Fat mass and obesity-associated protein downregulation trigger the activation of the sirtuin 1/forkhead box O1 signaling pathway, drive glycolysis, and promote the progression of renal cell carcinoma

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Objective: This study aims to investigate the role of fat mass and obesity-associated protein (FTO) in renal clear cell carcinoma (RCC), particularly its regulatory effects on glycolysis, cell proliferation, and sirtuin 1/forkhead box O1 (SIRT1/FOXO1) signaling pathway. Material and Methods: The messenger RNA and protein expression levels of FTO in human proximal tubular epithelial cells (human kidney 2 [HK-2]) and the RCC cell line A498 were determined by quantitative reverse transcription polymerase chain reaction and Western blot. FTO expression was downregulated by FTO short hairpin RNA and overexpressed using plasmids. Glycolysis levels were assessed by measuring glucose uptake, lactate secretion, extracellular acidification rate, and adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio. The effects of FTO on cell proliferation and cell cycle were evaluated through colony formation assays, 5-ethynyl-2'-deoxyuridine (EdU) staining, and flow cytometry. The SIRT1/FOXO1 signaling pathway was analyzed through Western blot, and FOXO1 pathway inhibitor (AS1842856) was used to further explore the role of SIRT1/FOXO1 in the FTO-mediated regulation of RCC. Results: FTO was downregulated in A498 cells compared with that in HK-2 cells. FTO downregulation markedly increased glucose uptake, lactate secretion, and the ATP/ADP ratio in A498 cells, and its overexpression inhibited these processes. FTO downregulation also promoted RCC cell proliferation, as evidenced by an increase in colony formation and the number of EdU-positive cells. Meanwhile, FTO overexpression suppressed the proliferation of these cells. Flow cytometry analysis revealed that FTO downregulation notably increased the proportion of cells in the S phase, and its overexpression increased the proportion of cells in the G0/G1 phase. Further analysis indicated that FTO downregulation activated the SIRT1/FOXO1 signaling pathway, and its overexpression inhibited this pathway. Treatment with the FOXO1 inhibitor AS1842856 significantly reversed the pro-glycolysis and pro-proliferation effects of FTO downregulation, supporting the role of the SIRT1/FOXO1 pathway in FTO-mediated regulation. Conclusion: FTO downregulation promotes glycolysis and proliferation in RCC cells by activating the SIRT1/ FOXO1 signaling pathway. Targeting the FTO and SIRT1/FOXO1 pathway may provide potential therapeutic strategies for the treatment of RCC.