Basic fibroblast growth factor induces VEGF expression in chondrosarcoma cells and subsequently promotes endothelial progenitor cells-primed angiogenesis
Huey-En Tzeng1, 2#, Po-Chun Chen3, 4#, Kai-Wei Lin3, Chih-Yang Lin3, Chun-Hao Tsai5, 6, Shao-Min Han7, Chieh-Lin Teng7, Wen-Li Hwang7, Shih-Wei Wang8 and Chih-Hsin Tang3, 9, 10*
1Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan
2Division of Hematology/Oncology, Taichung Veterans General Hospital, Taichung, Taiwan
3Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
4Department of Medical Research, Chung Shan Medical University Hospital, Chung Shan Medical University, Taichung, Taiwan
5Department of Medicine and Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan
6Department of Orthopedic Surgery, China Medical University Hospital, Taichung, Taiwan
7Division of Hematology/Oncology, Department of Medicine, Taichung Veterans General Hospital, Taichung, Taiwan
8Department of Medicine, Mackay Medical College, New Taipei City, Taiwan
9Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan
10Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan
# These authors contributed equally to this study and share first authorship *Corresponding authors
Chih-Hsin Tang, PhD
Graduate Institute of Basic Medical Science, China Medical University No. 91, Hsueh-Shih Road, Taichung, Taiwan
Tel: 886-4-22052121-7726 Fax: 886-4-22333641 E-mail: [email protected] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Keywords: Basic fibroblast growth factor, endothelial progenitor cells, angiogenesis, chondrosarcoma
Running title: bFGF promotes angiogenesis in chondrosarcoma
Abbreviations: endothelial progenitor cells, EPCs; basic fibroblast growth factor, bFGF; chick chorioallantoic membrane assay, CAM; FGF receptor, FGFR
Abstract
Chondrosarcoma, a common malignant tumor, develops in bone. Effective adjuvant therapy remains inadequate for treatment, meaning poor prognosis. It is imperative to explore novel remedies. Angiogenesis is a rate-limiting step in progression that explains neovessel formation for blood supply in tumor microenvironment. Numerous studies indicate endothelial progenitor cells (EPCs) promoting angiogenesis and contributing to tumor growth. Basic fibroblast growth factor (bFGF), a secreted cytokine, regulates biological activity including angiogenesis and correlates with tumorigenesis. However, the role of bFGF in angiogenesis-related tumor progression by recruiting EPCs in human chondrosarcoma is rarely discussed. Here, we found bFGF induced vascular endothelial growth factor (VEGF) expression via FGFR1/c-Src/p38/NF-κB signaling pathway in chondrosarcoma cells, thereby triggering angiogenesis of endothelial progenitor cells. Our in vivo data revealed tumor-secreted bFGF promoting angiogenesis in both mouse plug and chick chorioallantoic membrane assay (CAM). Xenograft mouse model data, due to bFGF-regulated angiogenesis, showed bFGF regulating angiogenesis-linked tumor growth. Finally, bFGF was highly expressed in chondrosarcoma patients than normal cartilage, positively correlating with VEGF expression and tumor stage. Our study offers a 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
novel therapeutic target for chondrosarcoma progression.
Introduction
Chondrosarcoma, a common malignant tumor developing in bone, shows refractory phenotype to chemo- and radiotherapy, posing a complicated challenge . Surgical resection remains the common mode of therapy for chondrosarcoma. Since adjuvant therapy remains inadequate and prognosis poor, we must explore novel remedies .
Angiogenesis, the state of cancer to promote neovessel formation, has been proposed as a regulator in tumor progression. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis of tumor progression . EPCs in peripheral blood were first discovered as CD34+/ VEGFR2+ mononuclear cells in 1997; they have the capacity to differentiate into an endothelial phenotype, express endothelial markers, and form new vessels at ischemia sites . A large number of reviews discuss the capacity of EPCs to participate in endothelial repair and neoangiogenesis due to their abilities of differentiating into endothelial cells . Researchers cite EPCs promoting angiogenesis-related growth , and targeting EPCs in search of anti-angiogenesis drugs.
Basic fibroblast growth factor (bGFG/FGF-2), a secreted cytokine, encodes heparin-binding proteins with growth, antiapoptotic, differentiation promotion and angiogenic activity. Its correlation with progression is certified in many cancers: e.g., expressed in oral squamous cell carcinoma (OSCC), correlated with lymph node metastasis and prognosis in OSCC . Expression of bFGF is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer . This secrete cytokine regulates biological activities, including angiogenesis, and expression of bFGF correlates with tumorigenesis .
Chondrosarcoma shows poor prognosis caused by absence of effective adjuvant therapy. Previous study indicates angiogenesis playing crucial roles in biological processes and rate-limiting steps in tumor progression, with VEGF as paramount factor in angiogenesis while participating in tumorigenesis . bFGF notably regulates MMP-13 expression and promotes human chondrosarcoma . Moreover, bFGF has 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114
been proposed to accelerate the growth of 6 tumor cell lines including SW1353 (chondrosarcoma) . In addition, in vivo results indicate that intralesional administration of mouse monoclonal anti-beta-FGF (DG-2) antibody significantly inhibited rat chondrosarcoma growth and vascularization . Studies indicate EPCs expediting both angiogenesis and angiogenesis-related growth . Whether bFGF promotes angiogenesis by affecting EPCs in human chondrosarcoma is still unclear. We show evidence of bFGF accelerating EPCs-primed angiogenesis by VEGF expression in chondrosarcoma cells: bFGF treatment inducing VEGF expression in chondrosarcoma cells and promoting EPC tube formation in a dose-dependent manner. Pretreatment with VEGF neutralized antibody dramatically reduced this effect. We also proved FGFR1/c-Src/p38/NF-κB signaling pathway’s involvement in VEGF-expression and EPCs-primed angiogenesis. Both CAM and mouse xenograft
in vivo models also revealed the role of bFGF in angiogenesis-related tumor growth.
Finally, correlation between bFGF, VEGF and tumor stage in clinical specimens was consistent with our in vitro and in vivo results, which suggest bFGF promoting angiogenesis by VEGF expression within chondrosarcoma microenvironment.
Materials and Methods Materials
Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for p-c-Src, c-Src, p-p38, p38, p-p65, p65, β-actin, CD31, CD34, CD133 and bFGF were purchased from Biotechnology (Santa Cruz, CA); VEGF antibody from Abcam (Cambridge, MA). Recombinant human VEGF was purchase from R&D Systems (Minneapolis, MN); Dulbecco’s modified Eagle’s medium, α-minimum essential medium (MEM), fetal bovine serum and all other cell culture reagents from Gibco-BRL Life Technologies (Grand Island, NY). ON-TARGETplus siRNAs were purchased from Dharmacon Research (Lafayette, CO), pSV-β-galactosidase vector and luciferase assay kit from Promega (Madison, WI), all other chemicals from Sigma-Aldrich (St Louis, MO).
Cell culture
Human chondrosarcoma cell line (JJ012) was donated by the laboratory of Dr. Sean P. Scully (University of Miami School of Medicine, Miami, FL). Cells were cultured in complete medium containing Dulbecco’s modified Eagle’s medium/α-MEM with 10% fetal bovine serum supplement.
Endothelial progenitor cells cell culture 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
Protocol for EPC culture was approved by the Institutional Review Board of Mackay Medical College, New Taipei City, Taiwan (reference number P1000002). All subjects gave informed written consent before enrolling. Peripheral blood (80 ml) was gleaned from healthy donors, mononuclear cells isolated by Ficoll-Paque PLUS centrifuge (Amersham Biosciences, Uppsala, Sweden), according to manufacturer’s instructions. CD34-positive mononuclear cells were isolated from mononuclear cell fraction by CD34 MicroBead kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany), their EPCs maintained and characterized as detailed previously . EPC were defined as UEA-1+, CD34+, KDR+ and CD31+ . Human CD34-positive EPCs were cultured in MV2 complete medium containing MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany), supplemented with 20% defined FBS (HyClone, Logan, UT). Cultures were seeded on 1% gelatin-coated plasticware and maintained at 37°C in humidified 5% CO2 atmosphere.
Western blot
Cellular lysates were prepared, proteins resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membrane. Blots were blocked with 4% BSA for 1 h at room temperature and then probed with antibodies (1:1000) for 1 h at room temperature. After three washes, blots were incubated with peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature and visualized by enhanced chemiluminescence, using X-OMAT LS film (Eastman Kodak, Rochester, NY). Data were quantified by computing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Quantitative real-time PCR
This qPCR analysis used Taqman® one-step PCR Master Mix (Applied Biosystems, Foster City, CA). Total cDNA (100mg) was added per 25-µl reaction with sequence-specific primers and Taqman® probes. Sequences for all target gene primers and probes were purchased commercially (β-actin as internal control) (Applied Biosystems, CA); qPCR assay was triplicated by StepOnePlus sequence detection system. Cycling conditions were 10-min polymerase activation at 95℃ followed by 40 cycles at 95℃ for 15 sec and 60℃ for 60 sec, threshold set above non-template control background and within linear phase of target gene amplification to calculate the cycle number at which transcript was detected (denoted as CT).
Reporter assay
Cells were transfected for 24 hr with NF-κB report plasmid by Lipofectamine 2000 (Invitrogen), as per manufacturer's recommendations, extracts prepared. Activities of 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190
luciferase and β-galactosidase were then measured. Transwell migration assay
This process used Transwell inserts (8-μm pore size; Costar, NY) in 24-well dishes. Chondrosarcoma cells were pretreated for 30 min with designated inhibitor or vehicle concentration (0.1% DMSO) and CM were collected after 24 h. EPCs were seeded in the upper Transwell chamber and 300 μl of CM placed in the lower chamber. Each experiment is performed with
Enzyme-linked immunosorbent assay
All cells were pretreated for 30 min with varying concentrations of inhibitors (PP2, SB203580, PDTC, or TPCK) or vehicle (0.1% DMSO) and then incubated with bFGF (30 ng/ml) for 24 h at 37°C. Medium removed was stored at -80°C, VEGF gauged by ELISA kits (Biocompare, San Jose, CA), as per manufacturer’s instructions.
Tube formation
Matrigel (BD Biosciences, Bedford, MA) was dissolved at 4°C, aliquots of 150 µl/well were added to 48-well plates, which were incubated at 37°C for 30 min. EPCs were resuspended at 5 × 104/100 µl in culture medium (50% EGM-MV2 medium and 50% CM from JJ012 cells), and added to wells. VEGF (20 ng/ml) or culture medium were used as positive and negative controls, respectively. After 6 h of incubation at 37°C, EPC tube formation was assessed by microscopy and each well photographed. Number of tube branches and total tube length were calculated by MacBiophotonics Image J software.
Animal Model and Imaging
Experimental procedures were approved by the Institutional Animal Care and Use Committee. Male nu/nu mice (6-8 weeks of age) were subcutaneously injected with 5× 105 cells suspended in 100 μl medium. Tumor growth, local invasion and metastasis were monitored by IVIS Imaging System.
Immunohistochemistry (IHC)
Human chondrosarcoma tissue array was purchased from Cybrdi (Rockville, MD) and Biomax (Rockville, MD). We explored 8 cases of normal cartilage, as well as 26 of Grade I, 12 of Grade II and 18 of Grade III chondrosarcoma. Sections (5-µm thick) of paraffin-embedded tissue were placed on glass slides, rehydrated, incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity, then blocked by 3% BSA incubation in PBS. Sections were incubated with the primary mouse polyclonal 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228
anti-human bFGF and VEGF antibody at 1:50 dilution and incubated at 4°C overnight. After three PBS washes, samples were incubated with a 1:50 dilution of biotin-labeled goat anti-mouse IgG secondary antibody, bound antibodies detected by ABC Kit (Vector Laboratories, Burlingame, CA). Slides were stained with chromogen diaminobenzidine, washed, counterstained with Delafield’s hematoxylin, dehydrated, treated with xylene, then mounted.
For in vivo bFGF, CD31, CD34 and CD133 IHC assays in the xenograft model, plug and tumor samples collected from sacrificed mice were fixed in 4% paraformaldehyde in PBS for at least 72 h, dehydrated in increasing concentrations of ethanol, then embedded in paraffin. Serial sections of 5-μm thickness were cut longitudinally and incubated with anti-bFGF (1:50), anti-CD31 (1:50), anti-CD34 (1:100) or anti-CD133 antibody (1:100) at 4°C overnight. After three PBS washes, samples were incubated with 1:50 dilution of biotin-labeled goat anti-mouse IgG secondary antibody, with bound antibodies detected by ABC Kit. The slides were stained with chromogen diaminobenzidine, washed, counterstained with Delafield’s hematoxylin, dehydrated, treated with xylene, then mounted. To quantify microvessel density (MVD), we evaluated the degree of microvessel coverage in 3 random fields per plug (200 x magnification). MVD was calculated using quantification of CD31-positive microvessels per field of view.
Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM). Statistical comparisons between 2 samples were performed using the Student’s t-test. Statistical comparisons of more than 2 groups were performed using one-way analysis of variance with Bonferroni’s post-hoc test. A p-value of less than 0.05 was considered statistically significant.
Results
bFGF regulates EPCs-primed angiogenesis by increasing VEGF expression in chondrosarcoma cells
Emerging evidence indicates that angiogenesis contributes to tumor metastasis as a vital step in tumor progression . VEGF is the most important angiogenic factor involved in angiogenesis and participating in tumorigenesis . To learn whether bFGF promotes angiogenesis by VEGF expression, we certified VEGF expression by bFGF treatment in chondrosarcoma cells (JJ012). Results averred bFGF increasing VEGF expression and secretion in JJ012 (Fig. 1A-C). Prior study indicates EPCs contributing to both angiogenesis and angiogenesis-related growth. The data 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266
confirmed that tumors recruiting EPCs to tumor microenvironment, followed by differentiation into endothelial cells and promoting vessel formation . To investigate whether bFGF-induced VEGF secretion in chondrosarcoma cells could recruit EPCs and following promote EPCs angiogenesis, we treated JJ012 cells with recombinant bFGF and collected condition media (CM) after 24 h. The JJ012-CM was accessed for EPCs migration assay and tube formation assay. Transwell migration assay indicated condition media (CM) collected from bFGF-treated JJ012 increasing EPC migration. Pretreatment with VEGF neutralized antibody negated the effect (Fig. 1D), suggesting EPCs recruited to tumor microenvironment by bFGF-regulated VEGF expression. We also examined angiogenic function of recruited EPCs, noting that CM collected from bFGF-treated chondrosarcoma cells increased EPC tube formation and inhibited by VEGF neutralized antibody treatment (Fig. 1E-F). Cell surface receptor is a key mediator to coordinate extracellular response into cells; previous studies indicate that bFGF could regulate cell signaling via FGF receptor 1 (FGFR1) . Transfection with FGFR1 siRNA inhibited bFGF-enhanced VEGF expression in chondrosarcoma and as well as EPCs-primed angiogenesis (Fig. 1G-I). These results point to bFGF promoting VEGF expression in chondrosarcoma cells, and regulate EPC-primed angiogenesis in chondrosarcoma.
bFGF promotes VEGF expression in chondrosarcoma cells and EPCs-primed angiogenesis by c-Src/p38 signaling pathway
Intracellular signaling pathways that activate transcription factors and subsequently up-regulate gene expression are crucial to cell biological functions. Earlier study cited c-Src and p38 signaling cascades involved in VEGF expression . We investigated the signaling pathway involved in bFGF-regulated VEGF expression. Pretreatment or transfection with c-Src and p38 inhibitors or siRNAs reversed bFGF-induced VEGF expression in chondrosarcoma cells (Fig. 2A-C). EPCs migration and tube formation induced by bFGF-treated JJ012 CM were abolished (Fig. 2D-E). Finally, bFGF pretreatment of chondrosarcoma cells (30 ng/ml) increased phosphorylation of c-Src and p38 signaling proteins (Fig. 2F); pretreatment with c-Src inhibitor (PP2) decreased p38 phosphorylation (Fig. 2G). These indicated that bFGF regulates VEGF expression and EPC-primed angiogenesis through c-Src/p38 signaling pathway. bFGF promotes VEGF expression in chondrosarcoma cells and EPC-primed angiogenesis by NF-κB signaling pathway
Plenty of reports document HIF-1α as the canonical transcription factor involved in VEGF expression , but other such factors bind to VEGF promoter. NF-κB is often activated in tumors and associated with progression of cancer. Prior report indicates 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
NF-κB involvement in upregulation of VEGF mRNA in breast cancer , suggesting possible regulation of VEGF expression by NF-κB in chondrosarcoma. Pretreatment or transfection with NF-κB pathway inhibitors or p65 siRNAs reversed bFGF-induced VEGF expression (Fig. 3A-C). As expected, EPCs migration and tube formation induced by bFGF-treated JJ012 CM were abolished (Fig. 3D-E); bFGF (30 ng/ml) pretreatment of chondrosarcoma cells increased while pretreatment with c-Src and p38 inhibitors impeded p65 phosphorylation (Fig. 3F-G). Luciferase reporter assay analyzed for NF-κB activation revealed that stimulation of JJ012 cells with bFGF increased luciferase reporter activity and negated by pretreatment with c-Src, p38 and NF-κB inhibitor or siRNA (Fig. 3 H-I). These data hint bFGF regulating VEGF expression in chondrosarcoma cells and EPC-primed angiogenesis via NF-κB activation.
Knockdown of bFGF expression decreases VEGF expression in chondrosarcoma cells and EPCs-primed angiogenesis
We saw bFGF promoted VEGF expression in chondrosarcoma and enhanced EPCs-primed angiogenesis. It is critical to pinpoint the role of bFGF in vivo. To confirm its regulatory role in VEGF expression, we utilized JJ012 cells stably expressing bFGF shRNA. Results showed expression level of bFGF was decreased in bFGF shRNA stable clone. Moreover, bFGF knockdown significantly reduced VEGF expression (Fig. 4A-B). CM collected from JJ012/control-shRNA promoted EPCs cell migration and tube formation but declined while incubating with CM collected from JJ012/bFGF-shRNA (Fig. 4C-D). Finally, the bFGF in vivo role was examined by chick embryo chorioallantoic membrane (CAM) assay. As expected, CM amassed from JJ012/control-shRNA cells obviously enhanced CAM angiogenesis, while bFGF-shRNA completely reduced angiogenesis in CAMs (Fig. 4E). For in vivo Matrigel plug formation assay by subcutaneous implantation in mice, Matrigel mixed with CM from JJ012/control-shRNA increased blood vessel growth; CM from JJ012/bFGF-shRNA starkly reduced neovascularization (Fig. 4F, first row). Vascular formation in Matrigel declined in CD31 IHC and hemoglobin content assay (Fig. 4F, second and third row); these indicate bFGF promoting angiogenesis through VEGF expression in vivo.
Knockdown of bFGF decreases angiogenesis-related tumor growth in vivo
To evaluate bFGF contributing to VEGF expression and EPC-primed angiogenesis in chondrosarcoma microenvironment, as well as promoting tumor growth, our experiments used bFGF shRNA stable cell line in a mouse xenograft model. Bioluminescence images indicated JJ012/control-shRNA profoundly inducing tumor 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342
mass formation, but knockdown of bFGF reduced tumor growth in mice (Fig. 5A-D). We quantified the level of angiogenesis by examining hemoglobin content of tumors to find knockdown of bFGF expression impeding chondrosarcoma-related angiogenesis in vivo (Fig. 5E). Hemoglobin content also positively correlated with tumor volume (Fig. 5F). Finally, IHC results of bFGF and CD31 certified bFGF-promoted angiogenesis (Fig. 5G). Moreover, detection of EPCs markers positive staining (CD-34 and CD-133) cells in control shRNA group but not bFGF shRNA group showed that bFGF recruited EPCs in chondrasarcoma microenvironment (Fig. 5G). In sum, data revealed bFGF promoting VEGF expression, recruiting EPCs, and promoting EPCs-primed angiogenesis and tumor growth in vivo.
bFGF highly expressed in chondrosarcoma, correlated with VEGF expression Our in vitro and in vivo results showed bFGF promoting EPCs-primed angiogenesis by regulating VEGF expression in chondrosarcoma cells. It is important to investigate correlation between bFGF and VEGF in clinical specimens. We tabulated bFGF and VEGF expression levels in specimens collected from human chondrosarcoma cases, using immunohistochemistry (IHC), to find bFGF and VEGF nearly undetectable in normal cartilage but associated with higher clinical pathologic Grade (Fig. 6 A-C). Quantitative data show bFGF expression correlating with VEGF expression in our specimens (Fig. 6D), suggesting bFGF linked with VEGF expression and tumor progression in chondrosarcoma.
Discussion
Unlike other mesenchymal malignancies like osteosarcoma and Ewing’s sarcoma, which saw dramatically longer survival with the advent of systemic chemotherapy, chondrosarcoma continues to show poor prognosis without effective adjuvant therapy . In the past decade, it has become a general concept that angiogenesis could serve as a therapeutic target for blocking cancer growth; VEGF has been implicated as a pivotal target of antiangiogenic therapy . Previous observation indicates that high microvessel density associated with higher histological grade and poor prognosis in chondrosarcoma , prompting search for means to prevent angiogenesis. We found evidence that bFGF promoted EPCs-primed angiogenesis by regulating VEGF expression in chondrosarcoma cells. Also, bFGF regulated VEGF expression through c-Src/p38/NF-κB signaling pathway in chondrosarcoma cells. The IHC results from clinical specimens of chondrosarcoma validated our findings. In sum, our clinical specimens, cell experiment and animal models suggest bFGF boosting VEGF expression and expediting EPCs-primed angiogenesis.
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As the pro-angiogenic function and tumor angiogenesis effects of bFGF, up-regulation of bFGF is discussed in various types of cancer such as OSCC, esophageal cancer, haematological malignancies, and glioblastoma . However, controversial findings were discussed in breast cancer. bFGF expression in breast cancer cells shows less malignant phenotype by decreasing motility and invasion . The other study also provides the evidence of pro-apoptosis effects of bFGF in breast cancer by regulating Bcl-2 family expression . In contract, some studies indicate that bFGF promote cell proliferation and treatment with bFGF antagonist peptide (P7) inhibits breast cancer cell growth . These observations maybe caused by cell type specific effects and heterogeneity of cancer cells. However, the role of bFGF in chondrasarcoma is poorly discussed.
Previous study indicates that bFGF regulates MMP-13 expression to promote chondrosarcoma progression . Moreover, bFGF increases proliferation of SW1353 chondrosarcoma cell line . Our in vivo results showed inhibition of tumor growth by bFGF knockdown maybe due to proliferation inhibition and decreasing angiogenesis-related tumor growth (Fig. 5A-D). In spite of this, the angiogenic regulation by bFGF shows novel therapeutic opportunity. A single angiogenic inhibitor is not sufficient to inhibit angiogenesis and tumor growth. The pivotal role of bFGF in chondrosarcoma may provide a combination treatment to inhibit bFGF and VEGF pathway.
Since EPCs were first described a decade ago, emerging evidence indicated their contribution to angiogenesis-mediated growth of certain tumors in mice and human . Recruitment of EPCs is regulated by growth factors, chemokines and cytokines secreted during tumor growth . One of them is VEGF; it modulates bone marrow microenvironment from a quiescent to a highly pro-angiogenic state, thus promoting mobilization of both vascular and hematopoietic progenitors to peripheral circulation, which are recruited to primary tumors or metastatic lesions . The role of bFGF, a pivotal component involved in angiogenesis, in EPC regulation is little discussed. We found tumor-secreted bFGF recruiting EPSs and promoting angiogenesis through VEGF expression in chondrosarcoma cells (Fig. 1A-F). It has been proposed that bFGF and VEGF are the most important tumor-secreted angiogenic cytokines in lung cancer , but the angiogenic cytokine involved in chondrasarcoma has not been discussed. Our report first certifies the role of these cytokines in angiogenesis, warranting investigation of their clinical significance in chondrasarcoma.
Cell surface receptor is an important mediator to coordinate extracellular response into cells. Yet the receptor activated by bFGF is little discussed. We classified FGFR1 as the binding receptor activated by bFGF treatment. Our result showed transfection with FGFR1 siRNA abolishing bFGF-induced VEGF expression in chondrosarcoma (Fig. 1G-J). These reveal a vital role of FGFR1 in bFGF activation. We have first 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418
discussed FGFR1 involvement in bFGF activation, meriting further examination to reveal clinical relevance. Likewise, downstream effectors and transcription factors of FGFR1 by bFGF activation prove critical for exerting cell biological functions. We found bFGF activating c-Src, p38 signaling proteins to regulate VEGF expression (Fig. 2). Previous studies indicated FGFRs eliciting different signal cascades: e.g., Src, PLC-γ, PI3K, Ras and MAPK signaling proteins . Our results showed bFGF promoting VEGF expression in chondrosarcoma via c-Src/p38 signal cascade. c-Src, non-receptor tyrosine kinase, is a co-transducer of mitogenic signals eliciting from numerous tyrosine kinase growth factor receptors, including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), colony stimulating factor-1 receptor (CSF-1R), and basic fibroblast growth factor receptor (bFGFR) . Prior study shows Src signaling protein highly activated in chondrosarcoma, playing a role in chemoresistance . Dasatinib, a small molecule tyrosine kinase inhibitor targeting Src, decreases viability in chondrosarcoma cell lines. Whether it can abolish bFGF-regulated angiogenesis in chondrosarcoma will be discussed in the future.
Clinical Perspectives
(i) VEGF and bFGF were most prominent tumor-related angiogenesis secreted factors produced by cancer cells. VEGF expression correlated with chondrosarcoma angiogenesis. Still, clinical correlation between bFGF and chondrosarcoma is unknown.
(ii) Higher bFGF expression associates with higher clinical pathologic stages: expression of bFGF correlates with VEGF expression in human chondrosarcoma specimens. We show bFGF promoting EPCs-primed angiogenesis by regulating VEGF expression in chondrosarcoma cells, with FGFR1/c-Src/p38/NF-κB signal pathway activation.
(iii) A concept of tumor neoangiogenesis has been verified. We cite evidence of bFGF-regulated VEGF expression and angiogenesis in chondrosarcoma via FGFR1. Recently, brivanib, a specific receptor tyrosine kinase inhibitor targeting key angiogenesis receptors VEGFR2, FGFR1 and -2, has been devised under clinical evaluation. Our findings of bFGF and VEGF may provide a novel target for brivanib as synergic inhibition drug.
Conflict of Interest: The authors declare that they have no competing interests Author contributions
H.E. Tzeng: conception and design, collection of data, data analysis and 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456
interpretation;P.C. Chen: data analysis and interpretation manuscript writing; K.W. Lin and C.Y. Lin: conception and design, collection of data, data analysis and interpretation; C.H. Tsai, S.M. Han, C.L. Tengand W.L. Hwang: provision of study material; S.W. Wang: provision of study material; C.H. Tang: conception and design, data analysis and interpretation, manuscript writing provision of study material
Funding
This study was supported by grant from the National Science Council of Taiwan (NSC 102-2632-B-039-001-MY3), China Medical University (CMU 103-S-06), Ministry of Science and Technology (MOST 103-2628-B-039-002) and Taichung Veterans General Hospital (TCVGH-1043701C).
Figure legends
Fig.1 bFGF regulates EPCs-primed angiogenesis by raising VEGF expression in chondrosarcoma. (A-C) JJ012 cells were incubated with bFGF (0–100 ng/ml) for 24 h, VEGF expression examined by qPCR, ELISA and western blot. (D-F) JJ012 cells were incubated with bFGF (0–100 ng/ml) for 24 h, medium collected as CM. EPCs were pre-treated for 30 min with IgG control antibody or VEGF antibody (1 μg/ml) and incubated with CM for 24 h, cell migration examined by Transwell assay (D). EPCs were incubated with CM for 6 h and cell capillary-like structure formation in EPCs was photographed and counted (E-F). (G-H) JJ012 chondrosarcoma cells were transfected with FGFR1 siRNAs for 24 h, followed by stimulation with bFGF (30 ng/ml) for 24 h. VEGF expression was examined by qPCR and ELISA. (I-J). JJ012 cells were treated as (G), and the medium was collected as CM. The EPCs were incubated with CM for 24 h and cell migration examined by Transwell assay (I). Moreover, EPCs were incubated with CM for 6 h and the cell capillary-like structure formation in EPCs examined by tube formation assay (J). Data are expressed as mean ± SEM *p<0.05 compared to control; #p<0.05 compared to bFGF-treated group. Fig.2 Src/p38 signaling pathway is involved in bFGF-promoted VEGF expression in chondrosarcoma cells and EPC-primed angiogenesis. (A-C) JJ012 cells were pre-treated with Src inhibitor PP2 (1 μM) or p38 inhibitor SB203580 (1 μM) for 30 min, or transfected with c-Src or p38 siRNAs for 24 h, followed by bFGF (30 ng/ml) stimulation for 24 h, with VEGF expression examined by qPCR, ELISA and western blot. (D-E) JJ012 cells were treated as (A), medium collected as CM. EPCs were incubated with CM for 24 h, cell migration examined by Transwell assay (D). EPCs were incubated with CM for 6 h; tube formation assay examined capillary-457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494
like structure formation in EPCs (E). (F) JJ012 cells were incubated with bFGF (30 ng/ml) for designated times, c-Src and p38 phosphorylation determined by western blot. (G) JJ012 cells were pre-treated with Src inhibitor PP2 (1 μM) for 30 min, followed by stimulation with bFGF (30 ng/ml) for 60 min, and p38 phosphorylation were determined by western blot. Data express mean ± SEM *p<0.05 compared with control; #p<0.05 compared with bFGF-treated group.
Fig.3 NF-κB transcription factor is involved in bFGF-promoted VEGF expression in chondrosarcoma cells and EPCs-primed angiogenesis. (A-C) JJ012 cells were pre-treated with NF-κB inhibitor PDTC (10 μM) or TPCK (1 μM) for 30 min, or transfection p65 siRNAs for 24 h, followed by stimulation with bFGF (30 ng/ml) for 24 h. VEGF expression was rated by qPCR, ELISA and western blot. (D-E) JJ012 cells were treated as (A), medium collected as CM. EPCs were incubated with CM for 24 h, cell migration tested by Transwell assay (D). EPCs were incubated with CM for 6 h; tube formation assay examined capillary-like structure formation (E). (F) JJ012 cells were incubated with bFGF (30 ng/ml) for designated times, p65 phosphorylation assessed by western blot. (G) JJ012 chondrosarcoma cells were pre-treated with Src inhibitor PP2 (1 μM) and p38 inhibitor SB203580 (1 μM) for 30 min, followed by stimulation with bFGF (30 ng/ml) for 24 h, with p65 phosphorylation determined by western blot. (H) JJ012 cells were transfected for 24 h with NF-κB promoter reporter plasmid, treated with Src inhibitor PP2 (1 μM), p38 inhibitor SB203580 (1 μM), NF-κB inhibitors PDTC (10 μM) or TPCK (1 μM) for 30 min, followed by r 24 h bFGF (30 ng/ml) stimulation, then analyzed for luciferase activity. (I) JJ012 cells were co-transfected for 24 h with NF-κB promoter reporter plasmid and c-Src, p38 or p65 siRNAs for 24 h, followed by stimulation with bFGF (30 ng/ml) for 24 h, and analyzed for luciferase activity. Data represent mean ± SEM *p<0.05 compared to control; #p<0.05 compared with bFGF-treated group.
Fig.4 Knockdown of bFGF expression in chondrosarcoma cells lowers VEGF expression and EPCs-primed angiogenesis. (A-B) The mRNA and protein expressions of bFGF and VEGF in JJ012/control shRNA and JJ012/bFGF shRNA were examined by qPCR, western blot and ELISA. (C-D) EPCs were incubated with CM collected from JJ012/control shRNA or JJ012/bFGF shRNA for 24 h, cell migration or tube formation examined by Transwell assay or photographed by a microscope. (E) Upper panel: PBS, VEGF, JJ012/control shRNA CM or JJ012/bFGF shRNA CM suspended in Matrigel were placed on chick chorioallantoic membranes. CAM arteriosus branches in groups were photographed on developmental Day 12. Lower panel: Quantification of the branch number in the CAM assay (n=7). (F) Mice 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532
were subcutaneously injected with Matrigel mixed with PBS, JJ012/control shRNA CM or JJ012/bFGF shRNA CM for seven days. Plugs excised from mice were photographed and stained with CD31, quantification of microvessel density (MVD) was showed as mean percentage of 3 random fields (n=5). Data represent mean ± SEM *p<0.05 compared to JJ012/control shRNA group.
Fig.5 Knockdown of bFGF expression decreases angiogenesis-related tumor growth in vivo. (A) JJ012/control shRNA and JJ012/bFGF shRNA cells were mixed with Matrigel and injected into flank sites of mice for 28 days, tumor growth monitored by IVIS Imaging System (n=6). Fluorescent imaging quantified data at Weeks 0-6 (B-E), after which mice were sacrificed. Tumors were resected, photographed by a microscope, measured for weight and volume, with hemoglobin quantified. Correlation between tumor volume and hemoglobin level appears in (F). Tumors were paraffin embedded, bFGF, CD31, CD34 and CD133 antibody used for IHC assay (G). Data are expressed as mean ± SEM *p<0.05 compared with JJ012/control shRNA group.
Fig.6 Correlation of bFGF, VEGF and tumor grades in human chondrosarcoma tissues. Tumor specimens were stained by immunohistochemical (IHC) with anti-bFGF and anti-VEGF antibodies, staining intensity scored 1-5. IHC quantified anti-bFGF and VEGF expression. (A) IHC photography. (B-D) Quantitative results and correlation among bFGF, VEGF and clincal grade of chondrosarcoma. (E) Diagrammatic model for the role of bFGF in chondrosarcoma tumor microenvironment. (1) bFGF induces VEGF expression and secretion in JJ012 chondrosarcoma through FGFR1/c-Src/p38/NF-κB signal pathway activation. (2) The bFGF-induced secretion of VEGF subsequently recruiting EPCs to chondrosarcoma tumor microenvironment and promoting neoangiogenesis.
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