Exploiting APC Function As a Novel Cancer Therapy

Send Orders for Reprints to [email protected] 90 Current Drug Targets, 2014, 15, 90-102 Exploiting APC Function as a Novel Cancer Therapy

Alyssa C. Lesko1, Kathleen H. Goss2 and Jenifer R. Prosperi1,3,*

1Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, 2Department of Surgery, University of Chicago, Chicago, IL 60637, 3Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN 46617, USA

Abstract: The Adenomatous Polyposis Coli (APC) tumor suppressor is most commonly mutated in colorectal cancers such as familial adenomatous polyposis (FAP); as well as many other epithelial cancers like breast, pancreatic, and lung cancer. APC mutations usually result in a truncated form of the protein lacking the carboxy-terminal region resulting in loss of function. Mutations in APC have been identified in early stages of cancer development making it a gatekeeper of tumor progression and therefore an ideal therapeutic target. APC is best known for its role as a negative regulator of the Wnt/ -catenin pathway. However, APC also mediates several other normal cell functions independently of Wnt/-catenin signaling such as apical-basal polarity, microtubule networks, cell cycle, DNA replication and repair, apoptosis, and cell migration. Given the vast cellular processes involving APC, the loss of these “normal” functions due to mutation can contribute to chemotherapeutic resistance. Several therapeutic treatments have been explored to restore APC function including the reintroduction of APC into mutant cells, inhibiting pathways activated by the loss of APC, and targeting APC- mutant cells for apoptosis. This review will discuss the normal functions of APC as they relate to potential treatments for patients, the role of APC loss in several types of epithelial cancers, and an overview of therapeutic options targeting both the Wnt-dependent and -independent functions of APC. Keywords: APC, cancer, targeted therapy, tumor suppressor

INTRODUCTION of APC as they relate to potential treatments for patients with APC-mutant tumors, will discuss the importance of APC in a Mutations in the Adenomatous Polyposis Coli (APC) tu- variety of epithelial-derived tumors, and finally will investi- mor suppressor gene are most commonly found in colorectal gate options for restoring APC function and therefore treat- cancers such as the inherited syndrome familial adenomatous ing APC-mutant tumors. polyposis (FAP), but have also been identified in many epithelial cancers including breast cancer ([1-3] and re- NORMAL FUNCTIONS OF THE APC TUMOR SUP- viewed in [4]). Most APC mutations are nonsense mutations, PRESSOR frequently created by frame-shifts, resulting in premature stop codons and a truncated gene product lacking the car- APC (by convention, the human and mouse genes are boxy-terminus of the protein. Patients with FAP inherit one APC and Apc, respectively, whereas the protein from all spe- germline mutation and develop tumors when another somatic cies is APC) is a large multifunctional protein made up of mutation is received resulting in loss of the wild-type APC 2843 amino acids and multiple binding domains. While the allele. In this way, APC mutation follows the classical ‘two- best-known role of APC is to act as a negative regulator of hit’ model. The disease presentation in patients with APC the Wnt/-catenin signaling pathway, there are many other mutation often depends on the location of the mutation. For normal functions of APC. Based on the ability of APC to example, mutations at the 5’ and 3’ ends of the coding se- bind a variety of protein partners, other activities of APC quence are associated with a weakened FAP phenotype, include mediating cell migration, regulation of apical-basal while extracolonic phenotypes are associated with mutations polarity, microtubule networks, cell cycle, DNA replication in other regions of the sequence ([5] and reviewed in [6]). and repair, and apoptosis (summarized in Fig. 1). These Importantly, APC mutations similar to those found in FAP functions of APC, including regulation of Wnt/-catenin have been identified in 50-80% of sporadic colon adenomas signaling and other non-Wnt related functions have been and adenocarcinomas (reviewed in [4, 7]). Since APC has recently reviewed elsewhere [9, 10]. Prior to discussion of been identified in the earliest stages of tumor progression targeting APC loss in cancer therapeutics, we will briefly [8], it has emerged as the ‘gatekeeper’ of colorectal cancer summarize some key functions of APC that may be targeted development. This review will describe the normal functions in cancer.

Association of APC with Polarity and Junctional Com- *Address correspondence to this author at the Department of Biochemistry plexes and Molecular Biology, Indiana University School of Medicine – South Bend, 1234 Notre Dame Ave, South Bend, IN 46617, USA; Role in Apical-Basal Polarity of Epithelial Cells: Some Tel.: 574-631-4002; Fax: 574-631-8932; Email: [email protected] non-gastrointestinal model systems have provided evidence

Fig. (1). APC protein structure. Specific APC functional domains are shown, and known binding partners are listed below the domain with which they interact. for the involvement of APC in regulating aspects of apical- uted to this function; however, the loss of epithelial polarity basal polarity and epithelial morphogenesis. For example, a and the disruption of tissue organization are normally associ- role for APC in overall epithelial organization is supported ated with more aggressive cancers ([19, 20] for review). by the homozygous germline deletion of Apc preventing de- Moreover, disrupted localization of adherens junction pro- velopment past embryonic day 6 [11]. Apc mutations in the teins is frequent in cancers [21], and components of the mature cochlea result in a decreased number of parallel mi- Scrib/Dlg/Lgl complex, protein partners of APC, have re- crotubule arrays, which are essential for epithelial polariza- cently been implicated in several cancers such as cervical tion [12]. Studies from our laboratory have also demon- and colorectal cancer [20, 22, 23]. Collectively, these data strated a role for APC in mammary epithelial cell polariza- support a role for APC in epithelial polarization that may be tion and morphogenesis as ApcMin/+ female mice, containing important for its function during tumor development. a mutant Apc allele, exhibited altered mammary epithelial Junctional Complexes: A potential role of APC in medi- polarity and overall integrity [13]. In addition, knockdown of ating cell-cell interactions and cell migration emerged APC in the Madin-Darby Canine Kidney (MDCK) model, a through the interaction of APC with -catenin at the adher- standard model for epithelial polarity, resulted in altered ens junction [24], and plakoglobin (-catenin), at the desmo- epithelial morphogenesis and polarity defects in 3D culture some [25, 26]. However, the APC/-catenin and - (J.R.P and K.H.G., submitted). In Drosophila neuroepithelial catenin/E-cadherin complexes were observed to be mutually cells, APC is recruited to the adherens junction, and mediates exclusive [24, 26, 27], and studies have largely focused on symmetric cell division along the planar axis through its in- APC’s regulation of the Wnt pathway through -catenin deg- teractions with microtubules by positioning the mitotic spin- radation. Although the role of APC in cellular adhesion, mo- dle [14]. Other roles for APC include the polarized distribu- tility, polarization, and morphogenesis has been overshad- tion of MUC1, a transmembrane glycoprotein that is fre- owed, there is evidence that unregulated APC levels are as- quently mislocalized in cancers, to the apical membrane of sociated with altered cell migration and adhesion. Manipula- differentiated mammary epithelial cells [13], and the binding tion of APC levels in mouse intestinal epithelial cells re- of APC to Striatin, a calmodulin-binding protein predomi- sulted in altered enterocyte migration along intestinal villi in nately expressed in the central nervous system, in cell-cell vivo [28-30]. Reintroduction of APC in SW480 (APC- junctions [15]. mutant) colorectal cancer cells caused changes in the local- The role of APC in maintaining apical-basal polarity is ization of adherens junction proteins, tighter cell-cell con- also conveyed in it’s importance in epithelial cell differentia- tacts, and an epithelial phenotype [31]. Localization of APC tion, as the establishment of polarity is required for differen- at cell-cell contacts has been shown to be dependent on - tiation. APC’s role in zebrafish intestinal differentiation in- catenin and VE-cadherin, as depletion of either -catenin or volves the transcriptional co-repressor carboxy-terminal VE-cadherin resulted in the disruption of cell-cell adhesion binding protein 1 (CtBP1), which interacts with APC to and the loss of APC from the lateral membrane [32]. Studies drive differentiation independent of -catenin through con- using wound healing assays also showed that the inhibition trolling the expression of enzymes like intestinal retinal de- of glycogen synthase kinase 3 (GSK3) and casein kinase I hydrogenases [16, 17]. A recent study showed that APC is (CK1) decreased wound closure, suggesting that cell mi- also required for apical extrusion of human bronchial epithe- gration is promoted by a shift of APC to cell protrusions lial cells [18]. The investigators further determined that api- resulting from the phosphorylation by GSK3 and CKI cal extrusion is not dependent on Wnt/-catenin signaling, [32]. but instead requires the carboxy-terminus of APC and micro- While APC likely mediates cell-cell interactions by regu- tubule binding. In addition, the introduction of the EB1 bind- lating levels of -catenin or plakoglobin directly, and - ing domain of APC to DLD-1 colorectal cells was sufficient catenin/TCF target genes specifically implicated in adhesion, to shift cells from basal extrusion to apical extrusion, sug- such as E-cadherin [33], other APC binding partners such as gesting that APC requires microtubule binding to mediate polarity complex proteins or the cytoskeleton may also play cell differentiation and migration through apical-basal polar- an important role. For example, Odenwald et al. showed that ity [18]. the localization of APC/-catenin at protrusion ends controls Despite the increased understanding about the role of cell migration and the maintenance of mesenchymal mor- APC in regulating apical-basal polarity, it is still unclear how phology [34]. APC knockdown prohibited -catenin localiza- much of the tumor suppressive activity of APC can be attrib- tion at the protrusions, but Wnt pathway activation was not 92 Current Drug Targets, 2014, Vol. 15, No. 1 Lesko et al. observed. The formation of these complexes was dependent Several other binding partners have been found to link on the microtubule cytoskeleton suggesting that microtu- APC with microtubules. Interaction with end-binding protein bules are required for APC’s role in cell-cell interactions, 1 (EB1), a plus-end binding protein that regulates microtu- migration, and morphology [34]. bule polymerization through a carboxy-terminal binding domain of APC, results in the localization of APC to microtu- APC Interaction with the Cytoskeleton bule distal ends [47, 48]. In addition to EB1, Kap3 and Kif3, members of the kinesin superfamily, interact with APC in Actin: APC localizes F-actin to the plasma membrane the armadillo repeat region to direct its transport to microtu- through its carboxy-terminal region, which also directly in- bule clusters at cell protrusions and the leading edge of mi- teracts with microtubules [35]. These data and the observa- grating cells [49]. mDia, a formin protein that stabilizes mi- tion that APC binds to EB1 (a microtubule-binding protein crotubules, binds the carboxy-terminus of APC in a complex that will be discussed in detail below) suggest that APC may with EB1 to promote fibroblast motility through Rho- be involved in regulating actin and microtubule dynamics induced stabilization [50]. KIF17, an anterograde kinesin [36]. Recent work demonstrates that APC promotes nuclea- recently shown to participate in localizing APC to the plus tion by recruiting actin monomers, and its synergistic activity ends of microtubules, is required for proper epithelial polari- with the formin mDia is involved in the assembly of actin zation and morphogenesis [51]. The nuclear pore complex filaments [37]. Although some studies suggest that APC di- protein importin-, interacts with the central region of APC rectly interacts with F-actin through the basic domain, it has where it can compete with -catenin, and also in two regions been shown that APC localization to the plasma membrane is of the carboxy-terminus, to regulate APC-mediated microtu- primarily mediated by the ARM domain in the amino- bule assembly and spindle integrity [52]. APC also interacts terminus [37]. These discrepancies warrant further investiga- with the nuclear pore complex proteins, Nup153 and tion to understand the interactions between APC and actin. Nup358, to regulate cell migration and microtubule localiza- Actin Effectors: Through its armadillo repeats APC in- tion [53, 54]. Finally Dlg promotes APC accumulation at teracts directly with the APC-stimulated guanine nucleotide plus ends of microtubules and the interaction of microtubules exchange factor (Asef)-1 and -2 to promote epithelial cell with the plasma membrane, suggesting it has an APC- motility and maintain cell morphology [38, 39]. However, in mediated role in microtubule dynamics [55]. MDCK epithelial cells and colorectal tumor cells, cell migra- Role in Front-Rear Polarity and Directional Cell Mi- tion was only induced by interaction between Asef-1 and the gration: APC’s association with actin and the microtubule truncated form of APC [38]. Some studies suggest that APC network is closely linked with its role in directional cell mi- may be a scaffold for a complex containing APC/Asef-1/- gration. APC expression is localized to puncta at the leading catenin despite a lack of direct interaction between -catenin edge or ends of cell protrusions in central nervous system and Asef-1 [39]. Asef-1 and-2 both function as guanine nu- cells, as well as subconfluent epithelial cells and some tumor cleotide exchange factors (GEF) for Rac [39], and binding cells in culture [34, 56, 57]. This localization depends on the with APC activates the GEF activity of both Asef-1 and -2 to microtubule cytoskeleton and is regulated by APC phos- promote tumor cell migration [40]. Unlike Asef-1, which phorylation [56, 57]. APC loss results in decreased microtu- only contains one binding domain for APC, Asef-2 contains bule stability, fewer cell protrusions and reduced cell migra- two binding sites for APC, the APC binding region and the tion [46, 58]; conversely, APC over-expression induces the Src-homology (SH) 3 domain. Interactions between APC formation of cell protrusions [58]. and the SH3 domain of Asef-2 have been shown to activate the GEF activity of Asef-2 [41]. It has been shown that the regulation of APC by common APC also modulates the actin cytoskeleton through direct cytoskeleton modulators is required for the polarization of migrating astrocytes [59]. The Rac effector Cdc42, a small binding of the actin cross-linking protein IQ motif containing Rho GTPase required to polarize the actin and microtubule GTPase activating protein (IQGAP1), an effector of Rac and networks during migration, phosphorylates and inactivates Cdc42, through its armadillo repeat domain [42]. IQGAP1 GSK3 promoting the association of APC with microtubule recruits APC to membrane ruffles and is involved in main- plus ends and the assembly of Dlg-containing puncta at the taining the Rho GTPases in an active state [43]. This complex promotes cell polarization and migration, and binds the plasma membrane [55, 59]. Centrosome orientation and polarized motility was severely disrupted in APC-mutant cells microtubule stabilizing protein, CLIP-170 [42], indicating lacking the carboxy-terminus, which contains the PDZ- that APC/IQGAP1 could link actin and microtubule net- binding domain and the binding domains for microtubules, works. EB1, Dlg, and Scrib [55, 59]. These data indicate an impor- Microtubules and Microtubule-Binding Partners: In tant role for APC in mediating microtubule polarization and addition to binding to the actin cytoskeleton, APC also inter- directed cell migration through its interactions with polarity acts, through the carboxy terminus, with microtubules to proteins like Dlg and Scrib. promote assembly [44] by increasing both the stability and APC also interacts with activated focal adhesion kinase the lifespan of the microtubules [45, 46]. Phosphorylation of (FAK) at the leading edge in migrating cells to regulate focal APC by GSK3 and Protein Kinase A (PKA) decreases its adhesion turnover, suggesting that APC may also impact cell interactions with microtubules and also strengthens binding motility by directly controlling cell-matrix interactions [60]. with -catenin [45]. These observations suggest that these The association of APC with FAK is biologically important interactions are differentially regulated and provide evidence as FAK activity regulates proliferation and is required for for mutually exclusive pools of APC with different cellular tumorigenesis in the intestinal epithelium of Apc-mutant functions [45]. Exploiting APC Function as a Novel Cancer Therapy Current Drug Targets, 2014, Vol. 15, No. 1 93 mice [61]. In the intestinal epithelium, APC-mediated FAK Additional regulation of APC during proliferation comes expression is Wnt-dependent [61]. Mammary tumors from from casein kinase 2 (CK2), which interacts with APC at the MMTV-PyMT;ApcMin/+ mice demonstrated increased phos- amino-terminus to promote nuclear translocation of APC by phorylated FAK, and increased signaling through Src and phosphorylation [80]. Interestingly, it is the carboxy-terminal JNK [62]. Although FAK signaling was enhanced, -catenin region that inhibits CK2 and promotes proliferation [81]. was not localized to the nucleus and TCF activity was not These findings suggest that full-length APC is needed for the detected, suggesting that the activation of FAK in this model APC/CK2 complex activity, although there is evidence that is independent of Wnt signaling [62]. Combined, these stud- shows no changes in proliferation in cells with loss of APC ies support a model in which the interaction between APC [58]. and FAK is important for the role of APC as a tumor sup- APC also binds proliferating cell nuclear antigen pressor; however, the mechanisms of FAK activation may be (PCNA), a marker for the G1/S phase and a co-factor of context-specific. DNA polymerase, hence it has roles in both DNA replication and repair [82, 83]. The APC binding site for PCNA occurs Cell Cycle Control and DNA Replication in the 15-amino acid repeat region of APC that contributes to During mitosis, APC localizes near the centrosomes and binding -catenin [84]. The binding site for topoisomerase is associated with the microtubule-organizing center to main- II to regulate cell cycle progression also involves the 15- tain chromatin structure [63]. Although it is expressed and amino acid repeat region. Functionally, cells over-expressing phosphorylated throughout the cell cycle, APC is transiently the APC fragment responsible for binding topoisomerase II hyperphosphorylated at the carboxy-terminus in M-phase by exhibit a G2 cell cycle arrest [84]. the cyclin-dependent kinase (CDK) complex, cyclin B/cdk1, Targeting the mitotic spindle checkpoint may present an which regulates its interaction with EB1 [64, 65]. Similarly important therapeutic approach for APC-mutant cancers. the cyclin A/cdk2 complex is required for mitotic entry, and Some chemotherapeutic agents, such as taxanes and vinca also associates with APC in G2/M [66]. The APC/EB1 com- alkaloids, work as spindle poisons by disrupting microtu- plex, along with the phosphorylation of APC by the Bub1 bules. Recent work demonstrated that low levels of taxol and Bub3 mitotic checkpoint kinases, provide stable kineto- caused APC-deficient cells in vivo to resist arrest due to chore microtubule attachment for proper chromosome Wnt-independent microtubule destabilization. However, high alignment [67, 68]. taxol concentrations failed to prevent arrest [85]. Another in There is evidence that APC plays a direct role in mitosis vitro study implicated a role for APC in response to low lev- through its association with the centrosome and microtubules els of mitotic arrest caused by nocodazole treatment. How- at the mitotic spindle to mediate all phases of the cell cycle. ever, similar to previous findings, APC loss had no impact APC functions in the cell cycle partially through the activa- on high levels of arrest [77, 86]. These data indicate that tion of the Wnt/-catenin pathway. Studies have shown that APC status may influence therapeutic efficacy of specific loss of APC in colorectal cancer cells can modulate cell cy- chemotherapies. cle progression; however, overexpressing APC results in cell Role in DNA Repair cycle arrest [69], and introduction of -catenin only partially rescued progression [70, 71]. In fibroblasts, the interaction of APC is involved in the inhibition of multiple DNA repair APC with Dlg is required for the APC-induced G1/S arrest pathways, such as single nucleotide base nucleotide repair independent of mediating -catenin degradation [72]. Further (SN-BER) where it interacts with DNA polymerase (Pol- studies showed that the direct binding of APC to A/T rich ), and long-patch base excision repair (LP-BER) where it sequences of DNA through the carboxy-terminal region of interacts with flap endonuclease 1 (Fen-1) [82, 87, 88]. APC APC inhibits DNA replication in the G1/S progression [73]; is also recruited to the site of DNA damage during double- however phosphorylation of APC by CDK1 and 2 allows stranded DNA break repair where it induces early response cells to progress though the G1/S phase [74]. to the damage by interacting with the DNA-dependent protein kinase catalytic subunit [89]. This activity is consistent The role of APC in chromosome segregation and cytoki- with APC’s role as the ‘gatekeeper’, as its mutation may nesis is critical, as embryonic stem cells from Apc-mutant slow DNA repair and increase mutations in surrounding cells mice and cells with APC depletion suffer from severe chro- to promote tumor progression [5, 89]. mosomal and mitotic spindle defects [75]. This is most likely an early consequence of APC loss since normal tissues iso- The loss or mutation of APC has also been associated lated from Apc-mutant mouse models exhibit aneuploidy and with increased ability to repair mutated DNA [82]. Specifi- severe mitotic defects, and the misorientation of spindles cally, APC interacts with Pol- and Fen-1 to block LP-BER, worsens as intestinal tumor development progresses [76, 77]. and treatment with a DNA alkylating agent methylmethane APC’s amino-terminus may be involved in mitosis as intro- sulfonate (MMS) did not damage cells with mutated APC duction of amino-terminal APC mutants into HCT-116 colo- [87, 88]. This observation is important as APC status could rectal cancer cells inhibits proliferation and mitosis by im- impact treatment of tumor cells with chemotherapeutic alky- pacting chromosomal stability through weakening the micro- lating agents such that cells with APC mutations may be tubule and kinetochore interactions [78]. The role of APC in more resistant than those with wild-type APC. Interestingly, APC expression is regulated by a variety of alkylating chromosomal instability has also been attributed to - catenin/TCF-mediated transcription as shown in studies with agents, including MMS [90], and carcinogens [91-93] suggesting that there may be a feedback loop that regulates APC APC mutant embryonic stem cells and DLD-1 colorectal levels and activity. cancer cells [79]. 94 Current Drug Targets, 2014, Vol. 15, No. 1 Lesko et al.

Association of APC with Apoptosis Regulators Interestingly, APC inactivation occurs at different fre- APC mutation has long been associated with changes in quency in specific breast cancer subtypes. For example, 50% cellular apoptosis [94-96]. In fact, reintroduction of full- of lobular carcinomas [131], and 70% of inflammatory breast length APC to colorectal cancer cells with truncated APC cancers exhibit promoter methylation and silencing of APC enhanced both basal apoptotic activity and drug-induced [133]. Similarly, subtype specificity has been observed with -catenin cytosolic and nuclear accumulation [135, 136]; apoptosis [97]. APC can induce apoptosis through regulation however, it is unclear whether the subtype specificity of APC of the Wnt target gene survivin, a member of the inhibitor of inactivation and -catenin nuclear accumulation are related. apoptosis family of pro-survival factors [98]; however, con- Exploring this relationship could lead to potential therapeutic trol of Wnt signaling by APC may not be sufficient to ac- treatments because it appears that APC functions separately count for APC’s role in apoptosis. For example, an increase from the Wnt/-catenin pathway to suppress tumor activity in apoptosis was observed during lactation in the mammary Min/+ [13, 137 for review]. Not only does the frequency of APC gland of Apc mice in the absence of Wnt pathway activa- inactivation help to determine the cancer phenotype, but it tion [13] and APC modulated apoptosis in a transcription- may also be associated with certain clinical parameters such free Xenopus extract assay [99]. as tumor stage and size, poor prognosis, and overall survival The context-dependent manner in which APC induces [126, 128-130]. apoptosis with both APC mutation or depletion and APC Although mutations and silencing of APC are most over-expression presents a challenge in identifying the role commonly found in colorectal cancer, loss of APC also oc- of APC in regulating apoptosis. As an example, apoptosis in Min/+ curs in several other epithelial cancers (reviewed in [4]). the mammary epithelium in Apc mice may be caused by Briefly, APC promoter methylation has been found in 95% a disruption in epithelial polarization [13]; while apoptosis in of lung cancers [138, 139], and 90% of prostate cancers Apc-mutant adult small intestine epithelium of mice may be [140-142]. APC mutations have been detected in 18% of a result of perturbed migration and differentiation [100]. pancreatic acinar cell carcinomas [143], and have been asso- Because APC’s regulation of apoptosis may be dependent on ciated with pancreatoblastomas [144, 145]. Recently, APC cancer type, methods triggering apoptosis in APC-deficient loss by missense or frameshift mutations has been identified cancers by introducing APC domains or other approaches in 16% of invasive urothelial cancers [146]. Biallelic dele- might prove to be a promising therapeutic strategy, but still tion has been detected in 33% of ovarian cancers [147], and need to be thoroughly examined for context-dependence in 17% of melanoma tumors [148-150]. These data collec- [101]. tively suggest that APC mutations likely contribute to the development of diverse types of cancer, suggesting that LOSS OF FUNCTIONAL APC IN HUMAN CANCERS treatments that specifically target APC status may be appli- cable to a variety of patients. Modifications of APC are most commonly found in hereditary and sporadic colorectal cancers. Depending on the TARGETING APC FOR CANCER THERAPEUTICS type of analysis performed or the subset of tumors investigated, 25%-88% of sporadic colorectal adenomas and 31%- Targeting the Wnt/-Catenin Pathway 83% of adenocarcinomas are a result of APC inactivation [102-114]. The majority of somatic mutations of APC in While studies have investigated targeting the upstream FAP and sporadic colorectal cancers occur in the mutation components of the Wnt/-catenin pathway, such as Wnt cluster region (MCR) in the middle portion of APC where - ligands and DVL (reviewed in [151]), these approaches catenin binding and down-regulation occurs (reviewed in [6, would have minimal effect in APC-mutant cancers. There- fore, potentially successful therapeutic treatments for these 115]). Interestingly, mutations in the MCR causing allelic tumors should target components of the Wnt pathway down- loss are often found in colorectal cancers; whereas mutations stream of APC, and will be the focus of this review (summa- outside the MCR are commonly found in breast cancers rized in Table 1). Many approaches have focused on sup- [106, 116]. APC loss can also be the result of hypermethyla- pressing -catenin expression by promoting its degradation. tion of the promoter and epigenetic silencing of APC, and For example, antisense-mediated suppression of -catenin has been observed in many gastrointestinal and non- expression blocked colorectal cancer cell tumorigenic prop- gastrointestinal tumor types [117-121]. erties in vitro and tumor development in Apc-mutant models Specifically in breast cancer, both epigenetic and genetic in vivo [152, 153]. Although these strategies target a critical alterations in APC have also been identified. APC mutations component of the Wnt/-catenin pathway, they are not tumor that regulate Wnt signaling and result in the accumulation of cell specific and deplete all pools of -catenin, including cytosolic and nuclear -catenin have been identified in breast membrane-associated -catenin required for cell-cell interac- cancer [122-124]; however, most APC mutations associated tions. with sporadic human breast cancer occur outside the MCR Other strategies have focused on preventing the transcrip- and function independently of the Wnt pathway to cause tional activity of -catenin/TCF complexes to inhibit pro- cancer progression [124-127]. While APC mutations occur in tumorigenic and proliferative Wnt/-catenin pathway targets. a subset of breast cancers, the most common method of APC In colorectal cancer cells with active Wnt/-catenin pathway, inactivation in breast cancer is promoter methylation [128- two small molecule inhibitors PKF115-584 and CGP049090 133]. Recent studies demonstrated that alterations of APC, have been developed and characterized that disrupt - both by deletion or methylation, occur more frequently in catenin/TCF binding and inhibit proliferation. However, the ER-/PR- breast cancers, and are associated with higher tumor mechanisms by which these compounds function are still grade and poor survival [134]. Exploiting APC Function as a Novel Cancer Therapy Current Drug Targets, 2014, Vol. 15, No. 1 95

Table 1.

Therapeutic Target Reference

Restoration of APC [71, 167, 168]

Antisense -catenin -catenin degradation [152, 153]

PKF115-584

CGP049090 Inhibits -catenin/TCF binding [154, 157]

FH535

ICG-001 Weakens -catenin and CBP interactions [155]

Inhibits interaction between TCF and -catenin StAx-35 [158] through overlapping binding site

MF-tricyclic Cox-2 inhibitor [161, 162] Celecoxib

EGFR inhibitor, successful in combination with Erlotinib + NSAIDs [162] celecoxib and other NSAIDs

Sulindac Cox-1/Cox-2 inhibitor [163, 164]

Aminoglycosides (tylosin) Induces read-through of premature stop codons [169] Macrolides causing truncation of APC

Dasatinib

AZD0530 Src inhibitor [170]

SKI-606

TAE226

PF-562,271 FAK inhibitor [171] 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)

Tumour necrosis factor-related apoptosis- Induces apoptosis in cancer cells (effective when [101] inducing ligand (TRAIL) used in combination with RAc)

Inhibits proliferation and carcinogenic All-trans-retinyl acetate (RAc) [101] transformation (effective with TRAIL)

Inhibit BER; APC-mutant cancers Alkylating agents- MMS, oxalipatan, cisplatin [173-175] may be resistant

Spindle poisons that disrupt microtubules to Vinca alkaloids- taxol, nocodazole, cause apoptosis; APC- mutant cancers may be [77, 85, 86] resistant

Vinorelbine Induces apoptosis in interphase [177] unclear, and treatment with these compounds resulted in observed, and this compound has been used extensively in cytotoxicity [154]. ICG-001, a small molecule that weakens preclinical studies [155]. Another small molecule inhibitor, the interaction of -catenin with CREB-binding protein FH535, prevents the interaction between -catenin and TCF (CBP) [155], a transcriptional co-activator, significantly and inhibits proliferation through a mechanism involving suppressed colorectal cancer cell growth by inhibiting the peroxisome proliferator-activated receptor (PPAR) [157]. expression of cell survival gene survivin [156]. Unlike other Recent studies demonstrated the use of a hydrocarbon- small molecule inhibitors significant toxicity in vivo was not stapled axin peptide sequence (StAx-35), which has an over- 96 Current Drug Targets, 2014, Vol. 15, No. 1 Lesko et al. lapping binding site on -catenin with TCF, was found to transcriptional activity, suppressed cell growth and tumori- inhibit Wnt pathway transcriptional activity and proliferation genicity, tighter cell-cell contacts, and a more adhesive morin colorectal cancer cells [158]. phology [71, 167, 168]. This therapeutic approach induced minimal toxicity [167], but has not been actively pursued. Cyclooxygenase-2 (COX-2), a regulator found to be associated with cell proliferation, differentiation, and tumori- Another approach to restore APC function in mutant cells is to use aminoglycosides and macrolides to induce read- genesis, has been identified as a downstream target of the through of the premature stop codon causing a truncated Wnt pathway through -catenin/TCF transcriptional activity. protein. Colorectal cancer cells containing a nonsense APC Specifically, down-regulation of APC resulted in increased mutation treated with aminoglycosides showed reduced tu- COX-2 levels in colorectal cancer cells [159]. Similarly, mor growth and oncogenic phenotypes [169]. Aminoglyco- Oshima et al. showed that loss of the COX-2 gene Ptgs2 in Min/+ the colorectal cells of Apc-mutant mice side treatment of Apc mice also reduced polyp size and 716 increased life span [169]. However, aminoglycosides and (Apc (+/) Ptgs2(/)) caused a decrease in polyp num- macrolides can be very toxic, and could cause normal cells to ber and size [160]. Therefore, inhibition of COX-2 presents a produce toxic aggregates due to the read-through of correctly promising modality to treat APC-mutant cancers. Both MF- placed stop codons. Although some aminoglycosides, such tricyclic and Celecoxib decreased polyp number in Apc- as tylosin, have proven to be non-toxic [169]; further inves- mutant mice modeling human FAP [161, 162]. Results with Celecoxib were further enhanced upon combination with the tigation into the mechanisms and toxicities of these compounds is needed. Nevertheless, restoration of APC through EGFR inhibitor, erlotinib, and polyps were decreased by gene therapy or read-through mechanisms may be successful 96% [162]. Because long-term treatment with Celecoxib can therapeutic techniques for APC-mutant cancers. lead to cardiovascular side effects, NSAIDs have replaced COX-2 inhibitors in combination with erlotinib and have Min/+ Targeting Pathways Regulated by APC produced similar results in Apc mice [162]. Likewise, the COX-1/COX-2 inhibitor sulindac has been shown to reduce FAK/Src Signaling: Focal adhesion kinase (FAK) is in- the number and size of intestinal tumors in FAP patients, volved in cell adhesion and migration, and recent studies ApcMin/+ mice, and other Apc-mutant models ([163] and re- suggest that loss of APC activates FAK signaling through viewed in [164]). However, long-term treatment with sulin- Wnt-dependent and Wnt-independent mechanisms to induce dac increased tumors in the cecum, and caused reoccurrence proliferation through its downstream effectors. APC- of colorectal cancer in patients due to drug-induced inflam- mediated FAK activation is Wnt-dependent in the intestinal mation [163]. Although most studies investigating COX-2 epithelial cells of Apc-mutant mice [61]. Mammary tumors inhibitors have been performed in colorectal models, there is from MMTV-PyMT;ApcMin/+ mice showed that loss of APC some evidence for its use in breast cancer. Although not di- resulted in a Wnt-independent increase of FAK, Src, and rectly studied in the context of mutant or wild-type APC, JNK phosphorylation [62]. These observations suggest that multiple COX-2 inhibitors have successfully inhibited pro- successful therapeutic treatments could inhibit proliferation liferation in human mammary cells, and decreased tumor by inhibiting FAK signaling through Src or JNK. PP2, a Src burden in animal models [165]. Other treatments have been inhibitor, resulted in decreased proliferation in cells from explored such as the use of NSAIDs and combining cele- MMTV-PyMT;ApcMin/+ tumors [62]. coxib with other drugs; however, the cardiovascular and gas- Treatments using Src inhibitors have not been explored trointestinal side effects of these approaches have stalled specifically in APC-mutant cancers; however, several Src clinical use [165]. Nevertheless, experiments with COX-2 inhibitors, such as dasatinib, AZD0530, and SKI-606 have inhibitors have produced positive results in treating both colorectal and breast cancer and warrant further development been found to decrease proliferation, invasion and metastasis in some breast cancers [170]. Interestingly, the observation to maximize efficacy while eliminating deleterious side ef- that Src inhibition was accompanied by the suppression of fects. FAK signaling makes Src inhibitors a potential therapeutic Although therapeutic treatments targeting the Wnt path- for APC-mutant cancers resulting in activated FAK signaling way may not be sufficient to attenuate cancer progression, [170]. Directly inhibiting FAK through small molecule in- studies have shown that the Wnt signaling pathway effects hibitors has also been recently studied, but little is known cancer cell’s sensitivity to toxicity [166]. Therefore, methods about how these inhibitors will impact APC-mutant tumors. targeting the downstream components of the Wnt pathway, Multiple FAK inhibitors (TAE226 + PF-562,271) which alone or in combination with other treatments may be suc- block the ATP binding site and inhibit FAK phosphoryla- cessful in treating cancer development. tion, inhibited both colon and breast cancer [171]. Direct inhibition of FAK activation via 1,2,4,5-benzenetetraamine Restoring APC Expression tetrahydrochloride (Y15) has also been shown to inhibit tu- Early approaches to inhibit Wnt/-catenin signaling fo- morigenesis in breast cancer cells [171]. Although these cused on restoring APC function via gene therapy. For ex- small molecule inhibitors have shown promise to attenuate ample, studies demonstrated the re-introduction of APC into cancer growth through inactivation of FAK; it is unknown the intestine of ApcMin/+ mice decreased tumor burden [167]. how these inhibitors affect cancers resulting from the loss of Similarly, other studies showed that the introduction of APC APC. Since FAK activation is regulated by many other fac- into colorectal cells expressing only the truncated form of tors in addition to APC, and FAK signaling is likely not the APC resulted in translocation of -catenin from the cyto- only pathway affected by APC mutation, combination thera- plasm and nucleus to the plasma membrane, decreased TCF pies may be required to target this pathway. Therefore, fur- Exploiting APC Function as a Novel Cancer Therapy Current Drug Targets, 2014, Vol. 15, No. 1 97 ther investigation is needed to understand the mechanisms of ATP-binding cassette sub-family G member 2/Breast cancer action. resistance protein (ABCG2/BCRP) have altered gene expression in Apc-mutant mouse mammary glands as MDR1 is up- Apoptosis: Recent studies have explored targeting APC- regulated, and ABCG2/BCRP is down-regulated [13]. Inter- deficient colorectal cells for apoptosis through the use of TNF-related apoptosis-inducing ligand (TRAIL) and all- estingly, COX-2 mediates resistance by up regulating MDR1 [176], suggesting APC loss may have an indirect role in trans-retinyl acetate (RAc) [101]. TRAIL induces apoptosis therapeutic resistance. Further investigation is needed to un- in cancer cells without affecting normal cell growth and RAc derstand the mechanisms by which APC mediates therapeu- decreases cell proliferation and inhibits carcinogenic trans- tic resistance through ATP-binding cassette transporters. formation [101]. Individually, the drugs had no effect on apoptosis of the ApcMin/+ cells; however, the combination of Microtubule Binding: Several drugs including taxol and the two drugs had promising effects [101]. RAc sensitized other vinca alkaloids disrupt microtubules and act as spindle APC-mutant intestinal adenoma cells to TRAIL to induce poisons to induce apoptosis when the mitotic cycle is ar- apoptosis in vitro. In ApcMin/+ mice activation of TRAIL by rested [85]. For example, taxol stabilizes microtubules and RAc decreased tumor growth and increased survival rate inhibits depolymerization, and nocodazole, a vinca alkaloid, [101]. This treatment was also found to be effective in short prevents microtubule polymerization [85]. However, the loss term doses, and did not harm normal or stem cell function of APC has been shown to cause resistance to these treat- [101]. The sensitivity of cells to TRAIL and RAc was de- ments [85]. In mouse intestinal cells APC loss caused resis- pendent on the role of APC in mediating Wnt/-catenin sig- tance to low levels of taxol independent of Wnt signaling naling [101]. Therefore, the treatment combination of [85]. These studies also reported that high levels of taxol TRAIL and RAc may not be suitable in cancers where APC were able to overcome the loss of Apc and resume mitotic acts independently of the Wnt pathway to induce tumor pro- arrest; however, these levels may be unattainable in patients gression. [85]. These data suggest that APC’s role at the mitotic spindle checkpoint effects cells’ response to certain chemothera- Resistance to Chemotherapeutics peutics and must be considered when investigating possible treatments for patients with APC-mutant tumors. DNA Repair: Many chemotherapeutic drugs act as alkylating agents to impair DNA base excision repair (BER) and Inhibition of APC through siRNAs decreased mitotic ar- promote apoptosis; therefore the role of APC to inhibit BER rest in taxol and nocodazole treated human osteosarcoma may help enhance these treatments. However, these treat- cells [77]. These studies also showed that APC-knockdown ments would not be as effective in APC-mutant cancers as cells had reduced apoptosis after treatment with either taxol loss of APC resulting in functional BER could decrease cel- or nocodazole, and that these effects were independent of lular response to treatment. For example, Apc knockdown in Wnt/-catenin pathway activation [77]. These findings sug- mouse embryonic cells caused a decrease in the number of gest that the loss of APC causes spindle defects to disrupt the apoptotic cells when treated with methylmethane sulfonate mitotic checkpoint, resulting in resistance to these treat- (MMS) to impair DNA replication [172]. Similar studies ments. Studies have also shown that vinorelbine, a drug simi- showed human colorectal cancer cells expressing a truncated lar to nocodazole, may be a possible treatment for patients APC protein, continued to grow after treatment with MMS; suffering from APC-mutant cancers because it induces apop- however, MMS inhibited proliferation of HCT-116 human tosis during interphase instead of during mitosis. APC loss in colorectal cancer cells with wild-type APC [82]. In addition, U2OS cells enhanced the effects of vinorelbine and resulted knockdown of APC in HCT-116 cells resulted in no response in increased cell death [177]. Furthermore, vinorelbine was to MMS treatment compared to cells with wild-type APC able to induce cell death in the intestinal epithelial cells of Min/+ expression [82]. These data suggest APC knockdown causes Apc mice, and therefore reduce tumor growth [177]. Be- resistance to alkylating agents through increased BER capac- cause the mechanisms by which APC loss causes increased ity. Other studies have shown that another alkylating agent, sensitivity to this drug are unknown, these positive results oxalipatan, had differential responses in wild-type and mu- warrant further investigation into the use of vinorelbine tant APC colorectal cells. Normal APC increased the disease treatments in cells lacking APC. control rate of the drug about 2.5 times more than cells with mutant APC [173]. Cisplatin also hinders DNA replication CONCLUSION and induces cell cycle arrest and apoptosis [174]; however Loss of function mutations and silencing of the APC tu- enhanced DNA repair can attenuate the effects of this drug, mor suppressor gene are commonly observed in several and studies have shown that APC increases cellular thera- epithelial cancers such as colorectal, breast, ovarian, pancre- peutic resistance [175]. Therefore, cells with mutant APC atic, and lung cancer. APC is best known for regulating the may be resistant to certain chemotherapeutic agents, espe- Wnt/-catenin pathway; however, APC has many Wnt- cially those that target BER, and levels of APC must be considered for effective treatment. independent roles, such as mediating the actin cytoskeleton, DNA replication and repair, and apoptosis, which influence ATP-Binding Cassette Transporters: ATP-binding cas- its tumor suppressor activity. Several therapeutic approaches sette transporters are involved in a variety of cellular proc- targeting both the Wnt-dependent and Wnt-independent esses including transmembrane transportation, translation functions of APC have been explored to treat APC-mutant elongation, and DNA repair; however they have also been cancers such as the reintroduction of APC, targeting the Wnt implicated in tumor resistance. Two ATP-binding cassette pathway downstream of the destruction complex, and inhib- transporters, multidrug resistance protein 1 (MDR1) and iting signaling pathways regulated by APC. However, levels 98 Current Drug Targets, 2014, Vol. 15, No. 1 Lesko et al. of APC must be considered when choosing therapeutic MDR1 = Multidrug resistance protein 1 strategies, as APC loss has been shown to contribute to ABCG2 = ATP-binding cassette sub-family G mem- therapeutic resistance through enhancing DNA repair, ATP- ber 2 binding cassette transporters, and disrupting the mitotic spindle. Taken together these studies have greatly contrib- REFERENCES uted to understanding the mechanisms by which APC mediates tumorigenesis, and have provided a promising founda- [1] Herrera L, Kakati S, Gibas L, Pietrzak E, Sandberg AA. Gardner syndrome in a man with an interstitial deletion of 5q. Am J Med tion for the development of therapeutics to treat APC-mutant Genet 1986; 25(3): 473-6. cancers. 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Received: September 24, 2013 Revised: October 10, 2013 Accepted: November 07, 2013

PMID: 24200292