Targeting heat shock protein 90 for the treatment of malignant pheochromocytoma.

Metastatic pheochromocytoma represents one of the major clinical challenges in the field of neuroendocrine oncology. Recent molecular characterization of pheochromocytoma suggests new treatment options with targeted therapies. In this study we investigated the 90 kDa heat shock protein (Hsp90) as a potential therapeutic target for advanced pheochromocytoma. Both the first generation, natural product Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), and the second-generation synthetic Hsp90 inhibitor STA-9090 (ganetespib) demonstrated potent inhibition of proliferation and migration of pheochromocytoma cell lines and induced degradation of key Hsp90 clients. Furthermore, ganetespib induced dose-dependent cytotoxicity in primary pheochromocytoma cells. Using metastatic models of pheochromocytoma, we demonstrate the efficacy of 17-AAG and ganetespib in reducing metastatic burden and increasing survival. Levels of Hsp70 in plasma from the xenograft studies served as a proximal biomarker of drug treatment. Our study suggests that targeting Hsp90 may benefit patients with advanced pheochromocytoma

Ganetespib inhibits proliferation of primary human pheochromocytoma cells.

<p>Bar graphs represent cell counts of tyrosine hydroxylase (TH)-positive first passage primary human pheochromocytoma cells treated with ganetespib. Control indicates untreated cells; vehicle indicates DMSO treatment; the numbers under each bar indicate the ganetespib concentration (micromolar).</p

Ganetespib inhibits tumor metastasis in a spontaneous metastatic pheochromocytoma model.

<p>A) Efficacy of ganetespib in an MTT-luc subcutaneous model of pheochromocytoma. Quantification of tumor growth was determined by luciferase measurement in an IVIS instrument. Data are plotted as mean photon counts (total flux) from regions of interest (ROI). Error bars represent standard deviations. B) Primary tumors excised from the two groups were compared. C and D) <i>Ex vivo</i> bioluminescence signals from lung and liver were compared between the two groups and graphed as total flux as a method of quantification of metastatic burden at the end of the experiment (*p<0.05).</p

17-AAG inhibits tumor metastasis in a luciferase metastatic pheochromocytoma model.

<p>A) Efficacy of 17-AAG in an MTT-luc experimental (tail vein) metastasis model. Representative images of animals (n = 6) treated three times a week with 17-AAG over a period of 5 weeks and a control group treated with vehicle alone. B) Quantification of metastatic tumor growth as determined by luciferase measurement in an IVIS instrument. Data are plotted as mean photon counts (total flux) from regions of interest (ROI). Error bars represent standard deviations. C) 17-AAG increases Hsp70 plasma levels <i>in vivo</i> (p = 0.035). D) Kaplan-Meier survival analysis: outcome as survival time was graphed and analyzed for statistical significance using GraphPad; control (vehicle alone; black line) and 17-AAG treated animal (red line) are represented as a function of percent survival. Using a log-rank (Mantel-Cox) test the two groups were statistically different at p<0.0001.</p

17-AAG and ganetespib inhibit cellular proliferation in pheochromocytoma cell lines. A and B) MPC cells, C and D) MTT cells, and E and F) PC12 cells were treated with different concentrations of 17-AAG or ganetespib (from 1 nM to 1 µM) and incubated for 48 hours.

<p>The graph represents the dose-response of log 10 concentrations of 17-AAG or ganetespib versus signal intensity in a thiazolyl blue formazan (MTT)-based assay.</p

Incubation with 17-AAG or ganetespib inhibits cellular migration in pheochromocytoma MTT cells.

<p>Inhibition of migration by 17-AAG-treated (A) or ganetespib -treated (B) MTT cells in a Transwell assay in the presence of serum. Mean number of migrated cells per microscopic field (at 10× magnification) was determined from triplicate wells and is represented in this graph: 17-AAG (C) and ganetespib (D); error bars represent the standard deviation of triplicate determinations.</p

Characterization of Hsp90 client proteins affected by 17-AAG and ganetespib.

<p>MTT cells were treated with different doses of 17-AAG (A) or ganetespib (B) (0.1, 0.3, and 1 µM 17-AAG for 20 hours; 10 nM and 100 nM ganetespib for 20 hours) and total cell lysates were analyzed by western blotting. Control cells (0) were treated with DMSO vehicle alone for the same time. 17-AAG and ganetespib induce PARP cleavage in pheochromocytoma MTT cells. MTT cells were cultured with the indicated amount of 17-AAG (C) or ganetespib (D) for 20 hours and total cell lysates were subjected to western blot for PARP and reblotted for actin.</p

Combination of SAHA and epirubicin in MTT cells.

<p>Combination of SAHA and epirubicin in MTT cells.</p