[CANCER RESEARCH 62, 338 –340, January 15, 2002] Advances in Brief NKX-3.1 Interacts with Prostate-derived Ets Factor and Regulates the Activity of the PSA Promoter 1 Hui Chen, Asit K. Nandi, Xiang Li, and Charles J. Bieberich 2 Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland 21250 Abstract The NKX-3.1 homeobox gene maps to human chromosome 8p21, a region that undergoes frequent loss of heterozygosity in prostate tumors. Loss of Nkx-3.1 function in mice leads to epithelial overgrowth. To further elucidate the molecular basis of NKX-3.1 function, a genetic screen for proteins that interact with NKX-3.1 was performed. Prostate-derived Ets factor (PDEF) was identified as a potential partner of NKX-3.1. Coim- munoprecipitation analyses demonstrated that NKX-3.1 and PDEF are physically associated in prostate epithelial cells. Cotransfection analyses demonstrated that NKX-3.1 can abolish the transcriptional activation function of PDEF on the prostate-specific antigen (PSA) promoter. These data identify PSA as a target gene for NKX-3.1 and provide new insights into the function of this candidate tumor suppressor. Introduction Loss of heterozygosity of chromosome 8p occurs in a majority of human prostate tumors (reviewed in Ref. 1). Loss of 8p12–21 appears to be an early event in the oncogenic process and is detected in prostatic intraepithelial neoplasia lesions. Although no bona fide tumor suppressor genes have been mapped to this region, a growing body of evidence points to the NKX-3.1 gene, which maps to 8p21, as a viable candidate (1). First described in the mouse as an androgen- regulated prostate-restricted gene encoding a homeodomain transcrip- tion factor, NKX-3.1 has come under increasing suspicion as a regu- lator of prostate cell growth and differentiation (2, 3). Unlike the canonical tumor suppressor p53, in tumors where one NKX-3.1 allele is lost, the protein-coding region of the remaining allele is not mutated (4). However, a recent immunohistochemical study demonstrated decreased accumulation of NKX-3.1 protein in tumors, and dimin- ished NKX-3.1 correlated with disease progression (5). Although these results are difficult to reconcile with the reported increase in NKX-3.1 mRNA in prostate tumors, it is possible that post-transcrip- tional regulation plays an important role in modulating NKX-3.1 levels (6). The strongest evidence that NKX-3.1 regulates prostate growth has emerged from phenotypic analyses of Nkx-3.1 knockout mice (7–9). Mice heterozygous for a null mutation in Nkx-3.1 display prostate epithelial hyperplasia and dysplasia, demonstrating that loss of a single allele is sufficient to alter growth control (7). Homozygous null Nkx-3.1 mutants show more profound epithelial hyperprolifera- tion in addition to defects in branching morphogenesis and secretory protein production (7). To date, no prostate tumors have been reported in Nkx-3.1 null mutants, suggesting that loss of Nkx-3.1 alone is not sufficient to induce prostate malignancy. To further understand the functions of NKX-3.1 in prostate biology, we have performed a screen to identify NKX-3.1-interacting proteins. In this report we demonstrate that NKX-3.1 interacts with PDEF, 3 a prostate-specific transcription factor that positively regulates PSA gene expression (10). We further demonstrate that expression of NKX-3.1 abrogates the transcriptional activation function of PDEF on PSA regulatory elements. These data provide new insights into the molecular basis of NKX-3.1 function and PSA gene regulation in prostate epithelial cells. Materials and Methods Yeast Two-Hybrid Screen. A full-length cDNA encoding the R52 allele of human NKX-3.1 (4) was cloned in frame into pLexA (Clontech Laborato- ries, Palo Alto, CA), which encodes a full-length LexA protein containing a well-defined DNA binding activity. Expression of the LexA-NKX-3.1 fusion protein was confirmed by Western blot analysis using an anti-LexA polyclonal antibody (Invitrogen, Carlsbad, CA). A human prostate MATCHMAKER cDNA library (Clontech Laboratories), cloned into pB42AD containing a bacterial transcriptional activation domain, was amplified on plates, and plasmid DNA prepared from the library was used to transform a clone of yeast, EGY48[p8op-lacZ], harboring the pLexA/NKX-3.1 bait plasmid. EGY48[p8op-lacZ] also carries a plasmid-based lacZ reporter gene and a chromosomal LEU2 reporter gene, both driven by a multimerized LexA operator site. This transformation yielded 10 7 cfu, and 100,000 independent clones were plated onto -His, -Trp, -Ura, -Leu plates. Eighty-one colonies grew and were patched onto -His, -Trp, -Ura, +X-gal plates. Three colonies that were blue in the presence of X-gal were plated onto +His plates to lose the bait plasmid; DNA prepared from these bait-negative derivatives was used to transform Escherichia coli strain DH10B. Sequence analysis of plasmids recovered from one EGY48 transformant revealed a nearly complete open reading frame encoding PDEF (10). The NH 2 -terminal coding region of PDEF was completed by PCR. Cotransfection and Coimmunoprecipitation. A full-length human PDEF cDNA was cloned in frame into pcDNA6/Myc-His (Invitrogen), which re- sulted in the addition of a COOH-terminal MYC epitope. A full-length NKX-3.1 (R52 allele) cDNA was generated by reverse transcription-PCR using RNA from LNCaP cells. A 10-amino acid HA epitope tag (YPYDVPDYAS) was added to the COOH terminus by PCR, and NKX-3.1/HA was cloned into pcDNA3 (Invitrogen). LNCaP cells grown in RPMI 1640 supplemented with 10% FBS were transiently transfected using LipofectAMINE Plus (Invitrogen) according to the manufacturer’s recommendations. Transfected cells were harvested after 48 h, and lysates were used for coimmunoprecipitation as described (11). PDEF was precipitated using either a mouse anti-c-myc mono- clonal antibody (clone 9E10) or a rabbit antimouse PDEF polyclonal antibody (X. Li and C. J. Bieberich, unpublished observations). Immunoprecipitates captured on protein G-Sepharose beads were denatured with SDS-PAGE loading dye, and supernatants were separated on duplicate 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes essentially as described (11). The blots were probed with the anti-c-myc monoclonal anti- body to detect MYC-tagged PDEF and with a rat anti-HA monoclonal anti- body (clone 3F10) to detect the HA-tagged NKX-3.1. PSA Reporter Gene Assays. The 5.3-kb PSA promoter (12) was inserted upstream of the luciferase gene in pGL3 (Promega, Madison WI) to generate Received 9/10/01; accepted 11/30/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1DK54067 2 To whom requests for reprints should be addressed, at Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland 21250. Phone: (410) 455-3125; Fax: (410) 455-3875; E-mail: bieberic@umbc.edu 3 The abbreviations used are: PDEF, prostate-derived Ets factor; AR, androgen recep- tor; PSA, prostate-specific antigen; X-gal, 5-bromo-4 chloro-3-indolyl--D-galactopyr- anoside 338 Research. on November 24, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from