PROJECT SUMMARY/ABSTRACT Cancer cells constantly fight and win a myriad of battles brought on by their surrounding microenvironment in the form of hypoxia, nutrient shortage, immunological attack and anti-cancer agents. Key to this resilience is the extraordinary ability of cancer cells to rapidly evolve their internal composition and organization through a combination of genetic, epigenetic and posttranslational mechanisms. However, after decades of studies, our knowledge of the mechanisms that mediate cancer cell remodeling remains fragmentary. Most importantly, a major challenge is to map the sequence of events that punctuate the life cycle of a tumor as it evolves from early to late stage disease within its host organism. The centerpiece of this proposal is the development of a novel genetically engineered mouse model (`iMAOS' - in vivo Mass spectrometry Analysis of Organelle Substrates), that enables tracking of changes in protein composition in a tumor cell as it progresses through sequential stages of malignant transformation within its host tissue. Specifically, iMAOS will allow us to test the role of lysosome-mediated degradation as a novel, previously unrecognized driving force in cancer proteome remodeling. Our recent work has challenged the long-held perception of the lysosome as a cellular `dead end' devoid of regulation and incapable of imparting substrate selectivity. Instead we have shown that autophagosomes (specialized organelles that mediate `self-catabolism') and lysosomes are subject to sophisticated transcriptional co-regulation in highly aggressive pancreatic ductal adenocarcinoma (PDA) cells, leading to increased tumorigenesis. Moreover, we found that lysosomes selectively digest vast quantities of protein in preference to lipid, carbohydrate or nucleic acid species in PDA cells. These results imply that a key function of the autophagy-lysosome system is to profoundly remodel the proteome of PDA cells during their transition from a normal to malignant state. However, identifying the protein species targeted for lysosome-mediated removal in a stage-specific manner, is unsuited to conventional proteomics-based approaches, which measure overall changes in cellular protein content at the cost of spatial and often temporal detail. In contrast, our new genetic model of pancreatic cancer will enable stage-specific and site-specific identification of proteins targeted for degradation through incorporation of cancer cell-specific fluorescent probes and affinity handles for organelle purification and quantitative proteomics analysis. We will capture alterations in the PDA proteome as it evolves in a living host in two dimensions 1) temporal analysis during the course of tumor progression from early to late stage disease and 2) spatial analysis comparing whole cell proteomic changes to those specific to an organelle compartment ? the lysosome ? using a single mouse model system. By systematically combining time-resolved lysosomal proteomics with high throughput functional screens, we will begin to dissect the key pathways and players that enable selective protein turnover during PDA development and thus identify new therapeutically relevant targets for drug development. More broadly, we anticipate that our elegant model system for simultaneous cell isolation and organelle profiling in vivo will be a powerful tool in the study of normal homeostatic processes and additional disease settings where alterations in proteostasis and lysosome function have been implicated. These include normal states of tissue differentiation and developmental, as well as in neurodegenerative disease, lysosome storage disorders, other cancers and aging, thus providing unprecedented insights into the role and regulation of lysosomal catabolism in vivo and highlighting the value and applicability of our proposal to the broader scientific community.