Nicotinamide Phosphoribosyl Transferase Is Increased in Osteosarcomas and Chondrosarcomas Compared to Benign Bone and Cartilage.

BACKGROUND/AIM: Primary bone neoplasms include osteosarcomas (OS), chondrosarcomas (CS), and giant cell tumors (GCT). Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide synthesis and is increased in multiple tumor types. In malignancies, NAMPT expression often correlates positively with tumor grade, chemotherapy resistance, and metastatic potential. MATERIALS AND METHODS: Tissue microarray was used to examine NAMPT expression in benign bone and cartilage, GCTs, OS, and different CS grades. RESULTS: For the first time, we showed that NAMPT expression was increased in GCTs and OS compared to benign bone, and in CS compared to benign cartilage. Its expression also increased with higher CS grade. CONCLUSION: Our data indicate that NAMPT plays a role in bone sarcomas and GCTs, and its higher expression may contribute to increased tumor aggressiveness.

Therapeutic effect of palbociclib in chondrosarcoma: implication of cyclin-dependent kinase 4 as a potential target.

BACKGROUND: Chondrosarcoma is a malignant cartilaginous neoplasm of the bone which resistant to radiation therapy and chemotherapy. Cyclin-dependent kinase 4 (CKD4) is highly expressed in human cancer, and palbociclib, the inhibitor of CDK4 has been used clinically under FDA approval for application in cancer therapeutic remedies. However, the level of CDK4 and the treatment possibility in chondrosarcoma require further exploration. Thus, we aim to investigate the level of CDK4 and accompanying therapeutic effects of palbociclib in chondrosarcoma. METHODS: We used immunohistochemistric analysis to evaluate human CDK4 productions in chondrosarcoma tissues. The inhibitory expression of CDK4 by siRNA or palbociclib on cell proliferation, invasion, migration, apoptosis and cycle arrest of chondrosarcoma were determined by MTT, wound healing, transwell and flow cytometry. CDK4/Rb signaling pathway were determined by western blot and Immunofluorescence assay. The inhibition effect of palbociclib on tumor growth within the bone were determined by bioluminescence imaging in vivo. RESULTS: CDK4 was found to express significantly in human chondrosarcoma samples. The enhanced levels of CDK4 were interlinked with malignant metastasis and undesirable prognosis of chondrosarcoma patients. CDK4 was also highly expressed in human chondrosarcoma cell lines and its inhibition by specific siRNA and palbociclib lead to a decrease in cell proliferation, accompanied by the phosphorylation of Rb. Furthermore, palbociclib also induced cell cycle arrest in G1 phase and decreased cell migration and invasion via CDK4/Rb signaling pathway. Administration of palbociclib in vivo could reduce tumor burden in chondrosarcoma. CONCLUSIONS: In summary, these data highlight CDK4 inhibitors, such as palbociclib, as potential promising therapeutics in the treatment of human chondrosarcoma.

Apoptotic effect of pyrroloquinoline quinone on chondrosarcoma cells through activation of the mitochondrial caspase­dependent and caspase­independent pathways.

Chondrosarcomas are malignant tumors of the bone that exhibit resistance to chemotherapy and radiation. Pyrroloquinoline quinone (PQQ) is a bacterial redox co­factor and antioxidant that has been found to induce apoptosis in various cancer cells. This study investigated the role of PQQ in cell apoptosis of chondrosarcoma cells and the underlying pathways involved. We confirmed that PQQ was cytotoxic to chondrosarcoma SW1353 cells by a cell cytotoxicity assay. Furthermore, flow cytometry showed that the number of apoptotic cells increased in a concentration­dependent and time­dependent manner following PQQ treatment, but this effect was not significant in normal cells. Co­immunoprecipitation assays showed that the binding of Smac to X­linked inhibitor­of­apoptosis protein (XIAP) was significantly increased and the binding of XIAP with caspase­3 was significantly decreased following PQQ treatment. This was accompanied by a decrease in the levels of caspase­1 and procaspase­3, as demonstrated by western blot analysis. Western blotting also showed that the level of cytochrome c in the mitochondria was decreased and its level in the cytoplasm was increased. These findings indicate the role of caspase­dependent apoptotic pathways in the effect of PQQ. Furthermore, the cytoplasmic and nuclear levels of apoptosis­inducing factor (AIF) were increased and its mitochondrial levels were decreased, and similar results were obtained for endonuclease G. Thus, the role of caspase­independent pathways was also demonstrated. Finally, in vivo tumor implantation experiments showed that PQQ was able to inhibit tumor growth in mice with chondrosarcoma. These findings demonstrated that PQQ induced apoptosis in human chondrosarcoma cells by activating mitochondrial caspase­dependent and caspase­independent pathways. Thus, the proteins involved in these pathways may have potential as antitumor treatment targets for chondrosarcoma.

Expression of MMP-9 decreases metastatic potential of Chondrosarcoma: an immunohistochemical study.

BACKGROUND: Chondrosarcoma is the second most common primary malignant bone tumor. Because of their heterogeneity, with differences in invasive and metastatic behavior, it is important to identify biological markers that will allow for a more accurate estimation of prognosis in patients with these tumors. Matrix metalloproteinases (MMP) play a crucial role in tumor progression, invasion and metastasis. The mechanism of tumor progression dependent of MMPs is complex and influences malignant transformation, angiogenesis and tumor growth at the primary and metastatic sites. The purpose of this study was to investigate immunohistochemicaly the influence of MMP-1, MMP-3, MMP-9 and MMP-13 expression on prognostic parameter in chondrosarcoma. METHODS: We investigated tissue samples of 28 patients with chondrosarcoma. Immunohistochemical staining to evaluate the expression of MMP-1, MMP-3, MMP-9 and MMP-13 was performed. Subsequently, the expression level was correlated with metastatic potential, histological grading and overall survival in patients with this neoplasm. RESULTS: In consideration of semi quantitative scoring 64% of chondrosarcoma were scored as positive for MMP-1, 46% for MMP-3, 61% for MMP-9. The specimens had shown no expression of MMP-13. High expression of MMP-9 was associated with better histological differentiation, decreased metastatic potential and favourable overall survival. No correlation was found for expression of MMP-1, MMP-3 or MMP-13. CONCLUSIONS: MMP-1, MMP-3 and MMP-9 are expressed in chondrosarcoma. Our findings suggest that the expression of MMP-9 is associated with clinical outcome parameters in chondrosarcoma.

Kinase Activity in Recurring Primary Skull Base Chordomas and Chondrosarcomas: Identification of Novel Pathways of Oncogenesis and Potential Drug Targets.

BACKGROUND: Chordomas and chondrosarcomas can occur in the skull base. Currently, 45% of chordomas and 56% of chondrosarcomas recur within 5 years of surgery. The role of adjuvant therapy is highly debated. No pharmacotherapies have been approved by the U.S. Food and Drug Administration for chordomas or chondrosarcomas. High propensity for recurrence and lack of definitive adjuvant therapy necessitate additional basic science research to identify molecular anomalies associated with recurrent disease. METHODS: We pooled tumor lysates from patients based on clinical criteria into 4 groups: primary chordomas, primary chordomas that recurred, primary chondrosarcomas, and primary chondrosarcomas that recurred. We used a peptide labeling method, isobaric tags for relative and absolute quantitation, to uniquely identify each tumor group. Phosphorylated peptides were identified and quantified via mass spectroscopy to determine and predict active kinases. RESULTS: Six groups of phosphorylated peptides were associated with primary tumors that later recurred. Specific kinases associated with primary chordomas that recurred were FES and FER. Specific kinases associated with primary chondrosarcomas that recurred were FES, FER, SRC family kinases, PKC, ROCK, and mitogen-activated protein kinase signaling (JNK, ERK1, p38). CONCLUSIONS: These data provide clinicians with a means to screen skull base chordomas and chondrosarcomas to help identify tumors with a propensity to recur. Many of these kinases can be efficaciously inhibited by Food and Drug Administration-approved drugs that have not yet been used in clinical trials for treatment of skull base chordomas or chondrosarcomas. Validation of kinases identified in this study may advance treatment options for patients with these tumors.

Diagnostic utility of IDH1/2 mutations to distinguish dedifferentiated chondrosarcoma from undifferentiated pleomorphic sarcoma of bone.

Histologically, it is nearly impossible to distinguish the dedifferentiated component of dedifferentiated chondrosarcoma from undifferentiated pleomorphic sarcoma (UPS) of bone when the low-grade cartilaginous component is absent. Previous studies have revealed that isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are present in a significant number of cartilaginous tumors including most conventional chondrosarcomas and dedifferentiated chondrosarcomas. These mutations have not been studied in UPSs of bone. We sought to investigate whether an IDH1 or IDH2 mutation signature could be used as a clinically diagnostic marker for the distinction of dedifferentiated component of chondrosarcoma from UPS of bone. Sixty-eight bone tumor cases, including 31 conventional chondrosarcomas, 23 dedifferentiated chondrosarcomas, and 14 UPSs of bone, were collected for IDH1/2 mutation analysis either using the Qiagen IDH1/2 RGQ PCR Kit or using whole-exome sequencing. IDH1/2 mutations were detected in 87% (20/23) of dedifferentiated chondrosarcomas and 30% (6/20) of conventional chondrosarcomas. No mutations were detected in the IDH1/2 codon 132 or codon 172 among 14 UPSs of bone. Identification of IDH1 or IDH2 mutations supports the diagnosis of dedifferentiated chondrosarcoma rather than UPS of bone while also providing some insight into the pathogenesis of these 2 lesions.

Icariin inhibits MMP­1, MMP­3 and MMP­13 expression through MAPK pathways in IL­1ß­stimulated SW1353 chondrosarcoma cells.

Osteoarthritis (OA) is the most common type of arthritis and is a leading cause of disability worldwide, resulting in pain, reduced quality of life and socioeconomic burden. Current therapies for OA focus on mitigating the symptoms of advanced disease, but novel therapeutic agents are needed to inhibit the processes leading to OA. The present study aimed to investigate the effects of Icariin on matrix metalloproteinase (MMP)­1, MMP­3 and MMP­13 expression in interleukin (IL)­1ß­stimulated human SW1353 chondrosarcoma cells, and to investigate the possible mechanism underlying the chondroprotective effects of Icariin. In the present study, IL­1ß was applied on SW1353 chondrosarcoma cells to mimic the microenvironment of osteoarthritis. The cells were treated with Icariin and mitogen­activated protein kinase (MAPK) signaling pathway activators or inhibitors. MMP­1, MMP­3, MMP­13, phosphorylated (P)­p38, P­c­Jun N­terminal kinase (JNK) and P­extracellular signal­regulated kinase (ERK) expression was assessed using reverse transcription­quantitative polymerase chain reaction, ELISA and western blot analysis. The results of the present study demonstrated that Icariin inhibited the expression of MMP­1, MMP­3, MMP­13, P­p38, P­ERK and P­JNK. Furthermore, it was revealed that the inhibition of p38 and ERK contributed to the inhibition of MMP­1 and MMP­3 by Icariin, whereas the inhibition of p38 and JNK contributed to the inhibition of MMP­13. The present results suggested that Icariin may have a chondroprotective effect, exerted through the inhibition of MMP­1, MMP­3 and MMP­13 via MAPK pathways. Therefore, Icariin may have potential as a novel therapeutic strategy for the treatment of osteoarthritis.

Amphiregulin enhances VEGF-A production in human chondrosarcoma cells and promotes angiogenesis by inhibiting miR-206 via FAK/c-Src/PKCδ pathway.

Chondrosarcoma is the second most common primary malignancy of bone after myeloma and osteosarcoma. Chondrosarcoma development may be linked to angiogenesis, which is principally elicited by vascular endothelial growth factor-A (VEGF-A). The expression of VEGF-A has been recognized as a prognostic marker in angiogenesis. Amphiregulin (AR), an epidermal growth factor receptor ligand, promotes tumor proliferation, metastasis and angiogenesis. However, the role of AR in VEGF-A expression and angiogenesis in human chondrosarcoma remains largely unknown. This current study shows that AR promoted VEGF-A production and induced angiogenesis of human endothelial progenitor cells. Moreover, AR-enhanced VEGF-A expression and angiogenesis involved the FAK, c-Src and PKCδ signaling pathways, while miR-206 expression was negatively mediated by AR via the FAK, c-Src and PKCδ pathways. Our results illustrate the clinical significance between AR, VEGF-A and miR-206, as well as tumor stage, in human chondrosarcoma. AR may represent a novel therapeutic target in the metastasis and angiogenesis of chondrosarcoma.

Adiponectin promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-27b through a CaMKII/AMPK/p38 signaling pathway in human chondrosarcoma cells.

Chondrosarcoma is the second most frequently occurring type of bone malignancy characterized by distant metastatic propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumour lymphangiogenesis and lymphatic metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. In recent years, adiponectin has also been indicated as facilitating tumorigenesis, angiogenesis and metastasis. However, the effect of adiponectin on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has remained largely a mystery. In the present study, we have shown a clinical correlation between adiponectin and VEGF-C, as well as tumour stage, in human chondrosarcoma tissues. We further demonstrated that adiponectin promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium from adiponectin-treated cells significantly induced tube formation and migration of human lymphatic endothelial cells. In addition, adiponectin knock down inhibited lymphangiogenesis in vitro and in vivo We also found that adiponectin-induced VEGF-C is mediated by the calmodulin-dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK) and p38 signaling pathway. Furthermore, the expression of miR-27b was negatively regulated by adiponectin via the CaMKII, AMPK and p38 cascade. The present study is the first to describe the mechanism of adiponectin-promoted lymphangiogenesis by up-regulating VEGF-C expression in chondrosarcomas. Thus, adiponectin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.

α-methylacyl-CoA racemase (AMACR) expression in chordomas differentiates them from chondrosarcomas.

AIMS: Chordomas and chondrosarcomas are malignant mesenchymal tumours with overlapping morphological and immunohistochemical (IHC) characteristics. Our aim was to evaluate the IHC expression of α-methylacyl-CoA racemase (AMACR/P504S), ß-catenin and E-cadherin in chordomas relative to chondrosarcomas and assess the utility of these markers for differential diagnosis. METHODS: Archival sections of 18 chordomas, 19 chondrosarcomas and 10 mature cartilage samples were immunostained and scored for AMACR, ß-catenin and E-cadherin and the relative differential capacity of each marker was calculated. In addition, AMACR mRNA level was assessed in 5 chordomas by RT-PCR and evaluated by comparative CT method. RESULTS: AMACR and ß-catenin stained 88.9% and 94.1% of the chordomas respectively, 21.1% and 10.5% of the chondrosarcomas correspondingly and none of the mature cartilage samples. E-cadherin stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Both AMACR and ß-catenin showed statistically significant difference between chordomas and chondrosarcomas (p < 0.001 for both), unlike E-cadherin. AMACR was detected at the mRNA level. CONCLUSIONS: AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. AMACR may serve as IHC marker of chordoma with differentiating ability comparable to that of ß-catenin.

Suppressed invasive and migratory behaviors of SW1353 chondrosarcoma cells through the regulation of Src, Rac1 GTPase, and MMP13.

Chondrosarcoma is the second frequent type of primary bone cancer. In response to stress to the endoplasmic reticulum, activation of eIF2α-mediated signaling is reported to induce apoptosis. However, its effects on invasive and migratory behaviors of chondrosarcoma have not been understood. Focusing on potential roles of Src kinase, Rac1 GTPase, and MMP13, we investigated eIF2α-driven regulation of SW1353 chondrosarcoma cells. In particular, we employed two chemical agents (salubrinal, Sal; and guanabenz, Gu) that elevate the level of eIF2α phosphorylation. The result revealed that both Sal and Gu reduced invasion and motility of SW1353 chondrosarcoma cells in a dose dependent manner. Live imaging using a fluorescent resonance energy transfer (FRET) technique showed that Sal and Gu downregulated activities of Src kinase as well as Rac1 GTPase in an eIF2α dependent manner. RNA interference experiments supported an eIF2α-mediated regulatory network in the inhibitory role of Sal and Gu. Partial silencing of MMP13 also suppressed malignant phenotypes of SW1353 chondrosarcoma cells. However, MMP13 was not regulated via eIF2α since administration of Sal but not Gu reduced expression of MMP13. In summary, we demonstrate that eIF2α dependent and independent pathways regulate invasion and motility of SW1353 chondrosarcoma cells, and inactivation of Src, Rac1, and MMP13 by Sal could provide a potential adjuvant therapy for combating metastatic chondrosarcoma cells.

Mutant IDH1 Dysregulates the Differentiation of Mesenchymal Stem Cells in Association with Gene-Specific Histone Modifications to Cartilage- and Bone-Related Genes.

Somatic mutations in the isocitrate dehydrogenase (IDH)1/2 genes endow encoding proteins with neomorphic activity to produce the potential oncometabolite, 2-hydroxyglutarate (2-HG), which induces the hypermethylation of histones and DNA. The incidence of IDH1/2 mutations in cartilaginous tumors was previously shown to be the highest among various types of tumors, except for those in the central nervous system. Mutations have been detected in both benign (enchondromas) and malignant (chondrosarcomas) types of cartilaginous tumors, whereas they have rarely been found in other mesenchymal tumors such as osteosarcomas. To address this unique tumor specificity, we herein examined the effects of IDH1 R132C, which is the most prevalent mutant in cartilaginous tumors, on the differentiation properties of human mesenchymal stem cells (hMSCs). The induction of the IDH1 R132C gene into MSCs markedly increased the amount of 2-HG and up-regulated global histone methylation. The induction of IDH1 R132C promoted the chondrogenic differentiation of hMSCs by enhancing the expression of SOX9 and COL2A1 genes in association with an increase in the active mark (H3K4me3), but disrupted cartilage matrix formation. On the other hand, IDH1 R132C inhibited expression of the ALPL gene in association with an increase in the repressive mark (H3K9me3), and subsequently inhibited the osteogenic properties of hMSCs and human osteosarcoma cells. Since osteogenic properties are an indispensable feature for the diagnosis of osteosarcoma, the inhibitory effects of IDH1 R132C on osteogenic properties may contribute to the lack of osteosarcomas with the IDH1 R132C mutation. These results suggested that IDH1 R132C contributed to the formation of cartilaginous tumors by dysregulating the chondrogenic and osteogenic differentiation of hMSCs via gene-specific histone modulation.

Glandular differentiation in dedifferentiated chondrosarcoma: molecular evidence of a rare phenomenon.

Epithelial glandular differentiation in dedifferentiated chondrosarcoma has not been described. Our patient was a 64-year-old man with a history of prostate cancer status post-radiation and hormonal therapy. On screening bone scan, he was found to have increased uptake in his right femoral shaft. Biopsy revealed intermediate-grade conventional chondrosarcoma. Subsequent femoral resection was remarkable for an intermediate-grade chondrosarcomatous component juxtaposed to an area composed of anastomosing nests and cords of malignant epithelial cells showing nuclear atypia and increased mitotic activity. A fibroblastic-appearing spindle cell population was intimately associated with the epithelial cells. The epithelial cells labeled with 34bE12, AE1/AE3, EMA, and Vimentin (both spindled and epithelial components) while being negative for prostate-specific antigen, prostate specific acid phosphatase, cytokeratin 20, thyroid transcription factor-1, and CDX2. The patient developed local recurrence 9 months after the initial resection but has had no metastatic disease and consistently undetectable prostate-specific antigen levels. Deep parallel sequencing of the dedifferentiated component showed a nonsynonymous mutation at exon 4 of IDH1 gene at codon R132 leading to a substitution of arginine, with serine confirming glandular differentiation in dedifferentiated chondrosarcoma.

The biology and management of cartilaginous tumors: a role for targeting isocitrate dehydrogenase.

Chondrosarcomas are rare mesenchymal neoplasms defined by the production of abnormal cartilaginous matrix. Conventional chondrosarcoma is the most common histology. The management of primary conventional chondrosarcoma generally is surgical with the possible addition of radiation therapy. Treatment of conventional chondrosarcoma is problematic in unresectable or metastatic disease because the tumors tend to be resistant to standard sarcoma chemotherapy regimens. Previous attempts at targeted therapy, including inhibitors of Hedgehog signaling, the mTOR pathway, and platelet-derived growth factor receptor (PDGFR) have been largely disappointing. However, heterozygous mutations in isocitrate dehydrogenase (IDH) enzymes recently have been identified in chondrogenic neoplasms, with mutations reported in approximately 87% of benign enchondromas, 70% of conventional chondrosarcomas, and 54% of dedifferentiated chondrosarcomas. The normal IDH protein continues to produce alpha-ketoglutarate (alpha-KG) whereas the mutant IDH protein converts KG to the oncometabolite 2-hydroxyglutarate (2-HG). Clinical trials of novel IDH inhibitors are ongoing, with evidence of early activity in IDH-mutant leukemias. IDH inhibitors show antitumor effects against IDH-mutant chondrosarcoma cell lines, supporting the inclusion of patients with chondrosarcoma with IDH mutations on IDH inhibitor clinical trials for solid tumors. Targeting IDH mutations may offer hope of a novel antineoplastic strategy not only for patients with chondrosarcomas, but also for other solid tumors with aberrant IDH activity.

Amphiregulin enhances alpha6beta1 integrin expression and cell motility in human chondrosarcoma cells through Ras/Raf/MEK/ERK/AP-1 pathway.

Chondrosarcoma is a malignant tumor that produces cartilage matrix. The most lethal aspect is its metastatic property. We demonstrated that amphiregulin (AR) is significantly upregulated in highly aggressive cells. AR silencing markedly suppressed cell migration. Exogenous AR markedly increased cell migration by transactivation of α6ß1 integrin expression. A neutralizing α6ß1 integrin antibody can abolish AR-induced cell motility. Knockdown of AR inhibits metastasis of cells to the lung in vivo. Furthermore, elevated AR expression is positively correlated with α6ß1 integrin levels and higher grades in patients. These findings can potentially serve as biomarker and therapeutic approach for controlling chondrosarcoma metastasis.

Histone deacetylase inhibitors repress chondrosarcoma cell proliferation.

PURPOSE: Due to the high resistance to conventional therapy, there is still no convincingly effective treatment for chondrosarcoma. As a promising new treatment strategy, histone deacetylase inhibitors (HDACIs) have been reported to induce cell arrest, apoptosis and differentiation in some kinds of malignancies, but how HDACi exert their effects on chondrosarcoma is not well understood yet. METHODS: We investigated the effects of HDACIs trichostatin A (TSA) and sodium valproate (VPA) on chondrosarcoma cells in vitro and in vivo. The cell proliferation and cell cycle were examined in two chondrosarcoma cell lines, SW1353 and JJ012, by MTS and flow cytometry assays, respectively. The in vivo effects of HDACIs were investigated by assessing the chondrosarcoma growth in a mouse xenograft model. RESULTS: Our results showed that TSA and VPA significantly repressed the proliferation of chondrosarcoma cells in a concentration-dependent manner. Flow cytometry indicated that TSA arrested the cell cycle in G2/M phase and VPA arrested the cell cycle in G1 phase. The tumor growth was markedly suppressed in mice treated with TSA and VPA. CONCLUSIONS: HDACIs significantly repress the proliferation of chondrosarcoma cells in vitro and in vivo. Our findings imply that HDACIs may provide a novel therapeutic target for the treatment of chondrosarcoma.

A novel monoclonal antibody SMab-2 recognizes endogenous IDH2-R172S of chondrosarcoma.

Isocitrate dehydrogenase 2 (IDH2) mutations have been reported in gliomas, osteosarcomas, cartilaginous tumors, giant cell tumors of bone, and acute myeloid leukemias. Although IDH2 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate (α-KG) in mitochondria, mutated IDH2 proteins possess the ability to change α-KG into the oncometabolite R(-)-2-hydroxyglutarate (2-HG). To date, several monoclonal antibodies (mAbs) specific for IDH2 mutations have been established, such as KMab-1 against IDH2-R172K, MMab-1 against IDH2-R172M, and WMab-1 against IDH2-R172W. Although a multi-specific mAb MsMab-1 reacted with IDH2-R172G and IDH2-R172S, a mono-specific mAb against IDH2-R172S has not been established. In this study, we established a novel mAb SMab-2, which recognizes IDH2-R172S but not with wild type IDH2 in ELISA. Although SMab-2 reacted with both IDH1-R132S and IDH2-R172S expressed in Escherichia coli, it reacted with only IDH2-R172S expressed in U-2 OS osteosarcoma cells. Furthermore, SMab-2 recognized endogenous IDH2-R172S protein expressed in SW1353 chondrosarcoma cells in Western blot and immunocytochemical analyses. SMab-2 is expected to be useful for diagnosis of IDH2-R172S-bearing tumors.

Regulation of chondrosarcoma invasion by MMP26.

The molecular mechanism underlying metastasis of chondrosarcoma (CS) remains unclarified. Here, we show that matrix metalloproteinase-26 (MMP26) level is significantly higher in the resected CS than in the adjacent healthy chondral tissue from the patients. To examine the role of MMP26 in CS invasion, we used a human CS line SW1353 and we either overexpressed or inhibited MMP26 in these cells. We found that overexpression of MMP26 in SW1353 cells increased cell invasiveness, while inhibition of MMP26 decreased cell invasiveness. To define the signal transduction cascades downstream of MMP26 activation, we applied specific inhibitors for PI3K, ERK/MAPK, JNK, and Wnt signaling, respectively, to the MMP26-overexpressing SW1353 cells. We found that only inhibition of Wnt signaling by either metformin or IWP-2 significantly decreased the effect of MMP26 on cancer cell invasion, possibly through increasing ß-catenin phosphorylation. Further, a strong correlation was detected between MMP26 levels and the ratio of phosphorylated/total ß-catenin in CS from the patients. Taken together, our study highlights MMP26-regulated Wnt signaling as a novel therapeutic target for CS.

Fluid shear promotes chondrosarcoma cell invasion by activating matrix metalloproteinase 12 via IGF-2 and VEGF signaling pathways.

Interstitial fluid flow in and around the tumor tissue is a physiologically relevant mechanical signal that regulates intracellular signaling pathways throughout the tumor. Yet, the effects of interstitial flow and associated fluid shear stress on the tumor cell function have been largely overlooked. Using in vitro bioengineering models in conjunction with molecular cell biology tools, we found that fluid shear (2 dyn/cm(2)) markedly upregulates matrix metalloproteinase 12 (MMP-12) expression and its activity in human chondrosarcoma cells. MMP-12 expression is induced in human chondrocytes during malignant transformation. However, the signaling pathway regulating MMP-12 expression and its potential role in human chondrosarcoma cell invasion and metastasis have yet to be delineated. We discovered that fluid shear stress induces the synthesis of insulin growth factor-2 (IGF-2) and vascular endothelial growth factor (VEGF) B and D, which in turn transactivate MMP-12 via PI3-K, p38 and JNK signaling pathways. IGF-2-, VEGF-B- or VEGF-D-stimulated chondrosarcoma cells display markedly higher migratory and invasive potentials in vitro, which are blocked by inhibiting MMP-12, PI3-K, p38 or JNK activity. Moreover, recombinant human MMP-12 or MMP-12 overexpression can potentiate chondrosarcoma cell invasion in vitro and the lung colonization in vivo. By reconstructing and delineating the signaling pathway regulating MMP-12 activation, potential therapeutic strategies that interfere with chondrosarcoma cell invasion may be identified.