Annals of Oncology 2: 169-182, 1991. C 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Review Expression of transforming growth factor alpha (TGFa) in breast cancer F. Ciardiello, N. Kim, M. L. McGeady, D. S. Liscia, T. Saeki, C. Bianco & D. S. Salomon Laboratory of Tumor Immunology and Biology, Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, U.S.A.

Summary. Transforming growth factor alpha (TGFa) is one growth factor that has been circumstantially implicated in regulating the autocrine growth of breast cancer cells. Expression of TGFa can be modulated by activated cellular protooncogenes such as ras and by estrogens. For example, the epidermal growth factor (EGF)responsive normal NOG-8 mouse and human MCF-10A mammary epithelial cell lines can be transformed with either a point-mutated c-Ha-ras protooncogene or with a normal or point-mutated c-neu (erbB-2) protooncogene. In ras transformed NOG-8 and MCF-10A cells but not in neu transformed cells there is a loss in or an attenuated response to the mitogenic effects of EGF. This response may be due in part to an enhanced production of endogenous TGFa that is coordinately and temporally linked to the expression of the activated ras gene and to the acquisition of transformation-associated properties in these cells. TGFa mRNA and TGFa protein can also be detected in approximately 50-70% of primary human breast tumors. In addition, approximately 2- to 3-fold higher levels of biologically active and immunoreactive TGFa can also be detected in the pleural effusions from breast cancer patients as compared with the TGFa levels in the serous effusions of noncancer patients. Overexpression of a full-length TGFa cDNA in NOG-8 and MCF-10A cells is capable of transforming these cells. Finally, expression of TGFa mRNA and production of biologically active TGFa protein is also found in normal rodent and human mammary epithelial cells. Key words: estrogens, mammary gland, neu, ras, transformation, TGFa Introduction Breast cancer is second only to lung cancer in its occurrence in women. In this respect, 1 out of 11 women will ultimately develop some form of breast cancer during her life [1,2]. Therefore, delineation of the genetic, molecular, and biochemical events that may be responsible for the initiation and transformation of mammary epithelial cells is crucial for understanding the etiology and pathogenesis of this disease. A number of genetic and environmental risk factors for breast cancer development have already been identified such as a family history of breast cancer, early menarche, marital status, nulliparity, increased dietary intake of saturated fats and subsequent development of obesity and late onset of menopause [2,3]. Several of these risk factors may indirectly relate to or influence the circulating levels of various mammotrophic hormones such as estrogen, progesterone, and prolactin. In breast cancer patients, approximately 50% of breast tumors contain estrogen receptors (ER) and/or progesterone receptors (PgR) of which half of these tumors will respond to some sort of adjunct endocrine therapy such as antiestrogens [2,4]. Estrogens are significant in the pathophysiology of the mammary gland since they are known to be essential for regulating the growth and differentiation of both

normal and malignant mammary epithelial cells and possibly surrounding stromal cells [5]. In addition, estrogens may function as tumor promoters in facilitating the development of breast cancer in mice or rats which have previously been exposed to chemical carcinogens such as dimethylbenz(a)anthracene (DMBA) or nitrosomethylurea (NMU) or viruses such as mouse mammary tumor virus (MMTV) [6-8]. It is still unclear as to the mechanism (s) by which estrogens can modify these cellular processes. One possible way by which estrogens might function as mitogens is through their ability to induce either locally within the mammary gland or systemically the synthesis and secretion of growth factors which could function in either an autocrine and/or a paracrine manner [5,9,10]. This possibility is particularly attractive since it is known that specific cellular protooncogenes have the capacity to code for proteins that are growth factors (e.g., c-sis/B chain of platelet-derived growth factor [PDGF]), proteins that are growth factor receptors (e.g., c-er£>Z?/epidermal growth factor [EGF] receptor [EGFR]) or proteins that may function in the intracellular signal transduction pathway for growth factors as coupling factors or effectors for growth factor receptors (e.g., c-ras, c-raf) or as transcriptional activating factors (e.g., c-fos and c-jun) [11-17]. Alternatively,

170 protooncogenes may indirectly regulate the level of expression of different endogenous cellular growth factors or their receptors or the response of cells to growth factors [18-21]. Protooncogenes have been demonstrated to be important in regulating various aspects of cellular growth and differentiation [11,13,22]. Activation of these genes by events such as point-mutation, amplification, insertion or rearrangements can lead to cellular transformation in vitro and to the development of tumors in vivo [23,24]. In fact, changes in the level of expression or alterations in the structure or copy number of protooncogenes probably are important at some stage in the pathogenesis of a number of different human malignancies [11,25,26]. In human breast cancer several different protooncogenes are known to be altered. These include protooncogenes such as c-Ha-ras (overexpression and deletions), c-myc (amplification and rearrangements), c-erbB (overexpression) c-erbB-2 (amplification and . overexpression) or int-2 (amplification) and the tumor suppressor Rb-1 gene (deletions and inactivation) [2736]. These alterations may have prognostic significance since some of these changes are associated with various clinical parameters in breast cancer such as axillary lymph node involvement, ER and/or PgR status, tumor size, local tumor recurrence, and poor prognosis. Mechanistically, the inappropriate expression or loss of expression of these protooncogenes or tumor suppressor genes may indirectly control the production of or the response of cells within the mammary gland to various growth factors. In this respect, several different growth factors or growth inhibitors such as EGF, transforming growth factor alpha (TGFa), TGFp1, insulinlike growth factor (IGF-I), IGF-H, basic fibroblast growth factors (bFGF), hst-l/K-FGF, A and B chains of PDGF, amphiregulin, mammary-derived growth inhibitor (MDGI), mammary derived growth factor-I (MDGF-I), mammastatin, p52 (cathepsin D) and pS2 have been shown to be associated with both normal and/or malignant rodent and human mammary epithelial cells [5,10,36-40]. These activities could be important in regulating the autocrine and/or paracrine growth of different populations of cells within the mammary gland and in participating in the neovascularization and desmoplasia which is observed during lactation and in various breast tumors. In fact, some of these growth factors are known to modulate the proliferation of normal and malignant mammary epithelial cells in vitro and in vivo [41-48]. One growth factor which plays a central role in regulating the proliferation and possible differentiation of mammary epithelial cells in vitro and in vivo is EGF. EGF is a potent mitogen for normal and malignant mammary epithelial cells and can stimulate the lobuloalveolar development of the mouse mammary gland in organ explants in vitro and following administration in vivo [41,43-45,49]. TGFa is a low molecular weight, 50 amino acid peptide (Mr, 5,600) which is functionally related to EGF and other members of the EGF fami-

ly of peptides [40,50,51]. TGFa is a potent mitogen for epithelial cells anf for fibroblasts and can function as an angiogenic factor in vivo for capillary endothelial cells [52]. TGFa can also cooperate with other growth factors such as TGF0 and bFGF to reversibly induce a number of biological properties in vitro which are generally associated with the transformed phenotype in nontransformed cells [53-55]. Although TGFa exhibits only a 30-40% amino acid sequence homology to EGF, its tertiary structure is sufficiently similar to EGF such that it can bind to and interact through the EGFR [50,56]. TGFa is initially synthesized as a high molecular weight, glycosylated membrane-associated precursor (Mr, 18,000-20,000). The precursor as well as other soluble high molecular weight meso forms of this growth factor are biologically active [57,58]. The genes for mouse, rat and human TGFa have been cloned and entirely sequenced. A major TGFa mRNA transcript of 4.8 kb and a minor 1.6 kb mRNA species can be detected in a number of rodent and human tumors and tumor cell lines [50,59,60]. In fact, in some tumors there appears to be a reasonable degree of concordance between the expression of TGFa mRNA and EGFR mRNA expression suggesting that a potential autocrine loop may exist in these tumors [6163]. In addition, overexpression of EGFRs by amplification may contribute to the onset or maintenance of the malignant phenotype by sensitizing tumor cells to low concentrations of locally derived EGF-like growth factors [35,63-65]. Conversely, in rodent fibroblasts such as rat-1 cells which possess a sufficient complement of functional EGFRs, overexpression of a transfected TGFa cDNA expression vector plasmid is capable of transforming these cells in vitro and in vivo [66]. Although TGFa was originally detected in tumor cells, it has more recently been found to be expressed in a limited number of normal fetal and adult tissues [67-72].

Transformation of mouse NOG-8 and human MCF10A mammary epithelial cells by ras, neu and TGFa

The role that TGFa may be performing in the initiation and progression of breast cancer in rodents and humans is just starting to be defined. Exogenous TGFa like EGF is capable of stimulating the proliferation of normal and malignant mammary epithelial cells [46,47, 72,73). In addition, TGFa is more potent than EGF in stimulating the lobulo-alveolar development of the mouse mammary gland in vivo [45]. Several years ago we initially observed that TGFa was produced in a series of well differentiated, rat mammary adenocarcinomas that could be induced at a relatively high frequency by treatment with either DMBA or NMU [74,75]. These tumors were of particular interest because the majority of them are estrogen-dependent in vivo and may therefore represent an appropriate animal model system which is analogous to a subset of

171 hormone-dependent human breast cancers [6,7]. It was also found that the level of expression of TGFa in these tumors could be enhanced either in vitro or in vivo with estrogens suggesting that the capacity of estrogen to function as a mitogen for mammary epithelial cells may be mediated in part through an increased expression of this and possibly other growth factors [75]. In addition, it had previously been shown that NMU- or DMBA-induced rat mammary tumors possessed a point-mutated c-Ha-ra5 protooncogene and that the DMBA-induced mammary tumors also contained an increased level of expression of c-Ha-ras mRNA and p21"" protein [7678]. Since transformation of rodent fibroblasts by a viral ras oncogene or by an activated ras protooncogene can lead to a loss in the growth responsiveness of these cells to exogenous EGF due to an increased production of endogenous TGFa, this may partially account for the refractoriness of primary cultures of DMBA-induced rat mammary tumor cells to the mitogenic effects of EGF and for the reduction of cell surface EGFRs found on these cells [19-21,43]. Relative Protein Levels

2 4 6 8 10 12 Days of Treatment with Dexamethasone Relative mRNA Levels

2 4 6 8 10 12 Days of Treatment with Dexamethasone

Fig. 1. Temporal summary of the biological effects following induction of point-mutated c-Hz-ras in NOG-8 ras cells. Cells were treated with 10"* M dexamethasone for the indicated time periods and subsequently analyzed for the indicated parameters. A. Phcnotypic changes observed in NOG-8 ras cells. p21"" Protein levels were detected by Western blotting and TGFa protein secreted into the CM was determined by RIA and by RRA. TGFR protein secreted into the CM was measured by RRA. B. Densitometric scan of Northern blot analysis of poly(A)+ RNA isolated from NOG-8 ras cells and hybridized to the appropriate 32P-labeled cDNA probes to detect specific mRNA transcripts.

To ascertain if TGFa production and secretion is enhanced in mammary epithelial cells which have been transformed by an activated ras gene and to determine if this response is an early or late event after the initiation of ras transformation, TGFa expression was examined in NOG-8 mouse mammary epithelial cells before and after transformation using an expression vector plasmid containing the human point-mutated c-Ha-ras protooncogene that had been placed under the transcriptional control of the MMTV-LTR promoter, a glucocorticoid responsive element [79]. NOG-8 cells seemed distinctly suited for such studies since these cells are a clonal, near diploid mouse mammary epithelial cell line which was originally derived from the polyclonal NMuMG mouse mammary epithelial cell line [80]. NOG-8 cells fail to grow in soft agar and are not tumorigenic in nude mice. In addition, these cells can be directly transfected by calcium phosphate precipitation. More importantly, these cells express approximately 105 EGFR sites/cell suggesting that they can mitogenically respond to EGF or TGFa. This is the case since exogenous EGF or TGFa can induce a 4- to 5-fold increase in the anchorage-dependent growth of these cells in serum-free medium and can facilitate the growth of these cells in soft agar as colonies [79]. Figure 1 demonstrates that following the addition of dexamethasone (dex) to NOG-8 cells that are carrying the MMTV-ras expression vector, NOG-8 ras cells, there is a rapid and sustained increase in both p21"" protein and c-Ha-ras mRNA which can first be detected within 1 to 3 hours after the addition of steroid. This increase in ras expression is reflected by a corresponding increase in the ability of these cells to clone in soft agar. After 5 to 8 days, NOG-8 ras cells exhibit a loss in density-dependent, contact inhibition of growth and show a 3- to 4-fold increase in their growth rate in serum-free medium. These phenotypic changes produced in the NOG-8 ras cells following overexpression of a point-mutated c-Ha-ras protooncogene may be partly mediated through an increase in the production and secretion of different endogenous growth factors such as has been demonstrated in ras transformed NRK and NIH-3T3 cells [20,21]. In this respect, TGFa is one growth factor which is consistently elevated in NIH-3T3 cells that have been transformed by a number of different oncogenes [21]. Conditioned medium (CM) was therefore analyzed for TGFa activity from NOG-8 ras cells that had been treated with dex. Before induction of ras, there were very low levels of biologically active or immunoreactive TGFa which could be detected in the CM as measured in a EGF/TGFa competitive radioreceptor assay (RRA) and in a specific TGFa radioimmunoassay (RIA), respectively. However, within 7 to 9 days after dex treatment, there is a 4- to 5-fold increase in the CM levels of TGFa which reaches a maximum within 12 days. Prior to this change in TGFa secretion, there is a 3- to 4-fold increase in the expression of a 4.8 kb TGFa mRNA transcript that occurs

172 within 1 to 2 days after dex treatment and which parallels the kinetics of c-Ha-ras mRNA expression. There is also an equivalent and simultaneous increase in TGFp mRNA expression, TGFp1 protein secretion and IGF-I protein production that occurs in NOG-8 ras cells after dex treatment suggesting that the synthesis of other growth factors may also be enhanced after ras induction in these cells. There are several additional changes that occur in these ras transformed NOG-8 mammary epithelial cells probably as a consequence of the increase in endogenous TGFa production. There is a gradual reduction in the number of cell surface EGFRs on these cells that occurs within 7 to 10 days after dex treatment and which is accompanied by a loss in the high affinity EGFR population. This reduction in EGFR expression is inversely related to the amount of TGFa which can be found in the CM from these cells. The reduction in EGFR receptor expression may be due to a TGFa-induced down-regulation in the number of EGFR expressed on these cells as a result of chronic occupation by secreted TGFa. In other words, the cells may become desensitized to the effects of exogenous growth factor due to the constitutive production of endogenous TGFa which could then supplant their requirement for growth factors such as TGFa or EGF. This is probably the case, since after 7 to 9 days of dex treatment the NOG-8 ras cells are no longer responsive to the mitogenic effects of either exogenous TGFa or EGF either in monolayer culture or in soft agar. There is also a gene dosage effect of p21"" expression since the amount of endogenous TGFa production in different clones of ras transformed NOG-8 cells is directly proportional to the level of p21 raI protein expression in these cells and to the degree of growth of these clones in soft agar [81]. Collectively, these results suggest that TGFa may be functioning as one important intermediary in the transformation pathway that is used by an activated ras gene in mammary epithelial cells and that this growth factor may subserve an autocrine function in regulating the proliferation of these ras transformed mammary epithelial cells. A change in the level of expression of endogenous TGFa appears to be unique to ras transformed NOG-8 cells since NOG-8 cells which have been transformed by a point-mutated rat c-neu (c-erbB-2) protooncogene do not exhibit any significant change in either the amount of TGFa which is secreted into the CM or in the level of EGFR expression as compared to nontransformed NOG-8 cells [81]. However, these neu transformed mammary epithelial cells exhibit a cloning efficiency in soft agar that is comparable to the ras transformed NOG-8 cells and like the ras transformed cells form locally invasive, undifferentiated carcinomas in nude mice. These results demonstrate that neu may be operating in part through a transformation pathway that is not used by ras at least with respect to the production of TGFa and to changes in EGFR expression. Furthermore, the results assume some clinical signifi-

cance since amplification and/or overexpression of the c-erbB-2 gene is found in approximately 10-30% of primary human breast rumors which in some cases is associated with a subset of more aggressive tumors in lymph node positive patients that generally have a poor prognosis [34]. The inappropriate overexpression of a transfected rat or human TGFa gene results in the partial or complete transformation of immortalized rodent fibroblasts [63,66,82]. Since the previous results have demonstrated that TGFa production increases in ras transformed mouse and rat mammary epithelial cells and since exogenous TGFa can induce or mimic in nontransformed NOG-8 cells some of the same phenotypic properties which are associated with transformation such as growth in soft agar, then overexpression of a TGFa cDNA in an expression vector plasmid in NOG-8 cells may therefore be sufficient to transform these cells. To test this thesis, an expression vector plasmid containing the full-length human TGFa cDNA under the transcriptional control of the SV40 early promoter was cotransfected with an SV40neo plasmid (pSV2neo) into NOG-8 cells [83]. Following growth in geneticin (G418) for several weeks, 180 G418 resistant colonies were isolated. Nine colonies were randomly selected, cloned, and expanded into cell lines. The CM from these clonal lines of TGFa transfected NOG-8 cells, NOG-8 TF, were analyzed for immunoreactive and bioactive TGFa (Table 1). NOG-8 cells were also transfected with just the pSV2 neo plasmid and several G418 resistant clones isolated. These neo transfected cell lines (TF Cl 1-3) like the parental NOG-8 cells secreted very low levels of TGFa. However, 3 (TF Cl 5,

Table 1. Biological properties of transfected TGF-a-expressing NOG-8 mouse mammary epithelial clones. Clones

NOG-8 TF Q 2 (pSV2neo) TFC15 TFC17 TFC1 10 TFC1 13 1

Soft agar growth* (no. of colonies/ dish)

0 (0/10) 4 (0/5) 1048(8/10) 1297(10/10) 1054 (5/5) 1103(0/5)

TGFa secreted1" (ng/ 108 cells/ 48 h) RIA

RRA

7 14 312 610 339 120

7 7 296 595 177 36

EGF receptors0 (total sites/cell)

95,000 91,000 12,000 16,000 44,000 41,000

Soft agar growth represents the average of quadruplicate determinations. The SD was less than 10%. Values in parentheses represent the number of tumors per number of animals injected with 5 x 106 cells s.c. b TGFa protein from concentrated CM was evaluated in a TGFaspecific radioimmunoassay (RIA) and in an EGF/TGFa radioreceptor assay (RRA). Values represent the average of quadruplicate determinations. The SD was less than 10%. c EGF binding sites per cell were calculated by Scatchard analysis from the specific binding isotherms using different concentrations of mouse '"I-EGF.

173 7, and 10) out of 6 TGFa cotransfected NOG-8 clonal cell lines that were assayed produced TGFa at levels which were approximately 10- to 80-fold higher than the levels found in the CM from the parental or pSV2neo transfected NOG-8 cells and 2- to 13-fold higher than the levels found in the ras transformed NOG-8 cells. In all of the clones, 95% of the TGFa activity was found in the CM. In most of the clones there was a reasonable concordance between the amount of immunoreactive TGFa that could be detected in the CM by RIA and the amount of bioactive TGFa as measured in an EGF RRA. Moreover, the level of unoccupied cell surface EGFRs on these different TGFa expressing NOG-8 clones appeared to be inversely related to the amount of TGFa that could be found in the CM from these clonal lines suggesting that a downregulation in the number of free unoccupied EGFRs was probably occurring due to the high levels of secreted TGFa. NOG-8 clones which were secreting high levels of TGFa such as TF clones 5, 7, and 10 exhibited cloning efficiencies in soft agar which were nearly equivalent to that observed for the ras or neu transformed NOG-8 cells. Cells from clones 5, 7, and 10 also exhibited growth rates in serum-free medium in the absence of exogenous EGF which were generally 4- to 6-fold higher than the growth rates of the nontransfected parental NOG-8 or neo transfected NOG-8 cells. In one clone, TF Cl 13, only 30% of the immunoreactive TGFa was biologically active, suggesting that the TGFa may be incompletely processed in these cells. In this respect, approximately 2/3 of the TGFa activity in the CM from the high secreting clones was present in a low molecular weight form (Mr, 6,000-7,000) whereas the remainder existed as larger species ranging from 18,000 to 25,000 Mr. The level of bioactive TGFa in the CM from TF Cl 13 cells was similar to the levels of TGFa detected in the CM from the ras transformed NOG-8 cells [79]. However, these cells were still able to grow in soft agar at a level which was comparable to that observed for clones 5, 7, and 10. In clones 5, 7, and 10, a specific 2.3 kb mRNA transcript could be detected which hybridized to a labeled human TGFa cDNA insert demonstrating that the plasmid cDNA was being transcribed and was using the correct polyadenylation signals from the hepatitis B antigen sequences within this plasmid [66]. Southern analysis of DNA isolated from TGFa expressing TF clones 5, 7, and 10 and digested with either Hind HI or Bam HI also demonstrated that multiple copies of the plasmid DNA had been integrated into these cell lines and in some cases rearranged suggesting that these effects might be responsible for the overexpression of the ectopic human TGFa mRNA which were found in these cell lines. If the secreted TGFa were functioning in some capacity as a bonafide external autocrine growth factor then it should be possible to attenuate the effect of this growth factor by either neutralizing its activity, by in-

hibiting its binding to the EGFR with an appropriate blocking antibody or by blocking its production. Addition of a neutralizing mouse monoclonal anti-TGFa antibody (2-20 |ig/ml), TAb 1, which was generated against the folded recombinant human TGFa peptide produced a 60-70% inhibition in the soft agar growth of some of the high TGFa producing NOG-8 clones, hi contrast, a non-neutralizing anti-TGFa antibody of the same isotype as TAb 1 was unable to significantly affect the growth of colonies in soft agar from these cell lines. Likewise, addition of an antineoplastic analog of cyclic AMP (cAMP), 8-Cl-cAMP (5-50 \iU), was able to reversibly inhibit the growth of TGFa transformed NOG-8 cells without significantly affecting the growth on nontransformed NOG-8 cells [84]. This growth inhibitory, cytostatic effect of 8-Cl-cAMP was due to its ability to selectively inhibit the production of TGFa in these cells by specifically blocking the transcription of TGFa mRNA. Finally, the in vitro transformation of NOG-8 cells which were secreting high levels of biologically active TGFa is also mirrored by the acquisition of a tumorigenic phenotype in vivo since these cells were capable of forming undifferentiated, locally invasive carcinomas in nude mice. Transformation of NOG-8 cells in vitro and in vivo could also be achieved when NOG-8 cells were infected with a replication defective, recombinant ecotropic retroviral expression vector containing the human TGFa cDNA [82]. These results demonstrate that a population of immortalized rodent mammary epithelial cells can be efficiently transformed by TGFa if these cells are intrinsically capable of responding mitogenically to EGF/TGFa, if they express a sufficient number of functional EGFRs (>105 EGFR sites/cell) and if a sustained synthesis and secretion of endogenous TGFa at a critical threshold level can be adequately maintained [63,82,83]. Like NOG-8 mouse mammary epithelial cells, some normal human mammary epithelial cells such as MCF10A can also be transformed in vitro and in vivo following transfection with a single activated protooncogene, such as a point-mutated human c-Ha-ras protooncogene, whereas others such as 184A1N4 cells require an additional activated protooncogene [73]. MCF-10A cells exhibit an absolute dependency upon EGF for their in vitro growth and express approximately 2.5 x 105 EGF receptor sites per cell. Transformed MCF-10A Ha-nw cells contain approximately a 5- to 10-fold increase in p21 rai protein and c-Ha-ras mRNA levels as compared with MCF-10A cells [85]. MCF10A Ha-ras cells grow in soft agar as colonies and form tumors in nude mice. MCF-10A Ha-ras cells also exhibit a 3- to 4-fold increase in their anchorage-dependent growth rate in serum-free medium and show a reduced mitogenic response to either exogenous EGF or TGFa as compared with MCF-10A cells. This may be due to an enhanced production of endogenous TGFa by these cells since MCF-10A Ha-ras transformed cells express a 4- to 8-fold increase in the levels of a 4.8 kb TGFa mRNA transcript and secrete

174 approximately 4- to 6-fold more TGFa protein as compared with the parental MCF-10A cells. Addition of either an anti-TGFa neutralizing monoclonal antibody (TAb 1) or an anti-human EGF receptor blocking monoclonal antibody (528) to the MCF-10A Ha-ras cells was able to produce a 50-80% inhibition in the anchorage-independent growth of these cells in soft agar, suggesting that part of the growth-promoting effects of an activated ras gene in these cells is mediated through TGFa. MCF-10A cells were also transfected with an expression vector containing the rat c-neu (c-erbB-2) protooncogene under the transcriptional control of the Moloney sarcoma virus LTR. MCF-10A c-neu cells that were overexpressing plg -crbB-2 w e r e ext o c ] o n e m so ft hibited an enhanced growth rate in serum-free medium at levels comparable to those observed in MCF-10A ras cells. Addition of an anti-c-erbB-2 monoclonal antibody (TAb 250) but not of TAb 1 or 528 antibodies inhibited the anchorage-independent growth of these cells in soft agar. Unlike MCF-10A Ha-ras cells, MCF-10A c-neu cells show no increase in TGFa secretion and no change in their responsiveness to exogenous EGF or TGFa. Although MCF-10A c-neu cells are transformed in vitro, they are not tumorigenic in normal or in y-irradiated nude mice. The human TGFa gene was then introduced into MCF-10A cells with a recombinant retroviral expression vector containing the neo gene and the human TGFa gene under the transcriptional control of a heavy metal inducible promoter, the mouse metallothionein-1 promoter, to ascertan if overexpression of TGFa in these cells is able to alter their growth properties or is capable of transforming these cells [85]. Fifteen G418resistant MCF-10A TGFa clones were isolated and expanded and were found to secrete between 7- to 25fold more bioactive TGFa than uninfected MCF-10A cells in response to cadmium treatment. Several of the clones which were secreting the highest levels of TGFa were able to form colonies in soft agar, exhibited an enhanced growth rate in serum-free medium, and showed a decreased mitogenic response to exogenous EGF as compared with uninfected MCF-10A cells. Growth of these clones in soft agar could be completely blocked by the TAb-1 anti-TGFa neutralizing antibody or the 528 anti-EGF receptor blocking monoclonal antibody, demonstrating that an autocrine-dependent transformation pathway had been established in these cells. However, none of the TGFa expressing clones were able to form tumors in normal or 7-irradiated nude mice. These results demonstrate that overexpression of this growth factor is able to transform in vitro immortalized human mammary epithelial cells that express a threshold level of functional EGF receptors. However, overexpression of TGFa or the normal c-neu protooncogene is not sufficient to elicit tumor formation in vivo suggesting that additional genetic changes are needed to complement these effects such as the activation of a

second protooncogene or loss of expression of a tumor suppressor gene. Expression of TGFa in primary human breast tumors and in pleural effusions from breast cancer patients

Secretion of TGFa protein and expression of TGFa mRNA has been detected in several human breast cancer cell lines [86-89]. The basal level of TGFa production is generally somewhat higher in ER negative, estrogen nonresponsive breast cancer cell lines such as MDA-MB-231 and MDA-MB-468 than in ER positive, estrogen responsive cell lines such as MCF-7, T47-D and ZR-75-1 [87,88,90]. A human mammary carcinosarcoma cell line, HS578T, is negative for TGFa mRNA expression. In ER positive cell lines, such as MCF-7 cells, production of 6,000 Mr and 30,000 Mr TGFa proteins can be enhanced several fold by treatment with physiological concentrations of 17p-estradiol (10"'° - 10"8 M) in cells which have been maintained under estrogen-deprived conditions for several days in phenol red-free medium in the presence of charcoal-stripped calf serum [86]. A similar 30,000 Mr TGFa species can be detected in ZR-75-1 and in MDA-MB-231 cells [91]. The 30,000 Mr protein probably represents a glycosylated precursor form for the 6,000 TGFa species. Both forms of TGFa can be secreted by breast cancer cells in vitro [86]. In T47-D and MCF-7 cells basal levels of TGFa mRNA expression can be reduced by treatment of these cells with antiestrogens such as tamoxifen [88,90,92]. No consistent relationship however could be established between growth rate and the level of TGFa expression at least in the T47-D cell line [92]. This is in contrast to the results observed in the immortalized but nontumorigenic 184A1N4 human mammary epithelial cell line which expresses TGFa mRNA and which secretes TGFa at a level which is equivalent to that observed in some of the breast cancer cell lines [73]. In these cells TGFa mRNA expression is highest in proliferating cells and drops as the cells become quiescent [72]. Furthermore, addition of exogenous EGF or TGFa can lead to an autoinduction of TGFa expression in these cells [72]. A similar inductive response has also been noted in MDA-MB-468 cells after treatment with either EGF or TPA [89]. Expression of EGFRs has been found in approximately 40-50% of primary human breast tumors [35]. In fact, a high level of EGFR expression is positively correlated with axillary lymph node involvement and with poor prognosis and negatively correlated with ER and PgR status [35]. Therefore, the production of EGF-like growth factors such as TGFa or amphiregulin in primary human breast tumors may be significant since these growth factors might be involved in regulating the proliferation of these tumor cells through an autocrine-dependent pathway involving the EGFR

175 system. Immunoreactive and bioactive TGFa can be found in 30-50% of primary human breast tumors of which 50% of the TGFa positive tumors had immunoreactive TGFa levels that exceeded the levels of growth factor that could be detected in benign breast lesions or in normal mammary tissues [87,93,94]. A biologically active 6,000 Mr TGFa species and an immunoreactive 30,000 Mr TGFa peptide can be detected in breast tumors [91,94]. In one study, a significantly higher content of immunoreactive 30,000 Mr TGFa could be detected in breast tumors that were ER and PgR positive [91]. However, other studies have failed to find such an association or have found higher levels of TGFa in ER and PgR negative tumors [86,95]. TGFa protein can also be found in approximately 50% of lymph node metastases [93]. Tamoxifen treatment led to a 10-fold reduction in the breast tumor content of TGFa [94]. There appears to be coexpression of both TGFa protein and EGFR protein in a subset of human breast tumors which is also reflected by coexpression of appropriate mRNA transcripts for these proteins [62, 93, 95, 96]. In fact, expression of a major 4.8 kb and in some cases a minor 2.2 kb TGFa mRNA species can be found in 40-70% of primary human breast tumors and in approximately 20-40% of nonmalignant breast tissue samples such as fibroadenomas, fibrocystic lesions, mammary dysplasias and reductive mammoplasties [62, 86, 87, 96]. Although no unique association has been discerned between TGFa mRNA expression and steroid receptor status [62,86], coexpression of both mRNA and protein for EGFR and TGFa may occur more frequently in ER-negative tumors [95,96]. A more extensive analysis was conducted on a small group of 18 histologic grade II or HI human breast tumors to ascertain if TGFa mRNA expression might be associated with any measurable prognostic parameter such as ER and/or PgR status, axillary lymph node involvement or subsequent patient relapse due to tumor recurrence [97]. Fifty percent of the tumors were found to express TGFa mRNA (Table 2). Fifty-six percent of the tumors were positive for both ER and PgR, whereas 61% were positive for one or more axillary lymph nodes. There was no significant association between TGFa mRNA expression and the presence or absence of measurable ER and/or PgR. In addition, no unique correlation could be discerned between axillary lymph node status of the breast cancer patients, relapse in these patients due to local recurrence of the primary tumor or to a distal metastasis and association of these parameters with TGFa mRNA expression in the primary tumor. Similar lack of association between TGFa mRNA expression and histologjcal grade, nodal status, tumor cellularity, desmoplasia, and patient survival has been found in a panel of 66 primary breast tumors [96]. Expression of c-Ha-ras mRNA has also been examined since a majority of human breast tumors express this protooncogene and since we had previously demonstrated that transformation of mouse NMuMG and NOG-8 and human MCF-10A mammary epithelial

cells with an activated c-Ha-nis protooncogene can lead to an increase in TGFa expression [29,78-80]. Sixty-one percent of the tumors were found to express c-Ha-ras mRNA [97]. However, there was no significant association between mRNA expression for this protooncogene and for TGFa. Collectively, these results should be viewed with some reservation since only a small number of tumors were analyzed in both of these studies [96,97]. Therefore, a larger cohort of tumors should be examined to determine if any of these clinical parameters significantly correlates with the expression of TGFa and/or EGFRs. To ascertain if expression of TGFa in these breast tumors might be due to any alterations in the structure of the TGFa gene, DNA that had been isolated from 79 breast tumors and in some cases from matched lymphocyte preparations, was digested with BamH 1 and analyzed by Southern blot analysis for any potential amplification or rearrangements of the TGFa gene. None of the tumors showed any evidence for gross amplification or major rearrangements of the TGFa gene when compared to the restriction endonuclease digestion pattern obtained from matched lymphocyte DNA samples. However, there were minor restriction fragment length polymorphisms (RFLPs) for BamH 1 that could be detected in some of the DNA samples between individual patients. No unique RFLPs were observed in matched DNA samples obtained from lymphocytes or from tumors of the same patient [97]. Since TGFa protein and TGFa mRNA are found in primary human breast tumors and in some metastases [86, 93, 97], then it is possible that this growth factor might be released from metastatic tumor cells and as such might be important as one factor in the progression of breast cancer. This seemed likely since urine and in some cases effusions obtained from patients with several different types of cancer such as breast, liver, bladder, kidney, and melanoma contain appreciable amounts of TGFa, TGF0 or bFGF which has been correlated with tumor burden and the presence of other tumor-associated antigens such as a-fetoprotein [98-102]. Pleural or ascitic effusions were obtained from 37 noncancer patients who were being treated for such diseases as hepatic cirrhosis, congestive heart failure, pulmonary infarction or peritonitis and from 63 cancer patients who had been diagnosed with breast, ovarian, uterine, stomach or colon cancer or with leukemias and lymphomas [97]. These samples were assayed for immunoreactive and bioactive TGFa and also for two other well characterized tumor-associated antigens, TAG-72 and carcinoembryonic antigen (CEA). As illustrated in figure 2A, the amount of immunoreactive TGFa in the effusion samples from the noncancer and cancer patients ranged from 0.2 to 26 ng/ml. Using an arbitrary cut-off value of 6 ng/ml, 19% of the noncancer patients effusions had TGFa levels that exceeded this level whereas 46% of the cancer patients effusions had TGFa levels that were above this level. In the 13 breast cancer patients (Fig. 2B), this

176 difference was even larger since 69% of these pleural effusions possessed TGFa levels greater than 6 ng/ml. In 90% of the cancer patient effusions equivalent amounts of immunoreactive and biologically active TGFa were detected. However, in effusions obtained from 72% of the noncancer patients, the levels of immunoreactive TGFa were generally 2- to 10-fold higher than the levels of bioactive peptide that could be detected using a competitive EGF/TGFa RRA demonstrating that the majority of this material is probably inactive. The levels of immunoreactive TAG-72 and CEA were also quantitated in these effusion samples since it has previously been shown that both antigens are present in the effusions of cancer patients at significantly higher levels than in the effusions from noncancer patients [97]. It also seemed worthwhile to determine if TGFa like these other two tumor antigens could be used prognostically to follow tumor relapse or recurrence. Eleven percent of the effusions from the noncancer patients were positive for either TAG-72 or CEA whereas 22% of the cancer patient effusions were positive for either antigen. Within these two antigen positive groups, TGFa at levels greater than 6 ng/ml was detected in 25% of the antigen positive effusions from the noncancer patients and in 64% of the antigen positive effusions from the cancer patient population. These results demonstrate that biologically active TGFa is present in the majority of effusions from cancer patients [97,102] and in particular from breast cancer patients at higher levels than are normally found in the effusions that result from other pathologic conditions, suggesting that this growth factor may be derived from resident metastatic tumor cells in these effusions.

This is particularly germane to breast cancer, since a number of breast cancer cell lines that were originally derived from pleural effusions are known to produce and secrete TGFa [86]. Expression of TGFa in normal rodent and human mammary epithelial cells

We had previously demonstrated that multiple isoelectric forms of TGFa could be detected at relatively high concentrations in samples of human milk and that the total level of biologically active TGFa in these samples varied between individual donors [10,103]. One of these TGFa-like species was initially designated as mammary-derived growth factor II (MDGF-II). MDGF-n had a Mr, 17,000, a pi of 4.0 and was immunologically related to authentic human TGFa but not to human EGF. In addition, MDGF-II was physiochemically similar to a breast tumor-derived TGFa activity [103]. These results suggested that TGFa might be synthesized and secreted by certain cells within the mammary gland and that it may subserve some normal function during the development of the mammary gland in vivo or for the growing neonate. In this respect, normal epithelial cells in vitro have been shown to express this growth factor. For example, primary cultures of proliferating normal rat and human mammary epithelial cells contain bona fide TGFa in their CM and express TGFa mRNA [72,73,87,104]. Moreover, several nontransformed human mammary epithelial cell lines also produce TGFa at levels which in some cases approach the levels of TGFa that are found in the CM from certain human breast cancer cell lines [72,73]. These results demonstrate that this growth

i

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i S

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Fig. 2. A. Immunoreactive TGFa activity in pleura] effusions and in ascitic fluids from 37 noncancer patients and 63 cancer patients. B. Distribution of immunoreactive TGFa activity in effusions and ascitic fluids from patients with various types of cancer (from \91\).

177 Table 2. TGFa mRNA expression in primary human breast carcinomas*. Patient

TGFa mRNA

c-Ha-ras mRNA

P-actin mRNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

PgR*

ER«

Histologic grade1" II

III ND

d

ND

m u II II

m in

II

m

Lymph node status 2

NK d 0 5 NK

3 0 11

0 4

m

¥

in U

0 i i

u n ii i

n

0

a

7 18

Relapsec

M(20) R(24) -(96) M(75) -(64) -(79) -(72) M(10) -(35) -(39) -(35) -(38) -(29) -(36) -(36) M(24) M(36) -(30)

* Receptors were considered negative (-) when they were less than 15 fmol/mg of protein, positive (+) when they ranged from 15 to 50 fmol/mg of protein, moderately positive (++) when they ranged from 50 to 150 fmol/mg of protein, and strongly positive (+++) when they were greater than 150 fmol/mg of protein. b The tumors were infiltrating ductal carcinomas classified in histologic grades according to Bloom et al. c R, local recurrence; M, distal metastasis; —, no relapse. The number in parentheses is the number of months postsurgery. d NK, not known; ND, not done. • From |97|.

factor is not restricted to malignant mammary epithelial cells in vitro. Expression of TGFa mRNA has been detected in vivo in the mouse, rat, and human mammary gland and biologically active TGFa has also been detected in these tissues [105,106]. The distribution and relative level of expression of TGFa mRNA was studied by in situ hybridization in virgin, pregnant and lactating rat and human mammary tissue using a labeled TGFa antisense RNA riboprobe [105]. Expression of TGFa

.

mRNA could be detected in 80-90% of the ductal and alveolar epithelial cells in the virgin, pregnant or lactating mammary gland in rats. In addition, during pregnancy approximately 10-15% of the surrounding stromal fibroblasts were also expressing very high levels of TGFa mRNA. There was a slight increase in TGFa mRNA expression in the epithelial cells from the pregnant gland as compared to the epithelial cells in the virgin gland. However, during lactation, there was a 2- to 3-fold increase in the levels of TGFa mRNA in the

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Fig. 3. Localization of TGFa mRNA expression in lactating mouse mammary gland by in situ hybridization. Paraffin-embedded sections were hybridized with a 33S-labeled TGFa antisense riboprobe (A) or with a corresponding 33S-labeled TGFa sense riboprobe (B). Magnification, X400.

178 mammary epithelium as compared to the epithelium in virgin or pregnant gland. A similar pattern of TGFa mRNA expression was observed in corresponding mouse mammary tissue obtained from similar developmental stages. As illustrated in figure 3A, lactating mouse mammary ductal epithelial cells expressed considerable amounts of TGFa mRNA as detected with an 35 S-labeled TGFa antisense riboprobe. In contrast, very little hybridization could be discerned with the homologous labeled TGFa sense riboprobe (figure 3B). We also had the opportunity to examine a small series of normal human mammary tissue obtained from nulliparous, parous and midpregnant women for TGFa mRNA expression by in situ hybridization [105]. In both nulliparous and parous mammary tissue, TGFa mRNA could be detected at a low but demonstrable level in the epithelial cells composing the ducts and lobules in tissue obtained from nulliparous or parous women. The level of TGFa mRNA was not significantly different between these two types of tissue. However, during pregnancy there was a 2-fold increase in the level of TGFa mRNA expression in the epithelium as compared to the level of TGFa mRNA expression in the epithelium from nonpregnant mammary tissue. As was found in the rat, a small fraction of stromal fibroblasts in the pregnant mammary gland were also found to be expressing moderate levels of TGFa mRNA. Collectively, these results demonstrate that TGFa mRNA is present in the normal mammary gland and that its expression increases during pregnancy and more dramatically during lactation. Bioactive TGFa has also been detected in the virgin, pregnant, and lactating rat mammary gland with a 5- to 6-fold increase in TGFa being found in the lactating gland supporting the variation in the level of TGFa mRNA expression as detected by in situ hybridization and suggesting that TGFa found in milk may originate directly from the mammary gland [103,106]. In addition, since exogenously administered EGF or TGFa can directly stimulate ductal morphogenesis and mammary end bud growth in vivo, these results further support the contention that cells within the mammary gland have the capacity to respond to these growth factors [44,45]. Locally derived TGFa from either stromal cells, myoepithelial cells or ductal and alveolar epithelial cells may therefore function in either an autocrine and/or paracrine fashion to initiate and to maintain the normal growth and development of the mammary gland in vivo.

Conclusions TGFa is one of several growth factors that are expressed in both normal and malignant mammary epithelial cells [5, 10, 62, 72, 73, 75, 80, 85-88, 91-97, 104]. It is not unreasonable to assume that TGFa probably has to cooperate with a number of these other activities for modulating the growth and/or differentia-

tion of different populations of cells that are found within the mammary gland [105,106]. Interaction between these different growth factors as well as their synthesis and distribution within the mammary gland may change during the normal or pathologic development of the mammary gland. In addition, estrogens are at least one class of mammotrophic hormones that can regulate the production of TGFa. Likewise, activation of specific protooncogenes such as ras can lead to a rapid and constitutive increase in the level of TGFa expression suggesting that there may be some overlap in the biochemical pathways that are used by hormones like estrogen and oncogenes such as ras [79, 80, 86, 87]. However, whereas overexpression of the TGFa can lead to the malignant transformation of immortalized mammary epithelial cells that are intrinsically responsive to EGF and to a phenotype that resembles ras transformed mammary epithelial cells [79-85], overproduction of TGFa alone is not sufficient by itself to induce the appearance of an estrogen-independent phenotype in estrogen-dependent human breast cancer cells [107]. Nevertheless, a portion of the estrogen stimulated growth of certain human breast cancer cell lines such as MCF-7 cells may be mediated in part through the synthesis and secretion of peptide growth factors such as TGFa since monoclonal anti-EGFR blocking antibodies or a synthetic TGFa peptide antagonist which does not interact with the EGFR are capable of partially inhibiting the estrogen-induced growth of MCF-7 cells [86,108]. Additionally, monoclonal antiTGFa neutralizing antibodies or monoclonal antiEGFR blocking antibodies are able to inhibit to varying degrees the growth of rodent and human mammary epithelial cells that have been transformed by an activated ras gene or by overexpression of the human TGFa gene [83,85]. Collectively, these results demonstrate that reagents that can either neutralize a growth factor or that can block growth factor binding to its cognate receptor may have potential clinical application in the treatment and management of breast cancer. This sort of approach may be particularly applicable to breast cancer and more specifically to EGF/TGFa and the EGFR system since EGFRs are expressed at a fairly high frequency in breast rumors and since higher levels of EGFR expression are observed on tumors that are generally more aggressive and that probably will be refractory to antiestrogens or to conventional chemotherapy [35]. Moreover, it has recently been demonstrated that radiolabeled monoclonal anti-EGFR antibodies can be used to localize and to image human breast cancer xenografts established from cells that overexpress the EGFR in nude mice [109]. In addition, some of these unmodified monoclonal anti-EGFR antibodies or antibodies that have been conjugated to toxins such as the ricin A chain are selectively cytotoxic both in vitro and in vivo for human breast cancer cells that express a high level of EGFRs [110-112].

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