ART-27, an Androgen Receptor Coactivator Regulated in Prostate Development and Cancer

Androgen receptor trapped clone-27 (ART-27) is a newly described transcriptional coactivator that binds to the N terminus of the androgen receptor (AR). Given the vital importance of AR signaling in prostate growth and differentiation, we investigated the role of ART-27 in these processes. Immunohistochemical studies indicate that ART-27 protein is expressed in differentiated epithelial cells of adult human prostate and breast tissue. In prostate, ART-27 is abundant in AR-positive prostate luminal epithelial cells, in contrast to the stroma, where cells express AR but not ART-27. The use of a rat model of androgen depletion/reconstitution indicates that ART-27 expression is associated with the elaboration of differentiated prostate epithelial cells. Interestingly, regulated expression of ART-27 in the androgen-sensitive LNCaP prostate cancer cell line inhibits androgen-mediated cellular proliferation and enhances androgen-mediated transcription of the prostate-specific antigen (PSA) gene. Consistent with a growth suppressive function, we show that ART-27 expression levels are negligible in human prostate cancer. Importantly, examination of ART-27 protein expression in early fetal prostate development demonstrates that ART-27 is detected only when the developing prostate gland has proceeded from a solid mass of undifferentiated cells to a stage in which differentiated luminal epithelial cells are evident. Thus, ART-27 is an AR cofactor shown to be subject to both cell type and developmental regulation in humans. Overall, the results suggest that decreased levels of ART-27 protein in prostate cancer tissue may occur as a result of de-differentiation, and indicate that ART-27 is likely to regulate a subset of AR-responsive genes important to prostate growth suppression and differentiation. Androgen receptor trapped clone-27 (ART-27) is a newly described transcriptional coactivator that binds to the N terminus of the androgen receptor (AR). Given the vital importance of AR signaling in prostate growth and differentiation, we investigated the role of ART-27 in these processes. Immunohistochemical studies indicate that ART-27 protein is expressed in differentiated epithelial cells of adult human prostate and breast tissue. In prostate, ART-27 is abundant in AR-positive prostate luminal epithelial cells, in contrast to the stroma, where cells express AR but not ART-27. The use of a rat model of androgen depletion/reconstitution indicates that ART-27 expression is associated with the elaboration of differentiated prostate epithelial cells. Interestingly, regulated expression of ART-27 in the androgen-sensitive LNCaP prostate cancer cell line inhibits androgen-mediated cellular proliferation and enhances androgen-mediated transcription of the prostate-specific antigen (PSA) gene. Consistent with a growth suppressive function, we show that ART-27 expression levels are negligible in human prostate cancer. Importantly, examination of ART-27 protein expression in early fetal prostate development demonstrates that ART-27 is detected only when the developing prostate gland has proceeded from a solid mass of undifferentiated cells to a stage in which differentiated luminal epithelial cells are evident. Thus, ART-27 is an AR cofactor shown to be subject to both cell type and developmental regulation in humans. Overall, the results suggest that decreased levels of ART-27 protein in prostate cancer tissue may occur as a result of de-differentiation, and indicate that ART-27 is likely to regulate a subset of AR-responsive genes important to prostate growth suppression and differentiation. Androgen steroid hormones direct the genetic program dictating prostate development and maturation in male development. They exert biological effects by binding to the androgen receptor (AR), 1The abbreviations used are: AR, androgen receptor; AF-1, activation function 1; AF-2, activation function 2; ART-27, androgen receptor-trapped clone-27; ARE, androgen response element; IRES, internal ribosome entry site; PB, prostatic bud; PCNA, proliferating cell nuclear antigen; PIN, prostatic intraepithelial neoplasia; TBP, TATA box-binding protein; GST, glutathione S-transferase; PSA, prostate-specific antigen; RT, reverse transcription; HA, hemagglutinin. a member of the steroid receptor family of transcription factors. Functional mapping of the androgen receptor shows that several regions are required for transcriptional activation (1Jenster G. van der Korput H.A. Trapman J. Brinkmann A.O. J. Biol. Chem. 1995; 270: 7341-7346Google Scholar, 2Chamberlain N.L. Whitacre D.C. Miesfeld R.L. J. Biol. Chem. 1996; 271: 26772-26778Google Scholar). These include a C-terminal domain, AF-2, as well as two regions in the N terminus, AF-1a and AF-1b. Recent evidence suggests that the AR cell- and promoter-specific transcriptional response is generated through interactions with regulatory proteins termed coactivators and corepressors with AF-1 and AF-2 (2Chamberlain N.L. Whitacre D.C. Miesfeld R.L. J. Biol. Chem. 1996; 271: 26772-26778Google Scholar, 3Cleutjens C.B. Steketee K. van Eekelen C.C. van der Korput J.A. Brinkmann A.O. Trapman J. Endocrinology. 1997; 138: 5293-5300Google Scholar, 4Scheller A. Hughes E. Golden K.L. Robins D.M. J. Biol. Chem. 1998; 273: 24216-24222Google Scholar, 5Hsiao P.W. Chang C. J. Biol. Chem. 1999; 274: 22373-22379Google Scholar, 6Kang H.Y. Yeh S. Fujimoto N. Chang C. J. Biol. Chem. 1999; 274: 8570-8576Google Scholar, 7Moilanen A.M. Karvonen U. Poukka H. Janne O.A. Palvimo J.J. Mol. Biol. Cell. 1998; 9: 2527-2543Google Scholar, 8Muller J.M. Isele U. Metzger E. Rempel A. Moser M. Pscherer A. Breyer T. Holubarsch C. Buettner R. Schule R. EMBO J. 2000; 19: 359-369Google Scholar, 9Aarnisalo P. Palvimo J.J. Janne O.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2122-2127Google Scholar, 10Fronsdal K. Engedal N. Slagsvold T. Saatcioglu F. J. Biol. Chem. 1998; 273: 31853-31859Google Scholar, 11Fujimoto N. Yeh S. Kang H.Y. Inui S. Chang H.C. Mizokami A. Chang C. J. Biol. Chem. 1999; 274: 8316-8321Google Scholar, 12Heinlein C.A. Ting H.J. Yeh S. Chang C. J. Biol. Chem. 1999; 274: 16147-16152Google Scholar). Androgen stimulation results in cell proliferation in both the developing prostate and the malignant prostate. Current therapy for metastatic prostate cancer includes injections of luteinizing hormone-releasing hormone (LHRH) analogues to pharmacologically lower testosterone levels (androgen ablation), treatment with anti-androgens such as flutamide or bicalutamide, to block testosterone binding to the AR, or a maximal androgen blockade in which both treatments are combined. While such therapies are effective in the short term, cancers treated in this fashion will inevitably progress in an androgen-independent fashion. An important determinant of AR transactivation function resides in the N terminus (1Jenster G. van der Korput H.A. Trapman J. Brinkmann A.O. J. Biol. Chem. 1995; 270: 7341-7346Google Scholar, 13Simental J.A. Sar M. Lane M.V. French F.S. Wilson E.M. J. Biol. Chem. 1991; 266: 510-518Google Scholar, 14Rundlett S.E. Wu X.P. Miesfeld R.L. Mol. Endocrinol. 1990; 4: 708-714Google Scholar). The AR N-terminal residues 142–485 have been shown to activate a minimal promoter construct in a cell-free transcription system and to selectively interact with the transcription factors TFIIF and the TATA-binding protein (TBP), suggesting direct contact with general transcription factors (15McEwan I.J. Gustafsson J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8485-8490Google Scholar, 16Reid J. Murray I. Watt K. Betney R. McEwan I.J. J. Biol. Chem. 2002; 277: 41247-41253Google Scholar). A growing list of proteins has also been reported to interact with the AR N terminus (15McEwan I.J. Gustafsson J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8485-8490Google Scholar, 17Lee D.K. Duan H.O. Chang C. J. Biol. Chem. 2000; 275: 9308-9313Google Scholar, 18Yamamoto A. Hashimoto Y. Kohri K. Ogata E. Kato S. Ikeda K. Nakanishi M. J. Cell Biol. 2000; 150: 873-880Google Scholar, 19Park J.J. Irvine R.A. Buchanan G. Koh S.S. Park J.M. Tilley W.D. Stallcup M.R. Press M.F. Coetzee G.A. Cancer Res. 2000; 60: 5946-5949Google Scholar, 20Hsiao P.W. Lin D.L. Nakao R. Chang C. J. Biol. Chem. 1999; 274: 20229-20234Google Scholar, 21Yu X. Li P. Roeder R.G. Wang Z. Mol. Cell. Biol. 2001; 21: 4614-4625Google Scholar, 22Beitel L.K. Elhaji Y.A. Lumbroso R. Wing S.S. Panet-Raymond V. Gottlieb B. Pinsky L. Trifiro M.A. J. Mol. Endocrinol. 2002; 29: 41-60Google Scholar). The clinical importance of investigating the biological role of coactivators that bind the AR N terminus is underscored by a recent report describing a patient diagnosed with complete androgen insensitivity, but exhibiting no mutation in AR. The study suggested that a coactivator interacting with the N-terminal region of AR, apparently essential for genital development, was lacking in this patient (23Adachi M. Takayanagi R. Tomura A. Imasaki K. Kato S. Goto K. Yanase T. Ikuyama S. Nawata H. N. Engl. J. Med. 2000; 343: 856-862Google Scholar). The work described in this article investigates the biological role of the newly described androgen receptor coactivator, androgen receptor trapped clone-27 (ART-27). ART-27 binds to the AR N terminus and facilitates receptor dependent transcriptional activation. The ART-27 clone was isolated from an androgen-stimulated LNCaP cell cDNA library in a two-hybrid screen designed to identify proteins that interacted with the AR-N terminus (24Markus S.M. Taneja S.S. Logan S.K. Li W. Ha S. Hittelman A.B. Rogatsky I. Garabedian M.J. Mol. Biol. Cell. 2002; 13: 670-682Google Scholar) and was previously cloned in an exontrapping experiment (25Schroer A. Schneider S. Ropers H. Nothwang H. Genomics. 1999; 56: 340-343Google Scholar). The ART-27 gene encodes a protein of 157 amino acids with a calculated molecular weight of 18 kDa. Functional characterization of ART-27 indicates that it is a nuclear protein that interacts with N-terminal rat AR amino acids 153–336, containing AF-1a and a part of AF-1b (24Markus S.M. Taneja S.S. Logan S.K. Li W. Ha S. Hittelman A.B. Rogatsky I. Garabedian M.J. Mol. Biol. Cell. 2002; 13: 670-682Google Scholar). Biochemical analysis of ART-27 indicates that it interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. In addition, ART-27 increases the transcriptional potency of AR when overexpressed in a variety of cultured mammalian cells. Mechanistically, ART-27 is likely to function as part of a transcription complex since analysis of ART-27 behavior in density gradient sedimentation of HeLa nuclear cell extracts indicates that it co-sediments as part of a larger complex (24Markus S.M. Taneja S.S. Logan S.K. Li W. Ha S. Hittelman A.B. Rogatsky I. Garabedian M.J. Mol. Biol. Cell. 2002; 13: 670-682Google Scholar). Although the mechanism by which ART-27 functions as a transcriptional activator is unclear, it is likely that ART-27 acts as an adapter to assemble protein complexes at androgen-responsive promoters. receptor coactivators and corepressors are likely important of the response of a hormone receptor in a cellular androgen stimulation results in cellular at of prostate development and malignant we investigated the role of ART-27 in prostate epithelial cells. of ART-27 construct was by ART-27 as a from and in ART-27 and ART-27 by the ART-27 a with an N-terminal or a with a C-terminal of of C-terminal has been previously described (24Markus S.M. Taneja S.S. Logan S.K. Li W. Ha S. Hittelman A.B. Rogatsky I. Garabedian M.J. Mol. Biol. Cell. 2002; 13: 670-682Google Scholar). C-terminal by of the a protein A by to the ART-27 was with and the protein by with the protein human used with of the of from or prostate was C-terminal ART-27 or AR AR in and in antigen in a in by treatment with and with with ART-27 or AR by with a from human of for to this for the of was by the of was for of by and the and in of Androgen from and The and two at In addition, at the the with testosterone of at and testosterone The of the that tissue androgen of cell a ART-27 gene in LNCaP cells. LNCaP cells with the and in to with and for activation by gene A LNCaP clone activation was with to of in B. and in the and of Cell and to analysis to which cell ART-27 was used for cell in in fetal The cells with and to fetal and was the of the was to and with cells for at and to and was isolated from ART-27 clone cells the to the was the C. are as T. S. M. T. S. J. 1999; I. Wang Garabedian M.J. Proc. Natl. Acad. Sci. U. S. A. Scholar). The for and are and for to in the transcription was at for for and of used for the of transcription was by at at and at for of used for reverse transcription by at at and at for ART-27 in of and the biological role of ART-27, that the cell and expression of ART-27 be in of is shown in with ART-27, and with ART-27 or the or used for shows that a the C-terminal of ART-27 overexpressed ART-27 and overexpressed ART-27 and as well as ART-27 protein and A protein these proteins and the of ART-27 of ART-27 an of ART-27 was expressed and with expression of ART-27 The results that ART-27 is the as the ART-27 and that the is of overexpressed and ART-27. the but not the ART-27 protein the cell type of ART-27 expression with AR ART-27 and AR with of human prostate and breast tissue. from tissue ART-27 and AR are expressed in of differentiated epithelial cells of human prostate tissue. AR is ART-27 to be in both the and the A and In breast AR and ART-27 are both expressed in of epithelial cells. AR to be abundant in the regions of the gland as the gland by the in in the part of the gland ART-27 protein to be abundant in both protein is expressed in epithelial cells of tissue is in epithelial cells of the a for in the of the the these show no that of the is to of the ART-27 C-terminal of the with only and indicates that the not to the the of ART-27 protein expression is epithelial examination of with the ART-27 in human prostate tissue shows epithelial cell and to in the of adult human prostate tissue the cell type of ART-27 protein analysis was from of human epithelial and cells M. H. 1998; Scholar). Consistent with the ART-27 is expressed in epithelial cells and expressed at at in cells ART-27 in studies in human prostate tissue that ART-27 and AR are both expressed in prostate epithelial cells, a rat model of androgen depletion/reconstitution was used to the effects of androgen-mediated prostate growth and ART-27 protein expression R. Cell Biol. 1995; Scholar). to the expression of ART-27 in human prostate rat prostate tissue that ART-27 protein is expressed in prostate luminal epithelial cells, but not in cells the to of and of the prostate for and in cellular proliferation and growth of the prostate. from the at the and from gland and used for The with ART-27 as well as proliferating cell nuclear antigen a for cellular protein that increases prostate and R. C.A. J. Chang C. M. Mol. Cell. Biol. 9: Scholar, J. J.M. C. P. J. M. J. Biol. Chem. 1998; 273: Scholar, S. G. C. A. S. J. Endocrinol. 2000; and as an internal expression is and expression is the of in the prostate of levels are with of the role of in this model and ART-27 protein is androgen but is abundant when are and Although it is not in this of the from the with ART-27 ART-27 in the from the in this the expression of ART-27 is associated with an of differentiated prostate epithelial cells and is with of epithelial cells of androgen designed to ART-27 may a role in prostate growth differentiation. of ART-27 in the LNCaP Cell Cell the effects of ART-27 prostate cancer cell growth we cell that express an of ART-27 the regulation of a promoter in androgen-sensitive LNCaP prostate cancer cells. LNCaP cells with the and for activation by gene A LNCaP clone activation was with to of shows expression of (ART-27) and ART-27 in of the in the and of and the as by ART-27 is in the of and ART-27 levels or ART-27 levels are in response to the or of and and that not regulate the transcription of ART-27. designed to at the of ART-27 androgen cell growth in LNCaP cells it was ART-27 protein levels are regulated by as be suggested by the of ART-27 protein levels in the rat prostate cell this ART-27 protein levels in LNCaP cells in the and of analysis of from androgen or androgen LNCaP prostate cancer cells is shown in The of ART-27 protein in cells is to the levels of ART-27 in LNCaP cells suggesting that not the levels of ART-27 of protein expression in response to is shown in treatment of an clone with of results in levels of ART-27 protein a to the described it was also that levels of AR expression are not by treatment of the cell with the of ART-27 androgen-mediated LNCaP cell was by of cellular and shows analysis of clone and clone in the and of and regulation of in response to shows a experiment in which was to the cells to is of in the of the cells are treated with or not In the of is the cellular as cells ART-27 show with that not at the and treatment with of an clone clone shows a the cells show in response to to In this expression of in the of also with the cells in the of In in clone the of is to levels in the to a it was shown that at the not LNCaP cell The decreased of LNCaP cell to in the of ART-27 gene expression is by the that we not to LNCaP cell that overexpressed ART-27 In these ART-27 was the that an internal ribosome entry to express the protein of (ART-27) the as a While of LNCaP when with no with suggesting that levels of ART-27 protein or LNCaP cell LNCaP cell growth suppression is to the of androgen-responsive genes in differentiated cell function, we the of gene transcription in the and of and by is an AR gene and of differentiated epithelial cells. shows the analysis of generated in for the gene or the protein gene as a and The ART-27 clone was in and treated with or for or as treated with to be with which ART-27 suppression of cell growth was and is used at to it was that of levels be shows that is in the and that the gene is in response to ART-27 with at both the and that this experiment not indicate the mechanism by which ART-27 enhances gene and are to ART-27 a direct with the that ART-27 a role in cellular and growth transcription of is of growth ART-27 in the results suggested that ART-27 inhibits androgen-mediated prostate cell we the expression of ART-27 protein in and malignant human prostate tissue through of tissue with ART-27 or with an AR show nuclear of prostate A and as previously in A and show that ART-27 AR in prostate cells Immunohistochemical of containing regions of epithelial indicate that ART-27 in prostate cancer is decreased by the in and to that in prostatic by in and AR of a shows in and malignant a for the of the tissue and A of AR and ART-27 expression in an prostate cancer tissue levels of AR and negligible levels of ART-27 A and expression in cancer in of prostate cancer tissue with or with are from shown in are from of the tissue A and show expression of AR and ART-27 in prostate cancer. and show expression of AR and ART-27 in are shown at ART-27 was in cancer ART-27 not be to tissue by since only that with in the The of differentiated with a differentiated In the of the malignant of the of differentiation. In containing prostatic intraepithelial a to be a to prostate ART-27 was the but and Thus, in ART-27 expression to be a in prostate in prostate cancer and prostate cancer cell we that ART-27 may a role in epithelial or proliferation role in to be ART-27 in of ART-27 protein in prostate cancer and suggests that ART-27 may a role in luminal epithelial cell differentiation. ART-27 protein expression developmental differentiation, we the of ART-27 protein in the region of the from which the prostate in human fetal tissue. through the region of a show a cell region in the which is the of the by the in A and to the are of epithelial cells that are by or tissue from the and the to the prostate or of a tissue with ART-27 shows ART-27 protein in the of the epithelial cells to the of the but not in cells or in the prostatic at of development with shows of the epithelial cells the as well as in cells, and epithelial cells in the region of the prostatic from the region of a was also with ART-27 the is larger at this in development only a of the is in the ART-27 is in the luminal cells to the of the a cell of cells to the is in contrast to the of AR protein of a with shows that AR is expressed in cells as well as in of epithelial cells to the of the The indicate cells that are for the of both ART-27 and AR. These results suggest that in the ART-27 is in differentiated cells with an to the in cells to the cell is by the that in tissue from the region of a is expression of ART-27 in the prostatic is expression in prostatic that have to a of the prostatic have a and nuclear expression of ART-27 is in of the prostatic Thus, expression of ART-27 with of prostate luminal epithelial cells in human fetal development. to the role of AR in prostate cell growth or suggest that effects are complex and dependent cellular B. M. Cancer Res. 2001; Scholar, M. I. P.W. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar, van M.A. van E. Scholar, A. A.M. J. Mol. Cell. Endocrinol. 1997; Scholar). Androgen receptor coactivators such as ART-27, are likely to AR and and proliferation of prostate cell gene transcriptional regulation is a for the development of cell that mechanism such is the expression of of the transcriptional to cell a of the transcription is to the cells of the and a subset of genes for development S. R. 2001; Scholar). The cell type of ART-27 protein fetal development suggests that ART-27 a subset of AR-responsive genes in cell AR is expressed in cell of the ART-27 is expressed in a cell to the In prostate, ART-27 is not in prostatic at but at that with the of luminal epithelial cells and the of a the of proteins expressed by the differentiated prostate are in the of a indicates cellular differentiation. Thus, ART-27 protein expression with prostate cell development. the prostate, ART-27 expression to to differentiated epithelial cells. to no expression is the of the or the the luminal of ART-27 nuclear is cells for AR. suggests a role for ART-27, dependent cell stage of cellular or ART-27 is in both and but in prostate the of ART-27 expression to be a in of ART-27 a prostate cancer cell line by results in decreased androgen-stimulated complete of growth to occur to express ART-27 the cell These are with the that ART-27 functions in or of an AR program of prostate cell and are by evidence that expression of ART-27 results in of gene transcription a gene expressed by differentiated prostate luminal epithelial cells. Androgen receptor coactivators and corepressors are likely to a role in androgen dependent and androgen-independent prostate cancer. The effective of prostate cancer is by androgen which prostate tissue with prostate to While the of is in the short term, prostate cancer cells inevitably to in a hormone and at this treatment are hormone androgen receptor mutation and of AR coactivators or corepressors to the AR S. D.M. Med. 2000; Scholar, 2002; 60: Scholar). The that steroid receptor coactivators be in cancer has been with the that the used to breast acts by the nuclear receptor and binding to A.M. Z. T. L. Gustafsson J.A. M. 1997; Scholar, L. Cell. 1998; 95: Scholar, K. V. R. V. F. E. S.M. G. Cell. 2000; Scholar). A recent report indicates that prostate cancers express levels of the steroid hormone transcriptional and coactivator receptor genes B. French F.S. Wilson E.M. Cancer Res. 2001; Scholar). In addition, in analysis of androgen receptor coactivator expression levels that expression of the coactivators and malignant prostate tissue P. X. K. J. Roeder R.G. Wang Z. J. 2002; Scholar). prostate cell growth have also been in studies by of the gene J. Y. M.J. 1998; Scholar). these effects are and suggest that that are required for androgen cell Although the AR N terminus has been shown to be for AR the expression of proteins has not been prostate cancer indicate that ART-27 expression is prostate cancer and may an important role in this is ART-27 is regulated in prostate cells or in prostate cancer. The cell type of ART-27 expression suggests that it is not regulated by since ART-27 is expressed in a AR. In addition, androgen treatment not ART-27 or protein in LNCaP cells and are no androgen response in the promoter of ART-27. Li and M. J. ART-27 protein expression may be regulated by of or growth factors in response to androgen A growing of evidence suggests that of steroid hormone the to biological Cell. 2002; Scholar). results are with the that the androgen receptor coactivator ART-27, is a cell and prostate epithelial cell differentiation, cellular of the Cancer for analysis for of the

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