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. Author manuscript; available in PMC: 2013 May 11.
Published in final edited form as: Oncogene. 2010 Jun 14;29(35):4865–4873. doi: 10.1038/onc.2010.222

The ubiquitous nature of cancer: the role of the SCFFbw7 complex in development and transformation

KM Crusio 1,2, B King 1,2, LB Reavie 1,2, I Aifantis 1,2
PMCID: PMC3651593  NIHMSID: NIHMS461140  PMID: 20543859

Abstract

The ubiquitin-proteasome system (UPS) is a multi-subunit pathway that allows for ubiquitin modification of proteins and leads to either degradation or other non-proteolytic processes such as trafficking or transcriptional activation. Given its role as a regulator of cellular homeostasis it is not surprising that members of the UPS are frequently aberrantly expressed in a number of disease states including cancer. This review will focus on one member of the UPS, the F-box protein, Fbw7 (also known as Sel-10, Ago, hCDC4) and mechanisms by which Fbw7 interacts with its substrates in the context of development and tumorigenesis will be discussed. In addition, antagonists of this pathway as well as current and future therapeutics for the UPS will be examined.

Keywords: Fbw7, ubiquitin-proteasome system, GSK-3 β, p53

The ubiquitin-proteasome system (UPS)

The ubiquitin-proteasome system (UPS) is considered the primary destruction mechanism of regulatory proteins within the eukaryotic cell and thus serves as a regulator of cellular homeostasis. Its function has been shown to be critical to adult stem cell quiescence, cell cycle, immune response, transcription, DNA damage repair and apoptosis (Schwartz and Ciechanover, 2009). It is thus not surprising that members of the UPS are frequently found mutated in a number of disease states including cancer. Studies of SCFFbw7, a well-characterized member of the UPS, offers insight as to how the UPS can function as a modulator of disease and offer novel druggable targets.

The UPS exerts its function through an enzymatic cascade that consists of E1, the ubiquitin-activating enzyme, E2, the ubiquitin-conjugating enzyme and E3, the ubiquitin-protein ligase (Schwartz and Ciechanover, 2009). Briefly, the E1 enzyme activates ubiquitin in an adenosine triphosphate-dependent manner by forming a thiol ester bond with ubiquitin, which is subsequently transferred to E2. The E2 enzyme, also known as ubiquitin-carrier protein, cooperates with the E3 ligase, which properly positions the target protein and allows the transfer of activated ubiquitin forming a covalent bond between the carboxy-terminus of ubiquitin and the ε-amino group of a lysine residue within the target protein. Ubiquitin has several acceptor lysines for ubiquitin conjugation, which can affect the length and thus function of the ubiquitin modification (Pickart and Eddins, 2004). Substrates can be modified with a single ubiquitin (monoubiquitination) or alternatively multi-monoubiquitined, or polyubiquitinated. It has been shown that ubiquitin chains extended from ubiquitin lysine 11 and 48 targets proteins to the 26S proteasome. The 26S proteasome is a large multi-subunit protease complex that is responsible for the proteolytic activity of the UPS. Chains extended through lysine 29 and 63 or monoubiquitination function as a nonproteolytic signal leading to the regulation of transcription factors, kinases and chromatin regulators (Mukhopadhyay and Riezman, 2007).

Among the members of this cascade, the E3 ligase provides substrate specificity. It has been estimated that several hundred E3 ligases exist in the human genome, although relatively few have been characterized (Pickart and Eddins, 2004). Two major classes of E3s have been identified, the HECT (homologous to E6AP C-terminus) domain and the RING-finger domain containing E3 ligases (for review, see Weissman (2001)). RING-finger domain containing E3 ligases consist of single and multi-subunit subcategories. Included within the multi-subunit subcategory are the cullin-RING ubiquitin ligases and the anaphase promoting complex/cyclosome ligases, both of which have been shown to have an important role in cell cycle progression (Skaar and Pagano, 2009). The SCF (SKP1/CUL1/F-box) complex is among the best studied multi-subunit RING-finger ligases. It contains a cullin family scaffolding protein, which binds a catalytic RING finger (RBX1), which recruits E2 and SKP1 that interacts with the F-box that lends substrate specificity to the complex (Figure 1).

Figure 1.

Figure 1

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Pathway of Fbw7-mediated degradation. A putative Fbw7 substrate either harbors a negatively charged amino acid in the +4 position or is phosphorylated by a kinase at this position. It is then phosphorylated by an additional priming kinase, which allows the interaction of the substrate with the SCFFbw7 complex. Fbw7 holds the substrate in close proximity to the E2 enzyme (ubc), which enables the covalent conjugation of ubiquitin. The addition of polyubiquitin targets the substrate for degradation by the proteasome. Alternatively, a deubiquitinating enzyme removes covalently bound ubiquitn from the substrate preventing its degradation effectively stabilizing the substrate. Data discussed in this review show that Fbw7 regulates the stability of proteins that have been associated with proliferation suggesting Fbw7-mediated proteasomal degradation is a mechanism important to the maintenance of cellular quiescence. Conversely, antagonists to this pathway lead to cellular proliferation.

Although ubiquitin modification can lead to irreversible proteolysis, it is important to note that ubiquitin modification is reversible. The removal of ubiquitin is mediated by a family of deubquitnating enzymes (DUBs) a majority of which are cysteine proteases (Song and Rape, 2008). It has been estimated that approximately 100 DUBs are encoded in the human genome. DUBs function to remove ubiquitin from targeted substrates to rescue proteins from degradation and to remove ubiquitin tags affectively ending any signaling role ubiquitin modification may have provided. In addition, DUBs remove ubiquitin as a protein enters the 26S proteasome thereby recycling ubiquitin. On the basis of catalytic domain homology DUBs can be divided into five subfamilies: ubiquitin-specific proteases (Usp), ovarian tumor-like proteases, JaMM/MPN metalloproteases, ubiquitin carboxy-terminal hydrolases and Machado–Jakob-disease proteases. It has been shown that DUBs have a central role in cell cycle regulation, DNA damage response and, depending on the context, can function as either tumor suppressors or oncoproteins (Love et al., 2007).

The Fbw7 E3 ubiquitin ligase

Among the RING-finger domain E3 ligases, SCFFbw7, is one of the better studied. Its F-box protein, Fbw7, has been shown to not only bind the SCF but to also dimerize with itself (Welcker and Clurman, 2007). Fbw7 possesses three isoforms (Fbw7-α, β and γ) each having their own promoter and which vary in the alternative splicing of their first exons. This leads to differences in the N-terminal end of the protein, which affects localization of the protein as well as tissue- and animal-specific expression. Fbw7-α localizes to the nucleus as well as being the isoform that is predominately expressed in the mouse. Fbw7-β localizes to the cytoplasm and is found primarily in the human brain. Fbw7-γ is expressed in the nucleolus and has been shown to be mainly expressed in human skeletal muscle (Spruck et al., 2002).

Fbw7 has several important domains that are essential for its function. As its name suggests, the two essential components, which promote Fbw7 function, are the F-box and WD40 domains. The F-box domain associates with the SCF complex through direct interaction with Skp1 (Bai et al., 1996). A second important protein-protein interaction is carried out by eight tandem repeats of the WD40 domain that form a β-propeller structure containing three critical arginine residues (R465, R479 and R505), which recognize a specific consensus phospho-motif within the target substrate that has been termed the Cdc4 phospho degron (Hao et al., 2007).

The primary function of Fbw7 in regulating cellular homeostasis has been elucidated in several different model systems. Fbw7 was first identified in yeast as a regulator of cell cycle-related proteins and named Cdc4 (Hartwell et al., 1973). In Caenorhabditis elegans, Sel-10 (homlog of Fbw7) was identified as a negative regulator of Lin-12 (homolog of Notch) and from these studies the mammalian homolog was identified and termed Fbw7 (Hubbard et al., 1997). Work in all of these systems has identified a handful of Fbw7-specific substrates including cyclin E, Notch, c-Myc, c-Jun, SREBP, presenilin and Aurora A (Hubbard et al., 1997; Gupta-Rossi et al., 2001; Koepp et al., 2001; Moberg et al., 2001; Oberg et al., 2001; Strohmaier et al., 2001; Li et al., 2002; Anand et al., 2003; Yada et al., 2004; Welcker et al., 2004a, b; Sundqvist et al., 2005; Wei et al., 2005; Thompson et al., 2007).

In this review, the interaction between Fbw7 and c-Myc will be used as an example to show how Fbw7 binds specific substrates. c-Myc is a transcription factor that is thought to bind and regulate expression of at least 15% of all genes, driving primarily cell growth, proliferation and differentiation (Palomero et al., 2006). c-Myc is also a well-known oncogene and is typically overexpressed in many different tumor types. Fbw7 has been shown to interact with c-Myc through its Cdc4 phospho degron, which is located in Myc Box1 within the transactivation domain (Yada et al., 2004; Welcker et al., 2004b).

For c-Myc to be efficiently degraded, Fbw7 binding requires at least two initial phosphorylation events within the Cdc4 phospho degron, one on a serine residue (S62 of c-Myc) and a second one involving a threonine residue located at c-Myc amino acid position 58 (T58, TPPLS) (Henriksson et al., 1993; Lutterbach and Hann, 1994). This two step priming event is required for degradation of most, if not all, Fbw7-specific substrates. Although phosphorylation of a central serine/threonine residue (T58 of c-Myc) is essential to enable Fbw7 binding, this phosphorylation event cannot occur without the existence of either a negatively charged amino acid at the +4 position to this site or a initial phosphorylation event occurring at the +4 site (that is, phosphorylation of S62 of c-Myc). It has been shown that S62 phosphorylation is required to prime T58 for the second phosphorylation event. This +4 residue within the Cdc4 phospho degron is conserved in all known Fbw7 substrates either as a negatively charged amino acid residue or by a second phosphorylation event (Welcker and Clurman, 2007). Together, S62 and T58 phosphorylation enable ubiquitination and c-Myc protein degradation (Sears et al., 2000). It is noteworthy that T58 phosphorylation must occur for Fbw7 to bind c-Myc. Conversely, S62 phosphorylation alone is insufficient to initiate degradation (Sears et al., 2000). Interestingly, T58 mutations in c-Myc are found in many human cancers, most notably human lymphomas (Bhatia et al., 1993; Yano et al., 1993). However, in the case of c-Myc, the role that S62 phosphorylation has in contributing to proteasomal-dependent degradation beyond its requirement for priming T58 phosphorylation has not been determined.

GSK3- β: a Fbw7 priming kinase

Although a two step priming event is a central process leading to proteasomal degradation of Fbw7 substrates not all the mediators of this process have been well delineated. It is known, however, that phosphorylation of c-Myc at T58 is mediated by glycogen synthase kinase 3 β (GSK3-β) (Welcker et al., 2004b). In fact, GSK3-β is the priming kinase for most known Fbw7 substrates with the exception of Notch and presenilin. In general, GSK3-β binding to many of its substrates occurs through the recognition of either the phosphorylation or negatively charged +4 residue (S62 on c-Myc) (Cohen and Frame, 2001). Several signaling pathways such as insulin and phosphatidylinositol 3 kinase/Akt have been observed to inhibit GSK3-β activity through phosphorylation of an N-terminal serine residue that renders GSK3-β inactive (Cohen and Frame, 2001). Therefore, it can be postulated that an inactive form of GSK3-β can lead to substrate stabilization by lack of Fbw7-mediated degradation among other cellular consequences (Figure 1). For these reasons, it is worth noting that the regulation of protein stability mediated by GSK3-β activity or Fbw7 could have a profound effect on cellular function, particularly stem cell function.

In a recent study, Bechard and Dalton showed that GSK3-β activity, regulated by phosphatidylinositol 3 kinase/Akt signaling, controls c-Myc stability and consequently murine embryonic stem cell (mESC) self-renewal and differentiation (Bechard and Dalton, 2009). Active phosphatidylinositol 3 kinase/Akt signaling sequestered GSK3-β in the cytoplasm by constitutively phosphorylating serine 9. Inactivation leading to GSK3-β hypo-phosphorylation of c-Myc at T58 confers c-Myc protein stability, which sustained mESC self-renewal. Conversely, when LIF (an essential factor for mESC self-renewal) was removed to initiate differentiation, phosphatidylinositol 3 kinase/Akt signaling was inhibited leading to dephosphorylation of GSK3-β and translocation to the nucleus in which T58 phosphorylation occurred and Fbw7-mediated proteasomal degradation could be initiated (Bechard and Dalton, 2009 and our unpublished data). These data suggest that the availability of priming kinases such as GSK3-β could add yet another level of complexity to the UPS. Thus, GSK3-β activity could be a limiting factor for protein degradation in addition to the availability or tissue specificity of the E3 ligase itself.

Recently, Fbw7 has been shown to bind and target m-Tor and c-Myb for degradation adding two additional well-studied proto-oncogenes to its list of substrates (Kanei-Ishii et al., 2008; Mao et al., 2008; Kitagawa et al., 2009). As many substrates of Fbw7 have been comprehensively discussed in other reviews (see Welcker and Clurman, 2008) we will briefly mention some of the latest additions.

mTor: a Fbw7 substrate

A few known functions of the mammalian target of rapamycin protein (mTor) are promotion of cell growth, proliferation and survival. The mTor protein is commonly overexpressed in different tumor types yet little is known regarding mTor regulation in either a normal or tumorigenic environment. Recently, Mao et al. identified a putative Fbw7 degron within mTor that facilitates Fbw7 binding, ubquitination and degradation. It is noteworthy that this work showed breast cancer cells lines harboring Fbw7 mutations showed increased sensitivity to the mTor inhibitor, rapamycin. This could implicate Fbw7 mutations as a responder context for patients who would be expected to be more sensitive to treatment with mTor inhibitors (Mao et al., 2008).

c-Myb: a Fbw7 substrate

c-Myb is a proto-oncogene involved in controlling the proliferation, differentiation and cell fate decisions of hematopoietic cells (Oh and Reddy, 1999). c-Myb is also deregulated in many human cancers, particularly, acute myeloblastic and lymphoblastic leukemias (Slamon et al., 1986; Siegert et al., 1990). In these tumors, it is thought that c-Myb overexpression leads to inhibition of terminal differentiation and enhanced proliferation. Until recently, little data have emerged describing how the levels of c-Myb are controlled. Two independent groups simultaneously identified Fbw7 as the E3 ligase responsible for the ubiquitination and proteasomal degradation of c-Myb. These data showed that Fbw7 binds c-Myb and facilitates ubiquitination through a phosphorylation-dependent event involving Thr-572, Ser-556 and Ser-528 (Kanei-Ishii et al., 2008; Kitagawa et al., 2009). However, both studies identify a different kinase that is responsible for priming c-Myb for Fbw7 binding. The discrepancy between studies regarding which kinase is responsible for priming c-Myb for proteasomal degradation has yet to be reconciled but perhaps suggests that one kinase can predominant depending on cell specificity or upstream signaling cascades.

Usp28: an Fbw7 antagonist?

The ubiquitin ligase activity of Fbw7 has been shown in a number of cancer cell lines to be antagonized through a deubiqutinating enzyme called Usp28 (Popov et al., 2007b). It has been suggested that Usp28 interacts directly with nuclear Fbw7-α. Like most deubiquitinating enzymes, Usp28 is a cysteine protease and it has been shown that it is able to remove ubiquitin from both c-myc and cyclin E, thus preventing their degradation. Usp28 function has been shown to have an important role within two important cellular pathways: DNA damage-induced apoptosis and proliferation.

In separate work it has been shown that in response to DNA damage Usp28 can regulate checkpoint mediators 53BP1 and Claspin (Zhang et al., 2006; Bassermann et al., 2008). In addition, in response to DNA damage, levels of c-myc decline because of enhanced proteasomal degradation through Fbw7. It has been suggested that Usp28 dissociates from Fbw7 in response to DNA damage thus allowing it to interact with members of the DNA damage pathway. This suggests that Usp28 in different cellular contexts is a mediator of proliferation (Popov et al., 2007a).

Usp28 is frequently overexpressed in breast adenocarcinomas and colon carcinomas (Popov et al., 2007b). It has been shown in vitro that Usp28 overexpression leads to an increase in c-myc stability. It has therefore been suggested that this overexpression leads to increased proliferation of varying tumor types. As Usp28 seems to have an antagonistic role within proteasomal regulation of Fbw7 substrates, many of which are oncoproteins, therapeutically silencing the activity of Usp28 may enhance Fbw7-mediated degradation and limit cell proliferation (Figure 1).

Fbw7: role in development

As Fbw7 has been shown to regulate important mediators of development and tumorigenesis, knockout mice were generated to study the effects of Fbw7 ablation on substrate stabilization in an in vivo environment. However, germline deletion resulted in embryonic lethality at d10.5–11.5 because of defects in vascular development. It is noteworthy that two independent groups observed a stabilization of Notch4 in the Fbw7 null embryos and showed that Fbw7 is indispensable for vascular development in mice (Tetzlaff et al., 2004; Tsunematsu et al., 2004). One of these studies concluded that additional Fbw7 substrates are stabilized in the knockout embryo (Notch1) and placental tissues (cyclin E) (Tetzlaff et al., 2004). Regardless of these discrepancies, these data infer that Fbw7 has an essential role in the developing embryo.

To study Fbw7 function in adult mice, conditional Fbw7 knockout mice were generated in several laboratories, which collectively have emphasized the importance of Fbw7, particularly the hematopoietic system. Our laboratory and others have shown that deletion of Fbw7 in hematopoietic stem cells (HSCs) results in the premature loss of the long-term HSC compartment. (Onoyama et al., 2007; Thompson et al., 2008). It was hypothesized that this loss is due to the aberrant cell cycle entry of LSKs (a population highly enriched in HSCs) as a consequence of Fbw7-mediated c-Myc protein stabilization (Matsuoka et al., 2008; Thompson et al., 2008; Reavie et al. 2010). Matsuoka et al. additionally showed that Fbw7 mediates c-Myc stabilization in HSCs eventually triggering p53-dependent apoptosis (Matsuoka et al., 2008). Using a c-Myc-eGFP knock-in strategy, we have shown that c-Myc protein was indeed stabilized in both the LT-HSC and LSK compartment of Fbw7 null mice (Reavie et al. 2010). Moreover, we observed a rescue of the Fbw7 null HSC phenotype when levels of c-Myc protein are reduced; assessed by a rescue in the percentage of HSCs, cell cycle status and in vitro colony-forming ability.

Not only is Fbw7 important for HSC biology, it has been observed to also influence the cell fate of progenitor and mature subsets in the hematopoietic compartment (Onoyama et al., 2007; Thompson et al., 2007). Using the Mx-1-cre system, Thompson et al. found that deletion of Fbw7 decreases the absolute number of early thymic progenitor population resulting in an overall reduction of total thymocytes. Onoyama et al. also observed a thymic phenotype when Fbw7 was deleted on cre expression driven by the LCK promoter thereby ablating Fbw7 in T-cell-committed progenitors. Immature T cells in mice lacking Fbw7 failed to exit the cell cycle at the double-positive stage (CD4+ CD8+) suggesting that in a wild-type cell Fbw7 effectively harnessed the cell cycle at this stage in T-cell development. It was hypothesized that failure to exit the cell cycle was mediated by c-Myc stabilization and not Notch, as ablation of Rbpj, a mediator of Notch signaling, did not rescue the Fbw7-deficient effects. Finally, deletion of Fbw7 in mature T cells was found to induce p53 expression leading to cell cycle arrest and apoptosis in CD4 or CD8 expressing T cells (Onoyama et al., 2007).

In contrast to adult HSCs that are mainly quiescent, self-renewing mESCs cycle rapidly and express relatively low levels of Fbw7 (Reavie et al. 2010). It is widely accepted that high levels of c-Myc are essential to sustain the in vitro self-renewal capacity of mESCs (Cartwright et al., 2005). On differentiation, we have shown that Fbw7 protein is upregulated whereas c-Myc protein is rapidly downregulated. Small interfering RNA/short hairpin RNA-mediated knockdown of Fbw7 lead to an accumulation of both phospho- and total c-Myc protein as cells differentiate (Bechard and Dalton, 2009; Reavie et al. 2010). The difference in the expression pattern of the Fbw7:c-Myc axis in mESC compared with adult HSCs raises the interesting point that the interaction of Fbw7 and c-Myc can promote opposing cellular functions (self-renewal versus differentiation) in different stem cell populations. These data together support the notion that Fbw7 and the UPS are key regulators of cell fate determination and may function differently different developmental contexts evolve.

Fbw7 as a tumor suppressor

As Fbw7 regulates the stability of an ever-growing list of oncoprotein substrates, it is perhaps not surprising that it is implicated in wide variety of human cancers (Tan et al., 2008). Fbw7 is located on chromosomal region 4q32, which is frequently lost in tumors (Spruck et al., 2002). In a comprehensive study of over 1500 human tumors, Akhoondi et al. found that, overall, 6% of tumors harbored mutations in the Fbw7 coding regions. Mutations were most frequently identified in cholangio-carcinoma (35%), T-cell acute lymphocytic leukemia (T-ALL: 31%), and tumors of colon (9%), endometrium (9%) or stomach (6%) (Akhoondi et al., 2007). Strikingly, nearly half (43%) of these were missense mutations that resulted in amino acid substitutions at key arginine residues within the WD40 domain (Arg465 and Arg479), which are shared by all three Fbw7 isoforms. The relatively low frequency of mutations found within the isoform-specific N-termini (6%) suggests that all three isoforms might collectively contribute to its tumor-suppressor function.

In hematological malignancies, Fbw7 mutations arise primarily in T-ALL. T-ALL is characterized by infiltration of the bone marrow, peripheral blood and secondary lymphoid organs with immature lymphoblasts that express T-cell surface markers (Ferrando, 2009). Activating mutations in NOTCH1 are predominant in T-ALL and were identified in approximately 50% of human T-ALL cell lines (Weng et al., 2004). NOTCH1 mutations tend to occur in either the HD domain, which allows signaling in the absence of ligand binding, or the PEST domain, which inhibits its phosphorylation and subsequent polyubiquitination by SCFFbw7 (Fryer et al., 2004). Either mutation results in accumulation of intracellular NOTCH1 and increased expression of NOTCH1 target genes such as Deltex1, Hes1 and c-Myc. Mutations in Fbw7 are the second most common genomic lesion in T-ALL and were identified in 30% of clinical samples (Maser et al., 2007). The role of Fbw7 mutations in T-ALL was discovered in two separate studies examining mechanisms of resistance to (γ)-secretase inhibitors, which block the secondary cleavage event that releases the intracellular signaling domain of Notch (O’Neil et al. 2007; Thompson et al., 2007). Similarly to activating mutations in Notch1, Fbw7 mutations lead to accumulation of intracellular Notch. However, the mechanism of resistance to (γ)-secretase inhibitors in Fbw7 mutant cell lines was mediated instead by stabilization of c-Myc. Therefore, it is likely that stabilization of Notch1 is the switch that drives hematopoietic progenitor cell fate specification toward the T-cell lineage (Pui et al., 1999), but it is c-Myc stabilization that allows the extensive proliferation of immature lymphoblasts.

Unlike many known tumor suppressors, Fbw7 tends to be mutated in only one allele and loss of heterozygosity does not seem to be required for oncogenic transformation. Given the nature of the Fbw7 mutations manifested in human cancers, it is still unclear whether Fbw7 is truly a haploinsufficient tumor suppressor or if mutant forms can exert a dominant-negative function. The observation that SCFFbw7 complexes can form homodimers lends to the hypothesis that mutant Fbw7 may also inhibit, in trans, the ability of wild-type complexes to efficiently polyubiquitinate their substrates. Although Fbw7−/− mice show embryonic lethality, Fbw7+/− on other develop but mice, the hand, normally show increased susceptibility to radiation-induced tumors (Mao et al., 2004). Therefore, a single wild-type allele is sufficient for the developmental role of Fbw7, but may be insufficient for its role as tumor suppressor under conditions of cellular stress.

Conditional knockout alleles have been generated in mice to study whether the loss of Fbw7 in somatic tissues can lead to enhanced cell proliferation and tumorigenesis (Onoyama et al., 2007; Thompson et al., 2008). Loss of Fbw7 in the thymus resulted in the expansion of the CD4+ CD8+ T lymphocyte population and led to the development of thymic lymphomas in approximately half of the animals studied (Onoyama et al., 2007). Although both Notch1 and c-Myc protein levels were increased in the tumor population, the proliferation of CD4+ CD8+ thymocytes was determined to be predominantly Myc-dependent. Ablation of Rbpj, an essential component of the Notch transcriptional complex, did not rescue the effects of Fbw7 deficiency in the thymus. The late age of disease onset and the fact that not all Fbw7 conditional knockout mice go on to develop lymphomas implies that additional genetic events may necessary for tumor formation. Furthermore, Fbw7 conditional knockout mice developed lymphomas with increased penetrance and reduced latency on the additional loss of p53 (Matsuoka et al, 2008). The synergistic effect of these genes had been previously observed in epithelial tumors induced in Fbw7+/− p53+/− mice (Mao et al. 2004).

The prevailing hypothesis for the genetic association of Fbw7 and p53 is that pathophysiological levels of oncoproteins, particularly c-Myc, Notch and cyclin E, accumulate in the absence of Fbw7, which can then trigger p53-dependent mechanisms of apoptosis or senescence (Figure 2). Oncogene-induced apoptosis or senescence is mediated by the induction of p19ARF, which inhibits Mdm2 and ultimately leads to the stabilization of p53 (Lowe and Sherr, 2003). Fbw7-deficient mouse embryonic fibroblasts prematurely senesce and have elevated expression of p16, p19ARF and p21, albeit decreased levels of p27Kip1 and p57Kip2 (Masuda et al. 2010). Cell cycle arrest in Fbw7-deficient mouse embryonic fibroblasts was rescued by the additional loss of p53 (Ishikawa et al., 2008). Accordingly, p19ARF and p53 are frequently mutated in lymphomas in which MYC is overexpressed (Eischen et al., 1999). In Burkitt’s lymphomas without p53 or p19ARF mutations, MYC itself is frequently mutated at residues P57 or T58, which are located in the conserved phosphodegron region bound by Fbw7 (Hemann et al., 2005). However, it was determined that T58A or P57S mutations did not greatly enhance the stability of c-Myc in these lymphomas, but hindered its ability to induce the expression of Bim, a pro-apopotic BH3-only protein. Similarly, overexpression of cyclin E has been shown to contribute to oncogenic transformation and genomic instability in the absence of p53 (Minella et al., 2002). Mice expressing mutant alleles of cyclin E that cannot be bound or ubiquitinated by SCFFbw7 show no phenotypic abnormalities on wild-type p53 background, but show a significant increase in tumor incidence on a p53 null background when compared with loss of p53 alone (Loeb et al., 2005; Smith et al., 2006). Although it is difficult to assess the complex and cell context-dependent roles of Fbw7 by overexpression of its substrates individually, these studies corroborate that the loss of p53-dependent tumor surveillance mechanisms is likely to be a necessary step in the transformation of Fbw7 mutant tumor-initiating cells. Whether this genetic association holds true for human tumors with Fbw7 mutations remains to be determined.

Figure 2.

Figure 2

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Proposed mechanism of malignant transformation mediated by Fbw7 mutations. Somatic missense mutations in any of the three critical arginine residues (Arg465, Arg479 or Arg505) within the WD40 domain of Fbw7 abrogate its interaction with Cdc4 phospho degron (CPD) motifs on target substrates. Phosphorylated substrates are no longer bound and polyubiquitnated by SCFFbw7 and accumulate in the nucleus. Accumulation of Fbw7 targets, such a c-Myc, cyclin E and Notch-1, can trigger oncogene-induced apoptosis and senescence, which is largely dependent on p53 status. Cells harboring mutations in Fbw7 are selectively pressured to inactivate p53 for continued proliferation and survival, ultimately leading to increased genomic instability and tumorigenesis.

There is increasing evidence to support the notion that loss of function mutations in Fbw7 could expedite the onset of additional genetic lesions that lead to malignancy. Targeted disruption of Fbw7 in karyotypically stable colorectal cancer cells resulted in a significant increase in micronuclei formation and the gain or loss of chromosomes (Rajagopalan et al., 2004). Aneuploidy induced by the loss of Fbw7 was attributed to the deregulation of both cyclin E and Aurora-A, which have both been previously implicated in chromosomal instability (Zhou et al., 1998; Spruck et al., 1999; Anand et al., 2003). Normally, cyclin E is degraded as cells progress through S phase to prevent the reinitiation of DNA replication and additional amplification of centrosomes before mitosis. In the absence of Fbw7, however, cyclin E is constitutively expressed throughout the cell cycle, resulting in a prolonged S phase. Aurora-A, in contrast, is normally active during later stages of the cell cycle and is critical for mitotic spindle formation and centrosome maturation (Barr and Gergely, 2007). Loss of Fbw7 has been shown to increase the incidence of endoreduplication and polyploidy in cells treated with chemotherapeutic agents that target the mitotic spindle (Finkin et al., 2008). Elevated levels of Aurora-A and cyclin E were required for this phenotype, but their overexpression alone was insufficient to override the tetroploidy checkpoint. More recently, centrosomal abnormalities have been directly linked to aneuploidy through the lagging of chromosomes during anaphase (Ganem et al., 2009). Centrosomal abnormalities are likely to occur in Fbw7-deficient cells, given the deregulation of both cyclin E and Aurora-A, but this association has yet to be studied directly. Nevertheless, there is strong evidence to suggest that mutations in Fbw7 can compromise genomic integrity and could, therefore, be a primary event leading to tumorigenesis.

Targeting UPS and beyond

As the majority of Fbw7 substrates are oncoproteins, it stands to reason that enhancing Fbw7-mediated degradation would be important goal of chemotherapeutics. However, development of agonists for F-box proteins is notoriously difficult because of the lack catalytic pockets for small molecule binding (Petroski, 2008). Given that Usp28 is catalytically active, is overexpressed in a number of tumor types and that it has an antagonistic role to Fbw7-mediated degradation, it lends itself as a putative therapeutic target. Studies have shown the efficacy of targeting DUBs with small molecules in various cancers (Love et al., 2007; Schwickart et al., 2010). It remains to be seen whether such small molecules can enhance Fbw7 degradation of oncoproteins in various tumor cells.

Given the critical role the UPS has in cellular homeostasis it may be inferred that enhancement or inhibition of one or many members of the UPS would be detrimental to the cell and not well tolerated as a chemotherapeutic. However, this is not the case. The ability to effectively silence the UPS and be well tolerated was first shown with the use of Bortezomib (Kane et al., 2003, 2007). Bortezomib (PS-341, Velcade Millennium Pharmaceuticals, Cambridge, MA, USA) is a reversible inhibitor of the catalytic activity of the 26S proteasome. It has been used successfully for the treatment of mantle cell lymphoma and multiple myeloma. The demonstration that Bortezomib, a universal inhibitor of the proteasome, could be used effectively suggests that more specific inhibitors of UPS could be developed as novel chemotherapeutics with an improved efficacy. One such example is MLN4924, a small molecular inhibitor of the NEDD8-activating enzyme an E1 enzyme of the neddylation pathway, which leads to the functionally necessary modification of the cullin family of scaffolding proteins (Soucy et al., 2009). Although retaining a relatively broad effect, initial studies have suggested that inhibition of NEDD8-activating enzyme is more specific to the cellular homeostasis of cancer cells then general proteasome inhibition suggesting it would be more effective and perform better in safety profiles.

Conclusions

The UPS has been previously shown to be an important mechanism in cellular homeostasis. In this review the role of Fbw7, a Fbox protein, has in the maintenance of this homeostasis was examined. Work summarized here shows that it is critical regulator of development in both fetal and adult tissue. Fbw7 exerts this affect by targeting of a number of proteins that are important to the proliferation of the cell. It is thus not surprising that Fbw7 has been shown to function as a tumor suppressor and is often mutated in a number of cancers. Owing to the affect Fbw7 mutations have on stabilizing cell proliferation proteins as well as how, in combination with p53 mutation, this could lead to genomic instability, it has been suggested that Fbw7 mutations can be a primary event on the path toward malignant transformation. Although known effective drugs that target the UPS discussed here inhibit degradation by either blocking the proteasome (Bortezomib) or cullin assembly (MLN4924), drugs that enhance Fbw7-mediated degradation would be advantageous in various malignancies. To that end, drug discovery within the UPS is still at its infancy but novel targets within the DUB family are particularly interesting because of the catalytic accessibility of DUBs as well as the antagonistic role they have within the UPS. Thus, recent work studying the UPS, and in particular Fbw7, has revealed many layers of complexity regulating cellular maintenance but also has offered many novel therapeutic targets, which can open new era of drug discovery.

Acknowledgements

We thank the members of the Aifantis Lab for advice and illuminating discussions. Supported by the National Institutes of Health (RO1CA133379, RO1CA105129, R21CA141399, RO1CA149655 and P30CA016087), the American Cancer Society (RSG0806801), the Edward Mallinckrodt Jr Foundation, the Irma T Hirschl Trust, the Alex’s Lemonade Stand Foundation (all to IA), NYU Molecular Oncology and Immunology/Ruth L Kirchstein Institutional Training Grant (5T32CA009161 to KC), NYU Cell and Molecular Biology/Ruth L Kirchstein Institutional Traning Grant (BK). IA is a Leukemia & Lymphoma Society Scholar and an Early Career Scientist at the Howard Hughes Medical Institute.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

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