The autophagy inducer trehalose stimulates macropinocytosis in NF1-deficient glioblastoma cells

BACKGROUND: Glioblastoma is a highly aggressive brain tumor. A big effort is required to find novel molecules which can cross the blood–brain barrier and efficiently kill these tumor cells. In this perspective, trehalose (α-glucopyranosyl‐[1→1]‐α‐d‐glucopyranoside), found in various dietary sources and used as a safe nutrient supplement, attracted our attention for its pleiotropic effects against tumor cells. METHODS: Human glioblastoma cell lines U373-MG and T98G were exposed to trehalose and analyzed at different time points. Cell proliferation was evaluated at medium term, and clonogenic capacity and cell morphology were evaluated at long term. Western blot was used to evaluate biochemical markers of autophagy (also measured in cells co-treated with EIPA or chloroquine), and mTOR, AMPK and ERK 1/2 signalling. Macropinocytosis was evaluated morphologically by bright-field microscopy; in cells loaded with the fluorescein-conjugated fluid-phase tracer dextran, macropinocytic vacuoles were also visualized by fluorescence microscopy, and the extent of macropinocytosis was quantified by flow cytometry. RESULTS: The long-term effect of trehalose on U373-MG and T98G cell lines was impressive, as indicated by a dramatic reduction in clonogenic efficiency. Mechanistically, trehalose proved to be an efficient autophagy inducer in macropinocytosis-deficient T98G cells and an efficient inducer of macropinocytosis and eventual cell death by methuosis in U373-MG glioblastoma cells, proved to be poorly responsive to induction of autophagy. These two processes appeared to act in a mutually exclusive manner; indeed, co-treatment of U373-MG cells with the macropinocytosis inhibitor, EIPA, significantly increased the autophagic response. mTOR activation and AMPK inhibition occurred in a similar way in the two trehalose-treated cell lines. Interestingly, ERK 1/2 was activated only in macropinocytosis-proficient U373-MG cells harbouring loss-of-function mutations in the negative RAS regulator, NF1, suggesting a key role of RAS signalling. CONCLUSIONS: Our results indicate that trehalose is worthy of further study as a candidate molecule for glioblastoma therapy, due to its capacity to induce a sustained autophagic response, ultimately leading to loss of clonogenic potential, and more interestingly, to force macropinocytosis, eventually leading to cell death by methuosis, particularly in tumor cells with RAS hyperactivity. As a further anticancer strategy, stimulation of macropinocytosis may be exploited to increase intracellular delivery of anticancer drugs.