MOLECULAR CARQNOGENESIS 6:43-52 (1992)

Additive Effects of c-erbB-2, c-Ha-ras, and Transforming Growth Factor-a Genes on In Vitro Transformation of Human Mammary Epithelial Cells Fortunato Ciardiello,' Marco Gottardis, Fulvio Basolo, Stefano Pepe, Nicola Normanno, Robert B. Dickson, A. Raffaele Bianco, and David 5. Salomon Cattedra di Oncologia Medica, I1 Facolta di Medicina e Chirurgia, Naples, Italy (FC,Se ARB), Vincent Lombard1 Cancer Research Center, Georgetown University Medical School, Washington, DC (MG, RBD), lstituto di Anatornia e Istologia Patologica Facolta h Medicina e Chirurgia, Pisa, Italy (FB), and Tumor Growth Factor Section, Laboratory of Tumor Immunology and Bioloqx National Cancer Institute, National Institutes of Health, Bethesda, Maryland (NN, DSS)

MCF-1OA cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression o f a human point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erbB-2) proto-oncogene, or the human transforming growth factor-a (TGF-a) gene in MCF-1OA cells leads t o in vitro transformation o f such cells. To ascertain whether the introduction o f t w o o f these genes into MCF-1OA human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF-1OA Ha-ras and MCF-1OA TGF-a cells with a recombinant retroviral vector containing the human c-erbB-2 proto-oncogene and the hygromycin-resistance gene. Ten MCF-1OA TGF-alc-erbB-2 (MCF-1OATE) and 10 MCF-1OA Ha-raslc-erbB-2 (MCF-1OA HE) hygromycin-resistant clones were randomly selected and expanded into cell lines. MCF-1OA TE and MCF-1OA HE clones expressed a 10-fold t o 40-fold increase in p185 erbB-2 protein levels compared w i t h parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum-free medium and showed a strongly reduced mitogenic response t o exogenous epidermal growth factor or TGF-a compared with MCF-1OA cells. Moreover, both MCF-1OATE and MCF-1OA HE clones exhibited a fivefold to 20-fold higher cloning efficiency in soft agar than MCF-1OA Ha-ras, MCF-1OA c-erbB-2, or MCF-1OA TGF-a clones. However, neither MCF-1OA TE nor MCF-1OA HE cells were able t o grow as tumors in vivo when they were injected into nude mice. These results suggest that c-Ha-ras, c-erb5-2, and TGF-a genes have an additive effect on the in vitro transformation o f an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation o f other proto-oncogenes or inactivation of a tumor suppressor gene may be necessary t o elicit a fully tumorigenic phenotype. o 1992 wiley-~iss,~ n c . Key words: Transformation, growth factors, mammary cells INTRODUCTION Human cancers generally arise from a series of multiple genetic alterationsin a set of specific regulatory genes that are normally involved in the control of cell growth and differentiation [I,2]. In this respect, activation or enhanced expression (or both) of several cellular proto-oncogenes, growth factor genes, and growth factor receptor genes, as well as deletions or inactivations of tumor suppressor genes, have been implicated to varying degrees in the development and the progression of breast cancer [I-41. Several experimental and clinical studies have focused on the role of some of these genes, such as c-Ha-ras, c-erbB (the epidermal growth factor receptor (EGFR) gene), c-erbB-2, and transforming growth factor-a (TGF-a), in mammary tumorigenesis [4-81. In this respect, spontaneously immortalized mouse mammary epithelial cell lines, such as NOG-8 and HC-11 cells, that possess sufficient threshold levels of functional EGFR can be transformed in vitro and in vivo by the overexpression of the point-mutatedactivated c-Haras or c-neu (the rat homologue of the human c-erbB-2 gene) proto-oncogenes or of the TGF-a gene [9-141. More0 7992 W\LEY-t/SS,1NC.

over, transgenic mice have been generated that develop stochastic clonal mammary tumors after the insertion of one of these genes into their germlines [ I 5-21 1. Clinically, overexpressionof c-Ha-ras mRNA and elevated levels of p21 ras protein have been found in 60-70% of human primary breast carcinomas and in some cases have been positively correlated with axillary lymph node metastases and a more advanced tumor stage [22-241. Amplification of the c-erb6-2 proto-oncogeneor overexpression of pl 85 erbB-2 protein or both have also been detected in approximately 25% of human primary breast cancers in which there is an association with a more aggressive disease [7,25,261. Enhanced levels of EGFR have been observed in 35-50% of human breast tumors, are gener'Corresponding author: Cattedra di Oncologia Medica, II Facolta di Medicina e Chirurgia, Via 5. Pansini 5, 80131 Naples, Italy. Abbreviations: TGF-a, transforming growth factor-a; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; RIA, radioimmunoassay; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-bufferedsaline; Mi-1 , metallothionein-1; CM, conditioned medium.

44

CfARDlELLO ETAL.

ally inverselycorrelated with estrogen-receptor status, and are an independent negative prognostic factor both in axillary lymph node-positive and -negative patients [27-301. Expressionof TGF-a, which is a physiological ligand for EGFR, occurs in approximately 50% of primary and metastatic human breast cancers and is generally associated with high levels of EGFR expression 13 1-34], MCF-1OA cells are a unique system for studying the mechanisms of mammary epithelial cell transformation by cellular proto-oncogenes and growth factor genes. These cells are a near-diploid cell line derived from a population of normal human luminal mammary epithelial cells [35,36]. MCF-1OA cells also express epithelial-specific keratins and the mammary-specificmilk-fat-globuleantigen, do not grow in soft agar, and are nontumorigenic in nude mice. We have previously shown that overexpressionof the activated point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erb6-2) proto-oncogene, or the TGF-a gene is able to induce the in vitro transformation of MCF-1OA human mammary epithelial cells (37,381. However, none of these genes alone is sufficient to elicit a tumorigenic phenotype in MCF-IOA cells. In the study presented here, we infected MCF-1OA Ha-ras and MCF-1OA TGF-a cells, which overexpress an activated c-Ha-ras proto-oncogeneand the TGF-a gene, respectively, with a recombinant retroviral expression vector containing the human c-erbB-2 proto-oncogene to ascertain whether coexpression of two of these genes is sufficient to initiate a fully malignant phenotype. MATERIALS AND METHODS Infection and Establishment of Cell Lines MCF-1OA cells and their transformed derivatives were grown in a 1 : 1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 (v/v) supplemented with 5% horse serum, 20 m M 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid, pH 7.4, 4 m M glutamine, 100 U/mL penicillin, 100 pg/mL streptomycin (GIBCO, Grand Island, NY), 100 ng/mL cholera toxin (List Biological Laboratories, Campbell, CA), 10 ng/mL EGF, 10 pg/mL insulin (Collaborative Research, Bedford, MA), and 0.5 pg/mL hydrocortisone (Sigma Chemical Co., St. Louis, MO) in a humidified atmosphere of 95% air and 5% C 0 2 at 37°C. The pN12 retroviral expression vector plasmid was generated by inserting a 4.5-kb Smal-Dral full-length human c-erbB-2 cDNA [I81 into the unique Clal site of the Moloney murine leukemia virus-based retroviral vector pNO2, kindly provided by Dr. M. L. McGeady (National Cancer Institute, Bethesda, MD). pN12 contains an internal SV40-early-regionpromoter that drives the expression of the hygromycin-resistancegene 139) and a mouse metallothionein-I (MT-1) promoter that controls the expression of the human c-erbB-2 cDNA [40]. PA317 amphotropic packaging cells [41] were transfected with the pN12 plasmid by the calcium-phosphate precipitation method, as previously described [121. After 3 wk of selection in hygromycin-B-containing medium (200 wg/mL; Sigma Chemical Co.), antibiotic-resistant PA317 cells were pooled, and cell-free supernatants were collected and filtered through 0.45-wm filters. The liters of the viral stocks

on mouse NIH 3T3 fibroblasts ranged from 1 x 1O5 to 5 x IO5 hygromycin-resistant colonies per milliliter. MCF-1OA TGF-alc-erbB-2(MCF-1OA TE cells) and MCF-1OA Ha-raslcerb5-2 (MCF-1OA HE) cells were generated by infecting MCF-1OA TGF-a CI 4 and MCF-1OA Ha-fJs CI 1 cells 1371, respectively, with the amphotropic recombinant retrovirus pN 12, as previously described [ 131. After hygrornycin selection (200 pg/mLfor 4 wk), 10 MCF-1OATEand 10 MCF-1OA HE individual clones were randomly selected and expanded into cell lines. All cell lines were grown in the continuous presence of 1 p M CdClz to maximally induce the expression of the c-erbB-2 gene through the MT-1 promoter [37]. Monolayer Growth Cells (2 x 104)were seeded in 12 multiwell cluster dishes (Costar, Cambridge, MA) in serum-containing medium. After 24 h, the cells were washed twice and incubated in PC-1 serum-free medium (Ventrex, Portland, ME) in the absence or the presence of 10 ng/mL human recombinant EGF (Collaborative Research) or 10 ng/mL human synthetic TGF-a (Bachem, Torrance, CA). After 4 d of treatment, the cells were trypsinized and counted with a hemocytometer. To determine the extent of basal growth in the absence of exogenous EGF or TGF-a, l o 4 cells were seeded in 12 multiwell cluster dishes in serum-containing medium and, after 24 h, were switched to PC-1 serum-free medium. The cells were trypsinized and counted each day for 5 d. Growth in Soft Agar Cells (2 x 104)were seeded in 1 mLof 0.3% Difco Noble agar (Difco, Detroit, MI) supplemented with DMEM and Ham's F12 medium containing 5% horse serum. This suspension was layered over 1 mL of a 0.8% agar-medium base layer in 35-mm dishes (Costar). After 18 d, the cells were stained with nitro blue tetrazolium (Sigma Chemical Co.). Colonies larger than 0.5 mm in diameter were counted using an Artek 880 colony counter (Artek Systems, Farmingdale, NY). To determine the effects of anti-TGF-a, anti-EGFR, and anti-erbB-2 monoclonal antibodies on anchorage-independent growth of MCF-1 OA TE and MCF-1OA HE clones, cells were plated in soft agar, as described above, in the absence or in the presence of different concentrations of the following antibodies: TAbl, an antihuman TGF-a neutralizing mouse monoclonal lgGl antibody [12,42]; TAb2, an antihuman TGF-a nonneutralizing mouse monoclonal lgG2a antibody [12]; 528, an antihuman EGFR-blockingmouse monoclonal lgG2a antibody [43]; or TAb250, an antihuman c-erb5-2 blocking mouse monoclonal IgG1 antibody that reacts with the extracellular domain of the p185 erbB-2 protein [44]. Preparation of Conditioned Medium and Radioirnmunoassay for TGF-a Conditioned medium (CM) was collected for a 48-h period from cells that were grown in PC-1 serum-free medium and concentrated using a C18 Sep-pak column (Waters Instruments, Rochester, MN), as previously described [lo].TGF-a protein in the CM was measured using a TGF-

TRANSFORMATION OF HUMAN MAMMARY EPITHELIAL CELLS

a-specific radiotmmunoassay(R1A)kit (Biotope, Seattle, WA), as previously described [lo]. Western Blot Analysis Protein lysates (50 p,g of total protein/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and reacted with either 21 N rabbit polyclonal anti-p185 erbB-2 antiserum [45] or with Y13-259 rat monoclonal anti-p21 ras antibody [22], as previously described [22]. Flow-Cytometric Analysis Cells were detached with a nonenzymatic cell dissociation solution (Sigma Chemical Co.) and incubated for 30 min at 4°C with either TAb250 anti-p785 erbB-2 mouse monoclonal antibody or with a nonimmune mouse lgGl mixture (Becton Dickinson, San Jose, CA). After being washed with phosphate-buffer saline (GIBCO) containing 0.5% bovine serum albumin (Sigma Chemical Co.), cells were incubated with fluorescein isothiocyanate-labeled goat antimouse antiserum (Techno Genetics, Milan, Italy) for 30 min at 4°C. Flow-cytometric analysis was performed with a FACScan fluorescence-activatedcell analyzer (Becton Dickinson). Forward- and side-scatter cell gating was performed to detect fluorescence only on intact living cells. For each analysis, a nonimmune mouse lgGl mixture was used as a control to measure nonspecific fluorescence emission. This value was subtracted from each histogram using Consort 30 software (Becton Dickinson) to determine specific immunofluorescent labeling. Tumorigenicity in Nude Mice Groups of six 4-wk-old female BALB/c nude (nu +/nu + ) mice were injected subcutaneously into the dorsal flank with 5 x 1O6 or with 10 x 1O6 of MCF-1OA cells or their derivatives that were resuspended in 200 p,L of phosphatebuffered solution (PBS) or in 200 p L of Matrigel (1 0 mg/mL; Collaborative Research) in the presence or the absence of recombinant human basic fibroblast growth factor (FGF) (200 ng/mL; Amgen, Thousand Oaks, CA). The animals were given drinking water containing 25 m M ZnS04 to activate the MT-1 promoter [46]. Mice were monitored for tumor appearance for a 4-mo period.

RESULTS MCF-1OA Ha-ras and MCF-1OA TGF-a clones were generated after transfection or infection with expression vectors containing the neomycin-resistancegene as selectable marker [37,38]. Therefore, to stably introduce a second gene such as the c-erb6-2 proto-oncogene into these cells, we constructed the pN12 expression vector plasmid by inserting the full-length human c-erb6-2 cDNA [I81 under the transcriptional control of the mouse MT-1 promoter into pNO2, a Moloney leukemia virus-based retroviralexpression vector. pNO2 was derived from the OT-I 529 expression vector plasmid [40] by replacing the neomycin-resistance gene with the gene for resistance to hygromycin, a non-cross-resistant antibiotic [39]. To generate a cell line that produces a replication-defective, recombinant am-

45

photropic retroviruscontaining the c-erb6-2 proto-oncogene and the hygromycin-resistancegene, PA317 packaging cells 141] were transfected with the pNl2 expression vector plasmid and selected in hygromycin-containing medium for 3 wk. pN12 viral stocks were used to infect MCF-1OA cells; MCF-1OA Ha-rasCI 1 cells, which overexpress an activated point-mutated human c-Ha-ras proto-oncogene; and MCF-1OA TGF-a CI 4 cells, which exhibit a 20-fold increase in the production of TGF-(Yprotein compared with MCF-1OA cells [371. We have previously shown that both MCF-1OA Ha-ras CI 1 and MCF-1OA TGF-0: CI 4 cells have the biological characteristics of in vitro transformed cells since they exhibit anchorage-independentgrowth and are able to proliferate under serum-free anchorage-dependent conditions [371. However, neither MCF-1OA Ha-ras nor MCF-1OA TGF-a are tumorigenic. After mass infectionof MCF-1OA, MCF-1 OA Ha-rasCI 1,and MCF-lOATGF-(~C14cellswith thepNl2 virus and selection in hygromycin-containing medium for 4 wk, one MCF-1OA c-erb6-2, 10 MCF-1OA HE, and 10 MCF-1OA TE hygromycin-resistantindividual colonies were randomly selected and expanded into cell lines. All clones were subsequently grown in the continuous presence of 1 p,M CdClz to maximally induce the expression of the c-erb6-2 gene through the MT-1 promoter [37]. To ascertain whether the hygromycin-resistant clones were able to express p l 8 5 erbB-2 protein, western blot analysis with the 21N rabbit anti-erbB-2 antiserum [45] was performed on equivalent amounts of protein extracts prepared from MCF-1OA cells; from MCF-1OA neo cells, which are MCF-1OA cells transfected with a plasmid containing only the neornycin-resistance gene [37]; from the 10 MCF-1OA HE clones; from the 10 MCF-IOATE clones; and from MCF-1OA c-erb6-2 CI 1 cells. As shown in Figure 1, a low but detectable level of p185 erbB-2 protein was observed in MCF-1OA and MCF-1OA neo cells, whereas MCF-1OA c-erb6-2 CI 1, MCF-1OA TE, and MCF-1OA HE clones exhibited a 10-fold to 40-fold increase in p l 8 5 erbB-2 protein levels. p l 8 5 erbB-2 IS probably a growth factor receptor for a novel ligand [47] with a tyrosine kinase activity that is localized on the cell membrane (481. To ascertain the cellular distribution of the p185 erbB-2 protein in the c-erb6-2infected cells, MCF-1OA cells, MCF-1OA c-erb6-2 Cl 1 cells, the 10 MCF-1OA TE clones, and the 10 MCF-1OA HE clones were incubated with TAb250, a mouse anti-erbB-2 monoclonal antibody [44], or with a nonimmune control mouse IgG 1 mixture; labeled with fluorescein isothiocyanate goat antimouse antiserum; and analyzed with a fluorescenceactivated cell analyzer. As illustrated in Figure 2, a 20- to 40-fold increase in the levels of cell-membrane-associated p l 8 5 erbB-2 protein can be detected in MCF-1OA c-erb6-2 CI 1, MCF-1OA TE CI 5 and CI 20, and MCF-1OA HE CI 2 and CI 9 cells compared with parental noninfected MCF-1OA cells. Similar levels of p l 8 5 erbB-2 protein could be detected in the other MCF-1OA TE and MCF-1OA HE clones (data not shown). Furthermore, fluorescence microscopy of cytospin preparations revealed a very intense membraneassociated p l 8 5 erbB-2-specific staining in all the c-erbS 2-infectedclonesascompared with MCF-1OAcontrolcells (data not shown).

46

ClARDlELLOETAL.

Figure 1. Western blot analysis with 21N rabbit polyclonal anti-pl85 erbB-2 antiserum for p185 erbB-2 protein expression in MCF-1OAcellsand derivatives: (A) MCF-1OA neo cells, MCF-1OA

c-erb8-2 CI 1 cells, and MCF-1OATEclones; (B) MCF-1OA HE clones. Fifty microgramstotal proteins/lane were separated by SDS-PAGE and transferred to nitrocellulose [22].

To determine whether c-erbB-2 overexpressioncould interfere with TGF-a production in the MCF-1OA clones, TGF-a levels were measured in the CM obtained from MCF-1OA cells, MCF-1OA c-erb5-2 CI 1 cells, MCF-1OA TGF-a CI 4 cells, and the 10 MCF-1OATE clones using a TGF-a-specific RIA [lo]. All MCF-1OA TE clones secreted TGF-a at a level comparable to that observed in MCF-1OATGF-a CI 4 cells (270-300 ng/l O8 cells/48 h). In contrast, very low levels of TGF-a(l2-15 ng/108cells/48h)wereproduced by MCF-1OA and MCF-1OA c-erbB-2 CI 1 cells. Furthermore, to assess if c-erb5-2 overexpression could affect p2 1 ras expression in the MCF-1OA HE clones, we performed western blot analysis with Y13-259 rat monoclonal anti-p21 ras antibody on cell extracts prepared from MCF-1OA cells, MCF-1OA c-erb5-2 cells, MCF-1OA Ha-ras CI 1 cells, and the 10 MCF-1OA HE clones. A fivefold to 10-fold increase in p21 ras protein levels was observed in MCF-1OA Ha-ras CI 1 and MCF-1OA HE clones compared with MCF-1OA and MCF-1OA c-erb5-2 CI 1 cells (data not shown). To determine whether overexpression of p l 8 5 erbB-2 protein in the MCF-1OA TE and MCF-1OA HE clones was functionally significant and could confer on these cells an advantage in anchorage-independent growth compared with MCF-IOA cells that have been transformed with a single oncogene, the ability of these cells to grow in soft agar was analyzed. As shown in Table 1, MCF-1OA TE and

MCF-1OA HE clones formed fivefold to 20-fold more colonies in soft agar than did MCF-1OA cells transformed with either Ha-ras, TGF-a, or c-erb5-2 alone. MCF-1OA cells exhibited a morphology in monolayer cultures that is typical of normal mammary epithelial cells; they appear flat and cuboidal and exhibit contact-inhibited growth (Figure 3A). No major morphological changes were observed in MCF-1OA cells transformed with either c-Haras, TGF-a, or c-erb6-2 alone (data not shown) or in any of the MCF-1OA TE clones (data not shown; see Figure 38 for a representative clone, i.e., MCF-1OATE CI 5). However, all MCF-1OA HE clones, as shown for MCF-1OA HE CI 9 cells in Figure 3C, appeared fibroblastic with spindleshaped cells. These cells lost contact inhibition of growth and started to grow as multilayers of cells. MCF-1OA cells possess approximately 250 000 EGFRdcell, are very sensitive to the mitogenic effects of either EGF or TGF-a, and require one of these two growth factors for their optimal growth in both serum-containing and serumfree medium [35,37]. In fact, MCF-1OA cells became quiescent after 4-5 d of growth in medium that was depleted of EGF or TGF-a (Figure 4A). In contrast, MCF-1OA Ha-ras CI 1, MCF-1OA TGF-a CI 4, MCF-1OA c-erbB-2 CI 1, MCF-1OA TE, and MCF-1OA HE clones exhibited a threefold to fourfold increase in their anchorage-dependent growth rate under serum-free conditions in the absence

47

TRANSFORMATION OF HUMAN MAMMARY EPITHELIAL CELLS

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RELATIVE FLUORESCENCE INTENSITY Figure 2. Flow-cytometric analysis of specific immunofluorescencelabelingof cell membrane-associated pl85erbB-2 protein with mouse TAb250 anti-pl85 erbB-2 monoclonal antibody. (A) MCF-1OA cells; (6) MCF-1OA c-efbB-2 CI 1 cells; (C) MCF-1OA TE CI 5 cells; (D)MCF-1OA TE CI 20 cells; (E) MCF-1OA HE CI 2

cells; (F) MCF-1OA HE CI 9 cells. For each analysis a nonimmune mouse lgGl mixture was used as a control to measure nonspecificfluorescence emission. This value was subtracted from each histogram using Consort 30 software (Becton Dickinson) to determine specific imrnunofluorescencelabeling.

of EGF or TGF-a. A fourfold to fivefold increase in cell growth was detectable in MCF-1OA cells or in MCF-1OA c-erbB-2 CI 1 cells that had been grown for 4 d in serum-free medium in the presence of 10 ngimL exogenous TGF-a (Figure 4B) or 10 ng/mL exogenous EGF (data not shown). However, MCF-1OAHa-rasCI 1 and MCF-1OATGF-aCI4cellsexhibited a significantly reduced mitogenic response to these growth factors; they showed only a twofold increase in their cell number after 4 d of growth in serum-free medium supplemented with TGF-a (Figure 45). Moreover, MCF-1OA TE and MCF-1OA HE clones, such as the MCF-1OA TE CI 20 and MCF-1OA HE CI 9 cells illustrated in Figure 48, showed an even more reduced response to these growth factors. In fact, they exhibited only a 40% increase in their growth under these growth conditions.

We have previously shown that specific anti-EGFR or antiTGF-a mouse monoclonal antibodies are able to significantly inhibit the growth in soft agar of MCF-1OA Ha-rds cells, thus suggesting that TGF-a is a secreted autocrine growth factor that is an important mediator in the ras transformation of MCF-1OA cells [ 3 7 ] . In fact, MCF-1OA Ha-ras clones express a fourfold to eightfold increase in TGF-a mRNA and protein levels compared with parental MCF-1OA cells. Anti-TGF-a or anti-EGFR antibodies were capable of totally inhibiting the growth in soft agar of MCF-1OA TGF-a clones, thus demonstrating that TGF-a was acting through an external autocrine pathway when overexpressed in MCF-1OA cells [371. Moreover, an antierbB-2 mouse monoclonal antibody could efficiently block the anchorage-independent growth of several MCF-1OA

48

ClARDlELLO ETAL.

Table 1. Anchorage-Independent Growth of MCF-1OA Cells*

Clones

Coloniesidish

MCF-1OA MCF-10Aneo MCF-10 A Ha-rds CI 1 MCF-IOATGF-aCI4 MCF-1OAc-erb5-2CI 1 MCF-10ATECI2 MCF-10 ATE CI 4 MCF-IOATECI 5 MCF-10 ATE CI 9 MCF-IOATECI 10 MCF-IOATE CI 1 1 MCF-10 ATE CI 15 MCF-IOATECI 17 MCF-IOATECI 19 MCF-lOATECI20 MCF-10 A HE CI 1 MCF-10 A HE CI 2 MCF-10 A HE CI 3 MCF-10AHECI4 MCF-10 A HE CI 5 MCF-10 A HE CI 6 MCF-10 A HE CI 7 MCF-10 A HE CI 8 MCF-10 A HE CI 9 MCF-IOAHECI 10

0 0 115 215 130 1625 1600 1890 2000 1680 1750 2080 1810 1750 2125 2400 2750 1835 1380 820 590 2395 1725 2700 2125

(24) (210) (215) (280) (245) (255) (265) (530) (225) (580) (270)

(240) (255) (265) (t85) (k45)

(525) (530) (215)

(585) (k90)

(k95) (270)

*Twenty thousand cells/35-mm dish were seeded in soft agar as described in Materialsand Methods. After 18 d, the cells were stained with nitro blue tetrazolium. Colonies larger than 0.5 mm in diameter were counted using an Artek 880 colony counter. Results represent the average ( I SD)of two separateexperiments,each performedin triplicate.

c-neu clones, whereas it had no effect on the growth in soft agar of MCF-1OA Ha-ras and MCF-1OA TGF-a clones (data not shown) [ 3 7 ] . To ascertain whether these antibodies were also able alone or in combination to interfere with the soft-agar cloning efficiencyof MCF-1OA cells that had been transformed with t w o oncogenes, MCF-1OA TE CI 5 and CI 20 or MCF-1OA HE CI 2 and CI 9 cells were plated in soft agar in the presence of different concentrations of TAbl, a mouse monoclonal anti-TGF-a-neutralizing antibody [ 121; 528, a mouse monoclonal anti-EGFR-blocking antibody (431; or TAb250, a mouse monoclonal anti-erbB-2-blocking antibody [44]. As shown in Figure 5, there was a specific dosedependent inhibition of colony formation in soft agar with a maximum inhibition of 50-70% using the highest dose tested of each antibody in both MCF-1OA TE and MCF-1OA HE clones. Furthermore, when combinations of low, suboptimal concentrations of all three antibodies were used, an additive inhibitory effect was observed upon the anchorageindependent growth of these cells. In contrast, a control antibody such as TAb2, a nonneutralizing anti-TGF-a mouse monoclonal antibody [ 12,371, had no effect on the growth insoftagarof MCF-1OATEand MCF-IOAHEclones. To determine whether the MCF-1OA TE and MCF-1OA HE clones that had a higher cloning efficiency in soft agar than MCF-1OA cells that have been transformed with a

Figure 3. Morphology of (A) MCF-1OA cells, ( 6 ) MCF-1OA TE CI 5 cells, and (C) MCF-1OA HE CI 9 cells in anchorage-dependent growth conditions in serum-containing medium.

singlegenearetumorigenic, 5 x I060r 10 x I06ceIIsfrom MCF-1OA, MCF-1OA neo, MCF-1OA c-erbB-2 CI 1, MU-1OA TGF-aCI4, MCF-1OA Ha-rasCI 1, MCF-IOATE CI 5, CI 9, CI 15, and CI 20, or MCF-1OA HE CI 2 and CI 9 cells were injected subcutaneously into the dorsal flank of nude mice. No tumors were detected in any of the animals during a 4-mo observation period. It has recently been shown that several human small-cell lung cancer cell lines and primary colon adenocarcinomas, when suspended in a reconstituted basement membrane and extracellular matrix such as Matrigel [49], are able to grow with a better efficiency as tumors in nude mice [50,511. For this reason, 10 x lo6 cells from MCF-IOA, MCF-1OA neo, MCF-1OA Ha-ras CI 1, MCF-IOATGF-aC14, MCF-IOAc-erbB2CI 1,MCF-1OA

TRANSFORMATION OF HUMAN MAMMARY EPlTHELlAL CELLS

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Figure 4. Anchorage-dependent growth of MCF-1OA cells under serum-free medium conditions. (A) Ten thousand cells/well were seeded in 12 multiwell cluster dishes. Twenty-four hours later, the cells were switched to PC-1 serum-free medium and counted with a hemocytometer every day for 5 d. 0 , MCF-1OA cells; A,MCF-IOA Ha-ras CI 1 cells; m, MCF-1OATGF-aCI 4 cells; A,MCF-1OA c-erbB-2 CI 1 cells; 0, MCF-1OA TE CI 20 cells; 0, MCF-1OA HE CI 9 cells. Values represent the average of two different experiments, each performed in triplicate; SD was less than 10%. (B) Twenty thousand cells/well were seeded in 12

multiwell cluster dishes. After 24 h, the cells were switched to PC-1 serum-free medium and grown for 4 d in the presence (hatched columns) or in the absence (open columns) of TGF-a (10 ng/mL) before being counted. Values represent the average ( 2SD) of two different experiments, each performed in quadruplicate. MCF-IOATECI 20 and MCF-1OA HE CI 9 cells are shown as representative examples; the anchorage-dependent growth properties of all the MCF-1OA TE and MCF-1OA HE clones were comparable.

TE CI 5, CI 9, CI 15, and CI 20, or MCF-1OA HE CI 2 and CI 9 cells were mixed with 200 KL of Matrigel (10 mg/mL) in the presence or absence of a potent angiogenetic factor such as recombinant human basic FGF (200 ng/mL) to facilitate neovascularization, then injected subcutaneously into nude mice. However, even under these conditions, no tumors were observed in any of the MCF-1OA clones that were tested

c-rnyc oncogenes, mammary tumors develop at a much higher frequency and with a shorter latency period than in transgenic animals that express either gene alone [I 51. Moreover, cooperation between other functional classes of oncogenes has also been described. In this regard, a synergistic interaction between p l 8 5 erbB-2 and EGFR, two tyrosine kinases that are growth factor receptors, has been shown in the transformation of NR6 Swiss 3T3 mouse fibroblasts [59]. Our study was undertaken to analyze the effects and the possible interactions between proto-oncogenes such as c-Ha-ras, €GFR, c-erb8-2, and TGF-w in the in vitro and in vivo transformation of normal human mammary epithelial cells. MCF-1OA cells are a unique model for these purposes because they are human untransformed, mammary epithelial cells that possess a functional complement of EGFR and are extremely sensitive to the mitogenic effects of EGF and TGF-cx[35,37]. In addition, these cells can be transformed in vitro after the introduction of one of these genes [35-381. This study is the first to demonstrate that the c-erbB-2 proto-oncogene in combination with an activated c-Ha-ras or the TGF-a gene had an additive effect in the in vitro transformation of human mammary epithelial cells. In fact, MCF-1OA TE and MCF-1OA HE clones have undergone a further step toward neoplastic transformation compared with MCF-1OA Ha-ras, MCF-1OA c-erb5-2, or MCF-1OA TGF-w cells. These cells grew with a significantly higher cloning efficiency in soft agar than MCF-1OA cells that had been transformed by a single oncogene.

DISCUSSION

Previous epidemiological, clinical, and experimental studies have suggested that tumor development and progression is a multistage process that involves alteration or activation of several growth regulatory genes, or both [1,52-541. In this respect, introduction of at least two oncogenes is necessary for the in vitro and in vivo transformation of primary cultures of rodent embryo fibroblasts [21. Generally, a nuclear oncogene, such as rnyc, f a , jun, mutant forms of p53, or adenovirus EIA, is required to cooperate with a cytoplasmic oncogene, such as ras, src, or polyoma middle-T antigen, to transform these cells [ 1,55,56]. Oncogene cooperation has been also observed in the transformation of some epithelial cells. For example, loss of differentiation and complete transformation of PC-rat thyroid epithelial cells can be obtained following the introduction of at least two oncogenes such as v-Harasand c-mycor polyoma middle T and c-rnyc(57,58]. Similar results have been obtained with in vivo experimental models. In transgenic mice that express both v-Ha-rasand

ClARDlELLO ETAL.

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Figure 5. Effects of antihuman neutralizing anti-TGF-a (TAbl), antihuman blocking anti-EGFR (528). and antihuman blocking anti-erbl-2 specific monoclonal antibodies on the anchorageindependent growth of (A) MCF-1OA TE CI 5 and (€3) CI 20 cells and of (C) MCF-IOA HE CI 2 and (D) CI 9 cells. Twenty thousand cells were plated in soft agar as described in Materials and Methods and treated with different concentrations of TAbl, TAb250, 528, and TAb2, a nonneutralizing anti-TGF-a antibody that was used as an internal negative control. Column a of each panel shows the results for cells treated with a combination of 10 pg/mL TAbl, 10 pg/mL TAb250, and 1 pg/rnL 528. Column b of each

panel shows the results for cells treated with a combination of 25 pg/mL TAbl, 25 pg/mL TAb250. and 5 pg/mL 528. Data are expressed as the percentage of cell growth in treated versus control dishes (number of colonies larger than 0.5 mm in diameter in the presence of the indicated treatment divided by number of colonies in applicable untreated control dishes). Cell growth for untreated controls was as follows: MCF-1OA TE CI 5, 1985 colonies/dish; MCF-1OA TE CI 20, 2210 colonieddish; MCF-1OA HE CI 2,2895 colonies/dish; MCF-1OA HE CI 9,2840 colonieddish. Values are the average (-c SD) of four determinations.

Moreover, MCF-1OA cells that overexpress both p l 8 5 erbB-2 and p21 ras or TGF-a have a greater reduction in their growth factor requirement compared with MCF-1OA cells that have been transformed with either c-Ha-ras or TGF-a. These cells can grow optimally in the absence of exogenous EGF or TGF-a because they produce high levels of endogenous TGF-a (data not shown), which may function in an autocrine fashion, because addition of antiEGFR-blocking or anti-TGF-a-neutralizing monoclonal antibodies was able to significantly decrease the ability of both MCF-1OA TE and MCF-1OA HE cells to grow in soft agar. Moreover, cotreatment with anti-EGFR, anti-TGF-a, and antierbB-2 monoclonal antibodies had an additive effect in inhibiting the anchorage-independent growth of MCF-1OA TE and MCF-1OA HE cells. Finally, MCF-1OA HE cells, but not MCF-1OATEcellsor MCF-IOAcellsthatwere transformed with a single gene, showed morphological changes that were similar to those observed in oncogene-transformed rodent fibroblasts. These experimental findings may be of some clinical relevance for the treatment of human breast cancer, since it has recently been shown that it is possible to identify subgroups of primary human breast carcino-

mas that overexpress either p21 ras and p185 erbB-2 or EGFR and p185 erbB-2, and that also have a very poor clinical outcome [60,61]. Introduction of two transforming genes such as c-erb5-2 and c-Ha-ras or c-erb5-2 and TGF-a into MCF-1OA human mammary epithelial cells is not, however, sufficient to render these cells tumorigenic. This contrasts with the results that have been recently obtained with the 184B5 human mammary epithelial cell line [621. Overexpression of the c-erbB-2 proto-oncogene alone in these cells was able to induce in vitro transformation and in vivo tumorigenesis at a low frequency. However, in contrast with MCF-1OA cells, which are a near-diploid, untransformed, spontaneously immortalized human mammary epithelial cell line, 18485 cells were originally obtained as an immortal cell line after treatment of a primary human mammary epithelial outgrowth with benzo[a]pyrene, a chemical carcinogen, and in culture have acquired an aneuploid karyotype [63]. The establishment of several MCF-1OA clones with different combinations of oncogenes may provide a useful model system for testing the effects of other genes that have been associated with breast cancer progression, inva-

TRANSFORMATION OF HUMAN MAMMARY EPITHELIAL CELLS

sion, and metastasis. It is conceivable that one or more additional genetic changes in other cellular proto-oncogenes or in tumor suppressor genes that have been associated with the development and the progression of breast cancer [3]may be required to elicit a fully malignant phenotype in MCF-1OA cells. In this respect, a genetic change may be necessary in the p53 tumor suppressor gene [64] since p53 mutations and deletions are two of the more common genetic alterations that have been found in human breast cancer [65]. Finally, MCF-1OA cells and their derivatives that have been transformed with c-Ha-ras, c-erbB-2, and TGF-a genes alone or in combination may represent a useful mammary epithelial cell model in which to study the effects of conventional antineoplastic drugs as well as of other agents such as specific monoclonal antibodies, antisense oligonucleotides, and inhibitors that interferewith oncogene, growth factor, and growth factor receptor action or with the intracellular signalling pathway that is induced following activation of these genes or with both. ACKNOWLEDGMENTS The authors thank Drs. H. Soule and J. Russo, Michigan Cancer Foundation, Detroit, MI, for kindly providing the MCF-1OA and MCF-1OA Ha-ras cells; Dr. M.L. McGeady, National Cancer Institute, Bethesda, MD, for the generous gift of the pNO2 expression vector plasmid and for helping to prepare the pN12 expression vector plasmid; Dr. T. Yamamoto, University of Tokyo, Japan, for the gift of the full-length human c-erbB-2 cDNA; Dr. J. Mendelsohn, Memorial Sloan Kettering Cancer Center, New York, NY, for kindly providing the 528 monoclonal antibody; Dr. B.C. Langton, Berlex Biosciences Inc., Alameda, CA, for the gracious gift of TAbl, TAb2, and TAb250 monoclonal antibodies; and Dr. W. Gullick, Imperial Cancer Research Fund, London, England, for generously providing the 21 N rabbit antiserum. The excellent technical assistance of Mr. S. Pedace is acknowledged. Part of this research was supported by grants from the Associazione ltaliana per la Ricerca suI Cancro. N.N. was partly supported by a fellowship from the Associazione ltaliana per la Ricerca sul Cancro. R.B.D. was supported by a grant from the American Cancer Society. Received December 31, 1991; revised February 19, 1992; accepted March 13, 1992.

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Additive effects of c-erbB-2, c-Ha-ras, and transforming growth factor-alpha genes on in vitro transformation of human mammary epithelial cells.

MCF-10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a hum...
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