Project Grant R01DK140310

THE LYSOSOMAL FASTING RESPONSE IN INTESTINAL STEM CELLS AND CANCER - PROJECT SUMMARY FASTING IMPROVES ORGANISMAL HEALTH AND TISSUE HOMEOSTASIS IN PART BY ENHANCING STEM CELL ACTIVITY. IN ADDITION, STEM CELLS SERVE AS A CELL-OF-ORIGIN IN MULTIPLE CANCER TYPES; HOWEVER, HOW FASTING INFLUENCES TISSUE STEM CELLS REMAINS POORLY UNDERSTOOD. HERE WE PROPOSE TO DISSECT THE ROLE OF LYSOSOMES, A DYNAMIC SIGNALING ORGANELLE THAT ORCHESTRATES AND EXECUTES KEY ASPECTS OF THE INTESTINAL STEM CELL FASTING RESPONSE AND TUMORIGENESIS. LYSOSOMES CONTROL METABOLISM IN RESPONSE TO NUTRIENT AVAILABILITY, AND THEY CONTRIBUTE TO PROTEIN AND CELLULAR HOMEOSTASIS THROUGH CLEARANCE OF AGGREGATION IN PART THROUGH ITS FUNCTION CALLED AUTOPHAGY; A CRITICAL NEED IN THE FIELD IS TO UNCOVER THE RELEVANT AUTOPHAGIC CARGOS AND METABOLITES TO UNDERSTAND THEIR REGULATORY ROLES IN CELLULAR AND ORGANISMAL PHYSIOLOGY. USING THE MAMMALIAN INTESTINE AS A PARADIGM, WE AIM TO UNDERSTAND HOW LYSOSOMES MAINTAIN INTESTINAL STEM CELLS (ISCS) AND CONTRIBUTE TO TUMORIGENESIS, AS INSIGHTS INTO THIS BIOLOGICAL PROCESS CAN HELP DEVELOP THERAPIES BENEFITING HUMAN HEALTH. HERE WE FOCUS ON TWO LARGELY REDUNDANT TRANSCRIPTION FACTORS THAT ARE IMPORTANT FOR LYSOSOMAL BIOGENESIS, CALLED TFE3 AND TFEB: OUR CENTRAL HYPOTHESIS IS THAT TFE3 AND TFEB ORCHESTRATE THE LYSOSOME FASTING RESPONSE, AND THEY PLAY A CENTRAL ROLE IN COORDINATING INTER-ORGANELLAR COMMUNICATION TO MAINTAIN ISCS UNDER THE FASTED STATE BY ENGAGING THE PPAR DRIVEN, MITOCHONDRIAL FATTY ACID OXIDATION (FAO) PATHWAY. IN AIM 1, WE WILL CHARACTERIZE THE INTESTINE-SPECIFIC DELETION PHENOTYPES OF TFE3 TFEB DOUBLE MUTANTS IN BOTH FED AND FASTED CONDITIONS AND FURTHER TEST THE NECESSITY AND SUFFICIENCY OF THESE TRANSCRIPTION FACTORS IN DRIVING THE ISC FASTING RESPONSE. IN AIM 2, WE WILL INVESTIGATE THE MECHANISM BY WHICH LYSOSOMES SUPPORT ISCS BY EMPLOYING MULTIPLE MOLECULAR APPROACHES. FIRST, WE WILL DEFINE THE TRANSCRIPTION NETWORK ORCHESTRATED BY TFE3 AND TFEB. NEXT, WE WILL ISOLATE LYSOSOMES FROM AD LIBITUM OR FASTED ISCS TO DISCOVER METABOLOMIC AND PROTEIN CONTENTS. THE ADVANCE OF OUR APPROACH IS THE USE OF A RAPID LYSOSOMAL PURIFICATION METHOD (LYSOIP) THAT WE RECENTLY ADOPTED TO ENABLE CELL-TYPE SPECIFIC METABOLOMIC AND PROTEOMIC PROFILING FROM IN VIVO TISSUES IN A CELL- TYPE SPECIFIC MANNER. FURTHER, WE WILL INTERROGATE THE REGULATORY RELATIONSHIP BETWEEN TFE3, TFEB AND MITOCHONDRIAL FAO, A PATHWAY THAT WE PREVIOUSLY IDENTIFIED AS ESSENTIAL FOR ISCS MAINTENANCE IN FASTED CONDITIONS. IN AIM 3, WE WILL FOCUS ON UNDERSTANDING THE ROLE OF LYSOSOMES IN EARLY INTESTINAL TUMORIGENESIS. TO THIS END, THE WELL-ESTABLISHED APC MODEL OF INTESTINAL TUMORIGENESIS AND ORTHOTOPIC TRANSPLANTATION MODELS WILL BE USED TO ASK IF TFE3 AND TFEB CAN IMPACT INTESTINAL TUMOR INITIATION OR PROGRESSION. IN ADDITION, WE WILL PERFORM LYSOIP FROM PRIMARY TUMORS TO UNCOVER THEIR LYSOSOMAL CONTENTS. FINALLY, TO INVESTIGATE WHETHER THE LYSOSOMAL FASTING RESPONSE CAN BE SYNERGISTICALLY UTILIZED WITH CONVENTIONAL CHEMOTHERAPY, WE WILL PERFORM INTERMITTENT FASTING (IF) ON INTESTINAL ADENOMAS AND ASK WHETHER ESTABLISHED TUMORS SUBJECTED TO IF REGIMEN ARE MORE SENSITIZED TO AUTOPHAGY INHIBITION. BROADLY, THE PROPOSED RESEARCH WILL SHED INSIGHTS INTO STEM CELL FUNCTION, TUMORIGENESIS, AND MECHANISMS OF LYSOSOMAL REGULATION IN THE PHYSIOLOGICAL CONTEXT.