Extending the benefits of tumor molecular profiling for all cancer patients requires a comprehensive analysis of tumor genomes across distinct patient populations worldwide. In this study, we perform deep next-generation DNA sequencing (NGS) from tumor tissues and matched blood specimens from over 10,000 patients in China by using a 450-gene comprehensive assay, developed and implemented under international clinical regulations. We perform a comprehensive comparison of somatically altered genes, the distribution of tumor mutational burden (TMB), gene fusion patterns, and the spectrum of various somatic alterations between Chinese and American patient populations. Here, we show 64% of cancers from Chinese patients in this study have clinically actionable genomic alterations, which may affect clinical decisions related to targeted therapy or immunotherapy. These findings describe the similarities and differences between tumors from Chinese and American patients, providing valuable information for personalized medicine.

Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N⁶-methyladenosine (m⁶A) modification plays a critical role in influencing RNA fate. However, the biological significance of m⁶A modification and its potential regulatory mechanisms in the development of OS remain unclear. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m⁶A levels. Western blotting, quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) were used to investigate METTL14 expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL14. RNA pull-down and RNA immunoprecipitation (RIP) assays were conducted to explore the specific binding of target genes and relevant m⁶A “readers”. RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of the downstream genes of METTL14. IHC and clinical data were applied to explore the clinical correlations of METTL14 and its downstream target genes with the prognosis of OS. We observed the abundance of m⁶A modifications in OS and revealed that METTL14 plays an oncogenic role in facilitating OS progression. MeRIP-seq and RNA-seq revealed that MN1 is a downstream gene of METTL14. MN1 contributes to tumor progression and all-trans-retinoic acid (ATRA) chemotherapy resistance in OS. Mechanistically, MN1 is methylated by METTL14, specifically in the coding sequence (CDS) regions, and this modification is recognized by the specific m⁶A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to prevent MN1 mRNA degradation and promote it translation efficiency. IHC showed that MN1 expression was positively correlated with METTL14 and IGF2BP2 expression in OS tissues. The METTL14-IGF2BP2-MN1 panel demonstrated more promising prognostic value for OS patients than any of these molecules individually. Our study revealed that METTL14 contributes to OS progression and ATRA resistance as an m⁶A RNA methylase by regulating the stability and translation efficiency of MN1 and thus provides both an underlying biomarker panel for prognosis prediction in OS patients. This work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864).

Osteosarcoma is the most common primary bone tumor in children and adolescents. Many patients with osteosarcoma always develop drug resistance to current chemotherapy regimens, which induces a poor prognosis. And cancer stem cells (CSCs) have been reported to possess the properties to self-renew and maintain the phenotype of tumor, which may lead to clinical treatment failure. Thus, it is an urgent task to develop several potentially useful therapeutic agents, which could target CSCs in osteosarcoma. This study aims to clarify the in vitro and in vivo anti-osteosarcoma effects of dioscin, the primary component derived from Discorea nipponica Makino, and its molecular mechanism of action. In this study, all the ten human osteosarcoma cell lines were sensitive to dioscin treatment in a dose- and time-dependent manner. Dioscin inhibits proliferation and induces cell cycle arrest as well as apoptotic cell death in osteosarcoma cells. More importantly, oral administration of dioscin (60 mg/kg) showed significant therapeutic effect on osteosarcoma growth without obvious side effects in vivo. In addition, dioscin possesses the ability to suppress stem-cell-like phenotype of osteosarcoma cells. Mechanistically, dioscin inhibits osteosarcoma stem-cell-like properties and tumor growth through repression of Akt/GSK3/β-catenin pathway. Moreover, β-catenin expression in osteosarcoma patients was associated with clinical prognosis. Conclusively, the present study provides comprehensive evidence for the inhibition of dioscin on osteosarcoma stem-cell-like properties and tumor growth through repression of Akt/GSK3/β-catenin pathway, which suggests dioscin as a promising therapeutic regimen. And β-catenin may be a potential therapeutic target as well as a significant prognostic marker for osteosarcoma patients in clinic.

Purpose: Agents extracted from natural sources with antitumor property have attracted considerable attention from researchers and clinicians because of their safety, efficacy, and immediate availability. Degalactotigonin (DGT), extracted from Solanum nigrum L., has anticancer properties without serious side effects. Here, we explored whether DGT can inhibit the growth and metastasis of osteosarcoma. Experimental Design: MTT, colony formation, and apoptosis assays were performed to analyze the effects of DGT on osteosarcoma cell viability in vitro. The migration and invasion abilities were measured using a Transwell assay. Animal models were used to assess the roles of DGT in both tumor growth and metastasis of osteosarcoma. Gli1 expression and function were measured in osteosarcoma cells and clinical samples. After DGT treatment, Gli1 activation and the phosphorylation status of multiple cellular kinases were measured with a luciferase reporter and phospho-kinase antibody array. Results: DGT inhibited proliferation, induced apoptosis, and suppressed migration and invasion in osteosarcoma cells. DGT, injected intraperitoneally after tumor inoculation, significantly decreased the volume of osteosarcoma xenografts and dramatically diminished the occurrence of osteosarcoma xenograft metastasis to the lungs. Mechanistically, DGT inhibited osteosarcoma growth and metastasis through repression of the Hedgehog/Gli1 pathway, which maintains malignant phenotypes and is involved in the prognosis of osteosarcoma patients. DGT decreased the activity of multiple intracellular kinases that affect the survival of osteosarcoma patients, including GSK3β. In addition, DGT represses the Hedgehog/Gli1 pathway mainly through GSK3β inactivation. Conclusions: Our studies provide evidence that DGT can suppress the growth and metastasis of human osteosarcoma through modulation of GSK3β inactivation–mediated repression of the Hedgehog/Gli1 pathway. Clin Cancer Res; 1–15. ©2017 AACR.

Background: Osteosarcoma is a malignant tumor with high ability to form invasion and metastasis. Identifying prognostic factor in osteosarcoma is helpful to select those patients for more aggressive management. Our study evaluated serum alkaline phosphatase (ALP) cooperating with matrix metalloproteinase-9 (MMP-9) as an important prognostic predictor for local recurrence and distant metastasis of osteosarcoma.Methods: 177 cases were included from the osteosarcoma patients treated at 1st Affiliated Hospital of Sun Yat-sen University (1999-2008). Pre-chemotherapy serum ALP (pre-ALP) were studied and correlated with tumor recurrence, lung metastasis and patient survival. MMP-9 protein in tumor tissues was detected by immunohistochemistry and correlated with pre-ALP level.Results: Pre-ALP were partitioned into normal, high, and very high groups, in each group the incidence of metastases was 12.2%, 21.2% and 34.6%, respectively (p = 0.007). In the three groups the mean disease-free survival (DFS) was 57 ± 3.15, 28 ± 3.57 and 14 ± 3.35 months, respectively (p < 0.001); overall survival (OS) was 92 ± 26.89, 39 ± 8.61 and 17 ± 5.07 months, respectively (p < 0.001). By multivariate analysis, elevated serum pre-ALP were associated with shorter DFS (p = 0.018) and OS (p = 0.031). If elevated ALP levels decreased after clinical treatment, the incidence of lung metastasis rate decreased (p = 0.028); DFS and OS were both prolonged (p < 0.001). Pre-ALP was also positively correlated with MMP-9 expression (p = 0.015) in tumor tissue.Conclusions: Pre-ALP was an independent prognostic factor for the survival of osteosarcoma patients in south China, and correlated with MMP-9 expression and lung metastasis. ALP can also serve as a prognostic marker for treatment, and merit large-scale validation studies.

Purpose: Malignancy of cancer cells depends on the active transcription of tumor-associated genes. Recently, unique clusters of transcriptional enhancers, termed super-enhancers, have been reported to drive the expression of genes that define cell identity. In this study we characterized specific super-enhancer-associated genes of osteosarcoma, and explored their potential therapeutic value. Experimental Design: Super-enhancer regions were characterized through chromatin immunoprecipitation sequencing (ChIP-Seq). RT-qPCR was used to detect the mRNA level of CDK7 in patient specimens and confirm the regulation of sensitive oncogenes by THZ2. The phosphorylation of the initiation-associated sites of RNA polymerase II (RNAP II) C-terminal repeat domain (CTD) was measured using Western blotting. Microarray expression analysis was conducted to explore transcriptional changes after THZ2 treatment. A variety of in vitro and in vivo assays were performed to assess the effects of CDK7 knockdown and THZ2 treatment in osteosarcoma. Results: Super-enhancers were associated with oncogenic transcripts and key genes encoding cell-type-specific transcription factors in osteosarcoma. Knockdown of transcription factor CDK7 reduced phosphorylation of the RNAPII CTD, and suppressed the growth and metastasis of osteosarcoma. A new specific CDK7 inhibitor, THZ2, suppressed cancer biology by inhibition of transcriptional activity. Compared with typical enhancers, osteosarcoma super-enhancer-associated oncogenes were particular vulnerable to this transcriptional disruption. THZ2 exhibited a powerful anti-osteosarcoma effect in vitro and in vivo. Conclusions: Super-enhancer-associated genes contribute to the malignant potential of osteosarcoma, and selectively targeting super-enhancer-associated oncogenes with the specific CDK7 inhibitor THZ2 might be a promising therapeutic strategy for patients with osteosarcoma.