It is well known that sex steroids play important roles in the development of invasive ductal carcinoma (IDC) of the human breast. However, biological significance of sex steroids remains largely unclear in ductal carcinoma in situ (DCIS), regarded as a precursor lesion of IDC, which is partly due to the fact that the intratumoral concentration of sex steroids has not been examined in DCIS. Therefore, in this study, we first examined the intratumoral concentrations of estradiol and 5α-dihydrotestosterone (DHT) using liquid chromatography/electrospray tandem mass spectrometry in DCIS. Intratumoral concentrations of both estradiol and DHT were threefold higher in DCIS than non-neoplastic breast tissues and estrogen-producing enzymes (aromatase, steroid sulfatase, and 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1)), and androgen-producing enzymes (17βHSD5 and 5α-reductase type 1 (5αRed1)) were abundantly expressed in DCIS by real-time PCR and immunohistochemical analyses. The intratumoral concentration of DHT was significantly lower in IDC than DCIS, while the expression of aromatase mRNA in carcinoma cells and intratumoral stromal cells was significantly higher in IDC than those in DCIS. Immunohistochemistry for sex steroid-producing enzymes in DCIS demonstrated that 5αRed1 immunoreactivity was positively correlated with Ki-67 labeling index and histological grade and was also associated with an increased risk of recurrence in patients with DCIS examined. Results of our study suggest that intratumoral concentrations of estradiol and DHT are increased in DCIS, which is possibly due to intratumoral production of these steroids. Therefore, estradiol and DHT may play important roles in the development of DCIS of the human breast.

Early growth responsive gene 3 (EGR3) is a zinc-finger transcription factor and plays important roles in cellular growth and differentiation. We recently demonstrated estrogen-mediated induction of EGR3 in breast carcinoma cells. However, EGR3 has not yet been examined in breast carcinoma tissues and its significance remains unknown. Therefore, in this study, we examined biological functions of EGR3 in the breast carcinoma by immunohistochemistry, in vitro study, and nude mouse xenograft model. EGR3 immunoreactivity was detected in carcinoma cells in 99 (52%) out of 190 breast carcinoma tissues and was associated with the mRNA level. EGR3 immunoreactivity was positively associated with lymph node status, distant metastasis into other organs, estrogen receptor α, or EGR3 immunoreactivity in asynchronous recurrent lesions in the same patients, and was negatively correlated with tubule formation. EGR3 immunoreactivity was significantly associated with an increased risk of recurrence and adverse clinical outcome by both uni- and multivariate analyses. Egr3-expressing transformant cell lines derived from MCF-7 Tet-Off cells (Eg-10 and Eg-11) significantly enhanced the migration and invasion properties according to the treatment of doxycyclin, but did not significantly change the cell proliferation. Moreover, Eg-11 cells injected into athymic mice irregularly invaded into the adjacent peritumoral tissues, although Clt-7, which was stably transfected with empty vector as a control, demonstrated a well-circumscribed tumor. Eg-11 cells were significantly associated with invasive components and less tubule formation in the xenograft model. These results suggest that EGR3 plays an important role in estrogen-meditated invasion and is an independent prognostic factor in breast carcinoma.

Nuclear factor erythroid 2-related factor 2 (NRF2 (NFE2L2)) is an important transcriptional activator involved in the cellular defense mechanisms against electrophilic and oxidative stress. Recent studies have demonstrated that the expression of NRF2 protein is upregulated in several human malignancies and is associated with worse prognosis in these patients. However, the pathological and clinical significance of NRF2 has remained largely unknown in breast cancer patients. Therefore, in this study, we immunolocalized NRF2 in 106 breast carcinoma cases. NRF2 immunoreactivity was mainly detected in the nucleus of the breast carcinoma cells and it was positive in 44% of the cases. NRF2 status was significantly associated with histological grade, Ki-67 labeling index, p62 immunoreactivity, and NAD(P)H:quinone oxidoreductase 1 (NQO1) immunoreactivity, and the results of multivariate analyses revealed that NRF2 status was an independent adverse prognostic factor for both recurrence and disease-free survival of the patients. Subsequent in vitro studies demonstrated that the expression of NRF2 significantly increased the proliferation activity of MCF7 and SK-BR-3 breast carcinoma cells. These results indicate that nuclear NRF2 protein plays important roles in the proliferation and/or progression of breast carcinoma, and nuclear NRF2 immunoreactivity is therefore considered a potent prognostic factor in breast cancer patients.

Sex steroids play important roles in the development of many human breast carcinomas, and aromatase inhibitors are used for the anti-estrogen therapy. Recent studies have demonstrated that aromatase suppressed 5α-dihydrotestosterone (DHT) synthesis in breast carcinoma cells, but intratumoral concentration of androgens and its significance have not been reported in the breast carcinoma patients treated with aromatase inhibitors. Therefore, we examined androgen concentrations in breast carcinoma tissues treated with exemestane, and further performed in vitro studies to characterize the significance of androgen actions. Intratumoral DHT concentration was significantly higher in breast carcinoma tissues following exemestane treatment (n=9) than those without the therapy (n=7), and 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) status was significantly altered to be positive after the treatment. Following in vitro studies showed that 17βHSD2 expression was dose dependently induced by both DHT and exemestane in T-47D breast carcinoma cells, but these inductions were not additive. DHT-mediated induction of 17βHSD2 expression was markedly suppressed by estradiol (E₂) in T-47D cells. E₂-mediated cell proliferation was significantly inhibited by DHT in T-47D cells, associated with an increment of 17βHSD2 expression level. These findings suggest that intratumoral androgen actions are increased during exemestane treatment. 17 β HSD2 is a potent DHT-induced gene in human breast carcinoma, and may not only be involved in anti-proliferative effects of DHT on breast carcinoma cells but also serve as a potential marker for response to aromatase inhibitor in the breast carcinoma patients.