Distinct E€Ects of PIAS Proteins on Androgen-Mediated Gene Activation

Oncogene (2001) 20, 3880 ± 3887 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Distinct eects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells Mitchell Gross1, Bin Liu1, Jiann-an Tan3, Frank S French3, Michael Carey2 and Ke Shuai*,1,2 1Division of Hematology-Oncology, Department of Medicine, University of California, Los Angeles, California, CA 90095, USA; 2Department of Biological Chemistry, University of California, Los Angeles, California, CA 90095, USA; 3The Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, NC 27599-7500, USA Androgen signaling in¯uences the development and The androgen receptor (AR) is a member of the growth of prostate carcinoma. The transcriptional nuclear receptor (NR) superfamily (Bentel and Tilley, activity of androgen receptor (AR) is regulated by 1996). NRs have conserved domain structures (Freed- positive or negative transcriptional cofactors. We report man, 1999). At the N-terminus is the ligand- here that PIAS1, PIAS3, and PIASy of the protein independent transcriptional activation domain (AF- inhibitor of activated STAT (PIAS) family, which are 1). The central domain contains two zinc ®nger expressed in human prostate, display distinct eects on structures that are involved in DNA binding (DBD). AR-mediated gene activation in prostate cancer cells. The C-terminal region is the ligand binding domain While PIAS1 and PIAS3 enhance the transcriptional (LBD). In addition, an essential ligand-dependent activity of AR, PIASy acts as a potent inhibitor of AR transactivation domain (AF-2) is localized in the C- in prostate cancer cells. The eects of PIAS proteins on terminus of the LBD. The binding of androgen to AR AR are competitive. PIASy binds to AR but does not triggers the contact of AR to its speci®c androgen aect the DNA binding activity of AR. An NH2- response elements (AREs) in the promoters of terminal LXXLL signature motif of PIASy, although not androgen-responsive genes and the interactions of required for PIASy ± AR interaction, is essential for the AR with other transcription cofactors. These interac- transrepression activity of PIASy. Our results identify tions allow the activation or repression of genes that PIASy as a transcriptional corepressor of AR and regulate development, dierentiation and maintenance suggest that dierent PIAS proteins have distinct eects of male reproductive functions (Trapman and Cleut- on AR signaling in prostate cancer cells. Oncogene jens, 1997). (2001) 20, 3880 ± 3887. The activity of NR is regulated by transcriptional coactivators and corepressors (Freedman, 1999; Hor- Keywords: prostate cancer; androgen signaling; protein witz et al., 1996; Torchia et al., 1998). These inhibitor of activated STAT coregulator complexes modulate NR-mediated gene activation by interaction with general transcription factors or by remodeling chromatin. Many coregula- Introduction tors of AR have been described, most of them are suggested to function as coactivators of AR (a detailed Prostate cancer is the most frequently diagnosed cancer description of these factors can be found at http:// in American men (Parker et al., 1997). Androgen plays www.mcgill.ca/androgendb/). Many nuclear coactiva- an important role in the development and growth of tors contain one or multiple copies of the a-helical prostate carcinoma (Kokontis and Liao, 1999). Andro- LXXLL signature motif (where L is Leucine and X is gen ablation is an eective treatment for advanced any amino acid residue). Biochemical studies have prostate cancer. Unfortunately, after a period of demonstrated that the LXXLL signature motif med- remission, a relapse occurs due to the acquisition of iates ligand-dependent coactivator-NR interactions or androgen-independent tumor growth. A better under- coactivator ± coactivator interactions (Heery et al., standing of the regulation of androgen signaling in 1997; Torchia et al., 1997). human prostate cancer cells will enhance our ability to In studies aimed at the understanding of STAT design a rational therapeutic strategy for prostate (Signal Transducer and Activator of Transcription) cancer treatment. signaling (Darnell, 1997), our laboratory has uncovered a novel protein family termed PIAS (Protein Inhibitor of Activated STAT), of which four members have been identi®ed ± PIAS1, PIAS3, PIASx (consisting of two *Correspondence: K Shuai, Division of Hematology-Oncology, 11- splicing variants: PIASxa and PIASxb), and PIASy 934 Factor Bldg, 10833 LeConte Avenue, Los Angeles, California, (Shuai, 1999; 2000). PIAS1 and PIAS3 interact with CA 9005-1678, USA Received 10 January 2001; revised 20 March 2001; accepted 26 Stat1 and Stat3, respectively. PIAS can block the DNA March 2001 binding activity of STAT and can inhibit STAT- PIAS proteins and androgen-mediated gene-activation M Gross et al 3881 mediated gene activation (Chung et al., 1997; Liu et al., 1998). Recently, two independent yeast two-hybrid screens using the DNA binding domain of AR as the bait have identi®ed ARIP3 (Androgen Receptor Interacting Protein 3), a rat homologue of human PIASxa,as well as PIAS1 as AR-interacting proteins (Moilanen et al., 1999; Tan et al., 2000). Subsequent studies suggest that ARIP3/PIASxa and PIAS1 can function as coactivators of AR. We have carried out a systematic analysis on the roles of various PIAS members of AR signaling in prostate cancer cells. We found that PIAS1, PIAS3 and PIASy are expressed in human prostate. PIASy, unlike other PIAS proteins, is a potent inhibitor of AR-mediated gene activation in prostate cancer cells. PIASy interacts with AR but does not aect the DNA binding activity of AR. Mutational analysis identi®ed an essential function of an LXXLL signature motif at the N-terminus of PIASy for the transrepression activity of PIASy. Our results suggest that PIASy is a transcriptional corepressor of AR. Thus, androgen signaling can be positively or nega- tively regulated by PIAS proteins in prostate cancer cells. Results PIAS1, PIAS3 and PIASy are expressed in human prostate To study the role of PIAS proteins in prostate cancer, we ®rst performed Northern blot analysis to determine which PIAS family members are expressed in human prostate. It has been shown previously that PIAS3 is ubiquitously expressed in a variety of human tissues including prostate (Chung et al., 1997). A human tissue blot was probed for the expression of PIAS1, PIASx and PIASy. Two messages with the sizes of 2.3 and Figure 1 Expression of PIAS in human tissues and cell lines. A 3.5 kb, probably resulting from dierent RNA proces- human tissue blot (Clontech) was hybridized with radiolabeled sing, were detected in various human tissues including human PIASy cDNA (a), human PIAS1 cDNA (b), or a 200 bp prostate when the blot was probed with PIASy cDNA sequence of the 3'-end of the human PIASxa cDNA (c), following (Figure 1a). Similarly, two major forms of PIAS1 manufacturer's instructions. (d) Immunoblot analysis of LNCaP cytoplasmic (Cyt) and nuclear (Nuc) extracts with PIASy, PIAS1 messages with the sizes of 2.6 and 7.4 kb were present and PIAS3 antibodies as indicated in prostate and other tissues (Figure 1b). However, PIASxa is mainly expressed in human testis (Figure 1c), consistent with the result obtained when ARIP3, a rat homologue of PIASxa, was used as the probe be detected in the nuclear fractions (Figure 1d). The (Moilanen et al., 1999) (expression of PIASxa was nuclear locatization of PIAS1 is consistent with the detectable in other tissues when the blot was over- pattern seen in the germinal epithelium of human testis exposed (data not shown)). A similar expression by immunohistochemistry using the same PIAS1 anti- pattern of PIASxb was also detected (data not shown). body (Tan et al., 2000). We conclude that PIAS1, PIAS3, and PIASy are expressed in human prostate tissue. Distinct effects of PIAS proteins on AR-mediated gene We next wanted to con®rm that PIAS proteins are activation in prostate cancer cells indeed expressed in prostate epithelial cells. LNCaP human prostate cancer cells were subjected to We examined the eects of PIAS1, PIAS3 and PIASy biochemical fractionation, and expression of PIASy, proteins on AR-mediated gene activation in the PIAS3, and PIAS1 was examined by Western blotting LNCaP cell line. LNCaP cells express a mutated AR using speci®c anti-PIAS antibodies (Chung et al., 1997; but androgen response appears to be normal in these Liu et al., 1998, 2001). PIASy, PIAS1 and PIAS3 can cells (McDonald et al., 2000). Expression vectors Oncogene PIAS proteins and androgen-mediated gene-activation M Gross et al 3882 encoding Flag-tagged PIAS1, PIAS3 or PIASy were PIASy does not influence AR DNA bindings cotransfected together with a wild-type AR expression vector and a luciferase reporter (PSA-E4 Luc) with PIAS1 and PIAS3 can block the DNA binding activity the androgen-responsive promoter of PSA (Prostate of STATs in vitro (Chung et al., 1997; Liu et al., 1998). Speci®c Antigen) (Figure 2). Transfected cells were We wished to determine if PIASy aects the DNA untreated or treated with R1881 (methyltrienolone, a binding activity of AR by electrophoretic mobility shift synthetic androgen). We found that in the absence of analysis (EMSA). An anity puri®ed His-tagged DNA PIAS proteins, R1881 induced a 10 ± 15-fold activation binding domain of AR (His-DBD) was incubated with of the PSA reporter. In the presence of an increasing 32P-labeled ARE oligonucleotide in the presence or amount of PIAS1, the R1881-induced luciferase absence of GST or GST-PIASy fusion proteins. The activity was dramatically enhanced, up to 70-fold when DNA binding ability of AR was not aected by either 2 mg of PIAS1 was employed (Figure 2a). PIAS3 GST or GST-PIASy at various concentrations used showed similar eects as PIAS1 (Figure 2b). Most (Figure 5a). The lack of a PIASy-AR-DBD supershift interestingly, unlike other PIAS proteins, PIASy caused band was probably due to the instability of such a a dose-dependent inhibition of R1881-induced lucifer- complex under the conditions used in these assays.

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