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J. Biol. Chem., Vol. 275, Issue 37, 28371-28379, September 15, 2000

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Müllerian Inhibiting Substance Inhibits Breast Cancer Cell Growth through an NFB-mediated Pathway^*

Dorry L. Segev, Thanh U. Ha, Trinh T. Tran, Mary Kenneally, Paul Harkin^§, Mira Jung^¶, David T. MacLaughlin, Patricia K. Donahoe, and Shyamala Maheswaran

From the  Pediatric Surgical Research Laboratories, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, the ^§ Department of Oncology, Queen's University, Belfast BT9 7AB, Northern Ireland, and the ^¶ Department of Radiation Medicine, Georgetown University Medical Center, Washington, D. C. 20057

Received for publication, May 25, 2000, and in revised form, June 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Müllerian inhibiting substance (MIS), a member of the transforming growth factor- superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G₁ phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IB expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFB signaling pathway was required for these processes. These results identify the NFB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MIS,¹ a member of the TGF- family of hormones, causes regression of the epithelial-mesenchymal unit of the Müllerian duct in the embryonic male urogenital ridge. In females the Müllerian duct autonomously differentiates into the uterus, Fallopian tubes, and upper vagina (1). The Müllerian ducts of both male and female embryos are responsive to MIS only during a critical period in development after which they lose sensitivity (1, 2). However, MIS is produced at high levels by Sertoli cells of the testis even after the regression of the Müllerian duct, decreases at adolescence, and is produced throughout adult life. In females, it is synthesized by postnatal granulosa cells of the ovary and can be detected in the female serum until menopause (3, 4). Cleavage of MIS by a kex-like enzyme is required to manifest MIS activity since a noncleavable mutant is devoid of biological function (5).

The MIS type II receptor gene, a highly conserved single transmembrane serine threonine kinase that is homologous to members of the TGF- family of type II receptors, encodes a 2.0-kilobase mRNA (6-8). It is expressed at high levels in the Müllerian duct, Sertoli cells, and granulosa cells of the embryonic and adult gonads and in the uterus (8). The developmental significance of the MIS type II receptor was demonstrated in MIS type II receptor null male mice, which develop normally but have a persistent Müllerian duct that forms a uterus and oviducts; this phenotype resembles that of MIS ligand-deficient mice (9-11). Female MIS type II receptor or MIS ligand-deficient mice are normal and fertile as young adults.

The detection of MIS in the serum of males and females even after the regression and differentiation of the Müllerian duct (3, 4) suggests that MIS might be a multifunctional hormone. MIS is a negative regulatory factor in fetal rat lung maturation, where it inhibits the production of pulmonary surfactant both in vitro and in vivo (12, 13). MIS also inhibits oocyte meiosis in vitro (14, 15). The presence of MIS type II receptor in non-Müllerian tissues such as the ovary, lung, and Leydig cells of the testis (16-18) and the occurrence of Leydig cell hyperplasia and Leydig cell tumors in receptor null male mice (11) also suggest that the biological action of MIS is not limited to the Müllerian duct. Furthermore, MIS has been shown to inhibit the growth of tumor cells in vitro and in vivo (19-22). Masiakos et al. (17), using ovarian epithelial cells derived from the ascites of ovarian cancer patients, correlated MIS type II receptor expression with MIS-mediated growth inhibition. Furthermore, exogenous MIS has been shown to inhibit metastases of the ocular melanoma cell line OM431 (23) and to block the growth of the vulvar epidermoid carcinoma cell line A431 and the OM431 cell line in vivo (21, 24).

Expression of MIS-related proteins including TGF-, activin, inhibin, and BMP ligands and their receptors has been demonstrated in human breast cancer cell lines and in breast cancer specimens (25-33). TGF- has been shown to inhibit the growth of numerous cell types in vitro, including both normal human mammary epithelial cells and breast cancer cell lines (34-36). The importance of the signaling pathways initiated by members of the TGF- superfamily in the control of breast cell proliferation is also demonstrated by the presence of genetic mutations or loss of expression of activin/TGF- receptors in human breast cancers (29, 37). However, MIS type II receptor expression and the effects of MIS-mediated signaling on growth and differentiation of the breast have not been investigated.

Recent studies have shown that TGF--mediated inhibition of breast cancer cell proliferation is associated with a decrease in NFB DNA binding activity (38). The signaling cascade associated with activation of the NFB pathway, initially described in association with inflammatory responses in the cellular immune system, is also induced by stress, growth arrest, and apoptosis in a wide variety of cell types (39, 40). The NFB family consists of transcriptional activators, including p65, p50, p52, and c-Rel, which share a Rel homology domain. These molecules form either homo- or heterodimers and bind to DNA in a sequence-specific manner. NFB in its inactive state exists in the cytosol bound to the inhibitory IB family of molecules. Activation of the pathway by extracellular signals leads to phosphorylation and degradation of IB with subsequent nuclear localization of NFB (39, 40). IEX-1 is an NFB-inducible immediate early gene that is induced by radiation, 12-O-tetradecanoylphorbol-13-acetate, Fas, and TNF- (41). Wu et al. recently demonstrated the existence of two IEX-1 splice variants, IEX-1S and IEX-1L. IEX-1L has an in-frame insertion of 37 amino acids resulting from the presence of the entire intronic sequence within the coding region of the IEX-1S transcript (42). The presence of functional binding sites for NFB and p53 in the IEX-1 promoter supports its role in regulation of growth, differentiation, and stress response (43).

In this report we demonstrate MIS type II receptor expression in normal breast and in human breast cancer cell lines, breast fibroadenomas, and tumors. MIS inhibits the growth of breast cancer cells through activation of the NFB signaling cascade, which is developmentally regulated in breast epithelial cells (44) and is aberrantly modulated in several cancers including breast tumors (45, 46). Thus, breast tissue might be an additional target for the action of the ovarian hormone MIS.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines, Reagents, Colony Inhibition Assays, Cell Cycle Analysis, and Caspase-3 Assays-- Human breast cancer cell lines T47D, MCF7, MDA-MB-468, and MDA-MB-231 and COS cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% female fetal bovine serum, glutamine, and penicillin/streptomycin. ZR 75-1 was grown in RPMI 1640 containing the same supplements. COS cells were transfected using Fugene 6 transfection reagent (Roche Molecular Biochemicals) as described in the manufacturer's guide to determine protein expression.

To measure inhibition of cell proliferation, monolayers of equal numbers of T47D cells were transfected using the calcium phosphate DNA precipitation technique with vector or CMV-driven expression constructs (see figure legends) along with 0.5 µg of a construct that encodes hygromycin resistance. Cells were selected in medium containing 100 µg/ml hygromycin (Roche Molecular Biochemicals). After 3-4 weeks, colonies were stained with crystal violet and counted. To test the growth inhibitory effect of exogenous rhMIS, cells were seeded in 60-mm dishes at 3 × 10⁴ cells/plate in the absence of MIS. MIS was added after 24 h, cultures were grown in the presence or absence of 35 nM MIS for 4 days, and cells were counted on the Coulter counter. To analyze the effect of MIS on cell cycle progression, cells were grown to 20% confluence and treated with 35 nM (5 µg/ml) MIS for 4 days. Cells were detached with phosphate-buffered saline/EDTA, fixed in 95% ethanol, stained for DNA content with propidium iodide, and analyzed by fluorescence-activated cell sorting. Caspase-3 activity was determined as described in the CLONTECH ApoAlert Caspase-3 Assay kits user manual. Briefly, T47D cells were treated with 35 nM rhMIS for 3 days and equal amounts of protein lysate were analyzed for caspase-3 activity. Caspase-3 levels in the cell were calculated based on a standard curve, and relative fold induction was estimated.

NFB Electrophoretic Mobility Shift Assays and CAT Assays-- T47D cells were grown to 70% confluence and treated with indicated concentrations of rhMIS for specified lengths of time. Cells were harvested in cold phosphate-buffered saline, resuspended in 1 ml of TKM 10:10:1 (10 mM Tris, pH 8.0, 10 mM KCl, and 1 mM MgCl₂), and lysed with 0.1% Triton X-100. Nuclei were pelleted by centrifugation at 5,000 rpm at 4 °C, and proteins were extracted in buffer containing 10 mM HEPES, pH 7.0, 350 mM NaCl, and 1 mM EDTA. 3 µg of protein was used in 25-µl binding reactions containing 10 mM HEPES, pH 7.0, 70 mM NaCl, 0.1% Triton X-100, and 4% glycerol. NFB oligonucleotides (Promega) were ³²P-end-labeled, and DNA protein complexes were resolved on 4% native polyacrylamide gels. Supershift experiments were performed by adding 0.1 µg of rabbit anti-p65 or -p50 antibodies (Santa Cruz) to the binding reactions.

The 2X-NFB-CAT reporter construct was a kind gift from Dr. R. Bravo (Bristol-Myers Squibb Pharmaceutical Research Institute). For CAT assays, T47D cells were transiently transfected by calcium phosphate DNA precipitation technique with 5 µg of 2X-NFB-CAT and 1 µg of human growth hormone (hGH) encoding plasmid. Untreated cells and cells treated with 35 nM MIS for 12 h were harvested, lysed, and analyzed by CAT-ELISA according to the protocol provided by Roche Molecular Biochemicals. Transfection efficiency was standardized using hGH-ELISA as described in the manual provided by Roche Molecular Biochemicals.

RNase Protection, Northern Blot, and PCR Analyses-- RNase protection assays were done using a 302-base pair antisense probe containing exon 11 (228 base pairs) and the 3'-untranslated region (74 base pairs) of the human MIS type II receptor cDNA. This receptor fragment contained within PvuII-XhoI restriction sites was cloned in reverse orientation into XhoI-EcoRV sites of pCDNA 3.1() plasmid. The resulting construct was sequenced to confirm the boundaries of the insert and was linearized with HindIII, and the antisense transcript was obtained using T7 polymerase (MAXIscript in vitro transcription kit, Ambion). RNase protection assays were done with 75 µg of total RNA isolated from T47D, MDA-MB-231, ZR 75-1, and COS-7 cells using the RPA III ribonuclease protection assay kit (Ambion). Briefly, RNA was hybridized with 375,000 cpm of radiolabeled probe overnight at 55 °C and digested with a mixture of RNase A and RNase T1 for 30 min at 37 °C. The protected fragments were precipitated and analyzed on a denaturing 6% polyacrylamide, 8 M urea sequencing gel. 75 µg of yeast tRNA were used as positive control for the function of RNase, and another sample containing the same amount of yeast tRNA was incubated without RNase to control for probe integrity. Sequencing reactions of the plasmid containing the antisense MIS type II receptor probe was electrophoresed on the same gel to estimate the sizes of the fragments.

RNA was isolated from cells using RNA STAT-60 total mRNA isolation kit (Tel-Test, Inc). Poly(A)⁺ RNA from T47D, MDA-MB-231, MCF7, and MDA-MB-468 was obtained using the FastTrack 2.0 mRNA isolation kit (Invitrogen) and poly(A)⁺ RNA from the adult human testis was purchased from CLONTECH. 4 µg of poly(A)⁺ RNA was separated on a formaldehyde gel, transferred to HyBond membrane (Amersham Pharmacia Biotech), and probed with human MIS type II receptor cDNA.

7.5 µg of total RNA isolated from either T47D or MDA-MB-231 cells was analyzed by Northern blot with radiolabeled IEX-1 or actin. IEX-1 probe for Northern analysis was derived by PCR amplification of T47D cDNA using the following primers: sense, 5'-TCCAGAGGACGCCCCTAACG-3'; and antisense, 5'-GTTCACAGAACATACTAGGC-3'. Primers for detecting the presence of IEX-1L and IEX-1S transcripts by PCR were: sense, 5'-TCCGGTCCTGAGATCTTCAC-3'; and antisense, 5'-CTCTTCAGCCATCAGGATCT-3'. cDNA derived from total RNA isolated from untreated and MIS-treated T47D and MDA-MB-231 cells, and 50 ng of DNA from CMV-driven IEX-1S and IEX-1L expression constructs were amplified with the primers above at an annealing temperature of 50 °C for 30 cycles. Primers for detecting MIS type II receptor expression in normal human breast, breast carcinomas, and cell lines were: sense, 5'-GCT GGC TTA TGC TCT TCT CCT TC-3'; and antisense, 5'-ACC TCG CAC TCT GTA GTT CTT TCG-3'. Total RNA isolated from tissue derived from normal human breast, two invasive ductal carcinomas, and a fibroadenoma of the breast (Tumor Bank, Massachusetts General Hospital) was converted to cDNA and amplified with the above primers by PCR. The MIS type II receptor in normal rat breast cDNA was amplified using the following primers: sense, 5'-CAGCCCTGGCTTATCCTCAGG; and antisense, 5'-GCCACACGCATGTCAGCCTGT-3' (which generates a 220-base pair fragment); sense, 5'-GTGTGTTCTTTGAGGCTCCTGGAGTT-3'; and antisense, 5'-CTCAGCAGCAGCACAAGGAACACT-3' (which produces a 410-base pair fragment).

Antibodies and Western Blot Analyses-- The rabbit MIS type II receptor antibody was generated by injecting animals with the peptide C-G-T-D-F-C-N-A-N-Y-S-H-L-P-P-S-G, which corresponds to amino acids 111-127. The antibody was purified over a protein A-Sepharose column before use. Horseradish peroxidase conjugated anti-rabbit antibodies were purchased from Amersham Pharmacia Biotech. Proteins were harvested in RIPA buffer (50 mM Hepes, pH 7.0, 150 mM NaC1, 0.1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), fractionated on SDS-polyacrylamide gels, transferred to Immobilon-P membrane, and blocked in Tris-buffered saline and Tween 20 (25 mM Tris, pH 7.4, 136 mM NaCl, 5 mM KCl, 0.1% Tween) containing 30% milk. The type II receptor antibody was used at a dilution of 1:3000 in 10% milk/Tris-buffered saline and Tween 20 solution. Blots were incubated with the primary antibodies for 2 h at room temperature, washed, and incubated with the appropriate horseradish peroxidase-conjugated antibodies. Bound antibodies were detected with ECL (Amersham Pharmacia Biotech).

COS cells transfected with 3 µg of FLAG-tagged rat MIS type II receptor construct using 9 µl of FUGENE 6 (Roche Molecular Biochemicals), and expression was detected by Western blotting using antibodies to the type II receptor or FLAG.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MIS Type II Receptor Expression in the Breast-- Expression of the MIS type II receptor mRNA in human breast cancer cell lines was analyzed by RNase protection assay and Northern blot analysis. RNase protection assay was performed with a 302-base pair radiolabeled antisense riboprobe containing part of exon 11 and the 3'-untranslated sequence of the human MIS type II receptor. The MIS type II receptor mRNA was present in the breast cancer cell lines T47D, MDA-MB-231, and ZR 75-1 (Fig. 1A, left panel). The protected fragment was approximately 93 base pairs shorter than the full-length probe due to the presence of unrelated vector sequence at both the 5' and 3' ends. COS-7, the SV-40 transformed kidney cell line derived from the African green monkey, did not express the MIS type II receptor mRNA (Fig. 1A, right panel). Yeast tRNA samples were hybridized with the probe and incubated with or without RNase to control for RNase activity and probe integrity, respectively. Northern blot analysis of poly(A)⁺ RNA isolated from T47D, MCF 7, MDA-MB-231, and MDA-MB-468 human breast cancer cell lines also demonstrated that the 2-kilobase MIS type II receptor mRNA was present in a pattern similar to that observed with poly(A)⁺ RNA derived from adult human testis (Fig. 1B). The presence of two transcripts is consistent with Northern analyses of RNA isolated from rat and mouse Leydig cell tumor lines (18) and human ovarian cancer cell lines, which express the MIS type II receptor (17). The presence of MIS type II receptor in breast cancer cell lines T47D and MDA-MB-231 was also confirmed by reverse transcription-PCR amplification and sequence analyses using primers specific for exons 1 and 5 (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   MIS type II receptor expression in the breast. A, MIS type II receptor mRNA expression in human breast cancer cell lines. Upper panel, total RNA isolated from T47D, MDA-MB-231, ZR 75-1, and COS cells was analyzed by RNase protection assay. The protected fragments after digestion with RNase were analyzed on a 6% denaturing polyacrylamide gel. 75 µg of yeast tRNA was hybridized with the probe and incubated with or without RNase to test the activity of RNases and probe integrity, respectively. A small aliquot of the probe was loaded directly on the gel to control for integrity of the probe. Lower panel, schematic representation of the MIS type II receptor antisense riboprobe used for RNase protection assay. UTR, untranslated region; nt, nucletide(s). B, 4 µg of poly(A)+ RNA isolated from human breast cancer cell lines T47D, MCF7, MDA-MB-231, MDA-MB-468 and from adult human testis purchased from CLONTECH were analyzed by Northern blot using a human MIS type II receptor cDNA probe. The positions of 18 S ribosomal RNA (open arrow), MIS type II receptor mRNA (closed arrow), and the shorter transcript (open arrow) are indicated. C, left panel, characterization of rabbit anti-MIS type II receptor antibody. Western blot analysis of total protein (100 µg) isolated from COS cells transfected with 2 µg of FLAG-tagged rat MIS type II receptor using a rabbit anti-MIS the type II antibody. Right panel, the blot was reanalyzed with a mouse anti-FLAG antibody. An equal amount of protein from untransfected COS cells was used as a negative control. D, MIS type II receptor protein expression in human breast cancer cell lines. Total protein (100 µg) from T47D, MDA-MB-231, and MDA-MB-468 cells were analyzed by immunoblotting with rabbit anti-MIS type II receptor antibody or with antibody preincubated with the cognate peptide overnight at 4 °C. A parallel blot was probed with the preimmune rabbit serum to control for antibody specificity. Ab, antibody. E, upper panel, expression of MIS type II receptor in normal breast and breast tumors. Total RNA isolated from normal breast, breast fibroadenoma, and two invasive breast ductal adenocarcinoma tissue samples was converted to cDNA and analyzed by PCR amplification using primers specific for exons 1 and 5. A DNA fragment of the expected size (582 base pairs) is shown. cDNA obtained from MDA-MB-231 cells is shown as a positive control. Lower panel, total RNA isolated from normal rat breast was converted to cDNA and analyzed using primers that span exons 2 and 4 (410 base pairs), and exon 11 and the 3'-untranslated region (220 base pairs) of the rat MIS type II receptor. The rat MIS type II receptor cDNA was used as a positive control.

The expression of the MIS type II receptor protein in breast cancer cell lines was demonstrated by Western blot analysis. COS cells transfected with a CMV-driven FLAG-tagged rat MIS type II receptor construct were analyzed by Western blot to characterize the rabbit MIS type II receptor antibody. The MIS type II receptor protein migrated with a molecular mass of approximately 63 kDa, as detected by the MIS type II receptor antibody, and was absent in cells transfected with vector (Fig. 1C, left panel). Re-analysis of the membrane with an anti-FLAG antibody displayed a protein of the same molecular weight in cells transfected with the receptor (Fig. 1C, right panel). Immunoblot of total cellular protein isolated from human breast cancer cell lines T47D, MDA-MB-231, and MDA-MB-468 using the rabbit MIS type II receptor antiserum demonstrated the presence of the 63-kDa endogenous receptor protein. This protein was absent upon preincubation of the antibody with an excess of the cognate peptide. Antibody specificity was also confirmed by analyzing the blots with preimmune rabbit serum (Fig. 1D).

We also detected expression of the MIS type II receptor mRNA in normal breast and in a breast fibroadenoma and two invasive ductal carcinomas using PCR amplification of cDNA generated from these tissues (Fig. 1E, upper panel). Primers specific for exons 1 and 5 demonstrated the presence of a DNA fragment of the expected size (582), the DNA sequence of which was identical to that of exons 1, 2, 3, 4, and 5 of the human MIS type II receptor (data not shown). MIS type II receptor in normal rat breast was detected by PCR analyses using primers that span exons 2 and 4 (Fig. 1E, lower panel) and exon 11 and the 3'-untranslated region (Fig. 1E, lower panel) of the rat MIS type II receptor. Sequence of the DNA fragments amplified by these primers was identical to the rat MIS type II receptor (data not shown).

We could not detect expression of the MIS ligand in culture supernatants obtained from either normal or cancer cell lines of the human breast by MIS-ELISA. Furthermore, PCR of cDNA derived from normal and tumor tissue obtained from human breast cancer patients did not amplify the MIS ligand. These observations suggests that, although breast epithelial cells are responsive to serum MIS, they do not produce detectable levels of the MIS ligand.

MIS-induced Growth Inhibition Requires Activation of NFB-- To study the functional properties of MIS type II receptor expression in breast tissue, we determined whether MIS alters the growth properties of the ER-positive breast cancer cell line T47D. We cotransfected T47D cells with a plasmid encoding hygromycin resistance and either CMV-vector (V), CMV-driven cleavable bioactive MIS (K2), or noncleavable inactive MIS (K6) (23). The concentration of wild type and mutant MIS in the medium was 15-20 ng/ml by MIS-ELISA. Expression of MIS in T47D cells resulted in 75% reduction of drug-resistant colonies compared with vector-transfected cells. The noncleavable inactive form of MIS did not affect the growth of T47D cells (Fig. 2A).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   MIS-mediated inhibition of T47D cell growth. A, monolayers of T47D cells were stably transfected with 0.5 µg of hygromycin resistance plasmid and 10 µg each of pCDNA vector (V), pCDNA-bioactive MIS (K2), or pCDNA-noncleavable inactive MIS (K6). Hygromycin-resistant colonies were stained with crystal violet after 3 weeks. Number of colonies per plate is represented as percentage of survivors with vector-transfected plates set at 100%. B, T47D were treated with rhMIS over a period of 6 days and cell numbers were calculated using a Coulter counter. The mean number of cells in the untreated plates was set at 100%. C, cell cycle analysis of MIS-treated T47D cells. Cells were treated with 35 nM MIS for 4 days, stained for DNA content with propidium iodide, and analyzed by flow cytometry. Cell cycle distribution of untreated cells grown for 0 and 4 days is also shown. D, induction of apoptosis by MIS. T47D cells were treated with 35 nM rhMIS for 3 days, and equal amounts of protein lysates were analyzed for caspase-3 activity. Caspase-3 levels were estimated based on a standard curve, and the relative fold induction was calculated.

Addition of exogenous rhMIS (recombinant human MIS) to proliferating cells also inhibited growth (Figs. 2B and 3E). In order to determine whether MIS-mediated inhibition of T47D cell growth involved changes in cell cycle distribution, cells were treated with 35 nM (5 µg/ml) rhMIS, fixed in 95% ethanol, stained for DNA content with propidium iodide, and analyzed by fluorescence-activated cell sorting. Treatment with rhMIS lead to a consistent 10-16% increase in the G₁ phase of the cell cycle that was not observed in untreated cells (Fig. 2C). Activity of caspase-3, an enzyme activated in cells undergoing apoptosis, was monitored to determine whether MIS induces programmed cell death in T47D cells. MIS induced a 3-fold induction in caspase-3 activity in T47D cells compared with untreated cells (Fig. 2D). MIS-mediated induction of apoptosis was also confirmed using annexin V-fluorescein isothiocyanate staining, which detects early stage apoptosis. A 3-fold increase in annexin V-positive T47D cells was seen following treatment of cells with MIS (data not shown). Thus, MIS inhibits the growth of breast cancer cell lines in vitro by interfering with cell cycle progression and inducing apoptosis.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   MIS-induced NFB pathway mediates growth inhibition. A, upper panel, MIS activates the NFB pathway. T47D cells were treated with 35 nM MIS for indicated periods of time, and 3 µg of nuclear proteins were analyzed by EMSA using a 32P-labeled NFB oligonucleotide probe. Antibody supershift experiments were done with samples treated with MIS for 6 h. The positions of the NFB DNA protein complexes (closed arrows) and supershifted complexes (open arrows) are indicated. Asterisk (*) represents the most rapidly migrat ing complex, which is competed with excess unlabeled oligonucleotides but remains unchanged with MIS treatment. Lower panel, the same samples described above were analyzed using a control OCT-1 oligonucleotide probe. B, upper panel, T47D cells were treated with either 35 nM bioactive rhMIS or 35 nM noncleavable biologically inactive rhMIS for 1 h. 3 µg of nuclear proteins were analyzed by EMSA using a 32P-labeled NFB oligonucleotide probe. Closed arrow indicates the position of the complex. Lower panel, T47D and COS-7 cells were treated with 35 nM MIS for 1 h and nuclear proteins were analyzed for NFB binding activity. Arrowhead indicates the position of the complex. C, NFB activated by MIS is functionally active. T47D cells were transiently transfected with 5 µg of 2X-NFB-CAT construct. Cells were treated with 35 nM MIS for 12 h, and CAT enzyme levels were estimated by ELISA. Changes in CAT enzyme levels are represented as fold induction. Transfection efficiencies were standardized by cotransfecting hGH and analyzing the levels of hGH in culture supernatants by hGH-ELISA. D, left panel, inhibition of NFB activation abrogates MIS-mediated inhibition of growth. Monolayers of T47D cells were stably transfected with 0.5 µg of hygromycin resistance plasmid and 1) pCDNA vector (V), 2) 5 µg of pCDNA encoding bioactive MIS (K2), 3) 5 µg of K2 and 2.5 µg of pCDNA encoding IB-DN, or 4) 2.5 µg of pCDNA encoding IB-DN. The amount of DNA transfected was equalized with pCDNA vector. Hygromycin-resistant colonies were stained with crystal violet after 3 weeks. The number of colonies per plate are represented as percentage of survivors. Number of colonies on the plate transfected with pCDNA vector is set at 100%. Right panel, IB-DN does not ablate CMV-driven MIS expression. T47D and COS cells were transiently transfected with 1.5 µg of K2 alone or 1.5 µg of K2 and 1.5 µg of IB-DN using Fugene 6. The total amount of DNA transfected was equalized with pCDNA vector. The culture supernatants were analyzed by MIS-ELISA after 48 h of transfection. E, left panel, stable expression of T47D cells with IB-DN abrogates MIS-mediated induction of NFB. T47D cells transfected with IB-DN were treated with 35 nM rhMIS for 1 h, and NFB binding activity was analyzed by gel shift. Right panel, T47D control cells and T47D cells transfected with IB-DN were treated with 35 nM rhMIS for 4 days and cell numbers were calculated using a Coulter counter. The mean number of cells in the untreated plates was set at 100%. F, MIS does not induce caspase-3 in T47D cells expressing IB-DN. Cells were treated with 35 nM MIS for 3 days, and caspase-3 activity was estimated. Levels of caspase-3 was estimated based on a standard curve, and the relative fold induction was calculated.

We next investigated whether inhibition of T47D cell growth by MIS involves regulation of the NFB pathway. Following treatment of T47D cells with 35 nM rhMIS, a robust induction of NFB binding activity was observed as early as 1 h after treatment and persisted up to 24 h (Fig. 3A, upper panel). Simultaneous addition of either rabbit anti-p50 or anti-p65 antibodies to the binding reaction demonstrated that the induced complexes predominantly consist of p50 and p65 subunits. Incubation of nuclear lysates with anti-c-Rel antibody did not supershift the complex, suggesting that c-Rel protein was not present in the complexes (data not shown). Although the fastest migrating form of the DNA protein complex was abolished with an excess of unlabeled NFB oligonucleotide, it remained unchanged upon treatment with MIS and was not supershifted with any of the antibodies described. The specificity of NFB induction by MIS is demonstrated by its failure to increase the levels of the transcription factor OCT-1 (octamer binding protein-1; Fig. 3A, lower panel). Exogenous addition of the noncleavable biologically inactive form of rhMIS, which did not inhibit T47D cell growth (Fig. 2A), failed to induce NFB binding activity (Fig. 3B, upper panel). Furthermore, MIS treatment did not induce NFB in COS-7 cells, which do not express the MIS type II receptor (Fig. 3B, lower panel) (17).

To correlate the increase in NFB binding activity with functional induction of NFB, reporter gene assays were carried out using a CAT construct regulated by a basic promoter driven by two upstream NFB binding sites (2X-NFB-CAT). T47D cells were transiently transfected with the 2X-NFB-CAT construct and treated with 35 nM rhMIS for 12 h. A 5-7-fold induction of CAT enzyme expression was seen in cells treated with MIS, suggesting that the NFB binding activity induced by MIS was functionally active (Fig. 3C).

Colony inhibition assays were carried out to determine whether ablation of the NFB pathway by the dominant negative allele of rat IB (IB-DN) could affect MIS-mediated inhibition of growth. Since serine residues at positions 32 and 36 of IB, which are phosphorylated in response to activation signals, are replaced by alanines in IB-DN, its expression abrogates activation of NFB (47). As seen previously, expression of MIS in T47D cells resulted in about 60% inhibition of colony growth compared with cells transfected with the CMV vector. Cotransfection of IB-DN rescued the cells from MIS-mediated growth inhibition, whereas expression of IB-DN alone did not significantly affect the growth of colonies (Fig. 3D, left panel). To ensure that expression of IB-DN did not inhibit the CMV promoter (48), which drives the expression of MIS ligand, MIS-ELISAs were performed following transient transfection of CMV-driven MIS construct (K2) alone or K2 and IB-DN constructs together into T47D or COS cells. MIS-ELISA performed with the culture supernatants after 48 h of transfection demonstrated that expression of the MIS ligand was not affected by coexpression of IB-DN (Fig. 3D, right panel).

To further confirm that activation of NFB was required for MIS-mediated inhibition of growth, IB-DN was stably transfected into T47D cells. Fig. 3E (left panel) demonstrates that induction of NFB binding activity is attenuated in T47D cell clones transfected with IB-DN. Although treatment of control cells with exogenous rhMIS inhibited growth by 64%, growth was unaffected in cells in which NFB activation was abrogated (Fig. 3E, right panel). This was consistent with the lack of MIS-mediated induction in caspase-3 activity in these cells (Fig. 3F). Thus, activation of the NFB pathway is required for MIS-mediated inhibition of growth.

MIS Induces IEX-1S-- Total RNA isolated from T47D cells treated with MIS was analyzed by Northern blot using a PCR-derived, radiolabeled IEX-1 probe, which hybridizes to both IEX-1S and IEX-1L transcripts. Fig. 4A illustrates an estimated 10-20-fold induction of IEX-1 mRNA following 1 h of MIS treatment. Induction was maximal at 1 h and decreased with time, but remained above basal levels even after 24 h of treatment. Radiolabeled oligonucleotide and PCR-derived probes specifically designed to detect IEX-1L did not hybridize to the IEX-1 transcript (data not shown). To confirm the absence of IEX-1L mRNA, PCR analysis was carried out using oligonucleotide primers which permit the detection of both transcripts. CMV-driven IEX-1L and IEX-1S cDNA expression constructs were used as positive controls. Analysis of PCR products revealed that T47D cells expressed IEX-1S mRNA alone (Fig. 4B). Expression of NFB-inducible anti-apoptotic genes for A20 and c-IAP-2 (49, 50) was not affected following treatment with MIS (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   MIS induces the expression of IEX-1S. A, T47D cells were treated with 35 nM MIS for indicated periods of time and 7.5 µg of total RNA was analyzed by Northern blot using a PCR-derived IEX-1 probe, which recognizes both IEX-1S and IEX-1L. Hybridization to actin is shown to control for loading. B, left panel, schematic representation of the IEX-1S and IEX-1L PCR fragments expected from the primers shown. Right panel, PCR products using IEX-1L and IEX-1S expression constructs and cDNAs derived from untreated and MIS-treated T47D RNA as templates. Closed arrows indicate the 220- and 330-bp fragments derived from IEX-1S and IEX-1L, respectively. Open arrows indicate the positions of the 200- and 300-base pair markers. C, two clones of T47D cells stably transfected with IB-DN were treated with MIS for 0 and 1 h. Upper panel, 7.5 µg of total cellular RNA was analyzed for induction of IEX-1; lower panel, hybridization to actin is shown as control for loading.

To determine whether induction of IEX-1S by MIS is mediated by activation of NFB, T47D cell clones expressing IB-DN were tested for activation of IEX-1S mRNA. Induction of IEX-1S mRNA following treatment with MIS was attenuated in T47D cell clones transfected with IB-DN (Fig. 4C).

Expression of IEX-1S Inhibits Growth-- To determine whether up-regulation of IEX-1S, a downstream effector of NFB activation, was responsible for the growth inhibitory effect of MIS, T47D cells were transfected with vector alone or CMV-driven IEX-1S, IEX-1L, or a mixture of IEX-1S and IEX-1L along with a hygromycin resistance plasmid. As seen in Fig. 5A, expression of IEX-1S resulted in 50% reduction of drug-resistant colonies compared with cells transfected with the vector. IEX-1L, on the other hand, did not inhibit colony growth. However, it did enhance the number of colonies in some experiments, probably by contributing to cell survival or increased cell proliferation (42). Cotransfection of both IEX-1S and IEX-1L also resulted in reduction of drug-resistant colonies, suggesting that the growth inhibitory effect of IEX-1S overcomes the effect of IEX-1L. Both proteins were comparably expressed following transient transfection into COS cells (Fig. 5B). These results suggest that MIS-mediated growth inhibition of T47D cells may be attributed in part to up-regulation of IEX-1S expression and that, although IEX-1L may be responsible for cell survival (42), IEX-1S contributes to the growth inhibitory pathway induced by NFB.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   IEX-1S expression inhibits T47D cell growth. A, T47D cells were stably transfected with 0.5 µg of hygromycin resistance plasmid and 12 µg of vector DNA (V) or CMV-driven IEX-1S or IEX-1L or a mixture of 6 µg each of IEX-1L and IEX-1S plasmids. Hygromycin-resistant colonies were stained with crystal violet after 3 weeks, and number of colonies is represented as percentage of survivors. B, COS cells were transiently transfected with 2 µg of CMV-driven FLAG-tagged IEX-1S or IEX-1L expression constructs. 50 µg of total protein was analyzed by Western blot analysis using a mouse anti-FLAG antibody (Sigma). Positions of IEX-1S and IEX-1L are indicated by an open arrow. The closed arrow probably represents internally initiated polypeptides.

MIS Activates the NFB Pathway in Estrogen Receptor-negative MDA-MB-231 Cells-- Constitutive activation of NFB has been reported in several ER-negative breast cancer cell lines and primary breast tumors. Hence, we investigated whether MIS activates the NFB pathway in the ER-negative breast cancer cell line MDA-MB-231. As previously reported (45, 46), the basal level of NFB activity was higher in MDA-MB-231 cells compared with the ER-positive T47D cell line (data not shown). Treatment with MIS for 1 h strongly induced NFB binding activity in MDA-MB-231 cells (Fig. 6A). Antibody supershift assays indicated that the DNA protein complex consisted of both the p50 and p65 subunits of NFB. As with T47D cells, treatment of MDA-MB-231 with MIS also resulted in the up-regulation of the IEX-1 mRNA (Fig. 6B). PCR analysis of the cDNAs obtained from MDA-MB-231 cells demonstrated the presence of both IEX-1L and IEX-1S transcripts (Fig. 6C). However, Northern blot analyses with IEX-1L-specific probes revealed undetectable levels of IEX-1L (data not shown), suggesting that MIS treatment of MDA-MB-231 cells resulted in selective up-regulation of IEX-1S mRNA. As with T47D cells, treatment of MDA-MB-231 cells with exogenous MIS inhibited growth by 50% (Fig. 6D).

View larger version (35K): [in this window] [in a new window]   Fig. 6.   MIS induces the NFB pathway and IEX-1S expression in the estrogen receptor-negative human breast cancer line MDA-MB-231. A, MDA-MB-231 cells were treated with 35 nM MIS for 0, 1, and 3 h and 3 µg of nuclear proteins were incubated with radiolabeled NFB probe and analyzed by EMSA. Antibody supershifts using 1 µl of rabbit anti-p50 and anti-p65 antibodies were done with the sample treated with MIS for 1 h. Positions of the complexes are indicated. B, total cellular RNA isolated from MDA-MB-231 cells treated with 35 nM MIS for 0, 1, and 3 h was analyzed by Northern blot for IEX-1 induction. Equal loading is demonstrated using hybridization to actin. C, analysis of PCR products using IEX-1L and IEX-1S expression constructs and cDNAs derived from untreated and MIS-treated MDA-MB-231 RNA as DNA templates. Closed arrows indicate the 220- and 330-bp fragments derived from IEX-1S and IEX-1L, respectively. Open arrows indicate the positions of the 200- and 300-base pair markers. D, MIS inhibits MDA-MB-231 cell growth. Cells were grown in the presence or absence of 35 nM exogenous MIS, and cells were counted using the Coulter counter. The number of cells in the untreated plates was set at 100%.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The importance of MIS in the regression of the Müllerian duct in male embryos is well established (1). High levels of MIS type II receptor mRNA are detected in the testis, ovary, and uterus, and lower levels have been detected in rat lungs (6, 8, 16). However, systematic analyses of other tissues and cell lines that may be responsive to MIS have not yet been performed. We have demonstrated expression of MIS type II receptor in normal breast and in human breast cancer cells using several different techniques. The presence of MIS type II receptor in the breast and the ability of MIS ligand to block proliferation of breast cancer cells in vitro suggest that MIS may have a physiological role in regulating the proliferation of mammary epithelial cells. MIS induces the NFB signaling pathway in both ER-negative and -positive breast cancer cell lines and abrogation of NFB activation ablates MIS-mediated growth inhibition, indicating that NFB-regulated gene expression is required for this process.

Constitutive activation of NFB has been reported in many tumors, including primary breast cancers and breast cancer cell lines (45, 46), associating it with progression of breast epithelial cells toward malignancy (51). Furthermore, NFB activation is developmentally regulated in the mammary gland of the mouse and maximal increase in DNA binding occurs during post-lactational involution, a period of extensive apoptosis. However, epithelial cells in which NFB is activated demonstrated normal nuclear morphology, suggesting that its activation was limited to the nonapoptotic population of mammary epithelial cells (44). Paradoxically, growth inhibition of breast cancer cells by MIS requires activation of the NFB pathway. A dual role for NFB in regulating cell growth and apoptosis has been reported in many cell systems (39, 40). Induction of apoptosis in prostate cancer cells by the soy isoflavone genistein is achieved from a decrease in NFB binding activity (52), but induction of apoptosis of these cells by the Sindbis virus requires activation of NFB (53). The influence of NFB activating stimulus on the resulting phenotype is also demonstrated in RKO cells, in which activation of NFB by p53 induces apoptosis while NFB induction by TNF- in the same cells results in an anti-apoptotic function (54). The developmentally specific, contradictory effects of NFB activation on growth are evident in pro-B cells in which overexpression of p65 results in G₁ arrest and apoptosis while being ineffective in immature and mature B cell lines (53). Furthermore, inactivation of NFB in mice (55, 56) and in cell lines in vitro (57, 58) overwhelmingly supports a role for NFB in cell survival. However, overexpression of p50 and p65 transgene in the stratified epithelium of the epidermis of mice demonstrates a growth inhibitory role for NFB (59). Thus the role of NFB on cell cycle regulation and apoptosis appears to be specified by cell type, stages of development and differentiation, and the inducing stimuli.

TGF-1 and activin A, polypeptides related to MIS, inhibit the growth of human breast cancer cell lines (31, 33, 34). The ability of TGF- to inhibit the growth of breast cancer cells is associated with a decrease in the NFB binding activity resulting from increased stability of the inhibitory protein IB (38). A similar decrease in NFB binding was also demonstrated with TGF--mediated apoptosis of murine hepatocyte and B cell lines (60, 61). In contrast, MIS-induced NFB binding activity was persistent up to 24 h in human breast cancer cell lines and ablation of the NFB pathway prevented MIS-mediated inhibition of growth. These results suggest that, although these two related ligands play a similar role in inhibiting the growth of breast cancer cells, they may utilize different molecular mechanisms involving either activation or repression of a distinct subset of genes that are modulated by NFB.

The exact mechanism by which NFB regulates growth and apoptosis is not well understood. Regulation of NFB-mediated cell cycle progression occurs through its physical and functional interaction with cyclins, cyclin-dependent kinases and their inhibitors, and the transcriptional coactivator p300 (62-64). The effect of NFB activation on apoptosis is mediated through regulation of genes such as A20, c-IAP-2, bcl-Xs, and IEX-1L, which have been shown to contribute to NFB-associated survival of cells (42, 49, 50, 65). We demonstrate that MIS-mediated activation of NFB does not induce anti-apoptotic target genes including IEX-1L, but selectively up-regulates IEX-1S. IEX-1S represents the first transcript identified to be regulated by MIS during tumor cell growth inhibition. Growth inhibition following overexpression of IEX-1S in T47D cells indicates that MIS-mediated up-regulation of IEX-1S is in part responsible for the growth inhibitory effect of MIS. Coexpression of IEX-1L and IEX-1S suggests that the growth inhibitory effect of IEX-1S may be functionally dominant. This is also supported by cell cycle analysis following transient transfection of IEX-1 expression constructs, which show that expression of IEX-1S, but not IEX-1L, significantly increased the fraction of cells in the G₁ phase with the effect of IEX-1S being dominant.² Wu et al. (42) recently demonstrated that NFB-induced expression of IEX-1L protected cells from TNF-- and Fas-mediated apoptosis; IEX-1S did not play a role in cellular resistance to apoptosis. Variation in the ratio between these two forms of IEX-1 during development or following treatment with various reagents is not known. Differential regulation and functional interaction between the two splice forms may be responsible for the different phenotypic effects seen following NFB induction with various growth factors and cytokines in different cell types. Interestingly, MIS robustly induces NFB nuclear localization in ovarian cancer cell lines without affecting the levels of IEX-1 mRNA,² which suggests the possibility of tissue specific gene regulation following NFB induction by MIS.

Mammary tissue undergoes proliferation, differentiation, and extensive remodeling during puberty and pregnancy. Ovarian hormones such as estradiol and progestins regulate these processes, and exposure to these ovarian hormones has been suggested to play a key role in neoplastic transformation of breast tissue. Progesterone- and estrogen-mediated signaling pathways in the breast have been extensively studied using both in vitro cell culture and animal model systems. The role of MIS in development and neoplastic transformation of the mammary gland, however, is poorly understood. The presence of MIS type II receptor in breast epithelial cells and the ability of MIS ligand to block proliferation of breast cancer cells in vitro suggest a role for serum MIS in growth regulation of the mammary epithelium. Further characterization of the functional significance of the presence of the MIS receptor in the breast depends on testing the effect of MIS on estrogen receptor-positive and -negative mammary carcinoma cell growth in vivo; such studies will help to determine whether MIS would be of potential therapeutic benefit in treatment of breast cancer.

	ACKNOWLEDGEMENTS

We thank Drs. Daniel Haber, Jeff Settleman, Liz Perkins, James Lorenzen, and Leif Ellisen for critically reading this manuscript. We also thank Dr. Rodrigo Bravo for 2X-NFB-CAT and IB-DN constructs.

	FOOTNOTES

^* This work was supported by Grant IRG-173H from the American Cancer Society and a breast cancer research grant from the Massachusetts Department of Public Health (and to S. M.), National Institutes of Health NCI Training Grant in Cancer Biology T32 CA 71345 and Resident Research Award from the American College of Surgeons (both to D. L. S.), and Grants HD32112 and CA17393 from the National Institutes of Health, NICHD and NCI, respectively (both to P. K. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Pediatric Surgical Research Laboratories, WRN1024, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114. Tel.: 617-724-6552; Fax: 617-724-7221; E-mail: maheswaran@helix.mgh.harvard.edu.

Published, JBC Papers in Press, June 28, 2000, DOI 10.1074/jbc.M004554200

² D. L. Segev and S. Maheswaran, unpublished data.

	ABBREVIATIONS

The abbreviations used are: MIS, Müllerian inhibiting substance; TGF-, transforming growth factor-; CMV, cytomegalovirus; hGH, human growth hormone; CAT, chloramphenicol acetyltransferase; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; bp, base pair(s); BMP, bone morphogenetic protein; TNF-, tumor necrosis factor-.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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