Notch Signaling and ERK Activation Are Important for the Osteomimetic Properties of Prostate Cancer Bone Metastatic Cell Lines
2004; Elsevier BV; Volume: 279; Issue: 5 Linguagem: Inglês
10.1074/jbc.m308158200
ISSN1083-351X
AutoresMajd Zayzafoon, Sarki A. Abdulkadir, Jay M. McDonald,
Tópico(s)Prostate Cancer Treatment and Research
ResumoProstate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment. Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment. Prostate carcinoma most commonly metastasizes to the lymph nodes and to bone with a 70-80% frequency as determined by autopsy studies. A unique characteristic of skeletal metastases of human prostate cancer is their ability to induce osteoblastic lesions, characterized by new woven bone formation and variable degrees of osteoclastic bone resorption (1Koutsilieris M. Anticancer Res. 1993; 13: 443-449Google Scholar). It has been proposed that factors produced by prostate cells, both normal and neoplastic, have the potential to stimulate new bone formation by effects on both osteoblasts and osteoclasts. Included in this group are parathyroid hormone-related peptide (2Iwamura M. di Sant'Agnese P.A. Wu G. Benning C.M. Cockett A.T. Deftos L.J. Abrahamsson P.A. Cancer Res. 1993; 53: 1724-1726Google Scholar), transforming growth factor-β (3Barrack E.R. Prostate. 1997; 31: 61-70Google Scholar), urokinase-type plasminogen activator (4Rabbani S.A. Xing R.H. Int. J. Oncol. 1998; 12: 911-920Google Scholar), bone morphogenetic proteins (5Bentley H. Hamdy F.C. Hart K.A. Seid J.M. Williams J.L. Johnstone D. Russell R.G. Br. J. Cancer. 1992; 66: 1159-1163Google Scholar, 6Harris S.E. Harris M.A. Mahy P. Wozney J. Feng J.Q. Mundy G.R. Prostate. 1994; 24: 204-211Google Scholar), and endothelin-1 (7Nelson J.B. Carducci M.A. Br. J. Urol. 2000; 85: 45-48Google Scholar). The production of these factors by prostate cancer cells, accompanied by the release of growth factors during osteolysis, is proposed to result in stimulating new woven bone formation by increasing the differentiation of osteoblasts in the bone. Interestingly, there is increasing evidence that prostate cancer bone metastases express genes and proteins typically associated with osteoblasts, including RANK ligand, osteoprotegerin (8Brown J.M. Corey E. Lee Z.D. True L.D. Yun T.J. Tondravi M. Vessella R.L. Urology. 2001; 57: 611-616Google Scholar), sialoproteins, osteopontin (9Jung C. Ou Y.C. Yeung F. Frierson Jr., H.F. Kao C. Gene (Amst.). 2001; 271: 143-150Google Scholar), Runx2 (Runt-related transcription factor 2) (10Lin D.L. Tarnowski C.P. Zhang J. Dai J. Rohn E. Patel A.H. Morris M.D. Keller E.T. Prostate. 2001; 47: 212-221Google Scholar), and osteocalcin (11Yeung F. Law W.K. Yeh C.H. Westendorf J.J. Zhang Y. Wang R. Kao C. Chung L.W. J. Biol. Chem. 2002; 277: 2468-2476Google Scholar). This phenomenon has been attributed to a "unique" ability of prostate cancer cells to acquire "osteoblast-like properties" upon metastasizing to the bone microenvironment, thus enabling them to grow and thrive in this highly restrictive environment (10Lin D.L. Tarnowski C.P. Zhang J. Dai J. Rohn E. Patel A.H. Morris M.D. Keller E.T. Prostate. 2001; 47: 212-221Google Scholar, 11Yeung F. Law W.K. Yeh C.H. Westendorf J.J. Zhang Y. Wang R. Kao C. Chung L.W. J. Biol. Chem. 2002; 277: 2468-2476Google Scholar). Cell fate determination, mediated by local cell-cell contact, plays a critical role during development of multicellular organisms. The Notch signaling pathway is an evolutionarily conserved mechanism utilized by organisms, ranging from worms through humans, involved in fate determination of various cell lineages. Notch belongs to the family of epidermal growth factor-like homeotic genes, which encode transmembrane proteins with variable numbers of epidermal growth factor-like repeats in the extracellular region. In vertebrates, four Notch genes have been described, notch1, -2, -3, and -4; these are highly related to each other and to the Drosophila Notch and C. elegans lin-12 (12Artavanis-Tsakonas S. Matsuno K. Fortini M.E. Science. 1995; 268: 225-232Google Scholar, 13Lardelli M. Williams R. Lendahl U. Int. J. Dev. Biol. 1995; 39: 769-780Google Scholar). There are also multiple Notch ligands in vertebrates that are homologous to the Drosophila ligands, Delta and Serrate (14Weinmaster G. Mol. Cell Neurosci. 1997; 9: 91-102Google Scholar). Delta homologs are called "Delta-like" ligands (Dll1 and -4), and the Serrate homologs are called "Jagged" (Jagged-1 and -2). Activation of Notch upon ligand binding is accompanied by proteolytic processing by γ-secretase that releases the intracellular domain of Notch from the membrane. The Notch intracellular domain then translocates into the nucleus and associates with the CSL (CBF-1 (RBP-Jκ)/Su(H)/Lag-1) family of DNA-binding proteins to form a transcriptional activator (15Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Google Scholar). Most of the Notch target genes encode transcription regulators, which, in turn, modulate cell fate by affecting the function of tissue-specific basic helix-loop-helix transcription factors or through other molecular targets, such as activating protein-1 (16Chu J. Jeffries S. Norton J.E. Capobianco A.J. Bresnick E.H. J. Biol. Chem. 2002; 277: 7587-7597Google Scholar), NF-κB (17Bellavia D. Campese A.F. Alesse E. Vacca A. Felli M.P. Balestri A. Stoppacciaro A. Tiveron C. Tatangelo L. Giovarelli M. Gaetano C. Ruco L. Hoffman E.S. Hayday A.C. Lendahl U. Frati L. Gulino A. Screpanti I. EMBO J. 2000; 19: 3337-3348Google Scholar), and most importantly, HES-1 (Hairy and Enhancer-of-split-1) (18McLarren K.W. Lo R. Grbavec D. Thirunavukkarasu K. Karsenty G. Stifani S. J. Biol. Chem. 2000; 275: 530-538Google Scholar). Since Notch ligands are predominantly cell membrane-associated, Notch signaling is thought to mediate interactions between contiguous cells at many sites in the human body, including bone, where it has been shown recently that Notch1 activation by Dll1 stimulates the differentiation of osteoblasts (19Tezuka K. Yasuda M. Watanabe N. Morimura N. Kuroda K. Miyatani S. Hozumi N. J. Bone Miner. Res. 2002; 17: 231-239Google Scholar). Osteoblasts, the bone-forming cells, are derived from mesenchymal stem cells after osteogenic differentiation. Under in vitro osteogenic conditions, isolated human mesenchymal stem cells (hMSC) 1The abbreviations used are: hMSChuman mesenchymal stem cellsERKextracellular signal-regulated kinaseOSE-2osteoblast-specific element 2MAPKmitogen-activated protein kinase. form mineralized aggregates or nodules and increase their expression of alkaline phosphatase and osteocalcin, which are important markers of osteoblast differentiation (20Bruder S.P. Jaiswal N. Ricalton N.S. Mosca J.D. Kraus K.H. Kadiyala S. Clin. Orthop. 1998; : 247-256Google Scholar). Many of the osteoblast-related genes are modulated by Runx2 binding to a specific DNA element in the regulatory region of these target genes. Runx2 is an essential transcription factor for the differentiation of osteoblasts from mesenchymal precursors and the regulation of bone matrix deposition by differentiated osteoblasts (21Ducy P. Dev. Dyn. 2000; : 247-256Google Scholar). This suggests that Runx2 regulates osteoblast gene expression and function at multiple levels. Recent studies have demonstrated that prostate cancer cells, similar to mature osteoblasts, are capable of expressing and secreting osteocalcin upon metastasizing to bone (11Yeung F. Law W.K. Yeh C.H. Westendorf J.J. Zhang Y. Wang R. Kao C. Chung L.W. J. Biol. Chem. 2002; 277: 2468-2476Google Scholar). In addition, it has been shown that the osteoblastic C4-2B prostate cancer cell line (LNCaP derivative cell line) (22Wu H.C. Hsieh J.T. Gleave M.E. Brown N.M. Pathak S. Chung L.W. Int. J. Cancer. 1994; 57: 406-412Google Scholar) has an increased expression of Runx2 (10Lin D.L. Tarnowski C.P. Zhang J. Dai J. Rohn E. Patel A.H. Morris M.D. Keller E.T. Prostate. 2001; 47: 212-221Google Scholar), whereas the osteolytic PC3 cell line expresses transcriptionally active Cbaf1 as shown by its ability to bind to OSE2 on the osteocalcin promoter (11Yeung F. Law W.K. Yeh C.H. Westendorf J.J. Zhang Y. Wang R. Kao C. Chung L.W. J. Biol. Chem. 2002; 277: 2468-2476Google Scholar). This suggests that Runx2 expression/activity could play a role in the modulation of gene expression in prostate cancer metastases. Interestingly, HES-1 (a downstream target of Notch signaling) can physically interact with Runx2 and potentiate its mediated transactivation in transfected cells (18McLarren K.W. Lo R. Grbavec D. Thirunavukkarasu K. Karsenty G. Stifani S. J. Biol. Chem. 2000; 275: 530-538Google Scholar). Based on this, we hypothesize that the activation of Notch in prostate cancer metastases in the bone microenvironment leads to a cascade of signaling events, which ultimately results in a unique pattern of gene expression similar to that seen in mature osteoblasts. human mesenchymal stem cells extracellular signal-regulated kinase osteoblast-specific element 2 mitogen-activated protein kinase. The aim of the present study was to examine the role of the Notch signaling pathway in the unique osteomimetic properties of prostate cancer bone metastases. We report that osteoblastic prostate cancer cell lines (C4-2B and MDA PCa 2b) specifically express notch1, and the Notch ligand, dll1, is expressed only in the C4-2B cell line. This expression pattern of Notch receptor and ligand leads to the expression of HES-1 transcription factor independent of ERK activation. Furthermore, we demonstrate that in vitro osteogenic induction of prostate cancer cells increases ERK phosphorylation and Runx2 expression and activation, independent of Notch activation. Finally, we demonstrate that Notch signaling and ERK activation in skeletal prostate cancer metastases is critical for Runx2 DNA binding and osteocalcin expression. Taken together, our results suggest that Notch activation plays a critical role in the ability of prostate cancer metastases to acquire "osteoblast-like" properties. Cell Culture and Osteoblastic Differentiation—The human prostatic cell lines (Table I) DU145, LNCaP, and MDA PCa 2b were purchased from the American Type Culture Collection (Manassas, VA). The C4-2B cell line was purchased from UroCor, Inc. (Oklahoma City, OK). In addition, we used hMSC provided by the University of Alabama National Institutes of Health-funded Research Core Center for Musculoskeletal Disease. DU145 and LNCaP were maintained in RPMI 1640 containing 10% fetal bovine serum. MDA PCa 2b was maintained in Kaighn's modification of Ham's F-12 medium supplemented with 25 ng/ml Cholera toxin, 10 ng/ml epidermal growth factor, 0.005 mm phosphoethanolamine, 100 pg/ml hydrocortisone, 45 nm selenious acid, 0.005 mg/ml insulin, and 20% fetal bovine serum. The C4-2B cell line was maintained in T medium (80% Dulbecco's modified Eagle's medium, 20% F-12 medium, 3 g/liter NaCO3, 5 μg/ml insulin, 13.6 pg/ml triiodothyronine, 5 μg/ml transferrin, 0.25 μg/ml biotin, 25 μg/ml adenine) supplemented with 5% fetal bovine serum. The hMSC were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. All cultures were supplemented with 100 units/liter penicillin G and 100 μg/ml streptomycin. For osteogenic induction, regular medium was supplemented with 10 mm β-glycerophosphate and 50 μm ascorbic acid-2-phosphate (23Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Google Scholar).Table IProstate cancer cell lines used in these studiesCell lineCharacteristicsReferenceC4—2BForms osteoblastic bone metastases in vivoDerived from LNCaP cells22Wu H.C. Hsieh J.T. Gleave M.E. Brown N.M. Pathak S. Chung L.W. Int. J. Cancer. 1994; 57: 406-412Google ScholarLNCaPIsolated from lymph nodes metastases.Metastasize to lymph nodes in vivo47Dumont P. Petein M. Lespagnard L. Tueni E. Coune A. In Vivo. 1993; 7: 167-170Google ScholarMDA PCa 2bIsolated from osteoblastic bone metastases.Forms osteoblastic bone metastases in vivo48Navone N.M. Olive M. Ozen M. Davis R. Troncoso P. Tu S.M. Johnston D. Pollack A. Pathak S. von Eschenbach A.C. Logothetis C.J. Clin. Cancer Res. 1997; 3: 2493-2500Google ScholarDU145Isolated from brain metastases49Stone K.R. Mickey D.D. Wunderli H. Mickey G.H. Paulson D.F. Int. J. Cancer. 1978; 21: 274-281Google Scholar Open table in a new tab RNA Extraction and RT-PCR—Total RNA was extracted using the TRIzol method as recommended by the manufacturer (Invitrogen). The yield and purity of RNA was estimated spectrophotometrically using the A260/A280 ratio. The quality of RNA was examined by gel electrophoresis. One microgram of RNA was reverse transcribed using M-MLV reverse transcriptase, and the equivalent of 10 ng was used for the PCRs. These were carried out in a final volume of 25 μl containing 0.2 mm dNTPs, 120 nm each primer, and 1 unit of Taq DNA polymerase. TaqMan real-time quantitative RT-PCR analysis was performed using the relative standard curve method with SYBRGreen (TaqMan PCR detector 5700; PerkinElmer Life Sciences). The expression of 18 S rRNA was used as control. The sequences for the specific primers used in this study were as follows: notch1, forward primer (5′-CACTGTGGGCGGGTCC-3′) and reverse primer (5′-GTTGTATTGGTTCGGCACCAT-3′) (24Shou J. Ross S. Koeppen H. de Sauvage F.J. Gao W.Q. Cancer Res. 2001; 61: 7291-7297Google Scholar); dll1, forward primer (5′-TGTGTGACGAACACTACTACGGAG-3′) and reverse primer (5′-GTGAAGTGGCCGAAGGCA-3′) (24Shou J. Ross S. Koeppen H. de Sauvage F.J. Gao W.Q. Cancer Res. 2001; 61: 7291-7297Google Scholar); HES-1, forward primer (5′-AGGCGGACATTCTGGAAATG-3′) and reverse primer (5′-CGGTACTTCCCCAGCACACTT-3′); runx2, forward primer (5′-GATGACACTGCCACCTCTGACTT-3′) and reverse primer (5′-AAAAAGGGCCCAGTTCTGAAG-3′); osteocalcin, forward primer (5′-CCCCTGCTTGTGACGAGCTA-3′) and reverse primer (5′-AATAGTGATACCGTAGATGCGTTTGT-3′); 18 S rRNA, forward primer (5′-CGCCGCTAGAGGTGAAATTCT-3′) and reverse primer (5′-CGAACCTCCGACTTTCGTTCT-3′). Whole Cell Protein Extraction—At the end of the study, cells were washed with chilled phosphate-buffered saline and centrifuged at 800 × g for 5 min at 4 °C and then resuspended in lysis buffer (50 mm Tris (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and 10% glycerol). A mixture of protease and phosphatase inhibitors consisting of 2 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 1 mm EGTA, 10 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and 0.1 mm β-glycerophosphate was added to the lysis buffer. Samples were then centrifuged at 14,000 rpm for 30 min at 4 °C, and the supernatant protein concentration was measured using the Bio-Rad DC protein assay (25Zayzafoon M. Botolin S. McCabe L.R. J. Biol. Chem. 2002; 277: 37212-37218Google Scholar). Western Blot Analysis—Whole cell or nuclear extracts were loaded (30 μg/lane) onto an SDS mini-PAGE system. Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane, Immobilon-P (Millipore Corp.), using a Bio-Rad wet transfer system. Protein transfer efficiency and size determination were verified using prestained protein markers. Membranes were then blocked with Blotto B (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature and subsequently incubated overnight with antibodies directed against Notch1, ERK, phosphorylated ERK, Runx2, and actin (Santa Cruz Biotechnology). Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an ECL detection kit (Amersham Biosciences). Calcium Measurement—Cells were lysed with double distilled H2O, accompanied by three freeze/thaw cycles. Calcium content was measured using a calcium detection kit (Arsenazo III; Sigma). Nuclear Protein Extraction—C4-2B cells were washed with chilled phosphate-buffered saline and centrifuged at 800 × g for 5 min at 4 °C. Nuclei were then isolated by detergent lysis of the cells using an Nonidet P-40 lysis buffer containing 10 mm Tris, 10 mm NaCl, 3 mm MgCl2, 0.5% Nonidet P-40, and 0.56 m sucrose. Nuclei were then treated with a hypotonic solution containing 10 mm HEPES, 1.5 mm MgCl2, and 10 mm KCl followed by a 30-min incubation at 4 °C in an extraction buffer containing 20 mm HEPES, 20% glycerol, 600 mm KCl, 1.5 mm MgCl2, and 0.2 mm EDTA. Nuclei were finally centrifuged at 14,000 rpm for 30 min at 4 °C, and the supernatant protein concentration was measured by the Bio-Rad DC Protein Assay. All solutions in this procedure contained a mixture of protease and phosphatase inhibitors similar to that used in the whole cell protein extraction. Electrophoretic Mobility Shift Assay—Four micrograms of nuclear extracts were incubated for 20 min at room temperature with a 32P-labeled oligonucleotide containing the human Runx2 consensus sequence. The oligonucleotide sequence used as a probe was 5′-CGCAGCTCCCAACCACATATCCTCT-3′ (26Ziros P.G. Gil A.P. Georgakopoulos T. Habeos I. Kletsas D. Basdra E.K. Papavassiliou A.G. J. Biol. Chem. 2002; 277: 23934-23941Google Scholar) (top strand), derived from the human osteocalcin promoter (-141 to -165), and contained an OSE2 motif (AACCACA) (27Ducy P. Karsenty G. Mol. Cell. Biol. 1995; 15: 1858-1869Google Scholar). Oligonucleotide with mutation in the Runx2 binding site was also used to confirm binding specificity. The mutant Runx2 sequence is 5′-CGCAGCTCCCAgaCACATATCCTCT-3′ (top strand). The double-stranded Runx2 probe was end-labeled using [γ-32P]ATP and T4 polynucleotide kinase according to standard protocols. DNA-protein complexes were then resolved by 5% native polyacrylamide gels. Gels were dried and exposed to x-ray film at -80 °C with an intensifying screen. For the supershift experiment, nuclear extracts (10 μg) were incubated with HES-1 antibody (Santa Cruz Biotechnology) in binding buffer for 45 min at room temperature. 32P-labeled oligonucleotide containing the human Runx2 consensus sequence was then added, and the mixture was further incubated for 30 min at room temperature. Immunohistochemistry and Human Tissue Samples—Human bone samples from four patients were obtained from the Surgical Pathology Department of the University of Alabama at Birmingham Hospitals and Clinics with the approval of the Institutional Review Board. Tissues were formalin-fixed and decalcified in EDTA before processing on a VIP tissue processor followed by paraffin embedding. Sections were cut at 5 μm for immunostaining. C4-2B cells were cultured on glass coverslips for 4 days and then formalin-fixed. Tissues were deparaffinized and rehydrated, followed by antigen retrieval using 10 mm citrate buffer, pH 6. Endogenous peroxidase activity was quenched using 1% hydrogen peroxide. Samples were then blocked for 1 h in Fc receptor blocker (Innovex Biosciences, Richmond, CA). Anti-Notch1 (Santa Cruz Biotechnology) or pancytokeratin (Bio-genex) was diluted in Fc Blocker solution and applied to the sections for overnight incubation at 4 °C. Biotin-conjugated secondary antibodies were then used, followed by incubation with avidin-biotin enzyme reagents. Finally, specimens were incubated in peroxidase substrate for 30 s. Tissues were counterstained in Gill's heamatoxylin for 10 s, dehydrated, mounted, and coversliped. Negative controls were processed alongside the examined tissue, but rabbit IgG was used instead of the primary antibody. At least 10 randomly selected microscopic fields were examined using a ×10 and ×40 objective. Photos were taken using a SPOT digital camera. Statistical Analysis—All statistical analyses were performed using the Microsoft Excel data analysis program for t test analysis or using SPSS statistical analysis program for analysis of variance with the Bonferroni test. Experiments were repeated at least three times unless otherwise stated. Values were expressed as a mean ± S.E. Given the importance of Notch activation in determining cell fate, we examined notch1 gene expressions in two human osteoblastic prostate cancer cell lines (C4-2B and MDA PCa 2b) and compared them with brain (DU145) and lymph node (LNCaP) metastatic prostate cancer cell lines (Table I). Fig. 1A demonstrates that notch1 gene expression is increased 5-fold in osteoblastic (C4-2B and PCa 2b) versus nonosteoblastic (LNCaP and DU145) prostate cancer metastatic cell lines. This increase in gene expression is accompanied by an increase in the amount of Notch1 protein (Fig. 1B), suggesting that Notch1 is specifically expressed in osteoblastic prostate metastases. Immunohistochemical studies further demonstrate the expression and distribution of Notch1 in C4-2B cells (Fig. 1C). Interestingly, Dll1 Notch ligand is only detected in the C4-2B cell line (Fig. 1D). The presence of both Notch1 and Dll1 in the same cell suggests that Notch signaling can be successfully activated in C4-2B cells without the need to co-culture them with Notch ligand-expressing cells. The expression of other Notch receptors (Notch2, -3, and -4) and ligands (Dll4 and Jagged-1 and -2) were also examined but showed no significant correlation between skeletal and nonskeletal metastatic cell lines (data not shown). In order to examine the expression of Notch1 in prostate cancer bone metastases in vivo, human clinical samples from patients who developed bone metastases from a primary prostate tumor were obtained from the Surgical Pathology Department of the University of Alabama at Birmingham Hospitals and Clinics. Hematoxylin and eosin staining (Fig. 2A) confirms the osteoblastic nature of the prostate cancer metastases, which is demonstrated by the degree of trabacular bone present in the tissue. Immunohistochemical staining for pancytokeratin confirms the epithelial origin of the metastatic cancer cells and clearly distinguished them from the bone mesenchymal cells (Fig. 2B). Similar to C4-2B cells, human osteoblastic prostate cancer metastatic cells express Notch1 (Fig. 2C). Although most of the positively stained cells show an extranuclear distribution, Notch1 is also detected intranuclearly. To examine the ability of prostate cancer metastases to function similar to osteoblasts and to form mineralized bone, we cultured LNCaP, MDA PCa 2b, and C4-2B cells with and without osteogenic medium, consisting of normal culture medium, supplemented with ascorbic acid and β-glycerophosphate. This method of osteogenic induction has been previously characterized and is widely used for inducing osteoblast differentiation in vitro (23Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Google Scholar). Cells were also treated with 100 nm Notch inhibitor (L-685,458), known to be a potent γ-secretase inhibitor (28Tian G. Sobotka-Briner C.D. Zysk J. Liu X. Birr C. Sylvester M.A. Edwards P.D. Scott C.D. Greenberg B.D. J. Biol. Chem. 2002; 277: 31499-31505Google Scholar), to determine the role of Notch signaling in bone formation by metastatic prostate cancer cells. After 10 days of osteogenic induction, the calcium content in cultures was measured. Fig. 3 demonstrates that both LNCaP cells (which do not express Notch1) and MDA PCa 2b cells (which do not express Dll1) fail to increase calcium deposition in culture. On the other hand, C4-2B cells, which form osteoblastic bone metastases in vivo (22Wu H.C. Hsieh J.T. Gleave M.E. Brown N.M. Pathak S. Chung L.W. Int. J. Cancer. 1994; 57: 406-412Google Scholar) and express both Notch1 and Dll1, increase calcium deposition 5-fold when cultured in osteogenic medium. This increase is completely abolished when Notch inhibitor (L-685,458) is added. We also used hMSC as a positive control, since these cells develop into osteoblasts when cultured in osteogenic medium (23Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Google Scholar). We demonstrate a 7-fold increase in calcium content when hMSC are induced to develop into osteoblasts, by the presence of osteogenic medium. Interestingly, Notch inhibitor is also effective in blocking this mineralization. Osteogenic induction induces the osteoblastic differentiation of preosteoblasts by increasing the activity of MAPK, which ultimately leads to an increase in Runx2 activation and DNA binding (28Tian G. Sobotka-Briner C.D. Zysk J. Liu X. Birr C. Sylvester M.A. Edwards P.D. Scott C.D. Greenberg B.D. J. Biol. Chem. 2002; 277: 31499-31505Google Scholar). In addition, osteoblastic cell differentiation is also positively regulated by Notch1 (19Tezuka K. Yasuda M. Watanabe N. Morimura N. Kuroda K. Miyatani S. Hozumi N. J. Bone Miner. Res. 2002; 17: 231-239Google Scholar). Therefore, we tested the effect of osteogenic induction on Notch signaling by examining the expression of HES-1, a known downstream target of Notch signaling, in C4-2B cells. Furthermore, to determine the specific role of both Notch and ERK signaling in HES-1 expression, we pharmacologically inhibited Notch signaling with L-685-458 or ERK signaling with U0126 (29Xiao G. Jiang D. Gopalakrishnan R. Franceschi R.T. J. Biol. Chem. 2002; 277: 36181-36187Google Scholar). As expected, LNCaP cells, which lack Notch1 receptor, did not express HES-1 (Fig. 4). In contrast, C4-2B cells, which express both Notch1 and its ligand, expressed HES-1, regardless of osteogenic induction. The inhibition of Notch signaling by γ-secretase inhibitor successfully inhibited HES-1 expression, whereas the ERK inhibitor, U0126, had no effect. To examine whether C4-2B cells would respond similarly to osteoblasts when cultured in osteogenic medium, we tested the activation of ERK in response to osteogenic induction while inhibiting the activation of either Notch or ERK signaling. Fig. 5, A and B, demonstrate that osteogenic induction increases ERK phosphorylation in C4-2B cells, as in osteoblasts, without any change in the total ERK levels. Notch signaling does not seem to play a role in this activation, since we show no significant difference in ERK activity when Notch signaling was inhibited for 24 h by 1000 nm L-685,458 (Fig. 5A). As expected, U0126 inhibited the activation of ERK in response to osteogenic induction (Fig. 5B). The activity of MAPK in osteoblasts ultimately leads to an increase in Runx2 expression and its activation/nuclear localization (30Xiao G. Jiang D. Thomas P. Benson M.D. Guan K. Karsenty G. Franceschi R.T. J. Biol. Chem. 2000; 275: 4453-4459Google Scholar). Fig. 6 demonstrates that LNCaP cells do not express Runx2, even under conditions of osteogenic induction. In contrast, C4-2B cells express basal levels of Runx2 mRNA and nuclear protein. Upon osteogenic induction, both mRNA and nuclear localization of Runx2 are incre
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