acquisition of hormone-independent growth in mcf-7 cells is … · cells. mcf7/miii and mcf7/lcci,...

[CANCER RESEARCH 53, 283-290. January 15, 1993]

Acquisition of Hormone-independent Growth in MCF-7 Cells Is Accompanied by

Increased Expression of Estrogen-regulated Genes but Without Detectable DNA Amplifications 1

N i l s B r i i n n e r , 2 V i v i a n e B o u l a y , A n t o n i o F o j o , C a r l E. Freter , M a r c E. L i p p m a n , a n d R o b e r t C l a r k e 3

Vincent T. Lombardi Cancer Research Center, Georgetown University Medical School, Washington, DC 20007 [N. B., V B., C. E. F, M. E. L., R. C.], and the Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 [A. E]

A B S T R A C T

A hormone-independent but hormone-responsive subpopulation (MCF7/ Mil l ) of the hormone-dependent MCF-7 human breast cancer cell line (R. Clarke et al., Proc. Natl. Acad. Sci. USA 86: 3649-3653, 1989) was further passaged in ovariectomized nude mice and re-established in vitro as the continuous cell line MCF7/LCCI. The lag time to the appearance of proliferating tumors in ovariectomized animals is significantly reduced in MCF7/LCCI when compared with MCF7/MIII cells. In gel denaturation/ renaturation analysis of tumor, genomic DNA does not reveal significant differences in the pattern of detectable DNA amplifications between par- ent MCF-7 cells and MCF7/LCCI cells. In the absence of estrogen, steady- state levels of phosphoinositol turnover are similar in both MCF-7 and MCF7/LCCI cells, but turnover is increased by estrogen only in MCF-7 cells. MCF7/MIII and MCF7/LCCI, but not MCF-7 cells, express a high baseline level of the estrogen-regulated pS2 mRNA. The baseline level of expression of progesterone receptor protein, but not mRNA, is higher in MCF7/LCCI when compared with either MCF-7 or early passage MCF7/ Mi l l cells. However, while the estrogen receptor is also an estrogen- regulated gene, MCF7/MIII and MCF7/LCC1 cells retain estrogen receptor levels equivalent to the parental MCF-7 cells. These data indicate that progression to hormone independence can occur without major gene amplifications or a high constitutive induction of phosphoinositide metabolism. Thus, DNA amplifications may be acquired during the early initiation and/or promotional events of carcinogenesis. Significantly, acquisition of a hormone-independent but responsive phenotype in human breast cancer is associated with perturbations in the expression of specific estrogen-regulated genes.

I N T R O D U C T I O N

Many breast tumors appear to follow a predictable clinical pattern, being apparently confined to local structures, responsive to endocrine manipulation, and/or sensitive to cytotoxic chemotherapy. However, there is an almost inevitable progression to a more malignant phenotype characterized by invasive/metastatic loci that ultimately become resistant to endocrine manipulation and chemotherapeutic interven- tion. It is the development of a metastatic, hormone-independent, and drug-resistant phenotype that is responsible for the high percentage of treatment failures among breast cancer patients. The ultimate acquisition of this phenotype may be a fundamental biological property of many human breast cancer cells (1, 2).

Received 10/3/91; accepted 10/26/92. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by USPHS Grants 1-R55-CA51782, 5-R01-CA58022 (R. C.), and 5-U01-CA51908 and Grant 1P30-CA51008 from the National Cancer Insti- tute (M. E. L., R. C.); Research Grant 90BW65 from the American Institute for Cancer Research (R. C.); Fellowships from the Danish Cancer Society and National Cancer Institute/European Organization for Reasearch on Treatment of Cancer Exchange Program (N. B.); and Fondation pour la Recherche Medicale, and L'Association pour la Recherche sur le Cancer (V. B.).

2 Current address: Finsen Laboratory, Strandboulevarden 49, 2100 Copenhagen, Den- mark.

3 To whom requests for reprints should be addressed, at Room S128A, Vincent T. Lombardi Cancer Research Center, Georgetown University Medical School, 3800 Reser- voir Road NW, Washington, DC 20007.

A major obstacle in the study of human breast cancer progression from hormone-dependent growth to hormone-independent growth has

been the lack of suitable model systems. There is no direct biological relationship between the established hormone-dependent and hor-

mone-independent breast cancer cell lines, other than their human breast origin. These cell lines were all derived from different women, at different times, and in some cases from different metastatic sites. In

the present study, we describe the phenotype of cells obtained from a further selection of a hormone-independent but hormone-responsive

subline (MCF7/MIII) of the hormone-dependent MCF-7 cells. MCF7/ MIII cells were originally isolated from an MCF-7 tumor following a physiologically relevant selection in an ovariectomized athymic nude mouse (3). MCF7/MIII cells no longer require hormonal supplementation to form proliferating tumors (4) and are more invasive and

metastatic in vivo than the parental MCF-7 cells (5). We hypothesized that an additional selection of MCF7/MIII cells in ovariectomized athymic mice would induce a cellular morphology indicative of fur-

ther malignant progression. Further selection of MCF7/MIII cells produced a hormone-independent variant (MCF7/LCC 1) that exhibits phenotypic characteristics that imply a more malignant phenotype

than that expressed by the parental MCF-7 and MCF7/MIII cells. The molecular events that enable breast cancer cells to acquire a

progressed phenotype remain unclear. For example, hormone-resistant cells can exhibit a partial or complete down-regulation of hormone receptors (6, 7), but loss of receptor expression is not essential for the development of resistance. Approximately 40% of human breast tumors expressing ER 4 are insensitive to the inhibitory effects of antiestrogen treatment (8, 9). Increased tumorigenicity (10, 11) and acquired drug resistance (12) are associated with the presence of amplified DNA sequences in some cancers. The amplification and/or overexpression of the cellular protooncogenes e r b B 2 (13, 14), myc

(15), hst, and int2 (16-18) are observed in subsets of breast cancer patients. Overexpression of the receptor for epidermal growth factor is

also associated with a more malignant phenotype in human breast cancer (19), and with the acquisition of drug resistance (20). Many of these oncogenes and growth factor receptors utilize perturbations in phosphoinositol metabolism as a signal transduction mechanism.

Since MCF-7, MCF7/MIII, and MCF7/LCC 1 cells are derived from a common lineage, they provide a novel model to study the process of

malignant progression in human breast cancer. Mechanistically, the acquisition of a more malignant hormone-independent phenotype (MCF7/MIII, MCF7/LCC 1) could be associated with perturbations in the regulation of gene expression. Thus, DNA amplifications (11, 21, 22), alterations in phosphoinositol metabolism, and/or perturbations in the expression of specific E2-regulated genes may be associated with the increased malignant potential exhibited by MCF7/MIII and MCF7/LCC 1 cells. We have previously indicated that progression to hormone independence may be associated with the altered expression

of specific hormone-regulated genes (23, 24). We now describe the

4 The abbreviations used are: ER, estrogen receptor; PGR, progesterone receptor; EGF-R, epidermal growth factor receptor; TGF, transforming growth factor; SF, serum- free media; IMEM, improved minimal essential medium; CCS-IMEM, improved minimal essential medium supplemented with steroid-stripped serum.

283

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ACQUISITION OF HORMONE INDEPENDENCE IN BREAST CANCER

utilization of MCF-7, MCF7/MIII , and MCF7/LCC1 cells to investi-

gate the potential roles of DNA amplifications, gene regulation, and

phosphoinosit ide-associated signal transduction in the acquisition of a

more malignant phenotype in human breast cancer cells. Our data

provide strong evidence in support of our hypothesis that altered gene

regulation, and not DNA amplification, is more important in the

acquisition of hormone independence (23, 24).

M A T E R I A L S A N D M E T H O D S

Cell Culture. MCF-7 human breast cancer ceils (passage >500) were originally provided by Dr. Marvin Rich (Michigan Cancer Foundation, Detroit, MI) and were routinely grown in IMEM containing phenol red (Biofluids, Rockville, MD) and supplemented with 5% fetal calf serum. MCF7/MIII and MCF7/LCC1 cells were routinely carried in IMEM without phenol red and supplemented with CCS. Serum was stripped of endogenous steroids by treatment with dextran-coated charcoal and sulfatase as described previously (25). SF-IMEM consisted of IMEM without phenol red supplemented with 4 mr, glutamine (final concentration), 0.02 r" N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid, trace elements (Biofluids), 2 lag/ml transferrin (Collabo- rative Research, Bedford, MA), and 2 lag/ml fibronectin (Sigma Chemical Co., St. Louis, MO). All cell cultures were maintained at 37~ in a humidified 5% CO2:95% air atmosphere.

For studies in CCS-IMEM (e.g., PGR determination, isolation of RNA for pS2 mRNA expression, phosphoinositol turnover), endogenous steroids were removed by washing cells 3 times in CCS-IMEM and refed CCS-IMEM for 5 days. For studies utilizing SF-IMEM, cells were plated in CCS-IMEM over- night, and the following day the media were changed to SF-IMEM. Passage of cells growing in SF-IMEM was facilitated by passing cell clumps 3 times through a 19-gauge hypodermic needle and seeding into SF-IMEM at a split ratio of 1:5. Viability of cells growing in SF-IMEM was routinely determined by estimating their ability to attach to the substratum and proliferate in CCS-IMEM.

Establishment of Cells from Tumors Growing in Athymic Nude Mice. Cells at passage 6 of the hormone-independent but responsive MCF-7 subline (MCF7/MIII) (3, 4) were harvested at 80% confluence using a cell scraper, and 2 • 106 cells were inoculated into each flank of 6-8-week-old ovariectomized NCr nu/nu athymic nude mice (Harlan Sprague-Dawley, Indianapolis, IN). Cell viability was determined by trypan blue dye exclusion. E2-supplemented animals received an E2 pellet (60-day release, 0.72 mg; Innovative Research, Toledo, OH) at the time of cell inoculation. Proliferating tumors in ovariectomized animals were excised, cut into 1-mm 3 pieces, and plated in 24-well dishes (Costar, Cambridge, MA) in CCS-IMEM. When confluent, cells were passaged at a split ratio of 1:1 into 25-cm 2 tissue culture flasks, and the cell population expanded at a split ratio of approximately 1:20. This cell population was designated MCF7/LCC 1.

Karyotype and Isozyme Analyses. To eliminate the possibilities of con- tamination with either mouse stromal tissue or other breast cancer cell lines, the MCF-7 origin of MCFTFLCC 1 cells was investigated by both isozyme and karyotype analyses. The polymorphic enzymes analyzed were adenylate kinase (EC 2.7.4.3), esterase D (EC 3.1.1.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glyoxalase (EC 4.4.1.5), lactate dehydrogenase, mitochondrial malic enzyme (EC 1.1.1.40), phosphoglucomutase- 1 (EC 2.7.5.1), and phosphoglucomutase-3 (EC 2.7.5.1). For karyotype analysis, 100 metaphase spreads were examined in each cell line. These analyses were performed by Dr. Ward Peterson (Children's Hospital of Michigan, Detroit, MI).

Cell Population Growth Assays. Anchorage-independent growth was estimated by the ability of cells to form colonies while suspended in an agar solution. Briefly, bottom layers of agar were prepared by allowing 1 ml CCS-IMEM containing 0.6% (w/v) agar (Difco, Detroit, MI) to solidify in 35-mm Petri dishes (Costar). MCF-7 (104) or MCF7/LCC1 cells (5 • 103) were suspended in 1.5 ml CCS-IMEM containing 0.6% (w/v) agar at 41 ~ This agar solution was placed over the solid bottom agar layer, allowed to solidify, and subsequently incubated for 10 days at 37~ in a humidified 5% CO2:95% air atmosphere. Colonies of 50 cells or more (approximately 60-~tm diameter) were counted using an Omicon 3600 image analysis system (Artek, Farmingdale, NY). Anchorage-dependent growth was determined as described previously (25). Cell populations were refed with the appropriate medium on every third day.

Hormone Receptor Assays. ER and PGR levels were measured by whole cell binding assay as described previously (26, 27). Specific binding to receptors was determined after incubation with increasing concentrations of [2,4,6,7-3H]E2 (specific activity, 99 Ci/mmol; Amersham, Arlington Heights, IL) or [3H]ORG 2058 (specific activity, 41 Ci/mmol; Amersham) in the absence or presence of a 200-fold excess of unlabeled ligand. Cells were incubated with 100 nM hydrocortisone for 30 rain at 37~ before estimation of PGR expression. Data analysis was performed using the "LIGAND" receptor binding program described by Munson & Rodbard (28).

RNase Protection Analysis. The PGR riboprobe was kindly provided by Dr. Leigh Murphy (University of Manitoba), linearized from a pSP73 vector following digestion with HindIII, and subsequently transcribed with SP6 polymerase. The riboprobe produces a 240-base pair protected fragment (29). Equivalent loading of RNA was confirmed by measuring the level of expression of 36B4, an E2-independent mRNA that codes for the human acidic ribosomal protein P0 (30). The 36B4 riboprobe was linearized from pBR 322 by EcoRI and transcribed with T7 polymerase. The 36B4 riboprobe produces a 220-base pair protected fragment. Sixty lag of total RNA were hybridized with 5 • l04 cpm of probe for 12-16 h at 50~ and digested with 40 lag/ml of RNase A and 2 lag/ml RNase T1 for 30 rain at 25~ Digestion was terminated following the addition of both 0.25 mg/ml proteinase K and 0.5% (w/v) SDS. The samples were precipitated in absolute ethanol after one extraction against phenoi:chloroform:isoamyl alcohol (25:24:1). Pellets were boiled in loading buffer, and one-half of the sample was electrophoretically fractionated in 6% polyacrylamide gels containing 8 r" urea. The gels were dried and exposed at -80~ to Kodak XAR5 film between two Chronex Quanta III intensifying screens.

In-Gel Denaturation/Renaturation Assay. Cellular DNA was extracted by standard procedures (31) from MCF-7 tumors growing in E2-supplemented animals, from MCF7/LCC1 tumors growing in untreated ovariectomized mice, and from MCF-7 cells growing in vitro. DNA from the Kb-V1 multidrug- resistant subline of KB cells that expresses an amplification of the P170 multidrug resistance gene and other genomic amplifications was included (32) to demonstrate the ability to identify amplifications. Following digestion with H/ndlII, DNA was purified by phenol:chloroform extraction and ethanol pre- cipitation. One lag of digested DNA was end-labeled with 32p, mixed with 15 lag of digested DNA, electrophoresed in a 1% (w/v) agarose gel, and subjected to 2 cycles of in-gel denaturation/renaturation and digestion with S 1 nuclease, as described previously (33). The DNA was transferred onto a nitrocellulose membrane, and autoradiography was performed at -70~ utilizing 2 Chronex Quanta III intensifying screens.

Phosphoinositide Metabolism. Phosphoinositide turnover was estimated as described by Freter et al. (34). Subconfluent monolayers (80%) of either 106 MCF-7 or MCFTFLCCI cells growing in CCS-IMEM were exposed to 10 laCi of [3H]myo-inositol (specific activity, 80 Ci/mmol; Amersham) and incubated for 24 h at 37~ Cells were harvested and resuspended in a solution containing 60 lal 0.25 r" HC1 and 1.4 ml (2:1) chloroform:methanol. A mixture of 0.5 ml chloroform and 0.5 ml H20 was added, the aqueous phase removed, and the volume adjusted to 10 ml (final volume) with H20 prior to application on a column containing 2 ml of prewashed Dowex-1 formate (Dowex-1 1X8-200; Sigma). Columns were first washed with 10 ml H20 to remove the [3H]inositol, and the glycerophosphatidylinositol, lysophosphatidylinositol, and inositoI-P~ stepwise eluted with l0 ml of 5 nr" sodium tetraborate and 30

sodium formate. Inositol-P2 was eluted following application of l0 ml of a solution containing 0.1 M formic acid and 0.2 r" ammonium formate. Inosi- tol-P3 was eluted using 10 ml of 0.1 r" formic acid and 1.0 M ammonium formate. Column fractions were subjected to liquid scintillation counting and the identity of radioactivity determined by comparison with 3H- or 14C-labeled standard inositol phosphates.

Northern and Southern Blot Analyses. Total RNA and genomic DNA were prepared from cells as described previously (31, 35). For Northern analysis, 25 lag were size-fractionated by electrophoresis in a 1.2% (w/v) agarose gel containing 2.2 M formaldehyde. The RNA was transferred to a nitrocellulose filter and hybridized with 32p-labeled cDNA probes at 58~ for 16 h in hybridization buffer [50% (v/v) formamide, 5X standard sodium- citrate, 5 mM sodium phosphate, 0.1% (w/v) sodium dodecyl sulfate, 1 m i EDTA, 0.05% (w/v) bovine serum albumin, 0.5% (v/v) Ficoll, 0.05% (w/v) polyvinylpyrrolidone, 200 lag/ml denatured salmon sperm DNA, pH 6.8]. The cDNAs for pS2 and 36B4 were kindly provided by P. Chambon (Institute

284




National de la Sante et de la Recherche Medicale/Centre National de la Re- cherche Scientifique, Strasbourg, France) and were labeled by the random

priming method as described previously (31). Following hybridization, filters

were washed with 3 changes of 0.1 • standard sodium-citrate for a total of 1

h at 65~ For Southern analysis, 50 ~g of genomic DNA digested with a 100-fold excess of EcoRI and 10 lag of each digest size-fractionated in 0.7%

agarose gels. The DNA was transferred to a nitrocellulose filter and hybridized with 32p-labeled pS2 cDNA probe at 58~ for 16 h in hybridization buffer (31 ). Following hybridization, filters were washed with 2 changes of buffer (5 mM

NaPO4, 1 rnM EDTA, 0.2% SDS, pH 7.0) for a total of 1 h at room temperature. For both Northern and Southern analyses, autoradiography was performed at -70~ using two Chronex Quanta III intensifying screens.

RESULTS

Origin of MCF7/LCC1 Cells. MCF7/LCC1 cells were isolated from a rapidly proliferating MCF7/MIII tumor in an ovariectomized

NCr nu~zu athymic nude mouse. To ensure that the MCF7/LCC 1 cells

were der ived f r o m M C F - 7 cells and were not con t amina t ed wi th

m o u s e s t romal cells, we p e r f o r m e d i s o e n z y m e and ka ryo type analy-

ses. The p o l y m o r p h i c e n z y m e s ana lyzed indica te the presence o f

adeny la t e k inase ( i so form 1), es terase D ( i soform 1), g lucose-6-phos-

pha te d e h y d r o g e n a s e ( i so form 1), g lyoxa lase ( i soforms 1 and 2),

lactate d e h y d r o g e n a s e ( h u m a n band 5), phosphoglucornutase-1 (iso-

fo rm 1), and p h o s p h o g l u c o m u t a s e - 3 ( i so form 1). The mi tochondr ia l

mal ic e n z y m e is absent. This pat tern is ident ical to that expressed by

both M C F 7 / M I I I and M C F - 7 cells (3, 4) ind ica t ing wi th a grea ter than

99% probabi l i ty that all cell l ines were de r ived f rom the same indi-

vidual .

In an a t tempt to fur ther con f i rm the der iva t ion o f the cells, a total

o f 100 each o f M C F - 7 , M C F 7 / M I I I , and M C F 7 / L C C 1 me taphase

spreads were examined . All c h r o m o s o m e s were de t e rmined to

be o f h u m a n origin. F ive ka ryo types were prepared, and the probable

or ig in o f 32 marke r c h r o m o s o m e s es tabl ished. The dis t r ibut ion o f 24

marke r c h r o m o s o m e s is s imi lar a m o n g all 3 cell l ines (Table 1).

C h r o m o s o m e marker M 10 is c o m m o n to both M C F - 7 and M C F 7 / M I I I

cells. Six marke r c h r o m o s o m e s are present in both M C F 7 / L C C I

and M C F 7 / M I I I cells, 3 are un ique to the parenta l M C F - 7 cell line,

whi le M 3 0 is un ique to M C F 7 / M I I I cells. Three marke r ch rom-

o s o m e s are present only in the M C F 7 / L C C 1 subline, 2 o f wh ich have

not been repor ted p rev ious ly [M31=del (1) (p21: ) and M32=t -

(5qter---)5pl l : :? : : lq21---) lqter)] . The cons iderab le s imilari t ies in

marke r c h r o m o s o m e pat terns s t rongly sugges t that M C F - 7 , M C F 7 /

MIII , and M C F / L C C 1 cells share a c o m m o n cel lular l ineage.

The c h r o m o s o m e n u m b e r range o f M C F - 7 (range, 5 8 - 9 8 ) is re-

duced to 65 -75 in M C F 7 / M I I I cells (4) and fur ther reduced to 6 6 - 7 3

in M C F 7 / L C C 1 cells. The progress ive reduct ion in c h r o m o s o m e num-

ber impl ies that the var ious subl ines m a y be der ived f rom subpopu-

lat ions present in their respect ive parental cell popula t ions . The loss o f

s o m e marke r c h r o m o s o m e s , the appearance o f others in 50% or more

o f the ka ryo types , and the p ro longed per iod required to isolate the

M C F 7 / M I I I cells (3) sugges t that these popu la t ions m a y be present as

a smal l percen tage o f the parental popula t ion . Cel ls were also exam-

ined for their i m m u n o r e a c t i v i t y to human , mouse , and rat antisera.

M C F - 7 , M C F 7 / M I I I , and M C F 7 / L C C 1 cells exhib i ted i m m u n o r e a c -

t ivi ty on ly wi th h u m a n antisera. All 3 l ines are free f rom con tamina-

t ion wi th M y c o p l a s m a species.

G r o w t h o f M C F - 7 , M C F 7 / M I I I , a n d M C F 7 / L C C 1 Cel l s in Athymic Nude M i c e . The parenta l M C F - 7 cells are total ly dependen t

upon es t rogen for tumor fo rma t ion in nude mice (Fig. l). In contrast ,

both passage 12 M C F ' / / M I I I and passage 4 M C F 7 / L C C 1 cells fo rm

prol i fe ra t ing tumors in un t rea ted ova r i ec tomized nude mice. However ,

the g rowth o f these tumors is s ign i f icant ly s t imula ted by E2 supple-

menta t ion . By compar ing M C F 7 / M I I I t umor growth wi th M C F 7 /

Table 1 Distribution ~f marker chromosomes in MCF7/MIII, MCF7/LCCI, and parental MCF-7 cells as performed by Dr. Ward Peterson (Children's Hospital of

Michigan, Detroit, M1)

Origin of marker chromosomes MI-M5 are reported by Nelson-Rees et aL (53) and M6--M30 by Whang-Peng et al. (54).

Marker MCF-7 MCF7/MIII MCF7/LCC 1

M 1 , a _ _

M2 - - - M2A - - - M3 - - + M4 - + + M5 + - - M5A - + + M6 + + + M6A * - - M7 + + + M7A * + + M8 * - - M9 + + + MI0 + + - MII + + + MI2 + + + MI3 - - - MI4 - - - MI5 - - - M15A - - - MI6 + + + MI6A - - - M17 - - - MI8 - - - M19 + - - M20 + - - M21 - + + M22 - - - M23 - - - M24 - - - M25 - + + M26 - * - M27 - - - M28 - + + M29 - + + M30 - + - M31 - - + M32 - - +

*, persent in <50% of karyotypes; -, absent; +, present in t>50 of karyotypes.

300

E E

200

<

0 ~ 1 O0 F--

0 ~ 0 MCF-7-F..2 �9 ~ �9 MCF-7 +E.2 Z ~ Z ~ Mill-F_2 �9 /

�9 ~ �9 Mill +E.2 / I - I - - i - I LCC-1 -E2 / �9 ~ � 9 LCC-1 +E2 �9 /

�9

i t - 1 , . ~ / A j z x

__r-~----I-'l I " / A z x _ _ ~ . . . & ~ z x m A 1 t " x

. - - -o o . 2 0 4 0 6 0 8'0

DAYS AFTER CELL INOCULATION

Fig. 1. Growth of MCF-7 (passage >500), MCFT/MllI (passage 12), and MCF7/LCCI cells (passage 4) in athymic nude mice. Cells (2 • 106) were inoculated into each flank of ovariectomized athymic nude mice with or without E2 supplementation (60-day E2 pellet inoculated s.c. at the time of tumor cell inoculation). A total of 24 sites were inoculated for each cell line, with 12 sites in E2-supplemented animals and 12 sites in non-E2-supplemented animals. Points, mean tumor size, and only measurements obtained from growing tumors were included. MCF7/MIII cells were passage 6, MCF7/LCC1 cells passage 4, and MCF-7 cells were passage >500. O, m, A, data obtained from E2- supplemented animals; �9 D, A, nonsupplemented animals.

L C C 1 tumors , it appears that the addi t ional in v ivo select ion did not

al ter the doub l ing t ime o f exponen t i a l ly prol i fera t ing unt rea ted or

es t rogen- t rea ted tumors , respec t ive ly (Table 2). However , the t u m o r

g rowth lag per iod as de t e rmined by the t ime f r o m cell inocula t ion to

285




Table 2 Tumor doubling times of MCF-7, MCF7/MIII, and MCF7/LCC1 cells determined from the data presented in Fig. 1

Data points represent the mean time (in days) taken for all tumors in each group to double in size. Statistical analysis was performed using a one-way analysis of variance with significance level at P < 0.01. The mean tumor doubling times of MCF7/MIII and MCF7/LCCI growing in the presence E2 are significantly different from the appropriate cells growing in the absence of E2. There is no significant difference between the tumor doubling times of MCF7/MIII and MCF7/LCC1 growing in the absence of E2. The tumor doubling times of MCF-7, MCF7/MIII, and MCF7/LCCI growing in the presence of E2 are equivalent.

Tumor doubling time (days)

Cell line -E2 +E2

MCF-7 _ a i 1.47 MCFT/MIII 47.57 12.92 MCFT/LCC 1 38.81 13.50

'~ -, no proliferating tumors.

the appearance of tumors decreased significantly (Fig. 1). This observation suggests that the additional passage through nude mice induced a further adaption or selection for in vivo growth. Phenotypic alterations in the in vivo growth induced by passage through nude mice remains stable following repeated passage in vitro. Following more than 30 passages in vitro, MCF7/MIII and MCF7/LCC 1 cells continue to express hormone-independent but responsive growth when reinoc- ulated into nude mice.

Growth of MCF-7, MCF7/MIII, and MCF7/LCC1 Cells in Vitro. We wished to determine whether the perturbations in in vivo

growth between MCF7/MIII and MCF/LCC-1 cells had resulted in altered in vitro growth properties. The basal rates of cell proliferation are equivalent in both sublines (Fig. 2). In contrast to the parental MCF-7 and MCF7/MIII cells, MCF7/LCC1 cells can be routinely maintained in SF-IMEM. MCF7/LCC1 cells grow initially as monolayer cultures but form floating aggregates after 1 or 2 passages in SF-IMEM. When replated in CCS-IMEM, the aggregates adhere to the plastic and cells grow out from the clusters. Following 7 passages (110 days) in SF-IMEM, 2 X l06 M CF7/LCC1 cells were inoculated into the peritoneal cavity of both ovariectomized and E2-supplemented nude mice. Solid proliferating tumors were formed on the surface of peritoneal structures, e.g., mesentery, but we did not observe the presence of malignant ascites.

Response of MCF7/LCCI Cells to Estrogens and Antiestrogens in Vitro. We wished to determine whether the further selection of MCF-7 cells could produce an altered response to either estrogens or antiestrogens. The results in Fig. 2 demonstrate the anchorage-independent response of MCF-7 and MCF7/LCC 1 cells to both E2 and the antiestrogen 4-hydroxytamoxifen. E2 is mitogenic in MCF-7 cells and reverses the inhibitory effects of 4-hydroxytamoxifen. While E2 alone is not mitogenic in MCF7/LCC1 ceils, it can reverse the inhibitory effects of the antiestrogen on anchorage-independent growth. MCF7/ LCC1 cells growing in the chemically defined SF-IMEM also do not respond mitogenically to E2. When growing as a monolayer in SF- IMEM, MCF7/LCC1 cells are significantly inhibited by the antiestrogen 4-hydroxytamoxifen, this inhibition being at least partly re- versed by E2 (data not shown). These growth responses to E2 and antiestrogens are similar to the effects of E2 and antiestrogens on both the anchorage-dependent and anchorage-independent proliferation of MCF7/MIII cells (25).

Gene Amplification in MCF-7 and MCF7/LCC1 Cells. The overexpression and/or amplification of a number of genes has been implicated in conferring a more malignant phenotype upon breast cancer cells. These include EGF-R (13, 14, 20, 29), myc (15), hst, and int-2 (17, 18). However, it is not clear whether these amplifications occur during carcinogenic initiation, promotion, or progression. We wished to determine whether amplifications in genomic DNA sequences were associated with the progression from a MCF-7 to

MCF7/LCC1 phenotype. Genomic DNA from MCF-7 and MCF7/ LCC1 growing in vivo was subjected to in-gel denaturation/ renaturation (Fig. 3). The ability of this technique to identify the presence of multiple amplifications is evident in the lane representing DNA obtained from the Kb-V 1 cell line. These are multi-drug-resistant HeLa cells that contain multiple DNA amplifications (36).

225

200

175

~ 150

v 125 E O = I00

E 75 o

5O o -6 25

0

i

0 E2 OHT E2+OHT 0 Ir2 OHT E2+OHT

Fig, 2. Effect of E2 and 4-hydroxytamoxifen on the anchorage-independent growth of MCF-7 and MCF7/LCC1 cells. Cells were suspended in CCS-IMEM containing 0.6% w/v agar and the appropriate concentrations of E2 (1 nM) and 4-hydroxytamoxifen (0.1 MM). Colonies of 60 Mm were counted electronically using an Omicron 3600 image analysis system. Open bars, MCF-7 cells;filled bars, MCFT/LCC1 cells. Data indicate mean - SD of 3 determinations.

kb

9.5

6.7

4.5

2.3

1.9

It. O

O O -J

u. > I

O .ta

Fig. 3. In-gel denaturation/renaturation of DNA extracted from MCF-7, MCF7/LCCI, and Kb-V1 multi-drug-resistant cells. Genomic DNA was obtained from the appropriate cell lines and subjected to in-gel denaturation/renaturation as described in "Materials and Methods."

286




Although we detected the presence of several apparent amplifications in both MCF-7 and MCF7/LCC1 cells, a comparison of MCF-7 and MCF7/LCC 1 tumors indicates no significant differences in the pattern of amplified sequences. This in-gel renaturation technique can detect as few as 8 copies of an amplified sequence (33). These observations strongly suggest that major genomic amplifications do not contribute significantly to the malignant progression from a hormone-dependent MCF-7 phenotype to a more aggressive hormone-independent MCF7/ LCCI phenotype.

Altered Expression of the E2-regulated ER, PGR, and pS2. The hormone-independent phenotype may be the result of the loss of hormonal regulation of previously hormone-regulated genes. For example, hormone-unresponsive breast cancer tumors and cell lines frequently fail to express detectable levels of ER and/or PGR. We examined the levels of expression of the E2-regulated ER, PGR, and pS2 genes. MCF-7, MCF7/MIII, and MCF7/LCC1 cells express equivalent amounts of ER. MCF7/LCC 1 cells appear to have a high constitutive (P < 0.05, Student's t test), although still E2-inducible, expression of PGR protein (Table 3). In contrast, we did not observe a significantly increased baseline expression of steady-state PGR mRNA in either MCF7/MIII or MCF7/LCC1 cells when compared with MCF-7 cells (Fig. 4). The increased protein levels may reflect either an increased rate of translation or perturbations in the kinetics of PGR recycling and degradation, in the hormone-independent vail-

Table 3 ER and PGR protein expression in MCF-7, MCF7/MIII, and MCF7/LCCI cells as determined by whole cell ligand binding assays

Data represent the mean • SD of 3 or more experiments. For E2 induction of PGR, cells were deprived of E2 for 4 days by growth in CCS-IMEM and subsequently treated with 1 nM E2 for 48 h. Binding of progestin to glucocorticoid receptors was eliminated by prior exposure to 100 nM hydrocortisone. The number and affinity of receptor binding was determined as indicated in "Materials and Methods."

PGR (sites/cell)

Cell line ER (sites/cell) -E2 +E2

MCF-7 120,536 _+ 20,003 9,841 • 4,683 47,522 • 12,005 MCF7/MIII 143,082 __ 37,472 27,096 _+ 9,358 90,328 • 951 MCF7/LCCI 112,272 • 19,440 36,766 • 12,451 81,657 __. 18,656

�9 o4 ..o ..o ILl O O o~ ~.1 + Ih,i, L _

iX. fl- 14.1 r,.. rr "r < + I tL

rn Z : ---- 0

ILl I11 + I

u.I O O I _1 _1

,.L ,,' ,,' o o

IE ~ IE

PGR

36B4

Fig. 4. RNase protection analysis of E2 induction of PGR mRNA expression. MCF-7, MCF7/MIII, and MCF7/LCC 1 mRNA was obtained from cells growing in vitro in both the absence (-E2) and presence (+E2) of 1 nM E2. The PGR probe produces a protected fragment of 240 base pairs, the 36B4 probe producing a protected fragment of 220 base pairs.

ants when compared with the hormone-dependent MCF-7 cells. Anal- yses of the PGR gene and protein expression are currently in progress and will be reported elsewhere.

Preliminary data from our laboratory indicated that the expression of pS2 may be constitutively activated in MCF7/MIII hormone-independent breast cancer cells (23, 24). Northern blotting with subse- quent hybridization to a radiolabeled pS2 eDNA probe indicates that the expression of pS2 mRNA in MCF-7 cells is E2-regulated both in

vitro and in vivo. E2-deprived MCF7/LCC1 (Fig. 5) and MCF7/MIII cells (data not shown) growing both in athymic nude mice and in vitro

exhibit a high baseline expression of this gene. MCF-7 cells selected in vitro (37) also exhibit an increased pS2 expression when growing without E2, but pS2 expression is inhibited by physiological concentrations of E2 (38). This may represent an E2-supersensitive phenotype. In contrast, the level of expression of pS2 mRNA appears to be further increased by E2 in both MCF7/MIII and MCF7/LCC1 cells growing in vivo (Fig. 5A). Southern analysis of genomic DNA from MCF-7 and MCF7/LCC 1 cells did not demonstrate an increase in the copy number of this gene (Fig. 5B), indicating that the increased pS2 mRNA expression in the absence of E2 is not the result of an amplification of the pS2 gene. There is no significant or reproducible induction/repression of pS2 mRNA by E2 when either MCF7/LCC1 (Fig. 5) or MCF7/MIII cells (data not shown) are grown in vitro. Thus, acquisition of a hormone-independent phenotype by MCF7/MIII and MCF7/LCC 1 cells is associated with an apparent constitutive expression in vitro and elevated expression in vivo of the E2-regulated pS2 mRNA. The ability of pS2 to predict response to endocrine therapy is equivalent to that of a combination of ER and PGR (39). We have recently demonstrated a significant increase in the expression of the E2-regulated cathepsin D enzyme in MCF7/LCC1 cells (5). Expres- sion of both pS2 and cathepsin D is associated with increased metastatic potential (5, 39).

Steady State Phosphatidyl Inositol Turnover in MCF-7 and MCF7/LCC1 Cells. E2 stimulates phosphatidyl inositol turnover in a variety of E2-responsive tissues, including mouse uterus (40) and MCF-7 cells (34). Furthermore, E2-regulated growth factors can mod- ulate the turnover of inositol phosphates in MCF-7 cells (34, 41). Since MCF7/LCC1 cells have become E2-independent and exhibit a high baseline expression of E2-regulated genes, we wished to determine whether genes utilizing the phosphatidyl inositol signal transduction pathway are potentially involved in the acquisition of hormone-independent growth. The total intracellular inositol phosphates were expressed as a ratio of either the total water-soluble or total lipid-soluble [3HI dpm at steady state conditions. The data in Table 4 clearly indicate no significant differences in the steady state turnover of inositol phosphates between MCF7/LCC 1 and MCF-7 cells when growing without E2 (P > 0.1). Inositol phosphate turnover is E2- responsive in MCF-7 cells, whether expressed as total intracellular inositol phosphate/water-soluble ratio (P < 0.01) or total inositol phosphate/lipid-soluble ratio (P < 0.001). However, E2 does not influence turnover in MCF7/LCC 1 cells (Table 4).

DISCUSSION

There are few model systems that facilitate the study of the process of acquired hormone resistance or acquisition of hormone-independent growth in initially hormone-dependent human breast cancer cells. Resistance to the antiestrogen LY 117018 in hormone-dependent MCF-7 human breast cancer cells has been induced by a stepwise selection in the presence of drug (6). However, stable resistance is accompanied by a loss of tumorigenicity in athymic nude mice (25). Long-term estrogen deprivation of estrogen-dependent MCF-7 human breast cancer ceils either in vitro or in vivo can result in the emergence

287




of a hormone-independent subpopulation e.g., MCF7/MIII cells (4, 37). Although being fully estrogen-independent and unresponsive to estrogen in vitro, the cells demonstrate estrogen responsiveness in vivo

(4). Cells selected in vivo can also acquire a more malignant phenotype as determined by increased invasive and metastatic potential (4, 5) and increased anchorage-independent growth (25). In an attempt to induce additional characteristics of the progressed phenotype, MCF7/ MIII cells were further passaged through athymic nude mice. The resulting cell population (MCF7/LCC1) exhibits a reduced time to tumor formation in vivo, and an ability to grow continually in a chemically defined serum-free medium when compared with the appropriate parental MCF7/MIII and MCF-7 cells.

Acquisition of a malignant phenotype in human breast tumors is often associated with the presence of DNA amplifications. Increased tumorigenicity (10, 11), drug resistance (12), and a metastatic phenotype correlate with the presence of amplified DNA sequences (21). In breast cancer, overexpression/amplification of EGF-R (13, 14, 20, 29), myc (15), hst, and int-2 (17, 18) are associated with a more malignant

A 1 2 3 4 5 6 7

- - 36B4

4-- pS2

B

| MCF-7

MCF71LCC1

Fig. 5. A, effect of E2 on the expression of pS2 mRNA. For in vitro analysis, cells were grown in CCS-IMEM and treated for 48 h with 1 nM E2. MCF7/LCCI tumors were grown in both the presence and absence of E2. MCF-7 tumors were established in the presence of E2 supplementation. Subsequently, the E2 pellet was removed, and 7 days later the tumor was excised and total RNA prepared as described (35). Northern blotting was performed as indicated in "Materials and Methods." Lane 1, MDA-MB-231 (ER-negative human breast cancer cell line); Lane 2, MCF7/LCCI +E2 in vitro; Lane 3, MCF7FLCCI -E2 in vitro; Lane 4, MCF7/LCC1 +E2 in vivo; Lane 5, MCF7/LCCI -E2 in vivo; Lane 6, MCF-7 +E2 in vitro; Lane 7, MCF-7 -E2 in vitro. B, Southern analysis of the pS2 gene in MCF-7 and MCF7/LCC 1 cells. Genomic DNA was digested with EcoRI, subjected to electrophoresis in agarose gels, and probed with a randomly primed pS2 cDNA.

Table 4 Steady-state turnover of [3H]myoinositol in MCF-7 and MCF7/LCCI cells

Ratios are based on the mean value of 3 determinations for each of total intracellular inositol phosphates, total water-soluble 3H dpm, and total lipid-soluble 3H dpm. The differences in total intracellular inositol phosphates/total water-soluble 3H dpndtotal lipid- soluble 3H dpm/total water-soluble 3H dpm values for MCF-7 versus MCF7/LCCI and MCF7/LCCI + and - E2 are not statistically significant (P < 0.1; Student's t test). For both total intracellular inositol phosphates/total water-soluble 3H dpm and total lipid- soluble 3H dpm/total water-soluble 3H dpm, MCF-7 + E2 is significantly different from MCF-7 - E2 (P < 0.01 and P < 0.001, respectively).

MCF-7 - E2 MCF-7 + E2 MCF7/LCC1 - E2 MCF7/LCCI + E2

IP/TOT a 0.0480_+0.017 0.118_+0.026 0.031_+0.003 0.025+0.003 IP/LIP 0.0185 + 0.002 0.042 +- 0.006 0.032 -+ 0.003 0.029 _ 0.002

a IP, total intracellular inositol phosphates; TOT, total water-soluble 3H dpm; LIE total lipid-soluble 3H dpm.

phenotype. We looked for gene amplifications by using an in-gel denaturation/renaturation assay. This technique can indicate the presence of genomic sequences amplified 8-fold or more (33). We did not detect any significant differences in the pattern of amplified sequences present in MCF-7 and MCF7/LCC1 cells, indicating that acquisition of tumorigenicity in the absence of E2 occurs in the absence of significant DNA amplification. This observation does not exclude the possibility that DNA amplifications could be important in the early stages of neoplastic transformation in human breast tissue, for example, during the initiation and promotional phases of carcinogenesis.

The levels of expression and secretion of various mitogenic growth factors and oncogenes, including the ras oncogene (42), TGF-c~ (41), and insulin-like growth factors (43, 44), are substantially increased following treatment with E2 in some hormone-dependent breast cancer cells. In contrast, hormone-independent cell lines frequently exhibit a high level of constitutive growth factor expression that is not hormone regulated (41). The ability to secrete high amounts of mitogenic autocrine growth factors may be responsible for hormone-au- tonomous growth (45). Many of these factors, including TGF-ot and TGF-/3, utilize phosphatidyl inositol as a substrate for transduction of hormone and growth factor signals initiated by receptor-ligand inter- actions (34). Estrogens increase secretion of the mitogen TGF-ct (41), while decreasing secretion of the inhibitory factor transforming growth factor-/3 (46), and antiestrogens induce the inverse response (41, 46). Moreover, a MCF-7 variant selected in the presence of increasing concentrations of the antineoplastic agent Adriamycin produces hormone-independent and hormone-unresponsive tumors in

vivo (20). These cells exhibit a 60-fold increase in the hydrolysis of phosphoinositides when compared with the parental cells (47). Over- expression of the v-H-ras oncogene can induce hormone-independent growth in some MCF-7 cells (48). Consequently, we determined the steady state turnover of [3H]myo-inositol in MCF-7 and MCF7/LCC1 cells. The data show that while MCF7/LCC1 cells have acquired hormone-independent growth in vitro, this growth is not accompanied by elevated steady state levels of inositol phosphate turnover. Fur- thermore, the ability of E2 to increase inositol phosphate turnover in the parental MCF-7 cells has been lost by MCF7/LCC1 cells. This suggests that the acquired estrogen independence of MCF7/LCC1 cells may require events in addition to any constitutive expression of oncogenes or peptide growth factors that can increase steady state phosphatidyl inositol turnover. Alternatively, these and/or other growth factors may contribute to hormone independence through the induction of signal transduction pathways independent of phosphoinositol metabolism.

We have previously hypothesized that the altered expression of specific E2-regulated genes may be responsible for the acquisition of hormone independence in previously hormone-dependent breast cancer cells (23, 24). The up-regulation of pS2 mRNA and PGR protein expression, but not ER protein expression or steady state phophsoi- nositol turnover, provides further evidence in support of this hypothesis. These perturbations are transcriptional (pS2) or possibly translational/posttranslational (PGR) rather than the result of amplification of the genes. The altered expression of other hormonally regulated genes, e.g., EGF-R may be more closely associated with the later stages of malignant progression, perhaps after loss of ER and/or PGR expression (4), since the expression of EGF-R is not altered in either MCF7/MIII (4) or MCF7/LCC1 cells (data not shown). E2 induction of pS2 but not PGR or EGF-R is a primary response to E2, being both detectable within 3 h of stimulation and resistant to the inhibitory effects of cycloheximide (49). These data support our initial hypothesis, and strongly suggest that the acquisition of a hormone- independent phenotype is associated with an altered regulation of

288




expression of specific hormone-regulated genes. The genes of greatest importance may be those that represent primary rather than secondary responses to E2.

A hormone-independent and hormone-unresponsive phenotype can be induced in MCF-7 cells by exposure to cytotoxic drugs (20, 26, 50). This phenotype is characterized by loss of ER and PGR expression, loss of sensitivity to tamoxifen, and a loss of dependence upon estrogen for tumor formation in athymic nude mice (20). However, many breast tumors appear to acquire a highly malignant phenotype without exposure to cytotoxic agents. Since the plasma estrogen levels in athymic nude mice are comparable to those in postmenopausal women (51, 52), we have applied potentially more "physiological" selective pressures. These pressures have produced cells with an in- termediate hormone-independent but hormone-responsive phenotype that may more closely reflect the "natural" progression of breast tumors (1, 2, 5). This progression can occur without major increases in gene copy number or steady state phosphoinositol turnover. How- ever, we have observed significant alterations in the regulation of expression of specific hormone-regulated genes. These observations strongly implicate perturbations in the hormonal regulation of previously regulated genes in the acquisition of hormone independence.

ACKNOWLEDGMENTS

The authors thank Dr. Ward D. Peterson (Chi ldren 's Hospi ta l o f Michigan,

Detroit , MI) for the i sozyme and ka ryo type analyses, and Dr. Michae l Johnson

and Michae l M c G a r v e y for assistance in per forming the Southern analyses.

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