1984.

184 Horst HA, Horny HP. Characterization and frequency distribu-

tion of lymphreticular infiltrates in axillary lymph node metas-

tases of invasive ductal carcinoma of the breast. Cancer 60: 3001,

1987.

185 Bonilla F, Alvarez-Mon M, Merino F, de la Hera A. IL-2 induces

cytotoxic activity in lymphocytes from regional axillary nodes of

breast cancer patients. Cancer 61: 629, 1988.

186 Skoog L, Humla S, Axelsson M, et al. Estrogen receptor levels

and survival of breast cancer patients. Acta Oncol26: 95, 1987.

187 Antoniades K, Spector H. Quantitative estrogen receptor values

and growth of carcinoma of the breast before surgical interven-

tion. Cancer 50: 793, 1982.

188 Naukkarinen A, Syrjanen KJ. Quantitative immunohistochemi-

cal analysis of mononuclear infiltrates in breast carcinomas: cor-

relation with tumor differentiation. J Path01 160: 217, 1990.

189 Levy SM. Herberman RB, Whiteside T, Sanzo K. Perceived so-

cial support and tumor estrogen/progesterone receptor status as

predictors of natural killer cell activity in breast cancer patients.

Psychosom Med: 52,73, 1990.

190 Underwood JC, Giri DD, Rooney N, Lonsdale R. Immunophen-

otype of the lymphoid cell infiltrates in breast carcinomas of low

oestrogen receptor content. Br J Cancer 56: 744, 1987.

191 Fulton AM, Loveless SE, Heppner GH. Mutagenic activity of

tumor associated macrophages in Salmoneflu typhimurium strains

TA98 and TAlOO. Cancer Res 44: 4308, 1984.

192 Screpanti I, Santoni A, Gulino A, Herberman RB, Frati L. Estro-

gen and antiestrogen modulation of mouse natural killer activity

and large granular lymphocytes. Cell Immunol 106: 191, 1987.

193 Seaman WE, Blackman MA, Gindhart TD, Roubinian JR, Loeb

JM, Talal N. /%Estradiol reduces natural killer cells in mice. J Im-

munol 121: 2193, 1978.

194 Hanna N, Schneider M. Enhancement of tumor metastases and

suppression of natural killer cell activity by fi-estradiol treatment.

J Immunol 130: 974. 1983.

195 Seaman WE, Talal N. The effect of 17/?-estradiol on natural kill-

ing in the mouse. In: Herberman RB, ed. Natural Cell-mediated

Immunity Against Tumors. New York: Academic Press, 1980;

765.

196 Shafie SM, Grantham FH. Role of hormones in the growth and

regression of human breast cancer cells (MCF-7) transplanted

into athymic nude mice. J Nat1 Cancer Inst 67: 5 1, 1981.

197 Welsh CW, Swim EL, McManus MJ, White AC, McGrath CM.

Estrogen induced growth of human breast cancer cells (MCF-7)

in athymic nude mice is enhanced by secretions from transplant-

able pituitary tumor. Cancer Lett 14: 309. 1981.

198 Gottardis MM, Wagner RJ, Borden EC, Jordan CV. Differential

ability of antiestrogens to stimulate breast cancer cell (MCF-7)

growth in vivo and in vitro. Cancer Res 49: 4765, 1989.

199 Vickers PJ, Dickson RB, Shoemaker R, Cowan KH. A multi-

drug-resistant MCF-7 human breast cancer cell line which exhib-

its cross-resistance to antiestrogens and hormone independent

tumor growth. Mol Endocrinol2: 886, 1988.

200 Screpanti I, Toniato E. Gulino A, Santoni A, Frati L. Estradiol

increases the sensitivity of MCF-7 human breast cancer cells to

natural killer cell activity. In: Bresciani F, King RJB, Lippman

ME, Raynaud J-P, eds. Progress in Cancer Research and Thera-

py. Vol. 35: Hormones and Cancer 3, New York: Raven Press,

1988; 270.

201 Mandeville R, Ghali SS, Chausseau JP. In vitro stimulation of

NK activity by an estrogen antagonist (Tamoxifen). Eur J Cancer

Clin Oncol20: 983, 1984.

202 Berry J. Green BJ, Matheson DS. Modulation of natural killer

cell activity by tamoxifen in stage I post-menopausal breast

cancer. Eur J Cancer Clin Oncol23: 517, 1987.

203 Garrigues HJ. Romero P, Hellstrom I, Hellstrom KE. Adherent

cells (macrophages?) in tumor-bearing mice suppress MLC

responses. Cell Immunol60: 109, 1981.

204 Stanbridge EJ. Use and validity of tumorigenicity assays in im-

mune-deficient animals. In: Immune-deficient Animals. Base]:

Karger, 1984; 196.

205 Stutman 0. Natural antitumor resistance in immune-deficient

mice. Exp Cell Res 52: 30, 1984.

206 Weisz P, Schrater AF. Lamm ME, Thorbecke GJ. Immunoglob-

ulin isotypes in plasma cells of normal and athymic mice. Cell Im-

muno144: 343. 1979.

207 Myers MJ. Peterson BH. Estradiol indiced alterations in the im-

mune system. I. Enhancement of IgM production. Int J Immuno-

pharmacol7: 207. 1985.

208 van den Berg, HW, Leahey WJ, Lynch M, Clarke R, Nelson J.

Recombinant human interferon alpha increases oestrogen recep-

tor expression in human breast cancer cells (ZR-75-l) and sensi-

tises them to the anti-proliferative effects of tamoxifen. Br J Can-

cer 55: 25, 1987.

209 Porzsolt F, Otto, AM, Trauschel B. Buck C, Wawer AW, Scho-

nenberger H. Rationale for combining tamoxifen and interferon

in the treatment of advanced breast cancer. J Cancer Res Clinical

Oncol 115: 465, 1989.

210 Bezwoda WR, Meyer K. Effect of a-interferon, 17gestradio1, and

tamoxifen on estrogen receptor concentration and cell cycle kin-

etics of MCF-7 cells. Cancer Res 50: 5387, 1990.

211 Goldstein D, Bushmeyer SM. Witt PL. Jordan VC, Borden EC.

Effects of type I and II interferons on cultured human breast cells:

interactions with estrogen receptors and with tamoxifen. Cancer

Res 49: 2698, 1989.

212 Kangas L, Nieminen A-L, Cantell K. Additive and synergistic ef-

fects of a novel antiestrogen toremifene (Fc-1157a) and human

interferons on estrogen responsive MCF-7 cells in vitro. Med Biol

63: 187, 1985.

213 Sheffield LG. Optimization and growth of mammary epithelia in

the mammary gland fat pad. J Dairy Sci 71: 2855, 1988.

214 Azzopardi JG. Problems in Breast Pathology. London: Saunders,

1979; 8.

215 Page DL, Anderson TJ. Diagnostic Histopathology of the Breast.

London: Churchill Livingston, 1987: 4.

216 McGrath CM. Augmentation of the response of normal mam-

mary epithelial cells to estradiol by mammary stroma. Cancer

Res 43: 1355, 1983.

217 Haslem SZ, Levely ML. Estrogen responsiveness of normal

mouse mammary cells in primary cell culture: association of

mammary fibroblasts with estrogenic regulation of progesterone

receptors. Endocrinology 116: 1835. 1985.

218 Reichmann E. Ball R. Groner B, Friis RR. New mammary epi-

thelial and fibrobastic cell clones in coculture form structures

competent to differentiate functionally. J Cell Biol 108: 1127.

1989.

219 Sappino AP, Schiirch W, Gabbiani G. Differentiation repertoire

21

of fibroblastic cells: expression of cytoskeletal proteins as marker

of phenotypic modulations. Lab Invest 63: 144, 1990.

220 Schor SL, Schor AM. Clonal heterogeneity in fibroblast pheno-

type: implications for the control of epithelial-mesenchymal inter-

actions. BioEssays 7: 200, 1987.

221 Eyden BP, Watson RJ, Harris M, Howell A. Intralobular stromal

fibroblasts in the resting mammary gland: ultrastructural proper-

ties and intercellular relationships. J Submicrosc Cytol Pathol 18:

397, 1986.

222 Wargotz ES, Weiss SW, Norris HJ. Myofibroblastoma of the

breast. Am J Surg Path01 11: 493, 1987.

223 Miller H, Schmidts HL, Sakuma T, Stutte HJ. Investigations of

stromal reactions in mammary carcinoma. In: Basler R, Hiibner

K, eds. Pathology of Neoplastic and Endocrine Induced Diseases

of the Breast. Stuttgart: Gustav Fisher, 1986; 166.

224 Van de Berg JS, Rudolph R, Woodward M. Growth dynamics

of cultured myofibroblasts from human breast cancer and non-

malignant contracting tissues. Plast Reconstr Surg 73: 605, 1984.

225 Oda D, Gown AM, Van de Berg JS, Stern R. The fibroblast-like

nature of myofibroblasts. Exp Mol Path01 49: 316, 1988.

226 Sappino AP. Skalli 0, Jackson B, Schiirch W, Gabbiani G.

Smooth-muscle differentiation in stromal cells of malignant and

non-malignant breast tissues. Int J Cancer 41: 707, 1988.

227 Brouty-Boye D, Raux H, Azzarone B, et al. Fetal myofibroblast-

like cells isolated from post-radiation fibrosis in human breast

cancer. Int J Cancer 47: 697, 1991.

228 Haslam SZ. Mammary fibroblast influence on normal mouse

epithelial cell responses to estrogen in vitro. Cancer Res 46: 310.

1986.

229 Li ML, Aggeler J, Farson DA, Hattier C, Hassell J, Bissell MJ.

Influence of a reconstitutes basement membrane and its compo-

nents on casein gene expression and secretion in mouse mammary

epithelial cells. Proc Nat1 Acad Sci USA 84: 136, 1988.

230 Ura H, Obara T. Yokata K, Shibata Y, Okamura K, Namika M.

Effects of transforming growth factor-/? released from gastric car-

cinoma cells on the conreaction of collagen-matrix gels contain-

ing fibroblasts. Cancer Res 5 I : 3550, 1991.

231 Clemmons DR, Shaw DS. Variables controlling somatomedin

production by cultured human fibroblasts. J Cell Physiol I1 5:

137, 1983.

232 Bronzert DA, Pantazis P, Antoniades HN, et al. Synthesis and

secretion of platelet-derived growth factor by human breast can-

cer cell lines. Proc Nat1 Acad Sci USA 84: 5763, 1987.

233 Ewton DZ, Falen SL, Florini JR. The type-II insulin-like growth

factor (IGF) receptor has low affinity for IGF-I analogs: pleio-

tropic actions of IGFs on myoblasts are apparently mediated by

the type I receptor. Endocrinology 120: 115. 1987.

234 Bauer EA. Cooper TW. Huang JS, Altmen J, Deuel TF. Stimula-

tion of in vitro human skin collagenase expression by platelet-de-

rived growth factor. Proc NatI Acad Sci USA 82: 4132, 1985.

235 Keller S, Schmid C, Zapf J, Froesch ER. Inhibition of insulin

degradation by insulinlike growth factors (IGF-I and IGF-II) in

human hepatoma (HepG2) cells. Acta Endocrinol 121: 279. 1989.

236 Nestler JE. Insulin-like growth factor II is a potent inhibitor of

the aromatase activity of human placental cytotrophoblasts. En-

docrinology 127: 2064, 1990.

237 Mathieu M. Vignon F, Capony F. Rochefort H. Estradiol down-

regulates the mannose-6-phosphate/insulin-like growth factor-II

receptor gene and induces cathepsin-D in breast cancer cells: a re-

ceptor saturation mechanism to increase the secretion of lysoso-

mal proenzymes. Mel Endocrinol 5: 815, 1991.

238 Huff KK. Kaufman D, Gabbay KH, Spencer EM, Lippman ME,

Dickson RB. Human breast cancer cells secrete an insulin-like

growth factor-I-related polypeptide. Cancer Res 46: 4613, 1986.

239 Karey KP, Sirbasku DA. Differential responsiveness of human

breast cancer cell lines MCF-7 and T47D to growth factors and

l’lp-estradiol. Cancer Res 48: 4083, 1988.

240 Stewart A, Johnson MD, May FEB. Westley BR. Role of insulin-

like growth factors and the type I insulin-like growth factor re-

ceptor in the estrogen-stimulated proliferation of human breast

cancer cells. J Biol Chem 265: 21172, 1990.

241 Ethier SP. Chiodino C. Jones RF. Role of growth factor synthesis

in the acquisition of insulin/insulin-like growth factor-l indepen-

dence in rat mammary carcinoma cells. Cancer Res 50: 5351.

1990.

242 Williams DW, Williams ED, Wynford-Thomas D. Loss of depen-

dence on IGF-I for proliferation of human thyroid adenoma

cells. Br J Cancer 57: 535, 1988.

243 Schofield PN, Lee A, Hill DJ. Cheetham JE, James D, Stewart

C. Tumour suppression associated with expression of human in-

sulin-like growth factor-II. Br J Cancer 63: 687, 1991.

244 Daughaday WH. Editorial: the possible autocrine/paracrine and

endocrine roles of insulin-like growth factors of human tumors.

Endocrinology 127: 1. 1990.

245 Chen Y. Rabinovitch PS. Platelet-derived growth factor, epider-

mal growth factor, and insulin-like growth factor I regulate spe-

cific cell-cycle parameters of human diploid fibroblasts in serum-

free culture. J Cell Physiol 140: 59. 1989.

246 Florini JR, Ewton Z, Roof SL. Insulin-like growth factor-I stim-

ulates terminal myogenic differentiation by induction of myogen-

in gene expression. Mel Endocrinol5: 7 18, 1991.

247 Spencer EM, Skover G. Hunt TK. Somatomedins: do they play

a pivotal role in wound healing? Prog Clin Biol Res 266: 103,

1988.

248 Skottner A. Kanje M, Jennische E, Sjogren J. Fryklund L. Tissue

repair and IGF-I. Acta Paediatr Stand 347: 110. 1988.

249 Jennische E. Skotter A. Hansson H-A. Dynamic changes in insu-

lin-like growth factor-I immunoreactivity correlate to repair

events in rat ear after freeze-thaw injury. Exp Mel Patho147: 193.

1987.

250 Osborne CK, Coronado EB, Kitten LJ, et al. Insulin-like growth

factor-II (IGF-II): a potential autocrineiparacrine growth factor

for human breast cancer acting via the IGF-I receptor: Mol En-

docrinol 3: 1701, 1989.

251 Rapraeger AC, Krufka A, Olwin BB. Requirement of heparin

sulfate for bFGF-mediated fibroblast growth and myoblast dif-

ferentiation. Science 252: 1705. 1991.

252 Colletta AA, Wakefield LM, Howell FV. et al. Anti-oestrogens

induce the secretion of active transforming growth factor beta

from human fetal fibroblasts. Br J Cancer 62: 405. 1990.

253 Miller FR. McEachern D, Miller BE. Growth regulation of

mouse mammary tumor cells in collagen gel cultures by diffusable

factors produced by normal mammary gland epithelium and stro-

mal fibroblasts. Cancer Res 49: 6091. 1989.

254 Adams EF. Newton CJ, Braunsberg H. Shaikh N. Effects of

human breast fibroblasts on growth and 17gestradiol dehydro-

genase activity of MCF-7 cells in culture. Breast Cancer Res

Treat 11: 165. 1988.

255 Hamada J, Takeichi N. Kobayashi H. Metastatic capacity and

intracellular communication between normal cells and metastatic

cell clones derived from a rat mammary carcinoma. Cancer Res

48: 5129. 1988.

256 Ren J, Hamada J. Takeichi N. Fujikawa S, Kobayashi H. Ultra-

structural differences in junctional intercellular communications

between highly and weakly metastatic clones derived from rat

mammary carcinoma. Cancer Res 50: 358, 1990.

257 Christensen L. Nielsen M, Andersen J, Clemmensen I. Stromal

22

fibronectin staining pattern and metastasizing ability of human breast carcinoma. Cancer Res 48: 6227, 1988.

258 Barsky SH, Gopalakrishna R. Increased invasion and spontaneous metastasis of BL6 melanoma with inhibition of the desmoplastic response in C57 BL/6 mice. Cancer Res 47: 1663, 1987.

259 Barsky SH, Gopalakrishna R. High metalloproteinase inhibitor content of human cirrhosis and its possible conference of metastasis resistance. J Nat1 Cancer Inst 80: 102, 1988.

260 Westley B, Rochefort H. A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell 20: 352, 1980.

261 Shimizu Y, Shaw S. Lymphocyte interactions with extracellular matrix. FASEB J 5: 2292, 1991.

262 van den Hooff A. Stromal involvement in malignant growth. Adv Cancer Res 50: 159, 1988.

263 Camps JL, Chang SM, Hsu TC, Freeman MR. Fibroblast mediated acceleration of human epithelial tumor growth in vivo. Proc Nat1 Acad Sci USA 87: 75. 1990.

264 Horgan K, Jones DL, Manse1 RE. Mitogenicity of human fibroblasts in vivo for human breast cancer cells. Br J Surg 74: 227, 1987.

265 Naor D. Coexistence of immunogenic and suppressogenic epi- topes in tumor cells and various types of macromolecules. Cancer Immunol Immunother 16: 1, 1983.

266 Enami J, Enami S, Koga M. Growth of normal and neoplastic mouse mammary epithelial cells in primary culture: stimulation by conditioned medium from mouse mammary fibroblasts. Gann 74: 845. 1983.

267 Milas L. Regulatory effects of tumor bed stroma in tumor progression and tumor response to therapy: influence of radiation. Cancer Bull 43: 25, 1991.

268 Barsky SH, Green WR, Grotendorst GR, Liotta LA. Desmoplas- tic breast carcinoma as a source of human myofibroblasts. Am J Path01 115: 329, 1984.

269 Regenass U, Geleick D, Curschellas E, Meyer T, Fabbro D. In vitro cultures of epithelial cells from healthy breast tissues and cells from breast carcinomas. Recent Results Cancer Res 113: 4,

270

271

272

273

274

275

276

277

1990. Skalli 0, Schiirch W, Seemayer T, et al. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intramediate filament proteins. Lab Invest 60: 275, 1989. El Nabout R, Martin M, Remy J, Kern P, Robert L, Laufma C. Collagen synthesis and deposition in cultured fibroblasts from subcutaneous radiation-induced fibrosis. Modification as a function of cell ageing. Matrix 9: 411, 1989. Berry J, Green BJ, Matheson DS. Modulation of natural killer cell activity in stage I postmenopausal breast cancer patients on low-dose aminogluthemide. Cancer Immunol Immunother 24: 72, 1987. Dexter DL, Calabresi P. Intraneoplastic diversity. Biochim Bio- phys Acta 695: 97. 1982. Osborne CK. Combined chemo-hormonal therapy in breast cancer: a hypothesis. Breast Cancer Res Treat 1: 12 1, 198 1. Lippman ME. Efforts to combine endocrine therapy and chemotherapy in the management of breast cancer: do two and two equal three? Breast Cancer Res Treat 3: 117, 1983. Sertoli MR, Scarsi PG. Rosso R. Rationale for combining chemotherapy and hormonal therapy in breast cancer. J Steroid Bio- them 23: 1097, 1985. Ballare C. Bravo AI, Laucella S. et al. DNA synthesis in estrogen receptor positive human breast cancer takes place preferentially in estrogen receptor-negative cells. Cancer 64: 842. 1989.

278

279

280

Bonadonna G. Does chemotherapy fulfill its expectations in cancer treatment? Ann Oncol 1: 11, 1990. Weichselbaum RR, Hellman S, Piro AJ, Nove JJ, Little JB. Pro- liferation kinetics of a human breast cancer cell line in vitro following treatment with 17fi-estradiol and I-B-o-arabinofuranosyl- cytosine. Cancer Res 38: 2339, 1978. Clarke R, van den Berg HW, Kennedy DG, Murphy RF. Oestro- gen receptor status and the response of human breast cancer cells to a combination of methotrexate and 17P-estradiol. Br J Cancer 51: 365, 1985.

281 Hug V, Johnston D, Finders M, Hortobagyi G. Use of growth stimulatory hormones to improve the in vitro therapeutic index of doxorubicin for human breast tumors. Cancer Res 46: 147, 196.

282

283

284

285

Bontenbal M, Sonneveld P, Foekens JA, Klijn JGM. Oestradiol enhances doxorubicin uptake and cytotoxicity in human breast cancer cells (MCF-7). Eur J Cancer Clin Oncol24: 1409, 1988. Toma S, Leonessa F, Coialbu T, Nicolo G, Rosso R. Effect of 17-beta-estradiol on doxorubicin cytotoxicity in human breast cancer cell culture. Anticancer Res 9: 303, 1989. Osborne CK, Kitten L, Arteaga CL. Antagonism of chemotherapy-induced cytotoxicity for human breast cancer cells by antiestrogens. J Clin Oncol7: 710, 1989. Markaverich BM, Medina D, Clark JH. Effects of combination estrogen:cyclophosphamide treatment on the growth of the MXT transplantable mammary tumor in the mouse. Cancer Res 43: 3298, 1983.

286 Emerman JT. Effects of hormonal modulation on cytotoxicity of chemotherapeutic agents in mouse mammary tumor cell cultures. Anticancer Res 8: 205, 1988.

287

288

Emerman JT, Siemiatkowski J. Effects of endocrine regulation of growth of a mouse mammary tumor on its sensitivity to chemotherapy. Cancer Res 44: 1327, 1984. Alabaster 0, Vonderhaar BK, Shafie SM. Metabolic modification by insulin enhances methotrexate cytotoxicity in MCF-7 human breast cancer cells. Eur J Cancer Clin Oncol 17: 1223. 1981.

289 Gross GE, Boldt DH, Osborne CK. Perturbation by insulin of human breast cancer cell cycle kinetics. Cancer Res 44: 3570, 1984.

290

291

Bontenbal M, Sieuwerts AM, Klijn JGM, et al. Effect of hormonal manipulation and doxorubicin administration on cell cycle kinetics of human breast cancer cells. B J Cancer 60: 688. 1989. Lippman ME, Cassidy J. Wesley J, Young RC. A randomized attempt to increase the efficacy of cytotoxic chemotherapy in metastatic breast cancer by hormonal synchronization. J Clin Oncol 2: 28, 1984.

292

293

294

Yang KP. Samaan NA. Enhancement of hormonal synchronization and 5-fluorouracil cytotoxicity in breast cancer cells by low concentrations of thymidine. Anticancer Res 7: 59, 1987. Brunner N, Bronzert D, Vindelov LL, Rygaard K, Spang-Thom- sen M, Lippman ME. Effect of growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro in nude mice. Cancer Res 49: 1515, 1989. Conte PF, Fraschini G, Alama A, et al. Chemotherapy following estrogen-induced expansion of the growth of human breast cancer. Cancer Res 45: 5926, 1985.

295 Conte PF. Alama A, Di Marco E, Canavese G, Rosso R, Nicolini A. Cytokinetic parameters of locally advanced human breast cancer treated with diethylstilbestrol and chemotherapy. Basic Appl Histochem 30: 227, 1986.

296 Conte PF, Alama A, Bertelli G, Canavese G. Chemotherapy with

23

estrogenic recruitment and surgery in locally advanced breast

cancer: clinical and cytokinetic results. Int J Cancer 40: 490, 1987.

297 Clarke R, van den Berg HW, Kennedy DG, Murphy RF. Reduc-

tion of the antimetabolic and antiproliferative effects of metho-

trexate by 17j%estradiol in a human breast carcinoma cell line

(MDA-MB-436). Eur J Cancer Clin Oncol 19: 19, 1983.

298 Goldenberg GJ. Froese EK. Antagonism of the cytocidal activity

and uptake of melphalan by tamoxifen in human breast cancer

cells in vitro. Biochem Pharmacol34: 763, 1985.

299 Haslam SZ, Shyamala G. Relative distribution of estrogen recep-

tors among epithelial, adipose and connective tissues of normal

mammary gland. Endocrinology 108: 825, 1981.

300 Goldstein LJ, Galski H, Fojo A. et al. Expression of a multidrug

resistance gene in human cancers. J Nat1 Cancer Inst 81: 116,

1989.

301 Salmon SE, Grogan TM, Miller T. Scheper R, Dalton WS. Pre-

diction of doxorubicin resistance in vitro in myeloma, lymphoma

and breast cancer by P-glycoprotein staining. J Nat1 Cancer Inst

81: 696. 1989.

302 Kacinski BM, Yee LD, Carter D, Li D, Kuo MT. Human breast

carcinoma cell levels of MDR-I (P-glycoprotein) transcripts cor-

relate in vivo inversely and reciprocally with tumor progesterone

receptor content. Cancer Commun 1: 1, 1989.

303 Kohono K, Sato S, Takano H. Matsuo, K, Kuwano M. The

direct activation of human multidrug resistance gene (MDRI) by

anticancer agents. Biochem Biophys Res Commun 165: 1415.

1989.

304 Ramu A, Glaubiger D, Fuks Z. Reverdl of acquired resistance to

doxorubicin in P388 murine leukemia cells by tamoxifen and

other triparanol analogues. Cancer Res 44: 4392, 1984.

305 DeGregorio MW, Ford JM. Benz CC, Wiebe VJ. Toremifene:

Pharmacological and pharmacokinetic basis of reversing multi-

drug resistance. J Clin Oncol 7: 1359, 1989.

306 Chatterjee M, Harris AL. Reversal of acquired resistance to

adriamycin in CHO cells by tamoxifen and 4-hydroxytamoxifen:

role of drug interaction with alpha 1 acid glycoprotein. Br J Can-

cer 62: 712, 1990.

307 Yang, C-PH, DePinho SG, Greenberger LM, Arceci RJ, Horwitz

SB. Progesterone interacts with P-glycoprotein in multidrug-res-

istant cells and in the endometrium of gravid uterus. J Biol Chem

264: 782, 1989.

308 Greenberg DA, Carpenter CL, Messing RO. Calcium channel an-

tagonist properties of the antineoplastic antiestrogen tamoxifen

in the PC12 neurosecretory cell line. Cancer Res 47: 70, 1987.

309 Lenaz G, Curatola G, Mazzanti L. Parenti-Castelli G. Biophysi-

cal studies on agents affecting the state of membrane lipids: bio-

chemical and pharmacological implications. Mol Cell Biochem

22: 3, 1978.

310 Ennis BW, Valverius EM, Bates SE. et al. Anti-epidermal growth

factor receptor antibodies inhibit the autocrine-stimulated

growth of MDA-468 human breast cancer cells. Mol Endocrinol

3: 1830. 1989.

311 Harris AL. The epidermal growth factor receptor as a target for,

therapy. Cancer Cells 2: 32 1, 1990.

312 Chaudhary VK, Fitzgerald S. Adhya S. Pastan 1. Activity of a

recombinant fusion protein between transforming growth factor

type alpha and Pseudomonas toxin. Proc Nat1 Acad Sci USA 84:

4538. 1987.

313 Heimbrook DC, Stirdivant SM. Ahern JD. et al. Transforming

growth factor alpha-Pseudomonas exotoxin fusion protein pro-

longs survival of nude mice bearing tumor xenografts. Proc Nat1

Acad Sci USA 87: 4697, 1990.

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Hormonal aspects of breast cancer: Growth factors, drugs and stromal interactions