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Wnt signaling from membrane to nucleus: β-catenin caught in a loop
The International Journal of Biochemistry & Cell Biology 44 (2012) 847–850
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Signalling networks in focus
Wnt signaling from membrane to nucleus: -catenin caught in a loop Cara Jamieson, Manisha Sharma, Beric R. Henderson ∗ Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia
a r t i c l e
i n f o
Article history: Received 19 January 2012 Received in revised form 28 February 2012 Accepted 1 March 2012 Available online 14 March 2012 Keywords: Beta-catenin Wnt signaling LEF-1 Nuclear retention Cancer
a b s t r a c t -catenin is the central nuclear effector of the Wnt signaling pathway, and regulates other cellular processes including cell adhesion. Wnt stimulation of cells culminates in the nuclear translocation of -catenin and transcriptional activation of target genes that function during both normal and malignant development. Constitutive activation of the Wnt pathway leads to inappropriate nuclear accumulation of -catenin and gene transactivation, an important step in cancer progression. This has generated interest in the mechanisms regulating -catenin nuclear accumulation and retention. Here we discuss recent advances in understanding feedback loops that trap -catenin in the nucleus and provide potential insights into Wnt signaling and the development of anti-cancer drugs. © 2012 Elsevier Ltd. All rights reserved.
1. Signaling network facts • -Catenin is a central mediator of the Wnt signaling pathway and controls the transcription of genes during both normal and malignant development. • Nuclear localization of -catenin is crucial to its role in Wnt signaling and cancer. • -catenin is retained in the nucleus through a LEF-1 dependent feedback loop which increases its concentration in the nucleus and overall transcriptional activity. • Inhibition of Wnt/-catenin signaling is a potential strategy for cancer therapy. • Further information on Wnt signaling can be found at http://www.stanford.edu/group/nusselab/cgi-bin/wnt/.
cytoplasm and nucleus (MacDonald et al., 2009) and is responsible for transducing canonical Wnt signals from plasma membrane to the nucleus. Nuclear -catenin is a hallmark of Wnt signaling and regulates diverse cellular processes in multiple cell types including stem cells (Tanaka et al., 2011) and neurons (Misztal et al., 2011). Deregulation of the Wnt pathway generates excessive nuclear catenin and inappropriate activation of Wnt target genes, leading to multiple diseases including cancer (MacDonald et al., 2009). The Wnt pathway is regulated by feedback loops both upstream at the membrane (Tanaka et al., 2011) and downstream in the nucleus. In this review we briefly outline the function of -catenin as a central effector of the Wnt signaling pathway and focus on how its nuclear accrual is regulated through nuclear retention mechanisms including a LEF-1 positive feedback loop, and the impact on malignant transformation.
2. Introduction -catenin is a member of the Armadillo (Arm) family and contains a central 12 Arm repeat domain (Fig. 1a) (Sharma et al., 2012), which acts as a platform for multiple protein interactions (Fig. 1a) giving rise to diverse cellular functions ranging from cell:cell adhesion at the plasma membrane to transcriptional activation in the nucleus (MacDonald et al., 2009). -catenin was first identified at adherens junctions where it links cadherins with the cytoskeleton to regulate the response to cell adhesion. In addition a small yet dynamic pool of -catenin shuttles rapidly between the
∗ Corresponding author. Tel.: +61 2 98459057; fax: +61 2 98459102. E-mail address: [email protected] (B.R. Henderson). 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2012.03.001
3. Functions 3.1. Wnt/ˇ-catenin signaling pathway The canonical Wnt/-catenin signaling pathway is conserved in evolution and controls processes including cellular proliferation, differentiation, motility, tissue maintenance (MacDonald et al., 2009), and cell fate specification and maintenance of pluripotency (Tanaka et al., 2011). Wnts are glycoprotein ligands of the Frizzled family of transmembrane receptors that modulate signaling to the nucleus through -catenin (MacDonald et al., 2009). In the absence of Wnt signaling, -catenin protein is maintained at low levels through degradation, predominantly by a multiprotein destruction complex comprising factors including tumor
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Fig. 1. (a) Schematic diagram of -catenin protein with binding sites. E-cadherin, LEF/TCF, APC and axin all compete for binding within the armadillo repeat region of -catenin. (b) Overview of the Wnt/-catenin signaling pathway. (i) Wnt off: In the absence of Wnt signaling, -catenin is degraded by a multi-protein destruction complex comprising APC, axin, CK1 and GSK-3. N-terminal phosphorylation of -catenin by this complex triggers -TrCP mediated ubiquitination and proteasomal degradation. (ii) Wnt on: The binding of Wnt ligand to Frizzled receptors at the plasma membrane lead to disassembly of the destruction complex, and stabilization of -catenin which accumulates and translocates to the nucleus where it interacts with members of the TCF/LEF-1 family. In the nucleus, -catenin recruits nuclear co-activators (e.g. BCL9 and pygopus) and converts TCF proteins into potent transcriptional activators to drive the transcription of target genes. (iii) Cancer: Mutations (*) in destruction complex members (APC and axin) or -catenin itself results in a constitutively active pathway. -catenin is no longer degraded by the destruction complex and accumulates in the nucleus to high levels, driving gene transcription. Fz, frizzled receptor; APC, adenomatous polyposis coli; CK1, casein kinase 1; GSK-3, glycogen synthase kinase 3-, -TrCP; -transducin repeat-containing protein; P, phosphorylation; Ub, ubiqutin; TCF, T-cell factor; LEF-1, lymphoid enhancer factor 1; BCL9, B-cell lymphoma 9.
suppressors adenomatous polyposis coli (APC) and axin, and the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK-3) (Fig. 1b). APC recruits -catenin to the destruction complex where it is phosphorylated at N-terminal serine and threonine residues by CK1 and GSK-3, marking it for ubiquitination and subsequent proteasomal degradation. In the presence of Wnt signaling, GSK-3 becomes inactivated (MacDonald et al., 2009) and -catenin is stabilized in a hypo-phosphorylated form that translocates to the nucleus to bind members of the T cell factor family of high motility group (HMG) proteins including LEF-1 (lymphoid enhancer factor 1), TCF-1 (T-cell factor), TCF-3 and TCF-4 (Fig. 1b). In conjunction with nuclear coactivators (B-cell lymphoma 9, pygopus, cyclin dependent kinase 8 (Najdi et al., 2011)), -catenin and TCFs form transcriptional complexes that activate specific Wnt target genes (MacDonald et al., 2009; Tanaka et al., 2011). The Wnt/-catenin pathway is activated by loss of function mutations in APC, axin and GSK-3 or gain of function mutations in -catenin (MacDonald et al., 2009), stimulating transcription of cancer-associated genes including cyclin D1, c-myc and urokinase plasminogen activator (Hiendlmeyer et al., 2004, Brabletz et al.,
2001) in colon cancer and up-regulation of glutamine metabolism genes such as glutamine synthetase in hepatocellular carcinoma (Cadoret et al., 2002; Loeppen et al., 2002). A non-canonical Wnt activation pathway also exists (Najdi et al., 2011). The mechanisms that regulate nuclear -catenin levels after Wnt signaling are therefore important for its pro-active role in cancer. 3.2. Nuclear retention of ˇ-catenin Immunohistochemical studies revealed a positive correlation between nuclear -catenin and advancing stages of human colorectal carcinogenesis (Wong et al., 2004). Moreover, nuclear -catenin has been detected at the invasive front of mesenchymelike tumors (Brabletz et al., 2001) and colorectal adenomas (Hao et al., 2001). It was earlier hypothesized that Wnt-induced nuclear -catenin accumulation was likely due to decreased nuclear export (Wiechens and Fagotto, 2001) and retention of -catenin (RosinArbesfeld et al., 2000). All four members of the TCF family (LEF-1, TCF-1, TCF-3 and TCF-4) contribute to the Wnt-induced nuclear localization of
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Fig. 2. Model of -catenin-LEF-1 positive feedback loop. In normal (unstimulated) cells -catenin shuttles rapidly in and out of the nucleus and is evenly distributed throughout the cell. In cancer cells, nuclear -catenin dominates due to the following events. (i) -catenin evades degradation and translocates to the nucleus. (ii) Nuclear -catenin binds to LEF-1 and TCF transcription complexes which can stimulate transcription of the LEF-1 promoter. (iii) Newly synthesized LEF-1 creates additional retention sites for -catenin within the nucleus creating a positive and reinforcing feedback loop. Red crosses represent potential therapeutic targets.
-catenin. Recent studies have shown that nuclear retention by LEF-1 dominates (Krieghoff et al., 2006; Jamieson et al., 2011) over enhancement of import and is often accompanied by a reduction in nuclear export rate (Jamieson et al., 2011). The role of LEF1 as a regulated nuclear anchor of -catenin is consistent with its high expression in colon cancer (Atcha et al., 2003). It was also shown that co-expression of TCF-4 (Krieghoff et al., 2006), sumoylation of TCF-4 (Yamamoto et al., 2003) or overexpression of TCF-3 (Wiechens and Fagotto, 2001) retained -catenin in the nuclear compartment. Of note, there are multiple spliced variants of TCF-1 each with different functions, and a switch from dominant negative to full-length isoforms is observed in carcinogenesis (Najdi et al., 2011). Importantly, -catenin complexes with LEF-1, TCF-4 and TCF-1 (Hovanes et al., 2001) to self-regulate transcription of the retention factors (in addition to cancer causing genes), thereby increasing the number of chromatin-associated retention sites available for -catenin. This feedback loop is discussed below. 4. Key molecules 4.1. The role of LEF-1 in establishing a positive feedback loop to retain ˇ-catenin in the nucleus The overexpression of LEF-1 is known to increase nuclear catenin levels (Behrens et al., 1996; Simcha et al., 1998; Henderson et al., 2002). LEF-1 can compete directly with APC and E-cadherin for -catenin binding (Fig. 1a) within the Arm repeat region (Orsulic et al., 1999), and dominates over APC to trap -catenin in the nucleus (Henderson et al., 2002; Jamieson et al., 2011). Wnt signaling not only stabilizes -catenin, but also stimulates the transcription and expression of LEF-1 (Kengaku et al., 1998; Jamieson et al., 2011; Hovanes et al., 2001; Atcha et al., 2003; Filali et al., 2002). Recently we proposed a two step mechanism for -catenin activation, wherein the Wnt-induced stabilization of -catenin itself triggers a feedback loop involving stimulation of LEF-1 transactivation, followed by progressive increases in -catenin nuclear retention (Fig. 2). In this scenario, -catenin/TCF1/TCF-4 complexes bind and transactivate the LEF-1 promoter (Hovanes et al., 2001; Atcha et al., 2003) generating newly synthesized LEF-1, which can then bind -catenin at chromatin, causing a self-amplifying increase in active -catenin transcriptional complexes (Jamieson et al., 2011). This leads to constitutive activation of LEF-1 and other genes. This feedback mechanism does not exclude the possibility of Wnt-regulated changes in LEF-1 stability. LEF-1 has also been reported, in combination with -catenin, to bind and stimulate its own promoter (Amen et al., 2007), and
perhaps with a higher affinity than TCF-1 observed in other promoters (Hebenstreit et al., 2008). Thus, LEF-1 may play a more dominant role than TCF-1 in its auto-regulation, providing one explanation for the elevated expression of LEF-1 in colon cancer cell lines and colorectal tumors (Atcha et al., 2003). Such auto-regulation is not without precedent, and was observed for SALL4/OCT4 proteins in transcriptional feedback regulation of embryonic stem cells (Yang et al., 2010). 5. Therapeutic implications There exist cellular protein inhibitors of Wnt/-catenin signaling including Inhibitor of -catenin and TCF (ICAT), Chibby (MacDonald et al., 2009) and Tax-interacting protein 1 (TIP1) (Kanamori et al., 2003) which act by preventing -catenin–TCF interactions, however these inhibitors are not sufficient to control the high levels of stabilized -catenin observed in cancer cells. Despite findings linking the activation of Wnt to accumulation of nuclear -catenin in cancers, only recently have attempts been made to manipulate the nuclear localization for therapeutic purposes. Several strategies targeted upstream Wnt ligand and related co-factors as a means to block Wnt signaling in cancer (Najdi et al., 2011). In terms of targeting downstream Wnt events, Lepourcelet et al. (2004) applied high throughput screening to identify small molecule inhibitors of -catenin–TCF-4 complexes that suppressed transcriptional activity, but these lacked specificity and inhibited -catenin interaction with APC and E-cadherin (Fig. 1a). Recent strategies have identified more specific inhibitors of -catenin–TCF-4 complexes (Wang et al., 2011; Gonsalves et al., 2011; Dehnhardt et al., 2010), and the plant-derived compound capsaicin was also found to interfere with -catenin–TCF-4 interactions and to lower transcriptional output (Lee et al., 2011). These compounds need optimization but may have therapeutic potential. In light of recent findings it would be interesting to see if these new compounds also inhibited binding to other TCF family members, particularly LEF-1 given the oncogenic nature of the LEF-1 feedback loop described above. Finding ways to not only block the interaction between LEF-1 and -catenin, but to disrupt its engagement to chromatin could be of therapeutic value. The antibiotic Streptonigrin was reported to directly block the complex formation of -catenin/TCF with DNA (Park and Chun, 2010). The ability to disrupt LEF-1–-catenin engagement to chromatin could cause the release and export of the entire -catenin–LEF-1 complex from the nucleus. In principle this is possible, as it was recently shown that -catenin–LEF-1 complexes were not retained in the nucleus when the chromatin-binding domain of LEF-1 was mutated (Sharma et al.,
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2012). The challenge will be to ensure that these agents are non toxic and highly specific for -catenin–LEF-1 complexes in cancer cells, leaving normal cellular processes intact. Furthermore, it will prove important to resolve which of the above mechanisms are invoked by the newest wave of candidate reagents that target nuclear -catenin action, such as the epidermal growth factor receptor antibody cetuximab, whose effective inhibition of MAPK signaling is thought to somehow reduce -catenin nuclear targeting and activity (Horst et al., 2012). Acknowledgments This work was supported by grants from the National Health and Medical research Council of Australia. We apologize to those whose work was not cited due to text restrictions. We thank Joshua Baldock for figures. References Amen M, Liu X, Vadlamudi U, Elizondo G, Diamond E, Engelhardt JF, et al. PITX2 and beta-catenin interactions regulate Lef-1 isoform expression. Mol Cell Biol 2007;27:7560–73. Atcha FA, Munguia JE, Li TW, Hovanes K, Waterman ML. A new beta-catenindependent activation domain in T cell factor. J Biol Chem 2003;278: 16169–75. Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, et al. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 1996;382:638–42. Brabletz T, Jung A, Reu S, Porzner M, Hlubek F, Kunz-Schughart LA, et al. Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc Natl Acad Sci USA 2001;98:10356–61. Cadoret A, Ovejero C, Terris B, Souil E, Levy L, Lamers WH, et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 2002;21:8293–301. Dehnhardt CM, Venkatesan AM, Chen Z, Ayral-Kaloustian S, Dos Santos O, Delos Santos E, et al. Design and synthesis of novel diaminoquinazolines with in vivo efficacy for beta-catenin/T-cell transcriptional factor 4 pathway inhibition. J Med Chem 2010;53:897–910. Filali M, Cheng N, Abbott D, Leontiev V, Engelhardt JF. Wnt-3A/beta-catenin signaling induces transcription from the LEF-1 promoter. J Biol Chem 2002;277:33398–410. Gonsalves FC, Klein K, Carson BB, Katz S, Ekas LA, Evans S, et al. An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway. Proc Natl Acad Sci USA 2011;108:5954–63. Hao XP, Pretlow TG, Rao JS, Pretlow TP. Beta-catenin expression is altered in human colonic aberrant crypt foci. Cancer Res 2001;61:8085–8. Hebenstreit D, Giaisi M, Treiber MK, Zhang XB, Mi HF, Horejs-Hoeck J, et al. LEF1 negatively controls interleukin-4 expression through a proximal promoter regulatory element. J Biol Chem 2008;283:22490–7. Henderson BR, Galea M, Schuechner S, Leung L. Lymphoid enhancer factor-1 blocks adenomatous polyposis coli-mediated nuclear export and degradation of betacatenin. Regulation by histone deacetylase 1. J Biol Chem 2002;277:24258–64. Hiendlmeyer E, Regus S, Wassermann S, Hlubek F, Haynl A, Dimmler A, et al. Betacatenin up-regulates the expression of the urokinase plasminogen activator in human colorectal tumors. Cancer Res 2004;64:1209–14.
Horst D, Chen J, Morikawa T, Ogino S, Kirchner T, Shivdasani RA. Differential WNT activity in colorectal cancer confers limited tumorigenic potential and is regulated by MAPK signaling. Cancer Res 2012., doi:10.1158/0008-5472.CAN-11-3222. Hovanes K, Li TW, Munguia JE, Truong T, Milovanovic T, Lawrence Marsh J, et al. Beta-catenin-sensitive isoforms of lymphoid enhancer factor-1 are selectively expressed in colon cancer. Nat Genet 2001;28:53–7. Jamieson C, Sharma M, Henderson BR. Regulation of beta-catenin nuclear dynamics by GSK-3beta involves a LEF-1 positive feedback loop. Traffic 2011;12:983–99. Kanamori M, Sandy P, Marzinotto S, Benetti R, Kai C, Hayashizaki Y, et al. The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells. J Biol Chem 2003;278:38758–64. Kengaku M, Capdevila J, Rodriguez-Esteban C, De La Pena J, Johnson RL, Izpisua Belmonte JC, et al. Distinct WNT pathways regulating AER formation and dorsoventral polarity in the chick limb bud. Science 1998;280:1274–7. Krieghoff E, Behrens J, Mayr B. Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention. J Cell Sci 2006;119:1453–63. Lee SH, Richardson RL, Dashwood RH, Baek SJ. Capsaicin represses transcriptional activity of beta-catenin in human colorectal cancer cells. J Nutr Biochem 2011. Lepourcelet M, Chen YN, France DS, Wang H, Crews P, Petersen F, et al. Smallmolecule antagonists of the oncogenic TCF/beta-catenin protein complex. Cancer Cell 2004;5:91–102. Loeppen S, Schneider D, Gaunitz F, Gebhardt R, Kurek R, Buchmann A, et al. Overexpression of glutamine synthetase is associated with beta-catenin-mutations in mouse liver tumors during promotion of hepatocarcinogenesis by phenobarbital. Cancer Res 2002;62:5685–8. MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 2009;17:9–26. Misztal K, Wisniewska MB, Ambrozkiewicz M, Nagalski A, Kuznicki J. WNT proteinindependent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons. J Biol Chem 2011;286:31781–8. Najdi R, Holcombe RF, Waterman ML. Wnt signaling and colon carcinogenesis: beyond APC. J Carcinog 2011;10:5. Orsulic S, Huber O, Aberle H, Arnold S, Kemler R. E-cadherin binding prevents betacatenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J Cell Sci 1999;112(Pt 8):1237–45. Park S, Chun S. Streptonigrin inhibits beta-catenin/TCF signaling and shows cytotoxicity in beta-catenin-activated cells. Biochim Biophys Acta 2010;1810:1340–5. Rosin-Arbesfeld R, Townsley F, Bienz M. The APC tumour suppressor has a nuclear export function. Nature 2000;406:1009–12. Sharma M, Jamieson C, Johnson M, Molloy MP, Henderson BR. Specific armadillo repeat sequences facilitate beta-catenin nuclear transport in live cells via direct binding to nucleoporins Nup62, Nup153 and RANBP2/Nup358. J Biol Chem 2012. Simcha I, Shtutman M, Salomon D, Zhurinsky J, Sadot E, Geiger B, et al. Differential nuclear translocation and transactivation potential of beta-catenin and plakoglobin. J Cell Biol 1998;141:1433–48. Tanaka SS, Kojima Y, Yamaguchi YL, Nishinakamura R, Tam PP. Impact of WNT signaling on tissue lineage differentiation in the early mouse embryo. Dev Growth Differ 2011;53:843–56. Wang W, Liu H, Wang S, Hao X, Li L. A diterpenoid derivative 15-oxospiramilactone inhibits Wnt/beta-catenin signaling and colon cancer cell tumorigenesis. Cell Res 2011;21:730–40. Wiechens N, Fagotto F. CRM1- and Ran-independent nuclear export of beta-catenin. Curr Biol 2001;11:18–27. Wong SC, Lo ES, Lee KC, Chan JK, Hsiao WL. Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer. Clin Cancer Res 2004;10:1401–8. Yamamoto H, Ihara M, Matsuura Y, Kikuchi A. Sumoylation is involved in betacatenin-dependent activation of TCF-4. EMBO J 2003;22:2047–59. Yang J, Gao C, Chai L, Ma Y. A novel SALL4/OCT4 transcriptional feedback network for pluripotency of embryonic stem cells. PLoS One 2010;5:e10766.
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Signalling networks in focus
Wnt signaling from membrane to nucleus: -catenin caught in a loop Cara Jamieson, Manisha Sharma, Beric R. Henderson ∗ Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia
a r t i c l e
i n f o
Article history: Received 19 January 2012 Received in revised form 28 February 2012 Accepted 1 March 2012 Available online 14 March 2012 Keywords: Beta-catenin Wnt signaling LEF-1 Nuclear retention Cancer
a b s t r a c t -catenin is the central nuclear effector of the Wnt signaling pathway, and regulates other cellular processes including cell adhesion. Wnt stimulation of cells culminates in the nuclear translocation of -catenin and transcriptional activation of target genes that function during both normal and malignant development. Constitutive activation of the Wnt pathway leads to inappropriate nuclear accumulation of -catenin and gene transactivation, an important step in cancer progression. This has generated interest in the mechanisms regulating -catenin nuclear accumulation and retention. Here we discuss recent advances in understanding feedback loops that trap -catenin in the nucleus and provide potential insights into Wnt signaling and the development of anti-cancer drugs. © 2012 Elsevier Ltd. All rights reserved.
1. Signaling network facts • -Catenin is a central mediator of the Wnt signaling pathway and controls the transcription of genes during both normal and malignant development. • Nuclear localization of -catenin is crucial to its role in Wnt signaling and cancer. • -catenin is retained in the nucleus through a LEF-1 dependent feedback loop which increases its concentration in the nucleus and overall transcriptional activity. • Inhibition of Wnt/-catenin signaling is a potential strategy for cancer therapy. • Further information on Wnt signaling can be found at http://www.stanford.edu/group/nusselab/cgi-bin/wnt/.
cytoplasm and nucleus (MacDonald et al., 2009) and is responsible for transducing canonical Wnt signals from plasma membrane to the nucleus. Nuclear -catenin is a hallmark of Wnt signaling and regulates diverse cellular processes in multiple cell types including stem cells (Tanaka et al., 2011) and neurons (Misztal et al., 2011). Deregulation of the Wnt pathway generates excessive nuclear catenin and inappropriate activation of Wnt target genes, leading to multiple diseases including cancer (MacDonald et al., 2009). The Wnt pathway is regulated by feedback loops both upstream at the membrane (Tanaka et al., 2011) and downstream in the nucleus. In this review we briefly outline the function of -catenin as a central effector of the Wnt signaling pathway and focus on how its nuclear accrual is regulated through nuclear retention mechanisms including a LEF-1 positive feedback loop, and the impact on malignant transformation.
2. Introduction -catenin is a member of the Armadillo (Arm) family and contains a central 12 Arm repeat domain (Fig. 1a) (Sharma et al., 2012), which acts as a platform for multiple protein interactions (Fig. 1a) giving rise to diverse cellular functions ranging from cell:cell adhesion at the plasma membrane to transcriptional activation in the nucleus (MacDonald et al., 2009). -catenin was first identified at adherens junctions where it links cadherins with the cytoskeleton to regulate the response to cell adhesion. In addition a small yet dynamic pool of -catenin shuttles rapidly between the
∗ Corresponding author. Tel.: +61 2 98459057; fax: +61 2 98459102. E-mail address: [email protected] (B.R. Henderson). 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2012.03.001
3. Functions 3.1. Wnt/ˇ-catenin signaling pathway The canonical Wnt/-catenin signaling pathway is conserved in evolution and controls processes including cellular proliferation, differentiation, motility, tissue maintenance (MacDonald et al., 2009), and cell fate specification and maintenance of pluripotency (Tanaka et al., 2011). Wnts are glycoprotein ligands of the Frizzled family of transmembrane receptors that modulate signaling to the nucleus through -catenin (MacDonald et al., 2009). In the absence of Wnt signaling, -catenin protein is maintained at low levels through degradation, predominantly by a multiprotein destruction complex comprising factors including tumor
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Fig. 1. (a) Schematic diagram of -catenin protein with binding sites. E-cadherin, LEF/TCF, APC and axin all compete for binding within the armadillo repeat region of -catenin. (b) Overview of the Wnt/-catenin signaling pathway. (i) Wnt off: In the absence of Wnt signaling, -catenin is degraded by a multi-protein destruction complex comprising APC, axin, CK1 and GSK-3. N-terminal phosphorylation of -catenin by this complex triggers -TrCP mediated ubiquitination and proteasomal degradation. (ii) Wnt on: The binding of Wnt ligand to Frizzled receptors at the plasma membrane lead to disassembly of the destruction complex, and stabilization of -catenin which accumulates and translocates to the nucleus where it interacts with members of the TCF/LEF-1 family. In the nucleus, -catenin recruits nuclear co-activators (e.g. BCL9 and pygopus) and converts TCF proteins into potent transcriptional activators to drive the transcription of target genes. (iii) Cancer: Mutations (*) in destruction complex members (APC and axin) or -catenin itself results in a constitutively active pathway. -catenin is no longer degraded by the destruction complex and accumulates in the nucleus to high levels, driving gene transcription. Fz, frizzled receptor; APC, adenomatous polyposis coli; CK1, casein kinase 1; GSK-3, glycogen synthase kinase 3-, -TrCP; -transducin repeat-containing protein; P, phosphorylation; Ub, ubiqutin; TCF, T-cell factor; LEF-1, lymphoid enhancer factor 1; BCL9, B-cell lymphoma 9.
suppressors adenomatous polyposis coli (APC) and axin, and the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK-3) (Fig. 1b). APC recruits -catenin to the destruction complex where it is phosphorylated at N-terminal serine and threonine residues by CK1 and GSK-3, marking it for ubiquitination and subsequent proteasomal degradation. In the presence of Wnt signaling, GSK-3 becomes inactivated (MacDonald et al., 2009) and -catenin is stabilized in a hypo-phosphorylated form that translocates to the nucleus to bind members of the T cell factor family of high motility group (HMG) proteins including LEF-1 (lymphoid enhancer factor 1), TCF-1 (T-cell factor), TCF-3 and TCF-4 (Fig. 1b). In conjunction with nuclear coactivators (B-cell lymphoma 9, pygopus, cyclin dependent kinase 8 (Najdi et al., 2011)), -catenin and TCFs form transcriptional complexes that activate specific Wnt target genes (MacDonald et al., 2009; Tanaka et al., 2011). The Wnt/-catenin pathway is activated by loss of function mutations in APC, axin and GSK-3 or gain of function mutations in -catenin (MacDonald et al., 2009), stimulating transcription of cancer-associated genes including cyclin D1, c-myc and urokinase plasminogen activator (Hiendlmeyer et al., 2004, Brabletz et al.,
2001) in colon cancer and up-regulation of glutamine metabolism genes such as glutamine synthetase in hepatocellular carcinoma (Cadoret et al., 2002; Loeppen et al., 2002). A non-canonical Wnt activation pathway also exists (Najdi et al., 2011). The mechanisms that regulate nuclear -catenin levels after Wnt signaling are therefore important for its pro-active role in cancer. 3.2. Nuclear retention of ˇ-catenin Immunohistochemical studies revealed a positive correlation between nuclear -catenin and advancing stages of human colorectal carcinogenesis (Wong et al., 2004). Moreover, nuclear -catenin has been detected at the invasive front of mesenchymelike tumors (Brabletz et al., 2001) and colorectal adenomas (Hao et al., 2001). It was earlier hypothesized that Wnt-induced nuclear -catenin accumulation was likely due to decreased nuclear export (Wiechens and Fagotto, 2001) and retention of -catenin (RosinArbesfeld et al., 2000). All four members of the TCF family (LEF-1, TCF-1, TCF-3 and TCF-4) contribute to the Wnt-induced nuclear localization of
C. Jamieson et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 847–850
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Fig. 2. Model of -catenin-LEF-1 positive feedback loop. In normal (unstimulated) cells -catenin shuttles rapidly in and out of the nucleus and is evenly distributed throughout the cell. In cancer cells, nuclear -catenin dominates due to the following events. (i) -catenin evades degradation and translocates to the nucleus. (ii) Nuclear -catenin binds to LEF-1 and TCF transcription complexes which can stimulate transcription of the LEF-1 promoter. (iii) Newly synthesized LEF-1 creates additional retention sites for -catenin within the nucleus creating a positive and reinforcing feedback loop. Red crosses represent potential therapeutic targets.
-catenin. Recent studies have shown that nuclear retention by LEF-1 dominates (Krieghoff et al., 2006; Jamieson et al., 2011) over enhancement of import and is often accompanied by a reduction in nuclear export rate (Jamieson et al., 2011). The role of LEF1 as a regulated nuclear anchor of -catenin is consistent with its high expression in colon cancer (Atcha et al., 2003). It was also shown that co-expression of TCF-4 (Krieghoff et al., 2006), sumoylation of TCF-4 (Yamamoto et al., 2003) or overexpression of TCF-3 (Wiechens and Fagotto, 2001) retained -catenin in the nuclear compartment. Of note, there are multiple spliced variants of TCF-1 each with different functions, and a switch from dominant negative to full-length isoforms is observed in carcinogenesis (Najdi et al., 2011). Importantly, -catenin complexes with LEF-1, TCF-4 and TCF-1 (Hovanes et al., 2001) to self-regulate transcription of the retention factors (in addition to cancer causing genes), thereby increasing the number of chromatin-associated retention sites available for -catenin. This feedback loop is discussed below. 4. Key molecules 4.1. The role of LEF-1 in establishing a positive feedback loop to retain ˇ-catenin in the nucleus The overexpression of LEF-1 is known to increase nuclear catenin levels (Behrens et al., 1996; Simcha et al., 1998; Henderson et al., 2002). LEF-1 can compete directly with APC and E-cadherin for -catenin binding (Fig. 1a) within the Arm repeat region (Orsulic et al., 1999), and dominates over APC to trap -catenin in the nucleus (Henderson et al., 2002; Jamieson et al., 2011). Wnt signaling not only stabilizes -catenin, but also stimulates the transcription and expression of LEF-1 (Kengaku et al., 1998; Jamieson et al., 2011; Hovanes et al., 2001; Atcha et al., 2003; Filali et al., 2002). Recently we proposed a two step mechanism for -catenin activation, wherein the Wnt-induced stabilization of -catenin itself triggers a feedback loop involving stimulation of LEF-1 transactivation, followed by progressive increases in -catenin nuclear retention (Fig. 2). In this scenario, -catenin/TCF1/TCF-4 complexes bind and transactivate the LEF-1 promoter (Hovanes et al., 2001; Atcha et al., 2003) generating newly synthesized LEF-1, which can then bind -catenin at chromatin, causing a self-amplifying increase in active -catenin transcriptional complexes (Jamieson et al., 2011). This leads to constitutive activation of LEF-1 and other genes. This feedback mechanism does not exclude the possibility of Wnt-regulated changes in LEF-1 stability. LEF-1 has also been reported, in combination with -catenin, to bind and stimulate its own promoter (Amen et al., 2007), and
perhaps with a higher affinity than TCF-1 observed in other promoters (Hebenstreit et al., 2008). Thus, LEF-1 may play a more dominant role than TCF-1 in its auto-regulation, providing one explanation for the elevated expression of LEF-1 in colon cancer cell lines and colorectal tumors (Atcha et al., 2003). Such auto-regulation is not without precedent, and was observed for SALL4/OCT4 proteins in transcriptional feedback regulation of embryonic stem cells (Yang et al., 2010). 5. Therapeutic implications There exist cellular protein inhibitors of Wnt/-catenin signaling including Inhibitor of -catenin and TCF (ICAT), Chibby (MacDonald et al., 2009) and Tax-interacting protein 1 (TIP1) (Kanamori et al., 2003) which act by preventing -catenin–TCF interactions, however these inhibitors are not sufficient to control the high levels of stabilized -catenin observed in cancer cells. Despite findings linking the activation of Wnt to accumulation of nuclear -catenin in cancers, only recently have attempts been made to manipulate the nuclear localization for therapeutic purposes. Several strategies targeted upstream Wnt ligand and related co-factors as a means to block Wnt signaling in cancer (Najdi et al., 2011). In terms of targeting downstream Wnt events, Lepourcelet et al. (2004) applied high throughput screening to identify small molecule inhibitors of -catenin–TCF-4 complexes that suppressed transcriptional activity, but these lacked specificity and inhibited -catenin interaction with APC and E-cadherin (Fig. 1a). Recent strategies have identified more specific inhibitors of -catenin–TCF-4 complexes (Wang et al., 2011; Gonsalves et al., 2011; Dehnhardt et al., 2010), and the plant-derived compound capsaicin was also found to interfere with -catenin–TCF-4 interactions and to lower transcriptional output (Lee et al., 2011). These compounds need optimization but may have therapeutic potential. In light of recent findings it would be interesting to see if these new compounds also inhibited binding to other TCF family members, particularly LEF-1 given the oncogenic nature of the LEF-1 feedback loop described above. Finding ways to not only block the interaction between LEF-1 and -catenin, but to disrupt its engagement to chromatin could be of therapeutic value. The antibiotic Streptonigrin was reported to directly block the complex formation of -catenin/TCF with DNA (Park and Chun, 2010). The ability to disrupt LEF-1–-catenin engagement to chromatin could cause the release and export of the entire -catenin–LEF-1 complex from the nucleus. In principle this is possible, as it was recently shown that -catenin–LEF-1 complexes were not retained in the nucleus when the chromatin-binding domain of LEF-1 was mutated (Sharma et al.,
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2012). The challenge will be to ensure that these agents are non toxic and highly specific for -catenin–LEF-1 complexes in cancer cells, leaving normal cellular processes intact. Furthermore, it will prove important to resolve which of the above mechanisms are invoked by the newest wave of candidate reagents that target nuclear -catenin action, such as the epidermal growth factor receptor antibody cetuximab, whose effective inhibition of MAPK signaling is thought to somehow reduce -catenin nuclear targeting and activity (Horst et al., 2012). Acknowledgments This work was supported by grants from the National Health and Medical research Council of Australia. We apologize to those whose work was not cited due to text restrictions. We thank Joshua Baldock for figures. References Amen M, Liu X, Vadlamudi U, Elizondo G, Diamond E, Engelhardt JF, et al. PITX2 and beta-catenin interactions regulate Lef-1 isoform expression. Mol Cell Biol 2007;27:7560–73. Atcha FA, Munguia JE, Li TW, Hovanes K, Waterman ML. A new beta-catenindependent activation domain in T cell factor. J Biol Chem 2003;278: 16169–75. Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, et al. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 1996;382:638–42. Brabletz T, Jung A, Reu S, Porzner M, Hlubek F, Kunz-Schughart LA, et al. Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc Natl Acad Sci USA 2001;98:10356–61. Cadoret A, Ovejero C, Terris B, Souil E, Levy L, Lamers WH, et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 2002;21:8293–301. Dehnhardt CM, Venkatesan AM, Chen Z, Ayral-Kaloustian S, Dos Santos O, Delos Santos E, et al. Design and synthesis of novel diaminoquinazolines with in vivo efficacy for beta-catenin/T-cell transcriptional factor 4 pathway inhibition. J Med Chem 2010;53:897–910. Filali M, Cheng N, Abbott D, Leontiev V, Engelhardt JF. Wnt-3A/beta-catenin signaling induces transcription from the LEF-1 promoter. J Biol Chem 2002;277:33398–410. Gonsalves FC, Klein K, Carson BB, Katz S, Ekas LA, Evans S, et al. An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway. Proc Natl Acad Sci USA 2011;108:5954–63. Hao XP, Pretlow TG, Rao JS, Pretlow TP. Beta-catenin expression is altered in human colonic aberrant crypt foci. Cancer Res 2001;61:8085–8. Hebenstreit D, Giaisi M, Treiber MK, Zhang XB, Mi HF, Horejs-Hoeck J, et al. LEF1 negatively controls interleukin-4 expression through a proximal promoter regulatory element. J Biol Chem 2008;283:22490–7. Henderson BR, Galea M, Schuechner S, Leung L. Lymphoid enhancer factor-1 blocks adenomatous polyposis coli-mediated nuclear export and degradation of betacatenin. Regulation by histone deacetylase 1. J Biol Chem 2002;277:24258–64. Hiendlmeyer E, Regus S, Wassermann S, Hlubek F, Haynl A, Dimmler A, et al. Betacatenin up-regulates the expression of the urokinase plasminogen activator in human colorectal tumors. Cancer Res 2004;64:1209–14.
Horst D, Chen J, Morikawa T, Ogino S, Kirchner T, Shivdasani RA. Differential WNT activity in colorectal cancer confers limited tumorigenic potential and is regulated by MAPK signaling. Cancer Res 2012., doi:10.1158/0008-5472.CAN-11-3222. Hovanes K, Li TW, Munguia JE, Truong T, Milovanovic T, Lawrence Marsh J, et al. Beta-catenin-sensitive isoforms of lymphoid enhancer factor-1 are selectively expressed in colon cancer. Nat Genet 2001;28:53–7. Jamieson C, Sharma M, Henderson BR. Regulation of beta-catenin nuclear dynamics by GSK-3beta involves a LEF-1 positive feedback loop. Traffic 2011;12:983–99. Kanamori M, Sandy P, Marzinotto S, Benetti R, Kai C, Hayashizaki Y, et al. The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells. J Biol Chem 2003;278:38758–64. Kengaku M, Capdevila J, Rodriguez-Esteban C, De La Pena J, Johnson RL, Izpisua Belmonte JC, et al. Distinct WNT pathways regulating AER formation and dorsoventral polarity in the chick limb bud. Science 1998;280:1274–7. Krieghoff E, Behrens J, Mayr B. Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention. J Cell Sci 2006;119:1453–63. Lee SH, Richardson RL, Dashwood RH, Baek SJ. Capsaicin represses transcriptional activity of beta-catenin in human colorectal cancer cells. J Nutr Biochem 2011. Lepourcelet M, Chen YN, France DS, Wang H, Crews P, Petersen F, et al. Smallmolecule antagonists of the oncogenic TCF/beta-catenin protein complex. Cancer Cell 2004;5:91–102. Loeppen S, Schneider D, Gaunitz F, Gebhardt R, Kurek R, Buchmann A, et al. Overexpression of glutamine synthetase is associated with beta-catenin-mutations in mouse liver tumors during promotion of hepatocarcinogenesis by phenobarbital. Cancer Res 2002;62:5685–8. MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 2009;17:9–26. Misztal K, Wisniewska MB, Ambrozkiewicz M, Nagalski A, Kuznicki J. WNT proteinindependent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons. J Biol Chem 2011;286:31781–8. Najdi R, Holcombe RF, Waterman ML. Wnt signaling and colon carcinogenesis: beyond APC. J Carcinog 2011;10:5. Orsulic S, Huber O, Aberle H, Arnold S, Kemler R. E-cadherin binding prevents betacatenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J Cell Sci 1999;112(Pt 8):1237–45. Park S, Chun S. Streptonigrin inhibits beta-catenin/TCF signaling and shows cytotoxicity in beta-catenin-activated cells. Biochim Biophys Acta 2010;1810:1340–5. Rosin-Arbesfeld R, Townsley F, Bienz M. The APC tumour suppressor has a nuclear export function. Nature 2000;406:1009–12. Sharma M, Jamieson C, Johnson M, Molloy MP, Henderson BR. Specific armadillo repeat sequences facilitate beta-catenin nuclear transport in live cells via direct binding to nucleoporins Nup62, Nup153 and RANBP2/Nup358. J Biol Chem 2012. Simcha I, Shtutman M, Salomon D, Zhurinsky J, Sadot E, Geiger B, et al. Differential nuclear translocation and transactivation potential of beta-catenin and plakoglobin. J Cell Biol 1998;141:1433–48. Tanaka SS, Kojima Y, Yamaguchi YL, Nishinakamura R, Tam PP. Impact of WNT signaling on tissue lineage differentiation in the early mouse embryo. Dev Growth Differ 2011;53:843–56. Wang W, Liu H, Wang S, Hao X, Li L. A diterpenoid derivative 15-oxospiramilactone inhibits Wnt/beta-catenin signaling and colon cancer cell tumorigenesis. Cell Res 2011;21:730–40. Wiechens N, Fagotto F. CRM1- and Ran-independent nuclear export of beta-catenin. Curr Biol 2001;11:18–27. Wong SC, Lo ES, Lee KC, Chan JK, Hsiao WL. Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer. Clin Cancer Res 2004;10:1401–8. Yamamoto H, Ihara M, Matsuura Y, Kikuchi A. Sumoylation is involved in betacatenin-dependent activation of TCF-4. EMBO J 2003;22:2047–59. Yang J, Gao C, Chai L, Ma Y. A novel SALL4/OCT4 transcriptional feedback network for pluripotency of embryonic stem cells. PLoS One 2010;5:e10766.