Author Correction: Human somatic cell mutagenesis creates genetically tractable sarcomas.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cross-species genomics identifies DLG2 as a tumor suppressor in osteosarcoma.

Leveraging the conserved cancer genomes across mammals has the potential to transform driver gene discovery in orphan cancers. Here, we combine cross-species genomics with validation across human-dog-mouse systems to uncover a new bone tumor suppressor gene. Comparative genomics of spontaneous human and dog osteosarcomas (OS) expose Disks Large Homolog 2 (DLG2) as a tumor suppressor candidate. DLG2 copy number loss occurs in 42% of human and 56% of canine OS. Functional validation through pertinent human and canine OS DLG2-deficient cell lines identifies a regulatory role of DLG2 in cell division, migration and tumorigenesis. Moreover, osteoblast-specific deletion of Dlg2 in a clinically relevant genetically engineered mouse model leads to acceleration of OS development, establishing DLG2 as a critical determinant of OS. This widely applicable cross-species approach serves as a platform to expedite the search of cancer drivers in rare human malignancies, offering new targets for cancer therapy.

LDOC1 regulates Wnt5a expression and osteosarcoma cell metastasis and is correlated with the survival of osteosarcoma patients.

Osteosarcomas are common bone malignancies in children and adolescents. LDOC1 (leucine zipper, down-regulated in cancer 1), a tumor suppressor, is down-regulated in many cancers. In this study, we investigated the role of LDOC1 in tumor metastasis and its prognostic significance in osteosarcomas. We established osteosarcoma cells stably expressing LDOC1, driven by an HIV-based lentiviral system. We investigated the impact of LDOC1 on migration and invasion abilities in these cells using a transwell assay. LDOC1-associated changes in expression of metastasis-promoting genes were analyzed with a quantitative real-time polymerase chain reaction primer array. A xenograft tumor model (n = 7 mice/group) was used to assess the effect of LDOC1 on osteosarcoma metastasis in vivo. The overall survival and disease-free survival of osteosarcoma patients (n = 74) were analyzed retrospectively based on immunohistochemical analysis of LDOC1 levels in tumors and Kaplan-Meier analysis. LDOC1-expressing osteosarcoma cells displayed decreased migration and invasion in vitro. The quantitative real-time polymerase chain reaction primer array data showed that increased LDOC1 expression up-regulated many metastasis-suppressor genes. In the xenograft model, micro-computed tomography imaging data indicated that increased LDOC1 expression is associated with weaker lung metastasis ability. The Wnt5a signaling pathway promotes osteosarcoma metastasis; LDOC1 expression decreased Wnt5a levels in osteosarcoma cells. Kaplan-Meier analysis showed that higher LDOC1 expression was associated with improved osteosarcoma patient overall survival and disease free survival (p = 0.022). Our data show that LDOC1 is a tumor suppressor in osteosarcoma, and that it regulates metastasis of osteosarcoma cells. Furthermore, LDOC1 might be a valuable prognostic marker in osteosarcomas.

Human somatic cell mutagenesis creates genetically tractable sarcomas.

Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis.

Critical role of heat shock protein 27 in bufalin-induced apoptosis in human osteosarcomas: a proteomic-based research.

Bufalin is the primary component of the traditional Chinese herb "Chan Su". Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s) is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX) sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27), decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27) were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.

Glycogen synthase kinase-3ß, NF-κB signaling, and tumorigenesis of human osteosarcoma.

BACKGROUND: Glycogen synthase kinase-3ß (GSK-3ß), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3ß in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent. METHODS: We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3ß expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5-8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3ß in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3ß inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3ß activity with overall survival. RESULTS: Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3ß formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3ß had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3ß resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3ß resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3ß inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3ß, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3ß and NF-κB (109.2 months). CONCLUSION: GSK-3ß activity may promote osteosarcoma tumor growth, and therapeutic targeting of the GSK-3ß and/or NF-κB pathways may be an effective way to enhance the therapeutic activity of anticancer drugs against osteosarcoma.

Salinomycin inhibits osteosarcoma by targeting its tumor stem cells.

Osteosarcoma is the most common primary bone tumor in children and adolescents and is typically associated with a poor prognosis. Tumor stem cells (TSCs) are presumed to drive tumor initiation and tumor relapse or metastasis. Hence, the poor prognosis of osteosarcoma likely results from a failure to target the osteosarcoma stem cells. Here, we have utilized three different methods to enrich TSCs in osteosarcoma and further evaluated whether salinomycin could selectively target TSCs in osteosarcoma. Our results indicated that sarcosphere selection, chemotherapy selection and stem cell marker OCT4 or SOX2 over-expression are all effective in the enrichment of TSCs from osteosarcoma cell lines. Further investigation found that salinomycin inhibited osteosarcoma by selectively targeting its stem cells both in vitro and in vivo without severe side effects, and the Wnt/ß-catenin signaling pathway may be involved in this inhibition of salinomycin. Taken together, we have identified that salinomycin is an effective inhibitor of osteosarcoma stem cells, supporting the use of salinomycin for elimination of osteosarcoma stem cells and implying a need for further clinical evaluation.

Enrichment of osteosarcoma stem cells by chemotherapy.

Osteosarcoma is the most common primary malignant bone cancer in children and adolescents. Emerging evidence has suggested that the capability of a tumor to grow is driven by a small subset of cells within a tumor, termed cancer stem cells (CSCs). Although several methods have been explored to identify or enrich CSCs in osteosarcoma, these methods sometimes seem impractical, and chemotherapy enrichment for CSCs in osteosarcoma is rarely investigated. In the present study, we found that short exposure to chemotherapy could change the morphology of osteosarcoma cells and increase sarcosphere formation in vitro, as well as increase tumor formation in vivo. Furthermore, methotrexate (MTX)-resistant U2OS/MTX300 osteosarcoma cells were larger in size and grew much more tightly than parental U2OS cells. More importantly, U2OS/MTX300 cells possessed a higher potential to generate sarcospheres in serum-free conditions compared to parental U2OS cells. Also, U2OS/MTX300 cells exhibited the side population (SP) phenotype and expressed CSC surface markers CD117 and Stro-1. Notably, U2OS/MTX300 cells showed a substantially higher tumorigenicity in nude mice relative to U2OS cells. Therefore, we conclude that chemotherapy enrichment is a feasible and practical way to enrich osteosarcoma stem cells.

[Wide resection and brachytherapy management of extremity soft tissue sarcoma close to neurovascular bundle].

OBJECTIVE: With the extremity soft tissue sarcoma close to neurovascular bundle, combined en bloc resection and brachytherapy or simple en bloc resection were performed to evaluate the treatment outcome of the combined en bloc resection and brachytherapy. METHODS: Retrospectively investigation was performed for the extremity soft tissue sarcoma close to neurovascular bundle between 2000 and 2009. Inclusion criteria were primary extremity soft tissue sarcoma, MRI showed that the reaction zone involved the main neurovascular bundle, and the reaction zone closed less than 1 cm to the main neurovascular bundle. 86 cases were included in the study. There were 41 men and 45 women. The average age was 38.5 years old (Range from 15 to 73). There were malignant fibrous histiocytoma, synovial sarcoma, fibrosarcoma, liposarcoma, clear cell sarcoma, epithelioid sarcoma, leiomyosarcoma, rhabdomyosarcoma and vascular sarcoma etc. The stage were IA (8), IIA (12), IIB (10), IIC (7), III (43) and IV (6). RESULTS: During an average follow-up of 53 months (range 24 - 102 months), the distant metastasis rate 32.56% (28/86) and the lymph node metastasis rate was 6.98% (6/86). The local recurrence rates was 13.95% (12/86). In the group of combined en bloc resection and brachytherapy with 38 cases, the local recurrence rates was 5.26% (2/38). Four cases had wound infection and six cases had wound delay healing. The MSTS functional score was 21.11 ± 1.79. In the group of simple en bloc resection with 48 cases, the local recurrence rates was 20.83% (10/48). One case had wound infection and four cases had wound delay healing. The MSTS functional score was 84.23% (26.11 ± 1.79). The local recurrence rates was significant different between. CONCLUSION: With the extremity soft tissue sarcoma close to neurovascular bundle, combined en bloc resection and brachytherapy could decrease the local recurrence rate.