Transactivation of the Epidermal Growth Factor Receptor by Heat Shock Protein 90 via Toll-like Receptor 4 Contributes to the Migration of Glioblastoma Cells

Abstract

Extracellular heat shock protein HSP90␣ was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90␣ favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90␣ rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca 2؉ appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90␣-induced Ca 2؉ oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P 2 inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl ␤-cyclodextrin impaired the Ca 2؉ rise induced by extracellular HSP90␣ combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90␣-induced Ca 2؉ signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90␣ without affecting Ca 2؉ oscillations observed with EGF. Extracellular HSP90␣ induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase C␦ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90␣ transactivates EGFR/ErbB1 through TLR4 and a PKC␦/c-Src pathway, which induces ATP release and cytosolic Ca 2؉ increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.Glioma ranges from slowly growing low grade tumors to rapidly growing high grade tumors, including anaplastic astrocytoma and glioblastoma (1, 2). High grade gliomas include anaplastic tumor cells, necrotic foci, and rich vascularity, due largely to the aberrant expression of angiogenic factors by tumor cells (3). Tumor cells are highly proliferative and invasive within the brain. Despite the development of various treatments, the life expectancy of patients remains poor (4).One of the molecules that could contribute to invasion is the stress or heat shock protein 90 (HSP90). In several tumor types, cell surface expression of HSP90 correlates with metastatic potential (5), and its inhibition with antibodies (6, 7) or with cell-impermeable inhibitors (8) reduces cell migration in vitro. Of the two HSP90 isoforms, only HSP90␣ has been described extracellularly, which argues against cell lysis as the source of extracellular HSP90 (8). The metalloprotease MMP-2 can be associated with extracellular HSP90␣ (8), which is favored by acetylation of the stress protein (9). Surface HSP90␣ also participates in extracellular matrix proteininduced c-Src/integrin association and reorganization of the actin cytoskeleton (10). The cell-impermeable inhibitor of HSP90, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG)-N-oxide, displays anti-invasive and anti-meta...

Renal cortical hexokinase and pentose phosphate pathway activation through the EGFR/Akt signaling pathway in endotoxin-induced acute kidney injury

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TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells

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