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.

We previously reported that our novel compound 3B-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (VN/124-1) is a potent 17A-hydroxylase/17,20-lyase (CYP17) inhibitor/antiandrogen and strongly inhibits the formation and proliferation of human prostate cancer LAPC4 tumor xenografts in severe combined immunodeficient mice. In this study, we report that VN/124-1 and other novel CYP17 inhibitors also cause down-regulation of androgen receptor (AR) protein expression in vitro and in vivo. This mechanism of action seems to contribute to their antitumor efficacy. We compared the in vivo antitumor efficacy of VN/124-1 with that of castration and a clinically used antiandrogen, Casodex, and show that VN/124-1 is more potent than castration in the LAPC4 xenograft model. Treatment with VN/124-1 (0.13 mmol/kg twice daily) was also very effective in preventing the formation of LAPC4 tumors (6.94 versus 2410.28 mm 3 in control group). VN/124-1 (0.13 mmol/kg twice daily) and VN/124-1 (0.13 mmol/kg twice daily) + castration induced regression of LAPC4 tumor xenografts by 26.55% and 60.67%, respectively. Treatments with Casodex (0.13 mmol/kg twice daily) or castration caused significant tumor suppression compared with control. Furthermore, treatment with VN/124-1 caused marked down-regulation of AR protein expression, in contrast to treatments with Casodex or castration that caused significant up-regulation of AR protein expression. The results suggest that VN/124-1 acts by several mechanisms (CYP17 inhibition, competitive inhibition, and down-regulation of the AR). These actions contribute to inhibition of the formation of LAPC4 tumors and cause regression of growth of established tumors. VN/ 124-1 is more efficacious than castration in the LAPC4 xenograft model, suggesting that the compound has potential for the treatment of prostate cancer.

Using the intratumoral aromatase xenograft model, we have observed that despite long-lasting growth inhibition, tumors eventually begin to grow during continued letrozole treatment. In cells isolated from these long-term letrozole-treated tumors (LTLT-Ca), estrogen receptor-A (ERA) levels were decreased, whereas signaling proteins in the mitogen-activated protein kinase cascade were up-regulated along with human epidermal growth factor receptor 2 (Her-2). In the current study, we evaluated the effect of discontinuing letrozole treatment on the growth of letrozole-resistant cells and tumors. The cells formed tumors equally well in the absence or presence of letrozole and had similar growth rates. After treatment was discontinued for 6 weeks, letrozole was administered again. Marked tumor regression was observed with this second course of letrozole treatment. Similarly, in MCF-7Ca xenografts, a 6-week break in letrozole treatment prolonged the responsiveness of the tumors to letrozole. To understand the mechanisms of this effect, LTLT-Ca cells were cultured in the absence of letrozole for 16 weeks. The resulting cell line (RLT-Ca) exhibited properties similar to MCF-7Ca cells. The cell growth was inhibited by letrozole and stimulated by estradiol. The expression of phosphorylated mitogen-activated protein kinase (MAPK) was reduced and ERA and aromatase levels increased compared with LTLT-Ca cells and were similar to levels in MCF-7Ca cells. These results indicate that discontinuing treatment can reverse letrozole resistance. This could be a beneficial strategy to prolong responsiveness to aromatase inhibitors for patients with breast cancer. [Cancer Res 2008;68(12):4518-24]

This study was carried out to determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer. We investigated the role of the androgen receptor and its relationship to other signal transduction proteins. A hormone-refractory prostate cancer cell line [high-passage LNCaP (HP-LNCaP)] was established in vitro. Cells were treated with inhibitors of mammalian target of rapamycin and tyrosine kinase receptors. Expression of these proteins and the androgen receptor were measured by Western immunoblotting. Analysis of the model and various treatments was also assessed through proliferation assays, luciferase activation assays, binding assays, and ELISA. Our novel antiandrogen, VN/124-1, effectively inhibited proliferation of hormone-resistant prostate cancer cell lines (HP-LNCaP), which were no longer sensitive to bicalutamide and had increased expression of the androgen receptor. Treatment with everolimus or gefitinib resulted in an increase in protein expression and activation of the androgen receptor. Conversely, inhibition of the androgen receptor resulted in increased expression of IGFR1B, pHER2, pmTOR, and pAkt. The addition of bicalutamide to everolimus or gefitinib inhibited cell proliferation in HP-LNCaP cells. However, the addition of VN/124-1 has proven to be superior to bicalutamide, and the combination was synergistic (P < 0.05) compared with either agent alone. This study suggests that compensatory cross-talk between the androgen receptor and various signaling pathways may account for decreased sensitivity to androgen receptor antagonists and the progression to hormone-resistant prostate cancer. Furthermore, these findings suggest that inhibition of both pathways may provide effective control in hormone-resistant prostate cancer and restore sensitivity to androgen antagonists in hormone-refractory patients.

Purpose: Aromatase inhibitors that block the synthesis of estrogen are proving to be superior to antiestrogens and may replace tamoxifen as first-line treatment for postmenopausal estrogen receptor (ER)^positive breast cancer patients. However, acquisition of resistance to all forms of treatments is inevitable and a major clinical concern. In this study, we have investigated the effects of long-term estrogen deprivation in the breast cancer xenograft model and whether sensitivity to antiestrogens can be restored in vivo. We also compared whether combining wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. Experimental Design: Long-term estrogen-deprived aromatase-transfected human ERpositive breast cancer cells (UMB-1Ca) were grown as tumors in ovariectomized athymic nude mice. Twelve weeks after inoculation, when tumors reached 300 mm 3 , animals were grouped and injected with vehicle, D 4 A, letrozole, tamoxifen, fulvestrant, wortmannin, tamoxifen plus wortmannin, and wortmannin plus fulvestrant. Tumor volumes were measured weekly. Results: Tumors of UMB-1Ca cells grew equally well with and without androstenedione, indicating the ability of the cells to proliferate in the absence of estrogen. The combination of wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. The combination of wortmannin plus fulvestrant was the most effective treatment that maintained tumor regression for a prolonged time. Conclusion: These results suggest that blocking both ER and growth factor receptor pathways could provide effective control over tumor growth of long-term estrogen-deprived human breast cancers.