FGF-2 Disrupts Mitotic Stability in Prostate Cancer Through the Intracellular Trafﬁcking Protein CEP57

Published OnlineFirst December 12, 2012; DOI: 10.1158/0008-5472.CAN-12-1857

Cancer Tumor and Stem Cell Biology Research

FGF-2 Disrupts Mitotic Stability in Prostate Cancer through the Intracellular Trafﬁcking Protein CEP57

Rolando Cuevas1, Nina Korzeniewski2, Yanis Tolstov2, Markus Hohenfellner3, and Stefan Duensing2,3

Abstract Malignant tumors with deregulated FGF-2 expression such as prostate cancer are also frequently aneuploid. Aneuploidy can be caused by cell division errors due to extra centrosomes and mitotic spindle poles. However, a link between FGF-2 overexpression and chromosome missegregation has so far been elusive. Here, we show that FGF-2 rapidly uncouples centrosome duplication from the cell division cycle in prostate cancer cells through CEP57, an intracellular FGF-2–binding and trafﬁcking factor. CEP57 was initially identiﬁed as a regulator of centriole overduplication in an RNA interference screen. We subsequently found that CEP57 rapidly stimulates centriole overduplication and mitotic defects when overexpressed and is required not only for FGF-2–induced centriole overduplication but also for normal centriole duplication. We provide evidence that CEP57 functions by modulating tubulin acetylation, thereby promoting daughter centriole stability. CEP57 was found to be overexpressed on the mRNA and protein level in a subset of prostate cancers, of which the vast majority also showed FGF-2 upregulation. Taken together, our results show an unexpected link between altered microenvironmental signaling cues such as FGF-2 overexpression and mitotic instability and provide a rationale for the therapeutic targeting of the FGF-2/FGFR1/CEP57 axis in prostate cancer. Cancer Res; 73(4); 1–11. 2012 AACR.

Introduction gene family consists of more than 20 members that encode Aneuploidy, that is, numerical whole chromosome imbal- secreted polypeptides. FGFs exert their activities mainly ances, is a frequent finding in most human cancers including through 4 conserved transmembrane tyrosine kinase recep- prostate cancer (1–3). Aneuploidy correlates with malignant tors (FGFR1-4). Downstream signaling events are complex progression, which is underscored by the finding that virtually and mainly consist of activation of the phosphoinositide all metastatic prostate cancers show such chromosomal aber- 3-kinase (PI3K)/AKT, mitogen-activated protein kinase rations. In addition, chromosomal imbalances have also been (MAPK), phospholipase Cg (PLCg), and STAT signaling implicated in the development of castration resistance, a pathways. FGFs are mitogenic, they increase cell survival hallmark of lethal prostate cancer (4). Many aneuploid tumors and migration and stimulate angiogenesis (9). harbor alterations in key signal transduction pathways but our There is compelling evidence that upregulation of FGF-2 knowledge of how aberrant signaling events contribute to (basic FGF) plays an important role in prostate carcinogenesis chromosomal instability is incomplete (5). and malignant progression (10). A major source of FGF-2 in the Alterations of fibroblast growth factor (FGF) signaling are prostate is the stroma cell compartment, in which FGF-2 is likewise very common in prostate cancer and other human secreted to function as a paracrine growth factor for prostate malignancies. Numerous in vitro and in vivo results under- epithelial cells, which express FGFRs. A switch from paracrine score a crucial role of FGFs and their receptors in carcino- to autocrine FGF-2 production by prostate cancer cells has genesis and malignant progression (6–8). The human FGF been reported (6). Knockout of FGF-2 was found to delay tumor progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) model (11). Whether and how aberrant FGF-2 expression can contribute to chromosomal instability in pros- Authors' Affiliations: 1Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; 2Section of Molecular Uroon- tate cancer is not known. cology, Department of Urology; and 3Department of Urology, University of Centrosomal protein 57 kDa (CEP57; also referred to as Heidelberg School of Medicine, Heidelberg, Germany translokin) specifically interacts with the 18 kDa isoform of Note: Supplementary data for this article are available at Cancer Research FGF-2 (12). CEP57 is essential for the intracellular transport of Online (http://cancerres.aacrjournals.org/). FGF-2 to the nucleus and the perinuclear compartment in a Corresponding Author: Stefan Duensing, Section of Molecular Urooncol- microtubule-dependent manner after internalization of the ogy, Department of Urology, University of Heidelberg School of Medicine, Medical Faculty Heidelberg, Im Neuenheimer Feld 517, D-69120 Heidel- FGFR1-bound protein (13). Although the precise function of berg, Germany. Phone: 49-6221-56-6255; Fax: 49-6221-56-7659; E-mail: nuclear FGF-2 is unclear, its nuclear localization was found to [email protected] be required for its mitogenic activity. CEP57 contains a cen- doi: 10.1158/0008-5472.CAN-12-1857 trosomal localization and multimerization domain in its N- 2012 American Association for Cancer Research. terminal half and a microtubule localization and stabilization

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domain in its C-terminal half (14). CEP57 binds microtubules PLK4-myc, or pcDNA-PLK4-D154-myc (both kindly provided and its ectopic expression was found to cause excessive by Erich Nigg, Biocenter, University of Basel, Basel, Switzer- microtubule bundling and the formation of basket-like micro- land) were used. Cells were transfected by lipofection using tubule structures (14). Recently, mutations in CEP57 have Fugene 6 (Roche) or the NEON transfection system (Life been identified as a cause of the autosomal-recessive mosaic Technologies) and protein expression was monitored by variegated aneuploidy syndrome, which is characterized by immunoblotting. DsRED- or cyan fluorescent protein (CFP)- mosaic aneuploidies, growth retardation, and cancer predis- encoding plasmids (Clontech) were cotransfected as transfec- position (15). tion markers. Cells were treated with Z-L3VS (Biomol) at a In most mammalian cells, microtubules are organized by 1 mmol/L concentration or 0.1% dimethyl sulfoxide (DMSO) cellular organelles known as centrosomes (16). Centrosomes as solvent control. Cells were treated with nocodazole at a normally consist of a pair of centrioles embedded in pericen- 2 mmol/L concentration dissolved in DMSO for time intervals triolar material (PCM). Their microtubule-organizing function indicated. Cells were treated with hydroxyurea at a 1 mmol/L involves the recruitment of g-tubulin ring complexes to the concentration dissolved in dH2O for 48 hours. Cells were treated PCM. Nondividing cells harbor a single centrosome, which with cycloheximide at a 30 mg/mL concentration dissolved must duplicate before mitosis. This process is tightly con- in dH2O for the time intervals indicated. LNCaP cells were trolled as extra centrosomes can have detrimental effects on treated with 10 ng/mL FGF-2 (Novus Biological) for the time mitotic fidelity through multipolar mitoses or aberrant spin- intervals indicated with dH2O used as solvent control. dle–kinetochore attachment (5). On a molecular level, the formation of a new (daughter) centriole in proximity to the Immunostaining preexisting (maternal) centriole is initiated by CEP152-medi- For immunofluorescence microscopic analyses, cells were ated recruitment of polo-like kinase 4 (PLK4) and involves the grown on 10-mm round coverslips, removed at subconfluent interactions of several additional centrosomal proteins includ- cell density and fixed in 4% paraformaldehyde/PBS for 15 ing hSAS-6, CPAP, CEP135, and CP110 (17–25). A number of minutes at room temperature, followed by washing in PBS human oncogenes and tumor suppressor proteins have been and permeabilization with 1% Triton-X-100 in PBS for 20 implicated in centrosome aberrations and there is evidence minutes. After blocking in 10% normal donkey serum (Jackson that acute centrosome overduplication can drive malignant Immunoresearch), cells were incubated with primary antibo- progression (26–29). Centrosome aberrations are a frequent dies overnight. The next morning, cells were warmed at 37C finding in cancer including prostate cancer, in which centro- for 2 hours, washed in PBS, and incubated with secondary some defects increase with malignant grade and stage and antibodies for 2 hours and mounted with 40,6-diamidino-2- correlate with aneuploidy (30, 31). phenylindole (DAPI)-containing mounting media (Vecta- Here, we show that stimulation of prostate cancer cells with shield; Vector Laboratories) to visualize nuclei. Cells were FGF-2 rapidly uncouples centriole duplication from the cell analyzed using an Olympus AX70 epifluorescence microscope division cycle in a CEP57-dependent manner. We found that or a Leica DM500 epifluorescence microscope. Primary anti- CEP57 itself promotes centriole overduplication and that bodies used were directed against g-tubulin (Sigma-Aldrich), CEP57 is a component of the PLK4-dependent centriole over- acetylated a-tubulin (Life Technologies), CAP350 (Bethyl), duplication pathway. We furthermore provide evidence that CEP57 (Sigma-Aldrich), CEP170 (Invitrogen), PLK4 (kindly CEP57 contributes to centriole amplification by stabilizing provided by Erich Nigg, Biocenter, University of Basel, Basel, daughter centrioles. CEP57 was found to be overexpressed in Switzerland), or hSAS-6 (Bethyl). Secondary antibodies used a subset of primary prostate adenocarcinomas on the tran- were conjugated with fluorescein isothiocyanate, Rhodamine scriptional and protein level and the majority of these tumors Red, or aminomethylcoumarin acetate. Tissue microarrays also overexpressed FGF-2. Taken together, our results provide (TMA) were obtained from US Biomax. Paraffin-embedded evidence for a novel link between FGF-2 and mitotic stability tissue specimens were deparaffinized and rehydrated by that involves the FGF-2 trafficking factor CEP57. Our results standard procedures (33). Antigen retrieval was conducted warrant further exploitation for the development of improved using citrate buffer (pH 6.0). Primary antibodies were direct- prognostic biomarkers and FGF-2/FGFR1/CEP57–targeted edagainstCEP57(Abcam)orFGF-2(SantaCruz)and therapies in prostate cancer. incubated overnight at 37C. The Histostain Plus Kit (Life Technologies) was used for immunodetection of primary Materials and Methods antibodies. Sections were counterstained with hematoxylin Cell culture, transfections, and drug treatments (Life Technology). Immunoblotting was conducted as pre- Human U-2 OS and LNCaP cells as well as mouse NIH 3T3 viously described (34). were obtained from American Type Culture Collection and maintained according to distributor's recommendations. U-2 siRNA OS cells were manipulated to stably express centrin-1-GFP An siRNA library targeting known centriolar proteins as (kindly provided by Michel Bornens, Institut Curie, Paris, described previously (35, 36) was obtained from Qiagen (2 France; ref. 32). Human pCMV6-CEP57 was obtained from different siRNAs per target; see Supplementary Table S1 for Origene. For transient transfections, plasmids encoding sequences). For the centriole overduplication screen, U-2 OS/ pCMV6-CEP57, mCEP57-Flag (kindly provided by Ko Momo- centrin-GFP cells were grown on coverslips in 12-well tissue tani, University of Virginia, Charlottesville, VA), pcDNA3- culture plates with 0.5 mL antibiotics-free Dulbecco's Modified

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Eagle's Medium. Cells were transfected with 3 mLof20mmol/L Supplementary Table S1). We identified 4 proteins that, when annealed RNA duplexes using oligofectamine (Life Techno- depleted, led to a reproducible reduction of the proportion of logy) transfection reagent. Twenty-four hours posttransfec- cells with centriole overduplication induced by Z-L3VS treat- tion, cells were treated with 1 mmol/L of the proteasome ment (34). An at least 20% reduction in 2 consecutive inde- inhibitor Z-L3VS and were microscopically analyzed 48 hours pendent screens was detected when cells were depleted of later. siRNA duplexes that yielded a more than 20% decrease of PLK4, hSAS-6, C4Orf15 (HAUS3), or CEP57. Whereas PLK4 the proportion of cells with overduplicated centrioles normal- and hSAS-6 have previously been implicated in centriole – fi ized to Z-L3VS treated, control siRNA-transfected cells in 2 overduplication (17, 22), HAUS3 has recently been identi ed independent rounds of screening were considered as positive as an important factor for structural centrosome integrity in hits. For confirmation of hits and further analyses, synthetic a similar RNA interference screen (37). We therefore focused RNA duplexes were obtained from Qiagen and cells cultured in on CEP57 and its potential role in centriole duplication 60-mm dishes with 2 mL of antibiotics-free media were trans- control. fected with 12 mLof20mmol/L annealed RNA duplexes. Confirmatory experiments showed a statistically significant Knockdown efficiency was monitored by immunoblotting and 1.8-fold reduction of the proportion of cells with centriole quantitative real-time PCR (qRT-PCR). overduplication (>4 per cell, >1 daughter at a single maternal centriole) from 62.7% in Z-L3VS–treated controls to 35.3% in – P Tissue qPCR analysis CEP57-depleted Z-L3VS treated U2-OS/centrin-GFP cells ( < The TissueScan Prostate Cancer cDNA Array III (Origene) 0.0001; Fig. 1A). Our definition of centriole overduplication containing 39 prostate cancer and 9 normal prostate cDNA ensures that only cells with aberrant synthesis of daughter samples from total RNA obtained from pathologist-verified centrioles are counted and not cells in which centrioles merely tissues, normalized and validated with b-actin in 2 sequential accumulate. Similar results were obtained with a different quantitative PCR (qPCR) analyses was used. cDNAs were resus- siRNA against CEP57. pended according to manufacturer's protocol. qPCR was A statistically significant 1.7-fold reduction of cells with conducted using specific primers to Cep57 (forward: 50-AAGCA- abnormal centrosome numbers (>2 per cell as detected by TGGTTCGGCATTCTTC-30,reverse:50-GGGAGCGTCGTAGAT- immunofluorescence for the PCM marker g-tubulin) was CACTATTA-30; Integrated DNA Technologies) and measured detected in murine CEP57 (mCEP57)-depleted NIH 3T3 fibro- P using the SsoFast EvaGreen Kit (Bio-Rad) according to the blasts treated with Z-L3VS from 40.9% in controls to 23.8% ( > manufacturer's protocol. Cycling conditions were 95C (30 sec- 0.0001; Fig. 1B). onds, activation), 95C(5seconds,denaturation),60C (10 sec- CEP57 was found to be present at the centrosome through- onds, annealing/extension) for 40 cycles on a Bio-Rad CFX96 out the cell division cycle and to colocalize with a marker of Real-Time System run on a C1000 Thermal Cycler (Bio-Rad). mature maternal centrioles, CEP170, and PLK4 (Supplemen- tary Fig. S1), respectively. We therefore tested next a possible Statistical analysis role of CEP57 in PLK4-induced centriole overduplication. Student two-tailed t test for dependent or independent Ectopic expression of PLK4 has previously been shown to samples, Fisher exact probability test or the x2 test were used effectively stimulate centriole overduplication with a charac- wherever applicable. P values less than 0.05 were considered teristic centriole multiplication or "centriole flower" phenotype statistically significant. (21, 34), in which multiple daughter centrioles form concur- rently at single mothers (Fig. 1C, left). This phenotype was Results significantly reduced by 1.6-fold from 47.8% in controls com- CEP57 is a novel regulator of centrosome duplication pared with 29.3% in PLK4-transfected cells in which CEP57 had To identify unknown regulators of centriole overduplication, been depleted (P < 0.0001; Fig. 1C, right). In contrast, knock- we used an siRNA library including genes identified as part of down of CEP57 in cells treated with hydroxyurea, which is the centrosome proteome by Andersen and colleagues (35; believed to stimulate centriole overduplication through a

Figure 1. CEP57 regulates centrosome duplication. A, left, centrioles in U-2 OS/centrin-GFP cells after treatment with either DMSO or Z-L3VS. Right, quantification of centriole overduplication after transfection with either control or CEP57 siRNA duplexes (72 hours) and treatment with Z-L3VS or DMSO (48 hours). B, quantification of NIH 3T3 cells with abnormal centrosome numbers following siRNA and drug treatment as described in A. C, left, U-2 OS/centrin-GFP cells with centriole overduplication after transfection with either empty vector (control) or PLK4 (48 hours). Mitochondrial DsRED was used as transfection marker. Right, quantification of cells with centriole overduplication after transfection with either control or CEP57 siRNA following transfection with empty vector (control) or PLK4. D, centriole overduplication in U-2 OS/centrin-GFP cells after siRNA transfection described in C and

treatment with either dH2O (control) or hydroxyurea (HU). E, immunofluorescence microscopic analysis of U-2 OS/centrin-GFP cells for CEP57 and hSAS-6. F, centrosomes in NIH 3T3 after transfection with murine CEP57 (mCEP57). Mitochondrial DsRED was used as transfection marker. G, quantification of 3T3 cells with more than 2 centrosomes (left) or more than 4 FOP dots (right) after transfection with either empty control vector (control) or mCEP57. H, left, centrioles in U-2 OS/centrin-GFP cells after transfection with control vector or CEP57. Middle, quantification of cells with centriole overduplication after transfection with empty vector (control) or CEP57, using centrin-GFP as centriole marker. Right, quantification of cells with centriole overduplication after transfection with empty vector (control) or CEP57, using FOP as centriole marker. I, left, mitotic NIH 3T3 cells after transfection with empty vector (control) or mCEP57. Middle, quantification of cells with bipolar, pseudobipolar, or multipolar spindle pole arrangement after transfection with control vector or mCEP57. Right, quantification of abnormal mitoses in metaphase or ana-/telophase in NIH 3T3 cells transfected with empty vector or mCEP57. J, quantification of U-2 OS/centrin-GFP cells with fully duplicated centrioles (n ¼ 4) following transfection with control or CEP57 siRNA. All scale bars indicate 10 mm. For quantifications, each bar represents mean and SE of at least 3 independent experiments.

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different mechanism (centriole reduplication in early S-phase), induced centriole overduplication as well as normal centriole had no effect on the proportion of cells with centriole overduplication. duplication (>4 centrioles per cell; Fig. 1D). To further corroborate that PLK4-induced centrin-GFP dots were in fact cen- CEP57 stabilizes daughter centrioles trioles, we costained PLK4-transfected cells and controls for We next sought to determine the mechanism through which CEP57 and hSAS-6, which associates with nascent procen- CEP57 participates in centriole duplication control. trioles (21). Centrin-GFP clearly colocalized with hSAS-6 dots It has been shown that centriole assembly can be reverted underscoring a true centriole overduplication (Fig. 1E). These by the microtubule-depolymerizing agent nocodazole with- results suggest that CEP57 participates in PLK4-induced cen- out affecting the initiation of centriole duplication (38). To triole overduplication but is not required for centriole over- explore a possible role of microtubule stability control in duplication during a prolonged S-phase arrest. CEP57-induced centriole overduplication, we transfected We next determined the effects of ectopically expressed U2-OS/centrin-GFP cells with either PLK4 alone or PLK4 CEP57 on centrosome numbers. Overexpression of mCEP57 in in combination with CEP57 (Fig. 2A, left). After 48 hours, NIH 3T3 fibroblasts (Fig. 1F) led to a significant 3.9-fold cells were treated with nocodazole for 30 or 60 minutes and increase of the proportion of cells with abnormal centrosome the percentage of cells in which centriole overduplication numbers (>2 per cell) from 5.1% to 20.1% when stained for was detectable was assessed. We found that cells transfected g-tubulin (P < 0.0001; Fig. 1G, left). This increase of centrosome with PLK4 alone showed a decline in the proportion of t aberrations was due to centriole overduplication as cells with cells with centriole overduplication from 40.8% at 0 to aberrant numbers (>4) of FGFR1 oncogene partner (FOP) dots 25.5% at t60 min nocodazole treatment (1.6-fold reduction; to visualize individual centrioles were significantly increased P < 0.0001; Fig. 2A, left). Cells transfected with PLK4 and by 4.1-fold from 6% in controls to 24.8% (P < 0.0001; Fig. 1G, CEP57 in combination showed more centriole overduplica- right). tion at t0 and the decrease of cells with centriole over- To test whether overexpression of CEP57 also causes cen- duplication was less pronounced from 54.4 at t0 to 47.6% t P triole overduplication in human cells, we ectopically expressed at 60 min (0.9-fold reduction; < 0.005; Fig. 2A, left). CEP57 in U-2 OS/centrin-GFP cells. Both endogenous and These results indicate that supernumerary daughter cen- overexpressed CEP57 were found to localize to the centrosome trioles are more resistant to nocodazole-induced microtubule in these cells with an occasional formation of basket-like depolymerization in the presence of CEP57. We next wanted to structures under conditions of overexpression as previously confirm that the stabilized structures were in fact centrioles described (Supplementary Fig. S2). We found a statistically and in particular daughter centrioles. We stained U-2 OS/ significant 11.0-fold increase of cells with genuine centriole centrin-GFP cells for the FOP-interacting protein CAP350, overduplication (>4 per cell, >1 daughter at a single maternal which localizes to centrioles and has been reported to stabilize centriole; Fig. 1H) from 0.7% in control-transfected cells to 7.7% procentrioles. We found CAP350 to localize preferentially to in CEP57-transfected cells (P < 0.0001; Fig. 1H, middle). This daughter centrioles surrounding a maternal centriole in increase was confirmed using FOP as centriolar marker with untreated cells (Supplementary Fig. S3). After 60-minute noco- 3.2% of controls showing more than 4 FOP dots and 9.8% of dazole treatment, there was an increased abundance of CEP57-transfected U-2 OS cells (3.1-fold; P < 0.001; Fig. 1H, CAP350 in CEP57-transfected cells in comparison with con- right). trols suggesting the presence of more aberrant daughter CEP57-induced supernumerary centrioles and centrosomes centrioles (Supplementary Fig. S3). were functional as they were associated with a significant We next wanted to explore the effect of CEP57 on normal increase of cells with supernumerary mitotic spindle poles centriole duplication and we transfected U2-OS/centrin-GFP leading to either a pseudobipolar mitotic spindle pole arrange- cells with empty vector or CEP57 and repeated the nocodazole ment or multipolar mitoses as evidenced by g-tubulin staining experiment but now determined the proportion of cells with (Fig. 1I, left and middle). CEP57-expressing cells with aberrant normally duplicated centrioles (3 or 4 per cell; Fig. 2A, right). mitoses (pseudobipolar or multipolar) were in fact able to We found that empty vector controls and CEP57-transfected enter later stages of the cell division cycle as evidenced by a cells showed the same proportion of cells with duplicated significant increase of cells with abnormal numbers of mitotic centrioles at t0 (62%). Control cells showed a gradual decrease P t P spindle poles in ana-/telophase ( < 0.05; Fig. 1I, right). of cells with duplicated centrioles to 51% at 60 min ( < 0.0001) Finally, we determined the effect of CEP57 depletion on and 33% at t180 min (P < 0.001). In CEP57-expressing cells, centrosome maintenance. We found that depletion of CEP57 in however, this decrease was noticeably attenuated (58% at t P t P U-2 OS/centrin-GFP cells by siRNA led to a reduction of 60 min, > 0.005; and 53% at 180 min, < 0.0005) suggesting centrioles and to a significant increase of cells with only 1 that the stabilizing effect of CEP57 in the presence of noco- centriole reaching 10.4% monocentriolar cells at 72 hours post- dazole may be more pronounced on aberrantly synthesized siRNA transfection (P < 0.0001). In addition, normal centriole rather than normal daughter centrioles, which is in line with a duplication was found to be impaired in CEP57-depleted cells previous report (38). as the proportion of cells with fully duplicated centrioles (n ¼ To further explore how CEP57 may affect daughter centriole 4) was found to be significantly reduced (Fig. 1J). stability, we stained empty vector or CEP57-expressing U2-OS/ Taken together, these results underscore that CEP57 is a centrin-GFP cells for acetylated a-tubulin after 180-minute novel regulator of centriole biogenesis and required for PLK4- treatment with nocodazole (Fig. 2B). CEP57-expressing cells

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nocodazole treatment in cells transfected with PLK4 and CEP57 compared with PLK4 alone (2.2-fold; P < 0.0001; Fig. 2C, left). We repeated this experiment with U-2 OS/centrin- GFP cells transfected with empty vector or CEP57 alone and found a significant increase of the abundance of acetylated a-tubulin associated with daughter centrioles after 180-minute in CEP57-transfected cells compared with controls (Fig. 2C, right). Finally, we sought to show biochemically that CEP57 enhances a-tubulin acetylation. Cells transfected with PLK4 alone or in combination with CEP57 were treated with nocodazole and analyzed by immoblotting for the abundance of acetylated a-tubulin. Expression levels of acetylated a-tubulin were higher in CEP57-expressing cells treated with nocodazole in comparison with controls (Supplementary Fig. S4). The finding that the detected increase was relatively modest is most likely related to the fact that CEP57 stabilizes mostly daughter centriolar a-tubulin and that microtubule bundling and basket formation was overall relatively rare in our experimental system (less than 5% of cells). These results show that CEP57-expressing cells can maintain a-tubulin acetylation in the presence of nocodazole as a mechanism of resistance to tubulin depolymerization. Taken together, these results suggest a role of CEP57 in promoting daughter centriole stability through posttransla- tional modification of a-tubulin.

FGF-2 disrupts centriole duplication control through CEP57 Given the role of CEP57 as intracellular trafficking protein for FGF-2 (12), we sought to explore next whether FGF-2 itself has an effect on centrosome duplication control. Human LNCaP prostate cancer cells, which express FGFR1, were treated with recombinant FGF-2 (rFGF-2) followed by an Figure 2. CEP57 stabilizes daughter centrioles. A, left, quantification of the analysis of centrosome numbers (Fig. 3A). We found that percentage of U-2 OS/centrin-GFP cells with overduplicated (n > 4) stimulation with 10 ng/mL of rFGF-2 led to a significant centrioles after transfection with either PLK4 alone or CEP57 and PLK4 increase of cells with supernumerary centrosomes as evi- (48 hours) followed by treatment with nocodazole for the indicated time intervals. Right, quantification of the percentage of U-2 OS/centrin-GFP denced by immunostaining for g-tubulin from 0.5% to 6.7% cells with normally duplicated centrioles (3 or 4) after transfection with after 8 hours (13.4-fold; P < 0.0001) and from 1.1% to 11% after empty control vector or CEP57 (48 hours) followed by treatment with 24 hours (10-fold; P < 0.0001; Fig. 3A). These aberrantly nocodazole for the indicated time intervals. B, immunofluorescence synthesized centrosomes were functional as rFGF-2 stimula- microscopic analysis of U-2 OS/centrin-GFP cells for acetylated a-tubulin after transient transfection with either empty vector or CEP57 tion of LNCaP cells caused an increase in cell division errors and treatment of cells with nocodazole for the indicated time intervals. (multipolar or pseudobipolar mitoses). Multipolar mitoses C, quantification of the ratios of acetylated a-tubulin and centrin-GFP increased 12-fold from 0.2% to 2.6% after rFGF-2 stimulation signals following the experimental procedure described in A. Each box/ (P < 0.05; Fig. 3B). Pseudobipolar mitoses also increased bar represents mean and SE of at least 3 independent experiments. significantly following rFGF-2 stimulation from of 0.5% to 4.4% (P < 0.001; Fig. 3B). were found to show a higher abundance of acetylated a-tubulin FGF-2 stimulation of U-2 OS/centrin-GFP cells also led to a at centrioles after nocodazole treatment than controls (com- modest increase of cells in S-phase (49.4% BrdUrd-positive t pare top right and bottom right in Fig. 2B at 180 min). This cells vs. 56.7% BrdUrd-positive cells after 24 hours rFGF-2 finding suggests that CEP57 increases tubulin acetylation to stimulation). At the same time, we detected an increase of stabilize daughter centrioles. cells with genuine centriole overduplication, that is, aberrant To assess this increase more accurately, we conducted daughter centriole assembly at maternal centrioles (Fig. 3C). image quantification of both acetylated a-tubulin and cen- Centriole overduplication increased from 0% to 2.2% after 8- trin-GFP using the UN-SCAN-IT software (Silk Scientific) and hour stimulation with rFGF-2 (P < 0.0005), from 0.5% to 4.7% calculated the ratio between the 2 values to account for after 24-hour stimulation with rFGF-2 (P < 0.0001), and from differences in centriole numbers. We found that acetylated 0.2% to 7.2% after 48-hour rFGF-2 stimulation (P < 0.0001), each a-tubulin was significantly more abundant after 60-minute in comparison with controls (Fig. 3C, right). These results show

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that FGF-2 can rapidly uncouple centriole duplication from the overduplication (0.3% in control-treated vs. 2.7% in FGF-2– cell division cycle. treated cells) in comparison with control-depleted cells (0.2% To determine the role of CEP57 in FGF-2–induced centro- vs. 11.2%; Fig. 3E). some overduplication in LNCaP cells, we used an siRNA Taken together, our results show that FGF-2 rapidly uncou- approach. Control and CEP57-depleted cells were treated with ples the cell cycle from the centriole duplication cycle in a 10 ng/mL rFGF-2 for 24 hours. A significant increase of cells CEP57- and FGFR1-dependent manner. with abnormal centrosome numbers was detected in control cells from 0.5% to 8.3% (16.6-fold; P < 0.0001), whereas FGF-2 CEP57 is overexpressed in prostate cancer was unable to stimulate centrosome overduplication in CEP57- CEP57 expression has not been analyzed in human cancer so depleted cells (1% vs. 2%; P > 0.05; Fig. 3D). far. To explore whether prostate cancer, in which disruption of We next analyzed the role of FGFR1 in FGF-2–induced FGF-2 expression is a frequent even (6, 8), may also show centrosome overduplication in LNCaP prostate cancer cells. altered CEP57 expression, we first conducted a qRT-PCR We found that siRNA-mediated depletion of FGFR1 signifi- analysis of 9 normal and 39 cancerous prostate samples cantly impaired the ability of FGF-2 to stimulate centrosome using a commercially available qPCR gene expression array

Figure 3. FGF-2 uncouples centrosome duplication from the cell division cycle through CEP57. A, left, centrosomes in LNCaP prostate cancer cells after stimulation with 10 ng/mL rFGF-2 for 24 hours or dH2O treatment. Right, quantiﬁcation of centrosome overduplication after stimulation with 10 ng/mL rFGF-2 or dH2O (control). B, left, mitotic LNCaP cells after stimulation with

10 ng/mL rFGF-2 for 24 hours or dH2 O (control). Right, quantification of multipolar or pseudobipolar mitoses after 24 hours stimulation with 10 ng/ mL rFGF-2 or dH2O (control). C, left, centrioles in U-2 OS/centrin-GFP cells after treatment with 10 ng/mL rFGF-2 for 24 hours or dH2O (control). Right, quantification of cells with centriole overduplication after stimulation with 10 ng/mL FGF-2 or dH2O (control). D, quantification of LNCaP cells with centrosome overduplication after transfection with either control siRNA or siRNA targeting CEP57 and stimulation with dH2O (ctrl.) or 10 ng/mL rFGF-2 for 24 hours. E, quantification of LNCaP cells with centrosome overduplication after transfection with either control siRNA or siRNA targeting FGFR1 and stimulation with dH2O (control) or 10 ng/mL rFGF-2 for 24 hours. All scale bars indicate 10 mm. For quantifications, each bar represents mean and SE of at least 3 independent experiments.

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ABBenign Prostate adenocarcinoma Amplification 8,000

7,000

6,000 CEP57 5,000

4,000

3,000

2,000 Relative fluorescence units Relative 1,000

0 26 28 30 32 34 36 38 40 FGF-2 Cycle number

CDE Diagnosis Staining CEP57 FGF-2 P < 0.05 P < 0.05 14 25 Normal (n = 10) Overexpression 0 3 (30%) Weak-moderate 9 (90%) 7 (70%) 12 Loss 1 (10%) 0 20 10 Hyperplasia (n = 19) Overexpression 0 5 (26.3%) 8 15 Weak-moderate 17 (89.5%) 12 (63.2%) Loss 2 (10.5%) 2 (10.5%) 6 10

Adenocarcinoma (n = 48) Overexpression 12 (25.0%) 40 (83.3%) 4 5 Weak-moderate 23 (47.9%) 8 (16.7%) 2 Loss 13 (27.1%) 0 % of cells with n > 4 centrioles % of cells with n > 4 FOP dots 0 0 P ≤ 0.01* P ≤ 0.0001* Control CEP57 CEP57 + + FGF-2 – + *Association between overexpression and prostate adenocarcinoma (Fisher exact probability test, two-tailed)

Figure 4. CEP57 is overexpressed in prostate cancer. A, qPCR analysis of 9 normal and 39 cancerous prostate specimens for CEP57 mRNA expression. Relative fluorescence units are given. Red amplification curves represent normal control tissue, blue amplification curves represent prostate cancer samples. B, immunohistochemical analysis of benign prostate specimens (left) and prostate adenocarcinomas (right) for CEP57 and FGF-2, respectively. Note the strong overexpression of both proteins in the prostate cancer specimens. Scale bar, 50 mm. C, overview of the immunohistochemical staining results for CEP57 and FGF-2 obtained from the TMA used in B. D, quantification of LNCaP cells with abnormal centriole numbers (>4 FOP dots per cell) after transfection with CEP57 or empty vector. E, quantification of LNCaP cells with abnormal centriole numbers (>4 centrin-GFP dots, plasmid was cotransfected) transiently transfected with CEP57 (24 hours) followed by stimulation with rFGF-2 (24 hours). Each bar represents mean and SE of at least 3 independent experiments.

containing normalized and validated cDNA samples (Fig. 4A). CEP57 staining (90% and 89.5%, respectively), an overexpres- The mean Cq for normal samples was 32.77 0.36 (SD). Of 39 sion of CEP57 was detected in 12 of 48 of prostate adenocar- prostate cancer samples, 2 tumors showed a Cq value greater cinomas (25%), whereas normal or hyperplastic tissue samples than mean plus 3 times SD (33.85 cycles) indicating reduced showed no overexpression of CEP57 (P 0.01). In a fraction of mRNA expression in comparison with controls (5.1%). A total prostate cancer samples, we also detected a loss of CEP57 of 15 prostate cancers showed a Cq below the mean minus 3 protein expression (27.1%). In addition, we conducted an times SD (31.69 cycles) indicating increased mRNA expression immunohistochemical analysis of an adjacent section of the (38.5%). These ﬁndings show CEP57 transcriptional deregula- same TMA for FGF-2. Most normal and hyperplastic tissue tion, most prominently upregulation, in a subset of human samples showed a weak to moderate protein expression (79% prostate cancers. and 63.2%, respectively), whereas 83.3% of the prostate ade- To further corroborate and extend these results, we con- nocarcinomas showed FGF-2 overexpression (P 0.0001). ducted an immunohistochemical analysis of normal (n ¼ 10), FGF-2 was predominantly found to be expressed by carcinoma hyperplastic (n ¼ 19), and cancerous (n ¼ 48) prostate speci- cells. Autocrine/paracrine production of FGF-2 has been mens using a TMA. In comparison with normal and hyper- reported for more advanced prostate cancer and most prostate plastic tissue specimens, which showed a weak to moderate cancers analyzed here had a T2 stage or higher. Importantly,

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11 of 12 prostate cancers with overexpression of CEP57 showed FGF-2/FGFR1 internalization and CEP57-dependent transport also an overexpression of FGF-2 (91.7%). of FGF-2 to the nuclear membrane (12), thereby shifting more To directly test the ability of CEP57 to induce centriole CEP57 to the centrosomal pool. Increased centrosomal CEP57 aberrations in prostate cancer cells, we transiently transfected would then lead to the stabilization of aberrant daughter LNCaP cells with CEP57. Both endogenous and overexpressed centrioles. Our finding that CEP57 promotes the posttransla- CEP57 were found to show a centrosomal staining pattern in tional modification of a-tubulin by acetylation at daughter LNCaP cells, again, with an occasional formation of basked centrioles (Fig. 2 and Supplementary Fig. S4) supports this structures in cells with overexpressed CEP57 (Supplementary notion. Once internalized, additional FGF-2–dependent sig- Fig. S5). When CEP57-transfected LNCaP cells were analyzed naling events, for example MAPK activation, are likely to for centriole numbers by immunofluorescence microscopy for contribute to centrosome overduplication (39). FOP, a significant increase from 5.2% of cells with more than 4 The question how a centriole-stabilizing protein may drive FOP dots per cell in controls to 10.0% was detected (1.9-fold; P < centriole overduplication remains to be elucidated. It has 0.05; Fig. 4D). In addition, we found that stimulation of CEP57- previously been shown that maternal centrioles can harbor overexpressing LNCaP cells with rFGF-2 led to a further more than one site of PLK4 recruitment without actual cen- increase of cells with abnormal centriole numbers from triole formation (38, 40) and that the stability of elongating 15.3% to 20.5% (P < 0.05; Fig. 4E). procentrioles is a highly regulated process (36, 38). It is hence Taken together, these data indicate that CEP57 is over- possible that CEP57 stabilizes procentrioles that normally expressed in a fraction of prostate adenocarcinomas on the would not become daughter centrioles. The importance of mRNA and protein level and, furthermore, that there is a high procentriole stability for centriole growth is underscored by coincidence of CEP57 and FGF-2 overexpression. In vitro previous studies that implicate CPAP as well as CAP350 in this recapitulation of CEP57 overexpression and FGF-2 stimulation process (24, 38) and results shown here add CEP57 to this in LNCaP prostate cancer cells led to a high frequency of group of microtubule-stabilizing proteins required for proper centriole aberration. centriole duplication. The fact that CEP57 overexpression results in an increase in the resistance of microtubules to Discussion nocodazole has also been observed for cytoplasmic microtu- There is compelling evidence from numerous in vitro as well bules (14). The precise molecular mechanisms of daughter as in vivo studies that aberrant FGF signaling plays a crucial centriole stabilization by CEP57 remain to be determined. role in prostate cancer development and progression (6, 8). Our finding that CEP57 is overexpressed in prostate cancer Results presented here provide additional insights into the is the first report to implicate CEP57 in a major human oncogenic activities of the FGF signaling axis. Our results malignancy. However, mutations in CEP57 have recently been provide the first evidence that a microenvironmental, secreted identified as a cause of a rare genetic disease associated with growth factor, FGF-2, can subvert mitosis, and hence promote cancer predisposition, mosaic-variegated aneuploidy syn- chromosomal instability. They show that the FGF-2–binding drome (MVA; ref. 15). The principal difference between the and trafficking protein CEP57 is a critical link in this process. finding reported here and the implication of CEP57 in MVA is Our data furthermore provide the first evidence of CEP57 the loss of function in the germline mutation and the signif- overexpression in a major human cancer, that is, prostate icant overexpression on the mRNA and protein level in pros- cancer. They also establish CEP57 as a novel regulator of tate cancer. To reconcile these findings, it is important to centriole duplication through centriole stability control, an emphasize that many proteins implicated in cancer can have understudied but important aspect of centrosome duplication. both, oncogenic or tumor suppressive activities depending on We initially identified CEP57 in an RNA interference screen the spectrum of their biologic functions, the cellular context for proteins involved in centriole overduplication. We subse- and the timing of their expression. Inherited loss of CEP57 quently discovered that CEP57 is critical for FGF-2–induced could promote cancer through the previously described role of centriole overduplication as well as normal centriole duplica- CEP57 role in proper microtubule-kinetochore attachment tion. Results showing the formation of extra centrioles at single and/or spindle pole integrity (41, 42). In contrast, its acute maternal centrioles underscore that CEP57 causes a genuine overexpression leads to a rapid uncoupling of the centrosome centriole duplication defect and not simply an accumulation of cycle from the cell division cycle and, subsequently, mitotic centrioles. This is further corroborated by our finding that defects as shown here. Continous adverse events such as the CEP57 is required for PLK4-induced centriole multiplication, a latter could ultimately cause chromosome missegregation in process in which multiple daughter centrioles form concur- viable cells, aneuploidy and enhanced clonal evolution and rently around single mothers (21, 34). Our results showing that tumor heterogeneity. Our data do not rule out that the massive FGF-2- and CEP57-induced centriole overduplication results in overexpression seen in a fraction of prostate adenocarcinomas a significant increase of defective mitoses underscores that (Fig. 4) may also have growth-suppressive properties for exam- CEP57-induced extra centrioles are functional and can mature ple, through impaired microtubule-dependent transport due to form mitotic spindle poles. to aberrant microtubule bundling induced by CEP57 (14). CEP57 has previously been shown to bind and bundle Collectively, our results provide a novel link between micro- microtubules and to localize to the centrosome through a environmental signaling cues such as FGF-2 and chromosomal C-terminal centrosomal localization domain (14). In the most instability in cancer. On the basis of the potentially oncogenic simplistic model, FGF-2 stimulation would trigger increased activities associated with acute FGF-2/CEP57 overexpression

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reported here, our results would argue in favor of FGF-targeted Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Hohenfellner, S. Duensing therapies, at least in a subset of patients with prostate cancer. Study supervision: M. Hohenfellner, S. Duensing Clearly, more detailed studies are warranted to dissect the role of CEP57 in prostate tumorigenesis and to fully exploit the Acknowledgments FGF-2/FGFR1/CEP57 axis for the development of improved The authors thank Ko Momotani, Michel Bornens, and Erich Nigg for sharing biomarkers and preventive as well as therapeutic approaches. important reagents. The authors also thank Geraldine Rauch for statistical advice. Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed. Grant Support This work was supported by the Medical Faculty Heidelberg of the University of Heidelberg School of Medicine, the Department of Urology of the University of Authors' Contributions Heidelberg School of Medicine and, in part, by a Research Scholar Grant from the Conception and design: R. Cuevas, S. Duensing American Cancer Society (S. Duensing). Development of methodology: R. Cuevas, S. Duensing The costs of publication of this article were defrayed in part by the Acquisition of data (provided animals, acquired and managed patients, payment of page charges. This article must therefore be hereby marked provided facilities, etc.): N. Korzeniewski, Y. Tolstov, M. Hohenfellner, S. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this Duensing fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Cuevas, N. Korzeniewski, S. Duensing Writing, review, and/or revision of the manuscript: R. Cuevas, Y. Tolstov, M. Received May 21, 2012; revised November 5, 2012; accepted November 25, 2012; Hohenfellner, S. Duensing published OnlineFirst December 12, 2012.

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FGF-2 Disrupts Mitotic Stability in Prostate Cancer through the Intracellular Trafficking Protein CEP57

Rolando Cuevas, Nina Korzeniewski, Yanis Tolstov, et al.

Cancer Res Published OnlineFirst December 12, 2012.

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