FTO deubiquitinated by USP7 influenced NEDD4 mRNA stability enhancing the chemoresistance of pancreatic cancer to gemcitabine by promoting cell proliferation and upregulating RRM1 expression

Abstract Background: Pancreatic cancer is the seventh leading cause of cancer mortality worldwide. It has been proven that the N6-methyladenosine (m6A) alteration is an important regulator of human cancer, however, it is unclear how FTO affects gemcitabine resistance in pancreatic cancer.Methods: Gemcitabine resistant pancreatic cancer cells and tissues were used to evaluated the expression level of FTO. The biological functions of FTO to gemcitabine resistant cells were investigated through cell counting kit-8, colony formation assay, flow cytometry and inhibitory concentration 50. Immunoprecipitation/MassSpectrometry, methylated RNA immunoprecipitation sequencing, RNA sequencing and RNA immunoprecipitation assays, RNA stability, luciferase reporter, and RNA pull down assays were performed to explore the mechanism of FTO in gemcitabine resistant pancreatic cancer cells.Results: FTO was substantially expressed in cells and tissues that were resistant to gemcitabine. Functionally, FTO enhanced gemcitabine resistance of pancreatic cancer in vitro. Depletion of FTO inhibited tumor growth of gemcitabine resistance cells in vivo. By immunoprecipitation/massSpectrometry we found FTO protein can be bound to USP7 and deubiquitinated by USP7 and lead to the upregulation of FTO. Mechanistically, FTO knockdown substantially reduced the expression level of NEDD4 in an m6A dependent manner. FTO enhanced the mRNA stability of NEDD4 through targeting its exonic regions. RNA pull down and RNA immunoprecipitation verified YTHDF2 was the reader of NEDD4. NEDD4 promoted the proliferation and chemoresistance of gemcitabine resistance cells. FTO knockdown markedly reduced RRM1 expression level in NEDD4 dependent manner, thus influence the chemosensitivity to gemcitabine in pancreatic cancer cells. Conclusion: We found FTO regulated gemcitabine resistance in pancreatic cancer by demethylated NEDD4 RNA in m6a-dependent manner which then influenced the RRM1 expression level. Meanwhile, we identified FTO protein level can be upregulated by USP7.