Poor Prognosis Of Osteosarcoma Patients Research Articles

Multi-omic validation of the cuproptosis-sphingolipid metabolism network: modulating the immune landscape in osteosarcoma.

The current understanding of the mechanisms by which metal ion metabolism promotes the progression and drug resistance of osteosarcoma remains incomplete. This study aims to elucidate the key roles and mechanisms of genes involved in cuproptosis-related sphingolipid metabolism (cuproptosis-SPGs) in regulating the immune landscape, tumor metastasis, and drug resistance in osteosarcoma cells. This study employed multi-omics approaches to assess the impact of cuproptosis-SPGs on the prognosis of osteosarcoma patients. Lasso regression analysis was utilized to construct a prognostic model, while multivariate regression analysis was applied to identify key core genes and generate risk coefficients for these genes, thereby calculating a risk score for each osteosarcoma patient. Patients were then stratified into high-risk and low-risk groups based on their risk scores. The ESTIMATE and CIBERSORT algorithms were used to analyze the level of immune cell infiltration within these risk groups to construct the immune landscape. Single-cell analysis was conducted to provide a more precise depiction of the expression patterns of cuproptosis-SPGs among immune cell subtypes. Finally, experiments on osteosarcoma cells were performed to validate the role of the cuproptosis-sphingolipid signaling network in regulating cell migration and apoptosis. In this study, seven cuproptosis-SPGs were identified and used to construct a prognostic model for osteosarcoma patients. In addition to predicting survival, the model also demonstrated reliability in forecasting the response to chemotherapy drugs. The results showed that a high cuproptosis-sphingolipid metabolism score was closely associated with reduced CD8 T cell infiltration and indicated poor prognosis in osteosarcoma patients. Cellular functional assays revealed that cuproptosis-SPGs regulated the LC3B/ERK signaling pathway, thereby triggering cell death and impairing migration capabilities in osteosarcoma cells. The impact of cuproptosis-related sphingolipid metabolism on the survival and migration of osteosarcoma cells, as well as on CD8 T cell infiltration, highlights the potential of targeting copper ion metabolism as a promising strategy for osteosarcoma patients.

Inhibition of miR-9 Combined With Cisplatin Targeting APE1 Against Angiogenesis in Osteosarcoma.

Osteosarcoma (OS) is a highly malignant tumor, and chemotherapy resistance suggests poor prognosis in OS patients. In this study, the authors discovered that miR-9 has a pro-angiogenic role in OS. The anti-angiogenic effects of cisplatin were greatly increased when miR-9 was suppressed in OS. In addition, the authors demonstrated that miR-9 plays a pro-angiogenic role by targeting apoptosis-inducing factor 1 (APE1) in OS. Importantly, our in vivo experiments showed that inhibition of miR-9 combined with cisplatin could suppress xenograft tumor growth by targeting APE1 and decreasing angiogenesis in OS. In summary, our results suggest that miR-9 plays a role as a tumor promoter, and inhibiting miR-9 and APE1 is a new strategy for inhibiting OS angiogenesis and chemotherapy resistance.

USP22 as a key regulator of glycolysis pathway in osteosarcoma: insights from bioinformatics and experimental approaches.

Osteosarcoma is the most common primary malignant bone tumor, but its pathogenesis remains unclear. Ubiquitin-specific processing peptidase 22 (USP22) is reported to be highly expressed and associated with tumor malignancy and prognosis in cancers. However, the role and mechanism of USP22 in osteosarcoma is not fully understood. This study aims to investigate the function and potential mechanism of USP22 in osteosarcoma using bioinformatics analysis combined with experimental validation. We first integrated transcriptomic datasets and clinical information of osteosarcoma from GEO and TCGA databases to assess the expression and prognostic value of USP22 in osteosarcoma. Then, differential expression analysis and weighted gene co-expression network analysis (WGCNA) were conducted to identify USP22-related co-expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore the biological functions and signaling pathways of USP22 co-expressed genes. To validate the accuracy of bioinformatics analyses, we downregulated USP22 expression in osteosarcoma cell line Sao-2 using siRNA and assessed its effect on cell proliferation, migration, invasion, apoptosis, and regulation of key signaling pathways. We found that USP22 was highly expressed in osteosarcoma tissues and correlated with poor prognosis in osteosarcoma patients. USP22 also showed potential as a diagnostic marker for osteosarcoma. In addition, 344 USP22-related co-expressed genes were identified, mainly involved in signaling pathways such as glycolysis, oxidative phosphorylation, spliceosome, thermogenesis, and cell cycle. The in vitro experiments confirmed the accuracy and reliability of bioinformatics analyses. We found that downregulation of USP22 could inhibit Sao-2 cell proliferation, migration, invasion, and induce apoptosis. Furthermore, downregulation of USP22 significantly reduced aerobic glycolysis levels in Sao-2 cells and inhibited the expression of key enzymes and transporters in aerobic glycolysis pathways such as HK2, PKM2, and GLUT1. USP22 plays a critical role in the occurrence, development, and prognosis of osteosarcoma. USP22 could influence Sao-2 cell proliferation, apoptosis, migration, and invasion by regulating the glycolysis pathway, thereby promoting osteosarcoma progression. Therefore, USP22 may be a potential therapeutic target for the treatment of osteosarcoma.

Abstract 1394: The CXCL10-CXCR3 axis as Jekyll and Hyde: A regulator of metastasis and immune response in osteosarcoma

Abstract The CXCL10-CXCR3 axis is known to promote tumor growth and metastasis via autocrine signaling, while it can also elicit anti-tumor responses by paracrine signaling. However, its roles are still elusive in osteosarcoma (OS), the most common malignant bone tumor in children. In this study, we utilized in vitro assays, mouse models, and gene expression analysis to characterize the roles of the CXCL10-CXCR3 axis in OS. To understand the autocrine signaling, we performed in vitro phenotypic assays on three OS cell lines with and without CXCL10. The results showed that the chemokine increased AKT phosphorylation and tumor cell migration. Using a CRISPR-based CXCR3 deletion mutant, we demonstrated that the lack of the receptor inhibited OS tumor growth and pulmonary metastasis in an orthotopic xenograft mouse model. The results indicate that the CXCL10-CXCR3 axis is sufficient and necessary to promote aggressive phenotypes in OS via autocrine signaling. Next, using gene expression datasets from two cohorts of OS patients, we showed that high expression of CXCL10 or its cognate receptor CXCR3 was associated with a better prognosis. Since CXCL10 is known to recruit CXCR3+ immune cells to fight against cancer, we further found that the expression of the T cell marker CD3D was associated with a better prognosis. These results suggest that the chemokine may also play a protective role in osteosarcoma by recruiting anti-tumor immune cells via paracrine signaling. Lastly, we have previously reported that a high circulating level of CXCL10 correlates with a poor prognosis in osteosarcoma patients. In this study, we demonstrated that increasing the circulating CXCL10 level in immunodeficient orthotopic xenograft mouse models of OS did not significantly promote the development of pulmonary metastases, suggesting circulating CXCL10 may exert its chemotactic effect mainly on immune cells, not on tumor cells. Taken together, we propose a model that tumor expression of CXCL10 promotes osteosarcoma growth and metastasis development via autocrine signaling. The increase of the local level of CXCL10 attracts CXCR3+ immune cells to the tumor site and promotes anti-tumor function via paracrine signaling. Metastatic cells colonize the lungs and cause tissue damage and inflammation, which increases the production of CXCL10 in the circulation. The higher circulating CXCL10 recruits T cells away from the primary tumor site and indicates the occurrence of metastasis and, hence, correlates with a worse outcome in patients. Our research highlights the importance of understanding the opposite effects of CXCL10 on tumor and immune cells as well as in the tumor microenvironment and circulation. Identification of the mechanistic differences in the CXCL10-CXCR3 signaling between tumor and immune cells will provide a novel therapeutic approach against tumor cells while promoting anti-tumor immune response in OS. Citation Format: Benjamin B. Gyau, Xiang Chen, Junyan Wang, Margaret A. Clement, Angela M. Major, Jun Xu, M. John Hicks, Tsz-Kwong Man. The CXCL10-CXCR3 axis as Jekyll and Hyde: A regulator of metastasis and immune response in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1394.

What role does GPR65 play in the progression of osteosarcoma? Its mechanism and clinical significance

BackgroundGPR65 is a pH-sensing G-protein-coupled receptor that acts as a key innate immune checkpoint in the human tumor microenvironment, inhibiting the release of inflammatory factors and inducing significant upregulation of tissue repair genes. However, the expression pattern and function of GPR65 in osteosarcoma (OS) remain unclear. The purpose of this study was to investigate and elucidate the role of GPR65 in the microenvironment, proliferation and migration of OS.MethodsRetrospective RNA-seq data analysis was conducted in a cohort of 97 patients with OS data in the TAEGET database. In addition, single-cell sequencing data from six surgical specimens of human OS patients was used to analyze the molecular evolution process during OS genesis. Tissues chips and bioinformatics results were used to verify GPR65 expression level in OS. MTT, colony formation, EdU assay, wound healing, transwell assay and F-actin assay were utilized to analyze cell proliferation and invasion of OS cancer cells. RNA-seq was used to explore the potential mechanism of GPR65’s role in OS.ResultsGPR65 expression was significantly low in OS, and subgroup analysis found that younger OS patients, OS patients in metastatic status, and overall survival and progression free survival OS patients had lower GPR65 expression. From ScRNA-seq data of GSE162454, we found the expression of GPR65 is significantly positively correlated with CD4 + T cells CD8 + T cells and OS related macrophage infiltration. Verification experiment found that silencing the expression of GPR65 in osteosarcoma cells U2OS and HOS could promote the proliferation and invasion process, RNA-seq results showed that the role of GPR65 in OS cells was related to immune system, metabolism and signal transduction.ConclusionThe low expression of GPR65 in OS leads to high metastasis rate and poor prognosis in OS patients. The suppression of immune escape and inhibition of proliferation may be a key pathway for GPR65 to participate in the progression of OS. The current study strengthens the role of GPR65 in OS development and provides a potential biomarker for the prognosis of OS patients.

Circular RNA CircSATB2 facilitates osteosarcoma progression through regulating the miR-661/FUS-mediated mRNA of ZNFX1

Circular RNAs (circRNAs) are a class of non-coding RNAs which take part in the regulation of the initiation and development of different types of cancer. Numerous studies have demonstrated that circRNAs are involved in the progression of osteosarcoma (OS) as well. Thus, we put our emphasis on the exploration of crucial circRNAs in the process of OS initiation and progression. Using RNA sequencing, we found that circSATB2 was highly expressed in OS tissues compared with adjacent normal tissues. Then, we confirmed the high expression of circSATB2 in OS cell lines and OS tissues and its high expression was related to poor prognosis of OS patients. Functional experiments exhibited that circSATB2 promoted OS proliferation and migration in vitro, primary OS model and OS lung metastasis model showed that circSATB2 aggravated OS progression in vivo. Mechanistically, circSATB2 was found to promote OS progression through sponging miR-661 and FUS regulating the mRNA of ZNFX1. Therefore, circSATB2 could act as a prognostic marker and a therapeutic target for osteosarcoma in the future.

METTL16 promotes osteosarcoma progression by downregulating VPS33B in an m6 A-dependent manner.

N6-methyladenosine (m6 A) is one of the main epitranscriptomic modifications that accelerates the progression of malignant tumors by modifying RNA. Methyltransferase-like 16 (METTL16) is a newly identified methyltransferase that has been found to play an important oncogenic role in a few malignancies; however, its function in osteosarcoma (OS) remains unclear. In this study, METTL16 was found to be upregulated in OS tissues, and associated with poor prognosis in OS patients. Functionally, METTL16 substantially promoted OS cell proliferation, migration, and invasion in vitro and OS growth in vivo. Mechanistically, vacuolar protein sorting protein 33b (VPS33B) was identified as the downstream target of METTL16, which induced m6 A modification of VPS33B and impaired the stability of the VPS33B transcript, thereby degrading VPS33B. In addition, VPS33B was found to be downregulated in OS tissues, VPS33B knockdown markedly attenuated shMETTL16-mediated inhibition on OS progression. Finally, METTL16/VPS33B might facilitate OS progression through PI3K/AKT pathway. In summary, this study revealed an important role for the METTL16-mediated m6 A modification in OS progression, implying it as a promising target for OS treatment.

Deubiquitinating enzyme USP10 promotes osteosarcoma metastasis and epithelial-mesenchymal transition by stabilizing YAP1.

Osteosarcoma (OS) is a fatal adolescent tumor, which is susceptible to remote metastases at an early stage, and its treatment remains a major challenge. ubiquitin-specific protease 10 (USP10) is primarily located in the cytoplasm and can therefore deubiquitinate various cytoplasmic proteins. However, the expression and mechanism of USP10 in OS remain ambiguous. The aim of this study was to explore how USP10 affects Yes-associated protein1 (YAP1) to influence the metastasis and epithelial-mesenchymal transition (EMT). Western blotting, qRT-PCR, and immunohistochemical (IHC) analyses were performed to evaluate USP10 and YAP1 levels. Using wound healing and transwell tests, the roles and molecular pathways of USP10 and YAP1 ability to migrate and invade of OS were investigated, and cell morphological alterations were examined using phalloidin staining. Our results indicated that USP10, a new type of deubiquitinating protease, is increased in OS tissues and cells contrasted with adjacent healthy tissues. Overexpression of USP10 correlated with tumor size, distant metastasis, and TNM stage, and was an independent factor of poor prognosis in OS patients. Also, USP10 expression is closely connected with the incident of OS metastasis and tumor size. Functional assays revealed that USP10 knockdown suppressed cell migrating and invading ability and inhibited the EMT of OS cells in vivo and in vitro. In addition, we showed that USP10 knockdown decreased the levels of YAP1, which is an important positive regulator of migration and invasion in many cancers. We also found a significant positive correlation between USP10 and YAP1 levels, further demonstrating that USP10-induced migration and EMT are based on YAP1 in OS cells. In a mechanistic way, USP10 stabilizes the expression of YAP1 by mediating its deubiquitination in OS cells. Together, this study showed that USP10 can directly interact with YAP1 to reduce ubiquitinated YAP1, thereby stabilizing its protein levels and affecting EMT and distant metastasis in OS cells.

Far upstream element-binding protein 1 confers lobaplatin resistance by transcriptionally activating PTGES and facilitating the arachidonic acid metabolic pathway in osteosarcoma.

Drug resistance is a major obstacle in cancer treatment and recurrence prevention and leads to poor outcomes in patients suffering from osteosarcoma. Clarification of the mechanism of drug resistance and exploration of effective strategies to overcome this obstacle could lead to clinical benefits for these patients. The expression of far upstream element-binding protein 1 (FUBP1) was found to be markedly elevated in osteosarcoma cell lines and clinical specimens compared with osteoblast cells and normal bone specimens. High expression of FUBP1 was correlated with a more aggressive phenotype and a poor prognosis in osteosarcoma patients. We found that overexpression of FUBP1 confers lobaplatin resistance, whereas the inhibition of FUBP1 sensitizes osteosarcoma cells to lobaplatin-induced cytotoxicity both in vivo and in vitro. Chromatin immunoprecipitation-seq and RNA-seq were performed to explore the potential mechanism. It was revealed that FUBP1 could regulate the transcription of prostaglandin E synthase (PTGES) and subsequently activate the arachidonic acid (AA) metabolic pathway, which leads to resistance to lobaplatin. Our investigation provides evidence that FUBP1 is a potential therapeutic target for osteosarcoma patients. Targeting FUBP1, its downstream target PTGES and the AA metabolic pathway may be promising strategies for sensitizing chemoresistant osteosarcoma cells to lobaplatin.

Abstract 1307: The effect of circulating CXCL10 on osteosarcoma metastasis using orthotopic xenograft mouse models

Abstract Osteosarcoma is the most common malignant bone tumor in children. Despite the use of chemotherapy and surgery, the survival of patients with metastasis is still dismal. We have previously reported that a high serum level of CXCL10 is associated with a poor prognosis and metastasis in osteosarcoma, suggesting that the chemokine may affect the development of metastasis. Nonetheless, the effect of endocrine signaling of CXCL10 is largely unknown. It is unclear if the prognostic effect of circulating CXCL10 is due to its metastasis-promoting effect or if CXCL10 is merely a biomarker. In this study, we employed orthotopic xenograft mouse models to examine the effect of circulating CXCL10 with respect to metastasis. We first injected luciferase-labeled 143B osteosarcoma cells into the tibia of NOD/SCID mice to generate an orthotopic xenograft mouse model. When the tumor was detectable by bioluminescence, we injected 1.5ug of CXCL10 protein or saline intravenously via the tail vein every day for two weeks. The development of pulmonary metastasis was monitored by bioluminescence. The mice were sacrificed at the endpoint when bioluminescence was detected in the lungs. The mouse lungs were then resected and examined by H&E staining for the detection of metastatic nodules. The mice in the experimental and control groups that developed lung metastases were compared. The results showed that no significant difference was observed between the two groups. The experiment was repeated one more time and similar results were observed. To investigate the influence of the cell line background, we analyzed another osteosarcoma cell line, MG63.3. No significant difference in metastasis development was observed between the mice injected with CXCL10 and the mice injected with saline. The results indicate that a higher level of circulating CXCL10 alone was not sufficient to promote pulmonary metastases in the orthotopic xenograft mouse models. We also showed that AKT phosphorylation was upregulated in the osteosarcoma cell lines incubated with CXCL10, ruling out that the CXCL10 protein used was biologically inactive. In summary, our results suggest that although an increased circulating level of CXCL10 is associated with a poor prognosis in osteosarcoma patients, it does not promote the development of pulmonary metastasis in xenograft mouse models. Circulating CXCL10 may only act as a prognostic biomarker, but not a metastatic driver. Alternatively, the CXCL10-CXCR3 axis may be necessary, but not sufficient, to cause metastasis. Citation Format: Xiang Chen, Margaret Clement, John Hicks, Tsz Kwong Man. The effect of circulating CXCL10 on osteosarcoma metastasis using orthotopic xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1307.

Abstract 6166: TBL1, a multifunctional transcriptional regulator, is highly expressed in osteosarcoma and correlates with activated beta catenin

Abstract Background: Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Two-thirds of OS patients are cured through chemotherapy and tumor resection. However, survival rates have plateaued over the past 30 years, especially for patients with relapsed and/or metastatic disease, with overall long-term outcomes of less than 30%. Transducin beta-like protein 1 (TBL1) protects beta-catenin from nuclear degradation and mediates Wnt targeted transcription by forming a TBL1-beta-catenin complex. Overexpression of TBL1 and nuclear beta-catenin are positively correlated to adverse clinicopathological features and poor prognosis for osteosarcoma patients. Functional experiments reveal that down-regulation of TBL1 and nuclear beta-catenin activity inhibit proliferation, migration, and invasion of osteosarcoma cells. The objective of this study was to determine TBL1 levels within a panel of OS patient-derived xenograft (PDX) tumors. Methods: TBL1 and beta-catenin protein levels were measured via western blot and immunohistochemistry in OS samples. We examined protein levels in OS PDX cell lines collected at distinct clinical stages of metastatic OS and OS primary tumor samples. TBL1, total beta-catenin, and activated beta-catenin levels were measured by immunohistochemical (IHC) staining in tissue microarray (TMA) derived from patient derived xenograft (PDX) tumor and clinical samples. Each tumor sample were formalin fixed, paraffin embedded, and spotted in triplicate on the TMA. Results: Beta-catenin and TBL1 protein were detected in OS samples, and we found higher TBL1 protein levels in cells derived from advanced disease stages. Assessment of TBL1 protein was expanded to OS tissue microarray where TBL1, total beta-catenin, and active beta-catenin were independently stained in a TMA. TBL1, total beta-catenin, and activated beta-catenin were present in 100% of OS PDX cores. Finally, a majority of OS cores were shown to have high expression for both TBL1 and active beta-catenin. Conclusion: Elevated TBL1 levels have been associated with metastatic disease and poor survival. We have measured TBL1 and activated beta-catenin protein levels in a variety of osteosarcoma samples and identified a large percentage of osteosarcoma that co-express both proteins. These results indicate that metastatic OS represents a TBL1 enriched patient population and is a candidate for therapeutics that target TBL1. Citation Format: Kimberly R. Holloway, Taku Yamamichi, Bikesh Nirala, Nino Rainusso, Jason T. Yustein, Stephen Horrigan. TBL1, a multifunctional transcriptional regulator, is highly expressed in osteosarcoma and correlates with activated beta catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6166.

Circ_0001060 Upregulates and Encourages Progression in Osteosarcoma

Circular RNA (circRNA) is involved in the occurrence and development of various cancers. To this day, the expression and mechanism of circRNA in osteosarcoma (OS) remain unclear. We previously found that circ_0001060 was highly expressed in OS tumor tissues. In this work, we identified that high level expression of circ_0001060 was significantly associated with late clinical stage, larger tumor volume, higher frequency of metastasis, and poor prognosis in OS patients. Furthermore, we confirmed that silencing circ_0001060 inhibited the proliferation and migration of OS cell. Using bioinformatics analysis, we built three circRNA-miRNA-mRNA regulatory modules (circ_0001060-miR-203a-5p-TRIM21, circ_0001060-miR-208b-5p-MAP3K5, and circ_0001060-miR-203a-5p-PRKX), suggesting that these signaling axes may be involved in the inhibitory effect of circ_0001060 on OS. To sum up, circ_0001060 is a novel tumor biomarker for OS as well as a potential therapeutic target.

Indoleamine 2,3-Dioxygenase: A Novel Immunotherapeutic Target for Osteosarcoma.

Tumour-emitted molecules induce immunosuppression in the tumour microenvironment. An immunosuppressive enzyme, indoleamine 2,3-dioxygenase (IDO/IDO1), facilitates immune escape in several malignant tumours, including osteosarcoma. Upregulation of IDO establishes a tolerogenic environment in the tumour and the tumour-draining lymph nodes. IDO-induced downregulation of effector T-cells and upregulation of local regulatory T-cells creates immunosuppression and promotes metastasis. Osteosarcoma is the most common bone tumour characterised by immature bone formation by the tumour cells. Almost 20% of osteosarcoma patients present with pulmonary metastasis at the time of diagnosis. The improvement in therapeutic modalities for osteosarcoma has been in a stagnant phase for two decades. Therefore, the development of novel immunotherapeutic targets for osteosarcoma is emergent. High IDO expression is associated with metastasis and poor prognosis in osteosarcoma patients. At present, only a few studies are available describing IDO's role in osteosarcoma. This review describes the prospects of IDO not only as a prognostic marker but also as an immunotherapeutic target for osteosarcoma.

Activation of PGRN/MAPK axis stimulated by the hypoxia-conditioned mesenchymal stem cell-derived HIF-1α facilitates osteosarcoma progression

BackgroundProgranulin (PGRN) is an important survival factor in the progression of multiple cancers. PurposeTo explore the effects and mechanisms of PGRN on malignant biological behavior of osteosarcoma (OS) cells and the effects of mesenchymal stem cells (MSCs) and the hypoxic microenvironment on PGRN alteration. Material and methodsThe expression pattern of PGRN in OS were evaluated in OS tissues and cell lines. Next, a loss-of-function assay investigated the function of PGRN on the proliferation, migration and cell death of OS cells. The activation of MAPK signaling in the process was examined by western blot and functional experiments accompanied by skatole. Additionally, we internally silenced hypoxia-inducible factor-1α (HIF-1α) in MSCs along with exogenously added HIF-1α (exo-HIF-1α) to explore how MSCs affect PGRN alteration and the malignant behavior of OS cells. ResultsAn aberrantly high expression of PGRN was observed in OS and associated with the poor prognosis of OS patients. PGRN knockdown repressed the proliferation, migration and induced cell death of OS cells, and activating MAPK pathway reversed these effects. Further evidence showed that MSCs regulated PGRN to mediate the malignant biological behavior of OS cells. Hypoxia enhanced HIF-1α expression in MSCs. HIF-1α silencing in MSCs under hypoxia suppressed the oncogenic effects of MSCs and reduced PGRN expression in OS cells, while the treatment of exo-HIF-1α reversed the depressive effects of HIF1α silencing on OS progression. ConclusionOverall, we concluded that PGRN, which was activated by the increase of hypoxic-MSCs-derived HIF-1α, promoted OS progression through the activation of MAPK signaling.

N6-methyladenosine reader YTHDF3 contributes to the aerobic glycolysis of osteosarcoma through stabilizing PGK1 stability.

N6-methyladenosine (m6A) modification is a pivotal transcript chemical modification of eukaryotics, which has been identified to play critical roles on tumor metabolic reprogramming. However, the functions of m6A-reading protein YTH N6-methyladenosine RNA-binding protein 3 (YTHDF3) in osteosarcoma is still unclear. This research planned to investigate the bio-functions and mechanism in osteosarcoma tumorigenesis. The aerobic glycolysis of osteosarcomacells were calculated byglucose uptake,lactate production analysis, ATP analysis andmetabolic flux analysis for extracellular acidification rate (ECAR).Molecular binding was identified byRIP-qPCR,RNA decay analysis. Results indicated that YTHDF3 is upregulated in the osteosarcoma tissue samples and cells, and closely correlated to the poor prognosis of osteosarcoma patients. Functionally, gain and loss-of-functional assays illustrated that YTHDF3 promoted the proliferation and aerobic glycolysis of osteosarcoma cells in vitro, and accelerated the tumor growth in vivo. Mechanistically, a m6A-modified PGK1 mRNA functioned as the target of YTHDF3, and YTHDF3 enhanced the PGK1 mRNA stability via m6A-dependent manner. In conclusion, these findings indicated that YTHDF3 functioned as an oncogene in osteosarcoma tumorigenesis through m6A/PGK1 manner, providing a therapeutic strategy for human osteosarcoma.

Hydroxysteroid Dehydrogenase Like 2 Promotes the Invasion and Migration of Osteosarcoma by Regulating Fibroblast Growth Factor Receptor 4

We aimed to explore the expression characteristics of HSDL2 in osteosarcoma (OS) as well as the underlying mechanism. A total of 42 OS patients’ tissue samples were collected. HSDL2 level was markedly higher in OS tissues as well as OS cell lines. Besides, patients with high HSDL2 expression had a higher incidence of distant metastasis and a lower overall survival rate. Furthermore, the ability of OS cells to proliferate, invade and metastasize was markedly reduced after HSDL2 knockdown; however, the overexpression of HSDL2 could markedly increase the proliferative, invasive and metastasis ability of OS cells. In addition, HSDL2 can target FGFR4. Furthermore, FGFR4 expression was markedly decreased after HSDL2 knockdown, besides, HSDL2 and FGFR4 expressions were positively correlated in OS tissues. In addition, the recovery experiments suggested that HSDL2 and FGFR4 had a mutual regulation, thereby jointly promoting the invasive and migration ability of OS. HSDL2 expression was markedly increased in OS tissues as well as OS cell lines, which was markedly related to distant metastasis along with poor prognosis of OS patients. Besides, HSDL2 may promote OS progression by increasing the FGFR4 expression.

The long non-coding RNA CRNDE promotes osteosarcoma proliferation and migration by sponging miR-136-5p/MRP9 axis.

BackgroundThe long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been found to be upregulated in several solid tumors. Whether CRNDE affects osteosarcoma (OS) and its underling mechanism remains unknown.MethodsTumor tissues and corresponding normal tissues were collected from 45 patients with OS. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied to determine lncRNA CRNDE level in the tissues. Participants were divided into a high CRNDE group and a low CRNDE group according to the median value of lncRNA CRNDE expression detected by in situ hybridization (ISH). The differences between high and low expression of lncRNA CRNDE in patients were compared clinically by chi-square test. Kaplan-Meier survival analysis was applied to analyze the relationship between lncRNA CRNDE expression and patient survival. Subsequently, silencing or overexpression of lncRNA CRNDE were performed in MG63 and 143B cell lines, qRT-PCR was applied to verify the expression of lncRNA CRNDE, miR-136-5p, and MRP9; dual-luciferase reporter assay was used to evaluate the targeting relationship between miR-136-5p, lncRNA CRNDE, and Cell Counting Kit-8 (CCK8), wound-healing, and Transwell assays were used to analyze for cell proliferation, migration, and invasion, respectively, and western blot was used to detect expression in cells.ResultsThe expression of CRNDE in OS tissues was higher than that in normal tissues. High lncRNA CRNDE expression was significantly associated with clinical stage, lung metastasis, and poor prognosis in OS patients. Additionally, overexpression of lncRNA CRNDE promoted proliferation and migration of OS cells. Bioinformatics analysis showed that lncRNA CRNDE competitively inhibited miR-136-5p through acting as a competitive endogenous RNA (ceRNA). It was also revealed that miR-136-5p is a binding target gene of lncRNA CRNDE and that MRP9 is involved in this process as a downstream target gene of miR-136-5p.ConclusionsThe lncRNA CRNDE promotes the proliferation and migration of OS cells by regulating the miR-136-5p/MRP9 pathway, and lncRNA CRNDE can be a significant marker of OS prognosis.

CEMIP Promotes Osteosarcoma Progression and Metastasis Through Activating Notch Signaling Pathway.

Cell migration inducing protein (CEMIP) has been linked to carcinogenesis in several types of cancers. However, the role and mechanism of CEMIP in osteosarcoma remain unclear. This study investigated the role of CEMIP in the progression and metastasis of osteosarcoma, CEMIP was found to be overexpressed in osteosarcoma tissues when compared to adjacent non-tumor tissues, and its expression was positively associated with a poor prognosis in osteosarcoma patients. Silencing CEMIP decreased osteosarcoma cells proliferation, migration, and invasion, but enhanced apoptosis in vitro, and suppressed tumor growth and metastasis in vivo. Mechanistically, CEMIP promoted osteosarcoma cells growth and metastasis through activating Notch signaling pathway, silencing CEMIP would reduce the protein expression and activation of Notch/Jagged1/Hes1 signaling pathway in vitro and in vivo, activation of Notch signaling pathway could partially reversed cell proliferation and migration in shCEMIP osteosarcoma cells. In conclusion, our study demonstrated that CEMIP plays a substantial role in the progression of osteosarcoma via Notch signaling pathway, providing a promising therapeutic target in osteosarcoma.

Circ-RANGAP1/MicroRNA-542-3p/Myosin Regulatory Light Chain Interacting Protein Axis Modulates the Osteosarcoma Cell Progression.

Objective This study is aimed at exploring the influence of circular RNA- (circRNA-) RANGAP1 targeting microRNA- (miR-) 542-3p/myosin regulatory light chain interacting protein (MYLIP) on the biological function of osteosarcoma (OS) cells. Methods Tumor tissues and normal tissues were collected from OS patients and circ-RANGAP1, miR-542-3p, and MYLIP expression was tested by RT-qPCR. The correlation between the clinicopathology/prognosis of patients with OS and circ-RANGAP1 expression was observed. Human OS cell line MG-63 was screened to determine the influences of circ-RANGAP1 and miR-542-3p on OS cell progression. The targeting relation of circ-RANGAP1, miR-542-3p, and MYLIP was probed. Results circ-RANGAP1 expression was elevated in tumor tissues from OS patients, which was correlated to the poor clinicopathology. circ-RANGAP1 expression was augmented in males or patients younger than 20 years old or patients with advanced OS. Higher circ-RANGAP1 expression indicated a poor prognosis in OS patients. After silencing circ-RANGAP1 or elevating miR-542-3p in MG63 cells, cell progression was limited. miR-542-3p downregulation reduced the therapeutic efficacy of silenced circ-RANGAP1. circ-RANGAP1 bound with miR-542-3p to target MYLIP. Conclusion Silenced circ-RANGAP1 boosts MYLIP expression via competitive binding of miR-542-3p to facilitate OS cell progression.

MiR-135a Targets SMAD2 to Promote Osteosarcoma Proliferation and Migration.

Osteosarcoma (OS) is an aggressive malignant neoplasm that commonly occurs in adults and adolescents. The objectives of this work were to verify the role of microRNA- (miR-) 135a in OS and determine whether it can regulate the growth and cellular migration of OS by targeting mothers against decapentaplegic homolog 2 (SMAD2). miR-135a and SMAD2 mRNA expression levels were measured using reverse transcription-quantitative PCR (RT-qPCR). Proliferation and migration of cells were studied using the Cell Counting Kit-8, EdU staining, and transwell invasion experiment. Additionally, a dual-luciferase reporter experiment was used to investigate the possible relationship between miR-135a and SMAD2's 3′-UTR. Immunohistochemistry was utilized to examine the expressions of SMAD2 and Ki67 in mouse tumor tissues to determine the influence of miR-135a on cancer progression in vivo. miR-135a was shown to be elevated in OS tissue samples as well as five cell lines. High expression levels of miR-135a were correlated with poor prognosis of OS patients. Cellular proliferation and migration were promoted by the upregulation of miR-135a with miR mimics; however, this effect was inhibited by SMAD2 overexpression. miR-135a was also shown to directly target the 3′-UTR of SMAD2. Animal experiments also demonstrated that miR-135a downregulation had an inhibitory effect on tumor growth in vivo. High expression levels of miR-135a promoted transplanted tumor development in vivo and the proliferation and migration of OS cells by targeting SMAD2. In summary, miR-135a may be a prospective therapeutic target for OS in the future.