MicroRNA-329-3p inhibits the Wnt/ß-catenin pathway and proliferation of osteosarcoma cells by targeting transcription factor 7-like 1.

An important factor in the emergence and progression of osteosarcoma (OS) is the dysregulated expression of microRNAs (miRNAs). Transcription factor 7-like 1 (TCF7L1), a member of the T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor family, interacts with the Wnt signaling pathway regulator ß-catenin and acts as a DNA-specific binding protein. This study sought to elucidate the impact of the interaction between miR-329-3p and TCF7L1 on the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches. MiR329-3p was significantly downregulated, while TCF7L1 was considerably up-regulated in all examined OS cell lines. Additionally, a clinical comparison study was performed using the TCGA database. Subsequently, the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments. When miR-329-3p was transfected into the OS cell line, the expression of TCF7L1 decreased, the proliferation of OS cells was inhibited, the cytoskeleton disintegrated, and the nucleus condensed to form apoptotic bodies. The expression of proteins that indicate apoptosis increased simultaneously. The cell cycle was arrested in the G0/G1 phase, and the G1/S transition was blocked. The introduction of miR-329-3p also inhibited downstream Cyclin D1 of the Wnt pathway. Xenograft experiments indicated that the overexpression of miR-329-3p significantly inhibited the growth of OS xenografts in nude mice, and the expression of TCF7L1 and c-Myc in tumor tissues decreased. MiR-329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo. Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7L1 by miR-329-3p. Summarizing these results, it can be inferred that miR-329-3p exerts anticancer effects in osteosarcoma by inhibiting TCF7L1.

Gait analysis of a patient after femoral nerve and malignant soft tissue tumor resections: a case report.

BACKGROUND: Malignant femoral soft tissue tumors are occasionally resected together with the femoral nerves, but this can cause loss of knee extensor muscle activity. To the best of our knowledge, no previous reports have detailed the gait analysis of such cases in combination with electromyography. Herein, we report the gait analysis of a patient who underwent left groin synovial sarcoma and left femoral nerve resection 12 years ago. CASE PRESENTATION: We analyzed the gait of a 38-year-old man who was able to walk unaided after the resection of a synovial sarcoma in the left groin together with the ipsilateral femoral nerve. The muscle activities of the affected medial (MH) and lateral hamstrings (LH), and lateral heads of the gastrocnemius (GL) were increased during 50-75% of the stance phase. The hip flexion angle of the affected limb was smaller, and the ankle plantar flexion angle of the affected limb was larger than that of the non-affected limb. This means that in the affected limb, the hip and ankle angles were adjusted to prevent knee collapse, and the MH, LH, and GL muscles contributed in the mid- and late-stance phases. Moreover, we found that the hamstring and gastrocnemius of the affected limb worked together to keep the ipsilateral knee extended in the mid-stance phase and slightly flexed in the late-stance phase. CONCLUSIONS: Patients capable of walking after femoral nerve resection may control their hamstrings and gastrocnemius muscles collaboratively to prevent ipsilateral knee collapse in the mid- and late-stance phases.

Sequential treatments with TRK inhibitors in a patient with NTRK fusion-positive sarcoma: A case report.

RATIONALE: Precision medicine and tumor-agnostic treatment strategies have recently been promoted for clinical use. One of the most successful treatments in patients with neurotrophic tyrosine receptor kinase (NTRK) fusion-positive tumors is targeting the tropomyosin receptor kinase (TRK) with an inhibitor. The TRK inhibitors, larotrectinib, and entrectinib, have been approved in many countries. Nevertheless, the most effective administration regimen for these TRK inhibitors is uncertain. To date, no reports have shown the efficacy of sequential treatment with larotrectinib and entrectinib in patients with NTRK fusion-positive tumors. In this report, we present a patient with NTRK fusion-positive sarcoma arising from the anterior mediastinum, with tumor progression after 4 months of entrectinib use. The patient took larotrectinib subsequently and maintained disease control for more than 21 months. PATIENT CONCERNS: A 48-year-old female visited a physician because she experienced difficulty in breathing and chest and back pain with no obvious cause 2 months ago. Computed tomography (CT)-guided biopsy was performed at a district general hospital, and histopathological examination revealed a small round cell tumor. She was referred to our hospital, and a second CT-guided biopsy was performed to confirm the pathological diagnosis. Considering the results of the histopathological examination, Ewing sarcoma was suspected, but a specific fusion gene was not detected due to poor quality specimens. DIAGNOSES: After 3 regimens of cytotoxic chemotherapy, biopsy was repeated, and specimens were analyzed using next-generation sequencing. The PHF20-NTRK1 fusion gene was detected, and the tumor was finally diagnosed as an NTRK fusion-positive sarcoma. INTERVENTIONS: She was administered the TRK inhibitor entrectinib, but the tumor started to grow after 4 months of medication, and she stopped taking entrectinib. After 1 cycle of cytotoxic chemotherapy, another TRK inhibitor, larotrectinib, was administered. OUTCOMES: Her stable disease was maintained for more than 21 months. Here, we have shown that sequential administration of both drugs can be effective. LESSONS: In the treatment of NTRK fusion-positive tumors, there are cases in which 2 approved first-generation TRK inhibitors can be used sequentially.

Tumor-suppressive microRNA-152 inhibits the proliferation of Ewing's sarcoma cells by targeting CDK5R1.

We elucidated the mechanism through which the reduced expression of miR-152 leads to the overexpression of its target cyclin-dependent kinase-5 activator 1 (CDK5R1) in Ewing's sarcoma (ES) cells and the role of this mechanism in the proliferation of ES cells. To explore possible oncogenic factors in ES, we conducted microarray-based investigation and profiled the changes in miRNA expression and their effects on downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). miR-152 was significantly downregulated, while cyclin-dependent kinase-5 activator 1 (CDK5R1) expression was significantly upregulated in all tested ES cells as compared to hMSCs. The overexpression of CDK5R1 led to the activation of CDK5, enabling the phosphorylation of retinoblastoma protein and persistent overexpression of CCNE. Moreover, miR-152 suppressed cell proliferation via cell cycle retardation, and its upregulation reduced tumor size and CCNE expression in tumor tissues. The overexpression of cyclin E (CCNE) has been detected in ES cells, but the detailed mechanisms have not been previously elucidated. These findings identify the miR152-CDK5R1 signaling axis as a critical mechanism for tumorigenesis that may serve as a new therapeutic target in Ewing's sarcoma. We believe that our results will aid in the development of effective treatment strategies for patients with ES.

The anti-oncogenic effect of 17-DMAG via the inactivation of HSP90 and MET pathway in osteosarcoma cells.

Heat shock protein (HSP) 90 plays a crucial role in correcting the misfolded three-dimensional structure of proteins, assisting them in folding into proper conformations. HSP90 is critical in maintaining the normal functions of various proteins within cells, as essential factors for cellular homeostasis. Contrastingly, HSP90 simultaneously supports the maturation of cancer-related proteins, including mesenchymal epithelial transition factor (MET) within tumor cells. All osteosarcoma cell lines had elevated MET expression in the cDNA array in our possession. MET, a tyrosine kinase receptor, promotes proliferation and an anti-apoptotic state through the activation of the MET pathway constructed by HSP90. In this study, we treated osteosarcoma cells with an HSP90 inhibitor, 17-demethoxygeldanamycin hydrochloride (17-DMAG), and assessed the changes in the MET signaling pathway and also the antitumor effect of the drug. The cell cycle in osteosarcoma cells administered 17-DMAG was found to be halted at the G2/M phase. Additionally, treatment with 17-DMAG inhibited cell proliferation and induced apoptosis. Inhibition of tumor cell proliferation was also observed in an in vivo model system, mice that were treated with 17-DMAG. Based on the results of this study, we were able to confirm that 17-DMAG promotes inhibition of osteosarcoma cell proliferation and induction of apoptosis by inhibition of MET, a protein highly expressed in osteosarcoma cells. This approach may be useful for the establishment of a new treatment strategy for patients resistant to the standard treatment for osteosarcoma.

Surrogacy analysis of intermediate end-points for overall survival in randomized controlled trials of rhabdomyosarcoma.

Treatment of malignant tumors, such as rhabdomyosarcoma (RMS), can improve overall survival (OS). It is time-consuming and expensive for patients to obtain benefits from randomized controlled trials (RCTs) with OS as the primary end-point. Therefore, another surrogate end-point is necessary; however, there is no report on the surrogacy analysis of RMS. In this study, we performed a systematic review of RCTs, involving patients with newly diagnosed RMS, and 11 RCTs were identified. We performed a meta-analysis to assess the surrogacy of intermediate end-points for OS. The correlations between surrogate end-points and OS were investigated using Spearman's rank correlation coefficient (ρ). The coefficient of determination (R2) was employed to measure the strength of the association. A total of 5183 patients were randomly allocated to 34 treatment groups. A marginal correlation (R2 = 0.281, ρ = 0.445) between the hazard ratios (HRs) for event-free survival (EFS) and OS was observed. In patients with localized RMS, the EFS HR had a weaker correlation with OS HR in the sensitivity analysis than that in the primary analysis. Overall, the surrogacy of EFS for OS cannot be confirmed.

Patellar dislocation following distal femoral replacement after extra-articular knee resection for bone sarcoma: A case report.

BACKGROUND: For the treatment of bone sarcoma in the distal femur, wide-margin resection and knee reconstruction with tumor endoprosthesis are standard therapies. Extra-articular knee resection is required in cases of tumor invasion of the knee joint; however, the incidence of complications, such as aseptic loosening, prosthesis infection, and implant failure, is higher than that following intra-articular knee resection. To the best of our knowledge, there are three reports of patellar dislocations after replacement of a tumor endoprosthesis. CASE SUMMARY: A 36-year-old man with no significant past medical history was admitted to our institution with continuous pain in his left knee for 4 mo. An open biopsy was performed, and the patient was diagnosed with a left distal femoral malignant bone tumor. Extra-articular knee resection and knee reconstruction with a tumor endoprosthesis were performed. Although the alignment of the tumor prosthesis was acceptable, knee instability was noticed postoperatively. The axial radiographic view of the patellar and computed tomography showed lateral patellar dislocation at 4 wk postoperatively. The patient had to undergo a lateral release and proximal realignment. He could perform his daily activities at 9 mo postoperatively. Radiography revealed no patellar re-dislocation. CONCLUSION: Proximal realignment may be considered during primary surgery if there is an imbalance in the forces controlling the patellar tracking.

Analysis of the signal cross talk via CCL26 in the tumor microenvironment in osteosarcoma.

Interaction with surrounding healthy cells plays a major role in the growth and metastasis of osteosarcoma. In this study, we hypothesized that humoral factors, which do not require direct contact with cells, are involved in the interaction between osteosarcoma and the surrounding cells. We identified the humoral factor involved in the association between tumor cells and surrounding normal cells using a co-culture model and investigated the significance of our findings. When human osteosarcoma cells (MG63) and human mesenchymal stem cells (hMSCs) were co-cultured and comprehensively analyzed for changes in each culture group, we found that the expression of chemokine (CC motif) ligand 26 (CCL26) was significantly enhanced. We also analyzed the changes in cell proliferation in co-culture, enhanced interaction with administration of recombinant CCL26 (rCCL26), reduced interaction with administration of anti-CCL26 antibodies, changes in invasive and metastatic abilities. CCL26 levels, motility, and invasive capability increased in the co-culture group and the group with added rCCL26, compared to the corresponding values in the MG63 single culture group. In the group with added CCL26 neutralizing antibodies, CCL26 level decreased in both the single and co-culture groups, and motility and invasive ability were also reduced. In a nude mice lung metastasis model, the number of lung metastases increased in the co-culture group and the group with added rCCL26, whereas the number of tumors were suppressed in the group with added neutralizing antibodies compared to those in the MG63 alone. This study identified a possible mechanism by which osteosarcoma cells altered the properties of normal cells to favorably change the microenvironment proximal to tumors and to promote distant metastasis.

Gait analysis of a patient who underwent complete resection of the patella and quadriceps femoris for soft tissue sarcoma.

The knee extension mechanism including the quadriceps femoris muscles and patella plays a crucial role in the stance phase of a normal gait cycle. We performed gait analysis of a patient who had undergone complete resection of the knee extension mechanism. An 8-month-old boy developed infantile fibrosarcoma of the right knee and underwent resection of the quadriceps femoris muscles, patella, and patellar tendon. The gait analysis performed at 8 years of age demonstrated that he could maintain the knee joint extension position during the stance phase. Increased muscle activities in the hamstring and gastrocnemius were observed. The results suggest that the hamstring and gastrocnemius muscles might play a role in maintaining the knee extension position during the stance phase. We suggest the importance of reinforcing these muscles in rehabilitation for patients who lost the knee extension mechanism.

Primary clear cell sarcoma of the femur: a unique case with RT-PCR and direct sequencing confirmation of EWSR1/ATF1 fusion gene.

BACKGROUND: It is very rare for clear cell sarcomas (CCS) to arise in the bone. During diagnosis, it is important to distinguish primary CCS of bone from bone metastasis of melanoma because this difference fundamentally changes the therapeutic options. Recently, characteristic fusion genes of CCS have been detected using reverse transcription polymerase chain reaction (RT-PCR) or direct sequencing which allowed to distinguish CCS from melanoma. However, there was no study applying these analyses with positive results. In this case, we describe the use of fusion gene analysis to diagnose a primary CCS of the bone. CASE PRESENTATION: A 36-year-old male presented with a four-months history of left knee pain. Magnetic resonance imaging showed a lesion in the left femoral medial epicondyle. Histological examination of the biopsy specimen revealed proliferating oval or rounded cells. These cells had clear cytoplasm arranged in fascicles or compact nests with frequent deposits of brown pigment. Furthermore, immunohistochemistry analysis revealed that tumor cells were positive for S-100 protein, HMB-45, Melan-A, and SOX10. It stained negative for CD34 and BRAF v600e. Conclusively, detection of the EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing confirmed that the lesion was a primary CCS of the bone. Wide-margin resection and reconstruction with a tumor endoprosthesis were performed. CONCLUSIONS: Herein, we diagnosed a rare case of primary CCS of the bone by detecting EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing. Since fluorescence-in situ hybridization (FISH) and RT-PCR could show false positive by mainly due to technical problems, it is better to perform direct sequencing to confidently diagnose the tumor as a primary CCS especially at very rare site such as bone.

Osteomyelitis of the Distal Phalanx of the Thumb due to Parvimonas micra and Fusobacterium nucleatum: A Case Report.

We herein report a case of osteomyelitis of the distal phalanx of the thumb of a 55-year-old man caused by Parvimonas micra and Fusobacterium nucleatum. Osteomyelitis often occurs in long bones and rarely occurs in the bones of the fingers. In addition, osteomyelitis of the finger frequently occurs after trauma or surgery, and blood-borne infection is very rare. P. micra and F. nucleatum, normal flora of the oral cavity, are very rare pathogenic bacteria of osteomyelitis except in periodontal disease, and there are no previous reports regarding the occurrence of osteomyelitis due to P. micra and F. nucleatum in the finger bones.

A meta-analytic evaluation of the correlation between event-free survival and overall survival in randomized controlled trials of newly diagnosed Ewing sarcoma.

BACKGROUND: In randomized controlled trials (RCTs) of adjuvant treatment for malignant tumors, event-free survival (EFS) is considered the most acceptable surrogate for overall survival (OS). However, even though EFS has repeatedly been selected as a primary endpoint in RCTs of Ewing sarcoma (ES), the surrogacy of EFS for OS has not been investigated. This study aimed to evaluate the correlation between EFS and OS in RCTs of chemotherapy for newly diagnosed ES using a meta-analytic approach. METHODS: We identified seven RCTs of newly diagnosed ES through a systematic review, and a meta-analysis was performed to evaluate the efficacy and adverse events associated with chemotherapy for previously untreated ES. The correlation between EFS and OS was investigated using weighted linear regression analysis and Spearman rank correlation coefficients (ρ). The strength of the correlation was evaluated using the coefficient of determination (R2). RESULTS: A total of 3612 patients were randomly assigned to 17 treatment arms in the eligible RCTs. The meta-analysis revealed that the hazard ratios for OS and EFS showed significantly better results in the experimental treatment groups with increasing toxicities. The correlation between the hazard ratios for EFS and OS was good (R2 = 0.747, ρ = 0.683), and the correlation tended to be more favorable in cases of localized ES (R2 = 0.818, ρ = 0.929). CONCLUSIONS: Overall, the trial-level correlation between EFS and OS was good for newly diagnosed ES and was very good in cases of localized disease. EFS may be a useful endpoint in RCTs of ES chemotherapy, and it is worth verifying using individual patient data.

Surrogate endpoints for overall survival in randomised controlled trials of localised osteosarcoma: A meta-analytic evaluation.

Event-free survival (EFS) is considered the most reliable surrogate endpoint for overall survival (OS) in randomised controlled trials (RCTs) of adjuvant therapies for malignant tumours. However, the surrogacy of intermediate endpoints such as EFS for OS in trials of patients with osteosarcoma has not been investigated to date. In this study, we investigated the correlation between OS and intermediate endpoints in RCTs of localised osteosarcoma. A systematic search identified 20 relevant RCTs. The correlations between the surrogate endpoints and OS were evaluated using weighted linear regression analyses and by calculating the Spearman rank correlation coefficients (ρ). The strength of the correlation was determined by calculating the coefficient of determination (R2). A total of 5,620 patients were randomly assigned to 45 treatment arms in the eligible 20 RCTs. The correlation between the hazard ratios for EFS and OS was moderate (R2 = 0.456, ρ = 0.440); this correlation tended to be weaker for patients with localised osteosarcoma excluding the patients with metastases. Overall, the trial-level correlation between the surrogate endpoints and OS was not robust in RCTs of osteosarcoma published to date. Hence, the suitability of the intermediate endpoints as surrogates for OS could not be confirmed.

Long-term disease control by trabectedin in a patient with dedifferentiated liposarcoma: A case report.

RATIONALE: Trabectedin is an antitumor drug considered to be effective for liposarcoma, leiomyosarcoma, and translocation-related sarcoma. Concerning liposarcoma subtypes, evidence of the efficacy of trabectedin for dedifferentiated liposarcoma (DDLPS) is poor, whereas it is known to have high efficacy against myxoid liposarcoma. Moreover, there are few reports of long-term trabectedin treatment of cases of DDLPS. Here, we present a case of advanced metastatic DDLPS that achieved long-term disease control by trabectedin treatment. PATIENT CONCERNS: A 68-year-old man presented with a mass in his back. Magnetic resonance imaging showed a tumorous mass in his back which exhibited low intensity on T1-weighted and high intensity on T2-weighted images. DIAGNOSIS: The mass was diagnosed as DDLPS by open biopsy. INTERVENTIONS: The patient underwent gemcitabine+docetaxel combination therapy followed by pazopanib and eribulin; all these therapies failed to halt disease progression. Subsequently, we changed the regimen to trabectedin as fourth-line therapy. OUTCOME: The patient achieved stable disease for approximately 18 months during 11 cycles of trabectedin therapy. LESSONS: Trabectedin should be considered as a treatment option for DDLPS even in cases of numerous failed prior chemotherapy regimens.

A meta-analysis of randomized controlled trials that compare standard doxorubicin with other first-line chemotherapies for advanced/metastatic soft tissue sarcomas.

OBJECTIVE: The standard treatment for patients with advanced/metastatic soft tissue sarcomas (ASTS) is systemic chemotherapy with doxorubicin. A previous meta-analysis of 8 randomized controlled trials (RCTs) demonstrated the superiority of single-agent doxorubicin over doxorubicin-based combination chemotherapy for ASTS. However, meta-analyses of all RCTs that compare doxorubicin to other single-agent or combination regimens as first-line treatments for ASTS are lacking. We conducted a systematic review and meta-analysis to evaluate the efficacy and toxicity of current primary treatments for ASTS. METHODS: Eligible studies were RCTs of first-line chemotherapies for ASTS comparing doxorubicin alone to other single agents or to combination therapies (experimental arm). Data from studies reporting hazard ratios (HR) and 95% confidence intervals (CI) for overall survival (OS) and progression-free survival (PFS) were pooled. Other time-to-event endpoints were extracted from the studies based on Kaplan-Meier estimates, and pooled odds ratios (OR) and 95% CI were calculated. RESULTS: Twenty-seven eligible RCTs comprising 6156 patients were identified. Overall, the 1-year OS (OR 0.88, 95% CI 0.79-0.99, P = 0.03) was significantly improved in the experimental arm over the doxorubicin-only arm; however, there was no significant difference in 2-year OS (OR 0.87, 95% CI 0.73-1.03, P = 0.11) or OS (HR 0.97, 95% CI 0.91-1.03, P = 0.28) between the two groups. PFS and other time-to-event endpoints were not significantly different between the two treatment arms. While incidences of overall severe adverse events were not significantly different (OR 1.20, 95% CI 0.88-1.65, P = 0.26), severe nausea/vomiting was significantly more frequent in the experimental arm (OR 1.90, 95% CI 1.27-2.83, P = 0.002). CONCLUSION: The efficacies of doxorubicin-only and experimental arm regimens were similar, although toxicities were more frequent in the experimental arms. Hence, doxorubicin monotherapy remains suitable as a standard first-line regimen for ASTS.

Surrogacy of intermediate endpoints for overall survival in randomized controlled trials of first-line treatment for advanced soft tissue sarcoma in the pre- and post-pazopanib era: a meta-analytic evaluation.

BACKGROUND: Overall survival is the true endpoint for most randomized controlled trials (RCTs) of malignant tumors, whereas progression-free survival (PFS) is considered the most reliable surrogate endpoint for overall survival (OS). The present study aimed to evaluate the correlation between surrogate endpoints and OS in randomized trials of first-line chemotherapy with doxorubicin (DOX), the standard treatment for advanced and metastatic soft tissue sarcomas (ASTS), using a meta-analytic approach. METHODS: In a systematic review, we identified RCTs of first-line chemotherapy for ASTS that compared single-agent doxorubicin (DOX) with other chemotherapy regimens, and were published in English during January 1974-December 2017. A meta-analysis was performed to evaluate the efficacy of first-line treatments for ASTS. Surrogacy of the intermediate endpoints for OS was investigated using weighted linear regression analysis. Correlation strength was examined using the coefficient of determination (R2). RESULTS: Twenty-seven randomized trials, comprising 6156 patients (3371 patients in the experimental arm and 2785 patients in the DOX arm) were identified. The hazard ratios for OS and PFS showed that the efficacy of treatment for ASTS was not significantly different between standard DOX and experimental treatments. The median OS was significantly prolonged in RCTs published after 2012 when pazopanib was approved for treating ASTS. The median PFS, however, did not differ significantly. The correlation between PFS and OS was moderate (R2 = 0.557), but better than that between OS and 3-month PFS, 6-month PFS, and response rate (R2 = 0.200, 0.073, and 0.278, respectively). The correlation between PFS and OS tended to be more favorable in RCTs published after 2012 (R2 = 0.586 and 0.459, respectively). CONCLUSIONS: The trial-level correlation between PFS and OS was only modest; it tended to be better in RCTs published after 2012. While the effective lines of chemotherapy and the introduction of new drugs prolonged OS but not PFS, PFS is a better surrogate than other intermediate endpoints in the first-line ASTS trials even in the post-pazopanib era. Although this does not negate the need for more reliable surrogate endpoints for OS.

Interaction between human osteosarcoma and mesenchymal stem cells via an interleukin-8 signaling loop in the tumor microenvironment.

BACKGROUND: Osteosarcoma (OS) is the representative primary malignant bone tumor with the highest incidence. It is known that malignant phenotypes of OS, such as proliferation, invasion, and metastasis, are significantly influenced not only by characteristics of the tumor itself, but also by the surrounding microenvironment. In other words, OS is considered to utilize cells in the vicinity of the tumor by changing the characteristics of these cells. Direct intercellular contact is believed to be important for this phenomenon. In the present study, we hypothesized that an interaction mediated by a humoral factor, requiring no cellular contact, might play a significant role in the progression of OS. METHODS: We developed a new co-culture model, using OS cells and mesenchymal stem cells (MSCs) without cellular contact, and found that both cell types expressed IL-8 at a high level, and FAK in OS cells was phosphorylated leading to an increase in the metastatic potential of the tumor in the co-culture condition. RESULTS: It was revealed that OS cells formed a loop of signal cross-talk in which they released IL-8 as a paracrine factor, stimulating MSCs to express IL-8, and received IL-8 released by MSCs to accelerate IL-8 expression in OS cells. Administration of anti-IL-8 antibody resulted in the inhibition of FAK expression, its downstream signaling, and the invasive potential of the OS cells, resulting in decrease in metastatic lesions. CONCLUSION: The present study might lead not only to the clarification of a new molecular mechanism of invasion and metastasis of OS, but also to the development of a new therapeutic strategy of blocking IL-8 in OS.

MicroRNA-181c prevents apoptosis by targeting of FAS receptor in Ewing's sarcoma cells.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that play important roles in multiple biological processes. Here, we show that miRNAs play an important function in the down-regulation of FAS expression in Ewing's sarcoma (ES) cells. METHODS: To identify and characterize possible oncogenic factors in ES, we employed a microarray-based approach to profile the changes in the expression of miRNAs and their target mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). RESULTS: MiRNA, miR-181c, was significantly up-regulated, whereas FAS receptor expression was significantly down-regulated in all tested ES cells compared with hMSCs. Introducing anti-miR-181c into ES cell lines resulted in an increased expression of FAS2. Additionally, anti-miR-181c prohibited cell growth and cell cycle progression in ES cells. Anti-miR-181c also promoted apoptosis in ES cells. Furthermore, the down-regulation of miR-181c in ES cells significantly suppressed tumor growth in vivo. CONCLUSIONS: These results suggest that unregulated expression of miR-181c could contribute to ES by targeting FAS. Reduction of miR181c increased expression of FAS. This proves that retardation of cell cycle progression removes apoptosis resistance, thereby repressing the growth of Ewing sarcoma. Since FAS signaling is involved in regulation of apoptosis and tumor proliferation, our findings might contribute to new therapeutic targets for ES.

MicroRNA-20b promotes cell proliferation via targeting of TGF-ß receptor II and upregulates MYC expression in Ewing's sarcoma cells.

Transforming growth factor-ß receptor II (TGFBR2) is implicated in various types of cancer. Most molecules involved in the TGF-ß pathway can be degraded by one or more microRNAs (miRNAs). In the present study, we show that miRNA plays an important role in downregulating TGFBR2 expression in Ewing's sarcoma (ES) cells. Microarray-based analyses revealed that the expression of miR-20b was significantly increased, whereas TGFBR2 and MYC were significantly downregulated and upregulated, respectively, in all ES cells compared to their expression in human mesenchymal stem cells (hMSCs). In ES cell lines, anti-miR-20b increased TGFBR2 expression and significantly decreased MYC expression, showing an inverse relationship with TGFBR2. The induction by anti-miR-20b further prohibited ES cell growth and cell cycle progression. Moreover, decreased miR-20b in ES cells significantly inhibited tumor growth in vivo. Taken together, these results suggest that miR-20b behaves as an oncogene in ES when its overexpression is unregulated by targeting TGFBR2. Because downstream TGFBR2 and TGF-ß signaling regulate cell cycle, apoptosis, and tumor proliferation via MYC, our findings may contribute to new targeted therapies for ES.

Dendritic cells combined with doxorubicin induces immunogenic cell death and exhibits antitumor effects for osteosarcoma.

The effects of dendritic cells (DCs) with low dose doxorubicin on the enhancement of the systemic immune response, including the effects on calreticulin (CRT) expression, heat shock protein 70 (HSP70) on the cell surface expression, and the enhancement of high mobility group box 1 (HMGB1) release from cancer cells, remain unclear. The present study investigated whether the combination of DCs and doxorubicin (ADM) induces immune cell death, and leads to tumor growth inhibition in a murine osteosarcoma model. To evaluate immune response activation in vivo, 4 groups of mice were established: i) untreated mice, ii) DC-treated mice, iii) ADM-treated mice, and iv) DC and ADM-treated mice. Immunological cell death and CRT, HSP70, and HMGB1 expression levels were higher in doxorubicin-treated cells than those in untreated or those treated with DCs alone. NF-κB expression was higher in the DCs after ligand activation using CRT, HSP70, or HMGB1 in vitro. Mice treated with DCs and ADM displayed an increased number of CD8+ T-lymphocytes within metastatic tumors and inhibition of metastatic growth. The expression of CRT and the release of HMGB1 from tumor tissues were increased in the ADM-treated groups. Treatment with DCs and ADM resulted in the highest serum interferon-γ levels. Combining ADM, which can induce immunogenic cell death, with DCs enhanced the systemic immune response. The findings of the present study provide further support for the continued development of antitumor agents that induce cell death and the immune response to target osteosarcoma.