Shidong Wang

Knockdown of long non-coding RNA HOTAIR increases miR-454-3p by targeting Stat3 and Atg12 to inhibit chondrosarcoma growth

Current practices for the therapy of chondrosarcoma, including wide-margin surgical resection and chemotherapy, are less than satisfactory. Recently, emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) have an essential role in the initiation and progression of tumors. As a typical lncRNA, HOTAIR is significantly overexpressed in various tumors. However, the function and potential biological mechanisms of HOTAIR in human chondrosarcoma remain unknown. Quantitative RT-PCR demonstrated that HOTAIR expression was upregulated in chondrosarcoma tissues and cell lines. High HOTAIR expression is correlated with tumor stage and poor prognosis. Functional experiments reveal that HOTAIR knockdown leads to growth inhibition of human chondrosarcoma cells in vitro and in vivo. In addition to cycle arrest and apoptosis, knockdown of HOTAIR inhibits autophagy, which favors cell death. Mechanistically, we demonstrated that HOTAIR induced DNA methylation of miR-454-3p by recruiting EZH2 and DNMT1 to the miR-454-3p promoter regions, which markedly silences miR-454-3p expression. Further analysis revealed that STAT3 and ATG12 are targets of miR-454-3p, initiate HOTAIR deficiency-induced apoptosis and reduce autophagy. Collectively, our data reveal the roles and functional mechanisms of HOTAIR in human chondrosarcoma and suggest that HOTAIR may act as a prognostic biomarker and potential therapeutic target for chondrosarcoma.

Inhibition of SOX2 induces cell apoptosis and G1/S arrest in Ewing's sarcoma through the PI3K/Akt pathway.

BackgroundEwing’s sarcoma is an aggressive bone and soft tissue tumor with a high incidence in children and adolescents. Due to its high malignancy and poor prognosis, identification of novel biomarkers for intervention therapies is necessary to improve outcome. The EWS/FLI1 fusion gene is a characteristic of Ewing’s sarcoma in most cases. Sex determining region Y-box 2 (SOX2) is a primary target of EWS/FLI1. It has been identified as an oncogene and linked to apoptotic resistance in several types of cancer. However, its role and regulatory mechanisms in Ewing’s sarcoma are largely unknown.MethodsWe systematically investigated the role of SOX2 in Ewing’s sarcoma cell lines, human tissue samples and xenograft models. The expression of SOX2 was detected in Ewing’s sarcoma samples by WB and IHC. siRNAs were used to knockdown EWS/FLI1 and SOX2 in A673 and RD-ES cell lines with the efficiencies tested by qRT-PCR and WB. The effect of SOX2 on cell cycle and apoptosis was determined by Flow cytometric and TUNEL assays. Akt overexpression was performed with plasmid. The protein expression of the corresponding factors was examined by WB analysis. Inhibition of SOX2 in vivo was performed by siRNA against SOX2 in xenograft models, and the protein expression of the regulators testified in vitro was examined in xenograft tumors by IHC and WB.ResultsThe results confirmed that SOX2 was highly expressed in Ewing’s sarcoma and was the target of EWS/FLI1. SOX2 advanced Ewing’s sarcoma cell survival and proliferation by regulating p21, p27 and cyclin-E to facilitate G1/S phase transition and mediating caspase-3, PARP via both extrinsic (Fas and caspase-8) and intrinsic (caspase-9, Bad, Bcl-2 and XIAP) apoptotic pathways to restrain cell apoptotsis. Additionally, SOX2 regulated the cell-cycle progression and apoptosis via activation of the PI3K/Akt signaling pathway. The mechanisms were proved both in vitro and in vivo.ConclusionsThe results demonstrate that SOX2 played a central role in promoting Ewing’s sarcoma cell proliferation in vitro and in vivo with the underlying mechanisms expounded. These findings suggest that SOX2 may serve as a potential biomarker for targeted intervention in Ewing’s sarcoma.

Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma

The cure rate of osteosarcoma has not improved in the past 30 years. The search for new treatments and drugs is urgently needed. Apatinib is a high selectivity inhibitor of vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase, exerting promising antitumoral effect in various tumors. The antitumor effect of Apatinib in human osteosarcoma has never been reported. We investigated the effects of Apatinib in osteosarcoma in vitro and in vivo. Osteosarcoma patients with high levels of VEGFR2 have poor prognosis. Apatinib can inhibit cell growth of osteosarcoma cells. In addition to cycle arrest and apoptosis, Apatinib induces autophagy. Interestingly, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells. Immunoprecipitation confirmed direct binding between VEGFR2 and signal transducer and activator of transcription 3 (STAT3). Downregulation of VEGFR2 by siRNA resulted in STAT3 inhibition in KHOS cells. VEGFR2 and STAT3 are inhibited by Apatinib in KHOS cells, and STAT3 act downstream of VEGFR2. STAT3 and BCL-2 were downregulated by Apatinib. STAT3 knockdown by siRNA reinforced autophagy and apoptosis induced by Apatinib. BCL-2 inhibits autophagy and was apoptosis restrained by Apatinib too. Overexpression of BCL-2 decreased Apatinib-induced apoptosis and autophagy. Apatinib repressed the expression of STAT3 and BCL-2 and suppressed the growth of osteosarcoma in vivo. To sum up, deactivation of VEGFR2/STAT3/BCL-2 signal pathway leads to Apatinib-induced growth inhibition of osteosarcoma.

Induction of the mesenchymal to epithelial transition by demethylation-activated microRNA-125b is involved in the anti-migration/invasion effects of arsenic trioxide on human chondrosarcoma

BackgroundIn addition to treating acute promyelocytic leukemia, arsenic trioxide (ATO) suppresses other solid tumors, including chondrosarcoma. However, the effects of ATO on metastasis in chondrosarcoma cells, and the underlying molecular mechanisms remain unclear.MethodsThe effects of ATO on the migratory and invasive capacities of chondrosarcoma cells were investigated by Wound healing, Transwell and EMT assays. The expression of miR-125b in human chondrosarcoma tissues and cell lines was detected by real-time PCR analysis. Bisulfite sequencing analysis (BSP) was used to detect the effects of ATO on the expression of miR-125b. The gain-of-function and loss-of-function experiments were performed on chondrosarcoma cell lines to investigate the effects of miR-125b on chondrosarcoma invasion, and to determine whether signal transducer and activator of transcription 3(Stat3) mediates these effects. Dual-luciferase reporter assay was used to identify whether Stat3 is a direct target of miR-125b.ResultsMiR-125b was significantly downregulated in human metastatic chondrosarcoma tissues and cell lines but not in non-metastatic chondrosarcoma tissues. ATO up-regulates the expression of miR-125b by the demethylation of DNA. ATO induces MET and attenuates the invasive capacities of chondrosarcoma cells through miR-125b. Stat3 was verified as a direct target of miR-125b, which is involved in ATO regulating EMT-associated traits.ConclusionsThese findings, for the first time, provides evidence that the miR-125b-mediated inhibition of Stat3 is involved in the ATO-induced attenuation of metastasis in chondrosarcoma cells.

PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse

BackgroundImmune checkpoint inhibitors have led to a breakthrough in solid tumor immunotherapy, but related studies on musculoskeletal tumors are few, especially for PD-L2.MethodsWe examined expression of three molecular effectors of the PD-1 axis in 234 patients with musculoskeletal tumors, including osteosarcoma, chondrosarcoma, synovial sarcoma, and giant cell tumor. Survival analyses and potential mechanisms were investigated in osteosarcoma per the Gene Expression Omnibus (GEO) and immunohistochemistry analyses. In vivo, humanized mice were used to evaluate the effect of nivolumab on osteosarcoma.ResultsPD-L1, PD-L2, and PD-1 expression levels were significantly different between the histologic types of the musculoskeletal tumors. For osteosarcoma, PD-L1 was negatively correlated with prognosis, while PD-1 had a negative correlation tendency with overall survival (OS). Meanwhile, PD-L2 had a positive correlation trend with OS. Nivolumab inhibited osteosarcoma metastasis in humanized mice by increasing CD4+ and CD8+ lymphocytes and the cytolytic activity of CD8 lymphocytes in the lung but did not affect primary osteosarcoma growth.ConclusionWe systematically detected the expression patterns of PD-L1, PD-L2, and PD-1 in musculoskeletal tumors for the first time and demonstrated the prognostic roles and underlying mechanisms of PD-1 axis in osteosarcoma. Furthermore, PD-1 blockade could effectively control osteosarcoma pulmonary metastasis in vivo. Therefore, the PD-1 axis may be a potential immunotherapeutic target for metastatic osteosarcoma.

BMPR2 promotes invasion and metastasis via the RhoA-ROCK-LIMK2 pathway in human osteosarcoma cells

Bone morphogenetic protein receptor 2 (BMPR2) has been identified in several types of cancer. However, its role in osteosarcoma is largely unknown. We systematically investigated the role of BMPR2 in osteosarcoma cell lines, human tissue samples and xenograft models. The relationship between BMPR2 expression and osteosarcoma patients’ survival was investigated by bioinformatics and clinical data. Wound healing assay and transwell assay were used to detect the changes of cell migration and invasion ability after BMPR2 transfection. In addition, downstream phosphoproteins were analyzed by iTRAQ-based phosphoproteomic analysis and verified by western blotting. In vivo, the effects of BMPR2 on the growth, formation and metastasis of 143B cells were observed by orthotopic transplantation of nude mice. Here, we demonstrated that BMPR2 expression was elevated in a majority of osteosarcoma tissues compared with normal bone tissue. Osteosarcoma patients with greater BMPR2 expressing level showed a poor overall survival. The depletion of BMPR2 in 143B cells markedly reduced the invasive capacity in vitro and metastatic potential in vivo. Mechanistically, we found that LIM domain kinase 2 (LIMK2) was phosphorylated and activated by BMPR2 and that this event was crucial for activation of the BMPR2-mediated signal pathway in osteosarcoma cells. Additionally, we demonstrated that BMPR2 could active LIMK2 through the RhoA/ROCK pathway and could also interact with LIMK2 directly. Taken together, our study revealed that BMPR2 functions as a prometastatic oncogene in vitro and in vivo with the activation of the RhoA-ROCK-LIMK2 pathway and may represent a potential therapeutic target for osteosarcoma.

BMPR2 and HIF1-α overexpression in resected osteosarcoma correlates with distant metastasis and patient survival

Objective Bone morphogenetic protein receptor 2 (BMPR2) and hypoxia-inducible factor 1-α (HIF1-α) existed abnormal expression in several types of cancer. However, their expressions and related roles in osteosarcoma are largely unknown. Methods To investigate the clinical significance of BMPR2 and HIF1-α in osteosarcoma, we analyzed their expression levels in 103 osteosarcoma specimens by immunochemistry. Meanwhile, we conducted a follow-up to examine the metastatic behavior and overall survival (OS) of osteosarcoma patients. Results Among 103 tissues, 61 cases had BMPR2-positive expression and 57 cases had HIF1-α positive expression. A significant correlation was noticed between BMPR2 and HIF1-α expression in osteosarcoma specimens (P=0.035). Receiver-operating characteristic (ROC) curves were calculated to investigate the predictive value of the two markers in tumor metastasis. By means of univariate and multivariate analysis, BMPR2 and HIF1-α expression, as well as higher tumor grade, were identified as significant risk factors for OS in patients with osteosarcoma. Kaplan-Meier survival analysis revealed that the patients with BMPR2 and HIF1-α positive expression had worse OS compared with patients with BMPR2-negative or HIF1-α-negative staining. Conclusions It can be concluded that BMPR2 and HIF1-α expression is highly correlated with metastatic behavior in patients with osteosarcoma and can serve as predictive markers for metastasis and OS of these patients.

Tumor-associated macrophages promote lung metastasis and induce epithelial-mesenchymal transition in osteosarcoma by activating the COX-2/STAT3 axis

Osteosarcoma (OS) is a common, malignant musculoskeletal tumor in young people. Neoadjuvant chemotherapy has improved the survival of osteosarcoma patients but with limited benefit due to metastasis. Tumor-associated macrophages (TAMs) are involved in various mechanisms of tumor biology, which include oncogenesis, drug resistance, and tumor immune escape, as well as tumor metastasis. In this study, we proved that TAMs possess the ability to promote OS cell migration and invasion by upregulating COX-2, MMP9, and phosphorylated STAT3 and to induce the epithelial-mesenchymal transition (EMT). This evidence has also been verified in a tumor-bearing animal model, and in OS patients. Furthermore, we observed the anti-metastasis effect of COX-2 inhibition by repressing COX-2 expression, EMT-activating transcription factors and the STAT3 pathway, both in vitro and in vivo. We propose that TAMs promote OS metastasis and invasion by activating the COX-2/STAT3 axis and EMT. These findings suggest that TAMs and COX-2 may be potential targets for future anti-metastasis therapy.

Correction to: PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse

The original article [1] contained an error in Table 1 whereby the ‘Positive’ column in the ‘PD-L1’ Tumor type group of columns was mistakenly included at the beginning of the ‘PD-L2’ Tumor type group of columns.

Can Aortic Balloon Occlusion Reduce Blood Loss During Resection of Sacral Tumors That Extend Into the Lower Lumber Spine

Background Although aortic balloon occlusion has been shown to reduce blood loss during sacral tumor resections, it has not been validated in larger sacral tumors involving the lower lumbar spine. If such an approach were shown to be associated with less blood loss, it might aid the tumor surgeon in resecting these difficult tumors. Questions/purposes (1) Is the use of aortic balloon occlusion associated with reduced blood loss in sacral tumor resections when the lower lumbar spine is also involved? (2) Does the use of the aortic balloon prolong total operating time? (3) What complications are associated with the use of a balloon? Methods We retrospectively studied all 56 patients diagnosed with sacral tumors involving the lower lumbar spine (L4, L5) who were treated surgically between 2004 and 2015 at our institute. During that time, 30 of the patients received aortic balloon occlusion therapy, whereas 26 of the patients did not. We generally used aortic balloon occlusion during procedures for hypervascular lesions (for example, giant cell tumors or metastatic renal cancers), primary malignant lesions, and recurrent lesions. We generally avoided use of aortic balloon occlusion in patients with anatomic defects of the aorta (aortic dissection or aneurysm was strictly contraindicated), renal artery bifurcation caudal to the L2 to L3 disc, age older than 70 years or younger than 12 years, history of Stage 2 hypertension [9], history of balloon use in previous surgeries, and presence of unstable plaque on abdominal CT. The demographic data, intraoperative blood loss, transfusion volume, operating time, and postoperative wound drainage between the two groups were collected and analyzed. Balloon-related complications were identified. Followup in terms of balloon-related complications was conducted in all 56 patients for at least 6 months after surgery. Results Intraoperative blood loss was determined to be less in patients treated with the balloon compared with those treated without the balloon (median volume, 2000 mL, range, 400-6000 mL versus 2650 mL, range, 550-6800 mL, respectively; median difference, 605 mL; 95% confidence interval [CI], 100-1500 mL; p = 0.035). Total operative time was not prolonged in the balloon group (including balloon insertion time) compared with those treated without it (median time, 215 minutes, range, 110-430 minutes versus 225 minutes, range, 115-340 minutes, respectively; median difference, 10 minutes; 95% CI, -40 to 30 minutes; p = 0.902). Balloon-related vascular complications included local hematoma at the puncture site in five patients, femoral artery spasm in three patients, lower limb ischemia in one patient, and femoral artery pseudoaneurysm in one patient. Acute kidney injury was found in two patients in the balloon group. Conclusions This study demonstrated that placement of the aortic balloon at a level just caudal to the renal artery bifurcation was associated with lower intraoperative blood loss and transfusion in lumbosacral tumor resections. However, procedure-specific complications were common and there was no benefit to total operative time. We suggest that the surgical procedures still need to be further refined to minimize complications. We also recommend that prospective studies be undertaken to confirm the efficacy of aortic balloon occlusion in surgery for lumbosacral tumors. Level of Evidence: Level III, therapeutic study.