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The oncoprotein HBXIP up-regulates Skp2 via activating transcription factor E2F1 to promote proliferation of breast cancer cells Fuqiang Xu a,b,d,1 , Xiaona You a,1 , Fabao Liu c , Xiaohong Shen b , Yuanqing Yao d,, Lihong Ye c,, Xiaodong Zhang a,a Department of Cancer Research, Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, PR China b School of Medicine, Nankai University, Tianjin 300071, PR China c Department of Biochemistry, Nankai University, Tianjin 300071, PR China d Department of Gynecology and Obstetrics, PLA General Hospital, Beijing 100853, PR China article info Article history: Received 11 October 2012 Received in revised form 7 January 2013 Accepted 15 January 2013 Keywords: HBXIP Skp2 E2F1 Cell proliferation Breast cancer abstract Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein. In this study, we found that the expres- sion levels of HBXIP were positively associated with those of S-phase kinase-associated protein 2 (Skp2) in clinical breast cancer tissues and cell lines. Moreover, we found that HBXIP was able to stimulate the promoter of Skp2 through binding to the 640/443 region in Skp2 promoter involving activating E2F transcription factor 1 (E2F1). Skp2 plays crucial roles in HBXIP-enhanced proliferation of breast cancer cells in vitro and in vivo. We conclude that HBXIP up-regulates Skp2 via activating E2F1 to promote pro- liferation of breast cancer cells. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The hepatitis B virus X-interacting protein (HBXIP), encoding a 18 kDa protein, was originally identified by its interaction with the C-terminus of the hepatitis B virus X protein (HBx) and located at human chromosome 1 p13.3 [1]. HBXIP inhibited the apoptosis induced by HBx in hepotoma cells [2]. HBXIP could form complex with survivin, an anti-apoptotic protein that was overexpressed in most human cancers [1,3], resulting in the suppression of apopto- sis through the mitochondrial/cytochrome pathway. HBXIP was also required for bipolar spindle formation and was a regulator of centrosome dynamics and cytokinesis in cells [4]. Our previous studies reported that HBXIP could promote cell proliferation and migration through S100A4 and IL-8 [5,6]. However, the mechanism by which HBXIP enhances the proliferation of breast cancer cells remains unclear. S-phase kinase-associated protein 2 (Skp2) belongs to the fam- ily of the F-box proteins. It was originally discovered by Beach and colleagues in 1995, because of its ability to interact with the cell cycle protein cyclin A [7]. Skp2 contains the N-terminal domain, F-box domain, and C-terminal leucine-rich repeats (LRRs) [8]. The Skp2 protein levels changes during the cell cycle, which is low in early G 1 phase, while it is high during G 1 /S transition [9]. This alter- ation in the Skp2 protein level during cell cycle progression is partly due to a change in its gene expression and protein stability [10]. Co-transfection of cyclin E and Skp2 synergistically promoted cell cycle progression in cultured primary hepatocytes in the ab- sence of mitogen or in the presence of growth inhibitors. Further- more, transfection of hepatocytes in vivo with cyclin E and Skp2 promoted abundant hepatocyte replication and hyperplasia of the liver [11]. Subsequent experiments revealed that Skp2 was in- volved in cell cycle progression. Overexpression of Skp2 was fre- quently observed in numerous human cancers, such as colorectal, gastric, breast, prostate, lung, sarcoma, ovarian and other cancers 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.01.029 Abbreviations: HBXIP, hepatitis B X-interacting protein; Skp2, S-phase kinase- associated protein 2; ChIP, chromatin immunoprecipitation; Co-IP, co-immunopre- cipitation; EMSA, electrophoretic mobility shift assay; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; E2F1, E2F transcription factor 1; FCS, fetal calf serum; RT-PCR, reverse-transcription polymerase chain reaction; IHC, immu- nohistochemistry; siRNA, small interference RNA; DAB, 3,3 0 -diaminobenzidine. Corresponding authors. Addresses: Nankai University, 94 Weijn Road, Tianjin 300071, PR China. Fax: +86 22 23501385 (X. Zhang, L. Ye), The General Hospital of the People’s Liberation Army, No. 28 Fuxing Road, Beijing 100853, PR China. Fax: +86 10 66938043 (Y. Yao). E-mail addresses: [email protected] (Y. Yao), [email protected] (L. Ye), [email protected] (X. Zhang). 1 These authors contributed equally to the work. Cancer Letters 333 (2013) 124–132 Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet

The oncoprotein HBXIP up-regulates Skp2 via activating transcription factor E2F1 to promote proliferation of breast cancer cells

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Cancer Letters 333 (2013) 124–132

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Cancer Letters

journal homepage: www.elsevier .com/ locate/canlet

The oncoprotein HBXIP up-regulates Skp2 via activating transcriptionfactor E2F1 to promote proliferation of breast cancer cells

0304-3835/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.01.029

Abbreviations: HBXIP, hepatitis B X-interacting protein; Skp2, S-phase kinase-associated protein 2; ChIP, chromatin immunoprecipitation; Co-IP, co-immunopre-cipitation; EMSA, electrophoretic mobility shift assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; E2F1, E2F transcription factor 1; FCS, fetalcalf serum; RT-PCR, reverse-transcription polymerase chain reaction; IHC, immu-nohistochemistry; siRNA, small interference RNA; DAB, 3,30-diaminobenzidine.⇑ Corresponding authors. Addresses: Nankai University, 94 Weijn Road, Tianjin

300071, PR China. Fax: +86 22 23501385 (X. Zhang, L. Ye), The General Hospital ofthe People’s Liberation Army, No. 28 Fuxing Road, Beijing 100853, PR China. Fax:+86 10 66938043 (Y. Yao).

E-mail addresses: [email protected] (Y. Yao), [email protected] (L. Ye),[email protected] (X. Zhang).

1 These authors contributed equally to the work.

Fuqiang Xu a,b,d,1, Xiaona You a,1, Fabao Liu c, Xiaohong Shen b, Yuanqing Yao d,⇑, Lihong Ye c,⇑,Xiaodong Zhang a,⇑a Department of Cancer Research, Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, Institute for Molecular Biology, College of Life Sciences,Nankai University, Tianjin 300071, PR Chinab School of Medicine, Nankai University, Tianjin 300071, PR Chinac Department of Biochemistry, Nankai University, Tianjin 300071, PR Chinad Department of Gynecology and Obstetrics, PLA General Hospital, Beijing 100853, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 October 2012Received in revised form 7 January 2013Accepted 15 January 2013

Keywords:HBXIPSkp2E2F1Cell proliferationBreast cancer

Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein. In this study, we found that the expres-sion levels of HBXIP were positively associated with those of S-phase kinase-associated protein 2 (Skp2)in clinical breast cancer tissues and cell lines. Moreover, we found that HBXIP was able to stimulate thepromoter of Skp2 through binding to the �640/�443 region in Skp2 promoter involving activating E2Ftranscription factor 1 (E2F1). Skp2 plays crucial roles in HBXIP-enhanced proliferation of breast cancercells in vitro and in vivo. We conclude that HBXIP up-regulates Skp2 via activating E2F1 to promote pro-liferation of breast cancer cells.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The hepatitis B virus X-interacting protein (HBXIP), encoding a18 kDa protein, was originally identified by its interaction withthe C-terminus of the hepatitis B virus X protein (HBx) and locatedat human chromosome 1 p13.3 [1]. HBXIP inhibited the apoptosisinduced by HBx in hepotoma cells [2]. HBXIP could form complexwith survivin, an anti-apoptotic protein that was overexpressed inmost human cancers [1,3], resulting in the suppression of apopto-sis through the mitochondrial/cytochrome pathway. HBXIP wasalso required for bipolar spindle formation and was a regulator

of centrosome dynamics and cytokinesis in cells [4]. Our previousstudies reported that HBXIP could promote cell proliferation andmigration through S100A4 and IL-8 [5,6]. However, the mechanismby which HBXIP enhances the proliferation of breast cancer cellsremains unclear.

S-phase kinase-associated protein 2 (Skp2) belongs to the fam-ily of the F-box proteins. It was originally discovered by Beach andcolleagues in 1995, because of its ability to interact with the cellcycle protein cyclin A [7]. Skp2 contains the N-terminal domain,F-box domain, and C-terminal leucine-rich repeats (LRRs) [8]. TheSkp2 protein levels changes during the cell cycle, which is low inearly G1 phase, while it is high during G1/S transition [9]. This alter-ation in the Skp2 protein level during cell cycle progression ispartly due to a change in its gene expression and protein stability[10]. Co-transfection of cyclin E and Skp2 synergistically promotedcell cycle progression in cultured primary hepatocytes in the ab-sence of mitogen or in the presence of growth inhibitors. Further-more, transfection of hepatocytes in vivo with cyclin E and Skp2promoted abundant hepatocyte replication and hyperplasia ofthe liver [11]. Subsequent experiments revealed that Skp2 was in-volved in cell cycle progression. Overexpression of Skp2 was fre-quently observed in numerous human cancers, such as colorectal,gastric, breast, prostate, lung, sarcoma, ovarian and other cancers

F. Xu et al. / Cancer Letters 333 (2013) 124–132 125

[12–23]. These observations suggest that Skp2 may contribute tothe development of human cancers. Accumulated evidence sug-gests that Skp2 displays a proto-oncogenic role in vitro and in vivo.

In the present study, we try to gain insight into the effect ofHBXIP on regulation of Skp2 in promotion of proliferation of breastcancer cells. Our data indicate that HBXIP is able to up-regulateSkp2 through E2F1 in breast cancer cells, resulting in the promo-tion of cell proliferation. Our findings provide insights into themechanisms by which HBXIP enhances the proliferation of breastcancer cells.

2. Materials and methods

2.1. Immunohistochemistry (IHC)

Breast cancer tissue array (No. 08C14), comprising 49 breast tumors, was pur-chased from Xi’an Aomei Biotechnology (Xi’an, China). Immunohistochemistry as-say was performed as described previously [6]. The slides were incubated withrabbit anti-HBXIP (Proteintech Group, Chicago, USA) (or rabbit anti-Skp2, BosterGroup, Wuhan, China) antibody at 4 �C for overnight. After incubation at room tem-perature for 30 min with biotinylated secondary antibody, the slides were incu-bated with streptavidin-peroxidase complex at room temperature for 30 min.Immunostaining was developed by using chromogen, 3,30-diaminobenzidine(DAB), and counterstained with Mayer’s hematoxylin. The staining levels of HBXIPand Skp2 were classified into three groups using a modified scoring method basedon the intensity of staining (0 = negative; 1 = low; 2 = high) and the percentage ofstained cells (0 = 0% stained; 1 = 1–49% stained; 2 = 50–100% stained). A multipliedscore (intensity score � percentage score) lower than 1 was considered to be a neg-ative staining (�), 1 and 2 were considered to be moderate staining (+), and 4 wasconsidered to be intense staining (++).

2.2. Cell lines, cell culture and patient samples

Breast cancer cell lines, MCF-7, SK-BR-3, LM-MCF-7 (a metastatic subclone fromthe MCF-7 breast cancer cell line) [6], MCF-7-HBXIP (a stable HBXIP transfected cellline of MCF-7) cells [6] were cultured in RPMI 1640 medium (Gibco, Grand Island,NY), 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 lg/ml streptomycin inhumidified 5% CO2 at 37 �C. Thirty breast cancer tissues utilized in this study wereimmediately obtained from Tianjin Cancer Hospital (Tianjin, China) after surgicalresection. Written consents approving the use of their tissues for research purposesafter the operation were obtained from each patient. The study protocol was ap-proved by the institute research ethics committee at Nankai University (Tianjin,China).

2.3. Plasmid construction and small interference RNA

(siRNA) pCMV-tag2B, pGL3-Basic vectors (Promega, Madison, WI, USA), pCMV-HBXIP were kept in our laboratory. The complete human Skp2 (GenBank accessionNo. NC_000005.9) gene was subcloned into the pCMV-tag2B vector to generate thepCMV-Skp2 construct. The 50-flanking region (from �1309 to + 235 nt) of Skp2 genewas inserted into the KpnI/XhoI site in the upstream of the luciferase gene in thepGL3-Basic vector, termed pGL3-Skp2 promoter. Mutant construction of Skp2 pro-moter, termed as pGL3-Skp2 promoter mut, carried a series substitution of nucleo-tides within E2F1 binding site. siRNAs duplexes targeting human HBXIP (Skp2 orE2F1) gene and siRNA duplexes with non-specific sequences using as negative con-trol (NC) were synthesized by RiboBio (Guangzhou, China) [4,24,25]. All primersand siRNA sequence were listed in Table S1.

2.4. Transfection

One day before transfection, cells were collected, and seeded into 6-well, 24-well or 96-well plates. Cells were transfected with plasmid or siRNAs using lipofect-amine 2000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’sprotocol.

2.5. RNA extraction, RT-PCR and real-time PCR

Total RNA of cells (or breast cancer tissues from patient tumors) was extractedusing Trizol reagent (Invitrogen). First-strand cDNA was synthesized by PrimeScriptreverse transcriptase (TaKaRa Bio, Dalian, China) and oligo (dT) following the man-ufacturer’s instructions. To examine the mRNA levels of HBXIP and Skp2, real-timePCR was performed by a Bio-Rad sequence detection system using double-strandedDNA-specific SYBR Premix Ex TaqTM II Kit (TaKaRa Bio) according to the manufac-turer’s instructions. Double-stranded DNA specific expression was tested by thecomparative Ct method using 2�DDCt [26]. All primers were listed in Table S1.

2.6. Western blot analysis

Western blotting was carried out with standard protocols. Primary antibodiesused were rabbit anti-Skp2 (Boster Group), rabbit anti-E2F1 (Proteintech Group),rabbit anti-HBXIP (Proteintech Group), mouse anti-p27Kip1 (Boster Group), rabbitanti-p21WAP1 (Boster Group), rabbit anti-cyclin E (Boster Group), mouse anti-cyclinA (Boster Group), rabbit anti-cyclin D1 (Boster Group), rabbit anti-caspase 3 (CellSignaling Technology, Boston, USA) and mouse anti-b-actin (Sigma, Aldrich, St.Louis, MO, USA). All experiments were repeated 3 times.

2.7. Luciferase reporter gene assays

For luciferase reporter gene assays, the breast cancer cells were transfectedwith plasmids encoding HBXIP (or siRNAs of HBXIP and E2F1) by lipofectamine2000. The luciferase activities were determined 48 h after transfection, and the re-sults are the average of 3 independent repeats. The luciferase activities in the celllysates were measured by a dual luciferase reporter assay kit (Promega), and theluciferase activity was normalized with renilla luciferase activity.

2.8. Chromatin immunoprecipitation assay (ChIP assay)

The ChIP assay was performed using the EpiQuikTM chromatin immunoprecip-itation kit from Epigentek Group Inc according to the published methods [5,27].Protein-DNA complexes were immunoprecipitated with HBXIP antibodies, withmouse IgG as a negative control antibody. DNA collect by these antibodies was sub-jected to PCR analysis, followed by sequencing. Amplification of soluble chromatinprior to immunoprecipitation was used as an input control.

2.9. Co-immunoprecipitation (Co-IP)

MCF-7 cells (2 � 106) were harvested and lysed in a lysis buffer (50 mM Tris–HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.3% Triton X-100, 1 mM protease inhibitorPMSF). The lysates were incubated with anti-HBXIP or anti-E2F1 antibody and pro-tein G conjugated agarose beads at 4 �C for 3 h. The precipitates were washed sixtimes with ice-cold lysis buffer, resuspended in phosphate-buffered saline (PBS),and resolved by SDS–PAGE followed by western blot.

2.10. Electrophoretic mobility shift assay (EMSA)

Nuclear protein extracts were prepared from MCF-7 cells and MCF-7-HBXIPcells. Probes were generated by annealing single strand oligonucleotides containingthe Skp2 promoter and labeling the ends with [c-32P] ATP using T4 polynucleotidekinase (New England Biolabs, Ipswich, USA). Binding reactions were performed onice for 1 h, in 10 ll mixtures containing 20 mM Hepes, 50 mM KCl, 0.5 mM DTT,0.05 mM EDTA, 5% glycerol, 0.5 lg poly (dI�dC), 0.05% NP-40, 1 mM MgCl2, 50 ngprobe and 1.5 lg nuclear extracts. Specificity of HBXIP-DNA or Skp2-DNA interac-tion was confirmed by competition or supershift with HBXIP or E2F1 antibody,respectively. For the antibody competitive or supershift experiment, 1 lg IgG,HBXIP or E2F1 antibody was added into the reaction mixture and incubated onice for 30 min before the DNA probe was added. For the competitive binding exper-iment of cold competitor, 500 ng unlabeled DNA was added after the initial incuba-tion for additional 30 min. The binding mixtures were then resolved on a native 6%polyacrylamide gel in 0.5 � TBE at 4 �C. The gel was dried and exposed to X-ray filmfor autoradiography.

2.11. MTT assays

For quantitative proliferation assays, MCF-7-HBXIP cells were seeded onto 96well plates (1000 cells/well) for 24 h before transfection and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) assays were used to as-sess cell proliferation every day from the first day until the third day aftertransfection. The protocol was described previously [28].

2.12. Cloning formation assays

For clonogenicity analysis, 48 h after transfection, 1000 viable transfected cellswere placed in 6-well plates and maintained in complete medium for 1 week. Col-onies were fixed with methanol and stained with methylene blue.

2.13. Flow cytometry analysis

Forty-eight hour after transfection, the cells (1 � 106) were harvested andwashed with cold PBS twice. Washed cells were fixed in 75% ethanol at 4 �C over-night. The fixed cells were rinsed twice with PBS and treated with propidium iodine(PI) solution including 50 lg/ml PI (Sigma) and 50 lg/ml RNaseA (Sigma) at 37 �Cfor 60 min. Stained cells were analyzed by a FACScan flow cytometer (Becton Dick-inson, Bedford, Mass) [29].

Fig. 1. The expression levels of HBXIP are positively associated with those of Skp2 in clinical breast cancer tissues. (A) The expression of HBXIP and Skp2 was examined byimmunohistochemical staining in breast cancer tissues using tissue microarray. Panel a, negative control; Panel b, breast cancer tissues with HBXIP-positive staining; Panel c,breast cancer tissues with Skp2-positive staining. (B) The relative expression levels of HBXIP and Skp2 in 30 breast cancer tissues were detected by real time-PCR (p < 0.001,r = 0.6916, Pearson’s correlation).

126 F. Xu et al. / Cancer Letters 333 (2013) 124–132

2.14. Animal transplantation

Nude mice were housed and treated according to guidelines established by theNational Institutes of Health Guide for the Care and Use of Laboratory Animals. Weconducted the animal transplantation according to the Declaration of Helsinki. Tu-mor transplantation in nude mice was performed as previously described [5].Briefly, cells were harvested and re-suspended at 2 � 107 per ml with sterile PBS.Groups of 4-week-old female BALB/c athymic nude mice (Experiment Animal Cen-ter of Peking, China) (each group, n = 6) were subcutaneously injected at the but-tocks with 0.2 ml of the cell suspensions. Tumor growth was measured after10 days since injection and then every 3 days. Tumor volume (V) was monitoredby measuring the length (L) and width (W) with calipers and calculated with theformula (L �W2/2). After 25 days, tumor-bearing mice were sacrificed, and the tu-mors were excised and measured.

2.15. Statistical analysis

Each experiment was repeated at least three times. Statistical significance wasassessed by comparing mean values (±SD) using a Student’s t-test for independentgroups or pairing Chi-Square for dependent groups and was assumed for p < 0.05(�), p < 0.01 (��) and p < 0.001 (���).

Table 1Cross tabulation analysis of HBXIP and Skp2 in primary breast cancer tissues.

HBXIP

Total Negative Positive

Skp2Negative 9 2 (22.22%) 7 (77.78%)Positive 40 9 (22.50%) 31 (77.50%)Total 49 11 (22.45%) 38 (77.55%)

The expression of HBXIP and Skp2 was immunohistochemically examined by tissuearray with 49 clinical breast cancer tissues. Pairing Chi-Square was used in thisstatistics analysis by SPSS, p > 0.05.

3. Results

3.1. The expression levels of HBXIP are positively associated with thoseof Skp2 in clinical breast cancer tissues

Our previous report showed that 75% clinical breast cancer tis-sues were positive for HBXIP by IHC staining [6]. It has been re-ported that Skp2 is overexpressed in breast cancer tissues andcell lines [14]. Thus, we supposed that Skp2 might be correlatedwith HBXIP in breast cancer. Then, we try to investigate the expres-sion correlation between HBXIP and Skp2 by IHC using tissue ar-rays which are from the same tissue paraffin block. Our datashowed that the positive rate of HBXIP was 77.55% (38/49) in clin-ical breast cancer tissue samples, in which the positive rate of Skp2was 81.58% (31/38) in the HBXIP-positive specimens (Fig. 1A).Pairing Chi-Square analysis showed that there was no significant

difference between the positive rate of HBXIP and that of Skp2 inthe tissues (p > 0.05, Table 1, Table S2), suggesting that the expres-sion of Skp2 is closely associated with that of HBXIP in breast can-cer tissues. In addition, we examined the mRNA levels of HBXIPand Skp2 in 30 fresh breast cancer tissues by real time-PCR. The re-sults revealed that the expression levels of Skp2 were positively re-lated to those of HBXIP in the same tissues (Fig. 1B). Therefore, weconclude that the expression levels of HBXIP are positively associ-ated with those of Skp2 in breast cancer tissues.

3.2. HBXIP up-regulates the expression of Skp2 in breast cancer cells

Next, we evaluated whether HBXIP was able to up-regulateSkp2 in breast cancer cell lines. To demonstrate the effect of HBXIPon Skp2 promoter, we cloned the promoter region of Skp2 (�1309/+235) into the pGL3-Basic plasmid. Luciferase reporter gene assaysshowed that HBXIP could increase the promoter activities of Skp2in MCF-7 (or SK-BR-3) cells in a dose-dependent manner (Fig. 2Aand Fig. S1A). Then, we observed that the activities of Skp2 pro-moter were decreased in MCF-7-HBXIP cells transiently transfec-ted with HBXIP siRNAs in a dose-dependent manner (Fig. 2B),

Fig. 2. HBXIP up-regulates the expression of Skp2 in breast cancer cell lines. (A, B) MCF-7 (or MCF-7-HBXIP) cells were co-transfected with Renilla luciferase plasmidcontaining Skp2 promoter with either pCMV or pCMV-HBXIP, (or either NC, negative control, or si-HBXIP). The luciferase activities were determined 48 h after transfection.Statistically significant differences are indicated: �p < 0.05, ��p < 0.01, Student’s t-test. (C, D) MCF-7 (or MCF-7-HBXIP) cells were transfected with either pCMV or pCMV-HBXIP(either NC or si-HBXIP). The mRNA and protein expression levels of HBXIP and Skp2 were determined by RT-PCR and western blot, respectively.

F. Xu et al. / Cancer Letters 333 (2013) 124–132 127

suggesting that HBXIP is capable of activating the Skp2 promoter inthe cells. Furthermore, we demonstrated that Skp2 was up-regu-lated by HBXIP in MCF-7, MCF-7-HBXIP or LM-MCF-7 cell lines atthe levels of mRNA and protein in a dose-dependent manner(Fig. 2C and D, Fig. S1B and C). To further validate that HBXIP isable to up-regulate Skp2, we observed the effect of HBXIP onp27Kip1, p21WAP1, cyclin E, cyclin A and cyclin D1, the substratesof Skp2 [8,30]. As shown in Fig. S2A, the expression levels of cyclinE, cyclin A and cyclin D1 were increased when MCF-7 cells weretransfected with HBXIP plasmid, while the expression levels ofp27Kip1 and p21WAP1 were decreased in the cells. Meanwhile, theopposite results were observed when MCF-7-HBXIP cells weretreated with HBXIP siRNAs. Thus, we conclude that Skp2 is up-reg-ulated by HBXIP in breast cancer cells.

3.3. HBXIP activates Skp2 promoter via transcription factor E2F1

Next, we try to investigate the underlying mechanism by whichHBXIP activates Skp2 promoter. In this study, IHC staining showedthat the expression of HBXIP could be observed in both cytoplasm

and nucleus in breast cancer tissues. Thus, we speculated thatHBXIP might be involved in the transcriptional regulation ofSkp2. To map the HBXIP binding site in Skp2 promoter, we randomdesigned a series of primer of Skp2 promoter fragments in 50-flank-ing region, including the fragment �776/�567, �640/�443 and�464/�250. Interestingly, ChIP assays showed HBXIP was able tooccupy the Skp2 promoter fragment �640/�443 (Fig. 3A), suggest-ing that the �640/�443 region of Skp2 promoter is the regulatorytarget sequence of HBXIP. Then, we used online promoter analysistool Search Promoter Site (http://alggen.lsi.upc.es/cgi-bin/pro-mo_v3/promo/promoinit.cgi?dirDB = TF8.3) to predict the putativetranscription factor binding sites in the �640/�443 promoter re-gion of Skp2. Strikingly, we observed an E2F1 binding site in the re-gion. Next, we evaluated whether HBXIP was able to interact withthe transcription factor E2F1 by Co-IP. We found that the anti-HBXIP antibody was able to immunoprecipitate the E2F1 in thecells, while the anti-E2F1 antibody could immunoprecipitate theHBXIP as well (Fig. 3B), indicating that HBXIP is able to bind toE2F1 directly or indirectly. We further examined whether HBXIPwas able to bind to the E2F1 binding site in 640/�443 region of

Fig. 3. HBXIP activates Skp2 promoter via transcription factor E2F1. (A) The interaction between HBXIP and promoter region of Skp2 was examined by ChIP assay. (B) Theinteraction between HBXIP and E2F1 was determined by Co-IP in MCF-7 cells. (C) EMSA with the addition of anti-HBXIP or anti-E2F1 antibodies was performed to examinethe binding of nucleus proteins to Skp2 promoter fragment. Nuclear protein extracts were prepared from MCF-7 cells and MCF-7-HBXIP cells. (D) The promoter activities ofSkp2 mediated by HBXIP were measured by luciferase reporter gene assay when MCF-7-HBXIP cells were transfected with E2F1 siRNAs. (E) The interference efficiency of E2F1was detected by western blot. (F) The promoter activities of Skp2 mediated by HBXIP were measured by luciferase reporter gene assay when the E2F1 binding site wasmutated. Statistically significant differences are indicated: �p < 0.05, ��p < 0.01, Student’s t-test.

128 F. Xu et al. / Cancer Letters 333 (2013) 124–132

Skp2 promoter by EMSA. Indeed, we observed an obvious interac-tion between the probe and proteins of nuclear extracts (Fig. 3Clane 2), which could be blocked by anti-HBXIP antibody (Fig. 3Clane 4), suggesting that HBXIP is able to bind to the segment. Wealso observed the interaction between the probe and proteins ofnuclear extracts could be blocked by anti-E2F1 antibody (Fig. 3Clane 5), suggesting that the transcription factor E2F1 is responsiblefor the HBXIP-DNA interaction. EMSA indicated that HBXIP couldinteract with Skp2 promoter through E2F1. Therefore, we pre-sumed that HBXIP may activate Skp2 promoter through E2F1.Luciferase reporter gene assays showed that the activities ofSkp2 promoter were decreased by E2F1 siRNAs in MCF-7-HBXIPcells in a dose-dependent manner (Fig. 3D), suggesting that HBXIPactivates the Skp2 promoter via transcription factor E2F1. Asshown in Fig. 3E, the RNA interference efficiency of E2F1 was con-firmed by western blot analysis. Meanwhile, HBXIP failed to workwhen the E2F1 binding site in Skp2 promoter was mutated byluciferase reporter gene assays (Fig. 3F). In addition, we observedthat the mRNA and protein levels of E2F1 were up-regulated intransiently HBXIP-transfected cell lines (MCF-7) and down-regu-lated in transiently HBXIP knockdown cell lines (MCF-7-HBXIP)in a dose-dependent manner (Fig. S3A and B), suggesting thatHBXIP is able to up-regulate E2F1 in breast cancer cells. Thus, weconclude that HBXIP activates Skp2 promoter activity throughbinding to the transcription factor E2F1.

3.4. Skp2 is responsible for HBXIP-enhanced proliferation of breastcancer cells in vitro

MTT assays showed that the treatment with Skp2 siRNAs abol-ished the enhanced proliferation of MCF-7-HBXIP cells mediatedby HBXIP in a dose dependent manner (Fig. 4A). Furthermore, wefound that HBXIP siRNAs decreased the proliferation of MCF-7-HBXIP cells in a time dependent manner, while the over-expres-sion of Skp2 was able to rescue the inhibition (Fig. 4B), suggestingthat Skp2 is involved in the HBXIP-enhanced proliferation of breastcancer cells. Then, we performed the clone formation assay toidentify the effect of Skp2 on HBXIP-enhanced proliferation ofbreast cancer cells. The results showed that the proliferation ofMCF-7-HBXIP cells could be inhibited by Skp2 siRNAs. HBXIP siR-NAs significantly decreased the proliferation of MCF-7-HBXIP cells,whereas the over-expression of Skp2 rescued the inhibition in-duced by HBXIP siRNAs (Fig. 4C). We are interested in the effectof HBXIP on cell cycle because Skp2 is involved in the regulationof cell cycle [31]. Then, the cell cycle was analyzed by flow cytom-etry analysis in MCF-7-HBXIP and LM-MCF-7 cell lines. As shownin Fig. 4D, our data displayed that Skp2 siRNAs resulted in thedecrease of S-phase MCF-7-HBXIP cells from 28.29% to 20.63% (orLM-MCF-7 cells from 30.21% to 17.93%, Fig. S4). While, the over-expression of Skp2 was capable of rescuing the inhibition ofS-phase cells mediated by HBXIP siRNAs, such as from 24.23% to

Fig. 4. Skp2 is responsible for HBXIP-enhanced proliferation of breast cancer cells in vitro. (A–D) MCF-7-HBXIP cells were transfected with NC, si-Skp2, si-HBXIP, or si-HBXIPand pCMV-Skp2. The cell proliferation was determined by MTT, colony formation assay and flow cytometry analysis, respectively. The images are representative of at leastthree independent experiments. Statistically significant differences are indicated: �p < 0.05, ��p < 0.01, ���p < 0.001, Student’s t-test.

F. Xu et al. / Cancer Letters 333 (2013) 124–132 129

32.21% in MCF-7-HBXIP cells (or from 21.87% to 34.36% in LM-MCF-7 cells, Fig. S4). Our data suggest that HBXIP promotes theproliferation of breast cancer cells through regulating cell cycleS-phase involving Skp2.

3.5. HBXIP promotes the tumor growth through Skp2 in vivo

To further validate the effect of HBXIP on tumor growth throughSkp2 in vivo, we performed tumor formation assay. We observedthat the volume and weight of tumors were highly enhanced inMCF-7-HBXIP group compared with MCF-7-pCMV control group(data not shown), whereas the Skp2 knockdown group displayeda significant decreased tumor growth (�p < 0.05, ��p < 0.01, Stu-dent’s t-test, Fig. 5A–C). Meanwhile, western blot analysis con-firmed that the expression levels of Skp2 were decreased by Skp2siRNAs in the tumor tissues from mice (Fig. 5D). Thus, we conclude

that HBXIP is capable of promoting growth of breast cancer cellsthrough Skp2 in vivo.

4. Discussions

Our studies show that HBXIP is a novel oncoprotein. HBXIP washighly expressed in breast cancer tissues and metastatic lymphnode tissues and significantly associated with the growth andmetastasis of breast cancer cells [6,32]. However, the underlyingmechanism is poorly understood. Skp2 has an established role intumors. Many studies have shown that the over-expression ofSkp2 is observed in a variety of human cancers, including colorec-tal cancer, gastric cancer, breast cancer, prostate cancer, sarcoma,ovarian cancer, lung cancer, pancreatic cancer and other cancers[12–23]. Therefore, we are interested in whether Skp2 is involvedin HBXIP-enhanced proliferation of breast cancer cells.

Fig. 5. HBXIP promotes growth of breast cells via Skp2 in vivo. (A) The growth curve of the tumors from mice transplanted with MCF-7-HBXIP cells treated with Skp2 siRNAsor control siRNAs. (B) The average weight of tumors. (C) The image of tumors. (D) The relative expression levels of Skp2 or HBXIP were detected by western blot analysis in thetumor tissues from mice, respectively. Statistically significant differences are indicated: �p < 0.05, ��p < 0.01, Student’s t-test.

130 F. Xu et al. / Cancer Letters 333 (2013) 124–132

In this study, we observed that the expression levels of HBXIPwere significantly correlated with those of Skp2 in breast cancertissues. Moreover, we identified that HBXIP was able to up-regu-late Skp2 in breast cancer cells. It has been reported that Skp2mediates the regulation of some cell cycle regulatory proteins thatmay contribute to cancer progression, including p27Kip1, p57Kip2,p21WAP1, cyclin E, cyclin A and cyclin D1 [8,30,33]. Skp2 is impor-tant for cell to enter S-phase. The Skp2 protein levels changes dur-ing the cell cycle, which is low in early G1 phase, while it is highduring G1/S transition [9]. In our study, we found that HBXIP wasable to increase the levels of cyclins (such as cyclin E, cyclin Aand cyclin D1) and decreased the levels of CDKIs (such as p27Kip1

and p21WAP1) in breast cancer cells. Therefore, it further supportsthat HBXIP is able to up-regulate Skp2 in the cells. It has been re-ported that Skp2 overexpression is correlated with tumor progres-sion such as stage and recurrence in human cancers [20], indicatingthat Skp2 may be important in cancer cell migration, invasion, andmetastasis. Accumulating evidence shows that Skp2 promotes can-cer cell growth. A study from Wan’s group has also shown thatoverexpression of Skp2 enhanced cell proliferation in normalbreast cell line MCF10A, while depletion of Skp2 reduced cellulargrowth in breast cancer cell line [34]. And other studies also foundthe similar results [35]. Accordingly, we sought to elucidate the

underlying mechanism by which HBXIP up-regulates Skp2. Wepreviously observed the nuclear localization of HBXIP in MCF-7cells [5], implying that HBXIP may be involved in the transcrip-tional regulation of Skp2.

Then, we predicted the putative transcription factor bindingsites in the �640/�443 promoter region of Skp2. Strikingly, we ob-served that there was an E2F1 binding site in the region. E2F1 isover-expressed in many cancers, such as breast cancer, ovariancancer, liver cancer, lung cancer and gastric cancer [36,37]. More-over, E2F1 protein stimulates tumor cell proliferation, and is inver-sely correlated with cancer patient’s survival [38–40].Interestingly, we found that HBXIP was able to bind to the Skp2promoter region which contains E2F1 binding site through E2F1transcription factor by ChIP, Co-IP and EMSA assays. We furtherdemonstrated that HBXIP activated Skp2 promoter through thetranscription factor E2F1, suggesting that HBXIP activates theSkp2 through binding to E2F1. In addition, we found that HBXIPwas able to up-regulate the expression of E2F1 (Fig. S3). Thus,we first report that the transcription factor E2F1 plays a role in reg-ulating Skp2 mediated by HBXIP in breast cancer cells. Our previ-ous report showed that HBXIP could up-regulate S100A4 and IL-8to promote the proliferation and migration of breast cancer cellsvia two pathways [5,6]. Consistent with above reports, our finding

F. Xu et al. / Cancer Letters 333 (2013) 124–132 131

suggests that HBXIP may serve as a co-activator of transcriptionfactors in breast cancer cells.

In function, we provided evidence that HBXIP promoted theproliferation of breast cancer cells via up-regulating of Skp2in vitro and in vivo. Interestingly, we found that HBXIP was ableto promote cells to enter into S-phase through Skp2. Meanwhile,we observed that HBXIP was able to up-regulate cyclin A, cyclinE and cyclin D1 in MCF-7 cells, and down-regulate p27Kip1 andp21WAP1 in MCF-7-HBXIP cells, which is consistent with abovefinding. In addition, it has been reported that Skp2 is involved inapoptosis [31]. Therefore, we examined the effect of HBXIP on cas-pase 3, a hallmark of apoptosis, in breast cancer cells. Our datademonstrated that caspase 3 was down-regulated by over-express-ing HBXIP in MCF-7 cells, and was up-regulated in MCF-7-HBXIPcells treated with HBXIP siRNAs (Fig. S2B), suggesting that HBXIPmay suppress apoptosis through Skp2. Accordingly, our data areconsistent with the report that the elevated Skp2 protein levelassociates with the progression of breast cancer [14].

In summary, our finding indicates that HBXIP promotes the pro-liferation of breast cancer cells, resulting in the increase of S-phasecells, through up-regulating Skp2, in which HBXIP activates thetranscription of Skp2 through interaction with transcription factorE2F1. Therefore, our finding provides new insight into the mecha-nism of HBXIP in promotion of growth of breast cancer cells. HBXIPmay serve as a target for the therapy of breast cancer.

Acknowledgements

This work was supported by grants from the National Basic Re-search Program of China (973 Program, Nos. 2011CB512113, and2009CB521702) and National Natural Science Foundation of China(Nos. 81071623, 81071624 and 81272217).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.canlet.2013.01.029.

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