Grant Buchanan

Androgen receptor inhibits estrogen receptor-alpha activity and is prognostic in breast cancer

There is emerging evidence that the balance between estrogen receptor-alpha (ER(alpha)) and androgen receptor (AR) signaling is a critical determinant of growth in the normal and malignant breast. In this study, we assessed AR status in a cohort of 215 invasive ductal breast carcinomas. AR and (ER(alpha)) were coexpressed in the majority (80-90%) of breast tumor cells. Kaplan-Meier product limit analysis and multivariate Cox regression showed that AR is an independent prognostic factor in (ER(alpha))-positive disease, with a low level of AR (less than median of 75% positive cells) conferring a 4.6-fold increased risk of cancer-related death (P = 0.002). Consistent with a role for AR in breast cancer outcome, AR potently inhibited (ER(alpha))transactivation activity and 17beta-estradiol-stimulated growth of breast cancer cells. Transfection of MDA-MB-231 breast cancer cells with either functionally impaired AR variants or the DNA-binding domain of the AR indicated that the latter is both necessary and sufficient for inhibition of (ER(alpha)) signaling. Consistent with molecular modeling, electrophoretic mobility shift assays showed binding of the AR to an estrogen-responsive element (ERE). Evidence for a functional interaction of the AR with an ERE in vivo was provided by chromatin immunoprecipitation data, revealing recruitment of the AR to the progesterone receptor promoter in T-47D breast cancer cells. We conclude that, by binding to a subset of EREs, the AR can prevent activation of target genes that mediate the stimulatory effects of 17beta-estradiol on breast cancer cells.

Androgen receptor coregulators and their involvement in the development and progression of prostate cancer

The androgen receptor signaling axis plays an essential role in the development, function and homeostasis of male urogenital structures including the prostate gland although the mechanism by which the AR axis contributes to the initiation, progression and metastatic spread of prostate cancer remains somewhat enigmatic. A number of molecular events have been proposed to act at the level of the AR and associated coregulators to influence the natural history of prostate cancer including deregulated expression, somatic mutation, and post‐translational modification. The purpose of this article is to review the evidence for deregulated expression and function of the AR and associated coactivators and corepressors and how such events might contribute to the progression of prostate cancer by controlling the selection and expression of AR targets.

Coordinating assembly and export of complex bacterial proteins

The Escherichia coli twin‐arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy‐transducing inner membrane. Complex cofactor‐containing Tat substrates, such as the model (NiFe) hydrogenase‐2 and trimethylamine N‐oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site‐directed mutagenesis, we highlight here this crucial ‘proofreading’ or ‘quality control’ activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor‐insertion activities of the TorA‐specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin‐arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.

Role of the Escherichia coli Tat pathway in outer membrane integrity

The Escherichia coli Tat system serves to export folded proteins harbouring an N‐terminal twin‐arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin‐arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin‐arginine signal sequences in the genomes of other Gram‐negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.

Contribution of the Androgen Receptor to Prostate Cancer Predisposition and Progression

Although prostate cancer is heterogeneous in its etiology and progression, androgen signaling through the androgen receptor (AR) appears to be involved in all aspects of the disease, from initiation to development of treatment resistance. Lifetime exposure to a constitutively more active AR, encoded by AR alleles as defined by two translated polymorphic microsatellites (CAG and GGC), results in a significant increase in prostate cancer risk. The AR gene is amplified or a target for somatic gain-of-function mutations in metastatic prostate cancer. Gain-of-function AR gene mutations may result in inappropriate activation of the AR, thereby contributing to the failure of conventional androgen-ablation treatments. In cases where no genetically altered receptors are observed, altered signaling through the AR, achieved by cross-talk with other signaling pathways (e.g. kinase-mediated pathways) and/or inappropriate expression of coregulatory proteins, may contribute to disease progression. Thus, the AR-signaling axis contributes to many aspects of prostate cancer, including initiation, progression and resistance to current forms of therapy. This recognition represents a paradigm shift in our understanding of the molecular mechanisms involved in progression of prostate cancer, and provides insight into novel AR-targeted therapies which ultimately may be more effective than current forms of androgen ablation.

Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of ≈25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains ≈15 mobile TatA complexes and a pool of ≈100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

Genomic Androgen Receptor-Occupied Regions with Different Functions, Defined by Histone Acetylation, Coregulators and Transcriptional Capacity

Background The androgen receptor (AR) is a steroid-activated transcription factor that binds at specific DNA locations and plays a key role in the etiology of prostate cancer. While numerous studies have identified a clear connection between AR binding and expression of target genes for a limited number of loci, high-throughput elucidation of these sites allows for a deeper understanding of the complexities of this process. Methodology/Principal Findings We have mapped 189 AR occupied regions (ARORs) and 1,388 histone H3 acetylation (AcH3) loci to a 3% continuous stretch of human genomic DNA using chromatin immunoprecipitation (ChIP) microarray analysis. Of 62 highly reproducible ARORs, 32 (52%) were also marked by AcH3. While the number of ARORs detected in prostate cancer cells exceeded the number of nearby DHT-responsive genes, the AcH3 mark defined a subclass of ARORs much more highly associated with such genes – 12% of the genes flanking AcH3+ARORs were DHT-responsive, compared to only 1% of genes flanking AcH3−ARORs. Most ARORs contained enhancer activities as detected in luciferase reporter assays. Analysis of the AROR sequences, followed by site-directed ChIP, identified binding sites for AR transcriptional coregulators FoxA1, CEBPβ, NFI and GATA2, which had diverse effects on endogenous AR target gene expression levels in siRNA knockout experiments. Conclusions/Significance We suggest that only some ARORs function under the given physiological conditions, utilizing diverse mechanisms. This diversity points to differential regulation of gene expression by the same transcription factor related to the chromatin structure.

Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis

The Escherichia coli Tat apparatus is a membrane‐bound protein translocase that serves to export folded proteins synthesized with N‐terminal twin‐arginine signal peptides. The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices. Sequence analysis identified conserved TatC amino acid residues, and the role of these side‐chains was assessed by single alanine substitution. This approach identified three classes of TatC mutants. Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat‐dependent protein export activity and thus represented residues essential for TatC function. Cross‐complementation experiments with class I mutants showed that co‐expression of D211A with either F94A or E103A regenerated an active Tat apparatus. These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the co‐existence of at least two TatC molecules within each Tat translocon. Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A. Class III mutants showed no significant defects in protein export. All but three of the essential and important residues are predicted to cluster around the cytoplasmic N‐tail and first cytoplasmic loop regions of the TatC protein.

Behaviour of topological marker proteins targeted to the Tat protein transport pathway

The Escherichia coli Tat system mediates Sec‐independent export of protein precursors bearing twin arginine signal peptides. Formate dehydrogenase‐N is a three‐subunit membrane‐bound enzyme, in which localization of the FdnG subunit to the membrane is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic face of the cytoplasmic membrane. However, the phenotypes of gene fusions between fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase) or lacZ (encoding β‐galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat‐dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat‐dependent membrane protein complexes.

Targeting Amino Acid Transport in Metastatic Castration-Resistant Prostate Cancer: Effects on Cell Cycle, Cell Growth, and Tumor Development

BACKGROUND L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. METHODS We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. RESULTS LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4-7 months of neoadjuvant hormone therapy (4-7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4-mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. CONCLUSION Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.