Direct anti-cancer effect of oncostatin M on chondrosarcoma
The cytokine Oncostatin M (OSM) is cytostatic, pro-apoptotic and induces differentiation of osteosarcoma cells into osteocytes, suggesting new adjuvant treatment for these bone-forming sarcomas. However, OSM systemic over-expression could lead to adverse side effects such as generalized inflammation, neoangiogenesis and osteolysis. We determine here the effect of OSM on chondrosarcoma, another primary bone sarcoma characterized by the production of cartilage matrix and altered bone remodelling. Chondrosarcomas are resistant to conventional chemotherapy and radiotherapy, and wide surgical excision remains the only available treatment. We found that OSM blocked the cell cycle in four of five chondrosarcoma cell lines, independently of p53 and presumably through the JAK3/STAT1 pathway. In two tested cell lines, OSM induced a hypertrophic chondrocyte differentiation, with an induced Cbfa1/SOX9 ratio and induced Coll10, matrix metalloproteinase 13 (MMP13) and RANKL expression. Adenoviral gene transfer of OSM (AdOSM) in the Swarm rat chondrosarcoma (SRC) model indicated that local intra-tumoral OSM over-expression reduces chondrosarcoma development not only with reduced tumor proliferation and enhanced apoptosis but also with enhanced RANKL expression, osteoclast formation and reduced bone volumes. Flu-like symptoms were induced by the AdOSM, but there was no effect on tumor angiogenesis. Therefore, OSM could be considered as a new adjuvant anti-cancer agent for chondrosarcomas. A local application of this cytokine is presumably needed to overcome the poor vascularization of these tumors and to limit the deleterious effect on other tissues. Its side effect on bone remodeling could be managed with anti-resorption agents, thus offering potential new lines of therapeutic interventions.
Chondrosarcomas, the second most frequent primary bone tumors after osteosarcomas, are relatively rare malignancies characterized by production of cartilage matrix. They have poor vascularity, are resistant to conventional chemotherapy and radiotherapy, and wide surgical excision remains the only available treatment with a 10-year survival between 30 and 80% depending on the grade.1–3 Poor survival rates are observed for dedifferentiated chondrosarcomas, for patients with disease recurrence or metastasis. Therefore, new therapeutic options are needed for these high risk patient groups.1–3 Recent studies identified potential new targets in chondrosarcoma such as hedgehog, p53, insulin-like growth factor, cyclin-dependent kinase 4 (cdk4), COX2, SRC or AKT.4–6 Their validation in preclinical animal models or human clinical trials is still sparse but new chemotherapeutic drugs will be certainly adopted in the near future. Other studies also described the potential of immunotherapy in combination with conventional treatment to eradicate primary bone tumors, mainly osteosarcomas or Ewing sarcomas. Not exclusively, these attempts used cytokines such as interleukin (IL)-2, IL-12, IL-17, IL-18, interferons, tumor necrosis factor (TNF)α or TRAIL (TNF-related apoptosis inducing ligand).7, 8 The major limitations of such approaches are the tumor resistance and unwanted side effects.
Chondrosarcoma cells are thought to derive not directly from cartilage cells but from bone mesenchymal stem cells (MSC), and are histologically and phenotypically related to the different stages of chondrocytes differentiation in the growth plate.1–4, 9 During skeletal development, long bones develop through endochondral ossification in the epiphyseal growth plate where MSC first differentiate into proliferating chondrocytes, enlarge, becoming hypertrophic, and die by apoptosis, allowing invasion of vascular sprouts, osteoblasts that form new bone and osteoclasts that resorb it.1, 2, 4, 10 The master chondrocytic transcription factor SOX9 is expressed by proliferating but not hypertrophic chondrocytes and induces expression of collagen type II (coll2), whereas Cbfa1 (Runx2) is expressed early in hypertrophic chondrocytes and induces expression of Coll10.10, 11 Matrix metalloproteinase 13 (MMP13) is selectively expressed in hypertrophic chondrocytes and degrades both fibrillar collagen and Aggrecan (Acan), allowing invasion of blood vessels.11, 12 Vascular invasion is facilitated by vascular endothelial growth factor (VEGF) expressed in hypertrophic chondrocytes.11 Thereafter, osteoclasts are recruited through enhanced expression of receptor activator for nuclear factor κ B ligand (RANKL) to resorb the remaining cartilage matrix.13, 14
Oncostatin M (OSM) is a pro-inflammatory cytokine of the IL-6 family which is composed of nine members such as IL-27 and leukemia inhibitory factor (LIF).15 OSM has cytostatic activities on sarcoma and carcinoma cell lines,15–17 activates endothelial cells,18 induces acute phase proteins production by liver as well as inflammatory and immune responses in various tissues such as skin, lung and bone/cartilage.15, 19, 20 In human, this pleiotropic cytokine can bind to the type I heterodimeric receptor composed of gp130 and LIF receptor (LIFR)21 or to the type II receptor composed of gp130 and OSMR.15 In contrast, murine OSM signals only through the gp130+OSMR complex.15 Binding of OSM to these receptors activates intracellular signalling proteins, mainly JAK1, JAK2, JAK3, STAT1, STAT3, the MAP kinase pathway as well as the PI3K/AKT cascade.15, 22
OSM is mainly produced by macrophages, neutrophils and T lymphocytes. Its levels are elevated in the synovial fluids of patients with rheumatoid arthritis (RA), contributing to cartilage and bone loss.18, 20, 23 Over-expression of OSM in mouse joints induces inflammation, cartilage and bone destruction, in association with increased RANKL synthesis in articular chondrocytes, synovial and inflammatory cells and thus enhanced osteoclast number.20, 23 OSM also induces RANKL expression in bone stromal cells or osteoblasts through STAT3.24, 25 In chondrocytes, OSM induces aggrecanases and collagenases such as MMP-1, MMP-3 and MMP-13, leading to extensive cartilage breakdown.18, 22 On RA synovial fibroblasts co-cultured with cartilage explants, OSM also enhances VEGF production.18 OSM is known to induce VEGF expression in various cancer cell types through activation of STAT326 and to increase endothelial cell tubule formation.18 Therefore, OSM may induce osteolysis and neoangiogenesis during articular inflammation or tumor development.
The effect of OSM or other IL-6 type cytokines on chondrosarcoma cells is poorly documented. In SW1353 chondrosarcoma cells, OSM upregulates expression of MMPs, cytokines, chemokines and extracellular matrix components.27 Because IL-6 serum levels are increased in adult primary bone sarcoma patients and correlate with poor survival, this cytokine may have an important role in the progression of osteosarcomas, chondrosarcomas, Ewing sarcomas and giant cell tumors of bone.15, 28 However, OSM or IL-6 block osteoblasts and osteosarcoma cells in the G0/G1 phase of the cell cycle by inducing expression of the cdk inhibitor p21WAF1.17, 29, 30 These cytokines do not induce apoptosis, but wild-type p53 osteosarcoma cells treated with OSM are particularly sensitive to apoptosis induced by staurosporine (STS), UV, TNFα or chemotherapeutic agents.29, 31 Indeed OSM, through activation of STATs and p53, increases the Bax/Bcl2 ratio which controls the mitochondrial apoptotic pathway.31 These effects on cell proliferation and apoptosis are concomitant with an induced terminal differentiation of osteoblasts or osteosarcoma cells into osteocyte-like cells.15, 32
Osteosarcomas and chondrosarcomas presumably derive from the same multipotent MSC which is able to differentiate into the osteoblastic or chondroblastic lineage.1, 9, 33 Therefore, we determine here the effect of IL-6 type cytokines on chondrosarcoma cell lines proliferation, apoptosis and differentiation in vitro and in vivo in the Swarm rat chondrosarcoma (SRC) model. OSM appeared as a major cytostatic cytokine inducing a hypertrophic-like differentiation but also with potential to enhance osteolysis.
Material and Methods
Cells and culture conditions
SRC cell line was established from tumor fragments of SRC, considered as a grade II chondrosarcoma (kind gift from P.A. Guerne, Geneva, Switzerland),34 after dissociation with collagenase 1 mg/ml (Roche diagnostics, Basel, Switzerland) and trypsine 0.12% (Lonza, Basel, Switzerland). SW1353 cell line, isolated from a human conventional grade II chondrosarcoma of the humerus, was purchased at American Type Culture Collection (ATCC, Manassas) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’sF12 (Lonza)(50:50) supplemented with 10% foetal bovine serum (FBS, Hyclone Perbio, Bezons, France). Human CAL78 cell line, isolated from a muscle reccurency of a dedifferentiated chondrosarcoma, was purchased at German collection of microorganisms and cell cultures (DSMZ, Braunschweig, Germany). Human OUMS27 cell line, isolated from a human conventional grade III chondrosarcoma of the humerus, was purchased at Health Science Research Resources Bank of Japan (HSRRB, Osaka, Japan). Human BCSCH03 cell line was isolated in the Unit of Cell and Gene Therapy (UTCG, Nantes hospital, France) from a dedifferentiated chondrosarcoma of the femur and was used between passage 25 and 42. Primary human chondrocytes, isolated from femur articulation (orthopedic service, CHU Nantes, France), were a kind gift from Atlantic Bone Screen (Nantes, France). Unless otherwise stated, all cells were cultured in DMEM supplemented with 10% FBS.
Viable cell quantification
For short-term studies, the number of viable cells was quantified using the Vialight plus kit (Lonza) or by XTT assay (Cell proliferation kit II, Roche) with similar results. Cells were plated into 96-well plates at a density of 1,000 cells per well and cultured 72 hr before adding the kit reagents. For the Vialight plus kit, the luminescence was read in a Tristar Luminometer (Berthold technologies, Wildbad, Germany). For the XTT assay, the absorbance was read at 490 nm in a Victor2 reader (Beckman Coulter, Paris, France). For long-term studies (15 days), cells were plated in duplicate into 24-well plates at a density of 10,000 cells per well. Every 3 or 4 days, cells were released with trypsin–ethylenediaminetetraacetic acid (EDTA) (Lonza) and living and dead cells were quantified by Trypan Blue exclusion. After counting, 10,000 cells were plated again.
Real-time polymerase chain reaction
Total RNA was extracted and subjected to cDNA synthesis as described Ref.17 The polymerase chain reaction (PCR) reaction mixture contained 20 ng reverse-transcribed total RNA, 300 nM forward and reverse primers and 8 μl SYBR green buffer (Bio-Rad, Marnes la Coquette, France) in a final volume of 10 μl. PCR were carried out in triplicate using the Chromo4 System (Bio-Rad). Primers (Supporting Information Table 2) were designed by Online Roche applied science software. Analyses were performed using the Vandesompele method.35
Experimental chondrosarcoma model
All animals for in vivo experimentations were housed under pathogen-free conditions in accordance with the institutional guidelines of the regional Ethical Committee for animal experiment (CEEA PdL06) and under the supervision of authorized investigators. Five-week-old male Sprague Dawley OFA (Oncins France souche A) rats (Charles River, L’Arbresle, France) were anesthetized by inhalation of an isoflurane–air mixture (3%, 0.3 l/min) before surgical procedures. For tumor induction, 2 × 2 × 2 mm3 fragments of SRC tumor were excised from a donor rat and transplanted into naive rats (in a intra-muscular approach in close contact to the left tibia).34 For fragment insertion, a 5-mm section was performed to open the muscle along the tibia, tumor fragment was transplanted, then muscular and subcutaneous wounds were sutured. The tumor volume was quantified by measuring two perpendicular diameters with a vernier caliper and calculated with the formula: (l2 × L)/2 (l, the smallest and L, the largest diameter).29, 34
In-vivo treatment with Adenovirus
Replication-deficient adenovirus encoding mouse OSM (AdOSM) has been described previously29, 36 and was produced, together with adenovirus encoding green fluorescent protein (AdGFP), in the vector facility of the INSERM U649 Laboratory (Nantes, France). Approximately 20 days after transplantation when tumor volumes reached 1,000 mm3 (considered as progressive tumors), rats were injected at three points in the tumor with either PBS, AdOSM or AdGFP at a dose of 1.109 pfu. Animals were weighed three times a week up to their sacrifice by CO2 inhalation. In a first long-time experiment, animals were sacrificed when suffering signs appeared or when tumor reached 15,000 mm3. In a second short-time experiment, animals were sacrificed before tumor relapse 9 days after adenovirus injection. All experiments with adenoviruses were approved by the French Ministry of Research (Commission de Génie Génétique n°4047).
Results were analysed with unpaired t-test using GraphPad InStat v3.02 software. Results are given as mean ± SD (for in vitro experimentations) or mean ± SEM (for in vivo experimentations) and results with p < 0.05 were considered significant. More information can be found in the Supporting Information Material and Methods.
OSM inhibits the proliferation of chondrosarcoma cells
Rat SRC cells were first treated with the nine cytokines in the IL-6 family (50 ng/ml for 3 days) and the number of viable cells was determined. OSM was the strongest inhibitor, followed by IL-27 and IL-6 when combined with its soluble receptor sIL-6R, the other cytokines being inactive (Fig. 1a). The inhibitory effect of OSM was maximal (50% inhibition) at 25 ng/ml, whereas a maximal inhibition (35%) was obtained with IL-6 at 50 ng/ml when combined with sIL-6R at 100 ng/ml (Fig. 1b). We then tested a panel of human chondrosarcoma cell lines and non transformed articular chondrocytes. We also used tumor cells isolated from a chondrosarcoma biopsy, the BCSCH03 cells, to extend our results to a relatively short-term culture of a human specimen. OSM significantly reduced the number of viable CAL78, OUMS27 and BCSCH03 chondrosarcoma cells, as well as the number of viable chondrocytes, whereas no effect was observed on SW1353 cells (Fig. 1c).
OSM reduces the number of viable chondrosarcoma cells. SRC cells were treated as indicated with different IL-6 type cytokines (50 ng/mL)(a) or with increasing concentration of murine OSM or sIL-6R in presence of 50 ng/ml of IL-6 (b) for 3 days and the number of viable cell was assessed using the Vialight kit. Assays were performed with n = 6 or 7 for each condition. c, Indicated chondrosarcoma cell lines were treated with murine or human OSM for 3 days (n = 4 for each condition), and the number of viable cell was assessed using the Vialight kit. Cartilage chondrocytes were also treated with OSM for 3 days (n = 7), and the number of viable cell was assessed by XTT assay. d, Indicated chondrosarcoma cell lines were treated with OSM for 15 days, and living cells were quantified by Trypan Blue exclusion. Results are expressed as the mean ± SD. *p < 0.05, **p < 0.0002 compared to the control without cytokine.
To discriminate between reduced proliferation and induced cell death, we treated the five chondrosarcoma cell lines with OSM during 15 days, with counting of the living and dead cells every 3–4 days. Chondrocytes could not be tested because of insufficient proliferative and survival capability. We never observed an OSM-induced cell death in these conditions, whereas the number of living cells was reduced in all cell lines except SW1353 cells (not shown and Fig. 1d). After 15 days of OSM treatment, the reduction of viable cell number reached 50% for OUMS27 cells, and 80% for SRC, CAL78 and BCSCH03 cells (Fig. 1d).
Indeed, 1 day of OSM treatment significantly blocked SRC cells in the S phase of the cell cycle (Fig. 2a). Longer treatment times (3 days until 15 days) indicated a stable S and G2/M blockade (not shown). On OUMS27, BCSCH03 and CAL78 cells, OSM had a significant effect after 3–7 days (Figs. 2b–2d), the cells being blocked in G0/G1 rather than in S or G2/M.
OSM induces cell cycle blockade. a, Cell cycle profile of SRC cells after 1 day of treatment with mOSM (50 ng/mL). The percentage of cells in G0/G1, S and G2/M is indicated. Percentages of cells in the different phases of the cell cycle for OUMS27 and BCSCH03 cells treated for 3 days with hOSM (b and d) or for CAL78 cells treated for 7 days with hOSM (c) are shown. e and f, Expression of cell cycle proteins in SRC, CAL78 and OUMS27 cells after 4, 24 or 96 hr of treatment with OSM was analyzed by Western blot. All presented results are representative of 2–3 independent experiments.
In accordance, OSM induced two cdk inhibitors in CAL78 cells, p21WAF1 and p27kip1, together with accumulation of G0/G1 markers like cyclin D1, the inactive phosphorylated form of cdk2 (P-cdk2) and Cdc25a (Fig. 2e). However, several markers of the G2/M checkpoint were modified with similar kinetics. The inhibitory kinase wee1 was induced, together with the inactive phosphorylated form of the phosphatase cdc25c, two events that presumably led to accumulation of inactive phosphorylated cdc2, the kinase necessary for entry into mitosis (Fig. 2e). In OUMS27 cells, we confirmed accumulation of G0/G1 proteins (p27kip1 but not p21WAF1) and G2/M proteins (wee1, P-cdc25c and P-cdc2) (Fig. 2f). The analysis in SRC cells, although limited to available antibodies to rat proteins, indicated an early induction of P-cdc2 at 24 hr, followed by an enhanced expression of p27kip1 but not p21WAF1 at 96 hr (Fig. 2e).
OSM sensitizes SRC cells to death
As previously described for osteosarcoma,31 we pretreated chondrosarcoma cells with OSM for 3 days and tested the cell death induced by the kinase inhibitor STS. We could detect a synergistic increase in cell death with the combination of OSM + STS in SRC cells (Supporting Information Fig. S1A) but not in SW1353, CAL78, OUMS27 or BCSCH03 cells (not shown). In SRC cells, OSM alone induced the phosphorylation and activation of p53, followed by accumulation of this protein (Supporting Information Fig. S1B). However, the pro-apoptotic protein Bax was not induced by OSM and the anti-apoptotic protein Bcl2 was not detected. The combination OSM + STS did not significantly enhance the activation of caspase 3 or 9 over the level observed with STS alone (Supporting Information Figs. S1C and S1D). SW1353 and OUMS27 cells are known to express a mutated p53,37, 38 that could explain their resistance to apoptosis induced by OSM + STS. We sequenced the entire p53 cDNA from CAL78 and BCSCH03 cells and also identified a mutated p53 (E285del, E286del for CAL78; S106del leading to a stop codon in EX4 for BCSCH03).
The cytostatic effect of OSM presumably depends on JAK3 and STAT1
OSM-resistant SW1353 cells expressed the transcripts for OSMR, LIFR and gp130 receptor subunits at similar levels as other cell lines (Supporting Information Fig. S2A) and responded to OSM by a similar level of activated STAT1, STAT3, ERK 1/2 and Akt (Fig. 3a). Thus, to identify the pathway leading to growth inhibition by OSM, we used a panel of kinase or transcription factor inhibitors (Fig. 3b). Only WHI-P131, an inhibitor of JAK3, completely prevented the reduction of SRC viable cells induced by OSM. In CAL78 cells, growth inhibition by OSM was also prevented by WHI-P131 (Fig. 3b). WHI-P131 (1 μM) reduced the activation of STAT1 by OSM in SRC cells, with unaffected STAT3 and ERK 1/2 activation (Fig. 3c). As previously described,22 WHI-P131 also reduced the activation of Akt by OSM, but a similar inhibition of Akt by the PI3K/Akt inhibitor LY294002 did not prevent growth inhibition by OSM. At 10 μM, WHI-P131 totally prevented the phosphorylation of STAT1, whereas activation of STAT3, ERK 1/2 and Akt by OSM was still detected.
OSM inhibits chondrosarcoma proliferation presumably through JAK3/STAT1. a, Activation of STAT1, STAT3, ERK and Akt in the five chondrosarcoma cell lines after 15 min of OSM treatment (50 ng/mL) was assessed by western blot. b, SRC cells (left panel) were pretreated for 30 min with UO126 (MEK/ERK inhibitor; 10 μM), LY294002 (PI3K/Akt inhibitor; 20 μM), SB203583 (p38 inhibitor; 10 μM), STAT3inhibitor (10 μM), Piceatannol (JAK1 inhibitor; 10 μM), AG490 (JAK2 inhibitor; 25 μM) and WHI-P131 (JAK3 inhibitor, 10 μM) and then with mOSM for 3 days. CAL78 cells (right panel) were similarly treated with OSM and WHI-P131 (1 or 10 μM). The number of viable cells was assessed using XTT Assay (n = 5 for each condition). Columns, mean; bars, SD; *p < 0.05, **p < 0.0002 compared to the control without OSM. c, SRC cells were pretreated for 30 min with UO126, LY294002 or WHI-P131 (1 or 10 μM) and then with mOSM for 15 min. Activation of STAT1, STAT3, ERK and Akt was assessed by Western blot.
OSM induces a hypertrophic-like phenotype
When tested for their migratory activity in a slit assay, none of the five chondrosarcoma cell lines were affected by OSM (not shown). In contrast, OSM significantly reduced the adhesion of all cell lines to the plastic (Fig. 4a) or to plastic coated with fibronectin, vitronectin, collagen I or matrigel (not shown). OSM also induced morphological changes in all cell lines tested, the cells being elongated and more refringent (Supporting Information Fig. S2B).
OSM inhibits chondrosarcoma cell adhesion and modifies expression of osteolytic, vascular and differentiation markers in SRC cells. a, Indicated chondrosarcoma cells were treated for 3 days with OSM (50 ng/mL), dissociated with EDTA and tested for adhesion to plastic for 10 min. Results are expressed as percentage of adherent cells in OSM-treated wells compared to control wells (n = 3 for each condition). b, SRC cells were treated for 2 days with OSM. mRNA expression of indicated genes was assessed by real-time polymerase chain reaction (PCR) (n = 3 for each condition). Columns, mean; bars, SD; *p < 0.05, **p < 0.0002 compared to the control without OSM. c, Proposed mechanisms of action of OSM on chondrosarcoma cells based on our study and references.15, 22, 25, 26, 31, 45 See the text for more descriptions.
Next, we analyzed the expression of several genes potently implicated in chondrosarcoma development (Fig. 4b; see also Fig. 4c for a schematic presentation). RANKL was induced by OSM in SRC cells, but its decoy soluble receptor osteoprotegerin was not. Coll1a1 (expressed in MSC and pre-chondrocytes) and SOX9 (expressed in pre-chondrocytes and differentiated chondrocytes) were reduced, whereas Cbfa1, Coll10a1, VEGFA and MMP13 (expressed in hypertrophic chondrocytes) were induced. Coll2a1 and aggrecan (Acan) (expressed in differentiated chondrocytes) were unaltered. Similar results were obtained with CAL78 cells, i.e., induced RANKL, MMP13 and Coll10a1 expression, whereas Coll1a1 and SOX9 expression was reduced (Supporting Information Fig. S3).
Adenoviral gene transfer of OSM in the SRC rat model limits tumor progression
A rat syngenic chondrosarcoma model was used, corresponding to the SRC cell line.34 In a first experiment, adenoviruses encoding GFP (AdGFP) or mOSM (AdOSM) were injected in the left contralateral tibial anterior muscle. We used sub-toxic doses of AdOSM (1 × 109 pfu) as determined previously.29 With this protocol, the tumor volume evolution was similar after AdGFP or AdOSM injections (not shown). We then tested intra-tumoral (i.t.) injections. AdOSM i.t. significantly reduced tumor progression compared to AdGFP (Fig. 5a) or PBS injected animals (not shown). In a first set of experiments, the mean tumor volume was reduced 2.5 fold 9 days after AdOSM injection (31 days post-implantation), at which time tumors started to relapse and reached the AdGFP tumor volume by the end of the experiment (long-term experiment). These results were confirmed in a second short-term experiment (Fig. 5b): six of seven tumors stopped growing during the first 4 days after AdOSM injection and by day 9 some tumors have regressed (n = 3) and some have reached a plateau or continued to progress (n = 4). This different behavior could be related to relatively important differences in tumor size at the time of AdOSM injection, tumor regression being observed with the smaller tumors. On average, these two experiments gave a 1.6 fold reduction in relative tumor progression at day 9 post-injection (Fig. 5c; p < 0.0001).
AdOSM limits SRC chondrosarcoma progression in vivo. SRC fragments were implanted in close contact to the right tibia of rats. Three weeks post-implantation, indicated adenoviruses (1 × 109 pfu) were injected into the tumor. a, Time course of the mean tumor volume from a long-time experiment (AdGFP group, n = 7; AdOSM group, n = 7). Adenoviruses were injected at day 22 and animals were sacrificed when suffering signs appeared or when tumor reached 15,000 mm3. b, Individual tumor volume of animals treated with AdGFP (n = 7) or AdOSM (n = 7) from a short-time experiment. Adenoviruses were injected at day 20 and all animals were sacrificed at day 29. c, Relative tumor progression at day 9 post-injection of adenoviruses (pooled animals from experiments presented in A and B; n = 14 or 12 in each group). Columns, mean; bars, SEM; †, dead animal; *p < 0.05, **p < 0.01 and ***p < 0.0001 between AdOSM and AdGFP group.
Fourteen percent of the rats died few days after i.t. injection of AdOSM and none with AdGFP, confirming the toxic effect of OSM.29 We also observed significant weight loss and different signs of generalized inflammation during the first week after AdOSM injection, as previously described.29 Very few animals developed pulmonary metastatic nodules even after OSM over-expression, excluding a significant analysis at this level. Overall survival was not modified by AdOSM, but again the effect on tumor volume was only transient (Fig. 5a).
Tumors from the short-term experiment 9 days post-injection were then extensively analyzed by histology (Fig. 6a). Alcian blue staining, revealing mainly aggrecan (Acan) in cartilage tissue, confirmed that SRC cells produce an abundant cartilaginous matrix. In AdGFP animals, large alcian blue positive tumor areas were visible, separated by alcian blue negative stromal areas (Fig. 6a, upper panels). Tumor areas were composed exclusively of chondrocytes-like tumor cells. In AdOSM animals, two types of tumors were observed. The larger tumors (mainly the one which continued to progress or plateaued) appeared identical to AdGFP tumors, whereas the smallest tumors (mainly the one which regressed) had a significant reduced alcian blue staining, with sparse and necrotic tumor cells. The stromal tissue appeared identical in all groups of tumors. Ki67 staining revealed proliferating tumor cells mainly at the periphery of AdGFP tumor areas, with identical numbers of positive tumors cells (15%) in the larger AdOSM tumors (Fig. 6a). In contrast, proliferating tumor cells were grossly absent in the smaller AdOSM tumors (< 2%). Similar results were obtained using the mitotic index which focuses on the G2/M checkpoint (not shown). TUNEL staining confirmed an extensive proportion of apoptotic tumor cells in this latter group (>80%), compared to the AdGFP or larger AdOSM tumors.
AdOSM induces histological modifications in the tumoral tissue and reduces the bone volume in the SRC in vivo model. SRC fragments were implanted in contact to the right tibia of rats. Around 3 weeks post-implantation, indicated adenoviruses (1 × 109 pfu) were injected into the tumor. a, Adenoviruses were injected at day 20 and all animals sacrificed at day 29 (short-time experiment). Tibia bearing tumors were fixed, included, sectioned and stained as described in Material and Methods section with alcian blue (cartilage matrix) or for Ki67 (proliferation), TUNEL (apoptosis) and RANKL. Stainings were done on sagittal sections. Because of contrasting results, the AdOSM group was subdivided into two sub-groups: large tumors (n = 4) and small tumors (n = 2)(see the Results section for more explanations). Positive tumor cells (in red) were quantified by manual counting (right panels). For TRAP staining, positive osteoclasts at the interface between bone and tumoral tissue (100-μm large) were quantified using the Qwin software. Results are expressed as the percentage of osteoclast surface. T, tumoral tissue; St, stromal tissue; B, bone. b, Mice were injected with adenoviruses at day 22 and sacrificed individually at the end point (long-term experiment). Tumor bearing tibias or control tibias without tumor were dissected and analyzed by microCT scan. Top: representative transversal sections are shown. Bottom: representative sagittal sections are shown. Note the ectopic bone neoformation mainly in the AdGFP group. Right panel: the total relative bone volume (BV/TV) is shown. Columns, mean; bars, SEM; *p < 0.05, **p < 0.01 between AdOSM and AdGFP group. [Color
figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
Effect of OSM on tumor induced bone remodeling and angiogenesis
We observed a significant increased RANKL expression in the larger AdOSM tumors compared to the AdGFP tumors, positive cells being almost exclusively of tumor origin (Fig. 6a). No RANKL expression can be detected in the smaller AdOSM tumors, presumably because tumor cells were dead. TRAP (tartrate-resistant acid phosphatase) staining revealed a significant increased osteoclast surface (OcS) at the interface between tumor/stromal areas and bone in the larger AdOSM tumors compared to the AdGFP or smaller AdOSM tumors (Fig. 6a, lower panels). In contrast, we did not observe modulation of osteoblasts at the tumor-bone interface (not shown).
To analyze the potential impact on bone, we performed micro-CT scans on tibia of rats from the long-term experiments where animals were sacrificed individually with similar tumor volume at end point (between day 45 and 55 post-implantation)(Fig. 6b). Development of SRC tumors was associated with an important periosteal bone neoformation, leading to a significant increased total bone volume (BV/TV). OSM over-expression reduced this ectopic bone formation to control level obtained with tumor free tibias. OSM-treated tibias were however not normal as they appeared twisted and less harmonious than tibias without tumor (Fig. 6b).
To study tumor angiogenesis, we first performed VEGF immunostaining. Positive cells were mainly tumors cells and OSM over-expression did not modified VEGF expression (Supporting Information Fig. S4A). Next, CD34 immunostaining revealed vascular vessels exclusively in stromal areas even after AdOSM injection. OSM over-expression did not induce vascularization in the short or long-term experiments (Supporting Information Figs. S4A and S4B, respectively). We observed a reduced vascularization in the small AdOSM tumors in the short-term experiment, but this effect could be related to the smaller size and necrotic aspect of these tumors rather than a direct inhibitory effect of OSM on angiogenesis.
We show here for the first time that within the IL-6 family, OSM is a major cytostatic factor in four of five chondrosarcoma cell lines. Because LIF is inactive and both human and murine OSM are active, the type II receptor (gp130+OSMR) appears implicated in growth inhibition by OSM. Moreover, this effect is observed on mutated p53 conventional grade III (OUMS27) or dedifferentiated chondrosarcomas (CAL78, BCSCH03) for which patient survival is below 30% at 10 years.3 Two other cytokines are able to reduce chondrosarcoma expansion but with lower efficiency: IL-6 in association with its soluble receptor and IL-27. The naturally occurring soluble version of IL-6R (sIL-6R) enables cells to respond to IL-6 in absence of membrane-associated IL-6R. Osteoblasts and osteosarcoma cells express relatively low levels of membrane IL-6R and addition of sIL-6R is required for maximum IL-6’s effects on these cells.15, 29 Chondrosarcoma cells express detectable levels of IL-6R transcripts but the corresponding membrane and soluble proteins are presumably also expressed at a low level. Only one previous report described that IL-6 enhances proliferation of SRC cells in serum free cultures.39 Our experiments were performed in presence of 10% FCS and decreasing the FCS percentage did not convert the growth inhibitory effect of OSM into a growth stimulatory one (not shown). To define the exact role of IL-6+sIL-6R and IL-27 in chondrosarcoma expansion, additional experiments are needed but we can envisage that OSM, IL-6 and IL-27, through recruitment of the shared gp130 receptor subunit, activate similar signaling pathways that lead to chondrosarcoma growth suppression.
Growth suppression of chondrosarcoma cells by OSM in vitro relies on cell cycle blockade (in G0/G1 or S/G2/M), and no cell death is observed even after a long-term treatment. Blockade in G0/G1 by OSM is well documented in several types of cancer, and depends on JAK/STAT activation and transcriptional induction of p21WAF1 and/or p27kip1 independently of p53, leading to inhibition of cdk2 and 4, the kinases necessary for progression into the S phase.30, 31, 40–42 This pathway is clearly active in chondrosarcoma cells but we also detected modifications of G2/M proteins, leading to accumulation of inactive cdc2, the kinase necessary for entry into mitosis.43 In SRC cells, this S/G2/M blockade seems to overwhelm the G0/G1 blockade, possibly because of a delayed induction of p27kip1 and no induction of p21WAF1. To the best of our knowledge, this is the first description of a S/G2/M blockade by an IL-6 type cytokine.
Only the JAK3 inhibitor WHI-P131 prevents growth inhibition by OSM and preferentially inhibits STAT1 activation, whereas the well described JAK2/STAT3 inhibitor AG490 has no effect. Although care should be taken with this kind of inhibitors, these results suggest an important role of the JAK3/STAT1 pathway. Similar data have been obtained with WHI-P131and AG490 in articular chondrocytes where OSM induces proteases expression through JAK3/STAT1.22 STAT1 is considered as a tumor suppressor important for cancer cell growth suppression44 and is known to delay S phase progression via inhibition of cdc2 and cdk2.43 STAT1-deficient chondrocytes are also defective in FGF-mediated growth inhibition45 and over-activation of the FGF receptor impairs growth of long bones, leading to dwarfism in a STAT1-dependent manner.44, 46 Interestingly, SW1353 cells are resistant to growth suppression by OSM, but do not have any abnormality in OSM receptor expression, signal transduction pathways, loss of adherence or morphological changes induced by OSM. Therefore, these cells could have a specific defect in induction of cell cycle inhibitors, as observed in IL-6-resistant melanomas.47 Accordingly, SW1353 cells are known to behave differently from other chondrosarcoma cell lines, they have a less cartilaginous appearance in vitro and a higher proliferation rate.6, 48
We previously described that OSM sensitizes WT p53 osteoblastic cells to the mitochondrial apoptotic pathway.29, 31 We observed similar results on SRC cells, in correlation with activation of p53 by OSM. However, OSM does not increase the Bax/Bcl2 ratio and does not activate caspase 9 and 3 in SRC cells, indicating that an alternate death pathway is activated. The four other chondrosarcoma cell lines are resistant to apoptosis induced by OSM+STS presumably because they have a mutated p53 gene. Interestingly, progression from low-grade toward high-grade chondrosarcoma is characterized by alterations of p53 and other proteins linked to cell survival such as AKT, HIF or SRC, with a dramatic decrease of patient survival from 80 to 30% at 10 years.1, 3, 4 Resistance to cell death induced by OSM in association with other death inducers could therefore participate in chondrosarcoma aggressiveness and poor survival.
In SRC and CAL78 cells, the Cbfa1/SOX9 ratio is increased by OSM, in correlation with induced hypertrophic marker expression (Coll10a1, RANKL, VEGF and MMP13). Because Coll2 and Acan, markers of differentiated chondrocytes, are not modulated by OSM, we suggest a pre-hypertrophic differentiation of chondrosarcoma cells. OSM also induces MMP13 expression in articular chondrocytes through the JAK3/STAT1 pathway, favoring cartilage breakdown in RA.22 Over-expression of OSM also induces growth plate damage23 and mice with altered gp130-STAT1/3 signaling have reduced bone size and premature growth plate closure.49 Therefore, the STAT1 dependent pathway observed in arthritic chondrocytes could be similar to the one implicated in hypertrophic chondrocyte differentiation as well as chondrosarcoma growth suppression by OSM. In addition, OSM induces VEGF expression in chondrosarcoma cells, possibly through STAT3 as described in other cells,26 and this could lead to increased angiogenesis. Induction of RANKL could lead to osteolysis, release of entrapped growth factors such as TGFβ or IGF-1, and thus to enhanced tumor proliferation. OSM also reduces the adherence of all chondrosarcoma cell lines, suggesting an enhanced metastatic potential. These proposed mechanisms of action of OSM on chondrosarcoma development are schematically presented in Figure 4c.
To complete our study, we used the better described and highly relevant chondrosarcoma model, the syngenic SRC model.34 This tumor is considered as a WT p53 grade II chondrosarcoma and it remains to determine the effect of OSM over-expression on mutated p53 conventional grade III or dedifferentiated chondrosarcomas in vivo. Local over-expression of OSM (i.t. injection of AdOSM) significantly reduces SRC tumor development transiently with reduced proliferation and enhanced apoptosis detected in two on six animals. We believe that the heterogeneity observed in Ki67 or TUNEL staining is linked to heterogeneity in tumor size and transduction efficiency, leading to tumors that regress, plateaued or still progress after AdOSM injection. Because apoptosis is observed in vivo but not in vitro, we can envisage that the effect of OSM is enhanced or acts in synergy with other factors present in the tumor microenvironment, such as the immune system, inflammation, osteolysis or extensive collagenous matrix. This complex 3-dimensional and hypoxic conditions could also allow a stronger effect on chondrosarcoma differentiation, reaching the terminal hypertrophic stage characterized by extensive apoptosis.10 After 9 days, the OSM-treated tumors grow very quickly, with kinetics faster than the GFP-treated ones. At this time point, we previously described that the OSM transgene expression subsides,29, 36 although a direct measurement of local and systemic mouse OSM levels is still needed.
OSM over-expression does not induce VEGF or tumor vascularization in the SRC model. This discrepancy with in vitro results could be explained by the relatively high level of VEGF expression in AdGFP tumors, possibly because other VEGF inducers are already present. Interestingly, vascularization of chondrosarcoma is limited to the stromal compartment, and tumor cells are entrapped in extensive collagenous matrix. This situation could explain why systemic over-expression of OSM in the SRC model (i.m. injection in the contralateral leg) does not reduce chondrosarcoma expansion, as observed with systemic chemotherapeutic drugs in the human clinic.1–3 The poor vascularity correlates also with poor lung metastatic potential in all treated rats, even if OSM decreases cell adhesion and thus could stimulate tumor spreading. In contrast, OSM over-expression significantly induces RANKL expression in chondrosarcoma cells, osteoclast formation at the tumor/bone interface and reduces the ectopic bone neoformation. Therefore OSM-induced bone resorption, but not tumor neoangiogenesis, could indirectly stimulate tumor proliferation especially at later time point.
In conclusion, OSM is directly cytostatic for high grade chondrosarcomas, independently of p53, and presumably through the JAK3/STAT1 pathway. Concomitantly, OSM induces a hypertrophic differentiation in chondrosarcomas. In a rat WT p53 chondrosarcoma model, OSM also enhances (i) cancer cell apoptosis and (ii) cancer-induced osteolysis, with potential indirect effects on cancer proliferation. OSM is therefore a double edge sword for chondrosarcomas: it is a promising adjuvant anti-cancer agent but it also possesses unwanted side effects on bone remodeling and generalized inflammation. A local application of this cytokine would overcome the poor vascularization/accessibility of these tumors and limit the deleterious effect on other tissues. Its combination with anti-resorption agents such as bisphosphonates would prevent bone destruction and tumor refueling,34 thus offering new lines of therapeutic interventions.
The authors thank the Vector Core of the University Hospital of Nantes (France) supported by the Association Française contre les Myopathies (AFM) for producing the adenovirus vectors, Sylvain Bercegeay and Soraya Saiagh (UTCG, Nantes Hospital) for the establishment of the BCSCH03 cell line. The authors are very grateful to Maria Cristina Cuturi, Claire Usal, Emmanuel Merieau and Ignacio Anegon (Inserm U643, Nantes, France) for animal care and their help in the experimental design. E.D. is a recipient from a fellowship from le Ministère de la Recherche.