Role of the ubiquitin ligase Fbw7 in cancer progression

NON-THEMATIC REVIEW

Role of the ubiquitin ligase Fbw7 in cancer progression

Yabin Cheng & Gang Li

Published online: 29 November 2011# Springer Science+Business Media, LLC 2011

Abstract Fbw7 is a member of F-box family proteins,which constitute one subunit of Skp1, Cul1, and F-boxprotein (SCF) ubiquitin ligase complex. SCFFbw7 targets aset of well-known oncoproteins, including c-Myc, cyclin E,Notch, c-Jun, and Mcl-1, for ubiquitylation and degrada-tion. Fbw7 provides specificity of the ubiquitylation ofthese substrate proteins via recognition of a consensusphosphorylated degron. Through regulation of severalimportant proteins, Fbw7 controls diverse cellular process-es, including cell-cycle progression, cell proliferation,differentiation, DNA damage response, maintenance ofgenomic stability, and neural cell stemness. As reducedFbw7 expression level and loss-of-function mutations arefound in a wide range of human cancers, Fbw7 is generallyconsidered as a tumor suppressor. However, the exactmechanisms underlying Fbw7-induced tumor suppressionis unclear. This review focuses on regulation network,biological functions, and genetic alteration of Fbw7 inconnection with its role in cancer development.

Keywords SCF. Fbw7 . Cancer progression

1 Introduction

The ubiquitin proteasome system is responsible for thedestruction of regulatory proteins within eukaryotic cell andis thus crucial in keeping cellular homeostasis [1]. This

proteolytic system attaches ubiquitin chains to substrateproteins rendering their subsequent degradation by 26sproteasome [2]. Various cellular processes are controlled bythe ubiquitin proteasome system, including cell cycle,proliferation, differentiation, stem cell quiescence, tran-scription, DNA damage repair, and apoptosis [3]. It is notsurprising that components of the ubiquitin proteasomesystem are commonly found lost or mutated in cancers, justlike F-box and WD repeat domain-containing 7 (alsoknown as Fbw7, Fbxw7, CDC4, AGO, and SEL10).

Fbw7 is a member of the F-box protein family which ischaracterized by the F-box motif, an approximately 40-amino acid region. Fbw7 serves as a substrate adaptor forthe Skp1–Cul1–F-box protein–Rbx1 (SCF) ubiquitin ligasecomplex and mediates the recognition and binding of thesubstrate proteins. SCFFbw7 degrades several proteins withimportant roles in cell growth, proliferation, differentiation,and survival [4]. Some of the Fbw7 substrates areextensively studied proteins in cell biology, including c-Myc, cyclin E, Notch, c-Jun, mammalian target ofrapamycin (mTOR), and Mcl-1 [5–10]. Notably, mostFbw7 substrates have been shown as oncogenes in multipletypes of human cancer [11]. Recent mouse model studiesshowed that loss-of-function mutations in Fbw7 causedboth hematopoietic and solid organ tumors in mice [12–14].Moreover, mutations in Fbw7 have been identified indiverse human cancers, including cholangiocarcinoma, Tcell acute lymphoblastic leukemia, pancreatic cancer,endometrial cancer, and colon cancer [15–18]. Therefore,Fbw7 is a general tumor suppressor in human cancers.

However, the exact molecular mechanism of Fbw7-associated tumor suppression remains unclear. Understand-ing the biological functions of Fbw7 and the regulationnetwork surround it is crucial for providing insights into themechanisms of Fbw7-mediated cancer and developing

Y. Cheng :G. Li (*)Department of Dermatology and Skin Science, Jack Bell ResearchCentre, Vancouver Coastal Health Research Institute,University of British Columbia,2660 Oak Street,Vancouver, B C V6H 3Z6, Canadae-mail: [email protected]

Cancer Metastasis Rev (2012) 31:75–87DOI 10.1007/s10555-011-9330-z

therapeutic strategies that target the Fbw7 pathway inhuman cancers. In this review, we focus on the majorfunctions and regulatory network of Fbw7, as well as itsrole in human cancer development.

2 Fbw7 structure and isoforms

F-box proteins are the variable substrate adapters for theSCF ubiquitin ligase complexes and dictate the substratespecificity of the ubiquitin ligase [19, 20]. F-box proteinsare divided into three classes on the basis of the presence ofWD40 repeats (Fbxw) or leucine-rich repeats (Fbxl), or theabsence of either of these domains (Fbxo) [21]. Normally,F-box proteins’ recognition of their substrates requires thephosphorylation of the substrate in a discreet degradationsequence (degron) in WD40 repeats or the leucine-richrepeats. As one F-box protein can recognize diversesubstrates, the SCF ubiquitin ligases have numerous targetsand multiple biological functions [21, 22] (Fig. 1).

Fbw7 is one of the well-studied members of F-boxprotein family. In mammals, Fbw7 gene is located withinchromosome 4 and encodes three transcripts (Fbw7α, β,and γ) as a result of alternative splicing of their first exons

[21]. These three isoforms have their own promoter andgenerate three protein isoforms with unique NH2 termini,which determines the expression pattern, subcellular distri-bution, as well as the functions of the protein [4]. Fbw7αlocalizes to the nuclear and is predominantly expressed inadult mouse. Comparing with other two isoforms, Fbw7αmRNA expression is much higher in most human cell linesand primary cells [23]. Grim et al. established isoform-specific Fbw7-null mutations in human cells and found thatthe Fbw7α isoform is responsible for the degradation ofmost Fbw7 substrates, including cyclin E, c-Myc, andSREBP1 [24]. Fbw7β is primarily cytoplasmic protein andmost abundant in brain and testis [23]. A recent studyshowed that Fbw7β contains a putative transmembranedomain in NH2 terminus region, and Fbw7β-deficient cellshave higher sensitivity to oxidative stress [25]. Fbw7γshows a nucleolar distribution and is expressed majorly inheart and skeletal muscle [23]. It has been reported thatFbw7γ isoform colocalized with c-Myc in the nucleolusand regulated its turnover [26]. Although one study showedthat Fbw7 protein isoforms interact with each other andregulate cyclin E degradation [27], more investigations arestill needed to confirm the cooperation status of eachisoform when SCFFbw7 exerts its functions. It is likely that

Fig. 1 Schematic diagram of the functions and regulation networks of Fbw7 in connection with cancer progression

76 Cancer Metastasis Rev (2012) 31:75–87

the transcription of each isoform is differentially regulated,and each isoform is responsible for different substrates andfunctions during protein degradation. However, the exactphysiological role and regulation mechanism of eachisoform remains unclear.

The structure of Fbw7 has been well studied, andseveral domains are found to be essential for Fbw7functions. F-box domain, which is the common featureof all F-box proteins, offers direct interaction withSkp1 in SCF complex [28]. Eight WD40 repeats areprotein–protein interaction domains that provide physio-logical connections with the substrate. The WD40 repeatsform an eight-bladed barrel-shaped β propeller structuretermed phosphodegron binding pockets, which recognizeand bind to substrates after they have been phosphory-lated within conserved phosphodegron motifs which aredefined as Cdc phosphodegrons (CPD) [29–32]. It hasbeen found that all Fbw7 substrates contain at least oneconserved CPD sequence (T/S) PXX(S/T/E) (Table 1), inwhich the T/S residue can be phosphorylated byglycogen synthase kinase 3 (GSK3) [4]. The motifsequence which localizes just before the F-box region,called D domain, is required for Fbw7 dimerization [33].Recent studies indicate that F-box proteins regulatesubstrate binding and ubiquitylation through forminghomodimeric or heterodimeric complexes [33, 34]. Thedimerization might largely depend on the degron strength

and is substrate-specific and provides a possibility ofprecise control of substrate degradation.

3 The regulation of Fbw7

Although much is known about the Fbw7 ubiquitin ligase-mediated protein degradation pathway and a number ofFbw7 substrates have been identified in the last decade, theregulation and upstream regulators of Fbw7 itself arepoorly understood. Here, we summarize several Fbw7regulators reported in recent literatures (Fig. 1).

Pawar et al. reported that the transcription factorCCAAT/enhancer binding protein-δ (C/EBP δ) directlyinhibits Fbw7 gene expression and induces Fbw7 oncogen-ic targets mTOR and Aurora A [35]. ChIP assay with MCF-7 breast cancer cells showed the endogenous C/EBP δbinds to the Fbw7α promoter. Consequently, C/EBP δenhances PI3K/AKT/mTOR pathway and promotes HIF-1αtranslation and activity, which is necessary for hypoxicadaptation and tumor metastasis [36]. This is the first reportshowing a correlation between Fbw7 and tumor metastasis,suggesting a potential value of clinical application of C/EBPδ or mTOR inhibitors [37] to prevent metastatic disease.Another regulator of Fbw7 is early region 1A (E1A), aproduct of human adenovirus type 5 [38]. This study for thefirst time showed E1A perturbs SCFFbw7-mediated protein

Table 1 Fbw7 substrates and major biological functions

Substrates Phosphorylationsites

CPD Biological functions of substrates Kinase(s) References

c-Jun T239/S242 GETPPLSPI Transcription factor, cell growth/proliferation

GSK3 [58]

c-Myb T572/S576 LMTPVSED Transcription factor, cell growth/proliferation

NIK/GSK3 [118, 119]

c-Myc T58/E62 LPTPDKEDD Transcription factor, cell growth/proliferation

GSK3 [5]

Cyclin E1/E2

T380/S384 T62/E66

LLTPPQSGKIPTPDKEDD

Cyclins, cell cycle CDK2,GSK3

[15, 56,65]

KLF5 S303 PPSPPSSE Transcription factor, cell proliferation ND [60]

Mcl-1 S159/T163 S121 DGSLPSTPPPAIMSPEEEL

Bcl-2 family, inhibits apoptosis GSK3 [9]

mTOR T631 LLTPSIHL Kinase, mTOR growth pathway ND [10]

Notch1 T2512/E2516 FLTPSPESP Transmembrane receptor, Notch signaling CDK8 [90, 91]

p63 S383 LPSVSQLINP Transcription factor, epithelialdifferentiation

GSK3 [129, 130]

Pgc-1 ND ND Transcription coactivator, metabolicpathways

GSK3,MAPK

[4]

Presenilin T116/E120 IYTPFTEDT Notch signaling ND [41]

Src3 ND ND Histone acetyltransferase, circadianrhythm

ND [4]

SREBP T426/S430 TLTPPPSD Transcription factor, sterol lipid synthesis GSK3 [4]

TGIF1 T235/T239 FNTPPPTP Transcription co-repressor ND [120]

CPD Cdc4 phosphodegron, ND not determined

Cancer Metastasis Rev (2012) 31:75–87 77

turnover through the inhibition of Fbw7 ubiquitin ligase.However, the mechanism by which E1A inhibits the activityof Fbw7 and the major determinant of the specificity remainunclear.

Presenilins (PS) are the catalytic components of γ-secretase, which is required for Notch1 activation [39, 40].It has been reported that Fbw7α targets Notch1 intracellulardomain (NICD) for degradation, positively regulates epi-dermal growth factor receptor (EGFR) by affecting ubiq-uitylation and stability of EGFR. In the meantime, PSnegatively modulates Fbw7 transcription, thus positivelyand negatively regulates EGFR and Notch signaling,respectively [41]. Using a novel epidermal conditional PS-deficient mouse model deleting PS in keratinocytes of thebasal layer of the epidermis, researchers found the micedeveloped epidermal hyperplasia associated with increasedexpression of both EGFR and Fbw7 and reduced NICDlevel in keratinocytes. This is the first study that showed amolecular regulation mechanism of Fbw7 expression attranscriptional level and indicated a crosstalk between PS,EGFR, and Notch signaling through Fbw7 during cellgrowth and skin carcinogenesis. Fbw7 serves as a centralmediator of the upstream PS and downstream EGFR andNotch signaling. In contrast with previous establishedtumor suppressor role, Fbw7 could function as an oncogeneas it promotes EGFR stability and signaling in epidermalcells. More study is required to determine the exact role ofFbw7 in epidermal cell transformation and to determinewhether Fbw7 affect EGFR degradation directly.

Another potential group of Fbw7 regulators are micro-RNAs. MicroRNAs are short RNA molecules with average22 nucleotides long, which bind to complementary sequen-ces on target mRNAs resulting in translational repression oftarget degradation and gene silencing [42]. Xu et al. foundthat overexpression of miR-223 could increase endogenouscyclin E protein and activity levels and increase genomicstability by significantly reducing Fbw7 mRNA level [43].This study provided first evidence that microRNA candirectly regulate the activity of SCFFbw7 ubiquitin ligase.Recently, another group identified miR-27a as a suppressorof Fbw7, and as a result, miR27a inhibits ubiquitylation anddegradation of the key Fbw7 substrate cyclin E [44]. Moreimportantly, their data demonstrated that miR-27a is theonly suppressor of Fbw7 during G1-S phase transition.Inhibition of Fbw7 by miR27a overexpression leads toabnormal cell-cycle progression and DNA replicationstress. These data confirmed the transcriptional regulationof Fbw7 by different type of regulators, which could affectFbw7 substrate degradation and functions.

Except the negative regulators of Fbw7 we mentionedabove, one positive regulator serum/glucocorticoid-inducible kinase 1 (SGK1) has been identified [45]. SGK1is an important regulator of multiple cellular processes,

including metabolism, cell proliferation, cell volume, cellsurvival, and differentiation [46, 47]. SGK1 is an inhibitorof γ-secretase [45], which is required for Notch1 activationby releasing the Notch1 intracellular domain (Notch1-IC)[40]. Mo et al. found that Notch-IC was able to form atrimeric complex with Fbw7 and SGK1; thus, SGK1promoted the protein degradation of Notch1-IC throughFbw7-dependent pathway. Furthermore, activated SGK1induces Notch1-IC protein degradation and ubiquitylationthrough phosphorylation of Fbw7 at serine 227 [45]. Thesedata indicate that activation of SGK1-mediated Fbw7phosphorylation could serve as a therapeutic strategy fordeveloping anti-leukemia drugs in T cell acute lymphoblas-tic leukemia (T-ALL) cells. Interestingly, another study alsofound that serine 227 was phosphorylated in a PI3K-dependent manner, but in particular Fbw7 alpha isoform[48]. This PI3K-dependent phosphorylation reduces Fbw7stability but promotes ubiquitylation of its two substrates, c-Myc and cyclin E. Moreover, it has been reported thatFbw7 is a substrate for PKC [49]. Serine 10 and 18 residuesin isoform-specific N-termini of Fbw7α are phosphorylatedin a PKC-dependent manner. Mutational analysis alsoindicates that phosphorylation of Fbw7α at serine 10, aswell as mutations in nearby residues at position 11 and 16,regulates its nuclear localization. All together, theseevidences suggest that several kinases serve as isoform-specific regulators of Fbw7 protein, and the activity ofFbw7 can be regulated in post-translational level.

4 Fbw7 and cancer progression

The mechanism of cancer initiation and development is notfully understood. Mutations that generate gain of functiononcoproteins and loss-of-function tumor suppressors arepossible basis of this disease. Sufficient evidences supportthat Fbw7 serves as a tumor suppressor through controllingthe degradation of several important oncoproteins withcentral role in cell-cycle progression, proliferation, and celldivision (Fig. 1). Therefore, understanding the biologicalfunctions regulated by Fbw7 is crucial for unveiling themechanisms of Fbw7-mediated cell transformation andcancer development.

4.1 Cell-cycle control

Cell cycle is driven by the activation of cyclin-dependentkinases (CDKs), the activity of which is modulated by thekey regulators such as cyclins and CDK inhibitors [50].Deregulated cell-cycle control results in developmentaldefects and is a fundamental cause of cancer. Ubiquitinproteasome system is responsible for the ubiquitylation andsubsequent degradation of most cell-cycle regulators. Two


major classes of ubiquitin ligases, the anaphase-promotingcomplex or cyclosome and the SCF protein complex, playimportant roles in cell-cycle progression [51–53].The F-boxprotein Fbw7 mediates the ubiquitylation and therebycontributes to the degradation of proteins that positivelyregulate cell cycle. Fbw7 was first identified in yeast as aregulator of cell-cycle-related proteins and named Cdc4[54]. In Caenorhabditis elegans, Sel10 (homolog of Fbw7)was identified as a negative regulator of Lin-12 (homologof Notch), and from these studies, the mammalian homologwas identified and termed Fbw7 [55]. It has been shownthat Fbw7 targets for degradation of various mammalianproteins that controls cell-cycle progression [4, 53],including cyclin E [56], c-Myc [57], c-Jun [7, 58], Notch[59], and KLF5 [60]. We will briefly discuss the mecha-nisms of Fbw7-mediated cell-cycle control through theseproteins.

4.1.1 Cyclin E

Cyclin E binds to G1 phase cyclin-dependent kinase Cdk2and controls the G1 to S phase transition in cell cycle,which is the rate-limiting step for proliferation [61, 62]. Asthe key regulator of cell-cycle machinery, the amount andkinase activity of cyclin E is tightly controlled by ubiquitin-mediated proteolysis. Deregulation of cyclin E has beenfrequently found in cancer, and elevated expression ofcyclin E leads to genomic instability and tumorigenesis[63]. It has been found that the p53 and Fbw7 pathwayscooperatively protect cells against cyclin E-induced ge-nomic instability in primary human cells. The p53 pathwayis activated by excess cyclin E expression and in turninhibits cyclin E activity by induction of p21Cip1 [64].Moreover, Minella et al. showed that p21 plays an essentialrole in suppressing cyclin E when Fbw7 is absent.

Cyclin E degradation is controlled by dual degrons: C-terminal CPD and N-terminal CPD. There are twophosphorylation sites in C-terminal CPD: threonine 380(T380) and serine 384 (S384). T380 is a CDK2 autophos-phorylation site, and phosphorylated T380 degron directlybinds to Fbw7 [15, 56]. Mutation of T380 disrupts cyclin Eubiquitylation in vivo and in vitro [6, 15]. In addition toCDK2, T380 is also phosphorylated by GSK3. S384 isanother phosphorylation site, which is uniquely phosphor-ylated by CDK2. S384 phosphorylation increases thestrength of the T380 degron and determines whether cyclinE can be degraded by monomeric of dimeric Fbw7. WhenS384 phosphorylation is inhibited, cyclin E degradationrequires Fbw7 dimers; when both T380 and S384 arephosphorylated, cyclin E is degraded efficiently by mono-meric Fbw7 [31, 33]. Cyclin E also contains an N-terminaldegron which is located on threonine 62 (T62) [65]. Crystalstructure study reveals that a T62 peptide makes fewer

contacts with Fbw7 than the T380 peptide does, butClurman and colleagues found that N-terminal degron alsohas a crucial role in regulating cyclin E activity in vivo. Thereason that cyclin E contains two CPDs is still unclear. Onepossible explanation is that degradation of cyclin E dependson the affinity and the extent of phosphorylation of degron.

4.1.2 c-Myc

The basic helix-loop-helix transcription factor c-Myc is akey regulator of exit and re-entry into the cell cycle [66].The expression level of c-Myc is increased in manymalignant tumors as a result of amplification or mutationof the c-Myc gene, and many c-Myc mutations affect thestability of the encoded protein [67–69]. Its turnover is thusthought to be a critical determinant in carcinogenesis. c-Myc is highly unstable and the half-life is only ∼30 min inproliferative cells [70]. It has been shown that c-Mycprotein is ubiquitylated and degraded by the ubiquitinproteasome system [71]. The phosphorylation region of c-Myc locates to the transactivation domain (TAD) [72]. Twoevolutionary conserved sequences, Myc box 1 (MB1) andMB2 in the TAD, have been shown not only to be involvedin the proteolysis of c-Myc but also involved in itstransactivation and oncogenic activities [68, 73]. c-Mycstability is mainly determined by the phosphorylation ofthreonine 58 (T58) and serine 62 (S62) in MB1 and T58phosphorylation requires prior phosphorylation at S62. Notsurprisingly, T58 and S62 residue mutations have beenfrequently found in human tumors [5].

At least four ubiquitin ligases have been identified thatubiquitylate c-Myc and regulate its turnover. Except Fbw7,few are known about the recognition of c-Myc by the otherubiquitin ligases. Degradation of c-Myc by Fbw7 has beenshown in controlling c-Myc stability in G1 phase [74].Recent study found that Fbw7 and another F-box protein β-TrCP (also known as Fbw1B) exert different effects on c-Myc stability in different cell-cycle phases. β-TrCP usesthe UbcH5 ubiquitin-conjugating enzyme to form hetero-typic polyubiquitin chains on c-Myc, and this ubiquityla-tion antagonizes Fbw7-mediated c-Myc turnover. Thisalternative ubiquitylation by β-TrCP is required for c-Myc-dependent acceleration of cell-cycle progression afterrelease from an arrest in S phase [75].

4.1.3 c-Jun

c-Jun is a major component of the AP1 transcription factor,the constitutive activation of which is found in varioustypes of human tumor cells [76]. The Jun N-terminal kinase(JNK) signaling pathway leading to c-Jun phosphorylationstimulates cell proliferation, as c-Jun−/− fibroblasts have aproliferation defect [77]. c-Jun induction is also required for


cell-cycle re-entry of UV-irradiated fibroblasts [78]. More-over, c-Jun deficiency in mouse fibroblasts also results in areduced ability to support keratinocyte proliferation inculture [79]. Fbw7 was found to antagonize c-Jun function,and the regulation of c-Jun by Fbw7 was initially reportedto be dependent on JNK phosphorylation of the c-Jun Nterminus [7]. Contradictorily, others found that JNK activitystabilizes Jun [80, 81]. Wei and coworkers subsequentlyidentified a CPD in the C-terminal of the protein, and thecentral T239 is phosphorylated by GSK3 after priming onS243 [58]. Interestingly, peptide-binding assays revealedthat only T239/S243 doubly phosphorylated peptides stablybound to Fbw7, whereas both monophosphorylated pep-tides failed to bind, further confirming the role of thenegative charging in forming contacts with the Fbw7WD40 repeats domain. The discrepancy between thesetwo reports is still not understood.

4.1.4 Notch

Notch proteins are a family of ligand-activated large(300 kDa) single-pass transmembrane heterodimeric tran-scription factors, the intracellular domains of whichtranslocate to the nucleus after cleavage by the γ-secretaseproteolytic complex, and this activates numerous cellularprograms, including cell fate decision, proliferation, andapoptosis [82, 83]. The Notch family is comprised of fourparalogs, termed Notch 1–4 that have high structuralsimilarity [84, 85]. Fbw7 was first identified in C. elegansas a negative regulator of Notch by genetic screening [8]. Ithas been shown that Notch1 and Notch4, as well aspresenilin, which are part of the γ-secretase proteolyticcomplex, are targets for ubiquitylation mediated by Fbw7[8, 86–88]. Fryer et al. showed that phosphorylation ofNotch in the PEST (rich in amino acids P, E, S, T) regionby cyclin C:csk8 leads to binding of Fbw7, resulting inturnover of the complex [89]. Other researchers havedefined a functional CPD within Notch, threonine 2512(T2512), which directs binding interactions between Notchand Fbw7 [90, 91].

Debates still exist on the functions of Notch in cell-cyclecontrol, cell proliferation, and the role in cancer. Theoncogenic role of Notch was first identified on human T-ALL. More than 50% of human T-ALLs bear mutations inNotch1, indicating a prominent role for Notch in this T cellmalignancy [92]. Experimentally, Notch1 can collaboratewith known oncogenes, including c-Myc, E2A-PBX1, andIkaros to induce T-ALL [93–95], although the completemolecular mechanism is still unclear. Furthermore, forcedexpression of the Notch cytoplasmic domain under theinfluence of the viral promoter can cause T cell neoplasia[96]. Moreover, overexpression of Notch ligand Dll4 alsoresults in the development of T cell leukemia and T cell

lymphoproliferative disease in different models [97, 98].Studies later on showed that all members of the Notchfamily have been implicated in cancer, including breast[99], medulloblastoma [100, 101], colorectal [101], mela-noma [102], and pancreatic cancer [103]. However, there ismounting evidence that Notch signaling can also functionas tumor suppressor. Recent data indicated that Notch cansuppress tumor formation in mouse skin, as well as agrowth inhibitor in keratinocytes, hepatocellular carcinoma,and small-cell lung cancer [104–107].

Onoyama et al. found that abnormal accumulation ofNotch1 and c-Myc and the consequential over-proliferationof thymocyte resulted in thymic lymphoma in mice withconditional inactivation of Fbw7 in the T cell lineage [108].These researchers also demonstrated that it was c-Myc, butnot Notch, responsible for the Fbw7 tumor suppressorfunction. This group showed reduced cell proliferation dueto over-accumulation of Notch in mouse embryonicfibroblasts, suggesting that Fbw7 may target differentsubstrates for degradation in different cell types andtherefore regulates cell-cycle progression in a cell type-specific manner [59].

4.1.5 Other substrates

Other than those well-known substrates related to cell cycleand cell proliferation regulated by Fbw7, some novelmolecules were identified recently. KLF5 (also known asIKLF and BTEB2) is a member of Krüppel-like family(KLF) transcription factors that play important roles inmultiple physiological and pathological processes, includ-ing stemness, inflammation, and atherogenesis [109].Accumulated evidence suggests that KLF5 promotesfibroblast, colon, bladder, and breast cell proliferation[110–112]. It has been shown that KLF5 promotes breastcell proliferation through directly upregulating the FGF-binding protein gene transcription [112]. More recently,inhibition of KLF5 by small interfering RNA using nano-particles has been shown to efficiently inhibit tumor growthin vivo [113]. These findings define KLF5 as an oncogenictranscription factor and a potential therapeutic target forinvasive breast cancer and other cancers. KLF5 is anunstable protein with a short half-life and can be degradedthrough the ubiquitin-dependent and ubiquitin-independentmechanisms [114]. Zhao et al. found that KLF5 trans-activation domain also contains two putative evolution-conserved CPD motifs, 303SPPSS and 323TPPPS, whichcould recruit Fbw7 upon phosphorylation. GSK3β kinase isinvolved in KLF5 S303 phosphorylation that is required forFbw7-mediated KLF5 degradation [60].

c-Myb is a transcription factor involved in controllingthe proliferation, differentiation, and cell fate decision ofhematopoietic cells [115]. c-Myb is also deregulated in


many human cancers, particularly, acute myeloblastic, andlymphoblastic leukemias [116, 117]. In these tumors, it isthought that c-Myb overexpression leads to inhibition ofterminal differentiation and enhanced proliferation. Untilrecently, limited data have emerged describing how thelevels of c-Myb are controlled. Recent studies identifiedFbw7 as the E3 ligase responsible for the ubiquitylation andproteasomal degradation of c-Myb. These data showed thatFbw7 binds c-Myb and facilitates ubiquitylation through aphosphorylation-dependent event involving Thr-572, Ser-556, and Ser-528 [118, 119]. However, these studiesidentified a different kinase that is responsible for primingc-Myb for Fbw7 binding. The reason behind the discrep-ancy between these studies regarding which kinase isresponsible for priming c-Myb for proteasomal degradationremains unclear, perhaps suggesting that one kinase ispredominant depending on cell specificity or upstreamsignaling cascades.

4.2 Fbw7 in DNA damage and cellular apoptosis

Fbw7 ubiquitin ligase has been shown to be importantin the control of chromosome stability and is crucial tocell differentiation and apoptosis through degradation ofthe downstream substrates [13]. p53 is a well-knowntumor suppressor protein that plays an essential role inconserving genome stability after DNA damage [120]. Itwas demonstrated that p53 regulates the expression of thecytoplasmic isoform Fbw7β [121]. Fbw7 might also bepositioned upstream of p53 in a signaling axis thatactivates the tetraploidy checkpoint in response to mitoticinhibitors [122]. Mao et al. found that radiation-inducedlymphomas from p53+/− mice, but not those from p53−/−

mice, have frequent loss of heterozygosity and a 10%mutation rate of Fbw7 gene. Fbw7+/− mice have greatersusceptibility to radiation-induced tumorigenesis, indicat-ing that Fbw7 is a p53-dependent, haplo-insufficienttumor suppressor gene [13]. We will discuss in detail thesubstrates of Fbw7 that are related to DNA damage andcellular apoptosis.

p63 is a p53-related protein, which has been shown toactivate p53 responsive genes and induce apoptosis incertain cell types [123]. It has been reported that p63 playsan important role in embryonic development using p63knockout mice. These mice show severe defects inepithelial stratification and fail to form epidermal appen-dages such as teeth, hair, and glands [124, 125]. However,the role of p63 in tumorigenesis appears to be opposite top53. For example, p63 is not mutated in tumors, which is inremarkable contrast to the high mutation status of p53 innumerous types of cancers. On the other hand, p63 is oftenoverexpressed and amplified in cancer, suggesting that p63provides cancer cells with selective advantage [126].

There are six major isoforms of p63 due to alternativetranscription start sites and splicing at the C-terminus [127],and they have several conserved regions common to p53family proteins, including the DNA-binding domain andthe oligomerization domain. ΔNp63 isoform lacks anamino-terminal exon that encodes a p53-like transactivationdomain and further splicing at the 3′ end generated differentC-termini, termed as ΔNp63α, β, and γ [128]. Inparticular, ΔNp63α isoform is abundantly expressed inembryonic ectoderm and highly proliferative basal cells ofmany adult epithelial tissues [126]. Rossi et al. havereported that the MDM2 nuclear localization signal isrequired for ΔNp63α nuclear export and subsequentdegradation by Fbw7 [129, 130]. By deletion and pointmutation analyses, these authors identified a phosphode-gron located in α and β tail of p63 that is required fordegradation. Furthermore, MDM2 or Fbw7 depletioninhibited degradation of endogenous ΔNp63α in cellsexposed to UV irradiation, adriamycin, or upon keratino-cyte differentiation [131]. The reason why only thecytoplasmic Fbw7β isoform expression was induced uponexposure to UV irradiation is still unclear. The physiolog-ical relevance and biological functions of each isoformwarrant more investigation.

Transforming growth factor-β (TGFβ) signaling regu-lates multiple cellular processes, including apoptosis andcell differentiation. Dysfunction of TGFβ signaling hasbeen implicated in various human disorders ranging fromvascular diseases to cancer [132, 133]. TGFβ signaling isnegatively regulated by the transcriptional repressor TGFβ-induced factor 1 (TGIF1), which is also recently reported tobe targeted for degradation by Fbw7 in a phosphorylation-dependent manner [134]. By inactivating TGIF1, Fbw7enhances TGFβ-dependent transcription. Accordingly, in-activation of Fbw7 results in the accumulation of phos-phorylated TGIF1 and attenuation of TGFβ-dependentgene transcription, cell proliferation, and migration. Thus,Fbw7 could be an important regulator of TGFβ signalingby targeting the transcriptional repressor TGIF1 fordegradation. Moreover, Fbw7 can control cellular apoptosisthrough regulating TGFβ signaling.

Both Notch and c-Jun have broad spectrum of functions invarious cellular processes. As mentioned before, the Notchsignaling pathway regulates neural differentiation and isknown to maintain neural stem cell character and to inhibitneurogenesis [77, 135]. Recent genetic and pharmacologicalrescue studies identified that c-Jun as a key molecule ofFbw7 in controlling progenitor cell survival. Fbw7 controlsneural stem cell differentiation and progenitor apoptosis viaNotch and c-Jun [136]. Nakayama et al. also found similareffects of Fbw7 and demonstrated that accumulation ofNotch1 and Notch3, as well as upregulation of Notch targetgenes in the mutant mice, was associated with neural stem


cell maintenance. Treating the cells with a pharmacologicalinhibitor of the Notch signaling pathway reversed the trendof neural progenitor cells skewing toward astrocytes ratherthan neuron in vitro [137]. Both studies indicate that Fbw7 isa pivotal regulator of maintenance and differentiation ofneural stem cells in the brain.

Mcl-1 is a pro-survival Bcl-2 family member, whichinhibits apoptosis by blocking the cell death mediators Baxand Bak (also known as Bak1) [138]. In 2011, two groupsindependently found that Mcl-1 is a novel substrate ofFbw7. Inuzuka et al. showed that Fbw7 governs cellularapoptosis by targeting Mcl-1 for ubiquitylation and de-struction upon phosphorylation by GSK3 [9]. They foundin T-ALL cell lines loss of Fbw7 resulted in Mcl-1overexpression. At the same time, T-ALL cell lines withdefective Fbw7 are particularly sensitive to the multi-kinaseinhibitor sorafenib but resistant to the Bcl-2 antagonistABT-737. Moreover, both Fbw7 reconstitution and Mcl-1depletion restore sensitivity to ABT-737, indicating Mcl-1as a relevant bypass survival mechanism that enablesFbw7-deficient cells to evade apoptosis. Similarly, anothergroup showed that the degradation of Mcl-1 was blocked inFbw7-deficient or loss-of-function mutated tumor cells, andMcl-1 regulated the sensitivity of tumor cells to anti-tubulinchemotherapy through Fbw7 [139]. These data indicate thatFbw7 regulates cellular response to DNA damage, as wellas the sensitivity to anti-cancer agents, suggesting promis-ing combination chemotherapy for cancer treatment.

4.3 Fbw7 and metastasis

In most types of human cancer, primary tumor mass mayinvade adjacent tissues and travel to distant sites and organswhere they can form new colonies. Tumor metastasisaccounts for 90% of human cancer deaths [140]. Theinvasion and metastasis requires expression or activation ofseveral classes of proteins, including cell–cell adhesionmolecules, integrins, and proteases [141–143].

Through regulating the expression of substrate proteins,Fbw7 suppresses tumor metastasis in certain types of cancer,such as breast cancer [36]. Recent studies showed that c-Myccan regulate the epithelial-to-mesenchymal transition (EMT)which is necessary for cellular invasion and migration bypromoting TGFβ-mediated activation of the SNAIL tran-scription factor [144]. Based on the previous reports, the roleof TGFβ is alternative in different stages of carcinogenesis.In early stage of tumor formation, TGFβ functions as atumor suppressor [145], but it will promote EMT andmetastasis during late stages of tumor development [134].This finding is contradictory with the notion that Fbw7promotes the degradation of negative regulator of TGFβ,TGIF1. Additionally, Balamurugan et al. reported that C/EBPδ directly inhibits Fbw7 gene expression, thus promot-

ing the mTOR/AKT/HIF-1 pathway, contributing to cellmigration and metastasis [36]. These studies demonstratethat the complexity of Fbw7-mediated signaling network andthe exact role of Fbw7 in tumor metastasis depend on thestages of cancer progression and type of cancer.

4.4 Fbw7 mutation

As a major regulator of a set of oncoproteins, Fbw7 werefound lost and mutated in various human cancers [11].Fbw7 gene localizes to 4q31.3, which is deleted in ∼30% ofhuman cancers [146]. By using a mammalian geneticscreen, Mao et al. found that Fbw7 loss cooperates p53 topromote tumor formation in mice, indicating that Fbw7 isp53-dependent, haplo-insufficient tumor suppressor gene[13]. Moreover, one study identified mutations in Fbw7 inboth human colorectal cancers and their precursor lesions,which result in chromosomal instability [147]. Thesefindings together suggest the tumor suppressor role ofFbw7 in human cancers.

Fbw7 status has been examined in numerous primaryhuman tumors. A genetic study of primary tumors showedthat the overall mutation rate in Fbw7 is approximately 6%,but the mutation rates are quite tumor type dependent [18].Cholangiocarcinoma and T-ALL harbor highest frequencyof mutations in 35% and 31%, respectively [18]. While intumors of stomach, colon, pancreas, and endometrium, only9–15% tumors contained Fbw7 mutations [17, 18, 23, 147].Interestingly, many tumor types including acute myeloidleukemia, breast, bladder, bone, liver, lung, and melanomashow rare (≤4%) or no mutations in Fbw7 gene [18, 148–151]. Notably, 6% of Fbw7 mutations are isoform-specific,suggesting each isoform may play a distinct role in humantumorigenesis. It remains to be determined whether Fbw7isoforms function cooperatively or separately in Fbw7-mediated protein ubiquitylation. Akhoondi et al. alsoidentified the two mutational hotspots within WD40 repeatsdomain: Arg465 and Arg479. These two residues account forapproximately 40% of all tumor-related mutations in Fbw7[18]. The point mutations occur in key residues disturbedthe formation of substrate binding interface, therefore resultin failure of substrate binding.

Fbw7 mutation was most extensively studied in T-ALLcompare with other tumor types. Notch pathway plays thecentral role in T-ALL development; 56% of T-ALL casesharbor Notch mutations [92]. Majority of mutations foundin Notch locus are located in two regions, the HD(heterodimerization domain 26%) and PEST (12.5%)domains. Mutations in HD domain induce ligand-independent proteolytic cleavage of Notch, which leads toactivation of Notch, while mutations in PEST domainincrease the half-life of NICD by blocking Fbw7 interac-tion. Fbw7 mutations were identified in 31% of T-ALL


patients, which are the second frequent genomic lesions inT-ALL [12]. O’Neil et al. show that Fbw7-mutated T-ALLcell lines cannot bind to the NICD, therefore causes NICDaccumulation [90]. NICD accumulation in turn stabilizesanother Fbw7 substrate c-Myc, both of which contribute toleukemia transformation. It seems that disturbed Notchdegradation by Fbw7 loss-of-function mutation is the majormechanism responsible for T-ALL pathogenesis.

Since GSK3 is the major priming kinase which mediatesthe phosphorylation of Fbw7 substrates, interruption ofGSK3 activity affects Fbw7 substrate stability. Severalactivated signaling pathways, such as Wnt and PI3K–Aktsignaling pathway, as well as reduced GSK3 activity werefound in many cancers [152]. In fact, phosphatase andtensin homolog (PTEN), which is an antagonist of the PI3Kpathway, is one of the most frequently inactivated tumorsuppressor genes, especially in T-ALL [12]. One compre-hensive study on 47 T-ALL patients indicated that 26 casesare with PTEN mutation, while five cases with Fbw7mutations [153]. Interestingly, this group also found thatPTEN transcripts positively correlated with c-Myc tran-script levels. The relationship between PTEN and Fbw7substrates still needs further investigation.

5 Conclusion

No doubt, Fbw7 is a crucial mediator in cancer development.Mounting evidence suggests that Fbw7 controls multiplecellular functions through regulation of several importantproteins, including c-Myc, cyclin E, c-Jun, Notch, and Mcl-1.Fbw7 is also a tumor suppressor, the regulatory network ofwhich is frequently disrupted in many human malignancies.Numerous mutations in Fbw7 and its substrates have beenidentified and proved to be related to cancers. Betterunderstanding of the structural and functional aspects ofFbw7 and its role in tumorigenesis will definitely be beneficialto the development of new anti-cancer therapies targetingFbw7 or its oncogenic substrates.

Acknowledgments This work was supported by Canadian Institutesof Health Research (MOP-84559, MOP-93810, and MOP-110974),Canadian Cancer Society Research Institute (2011-700714), andCanadian Dermatology Foundation to G. Li. Y. Cheng is a recipientof the trainee award from Canadian Institute of Health Research SkinResearch Training Centre and University of British ColumbiaGraduate Fellowship.

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Role of the ubiquitin ligase Fbw7 in cancer progression