HIF-1α Regulates CXCR4Expression During Hypoxia And Promotes Invasion And Metastases In Osteosarcoma

The malignant transformation of mesenchymal cells within the bone leads to thedevelopment of osteosarcoma(OS), but the genetic underpinnings of these events are notunderstood. From a clinical perspective, primary tumor management can be achievedsuccessfully in most patients. However, the development of metastasis to the lungsrepresents the most common cause of death in OS patients. A clearer understanding ofmetastasis biology is required to improve cancer mortality and improve outcomes. Somestudies have shown that the development and progression of OS is often associated withthe abnormal expression of some genes, which are essential to regulate apoptosis in OScells, so that the growth and metastasis of osteosarcoma is inhibited.Hypoxic microenvironment is one common feature of solid tumors, and HIF-1α iswidely present in mammals and humans under hypoxic conditions, a transcription factor,to adapt its core regulatory factors as tumor hypoxia microenvironment. After activationcan regulate transcription of many genes, tumor cells to maintain anaerobic metabolism,angiogenesis, promoting tumor survival, growth and metastasis have played a veryimportant role. CXCR4is an important signaling molecule, transport it and lymphocyte maturation ofhematopoietic cells. CXCR4and its ligand CXCL12(as is called stromal cell-derivedfactor or SDF-1) interaction activate multiple signal transduction pathways that regulatecell proliferation, survival, chemotaxis, migration and adhesion. Studies have shown: theinvasion of tumor hypoxia can increase through CXCR4, migration and adhesion.Based on HIF-1α and CXCR4showed high expression in many tumors, and is closelyassociated with tumor invasion and metastasis, we hypothesized that hypoxia by HIF-1αmay regulate CXCR4, makes osteosarcoma cells to adapt to hypoxic microenvironment tomaintain its survival, growth and metastasis potential to have an impact on the invasionand metastasis of osteosarcoma.Purpose：Cellular adaptation to hypoxic microenvironment is essential for tumorprogression and is largely mediated by HIF-1α through coordinated regulation of hypoxia-responsive genes. Chemokine SDF-1α and its unique receptor CXCR4have beenimplicated in organ-specific metastases of many cancers. In this study, we investigated theresponse of osteosarcoma cells to hypoxia and the expression of CXCR4and HIF-1α inhuman osteosarcoma specimens and explored the roles of CXCR4and HIF-1α in the cellmigration process.Materials and methods：We performed immunohistochemistry, immunocytochemistry,quantitative real-time PCR, Western blots and fluorescent reporter assays to evaluate thecorrelation between CXCR4and HIF-1α expression in human osteosarcoma specimens orSOSP-9607cells under normoxic and hypoxic conditions. Transwell assays were used toassess cell migration under different conditions. Exposure of SOSP-9607cells to hypoxicconditions resulted in significantly increased migration. When SOSP-9607cells weresubjected to hypoxic conditions, the mRNA and protein levels of CXCR4weresignificantly increased in a time-dependent manner. Moreover, siHIF-1α significantlydecreased the mRNA and protein levels of CXCR4under hypoxia, whereaspcDNA-HIF-1α significantly increased the mRNA and protein levels of CXCR4undernormoxia. A luciferase reporter gene study showed that siHIF-1α reduced pGL3-CXCR4luciferase activity. Furthermore, coexpression of HIF-1α and CXCR4was significantlyhigher in patients with distant metastasis compared with those without metastasis. Results：Exposure of SOSP-9607cells to hypoxic conditions resulted in significantlyincreased migration ability. When SOSP-9607cells were subjected to hypoxic conditions,the mRNA and protein levels of CXCR4were significantly increased in a time-dependentmanner. Moreover, siHIF-1α significantly decreased the mRNA and protein levels ofCXCR4under hypoxia while pcDNA-HIF-1α significantly increased the mRNA andprotein levels of CXCR4under normoxia condition. Luciferase reporter gene studyshowed that siHIF-1α reduced pGL3-CXCR4luciferase activity. Furthermore,coexpression of HIF-1α and CXCR4was significantly higher in patients with distantmetastasis compared to those without metastasis.Conclusions：Hypoxia-HIF-1α-CXCR4pathway played a crucial role during humanosteosarcoma migrations, and targeting of this pathway might represent a noveltherapeutic strategy for patients suffering from osteosarcoma.