Ovariectomized mice bearing tumor xenografts grown from aromatase-transfected estrogen receptor (ER)-positive human breast cancer cells (MCF-7Ca) were injected s.c. with 10 Mg/d letrozole for up to 56 weeks. Western blot analysis of the tumors revealed that ERs (ERA) were increased at 4 weeks but decreased at weeks 28 and 56. Expression of erbB-2 and p-Shc increased throughout treatment, whereas growth factor receptor binding protein 2 (Grb2) increased only in tumors proliferating on letrozole (weeks 28 and 56). In cells isolated from tumors after 56 weeks and maintained as a cell line (LTLT-Ca) in 1 Mmol/L letrozole, ERA was also decreased whereas erbB-2, adapter proteins (p-Shc and Grb2), and the signaling proteins in the mitogen-activated protein kinase (MAPK) cascade were increased compared with MCF-7Ca cells. Growth was inhibited in LTLT-Ca cells but not in MCF7Ca cells treated with MAPK kinase 1/2 inhibitors U0126, and PD98059 (IC 50 f25 Mmol/L). PD98059 (5 Mmol/L) also reduced MAPK activity and increased ERA to the levels in MCF-7Ca cells. Epidermal growth factor receptor kinase inhibitor, gefitinib (ZD1839) inhibited growth of LTLT-Ca cells (IC 50 f10 Mmol/L) and restored their sensitivity to tamoxifen and anastrozole. In xenografts, combined treatment with ER down-regulator fulvestrant and letrozole, prevented increases in erbB-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results indicate that blocking both ER-and growth factor-mediated transcription resulted in the most effective inhibition of growth of ER-positive breast cancer cells. (Cancer Res 2005; 65(12): 5380-9)

Estrogens are known to be important in the growth of breast cancers in both pre- and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also on innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs.

In this study, we investigated adaptive mechanisms associated with aromatase inhibitor (AI) resistance in breast cancer cells and show that sensitivity to AIs can be extended through dual inhibition of estrogen receptor (ER) and human epidermal receptor-2 (Her-2) signaling. We used human ER-positive breast cancer cells stably transfected with the aromatase gene (MCF-7Ca). These cells grow as tumors in nude mice and are inhibited by AIs. Despite continued treatment, tumors eventually become insensitive to AI letrozole. The cells isolated from these long-term letrozole-treated tumors (LTLT-Ca) were found to have decreased ERA levels. Our results suggest that LTLT-Ca cells survive estrogen deprivation by activation of Her-2/mitogen-activated protein kinase (MAPK) pathway. Here, we show that trastuzumab (antibody against Her-2; IC 50 = 0.4 mg/mL) was very effective in restoring the ERA levels and sensitivity of LTLT-Ca cells to endocrine therapy by down-regulation of Her-2/MAPK pathway and up-regulation of ERA. In contrast, trastuzumab was ineffective in the parental hormone-responsive MCF-7Ca cells (IC 50 = 4.28 mg/mL) and xenografts. By blocking Her-2, trastuzumab also up-regulates ERA and aromatase expression and hypersensitized MCF-7Ca cells to E 2 . We show that trastuzumab is beneficial in hormone-refractory cells and xenografts by restoring ER, implicating Her-2 as a negative regulator of ERA. In xenograft studies, the combination of trastuzumab plus letrozole is equally effective in inhibiting growth of MCF-7Ca tumors as letrozole alone. However, on the acquisition of resistance and increased Her-2 expression, the combination of letrozole plus trastuzumab provided superior benefit over letrozole or trastuzumab alone.

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side-effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. Here we show that a small-molecule bioprecursor prodrug, 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17β-estradiol in the brain after systemic administration, but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17β-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17β-estradiol treatment. Altogether, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side-effects of current hormone therapies.

Previous work has shown that androgens inhibit breast cancer cells and tumor growth. On the other hand, androgens can be converted to mitogenic estrogens by aromatase in breast cancer cells. Here, we report that androgens, such as the aromatizable androstenedione and the non-aromatizable 5A-dihydrotestosterone, inhibit MCF-7 cell proliferation. This effect is observed only in the absence or at a low concentration of estrogens and is evident in cells with low aromatase activity. Growth of a new aromatase stably transfected MCF-7 cell line (Ac1) was stimulated by conversion of androstenedione into estrogens and was sensitive to aromatase inhibitors. We show that blockade of the androgen receptor (AR) in these cells by the antiandrogen casodex or by the anti-AR small interfering RNA inhibited the antiproliferative effect of dihydrotestosterone and letrozole (aromatase inhibitor). We also show that suppression of the estrogen-induced antiapoptotic protein Bcl-2 may be involved in the antiproliferative effects of androgens and letrozole. These effects can be reversed by casodex. In conclusion, the results suggest that aromatase inhibitors may exert their antiproliferative effect not only by reducing the intracellular production of estrogens but also by unmasking the inhibitory effect of androgens acting via the AR. (Cancer Res 2006; 66(15): 7775-82)