TACC3 Deregulates the DNA Damage Response and Confers Sensitivity to Radiation and PARP Inhibition

ORIGINAL ARTICLE TACC3 deregulates the DNA damage response and confers sensitivity to radiation and PARP inhibition

G-H Ha1,2,5, J-L Kim1,2,5, A Petersson1,2,SOh2, MF Denning1,3, T Patel4 and E-K Breuer1,2

Deregulation of the transforming acidic coiled-coil protein 3 (TACC3), an important factor in the centrosome–microtubule system, has been linked to a variety of human cancer types. We have recently reported on the oncogenic potential of TACC3; however, the molecular mechanisms by which TACC3 mediates oncogenic function remain to be elucidated. In this study, we show that high levels of TACC3 lead to the accumulation of DNA double-strand breaks (DSBs) and disrupt the normal cellular response to DNA damage, at least in part, by negatively regulating the expression of ataxia telangiectasia mutated (ATM) and the subsequent DNA damage response (DDR) signaling cascade. Cells expressing high levels of TACC3 display defective checkpoints and DSB-mediated homologous recombination (HR) and non-homologous end joining (NHEJ) repair systems, leading to genomic instability. Importantly, high levels of TACC3 confer cellular sensitization to radiation and poly(ADP-ribose) polymerase (PARP) inhibition. Overall, our ﬁndings provide critical information regarding the mechanisms by which TACC3 contributes to genomic instability, potentially leading to cancer development, and suggest a novel prognostic, diagnostic and therapeutic strategy for the treatment of cancer types expressing high levels of TACC3.

Oncogene (2015) 34, 1667–1678; doi:10.1038/onc.2014.105; published online 28 April 2014

INTRODUCTION organisms have shown that the TACC family may interact with 16,39 The transforming acidic coiled-coil (TACC) protein family has an DNA damage-related molecules, suggesting a potential role important role in various cellular events, such as cell proliferation, for TACC3 in DNA damage response (DDR) signaling events. Upon growth, differentiation and gene regulation through a direct or DNA damage, cells rapidly initiate the DDR signaling network by indirect association with molecules involved in the centrosome– coordinating multiple cellular processes, including cell cycle 1–6 checkpoint control, and DNA repair and apoptosis, to maintain microtubule network, transcription and mRNA processing. The 40–43 third member of the TACC family, TACC3, was originally identified genomic integrity and prevent tumorigenesis. Therefore, fi as an interacting partner of aryl hydrocarbon receptor nuclear there is no doubt that ne-tuned regulation of DDR signaling translocator (ARNT)7 and subsequently of signal transducer and events is critical for preserving genomic integrity, and that activator of transcription 5 (STAT5)8 and Friend of GATA-1 (FOG- deregulation of genetic factors orchestrating these signaling events can contribute to cancer development. This led us to 1).9 TACC3 is abundantly expressed in peripheral blood leukocytes, – investigate whether high levels of TACC3 may alter the normal thymus, spleen and testis5,8,10 12 and is involved in the regulation – cellular response to DNA damage and thus contribute to genomic of centrosome–microtubule dynamics.13 15 Depletion of TACC3 instability. results in defects in kinetochore–microtubule attachment14 and 8,16 Herein, we demonstrate that TACC3 levels are elevated in a embryonic development in mice. Growing evidence suggests variety of cancer types, and its high expression disrupts normal that deregulation of TACC3 may be involved in oncogenic 17–22 DDR signaling events, at least in part, by negatively regulating the processes of certain types of solid tumors. In addition, data expression of ataxia telangiectasia mutated (ATM) and thus obtained from the Oncomine database (www.oncomine.org, 23 inhibiting the ATM-driven DDR signaling pathways. Moreover, Compendia Bioscience Inc, Ann Arbor, MI, USA) have shown high levels of TACC3 result in defects in DNA double-strand break that the expression of TACC3 is significantly elevated in various (DSB) repair and early G2/M cell cycle checkpoint activation, thus 6,24–36 types of human malignancies. We have previously reported elevating the frequency of chromosomal abnormalities. Impor- that TACC3 can induce epithelial–mesenchymal transition (EMT) tantly, cells expressing high levels of TACC3 display increased through activating phosphatidylinositol 3-kinase (PI3K)/Akt and sensitivity to ionizing radiation (IR) and poly(ADP-ribose) poly- extracellular signal-regulated protein kinase (ERK) signaling merase (PARP) inhibitors. Our results, thereby, identify a novel pathways36,37 and may be associated with cancer progression and function of TACC3 in the DDR signaling events that may metastasis.38 However, it still remains to be determined how contribute to genomic instability and tumorigenesis and provide TACC3 is directly linked to human cancer. Interestingly, high- TACC3 as a potential prognostic and diagnostic indicator and throughput screening data obtained from invertebrate model therapeutic target in cancer.

1Oncology Institute, Cardinal Bernardin Cancer Center, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA; 2Department of Radiation Oncology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA; 3Department of Pathology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA and 4Department of Molecular Pharmacology and Therapeutics Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA. Correspondence: Dr E-K Breuer, Department of Radiation Oncology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA. E-mail: [email protected] 5These authors contributed equally to this work. Received 3 September 2013; revised 28 February 2014; accepted 12 March 2014; published online 28 April 2014 DDR deregulation by TACC3 G-H Ha et al 1668 RESULTS was also observed in HEK293T, lung fibroblast WI-38 and skin Elevated levels of TACC3 lead to spontaneous DNA damage due to fibroblast NF560 expressing high levels of TACC3 (Supplementary impaired DNA DSB repair Figures 1a and b). To further verify the occurrence of DSBs in cells expressing high levels of TACC3, we performed the neutral pH To investigate the role of TACC3 in the DDR pathways, we stably 47 expressed control vector (pCEFL-HA) or HA-TACC3 (pCEFL-HA- comet assay, a method to detect DNA DSBs in single cells. TACC3) in U2OS cell lines (Figure 1a). U2OS cells were chosen Interestingly, we found that the average length of the comet tails because of their low degree of spontaneous DNA damage.44,45 To of cells expressing high levels of TACC3 was much longer than those of the control cells without exogenous stimuli (Figure 1c), study the induction of DSBs in the presence of high levels of fi TACC3, we monitored foci formation of phosphorylated H2AX on further con rming the effect of elevated TACC3 levels on DNA γ 46 damage. DSBs caused by various endogenous or exogenous Ser-139 ( H2AX), which is a reliable marker for DSBs. In U2OS 48–51 cells expressing high levels of TACC3, a significant number of cells sources are the most critical and serious lesions because γ unrepaired or misrepaired DSBs can cause cell death or genomic were positive for H2AX, whereas no foci formation was observed 52–54 in control cells (Figure 1b), suggesting that elevated levels of instability and tumor initiation. Strikingly, 6 h after exposure TACC3 lead to the accumulation of DNA DSBs. A similar result to 10 Gy of IR, the comet tails in cells expressing high levels of

*p<0.001 γH2AX DAPI Merge * 8 HA 7 * 6 5 H2AX

4 HA TACC3#8 3 2 foci per foci per cell TACC3 No. of 1 0 -actin TACC3#11

IR -15min6h * *p<0.05 ** **p <0.01 HA 1.2 * * - IR 1 + IR 15min 0.8 + IR 6h 0.6 TACC3#8 0.4 intact DNA 0.2 Cell fractions with 0 TACC3#11

- IR + IR

*p<0.001 γH2AX DAPI Merge γH2AX DAPI Merge * 40 * - IR HA 35 + IR 30 25 20 TACC3#8 15 H2AX foci per cell per foci H2AX

10 5 TACC3#11 0 No. of

Figure 1. Elevated levels of TACC3 lead to the accumulation of DSBs due to impaired DSB repair. (a) Western blot of TACC3 in U2OS cells stably expressing TACC3 (clones: TACC3 #8 and TACC3 #11) or control vector. U2OS cells were transfected with pCEFL-HA-TACC3 or pCEFL-HA and underwent G418 selection for 2 weeks. Cells were harvested and subjected to western blot analysis with antibodies against HA and TACC3. β- actin was used as a loading control. (b) Foci formation of γH2AX was assessed with immunofluorescence staining. Cells were fixed and stained with an anti-γH2AX antibody. Cells were then washed and stained with fluorescence-conjugated secondary antibodies. Nuclei were visualized with DAPI staining. Each value represents the mean ± s.d. of three independent experiments. (c) The neutral pH comet assay was performed. The percentage of cells with intact DNA (tail moment less than 1) was set at 1. At least 200 cells were scored in each sample. Each value represents the mean ± s.d. of three independent experiments. (d) Cells were treated with 10 Gy of IR or left untreated and, 1 h later, they were fixed and stained with an anti-γH2AX antibody. Nuclei were visualized with DAPI staining. Each value represents the mean ± s.d. of three independent experiments.

Oncogene (2015) 1667 – 1678 © 2015 Macmillan Publishers Limited DDR deregulation by TACC3 G-H Ha et al 1669 TACC3 remained elevated, whereas DNA repair was almost Elevated levels of TACC3 disrupt the normal cellular response to IR completed in control cells, as indicated by the reduction in comet DNA damage triggers the DDR pathways in cells to recruit multiple tail length (Figure 1c). A similar result was also observed in WI-38 DNA damage signaling proteins to the damaged sites,55 and these and NF560 expressing high levels of TACC3 (Supplementary Figure proteins form discrete nuclear foci.56,57 This recruitment event must 1c). Taken together, these data suggest that elevated TACC3 be in the correct order and come at the correct time to accurately levels lead to the accumulation of DSBs due to impaired DSB proceed to checkpoint activation and DNA repair.57,58 As in U2OS repair. cells expressing high levels of TACC3, DNA DSBs are accumulated

IR -+-+-+ IR -+ -+ -+ p-Chk1S345 p-ATMS1981 Chk1 ATM p-p53S15 p-Chk2T68 p53 Chk2 -actin

HA TACC3#8 TACC3#11

IR -+ - + - +

p-ATMS1981

DAPI

MDC1

DAPI

BRCA1

DAPI

53BP1

DAPI

Figure 2. Elevated levels of TACC3 negatively regulate the ATM-driven DDR pathway. (a) Cells were treated with 10 Gy of IR or left untreated. One hour after irradiation, cells were harvested, extracted and subjected to western blot analysis with antibodies against phospho-ATMS1981, ATM, phospho-Chk2T68, Chk2, phospho-Chk1S345, Chk1, phospho-p53S15 and p53. β-actin was used as a loading control. (b) Cells were treated with 10 Gy of IR or left untreated and, 1 h later, they were ﬁxed and stained with antibodies to the indicated proteins. Cells were then washed and stained with ﬂuorescence-conjugated secondary antibodies. Nuclei were visualized with DAPI staining.

© 2015 Macmillan Publishers Limited Oncogene (2015) 1667 – 1678 DDR deregulation by TACC3 G-H Ha et al 1670 *p<0.001 *p<0.05 **p<0.0001 ** **p<0.005 * * 1.2 ** 1.2 HA-TACC3 (g) 0 12345 1 1 0.8 0.8 ATM 0.6 0.6 HA 0.4 0.4

expression expression 0.2 0.2 -actin unit (fold change) unit (fold

0 Relative luciferase 0

Relative ATM mRNA * *p<0.05 **p<0.0001 ATM 1.2 ** -631 +352 1 A (-631 to -435) 0.8 B (-434 to -239) C (-238 to -43) 0.6 D (-42 to +154) 0.4 E (+155 to +352) 0.2 unit (fold change) Relative luciferase 0 B1 (-434 to -385) B2 (-389 to -340) B3 (-344 to -295) B4 (-299 to -239)

HA TACC3 #8 * *p<0.05 10 ATM - - + 8 HA ATM 6 HA-TACC3 -actin 4 *p<0.005 40 * 2 **p<0.001 Fold enrichment 0 30 ** - IR A BCDE

H2AX + IR 20 * 10 *p<0.005

foci per cell foci per 10 8 No. of HA 6 0 HA-TACC3 4 ATM - - + 2 HA TACC3 #8 Fold enrichment 0 Fold changes in B1 B2 B3 B4 H2AX foci 26.65 ± 6.04 2.39 ± 0.48 10.90 ± 5.28 (+IR/-IR) Figure 3. TACC3 negatively regulates ATM expression through transcriptional suppression, and re-expression of ATM partially rescues defective DDR events caused by high levels of TACC3. (a) HEK293T cells were transfected with increasing concentrations of HA-TACC3 plasmid. After 48 h of transfection, cells were harvested, extracted and subjected to western blot analysis with antibodies against ATM and HA. β-actin was used as a loading control. (b) The mRNA level of ATM was represented relative to β-actin transcripts. Each value represents the mean ± s.d. of three independent experiments. (c) Cells were transfected with the luciferase reporter vector containing ATM promoter or an empty promoter vector. After 24 h of transfection, cells were lysed and luciferase activity was measured. Each value represents the mean ± s.d. of three independent experiments. (d) Schematic representation of the ATM promoter. Indicated region of primers used for the ChIP–qPCR assays. Bar graphs showing fold enrichment over IgG. Each value represents the mean ± s.d. of three independent experiments. (e) Cells were transfected with the luciferase reporter vector containing ATM promoter, Δ − 434 to − 385 mutant or an empty promoter vector. After 24 h of transfection, cells were lysed and luciferase activity was measured. Each value represents the mean ± s.d. of three independent experiments. (f) U2OS cells expressing HA-TACC3 (clone TACC3 #8) and control vector were transfected with ATM or control plasmid. Forty-eight hours after transfection, cells were harvested and subjected to western blot analysis with an anti-ATM antibody. β-actin was used as a loading control. Foci formation of γH2AX was assessed with immunofluorescence staining. Cells were fixed and double-stained for antibodies against γH2AX and ATM. Cells were then washed and stained with fluorescence-conjugated secondary antibodies. γH2AX foci number was counted in only ATM-expressing cells. Nuclei were visualized with DAPI staining. Each value represents the mean ± s.d. of three independent experiments.

and DSB repair is impaired, we questioned whether high levels of (Supplementary Figure 1b). Next, we examined whether depletion TACC3 affect the DDR signaling events. To address this question, cells of TACC3 may have the opposite effect on γH2AX foci formation in were treated with 10 Gy of IR or left untreated, and γH2AX, which is response to IR. For this purpose, we expressed control or TACC3 short one of the earliest molecular markers of DDR,56 was detected with hairpin RNAs in U2OS cells (Supplementary Figure 2a). Intriguingly, immunofluorescence staining. As shown in Figure 1d, γH2AX foci depletion of TACC3 in U2OS cells facilitated γH2AX foci formation formation was significantly reduced in cells expressing high levels of following exposure to IR (Supplementary Figure 2b). Therefore, our TACC3 following exposure to IR. A similar result was also observed in findings suggest that elevated levels of TACC3 have a negative HEK293T,WI-38andNF560expressinghighlevelsofTACC3 impact on the early steps of the DDR pathways.

HR Mock HA TACC3 Mock shConshTACC3 SceGFP iGFP HA TACC3 -actin -actin I-Scel Bcgl *p<0.05 1.2 * * HR *p<0.05 1 3.5 3 0.8 2.5 Bcgl Bcgl 0.6 2 0.4 1.5 GFP expression 0.2 1 Fold change in

GFP positive cells 0.5 0 Fold change in

GFP positive cells GFP positive 0 FACS analysis HA+I-SceI shCon+I-Scel TACC3+I-SceI shTACC3+I-Scel

NHEJ HA TACC3HA TACC3kb I-Scel - 0.65 digestion -0.5 F I-Scel R BcgI I-SceI+Bcgl - 0.65 -0.5 HR NHEJ digestion - I-SceI + I-SceI

Bcgl Bcgl Bcgl * *p<0.05 PCR l) 1.2 1 Bcg

I-Scel digestion I-Scel + Bcgl digestion I+ 0.8

HR &NHEJ NHEJ Sce 0.6 0.4 0.65 kb 0.2 0.5 kb 0 product (I- HA TACC3 Fold change in uncut PCR

HA TACC3#8 TACC3#11

5 1×105 0.69 1×105 0.76 1×10 0.81

10000 10000 10000

0 Gy 1000 1000 1000

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5 0.16 5 0.42 1×10 1×10 1×105 0.39

10000 10000 10000

1000 1000 1000 3 Gy Phospho-histone H3 Phospho-histone 100 100 100 0 0 0

0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K DNA contents Figure 4. Elevated levels of TACC3 cause a defect in DSB repair and early G2/M checkpoint. (a) A schematic diagram of HR analysis. (b, c) Twenty-four hours after transfection of either HA-TACC3 or control vector (b) and short hairpin RNAs (shRNA) targeting TACC3 or non-target shRNA control (c) into U2OS cells stably expressing pDR-GFP, the I-SceI expression vector was transfected into these cells to generate a DSB within the Sce-GFP. Fluorescence-activated cell sorting (FACS) analysis was carried out to quantify HR-repaired GFP+ cells. Each value represents the mean ± s.d. of three independent experiments. (d) A schematic diagram of the PCR-based NHEJ analysis. (e) Twenty-four hours after transfection of either HA-TACC3 or control vector into U2OS cells stably expressing pDR-GFP, the I-SceI expression vector was transfected into cells to generate a DSB. The genomic DNA of control and I-SceI-transfected cells was isolated for PCR amplification, followed by digestion with I-SceI (HR and NHEJ) or I-SceI+BcgI (NHEJ). After agarose gel electrophoresis, the intensity of enzyme-resistant and enzyme-cleaved fragments was quantified using NIH imageJ software (http://rsb.info.nih.gov/nih-image). (f) Cells were irradiated with 3 Gy of IR or left untreated and then incubated for 1 h before fixation. Cells in mitosis were determined by staining with propidium iodide and phospho-histone H3 antibody followed by FITC-conjugated secondary antibody. The percentage of M-phase cells was determined using FACS.

© 2015 Macmillan Publishers Limited Oncogene (2015) 1667 – 1678 DDR deregulation by TACC3 G-H Ha et al 1672 Elevated levels of TACC3 lead to a failure in launching reduced the incidence of DSBs caused by high levels of TACC3, as ATM-mediated DDR pathways indicated by reduced γH2AX foci formation and slightly increased Within minutes after exposure to DNA damage, chromatin- γH2AX foci formation in response to IR (Figure 3f and Supplementary associated H2AX is phosphorylated on Ser-139 by ATM, Figure 3), suggesting that loss of ATM is one of the mechanisms ATM-Rad3-related or DNA-dependent protein kinase,59–61 forms involved in TACC3-induced DNA damage. However, as re-expression discrete nuclear γH2AX foci62,63 and subsequently recruits key of ATM was unable to completely restore normal DDR, other regulators of the DDR pathways.56,64–72 In response to IR, ATM is mechanisms must also contribute to the effect of TACC3 in DDR. auto-phosphorylated on Ser-1981 to initiate ATM signaling 73–75 pathways. Given that high levels of TACC3 reduced foci Elevated levels of TACC3 lead to impaired DSB repair and G2/M formation of γH2AX in response to IR (Figure 1d and checkpoint activation Supplementary Figure 1b), it is conceivable that TACC3 may directly To preserve genomic integrity, mammalian cells have evolved two or indirectly regulate ATM-driven pathways. Interestingly, after major DSB repair systems, homologous recombination (HR) and exposure to IR, cells expressing high levels of TACC3 failed to non-homologous end joining (NHEJ) under the guidance of cell phosphorylate and activate ATM due to a reduced total ATM cycle checkpoint regulators.53,76,77 Studies have shown the associa- expression, thus subsequently blocking the phosphorylation signal- tions of ATM with HR78,79 and NHEJ78–81 in the repair of DSBs; and ing cascade that leads to the activation of Chk1, Chk2 and p53 since our study indicates that cells expressing high levels of TACC3 (Figure 2a). In addition, cells expressing high levels of TACC3 fail to activate the ATM-driven DDR signaling pathways, we exhibited impaired foci formation of phospho-ATM, and thus hypothesized that elevated levels of TACC3 would lead to consequently failed to recruit its downstream DDR factors, including impairment of HR- and NHEJ-mediated DSB repair. We first mediator of DNA damage checkpoint 1 (MDC1), BRCA1 and p53- examined the efficiency of HR using the pDR-GFP (green fluorescent binding protein 1 (53BP1), to the damage sites (Figure 2b). protein) reporter system (Figure 4a).82,83 As shown in Figure 4b, cells To further assess the impact of TACC3 on ATM expression, we expressing high levels of TACC3 showed a significant decrease in transfected HEK293T cells with varying concentrations of TACC3 and HR-repaired GFP+ cells, indicating a defect in HR. Conversely, found that the endogenous level of ATM protein was reduced in a depletion of TACC3 increased HR (Figure 4c). In order to determine TACC3 dose-dependent manner (Figure 3a). Next, we sought to the role of TACC3 in NHEJ repair, we performed a PCR-based assay determine the molecular mechanisms by which TACC3 represses (Figure 4d).83–85 As shown in Figure 4e, elevated levels of TACC3 ATM expression. Importantly, we foundthatelevatedlevelsofTACC3 lead to impairment of NHEJ repair. These data support the notion decreased ATM mRNA levels (Figure 3b) by repressing its promoter that elevated levels of TACC3 negatively regulate ATM and thus activity (Figure 3c). Next, we carried out chromatin immunoprecipita- inhibit both the HR- and NHEJ-mediated DSB repair. – tion (ChIP) quantitative polymerase chain reaction (qPCR) assays to In response to DNA damage, cells activate checkpoint determine whether TACC3 could bind to the ATM promoter. As pathways86 to properly control cell cycle progression and thus showninFigure3d,TACC3associatedwiththeATMpromoterina provide enough time for DNA repair.87 Cells defective of ATM − − region between nucleotides 434 and 239 (B fragment) from the possess dysfunctional early G2/M checkpoints.88 To directly assess transcription start site. To narrow down the TACC3-binding site, the G2/M checkpoint activity in response to IR, we treated cells with ATM proximal promoter region was broken down into four 3 Gy of IR and labeled them with propidium iodide and a − − − − − fragments (B1: 434 to 385; B2: 389 to 340; B3: 344 to phospho-H3 antibody, a mitosis-specific marker.89,90 Whereas the − − − 295; B4: 299 to 239) of 50 bp with a 5 bp overlap. The mitotic population of control cells was significantly reduced in quantitative analysis of TACC3 binding by qPCR showed that TACC3 response to IR, a significant portion of cells expressing high levels fi − − speci cally binds to the ATM B1 fragment ( 434 to 385; Figure 3d). of TACC3 entered into mitosis (Figure 4f), indicating that cells Moreover, deletion of this region in the ATM promoter obliterated expressing high levels of TACC3 have a defective early G2/M TACC3-mediated downregulation of ATM promoter activity checkpoint. These data indicate a negative role for TACC3 in DNA − − (Figure 3e), demonstrating that the region of 434 to 385 is repair and checkpoint function. necessary for TACC3 to regulate ATM expression. These data suggest that TACC3 negatively regulates the ATM-mediated DDR signaling pathway, at least in part, through suppression of ATM gene expression. Elevated levels of TACC3 increase the frequency of chromosomal aberrations Impaired DDR pathways have been shown to be associated with TACC3-induced DNA damage is partially dependent on ATM genomic instability.91–93 As our findings clearly demonstrated that To further determine whether loss of ATM is responsible for the high levels of TACC3 disrupt the normal cellular response to TACC3-induced DNA damage, we re-expressed ATM in U2OS cells DNA damage, we examined whether elevated levels of TACC3 expressing high levels of TACC3 (Figure 3f), treated with 10 Gy of IR would increase chromosomal aberrations. Importantly, cytological and analyzed γH2AX foci formation. Re-expression of ATM partially analysis of metaphase chromosome spreads showed increased

Table 1. Elevated levels of TACC3 increase the frequency of chromosomal aberrations

Sample (n) Clone no. No. of cells analyzed % Normal diploid cells % Aneuploidy % Cells with breaks % Cells with fusions

HA (241) 1 79 87.3 12.7 7.6 2.5 2 77 88.3 11.7 6.5 3.9 3 85 83.5 16.5 8.2 2.4 Mean 80.3 ± 4.2 86.4 ± 2.5 13.6 ± 2.5 7.4 ± 0.9 2.9 ± 0.8 HA-TACC3 (245) 1 91 60.4 39.6 23.1 8.8 8 76 67.1 32.9 21.1 7.9 11 78 65.4 34.6 24.4 9 Mean 81.7 ± 8.1 64.3 ± 3.5 35.7 ± 3.5 22.9 ± 1.7 8.6 ± 0.6 Abbreviation: HA, hemagglutinin; TACC3, transforming acidic coiled-coil protein 3. Chromosomal abnormalities in metaphase chromosome spreads from the indicated clones.

Oncogene (2015) 1667 – 1678 © 2015 Macmillan Publishers Limited DDR deregulation by TACC3 G-H Ha et al 1673 chromosomal abnormalities (chromosome fusions, breaks and clinical significance of TACC3 in human cancer. The expression aneuploidy) in the presence of high levels of TACC3 (Table 1 and profiles of TACC3 were studied in eight solid tumors, including Supplementary Figure 4), indicating that elevated levels of TACC3 breast, colon, kidney, liver, lung, ovary, prostate and thyroid contribute to genomic instability. cancers, using the TissueScan Cancer Survey qPCR arrays. Interestingly, a statistically significant upregulation of TACC3 (Po0.05) was observed in breast, colon, liver and lung cancers Clinical significance of TACC3 in human cancer (Figure 5a). Although elevated levels of TACC3 were also found in As elevated levels of TACC3 negatively regulate the DDR pathways ovary, prostate and thyroid cancers, they were not statistically and thus lead to genomic instability, we sought to determine the significant. As the level of TACC3 was significantly elevated in

a 6 * *p<0.05 ** 5 **p<0.005 * 4 ** 3

TACC3 2 1 Relative of transcript 0 NCNCNCNCNCNCNCNC

Breast Colon Kidney Liver Lung Ovary Prostate Thyroid

* b Normal Cancer *p<0.001 3 n=100 2.5 2 n=10 1.5

Breast score 1 0.5

Staining intensity 0 Normal Breast Breast Cancer * 5 n=110 4 Lung 3 2 n=10 score 1 0 Staining intensity Staining Normal Lung Lung Cancer

1.0 c Breast Cancer 1.0 Lung Cancer 0.8 TACC3 low 0.8 TACC3 low

0.6 0.6 TACC3 high 0.4 0.4 TACC3 high Probability Probability 0.2 0.2 HR = 1.7 (1:49-1.93) HR = 1.7 (1:58- 1.84) logrank P = 2.2e-16 0.0 0.0 logrank P = 4e-09 0 5 10 15 0 50 100 150 200 Time (years) Time (months)

d Breast Lung e 1.8 r=-0.72401 3.5 r=0.70052 r=0.5-1341 P<0.05 3.0 1.6 P<0.0005 3.0 P<0.01 1.4 2.5 2.5 1.2 2.0 1.0 2.0 1.5 0.8

score 1.5 1.0 0.6 1.0 0.4 0.5 0.2 0.5 H2AX staining intensity staining H2AX ATM mRNA ATM mRNA expression ATMmRNA expression ATMmRNA 0.0 0.0 0.0 010200510 01234 TACC3 mRNA expression TACC3 mRNA expression TACC3 staining intensity score Figure 5. TACC3 is elevated in a variety of human cancer types. (a) Transcript expression of TACC3 in eight solid human cancers, including breast, colon, kidney, liver, lung, ovary, prostate and thyroid cancers, was determined using TissueScan Cancer Survey qPCR array analysis. (b) Representative immunohistochemical staining of TACC3 on breast (normal breast tissues (n = 10) and breast cancer tissues (n = 100)) and lung (normal lung tissues (n = 10) and lung cancer tissues (n = 110)) cancer tissue microarrays (left panel). Quantitative analysis of breast and lung cancer tissue microarrays showed that the expression of TACC3 is higher in cancer tissues than in normal tissues (right panel). (c) Kaplan–Meier survival curves of breast (left panel) and lung (right panel) cancer patients based on TACC3 expression. (d) Graphs showing an inverse correlation between TACC3 and ATM mRNA levels in breast (Pearson’s r = − 0.51341; Po0.01) and lung (Pearson’s r = − 0.72401; Po0.0005) cancers based on TissueScan Cancer Survey qPCR array analysis. (e) A graph showing a positive correlation between TACC3 and γH2AX staining intensity scores of breast cancer tissue microarrays (n = 75) (Pearson’s r = 0.70052; Po0.05).

© 2015 Macmillan Publishers Limited Oncogene (2015) 1667 – 1678 DDR deregulation by TACC3 G-H Ha et al 1674 breast (fold change: 3.04) and lung (fold change: 2.85) cancers MCF10A (Figure 5a), we decided to examine the protein expression of TACC3 using breast and lung cancer tissue microarrays (Supplementary Table 1). Consistent with the qPCR data, immunohistochemistry showed that the level of TACC3 protein was elevated in both breast and lung cancer tissues compared TACC3 – with corresponding normal tissues (Figure 5b). Moreover, Kaplan -actin Meier survival analysis (http://kmplot.com/analysis/)94 showed that high levels of TACC3 were signiﬁcantly associated with poor *p<0.05 survival in breast and lung cancer patients (Figure 5c). 100 In order to determine the role of TACC3 as a deregulator of DDR * signaling in human cancer, we examined mRNA expression of * TACC3 and ATM in TissueScan qPCR experiments and found an inverse correlation between TACC3 and ATM in breast (Pearson’s r = − 0.51341,Po0.01) and lung (Pearson’s r = − 0.72401, HA Po0.0005) cancers (Figure 5d). In addition, immunohisto- TACC3#1 chemistry on breast cancer tissue microarrays (Supplementary Survival fraction TACC3#2 Table 1) revealed a positive correlation between TACC3 and 10 γH2AX expression (Pearson’s r = 0.70052, Po0.05; Figure 5e). 0246810 Radiation Dose (Gy) Taken together, these data suggest that TACC3 functions as a deregulator of DDR signaling pathways and would serve as an important tumor marker in predicting survival outcomes for 120 * p<0.01 120 *p<0.01 patients with breast and lung cancers. 100 100 * * 80 * 80 * Elevated levels of TACC3 render cells more sensitive to IR and 60 60 PARP inhibition 40 HA 40 HA

fi (%) Cell growth TACC3#2 On the basis of our ndings that elevated levels of TACC3 cause 20 TACC3#2 Cell growth(%) 20 defects in HR-mediated DSB repair and checkpoint function, 0 0 together with studies demonstrating a significant association of 0 1 10 50 100 0 1 10 50 100 defective DDR with cellular sensitization to DNA-damaging AZD2281 (µM) NU1025 (µM) agents,95 we hypothesized that cells expressing high levels of Figure 6. Elevated levels of TACC3 render cells more sensitive to IR TACC3 would exhibit cellular hypersensitivity to radiation treat- a fi and PARP inhibition. ( ) Western blot of TACC3 in MCF10A cells ment. We rst established human mammary epithelial MCF10A stably expressing HA or HA-TACC3 (clones TACC3 #1 and TACC3 #2) cells stably expressing TACC3 and found that the levels of TACC3 as well as various breast cell lines. MCF10A cells were transfected in stable MCF10A clones expressing TACC3 (TACC3 #1 and TACC3 with pCEFL-HA or pCEFL-HA-TACC3 using Lipofectamine 2000. After #2) were comparable to those of a panel of breast cancer cell lines 24 h of transfection, cells were re-plated into medium containing (SUM159, MDA-MB-231, MDA-MB-468, BT549, BT474 and SKBR3) G418 (100 μg/ml). G418-resistant colonies were selected 3 weeks (Figure 6a). To examine the effects of radiation in the presence of later. Cells were harvested and subjected to western blot analysis high levels of TACC3, MCF10A cells stably expressing TACC3 were with an anti-TACC3 antibody. β-actin was used as a loading control. treated with various doses of radiation as indicated and subjected (b) MCF10A cells stably expressing HA-TACC3 or control vector were to clonogenic survival assays. As shown in Figure 6b, MCF10A cells seeded at various densities in six-well plates and treated with 0, 2, 4, 6, 8 or 10 Gy of IR. Clonogenic survival assays were performed, and stably expressing TACC3 displayed increased cellular sensitivity to survival curves were generated from a minimum of three IR. A similar result was also observed in U2OS cells expressing high independent experiments. (c) MCF10A cells stably expressing HA- levels of TACC3 (clones: TACC3 #8 and TACC3 #11; Supplementary TACC3 or control vector were exposed to the PARP inhibitors, Figure 5a). As PARP inhibitors have been shown to be selectively Olaparib (AZD2281) and NU1025. Cell viability was measured by lethal to cancer cells harboring dysfunctional HR,96,97 we MTT assay. examined the effect of PARP inhibitors in the presence of high levels of TACC3. Intriguingly, we found that MCF10A cells stably expressing TACC3 were more sensitive to PARP inhibitors, sensitive to PARP inhibition.98 However, whereas TACC3 decreases Olaparib (AZD2281) and NU1025 (Figure 6c). A similar result was both the Chk1 and Chk2 activities, Aurora A specifically targets also observed in U2OS cells expressing high levels of TACC3 Chk1 but not Chk2.98 At the moment, we cannot conclude that (Supplementary Figure 5b). These data suggest that elevated high levels of TACC3 and Aurora A disrupt normal DDR signaling levels of TACC3 confer vulnerability of cells to radiation and PARP events through the same pathway. inhibition. A growing body of evidence suggests that TACC3 may function as a transcriptional cofactor.9,99,100 For example, TACC3 regulates the transcriptional activation of hypoxia-inducible factor (HIF) via a DISCUSSION direct interaction with ARNT.99 Here, we demonstrated that TACC3 In this study, we showed that TACC3 is elevated in a variety of suppresses the expression of ATM, at least in part, through human cancer types and have proposed a model in which transcriptional repression. We were also able to demonstrate elevated levels of TACC3 disrupt the normal cellular response to TACC3 binding to the ATM promoter (−434 to − 385) and the DNA damage and thus lead to genomic instability and ultimately importance of this promoter region for TACC3 to regulate ATM cancer development. Importantly, high levels of TACC3 also promoter activity. The TFSERACH (http://www.cbrc.jp/research/db conferred increased cellular sensitivity to IR and PARP inhibitors. It /TFSEARCH.html) and TFBIND (http://tfbind.hgc.jp/) programs is quite interesting that Aurora A, known to be an upstream predicted E2F1, hepatocyte nuclear factor 4α (HNF4α), chicken molecule of TACC313 is also involved in the DDR signaling ovalbumin upstream promoter transcription factor II (COUP-TFII) pathways.98 Similar to TACC3, cells expressing high levels of and cyclic AMP response element binding protein (CREBP) to be Aurora A display defects in HR-mediated DSB repair and are more associated with this ATM promoter region. E2F1 has been shown

Oncogene (2015) 1667 – 1678 © 2015 Macmillan Publishers Limited DDR deregulation by TACC3 G-H Ha et al 1675 to elevate promoter activity, levels of mRNA and protein and kinase activity of ATM,101 raising the possibility that TACC3 may High levels of TACC3 attenuate its binding to the ATM promoter and thus negatively Loss of ATM and regulate ATM transcription. The region of the ATM promoter that other mechanisms binds to TACC3 also contains the CRE element, and a study has shown that depletion of CREBP reduces ATM mRNA expression.102 Interestingly, HNF4α interacts with CREBP103 or COUP-TFII.104 Defective DDR Therefore, it is possible that TACC3 may alter the regulation of ATM transcription via more than one transcription factor. In future studies, we will identify transcription factors involved in the Genomic instability Cellular sensitivity to TACC3-mediated ATM regulation and determine how TACC3 is DNA damaging agents recruited to the ATM promoter region. As the induction of DNA and PARP inhibitors damage caused by high levels of TACC3 was found to be only partially due to loss of ATM, we will further investigate the additional molecular mechanisms of the DNA damage induced Tumorigenesis by TACC3. In this study, we demonstrated that high levels of TACC3 Figure 7. Schematic representation of the effects of high levels of negatively regulate DNA repair and checkpoint function possibly TACC3. A depiction of the way in which high levels of TACC3 lead to through suppression of ATM. As ATM has an important role in DSB genomic instability but also implicate therapeutic potential. repair by initiating chromatin relaxation in response to DNA damage,105,106 in future studies, it would be interesting to study whether TACC3 is involved in the dysregulation of chromatin maintained in MEM (Hyclone) containing 10% FBS and 1% penicillin/ streptomycin solution. NF560 non-immortalized normal human skin relaxation and thus subsequently affects DSB repair. fi fi fi broblasts were kind gifts from Dr Mitchell F Denning (Loyola University Together, these ndings propose a signi cant role for TACC3 in Chicago) and maintained in DMEM containing 10% FBS and 1% penicillin/ clinical cancer management because cells exhibiting high levels of streptomycin solution. Cells were incubated under humidified conditions TACC3 display a phenotype of genomic instability, which is with 5% CO2 at 37 °C. considered to be an important step before becoming transformed An anti-TACC3 antibody was purchased from Santa Cruz Biotechnology and displaying abnormal morphological and physiological (Santa Cruz, CA, USA). Antibodies against HA, phospho-ATMS1981, ATM, features.93,107,108 Moreover, TACC3 is elevated in a variety of phospho-Chk2T68, Chk2, phospho-Chk1S345, Chk1, phospho-p53S15, p53, S10 human cancer types and notably, high levels of TACC3 are closely phospho-histone H3 and 53BP1 were purchased from Cell Signaling related to poor survival in patients with breast and lung cancers. Technology (Beverly, MA, USA). Antibodies against H2AX, mediator of DNA damage checkpoint 1 and BRCA1 were purchased from Abcam Our previous study suggested that elevated levels of TACC3 may γ β 36 (Cambridge, UK). Anti- H2AX and anti- -actin antibodies were purchased serve as a novel indicator of high metastatic potential. These from Millipore (Billerica, MA, USA) and Sigma-Aldrich (St Louis, MO, USA), current findings raise the possibility that the monitoring of TACC3 respectively. expression levels may allow us to better predict cancer progres- Olaparib (AZD2281) and ABT-888 were purchased from Selleckchem sion and patient survival and to manage the disease process (Houston, TX, USA), and NU1025 was obtained from Sigma-Aldrich. properly as well as to even prevent cancer development. We also showed that elevated levels of TACC3 increase cellular sensitivity Metaphase chromosome spreads to IR and PARP inhibitors possibly due to impaired HR and DDR Metaphase chromosome spreads were performed as described previously signaling pathways. Therefore, it is tempting to speculate that with modifications.109 Cells were treated with 0.1 μg/ml colcemid (Gibco, TACC3 would be a potential therapeutic target or a prognostic Gaithersburg, MD, USA), harvested, washed, centrifuged and treated with factor to predict therapeutic modalities and outcomes to DNA hypotonic solution (50 mM KCl) at 37 °C for 30 min. Cells were then fixed damaging agents or agents that target defective DNA repair. with freshly prepared methanol and acetic acid (3:1) at room temperature Overall, our study identifies a novel mechanism by which TACC3 for 30 min, centrifuged and washed. Fixation was repeated for 20 min at may contribute to genomic instability and cancer development room temperature. Chromosomes were spread on ice-cold slides, stained and provides new avenues of pursuit that are relevant to with DAPI for 10 min, washed, mounted with mounting medium and fl predicting prognosis and therapeutic interventions for solid observed under a uorescence microscope. tumors expressing high levels of TACC3 (Figure 7). TissueScan qPCR array The human TissueScan Cancer Survey cDNA array 96-I (OriGene MATERIALS AND METHODS Technologies Inc, Rockville, MD, USA) consisted of 96 tissues from eight solid tumors (breast, colon, kidney, liver, lung, ovary, prostate and thyroid). Cell culture, antibodies and PARP inhibitors qPCR was performed in 96-well PCR array plates using the iCycler iQ5 real- U2OS cells were maintained in McCoy's 5A medium (HyClone, Logan, UT, time PCR detection system (Bio-Rad, Hercules, CA, USA) and an iQ SYBR USA) supplemented with 10% fetal bovine serum (FBS; Gemini Bio- Green supermix (Bio-Rad). PCR amplification was carried out at 95 °C for Products, Woodland, CA, USA) and 1% penicillin/streptomycin solution 5 min and 30 s followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. For fi 110 (Thermo Fisher Scienti c, Waltham, MA, USA). MCF10A cells were data analysis, the ΔΔCt method was used. The sequences of the primer maintained in Dulbecco's Modified Eagle Medium (DMEM)/DMEM F12 pairs were as follows: TACC3 5′-GAACTGGGGAAGATCATGGA-3′ and (HyClone) containing 5% horse serum, 20 ng/ml epidermal growth factor, 5′-CTCTTCGTTCTTGCGGTAGC-3′111 and ATM 5'-CCAGGCAGGAATCATTCAG 0.5 μg/ml hydrocortisone, 100 ng/ml insulin, 100 ng/ml cholera toxin and -3' and 5'-CAATCCTTTTAAATAGACGGAAAGAA-3′.112 1% penicillin/streptomycin solution. SUM159 cells were maintained in Ham’s nutrient mixture F12 (HyClone) containing 5% calf serum, 5 μg/ml insulin, 1 μg/ml hydrocortisone and 1% penicillin/streptomycin solution. Tissue microarrays and immunohistochemistry HEK293 T, MDA-MB-231 and MDA-MB-468 cells were maintained in DMEM Breast (BC081120, BR1504) and lung (LC121) cancer tissue microarrays (HyClone) containing 10% FBS and 1% penicillin/streptomycin solution. were purchased from US Biomax (Rockville, MD, USA). Information for BT549, BT474 and SKBR3 cells were maintained in RPMI (HyClone) breast and lung cancer tissue microarrays is shown in Supplementary containing 10% FBS and 1% penicillin/streptomycin solution. Cell lines Table 1. Slides were deparaffinized, rehydrated, heat-treated for antigen were obtained from the American Type Culture Collection (ATCC; retrieval113 and incubated with an anti-TACC3 antibody for 1 h at room Manassas, VA, USA). WI-38 human embryonic lung fibroblasts were kindly temperature, followed by incubation with secondary biotinylated antibody provided by Dr Takeshi Shimamura (Loyola University Chicago) and and the Avidin Biotin complex (ABC) (Vector Laboratories, Burlingame, CA,

© 2015 Macmillan Publishers Limited Oncogene (2015) 1667 – 1678 DDR deregulation by TACC3 G-H Ha et al 1676 USA), following the manufacturer's instructions. After developing color protein expressed during murine embryonic development. Mech Dev 2000; 97: with diaminobenzidine, the slides were independently assessed by three 13–26. authors. The intensity of staining was recorded as follows: 0, negative 8 Piekorz RP, Hoffmeyer A, Duntsch CD, McKay C, Nakajima H, Sexl V et al. expression; 1+, weakly positive expression; 2+, medium positive expres- The centrosomal protein TACC3 is essential for hematopoietic stem cell function sion; and 3+, highly positive expression. Photomicrograph (magnification and genetically interfaces with p53-regulated apoptosis. EMBO J 2002; 21: x100) was taken by DP12 microscope (Olympus, Tokyo, Japan) equipped 653–664. with DP71 digital imaging system (Olympus). 9 Garriga-Canut M, Orkin SH. Transforming acidic coiled-coil protein 3 (TACC3) controls friend of GATA-1 (FOG-1) subcellular localization and regulates the association between GATA-1 and FOG-1 during hematopoiesis. J Biol Chem 2004; ChIP assay 279: 23597–23605. ChIP assays were performed using the EpiTect ChIP OneDay Kit (Qiagen, 10 Jung CK, Jung JH, Park GS, Lee A, Kang CS, Lee KY. Expression of transforming Valencia, CA, USA) in accordance with the manufacturer's instructions. acidic coiled-coil containing protein 3 is a novel independent prognostic marker fi Using a Fisher 60 Sonic dismembrator (Thermo Fisher Scienti c), chromatin in non-small cell lung cancer. Pathol Int 2006; 56:503–509. fi fi in formaldehyde- xed cellular lysates was subjected to ve sets of 11 Schuendeln MM, Piekorz RP, Wichmann C, Lee Y, McKinnon PJ, Boyd K et al. The 114 sonication for 20 s (output 7) with 1-min intervals. Immunoprecipitation centrosomal, putative tumor suppressor protein TACC2 is dispensable for normal fi was performed using antibodies speci c for TACC3 or with rabbit IgG and development, and deficiency does not lead to cancer. Mol Cell Biol 2004; 24: fi RNA polymerase II (Millipore) as controls. Puri ed ChIP DNA was subjected 6403–6409. to qPCR, and ChIP-qPCR data were analyzed using Excel-based EpiTecChIP 12 McKeveney PJ, Hodges VM, Mullan RN, Maxwell P, Simpson D, Thompson A et al. qPCR data analysis template (http://www.sabiosciences.com/ Characterization and localization of expression of an erythropoietin-induced chippcrarray_data_analysis.php). The sequences of the primer pairs were fi − − ′ ′ gene, ERIC-1/TACC3, identi ed in erythroid precursor cells. Br J Haematol 2001; as follows: A ( 631 to 435) 5 -GGGGTCCTAATTAAGTGTGG-3 and 112 – ′ ′ − − ′ : 1016 1024. 5 -GCCTCCCAGCTTACGAGT-3 ;B(434 to 239) 5 -AAAACCCCAA 13 Kinoshita K, Noetzel TL, Pelletier L, Mechtler K, Drechsel DN, Schwager A et al. AGCTTCCCTA-3′ and 5′-TGTTTGCTTTAACCTGAGTCT-3′;C(−238 to − 43) Aurora A phosphorylation of TACC3/maskin is required for centrosome- 5′-CAGCGACAGCTCCTGCG-3′ and 5′-CCTCATCCCCGCCCCTC-3′;D(−42 to dependent microtubule assembly in mitosis. J Cell Biol 2005; 170:1047–1055. +154) 5′-AGGGCGGGGAGGACGAC-3′ and 5′-CACGGTATGCCCATGCGC-3′; 14 Schneider L, Essmann F, Kletke A, Rio P, Hanenberg H, Wetzel W et al. The E (+155 to +352) 5′-CTCTGCGGCTGCTTGGC-3′ and 5′-TGCACTCGGAAGGT transforming acidic coiled coil 3 protein is essential for spindle-dependent CAAAG-3′;B1(−434 to − 385) 5′-AAAACCCCAAAGCTTCCCTA-3′ and ′ ′ − − ′ chromosome alignment and mitotic survival. J Biol Chem 2007; 282: 5 -GGACCTTTGAGGCCAAAG-3 ;B2( 389 to 340) 5 -GGTCCTTCTGTCCAG – CAT-3′ and 5′-GGACGCGGGATGGAGG-3′;B3(−344 to − 295) 5′-CGTCC 29273 29283. GCGCTTACCCAA-3′ and 5′-ATGTTGTTACCCTCGGAC-3′;B4(−299 to − 239) 15 Albee AJ, Wiese C. Xenopus TACC3/maskin is not required for microtubule sta- 5′-AACATGATCAAAACCACAGC-3′ and 5′-TGTTTGCTTTAACCTGAGTCT-3′; bility but is required for anchoring microtubules at the centrosome. Mol Biol Cell 19 – GAPDH 5'-TACTAGCGGTTTTACGGGCG-3′ and 5'-TCGAACAGGAGGAGCA 2008; :3347 3356. GAGAGCGA-3' (Millipore). 16 Lauffart B, Gangisetty O, Still IH. Evolutionary conserved interaction of TACC2/TACC3 with BARD1 and BRCA1: potential implications for DNA damage response in breast and ovarian cancer. Cancer Ther 2007; 5:409–416. Statistical analysis 17 Lauffart B, Howell SJ, Tasch JE, Cowell JK, Still IH. 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