Abstract Sox9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family of transcription factors, with known functions in proliferation, epithelial-mesenchymal transition (EMT), stem cell maintenance and senescence. Sox9 protein levels are elevated in several different cancer types, including lung cancer and osteosarcoma. Furthermore, Sox9 overexpression is associated with poor survival in both cancer types. We recently reported that Sox9 is a direct transcriptional target of the Notch signaling pathway and mediates Notch1-induced EMT in lung cancer (Capaccione et al., Oncotarget, 2014). Fbxw7 (F-box and WD repeat domain-containing 7) is a member of the SCFFbw7E3 ubiquitin ligase complexthat targets several oncogenic proteins for degradation, including Notch1. We hypothesized that Sox9 is targeted for degradation by SCFFbw7. Knocking down or overexpressingFbxw7 significantly increasedor decreased, respectively, endogenous Sox9 protein levels in lung cancer and osteosarcoma cell lines. In addition, overexpression of Fbxw7 resulted in active degradation of ectopic Sox9 protein levels, when assessed in combination with cycloheximide, and did so in a dose dependent-manner.Furthermore, both Sox9 and FBXW7 were detected in immunoblots after the reciprocal protein was immunoprecipitated. Because substrate degradation by SCFFbw7 is typically phosphorylation-dependent, frequently by glycogen synthase kinase (GSK)3-beta, we examined whether modulation of GSK3-beta could affect FBXW7-induced Sox9 degradation. Treatment of cells with the GSK3-beta inhibitor, CHIR99021, completely abolished Sox9 degradation by ectopically overexpressed FBXW7, in a dose-dependent manner. We next identified four putative phosphorylation sites within potential FBXW7 degron motifs in Sox9.Mass spectrophotometric analysis confirmed these sites within Sox9 are indeed phosphorylated.In summary, we found that the E3 ubiquitin ligase complex (SCFFbw7) targets Sox9 for ubiquitin-mediated degradation in a GSK3-beta-mediated Sox9 phosphorylation-dependent manner. As a result, this negative regulation of Sox9 by GSK3-beta/FBXW7 could lead to inhibition of Sox9-mediated proliferation, EMT and stemness. This new regulatory mechanism of Sox9 could have diagnostic and therapeutic implications for lung cancer, osteosarcoma, as well as other cancers. Citation Format: Xuehui Hong, Wenyu Liu, Hiroyuki Inuzuka, Lianxin Liu, Sharon R. Pine. Negative regulation of Sox9 by glycogen synthase kinase 3 beta phosphorylation and SCFFbw7-dependent ubiquitination in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1957. doi:10.1158/1538-7445.AM2015-1957