P01.22.B MEMBRANE-BOUND HEAT SHOCK PROTEINS: MHSP70 AS A TARGET FOR THERANOSTICS IN NEURO-ONCOLOGY

Abstract BACKGROUND Heat shock protein mHsp70 can be exposed on the surface of the plasma membrane of tumor cells. We demonstrate that mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells and can serve as a target for RAS70 peptide for tumor diagnostics. MATERIAL AND METHODS Protein expression on the cell membrane surface of tumor cell lines and primary brain tumors of adult and pediatric GBM patients was studied using FACS, live-cell confocal microscopy, and isolated lipid raft mass-spectrometry. Protein involvement in cell invasion and migration was assessed using low-molecular inhibitors (PES, JG-98) in vitro and in orthotopic glioma animal models. The target properties of the fluorescently-labeled peptide RAS70, which recognizes membrane-bound mHsp70, were assessed in primary patient tumors and in animal models. RESULTS Live-cell inverted confocal microscopy of brain tumor samples (n=32) showed mHsp70 overexpression on the membrane. Mass-spectrometry analysis of lipid rafts isolated from brain tumor cells confirmed the presence of the protein in HSP cluster, which in turn, during interactome analysis, was associated with proteins involved in cell migration and invasion (i.e., Rac1, RhoC, myosin-9). Application of small-molecule inhibitors of HSP70 (JG-98, PES) led to a significant decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of chaperone inhibitors in orthotopic brain tumors model in rodents significantly decreased tumor progression, which was accompanied by an increased overall survival. Data demonstrate that chaperone inhibitors, particularly JG-98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies. Apart from chaperone inhibitors we developed mHsp70-targeted peptide that could efficiently cross the blood-brain barrier accumulating in the glioblastoma cells in vivo. Subsequently, in the orthotopic GL261 mice and C6 rat glioma, intravenous administration of the fluorescently labeled peptide resulted in the tumor retention of agent (further confirmed by histological studies). Topical application of the mHsp70-targeted peptide (sprayed over the freshly dissected brain tissues) helped to delineate the tumors in neuro-oncological adult patients employing intraoperative fluorescent imaging system. CONCLUSION Membrane-bound mHsp70 could be used as a target for tumor theranostics. RAS70 peptide recognizing mHsp70 demonstrated high targeting properties which could be employed for the intraoperative visualization as well as for developing of a potential carrier for drug delivery.