The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of ∼6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational “hotspots,” which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer. [Cancer Res 2007;67(19):9006–12]

The ubiquitin-proteasome system regulates important cellular processes including differentiation and development, apoptosis, protein trafficking, the immune and inflammatory responses, cell cycle progression, and cellular division (1). The latter processes are primarily regulated by two ubiquitin ligases known as the anaphase-promoting complex (APC) and SCF. SCF ubiquitin ligases are composed of Cul1, Rbx1 (also called Roc1 or Hrt1), and Skp1 bound to a member of the F-box protein family, which provide substrate specificity. Approximately 65 F-box proteins exist in humans, each characterized by an ∼40-amino-acid F-box motif that associates with the SCF complex through Skp1 (1).

In S. cerevisiae, the F-box protein Cdc4 plays a critical role in cell cycle control by mediating the ubiquitin-dependent proteolysis of DNA replication protein Cdc6 and cyclin-dependent kinase (Cdk) inhibitors Far1 and Sic1 (2, 3). Similarly, the orthologue of Cdc4 in humans, designated Fbxw7 (also known as hCdc4, Fbw7, or SEL-10), mediates the ubiquitin-dependent proteolysis of several key regulatory proteins involved in cell division and cell fate determination, including cyclin E1, c-Myc, c-Jun, Notch, and Aurora-A (2). Furthermore, FBXW7 deficiency in mice has been shown to result in early embryonic lethality (day ∼10.5) with embryos exhibiting abnormalities in hematopoietic and vascular development (2).

Several lines of evidence suggest that Fbxw7 is a putative tumor suppressor in human tumorigenesis, including (a) the well-defined oncogenic potential of its putative substrates (described above); (b) localization of FBXW7 to chromosome 4q31.3, which is deleted in ∼30% of human cancers; (c) the finding that allelic loss of FBXW7 cooperates with p53 in tumorigenesis in mice (4); and (d) demonstration that targeted disruption of FBXW7 in cultured cells leads to an increase in genetic instability (5), a hallmark of human cancers.

In this study, we carry out an extensive genetic analysis of FBXW7 in primary human tumors of diverse tissue origin to determine its role as a putative tumor suppressor. We find that FBXW7 is mutated in a variety of human tumor types with an overall mutation frequency of ∼6%. Furthermore, we show that expression of an Fbxw7 mutant corresponding to one of the major mutational hotspots in primary tumors interferes with wild-type Fbxw7 function, suggesting a potential dominant negative mechanism of Fbxw7 inactivation. Our results show that FBXW7 is a general tumor suppressor in human tumorigenesis and provide insight into how Fbxw7 function is inactivated during tumorigenesis.

Tumor specimens. Primary tumor specimens (534 in total) were obtained from (a) Department of Obstetrics and Gynecology, Innsbruck Medical University, Innsbruck, Austria (breast and ovarian tumor specimens); (b) Sidney Kimmel Cancer Center, San Diego, CA (lung and prostate); (c) Department of Pathology and Gastroenterology, Uppsala University Hospital, Uppsala, Sweden (bile duct and liver); (d) Department of Oncology/Pathology, Karolinska Hospital, Stockholm, Sweden (hematologic malignancies and melanomas); and (e) Research Center for Gastrointestinal and Liver Disease, Talghani Hospital, Tehran, Iran (stomach, bladder, colon, and esophagus). This project was approved by the research ethics committees at the Karolinska Institute, Uppsala University Hospital, and Shahid Beheshti University of Medical Sciences. A total of 1,556 primary tumor specimens (our results pooled with previously published data) are included in our final analysis.

FBXW7 mutation screening. Screening for FBXW7 mutations was done as previously described (6).

Methylation analysis. Methylation status of mutational hotspot 1435-6 was determined by bisulfite sequencing as described (7). PCR amplifications were done with primers 5′-TGTGGAATGTAGAGATTGGAGAATGTATA-3′ and 5′-AAAAAATCCCAACCATAACAAAATTT-3′.

Antibodies. Antibodies used in this study include anti-Flag (Sigma), anti-Skp1 (Lab Vision), anti–cyclin E1 (HE12, Santa Cruz Biotechnology), anti–phospho-T380 cyclin E1 (Santa Cruz Biotechnology), anti-Myc (9E10, Santa Cruz Biotechnology), anti–glutathione S-transferase (GST; Santa Cruz Biotechnology), anti-actin (Santa Cruz Biotechnology), anti-p53 (DO-1, Santa Cruz Biotechnology), and anti-p21 (Ab-1, Calbiochem). Precipitation experiments were done using anti-Flag agarose (Sigma) or Glutathione-Sepharose (GE Healthcare). Immunohistochemical analysis of p21 and p53 was done as described (8). Analysis of ploidy was determined by Feulgen staining of histopathologic sections and image cytometry.

Plasmids and transfection. The full-length α-Fbxw7 cDNA was cloned into pFlag-CMV2 (Sigma) and GST expression vector pEBG. Expression plasmids for all FBXW7 mutants were generated using the GeneTailor Site-Directed Mutagenesis System (Invitrogen). Expression plasmid for 3× Myc-cyclin E1 was generated by cloning the full-length cyclin E1 cDNA into pcDNA3.1 (Invitrogen). Transfections were done using Lipofectamine 2000 (Invitrogen).

Molecular modeling. The molecular structure of α-Fbxw7 was created using the SWISS-MODEL program and viewed using the DeepView-SwissPDB-Viewer.

FBXW7 is mutated in a variety of primary human tumor types. To determine the role of FBXW7 as a tumor suppressor in human tumorigenesis, we genetically analyzed the complete coding region, including sequences unique to the three alternatively spliced Fbxw7 isoforms (α, β, and γ), in a variety of human tumor types. The results of this analysis, combined with previously reported data (917) for FBXW7 in primary tumors (1,556 specimens in total), are presented in Table 1. FBXW7 mutations were detected in tumors of diverse tissue origin, including those of the blood, breast, bile duct, colon, endometrium, stomach, lung, bone, ovary, pancreas, and prostate. The highest frequencies of mutations were observed in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), colon (9%), endometrium (9%), and stomach (6%). Overall, the frequency of FBXW7 mutations in all primary human cancers analyzed was ∼6% (87 of 1,556). DNA sequencing of matching negative biopsies from patients with stomach cancers showed that the FBXW7 mutations were exclusively in the tumor specimens, indicating that the mutations were somatic in nature.

Table 1.

FBXW7 mutations in primary human tumors

Tumor typeCurrent study, frequency (%)Previous studies, frequency (%)Nucleotide change*Amino acid change
Breast 1/122 (1)  A791G Q264R 
Bladder 0/20 (0)    
Cholangiocarcinoma 7/20 (35)  C1393T (×4) R465C 
   G1394A R465H 
   C1513T R505C 
   A1556G Y519C 
Colon 3/31 (10)  C1393T (×2) R465C 
   G1510A V504I 
  45/492 (9) C658T Q220X 
   C670T (×3) R224X 
   C832T (×4) R278X 
   C845A S282X 
   C907T Q303X 
   A936C R312S 
   C1099T R367X 
   C1177T (×3) R393X 
   G1190A G397D 
   G1268T G423V 
   C1313T S438F 
   G1338A W446X 
   C1393T (×7) R465C 
   G1394A (×2) R465H 
   G1436A (×7) R479Q 
   G1457A W486X 
   C1513T R505C 
   G1514C R505P 
   G1628A R543K 
   A1634G Y545C 
   C1745T (×4) S582L 
   C1787T S596F 
Endometrium§  9/102 (9) β-G67F V23I 
   G370T D124Y 
   C1099T R367X 
   2bp ins. 1110 376X 
   G1394A R465H 
   1bp ins. 1417 476X 
   G1436A (×2) R479Q 
   C1972T R658X 
Esophagus  0/27 (0)   
Leukemia  AML, 0/35 (0)   
 B-ALL, 0/20 (0)    
 B-CLL, 0/20 (0)    
 HCL, 0/20 (0)    
 T-ALL**, 8/26 (31)  G1394A (×4) R465H 
   C1435G(×2) R479G 
   G1436T R479L 
   C2065T R689W 
Liver 0/12 (0)    
Lung†† NSCLC, 1/38 (3) 0/50 (0) A32G K11R 
Melanoma 0/20 (0)    
Bone‡‡  1/47 (2) 3bp del., 424  
Ovarian§§ 0/32 (0) 2/111 (2) G734T S245I 
   G1411T E471X 
Prostate 1/83 (1)  3bp ins., 45 +CCT, +P 
Pancreas∥∥  1/11 (9) A1379G H460R 
Stomach¶¶ 8***,†††/52 (15)  C1393T (×4) R465C 
   G943A A315T 
   G1436T R479L 
   G1451C R484T 
   G1510A V504I 
  6/162 (4) G1318A D440N 
   C1393T R465C 
   G1947A W649X 
   1bp del, 1996 706X 
   G2021A R674Q 
   G2077A E693K 
Tumor typeCurrent study, frequency (%)Previous studies, frequency (%)Nucleotide change*Amino acid change
Breast 1/122 (1)  A791G Q264R 
Bladder 0/20 (0)    
Cholangiocarcinoma 7/20 (35)  C1393T (×4) R465C 
   G1394A R465H 
   C1513T R505C 
   A1556G Y519C 
Colon 3/31 (10)  C1393T (×2) R465C 
   G1510A V504I 
  45/492 (9) C658T Q220X 
   C670T (×3) R224X 
   C832T (×4) R278X 
   C845A S282X 
   C907T Q303X 
   A936C R312S 
   C1099T R367X 
   C1177T (×3) R393X 
   G1190A G397D 
   G1268T G423V 
   C1313T S438F 
   G1338A W446X 
   C1393T (×7) R465C 
   G1394A (×2) R465H 
   G1436A (×7) R479Q 
   G1457A W486X 
   C1513T R505C 
   G1514C R505P 
   G1628A R543K 
   A1634G Y545C 
   C1745T (×4) S582L 
   C1787T S596F 
Endometrium§  9/102 (9) β-G67F V23I 
   G370T D124Y 
   C1099T R367X 
   2bp ins. 1110 376X 
   G1394A R465H 
   1bp ins. 1417 476X 
   G1436A (×2) R479Q 
   C1972T R658X 
Esophagus  0/27 (0)   
Leukemia  AML, 0/35 (0)   
 B-ALL, 0/20 (0)    
 B-CLL, 0/20 (0)    
 HCL, 0/20 (0)    
 T-ALL**, 8/26 (31)  G1394A (×4) R465H 
   C1435G(×2) R479G 
   G1436T R479L 
   C2065T R689W 
Liver 0/12 (0)    
Lung†† NSCLC, 1/38 (3) 0/50 (0) A32G K11R 
Melanoma 0/20 (0)    
Bone‡‡  1/47 (2) 3bp del., 424  
Ovarian§§ 0/32 (0) 2/111 (2) G734T S245I 
   G1411T E471X 
Prostate 1/83 (1)  3bp ins., 45 +CCT, +P 
Pancreas∥∥  1/11 (9) A1379G H460R 
Stomach¶¶ 8***,†††/52 (15)  C1393T (×4) R465C 
   G943A A315T 
   G1436T R479L 
   G1451C R484T 
   G1510A V504I 
  6/162 (4) G1318A D440N 
   C1393T R465C 
   G1947A W649X 
   1bp del, 1996 706X 
   G2021A R674Q 
   G2077A E693K 

Abbreviations: B-CLL, B-cell chronic lymphocytic leukemia; B-ALL, B-cell acute lymphocytic leukemia; T-ALL, T-cell acute lymphocytic leukemia; HCL, hairy cell leukemia; NSCLC, non–small-cell lung carcinoma.

*

Nucleotide and codon position based on α-isoform transcript unless indicated.

Includes tumors analyzed in refs 5, 9.

Includes three tumors with multiple FBXW7 mutations.

§

Refs. 6, 10.

Ref. 11.

Ref. 12.

**

Malyukova et al., submitted for publication.

††

Ref. 13.

‡‡

Ref. 14.

§§

Ref. 15.

∥∥

Ref. 16.

¶¶

Ref. 17.

***

All mutations were confirmed as somatic.

†††

Restriction digest-based analysis showed five tumors with mutations had no LOH of the remaining FBXW7/hCDC4 allele.

Spectra of FBXW7 mutations reveal mutational “hotspots” and isoform-specific mutations. Of the mutations detected in FBXW7, the vast majority (94%) were single nucleotide changes, with the remainder (6%) being either small nucleotide deletions or insertions. Seventy-four percent of mutations introduced a single amino acid substitution (missense) in the Fbxw7 protein, whereas 26% induced a premature termination of translation (nonsense; Table 1). FBXW7 mutations were found to occur throughout the coding region, including the F-box motif, WD40 repeats, and two of three unique 5′ exons that determine the Fbxw7 isoforms (Fig. 1). Interestingly, isoform-specific mutations were found to represent ∼6% of FBXW7 mutations. Approximately 43% of all FBXW7 mutations occurred at two mutational hotspots positioned at codons Arg465 (nucleotides 1,393–1,394; 29%) and Arg479 (nucleotides 1,435–1,436; 14%). Additional minor mutational hotspots were detected at nucleotides 670 (Arg224; 3%), 832 (Arg278; 4%), 1,177 (Arg393; 3%), and 1,745 (Ser582; 4%). Molecular modeling revealed that codons Arg465, Arg479, and Ser582 are localized to the surface or lumen of the β-propeller structure of Fbxw7; Arg393 is at the rear of the β-propeller structure; and Arg278 is situated close to the isoform-specific NH2 terminus of Fbxw7.

Figure 1.

FBXW7 mutations in primary human tumors. A, spectra of FBXW7 mutations. Three 5′-exons (α, β, and γ) encoding the Fbxw7 isoforms are alternatively spliced to 10 common exons that encode the F-box domain (hatched box) and WD40 repeats (boxes I–VII). Each triangle represents an FBXW7 mutation found in a primary human tumor. Mutation type is indicated by color (dark gray, point mutations; light gray, deletions/insertions; black, C→T or G→A point mutations at CG dinucleotides). Mutational hotspots are observed at nucleotides 670, 832, 1,177, 1,393, 1,394, 1,436, and 1,745. *, chain terminating mutations (nonsense). B, molecular structure of Fbxw7 diagramming mutational hotspots in human tumors. The seven WD40 repeats of Fbxw7 form a β-propeller structure that is involved in substrate recognition. Amino acids that correspond to mutational hotspots are highlighted in yellow. Arg465, Arg479, Arg505, and Ser582 are localized to the surface or lumen of the β-propeller structure; Arg393 is located on the rear-face of the β-propeller; and Arg278 is located proximal to the F-box domain.

Figure 1.

FBXW7 mutations in primary human tumors. A, spectra of FBXW7 mutations. Three 5′-exons (α, β, and γ) encoding the Fbxw7 isoforms are alternatively spliced to 10 common exons that encode the F-box domain (hatched box) and WD40 repeats (boxes I–VII). Each triangle represents an FBXW7 mutation found in a primary human tumor. Mutation type is indicated by color (dark gray, point mutations; light gray, deletions/insertions; black, C→T or G→A point mutations at CG dinucleotides). Mutational hotspots are observed at nucleotides 670, 832, 1,177, 1,393, 1,394, 1,436, and 1,745. *, chain terminating mutations (nonsense). B, molecular structure of Fbxw7 diagramming mutational hotspots in human tumors. The seven WD40 repeats of Fbxw7 form a β-propeller structure that is involved in substrate recognition. Amino acids that correspond to mutational hotspots are highlighted in yellow. Arg465, Arg479, Arg505, and Ser582 are localized to the surface or lumen of the β-propeller structure; Arg393 is located on the rear-face of the β-propeller; and Arg278 is located proximal to the F-box domain.

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Mutation hotspots in FBXW7 correspond to methylated CG dinucleotides with signatures of deaminations of 5′-methylcytosine. Investigation of the mutational hotspots of FBXW7 revealed that all seven are centered on CG dinucleotides, potential sites of DNA methylation in humans. Interestingly, the prominent mutation type that occurs at each mutational hotspot was either a C→T or G→A transition within the CG dinuclotide, consistent with the spontaneous deamination of 5′-methylcytosine→thymine in DNA (18). Because 5′-methylcytosine has been shown to act as an endogenous mutagen (18), we analyzed the methylation status of DNA isolated from several primary tumor types to determine whether the cytosine corresponding to the mutation hotspot at nucleotides 1,435–1,436 (Arg479) was methylated in vivo. Indeed, bisulfite-based sequence analysis revealed that the cytosine was methylated in 18 of 18 primary tumors examined, including those of the ovary, breast, and endometrium (data not shown).

Functional analysis of Fbxw7 mutants reveals defects in localization and substrate binding. Substrate recognition by Fbxw7 is mediated through the interaction of the β-propeller surface formed by the WD40 repeats with the phosphodegron of the substrate protein (2, 3). To examine the consequence on substrate binding of the hotspot mutations, we expressed α-Fbxw7Arg465 and α-Fbxw7Arg479 in HEK 293T cells and assessed their ability to bind substrate cyclin E1. Whereas wild-type α-Fbxw7 readily bound cyclin E1 in vivo, mutants α-Fbxw7Arg465 and α-Fbxw7Arg479 did not (Fig. 2), despite their proper localization as assessed by immunofluorescence (data not shown). Interestingly, many FBXW7 mutations fall outside of the WD40 repeats and the effect of these on Fbxw7 function is unclear. To analyze this in more detail, we functionally tested mutant FBXW7+p16 identified in a prostate tumor specimen that contains a proline residue inserted at amino acid 16 of the α-isoform (Table 1). Expression of α-Fbxw7+p16 in HEK 293T cells showed that it was incapable of binding cyclin E1 substrate in vivo, although it could readily associate with the SCF core components (Fig. 2). Because residue 16 is in close proximity to the proposed nuclear localization signal of the α-isoform (2), we next tested whether α-Fbxw7+p16 is properly localized in cells. Immunofluorescence analysis showed that whereas wild-type α-Fbxw7 is almost exclusively localized to the nucleus in HEK 293T cells, α-Fbxw7+p16 is restricted to the cytoplasm (Fig. 2). These results show that the insertion of proline at residue 16 abolished the nuclear localization signal, preventing interaction with cyclin E1 substrate in the nucleus.

Figure 2.

Functional analysis of Fbxw7 mutants. A, effect of hotspot mutants Arg465 and Arg479 on binding to cyclin E1 substrate. HEK 293T cells were cotransfected with plasmids that express Myc-cyclin E1 and GST-α-Fbxw7 or mutants GST-α-Fbxw7R465C or GST-α-Fbxw7R479Q, and complexes precipitated and analyzed by Western blot analysis. B, functional analysis of α-isoform–specific mutant α-Fbxw7+p16 isolated from a prostate tumor specimen. HEK 293T cells were cotransfected with Myc-cyclin E1 and Flag-α-Fbxw7 or Flag-α-Fbxw7+p16 and extracts immunoprecipitated with anti-Flag antibodies. C, α-Fbxw7+p16 mutant mislocalizes to the cytoplasm. HEK 293T cells were transfected with plasmids that express Flag-α-Fbxw7 (left) or Flag-α-Fbxw7+p16 (right) and protein location was assessed by immunofluorescence with anti-Flag-FITC antibodies.

Figure 2.

Functional analysis of Fbxw7 mutants. A, effect of hotspot mutants Arg465 and Arg479 on binding to cyclin E1 substrate. HEK 293T cells were cotransfected with plasmids that express Myc-cyclin E1 and GST-α-Fbxw7 or mutants GST-α-Fbxw7R465C or GST-α-Fbxw7R479Q, and complexes precipitated and analyzed by Western blot analysis. B, functional analysis of α-isoform–specific mutant α-Fbxw7+p16 isolated from a prostate tumor specimen. HEK 293T cells were cotransfected with Myc-cyclin E1 and Flag-α-Fbxw7 or Flag-α-Fbxw7+p16 and extracts immunoprecipitated with anti-Flag antibodies. C, α-Fbxw7+p16 mutant mislocalizes to the cytoplasm. HEK 293T cells were transfected with plasmids that express Flag-α-Fbxw7 (left) or Flag-α-Fbxw7+p16 (right) and protein location was assessed by immunofluorescence with anti-Flag-FITC antibodies.

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FBXW7 mutants corresponding to a mutational hotspot in primary tumors act dominant negatively to inactivate Fbxw7 function. Recently, it was shown that isoforms of Fbxw7 can form homodimers and heterodimers in vivo (19), raising the possibility that mutants of Fbxw7 could interfere with wild-type Fbxw7 function in a dominant negative fashion. To explore this possibility, we cotransfected HCT116FBXW7−/− cells that have both FBXW7 alleles inactivated by targeted disruption (5) with plasmids that express Myc-cyclin E1, Cdk2, α-Fbxw7, and increasing amounts of α-Fbxw7R465C. The results of this experiment showed that coexpression of α-Fbxw7R465C with wild-type α-Fbxw7 resulted in a marked accumulation of cyclin E1 compared with expression of wild-type α-Fbxw7 alone (Fig. 3A). Similar results were observed for expression of α-Fbxw7R479Q, corresponding to the other major mutational hotspot in primary tumors, in HCT116FBXW7−/− cells and for both mutants in HEK 293T cells that contain wild-type FBXW7 alleles (data not shown). We next carried out cycloheximide-chase experiments to determine whether the increase in cyclin E1 level observed for α-Fbxw7R465C expression was due to increased protein stability. As expected, expression of wild-type α-Fbxw7 resulted in a decrease in cyclin E1 stability (half-life of ∼1.2 h) compared with expression of α-Fbxw7R465C or vector alone (half-life >3 h). In contrast, coexpression of α-Fbxw7 and α-Fbxw7R465C resulted in an increase in cyclin E1 half-life (>3 h), comparable to expression of α-Fbxw7R465C or vector alone (Fig. 3).

Figure 3.

Hotspot mutant Fbxw7R465C acts dominant negatively to inactivate wild-type Fbxw7 function. A, expression of α-Fbxw7R465C interferes with degradation of cyclin E1 by wild-type α-Fbxw7. HCT116Fbxw7−/− cells were cotransfected with plasmids that express Myc-cyclin E1, Cdk2, wild-type GST-α-Fbxw7, and increasing amounts of mutant GST-α-Fbxw7R465C. Lysates were subjected to Western blot analysis. Note the increase in cyclin E1 level in cells expressing wild-type α-Fbxw7 + α-Fbxw7R465C compared with wild-type α-Fbxw7 alone. An equal amount of total plasmid DNA was transfected in each lane. *, background band. B, coexpression of wild-type α-Fbxw7 + α-Fbxw7R465C leads to an increase in cyclin E1 half-life. HCT116Fbxw7−/− cells were cotransfected with plasmids that express wild-type α-Fbxw7 (wt), α-Fbxw7R465C, or wild-type α-Fbxw7 + α-Fbxw7R465C, and cyclin E1 half-life was determined by cycloheximide-chase experiments. Western blot for actin is shown as a loading control. C, graphical representation of results for cycloheximide-chase experiments.

Figure 3.

Hotspot mutant Fbxw7R465C acts dominant negatively to inactivate wild-type Fbxw7 function. A, expression of α-Fbxw7R465C interferes with degradation of cyclin E1 by wild-type α-Fbxw7. HCT116Fbxw7−/− cells were cotransfected with plasmids that express Myc-cyclin E1, Cdk2, wild-type GST-α-Fbxw7, and increasing amounts of mutant GST-α-Fbxw7R465C. Lysates were subjected to Western blot analysis. Note the increase in cyclin E1 level in cells expressing wild-type α-Fbxw7 + α-Fbxw7R465C compared with wild-type α-Fbxw7 alone. An equal amount of total plasmid DNA was transfected in each lane. *, background band. B, coexpression of wild-type α-Fbxw7 + α-Fbxw7R465C leads to an increase in cyclin E1 half-life. HCT116Fbxw7−/− cells were cotransfected with plasmids that express wild-type α-Fbxw7 (wt), α-Fbxw7R465C, or wild-type α-Fbxw7 + α-Fbxw7R465C, and cyclin E1 half-life was determined by cycloheximide-chase experiments. Western blot for actin is shown as a loading control. C, graphical representation of results for cycloheximide-chase experiments.

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Our results show that FBXW7 is a general tumor suppressor in human tumorigenesis. FBXW7 mutations were detected in primary human tumors of the blood, breast, bile duct, bone, colon, endometrium, stomach, lung, ovary, prostate, and pancreas, with an overall mutation frequency of ∼6%. Mutations were most frequent in tumors of the bile duct (35%), blood (T-cell acute lymphocytic leukemia, 31%), colon (9%), endometrium (9%), pancreas (9%), and stomach (6%), suggesting a potential tissue specificity of FBXW7 inactivation in human tumorigenesis.

FBXW7 mutations were found localized throughout the coding region of the gene including the isoform-specific 5′-exons, nuclear localization signal, F-box motif, and WD40 repeats. These mutations are predicted to adversely affect isoform-specific functions, subunit dimerization, Pin-1 association, localization, SCF assembly, and substrate recognition, highlighting the plethora of ways Fbxw7 can be functionally inactivated in tumors. Interestingly, ∼6% of FBXW7 mutations are isoform specific, suggesting that inactivation of an individual isoform could play a causative role in human tumorigenesis. This finding could reflect the potential of a mutant Fbxw7 isoform to act dominant negatively (see below) or that individual Fbxw7 isoforms may target a unique set of substrates for ubiquitination. In support of the latter hypothesis, Fbxw7 isoforms are differentially localized in mammalian cells (α-nuclear, β-cytoplasmic, and γ-nucleolar; ref. 20). Furthermore, ubiquitination of c-Myc has been shown to be mediated by γ-Fbxw7, which selectively binds substrate in the nucleolus (2). Additionally, ubiquitination of cyclin E1 is mediated through a cooperative effort of α- and γ-Fbxw7, in which α-Fbxw7 binds cyclin E1 and stimulates prolyl-isomerization by Pin-1, which then promotes efficient ubiquitylation by SCFγ-Fbxw7 (20). Indeed, one of the α-isoform–specific mutations detected in tumors (D124Y) was shown to be defective in supporting Pin-1–mediated isomerization of cyclin E1 but was not defective in ubiquitylation function (20). It is currently not understood whether other SCFFbxw7 substrates are ubiquitinated via an analogous cooperative mechanism of Fbxw7 isoforms.

Our analysis identified seven mutational hotspots in FBXW7, representing >60% of the total mutations. Two major hotspots located at nucleotides 1,393–1,394 (Arg465) and 1,435–1,436 (Arg479) account for ∼43% of all mutations. The amino acid residues corresponding to six of seven mutational hotspots in primary human tumors are evolutionarily conserved in flies, worms, and yeast (2), showing their potential importance in Fbxw7 function. Interestingly, all seven FBXW7 mutational hotspots involve CG dinucleotides, where the resultant mutation is primarily a C→T or G→A transition. Consistent with 5′-methylcytosine deamination as a mechanism for FBXW7 mutation, the cytosine nucleotide at hotspot position 1,435–1,436 (Arg479) was found to be methylated in all primary tumors analyzed. These data stress the potential importance of endogenous methylation in mutational inactivation of Fbxw7 in tumors, as has previously been postulated for the tumor suppressor p53 (18).

It is currently not understood what role, if any, other genetic and epigenetic mechanisms play in FBXW7 inactivation in human tumors. In many primary tumors and derived cell lines, FBXW7 mutations frequently occur without a concomitant loss or mutation of the remaining allele, suggesting that FBXW7 may not follow the classic “two-hit” model of tumor suppressor gene inactivation. It is possible that reduced expression of the wild-type allele might be sufficient to abrogate tumor suppressor activity or that FBXW7 mutations could act dominant negatively. The former possibility is supported by a study that showed that loss of a single FBXW7 allele can accelerate tumor development in p53+/− mice (4), suggesting FBXW7 is a p53-dependent haploinsufficient tumor suppressor. However, we analyzed FBXW7 and p53 mutations in gastric cancers and failed to detect cooperation between these proteins (Supplementary Table S1). It remains to be determined whether FBXW7 expression is reduced or silenced in tumors through epigenetic mechanisms such as promoter methylation.

Our data show that Fbxw7 mutants corresponding to the major mutational hotspots in primary tumors (Arg465 and Arg479) can act dominant negatively to abrogate wild-type Fbxw7 function. Coexpression of wild-type α-Fbxw7 and α-Fbxw7R465C was found to increase the steady-state level of cyclin E1 as well as its half-life, compared with expression of wild-type α-Fbxw7 alone. The different isoforms of Fbxw7 interact in vivo through association of their D-box motifs located in the NH2-terminal region of the protein, and this interaction enhances SCFFbxw7-associated ubiquitylation activity (19). If Fbxw7 predominantly functions as a homodimer or heterodimer in vivo, then expression of a single mutant allele may be sufficient to functionally inactivate its tumor suppressor function. This could explain why most primary tumors contain only a single mutant FBXW7 and retain a wild-type allele. Further study is needed to determine whether other Fbxw7 mutations also act dominant negatively and whether expression at physiologic levels is sufficient to exert the dominant negative effects on wild-type Fbxw7.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: University of California Tobacco-Related Disease Research Program, Swedish Cancer Society, the Swedish Research Council, the Swedish Institute, the Children Cancer Foundation, the Cancer Society of Stockholm, and Karolinska Institute Foundations.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Katayoun Aghajani, Mohsen Chiani, and Dr. Ali Mansouri for technical support in tumor specimen preparation. We also thank Bert Vogelstein for providing HCT116FBXW7−/− cells for this study.

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Supplementary data