The online version of this article (doi:10.1186/1476-4598-11-66) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
AG carried out major experiments including Western blotting with human normal and tumor tissue lysates, immunohistochemistry on TMA, analyses with conditioned medium (Western blotting and osteoclast differentiation), studies with inhibitors (αv and PKC) and SiRNA (Smad 5). AG also participated in the MS preparation, statistical analysis of the data, discussion and interpretation of results. WC generated CD44 knockdown stable PC3 cell lines. MAC conceived the study, confocal microscopy analysis of immunostained PC3 cells, RUNX2 knockdown experiments and manuscript preparation. All authors read and approved the final manuscript.
Bone loss and pathological fractures are common skeletal complications associated with androgen deprivation therapy and bone metastases in prostate cancer patients. We have previously demonstrated that prostate cancer cells secrete receptor activator of NF-kB ligand (RANKL), a protein essential for osteoclast differentiation and activation. However, the mechanism(s) by which RANKL is produced remains to be determined. The objective of this study is to gain insight into the molecular mechanisms controlling RANKL expression in metastatic prostate cancer cells.
We show here that phosphorylation of Smad 5 by integrin αvβ3 and RUNX2 by CD44 signaling, respectively, regulates RANKL expression in human-derived PC3 prostate cancer cells isolated from bone metastasis. We found that RUNX2 intranuclear targeting is mediated by phosphorylation of Smad 5. Indeed, Smad5 knock-down via RNA interference and inhibition of Smad 5 phosphorylation by an αv inhibitor reduced RUNX2 nuclear localization and RANKL expression. Similarly, knockdown of CD44 or RUNX2 attenuated the expression of RANKL. As a result, conditioned media from these cells failed to support osteoclast differentiation in vitro. Immunohistochemistry analysis of tissue microarray sections containing primary prostatic tumor (grade2-4) detected predominant localization of RUNX2 and phosphorylated Smad 5 in the nuclei. Immunoblotting analyses of nuclear lysates from prostate tumor tissue corroborate these observations.
Collectively, we show that CD44 signaling regulates phosphorylation of RUNX2. Localization of RUNX2 in the nucleus requires phosphorylation of Smad-5 by integrin αvβ3 signaling. Our results suggest possible integration of two different pathways in the expression of RANKL. These observations imply a novel mechanistic insight into the role of these proteins in bone loss associated with bone metastases in patients with prostate cancer.
Additional file 1: Figure S1. Analysis of the effects of SiRNA to RUNX2 on MMP9 and MMP2 RNA and protein levels (A-E) and revelation of major MMPs present in PC3 and LNCaP cells (F). A-D: We determined the effects of RUNX2 knockdown on the expression of MMP9 and MMP2 at mRNA (Figure S1-A) and protein levels (Figure S1D) in PC3 cells. Dose-dependent decrease in the levels of RUNX2 expression was observed in PC3 cells treated with SiRNA to RUNX2 at concentrations of 10, 20, and 50nM. The decrease was maximal (>90%) at 50nM RUNX2 SiRNA (A, lane 4). PC3 cells treated with scrambled RNAi (50nM) were used as control (A, lane 1). SiRNA to RUNX2 had very negligible effects on the changes in the levels of mRNA expression of MMP2 in PC3 cells (lane 6). GAPDH was used as internal control (Figure S1-B). A decrease in the expression of MMP9 at mRNA (Figure S1-A, lane 4) parallels with the MMP9 activity (~ 90kDa) in the conditioned medium isolated from cultures of PC3 cells treated with RUNX2 SiRNA (Figure S1-E, lane 3). MMP9 activity was determined by zymogram analysis. About 50μg membrane protein was used for the gelatin zymography to determine the activities of MMP9 (S1-E). As shown previously [Ref.28], only the active form of MMP-9 was observed in the conditioned medium (Figure S1-E, lanes 1-3). The activity of a recombinant MMP-9 protein containing pro- and active band was used as an identification marker (lane 4 in S1-E). Furthermore, the decrease in the protein levels of RUNX2 (~55kDa) in SiRNA to RUNX2 treated cells (Figure S1-C, lane 3) corresponds with a decrease in the total cellular protein levels of MMP 9 (Figure S1-D, lane 3) but not MMP 2 (~72kDa). MMP 2 levels remain the same in control untreated as well as scrambled RNAi and SiRNA to RUNX2 treated cells (Figure S1- D). These results imply that the RUNX2 is not a direct binding factor to induce transcriptional activation of MMP 2.F: Zymogram analysis with normal prostatic epithelial cells (HPR1) was used as a control (lane 4) for prostate cancer cells derived from lymph node (LNCaP, lane 2) and bone (PC3, lane 3) metastases. The activity of a recombinant MMP2 and MMP9 protein containing pro and active bands (indicated by arrows) were used as an identification marker (lane 1). LNCaP cells demonstrated MMP2 as a major metalloproteases where as MMP9 was observed as major MMP although MMP2 was observed at mRNA (Figure 1A) and protein levels (Figure S1-D and F) in PC3 cells. About 75μg total cellular protein was used for zymogram analysis as shown previously [ref.[ 28]. Method: Gelatin zymography: Conditioned media collected from various PC3 cell lines were concentrated approximately 10-fold) with a centricon concentrator (Amicon, Beverly, MA). Ten micrograms of concentrated media protein in 10-20 μl were mixed with gel loading buffer with no reducing agent (βME or DTT) and incubated at RT for 10-15 min. SDS-PAGE containing 0.1% gelatin was used for electrophoresis. Samples were loaded without heating with sample buffer. After electrophoresis, gels were incubated overnight in a buffer containing 50 mM Tris-HCl, pH 7.6, 5 mM CaCl 2, 1 μM ZnCl 2, and 1% Triton X-100. Triton was used to remove SDS from the gel. Gels were then stained with Coomassie brilliant blue for 2-3 h and destained with 7% acetic acid or water. Gelatinolytic activity was detected as clear bands in the background of blue staining [ref.[ 28]. (DOC 122 KB)
Additional file 2: Figure S2.Immunoblotting analysis for Smad 2, 3, 5 and 6 proteins in PC3 cells. About 50μg total cellular lysate protein was used for immunoblotting with antibodies to phospho-Smad (p-Smad) -2 (60kDa; lane 1), -3 (52 kDa; lane 2), -5 (60kDa; lane 3) and -6 (62kDa; lane 4). Blots were reprobed with an antibody to GAPDH after stripping. Phosphorylation of 2, 3, and 5 was observed in PC3 cells. However, Smad- 5 phosphorylation is significantly more than Smad-2 and 3 (lanes 1 and 2). Phosphorylation of Smad-6 is really negligible or not observed. (DOC 70 KB)12943_2012_1043_MOESM2_ESM.doc
Authors’ original file for figure 112943_2012_1043_MOESM3_ESM.tiff
Authors’ original file for figure 212943_2012_1043_MOESM4_ESM.tiff
Authors’ original file for figure 312943_2012_1043_MOESM5_ESM.tiff
Authors’ original file for figure 412943_2012_1043_MOESM6_ESM.tiff
Authors’ original file for figure 512943_2012_1043_MOESM7_ESM.tiff
Authors’ original file for figure 612943_2012_1043_MOESM8_ESM.tiff
Authors’ original file for figure 712943_2012_1043_MOESM9_ESM.tiff
Authors’ original file for figure 812943_2012_1043_MOESM10_ESM.tiff
Authors’ original file for figure 912943_2012_1043_MOESM11_ESM.tiff
Authors’ original file for figure 1012943_2012_1043_MOESM12_ESM.tiff
Authors’ original file for figure 1112943_2012_1043_MOESM13_ESM.doc
van der Gulden JW, Kiemeney LA, Verbeek AL, Straatman H: Mortality trend from prostate cancer in The Netherlands (1950–1989) 7. Prostate. 1994, 24: 33-38. 10.1002/pros.2990240108 CrossRef
Brawley OW: Prostate cancer epidemiology in the United States. World J Urol. 2012, 30: 195-200. 10.1007/s00345-012-0824-2 CrossRef
Carlin BI, Andriole GL: The natural history, skeletal complications, and management of bone metastases in patients with prostate carcinoma 1. Cancer. 2000, 88: 2989-2994. 10.1002/1097-0142(20000615)88:12+<2989::AID-CNCR14>3.0.CO;2-Q CrossRef
Sanchez-Sweatman OH, Orr FW, Singh G: Human metastatic prostate PC3 cell lines degrade bone using matrix metalloproteinases. Invasion Metastasis. 1998, 18: 297-305. 10.1159/000024522 CrossRef
Dougall WC: RANKL signaling in bone physiology and cancer. Curr Opin Support Palliat Care. 2007, 1: 317-322. 10.1097/SPC.0b013e3282f335be CrossRef
Hofbauer LC, Schoppet M: Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases12. JAMA. 2004, 292: 490-495. 10.1001/jama.292.4.490 CrossRef
Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T: Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998, 93: 165-176. 10.1016/S0092-8674(00)81569-X CrossRef
Zhang J, Dai J, Yao Z, Lu Y, Dougall W, Keller ET: Soluble receptor activator of nuclear factor kappaB Fc diminishes prostate cancer progression in bone. Cancer Res. 2003, 63: 7883-7890.
Miller RE, Roudier M, Jones J, Armstrong A, Canon J, Dougall WC: RANK ligand inhibition plus docetaxel improves survival and reduces tumor burden in a murine model of prostate cancer bone metastasis. Mol Cancer Ther. 2008, 7: 2160-2169. 10.1158/1535-7163.MCT-08-0046 CrossRef
Yonou H, Ochiai A, Ashimine S, Maeda H, Horiguchi Y, Yoshioka K: The bisphosphonate YM529 inhibits osteoblastic bone tumor proliferation of prostate cancer2. Prostate. 2007, 67: 999-1009. 10.1002/pros.20592 CrossRef
Schneider A, Kalikin LM, Mattos AC, Keller ET, Allen MJ, Pienta KJ: Bone turnover mediates preferential localization of prostate cancer in the skeleton. Endocrinology. 2005, 146: 1727-1736. 10.1210/en.2004-1211 CrossRef
Liao J, McCauley LK: Skeletal metastasis: Established and emerging roles of parathyroid hormone related protein (PTHrP). Cancer Metastasis Rev. 2006, 25: 559-571. CrossRef
Barnes GL, Hebert KE, Kamal M, Javed A, Einhorn TA, Lian JB: Fidelity of Runx2 activity in breast cancer cells is required for the generation of metastases-associated osteolytic disease. Cancer Res. 2004, 64: 4506-4513. 10.1158/0008-5472.CAN-03-3851 CrossRef
Brubaker KD, Vessella RL, Brown LG, Corey E: Prostate cancer expression of runt-domain transcription factor Runx2, a key regulator of osteoblast differentiation and function. Prostate. 2003, 56: 13-22. 10.1002/pros.10233 CrossRef
Selvamurugan N, Shimizu E, Lee M, Liu T, Li H, Partridge NC: Identification and characterization of Runx2 phosphorylation sites involved in matrix metalloproteinase-13 promoter activation. FEBS Lett. 2009, 583: 1141-1146. 10.1016/j.febslet.2009.02.040 CrossRef
Pratap J, Lian JB, Javed A, Barnes GL, van Wijnen AJ, Stein JL: Regulatory roles of Runx2 in metastatic tumor and cancer cell interactions with bone. Cancer Metastasis Rev. 2006, 25: 589-600. CrossRef
Kitazawa R, Mori K, Yamaguchi A, Kondo T, Kitazawa S: Modulation of mouse RANKL gene expression by Runx2 and vitamin D3. J Cell Biochem. 2008, 105: 1289-1297. 10.1002/jcb.21929 CrossRef
Hanai J, Chen LF, Kanno T, Ohtani-Fujita N, Kim WY, Guo W-H: Interacton and functional cooperation of PEBP2/CBF with Smads. J Biol Chem. 1999, 274: 31577-31582. 10.1074/jbc.274.44.31577 CrossRef
Javed A, Afzal F, Bae JS, Gutierrez S, Zaidi K, Pratap J: Specific residues of RUNX2 are obligatory for formation of BMP2-induced RUNX2-SMAD complex to promote osteoblast differentiation. Cells Tissues Organs. 2009, 189: 133-137. 10.1159/000151719 CrossRef
Ito Y, Zhang YW: A RUNX2/PEBP2alphaA/CBFA1 mutation in cleidocranial dysplasia revealing the link between the gene and Smad. J Bone Miner Metab. 2001, 19: 188-194. 10.1007/s007740170041 CrossRef
Lee KS, Kim HJ, Li QL, Chi XZ, Ueta C, Komori T: Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol. 2000, 20: 8783-8792. 10.1128/MCB.20.23.8783-8792.2000 PubMedCentralCrossRef
Weber GF, Ashkar S: Molecular mechanisms of tumor dissemination in primary and metastatic brain cancers. Brain Res Bull. 2000, 53: 421-424. 10.1016/S0361-9230(00)00379-8 CrossRef
Pecheur I, Peyruchaud O, Serre CM, Guglielmi J, Voland C, Bourre F: Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone. FASEB J. 2002, 16: 1266-1268.
Naor D, Sionov RV, Zahalka M, Rochman M, Holzmann B, Ish-Shalom D: Organ-specific requirements for cell adhesion molecules during lymphoma cell dissemination. Curr Top Microbiol Immunol. 1998, 231: 143-166. 10.1007/978-3-642-71987-5_9
Sy MS, Guo YJ, Stamenkovic I: Distinct effects of two CD44 isoforms on tumor growth in vivo. J Exp Med. 1991, 174: 859-866. 10.1084/jem.174.4.859 CrossRef
Gao AC, Lou W, Dong JT, Isaacs JT: CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome 11p13. Cancer Res. 1997, 57: 846-849.
Noordzij MA, Van Steenbrugge GJ, Schroder FH, Van Der Kwast TH: Decreased expression of CD44 in metastatic prostate cancer. Int J Cancer. 1999, 84: 478-483. 10.1002/(SICI)1097-0215(19991022)84:5<478::AID-IJC5>3.0.CO;2-N CrossRef
Tanne Y, Tanimoto K, Tanaka N, Ueki M, Lin YY, Ohkuma S: Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation. Arch Oral Biol. 2008, 53: 478-487. 10.1016/j.archoralbio.2007.12.007 CrossRef
Cao J, Singleton P, Majumdar S, Burghardt A, Bourguignon GJ, Halloran BP: Hyaluronan increases RANKL expression in mouse primary osteoblasts through CD44. A potential role in age-related bone loss. J Bone Miner Res. 2003, 18 (S2): S78-Ref Type: Abstract.
Ricciardelli C, Russell DL, Ween MP, Mayne K, Suwiwat S, Byers S: Formation of hyaluronan- and versican-rich pericellular matrix by prostate cancer cells promotes cell motility. J Biol Chem. 2007, 282: 10814-10825. 10.1074/jbc.M606991200 CrossRef
Dhir R, Gau JT, Krill D, Bastacky S, Bahnson RR, Cooper DL: CD44 Expression in Benign and Neoplastic Human Prostates. Mol Diagn. 1997, 2: 197-204. 10.1016/S1084-8592(97)80029-X CrossRef
Grier DG, Thompson A, Kwasniewska A, McGonigle GJ, Halliday HL, Lappin TR: The pathophysiology of HOX genes and their role in cancer. J Pathol. 2005, 205: 154-171. 10.1002/path.1710 CrossRef
Roccisana JL, Kawanabe N, Kajiya H, Koide M, Roodman GD, Reddy SV: Functional role for heat shock factors in the transcriptional regulation of human RANK ligand gene expression in stromal/osteoblast cells. J Biol Chem. 2004, 279: 10500-10507. CrossRef
Cao JJ, Singleton PA, Majumdar S, Boudignon B, Burghardt A, Kurimoto P: Hyaluronan increases RANKL expression in bone marrow stromal cells through CD44. J Bone Miner Res. 2005, 20: 30-40. 10.1359/JBMR.041014 CrossRef
Yu C, Yao Z, Dai J, Zhang H, Escara-Wilke J, Zhang X: ALDH activity indicates increased tumorigenic cells, but not cancer stem cells, in prostate cancer cell lines. In Vivo. 2011, 25: 69-76.
Mori K, Kitazawa R, Kondo T, Maeda S, Yamaguchi A, Kitazawa S: Modulation of mouse RANKL gene expression by Runx2 and PKA pathway. J Cell Biochem. 2006, 98: 1629-1644. 10.1002/jcb.20891 CrossRef
Yeung F, Law WK, Yeh CH, Westendorf JJ, Zhang Y, Wang R: Regulation of human osteocalcin promoter in hormone-independent human prostate cancer cells. J Biol Chem. 2002, 277: 2468-2476. 10.1074/jbc.M105947200 CrossRef
van der Deen M, Akech J, Wang T, FitzGerald TJ, Altieri DC, Languino LR: The cancer-related Runx2 protein enhances cell growth and responses to androgen and TGFbeta in prostate cancer cells. J Cell Biochem. 2010, 109: 828-837. PubMedCentral
Fowler M, Borazanci E, McGhee L, Pylant SW, Williams BJ, Glass J: RUNX1 (AML-1) and RUNX2 (AML-3) cooperate with prostate-derived Ets factor to activate transcription from the PSA upstream regulatory region. J Cell Biochem. 2006, 97: 1-17. 10.1002/jcb.20664 CrossRef
Chua CW, Chiu YT, Yuen HF, Chan KW, Man K, Wang X: Suppression of androgen-independent prostate cancer cell aggressiveness by FTY720: validating Runx2 as a potential antimetastatic drug screening platform. Clin Cancer Res. 2009, 15: 4322-4335. 10.1158/1078-0432.CCR-08-3157 CrossRef
Afzal F, Pratap J, Ito K, Ito Y, Stein JL, van Wijnen AJ: Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription. J Cell Physiol. 2005, 204: 63-72. 10.1002/jcp.20258 CrossRef
Lee KS, Hong SH, Bae SC: Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene. 2002, 21: 7156-7163. 10.1038/sj.onc.1205937 CrossRef
Selvamurugan N, Kwok S, Partridge NC: Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase-3 expression in human breast cancer cells. J Biol Chem. 2004, 279: 27764-27773. 10.1074/jbc.M312870200 CrossRef
Leboy P, Grasso-Knight G, D’Angelo M, Volk SW, Lian JV, Drissi H: Smad-Runx interactions during chondrocyte maturation. J Bone Joint Surg Am. 2001, 83-A (Suppl 1): S15-S22.
Ohyama Y, Tanaka T, Shimizu T, Matsui H, Sato H, Koitabashi N: Runx2/Smad3 complex negatively regulates TGF-beta-induced connective tissue growth factor gene expression in vascular smooth muscle cells. J Atheroscler Thromb. 2012, 19: 23-35. 10.5551/jat.9753 CrossRef
Tanikawa R, Tanikawa T, Hirashima M, Yamauchi A, Tanaka Y: Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway. Biochem Biophys Res Commun. 2010, 394: 317-322. 10.1016/j.bbrc.2010.02.175 CrossRef
Choo CK, Ling MT, Chan KW, Tsao SW, Zheng Z, Zhang D: Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames. Prostate. 1999, 40: 150-158. 10.1002/(SICI)1097-0045(19990801)40:3<150::AID-PROS2>3.0.CO;2-7 CrossRef
Gupta A, Lee BS, Khadeer MA, Tang Z, Chellaiah M, Abu-Amer Y: Leupaxin is a critical adaptor protein in the adhesion zone of the osteoclast. J Bone Miner Res. 2003, 18: 669-685. 10.1359/jbmr.2003.18.4.669 CrossRef
Desai B, Ma T, Zhu J, Chellaiah MA: Characterization of the expression of variant and standard CD44 in prostate cancer cells: identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface. J Cell Biochem. 2009, 108: 272-284. 10.1002/jcb.22248 CrossRef
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262 CrossRef
- Integrin αvβ3 and CD44 pathways in metastatic prostate cancer cells support osteoclastogenesis via a Runx2/Smad 5/receptor activator of NF-κB ligand signaling axis
Meenakshi A Chellaiah
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II