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Hedgehog signaling induces osteosarcoma development through Yap1 and H19 overexpression

Abstract

Osteosarcoma is one of the most common bone tumors. However, the genetic basis for its pathogenesis remains elusive. Here, we investigated the roles of Hedgehog (Hh) signaling in osteosarcoma development. Genetically-engineered mice with ubiquitous upregulated Hh signaling specifically in mature osteoblasts develop focal bone overgrowth, which greatly resembles the early stage of osteosarcoma. However, these mice die within three months, which prohibits further analysis of tumor progression. We therefore generated a mouse model with partial upregulated Hh signaling in mature osteoblasts and crossed it into a p53 heterozygous background to potentiate tumor development. We found that these mutant mice developed malignant osteosarcoma with high penetrance. Isolated primary tumor cells were mainly osteoblastic and highly proliferative with many characteristics of human osteosarcomas. Allograft transplantation into immunocompromised mice displayed high tumorigenic potential. More importantly, both human and mouse tumor tissues express high level of yes-associated protein 1 (Yap1), a potent oncogene that is amplified in various cancers. We show that inhibition of Hh signaling reduces Yap1 expression and knockdown of Yap1 significantly inhibits tumor progression. Moreover, long non-coding RNA H19 is aberrantly expressed and induced by upregulated Hh signaling and Yap1 overexpression. Our results demonstrate that aberrant Hh signaling in mature osteoblasts is responsible for the pathogenesis of osteoblastic osteosarcoma through Yap1 and H19 overexpression.

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References

  1. Sweetnam R . Osteosarcoma. Brit J Hosp Med 1982; 28: 116–121.

    Google Scholar 

  2. Dorfman HD, Czerniak B . Bone cancers. Cancer 1995; 75: 203–210.

    Article  CAS  Google Scholar 

  3. Berman SD, Calo E, Landman AS, Danielian PS, Miller ES, West JC et al. Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage. Proc Natl Acad SciUSA 2008; 105: 11851–11856.

    Article  CAS  Google Scholar 

  4. Walkley CR, Qudsi R, Sankaran VG, Perry JA, Gostissa M, Roth SI et al. Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease. Genes Dev 2008; 22: 1662–1676.

    Article  CAS  Google Scholar 

  5. Calo E, Quintero-Estades JA, Danielian PS, Nedelcu S, Berman SD, Lees JA . Rb regulates fate choice and lineage commitment in vivo. Nature 2010; 466: 1110–1114.

    Article  Google Scholar 

  6. Mutsaers AJ, Ng AJ, Baker EK, Russell MR, Chalk AM, Wall M et al. Modeling distinct osteosarcoma subtypes in vivo using Cre:lox and lineage-restricted transgenic shRNA. Bone 2013; 55: 166–178.

    Article  CAS  Google Scholar 

  7. Kasper M, Regl G, Frischauf AM, Aberger F . GLI transcription factors: mediators of oncogenic Hedgehog signalling. Eur J Cancer 2006; 42: 437–445.

    Article  CAS  Google Scholar 

  8. Rubin LL, de Sauvage FJ . Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 2006; 5: 1026–1033.

    Article  CAS  Google Scholar 

  9. Fults DW . Modeling medulloblastoma with genetically engineered mice. Neurosurg focus 2005; 19: E7.

    Article  Google Scholar 

  10. Raffel C, Jenkins RB, Frederick L, Hebrink D, Alderete B, Fults DW et al. Sporadic medulloblastomas contain PTCH mutations. Cancer Res 1997; 57: 842–845.

    CAS  PubMed  Google Scholar 

  11. Reifenberger J, Wolter M, Weber RG, Megahed M, Ruzicka T, Lichter P et al. Missense mutations in SMOH in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system. Cancer Res 1998; 58: 1798–1803.

    CAS  PubMed  Google Scholar 

  12. Wetmore C . Sonic hedgehog in normal and neoplastic proliferation: insight gained from human tumors and animal models. Curr Opin Genet Dev 2003; 13: 34–42.

    Article  CAS  Google Scholar 

  13. Fernandez LA, Northcott PA, Dalton J, Fraga C, Ellison D, Angers S et al. YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation. Genes Dev 2009; 23: 2729–2741.

    Article  Google Scholar 

  14. Lamar JM, Stern P, Liu H, Schindler JW, Jiang ZG, Hynes RO . The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proc Natl Acad SciUSA 2012; 109: E2441–E2450.

    Article  CAS  Google Scholar 

  15. Overholtzer M, Zhang J, Smolen GA, Muir B, Li W, Sgroi DC et al. Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc Natl Acad SciUSA 2006; 103: 12405–12410.

    Article  CAS  Google Scholar 

  16. Zhang X, George J, Deb S, Degoutin JL, Takano EA, Fox SB et al. The Hippo pathway transcriptional co-activator, YAP, is an ovarian cancer oncogene. Oncogene 2011; 30: 2810–2822.

    Article  CAS  Google Scholar 

  17. Xu MZ, Yao TJ, Lee NP, Ng IO, Chan YT, Zender L et al. Yes-associated protein is an independent prognostic marker in hepatocellular carcinoma. Cancer 2009; 115: 4576–4585.

    Article  CAS  Google Scholar 

  18. Muramatsu T, Imoto I, Matsui T, Kozaki K, Haruki S, Sudol M et al. YAP is a candidate oncogene for esophageal squamous cell carcinoma. Carcinogenesis 2011; 32: 389–398.

    Article  CAS  Google Scholar 

  19. Yagi R, Chen LF, Shigesada K, Murakami Y, Ito YA . WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator. EMBO J 1999; 18: 2551–2562.

    Article  CAS  Google Scholar 

  20. Zaidi SK, Sullivan AJ, Medina R, Ito Y, van Wijnen AJ, Stein JL et al. Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. EMBO J 2004; 23: 790–799.

    Article  CAS  Google Scholar 

  21. Hirotsu M, Setoguchi T, Sasaki H, Matsunoshita Y, Gao H, Nagao H et al. Smoothened as a new therapeutic target for human osteosarcoma. Molecular cancer 2010; 9: 5.

    Article  Google Scholar 

  22. Nagao H, Ijiri K, Hirotsu M, Ishidou Y, Yamamoto T, Nagano S et al. Role of GLI2 in the growth of human osteosarcoma. J Pathol 2011; 224: 169–179.

    Article  CAS  Google Scholar 

  23. Matouk IJ, Abbasi I, Hochberg A, Galun E, Dweik H, Akkawi M . Highly upregulated in liver cancer noncoding RNA is overexpressed in hepatic colorectal metastasis. Eur JGastroen Hepat 2009; 21: 688–692.

    Article  CAS  Google Scholar 

  24. Ponting CP, Oliver PL, Reik W . Evolution and functions of long noncoding RNAs. Cell 2009; 136: 629–641.

    Article  CAS  Google Scholar 

  25. Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 2007; 129: 1311–1323.

    Article  CAS  Google Scholar 

  26. Schmidt LH, Spieker T, Koschmieder S, Schaffers S, Humberg J, Jungen D et al. The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth. J Thorac Oncol 2011; 6: 1984–1992.

    Article  Google Scholar 

  27. Wang J, Liu X, Wu H, Ni P, Gu Z, Qiao Y et al. CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer. Nucleic Acids Res 2010; 38: 5366–5383.

    Article  CAS  Google Scholar 

  28. Pachnis V, Brannan CI, Tilghman SM . The structure and expression of a novel gene activated in early mouse embryogenesis. EMBO J 1988; 7: 673–681.

    Article  CAS  Google Scholar 

  29. Ulaner GA, Vu TH, Li T, Hu JF, Yao XM, Yang Y et al. Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site. Hum Mol Gen 2003; 12: 535–549.

    Article  CAS  Google Scholar 

  30. Mak KK, Bi Y, Wan C, Chuang PT, Clemens T, Young M et al. Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression. Dev Cell 2008; 14: 674–688.

    Article  CAS  Google Scholar 

  31. Venkatachalam S, Tyner SD, Pickering CR, Boley S, Recio L, French JE et al. Is p53 haploinsufficient for tumor suppression? Implications for the p53+/− mouse model in carcinogenicity testing. Toxicol Pathol 2001; 29: 147–154.

    Article  CAS  Google Scholar 

  32. Mak KK, Chen MH, Day TF, Chuang PT, Yang Y . Wnt/beta-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation. Development 2006; 133: 3695–3707.

    Article  CAS  Google Scholar 

  33. Zhang L, Ren F, Zhang Q, Chen Y, Wang B, Jiang J . The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. Dev Cell 2008; 14: 377–387.

    Article  CAS  Google Scholar 

  34. Zhang J, Ji JY, Yu M, Overholtzer M, Smolen GA, Wang R et al. YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway. Nat Cell Biol 2009; 11: 1444–1450.

    Article  CAS  Google Scholar 

  35. Jones KB, Salah Z, Del Mare S, Galasso M, Gaudio E, Nuovo GJ et al. miRNA signatures associate with pathogenesis and progression of osteosarcoma. Cancer Res 2012; 72: 1865–1877.

    Article  CAS  Google Scholar 

  36. Li N, Yang R, Zhang W, Dorfman H, Rao P, Gorlick R . Genetically transforming human mesenchymal stem cells to sarcomas: changes in cellular phenotype and multilineage differentiation potential. Cancer 2009; 115: 4795–4806.

    Article  CAS  Google Scholar 

  37. Mohseny AB, Szuhai K, Romeo S, Buddingh EP, Briaire-de Bruijn I, de Jong D et al. Osteosarcoma originates from mesenchymal stem cells in consequence of aneuploidization and genomic loss of Cdkn2. J Pathol 2009; 219: 294–305.

    Article  CAS  Google Scholar 

  38. Tang N, Song WX, Luo J, Haydon RC, He TC . Osteosarcoma development and stem cell differentiation. Clin Orthop Relat Res 2008; 466: 2114–2130.

    Article  Google Scholar 

  39. Molyneux SD, Di Grappa MA, Beristain AG, McKee TD, Wai DH, Paderova J et al. Prkar1a is an osteosarcoma tumor suppressor that defines a molecular subclass in mice. J Clin Invest 2010; 120: 3310–3325.

    Article  CAS  Google Scholar 

  40. Warzecha J, Gottig S, Chow KU, Bruning C, Percic D, Boehrer S et al. Inhibition of osteosarcoma cell proliferation by the Hedgehog-inhibitor cyclopamine. J Chemotherapy 2007; 19: 554–561.

    Article  CAS  Google Scholar 

  41. McClatchey AI, Saotome I, Mercer K, Crowley D, Gusella JF, Bronson RT et al. Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors. Genes Dev 1998; 12: 1121–1133.

    Article  CAS  Google Scholar 

  42. Zhang N, Bai H, David KK, Dong J, Zheng Y, Cai J et al. The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev Cell 2010; 19: 27–38.

    Article  CAS  Google Scholar 

  43. Galindo M, Pratap J, Young DW, Hovhannisyan H, Im HJ, Choi JY et al. The bone-specific expression of Runx2 oscillates during the cell cycle to support a G1-related antiproliferative function in osteoblasts. J Biol Chem 2005; 280: 20274–20285.

    Article  CAS  Google Scholar 

  44. Teplyuk NM, Galindo M, Teplyuk VI, Pratap J, Young DW, Lapointe D et al. Runx2 regulates G protein-coupled signaling pathways to control growth of osteoblast progenitors. J Biol Chem 2008; 283: 27585–27597.

    Article  CAS  Google Scholar 

  45. Lu XY, Lu Y, Zhao YJ, Jaeweon K, Kang J, Xiao-Nan L et al. Cell cycle regulator gene CDC5L, a potential target for 6p12-p21 amplicon in osteosarcoma. Molecular cancer research: MCR 2008; 6: 937–946.

    Article  CAS  Google Scholar 

  46. Sadikovic B, Thorner P, Chilton-Macneill S, Martin JW, Cervigne NK, Squire J et al. Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy. BMC cancer 2010; 10: 202.

    Article  Google Scholar 

  47. Andela VB, Siddiqui F, Groman A, Rosier RN . An immunohistochemical analysis to evaluate an inverse correlation between Runx2/Cbfa1 and NF kappa B in human osteosarcoma. J Clin Pathol 2005; 58: 328–330.

    Article  CAS  Google Scholar 

  48. Pratap J, Javed A, Languino LR, van Wijnen AJ, Stein JL, Stein GS et al. The Runx2 osteogenic transcription factor regulates matrix metalloproteinase 9 in bone metastatic cancer cells and controls cell invasion. Mol Cell Biol 2005; 25: 8581–8591.

    Article  CAS  Google Scholar 

  49. Won KY, Park HR, Park YK . Prognostic implication of immunohistochemical Runx2 expression in osteosarcoma. Tumori 2009; 95: 311–316.

    Article  CAS  Google Scholar 

  50. Ulaner GA, Yang Y, Hu JF, Li T, Vu TH, Hoffman AR . CTCF binding at the insulin-like growth factor-II (IGF2)/H19 imprinting control region is insufficient to regulate IGF2/H19 expression in human tissues. Endocrinology 2003; 144: 4420–4426.

    Article  CAS  Google Scholar 

  51. Arima T, Matsuda T, Takagi N, Wake N . Association of IGF2 and H19 imprinting with choriocarcinoma development. Cancer Genet Cytogen 1997; 93: 39–47.

    Article  CAS  Google Scholar 

  52. Matouk IJ, DeGroot N, Mezan S, Ayesh S, Abu-lail R, Hochberg A et al. The H19 non-coding RNA is essential for human tumor growth. PloS one 2007; 2: e845.

    Article  Google Scholar 

  53. Hibi K, Nakamura H, Hirai A, Fujikake Y, Kasai Y, Akiyama S et al. Loss of H19 imprinting in esophageal cancer. Cancer Res 1996; 56: 480–482.

    CAS  PubMed  Google Scholar 

  54. Tanos V, Ariel I, Prus D, De-Groot N, Hochberg A . H19 and IGF2 gene expression in human normal, hyperplastic, and malignant endometrium. Int J Gynecol 2004; 14: 521–525.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Drs. Tin-lap Lee and David John Wilmshurst for critical comments on the manuscript. This work is supported by the Seed Fund of the School of Biomedical Sciences, The Chinese University of Hong Kong (4620504), Early Career Scheme of the Research Grant Council of the Hong Kong Special Administrative Region (CUHK479012), Direct Grant for research (CUHK2041745) and the Endowment Fund Research Grant of the United College, The Chinese University of Hong Kong (CA11189).

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Chan, L., Wang, W., Yeung, W. et al. Hedgehog signaling induces osteosarcoma development through Yap1 and H19 overexpression. Oncogene 33, 4857–4866 (2014). https://doi.org/10.1038/onc.2013.433

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