Cyr61 silencing reduces vascularization and dissemination of osteosarcoma tumors

ABSTRACT Osteosarcoma is the most prevalent primary pediatric cancer-related bone disease. These tumors frequently develop resistance to chemotherapy and are highly metastatic, leading to

poor outcome. Thus, there is a need for new therapeutic strategies that can prevent cell dissemination. We previously showed that CYR61/CCN1 expression in osteosarcoma cells is correlated to

aggressiveness both _in vitro_ and _in vivo_ in mouse models, as well as in patients. In this study, we found that CYR61 is a critical contributor to the vascularization of primary tumor.

We demonstrate that silencing CYR61, using lentiviral transduction, leads to a significant reduction in expression level of pro-angiogenic markers such as VEGF, FGF2, PECAM and angiopoietins

concomitantly to an increased expression of major anti-angiogenic markers such as thrombospondin-1 and SPARC. Matrix metalloproteinase-2 family member expression, a key pathway in

osteosarcoma metastatic capacity was also downregulated when CYR61 was downregulated in osteosarcoma cells. Using a metastatic murine model, we show that CYR61 silencing in osteosarcoma

cells results in reduced tumor vasculature and slows tumor growth compared with control. We also find that microvessel density correlates with lung metastasis occurrence and that CYR61

silencing in osteosarcoma cells limits the number of metastases. Taken together, our data indicate that CYR61 silencing can blunt the malignant behavior of osteosarcoma tumor cells by

limiting primary tumor growth and dissemination process. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS

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2021 ANTISENSE MEDIATED BLOCKADE OF DICKKOPF 1 ATTENUATES TUMOR SURVIVAL, METASTASES AND BONE DAMAGE IN EXPERIMENTAL OSTEOSARCOMA Article Open access 13 January 2025 REFERENCES * Cheng YY,

Huang L, Lee KM, Li K, Kumta SM . Alendronate regulates cell invasion and MMP-2 secretion in human osteosarcoma cell lines. _Pediatr Blood Cancer_ 2004; 42: 410–415. Article  CAS  Google

Scholar  * Fromigue O, Hamidouche Z, Marie PJ . Blockade of the RhoA-JNK-c-Jun-MMP2 cascade by atorvastatin reduces osteosarcoma cell invasion. _J Biol Chem_ 2008; 283: 30549–30556. Article

  CAS  Google Scholar  * Xin ZF, Kim YK, Jung ST . Risedronate inhibits human osteosarcoma cell invasion. _J Exp Clin Cancer Res_ 2009; 28: 105. Article  Google Scholar  * Foukas AF,

Deshmukh NS, Grimer RJ, Mangham DC, Mangos EG, Taylor S . Stage-IIB osteosarcomas around the knee. A study of MMP-9 in surviving tumour cells. _J Bone Joint Surg Br_ 2002; 84: 706–711.

Article  CAS  Google Scholar  * Zhou Q, Zhu Y, Deng Z, Long H, Zhang S, Chen X . VEGF and EMMPRIN expression correlates with survival of patients with osteosarcoma. _Surg Oncol_ 2011; 20:

13–19. Article  CAS  Google Scholar  * Folkman J . Angiogenesis. _Annu Rev Med_ 2006; 57: 1–18. Article  CAS  Google Scholar  * Yang J, Yang D, Sun Y, Sun B, Wang G, Trent JC _et al_.

Genetic amplification of the vascular endothelial growth factor (VEGF) pathway genes, including VEGFA, in human osteosarcoma. _Cancer_ 2011; 117: 4925–4938. Article  CAS  Google Scholar  *

Lammli J, Fan M, Rosenthal HG, Patni M, Rinehart E, Vergara G _et al_. Expression of vascular endothelial growth factor correlates with the advance of clinical osteosarcoma. _Int Orthop_

2012; 36: 2307–2313. Article  Google Scholar  * Lee YH, Tokunaga T, Oshika Y, Suto R, Yanagisawa K, Tomisawa M _et al_. Cell-retained isoforms of vascular endothelial growth factor (VEGF)

are correlated with poor prognosis in osteosarcoma. _Eur J Cancer_ 1999; 35: 1089–1093. Article  CAS  Google Scholar  * Kaya M, Wada T, Akatsuka T, Kawaguchi S, Nagoya S, Shindoh M _et al_.

Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. _Clin Cancer Res_ 2000; 6: 572–577. CAS  Google Scholar  *

Hara H, Akisue T, Fujimoto T, Imabori M, Kawamoto T, Kuroda R _et al_. Expression of VEGF and its receptors and angiogenesis in bone and soft tissue tumors. _Anticancer Res_ 2006; 26:

4307–4311. CAS  Google Scholar  * DuBois S, Demetri G . Markers of angiogenesis and clinical features in patients with sarcoma. _Cancer_ 2007; 109: 813–819. Article  CAS  Google Scholar  *

Rastogi S, Kumar R, Sankineani SR, Marimuthu K, Rijal L, Prakash S _et al_. Role of vascular endothelial growth factor as a tumour marker in osteosarcoma: a prospective study. _Int Orthop_

2012; 36: 2315–2321. Article  Google Scholar  * Chen D, Zhang YJ, Zhu KW, Wang WC . A systematic review of vascular endothelial growth factor expression as a biomarker of prognosis in

patients with osteosarcoma. _Tumour Biol_ 2013; 34: 1895–1899. Article  CAS  Google Scholar  * Kreuter M, Bieker R, Bielack SS, Auras T, Buerger H, Gosheger G _et al_. Prognostic relevance

of increased angiogenesis in osteosarcoma. _Clin Cancer Res_ 2004; 10: 8531–8537. Article  Google Scholar  * Lau LF . CCN1/CYR61: the very model of a modern matricellular protein. _Cell Mol

Life Sci_ 2011; 68: 3149–3163. Article  CAS  Google Scholar  * Leu SJ, Lam SC, Lau LF . Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in

human umbilical vein endothelial cells. _J Biol Chem_ 2002; 277: 46248–46255. Article  CAS  Google Scholar  * Chen N, Leu SJ, Todorovic V, Lam SC, Lau LF . Identification of a novel integrin

alphavbeta3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in vascular endothelial cells. _J Biol Chem_ 2004; 279: 44166–44176. Article  CAS  Google Scholar  * Mo FE,

Muntean AG, Chen CC, Stolz DB, Watkins SC, Lau LF . CYR61 (CCN1) is essential for placental development and vascular integrity. _Mol Cell Biol_ 2002; 22: 8709–8720. Article  CAS  Google

Scholar  * Babic AM, Kireeva ML, Kolesnikova TV, Lau LF . CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. _Proc Natl Acad Sci USA_

1998; 95: 6355–6360. Article  CAS  Google Scholar  * Menéndez JA, Mehmi I, Griggs DW, Lupu R . The angiogenic factor CYR61 in breast cancer: molecular pathology and therapeutic

perspectives. _Endocr Relat Cancer_ 2003; 10: 141–152. Article  Google Scholar  * Jiang WG, Watkins G, Fodstad O, Douglas-Jones A, Mokbel K, Mansel RE . Differential expression of the CCN

family members Cyr61, CTGF and Nov in human breast cancer. _Endocr Relat Cancer_ 2004; 11: 781–791. Article  CAS  Google Scholar  * Sun ZJ, Wang Y, Cai Z, Chen PP, Tong XJ, Xie D .

Involvement of Cyr61 in growth, migration, and metastasis of prostate cancer cells. _Br J Cancer_ 2008; 99: 1656–1667. Article  CAS  Google Scholar  * Fromigue O, Hamidouche Z, Vaudin P,

Lecanda F, Patino A, Barbry P _et al_. CYR61 downregulation reduces osteosarcoma cell invasion, migration, and metastasis. _J Bone Miner Res_ 2011; 26: 1533–1542. Article  CAS  Google

Scholar  * Sabile AA, Arlt MJ, Muff R, Bode B, Langsam B, Bertz J _et al_. Cyr61 expression in osteosarcoma indicates poor prognosis and promotes intratibial growth and lung metastasis in

mice. _J Bone Miner Res_ 2012; 27: 58–67. Article  CAS  Google Scholar  * Wang L, Zhang Q, Chen W, Shan B, Ding Y, Zhang G _et al_. B7-H3 is overexpressed in patients suffering osteosarcoma

and associated with tumor aggressiveness and metastasis. _PLoS ONE_ 2013; 8: e70689. Article  CAS  Google Scholar  * Wen X, Liu H, Yu K, Liu Y . Matrix metalloproteinase 2 expression and

survival of patients with osteosarcoma: a meta-analysis. _Tumour Biol_ 2014; 35: 845–848. Article  CAS  Google Scholar  * Sternlicht MD, Werb Z . How matrix metalloproteinases regulate cell

behavior. _Annu Rev Cell Dev Biol_ 2001; 17: 463–516. Article  CAS  Google Scholar  * Oda Y, Yamamoto H, Tamiya S, Matsuda S, Tanaka K, Yokoyama R _et al_. CXCR4 and VEGF expression in the

primary site and the metastatic site of human osteosarcoma: analysis within a group of patients, all of whom developed lung metastasis. _Mod Pathol_ 2006; 19: 738–745. Article  CAS  Google

Scholar  * Pignochino Y, Grignani G, Cavalloni G, Motta M, Tapparo M, Bruno S _et al_. Sorafenib blocks tumour growth, angiogenesis and metastatic potential in preclinical models of

osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2, MCL-1 and ezrin pathways. _Mol Cancer_ 2009; 8: 118. Article  Google Scholar  * Wang CT, Lin CS, Shiau CW,

Chu PY, Hsiao CC, Chiang YL _et al_. SC-1, a sorafenib derivative, shows anti-tumor effects in osteogenic sarcoma cells. _J Orthop Res_ 2013; 31: 335–342. Article  Google Scholar  * Grignani

G, Palmerini E, Dileo P, Asaftei SD, D'Ambrosio L, Pignochino Y _et al_. A phase II trial of sorafenib in relapsed and unresectable high-grade osteosarcoma after failure of standard

multimodal therapy: an Italian Sarcoma Group study. _Ann Oncol_ 2012; 23: 508–516. Article  CAS  Google Scholar  * Wagner L, Turpin B, Nagarajan R, Weiss B, Cripe T, Geller J . Pilot study

of vincristine, oral irinotecan, and temozolomide (VOIT regimen) combined with bevacizumab in pediatric patients with recurrent solid tumors or brain tumors. _Pediatr Blood Cancer_ 2013; 60:

1447–1451. Article  Google Scholar  * Athanasopoulos AN, Schneider D, Keiper T, Alt V, Pendurthi UR, Liegibel UM _et al_. Vascular endothelial growth factor (VEGF)-induced up-regulation of

CCN1 in osteoblasts mediates proangiogenic activities in endothelial cells and promotes fracture healing. _J Biol Chem_ 2007; 282: 26746–26753. Article  CAS  Google Scholar  * Babkina IV,

Osipov DA, Solovyov YN, Bulycheva IV, Machak GN, Aliev MD _et al_. Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the sera of patients with primary

osteosarcomas. _Bull Exp Biol Med_ 2009; 148: 246–249. Article  CAS  Google Scholar  * Li C, Zhan C, Chen Y, Fu Q, Zhu XD, He DW _et al_. Analysis report for osteosarcoma expression profile.

_Eur Rev Med Pharmacol Sci_ 2013; 17: 2804–2809. CAS  Google Scholar  * Ren T, Qing Y, Dai N, Li M, Qian C, Yang Y _et al_. Apurinic/apyrimidinic endonuclease 1 induced upregulation of

fibroblast growth factor 2 and its receptor 3 induces angiogenesis in human osteosarcoma cells. _Cancer Sci_ 2014; 105: 186–194. Article  CAS  Google Scholar  * Odagiri H, Kadomatsu T, Endo

M, Masuda T, Morioka MS, Fukuhara S _et al_. The secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through integrin α5β1, p38 MAPK, and matrix metalloproteinases. _Sci

Signal_ 2014; 7: ra7. Article  Google Scholar  * Mintz MB, Sowers R, Brown KM, Hilmer SC, Mazza B, Huvos AG _et al_. An expression signature classifies chemotherapy-resistant pediatric

osteosarcoma. _Cancer Res_ 2005; 65: 1748–1754. Article  CAS  Google Scholar  * Maris JM, Courtright J, Houghton PJ, Morton CL, Gorlick R, Kolb EA _et al_. Initial testing of the VEGFR

inhibitor AZD2171 by the pediatric preclinical testing program. _Pediatr Blood Cancer_ 2008; 50: 581–587. Article  Google Scholar  * Kireeva ML, Lam SC, Lau LF . Adhesion of human umbilical

vein endothelial cells to the immediate-early gene product Cyr61 is mediated through integrin alphavbeta3. _J Biol Chem_ 1998; 273: 3090–3096. Article  CAS  Google Scholar  * Menendez JA,

Vellon L, Mehmi I, Teng PK, Griggs DW, Lupu R . A novel CYR61-triggered 'CYR61-alphavbeta3 integrin loop' regulates breast cancer cell survival and chemosensitivity through

activation of ERK1/ERK2 MAPK signaling pathway. _Oncogene_ 2005; 24: 761–779. Article  CAS  Google Scholar  * Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, Shattil SJ _et al_. A

mechanism for modulation of cellular responses to VEGF: activation of the integrins. _Mol Cell_ 2000; 6: 851–860. CAS  Google Scholar  * Wilkinson-Berka JL, Jones D, Taylor G, Jaworski K,

Kelly DJ, Ludbrook SB _et al_. SB-267268, a nonpeptidic antagonist of alpha(v)beta3 and alpha(v)beta5 integrins, reduces angiogenesis and VEGF expression in a mouse model of retinopathy of

prematurity. _Invest Ophthalmol Vis Sci_ 2006; 47: 1600–1605. Article  Google Scholar  * Tsou R, Isik FF . Integrin activation is required for VEGF and FGF receptor protein presence on human

microvascular endothelial cells. _Mol Cell Biochem_ 2001; 224: 81–89. Article  CAS  Google Scholar  * Huang Y, Lin Z, Zhuang J, Chen Y, Lin J . Prognostic significance of alpha V integrin

and VEGF in osteosarcoma after chemotherapy. _Onkologie_ 2008; 31: 535–540. Article  CAS  Google Scholar  * Gomes N, Legrand C, Fauvel-Lafève F . Shear stress induced release of von

Willebrand factor and thrombospondin-1 in HUVEC extracellular matrix enhances breast tumour cell adhesion. _Clin Exp Metastasis_ 2005; 22: 215–223. Article  CAS  Google Scholar  * North S,

Moenner M, Bikfalvi A . Recent developments in the regulation of the angiogenic switch by cellular stress factors in tumors. _Cancer Lett_ 2005; 218: 1–14. Article  CAS  Google Scholar  *

Estrada R, Li N, Sarojini H, An J, Lee MJ, Wang E . Secretome from mesenchymal stem cells induces angiogenesis via Cyr61. _J Cell Physiol_ 2009; 219: 563–571. Article  CAS  Google Scholar  *

Meyuhas R, Pikarsky E, Tavor E, Klar A, Abramovitch R, Hochman J _et al_. A Key role for cyclic AMP-responsive element binding protein in hypoxia-mediated activation of the angiogenesis

factor CCN1 (CYR61) in tumor cells. _Mol Cancer Res_ 2008; 6: 1397–1409. Article  CAS  Google Scholar  * Vilalta M, Dégano IR, Bagó J, Gould D, Santos M, García-Arranz M _et al_.

Biodistribution, long-term survival, and safety of human adipose tissue-derived mesenchymal stem cells transplanted in nude mice by high sensitivity non-invasive bioluminescence imaging.

_Stem Cells Dev_ 2008; 17: 993–1003. Article  Google Scholar  * Román I, Vilalta M, Rodriguez J, Matthies AM, Srouji S, Livne E _et al_. Analysis of progenitor cell-scaffold combinations by

in vivo non-invasive photonic imaging. _Biomaterials_ 2007; 28: 2718–2728. Article  Google Scholar  Download references ACKNOWLEDGEMENTS NH is a recipient of a PhD award from the Ministère

de la Recherche (Paris, France). This work was supported in part by Inserm (France), by Fondation de l’Avenir pour la Recherche Médicale Appliquée (Paris, France), by Red Temática de

Investigación Cooperativa (TERCEL, Spain) and SAF2012-33404 from MINECO (Spain). We thank the Department of Medical Biology and Pathology of Gustave Roussy Institute (Dr Adam; Villejuif,

France) for their contribution. AUTHOR INFORMATION Author notes * M Vilalta Present address: 8Current address: Division of Radiation and Cancer Biology, Department of Radiation Oncology,

Molecular Imaging Program at Stanford, Stanford University, Stanford, CA, USA., AUTHORS AND AFFILIATIONS * Inserm U981, Institut de cancérologie Gustave Roussy, Villejuif, France N Habel 

& O Fromigué * Gustave Roussy, Villejuif, France N Habel, O Bawa, P Opolon & O Fromigué * Université Paris Sud, Orsay, France N Habel, O Bawa, P Opolon & O Fromigué * Université

Paris Diderot, Paris, France N Habel * Cell therapy group, Institute for Advanced Chemistry of Catalonia (CSIC), Barcelona, Spain M Vilalta & J Blanco * Institut de Recherche Intégrée en

cancérologie à Villejuif (IRCIV), Laboratoire de pathologie expérimentale, Gustave Roussy, Villejuif, France O Bawa & P Opolon * Networking Biomedical Research Center on Bioengineering,

Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain J Blanco Authors * N Habel View author publications You can also search for this author inPubMed Google Scholar * M Vilalta View

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THIS ARTICLE Habel, N., Vilalta, M., Bawa, O. _et al._ Cyr61 silencing reduces vascularization and dissemination of osteosarcoma tumors. _Oncogene_ 34, 3207–3213 (2015).

https://doi.org/10.1038/onc.2014.232 Download citation * Received: 01 May 2014 * Revised: 10 June 2014 * Accepted: 24 June 2014 * Published: 28 July 2014 * Issue Date: 11 June 2015 * DOI:

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