Lysosomal Rerouting of Hsp70 Trafficking as a Potential Immune Activating Tool for Targeting Melanoma

Abstract

Tumor specific cell surface localization and release of the stress inducible heat shock protein 70 (Hsp70) stimulate the immune system against cancer cells. A key immune stimulatory function of tumor-derived Hsp70 has been exemplified with the murine melanoma cell model, B16 overexpressing exogenous Hsp70. Despite the therapeutic potential mechanism of Hsp70 transport to the surface and release remained poorly understood. We investigated principles of Hsp70 trafficking in B16 melanoma cells with low and high level of Hsp70. In cells with low level of Hsp70 apparent trafficking of Hsp70 was mediated by endosomes. Excess Hsp70 triggered a series of changes such as a switch of Hsp70 trafficking from endosomes to lysosomes and a concomitant accumulation of Hsp70 in lysosomes. Moreover, lysosomal rerouting resulted in an elevated concentration of surface Hsp70 and enabled active release of Hsp70. In fact, hyperthermia, a clinically applicable approach triggered immediate active lysosomal release of soluble Hsp70 from cells with excess Hsp70. Furthermore, excess Hsp70 enabled targeting of internalized surface Hsp70 to lysosomes, allowing in turn heat-induced secretion of surface Hsp70. Altogether, we show that excess Hsp70 expressed in B16 melanoma cells diverts Hsp70 trafficking from endosomes to lysosomes, thereby supporting its surface localization and lysosomal release. Controlled excess-induced lysosomal rerouting and secretion of Hsp70 is proposed as a promising tool to stimulate anti-tumor immunity targeting melanoma.

Cell surface localised Hsp70 is a cancer specific regulator of clathrin‐independent endocytosis

“…In agreement with our hypothesis, a remarkable increase in cluster size from small (⩽300 nm 2 , equivalent circular ø ∼20 nm) and medium (>300 nm 2 , <4000 nm 2 ) to large clusters (⩾4000 nm 2 , equivalent circular ø ∼70 nm) was observed as a result of Hsp70 overexpression. The larger cluster sizes of sHsp70 were also paralleled with a twofold increase in the surface density of Hsp70 on Hsp70 overexpressing cells compared to control (seeTable E1), matching our previous data acquired by flow cytometry[9].…”

“…Given that Hsp70 is frequently upregulated in tumour cells, where it is specifically present on the tumour cell surface, and that derailed endocytosis is a feature of cancer[8], we hypothesized that sHsp70 may have a general impact on the endocytic process. We previously showed that overexpression of Hsp70 in B16 mouse melanoma cells gave rise to the concentration of sHsp70[9]. Here we show for the first time, using the B16 model and human A375 melanoma cells, that high levels of Hsp70 facilitate CIE even at unstressed conditions.…”

“…To determine whether Hsp70 expression levels generally have an impact on the endocytic activity in unstressed cells, we used stably transfected tetracycline‐inducible systems for Hsp70 overexpression in mouse B16 and human A375 melanoma cells (Hsp70)[9]. Cells stably transfected with empty vector served as control (ctrl).…”

“…1D), indicating that substrate binding of Hsp70 may not be necessary for Hsp70‐mediated endocytosis. Since the Hsp70‐antibody adduct retains mobility of sHsp70[9], we next targeted sHsp70 by anti‐Hsp70 antibody (cmHsp70.1) immobilised on bead and well surfaces (see drawing inFig. 1E).…”

Key role of lipids in heat stress management

“…Membrane alterations, signal pathways from membranes to hsp genes and Hsps themselves play fundamental roles in the aetiology of several human diseases, such as cancer or type 2 diabetes [6]. For therapeutical purposes the membrane lipid structure can be directly modified by a membrane‐intercalating drug or by pharmacological modulation of enzymes involved in the metabolism of membrane lipids [5,6,11–13,75,77,140–144].…”