Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

Abstract Identifying novel therapeutic targets and drugs is crucial for treating triple‐negative breast cancer (TNBC). Bufalin, a key active ingredient of the traditional Chinese medicine HuaChansu , has been employed in tumor therapy. Here, SPR‐LC‐MS/MS is employed to characterize the targets of Bufalin and found that serine/threonine kinase 33 (STK33) possesses a strong binding affinity to Bufalin. Combining molecular docking, SPR analysis, and Biotin‐pulldown analysis, it is demonstrated that STK33 can bind Bufalin. Notably, STK33 is highly expressed in TNBC and is associated with poor prognosis in TNBC patients. STK33 knockdown inhibits TNBC cell growth both in vitro and in vivo. Mechanistically, STK33 phosphorylates and stabilizes CCAR1, which promotes tumor growth and metastasis, thereby driving tumor progression. Further analyses confirmed that Methionine 245 of STK33 is required for STK33‐Bufalin interaction, and Bufalin treatment promotes the degradation of STK33 protein by destroying the STK33‐HSP90 complex. Through in vitro, in vivo, and in patient‐derived TNBC organoids, it is observed that Bufalin inhibited the TNBC cell proliferation by targeting STK33. This study not only establishes Bufalin as a putative STK33 degrader to suppress TNBC but also identifies STK33 as a pro‐cancer factor in TNBC, presenting a potential therapeutic target for TNBC.