Multi-omics analysis identifies osteosarcoma subtypes with distinct prognosis indicating stratified treatment.

Osteosarcoma (OS) is a primary malignant bone tumor that most commonly affects children, adolescents, and young adults. Here, we comprehensively analyze genomic, epigenomic and transcriptomic data from 121 OS patients. Somatic mutations are diverse within the cohort, and only TP53 is significantly mutated. Through unsupervised integrative clustering of the multi-omics data, we classify OS into four subtypes with distinct molecular features and clinical prognosis: (1) Immune activated (S-IA), (2) Immune suppressed (S-IS), (3) Homologous recombination deficiency dominant (S-HRD), and (4) MYC driven (S-MD). MYC amplification with HR proficiency tumors is identified with a high oxidative phosphorylation signature resulting in resistance to neoadjuvant chemotherapy. Potential therapeutic targets are identified for each subtype, including platinum-based chemotherapy, immune checkpoint inhibitors, anti-VEGFR, anti-MYC and PARPi-based synthetic lethal strategies. Our comprehensive integrated characterization provides a valuable resource that deepens our understanding of the disease, and may guide future clinical strategies for the precision treatment of OS.

Inhibition of sphingolipid metabolism in osteosarcoma protects against CD151-mediated tumorigenicity.

Osteosarcoma is the most common primary bone tumor, with a poor prognosis owing to the lack of efficient molecular-based targeted therapies. Previous studies have suggested an association between CD151 and distinct consequences in osteosarcoma tumorigenicity. However, the potential of CD151 as a therapeutic target has not yet been sufficiently explored. Here, we performed integrated transcriptomic and metabolomic analyses of osteosarcoma and identified sphingolipid metabolism as the top CD151-regulated pathway. CD151 regulates sphingolipid metabolism primarily through SPTCL1, the first rate-limiting enzyme in sphingolipid biosynthesis. Mechanistically, depletion of CD151 enhanced c-myc polyubiquitination and subsequent degradation. c-myc is vital for the transcriptional activation of SPTLC1. Functionally, sphingolipid synthesis and the SPTLC1 inhibitor, myriocin, significantly suppressed the clonogenic growth of CD151-overexpression cells. Importantly, myriocin selectively restrained CD151-high expression tumor growth in preclinical patient-derived xenograft models. Collectively, these data establish that CD151 is a key mediator of sphingolipid metabolism and provide a new approach to developing novel CD151-based targeted therapies for osteosarcoma.

O-arm-guided percutaneous microwave ablation and cementoplasty for the treatment of pelvic acetabulum bone metastasis.

Objective: This study aims to evaluate the indications, safety, and efficacy of microwave ablation combined with cementoplasty under O-arm navigation for the treatment of painful pelvic bone metastasis. Methods: We retrospectively collected data from 25 patients with acetabulum bone metastasis who underwent microwave ablation combined with cementoplasty. All patients underwent percutaneous microwave ablation combined with cementoplasty under O-arm navigation. The postoperative follow-up included evaluations of pain, quality of life, function, the incidence of bone cement leakage, and the presence of perioperative complications. Pain and quality of life were evaluated using the visual analog scale (VAS) and the QLQ-BM22 quality of life questionnaire for patients with bone metastases, respectively. The functional scores were calculated using the MSTS93 scoring system of the Bone and Soft Tissue Oncology Society. Results: There were 10 males and 15 females with an average age of 52.5 ± 6.5 years, all 25 patients received percutaneous procedures, and no technical failure occurred. Major complications, including pulmonary embolism, vascular or nervous injury, hip joint cement leakage, and infection, were not observed in the current study. Pain regression was achieved in 24 of 25 patients. The mean VAS scores significantly decreased to 3.4 ± 1.0, 2.5 ± 1.2, and 1.2 ± 0.6 points at 1 week, 1 month, and 3 months after the procedure, respectively, compared with 7.0 points before the procedure (P < .05). The mean QLQ-BM22 score significantly decreased to 36.2 ± 4.9, 30 ± 5.6, and 25.4 ± 2.3 points at 1 week, 1 month, and 3 months after the procedure, respectively, compared with 55.8 points before the procedure (P < .05). The preoperative Musculoskeletal tumour society (MSTS) functional score of 25 patients was 18.5 ± 5.3 points, and MSTS score was 20.0 ± 3.0, 21.4 ± 4.9, and 22.8 ± 2.3 at 1 week, 1 month, and 3 months after the procedure, respectively (P < .05). The average bone cement injection volume was 8.8 ± 4.6 ml. Conclusion: The use of O-arm-guided percutaneous microwave ablation combined with cementoplasty for the treatment of pelvic metastases could quickly and significantly alleviate local pain, prevent pathological fracture, and improve the quality of life of patients with reduced complications.

CDK7/GRP78 signaling axis contributes to tumor growth and metastasis in osteosarcoma.

Osteosarcoma derives from primitive bone-forming mesenchymal cells and is the most common primary bone malignancy. Therapeutic targeting of osteosarcoma has been unsuccessful; therefore, identifying novel osteosarcoma pathogenesis could offer new therapeutic options. CDK7 is a subunit within the general transcription factor TFIIH. We aim to explore the new mechanism by which CDK7 regulates osteosarcoma and our studies may provide new theoretical support for the use of CDK7 inhibitors in the treatment of osteosarcoma. Here, we investigate the molecular mechanism underlying the association between CDK7 and GRP78 in osteosarcoma. Specifically, we find that an E3 ubiquitin ligase TRIM21 binds and targets GRP78 for ubiquitination and degradation, whereas CDK7 phosphorylates GRP78 at T69 to inhibit TRIM21 recruitment, leading to GRP78 stabilization. Notably, a CDK7-specific inhibitor, THZ1, blunts osteosarcoma growth and metastasis. Combination treatment with CDK7 and GRP78 inhibitors yield additive effects on osteosarcoma growth and progression inhibition. Thus, simultaneous suppression of CDK7 and GRP78 activity represents a potential new approach for the treatment of osteosarcoma. In conclusion, the discovery of this previously unknown CDK7/GRP78 signaling axis provides the molecular basis and the rationale to target human osteosarcoma.

Correlation between Patient-Derived Xenograft Modeling and Prognosis in Osteosarcoma.

OBJECTIVE: To retrospectively analyze and compare the relationship between the success rate of patient-derived xenograft (PDX) modeling of osteosarcoma and prognosis (3-year overall survival rate and disease-free survival rate) and incidence of lung metastasis. METHODS: The sample group consisted of 57 osteosarcoma patients with definite pathological diagnoses from Shanghai General Hospital from 2015-2017. PDX models in 57 patients were analyzed by retrospective analyses. Among the patients currently inoculated, 20 were tumorigenic in the PDX model, and 37 were nontumorigenic. According to the tumorigenicity of PDXs, the corresponding osteosarcoma patients were divided into two groups. The effects of clinically related indicators on the model were retrospectively compared. The patients were followed, and the 3-year survival, 3-year disease-free survival (DFS), and lung metastasis rates were collected. The relationship between the modeling success and patient prognosis was investigated. RESULTS: In the chemotherapy-treated group, the PDX modeling success rate was 17.4%, and in the nonchemotherapy group, the success rate was 47.1%. The success of PDX modeling was related to whether patients received chemotherapy. The success rate of PDX modeling is significantly reduced after receiving chemotherapy. The 3-year overall survival rate of the PDX-grafted group was 49.23%, and that of the PDX-nongrafted group was 65.71%. There was a significant difference between the two groups, showing a strong negative correlation between the 3-year survival rate and the success rate of the PDX model. The 3-year disease-free survival rate of the PDX-grafted group was 29.54%. The 3-year DFS of the PDX-nongrafted group was 50.34%. There was a significant difference between the two groups. Lower grafted rates indicate a higher DFS rate. The incidence of lung metastasis in the PDX-grafted group was 32.4%, and that in the nongrafted group was 13.1%. There was a significant difference between the two groups. The successful establishment of the PDX model indicates that patients are more likely to have lung metastases. CONCLUSIONS: The success of PDX modeling often indicates poor prognosis (low 3-year overall survival rate and disease-free survival rate) and a greater possibility of lung metastasis. Therefore, PDX modeling in osteosarcoma patients can accurately predict the prognosis of patients and the risk of lung metastasis in advance to help us develop better therapeutic strategies.

Do reverse total shoulder replacements have better clinical and functional outcomes than hemiarthroplasty for patients undergoing proximal humeral tumor resection using devitalized autograft composite reconstruction: a case-control study.

OBJECTIVE: To compare the efficacy and prognosis of reverse total shoulder arthroplasty (rTSA) with shoulder hemiarthroplasty (SHA) using devitalized autograft or allograft composite reconstruction after proximal humeral tumor resection. METHODS: We retrospectively reviewed patients who underwent SHA (32) and rTSA (20) for tumor resections of the proximal humerus from January 2014 to July 2020. The clinical results included duration of the operation, intraoperative blood loss, bone union, visual analog scale (VAS) score, shoulder range of motion (ROM), American Shoulder and Elbow Surgeons (ASES) shoulder score, recurrence, and overall survival. RESULTS: Fifty-two patients were followed up for a mean of 30 months. Thirty-two patients were SHA with allograft-prosthetic composite (APC) reconstructions, while other 20 were rTSA with devitalized autograft-prosthetic composite reconstructions. At the end of the follow-up, 2 recurrence, 3 postoperative infections, and 4 subluxations occurred among the SHA patients. Two patients in the rTSA group had postoperative anterior dislocation and underwent revision surgery with surgical mesh, and 2 (2/20) had grade II scapular notching. The mean VAS score of the shoulder was 1.5 ± 0.8 in the rTSA group and 2.3 ± 1.2 in the SHA group (p < 0.05). The mean active forward flexion of the shoulder joint was 50.6 ± 6.0 in the SHA group and 100 ± 7.6 in the rTSA group (p < 0.05). The ASES shoulder score was 78 ± 3.0 in the rTSA group and 52 ± 5.6 in the SHA group (p < 0.05). The overall 3-year survival rate of all patients was 60.0%, and patients in the rTSA group showed better survival in terms of the mean 3-year OS than patients in the SHA group (p = 0.04). CONCLUSION: rTSA with devitalized autograft-prosthetic composite can offer a reasonable reconstruction of the shoulder joint after Malawer type I tumor resection. Compared with patients who underwent SHA, patients who underwent rTSA present good outcomes, a better range of motion, better bone union, and no increase in instability rate in the mid-term.

Surgical Treatment of Sacral Metastatic Tumors.

OBJECTIVE: This study intends to retrospectively analyze the data of patients with sacral metastases in our center, and analyze the treatment methods and therapeutic effects of sacral metastases. METHODS: 73 patients with sacral metastases treated in our hospital from June 2013 to June 2019 were retrospectively analyzed. There were 54 cases of neurological symptoms, 42 cases of sacroiliac joint instability, 24 cases of lower limb muscle weakness and 19 cases of abnormal urination and defecation. Four patients with tumors below S3 underwent complete tumor resection, 23 patients with tumors above S3 and without sacroiliac joint instability underwent tumor curettage and nerve root lysis, 34 patients with tumors above S3 and sacroiliac joint instability underwent tumor curettage, nerve root release and screw rod reconstruction. 12 patients with multiple metastases underwent percutaneous radiofrequency ablation and sacroplasty. VAS was used to evaluate the preoperative and postoperative pain scores, and the postoperative pain relief, neurological function, bowel function, wound healing and complications were evaluated. RESULTS: There were no perioperative death, 8 cases of poor wound healing, 5 cases of nerve injury, postoperative sensory and motor loss of lower limbs. Cerebrospinal fluid (CSF) leak in 7 cases. The patients were followed up for 6-25 months (mean 12 months). The VAS scores of patients with pain symptoms were 7 points before operation and 1.44 points after operation, In 19 patients with abnormal urination and defecation function, 12 patients recovered to normal 3-6 months after operation, 5 cases had no significant change compared with preoperative, and 2 cases had aggravated symptoms; 17 cases of patients with lower limb muscle strength were significantly recovered after operation, and the average muscle strength was increased by 2 grades; 30 cases of patients with unstable sacroiliac joint got internal fixation had significantly pain relief. Pain symptoms of 9 patients were significantly relieved after percutaneous radiofrequency ablation. CONCLUSION: the operation of sacral metastases mainly adopts a relatively conservative surgical method, which can effectively improve the quality of life of patients with sacral metastases by retaining the nerve function and relieving the pain of patients, combining with radiofrequency ablation, sacroplasty and targeted drugs.

Erratum: Sarcoma-Targeting Peptide-Decorated Polypeptide Nanogel Intracellularly Delivers Shikonin for Upregulated Osteosarcoma Necroptosis and Diminished Pulmonary Metastasis: Erratum.

[This corrects the article DOI: 10.7150/thno.18299.].

Anlotinib, a novel small molecular tyrosine kinase inhibitor, suppresses growth and metastasis via dual blockade of VEGFR2 and MET in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, with highly aggressive behavior and early systemic metastasis. The survival rates for osteosarcoma remain unchanged over the past two decades. Studies aiming to find new or alternative therapies for patients with refractory osteosarcoma are urgently needed. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor (TKI), has exhibited encouraging clinical activity in NSLCC and soft tissue sarcoma, whereas its effect on osteosarcoma has not been studied. In our study, we investigated the anti-tumor activity and underlying mechanism of anlotinib in osteosarcoma. Various in vitro and in vivo models of human osteosarcoma were used to determine the anti-proliferative, anti-angiogenesis and anti-metastasis efficacy of anlotinib. Our results showed that anlotinib suppressed tumor growth and increased the chemo-sensitivity of osteosarcoma. In addition, anlotinib inhibited migration and invasion in osteosarcoma cells. Furthermore, in order to explore the anti-tumor mechanism of anlotinib, phospho-RTK antibody arrays were performed. These analyses confirmed that anlotinib suppressed the phosphorylation of MET, VEGFR2 and the downstream signaling pathway activation. Moreover, we demonstrated that anlotinib blocked hepatocyte growth factor (HGF)-induced cell migration, invasion and VEGF-induced angiogenesis. Notably, a 143B-Luc orthotopic osteosarcoma model further showed that anlotinib significantly inhibited growth and lung metastasis of implanted tumor cells. Our preclinical work indicates that anlotinib acts as a novel inhibitor of VEGFR2 and MET that blocks tumorigenesis in osteosarcoma, which could be translated into future clinical trials.

Glaucocalyxin A-induced oxidative stress inhibits the activation of STAT3 signaling pathway and suppresses osteosarcoma progression in vitro and in vivo.

Osteosarcoma (OS) is ranked as the most common primary bone malignancy in children and adolescents worldwide, and the 5-year overall survival rate of OS is not optimistic. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in tumor cell growth, proliferation, and anti-apoptosis in OS. Therefore, the discovery of novel molecular compounds that can effectively block STAT3 activation, is essential for the treatment of OS and improving prognosis. Here, we investigate whether Glaucocalyxin A (GLA), derived from Rabdosia japonica, exhibit the potential anticancer effects in OS. First of all, we identify that GLA potently suppressed cell proliferation, induced G2/M phase arrest and promoted substantial apoptosis in OS. Next, we conclude that GLA could induce Reactive oxygen species (ROS)-mediated oxidative stress via an imbalance of GSH and GSSG. Then, we elucidate for the first time that GLA could significantly inhibit both constitutive and IL-6-inducible activation of STAT3 (Tyr705) and JAK2, the upstream regulator of STAT3. Furthermore, we elucidate that the inhibition of STAT3 is mainly induced by ROS-mediated oxidative stress. Overall, our findings demonstrate that GLA could exhibit potent anticancer effects through effectively blocking the STAT3 signaling pathway, which was induced by ROS-mediated oxidative stress in OS in vitro and in vivo.

Rapid, large-scale stimulated Raman histology with strip mosaicing and dual-phase detection.

Two-color stimulated Raman scattering (SRS) microscopy with label-free mapping of lipid/protein distributions has shown promise in virtual histology. Despite previous demonstrations of SRS in tumor delineation and diagnosis, the speed and efficiency of the current technique requires further improvements for practical use. Here, we integrate parallel dual-phase SRS detection with strip mosaicing, which reduces the imaging time of a whole mouse brain section from 70 to 8 minutes. We further verified our method in imaging fresh human surgical tissues, showing its great potential for rapid SRS histology, especially for large scale, large quantity imaging tasks.

Evaluation of Quality of Life Using EORTC QLQ-BM22 in Patients with Bone Metastases after Treatment with Magnetic Resonance Guided Focused Ultrasound.

OBJECTIVE: To reveal the alterations in quality of life (QOL) in bone metastases patients after magnetic resonance guided focused ultrasound (MRgFUS). METHODS: This retrospective study enrolled 26 patients diagnosed with bone metastases. Patients had various primary malignant tumors and tumor lesions in different locations. All patients received MRgFUS for bone metastasis. Each focal spot sonication pulse that was applied to create energy deposition lasted 20 s and was performed at a frequency of 1.05 MHz. The visual analog scale (VAS) was used to measure pain level and the EORTC QLQ-BM22 was applied to evaluate QOL for 12 months. The lower the QLQ-BM22 score, the better the QOL of patients. RESULTS: The painful site subscale of the EORTC QLQ-BM22 was observed without significant change. Significant reductions in the functional subscales were observed after therapy compared with the baseline. The functional interference was reduced significantly during the first 12 months. From the 2-month time point onwards, the pain characteristics subscale also decreased significantly. VAS scores had decreased by 40.8% 1 month after the operation and had decreased 10.9% compared with VAS scores preoperation. Scores for pain characteristics decreased by 28.8% after the operation and the scores were still down by 10.8% 1 year after the treatment. VAS scores indicated a significant reduction in pain over the course of the research until the 12-month time point follow-up compared with the baseline. CONCLUSION: MRgFUS therapy improved the QOL of patients with bone metastasis by relieving bone pain.

Sarcoma-Targeting Peptide-Decorated Polypeptide Nanogel Intracellularly Delivers Shikonin for Upregulated Osteosarcoma Necroptosis and Diminished Pulmonary Metastasis.

PURPOSE: Osteosarcoma is the most common primary bone cancer and is notorious for pulmonary metastasis, representing a major threat to pediatric patients. An effective drug targeting osteosarcoma and its lung metastasis is urgently needed. DESIGN: In this study, a sarcoma-targeting peptide-decorated disulfide-crosslinked polypeptide nanogel (STP-NG) was exploited for enhanced intracellular delivery of shikonin (SHK), an extract of a medicinal herb, to inhibit osteosarcoma progression with minimal systemic toxicity. RESULTS: The targeted, loaded nanogel, STP-NG/SHK, killed osteosarcoma cells by inducing RIP1- and RIP3-dependent necroptosis in vitro. Necroptosis is a novel cell death form that could be well adapted as an efficient antitumor strategy, the main obstacle of which is its high toxicity. After intravenous injection, STP-NG/SHK efficiently suppressed tumor growth and reduced pulmonary metastasis, offering greater tumor necrosis and higher RIP1 and RIP3 upregulation compared to free SHK or untargeted NG/SHK in vivo. Additionally, the treatment with NG/SHK or STP-NG/SHK showed minimal toxicity to normal organs, suggesting low systemic toxicity compared to free SHK. CONCLUSION: The STP-guided intracellular drug delivery system using the necroptosis mechanism showed profound anti-osteosarcoma activity, especially eliminated lung metastasis in vivo. This drug formulation may have great potential for treatment of osteosarcoma.

HER4 promotes cell survival and chemoresistance in osteosarcoma via interaction with NDRG1.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. The abilities of chemotherapy resistance are major roadblock in the successful treatment of OS. The clarification of mechanism regarding cell survival during OS chemotherapy are important. Here, we examined HER4 expression by immunohistochemistry in a large series of OS tissues, and found HER4 expression correlated with tumor characteristics and patient survival rates. HER4 knockdown by shRNA inhibited OS cell growth and tumorigenesis, and induced cell senescence and apoptosis in vitro and in vivo. We demonstrated that HER4 expression upregulated in the adverse conditions, such as serum starvation and sphere culture. Moreover, HER4 knockdown cells became more sensitive in stressful conditions such as loss of attachment, cytotoxic agents or nutrition insufficiency. Mechanism studies revealed that HER4 interacted with NDRG1, and NDRG1 overexpression could antagonize HER4 knockdown-mediated cell growth and apoptosis in stressed conditions. There was a positive correlation between HER4 and NDRG1 immunoreactivity in OS patients. Together, our present study shows that HER4 and/or NDRG1 might play a critical role for the cell survival and chemo-resistance of OS, and could be used as potential therapeutic targets in OS.

Corrigendum: NDRG1 inhibition sensitizes osteosarcoma cells to combretastatin A-4 through targeting autophagy.

This corrects the article DOI: 10.1038/cddis.2017.438.

NDRG1 inhibition sensitizes osteosarcoma cells to combretastatin A-4 through targeting autophagy.

Combretastatin A-4 (CA-4), a tubulin-depolymerizing agent, shows promising antitumor efficacy and has been under several clinical trials in solid tumors for 10 years. Autophagy has an important pro-survival role in cancer therapy, thus targeting autophagy may improve the efficacy of antitumor agents. N-myc downstream-regulated gene 1 (NDRG1) is a significant stress regulatory gene, which mediates cell survival and chemoresistance. Here we reported that CA-4 could induce cell-protective autophagy, and combination treatment of CA-4 and autophagy inhibitor chloroquine (CQ) exerted synergistic cytotoxic effect on human osteosarcoma (OS) cells. Meanwhile, CA-4 or CQ could increase the expression of NDRG1 independently. We further performed mechanistic study to explore how CA-4 and CQ regulate the expression of NDRG1. Using luciferase reporter assay, we found that CA-4 transcriptionally upregulated NDRG1 expression, whereas CQ triggered colocalization of NDRG1 and lysosome, which subsequently prevented lysosome-dependent degradation of NDRG1. Further, we showed that knockdown of NDRG1 caused the defect of lysosomal function, which accumulated LC3-positive autophagosomes by decreasing their fusion with lysosomes. Moreover, NDRG1 inhibition increased apoptosis in response to combination treatment with CA-4 and CQ. Taken together, our study revealed abrogation of NDRG1 expression sensitizes OS cells to CA-4 by suppression of autophagosome-lysosome fusion. These results provide clues for developing more effective cancer therapeutic strategies by the concomitant treatment with CA-4 and clinical available autophagy inhibitors.

Arsenic sulfide induces apoptosis and autophagy through the activation of ROS/JNK and suppression of Akt/mTOR signaling pathways in osteosarcoma.

Osteosarcoma is a common primary malignant bone tumor, the cure rate of which has stagnated over the past 25-30 years. Arsenic sulfide (As2S2), the main active ingredient of the traditional Chinese medicine realgar, has been proved to have antitumor efficacy in several tumor types including acute promyelocytic leukemia, gastric cancer and colon cancer. Here, we investigated the efficacy and mechanism of As2S2 in osteosarcoma both in vitro and in vivo. In this study, we demonstrated that As2S2 potently suppressed cell proliferation by inducing G2/M phase arrest in various osteosarcoma cell lines. Also, treatment with As2S2 induced apoptosis and autophagy in osteosarcoma cells. The apoptosis induction was related to PARP cleavage and activation of caspase-3, -8, -9. As2S2 was demonstrated to induce autophagy as evidenced by formation of autophagosome and accumulation of LC3II. Further studies showed that As2S2-induced apoptosis and autophagy could be significantly attenuated by ROS scavenger and JNK inhibitor. Moreover, we found that As2S2 inhibited Akt/mTOR signaling pathway, and suppressing Akt and mTOR kinases activity can increase As2S2-induced apoptosis and autophagy. Finally, As2S2in vivo suppressed tumor growth with few side effects. In summary, our results revealed that As2S2 induced G2/M phase arrest, apoptosis, and autophagy via activing ROS/JNK and blocking Akt/mTOR signaling pathway in human osteosarcoma cells. Arsenic sulfide may be a potential clinical antitumor drugs targeting osteosarcoma.

Alternol, a natural compound, exerts an anti-tumour effect on osteosarcoma by modulating of STAT3 and ROS/MAPK signalling pathways.

Osteosarcoma (OS) is the most frequent primary malignant bone tumour. Alternol, a novel compound purified from microbial fermentation products exerts anti-tumour effects across several cancer types. The effect of alternol on human OS remains to be elucidated. We first evaluated the anti-tumour effect of alternol in several human OS cell lines in vitro and investigated its underlying mechanism. Alternol inhibited OS cell proliferation, migration and induced caspase-dependent apoptosis, G2/M cell cycle arrest in a dose and time-dependent manner. Moreover, alternol treatment inhibited signal transducer and activator of transcription-3 (STAT3) phosphorylation in 143B and MG63 human OS cells, as evaluated using a STAT3-dependent dual luciferase reporter system. Exposure to alternol resulted in excessive reactive oxygen species (ROS) generation and Jun amino-terminal kinases (JNK), extracellular signal-regulated kinases (ERK1/2) and p38 activation. Furthermore, alternol-induced cell death was significantly restored in the presence of the ROS scavenger, N-acetyl-l-cysteine (NAC) or a caspase inhibitor Z-VAD-FMK. NAC also prevented G2/M phase arrest and phosphorylation of mitogen-activated protein kinases (MAPK), but did not reverse STAT3 inactivation. Finally, alternol suppressed tumour growth in vivo in the nude mouse OS tibia orthotopic model. Immunohistochemistry revealed that alternol treatment resulted in down-regulation of phosph-STAT3 Tyr705 and up-regulation of cleaved caspase-3 and phosph-SAPK (Stress-activated protein kinases)/JNK expression. Taken together, our results reveal that alternol suppresses cell proliferation, migration and induces apoptosis, cell cycle arrest by modulating of ROS-dependent MAPK and STAT3 signalling pathways in human OS cells. Therefore, alternol is a promising candidate for developing anti-tumour drugs target OS.

CD151-mediated adhesion is crucial to osteosarcoma pulmonary metastasis.

CD151, a tetraspanin family protein involved in cell-cell and cell-extracellular matrix interaction, is differentially expressed in osteosarcoma cell membranes. Thus, this study aimed to investigate the role of CD151 in osteosarcoma metastasis. We analyzed CD151 expression in patient tissue samples using immunohistochemistry. CD151 expression was also silenced with shRNA in osteosarcoma cells of high metastatic potential, and cell adhesion, migration and invasion were evaluated in vitro and pulmonary metastasis was investigated in vivo. Mediators of cell signaling pathways were also examined following suppression of CD151 expression. Overall survival for patients with low versus high CD151 expression level was 94 vs. 41 months (p=0.0451). CD151 expression in osteosarcoma cells with high metastatic potential was significantly higher than in those with low metastatic potential (p<0.001). shRNA-mediated silencing of CD151 did not influence cell viability or proliferation; however, cell adhesion, migration and invasion were all inhibited (all p<0.001). In mice inoculated with shRNA-transduced osteosarcoma cells, the number and size of lung metastatic lesions were reduced compared to the mice inoculated with control-shRNA transduced cells (p<0.001). In addition, CD151 knockdown significantly reduced Akt, p38, and p65 phosphorylation as well as focal adhesion kinase, integrin ß1, p70s6, and p-mTOR levels. Taken together, CD151 induced osteosarcoma metastasis likely by regulating cell function through adhesion signaling. Further studies are necessary to fully explore the diagnostic and prognostic value of determining CD151 expression in osteosarcoma patients.