Interactions between progestins and heregulin (HRG) signaling pathways: HRG acts as mediator of progestins proliferative effects in mouse mammary adenocarcinomas

Interactions between progestins and heregulin (HRG) signaling pathways:HRG acts as mediator of progestins proliferative e�ects in mousemammary adenocarcinomas

Maria Eugenia BalanÄ a 1, Ruth Lupu2, Leticia Labriola1, Eduardo H Charreau1 andPatricia V Elizalde*,1

1Instituto de BiologõÂa y Medicina Experimental (IBYME), Obligado 2490, Buenos Aires 1428, Argentina; 2Lawrence BerkeleyNational Laboratories, University of California Berkeley, 1 Cyclotron Road, Building 934 Berkeley, California, CA 94710, USA

The present study addressed links between progestin andheregulin (HRG) signaling pathways in mammarytumors. An experimental model of hormonal carcinogen-esis, in which the synthetic progestin medroxyprogester-one acetate (MPA) induced mammary adenocarcinomasin female Balb/c mice, was used. MPA induced an invivo up-regulation of HRG mRNA expression inprogestin-dependent (HD) tumor lines. Mammary tumorprogression to a progestin-independent (HI) phenotypewas accompanied by a high constitutive expression ofHRG. The HRG message arose from the tumorepithelial cells. Primary cultures of malignant epithelialcells from a HD tumor line were used to investigateHRG involvement on cell proliferation. HRG induced apotent proliferative e�ect on these cells and potentiatedMPA mitogenic e�ects. Blocking endogenous HRGsynthesis by antisense oligodeoxynucleotides (ASODNs)to HRG mRNA inhibited MPA-induced cell growth,indicating that HRG acts as a mediator of MPA-inducedgrowth. High levels of ErbB-2 and ErbB-3 expressionand low ErbB-4 levels were found in HD cells.Treatment of these cells with either MPA or HRGresulted in tyrosine phosphorylation of both ErbB-2 andErbB-3. Furthermore, both HRG and MPA proliferativee�ects were abolished when cells were treated withASODNs to ErbB-2 mRNA, providing evidence for acritical role of ErbB-2 in HRG-induced growth. Finally,blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the completeinhibition of HRG proliferative e�ect, demonstratingthat a functional IGF-IR is required for HRG mitogenicactivity. These results provide the ®rst evidence ofinteractions between progestins and HRB/ErbB signaltransduction pathways in mammary cancer and the ®rstdemonstration that IGF-IR is required for HRGproliferative e�ects.

Keywords: progestin; heregulin; mouse mammarytumors; type I insulin-like growth factor receptor

Introduction

The Neu Di�erentiation Factor (NDF) or Heregulin(HRG) family includes a series of polypeptides sharingan epidermal growth factor-(EGF)-like motif that acts

as the receptor binding domain (Peles and Yarden,1993; Wen et al., 1994). According to the structure ofthis motif, two major subclasses of HRG variants, aand b, can be distinguished (Carraway and Cantley,1994). HRG exerts multiple physiological actionsthrough a unique combinatorial signaling. Twomembers of type I receptor tyrosine kinase family(RTKs-I), ErbB-3 and ErbB-4, mediate HRG functions(Carraway and Cantley, 1994). The other RTKs-Ifamily members, ErbB-1 (or EGF-R) and ErbB-2, arethe co-receptors (Pinkas-Kramarski et al., 1997).Activation of RTKs-I by HRG initiates signaltransduction cascades involved in cellular responsessuch as proliferation and di�erentiation. HRG treat-ment of breast cancer cells induces activation of theErk/MAP kinases (Marte et al., 1995a; Sepp-Lorenzinoet al., 1996), Jnk/SAP kinases (stress activated proteinkinases) (Amundadottir and Leder, 1998), p70/p85 S6kinase (Marte et al., 1995a) and phosphatidyl inositol3-kinase (PI-3K) (Sepp-Lorenzino et al., 1996; Amun-dadottir and Leder, 1998). Particularly interesting isthe role of ErbB-3 in PI-3K activation, since ErbB-3possesses the unique capacity among RTKs-I to bindthe p85 regulatory subunit of PI-3K (Carraway andCantley, 1994; Fedi et al., 1994; Graus-Porta et al.,1997). ErbB-3 ability to associate with PI-3K confersto this receptor the possibility of sharing signaltransduction pathways with insulin-like growth factors(IGFs), since activation of type I IGF receptor (IGF-IR) results in tyrosine phosphorylation of a substrateprotein, IRS-1, which in turn binds to and activatesp85 (Backer et al., 1993).

The biological e�ects of HRG exhibit variations thatdepend on cell type, HRG isoform, and cellularcomplements of RTKs-I available to make upfunctional heterodimers. Certain HRG isoforms arereported to induce growth-arrest and di�erentiation ofmammary epithelial cells, while other breast cellsrespond mitogenically (Yang et al., 1995; Marte etal., 1995b; Ram et al., 1995). Also, stimulatory(Holmes et al., 1992; Lewis et al., 1996) and inhibitorye�ects (Peles et al., 1992; Bacus et al., 1993; Culouscouet al., 1993) of HRG on the proliferation of breastcancer cell lines, expressing di�erent levels of ErbB-2,have been reported. In addition, a biphasic e�ect ofHRG on the proliferation of cells overexpressing ErbB-2 has been found. In the cell line BT-474 low doses ofHRG induce cell growth and higher doses inhibitproliferation (Grunt et al., 1995). Further support forHRG involvement in mammary carcinogenesis hasbeen provided by transgenic mice overexpressingHRGb; in these female mice, mammary adenocarcino-

*Correspondence: PV ElizaldeReceived 18 November 1998; revised 16 May 1999; accepted 16 May1999

Oncogene (1999) 18, 6370 ± 6379ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00

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mas arise in a stochastic fashion following severalpregnancies (Krane and Leder, 1996).

Links between HRG/ErbB-2 signaling pathway andestrogen receptor (ER) have been described. Treatmentof hormone-dependent breast cancer cell lines withHRG results in decreased ER expression at protein andRNA levels (Saceda et al., 1996; Pietras et al., 1995).However, whether HRG-induced down-regulation ofER is followed by induction (Pietras et al., 1995) orinhibition (Saceda et al., 1996) of ER activity remainscontroversial. On the other hand, treatment of breastcancer cells with estrogens results in decreasedexpression of ErbB-2 (Grunt et al., 1995).

The aim of the present study was to investigate theinteractions between progestins and HRG signalingpathways by using the well characterized model ofhormonal carcinogenesis in which the syntheticprogestin medroxyprogesterone acetate (MPA) in-duces mammary adenocarcinomas in virgin femaleBalb/c mice (Molinolo et al., 1987; Lanari et al.,1989). Most of the MPA-induced tumors are of ductalorigin, express high levels of ER and progesteronereceptor (PR) and are maintained by syngeneictransplants in MPA-treated mice, showing a proges-tin-dependent (HD) pattern of growth (Lanari et al.,1989). By transplantation into untreated mice, tumorvariants with an MPA-independent (HI) pattern ofgrowth have been generated. These HI tumors expressthe same ER and PR content as their parental HDcounterparts (Lanari et al., 1989). Finally, MPAinduces a small number of lobular mammarycarcinomas which lack ER and PR expression andevidence an MPA-independent (HI) behavior (Moli-nolo et al., 1987; Lanari et al., 1989).

We have found that in vivo HRG expression in HDtumors is under MPA control. Mammary tumorprogression to a hormone-independent phenotype isaccompanied by a high constitutive expression ofHRG. We have used primary cultures of epithelialcells from a HD tumor line to study the involvement ofHRG on cell growth, and show that HRG stimulatesHD cell proliferation and is able to potentiate MPAproliferative e�ects. MPA and HRG treatment of HDcells results in tyrosine phosphorylation of ErbB-2 andErbB-3. We blocked both HRG and ErbB-2 expressionusing antisense oligodeoxynucleotides (ASODN) anddemonstrated that HRG acts as mediator of MPA-induced proliferation and that ErbB-2 is essential inthis motogenic pathway. Finally, we showed, for the®rst time according to our knowledge, that a functionalIGFR-IR is required for HRG biological activity.

Results

Expression of HRG at the mRNA level in in vivo linesof MPA-induced mammary tumors

In order to obtain homologous probes for the analysisof heregulin expression in mouse mammary tumors,two fragments of the mouse HRG were cloned byusing PCR and cDNAs obtained from HD tumors ofour experimental model. Sequence analysis of thefragments, localized in the Ig domain (fragment 1)and the cytoplasmic tail domain (fragment 2), showeda high degree of homology with the rat (Wen et al.,

1992) (fragment 1: 89%; fragment 2: 92%) and human(Lupu and Lippman, 1993) (fragment 1: 83%;fragment 2: 89%) HRG genes (Figure 1).

HRG expression was studied performing RNAseprotection assays, using the fragment localized in the Igdomain of the gene as probe. This probe recognisesboth HRGa and b isoforms.

Three types of tumors from the MPA-induced modelof mammary carcinogenesis were included in thisstudy. The ®rst type were ductal hormone-dependent(HD) tumor lines that require MPA administration fortheir in vivo growth and express high levels of ER andPR content (Molinolo et al., 1987; Lanari et al., 1989).The second were their hormone-independent (HI)variants, that are able to grow in absence of MPAadministration. These HI ductal tumors, in spite of anMPA-independent pattern of growth, retain ER andPR expression (Molinolo et al., 1987; Lanari et al.,1989). The third type of tumors analysed were MPA-induced mammary carcinomas of lobular origin thatlack ER and PR expression and evidence a HIbehavior (Molinolo et al., 1987; Lanari et al., 1989).

HRG mRNA in vivo expression was found in ductalHD tumor lines C4, D5 and 53. A signi®cant increase(2 ± 5-fold) of HRG mRNA levels was detected intumors growing in MPA-treated mice, as comparedwith HD tumors growing in untreated animals (Figure2a,b). HRG message was also detected in the HIvariants of ductal origin, C4HI, D5HI, and 53HI, withsimilar levels to those observed in HD tumors growingin MPA-treated mice (Figure 2a,b). HRG mRNA wasnot found in HI lines of lobular histology 39, 55, 60and 52B (Figure 2a).

Cellular localization of HRG message

To identify the cellular origin of HRG, RNAs obtainedfrom primary cultures of epithelial and ®broblastic

Figure 1 Mouse HRG sequence. Nucleotide sequence of thecDNAs cloned fragments 1 and 2 and comparison betweenmouse, rat, and human HGF genes. Di�erences in HRGnucleotide sequence between species are indicated with boldletters. Dashes indicate gaps that were introduced for optimalalignment

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tumor cells were subjected to RNAse protection assays.HRG mRNA expression was limited to cells of

epithelial origin. Representative results in 59HD andC4HD tumors are shown in Figure 2c.

Involvement of HRG on the proliferation of primarycultures of C4HD tumor line

C4HD tumor line was selected to study HRGinvolvement in in vitro growth because it hasexhaustively been characterized as regard to its invitro proliferative behavior and response to MPA(Dran et al., 1995). C4HD epithelial cells proliferatevery slowly in absence of MPA treatment, retain PRexpression and the exquisite sensitivity to MPAproliferative e�ect shown by the in vivo tumor line(Dran et al., 1995). Therefore, all the characteristicsshowed by C4HD tumor in vivo growth are maintainedin this primary culture system. In addition, C4HD lineexpress the lower level of endogenous HRG among allHD lines studied, making it possible to detectbiological e�ects when HRG is exogenously added.

ErbB receptor expression

We ®rst investigated the cellular complements of ErbBreceptors expressed in C4HD cells. Western blots wereperformed on whole C4HD cell lysates with antibodiesto ErbB-2, ErbB-3 and ErB-4. As control for levels ofErbB-2 expression we used the human breast cancerMCF-7 cell line that contains low ErbB-2 levels. C4HDcells expressed signi®cantly higher levels of ErbB-2than MCF-7 cells (3 ± 5-fold), comparable with thoseconsidered as overexpression in human breast cancercell lines (Figure 3) (Lupu and Lippman, 1993). Highlevels of ErbB-3 expression were also found in C4HDcells. Our analysis of ErbB-4, using a panel of ErbB-4speci®c antibodies, indicated low ErbB-4 expressionlevels.

HRG stimulates the proliferation of epithelial cultures ofC4HD tumor line

HRGb1, at concentrations ranging from 0.02±200 ng/ml, was added to C4HD epithelial tumor cells andincorporation of [3H]thymidine was used as measure ofDNA synthesis and mitogenic activity. HRGb1 atconcentrations of 2 ng/ml and above signi®cantlyincreased [3H]thymidine uptake respect to cells grow-

A

B

C

Figure 2 HRG expression in mammary adenocarcinomasinduced by MPA. (a) HRG RNAse protection assay. Thirtymicrograms of total RNA from the tumors were hybridized withHRG and rpL32 probes as described in Materials and methods.HRG protected fragment is 333 b.p., and rpL32 protectedfragment is 76 b.p. MWM: molecular weight markers. +MPA:tumors growing in MPA-treated mice. C4, D5, 53 and C7 aretumors of ductal origin; 39, 55, 60 and 52B are hormone-independent tumors of lobular origin. (b) Quantitation of theamounts of HRG mRNA. Hybridization signals with HRG weredensitometrically scanned and normalized to rpL32 control andthe data from four separate experiments were pooled to give thehistogram which represents the mean+s.e.mean. (c) Cellularlocalization of HRG in HD tumors. Total RNA (40 mg) fromprimary cultures of epithelial (E) and ®broblastic (F) cells from59HD and C4HD tumors were hybridized with HrG and rpL32probes and subjected to RNAse protection assays

Figure 3 Expression of ErbB receptors in C4HD epithelial cells.Eighty mg of protein from C4HD cell lysates were electrophoresedand immunoblotted for ErbB-2, ErbB-3, and ErbB-4. MCF-7human breast cancer cells were used as control for ErbB-2expression levels

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ing in 2.5% ChFCS (Figure 4). Moreover, HRG (20 ±200 ng/ml) signi®cantly enhances MPA stimulatorye�ects (Figure 4).

Antisense oligodeoxynucleotides to HRG mRNA inhibitC4HD cells proliferation

Incubation of C4HD epithelial cells with antisenseoligodeoxynucleotides (ASODNs) to HRG mRNAresulted in a dose-dependent inhibition of MPA-mediated cell proliferation (Figure 5a). The additionof HRGb1 to C4HD cells treated with ASODNs fullyreverted growth inhibition (Figure 5a). Neither senseODNs (SODNs) at the same concentrations (Figure5a) nor scrambled oligos (data not shown) had anye�ect on cell growth. Growth of C4HD cells in 2.5%steroid-stripped fetal calf serum (ChFCS) was alsoinhibited by ASODNs (Figure 5a). There was noevidence of toxicity in response to ODNs asdetermined by loss of cells into the medium. Thee�ect of HRG ODNs on HRG expression wasassessed by immunoblotting C4HD cell lysates.Densitometric evaluation demonstrated that HRGlevels were reduced by 65 ± 76% after 4 mM ASODNstreatment, while SODNs did not reduce HRG levels(Figure 5b).

Tyrosine phosphorylation of ErbB family of receptors

In order to extend our observations that HRG mightact as a mediator of MPA proliferative e�ects, westudied the level of tyrosine phosphorylation of ErbBreceptors as a marker of their activation. Therefore,extracts from C4HD cells treated with either MPA orHRG were immunoprecipitated with anti ErbB-2 orErbB-3 antibodies and the phosphotyrosine content ofthese receptors was determined by performing Westernblotting with an anti-phosphotyrosine antibody.Figure 6 shows that while tyrosine phosphorylationof both receptors was undetectable in cells growing in2.5% ChFCS, MPA and HRG, after 48 h of

treatment, induced a strong phosphorylation of bothErbB-2 and ErbB-3. Increase of tyrosine phosphoryla-tion of ErbB-2 and ErbB-3 by HRG treatment wasseen as early as 10 min after the addition of HRG(data not shown).

Antisense oligodeoxynucleotides to ErbB-2 mRNAinhibit both HRG and MPA-induced proliferation ofC4HD cells

Incubation of C4HD cells with ASODNs to ErbB-2mRNA inhibited HRG-mediated proliferation (Figure7a). Since our results indicate that MPA-inducedgrowth of C4HD cells is mediated by HRG, throughthe activation of ErbB-2 and ErbB-3, we nextinvestigated whether ErbB-2 is a critical componentin this signaling pathway. Incubation of C4HD cellswith ASODNs to ErbB-2 mRNA resulted in a dose-dependent inhibition of MPA-mediated cell prolifera-tion. (Figure 7a). Sense ODNs had no e�ect on cellgrowth. (Figure 7a). The e�ect of ErbB-2 ODNs onprotein expression was assessed by immunoblottingC4HD cell lysates. Figure 7b shows the resultsobtained when ASODNs were used to inhibit MPA-induced proliferation. Densitometric evaluation de-monstrated that ErbB-2 levels were reduced by 68%after 2 mM ASODNs treatment, while SODNs did not

Figure 4 E�ects of exogenous HRG on the proliferation ofprimary cultures of C4HD cells. Primary cultures of epithelial cellsfrom C4HD tumors were incubated for 48 h in medium with 2.5%ChFCS supplemented with MPA 10 nM, HRG at variousconcentrations, and MPA+HRG. Incorporation of[3H]thymidine was used as a measure of DNA synthesis. b vs a, cvs a, d vs b, and d vs c: P50.001. Data are presented as mean+s.d.The experiment shown is representative of a total of three

Figure 5 Inhibition of C4HD cells proliferation by ASODNs toHRG RNA. (a) C4HD cells were incubated for 48 h in mediumwith 2.5% ChFCS supplemented with MPA 10 nM, ASODNs orSODNs to HRG mRNA, MPA 10 nM+ASODNs or SODNs toHRG mRNA, and MPA 10 nM+4 mM ASODN with the additionof 2 ng/ml of HRG. [3H]thymidine incorporation into DNA wasdetermined. Data are presented as mean+s.d. b vs a, c vs a, and dvs b: P50.001. The experiment shown is representative of a totalof four. (b) E�ect of HRG ASODNs on HRG synthesis. Eightymg of protein from C4HD cell lysates were electrophoresed andimmunoblotted for HRG. C4HD cells growing in 2.5% ChFCS(lane 1), 2.5% ChFCS+4 mM SODN (lane 2), 2.5%ChFCS+4 mM ASODN (lane 3), 10 nM MPA (lane 4), 10 nMMPA+4 mM SODN (lane 5), and 10 nM MPA+4 mM ASODN(lane 6). Densitometric analysis of HRG band expressed as apercentage of the control value (i.e. C4HD cells growing in 2.5%ChFCS or 10 nM MPA) is: 35% for cells growing in ChFCS and24% for cells growing in 10 nM MPA, treated with 4 mMASODN. No signi®cant di�erences in densitometric values ofHRG bands between control cells and cells treated with 4 mMSODN were found. This is an autoradiogram from arepresentative experiment of a total of four in which s.e.m. waswithin 10%

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reduce ErbB-2 levels. Similar inhibition of ErbB-2protein expression was obtained when ASODNs wereused to inhibit HRG-stimulated growth. When theninvestigated whether blocking ErbB-2 by ASODNshad any e�ect on the level of ErbB-3 or ErbB-4protein expression. Figure 7c,d shows that neitherErbB-3 nor ErbB-4 protein levels were a�ected byinhibition of ErbB-2 expression.

Antisense oligodeoxynucleotides to type I insulin-likegrowth factor receptor (IGF-IR) mRNA blockHRG-induced proliferation of C4-HD cells

We have previously demonstrated by using antisenseoligodeoxynucleotides to IGF-IR mRNA, that afunctional IGF-IR is required for MPA-inducedproliferation of HD cells (Elizalde et al., 1998).Therefore, we decided to investigate whether HRGalso required the presence of a functional IGF-Ireceptor for its mitogenic activity. Figure 8 showsthat treatment of C4HD cells with ASODNs to IGF-IR resulted in the complete inhibition of HRG-inducedproliferation of C4HD cells. Cells treated withASODNs showed 78% decrease in the number ofIGF-IR as measured by Scatchard plot analysis. Thenumber of receptors in control or SODNs-treatedC4HD epithelial cells was 48.6+4.1 fmol/mg protein,while in cells treated with the ASODNs it was10.5+1.1 fmol/mg protein. We further investigatedwhether abolishing IGF-IR had any e�ect on thelevel of ErbB-2, ErbB-3 or ErbB-4 protein expression.

Figure 6 Induction of ErbB-2 and ErbB-3 tyrosine phosphoryla-tion by MPA and HRG. Primary cultures of epithelial cells fromC4HD tumors were treated for 48 h with either 10 nM MPA or2 ng/ml HRG or left untreated growing in 2.5% ChFCS. Cellswere lysed, ErbB-2 and ErbB-3 were immunoprecipitated from200 mg of total proteins and immunocomplexes were subjcted toSDS±PAGE (6% gel) and analysed by Western blotting with ananti-P-Tyr mAb (a and c). Identical aliquots of each immuno-precipitate were subjected to immunoblot analysis with anti-ErbB-2 (b) or anti-ErbB-3 (d) antibodies to verify that nearly equalamount of immunoprecipitated proteins were loaded. W, Westernblot; IP, immunoprecipitation

Figure 7 Inhibition of HRG and MPA-induced proliferation ofC4HD cells by ASDNs to ErbB-2 mRNA. Primary cultures ofC4HD cells were incubated for 48 h in medium with 2.5%ChFCS supplemented with MPA 10 nM, HRG 20 ng/ml, andMPA or HRG+ASODNs or SODNs to ErbB-2 mRNA. Dataare presented as mean+s.d. b vs a, and c vs b, d vs a, e vs d:P50.001. The experiment shown in representative of a total offour (a). E�ect of ASODNs on ErbBs protein synthesis. Eighty mgof protein from C4HD cell lysates were electrophoresed andimmunoblotted for ErbB-2 (b) ErbB-3 (c), and ErbB-4 (d). C4HDcells growing in 10 nM MPA (lane 1), in 10 nM MPA+2 mMSODN (lane 2) and in 10 nM MPA+2 mM ASODN (lane 3).Densitometric analysis of ErbB-2 band expressed as a percentageof the control value (i.e. C4HD cells growing in 10 nM MPA) is:32% for cells treated with 2 mM ASODN. No signi®cantdi�erences in densitometric values of ErbB-2 bands betweencontrol cells and cells treated with 2 mM SODN were found. Thisis an autoradiogram from a representative experiment of a totalof four in which s.e.m. was within 10%

Figure 8 Inhibition of HRG-induced proliferation of C4HD cellsby ASODNs to IGF-IR RNA. Primary cultures of C4HDepithelial cells were incubated for 48 h in medium with 2.5%ChFCS supplemented with 20 ng/ml HRG, and 20 ng/mlHRG+40 mg/ml of ASODNs or SODNs to IGF-IR RNA.[3H]thymidine incorporation into DNA was determined. Dataare presented as mean+s.d. b vs a; c vs b: P50.001. Theexperiment shown is representative of a total of three (a). E�ectof IGF-IR ASODNS on ErbB-2 (b), ErbB-3 (c), and ErbB-4 (d)protein synthesis. Eighty mg of protein from C4HD cell lysateswere electrophoresed and immunoblotted for ErbBs. C4HD cellsgrowing in HRG 20 ng/ml (lane 1), in HRG+40 mg/ml ASODNs(lane 2), and in HRG+SODNs (lane 3)

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Figure 8b,c and d shows that inhibition of IGF-IR byASODNs did not a�ect the protein level of ErbBsreceptors.

Discussion

In this work, we have detected HRG expression in invivo, lines of the MPA-induced mammary tumor modelin Balb/c mice (Molinolo et al., 1987; Lanari et al.,1989). HRG message was con®ned to tumors of ductalorigin, ER and PR positive, since HRG mRNA wasnot found in tumors of lobular histology that lacksteroid hormone receptors. HRG mRNA expressionhas been studied in a small number of primary humanbreast carcinomas (Normanno et al., 1993, 1995) andno correlation between its expression, nodal status,EGF-R or ErbB-2 presence has been reported(Normanno et al., 1993, 1995). Other authors havedescribed an association between HRG expression andabsence of metastasis to the lymph nodes in humanbreast tumors (Bacus et al., 1993). HRG mRNAsynthesis was found to be very restricted in breastcancer cell lines (Normanno et al., 1993; Lupu andLippman, 1993) and was una�ected by 17b-estradiol inMCF-7 cells (Normanno et al., 1993).

We found that MPA induced an in vivo up-regulation of HRG mRNA levels in HD lines.Although to our knowledge this is the ®rst reportexploring progestin regulation of HRG synthesis inmammary cancer, Yang et al. (1995) have shown thatHRG expression in the mouse mammary gland isunder tight hormonal control. HRG is expressed inmesenchymal cells adjacent to the ductal epitheliumduring lobulo-alveolar development in pregnancy. NoHRG mRNA was measured during the virginal periodand expression sharply decreased during lactation andwas absent during involution (Yang et al., 1995). These®ndings, like ours, suggest that progesterone controlsthe synthesis of HRG. However, in our mammarytumor model, the source of HRG is the malignantepithelial cells, which also express HRG receptorsErbB-3 and ErbB-4 and the co-receptor ErbB-2. Thus,the switch from a paracrine role of HRG in normalmammary gland to an autocrine role in mammarycancer, could re¯ect a subversion of the normal growthcontrol mechanisms which results in a proliferativeadvantage for tumor cells.

Progression to a hormone-independent phenotype ofductal tumors was accompanied by a high constitutiveexpression of HRG. This ®nding is in accordance withprevious results (Pietras et al., 1995) demonstratingthat stable transfection of MCF-7 cells with a fulllength HRG b1 cDNA, conducting to overexpressionof HRG, enabled MCF-7 transfected cells to grow inathymic, ovariectomized mice without estrogen supple-mentation. Similarly, estrogen independent growth andantiestrogen resistance in vitro and in vivo, wereobtained when MCF-7 cells were transfected withHRG-b2 cDNA (Tang et al., 1996). In addition, it wasdemonstrated that either transfection of MCF-7 cellswith HRG cDNA or treatment of these cells withHRG elicited a signi®cant decrease in ER expression atthe protein and mRNA level (Pietras et al., 1995;Saceda et al., 1996). Prolonged exposure of MCF-7cells to HRG also resulted in a reduction of PR

content (Pietras et al., 1995). In this regard, it is worthnoting that in our in vivo model, progression to ahormone-independent phenotype, that is associatedwith a high constitutive expression of HRG, does notinvolve any change in either the number or the a�nityof ER or PR.

The in vitro studies of the e�ects of exogenous HRGon the growth of primary cultures of C4HD epithelialcells showed that HRG is able to induce a proliferativeresponse as potent as MPA on these cells. In addition,HRG potentiated MPA stimulatory e�ects. It remainsto be elucidated whether HRG proliferative e�ects andpotentiation of MPA-induced growth involves activa-tion of progesterone receptor, as has been reported forestrogen receptor (Pietras et al., 1995). HRG participa-tion as mediator of MPA-induced growth of C4HDcells was demonstrated by using an antisense strategyto HRG mRNA in which blocking endogenous HRGby ASODNs inhibited MPA-induced cell growth.

The biological e�ects of HRG may be attributable inpart to the cellular complements of class I RTKsavailable to make up functional heterodimers. InC4HD cells we found high levels of ErbB-2 and ErbB-3and very low levels of ErbB-4 expression. We havepreviously demonstrated that these cells lack ErbB-1(Lanari et al., 1989). Treatment of C4HD cells withHRG induced tyrosine phosphorylation of both ErbB-2and ErbB-3. Incubation with MPA, that induced an up-regulation of HRG levels, also resulted in tyrosinephosphorylation of ErbB-2 and ErbB-3. These resultsprovide further support for a role of HRG as mediator ofMPA-proliferative e�ects, since HRG produced inresponse to MPA could be the ligand responsible forErbB-2 and ErbB-3 phosphorylation.

Several reports have established that ErbB-2 is acritical component in mediating HRG-induced breastcancer cells growth. HRG stimulation of MCF-7 and T-47D cells growth was inhibited by anti-ErbB-2 mono-clonal antibodies (Lewis et al., 1996). A di�erentapproach, based on the intracellular retention of ErbB-2 by using a single-chain ErbB-2 antibody alsodemonstrated that stimulation of growth by HRG wasdramatically impaired in T-47D (Graus-Porta et al.,1995) and MCF-7 cells (Berlii et al., 1995). The resultspresented herein, demonstrating that HRG-inducedgrowth is abolished by ErbB-2 ASODNs indicate that,in our model system, ErbB-2 is essential in HRGsignaling pathway. We have also found that MPA-induced proliferation is inhibited by ASODNs to ErbB-2, providing further support for a role of HRG as amediator of MPA-induced growth. It has beenpreviously reported that stable transfection of thehuman breast cancer cell line AU565 with antisenseErbB-2 cDNA results in decreased ErbB-3 proteinexpression (Hamburger and Yoo, 1997). However, theresults we now show demonstrate that in C4HD cellsinhibition of ErbB-2 expression has no e�ect on proteinlevels of ErbB-3 or ErbB-4. Thus, it can be concludedthat blocking HRG and MPA response by ErbB-2ASODNs is the result of inhibiting the recruitment ofErbB-2 to a heterodimer complex with the othermembers of the ErbBs family, most likely with ErbB-3since ErbB-4 expression levels are extremely low, thatmediates HRG action in C4HD cells.

It has been previously found, in the humanmammary epithelial cell line MCF-10A, that HRG

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acts as a dual speci®city growth factor able to drivesignal transduction pathways that normally requireboth EGF and insulin-like growth factor-I (IGF-I)(Ram et al., 1995). One possible explanation for thisobservation is that in MCF-10A cells that expressErbB-2, ErbB-3 and EGF-R, stimulation by HRGresults in activation of signal transduction pathwaysthat are normally activated separately by EGF andIGF-I. This may occur by the formation ofheterodimers among ErbB family members that yieldboth EGF and IGF-I like signals. In this respect, ithas been well documented that ErbB-3 possesses theunique capacity among the ErbB kinase family tobind and activate the p85 regulatory subunit of PI-3K(Carraway and Cantley, 1994; Fedi et al., 1994;Graus-Porta et al., 1997). ErbB-3 has seven repeatsof the Tyr-X-X-Met motif in its carboxy-terminaldomain which is the p85 Src homology 2 (SH2)domain recognition sequence (Carraway and Cantley,1994). ErbB-3 ability to associate with PI-3K confersto this receptor the capacity to share signaltransduction pathways with IGFs, since activation oftype I IGF-R results in tyrosine phosphorylation of asubstrate protein IRS-1, which in turn binds to andactivates p85 (Backer et al., 1993). PI-3K activation inmammary tumor cells treated with HRG (Sepp-Lorenzino et al., 1996) or in mammary tumors thatarise in transgenic mice overexpressing HRG(Amundadotir and Leder, 1998) has been reported.We have recently demonstrated, by blocking type IIGFs receptor expression using antisense oligodeox-ynucleotides, that a functional type I IGF-R isessential for MPA-induced proliferation of C4HDcells (Elizalde et al., 1998). Therefore, herein we haveinvestigated whether HRG also requires a functionaltype I IGF-R for its mitogenic activity. The results weobtained blocking type I IGF-R and treating C4HDcells with HRG demonstrate, for the ®rst timeaccording to our knowledge, that malignant mam-mary epithelial cells that overexpress ErbB-2 andErbB-3 and express low levels of ErbB-4 cannotrespond to HRG with mitogenesis in the absence of afunctional type I IGF-R. This result excludes thepossibility that in cells with high ErbB-3 levels such asC4HD, HRG through the activation of ErbB-3 couldelicit signal transduction pathways shared with type IIGF-R, avoiding the necessity of an activated IGF-IR.A similar result has been reported showing that mouseembryo ®broblasts overexpressing EGF-R but lackingtype I IGF-R do not respond to EGF-inducedmitogenesis or transformation (Coppola et al., 1994).Our results are consistent with the hypothesis thatthere is an interdependence between growth factorreceptors and that type I IGF-R is downstream fromthe ErbB family of receptors, as predicted by thecompetence progression model of cell proliferation(Coppola et al., 1994). It is noteworthy that abolishingtype I IGF-R expression does not a�ect ErbBsreceptor expression at protein level. Thus, it remainsto be elucidated whether abolishing type I IGF-Ra�ects HrG capacity to activate ErbBs throughphosphorylation. In addition, recent ®ndings haveindicated the existence of a mitogenic pathway fortype I IGF-R which does not involve MAP kinasecascade or PI-3K activation (Barone and Courtneidge,1995; Sell et al., 1994; Lowe et al., 1997). Results from

Sell et al. (1994) have shown that this pathway is notshared with PDGF and EGF receptors.

In conclusion, our results provide evidence ofinteractions between progestins and HRG signalingpathways in mammary tumors and demonstrate that afunctional type I IGF-R is essential for the HRGproliferative e�ects in mammary tumor cells.

Materials and methods

Animals and tumors

Experiments were carried out in virgin female BALB/c miceraised at the National Academy of Medicine of Buenos Aires.All animal studies were conducted in accordance with thehighest standards of animal care as outlined in the NIH guidefor the Care and Use of Laboratory Animals. Hormone-dependent ductal tumors (C4HD, D5HD and 53HD)originated in mice treated with 40 mg MPA every 3 monthsfor a year, and have been maintained by serial transplanta-tion in animals treated with 40 mg s.c. MPA depot on theopposite ¯ank to tumor inoculum (Molinolo et al., 1987;Lanari et al., 1989). Their hormone independent counter-parts, C4HI, D5HI and 53HI, developed from HD tumorsgrowing in mice that were not treated with MPA,approximately a year after the inoculum (Molinolo et al.,1987; Lanari et al., 1989). These HI tumor lines have beenmaintained by serial transplantation in untreated femalemice. Tumor lines C4HD, D5HD and 53HD are of ductalorigin and express PR and ER (Molinolo et al., 1987; Lanariet al., 1989). Although their respective HI counterparts C4HI,D5HI and 53HI have developed the capacity to grow in theabsence of progestin treatment, they have retained PR andER (Molinolo et al., 1987; Lanari et al., 1989). HI lines 60,55, 52B and 39 originated in MPA-treated mice and havebeen maintained by syngeneic transplants in untreated virginfemale mice. They are of lobular origin and express EGF-Rbut not PR or ER (Lanari et al., 1989).

Primary cultures and cell proliferation assays

Primary cultures of epithelial cells or ®broblasts from C4HDand 59HD tumors, growing in MPA-treated mice, wereperformed as previously described (Dran et al., 1995). Inbrief, tumors were aseptically removed, minced and washedwith DMEM/F12 (Dulbecco's modi®ed Eagle's medium:Ham's F12, 1 : 1, without phenol red, 100 U/ml penicillinand 100 mg/ml Streptomycin). The tissue was suspended in5 ml of enzymatic solution [trypsin: 2.5 mg/ml, albumin:5 mg/ml and collagenase type II (Gibco±BRL, Gaithersburg,MD): 239 U/ml] in phosphate bu�ered saline (PBS) andincubated at 378C for 20 min, under continuous stirring. Theliquid phase of the suspension was then removed and theundigested tissue was incubated with fresh enzymatic solutionfor 20 min. Enzyme action was stopped by adding DMEM/F12+5% heat inactivated fetal calf serum (FCS) (Gen S.A.,Buenos Aires). Epithelial and ®broblastic cells were separatedas already described (Dran et al., 1995). Brie¯y, the cellsuspension was resuspended in 15 ml of DMEM/F12+10%FCS and allowed to sediment for 20 min. The upper 5 ml,corresponding to the ®broblastic fraction, was seeded in¯asks, and the cells were allowed to attach during 1 ± 2 hafter which the medium containing unattached cells wasremoved and replaced by fresh DMEM/F12+10% FCS. Thesedimented cells, corresponding to the epithelial enrichedfraction, were resuspended again in 15 ml of DMEM/F12+5% FCS and allowed to sediment for another20 min. The upper 15 ml were discarded and this procedurewas repeated until no ®broblasts were observed in thesupernatant. Cells were plated in culture ¯asks withDMEM/F12+5% steroid-stripped FCS (ChFCS) (Dran et

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al., 1995) and allowed to attach for 24 ± 48 h. Purity of theepithelial cultures was evaluated by cytokeratin staining. Cellswere incubated in DMEM/F12, (without phenol red, 100 U/ml penicillin, and 100 m/ml streptomycin), with 2.5% ChFCSin the presence or absence of MPA (10 nM and recombinanthuman HRGb1 (Neomarkers, Freemont, CA) at concentra-tions ranging from 0.02±200 ng/ml. After a 24 h incubation,50% of media was replaced by fresh media and cells wereincubated for another 24 h in the presence of 0.2 mCi of 3H-thymidine (NEN, Dupont, Boston MA, USA; speci®cactivity: 70 ± 90 Ci/mmol). Cells were then trypsinized andharvested. Assays were performed in octuplicate. Thedi�erences between control and experimental groups wereanalysed by ANOVA followed by Tukey t-test betweengroups. In formed experiments we demonstrated thatthymidine uptake correlates with the number of cells/well(Dran et al., 1995).

Antisense studies

HRG antisense (5'-AGA CAT CTC GCC GAA GA) andsense (5'-TCT TCG GCG AGA TGT CT) oligodeoxynucleo-tides (ODNs) correspond to the rat HRG mRNA translationstart (Yang et al., 1995). In addition, a scrambled sequence(5'-CAG TGC CAA CAA GCG AT) with the same overalloligodeoxynucleotides content as the HRG antisense oligowas used. ErbB-2 antisense (5'-GGC CGC CAG CTC CAT)and sense (5'-ATG GAG CTG GCG GCC) ODNs have beenpreviously described (Colomer et al., 1994). Type I IGR-Rantisense (5'-TCC TCC GGA GCC AGA CTT) and sense(5'-AAG TCT GGC TCC GGA GGA) ODNs correspond tocodons 21 ± 26 of the signal sequence of the subunit of type IIGF-R preceding the proreceptor sequence. ODNs werepurchased from CyberSyn (Lenni, PA, USA) or fromNucleiCo (Buenos Aires, Argentina).

The e�ect of ODNs was studied as follows: in a Falcon96-well microtitreplate, 0.2 ml/well of a suspension wereseeded at a concentration of 105 cell/ml. After attachment(24 ± 48 h), the cells were incubated for 48 h with medium(serum used in antisense studies was heated at 658C for30 min to inactivate exonucleases) containing the indicatedconcentrations of ODNs. The media were changed every 24 hadding fresh ODNs. After incubation, [3H]thymidine wasadded and incorporation was determined as described above.

Mouse heregulin cloning and sequencing

Total RNA isolated from tumors using the guanidiniumisothiocynate/phenol extraction method (Chomczynski andSacchi, 1987) was reverse transcribed using oligodT togenerate ®rst-strand cDNA (Perkin Elmer, Foster City, CA,USA) and used as a template with the followingoligonucleotides: 5'-GCGAATGGGCCTCACCACCCAA-AC (5' primer) and 5'-TTCGCTATGAGGAGAGTCT-CTGT (3' primer) in a 35-cycle polymerase chain reaction(PCR) ampli®cation. With this pair of ODN we identi®ed aPCR fragment of 387 b.p. localized in the cytoplasmicdomain of the gene. When the primers used were 5'-TGAAGAGCCAGGGTCAGCTGCAGG (5' primer) and5'-AGACTCTGAGGACACATAGG (3' primer), that am-plify the Ig domain of the gene, the fragment identi®ed was333 b.p. long. Both pairs of oligodeoxynucleotides werechosen on the basis of the rat HRG sequence (Wen et al.,1992). Fragments were cloned in a pCRII vector using theTA Cloning kit (Invitrogene Corp, San Diego, CA, USA)and subsequently sequenced. Sequences were obtained using[35S]dATP (Amersham International, UK) and Sequenaseversion 2.0 kit from US Biochemicals (United StatesBiochemical Corp, Cleveland, OH, USA) following themanufacturer's instructions. Both cDNA strands of thecloned fragments were sequenced using synthetic oligonu-cleotides as primers.

RNAse protection assay

The HRG probe was the 333 b.p. HRG cDNA fragmentcloned in a pCRII vector (Invitrogene) and linearized withHindIII to provide the antisense riboprobe. The rpL32 humancDNA clone (encoding ribosomal protein L32) (Young andTrowsdale, 1985) was provided by Dr R Rochford (The ScrippsResearch Institute, La Jolla, CA, USA). This recombinantconstructed in a pGem 4 vector (Promega, Madison, WI, USA)was linearized with EcoIR, providing a template for antisenseriboprobe protecting a 76 b.p. mRNA fragment. The antisense32P-labeled RNA probes were transcribed with T7 RNApolymerase following manufacturer's protocol (Promega).Thirty mg of total RNA isolated as described (Chomczynskiand Sacchi, 1987) were hybridized with 26105 c.p.m. of eachprobe in the conditions previously described (Guerra et al.,1996), followed by digestion with RNase A (Sigma). All RNAsamples were hybridized with HRG probe and rpL32 probe(used to correct for small variations in the amount of RNAloaded) simultaneously. Samples were extracted with phenol/chloroform/isoamyl alcohol (20 : 20 : 1) and precipitated with20 mg of tRNA (Sigma) and 2 v of absolute ethanol. The pelletswere resuspended in 5 ml of an 80% formamide loading bu�erand run on a 6% polyacrylamide sequencing gel with 8 M urea.Size markers were prepared by end labeling MspI-digestedfragments of pBR322 plasmid. Band intensities were quanti-tated by scanning multiple autoradiograms of variousexposures.

ErbBs and HRG Western blotting

In order to study expression of ErbB receptors, lysates wereprepared from C4HD cells growing in medium with 2.5%ChFCS. After washing in ice-cold PBS, cells were lysed inbu�er containing 20 mM Tris (pH 7.4), 0.1 M NaCl, 5 mM

MgCl2, 1% Nonidet P-40, 0.1% b-mercaptoethanol, 1 mM

phenylmethylsulfonyl¯uoride (PMSF), 10 mg/ml leupeptin,5 mg/ml pepstatin, 5 mg/ml aprotinin, 1 mM sodium orthova-nadate, 5 mM NaF, 20 mM sodium molybdate and 5 mM

sodium pyrophosphate. Cell lysates were centrifuged at40 000 g for 40 min at 48C and protein content in thesupernatant was determined using a Bio-Rad kit (Richmond,CA, USA). Proteins were solubilized in sample bu�er (60 mM

Tris-HCl, pH 6.8, 2% SDS, 10% glycerol and 0.01%bromophenol blue) and subjected to SDS ±PAGE on a 6%gel. Proteins were electroblotted on to nitrocellulose.Membrane was blocked with PBS, 0.1% Tween 20 (PBST)and immunoblotted with the following antibodies: ErbB-2rabbit polyclonal antibody Neu C-18 (Santa Cruz Biotech-nology, Santa Cruz, CA, USA), ErbB-3 rabbit polyclonalantibody C-17 (Santa Cruz Biotechnology), ErbB-4 rabbitpolyclonal antibody C-18 (Santa Cruz Biotechnology), andErbB-4/HER-4 Oncoprotein Ab-2 rabbit polyclonal antibody(Neomarkers, Freemont, CA, USA). After washing, themembranes were incubated with HRP-conjugated secondaryantibody (Amersham International, UK), Enhanced chemi-luminescence (ECL) was performed according to manufac-turer's instructions (Amersham).

For studying the e�ect of ASODs to ErbB-2 mRNA orto HRG mRNA on protein expression, lysates wereprepared as described above from cells growing in 2.5%ChFCS or 10 nM MPA and from cells growing in 10 nMMPA+ODNs. Rabbit polyclonal anti-HRG antibody,speci®c for HRG precursor (C-20, Santa Cruz Biotechnol-ogy) was used in HRG studies.

ErbB receptor tyrosine phosphorylation analysis

Lysates from C4-HD cells growing in 2.5% ChFCS, andfrom cells treated for 48 h with 10 nM MPA or 2 ng/ml ofHRG were prepared as described above. Immunoprecipita-tions were performed on 200 mg of total cell protein. All

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lysates were precleared with Protein A-Agarose (Santa CruzBiotechnology) and 2 mg of ErbB-2 Neu C-18 or ErbB-3 C17antibodies (Santa Cruz Biotechnology) were used. Immuno-precipitations were rocked 2 h at 48C and then theimmunocomplexes were captured by adding Protein A-Agarose and rocked for an additional 2 h. Beads werewashed three times with lysis bu�er, then boiled for 10 min insample bu�er and subjected to SDS ±PAGE on a 6% gel.Proteins were electroblotted on to nitrocellulose. Membraneswere blocked with PBS, 0.1% Tween 20 (PBST), 5% bovineserum albumin (Sigma) and 1% ovoalbumin (Sigma) and®lters were probed with mouse monoclonal Anti-P-Tyr PY-20(Santa Cruz Biotechnology). Proteins were visualized withHRP-conjugated secondary antibody, using ECL detection(Amersham). Identical aliquots of each immunoprecipitatewere subjected to immunoblot analysis with anti-ErbB-2 oranti-ErbB-3 antibodies to verify that nearly equal amounts ofproteins immunoprecipitated were loaded.

IGF-I receptor binding assay

Recombinant human IGF-I, kindly provided by Dr JamesMerryweather (Chiron, Emeryville, CA, USA), was iodinatedand puri®ed as descried (Guerra et al., 1996). Binding tointact cell monolayers from C4-HD epithelial cells, treatedwith ODNs, to IGF-IR was performed as described (Elizaldeet al., 1998). Assays were performed in triplicate and resultswere analysed by the Scatchard method (1949).

AcknowledgmentsThe authors thank Dr C Lanari for providing the tumorsand for advice with primary cultures assays, Dr AMIribarren for help with the HRG ASODN design, Dr E Balde Kier Jo�e for critical discussion and review of the workand Dr C Dosne Pasqualini for review of the manuscript.This work was supported by grants from the NationalScienti®c Council of Argentina, CONICET.

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