Cyr61 Silencing Reduces Vascularization and Dissemination of Osteosarcoma Tumors

SHORT COMMUNICATION Cyr61 silencing reduces vascularization and dissemination of osteosarcoma tumors

N Habel1,2,3,4, M Vilalta5,8, O Bawa2,3,6, P Opolon2,3,6, J Blanco5,7 and O Fromigué1,2,3

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.

Oncogene (2015) 34, 3207–3213; doi:10.1038/onc.2014.232; published online 28 July 2014

INTRODUCTION tumor-associated vascularization is essential for tumor growth 6 Osteosarcoma is a highly vascular and extremely destructive bone and progression, since it provides oxygen and nutrients. Thereby, malignancy that mainly affects children and young adults. It the expression of angiogenic markers frequently correlates with represents the most frequent pediatric cancer-related disease. clinical features in patients with osteosarcoma. For example, the Despite the introduction, during the 70 s, of aggressive multi- most important pro-angiogenic factor, VEGF, is overexpressed in agent chemotherapy in addition to tumor ablation surgery, the osteosarcoma.7 Several authors have reported a positive correla- long-term survival (45 years) increased from 10–60% for patients tion between VEGF expression and osteosarcoma tumor burden,8 – with localized primary tumors, but to o30% for patients the development of lung metastases9 11 and a poor prognosis – presenting metastases at initial diagnosis. Nowadays, knowledge with poor disease-free survival rate.8,12 14 In contrast, it was also of the mechanisms underlying osteosarcoma metastasis is quite reported that high microvessel density in the primary tumor limited. Thus, in order to improve the clinical outcomes for correlated with a good response to chemotherapy and a favorable patients with poor prognosis and to manage primary osteosar- prognosis.15 These conflicting data do not allow to clearly coma by preventing development of metastatic disease, it is establish a consensus on the association of VEGF expression in imperative to find new approaches to block the metastasis primary site with the prognosis of osteosarcoma patients. Some process. other markers or contributing molecules need to be identified for Degradation of the extracellular matrix, facilitated by the action a better characterization of blood vessel involvement in the of matrix metalloproteinases (MMPs), is a prerequisite of tumor metastatic features of osteosarcoma. invasion and metastasis in a variety of cancers including CYR61 (CCN1) belongs to the family of secreted matricellular osteosarcoma. MMP-2 and MMP-9 have been repetitively impli- proteins that includes connective tissue growth factor (CCN2), cated in osteosarcoma cell invasion,1–3 and increased expression NOV (CCN3) and WISP1-3 (CCN4-6). CYR61 is a cysteine-rich of membrane type-1 MMP correlated with decreased overall protein with diverse regulatory functions16 among which is a survival.4 A correlation was recently reported between the potent pro-angiogenic activity.17,18 Knockdown of CYR61 expres- expression of the extracellular MMP inducer EMMPRIN (CD147) sion in mice is associated with embryonic lethality due to 5 19 and vascular endothelial growth factor (VEGF) in osteosarcoma, placental vascular inefficiency and compromised blood vessels. suggesting a concomitant remodeling of extracellular matrix CYR61 was shown to promote tumor growth and vascularization and neo-formation or reorganization of blood vessels. The and cancer cell invasion and metastasis.20–23 Recently, it has been

1Inserm U981, Institut de cancérologie Gustave Roussy, Villejuif, France; 2Gustave Roussy, Villejuif, France; 3Université Paris Sud, Orsay, France; 4Université Paris Diderot, Paris, France ; 5Cell therapy group, Institute for Advanced Chemistry of Catalonia (CSIC), Barcelona, Spain; 6Institut de Recherche Intégrée en cancérologie à Villejuif (IRCIV), Laboratoire de pathologie expérimentale, Gustave Roussy, Villejuif, France and 7Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain. Correspondence: Dr O Fromigué, Inserm U981, Institut de cancérologie Gustave Roussy, 39 Rue Camille Desmoulins, F-94805 Villejuif, France. E-mail: [email protected] 8Current address: Division of Radiation and Cancer Biology, Department of Radiation Oncology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA, USA. Received 1 May 2014; revised 10 June 2014; accepted 24 June 2014; published online 28 July 2014 Cyr61 silencing reduces bone tumor angiogenesis N Habel et al 3208 reported that cysteine-rich protein 61 (CYR61/CCN1) is over- RESULTS AND DISCUSSION expressed in osteosarcoma compared with normal bone tissue, Anti-angiogenic effect of CYR61 silencing in osteosarcoma cells and that CYR61 protein level correlates with tumor In order to better characterize the effect of CYR61 silencing on 24,25 aggressiveness. Using genetically modified cell lines, we also tumor vascularization, a large panel of markers related to demonstrated that CYR61 silencing reduces cell motility in vitro angiogenesis was studied in cultured cells by quantitative reverse 24 and tumor cell dissemination to lungs in vivo. The present study transcriptase–PCR. We first confirmed that CYR61 was silenced by evaluates the influence of CYR61 expression level on vasculariza- a specific small hairpin RNA and this did not result in a tion of primary osteosarcoma tumor and occurrence of lung counterbalanced induction of CTGF/CCN2 expression (Figure 1a, metastases. Supplementary Figure 1).

a 1.5 b 1.5

1.0 1.0 * * * 0.5 * 0.5 mRNA Level mRNA Level

(Fold Change) * (Fold Change)

0.0 0.0 CTGF MMP9 MMP2 TIMP2 CYR61 MMP14 c 2.0

1.5

1.0 * * *

mRNA Level * (Fold Change) * 0.5 * * * 0.0 PGF KDR/ CD31 FGF2 FLT1/ PECAM/ VEGF -A ANGPT1 ANGPT2 VEGFR1 VEGFR2 TGFbeta1

de2.0 3.0 * * * * 1.5 2.0 1.0

1.0 mRNA Level mRNA Level (Fold Change)

(Fold Change) 0.5

0.0 0.0 ITGA5 ITGB1 ITGB3 ITGB5 ITGAV THSB1 SPARC Figure 1. CYR61 silencing reduces expression level of angiogenic markers in osteosarcoma K7M2 cells. K7M2 osteosarcoma cells (ATCC, Rockville, MD, USA) were cultured at 37 °C in humidified atmosphere containing 5% CO2 in air, in Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated fetal calf serum (PAN-Biotech, Aidenbach, Germany), 10 000 U/ml penicillin and 10 000 μg/ml streptomycin, refreshed three times a week. Cells were transduced using lentiviral particles (murine shCYR61 or control small hairpin RNA from Santa Cruz Biotechnology, Santa Cruz, CA, USA) and selected using puromycin dihydrochloride (Sigma-Aldrich) for 5 days. Viable cells were then routinely maintained in complete medium. Total RNA was isolated using TriZol Reagent (Invitrogen, Cergy Pontoise, France). RNA (3 μg) were reversed transcribed at 37 °C for 90 min using 300 U MMLV reverse transcriptase, 15 μg oligodT primers and 1 mM deoxynucleotide triphosphate in 30 μl total volume. Quantitative PCR was performed on ViiA7 Instrument (Applied Biosystems, Life Technologies, Saint Aubin, France) using the SYBR Green Master kit (ABGen, Courtabœuf, France) supplemented with 0.5 μM of specific primers (Table 1). Thermal conditions were: activation for 15 min at 95 °C, then 40 cycles of denaturation at 95 °C for 20 s, 58 °C annealing for 15 s and 72 °C extension for 15 s. The relative amount of RNA was calculated by the 2−ΔΔCt method. Results are expressed as fold change relative to control cells (mean ± s.d. *Po0.05 vs the control group) for CYR61 and CTGF (a), matrix metalloproteinases (b), pro-angiogenic markers (c), anti-angiogenic markers (d) and integrins (e). Comparisons were performed using the two-factor analysis of variance using the statistical package super-ANOVA.

Oncogene (2015) 3207 – 3213 © 2015 Macmillan Publishers Limited Cyr61 silencing reduces bone tumor angiogenesis N Habel et al 3209 Control Silenced Initial 14 days 21 days 28 days Initial 14 days 21 days 28 days

Relative light intensity - +

Control Silenced

5 2.0 Control Silenced 4 1.6

3 1.2

2 0.8 Tumor Weight (g) 1 * 0.4 * Bioluminescence (A.U.) * * 0 0.0 071421 28 Days Control Silenced

Figure 2. CYR61 silencing reduces primary osteosarcoma tumor growth. Control and silenced cells were transduced with lentiviral particles encoding the enhanced green fluorescent protein (eGFP) gene under the simian virus SV40 promoter and the luciferase gene (Promega Corporation, Madrid, Spain) under the cytomegalovirus promoter (see Vilalta et al.51). The highest eGFP-expressing clones were selected by flow cytometry. BALB/c homozygous nude mice (6 weeks old; Charles River, Barcelona, Spain) were randomized in two groups (n = 8 mice per group), acclimatized for 5 days and maintained in a pathogen-free environment throughout the experiment. All procedures were performed with the approval of the Cardiovascular Research Center ethical animal committee and the Government of Catalonia. Mice were injected intramuscularly with osteosarcoma cells (106 cells/20 μl phosphate-buffered saline) in both thighs. In vivo non-invasive bioluminescence imaging of living mice was performed at different time points, as described previously.52 Mice were inoculated with 50 μl of D-luciferin (100 mg/kg, Promega) and imaged 5 min after using a high-efficiency charge-coupled device camera (a). Bioluminescent signals were recorded and photon intensity in the tumor region was plotted as a function of time for control mice (black squares) and CYR61-silenced groups (open lozenges). Each data point represents mean ± s.d. (n = 8). *Po0.01 vs the control group (b). At day 28 after tumor cells injection, thigh muscles and lungs were harvested and fixed in formalin before paraffin embedding. Gross anatomical views of primary tumors from representative mice in the control and CYR61-silenced groups (c). Scatter dot plot specifying the tumor weight found at dissection. Lines indicate means ± s.d. (n = 8). *Po0.01 vs the control group (d). Comparisons were performed using the non-parametric Mann–Whitney test with Po0.05 considered to be significant.

The invasive process involves extracellular matrix CYR61 in coordinating MMPs activity. Such proteinases, degradation, mainly resulting from MMP2 activity.2,24,26,27 by remodeling the tumoral extracellular matrix, facilitate We conﬁrm that MMP2 messenger RNA level is downregulated cell motility, release of growth factors and promote when CYR61 expression is inhibited (Figure 1b). Moreover, angiogenesis.28 Our results thus suggest that targeting the observed downregulation of other related MMPs (MMP9 CYR61 may also interfere with tumor extracellular matrix and MMP14) and TIMP2 also points to an important role of remodeling.

5 Capillary density (A.U.) * 0 Control Silenced

c 35 2.5 2 0.8 30 2.0 )

2 25 0.6 1.5

µm 20

-3 * 0.4 * 15 1.0 lung tissue (x10 10 0.2 Metastasis size 0.5 * 5 Metastatic tissue area (% of total lung tissue) 0 0 0 Metastases number/mm Control Control Control Silenced Silenced Silenced d 3.0 1.4 2.5 1.2 1.0 2.0 0.8 1.5 lung tissue

0.6 2 1.0 0.4

0.5 /mm 0.2 Metastases number Metastatic tissue area (% of total lung tissue) 0 0 0 5 10 15 20 0 5 10 15 20 Capillary density (A.U.) Capillary density (A.U.) Figure 3. CYR61 silencing reduces primary tumor vasculature and lung metastasis occurence. Primary tumor tissues from animals intramuscularly injected with control or CYR61-silenced cells were cut in their center then each half paraffin-embedded side by side. Tissue sections (4 μm) were deparaffinized in xylene and rehydrated through a graded series of ethanol before staining with hematoxylin and eosin or immunostaining for CD34 expression (rat monoclonal anti-CD34 at 1:20, HyCult Biotechnology, TEBU, Le Perray en Yvelines, France). A careful scan of the tumor at × 100 magnification (Axiophot 1 microscope; Zeiss, Oberkochen, Germany) was used to evaluate tissue viability and to identify and exclude the pre-necrotic and necrotic regions. Nontumoral areas such as muscle fibers were also excluded. Several independent fields were selected disregarding any pre-existing mature vessels but reflecting the overall immunostaining of the tumor contained on the entire slide. CD34 immunostaining to detect endothelial cells and representative vessels (indicated arrow) (a). The CD34- positive cells were identified using the Photoshop software (Adobe Systems, Mountain View, CA, USA). Immunostaining intensity was calculated as the difference between cellular and background immunostainings and was designated as arbitrary units (a.u.). Barrs indicate means ± s.d. (n = 8). *Po0.01 vs the control group (b). Lung tissue sections (4 μm) were deparaffined in xylene and rehydrated through a graded series of ethanol before staining with hematoxylin and eosin. Metastatic lung nodules number and size were quantified from control and CYR61-silenced groups. Results are expressed as means ± s.d. *Po0.01 vs the control group (c). Spearman rank correlation coefficient relationship between capillary density in primary tumors and lung metastases surface (left panel) and number (right panel) (d).

Expression of VEGF in primary osteosarcoma samples correlates strategy is off-limits for the pediatric population and must be with the development of pulmonary metastases and poor adjusted to conﬁrm efﬁcacy in aggressive osteosarcoma, affecting prognosis.10,29 Although preclinical results show reduction of rather children. In osteoblastic cells, VEGF-mediated upregulation primary tumors following treatment with VEGF inhibitors,30,31 of CYR61 attracts endothelial cells and promotes angiogenesis.34 some patients will develop progressive disease.32,33 Such a In our model, we found that CYR61 silencing leads to a decrease in

Table 1. Primer sequences for quantitative PCR assays

Name Left primer sequence Right primer sequence

CYR61 5′-GGATCTGTGAAGTGCGTCCT-3′ 5′-CTGCATTTCTTGCCCTTTTT-3′ CTGF 5′-TGACCTGGAGGAAAACATTAAGA-3′ 5′-AGCCCTGTATGTCTTCACACTG-3′ MMP2 5′-TAACCTGGATGCCGTCGT-3′ 5′-TTCAGGTAATAAGCACCCTTGAA-3′ MMP9 5′-ACGACATAGACGGCATCCA-3′ 5′-GCTGTGGTTCAGTTGTGGTG-3′ MMP14 5′-AACTTCGTGTTGCCTGATGA-3′ 5′-CTTTGTGGGTGACCCTGACT-3′ TIMP2 5′-CGTTTTGCAATGCAGACGTA-3′ 5′-GGAATCCACCTCCTTCTCG-3′ ITGA5 5′-CACCATTCAATTTGACAGCAA-3′ 5′-TCCTCTCCCTTGGCACTGTA-3′ ITGB1 5′-TGGCAACAATGAAGCTATCG-3′ 5′-ATGTCGGGACCAGTAGGACA-3′ ITGAV 5′-GGTGTGGATCGAGCTGTCTT-3′ 5′-CAAGGCCAGCATTTACAGTG-3′ ITGB3 5′-GTGGGAGGGCAGTCCTCTA-3′ 5′-CAGGATATCAGGACCCTTGG-3′ ITGB5 5′-ACCTGCCAAGATGGCATATC-3′ 5′-CACGGACACTTCAAAGGATG-3′ FGF2 5′-CGGCTCTACTGCAAGAACG-3′ 5′-TGCTTGGAGTTGTAGTTTGACG-3′ ANGPT1 5′-CGGATTTCTCTTCCCAGAAAC-3′ 5′-TCCGACTTCATATTTTCCACAA-3′ ANGPT2 5′-CTCACCACCAGTGGCATCTA-3′ 5′-CCCACGTCCATGTCACAGTA-3′ VEGFR1 5′-GGCCCGGGATATTTATAAGAAC-3′ 5′-CCATCCATTTTAGGGGAAGTC-3′ VEGFR2 5′-ATTTCACCTGGCACTCTCCA-3′ 5′-AGGTGCTCAAAAACATCTTCG-3′ VEGFA 5′-AAAAACGAAAGCGCAAGAAA-3′ 5′-TTTCTCCGCTCTGAACAAGG-3′ PGF 5′-CTGGGTTGGCTGTGCATT-3′ 5′-GGCACCACTTCCACTTCTGT-3′ PECAM 5′-CGGTGTTCAGCGAGATCC-3′ 5′-ACTCGACAGGATGGAAATCAC-3′ TGFB1 5′-TGGAGCAACATGTGGAACTC-3′ 5′-GTCAGCAGCCGGTTACCA-3′ SPARC 5′-AGAGGAAACGGTCGAGGAG-3′ 5′-CTCACACACCTTGCCATGTT-3′ THBS1 5′-CACCTCTCCGGGTTACTGAG-3′ 5′-GCAACAGGAACAGGACACCTA-3′ 18S 5′-CGGCTACCACATCCAAGGAA-3′ 5′-GCTGGAATTACCGCGGCT-3′

VEGFA expression level (Figure 1c), indicating a two-way relation- the pro-angiogenic properties of CYR61 in endothelial cells are ship between CYR61 and VEGF. mainly driven through alphaVbeta3 integrin.17,18 In addition, Other main pro-angiogenic factors have also been reported in CYR61-enhanced breast cancer cells' invasive capacity and relation to aggressive primary bone tumor behavior. For example, agressiveness require both the concomitant overexpression of a correlation between serum PGF (placental growth factor) or FGFs alphaVbeta3 and a functional alphaVbeta3-dependent signaling (fibroblast growth factors) level and maximum tumor size was cascade.42,43 We may hypothesize that, in our osteosarcoma detected in patients with primary osteosarcoma.35 High expres- model, the surprising increase in alphaVbeta3 expression level sion of FGF2 and microvessel density are positively correlated with observed in CYR61-silenced cells could trigger a failure in the poor prognosis of osteosarcoma patients.36,37 As for VEGF, we CYR61/alphaVbeta3 autocrine loop, which would result in an show that CYR61 silencing in osteosarcoma cells strongly induction of integrin gene transcription. Alternatively, the decreases PGF and FGF2 expression levels (Figure 1c), confirming observed increase in alphaVbeta3 expression could result from a multifactorial inhibition in the tumor vascularization process. the observed upregulation of Thrombospondin-1 (Figure 1) We also find similarly coherent results regarding the expression induced by CYR61 silencing, as this extracellular matrix protein level of other pro-angiogenic factors already described to be has already been described to activate alphaVbeta3 integrin.47 38 linked to osteosarcoma metastasis such as angiopietin-1 and -2, This upregulation of alphaVbeta3 expression needs to be taken 39 platelet endothelial cell adhesion molecule or VEGF receptor into account in the development of further strategies targeting 40 tyrosine kinases 1 and 2. In addition, we find a marked increase CYR61, combining it, for example with specific alphaVbeta3 in the expression level of anti-angiogenic markers such as antagonist molecules. thrombospondin-1 and SPARC (Figure 1d) in CYR61-silenced cells, compared with control cells. Primary tumor development is dependent on the CYR61 Taken together, our results indicate that CYR61-silenced cells expression level are not able to recruit endothelial cells and to induce neo- angiogenesis in the developing tumor. Thus, targeting of CYR61 We first evaluated tumor cell proliferation and viability. Results could be a promising strategy to break the VEGF/CYR61 pro- from the BrdU incorporation assay and 3-(4,5-dimethylthiazol-2- angiogenic vicious circle and to exert a multifactorial inhibition in yl)-2,5-diphenyltetrazolium bromide test showed no significant the vascularization process and then restrain neo-vascularization difference between control and CYR61-silenced cells grown in of primary tumors. culture (Supplementary Figures 2A and B). Using the same K7M2 The biological functions of CCN proteins are mediated through murine osteosarcoma model, we injected both control and CYR61- interactions with cell surface receptors such as integrins.41 silenced cells, which were transduced for the constitutive Moreover, integrins are also important modulators of VEGF expression of a luciferase reporter, intramuscularly in BALB/c mice signaling. A functional connection was described between and monitored tumor growth by bioluminescence imaging. In all CYR61 and the integrin alphaVbeta3 for both angiogenesis and mice injected with control cells, tumor growth was progressive cancer progression.42,43 In addition, the blockade of integrins throughout the experiment (Figures 2a and b). In contrast, in mice alphaVbeta3 or alphaVbeta5 reduced VEGF and VEGF receptor injected with CYR61-silenced cells, tumor growth was strongly and expression, as well as angiogenesis.44,45 In osteosarcoma, expres- significantly repressed. Measurement of volume/weight of each sion of alphaV integrin also positively correlated with VEGF tumor at killing (day 28 post injection) confirmed that tumors expression.46 We observe that CYR61 silencing correlates with an established from CYR61-silenced cells were significantly smaller increased expression of alphaV and beta3 integrin subunits, than tumors arising from control cells (Figure 2c), with a mean whereas no effect was detectable on other integrins such as weight 71% lower than the control tumors (Po0.001; Figure 2d). alpha5, beta1 or beta5 (Figure 1e). A binding site for alphaVbeta3 These results demonstrate that CYR61 silencing affects osteosar- integrin has been clearly identified in the CYR61 sequence17,18 and coma tumor development in vivo.

© 2015 Macmillan Publishers Limited Oncogene (2015) 3207 – 3213 Cyr61 silencing reduces bone tumor angiogenesis N Habel et al 3212 Tumor-associated vascular development is dependent on CYR61 In summary, our results demonstrate that CYR61 produced by expression level osteosarcoma cells has a critical role in neo-angiogenesis and The fact that silencing CYR61 does not significantly affect cell extracellular matrix remodeling, and that CYR61-dependent proliferation in vitro, but impedes tumor growth in mice, points to vascularization of primary tumor modulates cells dissemination the involvement of complex higher-order mechanisms regulating to lungs. Our results thereby demonstrate that targeting CYR61 in tumor growth and aggresiveness. It is commonly agreed that in osteosarcoma cells significantly reduces tumor vascularization and tumors larger than 1–2mm3, passive diffusion of gases and affects osteosarcoma growth and metastatic potential in vivo. nutrients is not sufficient to sustain growth. The hypoxic microenvironment within the tumor promotes new vessels CONFLICT OF INTEREST formation.48 CYR61 is abundantly secreted by bone marrow- derived mesenchymal stem cells and contributes to the The authors declare no conflict of interest. angiogenesis-promoting activity of this secretome in vitro and 49 50 in vivo. Meyuhas et al. demonstrated that CYR61 transcription ACKNOWLEDGEMENTS is enhanced in hypoxic regions of tumors in vivo. We have previously shown that CYR61 is highly expressed in osteosarcoma NH is a recipient of a PhD award from the Ministère de la Recherche (Paris, France). ’ cell lines and tumor tissues,24 and that conditioned media from 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 CYR61-overexpressing cells facilitated endothelial cell tube 24 Cooperativa (TERCEL, Spain) and SAF2012-33404 from MINECO (Spain). We thank the formation in vitro. In the present study, macroscopic observation Department of Medical Biology and Pathology of Gustave Roussy Institute (Dr Adam; of tumors excised after bioluminescence imaging revealed that Villejuif, France) for their contribution. those derived from CYR61-silenced cells were significantly pale compared with control tumors, suggesting a general lack of vascular system (Figure 2c). AUTHOR CONTRIBUTIONS We analyzed the vascular structure of control and CYR61- NH and MV performed and analyzed experiments and wrote the report. OB and silenced tumor sections by anti-CD34 immunostaining (Figure 3a). PO performed and analyzed experiments. JB and OF designed, performed and Control tumors contained large and thick-walled vessels, whereas analyzed experiments, and wrote the report. CYR61-silenced tumors contained small and thin-walled capillaries and in some cases the CD34-positive signal was limited to single endothelial cells. These blood vessel patterns were homogeneous REFERENCES and independent of whether the samples came from central or 1 Cheng YY, Huang L, Lee KM, Li K, Kumta SM. Alendronate regulates cell invasion peripheral locations of the tumors. Microvessel density quantifica- and MMP-2 secretion in human osteosarcoma cell lines. Pediatr Blood Cancer tion confirmed a marked decrease (−76%, Po0.001 vs control) in 2004; 42:410–415. vessel density of CYR61-silenced tumors as compared with 2 Fromigue O, Hamidouche Z, Marie PJ. Blockade of the RhoA-JNK-c-Jun-MMP2 controls (Figure 3b). We confirmed that CYR61 protein level was cascade by atorvastatin reduces osteosarcoma cell invasion. J Biol Chem 2008; fi 283: 30549–30556. still signi cantly lower in silenced tumor cells at the end of the 3 Xin ZF, Kim YK, Jung ST. Risedronate inhibits human osteosarcoma cell invasion. experiment (day 28; Supplementary Figure 3A). These results J Exp Clin Cancer Res 2009; 28: 105. clearly indicate that the CYR61 protein level influences primary 4 Foukas AF, Deshmukh NS, Grimer RJ, Mangham DC, Mangos EG, Taylor S. Stage-IIB tumor vascularization in our K7M2 murine osteosarcoma model, osteosarcomas around the knee. A study of MMP-9 in surviving tumour cells. and that silencing CYR61 reduces blood vessel development in J Bone Joint Surg Br 2002; 84:706–711. primary osteosarcoma tumors. 5 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. Correlation between tumor-associated vasculature and pulmonary 6 Folkman J. Angiogenesis. Annu Rev Med 2006; 57:1–18. metastases occurence 7 Yang J, Yang D, Sun Y, Sun B, Wang G, Trent JC et al. Genetic amplification of the As metastatic spread is closely related to the capacity of tumor vascular endothelial growth factor (VEGF) pathway genes, including VEGFA, in cells to scape through the vascular system, we characterized human osteosarcoma. Cancer 2011; 117: 4925–4938. lung metastatic burden by hematoxylin–eosin–saffron staining 8 Lammli J, Fan M, Rosenthal HG, Patni M, Rinehart E, Vergara G et al. Expression of of paraffin-embedded sections (Supplementary Figure 3B). vascular endothelial growth factor correlates with the advance of clinical osteosarcoma. Int Orthop 2012; 36: 2307–2313. We observed a marked reduction in lung metastases number 9 Lee YH, Tokunaga T, Oshika Y, Suto R, Yanagisawa K, Tomisawa M et al. Cell- and size in mice bearing CYR61-silenced tumors, compared retained isoforms of vascular endothelial growth factor (VEGF) are correlated with with those bearing control tumors (Figure 3c), confirming our poor prognosis in osteosarcoma. Eur J Cancer 1999; 35:1089–1093. previous observations.24 Metastases occurrence also correlated 10 Kaya M, Wada T, Akatsuka T, Kawaguchi S, Nagoya S, Shindoh M et al. Vascular with capillary density in primary tumors (Figure 3d). Thus, a endothelial growth factor expression in untreated osteosarcoma is predictive of non-linear positive correlation exists between the blood vessels pulmonary metastasis and poor prognosis. Clin Cancer Res 2000; 6: 572–577. surface area (CD34-positive area) within primary tumors and both 11 Hara H, Akisue T, Fujimoto T, Imabori M, Kawamoto T, Kuroda R et al. Expression of the ratio of metastases surface to whole-pulmonary tissue, and the VEGF and its receptors and angiogenesis in bone and soft tissue tumors. Antic- ancer Res 2006; 26: 4307–4311. number of pulmonary metastatic foci (Spearman’s correlation fi 12 DuBois S, Demetri G. Markers of angiogenesis and clinical features in patients with coef cients of 0.782 and 0.709, respectively). Indeed, mice that sarcoma. Cancer 2007; 109:813–819. received control osteosarcoma cells developed vascularized 13 Rastogi S, Kumar R, Sankineani SR, Marimuthu K, Rijal L, Prakash S et al. Role of primary tumors and numerous pulmonary metastases, whereas vascular endothelial growth factor as a tumour marker in osteosarcoma: a mice that received CYR61-silenced cells developed poorly prospective study. Int Orthop 2012; 36: 2315–2321. vascularized primary tumors and small and scarce pulmonary 14 Chen D, Zhang YJ, Zhu KW, Wang WC. A systematic review of vascular endothelial metastases. These results indicate that CYR61-dependent vascu- growth factor expression as a biomarker of prognosis in patients with osteo- 34 – larization of primary tumor modulates osteosarcoma cells sarcoma. Tumour Biol 2013; : 1895 1899. 15 Kreuter M, Bieker R, Bielack SS, Auras T, Buerger H, Gosheger G et al. Prognostic dissemination to lungs, and the correlation between CYR61 level, relevance of increased angiogenesis in osteosarcoma. Clin Cancer Res 2004; 10: vessels density in primary tumors and number of pulmonary 8531–8537. metastases reinforces the involvement of angiogenesis in the 16 Lau LF. CCN1/CYR61: the very model of a modern matricellular protein. Cell Mol dissemination process. Life Sci 2011; 68: 3149–3163.

Oncogene (2015) 3207 – 3213 © 2015 Macmillan Publishers Limited Cyr61 silencing reduces bone tumor angiogenesis N Habel et al 3213 17 Leu SJ, Lam SC, Lau LF. Pro-angiogenic activities of CYR61 (CCN1) mediated 35 Babkina IV, Osipov DA, Solovyov YN, Bulycheva IV, Machak GN, Aliev MD et al. through integrins alphavbeta3 and alpha6beta1 in human umbilical vein Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the endothelial cells. J Biol Chem 2002; 277: 46248–46255. sera of patients with primary osteosarcomas. Bull Exp Biol Med 2009; 148: 18 Chen N, Leu SJ, Todorovic V, Lam SC, Lau LF. Identification of a novel integrin 246–249. alphavbeta3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in 36 Li C, Zhan C, Chen Y, Fu Q, Zhu XD, He DW et al. Analysis report for osteosarcoma vascular endothelial cells. J Biol Chem 2004; 279: 44166–44176. expression profile. Eur Rev Med Pharmacol Sci 2013; 17: 2804–2809. 19 Mo FE, Muntean AG, Chen CC, Stolz DB, Watkins SC, Lau LF. CYR61 (CCN1) is 37 Ren T, Qing Y, Dai N, Li M, Qian C, Yang Y et al. Apurinic/apyrimidinic endonu- essential for placental development and vascular integrity. Mol Cell Biol 2002; 22: clease 1 induced upregulation of fibroblast growth factor 2 and its receptor 3 8709–8720. induces angiogenesis in human osteosarcoma cells. Cancer Sci 2014; 105: 20 Babic AM, Kireeva ML, Kolesnikova TV, Lau LF. CYR61, a product of a 186–194. growth factor-inducible immediate early gene, promotes angiogenesis and 38 Odagiri H, Kadomatsu T, Endo M, Masuda T, Morioka MS, Fukuhara S et al. The tumor growth. Proc Natl Acad Sci USA 1998; 95: 6355–6360. secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through 21 Menéndez JA, Mehmi I, Griggs DW, Lupu R. The angiogenic factor CYR61 in breast integrin α5β1, p38 MAPK, and matrix metalloproteinases. Sci Signal 2014; 7: ra7. cancer: molecular pathology and therapeutic perspectives. Endocr Relat Cancer 39 Mintz MB, Sowers R, Brown KM, Hilmer SC, Mazza B, Huvos AG et al. An expression 2003; 10: 141–152. signature classifies chemotherapy-resistant pediatric osteosarcoma. Cancer Res 22 Jiang WG, Watkins G, Fodstad O, Douglas-Jones A, Mokbel K, Mansel RE. Differ- 2005; 65:1748–1754. ential expression of the CCN family members Cyr61, CTGF and Nov in human 40 Maris JM, Courtright J, Houghton PJ, Morton CL, Gorlick R, Kolb EA et al. Initial 11 – breast cancer. Endocr Relat Cancer 2004; :781 791. testing of the VEGFR inhibitor AZD2171 by the pediatric preclinical testing pro- 23 Sun ZJ, Wang Y, Cai Z, Chen PP, Tong XJ, Xie D. Involvement of Cyr61 in growth, gram. Pediatr Blood Cancer 2008; 50:581–587. 99 migration, and metastasis of prostate cancer cells. Br J Cancer 2008; : 41 Kireeva ML, Lam SC, Lau LF. Adhesion of human umbilical vein endothelial cells to – 1656 1667. the immediate-early gene product Cyr61 is mediated through integrin alphav- 24 Fromigue O, Hamidouche Z, Vaudin P, Lecanda F, Patino A, Barbry P et al. CYR61 beta3. J Biol Chem 1998; 273: 3090–3096. downregulation reduces osteosarcoma cell invasion, migration, and metastasis. 42 Menendez JA, Vellon L, Mehmi I, Teng PK, Griggs DW, Lupu R. A novel CYR61- 26 – J Bone Miner Res 2011; : 1533 1542. triggered 'CYR61-alphavbeta3 integrin loop' regulates breast cancer cell survival 25 Sabile AA, Arlt MJ, Muff R, Bode B, Langsam B, Bertz J et al. Cyr61 expression in and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway. osteosarcoma indicates poor prognosis and promotes intratibial growth and lung Oncogene 2005; 24:761–779. metastasis in mice. J Bone Miner Res 2012; 27:58–67. 43 Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, Shattil SJ et al. 26 Wang L, Zhang Q, Chen W, Shan B, Ding Y, Zhang G et al. B7-H3 is overexpressed A mechanism for modulation of cellular responses to VEGF: activation of the in patients suffering osteosarcoma and associated with tumor aggressiveness and integrins. Mol Cell 2000; 6:851–860. metastasis. PLoS ONE 2013; 8: e70689. 44 Wilkinson-Berka JL, Jones D, Taylor G, Jaworski K, Kelly DJ, Ludbrook SB et al. 27 Wen X, Liu H, Yu K, Liu Y. Matrix metalloproteinase 2 expression and survival of SB-267268, a nonpeptidic antagonist of alpha(v)beta3 and alpha(v)beta5 integ- patients with osteosarcoma: a meta-analysis. Tumour Biol 2014; 35:845–848. rins, reduces angiogenesis and VEGF expression in a mouse model of 28 Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. retinopathy of prematurity. Invest Ophthalmol Vis Sci 2006; 47:1600–1605. Annu Rev Cell Dev Biol 2001; 17:463–516. 45 Tsou R, Isik FF. Integrin activation is required for VEGF and FGF receptor protein 29 Oda Y, Yamamoto H, Tamiya S, Matsuda S, Tanaka K, Yokoyama R et al. CXCR4 and presence on human microvascular endothelial cells. Mol Cell Biochem 2001; 224: VEGF expression in the primary site and the metastatic site of human osteo- 81–89. sarcoma: analysis within a group of patients, all of whom developed lung 46 Huang Y, Lin Z, Zhuang J, Chen Y, Lin J. Prognostic significance of alpha V integrin metastasis. Mod Pathol 2006; 19:738–745. and VEGF in osteosarcoma after chemotherapy. Onkologie 2008; 31:535–540. 30 Pignochino Y, Grignani G, Cavalloni G, Motta M, Tapparo M, Bruno S et al. 47 Gomes N, Legrand C, Fauvel-Lafève F. Shear stress induced release of von Will- Sorafenib blocks tumour growth, angiogenesis and metastatic potential in ebrand factor and thrombospondin-1 in HUVEC extracellular matrix enhances preclinical models of osteosarcoma through a mechanism potentially involving 22 – the inhibition of ERK1/2, MCL-1 and ezrin pathways. Mol Cancer 2009; 8: 118. breast tumour cell adhesion. Clin Exp Metastasis 2005; :215 223. 31 Wang CT, Lin CS, Shiau CW, Chu PY, Hsiao CC, Chiang YL et al. SC-1, a sorafenib 48 North S, Moenner M, Bikfalvi A. Recent developments in the regulation of the 218 – derivative, shows anti-tumor effects in osteogenic sarcoma cells. J Orthop Res angiogenic switch by cellular stress factors in tumors. Cancer Lett 2005; :1 14. 2013; 31: 335–342. 49 Estrada R, Li N, Sarojini H, An J, Lee MJ, Wang E. Secretome from mesenchymal 219 – 32 Grignani G, Palmerini E, Dileo P, Asaftei SD, D'Ambrosio L, Pignochino Y et al. stem cells induces angiogenesis via Cyr61. J Cell Physiol 2009; : 563 571. A phase II trial of sorafenib in relapsed and unresectable high-grade osteo- 50 Meyuhas R, Pikarsky E, Tavor E, Klar A, Abramovitch R, Hochman J et al. A Key role sarcoma after failure of standard multimodal therapy: an Italian Sarcoma for cyclic AMP-responsive element binding protein in hypoxia-mediated activa- Group study. Ann Oncol 2012; 23:508–516. tion of the angiogenesis factor CCN1 (CYR61) in tumor cells. Mol Cancer Res 2008; 6 – 33 Wagner L, Turpin B, Nagarajan R, Weiss B, Cripe T, Geller J. Pilot study of : 1397 1409. vincristine, oral irinotecan, and temozolomide (VOIT regimen) combined with 51 Vilalta M, Dégano IR, Bagó J, Gould D, Santos M, García-Arranz M et al. bevacizumab in pediatric patients with recurrent solid tumors or brain tumors. Biodistribution, long-term survival, and safety of human adipose tissue-derived Pediatr Blood Cancer 2013; 60:1447–1451. mesenchymal stem cells transplanted in nude mice by high sensitivity non- 34 Athanasopoulos AN, Schneider D, Keiper T, Alt V, Pendurthi UR, Liegibel UM et al. invasive bioluminescence imaging. Stem Cells Dev 2008; 17: 993–1003. Vascular endothelial growth factor (VEGF)-induced up-regulation of CCN1 in 52 Román I, Vilalta M, Rodriguez J, Matthies AM, Srouji S, Livne E et al. Analysis of osteoblasts mediates proangiogenic activities in endothelial cells and promotes progenitor cell-scaffold combinations by in vivo non-invasive photonic imaging. fracture healing. J Biol Chem 2007; 282: 26746–26753. Biomaterials 2007; 28: 2718–2728.

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