DESTABILIZE THE GLIOMA CELL NICHE THROUGH HSP90-GRP78 NETWORK TARGETING

INTRODUCTION: Gliomas are the most common and lethal malignant tumor of the central nervous system representing 1.3% of all human cancers. Glioblastoma (GBM, WHO grade IV) is the most common astrocytic-derived brain tumor in adults, more frequent in older patients (mean age 55 years). They are characterized by a highly aggressive behavior and poor prognosis. Survival rates for gliomas are poor with a median survival that is not exceeding 14 months. Over the last years, the absence of adequate therapeutic options has led the scientific community to seek different molecular targets and bio-markers able to allow a more adequate prognostic and therapeutic stratification of patients. In fact, the absence of adequate therapeutic options makes GBM a fatal disease. A feature of gliomas is the presence of tumor necrotic foci surrounded by severely hypoxic pseudopalisading cells. This unfavorable microenvironment elicits in tumor cells compensatory pathways as autophagy and the unfolded-protein response (UPR). They are two interconnected pathways exploited by tumor cells to cope with low nutrient conditions, hypoxia or other microenvironment-generated stress. Moreover, evidences showed that tumor hypoxia fuel cell motility and preserve the so-called cancer stem cells niche (CSCs). Heat shock proteins (HSPs) are highly conserved proteins which are expressed in response to various forms of stress, in response to environmental challenges, and in specific stages of development and differentiation. Some HSPs are also expressed under non stress conditions and act as molecular chaperones. Thus, HSPs fulfill a variety of biologic functions, including the regulation of synthesis, folding, assembly, and degradation of different proteins. HSPs are constitutively expressed in human carcinomas or cancer cell lines of various histogenetic origins. The endoplasmic reticulum chaperone GRP78 is aberrantly overexpressed by human cancers and preliminary reports showed that targeting of GRP78 in glioma animal model can delay cancer growth. The high expression of HSPs in malignant glioma indicates a possible role of these proteins in resistance to cancer chemotherapy. They in fact, protect cells from many cell-damaging agents or conditions of environmental stress. AIM OF THE STUDY: Assess whether the HSP90-GRP78 axe expression in perinecrotic tumor areas and/or pseudo-palisading cells contributes in maintaining a pro-survival environment and whether it is regulated by hypoxic stimuli, plays roles in glioma cancer stem cell maintenance and in glioma cell motility. Define the contribution of molecular chaperones in glioma modulation of HSPGRP78 axe by specific drugs to provide targets for novel therapies useful to improve prognosis of patients affected by brain tumors. MATERIALS AND METHODS: Clinical series. A series of patients’ (n° 123) recruited at IRCCS Ca’ Granda between 2013-2016, with correlation of molecular profiles to clinicopathological characteristic were collected. From each patient the total RNA was isolated with Tizol® reagent (Thermo Fisher Scientific). For gene expression quantification Human TaqManAssays (Applied Biosystems) and reagents were used. Glioma cell lines. LN229, SW1088 and T98G were from ATCC and maintained as suggested by manufacturer. U251-HRE cell were a generous gift from Dr. Ottobrini. Gene knock-out experiments were carried out as transient transfection of short interfering RNA (Sigma Aldrich) using Lipofectamine3000 as transfection reagent (LifeTechnologies). Organotypic tissue cultures. Organotypic glioma tissue were generated through vibratome (VT1200 Leica Microsystems) serial cutting of fresh tumors, and slices were cultured on dedicated supports from Millipore, were cultured with or without the drugs: Temozolomide and/or Gamitrinibs. RESULTS: The expression of GRP78 and TRAP1 are increased in high-grade compared to lower grade or normal brain parenchyma and, high protein levels correlated to poor prognosis. Moreover, GRP78 appears to be enriched in perinecrotic areas of high-grade human gliomas where GRP78 overexpression correlates with HIF-1α expression. GRP78 targeting by siRNA or Gamitrinibs decreased the side population of LN229 cells as well as tumor cell motility. The hypoxia regulator HIF1α was decreased in TRAP1 knock-out cells respect to control. Lastly, an increase in apoptosis gene was evidenced after treatment with drugs. Finally, Gamitrinibs treatment of glioma organotypic cultures significantly affected tumor cell viability respect to Temozolomide. CONCLUSIONS: These data suggest that GRP78 is a poor prognostic marker for high grades gliomas and HSPs are enriched in peri-necrotic tumor areas sustaining a pro-survival environment. Moreover, these data suggest that GRP78 overexpression in brain tumors could sustain the stem cell population in stress conditions such as hypoxia. This study confirms the potential role of GRP78 as a molecular target in the treatment of malignant glioma.