Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases
2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.051276
ISSN1525-2191
AutoresRuth Schwaninger, Cyrill A. Rentsch, Antoinette Wetterwald, Geertje van der Horst, Rutger L. van Bezooijen, Gabri van der Pluijm, Clemens W.G.M. Löwik, Karin Ackermann, Walter Pyerin, Freddie C. Hamdy, George N. Thalmann, Marco Cecchini,
Tópico(s)Bone health and osteoporosis research
ResumoProstate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis. Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis. Prostate and mammary cancer are among the leading cancers diagnosed and the second leading cause of cancer death in men and women, respectively.1Jemal A Thomas A Murray T Thun M Cancer statistics, 2002.CA Cancer J Clin. 2002; 52: 23-47Crossref PubMed Scopus (2912) Google Scholar Both cancers show a high propensity to metastasize to bone. 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To test this hypothesis, we first evaluated the expression in vitro of extracellular BMP and Wnt antagonists, as well as the expression of osteoinductive and osteolytic cytokines, in a variety of CaP and CaM cell lines, which possess either osteolytic or osteoinductive potential in vivo. The pattern of expression was verified also in vivo for CaP cell lines xenografted into bone and in clinical samples of bone metastasis. We further investigated whether forced expression of the BMP antagonist noggin in an osteoinductive CaP cell line would abolish the osteoblast response in its experimental bone metastasis in vivo. Male CB17 SCID and BALB/c nude mice were purchased from Charles River France (L'Arbresle, France) and housed in individual ventilated cages according to the Swiss guidelines for the care and use of laboratory animals. Mice were 7 weeks old when used for the intraosseous implantation of tumor cells. For surgical manipulation, mice were anesthetized as described previously.28Wetterwald A van der Pluijm G Que I Sijmons B Buijs J Karperien M Lowik CW Gautschi E Thalmann GN Cecchini MG Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.Am J Pathol. 2002; 160: 1143-1153Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar Mice were sacrificed by CO2 euthanasia at the end of the observation period or at first signs of distress. Three CaP and one CaM clinical specimens of bone metastasis were obtained by radiography-guided biopsy, with prior informed consent of the patient and approval by the ethics committee of the University of Heidelberg–Mannheim, Germany. The CaM bone metastasis was classified as osteolytic whereas the three CaP bone metastases were classified as osteoblastic according to radiographical and histological criteria by a certified radiologist and a certified pathologist. Bone specimens were snap-frozen in liquid nitrogen immediately after surgical excision and embedded in precooled RNase-free water. Twelve-μm-thick cryosections were obtained with a tungsten carbide blade (C profile; Leica Microsystems, Bensheim, Germany) and the aid of the CryoJane tape-transfer system (CTS; Instrumedics Inc., Hackensack, NJ) in a cryomicrotome (CM 3050; Leica Microsystems) set at −24°C. Bone cryosections were transferred onto 4× adhesive-coated slides (Instrumedics) as described recently,29Eisenberger S Hoppe G Pyerin W Ackermann K High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections.Anal Biochem. 2004; 335: 260-266Crossref PubMed Scopus (7) Google Scholar fixed in 70% ethanol, stained with Mayer's hematoxylin (Sigma Diagnostics Inc., St. Louis, MO) and eosin Y (Merck, Darmstadt, Germany), and dehydrated by increasing grades of ethanol followed by xylene. The P.A.L.M. Robot Micro-Beam (P.A.L.M. Instruments, Bernried, Germany) was used to excise by laser capture microdissection ∼10,000 pure cancer cells from each clinical specimen of bone metastasis. The selection of purely neoplastic cells was in each case verified by a certified pathologist. We studied a range of human CaP and CaM cell lines, with different tumorigenic and metastatic potential, shown to induce either osteolytic or osteosclerotic bone metastases, as shown by others28Wetterwald A van der Pluijm G Que I Sijmons B Buijs J Karperien M Lowik CW Gautschi E Thalmann GN Cecchini MG Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.Am J Pathol. 2002; 160: 1143-1153Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 30Thalmann GN Anezinis PE Chang SM Zhau HE Kim EE Hopwood VL Pathak S von Eschenbach AC Chung LW Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer.Cancer Res. 1994; 54: 2577-2581PubMed Google Scholar, 31Wu TT Sikes RA Cui Q Thalmann GN Kao C Murphy CF Yang H Zhau HE Balian G Chung LW Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines.Int J Cancer. 1998; 77: 887-894Crossref PubMed Scopus (234) Google Scholar, 32Pettaway CA Pathak S Greene G Ramirez E Wilson MR Killion JJ Fidler IJ Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice.Clin Cancer Res. 1996; 2: 1627-1636PubMed Google Scholar, 33Yin JJ Mohammad KS Kakonen SM Harris S Wu-Wong JR Wessale JL Padley RJ Garrett IR Chirgwin JM Guise TA A causal role for endothelin-1 in the pathogenesis of osteoblastic bone metastases.Proc Natl Acad Sci USA. 2003; 100: 10954-10959Crossref PubMed Scopus (315) Google Scholar, 34Wang M Stearns ME Isolation and characterization of PC-3 human prostatic tumor sublines which preferentially metastasize to select organs in S.C.I.D. mice.Differentiation. 1991; 48: 115-125Crossref PubMed Scopus (111) Google Scholar, 35van der Pluijm G Sijmons B Vloedgraven H Deckers M Papapoulos S Lowik C Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases.J Bone Miner Res. 2001; 16: 1077-1091Crossref PubMed Scopus (106) Google Scholar and by us (A.W. and M.G.C., not shown) (Table 1). The osteolytic human CaP cell line PC-3 [American Type Culture Collection (ATCC)/LGC Promochem, Molsheim, France] and its isogenic clone PC-3M-Pro4, selected in vivo for enhanced metastatic potential32Pettaway CA Pathak S Greene G Ramirez E Wilson MR Killion JJ Fidler IJ Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice.Clin Cancer Res. 1996; 2: 1627-1636PubMed Google Scholar (kindly provided by Dr. I.J. Fidler, Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX), were grown in Dulbecco's modified Eagle's medium. The osteoinductive, androgen-dependent and non-bone metastatic human prostate cancer cell line LNCaP, and its isogenic variants C4-2 and C4-2B, androgen-independent and spontaneously metastasizing to bone after orthotopic implantation30Thalmann GN Anezinis PE Chang SM Zhau HE Kim EE Hopwood VL Pathak S von Eschenbach AC Chung LW Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer.Cancer Res. 1994; 54: 2577-2581PubMed Google Scholar (kindly provided by Dr. L. Chung, Winship Cancer Center, Emory University, Atlanta, GA) were grown in T-medium. The osteolytic human mammary cancer cell line MDA-MB-231 (ATCC) and its isogenic clone MDA-231B, selected after sequential passaging in vivo for bone-restricted metastatic potential28Wetterwald A van der Pluijm G Que I Sijmons B Buijs J Karperien M Lowik CW Gautschi E Thalmann GN Cecchini MG Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.Am J Pathol. 2002; 160: 1143-1153Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar were grown in Dulbecco's modified Eagle's medium. The osteoinductive human mammary cancer cell lines T-47D and ZR-75-1 were purchased from ATCC and cultured in RPMI 1640 medium. The mouse osteoblast-like cell line KS48336van der Horst G van Bezooijen RL Deckers MM Hoogendam J Visser A Lowik CW Karperien M Differentiation of murine preosteoblastic KS483 cells depends on autocrine bone morphogenetic protein signaling during all phases of osteoblast formation.Bone. 2002; 31: 661-669Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar was cultured routinely in phenol red-free minimum essential medium-α. All media were supplemented with 10% fetal bovine serum (BioWittaker, Verviers, Belgium). Cell lines were stimulated either with 10 ng/ml of recombinant human transforming growth factor (TGF)-β1 (R&D Systems Europe Ltd., Abingdon, UK) or 100 ng/ml of recombinant human BMP-2 (R&D Systems) or 100 ng/ml of recombinant human BMP-6 (kindly provided by Prof. S. Vukicevic, Genera, Croatia) for 6 or 24 hours.Table 1Cancer Cell Lines Used in This Study Listed According to Their Tissue Origin and Osteotropic EffectsProstate cancerMammary cancerOsteolyticPC-334Wang M Stearns ME Isolation and characterization of PC-3 human prostatic tumor sublines which preferentially metastasize to select organs in S.C.I.D. mice.Differentiation. 1991; 48: 115-125Crossref PubMed Scopus (111) Google ScholarMDA-MB-23135van der Pluijm G Sijmons B Vloedgraven H Deckers M Papapoulos S Lowik C Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases.J Bone Miner Res. 2001; 16: 1077-1091Crossref PubMed Scopus (106) Google ScholarPC-3M-Pro432Pettaway CA Pathak S Greene G Ramirez E Wilson MR Killion JJ Fidler IJ Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice.Clin Cancer Res. 1996; 2: 1627-1636PubMed Google ScholarMDA-231B28Wetterwald A van der Pluijm G Que I Sijmons B Buijs J Karperien M Lowik CW Gautschi E Thalmann GN Cecchini MG Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.Am J Pathol. 2002; 160: 1143-1153Abstract Full Text Full Text PDF PubMed Scopus (190) Google ScholarOsteoinductiveLNCaP31Wu TT Sikes RA Cui Q Thalmann GN Kao C Murphy CF Yang H Zhau HE Balian G Chung LW Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines.Int J Cancer. 1998; 77: 887-894Crossref PubMed Scopus (234) Google ScholarT-47D33Yin JJ Mohammad KS Kakonen SM Harris S Wu-Wong JR Wessale JL Padley RJ Garrett IR Chirgwin JM Guise TA A causal role for endothelin-1 in the pathogenesis of osteoblastic bone metastases.Proc Natl Acad Sci USA. 2003; 100: 10954-10959Crossref PubMed Scopus (315) Google ScholarC4-230Thalmann GN Anezinis PE Chang SM Zhau HE Kim EE Hopwood VL Pathak S von Eschenbach AC Chung LW Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer.Cancer Res. 1994; 54: 2577-2581PubMed Google ScholarZR-75-133Yin JJ Mohammad KS Kakonen SM Harris S Wu-Wong JR Wessale JL Padley RJ Garrett IR Chirgwin JM Guise TA A causal role for endothelin-1 in the pathogenesis of osteoblastic bone metastases.Proc Natl Acad Sci USA. 2003; 100: 10954-10959Crossref PubMed Scopus (315) Google ScholarC4-2B31Wu TT Sikes RA Cui Q Thalmann GN Kao C Murphy CF Yang H Zhau HE Balian G Chung LW Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines.Int J Cancer. 1998; 77: 887-894Crossref PubMed Scopus (234) Google ScholarThe osteolytic or the osteoinductive effect was confirmed in our laboratory for all the cell lines listed except for the T-47D cell line, which did not generate tumors after intraosseous implantation in tibiae of immunocompromised nu/nu mice. Open table in a new tab The osteolytic or the osteoinductive effect was confirmed in our laboratory for all the cell lines listed except for the T-47D cell line, which did not generate tumors after intraosseous implantation in tibiae of immunocompromised nu/nu mice. Cells were seeded at the density of 1.25 to 2.5 × 104 cells/cm2. After 1 day, the medium was replaced with serum-free medium, and the cells were cultured for a further 48 hours. The cell-conditioned media (CM) were centrifuged and stored in aliquots at −20°C for later use. The cell number was determined and, where necessary, serum-free medium was added to the CM to normalize for differences in cell density between samples. Cells were seeded at the density of 104 cells/cm2 and cultured for a total of 8 days. Cell proliferation was determined daily with a tetrazolium salt-based method (MTT assay, Cell Proliferation Kit I; Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer's protocol. Total RNA extraction from subconfluent cultures of the various cell lines and from intraosseous xenografts of parental, noggin-, and mock-transfected C4-2B/luc+ cells, and of PC-3 and PC-3M-Pro4 cells, was performed with RNeasy (Qiagen, Hombrechtikon, Switzerland). Reverse transcription was performed with M-MLV-RT (Promega, Wallisellen, Switzerland) and random primers (Roche Diagnostics). Human-specific real-time PCR (TaqMan) primers and probes (Applied Biosystems, Rotkreuz, Switzerland) are listed in Table 2. Extraction of total RNA from pure CaP or CaM cells, laser-microdissected from clinical specimens of bone metastasis, was performed according to the TriSpin method37Reno C Marchuk L Sciore P Frank CB Hart DA Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues.Biotechniques. 1997; 22: 1082-1086PubMed Google Scholar with several modifications, as previously described.29Eisenberger S Hoppe G Pyerin W Ackermann K High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections.Anal Biochem. 2004; 335: 260-266Crossref PubMed Scopus (7) Google Scholar cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Karlsruhe, Germany) and PCR amplified in Mx3000P (Stratagene, Amsterdam, The Netherlands) using QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturers' protocols. Sense and anti-sense human-specific noggin primers used were 5′-TGTGCAAGCCGTCCAAGT-3′ and 5′-GAGCACTTGCACTCGGAAAT-3′.Table 2Real-Time Primers and Probes Used in This StudyGenePrimers/probe*TaqMan gene expression assays were from Applied Biosystems.PTHrPHs_00174969_m1CSF-1Hs_00174164_m1RANKLHs_00243522_m1OPGHs_00171068_m1IL-8Hs_00184979_m1BMP-2Hs_00154192_m1BMP-3Hs_00609638_m1BMP-4Hs_00181626_m1BMP-6Hs_00233470_m1PDF (GDF-15)Hs_00171132_m1TGF-β1Hs_00171257_m1NogginHs_00271352_s1DANHs_00185054_m1GremlinHs_00171951_m1SOSTHs_00228830_m1DKK-1Hs_00183740_m1β-actinHs_99999903_m1GAPDHHs_99999905_m1GAPDH, glyceraldehyde-3-phosphate dehydrogenase.* TaqMan gene expression assays were from Applied Biosystems. Open table in a new tab GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Colony stimulating factor-1 (CSF-1) concentration in serum-free CM was determined by the Quantikine human CSF-1 immunoassay (R&D Systems), parathyroid hormone-related protein (PTHrP) by a two-site immunoradiometric assay (Nichols Institute Diagnostics, Bad Vilbel, Germany), and endothelin-1 (ET-1) by enzyme-linked immunosorbent assay (Biomedica, Wien, Austria). Protein secreted into the medium was normalized to the cell number at the end of the culture period. The full-length human noggin cDNA was excised from the plasmid pBSII SK+.hNG (kindly provided by Regeneron Pharmaceuticals, Inc., Tarrytown, NY) with KpnI and NotI and ligated into the pcDNA3.1/Hygro expression vector (Invitrogen) (pcDNA3.1/Nog). The C4-2B cell line was transfected with the luciferase (luc) expression vector pCMVluc28Wetterwald A van der Pluijm G Que I Sijmons B Buijs J Karperien M Lowik CW Gautschi E Thalmann GN Cecchini MG Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.Am J Pathol. 2002; 160: 1143-1153Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar with SuperFect transfection reagent (Qiagen). A luc-positive clone was selected and transfected with pcDNA3.1/Nog to generate C4-2B-Nog clones and with the original plasmid pcDNA3.1/Hygro to generate C4-2B-mock clones. Noggin expression in transfected C4-2B cells was determined in concentrated CM and in cell lysates prepared with radioimmunoprecipitation assay buffer supplemented with 1 mmol/L phenylmethyl sulfonyl fluoride (Sigma, Buchs, Switzerland). Protein concentration was determined by Dc protein assay (Bio-Rad, Reinach, Switzerland), and proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gels (30 μg of total protein per lane) and transferred on Hybond-P membranes (Amersham Biosciences, Otelfingen, Switzerland). Membranes were incubated with 40 ng/ml of a rat monoclonal antibody against the human native noggin protein38Gazzerro E Du Z Devlin RD Rydziel S Priest L Economides AN Canalis E Noggin arrests stromal cell differentiation in vitro.Bone. 2003; 32: 111-119Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar (RP57-16; kindly provided by Dr. A.N. Economides, Regeneron Pharmaceuticals, Inc.) and detected with an horseradish peroxidase-labeled anti-rat secondary antibody (1:1000; Amersham Biosciences). A mouse monoclonal anti-actin antibody (1:5000; Chemicon International, Juro Supply GmbH, Lucerne, Switzerland) was used as loading control. Immunoreactiv
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