Due to its malignant properties and invasion behavior, approximately 20%-30% of osteosarcoma patients present with lung metastasis at the initial diagnosis[21, 22]. Even with aggressive treatment, the prognosis of these patients remains poor. Thus, exploring the specific molecular mechanisms involved in the progression of osteosarcoma, and then guiding the development of promising therapeutic strategies targeting the molecular pathway, will contribute to ameliorating the prognosis of these patients.
In recent years, owing to the wide application of gene chips and high-throughput sequencing technology, it paves the way for our biological research. In the current study, raw data of two gene expression datasets were obtained to conduct bioinformatics analysis. As a result, a total of 364 DEGs were identified between osteosarcoma with lung metastasis and non-metastatic osteosarcoma, consisting of 268 upregulated DEGs and 96 downregulated DEGs. Subsequently, GO annotation, KEGG pathways enrichment analysis and PPI network construction were conducted to explore interactions between the DEGs. And the results suggest that the DEGs mainly involved in some important signaling pathways including MAPK signaling pathway, adherens junction, ECM-receptor interaction, focal adhesion. MAPK signal pathway, consisting of RAF1 (MAP kinase-kinase-kinase), MEK (MAP kinase-kinase), and ERK (extracellular signal-related kinase), plays a critical role in promoting tumor proliferation, migration, invasion and angiogenesis[24–27]. Wang et al. previously reported that the gene mutations associated with lung metastasis of osteosarcoma are mainly concentrated in the MAPK signaling pathway and another study also demonstrated macrophage migration inhibitory factor can promote osteosarcoma growth and lung metastasis through activating the MAPK signaling pathway. The extracellular matrix (ECM), composed of polysaccharides and fibrous proteins, is an important structural component of the tumor microenvironment. Previous studies have shown that dysregulation of the extracellular matrix-receptor interaction signaling pathway plays a vital role in regulating tumor invasion and metastasis. Not only that, focal adhesion (FA) promotes cell traction by associating the ECM bound to the transmembrane integrin molecule with the actin cytoskeleton, which also plays an important role in cell migration dynamics[31, 32]. Furthermore, we extracted the most key module from the PPI network based on MCODE score༞5 and found its significantly related to DNA replication, cell cycle, cell proliferation by DAVID analysis.
To conduct deeper research, the top 10 genes (SKP2, SMC2, CDC25C, SOCS3, PARPBP, DTL, ASPM, ECT2, CENPN, TOP2A) of DEGs were regarded as hub genes based on the predefined criteria. And the expression levels of hub genes were verified to be significantly elevated in the patients with lung metastasis from osteosarcoma. Meanwhile, the results of survival analysis indicated that SKP2 and ASPM were closely associated with poor prognosis of patients with osteosarcoma, which means SKP2 and ASPM may play vital roles in the carcinogenesis and progression of patients with osteosarcoma. SKP2 encodes a substrate recognition component of a SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex which mainly mediates the ubiquitination and subsequent proteasomal degradation of target proteins involved in cell cycle progression and signal transduction[33, 34]. At present, accumulating evidence demonstrates that SKP2 plays a vital role in promoting tumor invasion and metastasis. One hand, SKP2 could take part in the ubiquitination and degradation of E-cadherin to positively regulate cell migration. On the other hand, Skp2 could combine with Myc, Miz1 and p300 to form a transcriptional complex mediating the transcription of RhoA to participate in the invasion and metastasis of tumors. Furthermore, SKP2 has also been proven to enhance tumor invasion ability by promoting epithelial-mesenchymal transition (EMT) process and matrix metalloproteinase (MMP) expression[37, 38]. Based on these encouraging results, Zhang et al. found knockdown of Skp2 could result in RhoA and MMP-9 inhibition, thereby significantly reducing tumor cell invasion and lung metastasis in an orthotopic mouse model of osteosarcoma. Not only that, SKP2 has also been reported to exert an effect in DNA damage response, DNA repair, tumor angiogenesis and drug resistance[40, 41]. For example, Ding et al have demonstrated that SKP2 overexpression was also closely related to the acquisition of methotrexate-resistant and EMT properties in osteosarcoma. Due to the extensive carcinogenic effects of SKP2, research on the anti-tumor activity of SKP2 small molecule inhibitors is in full swing. Among them, flavokawain A (FKA), an effective apoptotic inducer and antiproliferative agent in the tumor, which could lead to down-regulation of SKP2, result in osteosarcoma growth retardation and metastasis inhibition. However, more preclinical and clinical trials are necessary to explore the application of the specific SKP2 inhibitors in the treatment of osteosarcoma with lung metastasis.
ASPM encodes a centrosomal protein that plays an essential part in maintaining the normal function of the mitotic spindle and regulating neurogenesis. Previous studies have prevailingly focused on the relationship between ASPM gene mutations and autosomal recessive primary microcephaly and abnormal neuronal differentiation. Nevertheless, except for its role in normal physiological regulatory functions, recent studies have also found that the upregulation of ASPM promotes the metastasis of tumors, including prostate cancer, endometrial cancer, melanomas, bladder cancer, clear cell renal cell carcinoma[45–49]. Pai et al. previously pointed out that overexpression of ASPM has energetically influence on prostate cancer cell proliferation and invasion, as it could enhance the Wnt-induced β-catenin transcriptional activity through interacting with disheveled-3, which is a cardinal upstream regulator of Wnt signaling. Likewise, Zhou et al. found that knockout of ASPM could inhibit tumor proliferation, migration and metastasis by restraining the Wnt/β-catenin pathway in endometrial cancer. Interestingly, ASPM was situated on chromosome 1q31, an enhancement area that was significantly correlated with invasion and metastasis of tumors[51, 52]. The above studies suggested that ASPM could give tumor an impetus to metastasize, but the role of ASPM in lung metastasis of osteosarcoma needs to be further explored.
Although no significant correlation was found between the other hub genes and prognosis in our study, they are still proven to be associated with the invasion and metastasis of tumors. TOP2A gene encodes a DNA topoisomerase that is essential in the regulation of DNA structure and transcription, and it is the direct molecular target of anthacyclines drugs. Previous investigations have demonstrated that TOP2A overexpression was involved in tumor proliferation, invasion, and angiogenesis in multiple malignancies including prostate cancer, pancreatic cancer, soft tissue sarcoma and osteosarcoma[53, 54]. And Nguyen et al. found that TOP2A overexpression was inclined to poor prognosis of osteosarcoma patients without ERBB2 co-amplification. Meanwhile, the therapeutic effect of pirarubicin for osteosarcoma patients with lung metastasis is also closely related to the expression level of TOP2A. The oncogene ECT2 is a guanine nucleotide exchange factor (GEF)of the Rho family members of small GTPases and essential for signal transduction pathways involved in the regulation of cytokinesis. Chen et al. showed that overexpression of ECT2 promoted the metastasis of osteosarcoma by regulating the EMT process. And the downregulation of ECT2 by siRNA and miR-223 could suppress osteosarcoma cell migration and invasion[58, 59]. Moreover, Qiu et al. found that Calycosin could inhibit osteosarcoma cell migration through restraining metastasis-associated IκBα/ECT2 signal pathway, but further research is needed to confirm the results. SOCS family proteins form part of a classical negative feedback system that regulates cytokine signal transduction through the JAK/STAT signaling pathway. Recent studies suggested that Circ_ANKIB1 inhibited the expression of SOCS3 and activated the JAK/STAT signaling pathway by regulating miR-19b, thereby promoting osteosarcoma growth and invasion. CDC25C encoded cell cycle regulatory protein that participated in regulating G2/M progression and mediating DNA damage repair. It has been reported that Evodiamine, Flavokawain B and Ludartin could inhibit proliferation, migration, and apoptosis of osteosarcoma cells by down-regulating the expression level of CDC25C[62–64]. At present, the relationship between the rest of hub genes (DTL, CENPN, PARPBP, SMC2) and osteosarcoma remains unclear and the specific role in lung metastasis deserves further exploration.
Based on the established miRNA-hub genes interaction network, we found that hsa-miR-340-5p was most closely associated with these hub genes. And the role of miR-340-5p in the carcinogenesis and progression of osteosarcoma has been reported. For example, miR-340-5p could negatively regulate Wnt/β-catenin signaling pathway through targeting STAT3 gene, thereby inhibiting the development of osteosarcoma. Meanwhile, overexpression of miR-340-5p could down-regulate the CTNNB1-mediated Notch signaling pathway, thereby suppressing osteosarcoma migration and invasion. However, the regulatory relationships between miR-340-5p and these hub genes in patients with lung metastasis of osteosarcoma need to be confirmed by further studies.
In the present study, key genes and critical signaling pathways which may be involved in the development of lung metastasis of osteosarcoma were identified via a comprehensive analysis of the two gene expression datasets, which may serve as diagnostic and therapeutic targets. Moreover, SKP2 and ASPM were found to be closely related to the poor prognosis of patients with osteosarcoma, which may present as potential prognostic biomarkers in the future. Meanwhile, the interaction network of miRNAs and hub genes illustrates the regulatory relationships of the hub genes and miRNA, and miR-340-5p which was most closely associated with these hub genes is reported to be involved in the progression of osteosarcoma. These results provide important ideas for a comprehensive understanding of cancer characteristics, however, further studies are needed to validate the current findings and elucidate the specific molecular mechanisms of these genes in osteosarcoma with lung metastasis.