Brett T. Marck

Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice

Hypoxia-inducible factor (HIF) transcription factors respond to multiple environmental stressors, including hypoxia and hypoglycemia. We report that mice lacking the HIF family member HIF-2α (encoded by Epas1) have a syndrome of multiple-organ pathology, biochemical abnormalities and altered gene expression patterns. Histological and ultrastructural analyses showed retinopathy, hepatic steatosis, cardiac hypertrophy, skeletal myopathy, hypocellular bone marrow, azoospermia and mitochondrial abnormalities in these mice. Serum and urine metabolite studies showed hypoglycemia, lactic acidosis, altered Krebs cycle function and dysregulated fatty acid oxidation. Biochemical assays showed enhanced generation of reactive oxygen species (ROS), whereas molecular analyses indicated reduced expression of genes encoding the primary antioxidant enzymes (AOEs). Transfection analyses showed that HIF-2α could efficiently transactivate the promoters of the primary AOEs. Prenatal or postnatal treatment of Epas1−/− mice with a superoxide dismutase (SOD) mimetic reversed several aspects of the null phenotype. We propose a rheostat role for HIF-2α that allows for the maintenance of ROS as well as mitochondrial homeostasis.

Resistance to CYP17A1 inhibition with abiraterone in castration resistant prostate cancer: Induction of steroidogenesis and androgen receptor splice variants

Purpose: Abiraterone is a potent inhibitor of the steroidogenic enzyme CYP17A1 and suppresses tumor growth in patients with castration-resistant prostate cancer (CRPC). The effectiveness of abiraterone in reducing tumor androgens is not known, nor have mechanisms contributing to abiraterone resistance been established. Experimental Design: We treated human CRPC xenografts with abiraterone and measured tumor growth, tissue androgens, androgen receptor (AR) levels, and steroidogenic gene expression versus controls. Results: Abiraterone suppressed serum PSA levels and improved survival in two distinct CRPC xenografts: median survival of LuCaP35CR improved from 17 to 39 days (HR = 3.6, P = 0.0014) and LuCaP23CR from 14 to 24 days (HR = 2.5, P = 0.0048). Abiraterone strongly suppressed tumor androgens, with testosterone (T) decreasing from 0.49 ± 0.22 to 0.03 ± 0.01 pg/mg (P < 0.0001), and from 0.69 ± 0.36 to 0.03 ± 0.01 pg/mg (P = 0.002) in abiraterone-treated 23CR and 35CR, respectively, with comparable decreases in tissue DHT. Treatment was associated with increased expression of full-length AR (ARFL) and truncated AR variants (ARFL 2.3-fold, P = 0.008 and ARdel567es 2.7-fold, P = 0.036 in 23 CR; ARFL 3.4-fold, P = 0.001 and ARV7 3.1-fold, P = 0.0003 in 35CR), and increased expression of the abiraterone target CYP17A1 (∼2.1-fold, P = 0.0001 and P = 0.028 in 23CR and 35CR, respectively) and transcript changes in other enzymes modulating steroid metabolism. Conclusions: These studies indicate that abiraterone reduces CRPC growth via suppression of intratumoral androgens and that resistance to abiraterone may occur through mechanisms that include upregulation of CYP17A1, and/or induction of AR and AR splice variants that confer ligand-independent AR transactivation. Clin Cancer Res; 17(18); 5913–25. ©2011 AACR.

Intratumoral De Novo Steroid Synthesis Activates Androgen Receptor in Castration Resistant Prostate Cancer and is Upregulated by Treatment with CYP17A1 Inhibitors

Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone. DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1-dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis.

Restoration of Norepinephrine and Reversal of Phenotypes in Mice Lacking Dopamine β‐Hydroxylase

Abstract: Mice with a targeted disruption of the dopamine β‐hydroxylase (DBH) gene are unable to synthesize norepinephrine (NE) and epinephrine. These mice have elevated levels of dopamine in most tissues, although the levels are only a fraction of those normally found for NE. It is noteworthy that NE can be restored to normal levels in many tissues after a single injection of the synthetic amino acid precursor of NE, l‐threo‐3,4‐dihydroxyphenylserine (DOPS). In other tissues, NE can be restored to normal levels after multiple injections of DOPS, whereas in the midbrain and cerebellum, restoration of NE is limited to 25–30% of normal. NE levels typically peak ∼5 h after DOPS administration and are undetectable by 48 h. Epinephrine levels are more difficult to restore. The elevated levels of dopamine fall modestly after injection of DOPS. S(−)‐Carbidopa, which does not cross the blood‐brain barrier, inhibits aromatic l‐amino acid decarboxylase and effectively prevents restoration of NE by DOPS in the periphery, while allowing restoration in the CNS. Ptosis and reductions in male fertility, hind‐limb extension, postdecapitation convulsions, and uncoupling protein expression in dopamine β‐hydroxylase‐deficient mice are all reversed by DOPS injection.

Intense Androgen-Deprivation Therapy With Abiraterone Acetate Plus Leuprolide Acetate in Patients With Localized High-Risk Prostate Cancer: Results of a Randomized Phase II Neoadjuvant Study

PURPOSE Cure rates for localized high-risk prostate cancers (PCa) and some intermediate-risk PCa are frequently suboptimal with local therapy. Outcomes are improved by concomitant androgen-deprivation therapy (ADT) with radiation therapy, but not by concomitant ADT with surgery. Luteinizing hormone-releasing hormone agonist (LHRHa; leuprolide acetate) does not reduce serum androgens as effectively as abiraterone acetate (AA), a prodrug of abiraterone, a CYP17 inhibitor that lowers serum testosterone (< 1 ng/dL) and improves survival in metastatic PCa. The possibility that greater androgen suppression in patients with localized high-risk PCa will result in improved clinical outcomes makes paramount the reassessment of neoadjuvant ADT with more robust androgen suppression. PATIENTS AND METHODS A neoadjuvant randomized phase II trial of LHRHa with AA was conducted in patients with localized high-risk PCa (N = 58). For the first 12 weeks, patients were randomly assigned to LHRHa versus LHRHa plus AA. After a research prostate biopsy, all patients received 12 additional weeks of LHRHa plus AA followed by prostatectomy. RESULTS The levels of intraprostatic androgens from 12-week prostate biopsies, including the primary end point (dihydrotestosterone/testosterone), were significantly lower (dehydroepiandrosterone, Δ(4)-androstene-3,17-dione, dihydrotestosterone, all P < .001; testosterone, P < .05) with LHRHa plus AA compared with LHRHa alone. Prostatectomy pathologic staging demonstrated a low incidence of complete responses and minimal residual disease, with residual T3- or lymph node-positive disease in the majority. CONCLUSION LHRHa plus AA treatment suppresses tissue androgens more effectively than LHRHa alone. Intensive intratumoral androgen suppression with LHRHa plus AA before prostatectomy for localized high-risk PCa may reduce tumor burden.

Induction of stereotypy in dopamine-deficient mice requires striatal D1 receptor activation

Motor stereotypies are abnormally repetitive behaviors that can develop with excessive dopaminergic stimulation and are features of some neurologic disorders. To investigate the mechanisms required for the induction of stereotypy, we examined the responses of dopamine-deficient (DD) mice to increasing doses of the dopamine precursor l-DOPA. DD mice lack the ability to synthesize dopamine (DA) specifically in dopaminergic neurons yet exhibit robust hyperlocomotion relative to wild-type (WT) mice when treated with l-DOPA, which restores striatal DA tissue content to ≈10% of WT levels. To further elevate brain DA content in DD mice, we administered the peripheral l-amino acid decarboxylase inhibitor carbidopa along with l-DOPA (C/l-DOPA). When striatal DA levels reached >50% of WT levels, a transition from hyperlocomotion to intense, focused stereotypy was observed that was correlated with an induction of c-fos mRNA in the ventrolateral and central striatum as well as the somatosensory cortex. WT mice were unaffected by C/l-DOPA treatments. A D1, but not a D2, receptor antagonist attenuated both the C/l-DOPA-induced stereotypy and the c-fos induction. Consistent with these results, stereotypy could be induced in DD mice by a D1, but not by a D2, receptor agonist, with neither agonist inducing stereotypy in WT mice. Intrastriatal injection of a D1 receptor antagonist ameliorated the stereotypy and c-fos induction by C/l-DOPA. These results indicate that activation of D1 receptors on a specific population of striatal neurons is required for the induction of stereotypy in DD mice.

Dihydrotestosterone Administration Does Not Increase Intraprostatic Androgen Concentrations or Alter Prostate Androgen Action in Healthy Men: A Randomized-Controlled Trial

CONTEXT Concern exists that androgen treatment might adversely impact prostate health in older men. Dihydrotestosterone (DHT), derived from local conversion of testosterone to DHT by 5α-reductase enzymes, is the principal androgen within the prostate. Exogenous androgens raise serum DHT concentrations, but their effects on the prostate are not clear. OBJECTIVE To determine the impact of large increases in serum DHT concentrations on intraprostatic androgen concentrations and androgen action within the prostate. DESIGN Double-blind, randomized, placebo-controlled. SETTING Single academic medical center. PARTICIPANTS 31 healthy men ages 35-55. INTERVENTION Daily transdermal DHT or placebo gel. MAIN OUTCOME MEASURES Serum and prostate tissue androgen concentrations and prostate epithelial cell gene expression after 4 wk of treatment. RESULTS Twenty-seven men completed all study procedures. Serum DHT levels increased nearly sevenfold, while testosterone levels decreased in men treated with daily transdermal DHT gel but were unchanged in the placebo-treated group (P < 0.01 between groups). In contrast, intraprostatic DHT and testosterone concentrations on d 28 were not different between groups (DHT: placebo = 2.8 ± 0.2 vs. DHT gel = 3.1 ± 0.5 ng/g; T: placebo = 0.6 ± 0.2 vs. DHT gel = 0.4 ± 0.1, mean ± se). Similarly, prostate volume, prostate-specific antigen, epithelial cell proliferation, and androgen-regulated gene expression were not different between groups. CONCLUSIONS Robust supraphysiologic increases in serum DHT, associated with decreased serum T, do not significantly alter intraprostatic levels of DHT, testosterone, or prostate epithelial cell androgen-regulated gene expression in healthy men. Changes in circulating androgen concentrations are not necessarily mimicked within the prostate microenvironment, a finding with implications for understanding the impact of androgen therapies in men.

Targeted Androgen Pathway Suppression in Localized Prostate Cancer: A Pilot Study

PURPOSE Ligand-mediated activation of the androgen receptor (AR) is critical for prostate cancer (PCa) survival and proliferation. The failure to completely ablate tissue androgens may limit suppression of PCa growth. We evaluated combinations of CYP17A and 5-α-reductase inhibitors for reducing prostate androgen levels, AR signaling, and PCa volumes. PATIENTS AND METHODS Thirty-five men with intermediate/high-risk clinically localized PCa were randomly assigned to goserelin combined with dutasteride (ZD), bicalutamide and dutasteride (ZBD), or bicalutamide, dutasteride, and ketoconazole (ZBDK) for 3 months before prostatectomy. Controls included patients receiving combined androgen blockade with luteinizing hormone-releasing hormone agonist and bicalutamide. The primary outcome measure was tissue dihydrotestosterone (DHT) concentration. RESULTS Prostate DHT levels were substantially lower in all experimental arms (0.02 to 0.04 ng/g v 0.92 ng/g in controls; P < .001). The ZBDK group demonstrated the greatest percentage decline in serum testosterone, androsterone, and dehydroepiandrosterone sulfate (P < .05 for all). Staining for AR and the androgen-regulated genes prostate-specific antigen and TMPRSS2 was strongly suppressed in benign glands and moderately in malignant glands (P < .05 for all). Two patients had pathologic complete response, and nine had ≤ 0.2 cm(3) of residual tumor (defined as a near-complete response), with the largest numbers of complete and near-complete responses in the ZBDK group. CONCLUSION Addition of androgen synthesis inhibitors lowers prostate androgens below that achieved with standard therapy, but significant AR signaling remains. Tissue-based analysis of steroids and AR signaling is critical to informing the search for optimal local and systemic control of high-risk prostate cancer.

Ganglioneuromas and renal anomalies are induced by activated RETMEN2B in transgenic mice

Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant syndrome characterized by the development of medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies and mucosal ganglioneuromas. MEN2B is caused by a specific mutation (Met918→Thr) in the RET receptor tyrosine kinase. Different mutations of RET lead to other conditions including MEN2A, familial medullary thyroid carcinoma and intestinal aganglionosis (Hirschsprung disease). Transgenic mice were created using the dopamine β-hydroxylase promoter to direct expression of RETMEN2B in the developing sympathetic and enteric nervous systems and the adrenal medulla. DβH-RETMEN2B transgenic mice developed benign neuroglial tumors, histologically identical to human ganglioneuromas, in their sympathetic nervous systems and adrenal glands. The enteric nervous system was not affected. The neoplasms in DβH-RETMEN2B mice were similar to benign neuroglial tumors induced in transgenic mice by activated Ras expression under control of the same promoter. Levels of phoshorylated MAP kinase were not increased in the RETMEN2B-induced neurolgial proliferations, suggesting that alternative pathways may play a role in the pathogenesis of these lesions. Transgenic mice with the highest levels of DβH-RETMEN2B expression, unexpectedly developed renal malformations analogous to those reported with loss of function mutations in the Ret gene.

Testosterone and dihydrotestosterone and incident ischaemic stroke in men in the Cardiovascular Health Study

Ischaemic stroke is a major cause of morbidity and mortality in elderly men. Our main objective was to examine whether testosterone (T) or dihydrotestosterone (DHT) was associated with incident ischaemic stroke in elderly men.