The coactivators CBP and SRC-1 in prostate cancer

Research project P 14709 The coactivators CBP and SRC-1 in prostate cancer Zoran CULIG 27.11.2000 Androgen receptor (AR) is expressed in nearly all prostate cancers including therapy-resistant ones. Its activity in target tissues depends on interactions with associated proteins, coactivators, and corepressors. In the present application we focus on expression and function of the promiscuous coactivators CBP and SRC- 1 in carcinoma of the prostate. We`ll investigate: a. how these coactivators modulate antagonist/agonist balance of the non-steroidal anti-androgens hydroxyflutamide and bicalutamide (Casodex) in the presence of either wild-type or mutant ARs, b. expression of endogenous CBP and SRC-1 in prostate cancer cell lines and clinical specimens, regulation of androgen-induced prostate cancer cell proliferative response, key cell cycle molecules and secretion by these coactivators, c. importance of the coactivators CBP and SRC-I for regulation of proliferation of prostate cells by interleukin- 6 and (e) inhibition of coactivator Function by ectopic expression of the nuclear corepressor N-CoR. To study modulation of antagonist/agonist balance of hydroxyflutamide and bicalutamide by the coactivators CBP and SRC-1, prostate cancer cells DU-145 will be transfected with an AR expression vector, the androgen- responsive reporter gene and a coactivator expression vector. Transfections will also be performed in sublines from LNCaP cells generated after long-term and intermittent androgen ablation in vitro. Expression of CBP and SRC-I mRNA and protein in prostate cancer cells will be investigated by serniquantitative PCR, in situ hybridization, Western blot and immunohistochemistry, respectively. To study the role of the coactivators in the regulation of proliferative and secretory responses in prostate cancer cells, the Tet-On system will be used. Expression of cyclin~dependentkinases, cell cycle inhibitors, prostate-specific antigen, human glandular kallikrein-2 and the horneobox gene NKX 3.1 will be investigated by Northern and Western blot. Interleukin-6 is a non-steroidal activator of the AR which undergoes functional transition from a paracrine growth inhibitor to an autocrine growth stimulator in prostate cancer. In interleukin-6-resistant prostate cancer cells, it will be investigated whether a regulated overexpression of a steroid receptor coactivator is sufficient for the reestablishment of growth inhibition by interleukin-6. To investigate whether the nuclear corepressors N-CoR and SMRT reverse coactivatormediated agonistic effects of anti-androgens, N-CoR and SI~MT cDNA will be overexpressed in prostate cancer cells. Endocrine therapy for prostate cancer is only palliative and it is thought that acquisition of agonistic properties of AR antagonists contributes to tumour progression. This project may provide explanations why AR antagonists switch to agonists. This information will be helpful for testing new antiandrogens for treatment of carcinoma of the prostate.

Prostate cancer is the most frequently diagnosed malignant tumor in male in the Western world. Androgen receptor (AR) is implicated in growth regulation of prostate cancers in all stages of the disease. Its expression in prostate cancer is in some cases increased due to amplification of the gene or stabilization of the protein. In the presence of mutated AR, receptor antagonists used in prostate cancer therapy switch to agonist thus contributing to accelerated tumour growth. It is speculated that there are other mechanisms involved in tumor progression towards therapy resistance, in particular alterations in expression or function of associated proteins, coactivators. Coactivators enhance AR functional activity in a ligand-dependent manner. In the present project, we focused on the coactivator CREB (cAMP-response element)-binding protein-binding protein (CBP) in prostate cancer. We showed that CBP potentiates AR activity in the presence of either androgen or a nonsteroidal AR antagonist. The effect of the drug hydroxyflutamide was more pronounced than that of another antagonist bicalutamide. We conclude that CBP preferentially enhances AR activity in the presence of hydroxyflutamide. This might be of importance for clinical situation because a paradoxical improvement in patients symptoms was observed after cessation of hydroxyflutamide from therapy protocols. We also demonstrated that CBP does not up-regulate AR expression in prostate cancer cells. CBP is expressed in prostate cancer cell lines and tissue specimens as demonstrated by immunohistochemistry. In specimens obtained from patients with advanced carcinoma of the prostate, CBP was detected mainly in high grade tumor areas. In LNCaP cells, CBP expression is down-regulated by androgen and interleukin-6 in a dose-dependent manner. This implies that up-regulation of CBP occurs during androgen ablation. This fact should be taken in consideration in discussion of endocrine therapy options in advanced prostate carcinoma.