Increased levels of COX-2 and prostaglandin E2 contribute to elevated aromatase expression in inflamed breast tissue of obese women

Abstract

Obesity is a risk factor for hormone receptor-positive breast cancer in postmenopausal women. Estrogen synthesis is catalyzed by aromatase, which is encoded by CYP19. We previously showed that aromatase expression and activity are increased in the breast tissue of overweight and obese women in the presence of characteristic inflammatory foci [crown-like structures of the breast (CLS-B)]. In preclinical studies, proinflammatory prostaglandin E(2) (PGE(2)) is a determinant of aromatase expression. We provide evidence that cyclooxygenase (COX)-2-derived PGE(2) stimulates the cyclic AMP (cAMP) → PKA signal transduction pathway that activates CYP19 transcription, resulting in increased aromatase expression and elevated progesterone receptor levels in breast tissues from overweight and obese women. We further demonstrate that a measure of in-breast inflammation (CLS-B index) is a better correlate of these biologic end points than body mass index. The obesity → inflammation → aromatase axis is likely to contribute to the increased risk of hormone receptor-positive breast cancer and the worse prognosis of obese patients with breast cancer.

Significance: We show that obesity-associated inflammatory foci in the human breast are associated with elevated COX-2 levels and activation of the PGE2 → cAMP → PKA signal transduction pathway resulting in increased aromatase expression. These findings help to explain the link among obesity, low-grade chronic inflammation, and breast cancer with important clinical implications.

Figure 1

Increased levels of aromatase are found in inflamed breast tissue. Crown-like structure of breast (CLS-B) is shown in panels A and B. A, H&E demonstrating an inflammatory focus containing macrophages surrounding an adipocyte (200×). B, Immunohistochemical stain of CD68 showing the same lesion (200×). In A and B, arrows indicate CLS-B. Levels of aromatase mRNA (C) and activity (D) are increased in inflamed breast tissue (CLS-B + vs. CLS-B −). Box plots are shown for subjects (n=30) varying in weight and breast inflammation status.

Figure 2

Increased levels of COX-2 mRNA, COX-2 protein and PGE₂ are found in inflamed breast tissue. Levels of COX-2 mRNA (A), COX-2 protein (B) and PGE₂ (C) were compared in breast tissue of normal weight vs. overweight/obese women and in breast tissue with or without evidence of inflammation (CLS-B + vs. CLS-B −). A, quantitative real-time PCR was used to determine COX-2 expression. B, immunoprecipitates were subjected to western blot analysis. Representative results (top of panel) for COX-2 and β-actin are shown vs. the CLS-B index for 17 cases. Densitometry (bottom of panel) was used to quantify COX-2 protein. C, PGE₂ was quantified by enzyme immunoassay. A-C, Box plots are shown for subjects (n=30) varying in weight and breast inflammation status.

Figure 3

Increased levels of cAMP and PKA activity are found in inflamed breast tissue. Levels of cAMP (A) and PKA activity (B) were compared in breast tissue of normal weight vs. overweight/obese women and in breast tissue with or without evidence of inflammation (CLS-B + vs. CLS-B −). Box plots are shown for subjects (n=30) varying in weight and breast inflammation status.

Figure 4

Levels of aromatase transcripts derived from promoters I.3 and II and progesterone receptor (PR) are increased in association with breast inflammation. Expression levels of promoter I.3 (A) and promoter II (B) specific aromatase transcripts were quantified by real-time PCR and compared in breast tissue of normal weight vs. overweight/obese women and in breast tissue with or without evidence of inflammation (CLS-B + vs. CLS-B −). C, Immunoprecipitates were subjected to western blot analysis. Representative results (top of panel) for PR and β-actin are shown vs. the CLS-B index for 16 cases. Densitometry (bottom of panel) was used to quantify PR protein. A-C, Box plots are shown for subjects (n=30) varying in weight and breast inflammation status.

Figure 5

PGE₂ derived from stearic acid (SA)-activated macrophages stimulates the cAMP→PKA pathway leading to increased aromatase transcription in preadipocytes. A, THP-1 cells were untreated or treated with control siRNA or siRNA to COX-2. Subsequently, the THP-1 cells were treated with vehicle (Control) or 10 µM SA for 24 hours. The abundance of COX-2 protein in cell lysates (inset) was determined by immunoblotting. β-actin was used as a loading control. Levels of PGE₂ in the medium were determined by enzyme immunoassay. B-H, preadipocytes were treated with THP-1 cell-derived CM for 24 hours prior to measurements of cAMP (B), PKA activity (C), relative aromatase expression (D), aromatase activity expressed as femtomoles/µg protein/minute (E), aromatase mRNA derived from promoters I.3 (F) and II (G), and PR protein levels (H). Columns, means (n=6); bars, SD. *, P < 0.05.