Regulation of androgen receptor activity by tyrosine phosphorylation.

The androgen receptor (AR) is essential for the growth of prostate cancer cells. Here, we report that tyrosine phosphorylation of AR is induced by growth factors and elevated in hormone-refractory prostate tumors. Mutation of the major tyrosine phosphorylation site in AR significantly inhibits the growth of prostate cancer cells under androgen-depleted conditions. The Src tyrosine kinase appears to be responsible for phosphorylating AR, and there is a positive correlation of AR tyrosine phosphorylation with Src tyrosine kinase activity in human prostate tumors. Our data collectively suggest that growth factors and their downstream tyrosine kinases, which are elevated during hormone-ablation therapy, can induce tyrosine phosphorylation of AR and such modification may be important for prostate tumor growth under androgen-depleted conditions.

Novel membrane-associated androgen receptor splice variant potentiates proliferative and survival responses in prostate cancer cells.

Progression from the androgen-sensitive to androgen-insensitive (or castration-resistant) stage is the major obstacle for sustained effectiveness of hormonal therapy for prostate cancer. The androgen receptor (AR) and its splice variants play important roles in regulating the transcription program essential for castration resistance. Here, we report the identification of a novel AR splice variant, designated as AR8, which is up-regulated in castration-resistant prostate cancer cells. AR8 is structurally different from other known AR splice variants because it lacks a DNA binding domain and therefore, unlikely functions as a transcription factor on its own. Immunofluorescence staining revealed that AR8 was primarily localized on the plasma membrane, possibly through palmitoylation of two cysteine residues within its unique C-terminal sequence. Mutation of these putative palmitoylation sites in AR8 led to loss of its plasma membrane localization. In addition, we demonstrated that overexpression of AR8 in prostate cancer cells promoted association of Src and AR with the EGF receptor in response to EGF treatment and enhanced tyrosine phosphorylation of AR. Conversely, specific knockdown of AR8 expression in prostate cancer cells compromised EGF-induced Src activation and AR phosphorylation. This effect was accompanied with attenuation of proliferation and increased apoptosis in prostate cancer cells cultured in androgen-depleted medium. We also showed that AR8 was required for optimal transcriptional activity of AR in response to treatment of both androgen and EGF. Taken together, our results demonstrate that the membrane-associated AR8 isoform may contribute to castration resistance by potentiating AR-mediated proliferative and survival responses to hormones and growth factors.

Inhibition of estrogen signaling activates the NRF2 pathway in breast cancer.

Exposure to higher levels of estrogen produces genotoxic metabolites that can stimulate mammary tumorigenesis. Induction of NF-E2-related factor 2 (NRF2)-dependent detoxifying enzymes (e.g., NAD(P)H-quinone oxidoreductase 1 (NQO1)) is considered an important mechanism of protection against estrogen-associated carcinogenesis because they would facilitate removal of toxic estrogens. Here, we studied the impact of estrogen-receptor (ER) signaling on NRF2-dependent gene transcription. In luciferase assay experiments using the 5-flanking region of the human NQO1 gene promoter, we observe that ERα ligand-binding domain (LBD) is required for estrogen inhibition of NQO1 promoter activity in estrogen-dependent breast cancer cells. Chromatin immunoprecipitation (ChIP) assay shows that estrogen recruits ERα and a class III histone deacetylase SIRT1 at the NQO1 promoter, leading to inhibition of NQO1 transcription. Inhibition of ERα expression by the antiestrogen shikonin reverses the inhibitory effect of estrogen on NQO1 expression. As a consequence, a chemoprevention study was undertaken to monitor the impact of shikonin on DNA lesions and tumor growth. Treatment of MCF-7 breast cancer cells with shikonin inhibits estrogen-induced 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA damage. NQO1 deficiency promotes estrogen-dependent tumor formation, and shikonin inhibits estrogen-dependent tumor growth in an NQO1-dependent manner in MCF-7 xenografts. These results suggest that estrogen-receptor signaling pathway has an inhibitory effect on NRF2-dependent enzymes. Moreover, shikonin reverses the inhibitory effects of estrogen on this pathway and may contribute to breast cancer prevention.

Anti-HER2 immunoliposomes for selective delivery of electron paramagnetic resonance imaging probes to HER2-overexpressing breast tumor cells.

Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 µM nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals.

Synergistic effect of a novel antiandrogen, VN/124-1, and signal transduction inhibitors in prostate cancer progression to hormone independence in vitro.

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 IGFR1beta, 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.

17alpha-Hydroxylase/17,20 lyase inhibitor VN/124-1 inhibits growth of androgen-independent prostate cancer cells via induction of the endoplasmic reticulum stress response.

Inhibitors of the enzyme 17alpha-hydroxylase/17,20 lyase are a new class of anti-prostate cancer agents currently undergoing preclinical and clinical development. We have previously reported the superior anticancer activity of our novel 17alpha-hydroxylase/17,20 lyase inhibitor, VN/124-1, against androgen-dependent cancer models. Here, we examined the effect of VN/124-1 on the growth of the androgen-independent cell lines PC-3 and DU-145 and found that the compound inhibits their growth in a dose-dependent manner in vitro (GI50, 7.82 micromol/L and 7.55 micromol/L, respectively). We explored the mechanism of action of VN/124-1 in PC-3 cells through microarray analysis and found that VN/124-1 up-regulated genes involved in stress response and protein metabolism, as well as down-regulated genes involved in cell cycle progression. Follow-up real-time PCR and Western blot analyses revealed that VN/124-1 induces the endoplasmic reticulum stress response resulting in down-regulation of cyclin D1 protein expression and cyclin E2 mRNA. Cell cycle analysis confirmed G1-G0 phase arrest. Measurements of intracellular calcium levels ([Ca2+]i) showed that 20 micromol/L VN/124-1 caused a release of Ca2+ from endoplasmic reticulum stores resulting in a sustained increase in [Ca2+]i. Finally, cotreatment of PC-3 cells with 5, 10, and 20 micromol/L VN/124-1 with 10 nmol/L thapsigargin revealed a synergistic relationship between the compounds in inhibiting PC-3 cell growth. Taken together, these findings show VN/124-1 is endowed with multiple anticancer properties that may contribute to its utility as a prostate cancer therapeutic.

Androgen receptor inactivation contributes to antitumor efficacy of 17{alpha}-hydroxylase/17,20-lyase inhibitor 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene in prostate cancer.

We previously reported that our novel compound 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (VN/124-1) is a potent 17alpha-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.

Aromatase inhibitors: are there differences between steroidal and nonsteroidal aromatase inhibitors and do they matter?

Aromatase inhibitors (AIs) are approved for use in both early- and advanced-stage breast cancer in postmenopausal women. Although the currently approved "third-generation" AIs all powerfully inhibit estrogen synthesis, they may be subdivided into steroidal and nonsteroidal inhibitors, which interact with the aromatase enzyme differently. Nonsteroidal AIs bind noncovalently and reversibly to the aromatase protein, whereas steroidal AIs may bind covalently and irreversibly to the aromatase enzyme. The steroidal AI exemestane may exert androgenic effects, but the clinical relevance of this has yet to be determined. Switching between steroidal and nonsteroidal AIs produces modest additional clinical benefits, suggesting partial noncrossresistance between the classes of inhibitor. In these circumstances, the response rates to the second AI have generally been low; additional research is needed regarding the optimal sequence of AIs. To date, clinical studies suggest that combining an estrogen-receptor blocker with a nonsteroidal AI does not improve efficacy, while combination with a steroidal AI has not been evaluated. Results from head-to-head trials comparing steroidal and nonsteroidal AIs will determine whether meaningful clinical differences in efficacy or adverse events exist between the classes of AI. This review summarizes the available evidence regarding known differences and evaluates their potential clinical impact.

Effects of novel retinoic acid metabolism blocking agent (VN/14-1) on letrozole-insensitive breast cancer cells.

Aromatase inhibitors are proving to be more effective than tamoxifen for postmenopausal estrogen receptor (ER)-positive breast cancer. However, the inevitable development of resistance to treatment is a concern. We investigated the effects of novel retinoic acid metabolism blocking agent, VN/14-1, in overcoming letrozole resistance in long-term letrozole cultured (LTLC) cells. Compared with MCF-7 cells stably transfected with aromatase (MCF-7Ca), LTLC cells were no longer sensitive to growth inhibition by aromatase inhibitors. The HER-2/phosphorylated mitogen-activated protein kinase (pMAPK) growth factor signaling pathways were activated, and ERalpha and coactivator amplified in breast cancer 1 (AIB1) were up-regulated approximately 3-fold in LTLC cells. VN/14-1 inhibited aromatase activity and growth values of in MCF-7Ca cells with IC(50) of 8.5 and 10.5 nmol/L, respectively. In human placental microsomes, aromatase activity was inhibited with IC(50) of 8.0 pmol/L. The IC(50) in LTLC cells was 0.83 nmol/L, similar to letrozole (IC(50), 0.3 nmol/L) in MCF-7Ca cells. LTLC cells were 10-fold more sensitive to growth inhibition by VN/14-1 than MCF-7Ca cells. VN/14-1 treatment effectively down-regulated ERalpha, AIB1, pMAPK, HER-2, cyclin D1, cyclin-dependent kinase 4 (CDK4), and Bcl2 and up-regulated cytokeratins 8/18, Bad, and Bax. Tumor growth of LTLC cells in ovariectomized nude mice was independent of estrogens but was inhibited by VN/14-1 (20 mg/kg/d; P < 0.002). Decreases in ERalpha, cyclin D1, CDK4, and pMAPK and up-regulation of cytokeratins, Bad, and Bax with VN/14-1 in tumor samples may be responsible for the efficacy of this compound in inhibiting LTLC cell growth in vitro and in vivo.

Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer.

This article describes the origins and evolution of "antiestrogenic" medicines for the treatment and prevention of breast cancer. Developing drugs that target the estrogen receptor (ER) either directly (tamoxifen) or indirectly (aromatase inhibitors) has improved the prognosis of breast cancer and significantly advanced healthcare. The development of the principles for treatment and the success of the concept, in practice, has become a model for molecular medicine and presaged the current testing of numerous targeted therapies for all forms of cancer. The translational research with tamoxifen to target the ER with the appropriate duration (5 years) of adjuvant therapy has contributed to the falling national death rates from breast cancer. Additionally, exploration of the endocrine pharmacology of tamoxifen and related nonsteroidal antiestrogen (e.g. keoxifene now known as raloxifene) resulted in the laboratory recognition of selective ER modulation and the translation of the concept to use raloxifene for the prevention of osteoporosis and breast cancer. However, the extensive evaluation of tamoxifen treatment revealed small but significant side effects such as endometrial cancer, blood clots and the development of acquired resistance. The solution was to develop drugs that targeted the aromatase enzyme specifically to prevent the conversion of androstenedione to estrone and subsequently estradiol. The successful translational research with the suicide inhibitor 4-hydroxyandrostenedione (known as formestane) pioneered the development of a range of oral aromatase inhibitors that are either suicide inhibitors (exemestane) or competitive inhibitors (letrozole and anastrozole) of the aromatase enzyme. Treatment with aromatase inhibitors is proving effective and is associated with reduction in the incidence of endometrial cancer and blood clots when compared with tamoxifen and there is also limited cross resistance so treatment can be sequential. Current clinical trials are addressing the value of aromatase inhibitors as chemopreventive agents for postmenopausal women.

Docetaxel followed by castration improves outcomes in LNCaP prostate cancer-bearing severe combined immunodeficient mice.

PURPOSE: Androgen ablation is the standard initial treatment for advanced prostate cancer; however, tumors eventually develop androgen independence and become incurable. Chemotherapy is commonly used after hormone treatment fails but has not shown significant survival benefit. Studies suggest that androgen ablation can select for a population of hormone-independent cells that are also relatively chemotherapy resistant. Thus, it may be therapeutically advantageous to target prostate cancer with chemotherapy before hormone ablation. This study was undertaken to determine the relative efficacy of such an approach in a preclinical model of prostate cancer. EXPERIMENTAL DESIGN: Severe combined immunodeficient mice bearing human LNCaP prostate tumors were treated with docetaxel and/or surgical castration applied singly, concurrently, or in different sequences. Treatment efficacy was determined by tumor volume and growth delay measurements. The extent of apoptosis in tumors in response to treatments was assessed via terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assays. In addition, Western blots were done to study the relative expression of Bcl-2 and Bax in the tumors. RESULTS: Docetaxel followed by castration showed the most potent antitumor effects. In contrast, with the exception of castration alone, castration followed by docetaxel produced the least antitumor activity. TUNEL assays confirmed that the density of apoptotic tumor cells was significantly greater for docetaxel followed by castration than for any other treatment. In tumors of mice treated with single modality therapies, Bax to Bcl-2 ratios decreased significantly after castration, whereas this ratio remained high after docetaxel treatment. CONCLUSION: A treatment sequence of docetaxel followed by hormone ablation may be more effective in treating prostate cancer than concurrent docetaxel/hormone therapy or hormone ablation followed by docetaxel.

Activation of mitogen-activated protein kinase in xenografts and cells during prolonged treatment with aromatase inhibitor letrozole.

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 microg/d letrozole for up to 56 weeks. Western blot analysis of the tumors revealed that ERs (ERalpha) 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 micromol/L letrozole, ERalpha 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 MCF-7Ca cells treated with MAPK kinase 1/2 inhibitors U0126, and PD98059 (IC(50) approximately 25 micromol/L). PD98059 (5 micromol/L) also reduced MAPK activity and increased ERalpha to the levels in MCF-7Ca cells. Epidermal growth factor receptor kinase inhibitor, gefitinib (ZD1839) inhibited growth of LTLT-Ca cells (IC(50) approximately 10 micromol/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.

Additive antitumor effect of aromatase inhibitor letrozole and antiestrogen fulvestrant in a postmenopausal breast cancer model.

Blocking estrogen receptors with antiestrogens and blocking estrogen synthesis with aromatase inhibitors are two strategies currently being used for reducing the effect of estrogen in postmenopausal estrogen receptor-positive breast cancer patients. To optimize these treatment strategies, we have investigated whether tumor progression can be delayed by combining the pure antiestrogen fulvestrant with the nonsteroidal aromatase inhibitor letrozole. These studies were done in ovariectomized, athymic mice bearing tumors of estrogen receptor-positive human breast cancer cells stably transfected with the aromatase gene (MCF-7Ca). Groups of mice with equivalent tumor volumes were injected s.c. daily with vehicle (control; n = 6), fulvestrant (1 mg/d; n = 7), letrozole (10 microg/d; n = 18), or letrozole (10 microg/d) plus fulvestrant (1 mg/d; n = 5). All treatments were effective in suppressing tumor growth compared with controls (P < 0.001). Tumor volumes of the fulvestrant-treated group had doubled in 10 weeks. After 19 weeks of letrozole (10 microg/d) treatment when tumors had nearly doubled in volume, mice (n = 18) were assigned to second-line therapy with letrozole (100 microg/d; n = 6), tamoxifen (100 microg/d; n = 6), or remained on letrozole treatment (10 microg/d; n = 6). However, tumors continued to increase in volume in these groups. Tumors of animals treated with the combination of letrozole plus Faslodex regressed over 29 weeks of treatment by 45%. Thus, the combination of letrozole plus fulvestrant was more effective in suppressing tumor growth than either letrozole or fulvestrant alone or sequential therapies with tamoxifen or a higher dose of letrozole (100 microg/d).

Signaling pathways of apoptosis activated by aromatase inhibitors and antiestrogens.

Aromatase inhibitors have recently been reported to be more effective than the antiestrogen tamoxifen (Tam) in treating breast cancer. Here, we studied the mechanisms and signaling pathways of cell growth, cell cycle progression, and apoptosis induced by three aromatase inhibitors: letrozole (Let), anastrozole, and 4-hydroxyandrostenedione in comparison with estrogen withdrawal (E2W) and antiestrogens Tam and faslodex. Estrogen-dependent human breast cancer cells stably transfected with aromatase (MCF-7Ca) were used. All treatments induced growth suppression and cell cycle arrest at the G(0)-G(1) phase that was associated with up-regulation of p53 and p21 protein and mRNA levels and down-regulation of cyclin D1 and c-myc mRNA. The apoptotic index was increased 4-7 fold, Bcl-2 protein expression decreased, Bax increased, and caspase-9, caspase-6, and caspase-7 were activated but not caspase-3 and caspase-8. Let and E2W caused regression of tumors of MCF-7Ca cells grown in nude mice and increased the number of cells undergoing apoptosis. In contrast, Tam and faslodex did not induce tumor regression and a lower number of apoptotic cells was detected. Cleavage of poly(ADP-ribose) polymerase was detected. Treatment with Let, Tam, or E2W resulted in a dose- and time-dependent increase in active caspase-7 and up-regulation of p53 and p21 protein. Although the mechanisms involved appeared to be similar for antiestrogens and aromatase inhibitors, the most significant effects occurred with Let, which were significantly greater than with E2W and consistent with marked effects of Let on tumor and cell growth.

Novel C-17-heteroaryl steroidal CYP17 inhibitors/antiandrogens: synthesis, in vitro biological activity, pharmacokinetics, and antitumor activity in the LAPC4 human prostate cancer xenograft model.

New chemical entities, steroidal C-17 benzoazoles (5, 6, 9 and 10) and pyrazines (14 and 15) were rationally designed and synthesized. The key reaction for synthesis of the benzoazoles involved the nucleophilic vinylic "addition-elimination" substitution reaction of 3beta-acetoxy-17-chloro-16-formylandrosta-5,16-diene (2) and benzoazole nucleophiles, while that for synthesis of pyrazines involved palladium-catalyzed cross-coupling reaction of 17-iodoandrosta-5,16-dien-3beta-ol (13) with tributylstannyl diazines. Some of the compounds were shown to be potent inhibitors of human CYP17 enzyme as well as potent antagonist of both wild type and mutant androgen receptors (AR). The most potent CYP17 inhibitors were 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (5, code named VN/124-1), 3beta-hydroxy-17-(5(1)-pyrimidyl)androsta-5,16-diene (15) and 17-(1H-benzimidazole-1-yl)androsta-4,16-dien-3-one (6), with IC(50) values of 300, 500 and 915 nM, respectively. Compounds 5, 6, 14 and 15 were effective at preventing binding of (3)H-R1881 (methyltrienolone, a stable synthetic androgen) to both the mutant LNCaP AR and the wild-type AR, but with a 2.2- to 5-fold higher binding efficiency to the latter. Compounds 5 and 6 were also shown to be potent pure AR antagonists. The cell growth studies showed that 5 and 6 inhibit the growth of DHT-stimulated LNCaP and LAPC4 prostate cancer cells with IC(50) values in the low micromolar range (i.e., <10 microM). Their inhibitory potencies were comparable to that of casodex but remarkably superior to that of flutamide. The pharmacokinetics of compounds 5 and 6 in mice were investigated. Following s.c. administration of 50 mg/kg of 5 and 6, peak plasma levels of 16.82 and 5.15 ng/mL, respectively, occurred after 30 to 60 min, both compounds were cleared rapidly from plasma (terminal half-lives of 44.17 and 39.93 min, respectively), and neither was detectable at 8 h. Remarkably, compound 5 was rapidly converted into a metabolite tentatively identified as 17-(1H-benzimidazol-1-yl)androsta-3-one. When tested in vivo, 5 proved to be very effective at inhibiting the growth of androgen-dependent LAPC4 human prostate tumor xenograft, while 6 was ineffective. Compound 5 (50 mg/kg/twice daily) resulted in a 93.8% reduction (P = 0.00065) in the mean final tumor volume compared with controls, and it was also significantly more effective than castration. To our knowledge, this is the first example of an antihormonal agent (an inhibitor of androgen synthesis (CYP17 inhibitor)/antiandrogen) that is significantly more effective than castration in suppression of androgen-dependent prostate tumor growth. In view of these impressive anticancer properties, compound 5 is a strong candidate for development for the treatment of human prostate cancer.

The effect of second-line antiestrogen therapy on breast tumor growth after first-line treatment with the aromatase inhibitor letrozole: long-term studies using the intratumoral aromatase postmenopausal breast cancer model.

PURPOSE: The aromatase inhibitors letrozole and anastrozole have been approvedrecently as first-line treatment options for hormone-dependent advanced breast cancer. Although it is established that a proportion of patients who relapse on first-line tamoxifen therapy show additional responses to aromatase inhibitors, it has not been determined whether tumors that acquire resistance to aromatase inhibitors in the first line remain sensitive to second-line therapy with antiestrogens. The aim of this study was to determine whether aromatase-transfected and hormone-dependent MCF-7Ca human breast cancer cells remain sensitive to antiestrogens after: (a) long-term growth in steroid-depleted medium in vitro; and (b) long-term treatment with the aromatase inhibitor letrozole in vivo. METHODS: In the first approach, a variant of the MCF-7Ca human breast cancer cell line was selected that had acquired the ability to grow in estrogen-depleted medium after 6-8 months of culture. Steroid-deprived UMB-1Ca cells were analyzed for aromatase activity levels, hormone receptor levels, and sensitivity to estrogens and antiestrogens in vitro and in vivo. In the second approach, established MCF-7Ca breast tumor xenografts were treated with letrozole 10 microg/day for 12 weeks followed by 100 microg/day for 25 weeks until tumors acquired the ability to proliferate in the presence of the drug. Long-term letrozole-treated tumors were then transplanted into new mice, and the effects of antiestrogens and aromatase inhibitors on tumor growth were determined. RESULTS: Steroid-deprived UMB-1Ca breast cancer cells continued to express aromatase activity at levels comparable with the parental cell line. However, compared with MCF-7Ca cells, UMB-1Ca cells expressed elevated levels of functionally active estrogen receptor. The growth of UMB-1Ca cells in vitro was inhibited by the antiestrogens tamoxifen and faslodex and tumor growth in vivo was inhibited by tamoxifen. In the second approach, the time for MCF-7Ca tumor xenografts to approximately double in volume after being treated sequentially with the increasing doses of letrozole was thirty-seven weeks. Long-term letrozole-treated tumors continued to express functionally active aromatase. When transplanted into new mice, growth of the long-term letrozole-treated tumors was slowed by tamoxifen and inhibited more effectively by faslodex. Tumor growth was refractory to the aromatase inhibitors anastrozole and formestane but, surprisingly, showed sensitivity to letrozole. CONCLUSIONS: Steroid-deprived UMB-1Ca human breast cancer cells selected in vitro and long-term letrozole-treated MCF-7Ca breast tumor xenografts remain sensitive to second-line therapy with antiestrogens and, in particular, to faslodex. This finding is associated with increased expression of functionally active estrogen receptor after steroid-deprivation of MCF-7Ca human breast cancer cells in vitro.

Effects of exemestane and tamoxifen in a postmenopausal breast cancer model.

PURPOSE: To optimize treatment strategies for postmenopausal breast cancer patients, we investigated the efficacy of the steroidal aromatase inhibitor exemestane alone or in combination with the antiestrogen tamoxifen in a xenograft model of postmenopausal breast cancer. We also determined the effects of these agents in sequential second-line therapy and the effect of the nonsteroidal aromatase inhibitor letrozole on tumors that progressed on the above treatments. EXPERIMENTAL: Aromatase-transfected human estrogen receptor-positive breast cancer cells (MCF-7Ca) were grown as tumors in ovariectomized athymic mice. Animals received subcutaneous injection with vehicle, tamoxifen, exemestane, tamoxifen plus exemestane, and letrozole. Tumor volumes were measured weekly. RESULTS: All treatments were effective initially in suppressing tumor growth as first-line therapy compared with vehicle treatment. Exemestane suppressed tumor growth to a greater extent than tamoxifen. However, the combination of tamoxifen plus exemestane was more effective than either drug alone. After tumor volumes doubled on initial treatment, the mice were crossed over to receive exemestane or tamoxifen. Tumor growth slowed briefly in mice treated with tamoxifen and crossed over to exemestane, but tumor growth continued unabated in those changed from exemestane to tamoxifen. However, letrozole was effective in both groups as third-line therapy for a limited period. Letrozole as initial single agent was the best overall treatment in terms of the degree of tumor suppression and the length of effectiveness of treatment. CONCLUSION: Exemestane was more effective in controlling tumor growth than tamoxifen. In addition, the combination of exemestane plus tamoxifen was clearly more effective than sequential use of these agents in the tumor model. However, the nonsteroidal aromatase inhibitor letrozole as first-line therapy was overall the most effective treatment in controlling tumor growth.

Discovery and development of Galeterone (TOK-001 or VN/124-1) for the treatment of all stages of prostate cancer.

In our effort to discover potent and specific inhibitors of 17α-hydroxylase/17,20-lyase (CYP17), the key enzyme which catalyzes the biosynthesis of androgens from progestins, 3ß-(hydroxy)-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (Galeterone or TOK-001, formerly called VN/124-1) was identified as a selective development candidate which modulates multiple targets in the androgen receptor (AR) signaling pathway. This drug annotation summarizes the mechanisms of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for galeterone, which has successfully completed phase II clinical development in men with castration resistant (advanced) prostate cancer (CRPC). Phase III clinical studies in CRPC patients are scheduled to begin in early 2015.

Three dimensional pharmacophore modeling of human CYP17 inhibitors. Potential agents for prostate cancer therapy.

We report here a molecular modeling investigation of steroidal and nonsteroidal inhibitors of human cytochrome P450 17alpha-hydroxylase-17,20-lyase (CYP17). Using the pharmacophore perception technique, we have generated common-feature pharmacophore model(s) to explain the putative binding requirements for two classes of human CYP17 inhibitors. Common chemical features in the steroid and nonsteroid human CYP17 enzyme inhibitors, as deduced by the Catalyst/HipHop program, are one to two hydrogen bond acceptors (HBAs) and three hydrophobic groups. For azole-steroidal ligands, the 3beta-OH group of ring A and the N-3 of the azole ring attached to ring D at C-17 act as hydrogen bond acceptors. A model that permits hydrogen bond interaction between the azole functionality on ring D and the enzyme is consistent with experimental deductions for type II CYP17 inhibitors where a sixth ligating atom interacts with Fe(II) of heme. In general, pharmacophore models derived for steroid and nonsteroidal compounds bear striking similarities to all azole sites mapping the HBA functionality and to three hydrophobic features describing the hydrophobic interactions between the ligands and the enzyme. Using the pharmacophore model derived for azole-steroidal inhibitors as a 3D search query against several 3D multiconformational Catalyst formatted databases, we identified several steroidal compounds with potential inhibition of this enzyme. Biological testing of some of these compounds show low to high inhibitory potency against the human CYP17 enzyme. This shows the potential of our pharmacophore model in identifying new and potent CYP17 inhibitors. Further refinement of the model is in progress with a view to identifying and optimizing new leads.

Novel retinoic acid metabolism blocking agents endowed with multiple biological activities are efficient growth inhibitors of human breast and prostate cancer cells in vitro and a human breast tumor xenograft in nude mice.

Novel retinoic acid metabolism blocking agents (RAMBAs) have been synthesized and characterized. The synthetic features include introduction of nucleophilic ligands at C-4 of all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid, and modification of terminal carboxylic acid group. Most of our compounds are powerful inhibitors of hamster liver microsomal ATRA metabolism enzyme(s). The most potent compound is methyl (2E,4E,6E,8E)-9-(3-imidazolyl-2,6,6-trimethylcyclohex-1-enyl)-3,7-dimethylnona-2,4,6,8-tetraenoate (5) with an IC(50) value of 0.009 nM, which is 666,667 times more potent than the well-known RAMBA, liarozole (Liazal, IC(50) = 6000 nM). Quite unexpectedly, there was essentially no difference between the enzyme inhibitory activities of the two enantiomers of compound 5. In MCF-7 cell proliferation assays, the RAMBAs also enhance the ATRA-mediated antiproliferative activity in a concentration dependent manner. The novel atypical RAMBAs, in addition to being highly potent inhibitors of ATRA metabolism in microsomal preparations and in intact human cancer cells (MCF-7, T47D, and LNCaP), also exhibit multiple biological activities, including induction of apoptosis and differentiation, retinoic acid receptor binding, and potent antiproliferative activity on a number of human cancer cells. Following subcutaneous administration to mice bearing human breast MCF-7 tumor xenografts, 6 (VN/14-1, the free carboxylic acid of 5) was well-tolerated and caused significant tumor growth suppression ( approximately 85.2% vs control, p = 0.022). Our RAMBAs represent novel anticancer agents with unique multiple mechanisms of action. The most potent compounds are strong candidates for development as therapeutic agents for the treatment of a variety of cancers.