Cystatin B Increases Autophagic Flux By Sustaining Proteolytic Activity Of Cathepsin B And Fuels Glycolysis In Pancreatic Cancer

OBJECTIVES: Pancreatic ductal adenocarcinoma(PDAC)is characterized by severe metabolic stress caused by desmoplasia and poor vascularity.Glycolysis and autophagy are activated during starvation to sustain cancer cell metabolism.Here,the mechanism by which cystatin B(CSTB)accelerates PDAC progression by promoting autophagy and glycolysis was explored.METHODS: The expression pattern of glycolysis signatures(GS)and autophagy signatures(AS)and their correlation with CSTB were analyzed using the GEO and TCGA datasets.The effects of CSTB on PDAC cell proliferation,glycolysis,and autophagy were determined by in vitro and in vivo assays.We assessed competitive binding to Cathepsin B(CTSB)between CSTB and Cystatin C(CSTC)via immunoprecipitation(IP)and immunofluorescence(IF).Ch IP-q PCR and luciferase reporter gene assays were used to unveil the mechanism underlying CSTB upregulation.The expression pattern of CSTB was examined in PDAC clinical samples and Kras G12D/+;Trp53R172H/+;Pdx1-Cre(KPC)mice.RESULT: GS and AS were enriched and closely associated in PDAC tissues.CSTB increased autophagic flux and provided substrates for glycolysis.CSTB knockdown attenuated the proliferation of PDAC cells.The LC-MS assay indicated that CSTB interacted with CTSB and contributed to the proteolytic activity of CTSB in lysosomes.IF and IP assays demonstrated that CSTB competed with CSTC to bind to CTSB.Deletion of the key sites of CSTB abolished the interaction between CSTB and CTSB.CSTB was highly expressed in PDAC due to H3K27 acetylation and SP1 activation.CONCLUSION: Competing with CSTC,CSTB increases autophagic flux by binding to CTSB and sustaining its proteolytic activity.Unobstructed autophagic flux facilitates the supply of substrates for biosynthesis and fuels glycolysis in PDAC.