A randomised controlled trial to evaluate the efficacy of a 6-month dietary and physical activity intervention for patients receiving androgen deprivation therapy for prostate cancer

PURPOSE: Treatment of prostate cancer with androgen deprivation therapy (ADT) is associated with an increased fat mass, decreased lean mass, increased fatigue and a reduction in quality of life (QoL). The aim of this study was to evaluate the efficacy of a 6-month dietary and physical activity intervention for prostate cancer patients receiving ADT, to help minimise these side effects.

METHODS: Patients (n = 94) were recruited to this study if they were planned to receive ADT for prostate cancer for at least 6 months. Men randomised to the intervention arm received a dietary and exercise intervention, commensurate with UK healthy eating and physical activity recommendations. The primary outcome of interest was body composition; secondary outcomes included fatigue, QoL, functional capacity, stress and dietary change.

RESULTS: The intervention group had a significant (p < 0.001) reduction in weight, body mass index and percentage fat mass compared to the control group at 6 months; the between-group differences were -3.3 kg (95 % confidence interval (95 % CI) -4.5, -2.1), -1.1 kg/m(2) (95 % CI -1.5, -0.7) and -2.1 % (95 % CI -2.8, -1.4), respectively, after adjustment for baseline values. The intervention resulted in improvements in functional capacity (p < 0.001) and dietary intakes but did not significantly impact fatigue, QoL or stress scores at endpoint.

CONCLUSIONS: A 6-month diet and physical activity intervention can minimise the adverse body composition changes associated with ADT.

IMPLICATIONS FOR CANCER SURVIVORS: This study shows that a pragmatic lifestyle intervention is feasible and can have a positive impact on health behaviours and other key outcomes in men with prostate cancer receiving ADT.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce anti-proliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G2/M arrest. G2/M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xL and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G2/M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-xL. Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOX-induced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer.

Favorable effect on acute and chronic graft-versus-host disease with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies for marrow transplantation from HLA-identical sibling donors for acquired aplastic anemia

Between August 1989 and November 2003, 33 patients at our center with acquired aplastic anemia underwent bone marrow transplantation (BMT) from HLA-identical sibling donors with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies (MoAb) for conditioning. The median age at BMT was 17 years (range, 4-46 years). Before BMT, 58% were heavily transfused (>50 transfusions), and 42% had previously experienced treatment failure with antithymocyte globulin-based immunosuppressive therapy. Unmanipulated bone marrow was used as the source of stem cells in all patients except 1. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine alone in 19 (58%) patients; 14 received anti-CD52 MoAb in addition to cyclosporine. The conditioning regimen was well tolerated without significant acute toxicity. Graft failure was seen in 8 patients (primary, n = 4; secondary, n = 4). Of those whose grafts failed, 4 survived long-term (complete autologous recovery, n = 2; rescue with previously stored marrow, n = 1; second allograft, n = 1). The cumulative incidence of graft failure and grade II to IV acute and chronic GVHD was 24%, 14%, and 4%, respectively. None developed extensive chronic GVHD. With a median follow-up of 59 months, the 5-year survival was 81% (95% confidence interval, 68%-96%). No unexpected early or late infectious or noninfectious complications were observed. We conclude that the conditioning regimen containing cyclophosphamide and anti-CD52 MoAb is well tolerated and effective for acquired aplastic anemia with HLA-matched sibling donors. The favorable effect on the incidence and severity of GVHD is noteworthy in this study and warrants further investigation.

Stoichiometric Molecularly Imprinted Polymers for the Recognition of Anti-Cancer Pro-drug Tegafur

Molecularly Imprinted Polymers (MIPs) targeting tegafur, an anti-cancer 5-fluorouracil pro-drug, have been prepared by stoichiometric imprinting using 2,6-bis(acrylamido)pyridine (BAAPy) as the functional monomer. Solution association between tegafur and BAAPy was studied by 1H NMR titration, which confirmed the formation of 1:1 complexes with an affinity constant of 574±15 M-1 ¬in CDCl3. Evaluation of the synthesised materials by HPLC and equilibrium rebinding experiments revealed high selectivity of the imprinted polymer for the pro-drug versus 5-fluorouracil and other competing analytes, with maximum imprinting factors of 25.3 and a binding capacity of 45.1 μmol g-1. The synthesised imprinted polymer was employed in solid-phase extraction of the pro-drug using an optimised protocol that included a simple wash with the porogen used in the preparation of the material. Tegafur recoveries of up to 96% were achieved from aqueous samples and 92% from urine samples spiked with the template and three competing analytes. The results demonstrate the potential of the prepared polymers in the pre-concentration of tegafur from biological samples, which could be an invaluable tool in the monitoring of patient compliance and drug uptake and excretion.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.

Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer

The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

Divergent androgen regulation of unfolded protein response pathways drives prostate cancer

The unfolded protein response (UPR) is a homeostatic mechanism to maintain endoplasmic reticulum (ER) function. The UPR is activated by various physiological conditions as well as in disease states, such as cancer. As androgens regulate secretion and development of the normal prostate and drive prostate cancer (PCa) growth, they may affect UPR pathways. Here, we show that the canonical UPR pathways are directly and divergently regulated by androgens in PCa cells, through the androgen receptor (AR), which is critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1α branch, but simultaneously inhibited PERK signaling. Inhibition of the IRE1α arm profoundly reduced PCa cell growth in vitro as well as tumor formation in preclinical models of PCa in vivo. Consistently, AR and UPR gene expression were correlated in human PCa, and spliced XBP-1 expression was significantly upregulated in cancer compared with normal prostate. These data establish a genetic switch orchestrated by AR that divergently regulates the UPR pathways and suggest that targeting IRE1α signaling may have therapeutic utility in PCa.

The ETS family member GABPα modulates androgen receptor signalling and mediates an aggressive phenotype in prostate cancer

In prostate cancer (PC), the androgen receptor (AR) is a key transcription factor at all disease stages, including the advanced stage of castrate-resistant prostate cancer (CRPC). In the present study, we show that GABPα, an ETS factor that is up-regulated in PC, is an AR-interacting transcription factor. Expression of GABPα enables PC cell lines to acquire some of the molecular and cellular characteristics of CRPC tissues as well as more aggressive growth phenotypes. GABPα has a transcriptional role that dissects the overlapping cistromes of the two most common ETS gene fusions in PC: overlapping significantly with ETV1 but not with ERG target genes. GABPα bound predominantly to gene promoters, regulated the expression of one-third of AR target genes and modulated sensitivity to AR antagonists in hormone responsive and castrate resistant PC models. This study supports a critical role for GABPα in CRPC and reveals potential targets for therapeutic intervention.

Maintaining and reprogramming genomic androgen receptor activity in prostate cancer

Prostate cancer treatment is dominated by strategies to control androgen receptor (AR) activity. AR has an impact on prostate cancer development through the regulation of not only transcription networks but also genomic stability and DNA repair, as manifest in the emergence of gene fusions. Whole-genome maps of AR binding sites and transcript profiling have shown changes in the recruitment and regulatory effect of AR on transcription as prostate cancer progresses. Defining other factors that are involved in this reprogramming of AR function gives various opportunities for cancer detection and therapeutic intervention.

Androgen-regulated metabolism and biosynthesis in prostate cancer

Metabolic changes are a well-described hallmark of cancer and are responses to changes in the activity of diverse oncogenes and tumour suppressors. For example, steroid hormone biosynthesis is intimately associated with changes in lipid metabolism and represents a therapeutic intervention point in the treatment of prostate cancer (PCa). Both prostate gland development and tumorigenesis rely on the activity of a steroid hormone receptor family member, the androgen receptor (AR). Recent studies have sought to define the biological effect of the AR on PCa by defining the whole-genome binding sites and gene networks that are regulated by the AR. These studies have provided the first systematic evidence that the AR influences metabolism and biosynthesis at key regulatory steps within pathways that have also been defined as points of influence for other oncogenes, including c-Myc, p53 and hypoxia-inducible factor 1α, in other cancers. The success of interfering with these pathways in a therapeutic setting will, however, hinge on our ability to manage the concomitant stress and survival responses induced by such treatments and to define appropriate therapeutic windows.

N-linked glycosylation supports cross-talk between receptor tyrosine kinases and androgen receptor

Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs). In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP) than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R) ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an important challenge in the future.

The androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man

The androgen receptor (AR) regulates prostate cell growth in man, and prostate cancer is the commonest cancer in men in the UK. We present a comprehensive analysis of AR binding sites in human prostate cancer tissues, including castrate-resistant prostate cancer (CRPC). We identified thousands of AR binding sites in CRPC tissue, most of which were not identified in PC cell lines. Many adjacent genes showed AR regulation in xenografts but not in cultured LNCaPs, demonstrating an in-vivo-restricted set of AR-regulated genes. Functional studies support a model of altered signaling in vivo that directs AR binding. We identified a 16 gene signature that outperformed a larger in-vitro-derived signature in clinical data sets, showing the importance of persistent AR signaling in CRPC.

Chromatin binding by the androgen receptor in prostate cancer

Alterations in transcriptional programs are fundamental to the development of cancers. The androgen receptor is central to the normal development of the prostate gland and to the development of prostate cancer. To a large extent this is believed to be due to the control of gene expression through the interaction of the androgen receptor with chromatin and subsequently with coregulators and the transcriptional machinery. Unbiased genome-wide studies have recently uncovered the recruitment sites that are gene-distal and intragenic rather than associated with proximal promoter regions. Whilst expression profiles from AR-positive primary prostate tumours and cell lines can directly relate to the AR cistrome in prostate cancer cells, this distribution raises significant challenges in making direct mechanistic connections. Furthermore, extrapolating from datasets assembled in one model to other model systems or clinical samples poses challenges if we are to use the AR-directed transcriptome to guide the development of novel biomarkers or treatment decisions. This review will provide an overview of the androgen receptor before addressing the challenges and opportunities created by whole-genome studies of the interplay between the androgen receptor and chromatin.

Global identification of androgen response elements

Chromatin immunoprecipitation (ChIP) is an invaluable tool in the study of transcriptional regulation. ChIP methods require both a priori knowledge of the transcriptional regulators which are important for a given biological system and high-quality specific antibodies for these targets. The androgen receptor (AR) is known to play essential roles in male sexual development, in prostate cancer and in the function of many other AR-expressing cell types (e.g. neurons and myocytes). As a ligand-activated transcription factor the AR also represents an endogenous, inducible system to study transcriptional biology. Therefore, ChIP studies of the AR can make use of treatment contrast experiments to define its transcriptional targets. To date several studies have mapped AR binding sites using ChIP in combination with genome tiling microarrays (ChIP-chip) or direct sequencing (ChIP-seq), mainly in prostate cancer cell lines and with varying degrees of genomic coverage. These studies have provided new insights into the DNA sequences to which the AR can bind, identified AR cooperating transcription factors, mapped thousands of potential AR regulated genes and provided insights into the biological processes regulated by the AR. However, further ChIP studies will be required to fully characterise the dynamics of the AR-regulated transcriptional programme, to map the occupancy of different AR transcriptional complexes which result in different transcriptional output and to delineate the transcriptional networks downstream of the AR.