volume 16 number 5 1988 estrogens and growth factors ... · unite d'endocrinologie cellulaire...

Volume 16 Number 5 1988 Nucleic Acids Research

Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breastcancer cells

Vincent Cavailles, Patrick Augereau, Marcel Garcia and Henri Rochefort*

Unite d'Endocrinologie Cellulaire et Moleculaire, U 148 INSERM, 60, rue de Navacelles, 34100Montpellier, France

Received January 12, 1988; Accepted February 11, 1988

ABSTRACTThe estrogen-induced 52K protein secreted by human breast

cancer cells is a lysosomal protease recently identified as apro-cathepsin D by sequencing several cDNA clones isolated fromMCF_ cells (Augereau et al., Mol. Endocr.). Using one of theseclones, we detected, in MCF7 cells, a 2.2 kb mRNA whose levelwas rapidly increased 4- to 10-fold by estradiol, but not byother classes of steroids. Other mitogens, such as epidermalgrowth factor and insulin, also induced the 2.2 kb mRNA in adose-dependent manner. Induction with epidermal growth factorwas as rapid but was 2- to 3-fold lower than with estradiol.Antiestrogens had no effect on the 52K-cathepsin-D mRNA in MCF?cells, but became estrogen agonists in two antiestrogen-resistant sublines R,7 and LY2. The use of transcription andtranslation inhibitors and nuclear run-on experiments indicatethat estradiol enhances transcription of the 52K-cathepsin-Dgene in MCF™ cells.

INTRODUCTION

A large proportion of human breast cancers is

characterized by its ability to exhibit metastasis and to be

regulated by estrogens (1). Estrogens stimulate growth of

metastatic breast cancer cell lines containing estrogen

receptors (MCF7, T4?D...) (2), following the induction of

several proteins (3 and ref. therein). The proteins that are

secreted, such as growth factors (4) and proteases (5), are

particularly interesting since they may stimulate tumor growth

and invasion by autocrine and paracrine mechanisms (6). They

are also generally produced, but not estrogen-regulated, in

estrogen-receptor negative cancers. We have extensively studied

a secreted 52K glycoprotein (7) which was found to be mitogenic

in vitro (8). The protein has recently been identified as a

URL Press Limited, Oxford, England. 1 9 0 3

Nucleic Acids Research

pro-cathepsin-D-like protease (52K-cath-D) that can degrade

extracellular matrix (9,10). The level of regulation of this

protease remained unknown, however, and the only gene (pS2)

(11), shown to be transcriptionally regulated by estrogens in

these cells (12), corresponds to a 7-10K protein of unknown

function. Using monoclonal antibodies and a 36-mer

oligonucleotide synthesized from the N-terminal sequence of the

protein, we have isolated from MCF_ libraries four cDNA clones

corresponding to a 2,039 bp coding sequence (13) that is more

than 99% identical to that of normal kidney cathepsin D (14).

In the present study, we used the 52K-9 cDNA clone to

analyze the hormonal regulation of the 52K-cath-D mRNA. We show

that estrogens, but not other steroids, rapidly induce

52K-cath-D mRNA by stimulating transcription. Moreover, it was

found that other mitogens such as epidermal growth factor (EGF)

or insulin can also rapidly increase the level of 52K-cath-D

mRNA.

MATERIALS AND METHODS

Cell culture

MCF7 cells (15) were obtained from the Michigan Cancer

Foundation and were routinely maintained in T75 flasks in

Dulbecco's modified Eagle's medium (DMEM) supplemented with

10 % fetal calf serum (Gibco) and 0.6 jig/ml bovine insulin

(Collaborative Research). To test the effect of hormones on RNA

accumulation, slightly confluent MCF_ cells were plated out in

T75 flasks (10-fold dilution) in the same medium for 2 days.

They were then stripped of hormones with 10 % serum treated

with dextran-coated charcoal in phenol-red-free DMEM. The

medium was changed every 2 days after two washes with

phosphate-buffered saline. After at least 5 days of withdrawal,

estradiol or other steroids were added to cells in an ethanol

solution (final concentration of ethanol 0.1 %) and solvent

alone was added to control cells. For stimulation by insulin

and mouse EGF (Collaborative Research), cells were cultured and

stripped under the same conditions but without Insulin.

The two antiestrogen-resistant variants of the MCF_ cell

line, R 2 7 (16) and LY2 (17), selected for their resistance to

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the growth inhibitory effects of tamoxifen and LY117018,

respectively, were obtained from Marc Lippman (National Cancer

Institute, Bethesda, Maryland, USA). R_7 cells were maintained

in DMEM with 10 % fetal calf serum treated with dextran-coated

charcoal, 0.6 ng/ml insulin, and 1 nM tamoxifen. They were

stripped of estrogens and tamoxifen by culturing for 14 days in

hormone-free medium, as in the case of MCF7 cells. During this

time, they were passaged once and the medium was changed every

3 days. LY2 cells were maintained in DMEM with 5%

charcoal-stripped calf serum and 0.6 ng/ml insulin. Stimulation

by estradiol (1 nM) or antiestrogen (1 |iM tamoxifen or its

high-affinity metabolite 1 nM 4-hydroxytamoxifen) was performed

as described (18).

RNA preparation, Northern blot analysis

Total RNA was extracted from MCF_ human breast cancer

cells by the method of Auffray and Rougeon (19). RNA was

electrophoresed on a 1% agarose formaldehyde denaturing gel and

then transferred to nitrocellulose. The double-stranded cDNAs32

in the vectors were P-labeled, using random primers (20), toq

a specific activity of 1 to 3 x 10 dpm per tig. Filters were

prehybridized for 24 h at room temperature and then hybridized

in 50% formamide for 3 days at 37°C (2 x 106 cpm/ml).

Hybridization solutions were prepared as described (18).

Washing was done in 2 x SSC, 0.1% SDS (1 x SSC is 150 mM NaCl,

15 mM sodium citrate) once for 20 min at room temperature and

twice for 1 h at 65°C, and the filters were autoradiographed

for 5 to 20 h at -70°C using intensifying screens. The amount

of each RNA was determined by densitometric scanning of

different exposures of the autoradiographs. The 36B4 cDNA,

which corresponds to an mRNA unaffected by estrogens in MCF_

cells (11), was used to correct for slight variations in the

amount of RNA loaded on each track. pS2 RNA, which is

transcriptlonally regulated by E. in MCF? cells (12), was used

as a positive control.

Measurement of protein synthesis and 52K-cath-D secretion

Inhibition of protein synthesis by cycloheximide was

estimated by measuring the incorporation of | S|methionine in

the same batches of cells used for RNA preparation. MCF_ cells

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cultured in the maintenance medium in 8-mm microwells were

treated or not with 50nM cycloheximide (Sigma) for 1 h, and

|35S|methionine (Amersham ; SA 800 Ci/mmol ; 10 UCi/well) was

then added to the culture medium. After 5 more hours, the

incorporation of radioactivity was determined by

trichloroacetic acid precipitation. The cycloheximide treatment

reduced radioactivity incorporation by 96-98%.

Immunoenzymatic assay of the secreted 52K-cath-D was

performed by a double-determinant solid-phase assay (21,22).

Nuclear run-on transcription assay

Nuclear transcription was performed according to Brown et

al. (12) with modifications. Nuclei were isolated from MCF7

cells treated or not with 10 nM estradiol and nascent RNA

transcripts initiated in vivo were elongated in vitro in the

presence of | P | UTP (400 Ci/mmole ; Amersham) for 45 min at

30°C. Labeled RNA was then extracted with phenol-chloroform

after DNAse and proteinase K treatment. The unincorporated

nucleotides were eliminated by precipitation twice with ethanol

and ammonium acetate. Seven- |ig of denatured plasmids were

spotted onto nitrocellulose filters using a BRL Dot blot

apparatus. We used 52K-9 cDNA to quantify the newly synthesized

52K-cath-D RNA, pS2 cDNA as a positive control of

transcriptional regulation by estradiol, 36B4 and C3 (23) cDNAs

(which correspond to poly A+ RNAs unaffected by estradiol in

MCF_ cells) as constant controls, and the M13 vector alone to

evaluate nonspecific hybridization. Prehybridization was done

for 2 days at 37°C in 50 mM NaP04, pH7, 750 mM NaCl, 50%

formamide, 0,5% SDS, 2 mM EDTA, 10X Denhart's, 1 tig/ml poly (A)

and 500 ug/ml denatured salmon sperm DNA. Hybridization was

done in the same solution for 4 days at 37°C, with the same

amount of labeled RNA (up to 2 x 10 cpm) from control or

Entreated cells. Filters were washed, RNAse A treated and

finally autoradiographed for 2 days. The relative intensity of

a spot evaluated by densitometric scanning was shown to be

proportional to the amount of sample hybridized (data not

shown).

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RESULTS

Effect of estradlol and growth factors on the levels of

52K-cath-D RNA

The 52K-9 cDNA probe corresponds to most of the coding

sequence of normal cathepsin D mRNA (Fig. la) and hybridized

with a 2.2 kb RNA of MCF? cells, which corresponds to the size

of the cathepsin D mRNA in normal tissue (14). The level of

this 2.2 kb RNA was increased 6- to 8-fold by estradiol (E2)

compared to the estrogen-independent 36B4 mRNA (Fig. lb and c).

The 0.6 kb pS2 mRNA (11) was also significantly increased by

estradiol as expected (Fig. lc). Similar results were obtained

with poly A+ RNA (not shown). The 52K-9 probe also detected a

less abundant 52K-cath-D RNA species of about 4.5 kb which was

also regulated by estradiol. The significance of this 4.5 kb

RNA is not known but it could correspond to a precursor of the

2.2 kb mRNA since its increase was transient and more rapid

(data not shown).

Other classes of steroid hormones, i.e. progestin (R5020)

and glucocorticoid (dexamethasone) increased the 2.2 kb mRNA by

no more than 10% (Fig. lb). The androgen dihydrotestosterone

was inactive at 10 nM but was active at micromolar

concentrations previously shown to induce the secreted

52K-cath-D protein via the estrogen receptor (7).

The 52K-cath-D mRNA level was, however, increased by

mitogens other than estradiol. EGF and insulin at

concentrations previously shown to stimulate the growth of MCF_

cells (24), increased the level of 52K-cath-D RNA 4- and

2-fold, respectively (Fig. lc). These effects were also

confirmed at the protein level by the increase in secreted and

cellular 52K-cath-D measured by immunoenzymatic assay (21)

(Fig. 3 and D. Derocq, unpublished experiments). Using

radiocompetition for the estrogen receptor, we checked that EGF

and insulin preparations contained no estrogen-like compounds

(not shown). Fig. lc also shows that both EGF and insulin

significantly induced the pS2 mRNA, which is also

estrogen-regulated in MCF- cells (11). The effect of EGF on the

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a

36 192

b kb

4.5-

22-

1.2-

c

3 1(

295

C E;

kb

4 5 -

12- —

12- «

0.6- •

C

u

-»7TOax

• - •

ft«ft# • •

E? Ins.

R5020

• •

ft^• fl

4 1EGF

t

w « J 5 2 K

• • * 3 6 M

OHT

}52K

I*36B4

>*PS2(nM)

Figure 1. Position of the 52K-9 cDNA clone (a) and effect ofsteroids (b) and growth factors (c) on the level of 52K-cath-DmRWA In MCF_ cells

a. The 52K-9 cDNA probe Isolated from MCF? cells isrepresented under cathepsin-D mRNA from normal kidney cells,according to Faust et al. (14). The open boxes stand for thecoding sequence and correspond to the signal sequence (1), thepro-sequence (2) and the sequence of the mature enzyme (3 and4). Coordinates of the coding sequence of normal cathepsin D(from 52 to 1287) and of the terminal nucleotides of the 52K-9clone are indicated. The internal deletion (dotted line from192 to 295) in this cDNA clone is a cloning artefact. Sequenceanalyses of 52K-9 and other clones (13) indicate a 99%homology with normal kidney cathepsin-D (14).

b. MCF_ cells were stripped of estrogens and thenincubated for 3 days either in the absence of hormone (C) orwith the indicated concentrations of estradiol (Ep),dexamethasone (Dex), a synthetic progestin (R5O2O), ordihydrotestosterone (DHT). Total RNA (40 tig) was analyzed byNorthern blotting as described in Materials and Methods.Hybridization was done with the 52K-9 cDNA to probe 52K-cath-DmRNAs and with 36B4 cDNA, which detects an RNA speciesunaffected by estradiol (11).

c. MCF_ cells were stripped of steroids for 7 days withoutadding insulin. They were then treated for 3 days with E p at10 nM, insulin (Ins) at 50 and 100 nM and EGF at 4 and 8 nM.Total RNA was analyzed as in b and also probed with pS2 cDNA,which corresponds to an estrogen-induced mRNA in MCF_ cells(11). The lengths of the RNA species detected are indicated inkilobases (kb).

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500 6 24 48 72

Hours of treatment

Figure 2. Time-course of the effect of estradlol and EGF on theaccumulation of 52K-cath-D mRNA.

Total RNA (40 y.g) was analyzed as described in Fig. lb.The levels of 52K-9 cDNA hybridized to 52K-cath-D RNA (2.2 kb)were determined by densitometric scanning. Values werecorrected for slight variations of hybridization in theconstant 36B4 RNA, and plotted as percentages of the maximumvalue.

a. Steroid-stripped MCF_ cells were cultured for theindicated times with (E2, *) 6r without (C, A ) 10 nM estradiol.

b. MCF? cells were stripped as described in Fig. lc andtreated (EGF, A ) or not (C,A ) with 4 nM EGF for the indicatedtimes.

level of 52K-cath-D mRNA was greater than that of insulin,

whereas both mitogens had a similar effect on pS2 mRNA,

suggesting a difference in the hormonal sensitivity of the

corresponding genes.

We then studied the induction of the 52K-cath-D RNA in

MCF? cells after treatment for different times with estradiol

and EGF. The level of the 2.2 kb 52K-cath-D RNA increased

rapidly within 2-6 h following the addition of estradiol and

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--8- 8E2

« C -12 -II -10 -9 -8 -7 -6o£ Log l i g a n d c o n c e n t r a t i o n ( M )

C 1.6 U 16

E G F c o n c e n t r a t i o n ( n M )

Figure 3. Effects of estradlol, tamoxifen and EGF on the levelsof 52K-cath-D mRNA and secreted protein.

MCF_ cells were treated for 3 days as in Fig. 2 withincreasing concentrations of estradiol (E ?), tamoxifen (T) (a)or EGF (b). The levels of 52K-cath-D mRlm (full symbols) weredetermined after hybridization with 52K-9 and 36B4 cDNAs asdescribed in Fig. 2. The amount of secreted 52K proteinaccumulated in the medium at the end of treatment (opensymbols) was determined using an immunometric assay (21,22).

Results are expressed as percentages of the maximum.

was nearly maximal after 24 h (Fig. 2a). This increase

anticipated that of the intracellular protein (25) and the

52K-cath-D protein secreted into the culture media, which

increased slowly for 16 h of treatment and more rapidly

thereafter (not shown). The half-maximal induction with EGF was

also obtained before 10 h of treatment but its effect at 2 h

was even higher than that of estradiol (Fig. 2b). Fig. 3 shows

that the induction of 52K-cath-D mRNA and the increase in the

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100

50

^ S2K-Coth.D mRNA• S«cret«d S2K-Cath.D

E2 T OHT T OHT T OHT

MCF7 R27 LY2

Figure 4. Effects of antlestrogens on the Induction of52K-cath-D RNA In MCF_ cells and in two antiestrogen-resistantvariants.

MCF_ cells and two antiestrogen-resistant variants, R 2 ?and LY2, were stripped of hormones and treated for 3 days wirn1 nM estradiol (E_), 1 nM tamoxifen (T), 1 nM OH-Tamoxifen(OHT) or with solvent alone. The levels of 52K-cath-D mRNAs( S ) and secreted 52K-cath-D protein (•) were determined asdescribed in Fig. 3.

Results are expressed as percentages of the inductionobtained with estradiol.

secreted 52K-cath-D were obtained at the same physiological

concentrations of estradiol and EGF. These effects are

consistent with a progressive occupation and activation of the

estrogen and EGF receptor sites, respectively.

Effect of antiestrogens on MCF_ cells and antiestrogen-

resistant variants

In MCF? cells, neither tamoxifen (Fig. 3a) nor hydroxy-

tamoxifen (not shown), which is its high-affinity metabolite,

stimulated 52K-cath-D mRNA accumulation while hydroxytamoxifen

antagonized the stimulation of 52K-cath-D mRNA by estradiol

(not shown). This is in full agreement with their lack of

agonistic effect on the expression of the secreted 52K-cath-D

protein (7), shown in parallel (Fig. 3a) and with the complete

inhibition of MCF_ cell growth by tamoxifen. By contrast, in

two antiestrogen-resistant variants, sublines R_7 and LY2, the

2.2 kb mRNA was significantly induced by tamoxifen (luM) and

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a

kb

2.2-

1.2-

CHX

E2

1 2 3 4

9 § # i -36B4

- - + +- + - +

bControl Estradiol

kb

2.2- m m m ** -52 K

i.2- m

0.6-

0 4 7 10 0 4 7 10Hours of Actinomycin D

Figure 5. Effects of translation and transcription inhibitors.a. MCF_ cells were treated as follows:Lane 1, control cells were incubated in stripped medium

for 5 days.Lane 2, cells were treated with 10 nM estradiol (Eo) for

8 h. d

Lanes 3 and 4, cells grown in stripped medium were treatedwith 50uM cycloheximide (CHX) for 9 h ; after the first hourof CHX treatment, estradiol (lane 4) or only ethanol (lane 3)was added to the cells.

b. MCF_ cells were stripped of estrogens and treated for 2days with 10 nM estradiol or with solvent alone (control).Cells were then treated with 5 n g/ml actinomycin D for theindicated times.

In both cases, the 2.2 kb cathepsin D mRNA level wasassayed in total RNA as described in Fig. lb.

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hydroxytamoxifen (1 nM) to a level that was similar or at least

40% of that obtained in parallel with estradiol (Fig. 4). The

same antiestrogens had little or no effect on the 2.2 kb mRNA

in wild-type MCF7 cells. The secreted 52K-cath-D assayed in

the same experiment was induced by antiestrogens in R27 cells

but not in LY2 cells, in accordance with previous studies

(18,26,27). The dissociated effects observed in LY2 cells, in

which the antiestrogens induced the 2.2 kb mRNA but not the

secreted 52K-cath-D is not yet explained, however they suggest

an additional effect of antiestrogens on protein maturation

and/or secretion.

Evidence for a regulation of 52K-cath-D gene transcription by

estradiol

To test whether the induction of 52K-cath-D mRNA is a

primary effect of estradiol, we first tested the effect of a

protein synthesis inhibitor. MCF_ cells were pretreated for 1 h

with 50 nM cycloheximide, which was found to reduce protein

synthesis by at least 96% (data not shown), before adding 10 nM

estradiol for 8 h with cyeloheximide still present. The

induction of 2.2 kb RNA was not significantly reduced (Fig. 5a)

suggesting that it does not depend on the induction of another

protein. We then analyzed the stability of 52K-cath-D mRNA in

the presence or absence of estradiol, while its synthesis was

blocked by actinomycin D. The level of 2.2 kb mRNA was stable

after 10 h of treatment with actinomycin D, in the absence of

hormone (control). Pretreatment for 48 h with estradiol did not

significantly affect this stability (Fig. 5b). In another

experiment, the increased level of 52K-cath-D mRNA produced by

11 h treatment with estradiol was totally abolished when

actinomycin D (5 ug/ml) was added together with the hormone

(not shown). This effect of actinomycin D suggested a

transcriptional regulation by estradiol.

We then studied the effect of estradiol on the

transcription of the 52K-cath-D gene in isolated nuclei.

Following treatment of cells with estradiol, the nascent mRNAs

initiated in vivo were elongated in vitro in isolated nuclei in

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op

the presence of | P|UTP. Labeled RNAs were then hybridized to

52K-9, pS2, or control cDNAs spotted on nitrocellulose. As

shown in Fig. 6a, 52K-cath-D gene transcription was increased

4-fold by incubation with fetal calf serum for 24 h and 2-fold

following 3 h of treatment by estradiol. Time-course

experiments indicated that the stimulation of 52K-cath-D gene

transcription was rapid (30 min), peaked at 1 h and remained

stable until 24 h. In the same experiments, pS2 gene

transcription was enhanced 4-fold 1 h after estradiol addition.

The transcription of two genes known to be unaffected by

estradiol in MCF? cells, i.e. 36B4 (Fig. 6b) and C3 (not shown)

was constant with time. These results indicate that estradiol

directly stimulates 52K-cath-D gene transcription.

DISCUSSION

The hormonal regulation of the 2.2 kb 52K-cath-D mRNA was

studied in MCF™ breast cancer cells and three sets of

information were obtained :

1) The regulation of 52K-cath-D mRNA by estradiol is at

least partly due to stimulation of transcription. We cannot

totally exclude a post-transcriptional effect of estradiol

since the maximal intensity of the transcription effect

(2-3-fold) was lower than the increased accumulation of mRNA

(4-10-fold). However, in our run-on experiments, estradiol

increased by only 4-fold, pS2 transcription which was

previously reported to be increased by 8-fold (12). Cathepsin D

is therefore, after the pS2 protein, the second example of a

gene transcriptionally regulated by estrogens in human breast

cancer cells. Three other mRNAs have been shown to accumulate

following estradiol treatment, i.e. progesterone receptor mRNA

(E. Milgrom, personal communication), and thymidine kinase and

dihydrofolate reductase mRNAs (28), but the mechanism of their

induction has not yet been determined. The absence of an effect

by cycloheximide indicates that no protein synthesis is

required for 52K-cath-D mRNA induction by estradiol, but an

indirect regulation of gene transcription via post-

translationally modified factors cannot be totally excluded.

Cloning of the 52K-cath-D gene will make it possible to

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52K-

36B4-

C FCS

2.5

0 1 3 5 24

Hours of treatment

Figure 6. Nuclear run-on experiments.a. Nuclei were isolated from MCF_ cells incubated in

stripped medium for 5 days (C) or treated with 10% fetal calfserum (FCS) for 3 days or with 10 nM estradiol for 3 h-(E_).Nascent RNA chains were elongated in the presence of | P|OTPand hybridized to 52K-9 and 36B4 cDNAs spotted in excess onnitrocellulose as described in Material and Methods.

b. Time course of the stimulation of 52K-cath-D genetranscription by estradiol. The nuclear run-on experiment wasperformed as described in a., following MCF_ cell treatmentwith 10 nM estradiol for increasing periods of time.Densitometric scanning of the level of newly synthesized52K-cath-D and 36B4 mRNAs is represented compared to the levelof stimulation at time 0.

determine this mechanism and to compare the estrogen-

responsive elements with those of other estrogen-regulated

genes. There are general differences between the regulation of

pS2 and 52K-cath-D in breast cancer cells. The expression of

1S15


pS2 only occurs in estrogen-receptor-positive cells, whereas

cathepsin D is also produced in estrogen-receptor-negative

breast cancer (13). Both genes are also regulated differently

in antiestrogen-resistant cells (see below).

2) The different effects of antiestrogens on the synthesis

of the 2.2 kb-cath-D mRNA in antiestrogen-sensitive and

-resistant cells confirm previous findings based on the

quantification of secreted 52K-cath-D (18,26). The discrepancy

observed in the LY2 subline, where antiestrogen stimulates

mRNA-cathepsin D accumulation but not 52K-cath-D secretion (27)

suggests an additional level of regulation for secretion. It

would thus appear that in the three antiestrogen-resistant

sublines (R2?, RTx6 and LY2 derived from MCF7 cells), the

estrogen receptor-antiestrogen complex is able to stimulate the

accumulation of 52K-cath-D mRNA, but is unable to do so in MCF?

wild-type cells. The effects of antiestrogens vary according to

different estrogen-induced responses. Progesterone receptors

are induced in both antiestrogen-sensitive and -resistant cells

by tamoxifen whereas pS2 mRNA and the 160K protein are not

affected in any of these cells (18). The induction of

52K-cath-D mRNA thus appears to be the only known response

associated with antiestrogen-resistant breast cancer. The

reasons for the induction of cath-D mRNA by tamoxifen in

resistant cells is unknown. Alterations during procedures for

subline selection may affect estrogen receptors or the

structure of estrogen-responsive elements.

3) The 52K-cath-D mRNA is also induced by other types of

mitogens, such as insulin and EGF, suggesting that its

regulation may be complex and controlled by different promoter

elements that are sensitive to different hormones. EGF-like or

IGFI growth factors have also been reported to be induced by

estrogens in the same cells (4) and these factors or other

mitogens might therefore mediate the induction of 52K-cath-D

mRNA by estrogens. However, this possibility is unlikely since

the rapid stimulation of 52K-cath-D gene transcription and the

resistance to cycloheximide, both of which suggest that the

regulation of 52K-cath-D mRNA by estrogens is not mediated by

growth factors.. The mechanism (direct or indirect) by which

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insulin or EGF operate has not yet been investigated. Further

studies are needed to determine the biological significance of

protease induction by growth factors. Moreover, induction of

52K-cath-D mRNA by mitogens such as estrogens and growth

factors is in agreement with clinical studies indicating that

tissue accumulation of this protease is associated with cell

proliferation (29). Another example of a growth-factor- induced

cathepsin involved in carcinogenesis is cathepsin-L (or major

excreted protein : MEP). In mouse BALB/c 3T3 cells, cathepsin-L

gene transcription increases after treatment with platelet-

derived growth factor (PDGF, but not EGF, IGF1 and insulin),

tumor promoter, or after transformation by oncogenes (30). In

contrast with the effect of estrogens on cathepsin-D mRNA, the

induction of cathepsin-L mRNA by PDGF requires de novo protein

synthesis.

Recently, using a different approach, Westley and May have

also shown that estradiol increases the level of cathepsin D

RNA in the ZR75-1 estrogen-receptor-positive cell line (31).

Their results are in accordance with the present results in

MCF? cells, but the mechanism of induction was not determined

and the effect of growth factors was not investigated.

Estrogens clearly induce the 52K-cath-D mRNA in human breast

cancer cells in culture, but their effect on normal mammary

cells or other estrogen target tissues has not been

demonstrated. Normal mammary cells in primary culture may not

contain a sufficient amount of estrogen receptors to stimulate

cathepsin D expression (G. Cavalie, F. Capony, unpublished

results). However, in rat uteri, which contain high level of

estrogen receptors, progestins rather than estradiol increase

cathepsin D activity and synthesis (32). Further studies will

be needed to determine whether estrogen induction of the

cathepsin D gene characterizes mammary cells or transformed

cells. Among the proteins shown to be regulated by estrogens at

the mRNA level in MCF cells (4,11,28,33), 52K-cath-D is the

first example of a protease that is transcriptionally regulated

by estrogens. This induction may be important in the process of

mammary carcinogenesis and metastasis since the resulting

pro-enzyme is also secreted in excess by breast cancer cells

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compared to normal cells (25 and F. Capony et al., in

preparation) and may therefore act either directly as a

classical growth factor or indirectly by its proteolytic

activity. The biological activity of this protease will be more

directly demonstrated by transfection experiments. These

investigations should increase our understanding of the role of

estrogens and of cathepsin D in mammary carcinogenesis, and

that of the mechanism by which estrogens and antiestrogens

regulate gene expression.

ACKNOWLEDGEMENTS

This research was supported by the "Institut National de

la Sante et de la Recherche Medicale", the "Centre National de

la Recherche Scientifique", the Faculty of Medicine of

Montpellier, and by a fellowship granted to V. Cavailles by the

"Ministere de la Recherche et de 1'Enseignement Superieur". We

are grateful to P. Chambon and J.M. Jeltsch for the cDNA

libraries and the pS2 and 36B4 probes, to F. Rougeon for oligo-

nucleotide synthesis, and to M. Egea and E. Barrie for typing

the manuscript. We thank D. Derocq and F. Depadova for

excellent technical assistance.

•To whom correspondence should be addressed.

REFERENCES

1. Banbury Reports 8, Hormones and Breast Cancer (1981)Pike.M.C, Siiteri.P.K. and Welsch.C.W. (eds), Cold SpringHarbor Laboratory, Cold Spring Harbor.

2. Llppman.M.E., Bolan.G. and Huff.K. (1976) Cancer Res. 36,4595-4601.

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volume 16 number 5 1988 estrogens and growth factors ... · unite d'endocrinologie cellulaire...

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