Autocrine and paracrine growth regulation of human breast cancer

J. sreroid Biochem. Vol. 24. No. I. pp. 147-154. 1986 0022-473 1186 $3.00 + 0.00 Printed in Great Britain Pergamon Press Ltd

AUTOCRINE AND PARACRINE GROWTH REGULATION OF HUMAN BREAST CANCER

MARC E. LIPPMAN, ROBERT B. DICKSON, ATTAN KASID. EDWARD GELMANN, NANCY DAVIDSON,

MARY MCMANAWAY, KAREN HUFF, DIANE BRONZERT, SUSAN BATES,

SANDRA SWAIN and CORNELIUS KNABBE Medical Breast Cancer Section, Medicine Branch, National Cancer Institute, Bethesda, MD 20205, U.S.A.

Summary-Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vim. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells. facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation. hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.

Data derived from epidemiologic and clinical observations as well as experimental evidence data has established the central role of estrogens in the genesis of human breast cancer [I]. When estrogenic stimulation does not occur (for example, in the case of primary ovarian failure), the incidence of breast cancer is only 1% of that in normal women. Normal breast development requires physiologic concen-

trations of estradiol. For nearly a century it has been known that some human breast cancers will regress following some for of endocrine therapy; however, by the time breast cancer has reached the stage of overt metastatic disease it retains the hormone-dependent phenotype of the normal breast approx. one-third of the time. The proportion of clinically-evident hormone-dependent primary breast cancers is unknown. As tumors evolve (particularly in the face of selective pressures exerted by systemic therapy), an increasing number become hormone independent. Multiple lines of investigation all suggest that estrogens are critically responsible for continued growth of these hormone-dependent breast cancers. Removal of sources of estrogens such as ovary and adrenal or therapy with antiestrogens results in worthwhile clinical responses. Response to these endocrine therapies is highly associated with the presence of specific cellular receptors for estrogens-a

point in favor of a direct interation of hormone with tumor. Administration of physiologic doses of estrogens to castrate or postmenopausal women can stimulate tumor proliferation in zko. While all of these data clearly demonstrate that estrogens are mitogens for human breast cancer (BC) they do not elucidate the exact mechanism of their action nor even prove that estrogens act directly on tumor cells to stimulate their growth.

In this review of some of our recent work we will present some information which supports the following three hypotheses:

I. Estrogens directly interact with human breast cancer (BC) cells and can directly alter gene expression at the level of messenger RNA concentrations. This results in increased activity of many enzymes involved in cell growth as well as increased activities of other intracellular proteins such as progesterone receptor.

II. One aspect of these effects on gene expression induced by estrogens results in alterations in specific proteins secreted by hormone- responsive BC cells. Some of these secreted proteins serve as autocrine and paracrine growth regulatory activities with multiple

147 sa 24,1&K

148 MARC E. LIPPMAN et al.

actions including: initiation of growth of quiescent cells; further stimulating growth of cells already in proliferative cycle; and finatty, induction of secretion of factors capable of causing reversible acquisition of the malignant phenotype in nontransformed cells.

Taken collectively, these growth factors are responsible for estrogen-mediated tumor progression.

XII. The mechanism by which hormone responsive breast cancer is converted to the hormone is by increased and nonho~~o~ally regulated (constituitive) secretion of a similar or identical collection of autocrine and paracrine growth factor activities.

In order to test these hypotheses we initially developed a suitable in ritro model system for human breast cancer. This preiiminary requirement must be satisfied because of two inherent difficulties in per- forming hormone manipulations in Z&J. First, in Z&Z it is impossible to alter the concentration of a hormone such as estradiol without altering the activities or concentrations of many other hormones or potential growth factors. Furthermore, if one hypothesizes that there is an unknown substance, secreted by some other tissue in the body which is under estrogenic control and which then alters breast cancer growth (a putative estromedin) [2,3]. it follows that an dn vitro system will not respond to estrogen directly. How- ever, once such putative estromedins were isolated, cells in culture would provide a suitable assay system. Second. even if a stimulatory or inhibitory response is observed in a tumor in oiw following a hormonal man~~ulat~on~ this may not represent a direct effect of the substance on the tumor cefls directly. An inter- vening or mediating effect on the immune system or supporting stroma or vasculature is just as plausible as a direct effect on the tumor cells and may be responsible for altered tumor behavior. In certain circumstances, such as androgen-induced mammary gland regression, an indirect mechanism involving effects on surrounding stroma has been shown to be the cause of hormonal responses &r oit~~ [4].

If one established an in u&a system which directly responded to estrogens (and antiestrogens) including direct assessment of the products regulated and the mechanisms involved an unequivocal validation of Hypothesis i would be possible. This also can provide part of the assay system for assessment of the growth- promoting properties of any secreted proteins (Wy- pothesis II and III). An ideal in aigro mode1 system would permit development of variant or mutant sell lines deficient or amplified with respect to growth factor production or response to further test the above hypotheses, Finally, in order to validate the causative role of secreted growth factors for in vim tumorigenesis it is essential to be able to reversibly transfer the in &TO system to an ipl ri~o milieu in which host interactions are integrated with the direct

responses of tumor ceils observed in aitro. In this review, our studies along these several lines of investigation will be summarized.

We have employed cloned lines of human BC cells. The human and mammary nature of these cells has been extensively discussed previously [S]. These cell lines respond to direct stimulation with physiologic concentrations of estradiol by increasing their rate of proliferation 16, 71. These results can be duplicated in defined medium devoid of unknown serum constituents [S]. Potent ant~~trogens are capable of inhibiting estrogenic stimulation. The effects of antiestrogens are specific in that only celi lines containing estrogen receptor respond, antiestrogen effects are prevented by simultaneous estrogen administration or reversed by subsequent estradiol administration. This latter effect has generated successful clinical trials in patients with breast cancer 19, 101. While some initial controversy arose because of difficulties of others in obtaining direct effects of estrogens on breast cell growth in rino [l 1,121; subsequently, nu- merous groups have succeeded in confirming that E, can serve as a proximate mitogen for human breast cancer [13-243. We do not wish to imply from this observation that estromedins are an impossibility. Rather that at best they play a supporting role. ~hys~olog~~ concentrations of estradiol have been shown to promote nucleoside incorporation into DNA. increase the thymidine labeling index, net DNA synthesis and cell number. Estradiol induces a large number of enzyme activities specifically involved in nucleic acid synthesis, including DNA polymerase, thymidine and uridine kinases, thy- midylate synthetase, carbamyl phosphate synthetase, aspartate transcarbamyla~, dihydroorotase and dihydrofolate reductase [X,26]. We have demonstrated that physiologic concentrations of estradiot stimulate DNA synthesis by both scavenger and cte nom bio- synthetic pathways. We have also shown that estrogens regulate both thymidine kinase and dihydrofolate reductase mRNA concentrations [27-29].

Aside from regulation of these essential growth regulatory enzymes, estrogens (and antiestrogens) alter the activity of sev*eral other proteins whose function remains unknown. These include progesterone receptor [303, plasminogen activator f31] and several secreted proteins including a 24 kdalton protein described by Ciocca et (r1.[32], 52 and 160 kdalton glycoproteins described by Westley and Rochefort[33] and a 7 kdalton protein identified initially by Sakowlew er al. by detection of an ~trog~n-indexed mRNA species using differential hybridization techniques [34]. The activity of B-2 mRNA has been shown to be regulated by estradiol at the level of transcription by direct assay. The exact function of these latter secreted proteins is unknown although several lines of experimental evidence have suggested that they do not function as major autostimulatory growth factors. Most notably, PS-2 mRNA and the 52 kdalton glycoprotein of Westley

Growth regulation of human breast cancer 149

and Rochefort are decreased by antiestrogens to the same extent in LY2 (a stable antiestrogen-resis~nt variant of MCF-7 cells). This suggests that their absence does not diminish ceil growth. Similarly, in Ii 3 (an MCF-7 cell variant we have isolated which is killed by physiologic concentrations of estradiol), PS-2 mRNA, as well as the 52 kdalton giycoprotein are induced normally.

We have however used related strategies of differential hybridization to identify several estrogen- induced genes. Our conditions of short-term estradiol stimulation prior to nucleic acid isolation (6 h) were designed to increase the likelihood of obtaining non- abundant genes induced at early time points by estradiol. Our preliminary results have been successful 1351. Thus, while estrogens may possibly affect many tissues of the body which may in turn indirectly alter breast cancer progression, direct effects of estrogens on isolated BC ceils including important growth regulatory effects are well- established, placing Hypothesis I on a firm footing.

We turn next to Hypothesis II-the regulation of growth through estrogenic regulation of secreted factors. Previous observations by Todaro and Sporn led them to propose that important aspects of growth control of tumors were mediated by cancer-cell- secreted factors which could either be autostimulatory (autocrine factors) or stimulatory of surrounding tissues such as connective tissue or vasculature (paracrine factors) [36]. Either of these classes of putative growth factors could be under endocrine control or released constituitively. Obvi- ously, endocrine stimulation of breast cancer growth need not necessarily involve such paracrine or autocrine mechanisms at all.

Three sets of observations suggested that such secreted growth factor activities could be important in the pathogenesis and progression of human breast cancer.

First, we and others have shown growth regulation and other phenotypic responses of BC cells to a long list of other trophic substances aside from estrogens. These include giucocorticoids, androgens, progestins, iodothyronines, vitamin D, retinoids, epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), calcitonin and prolactin [37]. This muiti- plicity of effector molecules suggested that some estrogenic responses in vivo could be modulated by effects of these other growth factors. Alternatively, this large number of exogenous hormones to which the BC cells responded suggested that similar or identical growth factors could be elaborated by the breast cancer itself.

Second, MCF-7 human breast cancer cells grown in vitro in cell culture have a qualitatively different response to estrogens from MCF-7 cells implanted in viuo in a nude mouse model system. Under the former set of conditions estrogens can directly stimulate proliferative rate but the ceils will grow in the absence of estradiol. When these cells are supplemented with

optimal concentrations of serum and insulin and other growth factors, estradiol will have little if any additional growth-promoting properties, This observation has incorrectly been interpreted by others as evidence against estrogenic responses of the BC cells. However, in z&o, in the ovariectomized athymic nude mouse, estradiol has been an absolute requirement for tumor growth [26,38,39]. Huseby et al. have further defined this system by showing that estradiol need not enter the systemic circulation in nude mice to promote MCF-7 cell tumor fo~ation; elevation of local estradiol concentration near the tumor was sufficient [40]. This suggested that although estrogens might be required to induce other mediating factors required by the tumor (or host) for tumorigenesis, the production and action of such factors is probably restricted to the local area of the tumor. These observations argue strongly against the view that estromedins (estrogen-induced products acting as endocrine factors) are of substantia1 importance in estrogen-evoked tumor formation since systemic administration of estradiol was not required.

Third, we observed that the initial growth rate of BC cells in culture was proportional to the number of cells plated [41]. While there are multiple in- terpretations of this finding, they are consistent with the production of autostimulatory growth factors. Work with 3T3 cells has also supported such a hypothesis [42]. In a series of preliminary experiments we found that conditioned medium prepared from human BC cell lines was capable of stimulating proliferation of identical or other BC ceil lines as compared with replenishment of cells with fresh, unconditioned medium. At the same time, Vignon et al. had also observed that MCF-7 cells showed enhanced stimulation by estradiol when medium was exchanged less frequently [43]. This also led them to hypothesize that estrogens acted, at least in part, by inducing secretion of autostimulatory growth factors which were diluted out by more frequent medium changes. These investigators also supported this hypothesis by crude conditioned medium experiments.

To identify and purify the components secreted by BC cells required both for autostimulation and in viva tumorigenesis it was necessary to develop a serum- free culture system which supported the growth of hormone-responsive and hormone-unresponsive human BC cell lines. We took advantage of previous work by Barnes and Sato[8, 131. We found that Richters Improved Minimal Essential Medium supplemented with 2mg/l transferrin and 2 mgjl ~bronectin supported cell growth for at least a week. Cells were grown in this medium with or without 10m9M estradiol for 4 days; subsequent 2-day collection periods in fresh identical medium were used as source of secreted protein activity. These collected media were harvested, filtered to remove cellular debris, dialyzed extensively against 1 M acetic acid, lyophilized and reconstituted in phosphate-buffered saline. This extraction methodology removes

150 MARC E. LIPPMAN et ul.

>99.98% of the residual estradiol as determined by studies with radiolabeled steroid or direct radioimmunoassay. This extract was the starting material for biological assays and purification procedures to be described. It was also the material used for in viva

infusions into ovariectomized athymic nude mice to stimulate tumor growth to be described later.

We initially examined BC cell line conditioned medium for production of IGF-I (somatomedin-C). We chose IGF-I because previous work had shown that some BC cell lines responded to IGF-I and had IGF-I receptors on their surface [44]. Using a sensitive and specific radioimmunoassay for IGF-I, all of the human BC cell lines we tested (MCF-7. ZR-75-1, MDA-MB-23 1, Hs578T) produced IGF-I immunoreactive material 1451. Secreted levels were as follows: MCF-7. 11.6 ng/ml 65 pg/pg DNA; ZR-75-1, 9.7 ng/ml, I03 pg!pg DNA; MDA-MB-23 1, 30.3 ng/ml 342 pg/pg DNA; Hs578T, 48.8 ng/ml, 534pg/pg DNA. The first two cell lines, both estradiol responsive, produced substantially less IGF-I than the latter two estrogen-independent cell lines. Estradiol did not stimulate IGF-I secretion, though glucocorticoids and antiestrogens. which both inhibit the growth of MCF-7 cells, strongly inhibit IGF-I secretion. All four cell lines respond to exogeneous authentic IGF-I with enhanced proliferation. Using acid-extracted medium, IGF-I migrates on a Biogel P60 column as a high molecular weight species but upon acid-ethanol extraction this high molecular weight immunoreactive material is converted to a lower molecular weight activity which comigrated with authentic IGF-I standard. In studies we have performed in collaboration with Kenneth Gabbay (Baylor College of Medicine) we have shown that a human cDNA probe for IGF-I identifies a single mRNA species identical in size to that seen in nontransformed human fibroblasts, thus confirming that human BC cells synthesize and secrete authentic IGF-I.

In studies with both unfractionated conditioned medium and after chromatography on Biogel P60 columns it was observed that conditioned medium derived from estrogen-treated hormone-responsive MCF-7 cells contained 3-8 times the autostimulatory activity of medium derived from nonestrogen-treated cells. Furthermore, since IGF-I is not estrogen induced in MCF-7 cells and since estrogen treatment was necessary for in oivo tumorigenesis with MCF-7 cells, it appeared likely that other activities were contributory to tumor growth.

Epidermal growth factor (EGF) has been shown to stimulate the growth of human BC cells (461. There- fore, we next examined these cell lines for the production of EGF-related peptides. We used A431 human carcinoma cells as a source of EGF receptor for radioreceptor assays of conditioned medium [47]. All of the human BC cell lines tested produced material which competed with iodinated EGF for binding to EGF receptor. None of the several peaks

of activity (30 kdalton to I’,) corresponded to the position of authentic iodinated EGF after cofrac- tionation on Biogel P60 columns. Depending on assay conditions estradiol induced a 4- to 6-fold increase in EGF receptor-reactive material in unfractionated conditioned medium. whereas after purification apparent fold of induction by estradiol rises considerably. It is not clear at this time whether this increase represents greater inactivation of EGF- related peptides in control cells during purification or removal of binding moieties of other inhibitory substances from estrogen-stimulated cells. Both EGF and transforming growth factor cc(TGFcc) are closely related peptides which apparently act through a single receptor (conventionally termed the EGF receptor). The principal peak of activity, ++ 30 kdalton however did not correspond to the reported molecular weight of sequenced TGFcr. While transforming growth factors may have other activities, they have been defined by their ability to induce anchorage- independent growth (colonies in soft agar) in nontransformed fibroblasts [47]. Using this clonogenic assay, MCF-7 cells had been reported to secrete transforming activity [48]. Therefore we assayed conditioned medium from human BC cell lines for TGF activity using normal rat kidney (NRK) fibroblast colony fo~ation in soft agar as an endpoint. At least two classes of TGF activity exist. We first employed assay conditions which predominantly measure TGFcr-like activity. TGFa-like peptides (by definition) compete with EGF for binding to EGF receptor. TGFa-like peptides contain sequence homology with EGF but are not recognized by antiEGF antibodies. AntiEGF receptor antibodies which also block EGF binding block TGFr activity 1491.

A second class of complementary TGF activity. TGFfi has been described [SO, 511. This peptide does not bind to EGF receptor. Both TGFcl and TGFjI are acid- and heat-stable peptides which are sullhydryl reagent sensitive. TGFa and TGFB are both required for anchorage-independent growth of NRK cells: however, in the assay conditions we employed, TGF$ activity was supplied so that we measured estrogen induction of either TGFa or an activity which could replace it. All BC cell lines tested except Hs578T produced colony-stimulating activity [.52]. Once again, hormone-independent (in this case MDA-MB- 23 1) cells produced substantially more TGFr activity than the hormone-responsive MCF-7 and ZR-75-l cells. Using this assay TGFa-like activity was induced up to 8-fold by estradiol treatment of MCF-7 cells. As was described for the autostimulatory activity in conditioned medium, fractionation of EGF receptor-reactive material leads to greater fold apparent induction of transforming activity by estradiol in hormone-responsive BC cells. Whether this activity is due to authentic TGFa or some alternative EGF receptor-reactive material is not as yet clear. Studies with a cDNA probe for human TGFa should help to

Growth regulation of human breast cancer 151

resolve whether the activity produced in MCF-7 cells is closely related to authentic TGFcr.

In order to assay TGFB we used A549 human carcinoma cells as a source of TGF/3 receptor for a radioreceptor assay [53]. In collaboration with Mi- chael Sporn and his colleagues at the NIH we have found that all of the human BC cell lines tested secrete TGFP receptor-reactive material. As with IGF-I, the two ho~one-unresponsive cell lines, Hs578T and MDA-MB-231, produced the highest concentrations of TGFB activity. An important point to be borne in mind is that although the TGFs are named (and defined) by their ability to induce anchorage-independent growth of nontransformed cell lines, this may not be their only important activity. For example, Hanauske et al. have recently shown that authentic TGFor can stimulate clonogenic growth of tumor samples of breast and other neoplasms[54] and, Sporn et al. and Halper and Moses have shown that TGFji is a growth inhibitor for certain established tumor cell lines including MCF-7 as well as having activity in bone resorption [5.5,56] and wound healing assay [57,58]. We have found that TGFfi activity as measured by radioreceptor assay in conditioned medium is increased by estrogen withdrawal in MCF-7 cells and stimulated still further by antiestrogen treatment. Glucocorticoids, which also inhibit cell growth also enhance TGFP secretion. Interestingly, a variant of MCF-7 which we have selected for growth resistance to antiestrogens secretes lower concentrations of TGFB and antiestrogens do not increase TGFP secretion. Whether or not this is the mechanism by which these cells escape antiestrogen inhibition is under investigation. Recently, in collaboration with Sporn and colleagues we have been able to show that a cDNA for human TGFfl identifies a single mRNA species of identical size to that found in other human tissues establishing that these human BC cells are a source of apparently authentic TGF&

Despite these data demonstrating TGFct-like and TGFfi activity, our column chromatography work still suggested substantial additional areas of autostimulatory activity produced by the cells. We therefore pursued the other possibility that the BC cells were producing other classes of growth factor activities.

Growth factors may be divided into at least two classes: competency and progression [57] defined operationally by their ability to stimulate quiescent contact inhibited, nontransformed fibroblasts. These cells will undergo a single wave of DNA synthesis if refed with fresh serum containing medium. If instead, serum derived from platelet poor plasma is used, no stimulation is seen. Reconstitution of platelet poor plasma with an extract derived from platelets led to growth stimulation. Platelet-derived growth factor (PDGF) the active moiety later purified from platelets was only required for 30 min after which it could be removed and the continuous presence of platelet-

poor plasma would be sufficient for subsequent DNA synthesis 24 h later. Much of the effect of the platelet- poor plasma could be replaced by EGF. Thus in such an assay system PDGF would be defined as a competency factor, EGF as a progression factor. Neither IGF-I, EGF or TGF can function as competency factors. We established an assay for competency factors produced by human BC cells using contact- inhibited fibroblasts and platelet-poor plasma. This assay was responsive to either exogenously-added PDGF or fibroblast growth factor (FGF) another competency factor. All of the human BC cell lines secreted competency activities. Studies aimed at identification of these activities are underway.

In preliminary work it appears that this competency activity is not stimulated by physiologic concentrations of estradiol in ho~one-responsive BC cells. Furthermore, basic FGF is not acid stable and probably would not be resistant to our extraction procedures. Whether or not the competency activity produced is PDGF will be established by radioimmunoassay and radioreceptor assay.

However. because the competency-related. secreted activity was not estrogen enhanced in hormone- responsive human BC cells, we believed that additional autostimulatory activities remained to be identified.

Moses and colleagues have recently described an adrenal carcinoma cell line SW-13 whose growth in soft agar is stimulated by conditioned medium from various cancer cell lines and extracts from primary tumors [58]. Its spectrum of response is such that FGF is strongly stimulatory whereas insulin, IGF-I. EGF, TGFLY and TGFB are inactive. We have employed this assay and found that BC cell lines are secreting a high molecular weight, acid-stable activity which is highly stimulatory of SW-13 colony formation. Whether our competency assay and SW-13 colony formation assay are scoring distinct or over- lapping growth factors is currently under study. Preliminary experiments suggest that peaks of competency activity and SW-13 growth stimulatory activity are distinct. This suggests that these may represent a separable class of carcinoma-acting transforming growth factor activity.

The studies summarized above clearly demonstrate that human BC cells secrete multiple, discrete growth factors at least some of which are clearly under estrogenic control. Obviously a great deal of effort will be required to completely characterize each of these separable growth stimulatory activities as well as other autocrine or paracrine activities which may be secreted as well.

Naturally, one would like to explore the physiologic significance (if any) of these observations. In order to pursue this issue, we took advantage of the observation, previously alluded to, that hormone- responsive MCF-7 cells would not form tumors in female, oophorectomized nude mice unless the animals were supplemented with estrogen pellets


[26,38,39]. Conditioned medium from MCF-7 cells treated or not with estradiol was centrifuged, acid extracted, extensively dialyzed and lyophilized; >99.98% of the estradiol is removed by this tech- nique. Reconstituted concentrated conditioned medium and conditioned medium derived from estrogen-treated cells were infused into female oophorectomized nude mice using Alzet mini- pumps. The equivalent of lOm1 of conditioned medium or conditioned medium from estrogen- treated cells was infused daily for 4 weeks from a middorsal S.C. location. The pumps were replaced with fresh pumps after 2 weeks; 2-5 x lo6 MCF-7 cells were injected into each of 4 different mammary fat pad locations in each mouse. Up to 0.5 cm tumors appeared at MCF-7 sites within 2 weeks. Tumors in animals infused with conditioned medium from nonestrogen-treated cdls appeared with a frequency of 1 I%, while conditioned medium from estrogen- treated cells supported tumors with a frequency of 25%. Animals infused with concentrated control medium had a tumor incidence of ~5%. Condi- tioned medium and conditioned medium from estrogen-treated animals stimulated tumor growth for 2-3 weeks after which time tumors usually declined in size. in contrast, animab treated with estradiol directly showed a continuous rise in tumor incidence to about 85% with continuous growth of all tumors. Reimplantation with pumps containing fresh conditioned medium did not prevent this regression in tumors induced by conditioned media. Histologic examination prior to regression revealed aden~arcinomas which were mo~hologicaiiy identical to tumors found in animals treated with an estrogen pellet instead of the conditioned medium. We do not as yet know why the adenocarcinomas induced by conditioned medium are not maintained and eventually regress. At least three nonmutually exclusive explanations are possible. Growth factors may not all be present in sufficient concentration when administered by an endocrine route. Alter- natively, during the purification of conditioned medium labile components may be eliminated. Finally, conditioned medium may possibly not replace some other action of estradiol on other tissues, for example natural killer cell activity. Conditioned medium from estrogen-treated animals had no effect on ovariectomized mouse uterine weight or histology. Conditioned medium from estrogen-treated animals was inactivated by trypsin treatment, reducing agents and heating at 56°C for 1 h. These observations together with direct measurements of estradiol concentration in the conditioned medium eliminate the possibility that the enhanced effects of conditioned medium from estrogen-treated animals was due to contaminating estradiol. The failure to see stimulation of uterine weight suggests that the peptides produced upon estrogen stimulation do not function as generalized estromedins but retain tumor specificity even when administered by an endocrine

route as opposed to their usual diffusion limited effects. These results also clearly suggest that a substantial fraction of estrogen-induced tumor growth is mediated at the level of alterations in secreted polypeptides growth factors. This has obvious potential therapeutic implications. For example, antibodies directed against either the secreted growth factors or their receptors might be expected to alter rates of tumor progression. The possibility of preparing synthetic growth factors with altered properties would also appear viable.

An additional implication of these observations is that a plausible mechanism by which breast tumours may become hormone independent is through loss of hormonal regulation of growth factor production. If acquisition of the ability to constitutively synthesize growth factors is an important m~hanism for loss of hormone dependence then therapies similar to those just mentioned might be equally effective for hormone-independent breast cancers.

Support for this viewpoint is provided by some recent experiments in which we have altered the hormone dependency of MCF-7 human BC cells by activated oncogenes introduced by DNA-mediated gene transfer (591. We transfected the cells with the ras oncogene derived from the Harvey sarcoma virus (v-rasH) by a coselection methodology using a bac- terial gene as a selectable gene marker. Stable transfected MCF-7 cells were obtained in which several copies of the v-rasH gene were integrated into the host cellular DNA. These cells (MCF-7,,) expressed v- ras’ mRNA and has detectably phosphorylated P21 (the protein which is the ras gene product). P21 was identified by direct immunoprecipitation (in studies done in collaboration with Douglas Lowy. NCI). MCF-7,,, (as compared with MCF-7 cells transected only with the selectable gene encoding the bacteria1 xanthinephosphoribosyl-transferase enzyme activity-MCF-7,,,) was essentially unresponsive to antiestrogens in vitro. These cells continue to have high-a~nity estrogen receptors which are functional in that addition of physiologic quantities of estradiol results in the expected induction of progesterone receptor. Unlike wild-type MCF-7 cells or MCF-‘7,,, which absolutely require estrogen supplementation for tumorigenesis in nude mice, MCF-7,,, formed tumors with about 85% efficiency in oophorectomized female athymic nude mice. Thus far, using MCF-7,, as a control, none of 10 innoculations have resulted in tumors in ovariectomized nude mice whereas 6 of 8 MCF-7ppl innocula grew into tumors in estradiol-supplemented animals. These data are similar to those obtained with wild-type MCF-7 in which 0 of 20 gave rise to tumors in ovariectomized animals and 8 of 12 innocula produced tumors in estrogen supplemented animals. The results with MCF-‘I,,, cells were very different: 36 of 42 innocula formed tumors in ovariectomized animals which is not different from the 10 tumors formed as a result of 12 tumor injections in estradiol-supplemented

Growth regulation of human breast cancer 1.53

animals. These effects are not due to adventitious estradiol in the system as uterine wet weight does not change in ovariectomized animals injected with MCF-7,,, cells nor does a sensitive and specific radio; immunoassay detect significant quantities of estradiol. The ras oncogene thus conferred hormone independence on the tumorigenecity of the MCF-7,,, C&S.

We have begun studies on the mechanism of the development of hormone independence by MCFJ,,. Conditioned medium prepared from MCF-7,,, con- tains 3- to d-fold increases in EGF receptor-reactive material and anchorage-independent growth- promoting activity for fibroblasts (TGFc(-like) as compared with wild-type cells. A single peak of EGF receptor-reactive material with transforming activity was eluted at an apparent molecular weight of about 30 kdaiton after acid gel chromatography of MCF- 7,,, conditioned medium. In addition, both IGF-I radioimmunoassayable material and TGFfi activity (as measured by radioreceptor assay) were also aug mented 3- to d-fold in MCF-7,,, cells. Thus ras gene activation appears to induce phenotypic activities and tumorigenic changes which are induced in wild-type MCF-7 cells only after estrogen stimulation.

In conclusion we have provided evidence that human BC cells secrete a collection of separable growth factors (IGF-I, TGFcc-like material, TGFB, a competency factor and an additional epithelial anchorage-independent growth-promoting activity). Some of these activities are estrogen inducible. Taken together, they are capable of promoting in z@va tumorigenesis of hormone-dependent BC ceIIs in nude mice though not to the same extent as estrogen treatment of the mice. Hormone-independent BC cells, whether spontaneously arising in culture or constructed by DNA-mediated gene transfer, appear

to secrete such growth factors in increased amounts. Such an externalized system of autocrine and paracrine cell regulation obviously suggests multiple inter- ventions which may eventually have clinical applica- bility in the control of neoplastic breast cancer

growth.

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Autocrine and paracrine growth regulation of human breast cancer

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