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FEBS Letters 584 (2010) 4253–4258

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Counteracting -induced HIPK2 downregulation restores HIPK2/ apoptotic signaling in cancer cells ⇑ ⇑⇑ Lavinia Nardinocchi a, , Rosa Puca a,b, David Givol c, Gabriella D’Orazi a,b,

a Department of Experimental Oncology, Molecular Oncogenesis Laboratory, National Cancer Institute ‘‘Regina Elena”, 00158 Rome, Italy b Department of Oncology and Experimental Medicine, School of Medicine, University ‘‘G. d’Annunzio”, 66100 Chieti, Italy c Department of Molecular Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel

article info abstract

Article history: Homeodomain-interacting -2 (HIPK2) is a crucial regulator of p53 apoptotic function Received 23 July 2010 by phosphorylating 46 (Ser46) in response to DNA damage. In tumors with wild-type p53, its Revised 21 August 2010 tumor suppressor function is often impaired by MDM2 overexpression that targets p53 for proteaso- Accepted 10 September 2010 mal degradation. Likewise, MDM2 targets HIPK2 for protein degradation impairing p53-apoptotic Available online 16 September 2010 function. Here we report that antagonised MDM2-induced HIPK2 degradation as well as p53 Edited by Gianni Cesareni ubiquitination. The zinc inhibitory effect on MDM2 activity leads to HIPK2-induced p53Ser46 phos- phorylation and p53 pro-apoptotic transcriptional activity. These results suggest that zinc deriva- tives are potential molecules to target the MDM2-induced HIPK2/p53 inhibition. Keywords: HIPK2 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. p53 transcriptional activity Zinc supplementation MDM2

1. Introduction [10]. Moreover, HIPK2-induced p53Ser46 pre- vents the MDM2-mediated p53 ubiquitination recovering p53 P53 is a potent tumor suppressor and one of the key play- apoptotic function [11]. Therefore, preserving HIPK2 function is ers in apoptosis signaling [1]. P53 activation is regulated through crucial for p53 apoptotic activity. specific posttranslational modifications, leading to p53 transcrip- MDM2 is not only a p53 inhibitor but also contributes to mod- tional activation of target [2]. Because of its critical function, ulate the p53-mediated biological outcomes (i.e., cell cycle arrest p53 is frequently targeted for inactivation and suffers disabling versus apoptosis) by regulating HIPK2. In response to non-lethal or deletions in about 50% of all malignant tumors. The DNA damage, p53-activated MDM2 inhibits the p53 apoptotic other half of human cancers express wild-type p53 protein that pathway by targeting HIPK2 at residue 1182 for proteasomal however, can be inactivated by deregulation of regulatory proteins degradation [12]. It is worth noting that the mdm2 gene is ampli- [3]. An important mechanism that regulates p53-apoptotic func- fied in a significant proportion of human tumor types, thereby tion involves MDM2 oncogene by means of its E3 ubiquitin ligase contributing to tumor progression by efficiently reducing the avail- activity that targets p53 for proteasomal degradation [4]. DNA- ability of a functional p53 as well as most likely the availability of damage-induced phosphorylation of its N-terminus contributes its apoptotic activator HIPK2. Several approaches have been tested, to p53 stability by preventing MDM2 from degrading it [5]. leading to the discovery of small molecules that can restore p53 Homeodomain-interacting protein kinase-2 (HIPK2) was identified function in tumor cells by targeting MDM2 through different as a p53-interacting protein [6,7] that specifically phosphorylates mechanisms [13]. This prompted us to evaluate whether it would p53 at Ser46 for transcriptional activation of pro-apoptotic factors be possible to restore p53 apoptotic function by reactivating HIPK2 such as p53AIP1, PIG3, Bax, Noxa, Puma and KILLER/DR5 [7–9],as in the presence of MDM2. well as for the repression of the anti-apoptotic factor galectin-3 2. Materials and methods

⇑ Corresponding author. 2.1. Cell culture and reagents ⇑⇑ Corresponding author at: Department of Experimental Oncology, Molecular Oncogenesis Laboratory, National Cancer Institute ‘‘Regina Elena”, 00158 Rome, Italy. Fax: +39 0652662505. Human embryo kidney 293 and human colon carcinoma HCT116 E-mail addresses: [email protected] (L. Nardinocchi), [email protected] (G. D’Orazi). cells were maintained in DMEM (Life Technology-Invitrogen), while

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.09.018 4254 L. Nardinocchi et al. / FEBS Letters 584 (2010) 4253–4258 human colon carcinoma RKO cells, human epithelial cervical cancer 2.5. Reverse transcription polymerase chain reaction (RT-PCR) cells HeLa, and human lung cancer H1299 (p53 null) cells were maintained in RPMI-1640 (Life Technology-Invitrogen), all supple- Cells were harvested in TRIzol reagent (Invitrogen) and total mented with 10% heat-inactivated fetal bovine serum plus gluta- RNA was isolated following the manufacturer’s instructions. The mine and antibiotics in humidified atmosphere with 5% CO2 at first strand cDNA and the semi-quantitative RT-PCRs were carried 37 °C. out essentially as described [14]. PCR products were run on a 2%

Treatments: cells were incubated with 100 lM ZnCl2, 200 lM agarose gel and visualized by ethidium bromide staining using Cobalt Chloride (CoCl2, hypoxia mimetic), 10 lmol/l Nutlin-3 UV light. The housekeeping glyceraldehydes-3-phosphate dehy- (Cayman), and 5-10 lmol/l inhibitor MG132 (Biomol drogenase (GAPDH) or aldolase-A (ald-A) mRNAs were used as Research Laboratories) for the indicated time. internal control.

2.2. Transient transfection and vectors 2.6. TUNEL assay and immunofluorescence

4 Cells were plated in 60 mm Petri dishes and, the day after, For TUNEL assay, 4 Â 10 cells were spun on a slide by cytocen- transfected with the modified version of the calcium phosphate trifugation and subsequently fixed in 4% paraformaldehyde for procedure or the cationic polymer LipofectaminePlus method 30 min at room temperature. The samples were permeabilized (Invitrogen), according to the manufacturer’s instructions. and then incubated in the TUNEL reaction mix for 1 h at 37 °C Expression vectors used were: wild-type HIPK2-Flag [7] and according to the manufacture’s instructions (Roche, Germany). mutant HIPK2-K1182R-Flag (MDM2-degradation resistant) [12], Cells were counter-stained with Hoechst 33 342 before analysis human HA-MDM2 (kindly provided by M. Oren, Weizmann Insti- with a fluorescent microscope (Zeiss). tute of Science, Rehovor, Israel), and pCAG3.1wtp53 (kindly pro- For indirect immunofluorescence, HeLa cells were transfected vided by E. Appella, NIH, Bethesda, MD, USA). with HIPK2-Flag expression vector and 24 h after transfection trea- ted with 100 lM ZnCl2 and 200 lM CoCl2 for 24 h and then fixed in 2.3. Western immunoblotting and ubiquitination methanol/acetone (1:1). Dried cells were blocked for 30 min at room temperature in phosphate-buffered saline containing 10% For Western immunoblotting, total cell extracts were (v/v) goat serum, incubated with primary anti-Flag antibody at prepared by incubating at 4 °C for 30 min in lysis buffer as 4 °C for 16 h and for 45 min with an appropriate fluorochrome- reported [11] and resolved by SDS–polyacrylamide gel electro- conjugated secondary antibody. Chromosomal DNA was visualized phoresis. Proteins were transferred to a polyvinylidene difluo- by DAPI staining, stained cells were mounted on glass slides and ride (PVDF) membrane (Millipore). Immunoblottings were examined using a confocal laser microscope (Nikon Eclipse). performed with: mouse monoclonal anti-p53 (DO1), rabbit poly- clonal anti-p53 (FL393), mouse monoclonal anti-MDM2 (Ab1), 2.7. Statistical analysis rabbit polyclonal anti-p21Waf1 (all from Santa Cruz Biotechnol- ogy), rabbit polyclonal anti-phospho-Ser46 (Cell Signaling Tech- All experiment unless indicated were performed at least three nology), rabbit polyclonal anti-HIPK2 (kindly provided by M.L. times. All experimental results were expressed as the arithmetic Schmitz, Justus-Liebig-University, Giessen, Germany), mouse mean and standard deviation (S.D.) of measurements was shown. monoclonal anti-Flag (M2, Sigma, Bio-Sciences), rat polyclonal Student’s t-test was used for statistical significance of the differ- anti-HA (Roche Diagnostic), mouse monoclonal anti-tubulin ences between treatment groups, determined as P < 0.05. (Immunological Sciences), and mouse monoclonal anti-Hsp70 (Stressgene). 3. Results P53 ubiquitination was performed essentially as described [11]. Briefly, H1299 cells were co-transfected with 1 lg pCAG3.1wtp53 3.1. MDM2-induced downregulation of HIPK2 and inhibition of and 4 lg HA-MDM2 expression vectors and 24 h later treated with p53Ser46 phosphorylation, are rescued by zinc treatment

ZnCl2 and 10 lM MG132 for, respectively 16 and 8 h. Equal amounts of total cell extracts were immunoprecipitated with In order to identify natural compounds that restore HIPK2 func- mouse monoclonal anti-p53 antibody (DO1) preadsorbed to pro- tion, the effect of zinc was investigated. 293 cells were co-transfec- tein G-agarose (Pierce). For endogenous HIPK2 ubiquitination as- ted with HIPK2-Flag or the K1182R-Flag mutant and MDM2-HA say, RKO cells were treated with ZnCl2 for 24 h, CoCl2 and 5 lM expression vectors and treated with zinc. As shown in Fig. 1, HIPK2 MG132 for 16 h. Equal amounts of total cell extracts were immu- expression induced p53Ser46 phosphorylation (lane 2) while noprecipitated with rabbit polyclonal anti-HIPK2 antibody pread- MDM2 co-transfection induced HIPK2 downregulation and abol- sorbed to protein G-agarose (Pierce). Immunocomplexes were ishment of p53Ser46 phosphorylation (lane 3); interestingly, the collected by centrifugation, separated by SDS–PAGE, and blotted MDM2-induced HIPK2 downregulation was completely rescued onto nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Immu- by zinc treatment with re-establishment of HIPK2-induced noreactivity was detected by enhanced chemiluminescence kit p53Ser46 phosphorylation and downregulation of MDM2 levels (ECL; Amersham). (lane 4). As expected, the MDM2 degradation-resistant HIPK2- K1182R mutant induced p53Ser46 phosphorylation (Fig. 1, lane 2.4. Luciferase assay 5) and was not downregulated by MDM2 (lane 6) nor its activity towards p53Ser46 phosphorylation was further affected by zinc Cells were co-transfected with expression vectors along with treatment (lane 7). the luciferase reporter gene driven by the p53-dependent promoter Noxa-luc (kindly provided by T. Taniguchi, University 3.2. Reactivation of HIPK2-induced p53 transcriptional and apoptotic of Tokyo, Japan) vector. Transfection efficiency was normalized activities with a co-transfected b-galactosidase plasmid. Luciferase activity was assayed on whole cell extract and the luciferase values The clear effect of zinc on HIPK2 stability and p53Ser46 phos- were normalized to b-galactosidase activity and protein phorylation in the presence of MDM2 prompted us to evaluate content. its biological consequences. First, the p53 responsive reporter L. Nardinocchi et al. / FEBS Letters 584 (2010) 4253–4258 4255

ZnCl2 ---+ --+ its activity, although it may not induce p53-dependent apoptosis HA-MDM2 - - + + - + + [15,16]. Nutlin-3 preserves MDM2 E3 ligase activity [15], thus it in- Flag-K1182R - - - - + + + duces HIPK2 downregulation inhibiting p53Ser46 apoptotic activ- Flag-HIPK2 - + + + - - - ity [17]. As shown in Fig. 3A, Nutlin-3 treatment markedly downregulated HIPK2 levels and induced p21Waf1 and MDM2, as Flag previously reported [17]. The effect of MDM2 antagonism was re- verted by zinc that neutralized the Nutlin-3-induced HIPK2 down- HA regulation (Fig. 3A). Next, H1299 cells were co-transfected with p53 and MDM2 expression vectors in the presence of MG132 and (p)Ser46 assayed by p53 immunoprecipitation. Co-transfection of p53 and MDM2 revealed a ladder of p53-specific high molecular weight p53 bands indicative of protein ubiquitination (Fig. 3B), while, zinc strongly reduced the extent of p53 ubiquitination (Fig. 3B, lower Tubulin panel). Finally, the inhibition of p53-induced Noxa-luc activity by MDM2 co-expression was significantly counteracted by zinc 1 2 3 4 5 6 7 (Fig. 3C). Fig. 1. MDM-2-dependent downregulation of HIPK2 levels and HIPK2-dependent p53Ser46 phosphorylation are rescued by zinc. 293 cells were co-transfected with 3.4. The hypoxia-dependent HIPK2 cytoplasmic delocalization and HIPK2-Flag or HIPK2-K1182R-Flag (2 lg) along with HA-MDM2 (6 lg) expression ubiquitination is counteracted by zinc vectors and 16 h after transfection treated with 100 lM ZnCl2 for 24 h. Anti-tubulin was used as protein loading control. We reported a chemical hypoxia model where HIPK2 downreg- ulation correlates with p53-induced MDM2 expression [18]. Indi- Noxa-luc activity was measured. The HIPK2-induced Noxa-luc rect immunofluorescence analysis of ectopic HIPK2 showed the activity was inhibited by MDM2 and efficiently restored by zinc usual localization as nuclear dots [7], while cobalt treatment (Fig. 2A). Consequently, HIPK2-induced p53 pro-apoptotic tran- strongly delocalized HIPK2 into the cytoplasm (Fig. 4A). Zinc pre- scription was inhibited by MDM2 but strongly relieved by zinc treatment, completely counteracted the cobalt effect on HIPK2 treatment (Fig. 2B). Finally, we observed a consistent inhibitory ef- subcellular localization, maintaining the nuclear dotted appear- fect of MDM2 on HIPK2-induced TUNEL positivity while MDM2 ance (Fig. 4A). Western immunoblotting showed that cobalt treat- was not able to inhibit the HIPK2-K1182R mutant, as reported ment induced MDM2 levels, that were downregulated by zinc [15]; of note, zinc relapsed the MDM2 inhibitory effect on HIPK2 supplementation (Fig. 4B). In agreement, MDM2 RNA levels, which activity and restored HIPK2-induced apoptosis (Fig. 2C). were induced by cobalt as reported [18], were reduced by zinc treatment. Finally, the cobalt-induced HIPK2 ubiquitination was 3.3. Zinc neutralizes the MDM2 ubiquitin ligase activity strongly inhibited by zinc treatment, in agreement with our hypothesis that the cobalt inhibition at least in part is affecting Then, we took advantage of Nutlin-3 molecule that binds the MDM2 ligase activity (Fig. 4C). MDM2 in the p53 pocket and inhibits p53 degradation restoring

A 1600 1200 * C 800 RLU 35 %)

400 ( 30 25 0 20 HIPK2 - + + + 15

MDM2 - - + + positivity 10 ZnCl2 -- -+ 5 Tunel 0 B HIPK2 - + + + - - - K1182R - - - - + + + ZnCl2 - - - + + MDM2 - - + + - MDM2 - - + + - + + HIPK2 - + + + + ZnCl2 - - - + - - + Bax

GAPDH 293

Fig. 2. Zinc treatment counteracts the MDM2-dependent HIPK2 inhibition and restores p53 apoptotic transcriptional activity. (A) 293 cells were co-transfected with HIPK2-

Flag (2 lg) and HA-MDM2 (6 lg) expression vectors along with Noxa-luc reporter plasmid and 16 h after transfection treated with ZnCl2 for 24 h before luciferase activity was assayed. Data are the mean ± S.D. of three independent experiments performed in duplicate. RLU: relative luciferase unit. *P < 0.005. (B) Total mRNAs were reverse transcribed from 293 cells treated as in (A), for semi-quantitative RT-PCR analysis. GAPDH was used as internal control. (C) 293 cells co-transfected with HIPK2-Flag or HIPK2-

K1182R-Flag (2 lg) along with HA-MDM2 (6 lg) expression vectors and 16 h after transfection treated with ZnCl2 for 24 h. Data are presented as percentage of TUNEL positive cells over Hoechst-stained nuclei. Standard deviation of three independent experiments is indicated. 4256 L. Nardinocchi et al. / FEBS Letters 584 (2010) 4253–4258

A C

ZnCl2 -- + --+ * * Nutlin-3 - + + -+ + 600 HIPK2 400 RLU 200 MDM2 0 p53 p53 + + + + MDM2-HA -+ + - ZnCl2 --+ + p21 HA Hsp70 p53 tubulin B

ZnCl2 --+ HA-MDM2 -+ + p53 + + +

IP: p53 IB: p53 p53-Ubn

HA

p53

Tubulin

1/10 Input

Fig. 3. The MDM2-induced p53 ubiquitination is counteracted by zinc. (A) RKO (left panel) and HCT116 (right panel) cells were treated with 10 lM Nutlin-3 and ZnCl2 for, respectively, 48 and 24 h. Equal amounts of total cell extracts were assayed for Western immunoblotting. Anti-tubulin was used as protein loading control. (B) For p53 ubiquitination, H1299 cells were co-transfected with CMVp53 (1 lg) and HA-MDM2 (4 lg) expression vectors and 16 h after transfection treated with ZnCl2 and 10 lM proteasome inhibitor MG132 for, respectively, 24 and 8 h. Equal amounts of total cell extracts were subjected to immunoprecipitation (IP) analysis with anti-p53 monoclonal antibody (DO1) and Western immunoblotting (IB) with anti-p53 polyclonal antibody (FL393). The position of the p53-ubiquitin (p53-Ubn) conjugates is indicated. The lower panel shows Western immunoblotting of 1/10 of total cell extracts (Input). Anti-tubulin was used as protein loading control. (C) H1299 cells were co-transfected with CMV- p53 (1 lg) and HA-MDM2 (4 lg) expression vectors along with Noxa-luc reporter plasmid and 16 h after transfection treated with ZnCl2 for 24 h before luciferase activity was assayed. Data are the mean ± S.D. of three independent experiments performed in duplicate. RLU: relative luciferase unit. *P < 0.001. The lower panel shows a Western immunoblotting of ectopic proteins. Anti-tubulin was used as protein loading control.

4. Discussion A1 (HMGA1) [24] or b4 integrin in breast cancers [25]. HIPK2 can be downregulated by several E3 ubiquitin ligases such as WD40- In the present study we disclosed a novel way to unlock the repeat/SOCS box protein WSB-1, the RING family ligases Siah-1 MDM2-p53 feedback loop and unveiled a way to counteract the and Siah-2 and, MDM2 oncogene [21]. Thus as p53 and MDM2 con- MDM2-induced HIPK2 downregulation that consequently restored stitute a reciprocal feedback loop, MDM2 and HIPK2 add an addi- also p53 apoptotic transcriptional activity. tional inhibitory arm to this loop to facilitate a more fine tuning HIPK2 is a multifunctional protein kinase that positively acti- of the regulation of apoptosis. vates p53 pro-apoptotic function in response to apoptotic stress We recently found that, upon chemical hypoxia, MDM2 is in- signals [7,19,20] and also exerts its antitumor activity by modulat- duced leading to HIPK2 downregulation and inhibition of p53 ing the transcription activity of molecules involved in tumor pro- apoptotic activity in response to drugs [18]. Interestingly, we gression, angiogenesis and chemoresistance, including HIF-1a found that zinc supplementation reverses the hypoxia-induced and b-catenin [21]. These findings suggest the potential role of HIPK2 downregulation which prompted us to further evaluate HIPK2 as cancer biomarker whose expression may contribute to the molecular mechanisms of reversible HIPK2 inactive/active p53 activity and the decision on anti-tumor therapies. Indeed we switch. Here, we showed that zinc relapsed the MDM2-induced previously showed a correlation between low HIPK2 expression HIPK2 downregulation, likely counteracting the MDM2 E3 ubiqui- and bad outcome in wild-type p53 expressing colon cancer tumors tin ligase activity. This finding was obtained upon MDM2/HIPK2 [14]. HIPK2 cytoplasmic localization may also affect p53 apoptotic co-transfection and by Nutlin-3 treatment (Fig. 4A), which has function, following overexpression of oncogenes such as CBFb- been shown to downregulate HIPK2 levels [17] without affecting SMMHC in a leukemogenesis model [22,23], high-mobility group MDM2 ubiquitin ligase activity [15]. The negative role of zinc on L. Nardinocchi et al. / FEBS Letters 584 (2010) 4253–4258 4257

B A ZnCl2 --+ ZnCl2 --+ CoCl2 -+ + CoCl2 -+ + HIPK2-Flag MDM2 MDM2 Hsp70 ald-A Mock protein RNA

C CoCl2

ZnCl2 --+ -+ + CoCl2 CoCl2 +

ZnCl2 300

HIPK2-Ubn ZnCl2 250

anti-Flag DAPI Merge 130 -HIPK2 IP: anti-HIPK2 IB: anti-HIPK2

Fig. 4. The hypoxia-dependent HIPK2 cytoplasmic delocalization and ubiquitination is counteracted by zinc. (A) HeLa cells were transfected with HIPK2-Flag and treated with

ZnCl2 and CoCl2 alone or in combination for, respectively, 24 and 16 h. HIPK2 localization was visualized by indirect immunofluorescence with anti-Flag antibody.

Overlapping localization in merged pictures is shown in yellow and chromosomal DNA was stained with DAPI. (B) RKO were treated with ZnCl2 and CoCl2 for, respectively, 24 and 16 h and then subjected to Western immunoblotting (left panel) or semi-quantitative RT-PCR analysis (right panel). Anti-Hsp70 was used as protein loading control and

GAPDH was used as internal RT-PCR control. (C) For endogenous HIPK2 ubiquitination, RKO were treated with ZnCl2 and CoCl2 for, respectively, 24 and 16 h in the presence of

MG132 for 16 h. Equal amounts of total cell extracts were subjected to immunoprecipitation (IP) analysis. The position of the HIPK2-ubiquitin (HIPK2-Ubn) conjugates is indicated.

MDM2 ubiquitin ligase activity was also observed on MDM2-in- Acknowledgments duced p53 ubiquitination (Fig. 3B) and indirectly on hypoxia-in- duced HIPK2 ubiquitination (Fig. 4C). Once HIPK2 is stabilised, We thank Drs. S. Soddu and C. Rinaldo (National Cancer Insti- it may inhibit MDM2 transcription and protein through p53- tute Regina Elena, Rome, Italy) for providing us the Nutlin-3 mole- dependent [11] or p53-independent mechanisms [26]; thus, here cule and for critical discussion. We greatly acknowledged the MDM2 downregulation was observed upon HIPK2 reactivation by support of Prof. ML Schmitz and Drs. L. de La Vega and R. Moreno zinc. Hence, as depicted in Fig. 5, zinc can interfere with the inter- (Justus-Liebig-University, Giessen, Germany) in the study of HIPK2 play between HIPK2, p53 and MDM2 favouring HIPK2 stabiliza- subcellular localization. This work was supported by the Italian tion and induction of p53 apoptotic activity with inhibition of Association for Cancer Research (AIRC) Grant. The FEBS Short-Term MDM2 function. However, as HIPK2 is a protein subjected to pro- Fellowship to L.N. is greatly acknowledged. L.N. is a recipient of a teasome degradation [21] it would be interesting to evaluate in Fellowship from L’Oreal-Italia-UNESCO for Women in Science. the future whether zinc can also inhibit other E3 ubiquitin ligases R.P. is a recipient of a Fellowship from the Italian Foundation for acting on HIPK2, in light of the finding that zinc may inhibit pro- Cancer Research (FIRC). teasome-dependent proteolysis [27].

HIPK2 References

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