Identification of BMP2 as an epigenetically silenced growth inhibitor in rhabdomyosarcoma

Wolf,Sebastian; Hagl,Beate; Kappler,Roland

doi:10.3892/ijo.2014.2312

Identification of BMP2 as an epigenetically silenced growth inhibitor in rhabdomyosarcoma

Authors:
- Sebastian Wolf
- Beate Hagl
- Roland Kappler
View Affiliations

Affiliations: Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University Munich, D-80337 Munich, Germany
Published online on: February 27, 2014 https://doi.org/10.3892/ijo.2014.2312
Pages: 1727-1735

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of infancy and although therapy has improved over the years, mortality is still fairly high. The establishment of new treatments has been hampered by the limited knowledge of the molecular mechanisms driving development of RMS. One characteristic of cancer cells is aberrant DNA methylation, which could lead to silencing of tumor suppressor genes. However, only a few epigenetically silenced genes have been described in RMS so far. We performed an expression profiling analysis of three RMS cell lines that were treated with the demethylating agent 5'-aza-2'-deoxycytidine (5-Aza‑dC) facilitating re-expression of epigenetically silenced genes. This treatment induced the gene BMP2 (bone morphogenetic protein 2) throughout all cell lines. Detailed methylation analysis of CpG sites in the BMP2 promoter region by bisulfite sequencing and methylation-specific PCR revealed that a high degree of DNA methylation is causatively associated with the suppression of BMP2 in RMS cells. Consequently, treatment of the RMS cell lines with 5-Aza-dC resulted in DNA demethylation of the BMP2 promoter, most prominently in alveolar RMS. Supplementation of recombinant human BMP2 (rhBMP2) led to a reduced viability of RMS cells. Altogether, these findings suggest that suppression of BMP2 by epigenetic silencing may play a critical role in the genesis of RMS, thereby providing a rationale for the development of a new treatment strategy for RMS.

View Figures

View References

1.	Paulino AC and Okcu MF: Rhabdomyosarcoma. Curr Probl Cancer. 32:7–34. 2008. View Article : Google Scholar
2.	Crist W, Gehan EA, Ragab AH, Dickman PS, Donaldson SS, Fryer C, Hammond D, Hays DM, Herrmann J, Heyn R, et al: The Third Intergroup Rhabdomyosarcoma Study. J Clin Oncol. 13:610–630. 1995.PubMed/NCBI
3.	Meyer WH and Spunt SL: Soft tissue sarcomas of childhood. Cancer Treat Rev. 30:269–280. 2004. View Article : Google Scholar : PubMed/NCBI
4.	Newton WA Jr, Soule EH, Hamoudi AB, Reiman HM, Shimada H, Beltangady M and Maurer H: Histopathology of childhood sarcomas, Intergroup Rhabdomyosarcoma Studies I and II: clinicopathologic correlation. J Clin Oncol. 6:67–75. 1988.PubMed/NCBI
5.	Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, Bridge JA, Crist WM, Triche TJ and Barr FG: PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children’s oncology group. J Clin Oncol. 20:2672–2679. 2002.PubMed/NCBI
6.	Loh WE Jr, Scrable HJ, Livanos E, Arboleda MJ, Cavenee WK, Oshimura M and Weissman BE: Human chromosome 11 contains two different growth suppressor genes for embryonal rhabdomyosarcoma. Proc Natl Acad Sci USA. 89:1755–1759. 1992. View Article : Google Scholar : PubMed/NCBI
7.	Barr FG: Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma. Oncogene. 20:5736–5746. 2001. View Article : Google Scholar : PubMed/NCBI
8.	Fredericks WJ, Galili N, Mukhopadhyay S, Rovera G, Bennicelli J, Barr FG and Rauscher FJ III: The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol Cell Biol. 15:1522–1535. 1995.PubMed/NCBI
9.	Herman JG and Baylin SB: Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 349:2042–2054. 2003. View Article : Google Scholar : PubMed/NCBI
10.	Harada K, Toyooka S, Maitra A, Maruyama R, Toyooka KO, Timmons CF, Tomlinson GE, Mastrangelo D, Hay RJ, Minna JD and Gazdar AF: Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines. Oncogene. 21:4345–4349. 2002. View Article : Google Scholar : PubMed/NCBI
11.	Wong IH, Chan J, Wong J and Tam PK: Ubiquitous aberrant RASSF1A promoter methylation in childhood neoplasia. Clin Cancer Res. 10:994–1002. 2004. View Article : Google Scholar : PubMed/NCBI
12.	Sugawara W, Haruta M, Sasaki F, Watanabe N, Tsunematsu Y, Kikuta A and Kaneko Y: Promoter hypermethylation of the RASSF1A gene predicts the poor outcome of patients with hepatoblastoma. Pediatr Blood Cancer. 49:240–249. 2007. View Article : Google Scholar : PubMed/NCBI
13.	Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM and Wang EA: Novel regulators of bone formation: molecular clones and activities. Science. 242:1528–1534. 1988. View Article : Google Scholar : PubMed/NCBI
14.	Lyons KM, Hogan BL and Robertson EJ: Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development. Mech Dev. 50:71–83. 1995. View Article : Google Scholar
15.	Duprez DM, Coltey M, Amthor H, Brickell PM and Tickle C: Bone morphogenetic protein-2 (BMP-2) inhibits muscle development and promotes cartilage formation in chick limb bud cultures. Dev Biol. 174:448–452. 1996. View Article : Google Scholar : PubMed/NCBI
16.	Narita T, Saitoh K, Kameda T, Kuroiwa A, Mizutani M, Koike C, Iba H and Yasugi S: BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression. Development. 127:981–988. 2000.PubMed/NCBI
17.	Chen D, Zhao M and Mundy GR: Bone morphogenetic proteins. Growth Factors. 22:233–241. 2004. View Article : Google Scholar
18.	Hollnagel A, Oehlmann V, Heymer J, Ruther U and Nordheim A: Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem. 274:19838–19845. 1999. View Article : Google Scholar : PubMed/NCBI
19.	Horvath LG, Henshall SM, Kench JG, Turner JJ, Golovsky D, Brenner PC, O’Neill GF, Kooner R, Stricker PD, Grygiel JJ and Sutherland RL: Loss of BMP2, Smad8, and Smad4 expression in prostate cancer progression. Prostate. 59:234–242. 2004. View Article : Google Scholar : PubMed/NCBI
20.	Soda H, Raymond E, Sharma S, Lawrence R, Cerna C, Gomez L, Timony GA, Von Hoff DD and Izbicka E: Antiproliferative effects of recombinant human bone morphogenetic protein-2 on human tumor colony-forming units. Anticancer Drugs. 9:327–331. 1998. View Article : Google Scholar : PubMed/NCBI
21.	Kawamura C, Kizaki M and Ikeda Y: Bone morphogenetic protein (BMP)-2 induces apoptosis in human myeloma cells. Leuk Lymphoma. 43:635–639. 2002. View Article : Google Scholar : PubMed/NCBI
22.	Zhao H, Ayrault O, Zindy F, Kim JH and Roussel MF: Post-transcriptional down-regulation of Atoh1/Math1 by bone morphogenic proteins suppresses medulloblastoma development. Genes Dev. 22:722–727. 2008. View Article : Google Scholar : PubMed/NCBI
23.	Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e452001. View Article : Google Scholar : PubMed/NCBI
24.	Williamson D, Missiaglia E, de Reynies A, Pierron G, Thuille B, Palenzuela G, Thway K, Orbach D, Lae M, Freneaux P, Pritchard-Jones K, Oberlin O, Shipley J and Delattre O: Fusion gene-negative alveolar rhabdomyosarcoma is clinically and molecularly indistinguishable from embryonal rhabdomyosarcoma. J Clin Oncol. 28:2151–2158. 2010. View Article : Google Scholar : PubMed/NCBI
25.	Missiaglia E, Dalai I, Barbi S, Beghelli S, Falconi M, della Peruta M, Piemonti L, Capurso G, Di Florio A, delle Fave G, Pederzoli P, Croce CM and Scarpa A: Pancreatic endocrine tumors: expression profiling evidences a role for AKT-mTOR pathway. J Clin Oncol. 28:245–55. 2010. View Article : Google Scholar : PubMed/NCBI
26.	Esteller M: Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum Mol Genet. 16(Spec No 1): R50–R59. 2007. View Article : Google Scholar
27.	Burbee DG, Forgacs E, Zochbauer-Muller S, Shivakumar L, Fong K, Gao B, Randle D, Kondo M, Virmani A, Bader S, Sekido Y, Latif F, Milchgrub S, Toyooka S, Gazdar AF, Lerman MI, Zabarovsky E, White M and Minna JD: Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst. 93:691–699. 2001. View Article : Google Scholar : PubMed/NCBI
28.	Dammann R, Li C, Yoon JH, Chin PL, Bates S and Pfeifer GP: Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet. 25:315–319. 2000. View Article : Google Scholar : PubMed/NCBI
29.	Morrissey C, Martinez A, Zatyka M, Agathanggelou A, Honorio S, Astuti D, Morgan NV, Moch H, Richards FM, Kishida T, Yao M, Schraml P, Latif F and Maher ER: Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma. Cancer Res. 61:7277–7281. 2001.PubMed/NCBI
30.	Yoon JH, Dammann R and Pfeifer GP: Hypermethylation of the CpG island of the RASSF1A gene in ovarian and renal cell carcinomas. Int J Cancer. 94:212–217. 2001. View Article : Google Scholar : PubMed/NCBI
31.	Dammann R, Yang G and Pfeifer GP: Hypermethylation of the cpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers. Cancer Res. 61:3105–3109. 2001.PubMed/NCBI
32.	Nakaoka T, Gonda K, Ogita T, Otawara-Hamamoto Y, Okabe F, Kira Y, Harii K, Miyazono K, Takuwa Y and Fujita T: Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2. J Clin Invest. 100:2824–2432. 1997. View Article : Google Scholar : PubMed/NCBI
33.	Lee SJ and McPherron AC: Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA. 98:9306–9311. 2001. View Article : Google Scholar : PubMed/NCBI
34.	Suryawan A, Frank JW, Nguyen HV and Davis TA: Expression of the TGF-beta family of ligands is developmentally regulated in skeletal muscle of neonatal rats. Pediatr Res. 59:175–179. 2006. View Article : Google Scholar : PubMed/NCBI
35.	Howe JR, Bair JL, Sayed MG, Anderson ME, Mitros FA, Petersen GM, Velculescu VE, Traverso G and Vogelstein B: Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis. Nat Genet. 28:184–187. 2001. View Article : Google Scholar : PubMed/NCBI
36.	Kodach LL, Wiercinska E, de Miranda NF, Bleuming SA, Musler AR, Peppelenbosch MP, Dekker E, van den Brink GR, van Noesel CJ, Morreau H, Hommes DW, Ten Dijke P, Offerhaus GJ and Hardwick JC: The bone morphogenetic protein pathway is inactivated in the majority of sporadic colorectal cancers. Gastroenterology. 134:1332–1341. 2008. View Article : Google Scholar : PubMed/NCBI
37.	Wen XZ, Akiyama Y, Baylin SB and Yuasa Y: Frequent epigenetic silencing of the bone morphogenetic protein 2 gene through methylation in gastric carcinomas. Oncogene. 25:2666–2673. 2006. View Article : Google Scholar : PubMed/NCBI
38.	Mahoney SE, Yao Z, Keyes CC, Tapscott SJ and Diede SJ: Genome-wide DNA methylation studies suggest distinct DNA methylation patterns in pediatric embryonal and alveolar rhabdomyosarcomas. Epigenetics. 7:400–408. 2012. View Article : Google Scholar : PubMed/NCBI
39.	Bird A: DNA methylation patterns and epigenetic memory. Genes Dev. 16:6–21. 2002. View Article : Google Scholar
40.	Esteller M: Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet. 8:286–298. 2007. View Article : Google Scholar : PubMed/NCBI
41.	Pregizer S and Mortlock DP: Control of BMP gene expression by long-range regulatory elements. Cytokine Growth Factor Rev. 20:509–515. 2009. View Article : Google Scholar : PubMed/NCBI
42.	Chandler RL, Chandler KJ, McFarland KA and Mortlock DP: Bmp2 transcription in osteoblast progenitors is regulated by a distant 3′ enhancer located 156.3 kilobases from the promoter. Mol Cell Biol. 27:2934–2951. 2007.PubMed/NCBI
43.	Jabbour E, Issa JP, Garcia-Manero G and Kantarjian H: Evolution of decitabine development: accomplishments, ongoing investigations, and future strategies. Cancer. 112:2341–2351. 2008. View Article : Google Scholar : PubMed/NCBI
44.	Cameron EE, Bachman KE, Myohanen S, Herman JG and Baylin SB: Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet. 21:103–107. 1999. View Article : Google Scholar : PubMed/NCBI
45.	Mossman D, Kim KT and Scott RJ: Demethylation by 5-aza-2′-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists. BMC Cancer. 10:3662010.
46.	Tachibana K, Takeda K and Shiraishi M: 5-Aza-2′-deoxycytidine reactivates the CDH1 gene without influencing the methylation of the entire CpG island or histone modification in a human cancer cell line. Proc Japan Acad. 80:342–348. 2004.
47.	Ghosh-Choudhury N, Woodruff K, Qi W, Celeste A, Abboud SL and Ghosh Choudhury G: Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation. Biochem Biophys Res Commun. 272:705–711. 2000. View Article : Google Scholar
48.	Beck SE, Jung BH, Fiorino A, Gomez J, Rosario ED, Cabrera BL, Huang SC, Chow JY and Carethers JM: Bone morphogenetic protein signaling and growth suppression in colon cancer. Am J Physiol Gastrointest Liver Physiol. 291:G135–G145. 2006. View Article : Google Scholar : PubMed/NCBI
49.	Wen XZ, Miyake S, Akiyama Y and Yuasa Y: BMP-2 modulates the proliferation and differentiation of normal and cancerous gastric cells. Biochem Biophys Res Commun. 316:100–106. 2004. View Article : Google Scholar : PubMed/NCBI
50.	Wong GA, Tang V, El-Sabeawy F and Weiss RH: BMP-2 inhibits proliferation of human aortic smooth muscle cells via p21^Cip1/Waf1. Am J Physiol Endocrinol Metab. 284:E972–E979. 2003.PubMed/NCBI
51.	Du Y and Yip H: Effects of bone morphogenetic protein 2 on Id expression and neuroblastoma cell differentiation. Differentiation. 79:84–92. 2010. View Article : Google Scholar : PubMed/NCBI
52.	Ogata T, Wozney JM, Benezra R and Noda M: Bone morphogenetic protein 2 transiently enhances expression of a gene, Id (inhibitor of differentiation), encoding a helix-loop-helix molecule in osteoblast-like cells. Proc Natl Acad Sci USA. 90:9219–9222. 1993. View Article : Google Scholar
53.	Zhang S, Fantozzi I, Tigno DD, Yi ES, Platoshyn O, Thistlethwaite PA, Kriett JM, Yung G, Rubin LJ and Yuan JX: Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 285:L740–L754. 2003.PubMed/NCBI
54.	Iantosca MR, McPherson CE, Ho SY and Maxwell GD: Bone morphogenetic proteins-2 and -4 attenuate apoptosis in a cerebellar primitive neuroectodermal tumor cell line. J Neurosci Res. 56:248–258. 1999. View Article : Google Scholar : PubMed/NCBI
55.	Sherr CJ and Roberts JM: CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13:1501–1512. 1999. View Article : Google Scholar : PubMed/NCBI