Abstract B052: Defining the lysosome proteome during tumor evolution

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.