Retinoic Acid Inhibits β-Catenin through Suppression of Cox-2

Abstract

Mutations in adenomatous polyposis coli (APC) underlie the earliest stages of colorectal carcinogenesis. Consequences of APC mutation include stabilization of β-catenin, dysregulation of cyclooxygenase-2 (COX-2) expression, and loss of retinoic acid production, events with poorly defined interactions. Here we showed that treatment of zebrafish expressing a truncated form of Apc with either retinoic acid or a selective COX-2 inhibitor decreased β-catenin protein levels and downstream signaling events. Interestingly, the destruction of β-catenin in apc mutant embryos following Cox-2 inhibition required the presence of truncated Apc. These findings support roles for retinoic acid and Cox-2 in regulating the stability of β-catenin following Apc loss. Furthermore, truncated Apc appears to retain the ability to target β-catenin for destruction, but only in the absence of Cox-2 activity. This novel function of truncated Apc may provide a molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer. Mutations in adenomatous polyposis coli (APC) underlie the earliest stages of colorectal carcinogenesis. Consequences of APC mutation include stabilization of β-catenin, dysregulation of cyclooxygenase-2 (COX-2) expression, and loss of retinoic acid production, events with poorly defined interactions. Here we showed that treatment of zebrafish expressing a truncated form of Apc with either retinoic acid or a selective COX-2 inhibitor decreased β-catenin protein levels and downstream signaling events. Interestingly, the destruction of β-catenin in apc mutant embryos following Cox-2 inhibition required the presence of truncated Apc. These findings support roles for retinoic acid and Cox-2 in regulating the stability of β-catenin following Apc loss. Furthermore, truncated Apc appears to retain the ability to target β-catenin for destruction, but only in the absence of Cox-2 activity. This novel function of truncated Apc may provide a molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer. Retinoids are important regulators of differentiation and cell proliferation. Induction of differentiation by retinoic acid has been observed in endothelial, melanoma, neuroblastoma, and lung cancer cells (1Hong W.K. Benner S.E. Lippman S.M. Leukemia (Baltimore). 1994; 8: 33-37PubMed Google Scholar). Retinoic acid has been shown to inhibit the growth of breast cancer cell lines and to reduce the average number and incidence of tumors in an animal model of breast cancer (2Anzano M.A. Byers S.W. Smith J.M. Peer C.W. Mullen L.T. Brown C.C. Roberts A.B. Sporn M.B. Cancer Res. 1994; 54: 4614-4617PubMed Google Scholar, 3Fitzgerald P. Teng M. Chandraratna R.A. Heyman R.A. Allegretto E.A. 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Retinoic acid mediates its effects by binding to its receptors, retinoic acid receptor (RAR), 4The abbreviations used are:RARretinoic acid receptorAPCadenomatous polyposis coliCOX-2cyclooxygenase-2TCFT-cell factorPGE2prostaglandin E2hpfhours post-fertilizationRTreverse transcriptionRXRretinoid X receptorGSK-3βglycogen synthase kinase-3βGFPgreen fluorescent protein. or retinoid X receptor (RXR), followed by heterodimerization and recognition of RAR element-containing promoters. In cells that express the transcription factor AP-1, retinoic acid regulates cell growth by inhibiting the formation of AP-1 complexes capable of DNA binding (8Li J.J. Dong Z. Dawson M.I. Colburn N.H. Cancer Res. 1996; 56: 483-489PubMed Google Scholar, 9Yang L. Munoz-Medellin D. Kim H.T. Ostrowski J. Reczek P. Brown P.H. Breast Cancer Res. Treat. 1999; 56: 277-291Crossref PubMed Google Scholar, 10Zhou X.F. Shen X.Q. Shemshedini L. Mol. Endocrinol. 1999; 13: 276-285Crossref PubMed Scopus (96) Google Scholar). In SW480 colon cancer cells, retinoic acid associates with its receptor RAR, and the complex sequesters β-catenin from the TCF transcription factor, thus preventing transcription of β-catenin/TCF target genes (11Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, RXR agonists induce degradation of β-catenin through a proteasome-dependent process (12Xiao J.H. Ghosn C. Hinchman C. Forbes C. Wang J. Snider N. Cordrey A. Zhao Y. Chandraratna R.A. J. Biol. Chem. 2003; 278: 29954-29962Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). retinoic acid receptor adenomatous polyposis coli cyclooxygenase-2 T-cell factor prostaglandin E2 hours post-fertilization reverse transcription retinoid X receptor glycogen synthase kinase-3β green fluorescent protein. β-Catenin is a multifunctional protein that transduces Wnt signals, mediates cell-cell adherens junctions through its interaction with E-cadherin, and stimulates cell proliferation. β-Catenin is regulated by, and forms part of, a multiprotein complex that also includes APC, axin, glycogen synthase kinase-3β (GSK-3β), and casein kinase 1 (13Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1310) Google Scholar, 14Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1118) Google Scholar, 15Fagotto F. Jho E. 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For example, impaired function of APC, which is observed in ∼85% of colorectal cancers, is accompanied by the stabilization and accumulation of β-catenin and the uncontrolled activation of cell proliferation programs. Interestingly, mutations in APC also result in decreased retinoic acid biosynthesis (26Jette C. Peterson P.W. Sandoval I.T. Manos E.J. Hadley E. Ireland C.M. Jones D.A. J. Biol. Chem. 2004; 279: 34397-34405Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 27Nadauld L.D. Sandoval I.T. Chidester S. Yost H.J. Jones D.A. J. Biol. Chem. 2004; 279: 51581-51589Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 28Nadauld L.D. Shelton D.N. Chidester S. Yost H.J. Jones D.A. J. Biol. Chem. 2005; 280: 30490-30495Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Prostaglandin E2 (PGE2), a cyclooxygenase-2 (COX-2) product, was recently shown to promote the stabilization of β-catenin in colon cancer cell lines by displacing GKS-3β from the β-catenin complex, thus preventing its phosphorylation and degradation (29Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science. 2005; 310: 1504-1510Crossref PubMed Scopus (775) Google Scholar, 30Shao J. Jung C. Liu C. Sheng H. J. Biol. Chem. 2005; 280: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). We previously showed that retinoic acid suppresses COX-2 expression in human cancer cell lines and in zebrafish embryos (31Eisinger A.L. Nadauld L.D. Shelton D.N. Peterson P.W. Phelps R.A. Chidester S. Stafforini D.M. Prescott S.M. Jones D.A. J. Biol. Chem. 2006; 281: 20474-20482Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Based on these findings, we hypothesized that retinoic acid antagonizes β-catenin signaling by regulating the levels of PGE2 generated by COX-2. In this study, we demonstrated that β-catenin is stabilized in three retinoic acid-deficient mutant zebrafish. In addition, supplementation of apc mutant zebrafish embryos with retinoic acid decreased β-catenin protein expression by a mechanism that required attenuation of Cox-2 expression and decreased accumulation of PGE2. Treatment of wild-type embryos with PGE2 led to stabilization of β-catenin, whereas reducing PGE2 levels in apc mutant zebrafish results in the degradation of β-catenin. These combined results establish a role for PGE2 as a key determinant of β-catenin signaling in vivo. Embryo Culture and Zebrafish Stocks—Wild-type, apc mutant, and nls mutant Danio rerio (zebrafish) were maintained on a 14:10-h light:dark cycle. Fertilized embryos were collected following natural spawnings and allowed to develop at 28.5 °C. Control and experimental embryos were raised in 0.003% phenylthiourea to inhibit pigment formation. Whole Mount in Situ Hybridization—Zebrafish embryos were fixed in sucrose-buffered 4% paraformaldehyde, rinsed in phosphate-buffered saline, dehydrated in methanol, and stored at -20 °C. The riboprobe for green fluorescent protein (GFP) was generated by linearization of pCRII (Invitrogen) containing GFP cDNA followed by in vitro transcription with SP6 or T7 RNA polymerase (Roche Diagnostics). The embryos were cleared in 70% glycerol/phosphate-buffered saline and photographed using an Olympus DP12 digital camera. Quantitative RT-PCR—We isolated RNA from embryos and cell lines using an RNeasy kit (Qiagen) and then synthesized single-stranded cDNA from 1 μg of total RNA using Superscript III (Invitrogen). PCR was performed using a Light Cycler instrument and software (version 3.5, Roche Diagnostics). The sequences of PCR primers used were as follows: GFP forward, 5′-CCAGATCCGCCACAACATCG-3′; reverse, 5′-GTCCATGCCGAGAGTGATCC-3′. We performed duplicate PCRs using the LightCycler FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics). The amplification conditions for GFP of at of at and of at for a total of was in Quantitative primers and conditions for amplification of and were previously (31Eisinger A.L. Nadauld L.D. Shelton D.N. Peterson P.W. Phelps R.A. Chidester S. Stafforini D.M. Prescott S.M. Jones D.A. J. Biol. Chem. 2006; 281: 20474-20482Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, F. C. O. V. B. Science. 2000; PubMed Scopus Google Scholar). and RNA from for the apc and were previously L.D. Sandoval I.T. Chidester S. Yost H.J. Jones D.A. J. Biol. Chem. 2004; 279: 51581-51589Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, T. S. E. M.A. S. A. PubMed Scopus Google Scholar). The and the were in and to a of For of was wild-type embryos at the to RNA for Cox-2 and were synthesized using to the Cox-2 and were embryos at the the role of retinoic acid in β-catenin expression, we embryos with acid in at for 1 with we the embryos with acid at and for 1 and then the of Cox-2 was by embryos at and with the selective COX-2 inhibitor were with PGE2 in for the in the the embryos were with in at and of the proteasome was by embryos with for and from zebrafish embryos in with the of a were at and then at for to the We protein in the the protein and then μg of protein to on to we the using for and then the with in The used this were from Cell and We then the using and a to the presence of PGE2 E2 levels were in wild-type and apc mutant zebrafish embryos using a PGE2 kit Retinoic of We previously that APC is also an of retinoic acid biosynthesis (26Jette C. Peterson P.W. Sandoval I.T. Manos E.J. Hadley E. Ireland C.M. Jones D.A. J. Biol. Chem. 2004; 279: 34397-34405Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 27Nadauld L.D. Sandoval I.T. Chidester S. Yost H.J. Jones D.A. J. Biol. Chem. 2004; 279: 51581-51589Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 28Nadauld L.D. Shelton D.N. Chidester S. Yost H.J. Jones D.A. J. Biol. Chem. 2005; 280: 30490-30495Abstract Full Text Full Text PDF PubMed Scopus (39) Google and that retinoic acid the expression COX-2 (31Eisinger A.L. Nadauld L.D. Shelton D.N. Peterson P.W. Phelps R.A. Chidester S. Stafforini D.M. Prescott S.M. Jones D.A. J. Biol. Chem. 2006; 281: 20474-20482Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Interestingly, has shown that a COX-2 product, the stabilization of β-catenin in colon cancer cell lines by displacing GKS-3β from the β-catenin destruction complex (29Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science. 2005; 310: 1504-1510Crossref PubMed Scopus (775) Google Scholar, 30Shao J. Jung C. Liu C. Sheng H. J. Biol. Chem. 2005; 280: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Based on these findings, we hypothesized that APC of retinoic acid and COX-2 may function to the stability of β-catenin and downstream this we apc mutant zebrafish embryos with retinoic acid and that this levels of β-catenin protein The of β-catenin were by levels of β-catenin target and L. A. F. Cancer Res. 2006; PubMed Scopus Google Scholar, Y. T. N. Koyama E. J. T. Y. Costantini F. S. M. M. M. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). the that the levels of β-catenin in apc mutant embryos from a of retinoic acid production, we the We the levels of β-catenin in zebrafish in which of the results in retinoic acid G. R. P.W. 2001; PubMed Google Scholar). We that nls embryos levels of β-catenin with wild-type a result that is with in apc These findings that retinoic acid β-catenin levels This was we a used as a of the of activation of β-catenin Dev. Biol. PubMed Scopus Google Scholar). We that of in embryos expression, β-catenin activation that Apc β-catenin levels by a mechanism that retinoic of Cox-2 for of β-Catenin retinoic acid Cox-2 expression in apc mutant zebrafish and that recently demonstrated that PGE2 stabilized β-catenin in human colon cancer cell lines (29Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science. 2005; 310: 1504-1510Crossref PubMed Scopus (775) Google Scholar, 30Shao J. Jung C. Liu C. Sheng H. J. Biol. Chem. 2005; 280: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (191) Google we Cox-2 and PGE2 as retinoic acid and β-catenin. with this we that of Cox-2 in apc mutant embryos β-catenin protein levels T. S. E. M.A. S. A. PubMed Scopus Google Scholar). inhibition of Cox-2 with the selective T. S. E. M.A. S. A. PubMed Scopus Google β-catenin expression with embryos activation of the β-catenin target genes and and the levels of β-catenin. These results demonstrated that the of Cox-2 was for and activation of β-catenin the function of Apc was of Cox-2 of β-Catenin in Cox-2 regulated the expression of β-catenin the function of Apc was we Cox-2 in wild-type zebrafish embryos by of and then the expression of β-catenin using This treatment in the of β-catenin with embryos of Cox-2 activation of β-catenin target as demonstrated by expression of of Cox-2 also expression of in zebrafish These that signaling through Cox-2 results in the of β-catenin and activation of target genes in wild-type zebrafish PGE2 β-Catenin in Zebrafish (29Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science. 2005; 310: 1504-1510Crossref PubMed Scopus (775) Google recently demonstrated that PGE2 the stability of the β-catenin degradation complex by the of GSK-3β from in human cell PGE2 a role in and following apc we the role for PGE2 on the stabilization and activation of β-catenin in apc mutant zebrafish We the levels of PGE2 in apc mutant zebrafish embryos and that the levels of PGE2 were in wild-type In addition, a receptor accumulation of β-catenin in apc mutant zebrafish that the levels of PGE2 the of β-catenin in vivo. of PGE2 was to β-catenin we wild-type zebrafish embryos with PGE2 and β-catenin protein levels in to wild-type embryos The stabilization of β-catenin was accompanied by activation of and treatment of embryos with PGE2 expression of with activation of β-catenin that the observed in PGE2 and subsequent in β-catenin on Apc we apc mutant embryos with a of Apc previously shown to β-catenin J. B. Cancer Res. 2000; Google Scholar). expression of β-catenin levels an that was by treatment with PGE2 These results that the of PGE2 on β-catenin expression downstream of Apc and a model PGE2 mediates stabilization and activation of β-catenin following Apc loss. Apc for β-Catenin of in APC are to formation of a β-catenin degradation we that retinoic acid or inhibition of Cox-2 loss of β-catenin protein expression in apc mutant zebrafish the presence of a truncated form of Apc. This that truncated Apc may retain ability to form a β-catenin degradation this we β-catenin degradation following Cox-2 inhibition required the of the we decreased PGE2 accumulation in apc using but also the effects of an inhibitor of the We that treatment the ability of to β-catenin that apc mutant embryos β-catenin through a proteasome-dependent process form of Apc was for degradation of β-catenin following Cox-2 we the expression of Apc in wild-type embryos using an Apc total levels of β-catenin, of Apc and levels of Cox-2 shown and A.L. Nadauld L.D. Shelton D.N. Peterson P.W. Phelps R.A. Chidester S. Stafforini D.M. Prescott S.M. Jones D.A. J. Biol. Chem. 2006; 281: 20474-20482Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). in to in apc mutant of PGE2 in Apc to reduce levels of β-catenin We this by of a of the the truncated form in apc mutant zebrafish. of Apc with on β-catenin protein In of Apc with with treatment by decreased β-catenin protein levels and expression of These that is required for the degradation of β-catenin in the presence of Cox-2 This is with results shown in and that inhibition of PGE2 with a Cox-2 inhibitor β-catenin to basal levels in the presence of truncated Apc. We previously that the APC is an of retinoic acid biosynthesis L.D. Sandoval I.T. Chidester S. Yost H.J. Jones D.A. J. Biol. Chem. 2004; 279: 51581-51589Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). that APC of retinoic acid biosynthesis through the and that this is of β-catenin L.D. Chidester S. Shelton D.N. T. Sandoval I.T. Peterson P.W. Manos E.J. Ireland C.M. Yost H.J. Jones D.A. S. A. 2006; PubMed Scopus Google Scholar, L.D. Phelps R. A. Sandoval I.T. Chidester S. Peterson P.W. Manos E.J. B. Jones D.A. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). the of retinoic acid biosynthesis appears of the of β-catenin, the may a number of that retinoic acid may the of β-catenin. in SW480 cells that β-catenin from the TCF transcription factor, thus preventing transcription of β-catenin/TCF target genes (11Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar). RXR agonists to the destruction of β-catenin through a proteasome-dependent process (12Xiao J.H. Ghosn C. Hinchman C. Forbes C. Wang J. Snider N. Cordrey A. Zhao Y. Chandraratna R.A. J. Biol. Chem. 2003; 278: 29954-29962Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The mechanism to the destruction of β-catenin following RXR activation is retinoic acid in the expression of Cox-2 downstream of a process that to decreased PGE2 accumulation (31Eisinger A.L. Nadauld L.D. Shelton D.N. Peterson P.W. Phelps R.A. Chidester S. Stafforini D.M. Prescott S.M. Jones D.A. J. Biol. Chem. 2006; 281: 20474-20482Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). the that the COX-2 product, stabilized β-catenin (29Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science. 2005; 310: 1504-1510Crossref PubMed Scopus (775) Google Scholar, 30Shao J. Jung C. Liu C. Sheng H. J. Biol. Chem. 2005; 280: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (191) Google we that retinoic acid may β-catenin by a mechanism that required attenuation of Cox-2 expression and decreased accumulation of PGE2. We this using three as follows: of Cox-2 expression, inhibition of Cox-2 and Cox-2 The results of a role for Cox-2 as a of β-catenin These findings and support a model retinoic acid suppresses β-catenin signaling by inhibiting the expression of (29Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science. 2005; 310: 1504-1510Crossref PubMed Scopus (775) Google recently the mechanism by which PGE2 β-catenin in colorectal showed that PGE2 the of GSK-3β from the β-catenin destruction This phosphorylation and degradation of β-catenin. the complex members were findings showed that the of Cox-2 was for stabilization of β-catenin in vivo. We that treatment of wild-type zebrafish embryos with PGE2 β-catenin protein levels and β-catenin target genes in vivo. reducing PGE2 levels in apc mutant zebrafish by inhibiting Cox-2 in degradation of β-catenin. These findings provide that PGE2 a role in the stabilization of β-catenin in and Cox-2 of β-catenin stabilization following apc also an APC and the of the β-catenin destruction demonstrated that APC including the in apc mutant result in a truncated protein that and that binding of APC and is for degradation of β-catenin M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 19Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). a number of that truncated forms of APC retain β-catenin binding S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, B. Albert I. Porfiri E. Munemitsu S. Polakis P. Cancer Res. 1997; 57: Google Scholar). apc mutant zebrafish a apc mutation that is in human polyposis and colorectal E. H. J. F. E. D. H. Nature. 2003; PubMed Scopus Google Scholar). provide a in experimental for the downstream to wild-type or truncated Apc E. H. J. F. E. D. H. Nature. 2003; PubMed Scopus Google Scholar). In this inhibition of PGE2 accumulation in embryos expressing this truncated form of Apc in the degradation of β-catenin. In of the Apc using an the destruction of β-catenin following inhibition of are with a model in which mutant Apc the ability to form a complex with β-catenin, but the of this complex is that degradation is impaired by levels of PGE2. is that this truncated form of Apc β-catenin binding to in its this was J. A. J. Mol. 16: PubMed Scopus Google that truncated APC regulates β-catenin in a cell This to result from in β-catenin In we shown that retinoic acid antagonizes β-catenin by regulating levels of Cox-2 and PGE2. These in with findings in colorectal cells and provide to support a role for PGE2 in the of the β-catenin destruction complex, accumulation of β-catenin, to the and activation of target that a truncated form of Apc the ability to target β-catenin for destruction by the but only in the absence of Cox-2 activity. These findings retinoic acid and Cox-2 as in the signaling and important the of APC mutation in colorectal cancer. destruction of β-catenin following inhibition of PGE2 biosynthesis may in the molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer. We of for the zebrafish.