(2012) 31, 1034–1044 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Identification of oncogenic microRNA-17-92/ZBTB4/specificity axis in breast

K Kim1, G Chadalapaka2, S-O Lee1, D Yamada3, X Sastre-Garau4, P-A Defossez3, Y-Y Park5, J-S Lee5 and S Safe1,2

1Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USA; 2Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA; 3CNRS UMR7216, Universite´ Paris 7, Baˆtiment Lamarck, Paris, France; 4Tumor Biology Department, Institut Curie, Paris, France and 5Department of Systems Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The human POK family members are Introduction factors with a POZ domain and zinc-fingers that act primarily as transcriptional repressors. Several members Transcription factors of the POK family are character- of this family are involved in oncogenesis and this ized by an N-terminal BTB/POZ domain, and a variable prompted us to assess whether expression levels of number of Kru¨ppel-like zinc-fingers (Perez-Torrado individual POK family members are associated with et al., 2006; Costoya, 2007). The encodes clinical outcomes in cancer. We have observed that over 40 members of this family, and several have ZBTB4 (zinc-finger and BTB domain containing 4) is causative roles in cancer (Perez-Torrado et al., 2006). downregulated in patients, and that its (also known as Pokemon, LRF or FBI-1) expression is significantly correlated with relapse-free controls cellular transformation (Pendergrast et al., survival. Further integrative analysis of mRNA and 2002; Maeda et al., 2005), and ZBTB29 (also known microRNA (miR) expression data from the NCI-60 cell as HIC-1) is a effector lost in many neuroblastomas lines revealed an inverse correlation between ZBTB4 and (Chen et al., 2003, 2005). ZBTB27 (also known as oncogenic miRs derived from the miR-17-92 cluster and BCL6) regulates lymphocyte survival and is crucially its paralogs. The experimental results using MDA-MB- involved in B-cell lymphoma (Kojima et al., 2001; 231 and MCF-7 human breast cancer cells confirm that Niu, 2002; Cattoretti et al., 2005). The functions of miRNAs derived from these clusters, containing miR-17- many other ZBTB are characterized poorly or 5p, miR-20a, miR-106a, miR-106b and miR-93, nega- not at all; this prompted us to examine whether the tively regulate ZBTB4 expression. Overexpression of expression of ZBTB is correlated with clinical ZBTB4 or restoration of ZBTB4 by using an antagomir outcome of cancer patients to identify new prognostic inhibit growth and invasion of breast cancer cells, and this and functional roles of ZBTB proteins in cancer. Here effect is due, in part, to ZBTB4-dependent repression of we report that ZBTB4 (zinc-finger and BTB domain the specificity protein 1 (Sp1), Sp3 and Sp4 genes, and containing 4) protein and mRNA levels are down- subsequent downregulation of several Sp-dependent onco- regulated in breast cancer patients and that its expres- genes, in part, through competition between ZBTB4 and sion has powerful predictive value for relapse-free Sp transcription factors for GC-rich promoter sequences. survival from this disease. These results confirm that ZBTB4 functions as a novel (miRs) exhibit sequence-specific interac- tumor-suppressor with prognostic significance for tions with 30-untranslated regions (UTRs) of mRNAs to breast cancer survival, and the oncogenic miR-17-92/ regulate their stability and translation and are crucial ZBTB4/Sp axis may be a potential therapeutic target. regulators of the cellular identity. Not surprisingly, Oncogene (2012) 31, 1034–1044; doi:10.1038/onc.2011.296; different miRs exhibit tumor-suppressive and oncogenic published online 18 July 2011 activities and these can be tumor context dependent (Volinia et al., 2006; Liu et al., 2010). Interaction of Keywords: ZBTB4; miR-17-92 cluster; breast cancer; miRs with 30-UTRs results in translational repression, prognostic; Sp transcription factors and POK family genes often have long and well- conserved 30-UTRs, suggesting their potential regulation by miRs, and our previous studies have demonstrated that ZBTB10 expression is suppressed by miR-27a in several cancer cell lines (Mertens-Talcott et al., 2007; Correspondence: Dr S Safe, Department of Veterinary Physiology and Jutooru et al., 2010). This led us to hypothesize that Pharmacology, Texas A&M University, 4466 TAMU, Veterinary ZBTB4 expression may also be regulated by miRs in Research Building 410, College Station, TX 77843-4466, USA. E-mail: [email protected] cancer cell lines and this possibility has been addressed Received 14 March 2011; revised 2 June 2011; accepted 7 June 2011; using publicly available mRNA and miR data sets. published online 18 July 2011 Analyses of these data indicate that ZBTB4 can be a ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1035 potential target of multiple miRs derived from the Regulation of ZBTB4 by miR-17-92 components of the miR-17-92 cluster and its paralogs. To identify miRs that can potentially negatively regulate POK proteins generally function as sequence-specific ZBTB4, the publicly available NCI-60 cell line mRNA transcriptional repressors (Tillotson, 1999; Filion et al., and miR expression profiling data sets were employed 2006; Jeon et al., 2008, 2009; Weber et al., 2008; Koh and the correlation between miR and mRNA expression et al., 2009; Sasai et al., 2010), and ZBTB4 binds several was analyzed by Pearson’s R correlation test (Figure 1c) types of genomic target sites, including certain GC-rich (Blower et al., 2007; Shankavaram et al., 2007). The sequences that bind specificity protein 1 (Sp1) (Filion results were then further integrated into the prediction et al., 2006; Weber et al., 2008; Sasai et al., 2010). Sp1, outcome from software algorithm programs of PicTar Sp3 and Sp4 are transcriptional activators that regulate (Krek et al., 2005) and TargetScan (Lewis et al., 2003) a number of prosurvival and proinvasion genes in breast and indicated that the ZBTB4 30-UTR can be poten- cancer cells (Abdelrahim et al., 2002, 2004, 2007; tially targeted by miRs derived from miR-17-92 cluster Chadalapaka et al., 2008, 2010; Jutooru et al., 2010). such as miR-17-5p (R ¼À0.582, Po0.00001) and miR- We posited that ZBTB4 might interfere with the 20a (R ¼À0.642, Po0.00001; Figure 1d and Supple- function of Sp1, Sp3 and Sp4 in breast cells and our mentary Table S2). As the miR-17-92 cluster and its results show that ZBTB4 indeed negatively regulates the paralogs contain previously described oncogenic miRs expression of Sp1, Sp3 and Sp4, and some of their and are frequently overexpressed in breast cancer and important target genes such as vascular endothelial metastatic breast cancer cells (Volinia et al., 2006; growth factor (VEGF), VEGF 1 (VEGFR1) Blenkiron et al., 2007), we focused on the roles of these and survivin (Abdelrahim et al., 2002, 2004, 2007; oncogenic miRs in regulating ZBTB4 expression. Chadalapaka et al., 2008, 2010; Jutooru et al., 2010). The 30-UTR of ZBTB4 has sequences that can The mechanism of ZBTB4-dependent repression of Sp1, interact with miR-17-5p and miR-20a, both of which Sp3, Sp4 and Sp-regulated genes involves competition have identical seed sequences (AAAGUG; Figures 2a between ZBTB4 and Sp proteins for binding to GC-rich and b). In cells transfected with 30-UTR-ZBTB4-luc, promoter sequences. luciferase activity was decreased after co-transfection Our results identify ZBTB4 as a new and important with an increasing amount of miR-17-5p or miR-20a prognostic factor in human breast cancer that is regulated mimics (Figure 2a) and this was also accompanied by by oncogenic miR-17-92 cluster and its paralogs. Re- decreased expression of ZBTB4. In contrast, transfec- storation of ZBTB4 by inhibiting expression of oncogenic tion of as-miR-17-5p or as-miR-20a increased activity miRs derived from miR-17-92 cluster and its paralogs (Figure 2b). Similar results were observed with antisense suppresses expression of Sp1, Sp3, and Sp4 and Sp- and mimics of miR-106a, miR-106b and miR-93 that are regulated pro-oncogenic gene products, resulting in paralogs (identical seed sequences) of miR-20a/miR17- inhibition of cancer cell proliferation and invasion. 5p; however, miR-27a did not affect activity (Supple- mentary Figure S3). In cells transfected with a 30-UTR- luc construct containing a wild-type (WT) or mutated Results (MT) core sequence (1836–1886 bp MT), the same miR mimics affected luciferase activity only in cells trans- Prognostic significance and expression of ZBTB4 fected with the wild-type construct (Figure 2c). As the in breast cancer patients miR-20a/miR-17-5p ratio was >8 in MCF-7 and MDA- The potential prognostic and functional roles of ZBTB MB-231 cells (data not shown), we used miR-20a as a transcription factors in breast cancer was examined by model. Transfection of as-miR-20a into MCF-7 or Kaplan-Meier survival analysis of two publicly available MDA-MB-231 cells induced ZBTB4 but not ZBTB10 breast cancer patient data sets (van de Vijver et al., 2002; mRNA, and this was accompanied by increased Oh et al., 2006) and, among this group of genes, only expression of the ZBTB4 protein (Figure 2d). ZBTB4 exhibited consistent clinical correlations in these These results establish that oncogenic miR-20a and two independent data sets. High ZBTB4 expression miR-17-5p negatively regulate ZBTB4 expression in correlated with longer relapse-free survival, whereas low breast cancer cells. ZBTB4 expression correlated with shorter relapse-free survival in two independent breast cancer patient cohorts (The Netherlands Cancer Institute (NKI) cohort ZBTB4 represses Sp (n ¼ 295); University of North Carolina (UNC) cohort ZBTB4 represses p21 expression in cancer cells (Weber (n ¼ 380); Figure 1a). Survival differences were not et al., 2008) and, in this study, the potential role of this observed for other ZBTB mRNAs (for example, gene as a repressor of Sp1, Sp3, Sp4 and Sp-regulated ZBTB2, ZBTB5, ZBTB7, ZBTB10, ZBTB29, ZBTB33 genes was investigated in breast cancer cells. Figure 3a and ZBTB38) expressed in these patients (Supplemen- illustrates that overexpression of ZBTB4 in MCF-7 and tary Figure S1). Expression of ZBTB4 protein was also MDA-MB-231 cells significantly decreased Sp1, Sp3 and determined in breast tumors and nontumor tissues Sp4 mRNA levels, and similar results were observed in (Figure 1b, Supplementary Table S1 and Supplementary these cells transfected with as-miR-20a (Figure 3b). Figure S2) and there was consistently increased ZBTB4 Both the Sp1 and Sp3 promoters contain GC-rich Sp- protein staining in nontumor vs tumor tissue from the binding sites (Nicolas et al., 2001; Tapias et al., 2008) same patients. and, in MCF-7 and MDA-MB-231 cells transfected with

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1036 513 798 514 199

NKI (N =295) UNC (N =380) ZBTB4 high ZBTB4 high N =147, 32 relapse N =190, 46 relapse 511 661 513 764

517 732 511 632

ZBTB4 low ZBTB4 low 514 782 514 834 N =148, 69 relapse N =190, 71 relapse p =1.81e-06 p =0.02

515 134 520 565 Relapse Free Survival Relapse Free Survival

Prediction Algorithm miRNAs highly correlated Analysis with ZBTB4 expression

miR-150 miR-20a miR-301 miR-96 miR-17-5p miR-133 miR-101 miR-106a miR-206 miR-32 miR-181 miR-106b miR-137 miR-93

Frequently overexpressed in cancer Figure 1 Identification of ZBTB4 as miR-17-5p/miR-20a target. (a) Breast cancer patients in the NKI (N ¼ 295) and UNC (N ¼ 380) cohorts, and lung cancer patients (N ¼ 149, GSE11969) were dichotomized by ZBTB4 expression level. Patients were divided into two groups, high and low, by median ZBTB4 expression values. (b) Immunostaining of ZBTB4 protein expression in nontumor (left) and tumor (right) tissue of breast cancer patients with invasive ductal carcinoma (also see Supplementary Table 1 and Supplementary Figure 1). (c) ZBTB4 expression is inversely correlated with multiple miRs from miR-17-92 cluster and its paralogs. Each cell in the heat map represents expression level of each gene. The red and green colors in cells stand for relatively high and low expression levels in log2 transformed scale. (d) Venn diagram displaying common set of miRs by integrating the predicted miRs targeting ZBTB4 by algorithm programs into miRs highly correlated with ZBTB4 expression by Pearson’s correlation analysis using NCI-60 miR and mRNA data sets. A full colour version of this figure is available at the Oncogene journal online.

pSp1For4 (À751 to À20 Sp1 promoter insert; Figure 3c) Confirmation that ZBTB4 repressed Sp mRNA levels or pSp3For5 (À417 to À38 Sp3 promoter; Figure 3d), in MCF-7 cells was determined by transfecting small co-transfection with ZBTB4 or as-miR-20a decreased interfering RNAs (siRNAs) against ZBTB4 (siZBTB4 I luciferase activity. Similar results were observed in and siZBTB4 II). Both siRNAs increased expression of MDA-MB-231 and MCF-7 cells transfected with a Sp1, Sp3 and Sp4 mRNA and proteins in MCF-7 cells GC-rich construct containing the À133 to þ 54 region (Supplementary Figures S5A and B), but not in MDA- of the VEGF promoter (Supplementary Figure S4). MB-231 cells that express very low endogenous levels of

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1037

Mimic CT miR-17-5p miR-20a Position 1859-1865

ZBTB4 ZBTB4 3'-UTR 5' - ...CAAAUCUUGUUUCUGGCACUUUA... Actin miR-20a 3' - GAUGGACGUGAUAUUCGUGAAAU

MCF-7 MCF-7 MDA-MB-231 ZBTB4 3'-UTR (1-2559 bp) ZBTB4 3'-UTR (1-2559 bp) ZBTB4 3'-UTR (1-2559 bp) 1.2 2.5 3 * 1 * 2 2.5 ** * 0.8 ** ** 2 1.5 * 0.6 1.5 RLU RLU RLU ** 1 0.4 1 0.5 0.2 0.5

0 0 0 miR-17-5p −++ +− − − As-miR-17-5p −+− As-miR-17-5p −+− miR-20a −− − − ++ + As-miR-20a −−+ As-miR-20a −−+

MCF-7 MCF-7 MDA-MB-231 1-2559 1836-1886 1836-1886 ZBTB4 bp bp bp 3'-UTR Full length WT MT As-CT As-miR-20a As-CT As-miR-20a ZBTB4 1.4 ZBTB4 Actin Actin 1.2 3 ZBTB4 1 * 3 ZBTB4 * 2.5 ZBTB10 ZBTB10 ** 2.5 0.8 ** ** ** 2 2 RLU 0.6 1.5 1.5 0.4 1 1 0.2 0.5 0.5 Relative mRNA amount 0 0 Relative mRNA amount 0 miR-17-5p −+−−+− −+− As-CT As-miR-20a As-CT As-miR-20a miR-20a −−+−−+ −−+ Figure 2 MiR-20a/miR-17-5p-dependent regulation of ZBTB4. Effects of miR-20a and miR-17-5p and their antagomirs on interactions with wild-type ZBTB4-30-UTR (a, b) and mutant (c) constructs. MCF-7 or MDA-MB-231 cells were transfected with miR- 20a and miR-17-5p mimics or antagomirs (100 nM) and wild-type or mutant constructs containing ZBTB4 30-UTR inserts linked to the luciferase gene, and after 48 h, luciferase activity was determined as described in the Materials and methods. (d) As-miR-20a induces ZBTB4 expression. MCF-7 or MDA-MB-231 cells were transfected with as-miR-20a (150 nM), and after 48 h, the expression of ZBTB4 protein and mRNA was determined as described in the Materials and methods. Results are means±s.e. (triplicate) and significant (Po0.05) induction (*) or inhibition (**) is indicated.

ZBTB4 (data not shown). The ZBTB4 siRNAs in- cells with as-miR-20a/as-Ctl, we observed binding of creased luciferase activity in MCF-7 and MDA-MB-231 Sp1 and pol II in cells transfected with as-Ctl, whereas cells transfected with pSp1For4 (Supplementary Figure as-miR-20a decreased Sp1 and pol II binding but S5C) and pSp3For5 (Supplementary Figure S5D), increased ZBTB4 binding to this region (Figures 4c confirming that ZBTB4 suppresses Sp1, Sp3 and Sp4 and d). The results demonstrate that ZBTB4 competi- mRNA expression in breast cancer cells. tively binds at GC-rich sites to displace Sp1 and thereby The mechanism of ZBTB4-dependent repression of decreases transactivation. Sp transcription factors was investigated using in vitro translated ZBTB4 and rSp1 in electrophoretic mobility shift assays. Incubation of a consensus GC-rich ZBTB4 and as-miR-27a repress Sp1, Sp3, Sp4 and oligonucleotide shows that ZBTB4 binds directly to Sp-regulated gene products the sequence, and binding is repressed after co-incuba- The effect of as-miR-20a and ZBTB4 on Sp1, Sp3 and tion with excess GC-rich oligonucleotide (Figure 4a). Sp4 protein expression was investigated in MDA-MB- Moreover, using the same electrophoretic mobility shift 231 and MCF-7 cells. Transfection of a ZBTB4 assay approach, the intensity of the rSp1-DNA complex expression plasmid (Figure 5a) or as-miR-20 is decreased after co-incubation with ZBTB4 or excess (Figure 5b) in MCF-7 and MDA-MB-231 cells down- GC-rich oligonucleotide (Figure 4b). Results of a regulated expression of Sp1, Sp3 and Sp4 proteins, and immunoprecipitation (ChIP) scanning analy- this was accompanied by downregulation of several Sp- sis (À2.5 to þ 2.0 kb) over the GC-rich region of the Sp1 dependent genes including VEGF, VEGFR1 and gene promoter showed an enhanced peak at À626 to survivin and enhanced poly(ADP-ribose) polymerase À386 (GC rich; Figure 4c). After transfection of MCF-7 (PARP) cleavage (Figure 5c). Moreover, RNA inter-

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1038 MCF-7 MDA-MB-231 MCF-7 MDA-MB-231 1.5 1.5 Empty 1.5 Empty 1.5 As-CT As-CT ZBTB4 ZBTB4 As-miR-20a As-miR-20a 1 1 1 1 ** ** ** ** ** ** ** ** ** ** ** 0.5 0.5 ** 0.5 0.5

0 0 0 0 Relative mRNA amount Sp1 Sp3 Sp4 Sp1 Sp3 Sp4 Relative mRNA amount Sp1 Sp3 Sp4 Sp1 Sp3 Sp4

pSp1For4-751 Luc =GC rich

MCF-7 MDA-MB-231 MCF-7 MDA-MB-231 1.2 1.2 1.2 1.2 1 1 1 1 ** 0.8 ** 0.8 0.8 ** 0.8 ** 0.6 0.6 0.6 0.6 RLU ** ** RLU ** 0.4 0.4 0.4 ** 0.4 0.2 0.2 0.2 0.2 0 0 0 0 ZBTB4 (g) 012 012 asMir-20a (nM) 0 0 100 150 100 150

pSp3For 5-417 Luc =GC rich

MCF-7 MDA-MB-231 MCF-7 MDA-MB-231 1.2 1.2 1.2 1.2

1 1 1 ** 1 ** 0.8 0.8 0.8 0.8 ** 0.6 0.6 0.6 ** RLU 0.6 ** **

RLU ** 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0 0 0 0 ZBTB4 (g) 012 012 asMir-20a (nM) 0 0 100 150 100 150 Figure 3 ZBTB4 and as-miR-20a suppress Sp1, Sp3 and Sp4 transcription. Decreased mRNA levels in cells transfected with ZBTB4 (a) or as-miR-20a (b). MCF-7 cells were transfected with ZBTB4 (2 mg) or as-miR-20a (100 nM) (and as-CT), and after 48 h, Sp1, Sp3 and Sp4 mRNA levels were determined as described in the Materials and Methods. As-miR-20a and ZBTB4 decreased luciferase activity in cells transfected with pSp1For4 (c) and pSp3For5 (d). MCF-7 and MDA-MB-231 cells were transfected with the different constructs (as indicated), ZBTB4 expression plasmid (0, 1 or 2 mg) and as-miR-27a (0, 100 or 150 nM) and, after 48 h, luciferase activity determined as described in the Materials and Methods. Results (a–d) are means±s.e. (triplicate) and significant (Po0.05) inhibition (**) is indicated.

ference studies in cells transfected with siSp1, siSp3 and Sp4, we re-examined the data sets for correlations siSp4 also shows that knockdown of Sp transcription between patient survival and expression of Sp1, Sp3, factors decreased VEGF, VEGFR1 and survivin protein Sp4, VEGFR1, survivin, cyclin D1 and VEGF. Among levels and induced PARP cleavage (Figure 5d). The role these mRNAs, only VEGF levels were prognostic of ZBTB4 in mediating the effects of as-miR-20a was factors for patient survival (Supplementary Figure investigated in MDA-MB-231 cells transfected with S6D). as-miR-20a alone or in combination with siZBTB4. As- MiR-20a and miR-17-5p also regulate cyclin D1 and miR-20a-induced downregulation of Sp1, Sp3 and Sp4 p21 expression in some cancer cell lines through 30-UTR proteins (Supplementary Figure S6A), Sp1 mRNA interactions (Yu et al., 2008; Inomata et al., 2009). (Supplementary Figure S6B) and luciferase activity in As both of these genes can also be regulated by Sp cells transfected with pSp1For4 construct (Supplemen- transcription factors and would therefore be affected by tary Figure S6C) were significantly inhibited after ZBTB4, we further investigated expression of cyclin D1 co-transfection with siZBTB4. As induction of ZBTB4 and p21 in MCF-7 and MDA-MB-231 cells. Cyclin D1 was associated with downregulation of Sp1, Sp3 and expression was decreased by knockdown of Sp1, Sp3

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1039 4 20 * 3 * 15 * 2 10 ** ** 1 5 ** ** Relative DNA Binding 0 Relative DNA Binding 0 ZBTB4 − + ++ ++ ZBTB4 −−+++− GC-rich oligo −− −++ rSp1 −++++ Competitor GC-rich oligo −−−−+ Competitor

EXON -2.5 Kb -2 -1 Kb 1 1 Kb 2 Kb Kb Sp1 = GC-rich Primer 123 4 567 891011 Primer 6 : -626 ~ -386 bp 10 As-CT POL II Input IgG POLII 8 As-miR-20a 6 As-CT 4 As-miR-20a

% of Input 2 0 Input IgG Sp1 1234567891011 As-CT 20 Sp1 As-CT As-miR-20a 15 As-miR-20a Input IgG ZBTB4 10 As-CT % of Input 5

0 As-miR-20a 1234567891011 Input IgG POLII 5 ZBTB4 As-CT GAPDH 4 As-miR-20a Negative 3 Control 2

% of Input 1 0 1234567891011 Figure 4 Competitive interactions of ZBTB4 and Sp1. Electrophoretic mobility shift assay (EMSA) analysis of ZBTB4 (a) and competitive ZBTB4/Sp1 binding (b). In vitro expressed ZBTB4, rSp1 alone or in combination were incubated with a GC-rich oligonucleotide (±excess competitor) and analyzed by EMSA as described in the Materials and methods. (c) ChIP scanning of the Sp1 promoter. Different primers were used for analysis of Sp1 binding on the Sp1 promoter (À2.5 to þ 2.0 kb) as described in the Materials and methods. (d) As-miR-20 recruitment of ZBTB4 to the Sp1 promoter. Cells were transfected with as-miR-20a or as-Ctl, and binding of pol II, Sp1 and ZBTB4 to the GC-rich À620 to À386 region of the Sp1 promoter was determined as outlined in the Materials and methods. In (a), significantly (Po0.05) increased binding (*) and inhibition of this binding (**) are indicated. and Sp4 in both cell lines and similar results were suppresses p21 (Filion et al., 2006). The effects of observed in cells transfected with ZBTB4 or as-miR-20a individual knockdown of Sp proteins were gene and cell (Figures 6a and b). These results indicate that cyclin D1 line specific (Figure 6c). The oligonucleotides used in is regulated by ZBTB4-Sp and not by direct interactions this study were highly specific for their respective targets with miR-20a. In MDA-MB-231 cells, both ZBTB4 in bladder cancer cells (Chadalapaka et al., 2008); overexpression and Sp1, Sp3 or Sp4 knockdown however, this specificity was not observed in breast decreased p21 expression, whereas minimal effects were cancer cells (Figure 6c), suggesting that in these cells observed in cells transfected with as-miR-20a. This Sp1, Sp3 and Sp4 are autoregulatory and this is suggests the possibility of opposing pathways in which consistent with their GC-rich promoter regions (Nicolas as-miR-20a directly enhances p21, whereas as-miR-20a et al., 2001; Tapias et al., 2008). activation of ZBTB4 and repression of Sp transcription factors decreases p21. As-miR-20a and knockdown of Sp1, Sp3 and Sp4 by RNA interference had minimal MiR-17-92: oncogenic or tumor suppressor-like activity? effects on p21 expression in MCF-7 cells, whereas The functional role of as-miR-20a and ZBTB4 on cell ZBTB4 inhibited p21 expression, and this is consistent proliferation was also investigated in MCF-7 and with previous studies showing that ZBTB4 directly MDA-MB-231 cells. Cells were transfected with ZBTB4

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1040 MCF-7 MDA-MB-231 higher levels of ZBTB4, miR-17-5p and miR-20a mimics ZBTB 4 (g) 012ZBTB 4 (g) 012 significantly enhanced invasion in a Boyden chamber Sp1 Sp1 assay (Supplementary Figure S7B). Sp4 Sp4 Sp3 Sp3 -Actin -Actin Discussion

MCF-7 MDA-MB-231 ZBTB4: a novel tumor suppressor, targeted by OncomiRs As-miR-20a (nM) 0100 150 As-miR-20a (nM)0 100 150 derived from miR-17-92 cluster and its paralogs Sp1 Sp1 As several POK proteins play a role in cancer, we Sp4 Sp4 - performed an unbiased analysis of gene expression data Sp3 Sp3 from breast cancer patients to determine which of these -Actin -Actin proteins are clinically associated with disease progres- sion or with patient survival. We found that the expression of one gene, ZBTB4, exhibited significant MCF-7 MDA-MB-231 MCF-7 MDA-MB-231  prognostic power; its expression was decreased in ZBTB 4 ( g) 012 012 As-miR-20a (nM) 0 100 150 0 100 150 VEGF VEGF tumors, and low expression correlated with shorter VEGFR1 VEGFR1 relapse-free patient survival (Figures 1a and b). Results Survivin Survivin of survival analysis from two independent breast cancer

C-PARP C-PARP patient data sets correlated with ZBTB4 expression with  -Actin -Actin patient outcomes (Figure 1a), and similar results were observed for lymphoma and lung cancer patients (data MCF-7 MDA-MB-231 not shown), confirming the prognostic significance of siCT siSp1 siSp3 siSp4 siCT siSp1 siSp3 siSp4 ZBTB4. There are over 40 POK proteins, and some of VEGF VEGF them, like ZBTB10, have previously been linked to VEGFR1 VEGFR1 breast cancer as inhibitors of several pro-oncogenic gene Survivin Survivin products (Mertens-Talcott et al., 2007); however, among C-PARP C-PARP several ZBTB mRNAs (Supplementary Figure S1), only -Actin -Actin ZBTB4 consistently exhibited a significant predictive Figure 5 Decreased Sp1, Sp3, Sp4 and Sp-regulated protein value in our data sets and, therefore, we hypothesized expression by ZBTB4 overexpression or endogenous miR-20a that ZBTB4 may play a unique role in cancer. ZBTB4 knockdown. Effects of ZBTB4 expression (a) and as-miR-20a (b) has two close paralogs, namely, Kaiso (ZBTB33) and on Sp1, Sp3 and Sp4 protein expression. MCF-7 or MDA-MB-231 cells were transfected with ZBTB4 (0, 1 or 2 mg) or as-miR-20a ZBTB38 (Perez-Torrado et al., 2006; Costoya, 2007), (0, 100 or 150 nM), and whole cell lysates were analyzed for Sp and they are also expressed in mammary tissue but proteins by western blots as described in the Materials and display no association with this disease (Supplementary methods. Effects of ZBTB4 (c) and as-miR-20a (d) on Sp-regulated Figure S1), suggesting that ZBTB4 represses a unique genes and PARP cleavage. Cells were transfected with either gene set that is not regulated by Kaiso or ZBTB38. ZBTB4 or as-miR-20a, and whole cell lysates were analyzed by western blots as described in the Materials and methods. However, the specificity among these genes is probably not dictated by their intrinsic DNA-binding capacities as they have similar targets, at least in vitro (Sasai et al., 2010), suggesting that other mechanisms may explain or empty vector and the effects on cell proliferation were why ZBTB4 exhibits unique activity. determined at 48 and 96 h after ZBTB4 overexpression (Figure 7a). A similar approach was used for as-miR- Regulation of ZBTB4 by members of the miR-17-92 20a (Figure 7b) and the results showed that ZBTB4 and cluster and its paralogs the antagomir decreased cell growth after 96 h. These The NCI-60 mRNA and miR expression profiles were results are consistent with a previous report showing used to correlate ZBTB4 and miR expression that Sp1 knockdown inhibited G0/G1 to S-phase (Figure 1c). These correlations coupled with analysis progression of MCF-7 cells (Abdelrahim et al., 2002). of miRs that are overexpressed in breast cancer and MDA-MB-231 (but not MCF-7) cells are highly target the 30-UTR of ZBTB4 identified a relatively invasive and metastatic, and the effects of ZBTB4 and small number of candidate miRs that regulate ZBTB4 as-miR-20a on MDA-MB-231 cell invasion were exam- (Figure 1d). MiR-20a and miR-17-5p are members ined using the Boyden Chamber invasion assay. The of the miR-17-92 cluster, consisting of six miRNAs results show that ZBTB4 and as-miR-20a decreased (miR-17-5p, miR-18a, miR-19a, miR-20a, miR-19b and invasion in MDA-MB-231 cells (Figure 7c), indicating miR-92), and have two closely related paralog clusters in that miR-20a-mediated suppression of ZBTB4 plays a different chromosomal locations (miR-106b-25: chro- role in the invasive phenotype. Overexpression of mosome 7, miR-106a-363: X). Several miR-20 mimic in MCF-7 or MDA-MB-231 cells did tumor-suppressor genes regulated by these oncogenic not significantly affect cell proliferation (Supplementary clusters are involved with cell survival, cell cycle Figure S7A); however, in MCF-7 cells that express progression and apoptosis (He et al., 2005; Dews

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1041 MCF-7 including survivin, VEGF and VEGFR1 (Figure 5). ZBTB 4 (g) As-miR-20a (nM) Using the GC-rich Sp1 promoter as a model, we show 012 0 100 150 siCT siSp1 siSp3 siSp4 that results of electrophoretic mobility shift assay and Cyclin D1 ChIP assay indicate that ZBTB4 competes with Sp1 for p21 binding to GC-rich promoter elements (Figure 4). Other -Actin members of the POK family such as ZBTB10 also bind GC-rich Sp-binding sites (Tillotson, 1999); however, MDA-MB-231 these genes do not show any clinical association with ZBTB 4 (g) As-miR-20a (nM) breast cancer in our study. ZBTB4 represses p21 012 0 100 150 siCT siSp1 siSp3 siSp4 expression in neuroblastoma cells (Pendergrast et al., Cyclin D1 2002), and this was also observed in breast cancer cells p21 (Figures 6a and b). A previous report showed that

-Actin ZBTB4 regulated p21 by interacting with the DNA- bound protein MIZ-1 (Weber et al., 2008). However, Sp1-binding sites are present in the p21 core promoter MCF-7 MDA-MB-231 (Koutsodontis et al., 2002) and, in light of our results, it siCT siSp1 siSp3 siSp4 siCT siSp1 siSp3 siSp4 Sp1 is now apparent that the regulation of p21 also involves Sp1 the recognition of these sites and displacement of Sp Sp4 Sp4 transcription factors by ZBTB4.

Sp3 Sp3 ZBTB4 was initially identified as a transcriptional repressor that binds methylated CGs (Perez-Torrado Sp3 Sp3 et al., 2006; Costoya, 2007), and subsequent studies -Actin -Actin demonstrate the versatility of ZBTB4, which can also bind nonmethylated GC-rich sequences containing Figure 6 Changes in cyclin D1 and p21 protein expression by Sp knockdown, ZBTB4 or as-miR-20a. Effects of as-miR-20a, Sp consensus Sp1-binding sequences (GGGCGG) (Sasai knockdown or ZBTB4 overexpression on p21 and cyclin D1 in et al., 2010). However, our results indicated that the MCF-7 (a) and MDA-MB-231 (b) cells. After transfection of the methylated DNA-binding activity of ZBTB4 is not indicated reagents (100 nM siSps) for 48 h, whole cell lysates were necessarily required, as the reporter plasmids were not analyzed by western blots as described in the Materials and methylated (Figure 3) but repressed after overexpression methods. (c) Effect of Sp protein knockdown of Sp-regulated genes on Sp protein expression. Cells were transfected with siCT of ZBTB4. It is possible that ZBTB4 binds its target sites (control), iSp1, iSp3 or iSp4 (100 nM) and, after 48 h, whole cell with more avidity when they are methylated, and DNA lysated were analyzed by western blots as outlined in the Materials methylation can inhibit Sp1 binding to its cognate sites. and methods. This suggests that the observed ZBTB4/Sp protein competition for binding sites may also be influenced by the methylation status of the DNA. et al., 2006; Fontana et al., 2008; Inomata et al., 2009; Liu et al., 2009; Li et al., 2011). MiR-20a and miR-17-5p MiR-17-92 as an oncogenic miR in breast cancer cells share the same seed sequences with miR-106a, miR-106b The complementary effects of ZBTB4 and miR-20a and miR-93, and mimics of these paralogs decrease antagomirs on downregulation of Sp transcription ZBTB4 expression in breast cancer cells and also factors and Sp-regulated genes (Figures 5 and 6), decrease luciferase activity in cells transfected with a coupled with their inhibition of MCF-7 and MDA- ZBTB4-30-UTR-luc construct (Figures 2a and c and MB-231 cell growth and invasion (MDA-MB-231 cells) Supplementary Figure S3). Expression patterns of the (Figure 7 and Supplementary Figure S7), are consistent miR paralogs in MCF-7 and MDA-MB-231 cells with several reports on the oncogenic activity of the demonstrated that miR-20a was one of the most highly miR-17-92 cluster and other paralogs (He et al., 2005). expressed miR in both cell lines and was used to further In breast cancer cells, some studies report that miR-17- investigate the regulation and consequences associated 92 exhibits tumor suppressor-like activity (Yu et al., with ZBTB4–miR-20a interactions. 2008, 2010); however, it was also reported that miR-17- 92 was associated with breast cancer cell growth, migration and invasion (Liu et al., 2009; Li et al., Competition between ZBTB4 and Sp proteins for GC-rich 2011). Differences in the conclusions of these studies sequences likely originate in the use of different cellular systems ZBTB4 and some other members of the POK family and methodologies. However, using the most appro- interact with methylated DNA, other cis-elements and priate assay, namely loss of function by antagomirs, we GC-rich sites that bind Sp transcription factors and clearly observe that miR-20a contributes to the trans- thereby inhibit transcription (Filion et al., 2006). The formed characteristics of two different breast cancer cell results in Figure 3 demonstrate that ZBTB4 and as-miR- lines (Figure 7) and this is because of perturbation of the 20a inhibit Sp1, Sp3 and Sp4 mRNA expression, and ZBTB4/Sp protein axis (Figure 7d) that is characterized this is accompanied by decreased expression of Sp1, Sp3 by decreased expression of Sp-regulated genes that play and Sp4 proteins and several Sp-regulated gene products critical roles in cancer cell growth, survival and

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1042

MCF-7MCF-7 MDA-MB-231 MDA-MB-231 5 Empty 5 10 10 As-CT Empty As-CT ZBTB4 4 4 As-miR-20a 8 ZBTB4 8 As-miR-20a 3 3 ** 6 6 ** 2 2 4 ** 4 ** 1 1 2 2

Relative Cell Growth 0 0 0 0 0 hr 48 hr 96 hr 0 hr 48 hr 96 hr 0 hr 48 hr 96 hr 0 hr 48 hr 96 hr

miR-17-92 MDA-MB-231 Empty ZBTB4 miR-106a-363 1.5 miR-106b-25

1 ** 0.5 ZBTB4

Cell Invasion 0 100 m Empty ZBTB4 Sp proteins MDA-MB-231 As-CT As-miR-20a 1.5

1 Sp-dependent 0.5 ** VEGF, Survivin, VEGFR1

Cell Invasion 0

As-CT

As-miR-20a ONCOGENSIS

Figure 7 ZBTB4 and as-miR-20 inhibit breast cancer cell growth and invasion. Inhibition of MCF-7 (a) and MDA-MB-231 (b) cell growth. Cells were transfected with ZBTB4 (2 mg) or as-miR-20a (150 nM), and their effects on cell growth after 48 or 96 h and cell invasion in a Boyden Chamber assay were determined as described in the Materials and methods. (c) Boyden chamber assay. This assay was carried out as described for cell proliferation (32 h) and cancer cell invasion was determined using a Boyden chamber as described in the Materials and methods. Schematic model to explain how miR-17-92/ZBTB4/Sp axis plays an important role in oncogenesis (d). Results are expressed as means±s.e. (triplicate) and significant (Po0.05) inhibition by ZBTB4 or as-miR-20a is indicated (**).

angiogenesis/metastasis (Abdelrahim et al., 2004, 2007; 2010). Antisense miRs, mimic miRs and their counterpart Jutooru et al., 2010). controls were purchased from Dharmacon (Lafayette, CO, In summary, results of this study demonstrate that the USA), and the ZBTB4 expression vector and empty vector mechanism of action of ZBTB4 as a transcriptional (pCMV-SPORT6) were from Open Biosystems (Huntsville, repressor is also due, in part, to its activity as a repressor AL, USA). MISSION siRNA was purchased from Sigma of Sp1, Sp3, Sp4 and Sp-regulated genes through Aldrich (St Louis, MO, USA) and control siRNA from Quiagen (Valencia, CA, USA), and MCF-7 and MDA-MB- competitive binding at GC-rich sites. MiR-20a, which 231 human breast cancer cell lines were obtained from the is a member of the miR-17-92 cluster, regulates ZBTB4 American Type Culture Collection (Manassas, VA, USA) expression and is part of a miR-17-92-ZBTB4-Sp (Mertens-Talcott et al., 2007). transcription factor network that determines the in- versely correlated expression of ZBTB4 and Sp1, both of Immunoblotting, transfection and luciferase assays which are positive and negative prognostic factors, Whole cell lysates were analyzed by western blots essentially as respectively, for survival/relapse-free survival of cancer described (Jutooru et al., 2010). Various constructs (0.25 mg), patients (Figure 1). MiR-20a-dependent suppression of b-galactosidase, siRNAs and other reagents and their corre- ZBTB4 allows for enhanced expression of Sp transcrip- sponding controls were transfected (Lipofectamine 2000, tion factors and Sp-regulated genes with pro-oncogenic Invitrogen, Carlsbad, CA, USA) and activity relative to b- activity, suggesting that drugs targeting miR-20a and galactosidase was determined. the oncogenic miR-17-92 cluster may have important clinical applications. Real-time PCR Real-time PCR was determined using total RNA extracts essentially as described (Mertens-Talcott et al., 2007; Jutooru et al., 2010), and PCR primers were purchased from Quiagen. Materials and methods Each gene value was normalized to the TATA-binding protein. Chemicals, antibodies, plasmids and reagents Antibodies against Sp1, Sp3, Sp4, VEGF and VEGFR1 were Cloning of 30-UTR ZBTB4 reporter construct purchased from Santa Cruz (Santa Cruz, CA, USA) and The entire 2559 bp 30-UTR of human ZBTB4 (2559 bp) was survivin and PARP from Cell Signaling Technology. ZBTB4 first amplified from human genomic DNA with the following antibody was generated in the Defossez laboratory (Paris, PCR primer set: 50-TATCTCGAGACCCTGGGGCTCAAT France) (Filion et al., 2006; Weber et al., 2008; Sasai et al., CCC-30 (sense) and 50-TATGCGGCCGCTCATTTGTCTTG

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1043 TTGGTTTATTGG-30 (antisense) and cloned into XhoI and upon ZBTB4 or as-miR-20a transfection. Cells were first NotI sites of psiCheck-2 vector (Promega, Madison, WI, transfected with either ZBTB4 or empty expression plasmid, or USA). The 30-UTR ZBTB4 reporter luciferase containing as-miR-20a or as-CT using Lipofectamine 2000 (Invitrogen). The either core seed sequence (1836–1886 bp WT) or mutated assay was carried out using the standard assay protocol and sequence (1836–1886 bp MT) was generated using the follow- invading cells were photographed and counted under a micro- ing primers: for 1836–1886 WT, 50-TCGAGAATTCCCAC scope and the relative number was calculated. AAATCTTGTTTCTGGCACTTTAGAAAAACTGCAAAAA AATACGTGC-30 (sense) and 50-GCGGCCGCACGTATT Cancer patient gene expression data TTTTTGCAGTTTTTCTAAAGTGCCAGAAACAAGATTT Normalized gene expression data from the NKI and UNC GTGGGAATTC-30 (antisense); and for 1836–1886 MT, 50- data were obtained from the public Stanford microarray site TCGAGAATTCCCACAAATCTTGTTTCTGGAAAGGTA (http://microarray-pubs.stanford.edu/wound_NKI) and from GAAAAACTGCAAAAAAATACGTGC-30 (sense) and 50- the UNC microarray database (http://genome.unc.edu). GGCCGCACGTATTTTTTTGCAGTTTTTCTACCTTTCC Normalized lung cancer cohort gene expression data and B- AGAAACAAGATTTGTGGGAATTC-30 (antisense). The cell lymphoma were obtained from Gene Expression Omnibus WT and MT core seed sequences are shown in bold. (accession numbers GSE11969 and GSE10866) (Takeuchi et al., 2006; Lenz et al., 2008). ChIP assay The ChIP assay was performed as previously described (Lee Statistical significance and survival analysis et al., 2010). Briefly, MCF-7 cells (2.5 Â 106) were seeded and Statistical significance was determined using Student’s t-test after 24 h, transfected with as-CT and As-miR-20a. After 48 h, (two sided). Gene clustering was performed using Cluster and cells were crosslinked by formaldehyde. An equal amount of Treeview programs (Eisen et al., 1998). Kaplan–Meier analysis fragmented chromatin was immunoprecipitated using Sp1, and log-rank test were applied to estimate patient survival. RNA polymerase II (Santa Cruz Biotech) and ZBTB4 (from Dr Defossez) antibodies. Rabbit immunoglobulin was used as Conflict of interest control. The primer sets for negative and positive (GAPDH (glyceraldehyde 3-phosphate dehydrogenase)) were also de- The authors declare no conflict of interest. scribed previously (Chadalapaka et al., 2010). Real-time PCR quantification was performed (see Supplementary Table S3 for the primer sequence for the ChIP assay) and the total input Acknowledgements was used as a control. The PCR-amplified product from primer set 6 was resolved on a 2% agarose gel in the presence This research was supported by the National Institutes of of ethidium bromide and visualized. Health (CA136571) and is gratefully acknowledged. PAD is supported by Association pour la Recherche sur le Cancer, Invasion assay Ligue contre le Cancer and Institut National du Cancer. DY Transwell plates (Costar, Cambridge, MA, USA; 8.0 mm pore was supported by a postdoctoral fellowship from Association size) coated with Matrigel were used to measure tumor invasion pour la Recherche sur le Cancer.

References

Abdelrahim M, Baker CH, Abbruzzese JL, Sheikh-Hamad D, Liu S, Chadalapaka G, Jutooru I, Chintharlapalli S, Papineni S, Cho SD et al. (2007). Regulation of vascular endothelial growth Smith III R, Li X et al. (2008). Curcumin decreases specificity factor receptor-1 (VEGFR1) expression by specificity proteins 1, 3 protein expression in bladder cancer cells. Cancer Res 68: and 4 in cells. Cancer Res 67: 3286–3294. 5345–5354. Abdelrahim M, Samudio I, Smith R, Burghardt R, Safe S. (2002). Chen WY, Wang DH, Yen RC, Luo J, Gu W, Baylin SB. (2005). Small inhibitory RNA duplexes for Sp1 mRNA block basal and Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53- estrogen-induced gene expression and cell cycle progression in dependent DNA-damage responses. Cell 123: 437–448. MCF-7 breast cancer cells. J Biol Chem 277: 28815–28822. Chen WY, Zeng X, Carter MG, Morrell CN, Chiu Yen RW, Abdelrahim M, Smith III R, Burghardt R, Safe S. (2004). Role of Sp Esteller M et al. (2003). Heterozygous disruption of Hic1 predis- proteins in regulation of vascular endothelial growth factor poses mice to a gender-dependent spectrum of malignant tumors. expression and proliferation of pancreatic cancer cells. Cancer Res Nat Genet 33: 197–202. 64: 6740–6749. Costoya JA. (2007). Functional analysis of the role of POK Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning transcriptional repressors. Brief Funct Genomic Proteomic 6: 8–18. MJ et al. (2007). MicroRNA expression profiling of human breast Dews M, Homayouni A, Yu D, Murphy D, Sevignani C, Wentzel E cancer identifies new markers of tumor subtype. Genome Biol 8: et al. (2006). Augmentation of tumor angiogenesis by a - R214. activated microRNA cluster. Nat Genet 38: 1060–1065. Blower PE, Verducci JS, Lin S, Zhou J, Chung JH, Dai Z et al. (2007). Eisen MB, Spellman PT, Brown PO, Botstein D. (1998). Cluster MicroRNA expression profiles for the NCI-60 cancer cell panel. analysis and display of genome-wide expression patterns. Proc Natl Mol Cancer Ther 6: 1483–1491. Acad Sci USA 95: 14863–14868. Cattoretti G, Pasqualucci L, Ballon G, Tam W, Nandula SV, Shen Q Filion GJ, Zhenilo S, Salozhin S, Yamada D, Prokhortchouk E, et al. (2005). Deregulated BCL6 expression recapitulates the Defossez PA. (2006). A family of human zinc finger proteins that pathogenesis of human diffuse large B cell lymphomas in mice. bind methylated DNA and repress transcription. Mol Cell Biol 26: Cancer Cell 7: 445–455. 169–181. Chadalapaka G, Jutooru I, Burghardt R, Safe S. (2010). Drugs that Fontana L, Fiori ME, Albini S, Cifaldi L, Giovinazzi S, Forloni M target specificity proteins downregulate epidermal growth factor et al. (2008). Antagomir-17-5p abolishes the growth of therapy- receptor in bladder cancer cells. Mol Cancer Res 8: 739–750. resistant neuroblastoma through p21 and BIM. PLoS One 3: e2236.

Oncogene ZBTB4 is regulated by microRNA-17-92 cluster KKimet al 1044 He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Mertens-Talcott SU, Chintharlapalli S, Li X, Safe S. (2007). The Goodson S et al. (2005). A microRNA polycistron as a potential oncogenic microRNA-27a targets genes that regulate specificity human oncogene. Nature 435: 828–833. protein (Sp) transcription factors and the G2-M checkpoint in Inomata M, Tagawa H, Guo YM, Kameoka Y, Takahashi N, Sawada MDA-MB-231 breast cancer cells. Cancer Res 67: 11001–11011. K. (2009). MicroRNA-17-92 down-regulates expression of distinct Nicolas M, Noe V, Jensen KB, Ciudad CJ. (2001). Cloning and targets in different B-cell lymphoma subtypes. Blood 113: 396–402. characterization of the 50-flanking region of the human transcription Jeon BN, Choi WI, Yu MY, Yoon AR, Kim MH, Yun CO et al. factor Sp1 gene. J Biol Chem 276: 22126–22132. (2009). ZBTB2, a novel master regulator of the p53 pathway. J Biol Niu H. (2002). The proto-oncogene BCL-6 in normal and malignant B Chem 284: 17935–17946. cell development. Hematol Oncol 20: 155–166. Jeon BN, Yoo JY, Choi WI, Lee CE, Yoon HG, Hur MW. (2008). Oh DS, Troester MA, Usary J, Hu Z, He X, Fan C et al. (2006). Proto-oncogene FBI-1 (Pokemon/ZBTB7A) represses transcription Estrogen-regulated genes predict survival in - of the tumor suppressor Rb gene via binding competition positive breast . J Clin Oncol 24: 1656–1664. with Sp1 and recruitment of co-repressors. J Biol Chem 283: Pendergrast PS, Wang C, Hernandez N, Huang S. (2002). FBI-1 can 33199–33210. stimulate HIV-1 Tat activity and is targeted to a novel subnuclear Jutooru I, Chadalapaka G, Abdelrahim M, Basha MR, Samudio I, domain that includes the Tat-P-TEFb-containing nuclear speckles. Konopleva M et al. (2010). Methyl 2-Cyano-3,12-dioxooleana-1,9- Mol Biol Cell 13: 915–929. dien-28-oate (CDDO-Me) decreases specificity protein (Sp) tran- Perez-Torrado R, Yamada D, Defossez PA. (2006). Born to bind: the scription factors and inhibits pancreatic tumor growth: role of BTB protein-protein interaction domain. Bioessays 28: 1194–1202. microRNA-27a. Mol Pharmacol 78: 226–236. Sasai N, Nakao M, Defossez PA. (2010). Sequence-specific recognition Koh DI, Choi WI, Jeon BN, Lee CE, Yun CO, Hur MW. (2009). A of methylated DNA by human zinc-finger proteins. Nucleic Acids novel POK family transcription factor, ZBTB5, represses transcrip- Res 38: 5015–5022. tion of p21CIP1 gene. J Biol Chem 284: 19856–19866. Shankavaram UT, Reinhold WC, Nishizuka S, Major S, Morita D, Kojima S, Hatano M, Okada S, Fukuda T, Toyama Y, Yuasa S et al. Chary KK et al. (2007). Transcript and protein expression profiles (2001). Testicular germ cell apoptosis in Bcl6-deficient mice. of the NCI-60 cancer cell panel: an integromic microarray study. Development 128: 57–65. Mol Cancer Ther 6: 820–832. Koutsodontis G, Moustakas A, Kardassis D. (2002). The role of Sp1 Takeuchi T, Tomida S, Yatabe Y, Kosaka T, Osada H, Yanagisawa K family members, the proximal GC-rich motifs, and the upstream et al. (2006). Expression profile-defined classification of lung enhancer region in the regulation of the human cell cycle inhibitor adenocarcinoma shows close relationship with underlying major p21WAFÀ1/Cip1 gene promoter. Biochemistry 41: 12771–12784. genetic changes and clinicopathologic behaviors. J Clin Oncol 24: Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ et al. 1679–1688. (2005). Combinatorial microRNA target predictions. Nat Genet 37: Tapias A, Ciudad CJ, Noe V. (2008). Transcriptional regulation of the 495–500. 50-flanking region of the human transcription factor Sp3 gene by Lee SO, Abdelrahim M, Yoon K, Chintharlapalli S, Papineni S, Kim NF-1, c-Myb, B-Myb, AP-1 and . Biochim Biophys Acta 1779: K et al. (2010). Inactivation of the orphan TR3/ 318–329. Nur77 inhibits pancreatic cancer cell and tumor growth. Cancer Res Tillotson LG. (1999). RIN ZF, a novel zinc finger gene, encodes 70: 6824–6836. proteins that bind to the CACC element of the gastrin promoter. Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H et al. (2008). J Biol Chem 274: 8123–8128. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med van de Vijver MJ, He YD, van’t Veer LJ, Dai H, Hart AA, Voskuil 359: 2313–2323. DW et al. (2002). A gene-expression signature as a predictor of Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. (2003). survival in breast cancer. N Engl J Med 347: 1999–2009. Prediction of mammalian microRNA targets. Cell 115: 787–798. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F et al. Li H, Bian C, Liao L, Li J, Zhao RC. (2011). miR-17-5p promotes (2006). A microRNA expression signature of human solid tumors human breast cancer cell migration and invasion through suppres- defines cancer gene targets. Proc Natl Acad Sci USA 103: sion of HBP1. Breast Cancer Res Treat 126: 565–575. 2257–2261. Liu H, D’Andrade P, Fulmer-Smentek S, Lorenzi P, Kohn KW, Weber A, Marquardt J, Elzi D, Forster N, Starke S, Glaum A et al. Weinstein JN et al. (2010). mRNA and microRNA expression (2008). Zbtb4 represses transcription of P21CIP1 and controls the profiles of the NCI-60 integrated with drug activities. Mol Cancer cellular response to p53 activation. EMBO J 27: 1563–1574. Ther 9: 1080–1091. Yu Z, Wang C, Wang M, Li Z, Casimiro MC, Liu M et al. (2008). Liu S, Goldstein RH, Scepansky EM, Rosenblatt M. (2009). Inhibition A cyclin D1/microRNA 17/20 regulatory feedback loop in control of rho-associated kinase signaling prevents breast cancer metastasis of breast cancer cell proliferation. J Cell Biol 182: 509–517. to human bone. Cancer Res 69: 8742–8751. Yu Z, Willmarth NE, Zhou J, Katiyar S, Wang M, Liu Y et al. (2010). Maeda T, Hobbs RM, Merghoub T, Guernah I, Zelent A, Cordon- microRNA 17/20 inhibits cellular invasion and tumor metastasis in Cardo C et al. (2005). Role of the proto-oncogene Pokemon in breast cancer by heterotypic signaling. Proc Natl Acad Sci USA 107: cellular transformation and ARF repression. Nature 433: 278–285. 8231–8236.

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

Oncogene