FBXO31 Is the Chromosome 16Q24.3 Senescence Gene, a Candidate Breast Tumor Suppressor, and a Component of an SCF Complex

Research Article

FBXO31 Is the Chromosome 16q24.3 Senescence Gene, a Candidate Breast Tumor Suppressor, and a Component of an SCF Complex

Raman Kumar,1 Paul M. Neilsen,1 Joanne Crawford,2 Ross McKirdy,1 Jaclyn Lee,1 Jason A. Powell,2 Zarqa Saif,1 Julie M. Martin,1 Marc Lombaerts,3 Cees J. Cornelisse,3 Anne-Marie Cleton-Jansen,3 and David F. Callen1

1Breast Cancer Genetics Group, Dame Roma Mitchell Cancer Research Laboratories, Department of Medicine, University of Adelaide and Hanson Institute, Institute of Medical and Veterinary Science Adelaide; 2Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital, North Adelaide, South Australia, Australia; and 3Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands

Abstract regions of LOH (2). These findings have subsequently been A BAC located in the 16q24.3 breast cancer loss of hete- substantiated by a pooled analysis of various published LOH studies rozygosity region was previously shown to restore cellular that confirmed band 16q24.3 as a region of preferential loss (1). All 104 genes in this LOH region were identified, and senescence when transferred into breast tumor cell lines. We have shown that FBXO31, although located just distal to this candidate genes were selected based on their known or predicted BAC, can induce cellular senescence in the breast cancer cell function as well as their expression in breast cancer cell lines as line MCF-7 and is the likely candidate senescence gene. determined by real-time reverse transcription-PCR (RT-PCR; ref. 3). FBXO31 has properties consistent with a tumor suppressor, Large variations in expression of FBXO31 and CBFA2T3 (also known because ectopic expression of FBXO31 in two breast cancer as MTG16) were observed in breast cancer cell lines similar to that cell lines inhibited colony growth on plastic and inhibited cell found for established tumor suppressor genes. Functional studies proliferation in the MCF-7 cell line. In addition, compared were consistent with CBFA2T3 as a breast cancer tumor suppressor with the relative expression in normal breast, levels of FBXO31 (4). Studies suggest that more than one tumor suppressor gene may were down-regulated in breast tumor cell lines and primary reside in regions of cancer LOH; for example, the 3p LOH region in small cell lung carcinoma contains several tumor suppressor genes tumors. FBXO31 was cell cycle regulated in the breast cell lines MCF-10A and SKBR3 with maximal expression from late G to (5). Therefore, we have continued to characterize additional 2 candidate tumor suppressor genes in the 16q24.3 region. early G1 phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of Previous studies showed that the microcell-mediated transfer of human chromosome 16q fragments caused senescence in human- cells at the G1 phase of the cell cycle. FBXO31 contains an F-box domain and is associated with the proteins Skp1, Roc-1, and mouse-immortalized cell lines (6). This senescence-associated and Cullin-1, suggesting that FBXO31 is a component of a SCF region was subsequently localized to a 360-kb yeast artificial ubiquitination complex. We propose that FBXO31 functions as chromosome (YAC) d792t2 mapping between the markers D16S498 a tumor suppressor by generating SCFFBXO31 complexes that and D16S476 (7) and more recently to an 85-kb BAC clone 346J21 target particular substrates, critical for the normal execution (8). Transfer of this BAC into several cell lines, including the human of the cell cycle, for ubiquitination and subsequent degrada- breast cancer line MCF-7, was shown to restore cellular senescence. These findings are consistent with the location of a senescence tion. (Cancer Res 2005; 65(24): 11304-13) gene within the BAC 346J21, and this gene may also function as a tumor suppressor gene. Mapping of the two previously identified Introduction potential tumor suppressor genes locates FBXO31 within the YAC Numerous cytogenetic and molecular studies of breast cancer d792t2, whereas CBFA2T3 is 1.7 Mb distal to this YAC. We report have identified frequent loss of heterozygosity (LOH) of the long mapping of the BAC 346J21 with respect to the FBXO31 gene and arm of chromosome 16 (1). We undertook detailed studies to define investigate if the properties of FBXO31 are consistent with a more precisely the region of chromosome 16 most frequently senescence and tumor suppressor gene. In addition, because affected by LOH and therefore the likely location of the breast FBXO31 encodes an F-box protein and other proteins with F-box cancer tumor suppressors. A total of 712 sporadic breast tumors domains have been shown to be variant components of SCF were screened, and it was established that a 2.4-Mb segment of the complexes (9), we have investigated whether FBXO31 mediates its chromosome 16q24.3 terminal band was one of the minimum function as part of an SCFFBXO31 E3 ubiquitin ligase complex.

Note: Z. Saif was on leave from the National Institute for Biotechnology and Materials and Methods Genetic Engineering, Faisalabad, Pakistan. Requests for reprints: David F. Callen, Breast Cancer Genetics Group, Dame Cell lines and antibodies. 184V and 48RS are finite life span human Roma Mitchell Cancer Research Laboratories, Institute of Medical and Veterinary mammary epithelial cells (HMEC) derived from two different patients, Science, Hanson Institute Building, Frome Road, Adelaide, South Australia 5000, whereas 184A1 is a nonmalignant immortally transformed cell line derived Australia. Phone: 618-8-222-3450; Fax: 618-8-222-3217; E-mail: david.callen@ from normal breast epithelium (10). These cell lines were grown in MCDB- imvs.sa.gov.au. I2005 American Association for Cancer Research. 170 medium (Invitrogen, Carlsberg, CA). GP-293 cells were purchased from doi:10.1158/0008-5472.CAN-05-0936 BD Biosciences (San Jose, CA). All other cell lines were purchased from the

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American Type Culture Collection (Manassas, VA) and were grown in the concentrations of 4 to 10 Â 103 cells/mL in selective media containing recommended media. Stable cell lines expressing FLAG-HA-FBXO31 were G418, and colonies were counted in triplicate wells after growth for a generated by G418 selection of 293T cells transduced with a pQCXIN-based further 2 to 3 weeks. The empty vector was used as a negative control. The retroviral construct. Antibodies used were rat anti-HA (Roche Diagnostics; transduction efficiency was monitored by immunofluorescence detection of Indianapolis, IN); mouse anti-myc and mouse anti-Cul-1 (Santa Cruz the myc-tag. Cellular senescence was assayed using a kit from Cell Signaling Biotechnology, Santa Cruz, CA); mouse anti-Skp1 (BD Biosciences); and Technologies (Beverly, MA) based on senescence-specific acidic rabbit anti-Skp1, rabbit anti-Roc-1, and rabbit anti–mitogen-activated h-galactosidase activity (12). Senescent cells stained intense blue after protein/extracellular signal-related kinase 2 (NeoMarkers, Fremont, CA). A overnight incubation at 37jC. Colonies of >30 cells containing >10 blue rabbit polyclonal antibody was raised against a 15-amino-acid synthetic staining cells were scored as senescent. Proliferation assays were peptide representing the COOH terminus (amino acids 525-539; QAF- determined on transduced cells selected for 10 to 14 days in the presence DEMLKNIQSLTS) of the FBXO31 protein conjugated to keyhole limpet of G418. These cells were plated at 10% to 20% confluence in 96-well plates hemocyanin. The polyclonal antibody was then affinity purified on and at various times samples (n = 6-8) assayed by incubating cells for Sepharose beads coupled with the same peptide. 2 hours with the CellTiter 96 AQueous One Solution Cell Proliferation Assay Plasmids. Appropriate protein-coding open reading frames (ORF) were (Promega, Madison, WI) and measuring absorbance at 490 nm. PCR amplified from adult brain marathon cDNA (BD Biosciences) and Cell synchronization. Cells were synchronized at G1-S phase using a cloned in-frame with the epitope tags into either pCMV-myc or pCMV-HA double thymidine block. Cells were grown in the presence of 2 mmol/L (BD Biosciences) mammalian expression vectors to generate clones thymidine (Sigma-Aldrich, St. Louis, MO) for 24 hours and then washed and expressing myc-FBXO31 and HA-Skp1 proteins. The sequence coding for grown in fresh medium without thymidine for 10 hours. Cells were cultured amino acids 51 to 109, containing the F-box domain, were deleted from the in the presence of 2 mmol/L thymidine for a further 16 hours and then pCMV-myc-FBXO31 construct using overlap PCR to generate pCMV-myc- released from the G1-S block by washing twice with fresh medium. Cells FBXO31DF. For retroviral-mediated ectopic expression studies, fragments collected at various time points following release from the second coding myc-FBXO31, myc-FBXO31DF, and myc-p53 were also cloned into thymidine block were lysed in 50 mmol/L Tris-HCl (pH 7.5), 250 mmol/L the pLNCX2 vector (BD Biosciences). For the cell cycle analysis, FBXO31, NaCl, 1% Triton X-100, 1 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L

FBXO31DF, and p53 coding fragments were cloned in-frame with the Na3VO4, 1 mmol/L DTT, 1Â protease inhibitors (Roche, Indianapolis, IN) on enhanced green fluorescent protein (EGFP) ORF into pEGFP-C1 (BD ice for 15 minutes. Lysed samples were clarified by centrifugation and then Biosciences) to generate constructs capable of expressing EGFP fusions. To assayed for protein concentration using bicinchoninic acid protein assay generate stable FBXO31 expressing cell lines, the FBXO31 ORF with FLAG reagent kit (Pierce, Rockford, IL). and hemagglutinin (HA) epitope tags was cloned into the pQCXIN retroviral Cell cycle analysis. MCF-7 or MDA-MB-468 cells were transfected with vector. The sequences of all constructs were confirmed by DNA sequencing. pEGFP-FBXO31, pEGFP-FBXO31DF, pEGFP-p53, or pEGFP-C1 using Human specimens. Primary breast tumors with known histology, stage, LipofectAMINE 2000 (Invitrogen). Cells were collected 24 and 48 hours and differentiation grade had 16q LOH status determined as reported after transfection and treated as described (13). Briefly, cells were pelleted previously (2). Patient material was obtained on approval of local medical (300 Â g for 5 minutes at 4jC), washed twice with cold PBS, resuspended in ethics committees. 500 AL of cold PBS, and then fixed for 1 hour at 4jC by adding of 500 AL Northern blot. Multiple Tissue Northern Blot (BD Biosciences) carrying fixation solution (2% w/v paraformaldehyde in PBS, pH 7.2). The fixed cells polyadenylated RNA from various human tissues was probed with a were pelleted, washed with cold PBS, resuspended in 1 mL of 70% ethanol 32P-labeled FBXO31 DNA fragment. The 224-bp DNA probe was generated added dropwise to the pellet while vortexing, and then incubated overnight by PCR amplification from the full-length clone using the following primers: at 4jC. The next day, the cells were pelleted and resuspended in 1 mL forward, 5V-CTTCACCGATATAGACAC and reverse, 5V-GGCCGTACATG- propidium iodide solution (40 Ag/mL with 100 Ag/mL RNase A) for CACTCCACTG and radiolabeled with a-32P-dCTP using the Megaprime 30 minutes at 37jC in the dark and analyzed on a FACScan flow cytometer DNA labeling system (Amersham Biosciences, Piscataway, NJ). (BD Biosciences, San Jose, CA), and the multivariate data were collected Real-timereversetranscription-PCR.FBXO31 expression was using CellQuest software (BDIS, Pittsburgh, PA). Cell cycle analysis of DNA determined by real-time RT-PCR with the forward primer: 5V-CCGGCGG- histograms was done using ModFit LT V2.0 (Verity, Topsham, ME) software. GAGGCAGGAGGAGT and reverse primer: 5V-GCGGCGGTAGGTCA- Western blot analysis. Cells were lysed in 150 mmol/L NaCl, 1% Triton- GGCAGTTGTCG. Using TRIzol (Invitrogen, Carlsbad, CA), total RNA X, 50 mmol/L Tris-HCl (pH 8) with complete protease inhibitor cocktail was isolated from pellets of cell lines grown to f80% confluence, or (Roche); sonicated; and centrifuged. Clarified cell lysates or immunopreci- paraffin blocks of breast tumors using 20 Â 20 Am tissue sections with pitated protein samples were resolved on SDS-PAGE and transferred on to at least 50% tumor. Two micrograms of total RNA were used as template Hybond-C Extra (Amersham Biosciences). Membranes were probed with for cDNA in a final volume of 20 AL. Each real-time RT-PCR reaction various primary antibodies and detected with appropriate horseradish (10-minute activation of the polymerase at 95jC, 45 cycles of 15 seconds peroxidase–conjugated secondary antibodies using enhanced chemilumi- at 95jC, 1 minute at 60jC, and signal detection at 60jC) contained 0.2 AL nescence detection system (Amersham Biosciences) using standard of this cDNA and used SYBR Green (qPCRTM Core kit, Eurogentec, protocols (14). Seraing, Belgium or Bio-Rad iQ Supermix) on a Bio-Rad iCycler (Bio-Rad, Coimmunoprecipitation and affinity purification. Plasmids (2 Ag Hercules, CA). The geometric mean expression of three housekeeping each) expressing either myc-FBXO31 or HA-Skp1 or both were transfected genes (HNRPM, CPSF6, and TBP) was used to normalize the expression into 5 Â 105 HEK293T cells in 35-mm wells. Twenty-four hours after of FBXO31 in breast tumor cDNA according to published methods (11). transfection, cells were harvested and lysed as for Western blot analysis. For cell line expression, the housekeeping gene cyclophilin A was used to Clarified lysates were incubated with either anti-myc or anti-HA- normalize the expression of FBXO31, because previous studies determined conjugated agarose beads (Sigma-Aldrich). Beads were washed twice with this was a transcript with minimal variation in such cell lines (3). 150 mmol/L NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, Cell-based assays. To generate amphotropic recombinant retroviruses, 50 mmol/L Tris-HCl (pH 8), then 20 mmol/L Tris-HCl (pH 7.5), and the GP2-293 cells were transfected with pVSV-G (BD Biosciences) and various protein complexes were eluted with 1Â protein-loading buffer (0.0625 mol/L gene constructs cloned into the pLNCX2 vector using LipofectAMINE 2000 Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 5% 2-mercaptoethanol; ref. 15). transfection agent (Invitrogen). Forty-eight hours after transfection, the Input and immunoprecipitated proteins were resolved by Western blot culture medium containing the retrovirus was collected, filtered, and used analysis, and membranes were immunoblotted with rat anti-HA, mouse to transduce the appropriate cell line at f50% confluence. Transduction anti-myc, mouse anti-Cullin-1, and rabbit anti-Roc-1 antibodies. HEK293T was enhanced by adding 8 Ag/mL polybrene and centrifuging plates at 1,250 and HEK293T cell lines stably expressing FLAG-HA-FBXO31 protein were rpm for 30 minutes. To determine the frequency of colony formation, after used to affinity purify the tagged FBXO31. Cells were lysed in 20 mmol/L

24 hours, the cells from each treatment were plated in six-well plates at Tris-HCl (pH 8), 200 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L EDTA, www.aacrjournals.org 11305 Cancer Res 2005; 65: (24). December 15, 2005

0.1% Tween 20, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1Â protease purchased and shown by PCR with STS primers derived from the inhibitors (Roche) on ice for 15 minutes. Clarified lysates were incubated cDNA sequence BX119888 to possess an interstitial deletion for 2 hours with anti-FLAG M2 agarose (Sigma-Aldich). The beads were extending at least 1.2 kb (Fig. 1A). These results were confirmed then washed with the lysis buffer, and the protein complexes were eluted by multiplex PCR with additional STSs present on the YAC (data with the same buffer containing 250 Ag/mL FLAG peptide (Roche). The not shown). The YAC d792t2 is a chimera because it also possesses inputs and eluates were analyzed by Western blotting with rat anti-HA, rabbit anti-Roc-1, mouse anti-Cullin-1, and mouse anti-Skp1 a segment of chromosome 11q13 (7). The BAC 346J21 only contains antibodies. 290 bp of the 3V end of FBXO31 with the majority of the gene positioned telomeric to this BAC. The genomic region encompassed by the BAC does not contain any predicted CpG islands and Results contains 44% low copy repeats as determined by RepeatMasker. Analysis of 16q24.3 senescence region. The relative positions The NCBI build 35.1 of the human genome sequence predicts three of the FBXO31 gene together with the YAC d792t2, the BAC 346J21, possible transcripts completely contained within the genomic and the various sequence-tagged sites (STS) used previously region of the BAC 346J21 (as defined by the location of the end to define the location of the BAC (8) are given in Fig. 1A. The sequences B15982 and B15983 from chromosome 16 base sequenced extremities of the BAC 346J21 are defined from the 85,835,220 to 85,920,731): LOC388305, LOC400552,and available end sequences with accession nos. B15982 and B15983 LOC440391. Based on the absence of introns for LOC400552 and available at the National Center for Biotechnology Information LOC440391, the lack of significant representation of any of the (NCBI). In BAC 346J21, the marker D16S3063 was reported to be three transcripts in expressed sequence tag (EST) libraries, and the distal to D16S3048 as determined from the mapping of STSs and absence of any motifs or species conservation of the predicted overlapping BAC clones (8). However, the order of markers as proteins, these are unlikely to represent functionally transcribed defined by the NCBI genomic sequence places D16S3063 proximal genes. Because well-defined genes within the confines of this BAC to D16S3048 and some 14 kb proximal to the STS 344A17.T7. The were absent, it was decided to further characterize the FBXO31 STS 346J21.T7, together with WI15838, WI12410, and the 3V end of gene, located within the YAC d792t2 and immediately adjacent to FBXO31 were all located within 200 bp. The YAC d792t2 was the BAC 346J21.

Figure 1. Location and tissue expression of the FBXO31 gene and presence of an F-box domain in the encoded protein. A, location of the FBXO31 gene. Positions of various markers are determined by analysis of the genomic sequence of this region of chromosome 16. Positions of the YAC d792t2 and the BAC 346J21 are shown (7, 8). B, Northern blot of polyadenylated RNAs from various human tissues showing a 3.6-kb FBXO31 transcript. Arrows, location of molecular weight markers. C, alignment of F-box domains from FBXO31 and other selected F-box proteins. Boxed, individual amino acids identical among all five F-box sequences; underlined, when present in three to four sequences. Nucleotide sequences are derived from Genbank, NCBI.

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Northern blot and in silico analyses. The presence of a 3.6-kb significant reduction (MDA-MB-468) compared with the vector RNA band on a Northern blot shows that FBXO31 expression is control (Fig. 3A-B). negligible in bone marrow, highly expressed in brain, and The effect of FBXO31 ectopic expression on the proliferation expressed at similar levels in the other 10 human tissues, including of MCF-7 cells was also determined by generating cultures of breast (Fig. 1B). FBXO31 mRNA contains a 1,620-bp ORF that is G418-resistant MCF-7 cells following retroviral transduction. transcribed from the genomic DNA with nine exons spanning f52 Growth curves of these cell lines showed that cells expressing kb. The FBXO31 transcript has an exceptionally short 23-base FBXO31 or p53 had a similar reduction in their proliferation 5V-untranslated region. In silico analysis of the EST database and (after 72 hours, 34% and 38%, respectively) compared with the 5V rapid amplification of cDNA ends (data not shown) failed to vector control (Fig. 3D). Cells expressing FBXO31DF had an identify any additional 5V transcript sequence. In addition, the intermediate 20% reduction of proliferation compared with the presence of a 2.48-kb CpG island (66% G + C and with 9% CpG) control. encompassing the first 350-bp exon of FBXO31 also supports the FBXO31 expression and mutation screening. To further 5V start of the transcript. FBXO31 encodes a 539-amino-acid investigate the possible role of FBXO31 as a tumor suppressor, protein with a predicted molecular mass of 61 kDa. The predicted the relative expression of FBXO31 was determined by real-time amino acid sequence of FBXO31 does not have significant RT-PCR in a panel of breast cell lines (Fig. 4A). Results showed homology to known proteins, except for a consensus F-box levels of expression in finite life span HMEC and nonmalignant domain at the amino terminus (Fig. 1C). The 40-amino-acid F-box immortalized cells that averaged 7.9 times greater than the domain is involved in protein-protein interactions and is present average expression breast cancer cell lines. The chromosome 16q in a large family of proteins (16). FBXO31 also contains six minimal LOH of the breast cancer cell lines has been determined (RxxL) destruction box (D-box) motifs that are hallmark of previously (19). The average relative expression of FBXO31 in proteins degraded via the anaphase-promoting complex/cyclo- the cell lines MCF-7 and BT20, without chromosome 16q LOH, some (APC/C; refs. 17, 18). was significantly higher than the cell lines T47-D, MDA-MB-231, Ectopic expression of FBXO31 induces cellular senescence in MDA-MB-468, and SKBR3 with chromosome 16 LOH (1.27 versus MCF-7. Colonies of MCF-7 growing in the presence of G418 after 0.58, P < 0.01). Based on the findings of reduced expression in retroviral transduction were stained for the presence of the breast cancer cell lines, expression was then assessed by real- senescence-specific acidic h-galactosidase activity (12). Senescent time RT-PCR in a panel of primary breast tumors selected to cells had intense blue staining with a large flattened morphology. have at least 50% tumor cells as estimated from H&E-stained The MCF-7 cell line has a spontaneous background of senescent sections. Presented in Fig. 4B is the relative FBXO31 expression cells usually evident as occasional blue staining cells (Fig. 2A). in tumors classified by analysis of polymorphic markers (2) Following several weeks of growth, 35% to 40% of colonies into those without chromosome 16q LOH and those with LOH ectopically expressing FBXO31 showed senescence compared with for the entire long arm of chromosome 16. There was a trend 5% to 7% of senescent colonies with vector alone (Fig. 2B-C; for lower FBXO31 expression in tumors with 16q LOH (10 of 15 Table 1). Colonies from cells transduced with FBXO31DF were tumors had expression of <50% of the average expression in similar to the vector alone, suggesting that the F-box motif of normal breast) compared with tumors without LOH of 16q (6 of FBXO31 is critical for this senescent function. In subsequent 11 tumors). experiments, MCF-7 cells ectopically expressing an EGFP-tagged Based on the observation of reduced FBXO31 expression in some FBXO31 showed that the presence of cell senescence was tumors, it was considered possible that the FBXO31 gene may correlated to the expression of FBXO31 (Fig 2D-I). In MCF-7 function as a tumor suppressor. Tumor-restricted mutations can colonies, senescent cells were frequent in regions with EGFP provide definitive evidence for a tumor suppressor gene (20). fluorescence, whereas there were few senescent cells in regions Accordingly, screening for the presence of mutations in FBXO31 without EGFP. was undertaken on DNA isolated from a panel of 24 breast cancer Alteration of FBXO31 expression effects growth of breast cell lines by single strand conformational analysis (SSCA). In cell lines. Because it was determined that ectopic expression of addition, DNA from 44 breast tumors with known LOH of the long FBXO31 could induce senescence in the breast cell line MCF-7, we arm of chromosome 16, and the DNA from the normal peripheral investigated if this gene could function more generally as a tumor blood lymphocytes of the same patients were also screened. suppressor. Ectopic expression of a tumor suppressor in a cancer Primers for SSCA were designed to bind within the intronic cell line would be expected to inhibit the ability of cells to initiate sequences such that coding and flanking intronic regions could be colonies on plastic and inhibit cell proliferation. FBXO31 was analyzed (data available upon request). No tumor specific ectopically expressed in the breast cancer cell lines MCF-7 and mutations were detected for FBXO31 both in breast cancer cell MDA-MB-468 by retroviral transduction and colony formation on lines and tumor DNA. plastic assessed. Both cell lines showed a reduction in colony FBXO31 is cell cycle regulated. Following the findings that formation, compared with the vector control, when transduced ectopic expression of FBXO31 suppresses growth, a fluorescence- with retroviruses expressing FBXO31 (Fig. 3A-B). This reduction in activated cell sorting–based cell cycle analysis was used to colony formation averaged 50% for MCF-7 from five independent determine if the mechanism of this growth suppression is due to experiments and 77% for MDA-MB-468 from six independent a specific effect on the cell cycle. Cell cycle analysis was done experiments (Fig. 3C). In all experiments, an expected severe on the breast cancer cell line MDA-MB-468 transiently express- inhibition in colony formation (generally 95% compared with the ing the EGFP-tagged proteins for 24 and 48 hours (Fig. 5). vector control) was observed in cells transduced with retrovirus Analysis of the asynchronous cell populations showed a 13% expressing the p53 protein. Ectopic expression of FBXO31DF, a increase in the proportion of EGFP-FBXO31-expressing G1 cells construct with a deletion of the F-box domain, showed an from 24 to 48 hours (Fig. 5A-B) compared with the 3% increase intermediate reduction in colony formation (MCF-7) or no observed in only EGFP-expressing G1 cells in the same time www.aacrjournals.org 11307 Cancer Res 2005; 65: (24). December 15, 2005

Figure 2. FBXO31 ectopic expression induces cellular senescence in MCF-7 cells. Staining of senescent cells in colonies of MCF-7 ectopically expressing empty vector (A) and FBXO31 (B and C). Colonies of MCF-7 stained for senescence (D, F, and H) and fluorescent images showing the ectopically expressing EGFP-FBXO31 (E, G, and I).

period (Fig. 5G-H). Ectopic expression of EGFP-FBXO31DF Specificity of the anti-FBXO31 antibody was confirmed by resulted in profiles similar to the control with a 3% increase Western blot analysis on protein lysates of MCF-10A and in G1 cells from 24 to 48 hours (Fig. 5C-D). However, when HEK293T cells transiently expressing myc-FBXO31. The protein EGFP-p53 was ectopically expressed, there was a 17% increase in samples showed a band at 78 kDa, suggesting that this antibody G1 cells from 24 to 48 hours (Fig. 5E-F). Similar results were could specifically detect endogenous and ectopically expressed observed in independent experiments and in the MCF-7 cell line FBXO31 (data not shown). FBXO31 levels at different stages of (data not shown). These observations suggest that a block in the the cell cycle were investigated in MCF-10A and the breast cell cycle at G1 is the probable cause of the observed negative cancer cell line SKBR3 by collecting samples at various time effect of FBXO31 ectopic expression on growth of breast cancer points following release from a double thymidine block. For both cell lines. cell lines, the variation in levels of FBXO31 at different times The cell cycle regulation of FBXO31 levels was then after release of the block shows that this protein is cell cycle investigated. The endogenous FBXO31 levels were determined regulated (Fig. 6A). The cell cycle synchronization was confirmed using an affinity-purified rabbit polyclonal anti-FBXO31 antibody. by change in the levels of cyclin B1. These levels were at a

Cancer Res 2005; 65: (24). December 15, 2005 11308 www.aacrjournals.org

Table 1. MCF-7 senescent colonies ectopically expressing FBXO31

2-wk growth 3-wk growth

No. colonies % Senescent No. colonies % Senescent

Senescent Total Senescent Total

FBXO31 27 78 34.6 26 65 40.0 FBXO31DF 2 80 2.5 5 38 13.2 Vector 5 93 5.4 4 56 7.1

maximum at 6 to 10 hours for MCF10A and 8 to 12 hours for profile indicate that levels of FBXO31 protein were at a SKBR3. Cell progression from mitosis is associated with maximum at late G2 to early G1 phase. decreased levels of cyclin B1. For both cell lines, the levels of FBXO31 exists as a component of SCFFBXO31 complex. FBXO31 were a maximum at similar times, from 8 to 16 hours Immunoprecipitation was used to determine if FBXO31 exists as after release (Fig. 6A). Comparison with the cyclin B1 cell cycle a component of the SCF ubiquitination complex. Reciprocal

Figure 3. Ectopic expression of FBXO31 reduced the growth characteristics of breast cancer cell lines MCF-7 and MDA-MB-468. The MCF-7 (A) and MDA-MB-468 (B) breast cancer cell lines were transduced with recombinant retroviruses expressing FBXO31, FBXO31DF, p53 (positive control), and empty vector (negative control). Number of colonies growing on plastic dishes in the presence of G418, 2 weeks after transduction. C, average effect of ectopic expression of FBXO31 on colony growth, compared with vector alone, from five independent experiments with MCF-7 and six experiments with MDA-MB-468. D, FBXO31 ectopic expression inhibited MCF-7 cell growth. Growth assays were initiated from cultures transduced with retroviruses expressing FBXO31, FBXO31DF, p53, and empty vector and selected for 14 days in media containing G418. www.aacrjournals.org 11309 Cancer Res 2005; 65: (24). December 15, 2005

Figure 4. FBXO31 expression as measured by real-time RT-PCR is reduced in breast cell lines and tumors. A, FBXO31 expression in various breast cell lines. B, FBXO31 expression in breast tumors with and without 16q LOH. Horizontal dashed line, at 50% of the average expression in the normal breast samples.

experiments on HEK293T lysates prepared from cells transfected detected in the reciprocal anti-HA immunoprecipitations done on with constructs either expressing myc-FBXO31, myc-FBXO31DF, the same lysates. To provide additional evidence for FBXO31 as a HA-Skp1, both myc-FBXO31 and HA-Skp1, or both myc-FBXO31DF component of an SCFFBXO31 complex, stable HEK-293T-based cell and HA-Skp1 proteins showed that FBXO31 specifically interacts lines were generated by retroviral transfer of FLAG-HA-FBXO31. with the Skp1 protein (Fig. 6B). The FBXO31-Skp1 interaction was The expression of the tagged FBXO31 protein was at near effectively eliminated when the F-box was deleted in cells endogenous levels (data not shown). Cell lysates from these stable transfected with the myc-FBXO31DF construct. Endogenous cell lines were affinity purified using anti-FLAG antibody, and the Cullin-1 and Roc-1 were also present in the immunoprecipitations presence of endogenous Skp1, Cullin-1, and Roc-1 proteins was from cells transfected with constructs expressing myc-FBXO31/ confirmed by Western blot analysis (Fig. 6C). Because Skp1, Cullin-1, HA-Skp1. The trace amounts of Cullin-1 and Roc-1 detected in anti- and Roc-1 constitute invariant components of the SCF complexes myc immunoprecipitations from cells transfected with myc- (21, 22), it is concluded that FBXO31, either ectopically expressed FBXO31DF and/or HA-Skp1 were considered to originate from or expressed in stable clones at near physiologic levels, exists as nonspecific interactions, because Cullin-1 and Roc-1 were not part of an SCFFBXO31 complex.

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Discussion observed that G418-selected cells with retrovirally induced Previous studies determined that the YAC d792t2, and subse- expression of FBXO31 have near physiologic levels of protein. quently the transfection of a retrofitted BAC 346J21, caused cellular Although the data presented here are consistent with FBXO31 senescence when transferred into cell lines, and this was inferred to being the previously identified cellular senescence gene SEN16 (8), be caused by a gene termed SEN16 (7, 8). Our analysis of the YAC the location of FBXO31 is immediately distal to the BAC 346J21. d792t2 showed an interstitial deletion was present in the region There are several possible explanations for the apparent absence encompassed by the BAC, whereas analysis of the genomic of the ORF of the FBXO31 gene in the BAC 346J21. First, the BAC sequence contained within the BAC 346J21 did not identify any may be rearranged and actually contain the FBXO31 gene. The potential candidate senescence genes. We chose to characterize the possibility of a rearranged clone is suggested by the reversal of gene FBXO31, as it was located within the YAC d792t2. FBXO31 the order of the markers D16S3063 and D16S3048 in the reported possessed properties consistent with the SEN16 senescence gene, physical mapping of BAC 346J21 (8). The second possibility is that because ectopic expression of FBXO31 in the breast cell line MCF-7 uncharacterized DNA rearrangements in the clones containing (one of the cell lines used in the SEN16 studies; ref. 8) resulted in an the BAC result in changes in endogenous FBXO31 expression. increase in the number of senescent colonies. In addition, colonies MCF-7 transfected with the retrofitted BAC 346J21 were identified of transduced MCF-7 showed the expression of FBXO31 was as rare G418-resistant colonies, but these could not be further correlated with the presence of regions of cellular senescence. characterized due to the absence of polymorphic markers unique In vitro systems using ectopic expression driven by highly active to the introduced BAC. Therefore, there is no information promoters can lead to nonphysiologic consequences. However, it is regarding whether the introduced DNA is integrated, the sites of unlikely that the observed functional consequences of FBXO31 integration and the copy number. Studies suggest that it is ectopic expression reflect nonphysiologic expression, because the extremely difficult to obtain stable cell lines with intact introduced senescence phenotype observed in MCF-7 cells (Fig. 2B-C) was DNA when transformed with large fragments of DNA, and similar to that previously reported (8). In addition, we have alternative vector systems have been specifically designed to alleviate this problem (23). Previously, based on breast cancer LOH studies, the chromosome band 16q24.3 was delineated as the smallest region of overlap and therefore the likely location of one or more breast cancer tumor suppressor genes (2). Therefore, the possibility was investigated that FBXO31 has a broader role as a breast tumor suppressor. The data presented suggest that this may be the case. The expression of the FBXO31 gene was reduced in both breast tumor cell lines and sporadic primary breast tumors compared with nonmalignant breast epithelium (Fig. 4A and B). In addition, there was a significantly reduced expression in breast cancer cell lines with 16q LOH compared with those without 16q LOH. Similarly, in primary tumor samples, there was a trend for a higher proportion of tumors showing reduced expression when LOH of 16q was present. However, complete elimination of FBXO31 expression was not observed in breast tumors, because the maximum reduction of the relative FBXO31 expression determined by RT-PCR was about 20% of the expression in normal breast. This suggests that down- regulation of FBXO31 is a likely scenario for function as a tumor suppressor but that residual levels of expression are retained. Additional studies were then undertaken to further investigate the potential function of FBXO31 as a tumor suppressor. Ectopic expression of FBXO31 in the breast cancer cell lines MCF-7 and MDA-MB-468 resulted in a moderate inhibition of colony growth on plastic and reduced the proliferation of MCF-7 cells. Cell cycle analysis of the breast cancer cell lines ectopically expressing FBXO31 was consistent with a block in the cell cycle at G1. Investigation of endogenous FBXO31 expression in synchronized MCF-10A and SKBR3 cells show FBXO31 protein levels are at a maximum from late G2 to early G1 phase (Fig. 6A). The timing of FBXO31 destruction is consistent with APC-mediated degradation (17). Cell cycle analysis showed that inhibition of growth of breast cancer cell lines by ectopic FBXO31 expression is likely to be caused by cells not progressing normally past G1 phase of the cell cycle (Fig. 5). These results are consistent with the analysis of replicative senescence in primary cells where entry into senescence Figure 5. FBXO31 ectopic expression blocks cells at G -G of the cell cycle. 0 1 is associated with the accumulation of cells at G1 phase (24). In Cell cycle curves of EGFP-positive asynchronous MDA-MB-468 cultures 24 and 48 hours after transfection with EGFP fusion constructs of FBXO31 (A-B), summary, the expression and functional studies and the role of FBXO31DF(C-D), p53 (E-F), and empty vector (G-H). FBXO31 in the cell cycle are consistent with a tumor suppressor www.aacrjournals.org 11311 Cancer Res 2005; 65: (24). December 15, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research role of FBXO31. Tumor haploinsufficiency for FBXO31 as a supported by a breast cancer gene expression microarray study, consequence of being in a region of frequent LOH in breast cancer which predicted the clinical outcome in breast cancer based on the is likely because tumors possess a low level of expression, and it is combined relative expression profiles of 70 genes (25). Represented predicted that the protein is still subject to cell cycle variation. It is in these 70 genes is FBX031 (denoted as contig 51464). suggested that FBXO31 haploinsufficiency contributes to the The predicted amino acid sequence of the FBXO31 protein does acquisition by tumor cells of escape from senescence and the not have significant homology to known proteins except for the normal controls of cell proliferation. These conclusions are further presence of a 40-amino-acid F-box domain at the amino acid terminus. F-box proteins can exist as part of SCF ubiquitin ligase complexes that are involved in diverse cellular functions, including signal transduction, control of G1-S progression, and orderly execution of the cell cycle (26, 27). Skp1, Cullin-1, and Roc-1 are invariant proteins of the SCF complex, whereas the F-box proteins that bind to Skp1 are the components that impart functional specificity. For example, Skp2 specifically binds phosphorylated p27 resulting in a critical role in the degradation of p27 and therefore a control of S phase entry of the cell cycle (28). Our results support the existence of a SCFFBXO31 complex. First, FBXO31 coimmunoprecipitated with Skp1, Cullin-1, and Roc1 (Fig. 6B) and copurified with FBXO31 in stable cell lines expressing tagged FBXO31 at near physiologic levels (Fig. 6C). Second, analysis of the nonmalignant breast cell line MCF-10A and the breast tumor cell line SKBR3 showed that FBXO31 is cell cycle regulated (Fig. 6A). Third, in silico analysis showed that FBXO31 protein has six minimal D-box (RxxL) motifs that are the hallmark of proteins degraded via the APC/C (17). FBXO31 shares all these features with the Skp2 protein present in the SCFSkp2-Cks1 complex (18), suggesting a functional similarity. It is therefore proposed that FBXO31 is likely to form a functional SCFFBXO31 complex that would recruit and ubiquitinate specific proteins for subsequent degradation. Most F-box proteins bind to their substrates through regions located COOH-terminal to the F-box motif (29). F-box proteins notated as ‘‘FBXO’’ do not have recognizable substrate binding domains; however, as analysis of FBXO proteins progresses, new substrate binding domains are being recognized. For example, Fbx7 has been shown to recruit a substrate; the region responsible was shown to be a proline-rich region and was found in two other FBXO proteins (30). The COOH-terminal part of FBXO31 contains a 175-amino- acid region that is unusually rich (25%) in glycine and arginine residues that may represent the substrate binding region. Regions rich in glycine and arginine have been implicated as protein binding domains, although such reported domains have a more defined glycine/arginine repeat structure than present in FBXO31 (30, 31). It is proposed that the substrates of the SCFFBXO31 complex that are ubiquitinated and subsequently degraded are critical cell cycle proteins, and we are presently working towards their identification.

Figure 6. FBXO31 is cell cycle regulated and is a component of the SCF complex. A, MCF-10A and SKBR3 cells were synchronized with a double thymidine block, harvested at the indicated time points after release from the block, and analyzed by Western blot analysis. Blots were probed with anti-FBXO31, anti-cyclin B1, and anti-MEK2 antibodies. B, HEK293T cells were transiently transfected with constructs expressing individually or combinations of myc-FBX031, myc-FBX031DF, or HA-Skp1 proteins as shown. Total cell lysates (lanes 1-5) were immunoprecipitated (lanes 6-10) with either anti-myc (top three blots) or anti-HA (bottom three blots) antibodies. Input (lanes 1-5) and immunoprecipitated (lanes 6-10) proteins were resolved by SDS-PAGE and probed with appropriate antibodies to detect FBXO31, FBXO31DF, Skp1, Cullin-1, and Roc-1 proteins. C, lysates from HEK293T and a stable HEK293T clone expressing FLAG-HA-FBXO31 were affinity purified using anti-FLAG M2-coated agarose beads. Input and affinity purified proteins were resolved by SDS-PAGE and probed with appropriate antibodies to detect FBXO31, Skp1, Cullin-1, and Roc-1 proteins.

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Acknowledgments 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 Received 3/21/2005; revised 8/26/2005; accepted 9/29/2005. with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: National Health and Medical Research Council of Australia grant We thank Dr. David Millband (Hanson Institute, Adelaide, Australia) for useful 207703 (D. Callen) and Faculty of Health Sciences and Department of Medicine, discussions and Dr. Martha Stampfer (Life Sciences Division, Lawrence Berkeley Nation- University of Adelaide and the Royal Adelaide Hospital. al Labs, Berkeley, CA) for generously providing the cell lines 184V, 48RS, and 184A1.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. FBXO31 Is the Chromosome 16q24.3 Senescence Gene, a Candidate Breast Tumor Suppressor, and a Component of an SCF Complex

Raman Kumar, Paul M. Neilsen, Joanne Crawford, et al.

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