Nitrated Hsp90 Supports Glioblastoma Multiforme Cell Survival and Migration

Abstract

Glioblastoma multiforme (GBM) is the most aggressive malignancy affecting the central nervous system. With no effective pharmacological options, patients undergo repeated surgeries to remove or debulk tumors. New targets for the development of safe and effective therapies are desperately needed. We and others have shown that solid tumors present high levels of protein tyrosine nitration, an oxidative post‐translational modification that occurs in pathological conditions involving oxidative stress but is absent in normal tissues. In many solid tumors tyrosine nitration correlates with poor prognosis, suggesting nitrated proteins may benefit tumor growth. We have previously shown that tyrosine nitration supports survival and migration of GBM cells. Here, we identified the first nitrated protein with pro‐tumorigenic activity, nitrated Heat shock protein 90 (Hsp90). Hsp90 is an essential molecular chaperone present in all cells. We showed that nitration of Hsp90 induces a gain‐of‐function that decreases mitochondrial metabolism in tumor cells. We detected endogenously nitrated Hsp90 (NO2Hsp90) in GBM‐patient derived cell culture models, and in 2D‐ and 3D‐ U87 cell culture models of GBM. To study the role of NO2Hsp90 in supporting GBM cell survival/proliferation, we prevented tyrosine nitration in U87 cells for 48 h to decrease the endogenous levels of nitrated proteins. In these conditions, cell viability decreases by ~25%, while the levels of nitrated proteins decrease by ~50%. We then intracellularly released NO2Hsp90 at levels comparable to those normally present in U87 cells (~1.5% of total cellular Hsp90 calculated by quantitative slot blot), and sustained prevention of endogenous tyrosine nitration for an additional 48 h. Cell viability was assessed at this final timepoint. If NO2Hsp90 is necessary and sufficient to support U87 cell survival/proliferation, intracellular release of the nitrated chaperone to cells containing decreased levels of nitrated proteins should rescue cell viability. Release of unmodified (UM) Hsp90 was used as control. Excitingly, the intracellular release of NO2Hsp90 but not UM Hsp90 significantly increased U87 cell survival/proliferation by ~50%. Intracellular release of NO2Hsp90 to untreated cells that contain endogenous NO2Hsp90 had no effect on cell proliferation but to our surprise, the extracellular release of NO2Hsp90 significantly increased cell numbers after 48 h incubation. This effect was significantly hindered by preventing endogenous tyrosine nitration, suggesting that intracellular nitrated proteins mediate the effect of extracellular NO2Hsp90. It is well described that extracellular Hsp90 (eHsp90) induces GBM motility and invasion. We detected eHsp90 in culture media conditioned by U87 cells by western blot. Using live transwell migration assays, we found that incubation with either extracellular UM‐ or NO2Hsp90 increased U87 cell migration versus control cells that did not receive protein. These results suggest that NO2Hsp90 plays multiple essential roles in GBM biology, posing NO2Hsp90 as a promising tumor‐directed therapeutic target.