PIASy, a member of the protein inhibitor of activated STAT (PIAS) family, represses the transcriptional activity of the androgen receptor (AR). In this report, we investigate the mechanism of PIASy-mediated repression of AR. We show that AR binds to the RING-finger like domain of PIASy. PIASy contains two transcriptional repression domains, RD1 and RD2. RD1, but not RD2, is required for PIASy-mediated repression of AR. We show that the RD1 domain binds HDAC1 and HDAC2 and that HDAC activity is required for PIASy-mediated AR repression. PIAS proteins possess small ubiquitin-related modifier (SUMO) E3 ligase activity. Conjugation of SUMO-1 to AR has been implicated in the regulation of AR activity. We examine if the SUMO ligase activity of PIASy is required for PIASy to repress AR. We show that a mutant PIASy, defective in promoting sumoylation, retains the ability to repress AR transcription. In addition, mutation of all the known sumoylation acceptor sites of AR does not affect the transrepression activity of PIASy on AR. Our results suggest that PIASy may repress AR by recruiting histone deacetylases, independent of its SUMO ligase activity.

Purpose: A better understanding of secreted proteins may lead to the discovery of new biomarkers, which, along with prostate-specific antigen (PSA), may be useful in the diagnosis and treatment of prostate cancer patients. Experimental Design: Conditioned medium was collected from LNCaP cells following stimulation with methyltrienolone (R1881), 17h-estradiol (estradiol), or interleukin-6 and analyzed for differential protein expression with surface-enhanced laser desorption/ionization-time of flight mass spectrometry. Quantitative reverse transcription-PCR, immunoblots, and ELISA were used to measure h-2-microglobulin (B2M) message and protein levels in cells, conditioned medium, and serum. Results: Surface-enhanced laser desorption/ionization-time of flight revealed that many peaks were induced or repressed following stimulation with R1881or estradiol. A peak of interest centered at 11.8 kDa was chosen for additional analysis. Immunodepletion identified the peak of interest as B2M. Reverse transcription-PCR and immunoblots confirmed that PSA and B2M were induced by R1881. However, unlike PSA, B2M was not increased on stimulation with estradiol or interleukin-6. Human B2M is identified in the serum of mice bearing human prostate cancer xenograft. B2M is expressed in human prostate cancer cell lines and tissues. Serum B2M levels are elevated in patients with metastatic, androgen-independent prostate cancer. Conclusions: B2M is a secreted protein expressed in prostate cancer, which is more specific for androgen stimulation than PSA under the conditions tested. Additional studies are warranted to explore if B2M is as useful marker for prostate cancer. Identification of proteins secreted from cancer cells in preclinical models may be a useful strategy for biomarker discovery.Most serum biomarkers routinely used in the diagnosis and treatment of cancer are cell surface or secreted proteins expressed by both benign and malignant tissue. As these proteins are generally found at low abundance, highly sensitive tests (such as ELISAs) are required to measure biomarker levels in peripheral blood. However, the limited sensitivity and specificity of these tests often restricts their clinical utility. For example, prostate-specific antigen (PSA) is an androgenregulated secreted protein that is expressed by both normal and malignant prostate epithelial cells, which is widely used as a serum marker for prostate cancer (1). Occult prostate cancers may be found in f25% of patients with PSA levels in the generally accepted reference range (V4 ng/mL; ref. 2). Most, but not all, patients with metastatic prostate cancer show elevated levels of PSA, which is often used as a marker to guide treatment decisions. However, the validity of PSA as a surrogate to predict overall survival in advanced prostate cancer remains controversial (3,4). Therefore, additional biomarkers may be useful in the diagnosis and treatment of patients with prostate cancer.Serum-based protein profiling with mass spectrometry (MS) is a promising technolo...