Oncogene (2008) 27, 366–377 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE The AP-1 transcription factor regulates breast cancer cell growth via cyclins and E2F factors Q Shen, IP Uray, Y Li, TI Krisko, TE Strecker, H-T Kim and PH Brown Breast Center, Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA The activating protein-1 (AP-1) transcription factor Introduction transduces growth signals through signal transduction pathways to the nucleus, leading to the expression of genes The activating protein-1 (AP-1) family of transcription involved in growth and malignant transformation in many factors consists of multiple Jun (cJun, JunB and JunD) cell types. We have previously shown that overexpression and Fos (cFos, FosB, Fra-1 and Fra-2) members (Angel of a dominant negative form of the cJun proto-oncogene, a and Karin, 1991). The Jun members either homodimer- cJun dominant negative mutant (Tam67), blocks AP-1 ize with Jun itself or heterodimerize with different Fos transcriptional activity, induces a G1 cell cycle block and members, whereas Fos members heterodimerize only inhibits breast cancer cell growth in vitro and in vivo.We with Jun members. Thus, Jun is the predominant found that AP-1 blockade byTam67 in MCF-7 breast partner to form AP-1 complexes such as Jun:Jun or cancer cells downregulates cyclin D1 transcriptional Jun:Fos dimers. The AP-1 complex converges multiple activitybyat least two mechanisms: bysuppressing trans- growth signals at the transcriptional level, making it a cription at the known AP-1 binding site (À934/À928) critical connecting node for many signal transduction and bysuppressing growth factor-induced expression pathways. We and other investigators have shown that through suppressing E2F activation at the E2F-responsive AP-1 regulates cellular proliferation, differentiation, site (À726/À719). AP-1 blockade also led to reduced apoptosis, oncogene-induced transformation and cancer expression of E2F1 and E2F2, but not E2F4, at the cell invasion (McDonnell et al., 1990; Szabo et al., 1991; mRNA and protein levels. Chromatin immunoprecipita- Brown et al., 1993). Proliferation of breast cells requires tion and supershift assays demonstrated that AP-1 signals from growth factors such as estrogen, epidermal blockade caused decreased binding of E2F1 protein to growth factor (EGF), transforming growth factor a the E2F site in the cyclin D1 promoter. We also found that (TGFa), heregulin and insulin-like growth factors Tam67 suppressed the expression of the E2F1 dimerizing (IGFs), and these factors activate AP-1 signaling. partner, DP1 and E2F-upregulated cell cycle genes Therefore, blockade of AP-1 complex may arrest multi- (cyclins E, A, B and D3) and enhanced the expression of ple growth signals important for breast cell proliferation E2F-downregulated cell cycle genes (cyclins G2 and I). and transformation. Reduced expression of other E2F-regulated genes was also We have previously shown that AP-1 blockade seen with AP-1 blockade and E2F suppression. Thus, the induced by expression of a specific AP-1 inhibitor (a AP-1 factor regulates the expression of cyclin D and E2F cJun dominant-negative mutant, Tam67) suppressed the (the latter in turn regulates E2F-downstream genes), growth of breast cancer cells induced by many growth leading to cell cycle progression and breast cancer cell factors such as estrogen, EGF, heregulin and IGF-1 proliferation. (Liu et al., 2002). We also demonstrated that AP-1 Oncogene (2008) 27, 366–377; doi:10.1038/sj.onc.1210643; blockade by Tam67 arrested cell cycle at the G1 phase published online 16 July 2007 (Liu et al., 2004). In addition, G1 cyclins were down- regulated at the mRNA and protein levels when breast Keywords: AP-1 factor; proliferation arrest; cyclin D1; E2F cancer cells were synchronized at the G2/M phase and factors; gene expression regulation; breast cancer cells released from synchronization by serum stimulation (Liu et al., 2004). However, the explicit mechanism by which AP-1 blockade regulates the cell cycle remains unclear. Potential consensus AP-1 binding sites or AP-1-like binding sites have been found in the promoter of the cyclin D1 gene (Herber et al., 1994). In the present study, we measured cyclin D1 expression at the mRNA and protein levels in MCF-7 cells synchronized at G0/G1 Correspondence: Dr PH Brown, Breast Center, Baylor College of phases, and determined that the consensus AP-1 binding Medicine, One Baylor Plaza, MS600, Houston, TX 77030, USA. E-mail: [email protected] site is responsible for maintaining basal cyclin D1 Received 18 August 2006; revised 22 May 2007; accepted 23 May 2007; promoter activity. We further demonstrated that an published online 16 July 2007 E2F site present in the cyclin D1 promoter was required AP-1 blockade downregulates cyclins and E2F1 Q Shen et al 367 for the Tam67-induced suppression of cyclin D1 We next mutated the E2F sites at À726/À719 and expression. We then demonstrated that AP-1 and E2F À139/À131 (Figure 2b). Mutation of the E2F site at factors bind to AP-1 and E2F binding sites in the cyclin À139/À131 resulted in a slight decrease in promoter D1 promoter. We also determined that E2F1 and E2F2, activity, while the Tam67-induced suppression remained and their dimerizing partner DP1 were downregulated the same. Mutation of the E2F site at À726/À719 by Tam67, as were many E2F downstream genes. Thus, caused loss of Tam67-induced suppression of promoter the AP-1 transcription factor regulates breast cancer cell activity (Figure 2b). As shown in Figure 2c, the growth via multiple mechanisms, including regulation of fragment containing the E2F site at À726/À719 was cyclin D1, E2F factors and their target genes. These ligated back to the shortest construct used that does not findings support that the AP-1 transcription factor is a have the E2F site at À726/À719, and the resulting critical regulator of breast cell proliferation, making this promoter construct restored the repression by Tam67. factor a potential target for the treatment and preven- These results confirm the importance of the E2F site at tion of breast cancer. À726/À719 in the AP-1 regulation of cyclin D1. Our studies demonstrate that AP-1 regulates cyclin D1 expression by at least two mechanisms: activation of basal cyclin D1 promoter activity via the AP-1 site and Results further by increasing E2F activity. Thus both the AP-1 and E2F factors play important roles in regulating AP-1 blockade by Tam67 downregulates cyclin D1 at the cyclin D1 expression in breast cancer cells. mRNA and protein levels in MCF-7 cells We have previously shown that AP-1 blockade by Tam67 reduced cyclins D1, D2, D3 and cyclin E at the Interaction of the AP-1 inhibitor, Tam67, with the mRNA and protein level in MCF-7 cells synchronized at promoter elements of cyclin D1 gene et al G2/M phase (Liu ., 2004). Here, we examined the We next performed chromatin immunoprecipitation effect of Tam67 on cyclin D1 expression in MCF-7 cells (ChIP) assays to demonstrate in vivo interaction between synchronized at G /G phase and released from 0 1 Tam67 and the AP-1 or E2F site within the cyclin D1 synchronization by serum stimulation. We confirmed promoter. Using cJun and cFos antibodies as positive that Tam67 expression suppressed cyclin D1 mRNA by controls, we found that cJun and cFos bound to the more than 50% in these cells (P 0.0004) (Figures 1a ¼ DNA fragments containing the AP-1 sites of either a and b). Tam67 reduced the expression of cyclin D1 positive control gene (collagenase/MMP-1) or the cyclin mRNA, while control cells still show a cyclic pattern D1 gene (Figure 3a). As shown using an anti-flag (Figure 1b). Tam67 also reduced the protein level of antibody in these ChIP experiments, flag-tagged Tam67 cyclin D1 (Figures 1c and d). Note that the maintained was found to bind the AP-1 sites of the control MMP-1 cyclic pattern of cyclin D1 protein expression still occurs and cyclin D1 promoters in doxycycline-untreated, thus suggesting that other mechanisms such as proteolysis Tam67-induced MCF-7 cells, but not in control cells still contribute to the degradation of cyclin D1. (Figure 3a). This suggests that Tam67 complex does bind to the AP-1 site in the cyclin D1 promoter, Tam67 suppresses cyclin D1 promoter activity supporting the conclusion from Figure 2 that AP-1 Since Tam67 suppressed cyclin D1 mRNA expression, maintains basal cyclin D1 promoter function. The ChIP we investigated whether the transcription factors that assays also show that the E2F1 protein, but not cJun or bind at the AP-1 site regulate cyclin D1 transcription. cFos, bound to the E2F site in the cyclin D1 promoter. Potential binding sites for AP-1, E2F, nuclear factor-kB To further verify the in vivo binding results, we (NF-kB) and Sp1 factors were found in the promoter performed in vitro electrophoretic mobility shift assay region of À2963 to þ 361 of the cyclin D1 promoter (EMSA) and supershift assays to determine direct or (Herber et al., 1994). To determine the cis-elements indirect interaction of Tam67 and E2F1 proteins to responsible for the Tam67-induced inhibition, we respective binding sites. We found that Tam67 is examined the promoter activity for cyclin D1 gene in capable of binding the AP-1 site in the cyclin D1 the presence and absence of Tam67 expression. We promoter (Figure 3b, lane 7), consistent with our results found that deletion of the AP-1 site at –934/À928 of reporter assays showing suppression of the basal reduced cyclin D1 promoter activity (in the absence of cyclin D1 promoter activity (Figure 2).