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Identification of Aryl Hydrocarbon Receptor As a Putative Wnt/ß

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Identification of Aryl Hydrocarbon Receptor As a Putative Wnt/ß [CANCER RESEARCH 64, 2523–2533, April 1, 2004] Identification of Aryl Hydrocarbon Receptor as a Putative Wnt/␤-Catenin Pathway Target Gene in Prostate Cancer Cells Dennis R. Chesire,1 Thomas A. Dunn,1 Charles M. Ewing,1 Jun Luo,1 and William B. Isaacs1,2 1Brady Urological Institute, 2Johns Hopkins Oncology Center, Johns Hopkins Medical Institutions, Baltimore, Maryland ABSTRACT (APC) gene product and Axin tumor suppressors, thereby permitting its nuclear accrual (11, 12). Once in the nucleus, ␤-catenin is proposed ␤ Recent genetic and functional analyses have implicated the wnt/ - to effect changes in gene expression by trans-activating promoter- catenin signaling pathway in prostate cancer (CaP) pathogenesis. Thus, bound transcription factors such as those of the T-cell factor/lymphoid there is much interest in understanding the consequences of wnt signaling in CaP; target gene expression is one important area of inquiry and is the enhancer factor (TCF/LEF) family (13, 14). ␤ focus of this report. Adenoviral-mediated overexpression of a mutant, The pervasive role of wnt/ -catenin-mediated gene expression in hyperactive form of ␤-catenin in CWR22-Rv1 CaP cells led to increased animal development and neoplasia, together with the realization that aryl hydrocarbon receptor (AhR, or dioxin receptor) and transmembrane one hallmark of cancer is tissue specificity, now forms the backdrop protein 2 RNA transcript expression, as detected by cDNA-microarray for current research of this pathway in CaP. Links between this signal analyses. Validating these results, reverse transcription-PCR assays dem- transduction pathway and prostate (normal and CaP) have only re- onstrated that in CWR22-Rv1 cells as well as in LAPC-4 CaP cells, cently come to light (1). Genetic analyses of prostate tumors have increased putative target gene RNA expression occurs with transient uncovered potentially oncogenic (i.e., selected) mutations in ␤-catenin ␤ overexpression of mutant -catenin, treatment of cells with lithium chlo- at a rate of approximately 5–10% (Refs. 11, 15–17). These alterations ride, or with wnt3a-conditioned medium, three distinct modes of experi- ␤ ␤ ␤ likely produce transcriptionally active -catenin, as mutation-positive mental wnt/ -catenin pathway activation. This -catenin-associated ex- ␤ pression of AhR and transmembrane protein 2 does not require de novo lesions manifest up-regulated nuclear and cytoplasmic -catenin lo- ␤ protein synthesis and may only involve a certain subset of CaP cell lines. calization (15, 16). Despite absence of direct -catenin mutation in Western and immunofluorescence analyses were undertaken to assess the certain, very different prostate tissues (normal rat prostate and ad- relationship between the wnt/␤-catenin-stimulated increase in AhR tran- vanced metastatic CaP), nuclear ␤-catenin has been observed, perhaps scripts and AhR protein expression; we provide evidence that an associ- arguing that various mechanisms can operate in prostate cells to ation exists whereby up-regulation of AhR RNA by wnt or ␤-catenin is stimulate elevated ␤-catenin stability (18, 19). One might infer from coupled with augmented AhR protein levels. Intriguingly, these studies these observations of nuclear ␤-catenin across this diverse group of also demonstrated that nuclear ␤-catenin staining may not be a sole prostate tissues that its ultimate downstream effects vary, depending ␤ deciding factor when predicting the status of wnt/ -catenin signaling in on the context. Cell biological evidence supports the hypothesis that CaP cells. Finally, the extent to which wnt signaling may synergize with an mutant ␤-catenin, or up-regulation of the ␤-catenin protein by other environmental agonist of AhR (2,3,7,8-tetrachlorodibenzo-p-dioxin) to po- tentiate AhR transcriptional activity was examined. Considering previous means, enhances gene expression in CaP cell lines (e.g., TCF/LEF- work linking AhR to processes of development and carcinogenesis, our dependent transcription; Refs. 15, 18–21). The potential relevance of data may highlight one particular role for wnt/␤-catenin signaling in wnt signaling to prostate physiology, possibly representing a prostate- prostate tumor biology. specific function, is additionally exemplified by novel reports of an interaction between the wnt/␤-catenin axis and androgen receptor; these pathways intersect leading to enhancement of ligand-dependent INTRODUCTION androgen receptor function and repression of ␤-catenin/TCF-dependent The etiology of prostate cancer (CaP) across the affected population transcription (1, 18, 20–27). It is necessary to dissect these genetic likely comprises a diverse array of selected growth and survival traits; and cellular phenomena in a more physiologically relevant setting therefore, multiple levels of complexity probably underlie the overall (i.e., in vivo); indeed, initial foundations have been laid recently using ␤ CaP phenotype. The wnt/␤-catenin signaling pathway has been de- transgenic models that conditionally target activated -catenin expres- ␤ scribed recently to be a potentially important contributor to this sion to the prostate, among other tissues, and directly associate - disease (see Ref. 1 and citations therein). Several normal functions catenin up-regulation to abnormal prostate growth (28, 29). ␤ have been ascribed to canonical wnt signaling and its pathway com- What are the wnt/ -catenin target genes in normal and transformed ponents, namely, the effector molecule ␤-catenin, in orchestrating prostate tissue? The answer to this question could provide rationale ␤ morphogenetic processes across a wide spatial (tissue/cell-specificity) behind the selection of ectopic -catenin activity in the pathogenesis and temporal (embryogenesis versus adult tissue homeostasis) spec- of certain CaPs; such data may additionally bridge the gap between ␤ trum (2–7). These observations tie wnt/␤-catenin signaling to modes classifying tumor genotype (e.g., -catenin mutation-positive) and of tissue-specific stem cell maintenance, proliferation, and differenti- applying this toward predicting tumor phenotype. In this study, we ation; quite significantly, analogous cellular properties are triggered in have approached this problem by applying cDNA-microarray tech- human cancer (1, 8–11). Possibly, the most overt, and likely the most nology (30) along with standard cell culture techniques. Our study influential, outcome of canonical wnt signaling is the activation of complements, but also differs from, recent work by Bierie et al. (29) ␤ ␤-catenin, which entails freeing ␤-catenin from post-translational investigating the effects of -catenin up-regulation on expression of degradation by a complex containing the adenomatous polyposis coli putative target genes in prostate. Of the several candidate genes we have scanned, certain focus will be drawn to the induction of one gene in particular, the aryl hydrocarbon receptor. Received 10/21/03; revised 12/17/03; accepted 1/20/04. Grant support: USPHS Grant CA58236. The costs of publication of this article were defrayed in part by the payment of page MATERIALS AND METHODS charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Cell Culture, Wnt3a-Conditioned Media, Plasmids, and Antibodies. Requests for reprints: William B. Isaacs, Department of Urology, Marburg 115, The Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21287. Phone: Cells were purchased from the American Type Culture Collection (DU145, (410) 955-2518; Fax: (410) 955-0833; E-mail: [email protected]. PC3, LNCaP, HEK-293, control-, or wnt3a-transduced mouse L cells) or were 2523 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. WNT/〉-CATENIN TARGET GENE EXPRESSION IN PROSTATE CANCER furnished to us by Dr. John Isaacs [Johns Hopkins University, Baltimore, MD; RNA Preparation and cDNA Microarray Analysis. Total RNA was ␤ CWR22-Rv1 (CW) and LAPC-4] and were incubated at 37°C/5% CO2/90% prepared from CW cells infected with either control or Del- -catenin- humidity in medium containing 10% FCS. Specific aspects for each cell line expressing adenoviruses using TRIzol reagent (Invitrogen). Cells were lysed in are listed in previous reports (18, 22). All media were purchased from Invitro- 8 ml of TRIzol, followed by 5 min of vortexing. Chloroform (0.2 volumes) was gen (Carlsbad, CA). Lithium chloride (LiCl) and 2,3,7,8-tetrachlorodibenzo- mixed with each preparation by vigorous shaking. Mixes were incubated for 3 p-dioxin (TCDD) were purchased from Sigma (St. Louis, MO). min and then spun at 10,000 ϫ g for 15 min at 4°C in a Beckman centrifuge. With minor modification, control- and wnt3a-conditioned media were pre- The aqueous portion of the phase separation was removed and gently mixed pared as described in a protocol provided by the American Type Culture with an equal volume of 70% ethanol. The mixtures were then entered into Collection with the purchase of the appropriate mouse L cells (31). Briefly, 1 RNeasy Midi columns (Qiagen, Valencia, CA) and centrifuged twice. The million mouse L fibroblasts (control or wnt3a cDNA-transduced) were plated columns were then processed according to the manufacturer’s directions. RNA to 100-mm plates in 10 ml of DMEM/10% FCS and incubated for 4 days. was eluted in 500 ␮l of diethyl pyrocarbonate-treated water and then concen- Medium was removed, clarified with a 0.45 ␮m Millex-HA syringe-driven
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