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C/Ebpδ Targets Cyclin D1 for Proteasome-Mediated Degradation Via Induction of CDC27/APC3 Expression

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C/Ebpδ Targets Cyclin D1 for Proteasome-Mediated Degradation Via Induction of CDC27/APC3 Expression C/EBPδ targets cyclin D1 for proteasome-mediated degradation via induction of CDC27/APC3 expression Snehalata A. Pawara,1,2, Tapasree Roy Sarkara,2, Kuppusamy Balamurugana, Shikha Sharana, Jun Wanga, Youhong Zhanga, Steven F. Dowdyb, A-Mei Huanga,3, and Esta Sternecka,4 aCenter for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201; and bDepartment of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego School of Medicine, La Jolla, CA 92093-0686 Edited* by George F. Vande Woude, Van Andel Research Institute, Grand Rapids, MI, and approved April 15, 2010 (received for review December 3, 2009) The transcription factor CCAAT/enhancer binding protein δ (C/EBPδ, breast tumor cell lines including MCF-7 (16). Addressing the CEBPD, NFIL-6β) has tumor suppressor function; however, the molecu- possibility of a causal relationship, we found that exogenous lar mechanism(s) by which C/EBPδ exerts its effect are largely unknown. C/EBPδ alone down-regulated endogenous cyclin D1 but not Here, we report that C/EBPδ induces expression of the Cdc27 (APC3) cyclin E2 in MCF-7 cells (Fig. 1A). An inverse correlation of C/ subunit of the anaphase promoting complex/cyclosome (APC/C), which EBPδ and cyclin D1 protein expression was also observed in MEFs results in the polyubiquitination and degradation of the prooncogenic from wild-type and Cebpd null mice (Fig. S1A). Next, we tested the cell cycle regulator cyclin D1, and also down-regulates cyclin B1, Skp2, role of target gene regulation by introducing an R198A mutation andPlk-1.InC/EBPδ knockout mouse embryo fibroblasts (MEF) Cdc27 into the DNA binding domain (C/EBPδ-R198A), which does levels were reduced, whereas cyclin D1 levels were increased even in not affect nuclear localization (17) but prevents DNA binding of the presence of activated GSK-3β. Silencing of C/EBPδ, Cdc27, or the C/EBPδ (Fig. S2A). This mutation abolished the ability of C/EBPδ APC/C coactivator Cdh1 (FZR1) in MCF-10A breast epithelial cells in- A creased cyclin D1 protein expression. Like C/EBPδ, and in contrast to to down-regulate cyclin D1 in MCF-7 cells (Fig. 1 ; see also C C fi cyclin D1, Cdc27 was down-regulated in several breast cancer cell lines, Figs. 2 and 3 ) and signi cantly diminished its ability to inhibit suggesting that Cdc27 itself may be a tumor suppressor. Cyclin D1 is cell growth (Fig. 1B). Surprisingly, there was only a minor effect of a known substrate of polyubiquitination complex SKP1/CUL1/F-box C/EBPδ on cyclin D1 mRNA levels, which was not significantly (SCF), and our studies show that Cdc27 directs cyclin D1 to alternative altered by the R198A mutation (Fig. 1C). Similar data were degradation by APC/C. These findings shed light on the role and regu- obtained in the untransformed MCF-10A human breast epithelial lation of APC/C, which is critical for most cellular processes. cells (Fig. 1D) and MEFs (Fig. S1B), suggesting that C/EBPδ down-regulates cyclin D1 primarily at the protein level. breast cancer | tumor suppressor | cell cycle | anaphase promoting complex | Cdh1/FZR1 C/EBPδ Regulates Genome Integrity Through Modulation of Cyclin D1 Protein Levels. Cyclin D1 levels decline at S-phase entry, which is he transcription factor CCAAT/enhancer binding protein δ necessary for orderly DNA replication to occur. Excess cyclin D1 T(C/EBPδ, CEBPD, NFIL-6β) is down-regulated in several during S-phase can lead to DNA damage and genomic instability types of tumors, including cervix, liver, and breast (1–5). In vitro, (18, 19). Because loss of C/EBPδ leads to genomic instability in C/EBPδ inhibits the growth of tumor cell lines, is associated primary MEFs (9), we asked whether this was due to the increased with G0 growth arrest, or induces differentiation (6–8), and pro- cyclin D1 protein level. Cells with damaged DNA exhibit histone motes genomic stability of mouse embryo fibroblasts (MEFs) (9). 2AX phosphorylation (γH2AX) and nuclear γH2AX staining foci C/EBPδ expression correlates with low proliferation and histo- mark sites of DNA damage (20, 21). Accordingly, immortalized logical grade in meningiomas (10) and was in a 70-gene signature C/EBPδ KO MEF cultures harbor more cells with increased num- predicting better survival of breast cancer patients (11). Despite bers of nuclear γH2AX foci than control cells and increased levels δ this overwhelming evidence that C/EBP is a tumor suppressor of γH2AX in cellular extracts (Fig. 2A). Knockdown of cyclin D1 to and inhibits cell growth, the molecular mechanism for this acti- levels as in wild-type MEFs significantly decreased γH2AX levels ity is largely unknown. (Fig. 2B). On the other hand, overexpression of cyclin D1 in WT Mice with a C/EBPδ gene deletion undergo delayed postlacta- MEFs phenocopied the elevated γH2AX levels of KO MEFs (Fig. tional mammary gland involution due to attenuated mammary epi- S1C). Ectopic wild-type C/EBPδ but not the R198A mutant reduced thelial cell (MEC) apoptosis. Gene expression analysis showed that δ cyclin D1 protein levels and H2AX phosphorylation in KO MEFs C/EBP induction during involution correlated with repression of C δ cyclin D1 expression (12). In vitro, C/EBPδ overexpression in (Fig. 2 ). These data suggest that C/EBP prevents DNA damage in a mouse MEC line accelerated the decline of cyclin D1 expression part by controlling cyclin D1 protein levels. upon serum withdrawal (6). Cyclin D1, the regulatory subunit of cyclin-dependent kinases 4 and 6, promotes the G1 toStransition of the cell cycle (13). In contrast to C/EBPδ, cyclin D1 is highly Author contributions: S.A.P., T.R.S., K.B., S.S., J.W., Y.Z., and E.S. designed research; S.A.P., > T.R.S., K.B., S.S., J.W., and Y.Z. performed research; S.F.D. and A.-M.H. contributed new expressed in 50% of breast tumors (13), and mouse models have reagents/analytic tools; S.A.P., T.R.S., K.B., S.S., J.W., Y.Z., and E.S. analyzed data; and established cyclin D1 as a key driver of mammary oncogenesis (14, S.A.P. and E.S. wrote the paper. 15). Cyclin D1 promotes cancer even by additional, cell cycle in- The authors declare no conflict of interest. ’ dependent functions (13). Thus, a cell s ability to control cyclin D1 is *This Direct Submission article had a prearranged editor. important to prevent tumor formation, and our understanding of how 1Present address: Department of Pharmaceutical Sciences, University of Arkansas for Med- cyclin D1 expression is regulated can provide valuable insight into the ical Sciences, Little Rock, AR 72205. mechanisms of oncogenesis and potential therapeutic avenues. 2S.A.P. and T.R.S. contributed equally to this work. 3Present address: Institute of Biochemistry, College of Medicine, Kaohsiung Medical Uni- Results versity, Kaohsiung 807, Taiwan. C/EBPδ Down-Regulates Cyclin D1 Protein Expression. It was repor- 4To whom correspondence should be addressed. E-mail: [email protected]. δ ted that C/EBP inhibits the growth of MCF-7 breast tumor cells This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (8) and that inhibition of cyclin D1 expression inhibits growth of 1073/pnas.0913813107/-/DCSupplemental. 9210–9215 | PNAS | May 18, 2010 | vol. 107 | no. 20 www.pnas.org/cgi/doi/10.1073/pnas.0913813107 Downloaded by guest on October 2, 2021 Fig. 1. C/EBPδ inhibits cyclin D1 expression and cell growth. (A) Western blot Fig. 2. Elevated levels of γH2AX in C/EBPδ-deficient MEFs are due to cyclin analyses of MCF-7 cells 24 h after transfection with vector or a wild-type or D1. (A) Representative Western blot analysis of total H2AX and Ser139 R198A mutant C/EBPδ expression construct as indicated. A Right is derived phosphorylated H2AX (γH2AX) in whole cell extracts of exponentially from one membrane with intermediate lanes deleted. Note the lower level of growing wild-type (WT) and C/EBPδ null (KO) MEFs and distribution of cells protein loading (β-actin) in the R198A lane. (B) MCF-7 cell survival after according to number of γH2AX foci in their nuclei. Data are from three in- transfection with the indicated C/EBPδ expression constructs or vector control dependent cell preparations per genotype with at least 100 cells counted and 2 weeks of drug selection. Living cells were quantified by spectropho- each (mean ± SEM). **, P < 0.01; ***, P < 0.001. (B) Western blot analysis of tometry of GIEMSA stain by OD at 630 nm. Data from seven independent MEFs 48 h after nucleofection with siRNA against cyclin D1 or nontargeting experiments are expressed relative to vector control (mean ± SEM). **, P < control siRNA as indicated. (C) Western blot analysis of whole cell lysates 0.005. (C) Q-PCR analysis of cyclin D1 mRNA levels in MCF-7 cells treated as in A. from WT or KO MEFs 48 h after transient transfection with vector (-) or Data (mean ± SEM) are from five independent experiments analyzed in trip- expression constructs for human wild-type C/EBPδ (WT) or the R198A mutant licates, normalized to actin, and relative to vector control. ***, P < 0.0001; (R198A). Transfection efficiency was estimated at 60% of the cultures based *, P < 0.05. The difference between the two C/EBPδ constructs was not sta- on a cotransfected GFP-expression construct. tistically significant. (D) MCF-10A cells were transiently transfected with a C/EBPδ expression construct or empty vector, and RNA and whole-cell pro- tein extracts were prepared 24 and 48 h later. Q-PCR analysis of cyclin hibition on cyclin D1 protein expression can be ruled out.
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