Previously, we reported that HDAC6 functions as an Hsp90 deacetylase and that inhibition of HDAC6 leads to Hsp90 hyperacetylation, its dissociation from p23, a co-chaperone, and a loss of chaperone activity (33). aromatase expression in LFS stromal cells. Consistent with these findings, levels of Hsp90 ATPase activity, Aha1, HIF-1, PKM2, and aromatase were increased in the mammary glands of p53 null wild-type mice. PKM2 and HIF-1 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-1 axis mediates the induction of aromatase in LFS. gene, catalyzes the synthesis of estrogens from androgens (1). In postmenopausal women, the adipose tissue becomes the main site of estrogen biosynthesis, and particularly, the breast adipose tissue is considered an important source of estrogens that drive the growth of hormone-dependent breast cancers. Consequently, it is important to elucidate the mechanisms that regulate the transcription of the gene. The expression of aromatase is tightly regulated, with transcription being under the control of several distinct tissue-selective promoters (2,C4). In normal breast adipose tissue, aromatase is expressed at low levels under the control of promoter I.4, whereas in obesity and cancer, the coordinated activation of the proximal promoters I.3 and promoter II (PII)3 causes a significant increase in aromatase expression (3,C5). The proximal promoters I.3 and PII are located close to each other, activated by stimulation of the cAMP PKA cAMP response element-binding protein (CREB) pathway (6, 7), and aided by many other regulators including CREB-regulated transcription co-activator 2 (CRTC2), p300, and hypoxia-inducible factor-1 (HIF-1) (8,C11). Several cytokines and tumor promoters, including prostaglandin E2, tumor necrosis factor-, and interleukin-1 stimulate aromatase expression (4, 12). In addition, its expression is regulated by oncogenes such as HER-2/neu and tumor suppressor genes including BRCA1, LKB1, Dexloxiglumide and p53 (9, 11, 13,C18). Germ line mutations in the gene, which encodes p53, lead to Li-Fraumeni Syndrome (LFS). Among women with LFS, the most common cancer is breast cancer, with the majority of breast cancers being hormone receptor-positive (19, 20). Aromatase expression has been shown to be increased in breast adipose stromal cells from LFS patients compared with non-LFS breast tissue (16). Recently, we showed that epithelial cells from LFS patients contained increased Hsp90 ATPase activity because of the increased expression of Aha1, a co-chaperone of Hsp90 (21, 22). Here, we extended these studies to breast adipose stromal cells and show that aromatase expression is increased in LFS wild-type stromal cells and that this increase is dependent on Hsp90 ATPase signaling involving Aha1, HIF-1, and PKM2. Consistent with these findings, levels of aromatase were increased in the mammary glands of p53 null wild-type mice. Taken together, this study provides new insights into the mechanism by which p53 regulates aromatase expression in stromal cells, which may be important for understanding the NMYC pathogenesis of estrogen-dependent breast cancer. Results Regulation of Aromatase by p53 Is Dependent on Hsp90 Initially, we compared levels of aromatase in stromal cells that were wild-type for p53 stromal cells from a LFS patient that expressed mutant p53. As shown in Fig. 1 (and wild-type stromal cells Dexloxiglumide (Fig. 1and and wild-type stromal cells (Fig. 4and = 6. **, 0.01; *** 0.001 compared with wild-type stromal cells (and = 6. *, 0.05; **, 0.01; ***, 0.001 Dexloxiglumide compared with vehicle-treated cells. Open in a separate window FIGURE 3. p53 regulates Hsp90 ATPase activity and aromatase expression. In and and = 6. *, 0.05; **, 0.01; ***, 0.001 compared with control siRNA-treated cells (and (in (= 6. ***, 0.001 compared with cells transfected with GFP siRNA. p53 Regulates Hsp90 ATPase Activity Leading to the Stabilization of HIF-1 and PKM2 HIF-1 is a client protein of Hsp90 and a known regulator of aromatase expression (26,C28). PKM2 is a co-activator of HIF-1-mediated gene expression (29,C32). Hence, we investigated whether these two proteins could be important for mediating the effects of p53 on aromatase. Levels of HIF-1 and Dexloxiglumide PKM2 were increased in LFS stromal cells (Fig. 5wild-type HCT116 cells (Fig. 5and and wild-type stromal cells, we investigated whether these differences were causally linked. Initially, we explored the possibility that HIF-1 and PKM2 were in a complex. As shown in Fig. 6promoter, ChIP assays were performed. ChIP assays revealed increased binding of both HIF-1 (Fig. 6and and and and and and and and = 6. **, 0.01; ***, 0.001 compared with wild-type cells (and and wild-type mice (Fig. 7, and =.