Nitration of Hsp90 Affects its Spatial Distribution and Promotes Schwannoma Cell Proliferation

Abstract

Neurofibromatosis Type 2 is a genetic disorder characterized by the development of schwannomas throughout the nervous system caused by loss of merlin tumor suppressor activity. Solid tumors develop in an oxidative environment due to production of oxidants such as peroxynitrite by tumor and infiltrating immune cells. We discovered peroxynitrite production and subsequent protein tyrosine (Y) nitration support schwannoma cell survival/proliferation. Further, Y nitration induces a metabolic reprogramming characterized by decreased oxidative phosphorylation activity, and increased glycolysis and glutaminolysis. Here, we identified the first nitrated protein supporting schwannoma cell proliferation, the molecular chaperone heat shock protein 90 (Hsp90). Out of five Y residues prone to nitration in Hsp90, we showed nitration at Y33 down‐regulates mitochondrial metabolism, while nitration at Y56 activates the ATP‐gated receptor P2X7, which increases glycolysis in tumor cells. We found schwannomas contain endogenous levels of Hsp90 nitrated at either position. Therefore, we hypothesized that nitrated Hsp90 (Hsp90NY) promotes schwannoma cell proliferation by regulating their metabolism. To investigate the role of Hsp90NY in proliferation, we increased the intracellular concentration of Hsp90NY in schwannoma cells by delivering Hsp90NY intracellularly at concentrations like those endogenously present in these cells (~6.5% of cellular Hsp90, calculated by quantitative dot blot). Doubling the intracellular concentration of Hsp90NYbut not wild‐type Hsp90 significantly increased cell proliferation 24 and 48 h post‐delivery. To establish the metabolic role of Hsp90NY, we delivered wild‐type or Hsp90NY into wild‐type Schwann cells (WT‐SC) and studied the metabolic changes by extracellular flux analysis 24 h post‐delivery. Hsp90NY decreased all parameters of mitochondrial activity, with no effect on the glycolytic rate. To test if Y33 and/or Y56 were responsible for the tumorigenic activity of Hsp90NY, we intracellularly delivered to WT‐SC either wild‐type Hsp90, or different site‐specific nitrated Hsp90 proteins produced by genetic code expansion: Hsp90 nitrated at Y33 (Hsp90NY33), at Y56 (Hsp90NY56), or at Y33 and Y56 simultaneously (Hsp90NY33+56). All three forms of Hsp90NY significantly increased WT‐SC proliferation at 24 and 48 h post‐delivery, suggesting Y33 and Y56 induce schwannoma cell proliferation independently. Using a three‐dimensional schwannoma cell culture model (tumoroids), we discovered Hsp90NY33colocalized with mitochondria and remained in the tumoroid periphery, while Hsp90NY56was also found in nuclei with a homogenous distribution throughout the tumoroid. Collectively, our results show that Hsp90, when nitrated at Y33 or Y56, gains a tumorigenic activity that supports schwannoma cell proliferation. In addition, residue‐specific nitration results in distinct localization, and potentially function, of the protein. This is the first nitrated protein shown to support tumor cell proliferation, and a potential tumor‐directed target for therapeutic development.