Chondrosarcoma Cell Migration Research Articles

Visfatin Promotes the Metastatic Potential of Chondrosarcoma Cells by Stimulating AP-1-Dependent MMP-2 Production in the MAPK Pathway.

Chondrosarcoma is a malignant bone tumor that is characterized by high metastatic potential and marked resistance to radiation and chemotherapy. The knowledge that adipokines facilitate the initiation, progression, metastasis, and treatment resistance of various tumors has driven several in vitro and in vivo investigations into the effects of adipokines resistin, leptin, and adiponectin upon the development and progression of chondrosarcomas. Another adipokine, visfatin, is known to regulate tumor progression and metastasis, although how this molecule may affect chondrosarcoma metastasis is unclear. Here, we found that visfatin facilitated cellular migration via matrix metalloproteinase-2 (MMP-2) production in human chondrosarcoma cells and overexpression of visfatin enhanced lung metastasis in a mouse model of chondrosarcoma. Visfatin-induced stimulation of MMP-2 synthesis and activation of the AP-1 transcription factor facilitated chondrosarcoma cell migration via the ERK, p38, and JNK signaling pathways. This evidence suggests that visfatin is worth targeting in the treatment of metastatic chondrosarcoma.

Abstract 4915: Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma cells

Abstract Chondrosarcomas are malignant bone tumors that commonly produce cartilaginous chondroid matrix and are associated with metastases in the lung, especially in patients with high-grade chondrosarcoma. Neurotrophins such as nerve growth factor (NGF) are recognized as being drivers of neurogenesis during development and regeneration, and their expression in human tumors can stimulate cancer cell growth. Importantly, the infiltration of nerves in the tumor microenvironment actively stimulates cancer cell mitogenesis, invasiveness and metastasis. Until now, it has been unclear as to the potential involvement of NGF in human chondrosarcoma metastasis. This study reports that NGF induces migration and invasion of chondrosarcoma cell lines JJ012 and SW1353 and significantly increasing LOX expression. Significant dose-dependent increases were observed in levels of LOX mRNA and protein expression in both cell lines. In immunohistochemistry analyses, levels of NGF and LOX expression in human chondrosarcoma cartilage correlated with tumor stage and were substantially higher than NGF and LOX expression in normal cartilage. NGF-induced stimulation of metastasis and increases in LOX expression were prevented by pretreatment of the cell lines with PI3K, Akt and mTOR inhibitors, or transfection with small interfering RNAs (siRNAs). Further analysis revealed that NGF induced PI3K/Akt/mTOR signaling and dose-dependently downregulated mature microRNA-149-5p (miR-149-5p), which targets the 3' untranslated region (3'-UTR) of LOX. Furthermore, JJ012 cells overexpressing NGF induced LOX expression and increased cell migration and invasion. In summary, NGF stimulates LOX expression and promotes chondrosarcoma cell migration and invasion by inhibiting miR-149-5p via the PI3K/Akt/mTOR signaling pathway. Citation Format: Anoop Thadevoos Louis, Chih-Yang Lin, Chih-Hsin Tang. Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4915.

Sphingosine-1-phosphate promotes PDGF-dependent endothelial progenitor cell angiogenesis in human chondrosarcoma cells.

The malignant bone tumors that are categorized as chondrosarcomas display a high potential for metastasis in late-stage disease. Higher-grade chondrosarcomas contain higher levels of expression of platelet-derived growth factor (PDGF) and its receptor. The phosphorylation of sphingosine by sphingosine kinase enzymes SphK1 and SphK2 generates sphingosine-1-phosphate (S1P), which inhibits human chondrosarcoma cell migration, while SphK1 overexpression suppresses lung metastasis of chondrosarcoma. We sought to determine whether S1P mediates levels of PDGF-A expression and angiogenesis in chondrosarcoma. Surprisingly, our investigations found that treatment of chondrosarcoma cells with S1P and transfecting them with SphK1 cDNA increased PDGF-A expression and induced angiogenesis of endothelial progenitor cells (EPCs). Ras, Raf, MEK, ERK and AP-1 inhibitors and their small interfering RNAs (siRNAs) inhibited S1P-induced PDGF-A expression and EPC angiogenesis. Our results indicate that S1P promotes the expression of PDGF-A in chondrosarcoma via the Ras/Raf/MEK/ERK/AP-1 signaling cascade and stimulates EPC angiogenesis.

ADAM8 promotes chondrosarcoma cell migration and invasion by activating the NF-κB/MMP-13 signaling axis.

ADAM8 is reported to promote extracellular matrix degradation to provide conditions for tumor metastasis. However, the underlying mechanism of ADAM8 in modulating chondrosarcoma (CHS) metastasis remains unclear. We used two human CHS cell lines SW1353 and HCS-2/8 to analyze the expression profiles of ADAM8 in CHS cells compared with the normal chondrocytes. An important proteolytic enzyme MMP-13 was detected as a marker for extracellular matrix degradation in chondrocytes. Then, by silencing or overexpressing ADAM8, the effects on cell migration and invasion in SW1353 and HCS-2/8, and the downstream signal transduction pathways were evaluated. ADAM8 and MMP-13 were highly expressed, and the NF-κB pathway was activated in SW1353 and HCS-2/8 cells. Silencing ADAM8 significantly reduced the ability of cell migration and invasion, and blocked the NF-κB signaling pathway through IκBα and p65 dephosphorylation, leading to reduced NF-κB transcription activity and decreased MMP-13 expression. ADAM8 overexpression promoted these processes, which, however, were reversed by an inhibitor Bay 11-7085. Our data showed a novel regulation mechanism for ADAM8 in promoting CHS migration and invasion by activating the NF-κB/MMP-13 signaling axis. Modulation of their levels may serve as potential targets in the treatment of CHS and even other cartilage diseases.

Abstract 156: Visfatin induces LOX expression and promotes tumor metastasis through the Src/Akt signaling pathway in human chondrosarcoma cells

Abstract Chondrosarcoma, a common primary bone malignancy that affects the joints and bones, is associated with metastases in the lung, especially in cases of high-grade chondrosarcoma. The protein lysyl oxidase (LOX) can contribute to tumor growth and metastasis, while the adipokine visfatin is pro-angiogenic and facilitates tumorigenesis as well as metastasis. However, it is unclear as to whether any association exists between visfatin and LOX expression in human chondrosarcoma metastasis. We have discovered that visfatin induces the migration, invasion and increased expression of LOX mRNA and protein in human chondrosarcoma cell lines (JJ012 and SW1353). Pretreatment of these cell lines with Src and Akt inhibitors, or transfecting them with small interfering RNAs (siRNAs), inhibited visfatin-induced chondrosarcoma metastasis and LOX expression. Thus, visfatin promotes chondrosarcoma metastasis by inducing LOX expression via the Src/Akt signaling pathway in the JJ012 cell line. microRNA analysis revealed that visfatin-induced Src/Akt signaling is responsible for downregulating mature miR-26b-5p, which targets the 3' untranslated region (3`UTR) of LOX but does not affect primary and precursor miR-26b-5p expression. This downregulation of miR-26b-5p expression by visfatin was dose-dependent. Pretreatment with Src and Akt inhibitors or transfection with siRNA reversed miR-26b-5p expression. The migration and invasion ability of JJ012 cells was reduced when they were transfected with the miR-26b-5p mimic. In summary, our data demonstrate that visfatin induces LOX expression and promotes chondrosarcoma cell migration and invasion through the Src/Akt signaling pathway, by inhibiting miR-26b-5p. Citation Format: Cheng-Chieh Yu, Chih-Yang Lin, Chih-Hsin Tang. Visfatin induces LOX expression and promotes tumor metastasis through the Src/Akt signaling pathway in human chondrosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 156.

COL1A2 is a TBX3 target that mediates its impact on fibrosarcoma and chondrosarcoma cell migration.

The developmentally important T-box transcription factor TBX3, is overexpressed in several cancers and contributes to tumorigenesis as either a tumour promoter or tumour suppressor. For example, TBX3 promotes cell proliferation, migration and invasion of chondrosarcoma cells but inhibits these processes in fibrosarcoma cells. This suggests that the cellular context influences TBX3 oncogenic functions, but the mechanism(s) involved has not been elucidated. COL1A2 encodes type I collagen and, like TBX3, plays important roles during embryogenesis and can act as either oncogene or tumour suppressor. Here we explore the possibility that COL1A2 may be a TBX3 target gene responsible for mediating its opposing oncogenic roles in chondrosarcoma and fibrosarcoma cells. Results from qRT-PCR, western blotting, luciferase reporter and chromatin immunoprecipitation assays show that TBX3 binds and activates the COL1A2 promoter. Furthermore, we show that TBX3 levels are regulated by AKT1 and that pseudo-phosphorylation of TBX3 at an AKT consensus serine site, enhances its ability to activate COL1A2. Importantly, we demonstrate that COL1A2 mediates the pro- and anti-migratory effects of TBX3 in chondrosarcoma and fibrosarcoma cells respectively. Our data reveal that the AKT1/TBX3/COL1A2 axis plays an important role in sarcomagenesis.

Amphiregulin enhances cell migration and resistance to doxorubicin in chondrosarcoma cells through the MAPK pathway.

Chondrosarcoma is characterized by the ability to produce extracellular matrix of cartilage. Curative surgical resection becomes difficult when high-grade chondrosarcoma metastasizes to other parts of the body. Doxorubicin (Dox) has been widely used clinically to treat many different types of cancers, including chondrosarcomas. However, chondrosarcoma is often resistant to chemotherapy and radiotherapy. New systemic treatment regimens are needed to improve the prognosis. Amphiregulin (AR) has been reported to be involved in cancer metastasis and drug resistance. The aim of the present study was to investigate the role of AR on cell migration and Dox sensitivity. To gain insight into the mechanisms used by AR to mediate its effects, we generated two chondrosarcoma cell lines, migration-prone JJ012 and Dox-resistant SW1353, to analyze the relationship between AR levels and cell functions. AR was highly expressed and secreted in mobile and drug-resistant cells. Next, we used the lentivirus-mediated RNAi system to knock down AR expression and found that AR silencing was significantly attenuated in cell migration and drug resistance. In addition, we used exogenous recombinant AR (r-AR) to confirm cell function and we introduced antibody array analysis to investigate pathways of AR activation. The results showed that AR can promote migratory activity and Dox resistance through activation of the MAPK pathway. Whereas the inhibition of the MAPK pathway has the potential to inhibit chondrosarcoma cell migration, p38 inhibition, and ERK activation may render cells more sensitive to Dox. These findings suggest that combining AR with MAPK blockade may effectively treat chondrosarcoma.

CCN6-mediated MMP-9 activation enhances metastatic potential of human chondrosarcoma

Chondrosarcomas are primary malignant bone tumors that have a poor prognosis. WNT1-inducible signaling pathway protein-3 (WISP-3, also termed CCN6) belongs to the CCN family of proteins and is implicated in the regulation of various cellular functions, such as cell proliferation, differentiation, and migration. It is unknown as to whether CCN6 affects human chondrosarcoma metastasis. We show how CCN6 promotes chondrosarcoma cell migration and invasion via matrix metallopeptidase-9 (MMP)-9 expression. These effects were abolished by pretreatment of chondrosarcoma cells with PI3K, Akt, mTOR, and NF-κB inhibitors or short interfering (si)RNAs. Our investigations indicate that CCN6 facilitates metastasis through the PI3K/Akt/mTOR/NF-κB signaling pathway. CCN6 and MMP-9 expression was markedly increased in the highly migratory JJ012(S10) cell line compared with the primordial cell line (JJ012) in both in vitro and in vivo experiments. CCN6 knockdown suppressed MMP-9 production in JJ012(S10) cells and attenuated cell migration and invasion ability. Importantly, CCN6 knockdown profoundly inhibited chondrosarcoma cell metastasis to lung. Our findings reveal an important mechanism underlying CCN6-induced metastasis and they highlight the clinical significance between CCN6 and MMP-9 in regard to human chondrosarcoma. CCN6 appears to be a promising therapeutic target in chondrosarcoma metastasis.

MicroRNA‑10b suppresses the migration and invasion of chondrosarcoma cells by targeting brain‑derived neurotrophic factor.

MicroRNAs (miRs) can lead to mRNA degradation or inhibit protein translation through directly binding to the 3'‑untranslational region (UTR) of their target mRNAs. Deregulation of miR‑10b has been reported to be associated with chondrosarcoma. However, the role of miR‑10b in chondrosarcoma cell migration and invasion, as well as the underlying mechanisms, has not been investigated. In the present study, it was demonstrated that miR‑10b was notably downregulated in the JJ012 and SW1353 chondrosarcoma cell lines compared with the TC28a2 normal chondrocyte line. Treatment with DNA demethylating agent 5‑aza‑2'‑deoxycytidine and histone deacetylase inhibitor 4‑phenylbutyric acid, or transfection with miR‑10b mimics promoted the expression of miR‑10b, which further suppressed the migratory and invasive capacities of JJ012 chondrosarcoma cells. Moreover, brain‑derived neurotrophic factor (BDNF) was identified as a novel target of miR‑10b, and its protein expression level was negatively regulated by miR‑10b in JJ012 cells. Furthermore, overexpression of BDNF reversed the inhibitory effect of miR‑10b upregulation on the migration and invasion of JJ012 cells. In addition, the data suggest that matrix metalloproteinase 1 (MMP1) may be involved in the miR‑10b/BDNF‑mediated chondrosarcoma cell migration and invasion in JJ012 cells. In conclusion, these findings suggest that miR‑10b/BDNF may serve as a potential therapeutic target for chondrosarcoma.

Abstract 828: Src plays a role in chemoresistance and migration in chondrosarcoma cell lines

Abstract Chondrosarcomas (CS) are malignant cartilage-forming tumors of bone. High grade chondrosarcomas show metastasis formation in 71% of cases, for which currently no treatment strategies exist. Kinome profiling revealed high GSK3β, AKT, and Src pathway activity. These kinases play a role in either cell survival, migration, or both. Our aim was to identify the roles of these pathways in chemoresistance and migration and investigate downstream effects while pinpointing the most important kinase in chondrosarcoma. We used 5 conventional chondrosarcoma cell lines and 3 dedifferentiated chondrosarcoma cell lines for the experimental procedures. Kinase inhibition was performed with enzastaurin (GSK3β and AKT inhibition) and dasatinib (Src inhibition). WST and Live cell imaging with AnnexinV staining (BD Pathway 855 Bioimager) were performed to investigate cell viability and apoptosis formation. Proliferation and migration assays were performed with the RTCA xCelligence System (Roche). Tissue microarrays (TMAs) were constructed containing 8 EC, 92 central CS (grade I n=42, grade II n=35, grade III n=14), 11 OC and 45 peripheral CS (grade I n=31, grade II n=11, grade III n=3). Overexpression of Src family members can lead to excess Nrf2 translocation from the nucleus. Immunohistochemistry was performed for the Src family members and GPX3, the target of the transcription factor Nrf2. Western blot was performed for Src family members, Nrf2, and GPX3. Inhibition of GSK3β and Akt with enzastaurin in chondrosarcoma cell lines was ineffective, also when combined with Src inhibition (dasatinib) or doxorubicin. Combination treatment of dasatinib with doxorubicin, however, showed synergistic loss in cell viability and apoptosis formation, with cleaved caspase3 activation, which was not observed after dasatinib single treatment. Migration assays in cell lines (n=6) revealed that at low concentrations of dasatinib (200nM), migration was successfully inhibited. TMA staining revealed sporadic expression of Yes and Lck (5%), expression of Src (57%) and abundant expression of Fyn (78%) throughout our panel of CS tissues. Fyn is described to be associated with metastasis formation and is a good candidate to play a role in chondrosarcoma progression. Overall pSrc (active Src) approached 100% in all grades. Western blot analysis showed nuclear localization of Nrf2 and restoration of GPX3 transcription after dasatinib treatment of cell lines. We show that Src family kinases rather than GSK3β and Akt kinases contribute to chemoresistance in chondrosarcoma. Src kinase inhibition successfully prepared cells for chemotherapy and acts synergistically with doxorubicin, and moreover was able to completely inhibit chondrosarcoma cell migration and restore GPX3 transcription. These results strongly indicate Src family kinases, and in particular Fyn, to be a potential target for the treatment of inoperable and metastatic chondrosarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 828. doi:1538-7445.AM2012-828

Involvement of the Soluble Urokinase Receptor in Chondrosarcoma Cell Mobilization

High levels of urokinase receptor (uPAR) in tissue and serum of patients with chondrosarcoma correlate with poor prognosis. First, we analyzed the uPAR levels in tissues and plasma of five patients affected by chondrosarcoma. Interestingly, very high levels of uPAR and its soluble forms (SuPAR) were found on tumor cell surfaces and plasma, respectively, of two patients with lung metastases. Therefore, to investigate the role of SuPAR in chondrosaromas, we generated a primary cell culture from a chondrosarcoma tissue overexpressing uPAR on cell surfaces. We found that chondrosarcoma-like primary culture cells release a large amount of SuPAR in the medium. In vitro, SuPAR elicits chondrosarcoma cell migration likely through its uPAR88-92 sequence, since the DII88-183 or DIIDIIR88-284 uPAR domains retain motogen effect whereas DI1-87 or DIII184-284 domains, both lacking the uPAR88-92 sequence, are ineffective. Chondrosarcoma cells cross matrigel in response to SuPAR, and their invasion capability is abrogated by RERF peptide which inhibits uPAR88-92 signalling. These findings assign a role to uPAR in mobilizing chondrosarcoma cells and suggest that RERF peptide may be regarded as a prototype to generate new therapeutics for the chondrosarcoma treatment.