KEAP1 Mutations Drive Tumorigenesis by Suppressing SOX9 Ubiquitination and Degradation

Abstract The transcription factor SOX9 is frequently amplified in diverse advanced‐stage human tumors. Its stability has been shown to be tightly controlled by ubiquitination‐dependent proteasome degradation. However, the exact underlying molecular mechanisms remain unclear. This work reports that SOX9 protein abundance is regulated by the Cullin 3‐based ubiquitin ligase KEAP1 via proteasome‐mediated degradation. Loss‐of‐function mutations in KEAP1 compromise polyubiquitination‐mediated SOX9 degradation, leading to increased protein levels, which facilitate tumorigenesis. Moreover, the loss of critical ubiquitination residues in SOX9, by either a SOX9 (ΔK2) truncation or K249R mutation, leads to elevated protein stability. Furthermore, it is shown that the KEAP1/SOX9 interaction is modulated by CKIγ‐mediated phosphorylation. Importantly, it is demonstrated that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the KEAP1/SOX9 interaction and its consequent degradation. Collectively, herein the findings uncover a novel molecular mechanism through which SOX9 protein stability is negatively regulated by KEAP1 to control tumorigenesis. Thus, these results suggest that mitigating SOX9 resistance to KEAP1‐mediated degradation can represent a novel therapeutic strategy for cancers with KEAP1 mutations.

. CUL3 governs the stability of SOX9 protein.  (G) Protein half-life assay was performed for the assessment of SOX9 stability in Huh7 cells 9 with KEAP1 knockout. 36 h after plasmid transfection, cells were treated with cycloheximide (CHX, 10 μg/ml) for the indicated time period before they were harvested for IB analyses.
Quantification of SOX9 levels relative to Vinculin was shown.
(H) Spearman's correlation analyses between SOX9, NRF2, and KEAP1 mRNA levels in The Cancer Genome Atlas (TCGA) data set of human lung carcinoma.
(I) IB analysis of KEAP1 and SOX9 protein levels in Huh7 and H1299 cells infected with the indicated lentiviral shRNAs for 72 h before harvesting.

(F) Colony formation assay of H1299 and Huh7 cells stably expressing SOX9 WT or SOX9
ΔDeg2 mutant with the endogenous SOX9 knocked down by shRNA. Data were presented as mean ± SEM of three independent experiments. *p<0.05, Student's t test.
(G) Tumor volume at the endpoint of the study was measured as indicated in Figure 6M. Data were presented as mean ± SEM, n = 5 per group. *p<0.05, **p<0.01, Student's t test. (E) IB analysis of SOX9 protein levels in Huh7 cells expressing the indicated casein kinases.

Immunoblotting (IB) and immunoprecipitation (IP)
For the preparartion of Whole-Cell Lysate (WCL), cells were lysed with IP lysis buffer

Colony formation assay
The indicated tumour cells (Huh7 and H1299) were plated in 24-well plates (200 cells per well) and maintained for up to 10-15 days until colonies were visible. Plates were washed with PBS and fixed with 10% acetic aids/10% methanol for 30 min, and then stained with 0.4% crystal violent/20% ethanol. After washing with distilled water, the plates were air-dried and the visible colonies were quantified and analysed as previously described. [2] Human clinical data analyses

In vivo ubiquitination assays
HEK293T cells with 80% confluence were co-transfected with HA-, 6×His-or GFP-tagged 18 ubiquitin and the desired constructs. Thirty-six hours post-transfection, cells were treated with 20 μM MG-132 for 6 h before they were harvested. Cells were lysed in IP lysis buffer containing freshly dissolved iodoacetamide and N-ethylmaleimide (5 mM each) to inhibit deubiquitinating enzymes. Immunoprecipitation was performed using antibodies against the tagged proteins. Immunoprecipitants were washed five times with IP lysis buffer before being resolved by SDS-PAGE and immunoblotted with antibodies against the tags on ubiquitin.

Immunofluorescence (IF) staining
Cells were grown on glass coverslips and fixed with 4% paraformaldehyde for 30 min at room temperature, washed three times with PBS and then permeabilized with 0.05% Triton X-100 for 10 min at room temperature. Following three PBS washes of 5 min each, the coverslips