Targeting the β-catenin nuclear transport pathway in cancer.

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Contents lists available at ScienceDirect

Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer

Review

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Targeting the ␤-catenin nuclear transport pathway in cancer

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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

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Keywords: ␤-Catenin Nuclear transport Cancer Nuclear envelope Wnt signaling

The nuclear localization of specific proteins is critical for cellular processes such as cell division, and in recent years perturbation of the nuclear transport cycle of key proteins has been linked to cancer. In particular, specific gene mutations can alter nuclear transport of tumor suppressing and oncogenic proteins, leading to cell transformation or cancer progression. This review will focus on one such factor, ␤-catenin, a key mediator of the canonical wnt signaling pathway. In response to a wnt stimulus or specific gene mutations, ␤-catenin is stabilized and translocates to the nucleus where it binds TCF/LEF-1 transcription factors to transactivate genes that drive tumor formation. Moreover, the nuclear import and accumulation of ␤-catenin correlates with clinical tumor grade. Recent evidence suggests that the primary nuclear transport route of ␤-catenin is independent of the classical Ran/importin import machinery, and that ␤catenin directly contacts the nuclear pore complex to self-regulate its own entry into the nucleus. Here we propose that the ␤-catenin nuclear import pathway may provide an opportunity for identification of specific drug targets and inhibition of ␤-catenin nuclear function, much like the current screening of drugs that block binding of ␤-catenin to LEF-1/TCFs. Here we will discuss the diverse mechanisms regulating nuclear localization of ␤-catenin and their potential as targets for anticancer agent development. © 2014 Elsevier Ltd. All rights reserved.

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1. Introduction The ability of proteins to shuttle into and out of the nucleus is now recognized as critical for many fundamental cellular processes such as cell cycle progression, but also for tissue or cell-type specific processes that require orchestrated nuclear localization of certain proteins [1,2]. The nuclear compartment is separated from the cytoplasm of eukaryotic cells by a double membrane permeated with nuclear pores, and most proteins larger than ∼40 kDa are actively imported into or exported out of the nucleus by transport receptors known as importins or exportins, respectively [3,4]. Defects in the nuclear entry or exit pathway of specific proteins can lead to their mislocalization, and this is often linked to a range of diseases including cancer [5–8]. In particular, known tumor suppressors such as BRCA1 [9], APC [10], p53 and others [6] become mislocalized within the cell as a consequence of gene mutations. Currently there is growing commercial interest in targeting the nuclear transport of specific proteins, or of the import/export process itself, in order to develop anticancer therapeutic agents [7,8,11]. In this review we

∗ Corresponding author at: Westmead Millennium Institute, Darcy Road, PO Box 412, Westmead, NSW 2145, Australia. Tel.: +61 2 9845 9057; fax: +61 2 9845 9102. E-mail address: [email protected] (B.R. Henderson).

focus on an oncogenic protein, ␤-catenin, that is a critical signaling molecule in the developmental Wnt signaling pathway and whose nuclear accumulation in response to gene mutations is directly linked to the onset of several cancers. We will describe the unique transport pathway of ␤-catenin, how its transport and stability are regulated by external stimuli, and the potential for targeting different steps of the ␤-catenin nuclear import path as a means to improve anticancer treatments.

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2. Nuclear transport of proteins

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2.1. Classical nuclear import and export pathways

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The nuclear envelope is permeated by thousands of pores, sites for the movement of molecules between nucleus and cytoplasm determined by large macromolecular assemblies known as nuclear pore complexes (NPC). The NPC comprises 30–50 different nucleoporin (Nup) proteins, which together form a structured gateway comprising 8 small filaments at the cytoplasmic face, a central channel with a mesh-like filter and a basket at the nuclear end [12]. A subset of nucleoporins contain recurring phenylalanine-glycine FxFG or GLFG dipeptides referred to as FG repeats [13–15]. The FG repeats play a crucial role in nuclear transport as they mediate interactions with transport receptors (importins and exportins) to

http://dx.doi.org/10.1016/j.semcancer.2014.04.012 1044-579X/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Jamieson C, et al. Targeting the ␤-catenin nuclear transport pathway in cancer. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.04.012

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Fig. 1. Transport of proteins across the nuclear pore complex (NPC): (i) In the nucleus, cargo proteins containing a nuclear export signal (NES) bind to CRM1 (export receptor) in the presence of RanGTP. CRM1 interacts with nucleoporins and transports protein substrates across the NPC. The complex dissociates in the cytoplasm where RanGTP is hydrolyzed to RanGDP. (ii) In the cytoplasm, proteins carrying a NLS signal bind to an importin-␣/importin-␤ complex. Importin-␤ facilitates passage through the NPC by contacting nucleoporins. The complex dissociates in the presence of RanGTP. Importin-␣ and ␤ are re-cycled back to the cytoplasm for the next round of import. (iii) Stabilized ␤-catenin shuttles between nucleus and cytoplasm independent of transport receptors and RanGTP. ␤-catenin binds to nucleoporins (Nup358 on cytoplasmic end, Nup62 in central channel and Nup98 and Nup153 on nuclear end) transiently and directly during translocation.

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facilitate movement of cargo proteins across the NPC while excluding other proteins [16]. Active protein transport occurs in three steps – docking of cargo–receptor complex at the cytoplasmic or nuclear face of the NPC, translocation through the NPC and disassembly and release of the complex at the other end (see Fig. 1). A classic nuclear import cycle begins in the cytoplasm where cargo proteins bind to importin-␤ through a short nuclear localization signal (NLS) sequence motif. Importin-␤ in turn binds importin-␣ to form a cargo–receptor complex [15,17,18]. The complex docks and interacts with cytoplasmic filament Nups (e.g. Nup358) and translocates through the central channel, and at the nuclear side importin-␣ binds to RanGTP, releasing the complex and freeing importin-␤ for another import cycle [16,19]. Nuclear protein export is facilitated by exportins such as CRM1, which binds as a complex with RanGTP to a consensus nuclear export sequence (NES) on cargo proteins, mediating translocation by transiently contacting Nup FG repeats at both ends and within the central channel of the NPC (Fig. 1). The cargo is released in the cytoplasm in the presence of RanGDP. The RanGTP-GDP gradient maintains directionality of transport across the NPC [20,21]. The actual translocation process is discussed in detail elsewhere [22].

2.2. Evidence for Ran/importin/exportin-independent transport of proteins A small number of proteins are found to enter or exit the nucleus through non-classical means. SMAD3 and SMAD4 are transcription regulators that shuttle between cytoplasm and nucleus

independently of importins and exportins. These proteins have a surface hydrophobic patch in the MH2 domain groove and were found to bind Nup214 and Nup153 directly. Mutations in hydrophobic amino acids reduced their ability to translocate into the nucleus [23]. The kinases MAPK and ERK2 were also shown to gain entry into the nucleus by direct binding to Nup214 and Nup153, respectively [24,25]. Moreover, nuclear entry of the transcription factor STAT1 was reported to correlate with in vitro binding to Nup 153 and 214 in a blot overlay assay [26]. Several proteins including ␤-catenin (discussed below) have been shown to exit the nucleus independent of CRM1 as revealed by lack of responsiveness to the CRM1 inhibitor, leptomycin B (LMB). Others include NF-kB [27], glucocorticoid receptor [28], and viral proteins such as human cytomegalovirus transactivator protein pUL69 [29], indicating that research into non-standard nuclear import/export may disclose a larger network of transported proteins than previously anticipated.

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3. The ␤-catenin transport pathway

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3.1. Wnt signaling triggers movement of ˇ-catenin to the nucleus

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Wnt signaling is a critical determinant of embryonic development, tissue development and cellular differentiation [30,31]. ␤-Catenin mediates a nuclear response to wnt ligand contact at the plasma membrane. In the absence of wnt proteins, ␤catenin is recruited into a destruction complex with the scaffolding proteins Axin and APC and kinases casein kinase 1 (CKI) and


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Fig. 2. ␤-Catenin regulation by phosphorylation and wnt signaling. (A) Schematic diagram of ␤-catenin protein with key serine (S), threonine (T) and tyrosine (Y) phosphorylation sites and protein interaction sites. CK1 phosphorylates ␤-catenin at S45, priming GSK-3␤ phosphorylation at S33, S37 and T41 sites targeting ␤-catenin for degradation. ␤-catenin interacts with multiple binding partners at adherens junctions (· · ·), cytoplasm (—) and nucleus (– –). E-cadherin, APC, Axin and TCF/LEF-1 all compete for binding within the armadillo repeat region of ␤-catenin. (B) Overview of the Wnt/␤-catenin signaling pathway. 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 ubiquitylation and proteasomal degradation. Wnt on: The binding of wnt ligand to Frizzled receptors at the plasma membrane lead to inhibition 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. Fz, frizzled receptor; LRP, low density lipoprotein receptor-related protein; Dvl; disheveled; 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; CBP, cyclic AMP response element binding protein; BCL9, B-cell lymphoma 9.

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glycogen synthase kinase-3␤ (GSK-3␤) [30,32,33]. CK1 primes GSK-3␤-dependent phosphorylation of ␤-catenin at T41, S37 and S33 (Fig. 2A). Phosphorylated ␤-catenin is then ubiquitinated by ␤-TrCP complex and degraded by proteasomes (Fig. 2B). Thus, cytoplasmic ␤-catenin is maintained at a low level. Binding of wnt protein to Frizzled receptor (or gene mutations that perturb the destruction complex) disrupts the destruction complex leading to stabilized ␤-catenin throughout the cell and increased translocation into the nucleus, where it binds to TCF/LEF-1 transcription factors and transactivates expression of genes involved in cell differentiation, proliferation, migration and matrix metalloproteases secretion [32]. The wnt signal also leads to increased retention of ␤catenin in the nucleus via a LEF1-dependent feedback loop [34,35]. While ␤-catenin is typically detected at the membrane in epithelial cells, nuclear staining has been reported in mesenchymal cells at the invasive front of tumors [36,37].

3.2. Evidence for Ran-independent import and export of ˇ-catenin ␤-Catenin comprises a flexible N-terminal domain of 140 amino acids, a structured central 12 armadillo (arm) repeat domain and a C-terminal transactivation domain (Fig. 2A). The ␤-catenin arm repeats mediate interaction with cytoskeletal (IQGAP1, ␣-catenin, E-cadherin), transcriptional (LEF-1, TCFs, ICAT, Chibby) and degradation complex (APC, Axin, GSK-3␤) proteins (http://www.stanford.edu/group/nusselab/cgi-bin/wnt/) (Fig. 2A). The N- and C-tails are unstructured [38] but possess a negative charge that may electrostatically interact with arm repeats and modulate affinity of arm binding partners [39,40]. ␤-Catenin has been known to enter the nucleus since 1996 [34,41] but does not possess a classical NLS or NES sequence. A number of studies showed that stabilized ␤-catenin (as seen in cancer cells) shuttles


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in and out of the nucleus independent of transport receptors. The first evidence came from in vitro studies showing that exogenous ␤-catenin could enter the nucleus of digitonin permeabilized cells without the addition of cytosolic factors [42]. The nuclear import of ␤-catenin competed with that of importin-␤ suggesting overlap in their transport route [42]. Using a similar assay ␤-catenin could enter or exit the nucleus independent of Ran-GTPase [43]. These studies showed that ␤-catenin docks at the nuclear envelope and enters the nucleus in a fashion similar to importin-␤ but without requirement of the importin–␣/␤ complex. In addition, the stabilized endogenous form of ␤-catenin was shown to rapidly exit the nucleus independent of CRM1 in SW480 colon cancer cells [44], as was an exogenous form of ␤-catenin after microinjection into the nucleus of Xenopus laevis oocytes [45]. More recent studies have analyzed GFP-tagged forms of ␤-catenin in living cells by fluorescent recovery after photobleaching (FRAP) assays, demonstrating very rapid kinetics of movement (t1/2 in seconds) between nucleus, cytoplasm and membrane [46–49]. 3.3. ˇ-Catenin nuclear transport is mediated by direct contact with the nuclear pore The ␤-catenin arm repeats 9–12 are structurally similar to the HEAT repeats of importin-␤ [50]. These helicoidal repeats provide importins and exportins the flexibility with which to bind cargo proteins and the FG repeats of Nups simultaneously, allowing for translocation of the complex through the NPC [16,51]. Earlier studies suggested that ␤-catenin could bind to yeast Nup1 [42], although the same group later retracted their findings [52]. The controversy was recently resolved when it was shown that the ␤catenin arm repeats 10–12 mediate rapid nuclear import/export of ␤-catenin in live cells, and that these same sequences bind directly to the FG repeats of NPC components in vitro [46]. ␤-catenin was found to bind directly to the outer NPC cytoplasmic filament protein Nup358, the central channel protein Nup62, and nuclear basket proteins Nup98 and Nup153 [46]. These findings support a model wherein ␤-catenin translocates through the NPC via a series of transient and sequential interactions with multiple Nups (see Fig. 1(iii)). In particular, the interaction with Nup358 appeared to be important as silencing of that nucleoporin slowed ␤-catenin import. Nup358 is a major constituent of the NPC cytoplasmic fibrils and acts as a docking platform to concentrate importin-␤ for nuclear entry [53,54]. By analogy, it is possible that Nup358 docks and concentrates ␤-catenin in vicinity of the NPC to increase its nuclear import/export rate. 3.4. Implications of ˇ-catenin–Nup interaction The ␤-catenin–NPC interaction has potential implications. First, it suggests that ␤-catenin acts as a specialized transport receptor. While the export activity and Nup-binding function of ␤-catenin are mainly contained within arm repeats 10–12, the majority of partners bind to arm repeats 1–8 [46]. Demonstrating proof of principle, GFP-tagged forms of ␤-catenin including just the arm domain were found to expedite nuclear export of a non-retained variant of LEF-1 [46]. Thus, ␤-catenin can transport specific cargo and when overexpressed in cancer cells may lead to mislocalisation of key tumor suppressors or oncogenes [55]. Alternately, in cancer cells the excess of ␤-catenin may compete with importin/exportin at the NPC to hinder normal nuclear transport pathways. In addition, nucleoporins have transport independent functions that might be affected by their interaction with ␤-catenin. For instance, mobile Nups such as Nup98 and Nup153 were found to associate with chromatin and to regulate active transcription by binding to RNA polymerase II [56–58]. These Nups were shown to regulate developmental and cell cycle related genes [56]. It is

possible that the binding of ␤-catenin to these Nups regulates their transcriptional function, or conversely that these Nups influence the transcriptional activity of ␤-catenin.

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In the canonical wnt pathway, ␤-catenin is the key mediator for transduction of signal from membrane to nucleus, where it triggers transcription of wnt target genes (see section 3.1 and Fig. 2B). In the absence wnt, ␤-catenin expression is tightly regulated [32]. The N-terminal phosphorylation of ␤-catenin (Section 3.1) tags it for ubiquitylation and degradation via the proteasome, keeping cellular levels low [30]. In response to wnt signals, or APC/Axin/␤catenin gene mutations, ␤-catenin accumulates and translocates to the nucleus where it binds TCF/LEF-1 and additional co-factors such as BCL9 (B-cell lymphoma 9), CBP (cyclic AMP response element binding protein) and pygopus, to activate wnt target genes [32,33]. The aberrant activation of wnt signaling in many cancers is linked to elevated levels of ␤-catenin and hyper-activation of TCF-dependent genes. This generally results from gene mutations affecting components of the ␤-catenin destruction complex, such as APC, Axin or ␤-catenin itself that prevent degradation [59,60]. The prime example is in colorectal cancer, where nearly 90% of cases harbor mutations in these key components leading to aberrant activation of the pathway [59–63]. The link between nuclear ␤-catenin and advancing stages of human colorectal carcinogenesis is strong [36,64–66] and is associated with a shorter survival of patients [67], making nuclear ␤-catenin an important target for new anti-cancer strategies. ␤-Catenin also mediates cell-to-cell adhesion at the membrane through interaction with ␣-catenin and E-cadherin, and these interactions are regulated by tyrosine phosphorylation (see Fig. 2A), which promotes its release from membrane retention [68]. For instance, phosphorylation at Y142 within the 1st arm repeat of ␤catenin by Fer, Fyn or Met kinases impairs the interaction between ␤-catenin and ␣-catenin, reducing the adhesive function of ␤catenin [69]. In addition, Y142 phosphorylation promotes nuclear sequestration of ␤-catenin through BCL9, promoting transcription of target genes [70]. Furthermore, c-Src-mediated phosphorylation of Y654, which resides within the transport-active sequence (arm repeats 10–12) of ␤-catenin, also shifts the balance of ␤catenin away from the membrane and toward the nucleus, in part by decreasing binding to E-cadherin and retention at the membrane [71,72]. However, a phospho-mimic mutation (Y654E) significantly enhanced ␤-catenin nuclear import [46], suggesting another link between Y654 phosphorylation and nuclear localization. Constitutive tyrosine activation of ␤-catenin and several protein tyrosine kinases including c-Met and pp60 (c-Src) are frequently observed in premalignant colorectal lesions [73], and the tyrosine kinase inhibitor Gleevac/STI-571 is currently undergoing various clinical trials with differing degrees of success (http://clinicaltrials.gov/ct2/home). Another kinase reported to alter the nucleo-cytoplasmic distribution of ␤-catenin is JNK. Rac1 activated JNK phosphorylates ␤-catenin at serine residues 191 and 605 in murine bone marrow stromal cells, and this was proposed to promote ␤-catenin nuclear localization [74]. 4.2. Regulation by binding partners Over the years many groups have observed how, when overexpressed, specific binding partners can influence the nuclearcytoplasmic distribution of ␤-catenin. For instance, overexpression of APC [34], Axin [75], Chibby [76], menin [77], p21-activated kinase


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4 (PAK4) [78], Kank [79] and leucine zipper tumor suppressor 2 (LZTS2)[80] can cause some degree of relocalization of ␤-catenin from nucleus to cytoplasm in a CRM1-dependent manner. Likewise, overexpression of Smad ¾ [81], Forkhead box M1 (FoxM1) [82], insulin receptor substrate 1 (IRS-1) [83], Mucin 1 (MUC-1) [84], BCL9 [85], androgen receptor [86] and LEF-1 [41] were reported to shift ␤-catenin to the nucleus. Some studies suggest ␤-catenin itself can import LEF-1 or TCF4 into the nucleus [46,87,88]. In most cases, the forced expression of ␤-catenin binding partners is sufficient to induce an artificial relocation through nuclear or cytoplasmic retention [48,49], and because ␤-catenin has an intrinsic and highly active transport ability of its own, no studies have yet managed to formally prove a chaperone activity of any ␤-catenin binding partner (including demonstration that they translocate through the NPC together). The role of retention is easier to demonstrate. ␣-catenin and E-cadherin sequester ␤-catenin to adherens junctions in epithelial cells [89]. Members of the TCF family, LEF-1, pygopus and BCL9 have all been implicated as anchors for ␤-catenin in the nucleus, making them attractive therapeutic targets [41,48,90–92]. Microinjection of TCF3 into X. laevis embryos resulted in a re-distribution of ␤catenin from the cytosol/membrane to the nucleus [93] and live cell microscopy revealed that exogenous TCF4, BCL9 and LEF-1 all recruit ␤-catenin to chromatin [48,49]. LEF-1 is the best characterized nuclear anchor of ␤-catenin and is discussed in more detail below.

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LEF-1 acts as a nuclear anchor for ␤-catenin, and its high expression in colon cancer cell lines and carcinomas correlates with increased nuclear ␤-catenin levels [94,95]. LEF-1 can compete directly with APC and E-cadherin for binding to the ␤-catenin Arm repeat region (Fig. 2A) [90], and TCF/LEF-1 has a higher affinity for ␤-catenin than unphosphorylated APC or Axin [40]. LEF-1 has also been reported to chaperone ␤-catenin into the nucleus [34]. In addition, photobleaching experiments in live cells showed that LEF-1 is responsible for reduced nuclear export and an increase in nuclear retention of ␤-catenin in response to wnt signaling [49]. LEF-1 gene transcription is aberrantly activated in 80% of colon tumors [96], and both ␤-catenin and LEF-1 accumulate to high levels in colon cancer cells [49,95]. LEF-1 also has a slightly higher binding affinity for ␤-catenin than does TCF4 [40,97]. The wnt mediated up-regulation of LEF-1 results in enhanced nuclear stabilization of ␤-catenin [90], which in turn drives expression of LEF-1 to engage a positive feedback loop [49,94,98–100]. As ␤-catenin stability and levels rise, it translocates to the nucleus, binds to LEF-1 at chromatin and transactivates wnt target genes including LEF-1 [49,101], with consequences for cellular transformation (Fig. 2B). Given the oncogenic nature of the LEF-1 feedback loop, therapeutic strategies that block both the interaction between ␤-catenin and LEF-1 and their association with chromatin could have therapeutic value as discussed below. 5. Therapeutic targeting of the Wnt/␤-catenin pathway The complexity of the wnt pathway offers multiple levels of therapeutic intervention (Fig. 3) and currently there is growing interest in targeting upstream wnt signaling events including wnt lipidation and secretion [102], ligand receptor interactions with LRP5/6 [103,104] and Frizzled receptor [105] and Disheveled–Frizzled interactions [106–108]. Some compounds have already reached clinical testing (Table 1) for their ability to inhibit wnt signaling in cancer. However, the inhibitors that target upstream membrane events may not be as effective in some

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Fig. 3. The Wnt/␤-catenin signaling pathway and its therapeutic targets. (A) Wnt ligands bind to Fz and LRP receptors on the cell surface triggering an intracellular signaling cascade, leading to the stabilization and nuclear translocation of ␤-catenin. In the nucleus, ␤-catenin binds to TCF/LEF-1 and multiple cofactors (including BLC9 and CBP) to modulate gene transcription. Possible therapeutic targets are indicated in brackets and drugs that target these stages are listed in Table 2. Red arrows indicate inhibition and green arrows indicate stabilization.

cancers as those targeting downstream wnt pathway endpoints in the nucleus. Targeting ␤-catenin selectively is difficult as it does not possess enzymatic activity; however ␤-catenin interacts with a multitude of binding partners at different subcellular sites, making small molecular inhibitors an attractive option. Ultimately, there are three key ␤-catenin regulatory events that promote tumorigenesis – stability in the cytoplasm, translocation to the nucleus and transactivation of wnt target genes. The potential for targeting these distinct events for therapeutic options is discussed below. 5.1. Targeting ˇ-catenin stability Drugs that promote destabilization of ␤-catenin either directly or through the destruction complex may be effective in controlling aberrant wnt signaling (see Table 2). For example, pyrvinium is a small molecular inhibitor identified by reporter based screening and shown to promote CK1 activation and subsequent ␤-catenin phosphorylation and degradation [109]. Currently, Axin stabilizing drugs are receiving attention, with one such agent IWR1-4 found to enhance activity of the ␤-catenin destruction complex by directly binding to and stabilizing Axin [102]. Using a similar approach, two additional small molecule wnt inhibitors, JW67 and JW74, were found to inhibit polyp formation in Apcmin mice by stabilizing Axin and enhancing ␤-catenin turnover [110]. However, the Axin stabilizers attracting the most excitement are tankyrase inhibitors. XAV939, the first tankyrase inhibitor identified by high throughput screens [111], can prevent tankyrases 1 and 2 from associating with Axin and promoting its turnover through poly-ADP ribosylation [111]. Recently, more potent tankyrase inhibitors have been identified that increase Axin2 accumulation in mouse tumor models and decrease Topflash reporter assays in colorectal cancer cell


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Table 1 Wnt inhibitors in clinical trials (clinicaltrials.gov). Compound

Target

Tumor

ClinicalTrials.gov Identifier

Clinical phase

Sponsor

LGK974 OMP-54F28 OMP-18R5 CWP232291 KPT-330 PRI-724

Porcupine Wnt ligands Frizzled receptors ␤-Catenin CRM1 CBP

Colorectal, breast, other Solid tumors Solid tumors AML Solid tumors Advanced solid tumors AML, CML Pancreatic

NCT01351103 NCT01608867 NCT01345201 NCT01398462 NCT01607905 NCT01302405 NCT01606579 NCT01764477

I I I I I I I/II I

Novartis OncoMed OncoMed JW Pharmaceutical NPM Pharma Prism Pharma

AML, acute myeloid leukemia; CML, chronic myeloid leukemia; CRM1, chromosome region maintenance 1; CBP, creb binding protein.

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lines [112]. G007-LK has also been reported to inhibit the growth of tumors in mice [113]. Two recent papers prompt caution in regard to the use of tankyrase inhibitors, with some evidence that ␤catenin activity is unresponsive to tankyrase inhibitors in colorectal cancer cells and in cells after prolonged wnt stimulation due to a protective effect by nuclear LEF-1 and BCL9-2 [95]. Tankyrase inhibition has also been linked to increased Wnt/␤-catenin signaling in the late streak of mouse embryos [114]. There are also safety considerations; these inhibitors can target ␤-catenin in normal tissues, such as the intestine, which may lead to dose limiting toxicity [113]. A few small compounds can promote ␤-catenin destabilization (Fig. 3 and Table 2). For instance, CWP232291 was active against wild-type and mutant ␤-catenin [115]. The antiproliferative effects demonstrated in various cell lines have seen this compound reach phase I clinical trials in acute myeloid leukemia (Table 1). The differentiation-inducing factors DIF-1 and -3 also inhibit cell proliferation by stimulating ␤-catenin degradation through GSK3␤ activation [116,117]. While other compounds can promote ␤-catenin turnover (see Table 2), their ability to selectively target cancer cells in patients remain to be determined. 5.2. ˇ-Catenin nuclear transport Nuclear transport is emerging as a potential pathway for targeting nuclear accumulation of ␤-catenin, although, few inhibitors have yet been identified (discussed below). 5.2.1. Targeting the nuclear pore complex One strategy would be to target the NPC directly, however very few compounds exist and achieving specificity would be challenging. Translocation through the nuclear pores can be blocked by monoclonal antibodies (mAb414 and RL2) which bind directly to the FG and FXFG dipeptide epitopes of the nucleoporins [118,119], or by wheat germ agglutinin (WGA) which binds directly to the NPC through the sugar-modified nucleoporins, essentially

blocking the channel [120]. These agents, however, were not designed to enter intact cells and their action would affect all nuclear transport. Despite these limitations, targeting specific subsets of nucleoporins may be feasible. ␤-catenin was shown to bind directly to the mobile Nups 98, 153 and 35 [46], implicated in transcription and disease [121]. Nup98 can transactivate genes involved in cell cycle and development either tethered to the NPC or in the nucleoplasm [56]. While speculative, it remains possible that disrupting the interaction between ␤-catenin and one or more of these specific Nups might impact not only on transport but also on cell proliferation. A more feasible strategy thus would be to select for small molecules that block the interaction between ␤-catenin and FG repeats (or other more specific sequences) of Nups 98, 153 or 358 to prevent the docking and nuclear translocation of ␤-catenin. 5.2.2. Targeting nuclear import pathway In recent years a number of nuclear import blockers were reported to target different aspects of the import pathway. Two peptide inhibitors (Bimax1 and 2) bind specifically to importin-␤ and prevent the release of protein cargo into the nucleus [122]. Other researchers identified a small peptidomimetic inhibitor (58H5-6) of importin-␤ mediated transport [123]. The structure of this inhibitor resembles the FxFG repeats found in nucleoporins suggesting it competes with the NPC for the FxFG binding pockets on importin-␤. This is particularly interesting as ␤-catenin (arm repeats) and importin ␤ (HEAT repeats) are structurally quite similar and thus 58H5-6 might conceivably affect import of ␤-catenin. This same group also identified Karyostatin 1A, which disrupts the interaction between importin-␤ and the Ran GTPase [124]. Karyostatin 1A did not interfere with importin-␤’s ability to bind to Nup153, however this does not exclude changes in the interaction of importin-␤ with other nucleoporins such as Nup358, Nup98 and Nup62, which also play an import role in ␤-catenin nuclear import. Similarly to Karyostatin, another small molecular inhibitor of importin-␤-RanGTP called Importazole has also been described [125]. Given that ␤-catenin has been proposed as a new type of

Table 2 Small molecular inhibitors of the Wnt/␤-catenin signaling.

(i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix) (x) (xi) (xii)

Target

Compound/s

References

Wnt secretion Fz receptors LRP co-receptors Dvl CK1 Tankyrase/Axin ␤-Catenin ␤-Catenin–nucleoporins ␤-Catenin–BCL9 ␤-Catenin–CBP ␤-Catenin–TCF TCF–Chromatin

IWP1 and 2 OMP-18R5 Antibodies FJ9, 3289–8625, NSC668036 Pyrvinium IWR, XAV939, JW67, JW74, Compound 22 and 49, G007-LK CWP232291, CCT031374, DIF1 and 3, Hexachlorophene, Isoreserpine

[102] [105] [103,104] [106–108] [109] [102,110–113] [116,117,150–153]

Triazole stapling peptides, SAH-BCL9 peptides ICG-001 iCRT-3,5,14, CPG049090, NC043, PKF115-584, PKF118-310, Celecoxib, Capsaicin Streptonigrin

[146,147] [144,145] [133,136–139,141] [140]

Fz, frizzled; LRP, low density lipoprotein receptor-related protein; Dvl; disheveled; CK1, casein kinase 1; BCL9, B-cell lymphoma 9; CBP, cyclic AMP response element binding protein; TCF, T-cell factor.


381 382 383 384 385 386 387 388 389 390 391 392 393 394

395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415



416 417 418

419

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

transport receptor [87,88] then similar strategies as those used above may help identify small molecular inhibitors of ␤-catenin nuclear import.

5.3. Transactivation Despite a range of proteins known to prevent ␤-catenin-LEF1/TCF interactions, such as ICAT [126], Chibby [76], Duplin [127], Fragile histidine triad [128], Hydrogen peroxide-induced clone 5 [129] and Tax-interacting protein 1 [130], these regulators are not able to prevent the nuclear accumulation of ␤-catenin observed in cancer. The selection of small artificial inhibitors of the nuclear interaction between ␤-catenin and LEF-1/TCFs has been possible but often suffers from lack of specificity, due to striking overlap in the binding site for these proteins with other ␤-catenin partners such as E-cadherin, APC, axin, and ICAT which all bind within the central arm repeat motif (Fig. 2a) [72,126,131,132]. Thus, any compound intended to disrupt the LEF-1/TCF-␤-catenin interaction will likely interrupt the overlapping binding interactions of ␤-catenin [133]. Indeed perturbation of the ␤-catenin–cadherin binding in normal intestinal epithelium in mice leads to intestinal inflammation and neoplasia [134]. Despite the challenge, von Kries et al., reported that substituting H460A in ␤-catenin selectively hinders binding to LEF-1 without impairing binding to either APC or Axin, suggesting that an appropriate compound may selectively inhibit binding to LEF-1/TCF complexes only [135]. More recent strategies have identified more specific inhibitors of ␤-catenin–TCF-4 complexes including iCRT-3,5,14 and NC043 [136–138] (see Fig. 3 and Table 2), and the plant-derived compound capsaicin was also found to interfere with ␤-catenin–TCF-4 interactions and to lower transcriptional output [139]. Given the important role LEF-1 in regulation of ␤-catenin (Section 4.3) it would be interesting if the above compounds also inhibit ␤-catenin–LEF-1 complexes. Other therapeutic strategies include preventing ␤-catenin–LEF-1 complexes from binding to chromatin which in theory may be possible. Streponigrin has been reported to inhibit the ␤-catenin/TCF complex binding to DNA [140] and celecoxib induces the degradation of TCF [141–143] and is used clinically in the treatment of FAP. While none of these ␤-catenin–TCF small molecule inhibitors have been used in the clinic due to dosage and toxicity issues, there is hope of future progress as biotechnology improves and off-target effects are reduced. Further targets include blocking ␤-catenin interactions with essential transcriptional co-factors such as CBP and BCL9, which bind at sites outside of the central arm domain and thus are not subject to the same specificity issue described above. High throughput screening identified ICG-001 as a potential inhibitor of ␤-catenin–CBP binding and further experiments showed decreased tumor volume in SW620 xenograft mouse models and a reduction in polyp formation in Apcmin mice [144,145]. Recently, a more potent and specific ␤-catenin–CBP antagonist, PRI-724, was identified and is currently undergoing clinical testing (Phase I) in patients with solid tumors (Table 1). Antagonists for BCL9–␤catenin have also been reported. Preliminary hydrocarbon stapling produced cell permeable peptides of BCL9 with enhanced ␤-catenin binding affinity and reduced transcriptional output by competing out native BCL9–␤-catenin complexes [146,147]. Carnosate, a natural compound with bioactivity, has also been reported to destabilize active-␤-catenin in colorectal cancer cells and mediate a reduced tumor burden in Apcmin mice by preventing the interaction between BCL9 and ␤-catenin [148]. There are also additional nuclear-localized transcriptional co-activators of the Wnt pathway such as CREB, BRG1, Pygopus, Hyrax and components of the Mediator complex that might also be amenable to molecular inhibition.

7

6. Conclusions and future perspectives The nuclear targeting of ␤-catenin is a regulated process, involving association and co-operativity between many proteins including nucleoporins and transcription factors that work synergistically to ensure nuclear persistence of ␤-catenin. Currently there are a plethora of interventional approaches being explored to reduce wnt/␤-catenin signaling output (Fig. 3). These range from inhibition of wnt secretion and receptor-ligand interactions at the cell surface, to Axin stabilizers and ␤-catenin destabilizers in the cytoplasm, to disruption of TCF–␤-catenin complex formation and activation of target genes in the nucleus. Drugs designed to disrupt LEF-1–␤-catenin transcription factor complexes hold great promise for treating cancers stemming for aberrant Wnt signaling, particularly due to the oncogenic nature of the LEF-1–␤-catenin feedback loop. We also believe that targeting the nuclear transport of ␤-catenin holds therapeutic potential and deserves further investigation. The ability to regulate the nuclear transport, including nuclear exclusion of ␤-catenin or its enhanced nuclear export to limit the large amount of nuclear ␤-catenin observed in multiple cancers, may have implications for anti-cancer therapies. Given that wnt signaling is required for normal homeostasis, a major challenge common to strategies that target any developmental signaling pathway, is how to develop therapeutic inhibitors that suppress cancer progression while leaving normal tissues minimally perturbed. In a recent review [149] our attention is drawn to the safety concerns that could potentially arise from long-term inhibition of wnt signaling in patients, including toxic side effects. For example, damage to intestinal mucosa, reduced bone density and other pathologies associated with regenerative tissues, should be anticipated from our understanding of wnt signaling [102,149]. Thus the challenge for researchers is to develop inhibitors that exert their anticancer effect with minimal impact to normal tissue. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was partly funded by grants from Cancer Council New Q2 South Wales, Australia. BRH is supported by a research fellowship Q3 from Cancer Institute NSW, Australia. References [1] Nigg EA. Nucleocytoplasmic transport: signals, mechanisms and regulation. Q4 Nature 1997;386:779–87. [2] Gorlich D. Transport into and out of the cell nucleus. EMBO J 1998;17:2721–7. [3] Gorlich D, Dabrowski M, Bischoff FR, Kutay U, Bork P, Hartmann E, et al. A novel class of RanGTP binding proteins. J Cell Biol 1997;138:65–80. [4] Terry LJ, Wente SR. Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex. J Cell Biol 2007;178:1121–32. [5] Fabbro M, Henderson BR. Regulation of tumor suppressors by nuclearcytoplasmic shuttling. Exp Cell Res 2003;282:59–69. [6] Kau TR, Way JC, Silver PA. Nuclear transport and cancer: from mechanism to intervention. Nat Rev Cancer 2004;4:106–17. [7] Turner JG, Dawson J, Sullivan DM. Nuclear export of proteins and drug resistance in cancer. Biochem Pharmacol 2012;83:1021–32. [8] Nguyen KT, Holloway MP, Altura RA. The CRM1 nuclear export protein in normal development and disease. Int J Biochem Mol Biol 2012;3:137–51. [9] Henderson BR. Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking. Bioessays 2005;27:884–93. [10] Brocardo M, Henderson BR. APC shuttling to the membrane, nucleus and beyond. Trends Cell Biol 2008;18:587–96. [11] Chahine MN, Pierce GN. Therapeutic targeting of nuclear protein import in pathological cell conditions. Pharmacol Rev 2009;61:358–72. [12] Ptak C, Aitchison JD, Wozniak RW. The multifunctional nuclear pore complex: a platform for controlling gene expression. Curr Opin Cell Biol 2014;28C:46–53.


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β-catenin pathway in hematological malignancies.

β-catenin signaling pathway.

β-catenin pathway in hepatocellular carcinoma treatment.

E-cadherin pathway.

Targeting ion transport in cancer.

TCF pathway.

β-catenin signaling pathway in liver cancer stem cells and hepatocellular carcinoma cell lines with FH535.

β-catenin pathway in cervical cancer.

Targeting nucleocytoplasmic transport in cancer therapy.

Targeting the TGFβ pathway for cancer therapy.

Rosamines targeting the cancer oxidative phosphorylation pathway.

Targeting the ubiquitin pathway for cancer treatment.

Cancer therapeutics: Targeting the apoptotic pathway.

β-catenin pathway in endometriosis: a potentially effective approach for treatment and prevention.

Safely targeting cancer stem cells via selective catenin coactivator antagonism.

Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

miR-223 promotes colon cancer by directly targeting p120 catenin.

MET pathway in gastric cancer.

Targeting the indoleamine 2,3-dioxygenase pathway in cancer.

Targeting the relaxin hormonal pathway in prostate cancer.

Targeting the sumoylation pathway in cancer stem cells.

Novel drugs targeting the androgen receptor pathway in prostate cancer.

β-catenin pathway in end-stage idiopathic pulmonary arterial hypertension.

Targeting the WNT Signaling Pathway in Cancer Therapeutics.