Effects of adjuvant exemestane versus anastrozole on bone mineral density for women with early breast cancer (MA.27B): a companion analysis of a randomised controlled trial.

BACKGROUND: Treatment of breast cancer with aromatase inhibitors is associated with damage to bones. NCIC CTG MA.27 was an open-label, phase 3, randomised controlled trial in which women with breast cancer were assigned to one of two adjuvant oral aromatase inhibitors-exemestane or anastrozole. We postulated that exemestane-a mildly androgenic steroid-might have a less detrimental effect on bone than non-steroidal anastrozole. In this companion study to MA.27, we compared changes in bone mineral density (BMD) in the lumbar spine and total hip between patients treated with exemestane and patients treated with anastrozole. METHODS: In MA.27, postmenopausal women with early stage hormone (oestrogen) receptor-positive invasive breast cancer were randomly assigned to exemestane 25 mg versus anastrozole 1 mg, daily. MA.27B recruited two groups of women from MA.27: those with BMD T-scores of -2·0 or more (up to 2 SDs below sex-matched, young adult mean) and those with at least one T-score (hip or spine) less than -2·0. Both groups received vitamin D and calcium; those with baseline T-scores of less than -2·0 also received bisphosphonates. The primary endpoints were percent change of BMD at 2 years in lumbar spine and total hip for both groups. We analysed patients according to which aromatase inhibitor and T-score groups they were allocated to but BMD assessments ceased if patients deviated from protocol. This study is registered with ClinicalTrials.gov, NCT00354302. FINDINGS: Between April 24, 2006, and May 30, 2008, 300 patients with baseline T-scores of -2·0 or more were accrued (147 allocated exemestane, 153 anastrozole); and 197 patients with baseline T-scores of less than -2·0 (101 exemestane, 96 anastrozole). For patients with T-scores greater than -2·0 at baseline, mean change of bone mineral density in the spine at 2 years did not differ significantly between patients taking exemestane and patients taking anastrozole (-0·92%, 95% CI -2·35 to 0·50 vs -2·39%, 95% CI -3·77 to -1·01; p=0·08). Respective mean loss in the hip was -1·93% (95% CI -2·93 to -0·93) versus -2·71% (95% CI -4·32 to -1·11; p=0·10). Likewise for those who started with T-scores of less than -2·0, mean change of spine bone mineral density at 2 years did not differ significantly between the exemestane and anastrozole treatment groups (2·11%, 95% CI -0·84 to 5·06 vs 3·72%, 95% CI 1·54 to 5·89; p=0·26), nor did hip bone mineral density (2·09%, 95% CI -1·45 to 5·63 vs 0·0%, 95% CI -3·67 to 3·66; p=0·28). Patients with baseline T-score of -2·0 or more taking exemestane had two fragility fractures and two other fractures, those taking anastrozole had three fragility fractures and five other fractures. For patients who had baseline T-scores of less than -2·0 taking exemestane, one had a fragility fracture and four had other fractures, whereas those taking anastrozole had five fragility fractures and one other fracture. INTERPRETATION: Our results demonstrate that adjuvant treatment with aromatase inhibitors can be considered for breast cancer patients who have T-scores less than -2·0. FUNDING: Canadian Cancer Society Research Institute, Pfizer, Canadian Institutes of Health Research.

Berries and ellagic acid prevent estrogen-induced mammary tumorigenesis by modulating enzymes of estrogen metabolism.

To determine whether dietary berries and ellagic acid prevent 17beta-estradiol (E(2))-induced mammary tumors by altering estrogen metabolism, we randomized August-Copenhagen Irish rats (n = 6 per group) into five groups: sham implant + control diet, E(2) implant + control diet (E(2)-CD), E(2) + 2.5% black raspberry (E(2)-BRB), E(2) + 2.5% blueberry (E(2)-BB), and E(2) + 400 ppm ellagic acid (E(2)-EA). Animals were euthanized at early (6 wk), intermediate (18 wk), and late (24 wk) phases of E(2) carcinogenesis, and the mammary tissue was analyzed for gene expression changes using quantitative real-time PCR. At 6 weeks, E(2) treatment caused a 48-fold increase in cytochrome P450 1A1 (CYP1A1; P < 0.0001), which was attenuated by both BRB and BB diets to 12- and 21-fold, respectively (P < 0.001). E(2) did not alter CYP1B1 levels, but both berry and EA diets significantly suppressed it by 11- and 3.5-fold, respectively, from baseline (P < 0.05). There was a 5-fold increase in 17beta-hydroxysteroid dehydrogenase 7 (17betaHSD7), and this was moderately abrogated to approximately 2-fold by all supplementation (P < 0.05). At 18 weeks, CYP1A1 was elevated by 15-fold in E(2)-CD and only E(2)-BB reduced this increase to 7-fold (P < 0.05). Catechol-O-methyltransferase expression was elevated 2-fold by E(2) treatment (P < 0.05), and all supplementation reversed this. At 24 weeks, CYP1A1 expression was less pronounced but still high (8-fold) in E(2)-treated rats. This increase was reduced to 3.2- and 4.6-fold by E(2)-BRB and E(2)-EA, respectively (P < 0.05), but not by E(2)-BB. Supplementation did not alter the effect of E(2) on steroid receptors. The diets also significantly suppressed mammary tumor incidence (10-30%), volume (41-67%), and multiplicity (38-51%; P < 0.05). Berries may prevent mammary tumors by suppressing the levels of E(2)-metabolizing enzymes during the early phase of E(2) carcinogenesis.

Tumor aromatase expression as a prognostic factor for local control in young breast cancer patients after breast-conserving treatment.

INTRODUCTION: We sought to determine whether the levels of expression of 17 candidate genes were associated with locoregional control after breast-conserving treatments of early-stage breast cancers in young, premenopausal women. METHODS: Gene expression was measured by using RT-PCR in the breast tumors of a series of 53 young (younger than 40 years), premenopausal patients. All treatments consisted of primary breast-conserving surgery followed by whole-breast radiotherapy (+/- regional lymph nodes) with or without systemic treatments (chemotherapy +/- hormone therapy). The median follow-up was 10 years. RESULTS: The 10-year locoregional control rate was 70% (95% CI, 57% to 87%). In univariate analysis, no clinical/pathologic prognostic factors were found to be significantly associated with decreased locoregional control. Expression of three genes was found to be significantly associated with an increased locoregional recurrence rate: low estrogen-receptor beta, low aromatase, and high GATA3. Two others were associated with only a trend (P < 0.10): low HER1 and SKP2. In multivariate analysis, only the absence of aromatase was significantly associated with an increased locoregional recurrence rate (P = 0.003; relative risk = 0.49; 95% CI 0.29 to 0.82). CONCLUSIONS: Recent data give credit to the fact that breast cancer in young women is a distinct biologic entity driven by special oncogenic pathways. Our results highlight the role of estrogen-signaling pathways (mainly CYP19/aromatase, GATA3, and ER-beta) in the risk of locoregional recurrence of breast cancer in young women. Confirmation in larger prospective studies is needed.

Dietary genistein negates the inhibitory effect of letrozole on the growth of aromatase-expressing estrogen-dependent human breast cancer cells (MCF-7Ca) in vivo.

Genistein (GEN), a soy isoflavone, stimulates growth of estrogen-dependent human tumor cells (MCF-7) in a preclinical mouse model for postmenopausal breast cancer. Antiestrogens and aromatase inhibitors are frontline therapies for estrogen-dependent breast cancer. We have demonstrated that dietary GEN can negate the inhibitory effect of tamoxifen. In this study, we evaluated the interaction of dietary GEN (at 250-1000 p.p.m. in the American Institute of Nutrition 93 growth diet) and an aromatase inhibitor, letrozole (LET), on the growth of tumors in an aromatase-expressing breast cancer xenograft model (MCF-7Ca) in the presence and absence of the substrate androstenedione (AD). Dietary GEN (250 and 500 p.p.m.) or implanted AD stimulated MCF-7Ca tumor growth. Implanted LET inhibited AD-stimulated MCF-7Ca tumor growth. In the presence of AD and LET, dietary GEN (250, 500 and 1000 p.p.m.) reversed the inhibitory effect of LET in a dose-dependent manner. Uterine wet weight, plasma estradiol (E(2)) levels (enzyme-linked immunosorbent assay) and total plasma GEN and LET levels (liquid chromatography-electrospray/tandem mass spectrometry) were measured. Ki-67 (cellular proliferation), aromatase and pS2 protein expression in tumors were evaluated using immunohistochemical (IHC) analysis. In conclusion, dietary GEN increased the growth of MCF-7Ca tumors implanted in ovariectomized mice and could also negate the inhibitory effect of LET on MCF-7Ca tumor growth. These findings are significant because tumors, which express aromatase and synthesize estrogen, are good candidates for aromatase therapy dietary and GEN can reverse the inhibitory effect of LET on tumor growth and adversely impact breast cancer therapy. Caution is warranted for consumption of dietary GEN by postmenopausal women with estrogen-dependent breast cancer taking LET treatment.

Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

Carcinogenesis of hormone-related cancers involves hormone-stimulated cell proliferation, which increases the number of cell divisions and the opportunity for random genetic errors. In target tissues, steroid hormones are interconverted between their potent, high affinity forms for their respective receptors and their inactive, low affinity forms. One group of enzymes responsible for these interconversions are the hydroxysteroid dehydrogenases, which regulate ligand access to steroid receptors and thus act at a pre-receptor level. As part of this group, the 17beta-hydroxysteroid dehydrogenases catalyze either oxidation of hydroxyl groups or reduction of keto groups at steroid position C17. The thoroughly characterized 17beta-hydroxysteroid dehydrogenase type 1 activates the less active estrone to estradiol, a potent ligand for estrogen receptors. This isoform is expressed in gonads, where it affects circulating levels of estradiol, and in peripheral tissue, where it regulates ligand occupancy of estrogen receptors. Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 are thus highly interesting potential therapeutic agents for the control of estrogen-dependent diseases such as endometriosis, as well as breast and ovarian cancers. Here, we present the review on the recent development of inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 published and patented since the previous review of 17beta-hydroxysteroid dehydrogenase inhibitors of Poirier (Curr. Med. Chem., 2003, 10, 453). These inhibitors are divided into two separate groups according to their chemical structures: steroidal and non-steroidal 17beta-hydroxysteroid dehydrogenase type 1 inhibitors. Their estrogenic/ proliferative activities and selectivities over other 17beta-hydroxysteroid dehydrogenases that are involved in local regulation of estrogen action (types 2, 7 and 12) are also presented.

Aromatase inhibitors in early hormone receptor-positive breast cancer : what is the optimal initiation time for the maximum benefit?

Breast cancer is common, affecting one in nine women worldwide. As stipulated by the St Gallen consensus guidelines, hormone therapy is an integral part of treatment for hormone-responsive disease. Previously, this has been with tamoxifen; however, as a result of a number of recent studies, aromatase inhibitors are now competing for use as first-line agents. In addition, there is as yet no firm consensus as to when and how these drugs should be used within the adjuvant setting. This article reviews the use of aromatase inhibitors in early stage hormone-positive breast cancer. It describes the evidence from the studies involving the aromatase inhibitors in an upfront, switch and extended setting. It further discusses the mathematical models proposed to determine the optimum timing of initiation. In light of the ongoing research into predictive biomarkers, this review then concentrates on whether future focus should be on more individualized treatment strategies than the optimum timing of aromatase inhibitors.

Efficacy of sulforaphane is mediated by p38 MAP kinase and caspase-7 activations in ER-positive and COX-2-expressed human breast cancer cells.

Sulforaphane is an antioxidant and a potent stimulator of natural detoxifying enzyme and associated with lowered risk of cancer that is associated with the consumption of cruciferous vegetables. The chemopreventive effects of SFN was investigated using the MCF-7 human breast cancer cells and the M13SV1-immortalized human breast luminal epithelial cells. Sulforaphane reduced proliferation in MCF-7 cells and inhibited cyclooxygenase-2 expression in M13SV1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The chemopreventive effects of sulforaphane were associated with p38 mitogen-activated protein kinase suggest its important role in cell survival/apoptosis regulation and stabilization of cyclooxygenase-2. Sulforaphane upregulates p38 in MCF-7 cells and prevented TPA-reduced phosphorylation of p38 in M13SV1 cells, but activated caspase-7 associated with apoptosis in MCF-7 cells. These results suggest that sulforaphane may be an alternative candidate for targeted prevention of ER-positive and cyclooxygenase-2-induced phenotypes and breast cancer.

Estradiol as an anti-aromatase agent in human breast cancer cells.

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.

Apoptosis of human prostate androgen-dependent and -independent carcinoma cells induced by an isopropanolic extract of black cohosh involves degradation of cytokeratin (CK) 18.

BACKGROUND: The inhibitory effects of black cohosh extracts (Cimicifuga syn. Actaea racemosa L.) on the proliferation of human breast cancer cells were reported recently. In this study, we turned examined another hormone-dependent, epidemiologically important tumor disease, prostate cancer. The cell growth inhibitory effect of an isopropanolic extract of black cohosh (iCR) on androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells was investigated. MATERIALS AND METHODS: The cytotoxic effect of the extract was determined by WST-1 assay. Apoptosis was determined by the appearance of apoptotic morphology, annexin V-FITC adherence and caspase activation. Cytokeratin (CK) 18 degradation was identified with M30 monoclonal antibody. RESULTS: Regardless of their hormone sensitivity, the growth of prostate cancer cells was significantly and dose-dependently down-regulated by iCR. The drug concentration producing 50% cell growth inhibition in all cell lines after 72h lay between 37.1 and 62.7 microg/ml. Increases in the level of M30 antigen of approximately 1.8-, 5.9- and 5.3-fold over untreated controls were observed in black cohosh-treated PC-3, DU 145 and LNCaP cells, respectively, with the induction of apoptosis being dose- and time-dependent. CONCLUSION: Black cohosh extract kills human hormone-responsive or-unresponsive prostate cancer cells by induction of apoptosis and activation of caspases. This finding suggests that the cell's hormone responsive status is not an important determinant of the response to the extract and that iCR extract may represent a novel therapeutic approach for the treatment of prostate cancer.

Aromatase inhibitors in the treatment of breast cancer.

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.

Inhibition of cyclooxygenase (COX)-2 expression by Tet-inducible COX-2 antisense cDNA in hormone-refractory prostate cancer significantly slows tumor growth and improves efficacy of chemotherapeutic drugs.

PURPOSE: Overexpression of the proinflammatory enzyme cyclooxygenase (COX)-2 is associated with the progression of various malignancies; the role of COX-2 in prostate cancer is less clear. The significance of COX-2 in prostate cancer growth and response to chemotherapy was investigated in an androgen-refractory prostate cancer cell line using a Tet-inducible antisense COX-2 expression system. EXPERIMENTAL DESIGN: An antisense COX-2 cDNA construct under the control of a doxycycline-inducible promoter was transfected into a prostate cancer cell line, PC-3ML. Modulations of cell growth, apoptosis, and chemosensitivity in the presence or absence of doxycycline were analyzed. Tumor incidence, growth rate, and response to two cytotoxic drugs, COL-3 [chemically modified tetracycline-3-(6-demethyl-6-deoxy-4-dedimethylamino-tetracycline)] and Taxotere (docetaxel), were investigated in tumor xenografts. Apoptotic incidences and tumor microvessel density in tumors were determined by immunohistochemistry. RESULTS: Conditional suppression of COX-2 in PC-3ML caused reduced cell proliferation, decreased levels of phosphorylated AKT, G(0)-G(1) arrest, and increased apoptosis and caspase-3 activity. Suppression of COX-2 increased Bax protein and decreased Bcl-x(L) protein in vitro. COX-2 antisense-expressing PC-3ML tumors showed a 57% growth delay compared with nontransfected or vector controls. Oral administration of COL-3 (40 mg/kg, oral gavage) or Taxotere (2.3 mg/kg, intraperitoneally; 3x per week) in tumor-bearing mice further slowed tumor growth (65% and approximately 94%, respectively). Compared with the control group, the occurrence of apoptosis in antisense COX-2 tumors was eight times higher, and the tumor microvessel density was three times lower. CONCLUSIONS: These results provide direct evidence that constitutive expression of COX-2 in prostate cancer has both angiogenic and cytoprotective functions. Suppression of tumor cell COX-2 is sufficient to enhance chemotherapy response in prostate cancer.

Structure-function studies of aromatase and its inhibitors: a progress report.

The utilization of computer modeling, site-directed mutagenesis, inhibition kinetic analysis and reaction metabolite analysis allows us to better understand the structure-function relationship between aromatase and its inhibitors. Our results have helped in determining how steroidal and nonsteriodal aromatase inhibitors bind to the active site of the enzyme. This information has also aided in the understanding of the reaction mechanism of aromatase. Furthermore, our structure-function studies of aromatase have generated important information for predicting how environmental chemicals interact with the enzyme. During the last 2 years, a new aromatase computer model based on the X-ray structure of rabbit cytochrome P450 2C5 has been generated and used to evaluate the results obtained from new aromatase mutants produced in this laboratory. In addition, we have succeeded in the expression and purification of functionally active aromatase using an Escherichia coli expression method. The catalytic properties of this recombinant aromatase are similar to those properties exhibited by the human placental aromatase preparation and the mammalian cell-expressed enzyme. The E. coli expressed aromatase will be very useful for further structure-function studies of aromatase. Our laboratory has also evaluated the growth-inhibiting activity of aromatase inhibitors in estrogen receptor-positive breast cancer using three-dimensional cell cultures of aromatase-over expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro). Our results demonstrate that these three-dimensional cultures are valuable approaches to assess the growth-inhibiting activity of aromatase inhibitors. Finally, we have identified several phytochemicals to be potent inhibitors of aromatase. To demonstrate the impact of the phytochemicals on estrogen formation in vivo, we showed that the intake of anti-aromatase chemicals from red wine was capable of suppressing MCF-7aro-mediated tumor formation in nude mice and aromatase-induced hyperplasia in a transgenic mouse model in which aromatase is over-expressed in the mammary tissue.

The intratumoral aromatase model: studies with aromatase inhibitors and antiestrogens.

Aromatase inhibitors have now been approved as first-line treatment options for hormone-dependent advanced breast cancer. When compared to tamoxifen, these aromatase inhibitors provide significant survival and tolerability advantages. However, the optimal use of an aromatase inhibitor and tamoxifen remains to be established. To date, the intratumoral aromatase xenograft model has proved accurate in predicting the outcome of clinical trials. Utilizing this model, we performed long-term studies with tamoxifen and letrozole to determine time to disease progression with each of the treatment regimens. Aromatase-transfected MCF-7Ca human breast cancer cells were grown as tumor xenografts in female ovariectomized athymic nude mice in which androstenedione was converted to estrogen and stimulated tumor growth. When tumor volumes were approximately 300 mm3, the animals were grouped for continued supplementation with androstenedione only (control) or for treatment with letrozole 10 microg per day (long-term), tamoxifen 100 microg per day (long-term), letrozole alternating to tamoxifen (4-week rotation), tamoxifen alternating to letrozole (4-week rotation), or a combination of the two drugs. Tumors of control mice had doubled in volume in 3-4 weeks. In mice treated with tamoxifen and the combination, tumor doubling time was significantly shorter (16 and 18 weeks, respectively) than with letrozole (34 weeks). Furthermore, alternating letrozole and tamoxifen treatment every 4 weeks was less effective than letrozole alone. Tumors doubled in 17-18 weeks when the starting treatment was tamoxifen and in 22 weeks when the starting treatment was letrozole. Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole (10 microg per day). However, when mice with letrozole-resistant tumors were switched to antiestrogen treatment, tumors did not respond to tamoxifen (100 microg per day) or faslodex (1 mg per day). This suggests that advanced breast cancers treated with letrozole may be insensitive to subsequent second-line hormonal agents. Thus, although letrozole was determined to be an effective second-line treatment option for tumors progressing on tamoxifen, antiestrogen therapy does not appear to be effective for tumors progressing on letrozole. However, response to second-line treatment was observed in a model where tumors that had progressed on letrozole were transplanted to new mice. These tumors had been allowed to grow in the presence of supplemented androstenedione but absence of letrozole. This suggests that resistance to letrozole may be reversible, allowing tumors to respond to subsequent antiestrogens and letrozole.

Protein kinase cepsilon has the potential to advance the recurrence of human prostate cancer.

Prostatic epithelial cells that are capable of surviving in the absence of androgenic steroids were found to express protein kinase Cepsilon (PKCepsilon), an oncogenic protein capable of promoting autocrine cell-signaling events. Gene transfer experiments demonstrated that PKCepsilon overexpression was sufficient to transform androgen-dependent LNCaP cells into an androgen-independent variant that rapidly initiated tumor growth in vivo in both intact and castrated male nude mice. This transformation was associated with an accelerated rate of androgen-independent LNCaP cell proliferation, resistance to apoptosis, hyperphosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase and transcriptional repressor protein retinoblastoma, and increased expression of E2F-1 and other 5'-cap-dependent mRNAs, including the G(1) cyclins, c-myc, and caveolin-1. Coimmunoprecipitation experiments indicated that PKCepsilon was associated with members of the extracellular signal-regulated kinase signaling cascade and the scaffolding protein caveolin-1. Caveolin-1, produced by LNCaP cells overexpressing PKCepsilon, was released into the medium, possibly through a Golgi-independent route, and significant growth inhibition was observed when these cells were cultured in the presence of an anti-caveolin-1 antiserum. Finally, antisense experiments established that endogenous PKCepsilon plays an important role in regulating the growth and survival of androgen-independent prostate cancer cells. This study provides several independent lines of evidence supporting the hypothesis that PKCepsilon expression may be sufficient to maintain prostate cancer growth and survival after androgen ablation.

Role of androgens in the growth of endometrial carcinoma: an in vivo animal model.

OBJECTIVE: We sought to create an animal model for the development of endometrial cancer in women with androgen excess. We examined the effects of estradiol and androgen, both alone and as precursors to estrogen biosynthesis on human endometrial cancers transplanted into a nude mouse model. STUDY DESIGN: We transplanted an estrogen-responsive, well-differentiated, established human endometrial carcinoma, EnCa-101, subcutaneously into athymic male nude mice. We established, first, that aromatase was expressed in this cell line, inducible by estrogen. We measured the growth of the tumor in the various groups weekly with Vernier calipers. We examined the effects of estradiol and androgens, both aromatizable and nonaromatizable, on tumor growth. RESULTS: Estrogen-supplemented tumors showed the greatest rate of growth and were significantly greater than the growth rate in castrate mice. Androgen-supplemented tumors showed a growth rate similar to that of tumors without significant hormonal exposure (castrate mice). Dihydrotestosterone had no effect on tumor growth in comparison with an agonadal state. CONCLUSIONS: Aromatizable and nonaromatizable androgens have little growth-promoting effect on a well-differentiated endometrial carcinoma. Estradiol is the most potent growth stimulus in our model.

Use of aromatase inhibitors in breast carcinoma.

Aromatase, a cytochrome P-450 enzyme that catalyzes the conversion of androgens to estrogens, is the major mechanism of estrogen synthesis in the post-menopausal woman. We review some of the recent scientific advances which shed light on the biologic significance, physiology, expression and regulation of aromatase in breast tissue. Inhibition of aromatase, the terminal step in estrogen biosynthesis, provides a way of treating hormone-dependent breast cancer in older patients. Aminoglutethimide was the first widely used aromatase inhibitor but had several clinical drawbacks. Newer agents are considerably more selective, more potent, less toxic and easier to use in the clinical setting. This article reviews the clinical data supporting the use of the potent, oral competitive aromatase inhibitors anastrozole, letrozole and vorozole and the irreversible inhibitors 4-OH androstenedione and exemestane. The more potent compounds inhibit both peripheral and intra-tumoral aromatase. We discuss the evidence supporting the notion that aromatase inhibitors lack cross-resistance with antiestrogens and suggest that the newer, more potent compounds may have a particular application in breast cancer treatment in a setting of adaptive hypersensitivity to estrogens. Currently available aromatase inhibitors are safe and effective in the management of hormone-dependent breast cancer in post-menopausal women failing antiestrogen therapy and should now be used before progestational agents. There is abundant evidence to support testing these compounds as first-line hormonal therapy for metastatic breast cancer as well as part of adjuvant regimens in older patients and quite possibly in chemoprevention trials of breast cancer.

Biological effects of stable overexpression of aromatase in human hormone-dependent breast cancer cells.

Aromatase is a key enzyme in the conversion of androstenedione and testosterone to oestrone and oestradiol. Intratumoral aromatase activity is expressed by around 70% of breast carcinomas, but it is not clear what effect this has on the tumour phenotype. To address this question we expressed human aromatase in hormone-dependent MCF-7 breast cancer cells. Clone Arom. 1 expressed aromatase at 1,000 times the endogenous level in wild-type (WT) cells. Clone Arom. 2 incorporated the expression construct but did not express aromatase at levels above WT. There was no morphological difference between the two clones and WT, all three cell lines expressed oestrogen receptor at equivalent levels, and all manifested a mitogenic response to oestradiol. In steroid-depleted medium Arom. 1 cells showed significant growth enhancement over WT and Arom. 2, and this growth advantage was increased by exogenous androstenedione or testosterone. Both the enzyme activity and androgen-stimulated growth of Arom. 1 cells were completely reversible by aromatase inhibitor CGS 16949A. The Arom. 1 cell line may contribute to the development of an in vivo model of intratumoral aromatase, to study the biological significance of this phenomenon.