Chondrosarcoma-derived Mutation in IDH1 Promotes Tumorigenesis by Inducing HIF-1α

Abstract

IDH mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into an oncometabolite, D-2-hydroxyglutarate (D-2HG). IDH mutations occur in more than 50% of patients with conventional or dedifferentiated chondrosarcoma. However, the function of these mutations in the pathogenesis of chondrosarcoma is still unclear. In this study, we aimed to establish a mutant IDH1 knockout model in a chondrosarcoma cell line and determine the role of mutant IDH1 in this malignancy. In our study, we employed a human chondrosarcoma cell line, JJ012 which harbors an endogenous IDH1R132G mutation. As a control, we also used an immortalized chondrocyte cell line, C28 which expresses wildtype IDH1 only. IDH1 was knocked out via CRISPR/Cas9 technology. Quantitative analyses of D-2HG were conducted by tandem mass spectrometry. RNA-Seq analysis was performed to identify differentially expressed genes between the mutant IDH1 knockout (KO) cells and the parental cells. RNA-Seq data were validated by real time PCR. Colony-forming ability of KO cells under hypoxia and normoxia conditions was evaluated using soft agar assay. The effect of mutant IDH1 on chondrosarcoma growth was analyzed in murine xenograft models. The expression of HIF1-1α and its targeted genes were examined in cells and tumor tissues by western blot and Immunohistochemistry (IHC). Mutant IDH1 was successfully knocked out in JJ012 cells. Single cell-derived KO clones were then identified and developed. As expected, D-2HG production is almost completely depleted in these KO clones. Using these genetically modified cells, we found that knockout of mutant IDH1 remarkably inhibited colony formation by 40% under normoxia condition compared to the parental control (p<0.05). Intriguingly, we observed that the HIF1-α levels were significantly reduced in the JJ012 KO cells compared with the parental cells. Moreover, RNA-seq data showed a widespread downregulation of HIF1-1α targeted genes such as VEGFA, VEGFC, SLC2A3, EDN1 in response to the knockout (FDR-adjusted p value < 0.01). These data were confirmed by qRT-PCR. The soft-agar colony formation assay further showed a significantly increased colony number and diameter for JJ012 KO cells under hypoxia condition compared with those under normoxia condition (P<0.05). Furthermore, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation in the murine xenograft model. Immunoblotting and IHC revealed that HIF-1α and its targeted genes were dramatically downregulated in the KO cell-derived tumors. We found that mutant IDH1 contributes to tumorigenicity of chondrosarcoma cells, at least partially through the upregulation of HIF1-α. This suggests that targeting HIF1-α signaling pathway is promising for combinatorial regimens with mutant IDH inhibitors for patients with IDH1-mutant chondrosarcoma.