The Deubiquitinating Enzyme USP9X Regulates the Protein Stability of the FAM49B in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is the predominant histological subtype of kidney cancer, characterized by a paucity of therapeutic targets and a dismal prognosis in advanced stages. Although family with sequence similarity 49 member B (FAM49B) has been implicated in tumor progression with context-dependent roles, its function in ccRCC remains undefined. Herein, we elucidated the expression profile and regulatory mechanisms of FAM49B by utilizing integrated bioinformatics, molecular biology, and proteomic approaches. Analysis of TCGA and CPTAC data sets revealed a significantly upregulated FAM49B protein in ccRCC tissues that positively correlated with pathological grade, while the mRNA level was unchanged. Mechanistically, FAM49B undergoes proteasomal degradation, whereas the deubiquitinating enzyme USP9X directly binds to and stabilizes FAM49B. Pharmacological inhibition or knockdown of USP9X notably reduces FAM49B protein abundance without altering its transcription. Quantitative proteomic and affinity mass spectrometry identified ATP1B1 as a critical downstream effector of FAM49B. Western blotting (WB) analysis verified that silencing FAM49B downregulated ATP1B1 in ccRCC cells, while coimmunoprecipitation assays established their direct physical interaction. Consistent with FAM49B expression profiles, ATP1B1 protein levels were elevated in high-grade ccRCC specimens. Collectively, these findings delineate a USP9X-FAM49B-ATP1B1 signaling axis that drives ccRCC progression, representing a promising diagnostic biomarker and potential therapeutic target.