CANCER RESEARCH57. 5022-5027, November 15, 1997J

Advances in Brief

Growth Regulation of Human Prostate Cancer Cells by Bone

Morphogenetic Protein-2'

Hisamitsu Ide, Teruhiko Yoshida, Nobuyuki Matsumoto, Kazunori Aoki, Yukio Osada, Takashi Sugimura, andMasaaki Terada2

Genetics Division, National Cancer Center Research Institute, 5-1-1 Tsuk:ji, Chuo-ku. Tokyo 104 (H. I., T. Y., N. M., K. A., T. S., M. T.], and Department of Urology, MiyazakiMedical College, 5200 Kiyotake, Miyazaki 889-16 (H. 1., 1'.0.]. Japan

Abstract

Bone morphogenetic proteins (BMPs) belong to the transforminggrowth factor-@3(TGF-@3)family and have been identified as factors thatstimulate bone formation in vivo. They turned out to be multifunctionalmolecules regulating the growth, differentiation, and apoptosis in varioustarget cells. Some BMPs and their receptors (BMPRs) are expressed onprostate cancer cells. We have reported previously that BMPR-IB mRNA

expression is highest in the prostate, a characteristic that is not shared bythe other BMPRS, BMPR-IA and BMPR-II. However, the amounts ofBMPR-IB mRNA were significantly low in prostate tissues after androgenwithdrawal therapy. They were also low in prostate cancer cell lines.Semiquantitative RT-PCR showed that BMPR-IB mRNA was inducedby androgen in the androgen-sensitive human prostatic cancer cell lineLNCaP, whereas the expression of BMPR-IA and BMPR-II mRNAs wasnot affected by androgen. When the recombinant human BMP-2 wasadded to the LNCaP cells in the presence of androgen, cell growth was

inhibited. In contrast, the growth rate was Increased by the addition of thesame ligand when the cells were cultured in the absence of androgen;

under this condition, the amounts of BMPR-IB mRNA were decreasedsignlflcantiy. These observations showed that the amounts of BMPR-IB,but not those of BMPR-IA, were regulated by androgen and furthersuggest that BMPR-IA and BMPR-IB differentially modulate prostatecancer cell growth in response to BMP under different hormonal conditions; BMPR-IA elicits growth stimulation, and BMPR-IB conveys anegative regulatory signal in response to BMP-2.

Introduction

The most favored site of metastasis of prostate cancer is bone,where new bone formation is often induced at the metastatic foci (1,2). However, molecular mechanisms involved in bone metastasisremain unsolved. The seed and soil theory permits metastatic tumorgrowth in certain organs because of the enhanced adhesion, chemotaxis, and growth at these sites (3—5).The selective spread of prostatecancer to bone, together with the consistent ability of prostate cancerto incite an osteoblastic reaction, suggests that there are bidirectionalstimulatory paracrine pathways between prostate cancer cells andosteoblasts and bone stromal cells (6).

BMPs3 were originally extracted from bone as factors that induce

Received 8/26/97; accepted 10/10/97.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported in part by a Grant-in-Aid for the Second Term Compre

hensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare ofJapan; by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare ofJapan and from the Ministry of Education, Science, Sports and Culture of Japan; and bythe Bristol-Myers Squibb Foundation. H. I. is an awardee of a Research ResidentFellowship from the Foundation for Promotion of Cancer Research.

2 To whom requests for reprints should be addressed.

3 The abbreviations used are: BMP, hone morphogenetic protein; BMPR, BMP recep

tor; RT-PCR, reverse transcription PCR; rhBMP-2, recombinant human BMP-2; TGF,transforming growth factor, PSA, prostate-specific antigen; FBS, fetal bovine serum;MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)@2@(4@sulfophenyl)@2Jgtetrazolium; EGF, epidermal growth factor.

cartilage and bone formation in vivo. The amino acid sequence revealed that they belong to the TGF-f3 superfamily (7, 8). A number ofstudies have demonstrated that BMPs stimulate proteoglycan synthesis in chondroblasts as well as alkaline phosphatase activity, collagen

synthesis, and osteocalcin expression (9—11). In addition to the functions as a possible bone-inducing factor, BMPs have been shown toplay important roles during vertebrate organogenesis (12). Further

more, BMPs were implicated in the chemotaxis of monocytes (13),migration of osteoblasts (14), and differentiation of neural cells (15).BMPs and their receptors are widely distributed not only in the boneand cartilage but in other tissues. BMPs may constitute anotherexample of a growth/differentiation factor family that shows a diversefunction, depending on the target cells.

Three BMPRs have been identified: two type I receptors (IA andIB) (16, 17) and one type II receptor (18—20).The three molecules aremembers of the TGF-!3 transmembrane serine-threonine kinase receptor family. Among 15 BMP family members known to date, at leastBMP-2, BMP-4, BMP-7, and growth differentiation factor-S wereshown to bind to BMPRs (21—23).It has also been reported that someof the BMPs can bind activin type I and II receptors (24). Effectiveligand binding and signal transduction of BMPs are dependent on theoptimal cooperation of one of the two type I receptors and the singletype II receptor, which together form the necessary heteromeric receptor complexes (18—20).

Recently, we reported the cloning of the human BMPR-IB cDNAand the expression of BMPRs in various human tissues and cell lines.Among various human tissues, the highest amounts of BMPR-IBmRNA were found in the prostate. Although the level of mRNA ofBMPR-IA and BMPR-II was similar among cancerous and noncancerous prostate tissues, BMPR-IB expression was significantly low incancerous and noncancerous prostate tissues obtained from the patients who received androgen withdrawal therapy. BMPR-IB mRNAexpression was also low in the prostate cancer cell lines (25). Although the expression of some BMPs and BMPRs was detected innormal and cancerous prostate tissues and in some prostate cancer celllines, the biological significance of BMPs in the development ofprostate cancer remains to be established (26, 27).

In this study, we demonstrate that among the three BMPRS, only

BMPR-IB expression was up-regulated by androgen in an androgensensitive prostate cancer cell line, LNCaP. Culture of LNCaP cellswith the rhBMP-2 in the presence of androgen resulted in the upregulation of BMPR-IB mRNA and growth inhibition of the cells.Culture of LNCaP cells with rhBMP-2 in the absence of androgen,however, led to the down-regulation of BMPR-IB mRNA and growthstimulation. This is the first report that shows that the growth ofprostate cancer cells was regulated by BMP, and that the growth wasnegatively regulated by BMP in androgen-sensitive prostate cancercells through induction of BMPR-IB.

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BIFUNCTIONAL GROWTH EFFECTS OF rhBMP-2 IN LNC*P CELL LINE

C.2.5

EC0

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of the mRNA expression. The expected sizes of the PCR products and the

primer sequences used are as follows: hBMPR-IA, 1401 bp, 5'-GCATAACTAATGGACATfGCT-3' and 5'-TAGAG1TFCTCCTCCGATGG-3';hBMPR-IB, 634 bp, 5'-GCAGCACAGACGGATATFGT-3' and 5'-TFTCATGCCTCATCAACACF-3'; hBMPR-Il, 694 bp, 5'-ACGGGAGAGAA

GACGAGCCT-3' and 5'-CTAGATCAAGAGAGGGTFCG-3'; BMP-2, 671bp, 5'-TCATAAAACCTGCAACAGCCAACTCG-3' and 5'-GCTGTACTAGCGACACCCAC-3'; PSA, 540 bp, 5'-GGTCGQCACAGCCTGTFTCA-3' and 5'-CCACGATGGTGTCCTFGATC-3'; f3-actin,313bp, 5'-GACTACCTCATGAAGATCCT-3' and 5'-GCGGATGTCCACGTCACACT-3'.These primers were designed from the sequences of human BMPR-IA (16),human BMPR-IB (25), human BMPR-Il (20), BMP-2 (7), PSA (29), and@3-actin (30). The amplification reactions were performed with an initial

incubation step at 95°Cfor 3 mm followed by 30 cycles each at 95°Cfor 45 s,60°Cfor I mm, and 72°Cfor 1 mm 30 s. These cycles were followed by afinal incubation step at 72°Cfor 10 mm. The samples were subjected toelectrophoresis in 2% agarose gel and stained with ethidium bromide. The

RT-PCR analysis was repeated at least twice for each of the two independentexperiments.

Cell Growth Assay. In RPMI 1640 containing 10%FBS, LNCaP,PC-3,TSU-PR1, and DU-l45 were seeded at 5000, 1000, 1000, and 1000cells/well,respectively, into 96-well plates and cultured for 24 h. In serum-free conditions, these cells were seeded at 5000 cells/well into 96-well plates andcultured for 24 h. Then the same volume of RPM! 1640 was added either withor without 200 ng/ml rhBMP-2. In androgen challenge tests, LNCaP cells wereseeded at 5000 cells/well and cultured in a serum-free RPMI 1640 for 3 daysfollowed by the addition of the same volume of RPM! 1640 containing 200

ng/ml rhBMP-2 either with or without 2 nMRl88l. The final concentrations

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@ 1.1

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2 3 4 5 6Time(Days)

PC-3

b. d.2.5

I 1O%FBSDU145

1O%FBS

1.5

0.50.5

Fig. 1. The effect of rhBMP-2 on the proliferation of four human prostate cancer cell lines: a, LNCaP; b, PC-3; c, TSU-PR1; and d, DU-l45. The cells were grown in RPMI 1640supplemented with 10% FBS. Each cell was treated with rhBMP-2 at 100 ng/ml at day 0 and subjected to the MTS assay every day for 6 or 4 consecutive days. In the MTS assay,the absorbance at 490 nm is correlated with the number of living cells. Open and closed circles are cells not treated and treated with rhBMP-2, respectively.

Materials and Methods

Cell Lines and Cell CuliUre. Thehumanprostatecancercell lines LNCaP,PC-3, and DU-l45 were obtained from the American Type Culture Collection(Rockville, MD). The TSU-PR1 cell line was provided by Dr. Shori Kanoh(The University of Tsukuba, Tsukuba, Japan). All of the cell lines wereroutinely maintained in RPM! 1640supplemented with 10%FBS, 100units/mlpenicillin, and 100 mg/mI streptomycin. For the steroid stimulation analysis,LNCaP cells were cultured in a serum-free medium for 3 days prior to theaddition of synthetic androgen Rl88l (New England Nuclear, Boston, MA) orl7@-estradiol (Sigma Chemical Co.).

RNA Preparation and Semiquantitadve RT-PCR. Androgen dependence of BMPR mRNA expression was examined by semiquantitative RT-PCRbased on the comparison with an internal reference, @-actinexpression. Theanalyses used four prostate cancer cell lines that express (LNCaP) or do notexpress (PC-3, TSU-PR1, and DU-l45) androgen receptor (Ref. 28; data notshown). The prostate cancer cell lines were stimulated by various concentrations of R188l (0—1saM)or l7@-estradiol(0—100aM) for 24 h. Total RNAswere extracted by ISOGEN (Nippongene, Tokyo, Japan) according to theprotocol recommended by the manufacturer. Likewise, total RNAs were cx

tracted from LNCaP cells that were stimulated by the combination of 100ng/ml rhBMP-2 (Genetics Institute, Inc., MA) with or without 1 flMRl88l for48 h. The rhBMP-2 protein was donated by Yamanouchi Pharmaceutical Co.,Ltd. (Tokyo, Japan).

A lO-@gportioneach of totalRNAs fromthe prostatecancercell lines wasreverse-transcribed by random primer and Super-Script reverse transcnptase(Life Technologies, Inc.) in a volume of 40 p1. The resulting cDNA (0.5 ml)

was subjected to PCR with the primers described below. The RT-PCR reaction

in the exponentially amplifying cycle allowed semiquantitative comparison

TSU-PR11O%FBS

2 3 4Time(Days)

2lime (Days)

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rhBMP-2 on the growth of LNCaP cells may be related to the statusBMP 2@ BMPR expression,which was examinedby semiquantitativeRT

+ - PCR. Fig. 3 shows that the BMPR-IB mRNA was up-regulated by

androgen in LNCaP cells, which express the androgen receptor, butnot in the PC-3 cells, which do not express the androgen receptor. Incontrast, the levels of BMPR-IA and BMPR-ll mRNA were notaltered by androgen in the androgen receptor-positive LNCaP cells orin the androgen receptor-negative PC-3 cells. The same results wereobserved in other androgen receptor-negative cell lines, DU-145 andTSU-PR1 (data not shown). The androgen dependence of the expression of the ligand, BMP-2, was examined by the same semiquantitative RT-PCR. The BMP-2 mRNA expression was not changed by 1aM R1881 in LNCaP or PC-3 cells (Fig. 3). These results suggest thatonly BMPR-IB expression is uniquely regulated by androgen in theBMPIBMPR system and contributes to the growth effect of rhBMP-2in the androgen-sensitive LNCaP cells.

The induction of the BMPR-IB mRNA in the LNCaP cells wasdose dependent. When examined under various concentrations ofR1881 (0—1pM) and 17@3-estradiol (0—100 mvi), the amounts ofBMPR-IB mRNA were increased over the 0—1nr@trange of R1881(Fig. 4a). The PSA was used as a positive control, because itsexpression has been shown to be induced by androgen (33). Interestingly, both PSA and BMPR-IB mRNA were up-regulated by @3-estradiol in the semiquantitative RT-PCR analysis (Fig. 4b). It hasbeen shown that 173-estradiol binds to the androgen receptor inLNCaP cells, because the cells carry a mutation in the androgenbinding domain of the androgen receptor, resulting in the alterationof the ligand-binding specificity and steroid-induced transactivation(34, 35).

Regulation of the Amounts of BMPR mRNAs by Androgen andrhBMP-2. The status of BMPR expression in the LNCaP cells showing the opposite growth response to BMP-2 (Fig. 2) was examined bysemiquantitative RT-PCR. RNAs were extracted from aliquots of thecells analyzed in Fig. 2, where the LNCaP cells in a serum-freemedium were treated with 100 ng/ml rhBMP-2 and/or 1 ni@iR1881.When only rhBMP-2 was added to the medium, the levels of themRNA of BMPRs were not changed on day 2. The addition of thesynthetic androgen up-regulated the BMPR-IB expression (Fig. 5,fifth lane from left), although the degree of the induction was lowerthan that stimulated by 1 ni@iR1881 only (Fig. 5, fourth lane from left).By contrast, the expression of BMPR-IA and BMPR-fl was unaffected by any combination of rhBMP-2 and R1881. The same resultswere obtained on day 5 (data not shown).

Discussion

Tumor progression to the stage of metastasis may result partly fromthe selection of certain primary tumor cell clones that are competentfor survival, invasion, and growth at secondary sites and may beregulated through the action of the growth factors available there.Prostate cancer frequently metastasizes to the bone. BMPRs werefound expressed in the prostate cancer tissues and prostatic cancer celllines (25), whereas BMPs were abundant in the bone matrix (8). Inthis report, we showed that rhBMP-2 inhibits the growth of androgensensitive LNCaP cells, but not that of androgen-insensitive prostatecancer cells (PC-3, TSU-PR1, and DU-145), in a medium containing10% FBS (Fig. 1). Interestingly, the growth effect of rhBMP-2 onLNCaP cells shifted from stimulatory to inhibitory action by theaddition of androgen in the serum-free medium (Fig. 2). The semiquantitative RT-PCR analyses showed that only the BMPR-IB mRNAexpression among three BMPRS was induced by the androgen stimulation, resulting in the up-regulation of BMPR-IB (Figs. 3 and 5).Because the signaling specificity could occur through interaction of

5024

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Serum-free1.3

1.2

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Time (Days)

105

0.95

0.85

0.75

Serum-free + androgen

0.85

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3 4 5 6

Time (Days)

Fig. 2. The effect of rhBMP-2 on the proliferation of LNCaP cells with or withoutandrogen. LNCaP cells were cultured in RPMI 1640 only (a) or with 1 nsi Rl881 addedto RPM! 1640 medium (b). Each cell was treated with 100 ng/ml of rhBMP-2, and thenumber of live cells was determined by MTS assay every day for 6 consecutive days.Open and closed circles are cells not treated and treated with rhBMP-2, respectively.

of rhBMP-2 and Rl881 were 100 ng/ml and 1 nM, respectively. The growthwas assessed by MTS cell proliferation assay using tetrazolium compound,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (Promega Corp.) as described (31). Each day, the tetrazoliumcompound was added, and after 90 mm of incubation at 37°C,absorbance at490 nm was measured on Microplate reader (Bio-Rad).

Results

Effects of rhBMP-2 on the Growth of Prostate Cancer CellLines. As shown in Fig. 1, the growth of the androgen-sensitiveLNCaP cells was inhibited by the addition of 100 ng/ml of rhBMP-2in the presence of 10% FBS. This growth inhibition was not observedat the lower concentrations of rhBMP-2 at 1 and 10 ng/ml (data notshown). For the androgen-insensitive prostate cancer cell lines PC-3,TSU-PR1, and DU-145, addition of rhBMP-2 did not lead to changeof growth rates (Fig. 1). However, when the LNCaP cells were grownin a serum-free medium, addition of the BMP-2 resulted in growthstimulation (Fig. 2a). Because some androgemc activity is known tobe present in the medium supplemented with 10% FBS (32), thisdifferential growth modulation might depend on the existence ofandrogen. In fact, Fig. 2b showed that the growth of LNCaP cells wasinhibited, when both the 1 nM R1881 and 100 ng/ml rhBMP-2 wereadded to the serum-free medium.

Regulation of BMPRS and BMP-2 mRNA Levels by Androgen.We have reported previously that the mRNA expression of BMPR-IBis regulated by androgen. Therefore, the bifunctional effect of

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BIFUNCFIONAL GROWTh EFFECTS OF rhBMP-2 IN LNCaP CELL LINE

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Noandrogen R1881i nM

I iF •1M10151820222530351015182022253035

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..4— 694 bp(BMPR-II)

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,*— 313bp

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671bp@*—(BMP-2)

Fig. 3. Androgen dependence ofmRNA expression of BMPRS in prostate cancer cells. Semiquantitative RT-PCR using androgen-sensitive LNCaP cells or androgen-insensitive PC-3cells is shown. The cells were maintained in serum-free medium for 3 days before the addition of 1 ElMRl88l for 24 h.

Fig. 4. Dose-dependent induction of BMPR-IBmRNA expression in LNCaP cells. LNCaP cellswere cultured in serum-free medium for 24 h conmining various concentrations of R188l (0—1pi@i;a) or l7@-estradiol (0—100nM; b). The RT-PCRreaction in the exponentially amplifying cycles allowed semiquantitative comparison of gene expression. Only the PCR products at representative cydes were shown: BMPR-IB, cycle 26; PSA, cycle30; and @-actin,cycle 22.

INN@ ø@ .i@.@

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a. R1881

BMPR-IB

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b.17/3-estradiol

BMPR-IB

PSA

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BIFUNCTIONALGROWFHEFFECI@SOF thBMP-2IN LNCaPCELLLINE

a bifunctionality of BMP-2, another member of the TGF-@ family.Moreover, our data suggest that the level of the BMPR-IB expression regulated by androgen is a molecular mechanism for the

bifunctional growth effects of BMP-2.It has been demonstrated that BMP-2, BMP-4, BMP-7, and growth

differentiation factor-5 can bind to BMPRs, which require the cooperation of one of the two type I receptors and the type II receptor foroptimal ligand binding and signal transduction (21—23).It was alsoshown that BMP-2 binding was more efficient to the BMPR-IB andBMPR-II complex than to the BMPR-IA and BMPR-ll complex (18).In addition to that, heterodimers of BMP-2 and BMP-7, and BMP-4and -7 have much higher activity than either homodimer (12, 44). Inthe prostate tissues and some prostate cancer cell lines, at leastBMP-2, BMP-4, and BMP-7 mRNAs were detected (26, 27).@Thecomplex nature of the varied affinity and specificity of BMP ligandsto their receptors implicates a significant functional diversity of theBMP/BMPR system in the prostatic cells in vivo. Which receptorcombinations occur in vivo, whether different type I receptors triggerdistinctive downstream pathways, and whether competitive interac

tion for receptor binding between ligands occurs in vivo are a few ofthe many questions to be answered regarding the BMPIBMPR system.Moreover, the complexity of the multihormonal control of the prostatic cell growth has been documented by the presence of otherandrogen-induced changes of the mRNA and protein expression ofgrowth factors and receptors; androgen could increase TGF-a andepidermal growth factor (EGF) receptor expression in an androgendependent human prostate cancer cell line ALVA1O1 (45). Androgenwithdrawal such as in the rat castration model resulted in decreasedEGF, insulin-like growth factor (IGF), and basic fibroblast growthfactor, whereas TGF-f3, EGF receptor, insulin-like growth factorreceptor, and TGF-f3 receptor were increased (46, 47). In contrast tothose receptors, BMPR-IB mRNA expression was uniquely downregulated following androgen ablation.

For over the last 50 years, androgen ablation has been the standardtreatment for prostate cancer. In most of the cases, however, prostatecancer cells eventually lose androgen dependence and metastasize tothe bone, where the cancer induces new bone formation (2). Severaldifferent and sometimes conflicting molecular mechanisms have beenproposed for the loss of androgen dependence in advanced prostatecancers, suggesting multiple modes of the hormone independence,

including the loss of androgen receptor expression and amplificationor mutation of the androgen receptor gene (48—51). However, it has

been an enigma why the skeletal metastases are often the only sites ofdisease progression at the time of failure of endocrine therapy (6). Ourstudy suggests that one of the possible mechanisms involves BMP-2acting as a positive growth regulator under the condition where theandrogenic signal has been ablated either by therapy or by the changeof the cellular signaling circuitry, such as the loss of androgenreceptor expression. Further study is required to elucidate the in situexpression of BMPs and BMPRs in prostate cancer tissues and theirmetastatic bone sites.

Acknowledgments

We thank Dr. T. Kakizoe (Director, National Cancer Center Central Hospital) for valuable discussions.

References

1. Clarke, N. W., McClure, J., and George, N. J. Morphometric evidence for honeresorption and replacement in prostate cancer. Br. J. Urol., 68: 74—80, 1991.

2. Scher,H. I., andYagoda,A. Bonemetastases:pathogenesis,treatment,andrationalefor use of resorption inhibitors. Am. J. Med., 82: 6—28,1987.

4 Unpublished data.

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BMPR-IA

BMPR-IB

BMPR-ll@

$ -actin

Fig. 5. Regulation of mRNA expression of BMPRS by rhBMP-2 and androgen. LNCaPcells were cultured in the serum-free RPM! 1640 for 48 h in the presence of rhBMP-2 (100ng/ml) with or without I flMR188l. The RT-PCR reaction in the exponentially amplifyingcycles allowed semiquantitative comparison of gene expression. Only the PCR productsat representative cycles were shown: BMPR-LA and II, cycle 25; BMPR-IB, cycle 28; and@3-actin,cycle 20.

BMPR-II with one of the two type I receptors, it is possible thatgrowth signaling of BMP-2 through BMPR-IB is growth suppressiveand that through BMPR-IA is growth stimulative. It is possible thatthe BMP-2 affects the prostatic cells in different ways, depending on

the balance of the BMPR-IB and BMPR-IA expression on the cells;BMP-2 may stimulate prostatic cell proliferation in an androgendeprived condition, whereas the same ligand may induce growthsuppression and promote differentiation in the presence of androgen

in vivo.

Several members of the TGF-j3 superfamily are primarily knownas growth inhibitors of various cell types (36—38), whereas at least

the archetype of the family, TGF-f3, is known to either inhibit orstimulate growth, depending on the target cells and their conditions(39). The molecular basis for the bifunctionality is yet to beestablished, although many studies have focused attention on the

difference in the receptor species; ligand binding assay and affinitylabeling of the receptor have demonstrated alterations of the receptor subtype expressed on the cell surface, depending on the cell

density (40), cell cycle phases (41), or ligand concentration (42).

More recently, it has been demonstrated that TGF-/3 stimulated theproliferation of adult lung fibroblasts but inhibited the growth offetal lung fibroblasts. Because a dominant-negative form of TGF-/3

type II receptor blocked not only TGF-@-induced mitogenic actionupon adult lung fibroblasts but also TGF-j3-induced growth inhibition of fetal lung fibroblasts, TGF-j3 type I receptors might beinvolved in the mechanism of multiple effects of TGF-fJ (43). Toour knowledge, the present study is the first report to demonstrate

—@ —

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3. Paget, S. The distribution of secondary growths in cancer of the breast. Lancet, 1:571—573,1889.

4. Hart, I. R., and Saini, A. Biology of tumour metastasis [see comments]. Lancet, 339:1453-1457, 1992.

5. Orr, F. W., Sanchez-Sweatman, 0. H., Kostenuik, P., and Singh, G. Tumor-boneinteractions in skeletal metastasis. Clin. Orthop., 312: 19—33,1995.

6. Koutsilieris, M. Osteoblastic metastasis in advanced prostate cancer. Anticancer Rca.,13: 443—449, 1993.

7. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L M., Whitters, M. J., Kriz, R. W.,Hewick, R. M., and Wang, E. A. Novel regulators of bone formation: molecularclones and activities. Science (Washington DC), 242: 1528—1534,1988.

8. Wozney, J. M. The bone morphogenetic protein family and osteogenesis. Mol.Reprod. Dcv., 32: 160—167,1992.

9. Vukicevic, S., Luyten, F. P., and Reddi, A. H. Stimulation of the expression ofosteogenic and chondrogenic phenotypes in vitro by osteogenin. Proc. Nati. Acad.Sci. USA, 86: 8793—8797, 1989.

10. Reddi, A. H. Bone and cartilage differentiation. Curr. Opin. Genet. Dcv., 4: 737—744,1994.

11. Yamaguchi, A., Ishizuya, T., Kintou, N., Wada, Y., Katagiri, T., Wozney, J. M.,Rosen, V., and Yoshiki, S. Effects of BMP-2, BMP-4, and BMP-6 on osteoblasticdifferentiationofbonemarrow-derivedstromalcelllines,ST2andMC3T3—G2IPA6.Biochem. Biophys. Res. Commun., 220: 366—371,1996.

12. Hogan, B. L. Bone morphogenetic proteins: multifunctional regulators of vertebratedevelopment. Genes. Dcv., 10: 1580—1594,1996.

13. Cunningham, N. S., Paralkar, V., and Reddi, A. H. Osteogenin and recombinant bonemorphogenetic protein 2B are chemotactic for human monocytes and stimulatetransforming growth factor beta 1 mRNA expression. Proc. Natl. Acad. Sci. USA, 89:11740—11744,1992.

14. LAnd, M., Eriksen, E. F., and Bunger, C. Bone morphogenetic protein-2 but not bonemorphogenetic protein-4 and -6 stimulates chemotactic migration of human osteoblasts, human marrow osteoblasts, and U2-OS cells. Bone, 18: 53—57,1996.

15. Paralkar, V. M., Weeks, B. S., Yu, Y. M., Kleinman, H. K., and Reddi, A. H.Recombinant human bone morphogenetic protein 2B stimulates PC12 cell differentiation: potentiation and binding to type IV collagen. J. Cell. Biol., 119: 1721—1728,1992.

16. ten Dijke, P., Ichijo, H., Franzen, P., Schulz, P., Saras, J., Toyoshima, H., Heldin,C. H., and Miyazono,K. Activinreceptor-likekinases:a novelsubclassof cellsurface receptors with predicted serine/threonine kinase activity. Oncogene, 8: 2879—2887, 1993.

17. ten Dijke, P., Yamashita, H., Ichijo, H., Franzen, P., Laiho, M., Miyazono, K., andHeldin, C. H. Characterization of type I receptors for transforming growth factor-betaand activin. Science (Washington DC), 264: 101—104,1994.

18. Liu, F., Ventura, F., Doody, J., and Massagué,J. Human type 11 receptor for bonemorphogenic proteins (BMPs): extension of the two-kinase receptor model to theBMPs. Mol. Cell. Biol., 15: 3479—3486,1995.

19. Nohno, T., Ishikawa, T., Saito, T., Hosokawa, K., Noji, S., Wolsing, D. H., andRosenbaum, J. S. Identification of a human type II receptor for bone morphogeneticprotein-4 that forms differential heteromeric complexes with bone morphogeneticprotein type I receptors. J. Biol. Chem., 270: 22522—22526, 1995.

20. Rosenzweig, B. L, Imamura, T., Okadome, T., Cox, G. N., Yamashita, H., ten Dijke,P.,Heldin,C. H.,andMiyazono,K.Cloningandcharacterizationof a humantypeIIreceptor for bone morphogenetic proteins. Proc. Nail. Acad. Sci. USA, 92: 7632—7636,1995.

21. ten Dijke, P., Yamashita, H., Sampath, T. K., Reddi, A. H., Estevez, M., Riddle, D. L.,Ichijo, H., Heldin, C. H., and Miyazono, K. Identification of type I receptors forosteogenic protein-l and bone morphogenetic protein-4. J. Biol. Chem., 269: 16985—16988, 1994.

22. Koenig, B. B., Cook, J. S., Wolsing, D. H., Ting, J., Tiesman, J. P., Correa, P. E.,Olson, C. A., Pecquet, A. L., Ventura, F., Grant, R. A., et al. Characterization andcloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells. Mol. Cell. Biol., 14:5961—5974, 1994.

23. Nishitoh, H., Ichijo, H., Kimura, M., Matsumoto, T., Makishima, F., Yamaguchi, A.,Yamashita, H., Enomoto, S., and Miyazono, K. Identification of type I and type IIserine/threonine kinase receptors for growth/differentiation factor-S. J. Biol. Chem.,271: 21345—21352, 1996.

24. Yamashita, H., ten Dijke, P., Huylebroeck, D., Sampath, T. K., Anduies, M., Smith,J. C., Heldin, C. H., and Miyazono, K. Osteogenic protein-i binds to activin type IIreceptors and induces certain activin-like effects. J. Cell. Biol., 130: 217—226,1995.

25. Ide, H., Katoh, M., Sasaki, H., Yoshida, T., Aoki, K., Nawa, Y., Osada, Y., Sugimura,T., and Terada, M. Cloning of human bone morphogenetic protein receptor type lB(BMPR-IB) and its expression in prostate cancer in comparison with other BMPRs.Oncogene. 14: 1377—1382,1997.

26. Bentley, H., Hamdy, F. C., Hart, K. A., Seid, J. M., Williams, J. L., Johnstone, D., andRussell, R. G. Expression of bone morphogenetic proteins in human prostatic adenocarcinoma and benign prostatic hyperplasia. Br. J. Cancer, 66: 1159—1163, 1992.

27. Harris, S. E., Harris, M. A., Malay, P., Wozney, J., Feng, J. Q., and Mundy, G. R.Expression of bone morphogenetic protein messenger RNAs by normal rat and humanprostate and prostate cancer cells. Prostate, 24: 204—211, 1994.

28. Tilley, W. D., Wilson, C. M., Marcelli, M., and McPhaul, M. J. Androgen receptor

gene expression in human prostate carcinoma cell lines. Cancer Res., 50: 5382—5386,1990.

29. Lundwall, A. Characterization of the gene for prostate-specific antigen, a humanglandular kallikrein. Biochem. Biophys. Res. Commun., 161: 1151—1159, 1989.

30. Nakajima-Iijima, S., Hamada, H., Reddy, P., and Kakunaga, T. Molecular structure ofthe human cytoplasmic beta-actin gene: interspecies homology of sequences in theintrons. Proc. Nail. Acad. Sci. USA, 82: 6133—6137, 1985.

31. Ozaki, K., Yoshida, T., Ide, H., Saito, I., Ikeda, Y., Sugimura, T., and Terada. M. Useof von Willebrand factor promoter to transduce suicidal gene to human endothelialcells, HUVEC. Hum. Gene Ther., 7: 1483—1490,1996.

32. Kokontis, J., Takakura, K., Hay, N., and Liao, S. Increased androgen receptor activityand altered c-myc expression in prostate cancer cells after long-term androgendeprivation. Cancer Res., 54: 1566—1573,1994.

33. Young, C. Y., Montgomery, B. T., Andrews, P. E., Qui, S. D., Bilhartz, D. L., andTindall, D. J. Hormonal regulation of prostate-specific antigen messenger RNA inhuman prostatic adenocarcinoma cell line LNCaP. Cancer Res., 51: 3748—3752,1991.

34. Veldscholte, J., Ris-Stalpers, C., Kuiper, G. G., Jenster, G., Berrevoets, C., Claassen,E., van Rooij, H. C., Trapman, J., Brinkmann, A. 0., and Mulder, E. A mutation inthe ligand binding domain of the androgen receptor of human LNCaP cells affectssteroid binding characteristics and response to anti-androgens. Biochem. Biophys.Rca. Commun., 173: 534—540,1990.

35. Kokontis, J., Ito, K., Hiipakka, R. A., and Liao, S. Expression and function of normaland LNCaP androgen receptors in androgen-insensitive human prostatic cancer cells.Altered hormone and antihormone specificity in gene transactivation. Receptor, 1:271—279,1991.

36. McCarthy, S. A., and Bicknell, R. Inhibition of vascular endothelial cell growth byactivin-A. J. Biol. Chem., 268: 23066—23071, 1993.

37. Wallen,J. W.,Cate,R. L., Kiefer,D. M.,Riemen,M. W., Martinez,D., Hoffman,R. M., Donahoe, P. K., Von Hoff, D. D., Pepinsky, B., and Oliff, A. Minimalantiproliferative effect of recombinant Mtlllerian inhibiting substance on gynecological tumor cell lines and tumor explants. Cancer Res., 49: 2005-201 1, 1989.

38. Matzuk, M. M., Finegold, M. J., Su, J. G., Hsueh, A. J., and Bradley, A. Alpha-inhibinis a tumour-suppressor gene with gonadal specificity in mice. Nature (Lond.), 360:313—319,1992.

39. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stem, D. F., andSpans, M. B. Type beta transforming growth factor: a bifunctional regulator ofcellular growth. Proc. Nail. Acad. Sd. USA, 82: 119—123,1985.

40. Goodman, L. V., and Majack, R. A. Vascular smooth muscle cells express distincttransforming growth factor-beta receptor phenotypes as a function of cell density inculture. J. Biol. Chem., 264: 5241—5244,1989.

41. Vivien, D., Redini, F., Galera, P., Lebrun, E., Loyau, 0., and Pujol, J. P. Rabbitarticular chondrocytes (RAC) express distinct transforming growth factor-beta receptor phenotypes as a function of cell cycle phases. Exp. Cell Res., 205: 165—170,1993.

42. Myoken, Y., Kan, M., Sato, 0. H., McKeehan, W. L., and Sato, J. D. Bifunctionaleffects of transforming growth factor-beta (TGF-beta) on endothelial cell growthcorrelate with phenotypes of TGF-beta binding sites. Exp. Cell Res., 191: 299—304,1990.

43. Zhao, Y., and Young, S. L. Requirement of transforming growth factor-beta (TGFbeta) type II receptor for TGF-beta-induced proliferation and growth inhibition.J. Biol. Chem., 271: 2369—2372, 1996.

44. Aono, A., Hazama, M., Notoya, K., Taketomi, S., Yamasaki, H., Tsukuda, R., Sasaki,S., and Fujisawa, Y. Potent ectopic bone-inducing activity of bone morphogeneticprotein-4/7 heterodimer. Biochem. Biophys. Rca. Commun., 210: 670—677, 1995.

45. Liu, X. H., Wiley, H. S., and Meikle, A. W. Androgens regulate proliferation ofhuman prostate cancer cells in culture by increasing transforming growth factor-alpha(TGF-alpha) and epidermal growth factor (EGF)ITGF-alpha receptor. J. Clin. Endocrinol. Metab., 77: 1472—1478,1993.

46. Steiner, M. S. Review of peptide growth factors in benign prostatic hyperplasia andurological malignancy. J. Urol., 153: 1085—1096,1995.

47. Fiorelli, G., Dc Bellis, A., Longo, A., Giannini, S., Natali, A., Costantini, A.,Vannelli, G. B., and Serio, M. Insulin-like growth factor-I receptors in humanhyperplastic prostate tissue: characterization, tissue localization, and their modulationby chronic treatment with a gonadotropin-releasing hormone analog. J. Clin. Endocrinol. Metab., 72: 740—746,1991.

48. Koivisto, P., Kononen, J., Pahnberg, C., Tammela, T., Hyytinen, E., Isola, J.,Trapman, J., Cleutjens. K., Noordzij, A., Visakorpi, T., and Kallioniemi, 0. P.Androgen receptor gene amplification: a possible molecular mechanism for androgendeprivation therapy failure in prostate cancer. Cancer Res., 57: 314—319, 1997.

49. Visakorpi, T., Hyytinen, E., Koivisto, P., Tanner, M., Keinanen, R., Palmberg, C.,Palotie, A., Tammela, T., Isola, J., and Kallioniemi, 0. P. In vivo amplification of theandrogen receptor gene and progression of human prostate cancer. Nat. Genet., 9:401—406,1995.

50. Gaddipati, J. P., McLeod, D. G., Heidenberg, H. B., Sesterhenn, I. A., Finger, M. J., Moul,J. W., and Srivastava, S. Frequent detection of codon 877 mutation in the androgenreceptor gene in advanced prostate cancers. Cancer Res., 54: 2861-2864, 1994.

5 1. Taplin, M. E., Bubley, G. J., Shuster, T. D., Frantz, M. E., Spooner, A. E., Ogata. G. K.,Keer, H. N., and Balk, S. P. Mutation of the androgen-receptor gene in metastaticandrogen-independentprostate cancer. N. Engl. J. Med., 332: 1393-1398, 1995.

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1997;57:5022-5027. Cancer Res   Hisamitsu Ide, Teruhiko Yoshida, Nobuyuki Matsumoto, et al.   Morphogenetic Protein-2Growth Regulation of Human Prostate Cancer Cells by Bone

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