SUMMARY Hypoxic stress and hypoxia-inducible factors (HIFs) play important roles in a wide range of tumors. We demonstrate that SPOP, which encodes an E3 ubiquitin ligase component, is a direct transcriptional target of HIFs in clear cell renal cell carcinoma (ccRCC). Furthermore, hypoxia results in cytoplasmic accumulation of SPOP which is sufficient to induce tumorigenesis. This tumorigenic activity occurs through the ubiquitination and degradation of multiple regulators of cellular proliferation and apoptosis, including the tumor suppressor PTEN, ERK phosphatases, the pro-apoptotic molecule Daxx and the Hedgehog pathway transcription factor Gli2. Knockdown of SPOP specifically kills ccRCC cells, indicating that it may be a promising therapeutic target. Collectively, our results indicate that SPOP serves as a regulatory hub to promote ccRCC tumorigenesis.

Both 5-methylcytosine (5mC) and its oxidized form 5-hydroxymethylcytosine (5hmC) have been proposed to be involved in tumorigenesis. Because the readout of the broadly used 5mC mapping method, bisulfite sequencing (BS-seq), is the sum of 5mC and 5hmC levels, the 5mC/5hmC patterns and relationship of these two modifications remain poorly understood. By profiling real 5mC (BS-seq corrected by Tet-assisted BS-seq, TAB-seq) and 5hmC (TAB-seq) levels simultaneously at single-nucleotide resolution, we here demonstrate that there is no global loss of 5mC in kidney tumors compared with matched normal tissues. Conversely, 5hmC was globally lost in virtually all kidney tumor tissues. The 5hmC level in tumor tissues is an independent prognostic marker for kidney cancer, with lower levels of 5hmC associated with shorter overall survival. Furthermore, we demonstrated that loss of 5hmC is linked to hypermethylation in tumors compared with matched normal tissues, particularly in gene body regions. Strikingly, gene body hypermethylation was significantly associated with silencing of the tumor-related genes. Downregulation of IDH1 was identified as a mechanism underlying 5hmC loss in kidney cancer. Restoring 5hmC levels attenuated the invasion capacity of tumor cells and suppressed tumor growth in a xenograft model. Collectively, our results demonstrate that loss of 5hmC is both a prognostic marker and an oncogenic event in kidney cancer by remodeling the DNA methylation pattern.