Oncogene (2009) 28, 2894–2902 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE KLF4 suppresses estrogen-dependent breast cancer growth by inhibiting the transcriptional activity of ERa

K Akaogi1, Y Nakajima1, I Ito1, S Kawasaki1, S-h Oie1, A Murayama1,2,3, K Kimura1 and J Yanagisawa1,2

1Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan; 2TARA Center, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan and 3PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan

Kruppel-like factor 4 (KLF4) is a transcription factor that et al., 1999, 2000; Nickenig et al., 2002; Chen et al., participates in both tumor suppression and oncogenesis. 2003). This indicates that KLF4 regulates the expression To determine the association of KLF4 with tumorigenesis, of a set of cell-cycle genes to coordinately inhibit cellular we integrated data assembled in the Oncomine database proliferation. in vivo, KLF4 is essential for maintaining and discovered a decrease in KLF4 gene transcripts in terminally differentiated epithelial cells in the lung, skin breast cancers. Further analysis of the database also and gastrointestinal tract (Shields et al., 1996; Segre showed a correlation between KLF4 expression and et al., 1999; Jaubert et al., 2003; Blanchon et al., 2006; estrogen receptor-a (ERa) positivity. Knockdown of Patel et al., 2006). In cultured cells, KLF4 expression is KLF4 in MCF-7 cells elevated the growth rate of these temporally associated with conditions that promote cells in the presence of estrogen. Therefore, we examined growth arrest, such as serum deprivation, contact the interaction between KLF4 and ERa, and found that inhibition and DNA damage, and constitutive KLF4 KLF4 bound to the DNA-binding region of ERa. KLF4 expression inhibits DNA synthesis (Garrett-Sinha thus inhibits the binding of ERa to estrogen response et al., 1996; Shields et al., 1996; Stone et al., 2002). elements in promoter regions, resulting in a reduction in g-Irradiation increases KLF4 mRNA levels in a ERa target gene transcription. Earlier studies have p53-dependent manner and similarly correlates with reported that KLF4 is transcriptionally activated by p53 increased expression of p21 (Yoon et al., 2003). It was following DNA damage. We also showed that activation further determined that KLF4 transactivates the p21 of p53 decreased the transcriptional activity of ERa by promoter by binding to a specific Sp-1-like cis-element elevating KLF4 expression. Our studies discovered a novel in the proximal region of the promoter (Zhang et al., molecular network between p53, KLF4 and ERa. As both 2000). Activation of p21 expression following DNA p53 and ERa are involved in cell growth and apoptosis, damage causes cell-cycle arrest at both the G1-S and these results may explain why KLF4 possesses both tumor G2-M transition points (Bissonnette and Hunting, 1998; suppressive and oncogenic functions in breast cancers. Winters et al., 1998). In addition to its effects on p21 Oncogene (2009) 28, 2894–2902; doi:10.1038/onc.2009.151; expression, KLF4 has been reported to inhibit expres- published online 8 June 2009 sion of cyclin D1 and cyclin B, which promote progression through the G1-S and G2-M boundaries, Keywords: breast cancer; estrogen receptor; KLF4; p53 respectively (Shie et al., 1999, 2000; Yoon and Yang, 2004). Thus, KLF4 has the potential to suppress tumor growth. It was also shown that KLF4 suppresses p53 Introduction expression by directly acting on its promoter, thus providing resistance to DNA damage-induced apoptosis Kruppel-like factor 4 (KLF4/GKLF/EZF) encodes a (Wei et al., 2007). Depleting KLF4 from breast cancer transcription factor that is associated with both tumor cells restores p53 levels and causes p53-dependent suppression and oncogenesis (Evans and Liu 2008). apoptosis (Rowland et al., 2005). In addition to p53 Several lines of evidence indicate that KLF4 is an suppression, KLF4 has been reported to suppress important regulator of cell proliferation. Among the apoptosis through the direct downregulation of the KLF4-regulated cell-cycle genes, many upregulated Bax promoter (Ghaleb et al., 2007). Thus, the oncogenic genes inhibit proliferation, whereas genes that promote potential of KLF4 appears to lie in its ability to inhibit proliferation are repressed (Mahatan et al., 1999; Shie apoptosis. Therefore, KLF4 could act as an oncogene in genetic contexts in which KLF4 inhibits apoptosis but does not induce cell-cycle arrest. Correspondence: Professor J Yanagisawa, Graduate School of Life As KLF4 has pivotal roles in cell-cycle arrest and and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, inhibition of apoptosis, it is logical to assume that KLF4 Tsukuba Science City, Ibaraki-ken 305-8572, Japan. E-mail: [email protected] would have both tumor-suppressive functions and Received 26 January 2009; revised 3 April 2009; accepted 16 April 2009; oncogenic potential. Consistent with this notion, published online 8 June 2009 KLF4 is downregulated in many human cancers, KLF4 suppresses estrogen-dependent breast cancer K Akaogi et al 2895 including esophageal, gastric, colorectal, bladder and database, which provides publicly available data sets prostate cancers, whereas it is overexpressed in primary on cancer gene expression. Nine of the 11 data sets, breast ductal carcinomas and oral squamous cell which contain gene chip profiles classified as normal or carcinomas (Foster et al., 2000; Wang et al., 2002; breast carcinoma tissues, showed that KLF4 mRNA Ohnishi et al., 2003; Luo et al., 2004). levels were lower in breast carcinomas than in normal Breast cancer is typically a hormone-dependent tissues. We also compared the correlation between tumor in which exposure to estrogen increases breast KLF4 mRNA levels and the breast cancer tumor grade cancer incidence and proliferation (Key and Pike, 1988; using data sets containing gene chip profiles classified by Henderson et al., 1988; Pike et al., 1993; Dorgan tumor grade. Fifteen of the 20 data sets showed an et al., 1996). Estrogens mediate their effects after inverse correlation between KLF4 expression levels and binding to the estrogen receptors (ERs), ERa and tumor grade. Representative results of two independent ERb, which are members of the nuclear receptor data sets characterized by large population sizes (Miller superfamily that function as ligand-induced transcrip- et al., 2005; Ivshina et al., 2006; Richardson et al., 2006; tion factors (McKenna 2002; Mangelsdorf et al., 1995; Finak et al., 2008) are shown in Figure 1a (KLF4 Chambon, 1996; Tateishi et al., 2006). On binding expression levels in normal versus breast carcinoma; estrogen, the receptor ligand-binding domain undergoes P ¼ 5.4EÀ4, 7.0EÀ3) and 1b (KLF4 expression levels a characteristic conformational change that induces in breast cancers classified by grade; P ¼ 8.0EÀ11, receptor dimerization. The dimer then binds DNA to 2.6EÀ10). Earlier reports have shown that 70% of stimulate target gene expression. ERa activity is breast carcinomas have elevated KLF4 expression and stimulated by two distinct regions, the activation that increased nuclear staining for KLF4 is associated function-1 and -2 domains; the latter domain is located with a more aggressive phenotype (Foster et al., 2000; in the ligand-binding domain and exerts ligand-dependent Pandya et al., 2004). Inconsistencies between the results transcriptional activity (Tora et al.,1989).Crystal obtained from the Oncomine database and earlier structure analysis of the ERs and other nuclear receptors reports may be due to the genetic or epigenetic context revealed 12 conserved helices within their ligand-binding of breast cancer tissues. domains (Shiau et al.,1998).Helix12,theC-terminal Breast cancer typically depends on hormones. Estro- helix, forms the critical core (AD core) for the activation gen binds to the ERa, which belongs to the nuclear function–2 function of the receptor. This region has an receptor superfamily, and increases the incidence and important role in binding co-activators to the ligand- proliferation of breast cancer. Furthermore, almost 60% bound receptor (Heery et al., 1997). of breast cancer tissues have higher ERa protein levels Here, we performed an extensive analysis of the than normal tissues. Recently, several lines of evidence Oncomine database and showed that KLF4 expression showed that the KLF family proteins are associated and was associated with breast cancer tumorigenesis. Ana- interact with nuclear receptors (Shindo et al., 2002; lysis of the database also showed a correlation between Oishi et al., 2008). Thus, we further examined the KLF4 expression and ERa-positive breast cancers. relationship between KLF4 and ERa in breast cancer in vitro experiments using the ERa-positive breast tissues. We compared the correlation between KLF4 cancer cell line MCF-7 revealed that estrogen-dependent expression levels and ERa positivity using Oncomine cell growth was significantly enhanced by knockdown of data sets containing gene chip profiles classified by ERa KLF4. Coimmunoprecipitation experiments revealed positivity (Minn et al., 2005; Ivshina et al., 2006). As that KLF4 binds to the DNA-binding region of ERa shown in Figure 1c, there was a significant correlation and inhibits the binding of ERa to estrogen response between KLF4 expression levels and ERa positivity. elements (EREs) in promoter regions to reduce the These results raise the possibility that KLF4 and ERa transcription of ERa target genes. We also showed that coordinately regulate breast cancer progression. activation of p53 decreased ERa transcriptional activity To investigate the relationship between KLF4 and by elevating KLF4 expression. These results revealed the ERa in breast cancer growth, we used KLF4 small existence of a novel molecular network that includes interfering RNAs (siRNAs) to knock down KLF p53, KLF4 and ERa. As both p53 and ERa are involved expression in the ERa-positive breast cancer cell line, in cell growth and apoptosis, the genetic and epigenetic MCF-7. Both KLF4 siRNAs (siRNA nos. 1 and 2) status of these factors may determine whether KLF4 effectively reduced KLF4 mRNA and protein levels acts as a tumor suppressor or oncogene in breast (Figure 2a and b) in MCF-7 cells. Using these siRNAs, cancers. we examined the effect of KLF4 knockdown on the growth rate of MCF-7 cells. In the absence of estrogen, KLF4 knockdown did not affect the growth rate of Results and discussion MCF-7 cells (Figure 2c). By contrast, in the presence of estrogen, the growth rate of MCF-7 cells increased and Several lines of evidences indicate that Kruppel-like this estrogen-dependent enhancement of cell growth was factor 4 (KLF4/GKLF/EZF) regulates tumor progres- further potentiated by KLF4 knockdown (Figure 2c). sion. To confirm the relationship between KLF4 These observations indicate that KLF4 suppresses expression levels and breast cancer progression, we estrogen-dependent breast cancer growth. compared the expression levels of KLF4 in normal Estrogen enhances breast cancer growth by inducing tissues and breast carcinomas using the Oncomine transcriptional activity of ERa. Thus, we next examined

Oncogene KLF4 suppresses estrogen-dependent breast cancer K Akaogi et al 2896 Richardson Finak KLF4 P-value= 5.4E-4 KLF4 P-value= 7.0E-3 2.0 2.0 1.5 1.5 1.0

0.5 1.0 0.0

0.5 -0.5

-1.0 0.0 -1.5 Normalized expression units Normalized expression units -0.5 -2.0 Normal Breast Normal Breast Breast Carcinoma Breast Carcinoma (n=7) (n=40) (n=6) (n=57)

Miller Ivshina KLF4 P-value= 8.0E-11 KLF4 P-value= 2.6E-10 1.5 2.0

1.5 1.0

1.0 0.5 0.5

0.0 0.0 Normalized expression units Normalized expression units -0.5 -0.5 Grade1 Grade2 Grade3 Grade1 Grade2 Grade3 (n=67) (n=128) (n=57) (n=68) (n=126) (n=55)

Minn Ivshina KLF4 P-value= 4.0E-3 KLF4 P-value= 5.0E-3 2.5 2.0 2.0 1.5 1.5 1.0 0.5 1.0 0.0 0.5 -0.5 -1.0 0.0

Normalized expression units -1.5 Normalized expression units

-2.0 -0.5 ERα negative ERα positive ERα negative ERα positive (n=42) (n=57) (n=34) (n=211) Figure 1 KLF4 expression decreases as breast cancers progress. (a) Decreased expression of KLF4 in tumor tissues in two independent studies on human breast cancers (Richardson et al., 2006; Finak et al., 2008). Data and statistics were obtained from www.oncomine.org. (b) Inverse correlation between KLF4 expression and tumor stage in two independent studies on human breast cancers (Miller et al., 2005; Ivshina et al., 2006). (c) Enhanced expression of KLF4 in ERa-positive human breast cancers (Minn et al., 2005; Ivshina et al., 2006).

the effect of KLF4 on the transcriptional activity of transcription of these reporter plasmids (Figure 3a, ERa. Luciferase reporter plasmids containing EREs compare lanes 3 and 5). This estrogen-stimulated were transfected into MCF-7 cells with or without KLF4 enhanced transcription was inhibited by KLF4 expres- expression plasmids. Estrogen treatment enhanced the sion in a dose-dependent manner (Figure 3a, lanes 6

Oncogene KLF4 suppresses estrogen-dependent breast cancer K Akaogi et al 2897 MCF-7 Cell Growth a MCF-7 MCF-7 bc si-cont. + DMSO KLF4 mRNA ERα mRNA MCF-7 si-cont. + E2 si-KLF4 #1 + DMSO 1.2 1.2 KLF4 0.8 si-KLF4 #1 + E2 si-KLF4 #2 + DMSO 1.0 1.0 si-KLF4 #2 +E2 α 0.8 0.8 ER 0.6 0.6 0.6 βactin 0.4 0.4 Relative Quantity (fold) Relative Quantity (fold) 0.2 0.2 0.4 si-cont. 0 0 OD (570nm) si-KLF4 #1 si-KLF4 #2 si-cont. si-cont. 0.2 si-KLF4 #1 si-KLF4 #2 si-KLF4 #1 si-KLF4 #2

0 01234 Days Figure 2 KLF4 reduces estrogen-dependent growth of breast cancer cells. (a, b) Knockdown of endogenous KLF4 expression. (a) Total RNA was isolated from control (si-cont.) or KLF4-specific (si-KLF4) small interfering RNA (siRNA)-treated MCF-7 cells, and KLF4 and ERa mRNA levels were quantified using real-time reverse transcription–PCR (RT–PCR). (b) MCF7 cells were transfected with control siRNA (si-cont.) or KLF4-specific siRNA (si-KLF4 nos. 1, 2). (c) Inhibition of E2-stimulated cell growth by KLF4. MCF-7 cells treated with control siRNA (si-cont.) or KLF4-specific siRNA (si-KLF4 nos. 1, 2) were seeded on 96-well plates at a density of 4000 cells per well in the presence of dimethylsulfoxide (DMSO) or estrogen (E2; 10À8 M). At the indicated time points, cell numbers were determined using an MTT assay. Bars represent means±s.d. (n ¼ 3).

MCF-7 MCF-7 MCF-7 MCF-7 ERE-luc ERE-luc pS2 mRNA pS2 mRNA

DMSO 35 DMSO 20 E2 E2 DMSO DMSO 30 2.0 E2 3.5 E2 15 25 3.0 20 1.5 2.5 (fold) (fold) 10 15 1.0 2.0 10 5 1.5 Transcriptional activity Transcriptional activity 5 0.5 1.0 0

0 Relative Quantity (fold)

Relative Quantity (fold) 0.5 1 2 3 4 5 6 7 1452 3 pGL3-basic pGL3-basic +-- - - 0 0 + + - - - - - 1 2 3 4 132 4 ERE-luc- + + + + ERE-luc - - + + + + + cont. + - + - si-cont. + + - - si-cont. + + +- - KLF4 - ++ - ++ - + ++ KLF4 - + - + si-KLF4 - - #1 #2 si-KLF4 - - -#1#2

Figure 3 KLF4 reduces ERa-dependent transcription. (a-d) Inhibition of ERa-dependent transcription by KLF4. (a) Expression plasmids encoding various amounts of KLF4 ( þ , 50 ng; þþ, 350 ng) were co-transfected into MCF-7 cells with the pGL3-basic control plasmid (pGL3-basic) or 4 Â ERE-TATA-luc reporter plasmid containing four tandem E2 response elements (ERE-luc) and cultured in the absence (dimethylsulfoxide (DMSO)) or presence of estrogen (E2; 10À8 M). Cell extracts derived from these cells were examined using luciferase assays. Bars represent means þ s.d. (n ¼ 3). (b) A control (pGL3-basic) or luciferase reporter plasmid containing four tandem E2 response elements (ERE-luc) was transfected into MCF-7 cells that were treated with control (si-cont.) or KLF4-specific (si-KLF4) small interfering RNA (siRNA). Cell extracts derived from cultures treated with or without estrogen (E2; 10À8 M) were examined by luciferase assays. Bars represent means þ s.d. (n ¼ 3). (c) Total RNA was isolated from MCF-7 cells transfected with control (cont.) or expression plasmids of KLF4 (KLF4), and the mRNA levels for pS2, which is an endogenous ERa target gene, were quantified by real-time reverse transcription–PCR. Bars represent means þ s.d. (n ¼ 3). (d) Total RNA was isolated from control (si-cont.) or KLF4-specific (si-KLF4) siRNA-treated MCF-7 cells, and mRNA levels for pS2 were quantified by real-time RT–PCR. Bars represent means þ s.d. (n ¼ 3). and 7). By contrast, siRNA-mediated knockdown of inhibited estrogen-induced pS2 mRNA expression endogenous KLF4 in MCF-7 cells enhanced estrogen- (Figure 3c, lane 4). On the other hand, KLF4 knock- dependent transcription in transient transcription assays down further increased pS2 mRNA levels (Figure 3d, with luciferase reporter plasmids (Figure 3b, lanes 4 lanes 3 and 4). These results show that KLF4 inhibits and 5). In MCF-7 cells, estrogen treatment increased the transcriptional activity of ERa. the mRNA levels of pS2, an endogenous ERa target To study the molecular mechanism for the inhibition gene (Figure 3c, lane 3), and overexpression of KLF4 of ERa-dependent transcription by KLF4, we examined

Oncogene KLF4 suppresses estrogen-dependent breast cancer K Akaogi et al 2898 the binding of KLF4 to ERa. In coimmunoprecipitation assay using an ERa-specific antibody. The ChIP assay experiments, KLF4 associated with ERa in an estrogen- confirmed that ERa bound to the promoter region of dependent manner (Figure 4a and b). Next, to identify the endogenous pS2 gene in MCF-7 cells in an estrogen- the regions of ERa responsible for KLF4 binding, we dependent manner (Figure 4d, lane 4). The levels of ERa generated several ERa truncation mutants. Coimmuno- bound to the pS2 promoter region were decreased by precipitation experiments indicated that the DNA- KLF4 overexpression (Figure 4d, lane 8). By contrast, binding domain of ERa was necessary for KLF4 knockdown of endogenous KLF4 increased the levels of binding (Figure 4c). These results raised the possibility ERa bound to the pS2 promoter region (Figure 4e, lane that the binding of KLF4 to the DNA-binding region 8). To confirm that KLF4 does not bind to the promoter of ERa may abrogate the recruitment of ERa to the region of the pS2, we conducted ChIP assay using a EREs in the promoter region and thereby decrease KLF4-specific antibody. The ChIP assay revealed that ERa-dependent transcription. To test this hypothesis, KLF4 bound to the promoter region of the Lefty1 gene we performed a chromatin immunoprecipitation (ChIP) (Figure 4f, lane 2), as reported earlier (Nakatake et al.,

I.P. : αFLAG (ERα) KLF4

293 ERα (WT) I.P. : αFLAG (ERα) MCF-7 DBD LBD α I.P. ER (ABC)& KLF4 ERα (WT) ERα (AB) Total extracts α α A/B C DFE α ER ER IgG IgG ER KLF4 α α α α Total extracts α ERα (WT) KLF4 ERα (ABC) KLF4 I.B. ERα (ABC)& αERα ERα (AB) ERα 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 1 2 3 4 5 E2 --+-- - - + --+ E2 - + - + ERα (AB) E2 - - - - + HA-KLF4 -+-+- + ++ -++ KLF4 - + - + + FLAG-ERα (WT) -+-+-+ ----- FLAG-ERα - - + + + FLAG-ERα (ABC) -----+++ --- FLAG-ERα (AB) ------+++

MCF-7 MCF-7 MCF-7 ChIP Assay ChIP Assay ChIP Assay

DMSO DMSO DMSO E2 E2 E2 40 120 25

20 30 80 15 20 10

40 DNA bound (fold)

pS2 DNA bound (fold) 10 pS2 DNA bound (fold) 5

0 0 0 123 4 567 8 1 2 3 4 5 6 7 8 1 2 345 6 IgG αERα IgG αERα IgG αERα IgG αERα I.P. IgG αKLF4 IgG αKLF4 I.P. I.P. cont. KLF4 si-cont. si-KLF4 Target genes Lefty1 pS2 Figure 4 KLF4 interacts with ERa and inhibits its binding to the pS2 gene promoter in the presence of E2. (a, b) Interaction between KLF4 and ERa in the presence of E2. (a) Extracts of 293 cells that were transfected with HA-KLF4 and/or FLAG-ERa and cultured with or without estrogen (E2; 10À8 M) were immunoprecipitated with an anti-FLAG antibody and then immunoblotted with an anti- HA (KLF4; top panel) or anti-FLAG (ERa; second panel) antibody. Alternatively, total cell extracts were immunoblotted with an anti-HA (KLF4; third panel) or anti-FLAG (ERa; fourth panel) antibody. (b) Cell extracts were prepared from MCF-7 cells cultured in the absence or presence of estrogen (E2; 10À8 M), immunoprecipitated with a control or anti-ERa antibody and then immunoblotted with the indicated antibodies. (c) KLF4 binds to the C region of ERa. The left panel shows a series of ERa truncation mutants. Expression plasmids encoding HA-KLF4 and the FLAG-ERa truncation mutants were transfected into 293 cells. After culturing the transfected cells in the presence or absence of estrogen (E2; 10À8 M), cell extracts were prepared, immunoprecipitated with an anti- FLAG antibody and then immunoblotted with the indicated antibodies (upper three panels). Alternatively, whole cell extracts were immunoblotted with the indicated antibodies (lower three panels). (d, e) Attenuation of ERa binding to pS2 DNA by KLF4. (d) MCF- 7 cells were treated with control (cont.) or expression plasmids of KLF4 (KLF4) and then cultured in the presence or absence of estrogen (E2; 10À8 M). Chromatin immunoprecipitation (ChIP) assays were performed using the indicated antibodies. Immunopre- cipitated DNA was analysed using quantitative PCR and pS2 primers. Samples were normalized to the IgG DNA. (e) MCF-7 cells were treated with control (si-cont.) or KLF4-specific (si-KLF4) RNAi and then cultured in medium with or without estrogen (E2; 10À8 M). ChIP assays were performed using the indicated antibodies. Immunoprecipitated DNA was analysed using quantitative PCR and pS2 primers. Samples were normalized to the IgG DNA. (f) KLF4 does not bind to pS2 DNA. MCF-7 cells were cultured with or without estrogen (E2; 10À8 M). ChIP assays were performed using the indicated antibodies. Immunoprecipitated DNA was analysed by quantitative PCR using Lefty1 and pS2 primers. Samples were normalized to the IgG DNA.

Oncogene KLF4 suppresses estrogen-dependent breast cancer K Akaogi et al 2899 2006). On the other hand, KLF4 did not bind to the We next examined the effect of siRNA-mediated promoter region of pS2 gene (Figure 4f lanes 5 and 6). knockdown of endogenous p53 in MCF-7 cells on KLF4 These results indicate that KLF4 associates with the levels. The siRNA for p53 effectively reduced p53 DNA-binding region of ERa and inhibits ERa from protein and mRNA levels. Treating MCF-7 cells with binding to the promoter region of target genes. p53 siRNA also decreased KLF4 protein and mRNA Recent reports have shown an interaction between levels, suggesting that p53 regulates the transcription of KLF4 and p53. KLF4 is transcriptionally activated by p53 KLF4 (Figure 5b, c). Earlier reports have shown that following DNA damage and causes cell-cycle arrest by estrogen induces accumulation of wild-type p53 protein inducing transcription of p21. Conversely, KLF4 sup- through ERa (Okumura et al., 2002). Moreover, we and presses the expression of p53 by directly acting on its other groups have shown that p53 enhances KLF4 promoter, thereby causing resistance to DNA damage- expression (Figures 5b and c; Zhang et al., 2000; Yoon induced apoptosis. Therefore, we compared KLF4 expres- et al., 2003; Yoon and Yang, 2004). It is reasonable that sion levels between wild-type and mutant p53-expressing ERa-positive tumors have higher levels of KLF4 than breast cancers using the Oncomine data sets. As shown in ERa-negative tumors (Figure 1c). Figure 5a, the expression levels of KLF4 in breast cancers Given that KLF4 suppresses the transcriptional expressing wild-type p53 were higher than those in breast activity of ERa (Figure 3), it was possible that p53 cancers expressing a mutated p53 (Miller et al., 2005; knockdown enhances the transcriptional activity of Ivshina et al., 2006). These results confirm an interaction ERa. Thus, we transfected a reporter plasmid contain- between KLF4 and p53 in breast cancer tissues. ing ERE into MCF-7 cells with control or p53 siRNA.

MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 KLF4 protein Miller Ivshina p53 mRNA KLF4 mRNA ERα mRNA KLF4 P-value= 9.5E-8 KLF4 P-value= 7.5E-7 p53 1.2 2.0 2.0 1.2 1.2 1.2 1.0 1.5 1.0 1.0 1.0 KLF4 1.0 0.8 0.8 0.8 1.0 0.8 α 0.5 ER 0.6 0.6 0.6 0.6 0.5 0.4 0.0 0.4 0.4 0.4 βactin 0.0 Relative Quantity (fold)

Relative Quantity (fold) Relative Quantity (fold) Relative Quantity (fold) 0.2 0.2 0.2 0.2 Normalized expression units Normalized expression units -0.5 -0.5 p53 wild Type p53 mutant p53 wild Type p53 mutant 0 0 0 0 si-p53 (n=179) (n=72) (n=189) (n=58) si-cont. si-p53 si-cont. si-p53 si-p53 si-p53 si-cont. si-cont. si-cont.

MCF-7 MCF-7 MCF-7 ERE-luc MCF-7 ERE-luc pS2 mRNA p53 DMSO DMSO 16 25 E2 7 E2

6 KLF4 20 12 5 15 ERα 8 4 (fold) 10 3 βactin 4

5 2 Transcriptional activity

Relative Quantity (fold) -UV +UV

Transcriptional activity (fold) 1 0 0 142 3 146782 3 5 9 0 pGL3-basic +++ ------si-cont. si-cont. si-KLF4 pGL3-basic +-- - si-KLF4 ERE-luc - + ++ ERE-luc -++- -+ ++ +

si-cont.+ + + - si-p53+E2 UV - + - -++- ++ si-cont.+E2

si-p53 - - -+ si-cont.+DMSO si-KLF4 --+--+--+

Figure 5 p53 reduces ERa-dependent transcription by upregulating KLF4 transcription. (a) Enhanced expression of KLF4 in wild- type p53 tumor tissues in two independent studies of human breast cancer (Miller et al., 2005; Ivshina et al., 2006). Data and statistics were obtained from www.oncomine.org. (b, c) Decreased KLF4 protein levels by p53 knockdown. (b) Total RNA was isolated from control (si-cont.) or p53-specific (si-p53) siRNA-treated MCF-7 cells, and p53, KLF4 and ERa mRNA levels were quantified by real- time revere transcription (RT)–PCR. (c) Cell extracts were prepared from MCF-7 cells that were transfected with control small interfering RNA (siRNA) (si-cont.) or p53-specific siRNA (si-p53), and immunoblotted with the indicated antibodies (left panel). KLF4 protein levels were quantified. Bars represent means þ s.d. (n ¼ 3) (right graph). (d, e) Stimulation of ERa-dependent transcription by p53 knockdown. (d) A control (pGL3-basic) or luciferase reporter plasmid containing four tandem E2 response elements (ERE-luc) was transfected into MCF-7 cells that were treated with control (si-cont.) or p53-specific (si-p53) siRNA. Cell extracts derived from these cultures that were treated with or without estrogen (E2; 10À8 M) were examined using luciferase assays. Bars represent means þ s.d. (n ¼ 3). (e) Total RNA was isolated from control (si-cont.) or p53-specific (si-p53)-treated MCF-7 cells and pS2 mRNA levels were quantified by real-time RT–PCR. Bars represent means þ s.d. (n ¼ 3). (f) Increased KLF4 protein levels by p53. MCF-7 cells treated with control (si-cont.) or KLF4-specific (si-KLF4) siRNA were UV irradiated (30 J/m2), and the cell extracts were immunoblotted with the specified antibodies. (g) p53-induced reduction of ERa-dependent transcription through KLF4. A control (pGL3-basic) or luciferase reporter plasmid containing four tandem E2 response elements (ERE-luc) was transfected into MCF-7 cells that were treated with control (si-cont.) or KLF4-specific (si-KLF4) siRNA and then either left untreated or exposed to UV irradiation (30 J/m2). Cell extracts derived from these cultures were examined using luciferase assays. Bars represent means þ s.d. (n ¼ 3).

Oncogene KLF4 suppresses estrogen-dependent breast cancer K Akaogi et al 2900 Knockdown of p53 by siRNA did not affect ERa Estrogen protein and mRNA levels (Figures 5b and c). However, the transcriptional activity of the reporter plasmids that Cellular stress was increased by estrogen treatment was further potentiated by p53 knockdown (Figure 5d). Reverse transcription–PCR analysis showed that pS2 mRNA levels were upregulated by estrogen treatment and p53 α further increased by knockdown of p53 (Figure 5e). ER ERα Next, we examined the effect of ultraviolet (UV) irradiation on the transcriptional activity of ERa,asUV KLF4 irradiation induces the accumulation and activation of KLF4 p53. We performed immunoblotting using a p53-specific antibody and confirmed the accumulation of p53 protein levels (Figure 5f). Similar to the elevated p53 protein ERE levels, UV irradiation also increased KLF4 protein levels Estrogen-responsive (Figure 5f). Treating the cells with the KLF4 siRNA oncogenes effectively decreased KLF4 protein levels but did not Breast cancer affect p53 protein levels (Figure 5f). In a transient proliferation transcription assay using reporter plasmids bearing Figure 6 A model of KLF4-mediated inhibition of estrogen- EREs, the transcriptional activity of ERa induced by dependent breast cancer proliferation. See the text for details. estrogen treatment was inhibited by UV irradiation (Figure 5g, compare lanes 7 and 8). Knockdown of KLF4 pathways that are associated with tumor suppres- endogenous KLF4 in MCF-7 cells partly recovered the sion or oncogenesis (Supplementary Figure 1). Thus, transcriptional activity of ERa that was suppressed by whether KLF4 acts as a tumor suppressor or oncogene UV irradiation (Figure 5g, compare lanes 8 and 9). These is likely due to differences in the cell context, such as the results show that p53-dependent suppression of ERa expression patterns of ERa, p21 and p53 (Supple- transcriptional activity was mediated by KLF4. mentary Figure 1). As the expression of these genes is Several lines of evidence suggest that there is cross-talk regulated by genetic and epigenetic mechanisms, genetic between p53 and ERa.ERa binds directly to p53 and and epigenetic alterations may determine whether KLF4 represses its function Liu et al., 2006). p53 and ERa acts as a tumor suppressor or oncogene in breast dimers interfere with the ability of ERa to bind EREs and cancers. Therefore, the differences between our results thereby inhibit ERa-dependent transcription Yu et al., obtained from the Oncomine database (Figure 1a and b) 1997). Consistent with the earlier reports, our observa- and earlier reported data (Foster et al., 2000; Pandya tions indicate that the accumulation and activation of p53 et al., 2004) may be due to the differences in the abrogate the transcriptional activity of ERa. The result expression pattern of ERa, p21 or p53. that KLF4 knockdown recovered p53-mediated suppres- sion of ERa transcriptional activity suggests that, in addition to the direct inhibition of ERa transcriptional Materials and methods activity by p53, there is another pathway of cross-talk between p53 and ERa that involves KLF4 (Figure 6). Cell culture and transfection The results from the Oncomine database indicated MCF-7 human breast cancer cells that express wild-type p53 that the KLF4 mRNA levels were lower in breast and 293 human kidney epithelial cells were maintained in carcinomas than in normal tissues. In addition, the Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Corpora- Oncomine analysis revealed that KLF4 expression levels tion, St Louis, MO, USA). All media were supplemented with were negatively correlated with the tumor grade. On the 10% fetal bovine serum and penicillin-streptomycin mixed other hand, Ruppert’s group showed that the mRNA solution (Nakarai tesque, Kyoto, Japan). Twenty-four hours and protein levels of KLF4 were higher in breast cancer before transfection, the medium changed to phenol red-free tissues than in normal breast tissues (Foster et al., 2000; Dulbecco’s modified Eagle’s medium containing 4% charcoal- Pandya et al., 2004). stripped fetal bovine serum. The proliferation of cultured cells was measured by MTT assay using the MTT Cell Count Kit Rowland et al. (2005) recently reported that KLF4 CIP1 (Nakarai Tesque). Transfection was performed with PerFectin simultaneously represses p53 levels and induces p21 , Transfection Reagent (Genlantis, San Diego, CA, USA), the latter of which represents the dominant response, Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) and resulting in cell-cycle arrest. In the absence of p21CIP1 or TransFast Transfection Reagent (Promega, Madison, WI, the presence of RASV12-cyclin-D1 signaling, KLF4 acts USA). as a cell-cycle promoter by suppressing p53 expression. Here, we showed that KLF4 inhibits ERa target gene Expression vectors, antibodies transcription in an estrogen-dependent manner and Complementary DNAs encoding full-length and various abrogates breast cancer cell growth. Moreover, p53 truncated forms of ERa were amplified by PCR and subcloned suppresses estrogen-dependent tumor growth by in- into the pcDNA3 plasmid (Invitrogen) containing sequences creasing KLF4 levels. Our data, along with earlier coding for FLAG sequences. Similarly, full-length KLF4 reports, indicate that there are at least three distinct complementary DNA was cloned into the pCMV5 plasmid

Oncogene KLF4 suppresses estrogen-dependent breast cancer K Akaogi et al 2901 containing sequences coding for HA sequence. Mouse ChIP and real-time PCR detection anti-FLAG-M2 (Sigma-Aldrich Corporation), anti-b-Actin Chromatin immunoprecipitation assay was performed (Sigma-Aldrich Corporation) and anti-human-p53 (Santa according to the published procedure (Murayama et al., Cruz Biotechnology, Santa Cruz, CA, USA) monoclonal 2008). The primers for real-time PCR are forward: TATGAA antibodies and rabbit anti-human-ERa (Santa Cruz Biotech- TCACTTCTGGAGTG A; reverse: GAGCGTTAGATAACA nology) and anti-human-KLF4 (Abcam, Cambridge Science TTTGCC for pS2 gene upstream region. Forward: GCCCAG Park, Cambridge, UK) polyclonal antibodies were used ACAGACTTGCCCTATC; reverse: CAGGCAGCTGGATG according to the manufacturers’ instructions. TGCTGA for Lefty1gene upstream region.

Luciferase reporter assay A4Â ERE-TATA-luc reporter plasmid has been described Real-time reverse transcription–PCR earlier (Tateishi et al., 2006). Twenty-four hours after Real-time reverse transcription–PCR was performed essen- transfection, we replaced the culture medium with fresh tially as described earlier (Murayama et al., 2008). Tissues and medium containing ligands. Luciferase assays were performed cells were homogenized in 1 ml of Isogen and total RNA was on cell extracts according to the manufacturer’s protocol extracted according to the instruction manual (Nippon gene, (Promega). Individual transfections, each of which was Tokyo, Japan). Complementary DNA was synthesized from performed in three wells, were repeated at least three times. total RNA using Revatra Ace reverse transcriptase (Toyobo, Osaka, Japan) and oligo dT primer. Real-time PCRs were RNA interference performed to amplify fragments representing the indicated For transfection of siRNAs, cells at 30–50% confluency were mRNA expression. The primer sequences are as follows: GAPDH forward primer: 50-ATCGTCCACCGCAAATG transfected with 20 nm of siRNA using LipofectAmine RNAi 0 max (Invitrogen) according to the manufacturer’s protocol. All CTTCTA-3 GAPDH reverse primer: 50-AGCCATGCCAATCTCATCT siRNAs were purchased from Invitrogen. The siRNA duplexes 0 0 TGTT-3 KLF4 no. 1; 5 -UGAGAUGGGAACUCUUUGUGUAG 0 0 0 KLF4 forward primer: 5 -GCCCCTCGGGCGGCTTCGT GU-3 , KLF4 no. 2; 5 -AUCGUUGAACUCCUCGGUCU 0 0 GGCCGAGCTC-3 CUCUC-3 , and p53; Validated Stealth RNAi. Stealth RNAi 0 negative control was used as a negative control. KLF4 reverse primer: 5 -CGTACTCGCTGCCAGGG GCG-30 pS2 forward primer: 50-TGCTGTTTCGACGACACC Coimmunoprecipitation and immunoblotting GTT-30 Cells were lysed in TNE buffer (10 mm Tris-HCl (pH 7.8), pS2 reverse primer: 50-AGGCAGATCCCTGCAGA 1% Nonidet P-40 (NP-40), 0.15 M NaCl, 1 mM EDTA, 1 M AGT-30 phenylmethylsulfonyl fluoride, 1 g/ml aprotinin). Extracted p53 forward primer: 50-ATCGAGATGTTCGCTCG proteins were immunoprecipitated with antibody-coated pro- TGC-30 tein G Sepharose (GE Healthcare Bio-Sciences, Piscataway, p53 reverse primer: 50-TTTAAAGGCAGACACTGAGT NJ, USA) or anti-FLAG M2 agarose (Sigma-Aldrich Cor- CAG-30 poration) beads. Bound proteins were separated by sodium dodecyl sulfate–PAGE, transferred to polyvinylidine difluoride membranes (Millipore, Billerica, MA, USA) and detected with appropriate primary antibodies and horseradish peroxidase- conjugated secondary antibodies. Specific proteins were Conflict of interest visualized using an enhanced chemiluminescence western blot detection system (GE Healthcare Bio-Sciences). The authors declare no conflict of interest.

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

Oncogene