Antiandrogens act as selective androgen receptor modulators at the proteome level in prostate cancer cells.

Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context.

FUS/TLS is a novel mediator of androgen-dependent cell-cycle progression and prostate cancer growth.

Progression of prostate cancer is highly dependent upon the androgen receptor pathway, such that knowledge of androgen-regulated proteins is vital to understand and combat this disease. Using a proteomic screen, we found the RNA-binding protein FUS/TLS (Fused in Ewing's Sarcoma/Translocated in Liposarcoma) to be downregulated in response to androgen. FUS has recently been shown to be recruited by noncoding RNAs to the regulatory regions of target genes such as cyclin D1, in which it represses transcription by disrupting complex formation. Here we show that FUS has some characteristics of a putative tumor suppressor, as its overexpression promoted growth inhibition and apoptosis of prostate cancer cells, whereas its knockdown increased cell proliferation. This effect was reproducible in vivo, such that increasing FUS levels in tumor xenografts led to dramatic tumor regression. Furthermore, FUS promoted conditions that favored cell-cycle arrest by reducing the levels of proliferative factors such as cyclin D1 and Cdk6 and by increasing levels of the antiproliferative Cdk inhibitor p27. Immunohistochemical analysis revealed that FUS expression is inversely correlated with Gleason grade, demonstrating that patients with high levels of FUS survived longer and were less likely to have bone metastases, suggesting that loss of FUS expression may contribute to cancer progression. Taken together, our results address the question of how androgens regulate cell-cycle progression, by demonstrating that FUS is a key link between androgen receptor signaling and cell-cycle progression in prostate cancer.

Manipulating prohibitin levels provides evidence for an in vivo role in androgen regulation of prostate tumours.

Current hormonal therapies for prostate cancer are effective initially, but inevitably tumours progress to an advanced, metastatic stage, often referred to as 'androgen independent'. However, the androgen receptor (AR) signalling pathway is still key for their growth. It is speculated that tumours escape hormonal control via reduction in corepressor proteins. Manipulating such proteins is thus a potential therapeutic strategy to halt or even reverse tumour progression. We aimed to elucidate the effects of altering levels of the AR corepressor and androgen-target protein prohibitin (PHB) on prostate tumour growth. Prostate cancer cells incorporating an integrated androgen-responsive reporter gene and stably expressing vectors to inducibly overexpress or knockdown PHB were generated and used to assess effects on androgen signalling (by real time imaging) and tumour growth both in culture and in vivo. PHB overexpression inhibited AR activity and prostate-specific antigen (PSA) expression as well as androgen-dependent growth of cells, inducing rapid accumulation in G(0)/G(1). Conversely, reduction in PHB increased AR activity, PSA expression, androgen-mediated growth and S-phase entry. In vivo, doxycycline-induced PHB regulation resulted in marked changes in AR activity, and showed significant effects upon tumour growth. Overexpression led to tumour growth arrest and protection from hormonal starvation, whereas RNAi knockdown resulted in accelerated tumour growth, even in castrated mice. This study provides proof of principle that i) reduction in PHB promotes both androgen-dependent and 'androgen-independent' tumour growth, and ii) altering AR activity via increasing levels or activity of corepressors is a valid therapeutic strategy for advanced prostate cancer.

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells.

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.

The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian cancer cell lines.

BACKGROUND: The cyclin D1 proto-oncogene is an important regulator of G1 to S-phase transition and an important cofactor for several transcription factors in numerous cell types. Studies on neonatal cardiomyocytes and postmitotic neurons indicate that the activity of cyclin D1 may be regulated through its cytoplasmic sequestration. We have demonstrated previously, that TSA induces the ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells. Additional studies were initiated in order to further investigate the effect of TSA on cyclin D1 regulation using sub-cellular fractionation techniques. RESULTS: Our studies revealed cyclin D1 to be localized predominantly within the cytoplasmic fraction of all cell lines tested. These observations were confirmed by confocal microscopy. GSK3beta was found to be localized within both the nucleus and cytoplasm throughout the cell cycle. Inhibition of GSK3beta or CRM1-dependent nuclear export resulted in only modest nuclear accumulation, suggesting that the cytoplasmic localization of cyclin D1 results from the inhibition of its nuclear import. CONCLUSION: We have shown by several different experimental approaches, that cyclin D1 is in fact a predominantly cytoplasmic protein in mammalian cancer cell lines. Recent studies have shown that the cytoplasmic sequestration of cyclin D1 prevents apoptosis in neuronal cells. Our results suggest that cytoplasmic sequestration may additionally serve to regulate cyclin D1 activity in mammalian cancer cells.

Androgen receptor is targeted to distinct subcellular compartments in response to different therapeutic antiandrogens.

PURPOSE: Antiandrogens are routinely used in the treatment of prostate cancer. Although they are known to prevent activation of the androgen receptor (AR), little is known about the mechanisms involved. This report represents the first study of the localization of wild-type AR following expression at physiologic relevant levels in prostate cells and treatment with androgen and antiandrogens. EXPERIMENTAL DESIGN: We have characterized a cellular model for prostate cancer using in situ cellular fractionation, proteomics, and confocal microscopy and investigated the effect of antiandrogens in clinical use on the subcellular localization of the AR. RESULTS: Different antiandrogens have diverse effects on the subcellular localization of the AR. Treatment with androgen results in translocation from the cytoplasm to the nucleoplasm, whereas the antiandrogens hydroxyflutamide and bicalutamide lead to reversible association with the nuclear matrix. In contrast, treatment with the antiandrogen cyproterone acetate results in AR association with cytoplasmic membranes and irreversible retention within the cytoplasm. In addition, we demonstrate that AR translocation requires ATP and the cytoskeleton, regardless of ligand. CONCLUSIONS: These results reveal that not all antiandrogens work via the same mechanism and suggest that an informed sequential treatment regime may benefit prostate cancer patients. The observed subnuclear and subcytoplasmic associations of the AR suggest new areas of study to investigate the role of the AR in the response and resistance of prostate cancer to antiandrogen therapy.

Androgens target prohibitin to regulate proliferation of prostate cancer cells.

Proteins involved in the growth response of prostate cancer cells to androgen were investigated by comparing the proteomes of LNCaP cells treated with vehicle or androgen. Whole-cell lysates were separated by two-dimensional PAGE, and HPLC-MS/MS was used to identify androgen-regulated proteins. Prohibitin, a protein with cell-cycle regulatory activity, was shown to be downregulated by 50% following androgen stimulation. Western blot and reverse transcription-PCR experiments confirmed the result and showed that regulation occurs at the level of transcription. To determine the importance of prohibitin in androgen-stimulated growth, we used transient transfection to overexpress the protein and RNA interference to knock down the protein. Subsequent FACS analysis showed that cells with reduced levels of prohibitin showed a slight but reproducible increase in the percentage of population in cell cycle, while cells with increased prohibitin levels showed a clear reduction in the percentage entering cell cycle, following dihydrotestosterone stimulation, when compared to untransfected controls. Confocal microscopy showed localization of prohibitin in the nucleus as well as the mitochondria of LNCaP cells. It therefore seems that the regulation of prohibitin is a vital part of the cellular growth response to androgen stimulation in LNCaPs and prohibitin may have a nuclear regulatory role in cell-cycle progression.