Abstract 1093 The role of heat-induced lipid changes in the plasma membrane localization of HSPA1A

Heat shock protein A1A (HSPA1A) translocates to the plasma membrane (PM) of cancer cells, enhancing tumor aggressiveness and resistance to therapeutic interventions. Consequently, targeting the localization of membrane-bound HSPA1A (mHSPA1A) holds significant therapeutic potential. Although the role of PM-localized phosphatidylserine (PS) and phosphatidylinositol-4-phosphate PI(4)P) in facilitating mHSPA1A translocation is well established, the triggers initiating this movement remain elusive. Our hypothesis centers on the notion that stress-induced alterations in cellular lipids drive the localization of mHSPA1A. To test this hypothesis, we (i) monitored the movement of HSPA1A to the PM in response to heat shock using confocal microscopy and western blotting; (ii) determined changes in phospholipid composition, employing biosensor imaging and lipidomics; (iii) assessed whether the manipulation of PM phospholipids had an impact on HSPA1A localization; and (iv) explored the relationship between HSPA1A and the patterns of PM phospholipids. Our findings revealed a complex interplay between HSPA1A and the PM, with maximal embedding in PS and PI(4)P-enriched membranes occurring at the PM approximately 8 hours post-heat shock. Additionally, our experiments demonstrated that the inhibition of PS and PI(4)P biosynthesis, achieved through fendiline and GSKA1 or RNAi targeting key biosynthetic enzymes, significantly reduced HSPA1A PM localization at the 8-hour time point. Furthermore, the alterations in PS and PI(4)P levels at the PM, which initially increased in response to heat shock but declined by the 8-hour mark, suggest a central regulatory role for these lipids in governing the dynamic movement patterns of HSPA1A to the PM. While ongoing investigations aim to establish causality, our present findings underscore the intricate interplay between altered phospholipid dynamics and the presence of mHSPA1A, both of which exert substantial influence on key oncogenic processes within cancer cells. Funds from NIH supported this project