PROJECT SUMMARY HSPA1A is a stress-inducible seventy-kilodalton heat shock protein that plays essential roles in tumor cell survival. This protein also localizes at the cell-surface and the extracellular medium of stressed and cancer cells. HSPA1A-membrane positive tumors are resistant to radiation therapy, show increased invasiveness, and develop distant metastasis, while membrane-bound and extracellular HSPA1A exert both immunostimulatory and immunosuppressive functions. Recent research revealed that HSPA1A's membrane localization depends on its selective interaction with specific lipids and the protein's extracellular transport is mediated by several mechanisms including exosomes, secretory lysosomes, and lipid rafts. However, the trafficking of HSPA1A towards the cell surface, the relationship between membrane-bound and extracellular HSPA1A, and the specific lipid or protein modifications that trigger HSPA1A's re-localization remain unknown. To answer these fundamental questions, we propose to characterize the trafficking mechanism of HSPA1A's membrane translocation by determining the mechanism by which lipid interactions recruit HSPA1A to endosomal pathway. This objective will be achieved by determining the re-localization pattern of HSPA1A in different subcellular compartments after heat shock using subcellular fractionation and imaging, and depleting specific endosomal lipids with drugs or lipid-biosensors. Furthermore, we will determine whether HSPA1A's extracellular transport depends on its membrane localization and binding to specific lipids. To this end, we will delineate the relationship between membrane-bound and extracellular HSPA1A, and then measure extracellular HSPA1A in the presence or absence of the endo/exosomal pathway inhibitors and lipid-biosensors. Lastly, we will characterize the lipid composition alterations and post-translational modifications on HSPA1A that affect its membrane localization. To determine these changes, we will characterize the dynamics of the lipid composition in stressed-cells, using a targeted lipidomics approach, and identify the differentially expressed lipid modifying genes. Additionally, we will determine whether specific post-translational modifications on HSPA1A are responsible for the increased membrane-bound HSPA1A using a targeted proteomics approach. This proposal will provide fundamental knowledge that will form the basis for future interventions to control membrane- associated and extracellular HSPA1A aiming to decrease tumor survival and increase the immune response. Furthermore, this project will train multiple non-traditional and first-generation undergraduate and master level students, and will prepare them for diverse careers in science.

PROJECT NARRATIVE The research described in this proposal focuses on understanding how HSPA1A localizes at the cell surface and outside of stressed and cancer cells. The characterization of this mechanism will allow devising of new ways to render these cells sensitive to conventional therapies and activate the immune system against them.