Abstract 1258: 27-Hydroxycholesterol impairs lysosomal integrity in myeloid immune cells, resulting in enhanced secretion of cancer promoting extracellular vesicles

Background: Breast cancer is the most common cancer among women in the United States, with elevated circulating cholesterol as an established risk factor for the onset and progression to metastatic disease. 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. Methods and Results: Immunofluorescence microscopy experiments revealed that 27HC increases the size and number of CD63+ multivesicular bodies (MVBs) but not 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 did observe 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 stained the lysosomes with a pH sensing fluorescent dye and observed a decrease in fluorescent intensity, indicating an increase in lysosomal pH. In addition, treatment with 27HC increased levels of cathepsin B in cells. Treatment of cells with Bafilomycin A1, a lysosomal proton pump inhibitor phenocopies the 27HC mediated increase in EV secretion and co-treatment of cells with Bafilomycin A1 and 27HC has the same effect. 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. Conclusion: 27HC increases the size of multivesicular bodies, impairs lysosomal integrity, and alters levels of ISG- and NEDDylated proteins in cells. These data suggest that 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). Citation Format: Anasuya Das Gupta, Natalia Krawczynska, Hashni Epa Vidana Gamage, Hannah Kim, Jaena Park, Janet E. Sorrells, Stephen A. Boppart, Erik R. Nelson. 27-Hydroxycholesterol impairs lysosomal integrity in myeloid immune cells, resulting in enhanced secretion of cancer promoting extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1258.