Inhibiting Heat Shock Protein 90 (HSP90) Limits the Formation of Liver Cysts Induced by Conditional Deletion of Pkd1 in Mice

Polycystic liver disease (PLD) occurs in 75-90% of patients affected by autosomal dominant polycystic kidney disease (ADPKD), which affects 1: 400-1,000 adults and arises from inherited mutations in the PKD1 or PKD2 genes. PLD can lead to bile duct obstructions, infected or bleeding cysts, and hepatomegaly, which can diminish quality of life. At present, no effective, approved therapy exists for ADPKD or PLD. We recently showed that inhibition of the molecular chaperone heat shock protein 90 (HSP90) with a small molecule inhibitor, STA-2842, induced the degradation of multiple HSP90-dependent client proteins that contribute to ADPKD pathogenesis and slowed the progression of renal cystogenesis in mice with conditional deletion of Pkd1. Here, we analyzed the effects of STA-2842 on liver size and cystic burden in Pkd(-/-) mice with established PLD. Using magnetic resonance imaging over time, we demonstrate that ten weeks of STA-2842 treatment significantly reduced both liver mass and cystic index suggesting selective elimination of cystic tissue. Pre-treatment cystic epithelia contain abundant HSP90; the degree of reduction in cysts was accompanied by inhibition of proliferation-associated signaling proteins EGFR and others, and induced cleavage of caspase 8 and PARP1, and correlated with degree of HSP90 inhibition and with inactivation of ERK1/2. Our results suggest that HSP90 inhibition is worth further evaluation as a therapeutic approach for patients with PLD

Changes in protein expression and activation of heat shock proteins associated with HSP90 inhibition.

<p>(<b>A</b>) Quantification of HSP90α and HSP70 expression levels among the <i>Pkd1</i>^–/– groups. <i>n</i> = 6–8 mice. * indicates comparisons to <i>Pkd1</i>^–/–, vehicle-treated mice: **, <i>P</i>≤0.01; ***, <i>P</i>≤0.001. (<b>B–E</b>) Negative correlations between HSP70, HSP90α expression in the drug treatment groups and (<b>B, D</b>) cyst volume/region of interest (ROI) (<i>P</i> = 0.0046 for HSP70 and <i>P</i> = 0.0136 for HSP90) and (<b>C, E</b>) liver weight/body weight (lw/bw) ratios (<i>P</i> = 0.0141 for HSP70 <i>P</i> = 0.0138 for HSP90). Dots represent individual mice; lines represent linear regression functions. <i>n</i> = 6–8 mice.</p

HSP90α is upregulated in epithelial cells lining liver cysts.

<p>(<b>A, B</b>) Representative hematoxylin stained liver sections with immunohistochemical detection of HSP90α (brown) from three (<b>A</b>) wild type (wt) and (<b>B</b>) <i>Pkd1</i>^–/– mice. bd  =  bile duct, pv  =  portal vein, c  =  cyst. Scale bars  = 100 µm; magnification, 20x.</p

HSP90 inhibition reduces late stages of liver cyst formation in conditional <i>Pkd1</i>^–/– mice.

<p>(<b>A</b>) Schematic outlining the timetable of the study. (<b>B</b>) Representative MRI images of mice, imaged at 0, 5, and 10 weeks of treatment with vehicle or drug (STA-2842) in <i>wt</i> (+) or <i>Pkd1</i>^–/– (–) mice. Scale bars  = 0.5 cm. (<b>C</b>) Cyst volume estimated as a percentage of hepatic tissue at 0, 5, and 10 weeks of treatment of <i>Pkd1</i>^–/– or wt mice. <i>n</i> = 6–8 mice. * indicates comparisons to <i>Pkd1</i>^–/–, vehicle-treated mice: *, <i>P</i>≤0.05. All data were graphed as mean ± standard error of the mean (SEM). No cysts were detected in any wt mice.</p

Changes in protein expression and activation of signaling proteins associated with HSP90 inhibition.

<p>(<b>A</b>) Differences in expression of phosphorylated Y¹⁰⁶⁸ and Y¹¹⁷³ EGFR and total EGFR normalized to actin, and Y¹⁰⁶⁸ and Y¹¹⁷³ EGFR normalized to total EGFR, following treatment with the indicated doses of STA-2842. *, P<0.05; **, P<0.01. (<b>B</b>) Insignificant differences in expression of phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 or total ERK1/2, normalized to tubulin, and phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 normalized to total ERK1/2, following treatment with the indicated doses of STA-2842; differences are not significant (<b>C</b>) Positive correlations between the ratio of phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 normalized to total ERK1/2 expression in the drug treatment groups and (left) cyst volume/region of interest (ROI) (<i>P</i> = 0.174) and (right) liver weight/body weight (lw/bw) ratio (<i>P</i> = 0.040). Dots represent individual mice; lines represent linear regression functions. <i>n</i> = 6–8 mice. All data graphed as mean ± standard error of the mean (SEM). (<b>D</b>) Expression of phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 in cystic epithelia (left) or total liver (right) quantified from immunohistochemical staining of liver sections from <i>Pkd1</i>^–/– mice treated with vehicle or 100 mg/kg STA-2842 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114403#pone.0114403.s004" target="_blank">Fig S4</a>). **, P<0.01. (<b>E</b>) Differences in expression of phosphorylated phS²³⁵/S²³⁶ S6 or total S6, normalized to tubulin, and phosphorylated phS²³⁵/S²³⁶ S6 normalized to total S6, following treatment with the indicated doses of STA-2842; *, P<0.05. <i>P-</i>values for comparisons between <i>Pkd1</i>^–/– vehicle- versus 100 mg/kg-treated groups bordering on significant (<i>P</i> = 0.060). (<b>F</b>) Differences in expression of phosphorylated phS⁴⁷³ AKT or total AKT, normalized to tubulin, and phosphorylated phS⁴⁷³ AKT normalized to total AKT, following treatment with the indicated doses of STA-2842; *, P<0.05.</p

Histopathological development of cysts.

<p>(<b>A</b>) Representative livers collected from <i>Pkd1</i>^–/– or <i>wt</i> mice treated with vehicle or STA-2842 at the indicated doses, at 6.5 months of age. (<b>B</b>) Liver weights (lw) to body weight (bw) ratio of in <i>wt</i> (+) or <i>Pkd1</i>^–/– (–) mice after 10 weeks of treatment with STA-2842 (50, 100) or vehicle (–). <i>n</i> = 5–8 mice. * indicates comparisons to <i>Pkd1</i>^–/–, vehicle-treated mice; # indicates comparisons to <i>wt</i> vehicle-treated mice: *, <i>P</i><0.05; ^##, <i>P</i><0.01; ^###, <i>P</i><0.001; ^####, <i>P</i><0.0001. (<b>C</b>) Representative H&E stained liver sections after 10 weeks of treatment with vehicle or drug in <i>wt</i> (+) or <i>Pkd1</i>^–/– (–) mice, indicating extent of cystogenesis. Scale bars  = 100 µm; magnification  = 10x. (<b>D</b>) Cystic indices quantified as a percentage of grid intersections that cross cysts on H&E slides. <i>n</i> = 6–8 mice. * indicates comparisons to <i>Pkd1^–/–</i>, vehicle-treated mice: *, <i>P</i><0.05. All data graphed as mean ± standard error of the mean (SEM). (<b>E</b>) Relative change in cyst burden by drug dose (6 month value divided by 4 month value), with simple linear fits of the relationship shown. None of the slopes were statistically significant (p>0.11 in all three cases). Since the range of values differed among the three doses, the X-axis is drawn on the log scale to better depict the relationships. <b>F</b>. Relative expression of cleaved PARP, normalized to total PARP, following treatment of <i>Pkd1</i>^–/– (–) mice with the indicated doses of STA-2842. **, <i>P</i><0.01 to vehicle-treated mice; ^##, P<0.01 relative to mice treated with 50 mg/kg STA-2842. <b>G</b>. Relative expression of cleaved caspase 8, normalized to GAPDH, following treatment of <i>Pkd1</i>^–/– (–) mice with the indicated doses of STA-2842. *, <i>P</i><0.05, ***, P<0.001 in reference to vehicle-treated mice.</p