Oncogene (2008) 27, 6385–6395 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Pituitary tumor transforming 1 regulates Aurora kinase A activity

Y Tong, A Ben-Shlomo, C Zhou, K Wawrowsky and S Melmed

Department of Medicine, Cedars-Sinai Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Pituitary tumor transforming gene 1 (PTTG1),a pathogenesis of estrogen-induced rat prolactinomas transforming gene highly expressed in several cancers,is (Heaney et al., 1999), and has been suggested as a a mammalian securin regulating both G1/S and prognostic marker for thyroid, breast (Solbach et al., G2/M phases. Using protein array screening,we showed 2004)and colon cancer invasiveness (Heaney et al., PTTG1 interacting with Aurora kinase A (Aurora-A), 2000). PTTG and HTLV-1 Tax exhibit a cooperative and confirmed the interaction using co-immunoprecipita- transforming activity (Sheleg et al., 2007), whereas small tion,His-tagged pull-down assays and intracellular interfering RNA (siRNA)directed against PTTG immunofluorescence colocalization. PTTG1 transfection suppressed lung cancer growth in nude mice (Kakar into HCT116 cells prevented Aurora-A T288 autopho- and Malik, 2006)and was also proposed as a subcellular sphorylation,inhibited phosphorylation of the histone H3 therapy for ovarian cancer (El-Naggar et al., 2007). Aurora-A substrate and resulted in abnormally condensed Overexpressed PTTG1 results in chromatin. PTTG1-null cell proliferation was more instability and aneuploidy, which has been suggested sensitive to Aurora-A knock down and to Aurora kinase as a mechanism underlying PTTG1 transforming Inhibitor III treatment. The results indicate that PTTG1 activity (Wang and Melmed, 2000; Yu et al., 2003). and Aurora-A interact to regulate cellular responses to PTTG1 inhibits transcriptional activity (Bernal anti-neoplastic drugs. PTTG1 knockdown is therefore a et al., 2002), and p53 stabilization is uncoupled by the potential approach to improve the efficacy of tumor loss of PTTG1 (Bernal and Hernandez, 2007). PTTG Aurora kinase inhibitors. interacts with Ku, the regulatory subunit of DNA- Oncogene (2008) 27, 6385–6395; doi:10.1038/onc.2008.234; dependent protein kinase (Chien and Pei, 2000; Pei, published online 28 July 2008 2000, 2001; Wang and Melmed, 2000), further indicating a role for the protein in DNA damage repair. PTTG1 Keywords: PTTG1; Aurora kinase A; drug response induces genetic instability in colorectal cancer cells by inhibiting double-stranded DNA repair activity (Kim et al., 2007), activates c-Myc (Pei, 2001) and basic fibroblast growth factor (bFGF)(Chien and Pei, 2000) Introduction and promotes tumor angiogenesis (Zou et al., 1999; Kim et al., 2006). Dysregulated PTTG1 likely prevents Pituitary tumor transforming gene 1 (PTTG1), isolated mitotic exit as doubly mutant Cdc20 and PTTG1 from rat pituitary tumor cells (Pei and Melmed, 1997), embryos were unable to maintain metaphase arrest (Li was subsequently identified as a securin protein (Zou et al., 2007). Although non-homologous end joining is et al., 1999), which binds separase, inhibits cohesin intact in securin-deficient cells, the process occurs cleavage and facilitates sister chromatid separation. through aberrant end processing (Bernal et al., 2008). PTTG1 is required for several cellular processes (Vlo- Furthermore, securin-deficient cells exhibit enhanced tides et al., 2007), including human fetal brain develop- sensitivity to DNA damage-inducing agents (Bernal ment (Boelaert et al., 2003), telencephalic neurogenesis et al., 2008). Using a ChIP (chromatin immunoprecipi- (Tarabykin et al., 2000)and rat liver regeneration tation)-on-Chip assay, PTTG1 was shown to bind (Akino et al., 2005). PTTG1-null mice exhibit testicular, multiple gene promoters (Tong et al., 2007), and PTTG1 pituitary, and pancreatic beta cell hypoplasia (Wang interacts with Sp1 to promote the G1/S transition (Tong et al., 2001b, 2003)and male-selective insulinopenic et al., 2007). This action may also contribute to PTTG1- diabetes (Wang et al., 2003). PTTG1 is abundantly induced cell transformation. To further investigate expressed in most cancers and PTTG1 levels correlate PTTG1 functions, we screened an array of 5000 with tumor development and size (Saez et al., 1999; for PTTG1 interactions and showed that PTTG1 McCabe et al., 2003). PTTG1 is induced early in the interacts with Aurora kinase A (Aurora-A), and also suppresses Aurora-A activity. Aurora-A is activated by Correspondence: Dr S Melmed, Department of Medicine, Cedars- TPX-2, Ajuba and Bora (Eyers et al., 2003b; Hirota Sinai Medical Center, University of California, Los Angeles, 8700 et al., 2003; Hutterer et al., 2006), suppressed by its N- Beverly Blvd, Academic Affairs, Room 2015, Los-Angeles, CA 90048, terminal domain (Zhang et al., 2007)and its oncogenic USA. activity suppressed by p53 (Chen et al., 2002). Our E-mail: [email protected] Received 1 April 2008; revised 15 May 2008; accepted 17 June 2008; findings thus add knowledge to mechanisms for Aurora- published online 28 July 2008 A regulation. PTTG1 and Aurora kinase A Y Tong et al 6386 Results (data not shown). Thus, in vivo PTTG1 binding to Aurora kinase A appears not to involve separase, PTTG1 interacts with Aurora-A in vitro and in vivo a known protein partner for PTTG1. Furthermore, PTTG1 is shown to interact with more than 700 gene PTTG1 also appears to direct the centrosomic location promoters (Tong et al., 2007). Here we show PTTG1 of separase during mitosis, besides inhibiting separase specifically interacts with several proteins (Supple- activity. Centrosomic separase accumulation may mentary data 1). Thus, we built a PTTG1 interaction be important for separase activation as the duration network by combining the results. With this assay, of accumulation approximates the time of separase we confirmed the reported interaction of PTTG1 and activation. ribosomal protein S10 (Pei, 1999), with a Z-score of 3.34 (Figure 1a). PTTG1 was shown to bind specifically to Aurora-A with a Z-score of 4.90, indicative of a high PTTG1 inhibits Aurora-A phosphorylation of histone H3 probability of true interaction (Figure 1a). Using co- To assess whether PTTG1 serves as a substrate immunoprecipitation and His-tag pull-down assay to competitor for Aurora-A activity, we tested whether confirm this interaction (Figure 1b), and using Aurora-A PTTG1 inhibits phosphorylation of the histone H3 antibody, we readily co-immunoprecipitated PTTG1 substrate. Aurora-A is an arginine-directed kinase protein from whole cell extracts. Aurora-A protein was (Ohashi et al., 2006)modulating spindle function and also detected in the immunoprecipitated complex using chromatin condensation, and which phosphorylates PTTG1 antibody, indicating an in vivo interaction. In histone H3 (Crosio et al., 2002), crucial for the onset His-Tag pull-down assays, wherein expressed Aurora-A, of mitosis in the early G2 phase (Pascreau et al., 2003). PTTG1 and their respective fragments served as bait, Non-phosphorylated histone H3 participates in chro- their respective counterparts were captured. As shown in matin compaction, and during mitosis, Ser10 phosphor- Figure 1c, His-tagged Aurora-A and fragment 1 co- ylation weakens histone tail–DNA interactions and precipitated with PTTG1, whereas His-tagged PTTG1 favors DNA–polyamine binding, thus enabling the and fragment 1 also pulled down Aurora-A. Thus, formation of highly compacted mitotic PTTG1 and Aurora-A appear to interact through their (Prigent and Dimitrov, 2003). Similar to the histone H3 respective N termini. Ser10 motif (Crosio et al., 2002), the PTTG1 protein sequence contains a putative Aurora-A consensus motif (RXS/T)(Cheeseman et al., 2002)in the basic N- PTTG1 colocalizes with centrosomic and spindle Aurora-A terminal domain (Figure 1d). We assayed cell-free Intracellular Aurora-A localization is important for kinase activity and as shown in Figure 3e, when mitotic control and spindle formation, and intracellular Aurora-A was omitted from the reaction, endogenous Aurora-A locations undergo striking changes during phosphorylated histone H3 was low, whereas the the cell cycle. In late G1/early S phase, Aurora-A is presence of Aurora-A enabled histone H3 phosphoryla- found in pericentriolar centrosome material, and during tion. PTTG1 appeared to directly inhibit Aurora-A prophase. The centrosomic association continues at the activity, as evidenced by dose-dependent inhibition of mitotic poles and, subsequently at metaphase, resides in Aurora-A histone H3 phosphorylation. adjacent spindle microtubules (Li and Li, 2006). Owing We then tested whether PTTG1 inhibits Aurora-A to lack of a sufficiently robust PTTG1 antibody for activity in vivo. The phosphorylation state of histone H3 intracellular immunofluorescent location, we monitored determines chromatin condensation and de-condensa- intracellular PTTG1 location by using green fluorescent tion (Crosio et al., 2002), and Aurora-A action depends protein (GFP)-tagged PTTG1. As shown in Figure 2a, on T288 autophosphorylation in the activation loop PTTG1-GFP colocalized with Aurora-A in the spindle (Walter et al., 2000). As shown in Figure 3b, PTTG1- and centrosome during metaphase. PTTG1 inhibits GFP overexpression in HCT116 cells resulted in separase proteolytic activity (Hornig et al., 2002; decreased phosphorylation of both Aurora-A and Waizenegger et al., 2002), and when degraded, releases histone H3 as demonstrated by western blot. Reduced separase histone H3 Ser10 phosphorylation levels also correlated at the metaphase to anaphase transition. As separase with chromatin abnormalities, as shown in Figure 3c. cleaves cohesin and promotes sister chromatid separa- Next, we tested whether separase inhibition induces tion (Hornig et al., 2002), we tested separase and similar effects as those observed with overexpressed Aurora-A localization in HCT116 cells. As shown in PTTG1. Although separase was reported to impact Figure 2a, separase and Aurora-A colocalized mainly to Aurora kinase B (Pereira and Schiebel, 2003), reduction the centrosome, clearly distinct from PTTG1 and of separase levels caused aneuploidy but had little effect Aurora-A colocalization, suggesting that PTTG1 on the spindle, Aurora-A distribution or chromatin and Aurora-A interact independently of separase. condensation (data not shown), conforming with pre- Furthermore, separase and Aurora-A centrosome colo- vious reports (Waizenegger et al., 2002). These observa- calization was not evident in cells devoid of PTTG1 tions also indicate that PTTG1 action on Aurora-A and (Figure 2b), further indicating the PTTG1 requirement chromatin occurs independently of effects on separase. for centrosomic separase location. When separase The small-molecule Aurora-A inhibitor, ZM 447439, levels were knocked down by specific RNA inter- retards the progression of chromosome condensation, ference, PTTG1 and Aurora-A colocalization persisted leading to a disorganized spindle (Gadea and Ruder-

Oncogene PTTG1 and Aurora kinase A Y Tong et al 6387

1

IP Neg IgG Anti-PTTG Input PTTG1 (Red) Z-score

BSA Detection Aurora-A

RPS10 3.34 a-A G

IP Neg Ig Anti-Auror Input Aurora-A 4.90 Detection PTTG1 Duplicate

.F1 .F2 a-A AurkA and its fragments Binding to PTTG1 Neg Auror Aurora-A Aurora-A AurkA.F1 1 128 + AurkA.F2 121 403 – PTTG1 AurkA 1 403 +

PTTG1 and its fragments Binding to Aurora PTTG1.F1 1 120 + Neg PTTG1 PTTG1.F1 PTTG1.F2 PTTG1.F2 101 202 – PTTG1 1 202 + Aurora-A

Aurora N C kinase A

Interaction PTTG1 N C

A box Transactivating DNA binding

Kinase Aurora kinase A Ken box SH-3 binding D box domain substrate motif Figure 1 Pituitary tumor transforming gene 1 (PTTG1)interacts with Aurora kinase A (Aurora-A).( a)PTTG1 was biotin-labeled and incubated with the protein microarray. Attached biotinylated PTTG1 was probed with Streptavidin-Alexa Fluor 647 conjugate. The results show that PTTG1 binds Aurora-A and ribosomal protein S10 (RPS10)but not bovine serum albumin (BSA).( b)Aurora-A antibody was used to co-immunoprecipitate PTTG1 and PTTG1 antibody used to co-immunoprecipitate Aurora-A, respectively. Western blots show that PTTG1 co-immunoprecipitated with Aurora kinase A. (c)His-Tag Aurora-A, PTTG1 and their fragments were expressed using the TNT Quick Coupled Transcription/Translation system. Aurora-A and its fragments were used to pull down PTTG1, or PTTG1 and its fragments were used to pull down Aurora-A. The results show that PTTG1 protein binds Aurora-A and its fragment 1 and Aurora-A binds to PTTG1 and its fragment 1, suggesting that the two proteins interact through their respective N termini. (d)Aurora-A and PTTG1 interaction domains. man, 2005). As shown in Figure 3a, PTTG1 over- we investigated the time course of PTTG1 and expression caused unequal PTTG1 and Aurora-A Aurora-A degradation during mitosis (Figure 3d), and distribution, suggestive of spindle disorganization. Also, PTTG1 degraded about 60 min before Aurora-A. Thus, transfectants overexpressing PTTG1-GFP contain ab- PTTG1 appears to inhibit Aurora-A activity, and normal chromatin, likely reflecting the interference with PTTG1 degradation may release Aurora-A for cell chromatin condensation and de-condensation cycle activity. Unlike results shown in HeLa cells (Figure 3c). Taken together, these results confirm that (Lindon and Pines, 2004), PTTG1 degradation starts PTTG1 inhibits Aurora-A activity in vivo. As both at 4.5 h and Aurora-A at 5.5 h, suggesting that HCT116 PTTG1 and Aurora-A are substrates of the adenoma- cell mitosis might be delayed upon synchronization tous polyposis coli (APC)degradation pathway, treatments.

Oncogene PTTG1 and Aurora kinase A Y Tong et al 6388 PTTG1-GFP Aurora-A DNA Merged

WT

Separase Aurora-A DNA Merged

PTTG –/–

WT

PTTG1-GFP Aurora-A DNA Merged

WT

Figure 2 Cellular location of Pituitary tumor transforming gene 1 (PTTG1). PTTG1-GFP was used to identify intracellular PTTG1 location. Aurora kinase A (Aurora-A)and separase localization was detected by antibodies as indicated in Materials and methods. Immunofluorescence shows (a)in HCT116 cells PTTG1 colocalizes with Aurora-A on the spindle and centrosome in HCT116 cells as indicated by the white arrow. (b)Separase is not detected in the centrosome (white arrow)in PTTG1 À/À HCT116 cells. (c)Separase is localized on the centrosome area (white arrow)in HCT116 cells. ( c)PTTG1 overexpression caused abnormal chromatin structure (white arrow). Scale bar ¼ 5 mm.

PTTG1–Aurora-A regulates Aurora kinase inhibitor III to Aurora-A knock down, with 62% remaining viable and doxorubicin response compared with 88% of WT cells (Po0.05)(Figure 4a). Aurora-A regulates mitotic entry at the G2 checkpoint Replication of PTTG1À/À cells treated with 12.5 mM (Marumoto et al., 2002), and abrogating DNA damage- Aurora kinase inhibitor III Cyclopropanecarboxylic induced G2 checkpoints may induce topoisomerase- acid-(3-(4-(3-trifluoromethyl-phenylamino)-pyrimidine- based drug sensitivity (Vogel et al., 2005, 2007). 2-ylamino)-phenyl)-amide (Figure 4b) was more sensi- As PTTG1 modulates Aurora-A activity, we tested tive than WT responses, with 56% remaining viable as whether PTTG1 modulates responses to antineoplastic compared to 73% of WT cells treated for 48 h (Po0.05). drugs whose efficacy relies on a functional G2 Archorage-dependent colony formation by PTTG1À/À checkpoint. cells was abrogated as compared with WT colonies in Aurora-A is overexpressed in a variety of tumor cell the presence of Aurora kinase inhibitor III (Figure 4c). lines and acts to transform fibroblasts and format We then either re-introduced PTTG1 into PTTG1À/À multipolar mitotic spindles associated with genomic HCT116 cells or transfected PTTG1 siRNA into WT instability (Zhou et al., 1998). As several Aurora-A cells to investigate the PTTG1 role in the drug res- inhibitors exhibit antineoplastic effects (Mountzios ponse. As shown in Figures 4d and e, knock down of et al., 2008), we tested the effects of Aurora-A siRNA PTTG1 increased HCT116 cell sensitivity to Aurora knock down in HCT116 wild-type (WT)and PTTG1 À/À kinase inhibitor III and overexpressed PTTG1 rescued cells. As shown by western blot (Figure 4a), the sensitivity of PTTG1À/À cells. Other less-specific RNA interference efficiently knocked down Aurora Aurora kinase inhibitors, including Aurora kinase kinase A expression in both WT and PTTG1À/À Inhibitor II (4-(40-benzamidoanilino)-6,7-dimethoxyqui- cells, but PTTG1À/À cells were more sensitive nazoline), Aurora kinase/CDK inhibitor 4-(5-amino-1-

Oncogene PTTG1 and Aurora kinase A Y Tong et al 6389 PTTG1-GFP Aurora-A

C PTTG1 pAurora-A T288

Aurora-A

pHistone H3 Ser10

Histone H3

Time 3.5 4 4.5 5 5.5 6 h PTTG1-GFP pHistone H3 DNA Merged PTTG1 Aurora-A Actin C

PTTG1(µg) Aurora-A C1C2 0.5 1 2 Inhibitor Histone H3 Ser10

Histone H3 PTTG1-GFP transfected GST-His (µg) Aurora-A C1C2 0.5 1 2 Inhibitor Histone H3 Ser10

Histone H3 Figure 3 Pituitary tumor transforming gene 1 (PTTG1)attenuates Aurora kinase A (Aurora-A)function. ( a)Overexpressed PTTG1 caused abnormal spindle formation as evidenced by unevenly distributed PTTG1-GFP and Aurora-A (white arrows). (b)Western blot showing that overexpressed PTTG1 suppressed Aurora-A T288 autophosphorylation and histone H3 Ser10 phosphorylation in HCT116 cells compared with controls. (c)Overexpressed PTTG1 repressed histone H3 Ser10 phosphorylation as shown by immunofluorescence (white arrows). Scale bar ¼ 5 mm. (d)Degradation of mitotic proteins. HCT116 cells were synchronized with 400 ng/ml nocodazole for 12 h and mitotic cells collected by shake off, washed and cultured in fresh medium at 37 1C to allow progression of G2/M, and cells harvested at the indicated time points. Western blot showed that PTTG1 degraded 4.5 h and Aurora-A 5.5 h after cells were released from G2/M block. (e) In vitro kinase assay. Aurora-A, histone H3 and increasing PTTG1 or GST-His levels (0.5, 1, 2 mg)were brought to a 30 ml volume reaction system as described in Materials and methods. After incubations at 37 1C for 20 min, reactions were halted and analysed by western blotting using histone H3 ser10 antibody. PTTG1 dose-dependently inhibits Aurora-A phosphorylation of histone H3 substrate, whereas GST-His has no effect at the same concentration. C1 is a control without Aurora-A; C2 is a positive control (without Aurora-A inhibitor).

(2,6-difluorobenzoyl)-1H-[1,2,4]triazol-3-yl-amino)-ben- with 55% of treated WT HCT116 cells. This zenesulfonamide, were more effective in suppressing result was confirmed by colony formation assays, HCT116 PTTG1À/À cells (Supplementary data 2). showing that after treatment with 0.16 mM doxorubicin, PTTG1À/À cells are sensitive to stress and enter the 36% of PTTG1À/À colonies survived as compared with G2/M phase prematurely (Bernal et al., 2008). More- 74% of WT colonies (Figure 5b). As shown in Figure 5c, over, G2 DNA damage checkpoint abrogation renders overexpressing PTTG1 in PTTG1À/À cells rescued cells more sensitive to DNA damage-inducing drugs tumor cell susceptibility to doxorubicin, whereas knock (Vogel et al., 2005, 2007). Our results suggest that in the down of PTTG1 in HCT116 WT cells enhanced cell absence of PTTG1, Aurora-A activity is enhanced, sensitivity to doxorubicin treatment. We then tested the likely disrupting the G2 checkpoint, with subsequently requirement for Aurora-A in response to DNA- enhanced sensitivity to DNA damage-inducing drugs. damaging agents by using Aurora-A inhibitor or To test whether PTTG1À/À cell sensitivity to DNA siRNA. Low-dose (3 mM)Aurora kinase inhibitor III damage is due to attenuated G2 checkpoint control exhibited similar proliferation inhibitory effects on both related to a PTTG1–Aurora-A interaction, we used WT and PTTG1À/À cell proliferations (Figure 4b). After the DNA damage agent doxorubicin to treat WT and preincubation with 3 mM Aurora kinase inhibitor III, PTTG1À/À HCT116 cells. As shown in Figure 5a, WT and PTTG1À/À cells exhibited a similar response we confirmed that PTTG1À/À cell proliferation was more to doxorubicin (Figure 5d), suggesting that abrogated sensitive to 1.25 mM doxorubicin (Bernal et al., 2008); G2 PTTG1–Aurora-A may underlie PTTG1À/À cell 27% of PTTG1À/À cells remained viable as compared sensitivity to doxorubicin. These results were further

Oncogene PTTG1 and Aurora kinase A Y Tong et al 6390 C SiRNA 100 Aurora-A 80 ∗ WT 60 Actin 40 Aurora-A –/– 20

PTTG Viable cells (%) Actin 0 HCT116 WT PTTG–/– Genotype

80 100 ∗ 80 ∗ 60 ∗ ∗ 60 40 ∗ 40 HCT116 WT 20

Viable cells (%) 20

PTTG–/– Colony formation (%) 0 0 3.13 6.25 12.50 25.00 50.00 HCT116 WT PTTG–/– Drug concentration (µM) Genotype

80 P>0.05

Plasmid siRNA 60 WT –/– WT –/– Cell type P>0.05 40 PTTG1 20

Actin Viable cells (%)

0 WT+PTTG1 KO+PTTG1 WT+PTTG1 KO+PTTG1 siRNA siRNA Conditions Figure 4 Pituitary tumor transforming gene 1 (PTTG1)regulates cell response to Aurora kinase inhibition. ( a)PTTG1 À/À cell proliferation was attenuated in response to Aurora kinase A (Aurora-A)siRNA (mean ±s.d., n ¼ 3. *Po0.05 vs WT (wild type)). Cells were seeded into 96-well plates, cultured overnight and then transfected with siRNAs (100 nM). After 72 h, premixed WST-1 cell proliferation reagent (10 ml)was added to each well. Cells were incubated for a further 4 h and shaken thoroughly for 1 min before measurement. (b)PTTG1 À/À cell growth is more sensitive to Aurora kinase inhibitor III (mean±s.d., n ¼ 3. *Po0.05 vs WT). Cells were seeded into 96-well plates, cultured overnight and then treated with drug as indicated for 48 h. Viable cells were measured using premixed WST-1 cell proliferation reagent. (c)Anchorage-dependent colony formation assay was performed as described in Materials and methods. PTTG1À/À cells were more sensitive to Aurora-A inhibitor III than WT cells (mean±s.d., n ¼ 3. *Po0.05 vs WT). (d)Overexpressed PTTG1 rescued PTTG1 À/À sensitivity to Aurora kinase inhibitor III (12.5 mM)(mean ±s.d., n ¼ 3. P>0.05 vs WT); PTTG1 knockdown enhanced WT cell sensitivity to Aurora kinase inhibitor III (12.5 mM)(mean ±s.d., n ¼ 3. P>0.05 vs PTTG1À/À). PTTG1À/À, PTTG1-null or knockout (KO)cells.

confirmed by knocking down Aurora-A expression drug treatment. Although phosphorylated PTTG1 may levels and showing similar responses to doxorubicin act with Aurora-A at the DNA damage-induced G2 upon Aurora-A siRNA transfection as those observed in check point, several phosphorylation-deficient PTTG1 PTTG1À/À cells (Figure 5e). mutants tested did not impair the PTTG1–Aurora-A interaction (data not shown). PTTG1–Aurora-A interaction is not related to impaired phosphorylation As Aurora-A activity is inhibited by DNA damage Discussion (Krystyniak et al., 2006), we tested Aurora-A activity in HCT116 WT and PTTG1À/À cells treated with doxor- Our results indicate that PTTG1 directly inhibits ubicin. Immunoprecipitation and the measurement of Aurora-A activity both in vitro and in vivo. However, kinase activity showed that after doxorubicin treatment, PTTG1 may also act in concert with other proteins to Aurora-A activity was higher in PTTG1À/À than in WT inhibit Aurora-A activity, as PP2A binds to PTTG1 cells (Figure 6a). We then tested PTTG1 responses to throughout the cell cycle, stabilizes PTTG1 by depho- DNA damage (Figure 6b)and showed enhanced sphorylation (Gil-Bernabe et al., 2006)and also depho- PTTG1 phosphorylation induced by DNA-damaging sphorylates and inactivates Aurora-A (Eyers et al.,

Oncogene PTTG1 and Aurora kinase A Y Tong et al 6391 100

80

60 100 HCT116 WT ∗ PTTG–/– 40 80 ∗ ∗ 60 ∗ 20 Colony formation (%) 40 HCT116 WT PTTG–/–

Viable cells (%) 20 Genotype

0.16 0.31 0.63 1.25 2.50 µ Control Drug concentration ( M) 80 P<0.05 Pretreated Plasmid siRNA 60 P>0.05 WT –/– WT –/– Cell type 40 PTTG1

20 Actin Viable cells (%)

0 WT PTTG–/– WT PTTG–/– 60 P >0.05 Genotype

Control 40 P >0.05 P<0.05 75

20 60

Viable cells (%) Pretreated 45 P >0.05 WT+PTTG1 KO+PTTG1 WT+PTTG1 KO+PTTG1 30 siRNA siRNA Viable cells (%) Conditions 15 0 WT PTTG–/– WT PTTG–/– Genotype Figure 5 Pituitary tumor transforming gene 1 (PTTG1)regulates cell response to doxorubicin. ( a)PTTG1 À/Àcell growth was more sensitive to doxorubicin (mean±s.d., n ¼ 3; *Po0.05 vs WT (wild type)). Cells were seeded into 96-well plates, cultured overnight and treated with test drug for 48 h. Premixed WST-1 cell proliferation reagent (10 ml)was used to measure viable cells. ( b)Results of colony formation assay after Aurora kinase inhibitor III treatment (mean±s.d., n ¼ 3. *Po0.05 vs WT). (c)Overexpressed PTTG1 rescued PTTG1À/À doxorubicin sensitivity (mean±s.d., n ¼ 3. P>0.05 vs WT); PTTG1 knockdown enhanced WT cell sensitivity to doxorubicin (mean±s.d., n ¼ 3; P>0.05 vs PTTG1À/À).(d)Low-dose (3 mM)Aurora kinase inhibitor III treatment abolished the different response of WT and PTTG1À/À HCT116 cells to 0.63 mM doxorubicin (mean±s.d., n ¼ 3). (e)Aurora kinase A siRNA abolished the differences in WT and PTTG1À/À cell responses to 0.63 mM doxorubicin (mean±s.d., n ¼ 3).

2003a; Horn et al., 2007). Whether PTTG1 facilitates sites (Xue et al., 2008). Whether Aurora-A phosphor- Aurora-A dephosphorylation by targeting PP2A to ylates the putative sites needs further investigation. Aurora-A requires further investigation. PTTG1À/À HCT116 cells exhibited enhanced sensitivity PTTG1 overexpression inhibited histone H3 phos- to Aurora-A siRNA, several Aurora kinase inhibitors phorylation and chromatin condensation. Abnormal and to doxorubicin. Aurora-A overactivity in PTTG1À/À chromatin structure was also observed in a PTTG1 HCT116 cells might be the reason for the cell more transgenic mouse model (Donangelo et al., 2006)where sensitive to Aurora kinase inhibitor III according to heterochromatin was less abundant in PTTG1-GFP similar reports (Ikezoe et al., 2007). Aurora-A over- overexpressing cells, suggesting the inhibition of chro- expression could cause drug resistance under matin condensation. However, whether these in vivo specific conditions (Yang et al., 2006; Sun et al., 2007). observations are related to Aurora-A is as yet unclear. In our experiments, Aurora-A overactivity in the Using algorithm to analyse the PTTG1 sequence further absence of PTTG1 more likely promotes cell death identifies several putative Aurora-A phosphorylation similar to reported (Zhang et al., 2004, 2008). The notice

Oncogene PTTG1 and Aurora kinase A Y Tong et al 6392 C1 C2 WT –/– Hernandez, 2007). PTTG1 regulates Ku heterodimer C DOX Ser 10 binding to DNA at double-strand break sites, and PTG1 intracellular PTTG1 suppresses DNA damage repair by Histone H3 binding to (Kim et al., 2007). Although non- Actin GST Aurora-A homologous end joining is quantitatively normal in Aurora-A securin-deficient cells, this occurs through aberrant end processing (Bernal et al., 2008), which may also account Stress for cell susceptibility to genotoxic stress. Thus, PTTG1 signaling after DNA damage could release Ku, translo- DNA damage cating from the nucleus to the cytoplasm, allowing Ku P53 Ku70 binding to DNA, and enabling damage repair. The Nucleus PTTG1 accumulation of cytoplasmic PTTG1 in turn inhibits Aurora-A from exerting G2 checkpoint control (Figure 6c). PTTG1 PTTG1 has been proposed as a prognostic marker for Cytoplasm Aurora-A differentiated thyroid cancer, lymph node invasion and breast cancer recurrence (Solbach et al., 2004), and G2 M colon cancer invasiveness (Heaney et al., 2000). Our Figure 6 Pituitary tumor transforming gene 1 (PTTG1)and DNA results show that PTTG1 plays a role at the G2 damage pathway. (a)Aurora kinase A (Aurora-A)activity was less checkpoint by interacting with Aurora kinase A and inhibited after DNA damage treatment in PTTG1À/À cells. Lane regulating responses to Aurora kinase inhibitors and C1, positive control with Aurora-A; lane C2, negative control DNA damage agents, suggesting that PTTG1 may act as without Aurora-A; lane WT, Aurora-A co-immunoprecipitated a marker for predicting chemotherapy responses. The from HCT116 WT cells after doxorubicin treatment; LaneÀ/À, Aurora-A immunoprecipitated from HCT116 PTTG1À/À cells after results also suggest that PTTG1 downregulation might doxorubicin treatment. Aurora-A activity is higher in PTTG1À/À enhance Aurora kinase inhibitor efficacy. than in WT HCT116 cells. (b)Increased phosphorylated PTTG1 (arrowhead, shifted band)after 24 h of 5 mM doxorubicin treat- ment. (c)Proposed signaling pathway for PTTG1 action at the G2 DNA damage checkpoint. Materials and methods

Protein microarray screening The human Protoarray nc3.0 study was performed as of cell death and related mechanisms remain to be described by the manufacturer (Invitrogen, Carlsbad, CA, elucidated. USA). PTTG1 protein was biotinylated using biotin-XX Pretreatment with low-dose Aurora kinase inhibitor sulfosuccinimidyl ester, purified by gel filtration, assessed by III or siRNA also abolished differences between WT western blot and appropriate protein aliquots used to and PTTG1À/À cell responses to doxorubicin, suggesting probe a protein microarray (Protoarray nc-v3, Invitrogen) alternative mechanisms for cell survival after different and attached biotinylate PTTG1 probed with Streptavidin- treatments. PTTG1 nuclear–cytoplasmic translocation Alexa Fluor 647 conjugate. Arrays were scanned by upon DNA damage (Kim et al., 2007)may underlie GenePix 4000B (Molecular Devices Corporation, Sunnyvale, PTTG1 suppression of Aurora-A. Unlike results ob- CA, USA), and images analysed using ProtoArray Prospector (Invitrogen). tained after ultraviolet and etoposide treatment (Kim et al., 2007; Lai et al., 2007), HCT116 cells treated with 5 mM doxorubicin for 24 h showed increased PTTG1 levels and phosphorylation, which might be due to Poly-His pull-down assays, in vitro transcription and co- immunoprecipitation different treatments or doses employed. TNT Quick Coupled Transcription/Translation system Although PTTG1 phosphorylation is enhanced after (Promega, Madison, WI, USA)was used to generate His– DNA damage, impaired phosphorylation caused by the PTTG1 and His–Aurora-A fusion proteins. The pull-down S181A mutation did not alter the interaction. After assay was performed with ProFound Pull-Down PolyHis DNA damage, extranuclear PTTG1 translocation likely Protein:Protein Interaction Kit (Pierce, IL, USA). Briefly, enables DNA damage repair (Kim et al., 2007), and also fusion proteins acting as bait were adsorbed to immobilized prevents cytoplasmic Aurora-A from exerting a G2 cobalt chelate gels. Prey proteins were biotin-lysine-labeled check point action, allowing time for DNA repair. during synthesis, and incubated with bait proteins for 1 h at However, the kinase responsible for PTTG1 phosphor- 4 1C. Bound proteins were eluted and analysed by SDS–PAGE ylation upon DNA damage is not known yet. Pds1 (a (polyacrylamide gel electrophoresis)and detected by Trans- cend Non-Radioactive Translation Detection Systems PTTG1 analog)is phosphorylated by Chk1 on nine sites (Promega, Madison, MI, USA). Co-immunoprecipitation upon DNA damage in yeast (Wang et al., 2001a), was carried out with a ProFound Mammalian Co-Immuno- suggesting that Chk1 might be one of the candidates. precipitation Kit. Control IgG and PTTG1 polyclonal anti- PTTG1 is a negative regulator of p53 transcriptional body were conjugated to Antibody Coupling Gel and activity (Bernal et al., 2002). p53 stabilization incubated overnight with HCT116 cell lysates in a in the absence of PTTG1 may also increase cell rotating platform. Immunoprecipitates were eluted and susceptibility to genotoxic stress (Bernal and analysed by western blotting.

Oncogene PTTG1 and Aurora kinase A Y Tong et al 6393 Immunofluorescence microscopy anti-rabbit or anti-mouse (GE Healthcare, Piscataway, NJ, Cells grown on coverslips were fixed using 4% paraformalde- USA)antibodies were conjugated to horseradish peroxide to hyde in phosphate buffer pH 7.4 for 20 min, permeabilized reveal immunocomplexes by enhanced chemiluminescence with 0.2% Triton X-100 in phosphate-buffered saline (PBS) (Pierce). for 10 min, and incubated with 1% bovine serum albumin (BSA)for 1 h. The following primary antibodies were included: Cell proliferation assay polyclonal Aurora-A (Santa Cruz Technology, Santa Cruz, Cell proliferation was evaluated using WST-1 cell proliferation CA, USA), Phospho-histone (Ser10) (Cell Signaling, Boston, reagent (Clontech, Mountain View, CA, USA). Briefly, 90 ml MA, USA)and monoclonal separase (Novus, Littleton, CO, cells were seeded into 96-well plates and cultured overnight, USA)antibodies. Slides were incubated overnight at 4 1C with treated with drugs for 48 h and 10 ml of premixed WST-1 cell primary antibodies suspended in BSA 0.1% containing PBS. proliferation reagent was added. Cells were incubated for a Coverslips were incubated with the relevant Alexa Fluor 488 further 4 h, shaken and absorbance measured at 450 nm using or Alexa Fluor 568 secondary antibodies for 2 h and with a Victor 3 multiwell plate reader (Perkin Elmer, Waltham, TO-PRO-3 (Invitrogen)for DNA detection for 20 min, then MA, USA). Cell viability rate was calculated for each well as washed with PBS-BSA 0.1%, dried at room temperature A450-treated cells/A450 control cells  100% (A450: OD value at protected from light and mounted using Prolong Gold 450 nm). (Invitrogen). Images were obtained using a Leica TCS SP confocal Anchorage-dependent colony formation assay microscope. Ar laser 488 and ArKr laser 568 were used for the Briefly, 20 000 cells were plated in a six-well culture dish, detection of Alexa fluorophores 488 and 568, respectively. The allowed to attach overnight and treated with depicted drug exclusion of autofluorescence was achieved by imaging with a concentrations. Cells were cultured in standard culture narrow spectral detection window with the pinhole set to 1.0 medium containing indicated treatments for 12 days and Airy unit (AU)for optimal resolution. Depicted images were medium re-freshened every 3 days. Colonies were stained with derived from maximum intensity projections of confocal 0.5% crystal violet in methanol/acetic acid (3:1)and those stacks. composed of >50 cells were counted.

Protein degradation during mitosis siRNA and transfection Mitotic cells for degradation sequence assessment were Aurora kinase A siRNA (AM51331, sense: GGCAACCA prepared by a modified synchronization regime. Cells were GUGUACCUCAUtt; anti-sense: 50-AUGAGGUACACUG released from aphidicolin (10 ng/ml)(Sigma Aldrich, St Louis, GUUGCCtg-30)and PTTG1 siRNA (16706, sense: 5 0-GUC MO, USA)into medium containing 400 ng/ ml of nocodazole UGUAAAGACCAAGGGAtt-30; anti-sense: 50-UCCCUUG (Sigma Aldrich)and incubated for 12 h before harvesting by GUCUUUACAGACtt-30)were purchased from Ambion shake off. Mitotic cells were washed three times in ice-cold (Austin, TX, USA). Transfections were carried out with 1 PBS and replaced in 37 C prewarmed medium. Mitotic cells lipofectamine 2000 according to protocol. were collected for analysis. Statistics In vitro kinase activity assay Analysis was performed by a standard two-tailed Student’s Assays were performed using a modified Aurora-A assay kit t-test and results plotted as mean±s.d. from Cell Signaling. Histone H3 was purchased from Promega and control His-GST protein from Upstate (Billerica, MA, USA). His-PTTG1 was expressed in insect cells and purified by Abbreviations the Caltech Protein Expression Center. Aurora-A and substrate histone H3 were brought to a 30 ml volume contain- Aurora-A, Aurora kinase A; PTTG1, pituitary tumor trans- ing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2,5mM, b- forming gene 1. glycerophosphate, 0.1 mM Na3VO4, 20–200 mM ATP, 1 mg histone 3, 10 U GST-Aurora-A and increasing PTTG1 levels Acknowledgements or control proteins (0.5, 1 and 2 mg). Reaction mixtures were incubated at 37 1C for 30 min, reactions stopped by the addition of 10 ml4Â NuPAGE LDS sample buffer and PTTG1 WT and KO HCT116 cells were kindly provided by reaction products denatured for 10 min at 95 1C. Proteins were Dr Bert Vogelstein, Johns Hopkins University. PTTG1-GFP separated by standard SDS–PAGE and analysed by western and PTTG1-PCDNA3.1 plasmids were kindly provided by Dr blotting. Run Yu. Supported by NIH Grant CA 75979 (SM), T32 DK007770, and The Doris Factor Molecular Endocrinology Laboratory. Western blot Protein extracts were resolved by NuPAGE 4–12% Bis-Tris Gel (Invitrogen). Samples were electroblotted onto polyviny- Disclosure lidene fluoride (PVDF)membrane (Invitrogen),and mem- branes blocked and incubated with primary antibody. Donkey The authors state no conflict of interest.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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