Comprehensive Analysis of FBXO43 Expression and Its Prognostic and Therapeutic Implications in Osteosarcoma

MicroRNAs (miRNAs) are a class of small noncoding RNAs that are implicated in various important biological processes by regulation of gene expression. Aberrant miRNA expression is suggested to be associated with various human disorders including cancer. In this study, we studied if miRNAs can be used as biomarkers in osteosarcoma (OS). OS is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, OS patients who respond poorly towards chemotherapy or develop relapses have a dismal outcome. Thus, it is of clinical significance if a biomarker approach can be developed to monitor tumor burden and detect early relapses in the patients, so that they can be treated as early as possible to improve the survival. To achieve this goal, we performed miRNA profiling on a cohort of OS plasma samples collected from the Texas Children's Hospital. miR-21 and other miRNAs were identified to be elevated in OS patients relative to a group of healthy controls. Circulating miR-21 has been detected in other types of cancer and in the tumor it is known to negatively regulate tumor suppressor genes involved in proliferation, apoptosis and invasion; however, its role in OS is still unclear. Hence, we first validated that miR-21 was elevated in a set of independent osteosarcoma plasma samples but not in the control samples. Using serial plasma samples from a group of patients, we found that the circulating miR-21 level was lower in the posttreatment plasma samples than in the pretreatment samples. These results suggest that circulating miR-21 may correlate with tumor burden of the patients. In addition, miR-21 was expressed in many OS tumors as detected by in situ hybridization on an OS tissue microarray. When tested on two OS cell lines, miR-21was significantly increased in the culture media between 24 and 48 hours after plating in a cell number dependent manner, suggesting that miR-21 could be released from the tumor cells. Taken together, our results suggest that miR-21 is expressed in OS and its level in plasma may reflect tumor burden in OS patients. Citation Format: Manjula Nakka, Yiting Li, Colin McGee, Ching Lau, Tsz Kwong Man. Characterization of miR-21 as a potential circulating biomarker for osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5228. doi:10.1158/1538-7445.AM2014-5228

Background/Aims: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. However, the molecular mechanisms regulating osteosarcoma tumorigenesis and progression are still poorly understood. Circular RNAs (circRNAs) have been identified as microRNA sponges and are involved in many important biological processes. This study aims to investigate the global changes in the expression pattern of circRNAs in osteosarcoma and provide a comprehensive understanding of differentially expressed circRNAs. Methods: Microarray based circRNA expression was determined in osteosarcoma cell lines and compared with hFOB1.19, which was used as the normal control. We confirmed the microarray data by real time-qPCR in both osteosarcoma cell lines and tissues. The circRNA/microRNA/mRNA interaction network was predicted using bioinformatics. Gene Ontology analysis and 4 annotation tools for pathway analysis (KEGG, Biocarta, PANTHER and Reactome) were used to predict the functions of differentially expressed circRNAs. Results: We revealed a number of differentially expressed circRNAs and 12 of them were confirmed, which suggests a potential role of circRNAs in OS. Among these differentially expressed circRNAs, hsa_circRNA_103801 was up-regulated in both osteosarcoma cell lines and tissues, while hsa_circRNA_104980 was down-regulated. The most likely potential target miRNAs for hsa_circRNA_103801 include hsa-miR-370-3p, hsa-miR-338-3p and hsa-miR-877-3p, while the most potential target miRNAs of hsa_circRNA_104980 consist of hsa-miR-1298-3p and hsa-miR-660-3p. Functional analysis found that hsa_circRNA_103801 was involved in pathways in cancer, such as the HIF-1, VEGF and angiogenesis pathway, the Rap1 signaling pathway and the PI3K-Akt signaling pathway, while hsa_circRNA_104980 was related to some pathways such as the tight junction pathway. Conclusions: This study has identified the comprehensive expression profile of circRNAs in osteosarcoma for the first time. And the ceRNA network prediction and bioinformatics functional analysis could provide a comprehensive understanding of hsa_circRNA_103801 and hsa_circRNA_104980, which may be involved in the initiation and progression of osteosarcoma. The present study indicates that circRNAs may play important roles in osteosarcoma and thus serve as biomarkers of osteosarcoma diagnosis and treatment.

Background: Osteosarcoma (OS) is an aggressive cancer of the bone with a poor prognosis. OS often involve mutations of tumor suppressors RB1 and TP53. One putative proto-oncogene in OS is SKP2, which codes for a substrate-recognition factor of the SCF E3 ubiquitin ligase. We have demonstrated that germline knockout of SKP2 in a pre-clinical transgenic mouse model of OS doubly deficient in Rb1 and Trp53 improved survival, delayed tumorigenesis, reduced malignant cell proliferation, slowed tumor growth, diminished stemness, and induced apoptosis. However, the role that SKP2 plays in OS progression remains unclear. Method: We performed bulk RNA-sequencing on pre-clinical transgenic mouse models of OS. To model OS in mice, we generated a conditional double-knockout line of Trp53 and Rb1 mutations induced in cells of the bone-forming osteoblast lineage via Cre-Lox recombination (“DKO”: Osx1-Cre;Rb1lox/lox;p53lox/lox). We compared this with a triple-knockout model, which additionally features a germline Skp2 knockout (“TKO”: Osx1-Cre;Rb1lox/lox;p53lox/lox;SKP2-/-). We compared tumors from the TKO and DKO models via bulk RNA-sequencing (n=3 per group) to study differential expression and immune infiltration. We used immunohistochemistry (IHC) and quantification to validate differences in immune markers between genotypes. Using the transcriptomic results from the mouse models, we analyzed a human OS patient cohort of 83 participants (NCI TARGET OS) with RNA-seq and clinical data via survival analysis to infer the impact of modulating SKP2 in OS patients. Result: We found a large difference in gene expression between the TKO and DKO models. Surprisingly, we observed a dramatic increase in the expression of genes related to immune microenvironment infiltration in TKO tumors compared to DKO. Macrophage transcriptomic signatures were the strongest signal detected (P<0.001), but significant increases in B cells, endothelial cells and T cell signatures were also observed. IHC also showed macrophage markers were higher in TKO (P = 0.08). In the NCI TARGET OS cohort, high expression of genes up-regulated in TKO was significantly correlated with 5-year overall survival (p=0.02). This effect was driven by macrophage transcriptomic signatures. This finding was supported by an immune microenvironment deconvolution analysis, which showed that macrophages and T cells were associated with improved overall survival in the patient cohort (p=0.01). We are currently performing single-cell RNA-sequencing analysis to study immune infiltration and cellular heterogeneity in more detail. Conclusion: Taken together, our new findings indicate that SKP2 modulation in OS may induce a vigorous anti-tumor immune activation especially in the form of macrophage and lymphocyte infiltration into the tumor microenvironment. Citation Format: Alexander Ferrena, Jichuan Wang, Ranxin Zhang, Rui Yang, David Geller, Bang Hoang, Deyou Zheng. SKP2 knockout induces macrophage infiltration in p53/Rb1 null transgenic mouse models of osteosarcoma and drives gene expression correlated with improved survival in patients [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 553.

LncRNA AWPPH promotes osteosarcoma progression via activation of Wnt/β-catenin pathway through modulating miR-93-3p/FZD7 axis

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Increasing evidence suggests that aberrant expression of circRNAs is associated with the occurrence and progression of many cancers. Here, we investigated the role of circNRIP1 in osteosarcoma and explored its possible underlying mechanisms. Three pairs of osteosarcoma tissues and adjacent normal tissues were applied to the detection of altered expression of circRNAs through circRNAs microarray. And the level of circNRIP1 expression was elevated in osteosarcoma tissues. Compared with that in adjacent normal tissue, circNRIP1 expression level was obviously elevated in 100 osteosarcoma tissues. Besides, circNRIP1 knockdown inhibited proliferation and migration, promoted apoptosis of osteosarcoma cells. Bioinformatic analysis demonstrated circNRIP1 contributed to FOXC2 expression by sponging miR-199a. Furthermore, METTL3 elevated circNRIP1 expression level via m6A modification. In short, METTL3-induced circNRIP1 exerted an oncogenic role in osteosarcoma by sponging miR-199a, which may provide new ideas for the treatment of osteosarcoma.

Osteosarcoma (OS) is the most frequent primary malignancy in bone, and commonly occurs in children and adolescents. The aim of this study was to assess the aberrant expression of miR-1274a in OS patients, and to evaluate the role of miR-1274a as a prognostic biomarker and tumor suppressor in OS progression. miR-1274a expression was estimated using quantitative real-time polymerase chain reaction. The Kaplan-Meier method and Cox regression analysis were used to evaluate the prognostic value of miR-1274a in OS. Gain- and loss-of-function in vitro experiments were used to explore the functional role of miR-1274a in OS progression. A target gene of miR-1274a was analyzed using a dual-luciferase reporter assay. miR-1274a expression was decreased in OS tissues and associated with distant metastasis and clinical stages in OS patients. Low miR-1274a could predict poor overall survival and disease-free survival in OS. The overexpression of miR-1274a could inhibit OS cell proliferation, migration and invasion. Additionally, ADAM9 was demonstrated to serve as a direct target of miR-1274a in OS cells. In conclusion, reduced miR-1274a predicts poor prognosis and serves as a potential tumor suppressor in OS. ADAM9 is a target of miR-1274a, which may mediate the functional role of miR-1274a in OS progression.

miR-491 Inhibits Osteosarcoma Lung Metastasis and Chemoresistance by Targeting αB-crystallin

A Glycerophospholipid Metabolism-Based Prognostic Model Guides Osteosarcoma Therapy.

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.

Osteosarcoma (OS), a bone tumor with high ability of invasion and metastasis, has seriously affected the health of children and adolescents. Many studies have suggested a connection between OS and the epithelial-mesenchymal transition (EMT). We aimed to integrate EMT-Related genes (EMT-RGs) to predict the prognosis, immune infiltration, and therapeutic response of patients with OS. We used consensus clustering to identify potential EMT-Related OS molecular subtypes. Somatic mutation, tumor immune microenvironment, and functional enrichment analyses were performed for each subtype. We next constructed an EMT-Related risk signature and evaluated it by Kaplan-Meier (K-M) analysis survival and receiver operating characteristic (ROC) curves. Moreover, we constructed a nomogram to more accurately predict OS patients' clinical outcomes. Response effects of immunotherapy in OS patients was analyzed by Tumor Immune Dysfunction and Exclusion (TIDE) analysis, while sensitivity for chemotherapeutic agents was analyzed using oncoPredict. Finally, the expression patterns of hub genes were investigated by single-cell RNA sequencing (scRNA-seq) data analysis. A total of 53 EMT-RDGs related to prognosis were identified, separating OS samples into two separate subgroups. The EMT-high subgroup showed favourable overall survival and more active immune response. Significant correlations were found between EMT-Related DEGs and functions as well as pathways linked to the development of OS. Additionally, a risk signature was established and OS patients were divided into two categories based on the risk scores. The signature presented a good predictive performance and could be recognized as an independent predictive factor for OS. Furthermore, patients with higher risk scores exhibited better sensitivity for five drugs, while no significant difference existed in immunotherapy response between the two risk subgroups. scRNA-seq data analysis displayed different expression patterns of the hub genes. We developed a novel EMT-Related risk signature that can be considered as an independent predictor for OS, which may help improve clinical outcome prediction and guide personalized treatments for patients with OS.

Background: Abnormal expression of Zinc finger (ZNF) genes is commonly observed in osteosarcoma (OS), the most prevalent malignant bone tumor in children and teenagers. This project focused on the role of ZNF560 in the progress of OS. Methods: The published datasets including TCGA-SARC and GSE99671 was utilized to screen out the abnormal expression of ZNF560 and associated gene patterns in sarcoma and OS tissues. Prognosis value of ZNF560 was identified in TCGA-SARC and OS cohorts. In order to manipulate ZNF560 expression in HOS and MG63 osteosarcoma (OS) cells, genetic strategies such as shRNA constructs were utilized. The expression patterns of ZNF560 were analyzed through techniques such as immunohistochemistry, Western blotting, and qRT-PCR. Results: By analyzing data from both the GEO and the Cancer Genome Atlas (TCGA) databases, increased expression of ZNF560 in OS tissues was verified, which was significantly associated with poorer outcomes in osteosarcoma patients both in TCGA-SARC and our own OS cohorts. Additionally, downregulation of ZNF560 resulted in decreased cell viability, fewer colonies, and induced apoptosis of osteosarcoma cells. Moreover, ZNF560 was found to be essential for migration of human osteosarcoma HOS and MG63 cells. Conclusion: Collectively, these findings suggest that ZNF560 has the potential to serve as a predictive biomarker for osteosarcoma.

Emerging evidence indicates that circRNAs are broadly expressed in osteosarcoma (OS) cells and play a crucial role in OS progression. Recently, cancer-specific circRNA circPRKAR1B has been identified by high-throughput sequencing and is recorded in publicly available databases. Nevertheless, the detailed functions and underlying mechanisms of circPRKAR1B in OS remains poorly understood. By functional experiments, we found that circPRKAR1B enhanced OS cell proliferation, migration, and promotes OS epithelial–mesenchymal transition (EMT). Mechanistic investigations suggested that circPRKAR1B promotes OS progression through sponging miR-361-3p to modulate the expression of FZD4. Subsequently, we identified that EIF4A3 promoted cirPRKAR1B formation through binding to the downstream target of circPRKAR1B on PRKAR1B mRNA. Further rescue study revealed that overexpression of the Wnt signalling could impair the onco-suppressor activities of the silencing of circPRKAR1B. Interestingly, further experiments indicated that circPRKAR1B is involved in the sensitivity of chemoresistance in OS. On the whole, our results demonstrated that circPRKAR1B exerted oncogenic roles in OS and suggested the circPRKAR1B/miR-361-3p/FZD4 axis plays an important role in OS progression and might be a potential therapeutic target.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Although activator of HSP90 ATPase activity 1 (AHA1) is reported to be a potential oncogene, its role in osteosarcoma progression remains largely unclear. Since metabolism reprogramming is involved in tumorigenesis and cancer metastasis, the relationship between AHA1 and cancer metabolism is unknown. In this study, we found that AHA1 is significantly overexpressed in osteosarcoma and related to the prognosis of osteosarcoma patients. AHA1 promotes the growth and metastasis of osteosarcoma both in vitro and in vivo. Mechanistically, AHA1 upregulates the metabolic activity to meet cellular bioenergetic needs in osteosarcoma. Notably, we identified that isocitrate dehydrogenase 1 (IDH1) is a novel client protein of Hsp90-AHA1. Furthermore, the IDH1 protein level was positively correlated with AHA1 in osteosarcoma. And IDH1 overexpression could partially reverse the effect of AHA1 knockdown on cell growth and migration of osteosarcoma. Moreover, high IDH1 level was also associated with poor prognosis of osteosarcoma patients. This study demonstrates that AHA1 positively regulates IDH1 and metabolic activity to promote osteosarcoma growth and metastasis, which provides novel prognostic biomarkers and promising therapeutic targets for osteosarcoma patients.

In this comprehensive study, we explored the molecular landscape C-X-C chemokine receptor (CXCR) family genes (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, and CXCR7) in osteosarcoma (OS) by scrutinizing the expression profiles and functional implications using Bioinformatics analyses and molecular experiments. We found significant up-regulation of these genes in OS cell lines compared to control cell lines, as assessed by RT-qPCR, with high diagnostic potential demonstrated by receiver operating characteristic (ROC) curve analysis. Cross-validation using the GSE12865 dataset revealed consistent up-regulation of CXCR family genes in OS samples, alongside decreased promoter methylation in tumor samples compared to normal tissues, as confirmed by the UALCAN database. Mutational analysis, conducted using data from 237 OS samples from the cBioPortal database, revealed minimal mutations in CXCR1 and CXCR2, with no alterations in CXCR3, CXCR4, CXCR5, and CXCR7. Copy number variation (CNV) analysis showed some level of amplification in CXCR1 and CXCR2, but no significant alterations in the copy numbers of the other genes. Survival analysis using meta-analysis across multiple independent studies showed that the expression of some CXCR genes were significantly associated with poor patient survival. Further exploration of the transcriptional regulation of CXCR genes using the ENCORI database revealed an intricate miRNA-mRNA network involving miR-130a, miR-146a, miR-155, miR-21, and miR-7, which regulate the expression of these genes. Elevated expression of these miRNAs in OS samples was validated by RT-qPCR, with promising diagnostic potential highlighted by ROC analysis. Additionally, the immunological analysis revealed a positive correlation between the expressions of CXCR genes and immune cell types, including macrophages and T cells, and CXCR genes were found to enhance drug responsiveness in OS patients. Gene enrichment analysis identified critical biological processes and pathways, such as chemokine-mediated signaling and immune response, linked to the CXCR family. Knockdown of CXCR1 in HOS and MG-63 cells confirmed that CXCR1 plays a crucial role in cell proliferation, colony formation, and migration. CXCR1 knockdown significantly reduced cell proliferation and colony formation, while enhancing cell migration, underscoring its functional importance in OS progression. Overall, our findings suggest that the CXCR family genes are potential diagnostic and prognostic markers in OS, with implications for therapeutic targeting and further investigation into their role in OS pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s41065-025-00569-3.

Long noncoding RNAs (lncRNAs) play critical roles in osteosarcoma (OS) progression. LncRNA DSCAM-AS1 has been reported to function as a tumor promoter in various cancers. However, the potential mechanism of DSCAM-AS1 in OS remains rarely know. The expression levels of DSCAM-AS1 and miR-101 were detected by RT-qPCR. The correlation between DSCAM-AS1 and miR-101 expression was analyzed by Pearson's correlation. Kaplan-Meier analysis was used to assess the overall survival rate. Cell viability and invasion were assessed by MTT assay and transwell assays, respectively. A Luciferase reporter assay was used to identify the relationship between DSCAM-AS1 and miR-101. In the present study, it was demonstrated that DSCAM-AS1 expression was significantly upregulated in OS tissues and cells and high expression of DSCAM-AS1 predicted poor prognosis in OS patients. In addition, the silencing of DSCAM-AS1 suppressed the viability and invasion of OS cells, while DSCAM-AS1 overexpression promoted cell viability and invasion. Furthermore, we found that DSCAM-AS1 inhibited miR-101 expression by direct interaction and DSCAM-AS1 promoted OS progression by sponging miR-101. In addition, miR-101 expression was negatively correlated with DSCAM-AS1 expression. Patients with low miR-101 expression had a shorter overall survival time compared with those with high miR-101 expression. The present study demonstrated that DSCAM-AS1 accelerated OS cell progression by sponging miR-101, which might provide a new sight in the treatment of OS.