THU482 The Cholesterol Metabolite, 27-hydroxycholesterol, Enhances The Secretion Of Cancer Promoting Extracellular Vesicles By Impairing Lysosomal Integrity

Disclosure: A. Das Gupta: None. N. Krawczynska: None. H. Vidana Gamage: None. H. Kim: None. J. Park: None. J.E. Sorrells: None. S.A. Boppart: None. E.R. Nelson: None. Breast cancer is a highly prevalent cancer among women and mortality is typically associated with metastatic disease. Elevated circulating cholesterol has been associated with breast cancer recurrence in pre-clinical as well as clinical models. The cholesterol metabolite, 27-hydroxycholesterol (27HC), has been identified to establish an immunosuppressive tumor microenvironment via Liver X Receptor (LXR) in myeloid immune cells. Interestingly, 27HC treatment of different myeloid immune cell types results in increased secretion of extracellular vesicles (EVs) with altered cargo. Importantly, EVs from 27HC treated myeloid cells promote tumor growth and metastasis in murine models of breast cancer. Given that these altered EVs are involved in cancer progression, it is imperative that we understand how their biogenesis and secretion are regulated. The formation of EVs involves maturation of endosomes to form multivesicular bodies containing intraluminal vesicles. These MVBs are either targeted to the lysosomes for degradation or directed towards the plasma membrane to release EVs. Immunofluorescence microscopy experiments revealed that 27HC increases the size and number of CD63+ multivesicular bodies (MVBs) but does not alter EEA1+ endosomes in RAW 264.7 cells. Quantitative PCR analysis revealed no significant changes in mRNA expression of genes associated with EV biogenesis machinery. However, we observed that 27HC increased the size and number of LAMP1+ lysosomes. This led to the hypothesis that 27HC increases EV secretion by skewing MVBs away from lysosomal degradation and towards secretion as EVs by i) impairing lysosomal integrity, and/or ii) altering the post-translational modifications on the MVBs. To study the effect of 27HC on lysosomal function, we found that 27HC increased in lysosomal pH, suggesting a loss of lysosomal integrity. Treatment of cells with a lysosomal proton pump inhibitor phenocopies the 27HC mediated increase in EV secretion. Post-translational modifications such as ISGylation and NEDDylation have been previously implicated in modulating EV secretion and cargo, and we have observed that 27HC decreases overall ISGylation and NEDDylation of proteins in cells. Thus, 27HC increases the size of multivesicular bodies, impairs lysosomal integrity, and alters levels of ISG- and NEDDylated proteins in cells, and a combination of these modulations leads to the increase in EV secretion. Collectively, our studies reveal novel mechanisms by which EV secretion is modulated by 27HC. By extension, this axis represents a novel avenue for intervening in EV secretion and ultimately in the development of breast cancer therapeutics. Funding: Department of Defense Breast Cancer Research Program Era of Hope Scholar Award (BC200206), and National Cancer Institute (R01CA234025). Presentation: Thursday, June 15, 2023