Estradiol, the most potent endogenous estrogen, is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase. Aromatase is present in breast tissue, and intratumoral aromatase is the source of local estrogen production in breast cancer tissues. Inhibition of aromatase is an important approach for reducing growth-stimulatory effects of estrogens in estrogen-dependent breast cancer. Steroidal inhibitors that have been developed to date build upon the basic androstenedione nucleus and incorporate chemical substituents at varying positions on the steroid. Nonsteroidal aromatase inhibitors can be divided into three classes: aminoglutethimide-like molecules, imidazole/triazole derivatives, and flavonoid analogs. Mechanism-based aromatase inhibitors are steroidal inhibitors that mimic the substrate, are converted by the enzyme to a reactive intermediate, and result in the inactivation of aromatase. Both steroidal and nonsteroidal aromatase inhibitors have shown clinical efficacy in the treatment of breast cancer. The potent and selective third-generation aromatase inhibitors, anastrozole, letrozole, and exemestane, were introduced into the market as endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies. These agents are currently approved as first-line therapy for the treatment of postmenopausal women with metastatic estrogen-dependent breast cancer. Several clinical studies of aromatase inhibitors are currently focusing on the use of these agents in the adjuvant setting for the treatment of early breast cancer. Use of an aromatase inhibitor as initial therapy or after treatment with tamoxifen is now recommended as adjuvant hormonal therapy for a postmenopausal woman with hormone-dependent breast cancer.

Estradiol is biosynthesized from androgens by the aromatase enzyme complex. Previous studies suggest a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Our hypothesis is that higher levels of COX-2 expression result in higher levels of prostaglandin E2, which, in turn, increases CYP19 expression through increases in intracellular cAMP levels. This biochemical mechanism may explain the beneficial effects of nonsteroidal antiinflammatory drugs on breast cancer. The effects of nonsteroidal antiinflammatory drugs, COX-1 and COX-2 selective inhibitors on aromatase activity and expression were studied in human breast cancer cells. The data from these experiments revealed dose-dependent decreases in aromatase activity after treatment with all agents. Real-time PCR analysis of aromatase gene expression showed a significant decrease in mRNA levels when compared with control for all agents. These results were consistent with enzyme activity data, suggesting that the effect of COX inhibitors on aromatase begins at the transcriptional level. Exon-specific real-time PCR studies suggest that promoters I.3, I.4, and II are involved in this process. Thus, COX inhibitors decrease aromatase mRNA expression and enzymatic activity in human breast cancer cells in culture, suggesting that these agents may be useful in suppressing local estrogen biosynthesis in the treatment of hormone-dependent breast cancer.

STAT5 consists of two proteins, STAT5A/B, that impact mammary cell differentiation, proliferation, and survival. In normal development, STAT5 expression and activity are regulated by prolactin signaling with JAK2/ELF5, EGF signaling networks that include c-Src, and growth hormone, insulin growth factor, estrogen, and progesterone signaling pathways. In cancer, erythropoietin signaling can also regulate STAT5. Activation levels are influenced by AKT, caveolin, PIKE-A, Pak1, c-Myb, Brk, beta-integrin, dystroglycan, other STATs, and STAT pathway molecules JAK1, Shp2, and SOCS. TGF-β and PTPN9 can downregulate prolactin- and EGF-mediated STAT5 activation, respectively. IGF, AKT, RANKL, cyclin D1, BCL6, and HSP90A lie downstream of STAT5.

Aromatase is a particularly attractive target in the treatment of estrogen receptor positive breast cancer. Aromatase levels in breast cancer cells are enhanced by prostaglandins and reduced by COX inhibitors. The synthesis and biological evaluation of a novel series of sulfonanilide analogues derived from the COX-2 selective inhibitor NS-398 are described. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose- and time-dependent manner. The effect of these compounds on COX-2 inhibition is investigated in breast cancer cells as well. Structure-activity analysis does not find a correlation between aromatase suppression and COX-2 inhibition. Microsomal aromatase inhibition studies rule out the possibility of direct enzyme inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogues decrease aromatase gene transcription in SK-BR-3 cells. These studies suggest that the novel sulfonanilide compounds suppress aromatase activity and transcription in SK-BR-3 breast cancer cells independent of COX-2 inhibition.

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