Canagliflozin as a Potential Preclinical Therapy for Tuberous Sclerosis Complex: Inhibition of Tsc2−/− Cell Proliferation via Cell Cycle Arrest and Mitochondrial Dysfunction

Juan Ye,1,2,* Boyuan Liu,3,* Jing Wu,4,* Hongliang Gao,5 Qingyun Wei,2 Kelei Su,2 Kai Zheng,6 Xuening Dai,3 Tao Xu,3 Yuqi Wang,3 Shuangchi Liu,7 Xing Peng,3 Liming Gou,4 Yinjuan Zhao,8,* Bin Xue1,4,7,9,10 1Medical School, Nanjing University Nanjing, Jiangsu, 210023, People’s Republic of China; 2Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210028, People’s Republic of China; 3Sir Run Run Hospital, Nanjing Medical University, Nanjing, Jiangsu, 211166, People’s Republic of China; 4Translational Medicine Research Center, Children’s Hospital of Nanjing Medical University, Nanjing, 210000, People’s Republic of China; 5Department of Pathophysiology, Wannan Medical college, Wuhu, 241002,People’s Republic of China; 6Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, 210029, People’s Republic of China; 7Department of General Surgery, the Affiliated Changzhou Second People’s Hospital of Nanjing Medical University, Changzhou, 213003, People’s Republic of China; 8Collaborative Innovation Center of Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, 210037, People’s Republic of China; 9NHC Key Laboratory of Antibody Technique, Nanjing Medical University, Nanjing, People’s Republic of China; 10Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, 211166, People’s Republic of China*These authors contributed equally to this work: Juan Ye, Boyuan Liu, Jing WuCorrespondence: Bin Xue, Email [email protected] Yinjuan Zhao, Email [email protected]: Canagliflozin (Ca), a sodium-glucose cotransporter 2 (SGLT2) inhibitor traditionally used for type 2 diabetes, has shown potential in the treatment of lymphangiomatosis (the pulmonary lesion phenotype of TSC). However, its effects on Tsc2−/− cells, a key feature of tuberous sclerosis complex (TSC), have not been previously explored. This preclinical study aimed to investigate Ca’s inhibitory mechanisms on Tsc2−/− cell proliferation and its therapeutic potential in TSC-related lesions.Methods: The effects of Ca on Tsc2−/− cells were evaluated using in vitro cellular assays, including proliferation, cell cycle, and mitochondrial function analyses, as well as proteomics. In vivo, a mouse xenograft model was employed to assess tumor growth inhibition and safety profile. Comparative studies with other SGLT2 inhibitors were conducted to identify compound-specific mechanisms.Results: Ca significantly inhibited Tsc2−/− cell proliferation in a dose-dependent manner, inducing G1 phase cell cycle arrest and impairing mitochondrial function, as evidenced by reduced membrane potential and ATP production. Proteomic analysis revealed mitochondrial protein alterations, and Ca-induced ROS accumulation promoted apoptosis. In vivo, Ca (100 mg/kg/day) effectively suppressed tumor growth without significant adverse effects. Notably, Ca’s effects were unique compared to other SGLT2 inhibitors, indicating mechanisms independent of SGLT2 inhibition.Conclusion: Ca inhibits Tsc2−/− cell proliferation through dual mechanisms of cell cycle arrest and mitochondrial impairment, demonstrating significant therapeutic potential for TSC-related lesions. These findings highlight Ca as a promising alternative to current mTOR inhibitors, warranting further investigation into its molecular targets and clinical applications.Keywords: canagliflozin, rare diseases, mitochondrial impairment, Tsc2 gene