Hsp90 and PKM2 Drive the Expression of Aromatase in Li-Fraumeni Syndrome Breast Adipose Stromal Cells

Abstract

Li-Fraumeni syndrome (LFS) patients harbor germ line mutations in the TP53 gene and are at increased risk of hormone receptor-positive breast cancers. Recently, elevated levels of aromatase, the rate-limiting enzyme for estrogen biosynthesis, were found in the breast tissue of LFS patients. Although p53 down-regulates aromatase expression, the underlying mechanisms are incompletely understood. In the present study, we found that LFS stromal cells expressed higher levels of Hsp90 ATPase activity and aromatase compared with wild-type stromal cells. Inhibition of Hsp90 ATPase suppressed aromatase expression. Silencing Aha1 (activator of Hsp90 ATPase 1), a co-chaperone of Hsp90 required for its ATPase activity, led to both inhibition of Hsp90 ATPase activity and reduced aromatase expression. In comparison with wild-type stromal cells, increased levels of the Hsp90 client proteins, HIF-1α, and PKM2 were found in LFS stromal cells. A complex comprised of HIF-1α and PKM2 was recruited to the aromatase promoter II in LFS stromal cells. Silencing either HIF-1α or PKM2 suppressed aromatase expression in LFS stromal cells. CP-31398, a p53 rescue compound, suppressed levels of Aha1, Hsp90 ATPase activity, levels of PKM2 and HIF-1α, and aromatase expression in LFS stromal cells. Consistent with these in vitro findings, levels of Hsp90 ATPase activity, Aha1, HIF-1α, PKM2, and aromatase were increased in the mammary glands of p53 null versus wild-type mice. PKM2 and HIF-1α were shown to co-localize in the nucleus of stromal cells of LFS breast tissue. Taken together, our results show that the Aha1-Hsp90-PKM2/HIF-1α axis mediates the induction of aromatase in LFS.

Keywords: heat shock protein 90 (Hsp90); hypoxia-inducible factor (HIF); p53; pyruvate kinase; signal transduction.

FIGURE 1.

Hsp90 is important for the p53-mediated increase in aromatase levels in LFS stromal cells. A–C, WT and LFS stromal cells were used. In D–I, LFS cells were treated with the indicated concentrations of 17-AAG (D–F) or PU-H71 (G–I) for 24 h. In A, F, and I, aromatase activity was measured. In B, E, and H, aromatase mRNA levels were measured. In C, D, and G, Hsp90 ATPase activity was measured. A–I, means ± S.D. (error bars) are shown, n = 6. **, p < 0.01; *** p < 0.001 compared with wild-type stromal cells (A–C) and in D–I, compared with vehicle-treated cells.

FIGURE 2.

Reactivation of p53 leads to inhibition of aromatase activity in LFS stromal cells. In A–D, LFS stromal cells were used. In A, LFS stromal cells were transfected with 1.8 μg of p53-luciferase construct and 0.2 μg of psv-β-galactosidase construct. 24 h after transfection, the cells were treated with the indicated concentrations of CP-31398. 24 h later, the cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-galactosidase activity. Inset, LFS cells were also treated with the indicated concentrations of CP-31398, lysates were subjected to Western blotting, and the blots were probed as indicated. In B–D, the cells were treated with the indicated concentrations of CP-31398 for 24 h. In B, Hsp90 ATPase activity was measured. In C and D, levels of aromatase mRNA and activity were measured in cell lysates. A–D, means ± S.D. (error bars) are shown, n = 6. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with vehicle-treated cells.

FIGURE 3.

p53 regulates Hsp90 ATPase activity and aromatase expression. In A–C, wild-type stromal cells were transfected with 2 μg of siRNA to GFP (control siRNA) or p53. 48 h after transfection, the cells were harvested, and levels of Hsp90 ATPase activity (A), aromatase mRNA (B), and aromatase activity (C) were measured. In D and E, HCT116 cells wild-type or null for p53 were used. The cells were harvested and Hsp90 ATPase activity (D) and aromatase expression (E) were measured. In F–H, EB-1 cells were used. In F, the cells were transfected with 1.8 μg of p53-luciferase construct and 0.2 μg of psv-β-galactosidase construct for 24 h. In F–H, EB-1 cells were treated with the indicated concentrations of ZnCl₂ for 24 h. Inset, EB-1 cells were treated with the indicated concentrations of ZnCl₂ for 24 h, and the lysates were subjected to Western blotting. In F, the cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-galactosidase activity. In G and H, levels of Hsp90 ATPase activity and aromatase mRNA levels were measured, respectively. In A–H, means ± S.D. (error bars) are shown, n = 6. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with control siRNA-treated cells (A–C), wild-type cells (D and E), or vehicle-treated cells (F–H).

FIGURE 4.

Aha1 is important for p53-mediated regulation of aromatase in LFS stromal cells. A, levels of Aha1 and β-actin in WT and LFS stromal cells were determined in cell lysates by immunoblotting. In B (left panel) and D–F, cells were transfected with 2 μg of siRNA to GFP (control siRNA), p53 (B), or Aha1 (D–F). 48 h after transfection, the cells were harvested, and levels of Aha1 protein (B, left panel; and inset in D), Hsp90 ATPase activity (D), and aromatase mRNA (E), and aromatase activity (F) were measured. In B (right panel), lysates were prepared from p53 wild-type and p53 null HCT116 cells and subjected to Western blotting. In C, LFS cells were treated with the indicated concentrations of CP-31398 for 24 h. The cell lysates were prepared and subjected to Western blotting. The blots were probed as indicated. In D–F, means ± S.D. (error bars) are shown, n = 6. ***, p < 0.001 compared with cells transfected with GFP siRNA.

FIGURE 5.

HIF-1α and PKM2 are client proteins of Hsp90. A, cell lysates from wild-type and LFS stromal cells (left panel) and HCT116 cells (right panel) were subjected to Western blotting, and the blots were probed as indicated. In B–D, immunoprecipitation experiments were performed. In B, cell lysates were prepared from wild-type and LFS stromal cells; in C, LFS stromal cells were treated with 20 μm CP-31398 for 3 h, and cell lysates were prepared; and in D, wild-type cells were transfected with 2 μg of siRNA to GFP (control siRNA) or p53. 48 h after transfection, the cells were harvested. In B–D, Hsp90 was immunoprecipitated with control IgG or antibody to Hsp90. Immunoprecipitates were subjected to Western blotting, and the blots were probed as indicated. Blank, total cell lysates were subjected to Western blotting and probed as indicated. In E and F, LFS stromal cells were treated with the indicated concentrations of 17-AAG and PU-H71 for 24 h. In G, LFS stromal cells were transfected with 2 μg of siRNA to GFP (control siRNA) or Aha1. 48 h after transfection, cells were harvested, and lysates were prepared. In H, LFS cells were treated with the indicated concentrations of CP-31398 for 24 h. The cells were harvested, and lysates were prepared. In I, EB-1 cells were treated with the indicated concentrations of ZnCl₂ for 24 h, and cell lysates were prepared. In E–I, the cell lysates were subjected to Western blotting, and the blots were probed with the indicated antibodies.

FIGURE 6.

HIF-1α and PKM2 are important for the p53-mediated regulation of aromatase. In A, cell lysates prepared from WT and LFS stromal cells were either subjected to Western blotting (Blank) or immunoprecipitated with control IgG, antibody to PKM2, or antibody to HIF-1α and then subjected to Western blotting. In B, lysates were prepared from wild-type and LFS cells. Cytosolic and nuclear fractions were isolated, and 75 μg of total protein and equal volumes of cytosolic and nuclear proteins were subjected to Western blotting, and the blots were probed as indicated. In C and D, ChIP assays were performed. Chromatin fragments were immunoprecipitated with antibodies against HIF-1α (C) or PKM2 (D), and the aromatase promoter was amplified by real time PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the aromatase promoter. This promoter was not detected when normal IgG was used or when antibody was omitted from the immunoprecipitation step. In E and H, LFS stromal cells were transfected with 0.9 μg of aromatase promoter construct and 0.2 μg of psvβ-gal constructs. 24 h later, the cells also received either 0.9 μg of control siRNA, HIF-1α siRNA (E), or PKM2 siRNA (H). 48 h after transfection, the cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-galactosidase activity. In F, G, I, and J, LFS stromal cells were transfected with 2 μg of GFP siRNA (control), siRNA to HIF-1α (F and G), or siRNA to PKM2 (I and J). In F and I, 48 h after transfection, the cell lysates were subjected to Western blotting (insets), and the blots were probed as indicated. In F and I, total RNA was isolated, and aromatase mRNA levels were measured. In G and J, aromatase activity was measured in cell lysates. In C–J, means ± S.D. (error bars) are shown, n = 6. **, p < 0.01; ***, p < 0.001 compared with wild-type cells (C and D); cells that were transfected with GFP siRNA (E–J).

FIGURE 7.

Levels of aromatase are increased in mammary glands of p53 null mice. Mammary gland tissue from female p53 wild-type and p53 null mice was used. A, lysates were subjected to Western blotting and probed as indicated. B, tissue lysates were used to measure Hsp90 ATPase activity. C, lysates were subjected to Western blotting and probed as indicated. D, total RNA was prepared, and poly(A) RNA was isolated. Relative expression of aromatase was quantified by real time PCR. The values were normalized to levels of β-actin. E, tissue lysates were used to measure aromatase activity. In B, D, and E, means ± S.D. (error bars) are shown, n = 6. ***, p < 0.001 compared with p53^+/+ mice.

FIGURE 8.

PKM2 is detectable in the nucleus of LFS stromal cells and is positively correlated with HIF-1α and aromatase. PKM2, HIF-1α, and aromatase immunoreactivity and localization were examined in adipose stromal cells from normal breast tissue and breast tissue from LFS patients using immunofluorescence and confocal microscopy. A, PKM2 (green) is detectable in the cytoplasm of stromal cells from normal breast tissue (white arrows). In LFS breast adipose stromal cells, PKM2 (B, C, and E; green) and HIF-1α (C; red) are detectable in the cytoplasm and nucleus. Co-localization of PKM2 and HIF-1α is visible in the nucleus of LFS stromal cells (C; white arrows), and the relative fluorescence intensity for HIF-1α is positively correlated with nuclear PKM2 (D). E, PKM2 (green) and aromatase (red) also co-localize in adipose stromal cells from LFS (arrows). F, aromatase immunofluorescence staining is positively correlated with nuclear PKM2 in these cells. Insets in A and B are two-channel images where Hoescht nuclear staining is in blue and PKM2 in green. Images presented (LFS6) are representative of samples examined (n = 7/group). Scale bars in A and B represent 25 μm. Arom, aromatase.

FIGURE 9.

Signaling pathway by which p53 regulates aromatase in LFS stromal cells. A, in stromal cells with wild-type p53, Aha1 is suppressed leading to the decreased ATPase activity of Hsp90 and the decreased stabilization of HIF-1α and PKM2. B, mutation or loss of p53 leads to an increase in Aha1 expression, which leads in turn to enhanced Hsp90 ATPase activity. This results in stabilization of HIF-1α and PKM2, their increased binding to the CYP19A1 promoter, and the up-regulation of aromatase expression.