In recent years, the number of studies based on public databases such as GEO and TARGET has increased. These are useful to mine potential biomarkers for osteosarcoma. For example, Dai et al. (20)screened candidate genes to predict the chemotherapy resistance response of osteosarcoma by miRNA-mRNA interaction network. Jiang et al. (21) identified autophagy and immune-related gene markers TRIM68, PIKFYVE, and DYNLL2, which could predict the prognosis of osteosarcoma. In the present study, using the GSE42352 dataset and by differential WGCNA analysis, we screened autophagy-related modular genes and construct a PPI network. A key gene, CXCR4, was identified by MCODE and Closeness algorithms. 222 autophagy-related genes were obtained from the online database. We performed concordance analysis for samples in GSE42352, and four subgroups were obtained; CXCR4 was identified in all four subgroups. Using the validation dataset GSE14359, we showed that CXCR4 was indeed highly expressed in osteosarcoma as compared to normal samples. Niu et al.(22) have also identified CXCR4 as a possible therapeutic agent to inhibit the progression of osteosarcoma by bioinformatic analysis.
Chemokine receptor-4 (CXCR4) is a specific receptor for chemokine stromal cell-derived factor-1 (CXCL12)(23). There is increasing evidence that CXCR4 plays a crucial role in osteosarcoma progression and metastasis(24, 25). In addition, CXCR4 is associated with poor survival in patients with osteosarcoma and is considered to be an important clinical prognostic indicator(26). In the present study, the module selected for WGCNA analysis showed the highest significant correlation with autophagy. Autophagy, a highly evolutionarily conserved cellular homeostatic dynamic recycling system, is used for cytoplasmic recycling, degradation, and reuse(27). Autophagy selectively degrades long-lived proteins, removes dysfunctional organelles under basal conditions, and promotes cell survival(28). It has been recognized as a cytoprotective process that promotes chemoresistance in osteosarcoma. Several studies have focused on autophagy inhibition in osteosarcoma chemo-sensitization(29). Coly et al.(30) found that, in HEK293 and U87 cells, activation of CXCR4 leads to a decrease in the number of autophagosomes, suggesting that CXCR4 exerts its anti-autophagic effect by regulating calcium-activating enzymes and preventing pre-autophagosomal vesicle formation. In the present study, our in vitro assays also showed that CXCR4 could affect the progression of osteosarcoma by regulating the PI3K-Akt-mTOR signaling pathway. The main regulator of autophagy is mTORC1; mTORC2 also contributes to the autophagic control through AKT pathway regulation(31). Under normal conditions, mTORC1 directly inactivates the ULK1/2 protein complexes. These complexes are critical and essential regulators of cellular pathways that control the initiation of gene translation and ribosome biogenesis and exhibit important monitoring roles for cellular metabolism, lipolysis, and autophagy (32, 33). In general, there is a negative interaction between autophagy and apoptosis, as autophagy prevents apoptosis induction and apoptosis-associated caspase activation inhibits the autophagic process. Some studies also show that the negative correlation between CXCR4 and autophagy is dependent on PI3K/AKT/mTOR signaling pathway (34).
In the present study, by single gene enrichment analysis, we showed that CXCR4 is mainly enriched in arachidonic acid metabolism, natural killer cell-mediated cytotoxicity, and IL-6/JAK/STAT3 signaling pathway in osteosarcoma. Previous studies(35) have shown that CXCR4 is itself involved in NK cell-mediated cytotoxicity in chronic granulocytic leukemia. IL-6/JAK/STAT3 is a stress-related inflammatory signaling pathway, with rapid response. Dysregulated cytokine signaling contributes to cancer development (36). In addition, the STAT family of transcription factors can be regulated by other kinases, such as mTOR, MAPK which phosphorylate Ser727 of STAT3 for sustained activation (37). STAT3 is potentially pro-oncogenic (a proto-oncogene) and is consistently expressed across several cancers. In the pathogenesis of atherosclerosis, CXCR4 is involved in chronic inflammation of the arterial wall and is characterized by a chemokine-mediated inward flow of leukocytes(38). Chronic inflammation and local infiltration of CXCR4-expressing immune cells can promote esophageal carcinogenesis(39). Activating downstream cascades involving JAK/STAT, PI3K/Akt, MAPK, JNK pathway considered important targets for development of new therapeutic strategies, and the activation can be triggered by the binding between CXCR4 and its ligand CXCL12. These critical points control stemness, chemotaxis and cell survival, proliferation, migration(40). CXCL12(41, 42) is a homeostatic chemokine produced by mesenchymal stem cells,which binds to chemokine receptors to regulate hematopoietic stem cell (HSC) transport and secondary lymphoid tissue structure. CXCR4 overexpression has been found in many tumors(43). There are evidences that CXCR4/CXCL12 axis activates multiple downstream pathways and plays an important role in tumor progression(41, 44).Besides, previous study have found that the overwhelming majority of metastases from osteosarcoma are to the lung, a site that expresses high levels of the ligand CXCL12(45).
Taken together, our results systematically demonstrated high CXCR4 expression in osteosarcoma. We speculated that it could induce autophagic cell death by regulating the PI3K/AKT/mTOR signaling pathway. Indeed, functional enrichment analysis showed that CXCR4 in inflammatory, immune-related contexts could provide new strategies for gene-targeted therapy for osteosarcoma.