ANKHD1 Silencing Inhibits Stathmin 1 Activity, Cell Proliferation and Migration of Leukemia Cells

Biochimica et Biophysica Acta 1853 (2015) 583–593

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Biochimica et Biophysica Acta

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ANKHD1 silencing inhibits Stathmin 1 activity, cell proliferation and migration of leukemia cells

João Agostinho Machado-Neto a, Mariana Lazarini a, Patricia Favaro a,1, Paula de Melo Campos a, Renata Scopim-Ribeiro a, Gilberto Carlos Franchi Junior b, Alexandre Eduardo Nowill b, Paulo Roberto Moura Lima a, Fernando Ferreira Costa a, Serge Benichou c, Sara Teresinha Olalla Saad a, Fabiola Traina a,⁎,2 a Hematology and Hemotherapy Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas 13083-878, São Paulo, Brazil b Integrated Center for Childhood Onco-Hematological Investigation, University of Campinas, Campinas 13083-878, São Paulo, Brazil c INSERM U1016, Institut Cochin, Paris, France article info abstract

Article history: ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions Received 11 September 2014 through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role Received in revised form 29 November 2014 in leukemia cells, we performed a yeast two-hybrid system screen and identiﬁed SIVA, a cellular protein known Accepted 10 December 2014 to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was conﬁrmed by Available online 16 December 2014 co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing pro- Keywords: ANKHD1 motes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 si- SIVA1 lencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the Stathmin 1 inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell pro- Acute leukemia liferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, Cell proliferation but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Ankyrin Repeat and KH Domain Containing 1 protein, ANKHD1, was initially identiﬁed, in humans, as a cytoplasmic protein that is highly Acute leukemia represents a group of clonal hematopoietic stem cell expressed in acute leukemia cells, compared to normal hematopoietic disorders characterized by alterations in the essential pathways of cell cells [3]; however functional studies of ANKHD1 in leukemia cells are physiology including apoptosis, proliferation and genome instability, still lacking. A hallmark of ANKHD1 is the presence of 20 ankyrin- which may be caused by deregulation of signal transduction pathways repeat domains. Each repeat consists of about 33 amino acids that fold that involve the coordinated action of many different proteins [1,2]. into a β-turn, followed by two antiparallel α-helices, which function The identiﬁcation of overactive proteins in leukemia cells, compared to mediate protein–protein interactions [4]. Ankyrin-repeat proteins to normal hematopoietic cells, as well as the understanding of the mo- are found in all species and are fundamental to biological processes, in- lecular and cellular basis of the disease may provide new therapeutic cluding cell cycle control, transcriptional regulation, differentiation, ap- opportunities. optosis, cytoskeletal organization, and may be involved in the development of neoplasia [5]. In solid tumors, ANKHD1 has been found to be associated with YAP1 and negatively impacts the prognosis

⁎ Corresponding author at: Hematology and Hemotherapy Center, University of of patients with breast cancer [6]. However, YAP1 is not expressed in Campinas, Rua Carlos Chagas, 480, CEP 13083-878, Campinas, SP, Brazil. Tel.: +55 19 leukemia cells [7,8], and the role of ANKHD1 in leukemia remains 3521 8734; fax: +55 19 3289 1089. unknown. E-mail addresses: [email protected], [email protected] (F. Traina). The presence of multiple ankyrin repeats suggests a role for ANKHD1 1 Department of Biological Sciences, Federal University of São Paulo, Diadema, 09913- as a scaffolding protein, bringing together a number of signaling mole- 030, São Paulo, Brazil. 2 Department of Internal Medicine, University of São Paulo at Ribeirão Preto Medical cules. To obtain new insight into the signaling properties of ANKHD1 School, Ribeirão Preto, São Paulo, Brazil. and to identify novel interacting proteins that mediate its functions in

http://dx.doi.org/10.1016/j.bbamcr.2014.12.012 0167-4889/© 2014 Elsevier B.V. All rights reserved. 584 J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593 leukemia cells, we used the ankyrin-repeat domain of ANKHD1 as bait The SIVA gene is a transcriptional target of p53 that was initially de- in a yeast two-hybrid (Y2H) assay and identiﬁed the proapoptotic pro- scribed as a proapoptotic protein acting in both extrinsic and intrinsic tein, SIVA. apoptotic pathways [9,10]. In acute lymphoid leukemia cell lines, both J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593 585

Fig. 2. Efﬁcient ANKHD1 and SIVA silencing in U937 and Jurkat leukemia cell lines. U937 or Jurkat leukemia cells were transduced with lentivirus-mediated shRNA control (shControl), lentivirus-mediated shRNA targeting ANKHD1 (shANKHD1) or SIVA (shSIVA). ANKHD1 mRNA (A) and protein (B) expression in shANKHD1 cells relative to the shControl cells. SIVA1 mRNA (C) and protein (D) expression in shSIVA cells relative to the shControl cells. The antibodies used for immunoblotting (IB) are indicated. Images are representative of at least three independent lentivirus-mediated shRNA transduction experiments; Western blot analyses were performed in duplicate for each lentivirus-mediated shRNA transduction.

isoforms of SIVA (SIVA1 and SIVA2) play an important role in the apo- GTCTTGCTCGAGATGTG-3′;RV:5′-TCCAGCAGGTCAGCAAAGAAT-3′). ptotic pathway induced through the CD27 antigen [11]. SIVA1 binds to Relative gene expression was calculated using the equation 2−ΔΔCT BCL-XL, BCL2 and XIAP, negatively regulates the anti-apoptotic NFκB [18]. A negative ‘No Template Control’ was included for each primer and positively regulates the proapoptotic JNK functions [12–14].In pair. The dissociation protocol was performed at the end of each run solid tumors, SIVA1 binds to Stathmin 1 and is essential to the Stathmin to check for non-specific amplification. All samples were tested in 1/CaMKII interaction and CaMKII phosphorylates Stathmin 1 at serine triplicate. 16, decreasing Stathmin 1's activity [15]. Stathmin 1 phosphorylation at specific serine sites reduces its affinity with alpha/beta-tubulin and 2.2. Plasmids leads to microtubule stability [16], which can be evidenced by alpha- tubulin acetylation [15]. The plasmids pEGFPC1, pEGFPC1-SIVA1, and pEGFPC1-SIVA2 were Thereby, Stathmin 1 regulates microtubules dynamics by promoting kindly provided by Dr. Serge Benichou (Institut Cochin, Université depolymerization of microtubule or preventing polymerization of tubu- Paris Descartes, Paris, France) [19]. The pcDNA3-3HA-ANKHD1 plasmid lin heterodimers, and is important for accurate chromosome segregation, was kindly provided by Dr. Nahum Sonenberg (Biochemistry Dept., Mc- cell division, proliferation and migration [17]. We, thus, hypothesized Gill University, Montreal, Canada) [20]. that ANKHD1 could be involved in leukemogenesis through its interaction with SIVA. 2.3. Yeast two-hybrid assay

2. Materials and methods The Matchmaker Two-Hybrid System 3 (Clontech, Palo Alto, CA, USA) was used to screen a human bone marrow cDNA library (in 2.1. Quantitative polymerase chain reaction pACT2) with pGBKT7-ANKHD1 (aa 1130–1243) as the bait. The choice of the ANKHD1 bait prioritized the inclusion of sequences encoding an- Quantitative PCR (qPCR) was performed with an ABI 7500 Sequence kyrin domain, and the exclusion of aminoacid regions with very nega- Detector System (Applied Biosystems, Foster City, CA, USA) with specif- tive charges to avoid self-activation of the bait [21,22]. Peptide ic primers for ANKHD1 (FW: 5′-CCTGCTTGGAACCCTATGATAAA-3′;RV: sequence analysis was performed using Gene Runner (http://www. 5′-CGTGCCAGGCCAAATCTG-3′), SIVA1 (FW: 5′-TCTTCGAGAAGACCAA generunner.com). Co-transformation of the bait and prey plasmids GCG-3′; RV: 5′-TGCCCAAGGCTCCTGATC-3′)andHPRT (FW: 5′-GAAC was performed according to the lithium-acetate transformation

Fig. 1. ANKHD1 is a binding partner of SIVA. (A) Schematic representation of the primary protein structures, the ANKHD1-bait and SIVA-preys identified in our yeast two-hybrid (Y2H) screen; ANKRD, ankyrin repeat domain; KH, K homology domain; SAH, Amphipathic helical region; DDRH, death domain homology region; and ZF, zinc finger-like domain. The alignments of the amino acid sequence obtained from Y2H clone and from the SIVA1 and SIVA2 proteins were performed by Multalin software (http://bioinfo.genopole-toulouse.prd.fr/multalin); the regions containing similarity are indicated in red. (B) Immunoprecipitation (IP) and immunoblotting (IB) of HEK293T lysates overexpressing HA-ANKHD1 and GFP, GFP-SIVA1 or GFP- SIVA2 with specific antibodies. Western blots and IP images are representative of at least two independent co-transfections assays. (C) Immunoprecipitation (IP) and immunoblotting (IB) of U937 cells with anti-ANKHD1 or anti-SIVA antibody. Total protein extract (input) and isotype IgG antibody were used as controls. Two replicates of immunoprecipitation and immunoblotting are illustrated. (D) Confocal analysis of U937 and Jurkat cells displaying SIVA (green), ANKHD1 (red) and DAPI (blue) staining; MERGE shows the overlapped images. Colocalization analysis was performed with the “colocalization finder” plugin of ImageJ NIH software and shows merged images of ANKHD1 and SIVA; the colocalized points are visualized in white. The correlation coefficient (r) values are indicated. Confocal analyses of cells were performed in at least three independent experiments. 586 ..McaoNt ta./Bohmc tBohsc ca15 21)583 (2015) 1853 Acta Biophysica et Biochimica / al. et Machado-Neto J.A. – 593

Fig. 3. ANKHD1 and SIVA silencing have opposing effects on leukemia cell migration and Stathmin 1 activation. Transwell migration assay of U937 and Jurkat leukemia cells submitted to shControl, shANKHD1 (A) or shSIVA (B) in the presence of 0.5% BSA, 10% FBS or 200 ng/mL CXCL12, the results are mean ± SD of at least three independent experiments; *p b 0.05, **p b 0.01; Student t test. Western blot analysis for Stathmin 1 phosphorylation and alpha-tubulin acetylation in U937 and Jurkat cells transduced with shControl, shANKHD1 (C) or shSIVA (D). Immunoprecipitation (IP) of SIVA and immunoblotting (IB) with anti-Stathmin 1 in shControl and shANKHD1 cells; total protein extract (input) was used as control (E). Western blot analysis for Stathmin 1 and alpha-tubulin acetylation in shControl and shSTMN1 total extracts (F). The leukemia cell lines used are indicated. Western blot and IP images are representative of at least two replicates. J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593 587 protocol, using yeast AH109 under high stringency conditions. Positive 2.8. Migration assays clones were confirmed by an X-gal filter assay [23] and the inserts were identified by PCR and sequencing. Two independent co- Migration assays were performed in duplicate using 8 μm pore size transformations of the bait and prey plasmids were performed. transwells (Corning Costar; Cambridge, MA, USA). The transwells were initially washed twice with PBS and blocked for 1 h at 37 °C with 0.5% BSA in RPMI and the lower compartment was filled with 500 μL 2.4. Lentiviral vectors 0.5% RPMI containing 10% FBS, 0.5% BSA plus 200 ng/mL CXCL12 or 0.5% BSA. Cells (1 × 105 in 100 μL) were applied to the upper compart- The preparations of lentiviral vectors expressing the human ment and allowed to migrate for 24 h. The migrated cells were counted short hairpin RNA (shRNA) target ANKHD1 (shANKHD1) or LacZ and expressed as a percentage of the input. (shControl) were performed using the BLOCK-It Lentiviral RNAi Expression System (Invitrogen; Carlsband, CA, USA), as previously described [24]. Lentivirus-mediated shRNA targeting SIVA (shSIVA; 2.9. Methylthiazoletetrazolium (MTT) assay sc-37385-V), Stathmin 1 (shSTMN1; sc-36127-V) or shRNA nonspe- cific control (shControl; sc-108080) were from Santa Cruz Biotech- Cell growth/viability was measured by MTT assay as previous de- nology (Santa Cruz, CA, USA), following the manufacturer's scribed [24]. Cells were serum-starved in 0.5% FBS for 12 h. A total of instruction. 5×104 cells per well were then plated in a 96-well plate in RPMI 10% FBS. In brief, 10 μL of a 5 mg/mL solution of MTT was added to the wells and incubated at 37 °C for 4 h. The reaction was stopped using 2.5. Cell culture, transfection and transduction 100 μL 0.1 N HCl in anhydrous isopropanol. Cell growth/viability was evaluated by measuring the absorbance at 570 nm, using an automated U937, Jurkat and HEK293T cells were obtained from ATCC, Philadel- plate reader. All conditions were tested in six replicates. phia, PA, USA. Cells were cultured in appropriate medium containing 10% fetal bovine serum (FBS) and glutamine with penicillin/streptomy- 2.10. Colony formation assay cin and amphotericin B, and maintained at 37 °C, 5% CO2. HEK293T cells were transiently co-transfected with 8 μg of the DNA plasmid of HA- ANKHD1, and GFP, GFP-SIVA1 or GFP-SIVA2 using a calcium phosphate Colony formation was carried out in semisolid methyl cellulose 3 procedure. After 24 h, the cells were harvested and total extract was medium. Cells (1 × 10 cell/mL) were seeded in MethoCult 4230 prepared and submitted to immunoprecipitation and Western blot. (StemCell Technologies Inc.; Vancouver, BC, Canada). Colonies U937 and Jurkat cells were transduced with lentivirus-mediated were detected after 10 days of culture by adding 1 mg/mL of MTT re- shRNA control, lentivirus-mediated shRNA targeting ANKHD1 or SIVA; agent and scored by Image J quantification software (U.S. National named shControl, shANKHD1 and shSIVA cells, respectively. In addition, Institutes of Health). for the xenograft model of tumorigenesis studies, U937 cells were also 5 transduced with both shANKHD1 and shSIVA. Briefly, 2 × 10 cells 2.11. Apoptosis assays were transduced with lentivirus by spinoculation at a multiplicity of in- fection of equal to 3 and selected by blasticidin (10 μg/mL) for the For apoptosis evaluation, cells were seeded on 12-well plates and Invitrogen system and/or by puromycin (1 μg/mL) for the Santa Cruz evaluated at 0, 3, 6 and 12 h after UV exposition (40 J/m2). ShControl Biotechnology system. or shSIVA U937 cells were also treated with daunorubicin (1 and 2 μM; Actavis Italy S.p.A, Milan, Italy) for 48 h. Cells were then washed 2.6. Western blot analysis and immunoprecipitation twice with ice cold PBS and resuspended in binding buffer containing 1 μg/mL PI and 1 μg/mL APC or FITC labeled Annexin-V. All specimens Equal amounts of protein were used for total extracts or for immu- were analyzed on a FACSCalibur after incubation for 15 min at room noprecipitation with specificantibodiesfollowedbySDS–PAGE and temperature in a light-protected area. Ten thousand events were ac- Western blot analysis with the indicated antibodies and the ECL West- quired for each sample. ern Blot Analysis System (Amersham Pharmacia Biotech, UK). Antibod- ies against ANKHD1 (sc-160960), SIVA (sc-7436), GFP (sc-8334), HA 2.12. Xenograft model of tumorigenesis in NOD/SCID mice (sc-805), p-Stathmin 1 (sc-12948-R), Stathmin 1 (sc-55531), alpha tubulin (sc-5286), BCL-XL (sc-8392), BAX (sc-20067), BCL2 (sc-492), Mice were provided by the University of Campinas Central Breed- YAP1 (sc-101199), SHP2 (sc-280), JAK2 (sc-294), STAT3 (sc-7179), ing Center (Campinas, SP, Brazil). Experimental groups consisted of STAT5 (sc-835) and Actin (sc-1616) were from Santa Cruz Biotechnolo- 6–8 week-old female non-obese diabetic/severe combined immuno- gy. Antibodies against p-JNK (44690G), JNK (446826), p-ERK1/2 deficiency (NOD/SCID) mice, injected with 1 × 107 U937 cells (44654G) and ERK1/2 (700012) were from Invitrogen, the antibodies (resuspended in 100 μL of PBS) that were either shControl, against p-YAP1 (#4911S), p-SHP2 (#3751S), p-JAK2 (#3774S), p- shANKHD1, shSIVA, or both shANKHD1 and shSIVA transduced. STAT3 (#9131S), and p-STAT5 (#9359S) were from Cell Signaling Tech- Cells were implanted into the dorsal sub cutis of mice. After nology (Danvers, MA, USA), the antibody against ANKHD1 (A303-306A) 12 days, tumors were excised, measured and weighed. Tumor mea- was from Bethyl Laboratories (Montgomery, TX, USA), and the antibody surements were converted to tumor volume (V) using the formula against acetyl-alpha tubulin (ab24610) was from Abcam (Cambridge, (V = W2 × L × 0.52), where W and L are the smaller and larger diam- MA, USA). eters, respectively [25]. All experiments were approved by the Ethics Committee of the University of Campinas. 2.7. Confocal immunofluorescence microscopy 2.13. Statistical analysis Confocal imaging was carried out using primary antibodies against ANKHD1 or SIVA (diluted 1:100), as previously described [3].The Statistical analyses were performed using GraphPad Instat 5 “colocalization finder” plugin of Image J quantification software (U.S. (GraphPad Software, Inc., San. Diego, CA, USA). For comparisons, National Institutes of Health, Bethesda, MD, USA) was used for the anal- Student's t-test or Mann–Whitney test were used, as appropriate. A p- ysis of ANKHD1 and SIVA colocalization. value b0.05 was considered as statistically significant. 588 J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593

3. Results U937 cells (p b 0.05), but not in Jurkat cells. Similar results were observed in shControl and shSIVA U937 cells following daunorubicin treatment 3.1. ANKHD1 binds to SIVA (Supplementary Fig. 1). In addition, in U937 cells, knockdown of SIVA resulted in the downregulation of JNK phosphorylation and BAX expression, In order to identify interaction partners of the ANKHD1 protein, and upregulation of BCL-XL expression (Fig. 4), indicating a lower sensi- we performed a Y2H screen of a human bone marrow cDNA library bility to apoptosis, but no changes in BCL2 protein levels were observed. using a fragment of ANKHD1 containing an ankyrin-repeat domain and encompassing the region comprised between aa 1130 and 1243 3.4. ANKHD1 modulates leukemia cell growth and clonogenicity as a bait. Among different clones, we identified one coding for the C- terminal of both SIVA isoforms (Fig. 1A). The association of ANKHD1 Stathmin 1 regulates microtubule dynamics, which is an impor- fi with SIVA1 and SIVA2 was con rmed in immunoprecipitation experi- tant mechanism involved in cell proliferation. We investigated the ments using HEK293T cells co-expressing HA-ANKHD1 in combination role of ANKHD1 and SIVA in cell growth and clonogenicity in U937 with GFP-SIVA1 or GFP-SIVA2 proteins. Protein extracts were immuno- and Jurkat leukemia cells in vitro. Cell growth and clonal growth precipitated with antibody against GFP and immunoblotted with anti- were significantly reduced in shANKHD1 cells, when compared body against HA. HEK293T cells co-expressing HA-ANKHD1 and GFP with shControl (p b 0.05). SIVA silencing did not modulate cell were used as a negative control (Fig. 1B). The interaction between en- growth and clonogenicity (Fig. 5). These data indicate that fi dogenous proteins was veri ed by immunoprecipitation and immuno- ANKHD1 has an important role in the cell growth of leukemia cells blotting of the human leukemia cell line U937 with ANKHD1 and SIVA in vitro, but the effect of SIVA on cell proliferation remains unclear antibodies (Fig. 1C). Finally, confocal microscopy analysis revealed co- and may vary in different cell types [29]. localization of the endogenous ANKHD1 and SIVA proteins in the cyto- plasm of U937 and Jurkat human leukemia cells (Fig. 1D). 3.5. ANKHD1 and SIVA have opposing effects on tumor growth of leukemia cells in vivo 3.2. ANKHD1 silencing inhibits cell migration and Stathmin 1 activity in leukemia cells Xenograft tumor formation is a multistep process that involves apoptosis and cell growth. Based on our findings regarding the role of U937 and Jurkat cells were stably transduced with lentivirus- ANKHD1 and SIVA in leukemia cells in in vitro assays, we analyzed the mediated shRNA targeting ANKHD1 (shANKHD1), SIVA (shSIVA) or an effects of SIVA and ANKHD1 silencing on tumor formation of leukemia appropriate control (shControl), and efficiently silenced for ANKHD1 cells in vivo. and SIVA (Fig. 2). U937 cells stably transduced with shANKHD1, shSIVA, both Transwell migration assay revealed that ANKHD1 silencing decreased shANKHD1 and shSIVA, or shControl, were implanted into female NOD/ the migration capacity of U937 and Jurkat cells upon FBS or CXCL12 stim- SCID mice by subcutaneous injection, and tumors were allowed to grow ulation (p b 0.05; Fig. 3A). In contrast, a significant enhancement of mi- for 12 days. ANKHD1 and SIVA had opposing effects on tumor growth gration was observed in shSIVA cells, when compared to shControl in vivo. While ANKHD1 silencing resulted in a significant reduction of cells in U937 and Jurkat cells (p b 0.05; Fig. 3B). tumor growth of leukemia cells in vivo (p b 0.05, Fig. 6A), SIVA knock- ANKHD1 knockdown resulted in Stathmin 1 inactivation, as revealed down potentiated tumor growth of leukemia cells (p b 0.05; Fig. 6B). In- by phosphorylation, and alpha-tubulin acetylation (Fig. 3C), indicating a terestingly, the respective effects of ANKHD1 and SIVA on the tumor stabilization of tubulin microtubules in agreement with the lower mi- growth of leukemia cells in vivo were abrogated when both proteins gration capacity of shANKHD1-transduced cells. In contrast, SIVA were depleted simultaneously (Fig. 6C), indicating that both proteins knockdown resulted in Stathmin 1 activation, as reflected by the de- may be involved in the same signaling pathway; in addition, the pheno- crease in phosphorylation on Ser16, and alpha-tubulin deacetylation type effect of ANKHD1 silencing depends on the presence of SIVA. compared to shControl cells (Fig. 3D). We then investigated whether ANKHD1 modulates the SIVA/ 3.6. ANKHD1 silencing does not modulate the Hippo, SHP2 and JAK2/STAT Stathmin 1 interaction. In U937 leukemia cells, ANKHD1 silencing result- signaling pathways in leukemia cells ed in increased SIVA/Stathmin 1 association (Fig. 3E), indicating that the presence of ANKHD1 inhibits the SIVA/Stathmin 1 interaction and con- Since previous studies have reported that ANKHD1 and/or its tributes to Stathmin 1 activation. Notably, in leukemia cells, we found ortholog protein mask (multiple ankyrin repeats single KH domain) is that ANKHD1 silencing presented a similar phenotype as previously de- involved in YAP1 (ortholog of Yorkie; yki) [6,24,30,31],SHP2(ortholog scribed for Stathmin 1 silencing with regard to cell proliferation, of Corkscrew; csw) [3,32] and JAK2 (ortholog of Hopscotch; hop) [33] clonogenicity, apoptosis [26–28] and microtubule dynamics (Fig. 3F). signaling, we verified the activation of these signaling pathways in leukemia cells submitted to ANKHD1 silencing. However, in U937 and 3.3. ANKHD1 does not modulate UV-induced apoptosis in leukemia cells Jurkat cells, we found that these signals are not activated, and not modulated by ANKHD1 silencing (Fig. 7). In agreement with recent studies Using a UV radiation-induced apoptosis model, ANKHD1 silencing did reported by our group and others, YAP1 is expressed at very low or un- not modulate apoptosis in U937 and Jurkat leukemia cells, nor the expres- detectable levels in leukemia cells [7,8], Taken together, we conclude sion of apoptosis-related proteins, BCL-XL, BAX, BCL2 and p-JNK (Fig. 4). A that the effects of ANKHD1 silencing on leukemia cell growth and significant reduction in the apoptotic response was observed in shSIVA clonogenicity are not associated with Hippo, SHP2 or JAK2/STAT signal- following UV radiation exposure, when compared to shControl cells in ing pathways.

Fig. 4. ANKHD1 does not modulate UV-induced apoptosis in leukemia cells. Apoptosis was detected by ﬂow cytometry in U937 and Jurkat cells transduced with shControl, shANKHD1 (A– B) and shSIVA cells (C–D) after UV radiation exposition (40 J/m2) using Annexin-V/PI staining method and a representative dot plot is illustrated. Bars indicate the relative number of (%) viable (Annexin-V−/PI−), apoptotic (Annexin-V+/PI−) or necrotic cells (Annexin-V+/PI+ plus Annexin-V−/PI+); and represents the mean of at least three independent experiments; *p b 0.05, **p b 0.01, ***p b 0.001; Student t test. Western blot analysis for the expression of BCL-XL, BAX, BCL2 and phospho-JNK (p-JNK) in total cell extracts from shControl, shANKHD1 (E) and shSIVA cells (F); membranes were reprobed with the antibody for detection of the respective total protein or actin, and developed with the ECL Western Blot Analysis System. The leukemia cell lines used are indicated. Western blot images are representative of at least three replicates. J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593 589 590 J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593

Fig. 5. ANKHD1, but not SIVA, modulates leukemia cell growth in vitro. Cell growth was determined by MTT assay at 48 h of incubation of shANKHD1 (A) or shSIVA cells (B) and normalized by the corresponding shControl cells. Results are shown as mean ± SD of six replicates and are representative of at least three independent experiments; *p b 0.05, **p b 0.01; Mann– Whitney test. Colonies containing viable cells were detected by MTT after 8 days of culture of shANKHD1 (C) or shSIVA cells (D) and normalized by the corresponding shControl cells. Re- sults are shown as mean ± SD of at least three independent experiments performed in triplicate; **p b 0.01, ***p b 0.001; Student t test.

4. Discussion understanding of the complex signaling network of the Stathmin 1 pathway in leukemia could add new insights in this field. To explore the possibility that the ANKHD1 protein plays a role in leu- We confirmed the inhibitory effect of SIVA on Stathmin 1 activation kemia processes, our aim was to identify cellular ANKHD1-interacting with a consequently increased cell migration of leukemia cells [15].In- proteins that could drive our investigation towards a specificsignaling terestingly, ANKHD1 had an opposite effect on Stathmin 1 and migra- pathway. Using an ankyrin-repeat domain of ANKHD1, we were able to tion, since ANKHD1 silencing resulted in Stathmin 1 inactivation, identify SIVA as a binding partner of ANKHD1. This finding prompted alpha-tubulin acetylation and reduced cell migration. Li et al. reported us to evaluate the role of ANKHD1 in important cellular processes that SIVA overexpression resulted in a similar phenotype to that ob- known to be modulated by SIVA; migration, apoptosis, and proliferation served by us following ANKHD1 silencing [15]. These opposing effects [34].Ourfirst hypothesis was that ANKHD1 could modulate apoptosis, as suggest that ANKHD1 binds to SIVA and inhibits its function in the SIVA has been well established as a proapoptotic protein [34,35]. Stathmin 1 pathway. These data are supported by our findings that, in We confirmed that SIVA silencing reduced irradiation-induced apoptosis U937 leukemia cells, ANKHD1 silencing enhances the SIVA/Stathmin 1 in U937 myeloid leukemia cells, downregulated the proapoptotic interaction and that the phenotype effect of ANKHD1 silencing on proteins p-JNK and BAX, and upregulated the anti-apoptotic protein in vivo tumor growth depends on the presence of SIVA. BCL-XL, as previously described in other cancer cell lines [12–14,36,37]. Although we observed an opposite effect of ANKHD1 and SIVA on However, ANKHD1 silencing did not change the percentage of apoptotic migration, Stathmin 1 pathway and xenograft tumor growth, we were cells nor the expression of apoptotic proteins. Our data are still limited to not able to document a potential role of SIVA on leukemia cell prolifer- enable us to justify why ANKHD1 and SIVA interact; however, results ation in vitro. The effect of Stathmin 1 on proliferation has been well de- suggest that only SIVA plays a role in the apoptotic process in vitro.It fined and low levels of Stathmin 1 stabilize the mitotic spindle, prevent is well recognized that leukemia cells have complex signaling path- mitosis and inhibit proliferation in tumors and leukemia cells [27,41, ways [2] and different partners of each protein may define specific 43]. Accordingly, we found that ANKHD1 silencing resulted in Stathmin functions. 1 inhibition and decreased in vitro leukemia cell proliferation and xeno- We then investigated a possible role for ANKHD1 in another signal- graft tumor growth. In Drosophila, recent studies also found that the ing pathway known to be modulated by SIVA. It has recently been re- orthologous protein of ANKHD1, mask, is essential for cell proliferation ported that SIVA1 suppresses the epithelial–mesenchymal transition and tissue growth [6,30], corroborating our findings in human cells. and metastasis of solid tumor cells by inhibiting Stathmin 1 and stabiliz- SIVA silencing resulted in increased in vivo tumor growth, but did not ing microtubules [15]. Stathmin 1 is a microtubule destabilizing protein modulate in vitro proliferation and colony formation. Two major points with a key role in cell cycle progression and cell migration that is up- need to be discussed here: i) First, why were the opposing effects of regulated in several cancers, including acute leukemia [16,38–40]. ANKHD1 and SIVA silencing observed only in vivo, but not in vitro as- Therefore, therapeutic modalities designed to interfere with Stathmin says? ii) Secondly, how do we justify our results regarding the effect 1 expression to inhibit cell proliferation, angiogenesis and cell migration of SIVA silencing on proliferation, compared to the work of Du et al. are of interest in several types of cancer [16,41–43], and a better [29]? J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593 591

Fig. 6. ANKHD1 and SIVA have opposing effects on xenograft tumor formation in NOD/SCID mice. Tumor measurement (volume and weight) at 12 days after subcutaneous injection of shControl, shANKHD1 (A), shSIVA (B), or both shANKHD1 and shSIVA cells (C) in female NOD/SCID mice. Tumor volume (V) was calculated using the formula (V=W2 × L × 0.52), where W and L are the smaller and larger diameters, respectively. Number of animals is indicated; *p b 0.05, **p b 0.01; Mann–Whitney test. (D) Potential model for the role of ANKHD1 and SIVA in leukemia cells; 1: SIVA is an adaptor protein that promotes Stathmin 1 phosphorylation and inactivation, and negatively regulates leukemia cell migration; 2: ANKHD1 binds to SIVA and leads to Stathmin 1 activation, tubulin depolymerization, increased cell migration and proliferation; 3: SIVA induces apoptosis by upregulation of the proapoptotic proteins BAX and p-JNK and downregulation of the anti-apoptotic protein BCL-XL. Abbreviations: Ac: acetylation; P: phosphorylation. Figure was produced using Servier Medical Art (http://www.servier.com/Powerpoint-image-bank).

A previous report showed that SIVA knockdown resulted in a signif- Supplementary data to this article can be found online at http://dx. icant reduction of in vitro and in vivo proliferation in cancer cells wild- doi.org/10.1016/j.bbamcr.2014.12.012. type for TP53, colon carcinoma (HCT116) and osteosarcoma (U2OS) cells, but not in TP53 null cancer cells [29].InAML,TP53 mutation is fre- Competing interests quent and it is associated with worse prognosis and survival [44]. All leukemia cell lines used in our studies present a TP53 mutation (inactive The authors declare that they have no competing interests. form) [45,46]. Thus, as indicated by Du et al., the effects of SIVA on proliferation and tumor growth vary according to the mutation status of Acknowledgments TP53. In the leukemia cells lacking the wild-type TP53, the effect of SIVA silencing on tumor growth may be related to increased resistance The authors would like to thank Dr Nicola Coran and Raquel S Foglio to apoptosis, or also due to Stathmin 1 activation. for English revision, Tereza Salles and Mariana O Baratti from In conclusion, we observed that ANKHD1 binds to SIVA and leads to Hemocentro-Unicamp, and Alexandre J C Quaresma and Jörg Kobarg Stathmin 1 activation, tubulin depolymerization, increased cell migra- from Laboratório Nacional de Biociências (LNBio) for their valuable tion and proliferation (Fig. 6D), possibly due to SIVA inhibition. technical assistance. We also thank the National Institute of Science ANKHD1 may be an oncogene and participate in the leukemia cell and Technology for Photonics Applied to Cell Biology (INFABIC) for phenotype. providing access to equipment and assistance. This work was supported 592 J.A. Machado-Neto et al. / Biochimica et Biophysica Acta 1853 (2015) 583–593

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