RBCK1 Drives Breast Cancer Cell Proliferation by Promoting Transcription of Estrogen Receptor Α and Cyclin B1

Home , ARNTL

Published OnlineFirst January 26, 2010; DOI: 10.1158/0008-5472.CAN-09-2674 Published Online First on January 26, 2010 as 10.1158/0008-5472.CAN-09-2674

Tumor and Stem Cell Biology Cancer Research RBCK1 Drives Breast Cancer Cell Proliferation by Promoting Transcription of Estrogen Receptor α and Cyclin B1

Nina Gustafsson1, Chunyan Zhao1, Jan-Åke Gustafsson1,2, and Karin Dahlman-Wright1

Abstract Cell cycle regulatory pathways in breast cancer are incompletely described. Here, we report an important role in estrogen receptor α (ERα)–positive breast cancer cells for the protein kinase C1 (PKC1)–interacting protein RBCK1 in supporting cell cycle progression by driving transcription of ERα and cyclin B1. RBCK1-

depleted cells exhibited increased accumulation in G2-M phase of the cell cycle, decreased proliferation, and reduced mRNA levels for ERα and its target genes cyclin D1 and c-myc. Chromatin immunoprecipitation revealed that ERα transcription is associated with RBCK1 recruitment to the ERα promoter, suggesting that

transcriptional regulation is one mechanism by which RBCK1 affects ERα mRNA levels. G2-M phase arrest was mediated independently from reduced ERα levels, instead associated with transcriptional inhibition of the key

G2-M regulator cyclin B1. In breast tumor samples, there was a positive correlation between levels of RBCK1, ERα, and cyclin B1 mRNA levels. Our findings suggest that RBCK1 regulates cell cycle progression and proliferation of ERα-positive breast cancer cells by supporting transcription of ERα and cyclin B1. Cancer Res; 70(3); 1265–74. ©2010 AACR.

Introduction 90% of breast tumors have an altered expression of one or several regulatory proteins in the G1-S phase transition (9, 10). The cell cycle consists of several controlled phases that Altered expression of cell cycle regulators in breast cancer lead to DNA replication and cell division. The progression has been best described and studied for the G1-S phase tran- of these phases is strictly controlled by the timely expression sition. However, key regulators of the G2-M phase, such as and activation of activating and inhibitory proteins, such as cyclin B1, Polo-like kinase 1 (Plk-1; ref. 11), and the cdc25B different cyclins and their associated cyclin-dependent ki- (12) phosphatase, are all overexpressed in breast cancer nases (CDK; ref. 1). Alterations in the expression of cell cycle cells (13–15). regulatory proteins are commonly found in many cancers (2). RING finger–containing E3 ubiquitin ligases have emerged Estrogens promote mammary epithelial cell proliferation as important proteins in breast cancer, in many cases dis- and growth of estrogen-dependent breast cancer cells by playing altered expression levels (16), and are therefore in- stimulating the expression of genes encoding cell cycle regu- teresting diagnostic markers and drug targets (17). The latory proteins (3). The biological effects of estrogens are me- Oncomine database3 identifies studies in which the E3 ubi- diated through binding to two estrogen receptor (ER) quitin ligase RBCC protein interacting with protein kinase C1 isoforms, ERα and ERβ, which are members of the nuclear (RBCK1; Genbank accession NP_112506; ref. 18) has an elev- receptor superfamily of transcription factors (4, 5). Estrogen- ated mRNA expression in breast cancer compared with nor- ic control of the cell cycle in breast cancer cells has been well mal breast tissue (P < 0.001; ref. 19) and elevated mRNA α described for the G1-S phase transition (6). ER increases the levels in ERα-positive human breast cancer compared with myc expression of cyclin D1 and c- in the presence of estradiol. ERα-negative human breast cancer tissue (P < 0.001; ref. 20). Both of these factors promote cell cycle progression by de- The human RBCK1 gene product has a predicted length of creasing the association between the cyclin E/CDK2 complex 510 amino acids. The NH2-terminal portion (1–250) of this Cip1/WAF1 Kip1 and the two CDK inhibitors p21 and p27 , an essen- protein contains an ubiquitin-like domain and a RanBP2- tial step for successful G1-S phase transition (7, 8). More than type zinc finger domain. Residues 282 to 332 comprise a C3HC4-type RING zinc finger domain, a domain that has Authors' Affiliations: 1Department of Biosciences and Nutrition, been implicated in protein-protein interactions and DNA Karolinska Institute, Huddinge, Sweden and 2Center for Nuclear binding (Supplementary Fig. S1A). RBCK1 possess an auto- Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, Texas ubiquitination activity (18), a characteristic of E3 ubiquitin Note: Supplementary data for this article are available at Cancer ligases, through its RING-between rings-RING (RBR; ref. 21) Research Online (http://cancerres.aacrjournals.org/). domain in the COOH-terminal portion of the protein (282– Corresponding Author: Nina Gustafsson, Department of Biosciences 493). Bioinformatics further reveals that the human RBCK1 and Nutrition, Karolinska Institute, Novum, S-141 57 Huddinge, Sweden. Phone: 46-8-608-9145; Fax: 46-8-774-5538; E-mail: [email protected]. doi: 10.1158/0008-5472.CAN-09-2674

www.aacrjournals.org 1265

Gustafsson et al.

protein is homologous to E3 ligases involved in cell cycle reg- was used to produce standard curves as 2-fold serial dilu- ulation such as CUL9, RNF144b/p53RFP, and RNF14/ARA54 tions; Ct values were converted to nanograms; and RPLP0 (Supplementary Fig. S1B; refs. 22–24). and GAPDH were used as reference genes. Primer sequences RBCK1 was originally identified as a protein kinase C are given in Supplementary Table S1. For the analysis of (PKC) subtypes η (25), β (26), and ζ (27) interacting protein. ERα-specific transcripts, primer sequences have been pub- In rat cardiac cells, RBCK1 plays a role in PKCβ-dependent lished before (34). cell growth (26). Interestingly, the PKC family of isozymes Chromatin immunoprecipitation. MCF-7 cells were seed- has higher activity in breast cancer compared with normal ed in 150-mm dishes and were transfected with siRNA breast tissue (28). All the PKC subtypes interacting with against RBCK1 or siControl after 24 h. Chromatin was pre- RBCK1 are important for progression into the S phase in pared 72 h posttransfection as previously described (35). breast cancer cells and for estrogen signaling (29–32). Chromatin fractions were immunoprecipitated with 1 μgof Based on the role of RBCK1-interacting proteins in breast the indicated antibodies, and the immune complexes were cancer and estrogen signaling, exhibiting homology to cell recovered using protein A/G-Sepharose (50% slurry; Phar- cycle regulatory proteins together with overexpression in macia) and processed as described by Matthews and col- ERα-positive breast cancer, we hypothesized that RBCK1 leagues (35). The antibodies used were RBCK1 (Santa might have a regulatory function in cell cycle progression Cruz Biotechnology) and mouse anti-human IgG (Santa in breast cancer cells. Cruz Biotechnology). Immunoprecipitated DNA was quantified by RT-PCR. Input DNA was used to produce standard curves as 2-fold serial dilutions and chromatin immuno- Materials and Methods precipitation (ChIP) data were converted to percentages of total input. Primer sequences are given in Supplemen- Cell culture. Original MCF-7 human epithelial breast can- tary Table S1. cer cells developed at the Michigan Cancer Foundation (33) Quantification of cell viability. MCF-7 cells were seeded were kindly provided by Dr. Robert P.C. Shiu (University of into 96-well plates 24 h before siRNA transfections. Metabolic Manitoba, Winnipeg, Manitoba, Canada). Cells were cultured activity was measured with WST-1 cell proliferation reagent in DMEM (Invitrogen) supplemented with 10% fetal bovine (Roche), and the number of viable/proliferating cells was serum and 1% penicillin/streptomycin (Invitrogen). Cells quantified at 450 nm using a plate reader (Powerwave X were incubated at 37°C in 5% CO2.A10μmol/L stock solu- Select, Bio-Tek Instruments, Inc., KC4). tion of 17β-estradiol (E2; Sigma) was prepared in ethanol. Ex- Fluorescent-activated cell sorting. The experimental out- periments evaluating E2 treatment, FCS with dextran-coated lines are described in detail in the supplementary materials. charcoal, and DMEM without phenol red were used. Ethanol Cells were harvested and fixed in 70% ethanol for 30 min on was used as vehicle control. ice followed by staining with propidium iodide. Staining Small interfering RNA. The individual siRNA constructs was measured using flow cytometry by the fluorescence in- are as follows: RBCK1, Individual Stealth Select siRNA referred tensity (FL-1, 530 nm) of 10,000 cells, data acquisition was to in this study as siRBCK1 oligo 1 and 2, respectively; ERα, done on a BD LSR II flow cytometer (BD Biosciences), and Individual Stealth Select siRNA, referred to in this study as data were analyzed using the software supplied by the siERα oligo 1 and 2, respectively; and control siRNA, Individual manufacturer. Stealth Select siRNA called siControl (Invitrogen). siRNA Human breast tumor samples. The 13 breast tumor sam- transfections were carried out using a final concentration of ples in this study have been previously described (36). The 50 nmol/L oligo (at 40–60% confluence) by using INTERFERin ethical committee of the Karolinska Institute approved transfection reagent (Polyplus Transfection) according to the studies. the manufacturer's recommendation. Statistics. All experiments were carried out in triplicates. Western blot analysis. Cells were seeded into six-well Student's t test, Pearson correlation coefficient, or multiple plates 24 h or into 10-cm plates 48 h before transfection, regression analysis (ANOVA) was used for group compari- and then lysed 72 h posttransfection in Laemmli sample sons. A P value of <0.05 was considered significant. loading buffer. For immunoblotting, the primary antibodies used were as follows: monoclonal mouse β-actin antibody (Sigma Aldrich), polyclonal goat RBCK1, polyclonal rabbit Results cyclin B1, and ERα antibody (Santa Cruz Biotechnology). RNA isolation and quantitative real-time PCR. MCF-7 RNAi depletion of RBCK1 reduces cell proliferation by cells were seeded into six-well plates 24 h before siRNA trans- inducing G0-G1 and G2-M cell cycle arrest. RBCK1 expression fection. Total RNA was extracted using the RNeasy kit (Qia- in the human breast cancer cell line MCF-7 was shown by gen). Two micrograms of total RNA were reverse transcribed immunoblotting and RT-PCR (Fig. 1A). To determine the into cDNA using SuperScript III reverse transcriptase (Invi- role of RBCK1 in cell proliferation and control of the cell trogen) with random hexamers. Reverse transcription-PCR cycle, we used siRNAs to decrease RBCK1 levels in MCF-7 (RT-PCR) measurements were performed in triplicate in a cells as shown in Fig. 1A. Cells transfected with either of 7500 ABI Real-time PCR thermocycler (Applied Biosystems) two RBCK1 siRNAs grew markedly slower than cells trans- using the SYBR Green reagent (Applied Biosystems). cDNA fected with siRNA control (data not shown). Furthermore,

1266 Cancer Res; 70(3) February 1, 2010 Cancer Research

Role of RBCK1 in Cell Cycle Progression and Breast Cancer

Figure 1. RBCK1 knockdown inhibits the viability of MCF-7 cells and induces G1 and G2-M arrest. A, siRNA-mediated knockdown of RBCK1 reduces RBCK1 mRNA and protein levels 72 h after transfection. Cells were transfected with RBCK1 siRNA duplex 1, 2, or siControl. RBCK1 mRNA levels were determined by quantitative RT-PCR with each mRNA quantification performed in triplicate. ***, P < 0.001 for siRBCK1 versus siControl (siCtrl). RBCK1 protein levels were determined by immunoblot, and β-actin was used as loading control. A representative of three independent experiments is shown. B and C, MCF-7 cells were transfected with either a 1:1 mixture of RBCK1-targeting siRNA duplex 1, 2, or siControl. B, RBCK1 depletion reduces proliferationin MCF-7 cells. WST-1 assays of cell metabolic activity were carried out at the indicated time points after siRNA transfection. ***, P < 0.001 for siRBCK1 versus siControl. C, RBCK1 knockdown induces arrest in the G2-M phase and inhibits the E2-mediated progression into the S phase. Cells were treated as described in Supplementary Fig. S2B. The proportion of MCF-7 cells in the S, G2-M, and G0-G1 phases as measured by FACS. ***, P < 0.001 for siRBCK1 versus siControl. Columns, mean of three independent experiments; bars, SD.

we observed that RBCK1-depleted cells displayed a time- Experiments performed in T-47D breast cancer cells showed dependent decrease in proliferation compared with cells similar results (data not shown). transfected with siRNA control (Fig. 1B). Reduction of RBCK1 levels reduces the levels of ERα, Next, we determined the effect of RBCK1 depletion on potentially by reduced recruitment of RBCK1 to the E2-dependent cell cycle progression. The estrogen-induced ERα promoter. As RBCK1 depletion reduced E2-stimulated entry into the S phase was completely abolished in the cell proliferation, we investigated the effects of RBCK1 deple- RBCK1-depleted cell population (Supplementary Fig. S2A tion on ERα levels. RBCK1-depleted cells showed a decrease and B; Fig. 1C). Additionally, we observed a significantly in ERα protein levels both in vehicle- and E2-treated cells higher proportion of cells in the G2-M phase after RBCK1 (Supplementary Fig. S2A; Fig. 2A), suggesting that the depletion in both the E2- and vehicle-treated cell popula- observed inhibition of E2-dependent entry into the S phase tions (Supplementary Fig. S2B; Fig. 1C). This was associated after RBCK1 depletion is due to reduced ERα levels. with a decrease in the number of cells in the G0-G1 phase We further investigated the effects of RBCK1 depletion on (Supplementary Fig. S2B; Fig. 1C). These data suggest that the mRNA levels of ERα and ERα target gene mRNA levels. RBCK1 depletion desensitizes the cells to estrogen, de- RBCK1-depleted cells had significantly decreased ERα mRNA creasing the E2-dependent entry into the S phase, and addi- levels for both vehicle- and E2-treated cells (Fig. 2B). Im- tionally causes an E2-independent arrest in the G2-M phase. portantly, RBCK1 depletion reduced the expression of the

www.aacrjournals.org Cancer Res; 70(3) February 1, 2010 1267

Gustafsson et al.

Figure 2. RBCK1 depletion affects ERα levels and signaling in MCF-7 cells. A, RBCK1 depletion significantly reduces ERα protein. Cells were treated as described in Supplementary Fig. S2B. RBCK1 and ERα protein levels as determined by immunoblot; β-actin was used as loading control. Shown is a representative of three independent experiments. B, RBCK1 depletion significantly reduces ERα mRNA and ERα target genes. Cells were transfected with RBCK1 siRNA duplex 1, 2, or siControl for 72 h and were stimulated with 10 nmol/L E2 or vehicle for the last 4 h before RNA extraction. ERα and ERα target genes [pS2, cyclin D1 (cyc D1), and c-myc] mRNA levels were determined by quantitative RT-PCR. *, P < 0.05; **, P <0.01forallsiRBCK1 treatments versus siControl. C, RBCK1 is recruited to the ERα promoter B, and RBCK1 depletion inhibits the expression of ERα mRNA (A) and ERα mRNA (B). Left, quantitative RT-PCR analysis of cell extracts 72 h after transfection with RBCK1 siRNA duplex 1, 2, or siControl. ***, P < 0.001 for siRBCK1 treatments versus siControl. Right, the recruitment of RBCK1 was examined by ChIP assays in the siControl- and siRBCK1- transfected cells. Immunoprecipitated DNA with the RBCK1 antibody (white columns) or IgG control (black columns) was quantified by RT-PCR. Data are expressed as percentages of input. Columns, mean of three independent experiments; bars, SD. D, mRNA levels of RBCK1 and ERα display a positive correlation in breast tumor samples (n = 13). RBCK1 and ERα mRNA levels were quantified by RT-PCR and normalized to RPLP0.

ERα-responsive genes pS2 (37), cyclin D1, and c-myc, show- pressedinMCF7cells,withERα transcripts derived from ing a decrease in ERα signaling (Fig. 2B). Decreased ERα sig- promoter B showing the highest expression as previously de- naling was also observed in T-47D cells under similar scribed (39). Transfection with siRNA-targeting RBCK1 re- conditions (data not shown). sulted in significantly decreased transcripts derived from Seven promoters have been identified for the ERα gene promoters A and B, whereas transcripts derived from pro- (38). We found ERα transcripts derived from promoters A, moter E2 was not changed (Fig. 2C, left). To assay the mech- B, and E2 (referred to as ERα mRNA A, B, and E2) to be ex- anism behind the promoter-specific downregulation of ERα,

1268 Cancer Res; 70(3) February 1, 2010 Cancer Research

Role of RBCK1 in Cell Cycle Progression and Breast Cancer

we investigated if endogenous RBCK1 associates with the between RBCK1 and ERα mRNA levels (r2 =0.31;P <0.05; ERα promoters. We performed ChIP assays to detect the re- Fig. 2D). Taken together, our data suggest that RBCK1 recruitment of RBCK1 to promoters A, B, and E2, respectively. cruitment to the ERα promoter regulates ERα expression, RBCK1 was recruited to the ERα promoter B and the recruit- and thereby estrogen signaling, in breast cancer cells. ment was reduced after RBCK1 depletion (Fig. 2C, right). ERα depletion does not affect the progression of the However, we could not observe the recruitment of RBCK1 G2-M phase. To directly determine the importance of re- to promoters A and E2 (Fig. 2C, right). duced ERα expression and signaling, which was observed up- α The determination of RBCK1 and ER mRNA levels in on RBCK1 depletion, for the S phase and G2-M phase in ductal breast cancers showed a strong positive correlation MCF-7cells,weperformedasiRNA-mediatedknockdown

Figure 3. ERα depletion affects G1-S progression, but not G2-M progression, in MCF-7 cells. A and B, cells were transfected with ERα siRNA duplex 1, 2, or siControl (siCtrl) for 72 h. A, siRNA-mediated knockdown of ERα significantly reduces ERα mRNA levels. Quantitative RT-PCR analysis of total RNA with each mRNA quantification performed in triplicate. **, P < 0.01 for siRBCK1 versus siControl. B, ERα depletion significantly reduces ERα protein levels.

β-Actin was used as loading control. C, ERα knockdown induces G1 arrest. Cells were treated as described in Supplementary Fig. S3B. The proportion of MCF-7 cells in the S, G2-M, and G0-G1 phases as measured by FACS. ***, P < 0.001 for siRBCK1 versus siControl. Columns, mean of three independent experiments; bars, SD. D, ERα depletion significantly reduces ERα protein to similar levels as in RBCK1-depleted cells. Cells were treated as described in Supplementary Fig. S3B, and ERα protein levels were determined by immunoblot; β-actin was used as loading control. Shown is a representative of three independent experiments.

www.aacrjournals.org Cancer Res; 70(3) February 1, 2010 1269

Gustafsson et al.

of ERα.ERα mRNA and protein levels were reduced 72 hours posttransfection (Fig. 3A and B). As for RBCK1-depleted cells, the estrogen-induced progression into the S phase was abolished in ERα-depleted cells (Supplementary Fig. S3A and B; Fig. 3C), with a corresponding increase in the number of cells in the G0-G1 phase (Fig. 3C). These results are in agreement with previous studies about the role of ERα in cell cycle progression (6). Additionally, we observed no significant differ- ence in the number of cells in the G2-M phase (Fig. 3C). ERα protein levels were reduced in vehicle- and E2-treated cells (Supplementary Fig. S3A; Fig. 3D) to similar levels as in RBCK1-depleted cells (Fig. 1D). These data are consistent with that the inhibitory effect of RBCK1 knockdown on the G1-S phase progression in MCF-7 cells is related to the inhibition of ERα signaling while the RBCK1-induced G2-M cell cycle arrest occurs in an ERα-independent manner. RBCK1 depletion affects the expression of genes crucial for mitotic entry. The frequency of double nuclei in RBCK1- depleted cells was the same as in control cells (data not shown). This indicates that the arrest in the G2-M phase due to RBCK1 depletion occurs in the G2 or in the very early M phase (40). To pursue a potential mechanism for the G2-M arrest upon RBCK1 depletion, we analyzed the mRNA levels of genes crucial for the transition from the G2 to the M phase: cyclin B1, Plk-1, cdc25B, and cyclin B2. After 72 hours of knockdown of RBCK1, we found cyclin B1, Plk-1, and cdc25B, but not cyclin B2, mRNA levels to be decreased both in vehicle- and E2-treated cells (Fig. 4). Thus, decreased levels of cyclin B1, Plk-1, and cdc25B are associated with the observed G2-M arrest upon RBCK1 depletion. Downregulation of cyclin B1 precedes G2-M arrest in RBCK1-depleted cells. Because the expression of several genes important for G2-M progression was affected after RBCK1 depletion, we conducted a time course study to investigate the series of events from RBCK1 depletion to G2-M arrest. MCF-7 cells were synchronized in minimal medium for 24 hours and were released by changing to a regular medium, at which point the cells were transfected with RBCK1 siRNA or control siRNA (Supplementary Fig. S4). The cells were analyzed at regular intervals for the distribution of cells in the different cell cycle phases and for the expression of selected mRNAs. We observed an increase in the proportion of cells in the G2-M phase already after 48 hours in the RBCK1 siRNA–transfected cell populations, increasing further at 72 hours (Fig. 5A). RBCK1 mRNA levels started to decrease 24 hours posttransfection of RBCK1 siRNA relative to control (Fig. 5B). No- tably, the reduction of cyclin B1 mRNA occurred at 36 hours in the RBCK1-depleted cells (Fig. 5B), whereas the accumulation of cells in the G2-M phase was first observed at 48 hours. Thus, the downregulation of cyclin B1 precedes the G2-M arrest. The levels of Plk-1 and cdc25B mRNAs were not decreased until at 72 hours in RBCK1-depleted cells (Fig. 5B). Altogether, our Figure 4. RBCK1 depletion reduces the expression of genes crucial for results suggest that downregulation of cyclin B1 could be G2-M progression in MCF-7 cells. Cells were transfected with siRBCK1 oligo 1 and 2 or siControl (siCtrl) for 72 h and were treated with E2 4 h before the target responsible for the G2-M arrest induced by RBCK1 RNA isolation. mRNA levels of cyclin B1, cdc25B, cyclin B2, and Plk-1 silencing. Immunoblot analysis confirmed decreased cyclin as measured by quantitative RT-PCR with each mRNA quantification B1 protein levels in RBCK1-depleted cells compared with performed in triplicate. *, P < 0.05; **, P < 0.01 for siRBCK1 versus control cells (Fig. 5C). To initiate studies to reveal a potential siControl. Columns, mean of three independent experiments; bars, SD.

1270 Cancer Res; 70(3) February 1, 2010 Cancer Research

Role of RBCK1 in Cell Cycle Progression and Breast Cancer

Figure 5. Time course study of the effects of RBCK1 siRNA treatment in MCF-7 cells. A and B, cells were treated as described in Supplementary Fig. S4.

A, the proportion of cells in the G2-M phase at the indicated time points after siRNA transfection. *, P < 0.05 for siRBCK1 versus siControl. B, fold change of siRNA RBCK1 or control cells over time point zero. *, P < 0.05; **, P < 0.01 for siRBCK1 versus siControl. Points, mean of one independent time course experiment that was repeated twice; bars, SD. C, RBCK1 depletion significantly reduces cyclin B1 protein levels in MCF-7 cells. Western blot analysis of cell extracts 72 h after transfection with either a 1:1 mixture of siRBCK1 oligo 1 and 2 or siControl. β-Actin was used as loading control. Transfection experiments were repeated thrice with similar results. D, mRNA levels of RBCK1 and cyclin B1 display a positive correlation in breast tumor samples (n = 13). RBCK1 and cyclin B1 mRNA levels were quantified by RT-PCR and normalized to RPLP0. mechanism for the reduction of cyclin B1 mRNA, we inves- Discussion tigated RBCK1 recruitment to the cyclin B1 promoter by performing ChIP assays. However, no recruitment of RBCK1 This study identifies a role for RBCK1 in the regulation of could be observed, suggesting that RBCK1 affects cyclin B1 cell cycle progression and proliferation in breast cancer cells. mRNA expression indirectly (data not shown). Three observations led us to investigate the role of RBCK1 in Furthermore, we assayed mRNA levels of RBCK1 and breast cancer cells. First, RBCK1 had originally been de- cyclin B1 in tumor samples. We observed a positive cor- scribed as a protein associated with PKCs (25–27), proteins relation (r2 = 0.34, P < 0.05) between RBCK1 and cyclin B1 known to be important for cell cycle progression through the mRNA expression in the tumors (Fig. 5D), consistent with G1-S phase in breast cancer cells (29–32); second, RBCK1 the observations in MCF-7 cells. mRNA is elevated in breast cancer samples (19, 20); and

www.aacrjournals.org Cancer Res; 70(3) February 1, 2010 1271

Gustafsson et al.

third, RBCK1 is a member of the RBR family of proteins (21), which includes several proteins involved in cell cycle progression (22–24, 41). RBCK1 depletion led to a decrease in MCF-7 cell proliferation (Fig. 1B). Subsequent fluorescence-activated cell sorting (FACS) analysis of RBCK1-depleted cells revealed a marked increase in the number of cells in the G2-M phase (Fig. 1C). Additionally, E2-dependent progression into the S phase, which requires ERα, was significantly reduced in RBCK1- depleted cells (Fig. 1C). ChIPs revealed that, in MCF-7 cells, RBCK1 is recruited to the major ERα promoter (Fig. 2C). RBCK1 depletion resulted in reduced recruitment to the promoter and reduced ERα expression (Fig. 2A and C), suggesting that RBCK1 might act as a novel transcription factor or transcriptional cofactor for regulation of ERα.Al- though RBCK1 depletion also reduced ERα transcripts derived from promoter A, we could not observe any recruitment of RBCK1 to promoter A. However, because the distance between ERα promoters A and B is short, it is possible that RBCK1 binding to promoter B also regulates transcription from promoter A. Figure 6. Potential mechanism of regulation of cell cycle progression and RBCK1 has previously been shown to shuttle between the proliferation by RBCK1. Left side, RBCK1 binds to the ERα promoter α nucleus and cytoplasm (42) and has been suggested to be a acting as a possible transcriptional coregulator affecting ER expression, subsequently ERα signaling and entry into the S phase. Right side,

potential transcriptional activator because it can bind DNA RBCK1 also affects the expression of genes crucial for the G2-M through its RING finger (43), supporting the possibility that transition, in particular cyclin B1. RBCK1 might act as a transcription factor. The other RBR family proteins that we found to be homologous to RBCK1 are also E3 ligases with transcriptional activity; RNF14/ shown); this is expected because c-myc is a transcriptional ARA54 regulates cell cycle progression through affecting cyclin repressor of p21Cip1/WAF1 (47) and RBCK1 depletion leads D1 expression in colon cancer cells (24), and RNF31/ZIBRA to decreased levels of c-myc (Fig.2B).Itisunclearwhy has been reported to repress transcription through direct RBCK1 affects p21 differently in these two studies, but it binding to promoters (44). might reflect the use of different cell lines as model systems. As inhibition of ERα reproduces the effects observed with The RBCK1 homologues and the E3 ubiquitin ligases inhibition of RBCK1 in the S phase but not the G2-M phase CUL19, RNF144b, and RNF14 regulate cell cycle progression (Fig. 3C), we hypothesize that RBCK1 inhibition affects (22–24), and further investigation will reveal whether RBCK1 cell proliferation by two distinct pathways: one that acts is a de facto E3 ubiquitin ligase and whether this activity is through ERα and one that acts independently of ERα required for cell cycle regulation. (Fig. 6). The E2-dependent regulation of ERα target genes de- PKC isoforms β and η have been shown to interact with creased upon RBCK1 depletion, including cyclin D1 and c-myc RBCK1 and the β isoform phosphorylates RBCK1, thereby (Fig. 2B), which are known to have key roles in the G1-S tran- preventing its self-ubiquitination (18) leading to increased le- sition (6). vels of RBCK1. Previous work in breast cancer cells has β η The RBCK1 siRNA–induced increase of cells in the G2-M shown that active PKC and , as well as estrogen through phase could be due to downregulation of cyclin B1 (Fig. 4) ERα, increase the expression of cyclin D1 (30, 32). One pos- because downregulation of this protein precedes G2-M arrest sible mechanism of PKC regulation of cyclin D1 expression is (Fig. 5A). The activation of the CDK1/cyclin B1 complex is a PKC-mediated phosphorylation of RBCK1, which leads to its α critical step in the transition from the G2 to the M phase (1), stabilization, thereby increasing the levels of ER and cyclin which is regulated by the Plk-1 kinase and the cdc25B phos- D1. Further work will be needed to elucidate RBCK1 function phatase (11, 45). However, Plk-1 and cdc25B seemed to be in the context of PKC signaling in ERα-dependent breast downregulated as a consequence of the G2-M arrest (Fig. 5B). cancer cells. Interestingly, RBCK1 does not seem to regulate cyclin B1 ex- In agreement with the effect of RBCK1 depletion on ERα and pression through recruitment to the cyclin B1 promoter, as cyclin B1 levels in breast cancer cell lines, we found that RBCK1 seems to be the case for regulation of ERα. mRNA correlates with ERα (Fig. 2D) and cyclin B1 (Fig. 5D) During the final preparation of this article, a global study expression in breast cancer tumors. To further extend the anal- by Mullenders and colleagues (46) identified that RBCK1 de- ysis of breast cancer samples, we analyzed publicly available pletion might lead to failed induction of p21Cip1/WAF1 expres- microarray data to investigate correlations between RBCK1 sion when inducing cell cycle arrest in the human fibroblast and ERα expression. Gene expression–profiling studies per- cell line BJtsLT. However, we found that RBCK1 depletion in- formed on breast cancer tissues were selected from Oncomine. creased p21Cip1/WAF1 expression in MCF-7 cells (data not Multiple regression analysis revealed a positive and significant

1272 Cancer Res; 70(3) February 1, 2010 Cancer Research

Role of RBCK1 in Cell Cycle Progression and Breast Cancer

correlation between RBCK1 and ERα mRNA expression for Acknowledgments the investigated studies (Supplementary Table S2). Therapy using antiestrogens is the mainline treatment of We thank Nina Heldring for the critical reading of the manuscript and Eric ERα-positive breast cancer and reduces the death rate in pa- W-F. Lam from Charing Cross Hospital, London, for providing the breast tumor tissue samples. tients by 30% (48). Interestingly, cyclin B1 levels tend to be increased in breast tumors (13) and have been suggested as a prognostic factor for breast cancer (49). It is thus of interest Grant Support to find upstream regulators of ERα and cyclin B1 expression; indeed, transcriptional regulation by RBCK1 might provide a The Swedish Cancer Fund (J-Å. Gustafsson and K.D. Wright). N. Gustafsson was supported in part by a Ph.D. fellowship, KID-medel, from the Karolinska target for affecting the expression of these genes. Institute. The costs of publication of this article were defrayed in part by the payment Disclosure of Potential Conflicts of Interest of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J-Å. Gustafsson is shareholder of KaroBio AB and consultant of KaroBio AB Received 7/17/09; revised 11/20/09; accepted 11/24/09; published and Bionovo. The other authors disclosed no potential conflicts of interest. OnlineFirst 1/26/10.

References 1. Nurse P. A long twentieth century of the cell cycle and beyond. Cell 19. Richardson AL, Wang ZC, De Nicolo A, et al. X chromosomal ab- 2000;100:71–8. normalities in basal-like human breast cancer. Cancer Cell 2006;9: 2. Sherr CJ. Cancer cell cycles. Science (New York) 1996;274:1672–7. 121–32. 3. Pettersson K, Gustafsson JA. Role of estrogen receptor β in estrogen 20. Desmedt C, Piette F, Loi S, et al. Strong time dependence of the 76- action. Annu Rev Physiol 2001;63:165–92. gene prognostic signature for node-negative breast cancer patients 4. Dahlman-Wright K, Cavailles V, Fuqua SA, et al. International Union of in the TRANSBIG multicenter independent validation series. Clin Pharmacology. LXIV. Estrogen receptors. Pharmacol Rev 2006;58:773–81. Cancer Res 2007;13:3207–14. 5. Gronemeyer H, Gustafsson JA, Laudet V. Principles for modulation 21. Marin I, Ferrus A. Comparative genomics of the RBR family, including of the nuclear receptor superfamily. Nat Rev 2004;3:950–64. the Parkinson's disease-related gene parkin and the genes of the ar- 6. Doisneau-Sixou SF, Sergio CM, Carroll JS, Hui R, Musgrove EA, iadne subfamily. Mol Biol Evol 2002;19:2039–50. Sutherland RL. Estrogen and antiestrogen regulation of cell cycle pro- 22. Nikolaev AY, Li M, Puskas N, Qin J, Gu W. Parc: a cytoplasmic gression in breast cancer cells. Endocr Relat Cancer 2003;10:179–86. anchor for p53. Cell 2003;112:29–40. 7. Perez-Roger I, Solomon DL, Sewing A, Land H. Myc activation of 23. Ng CC, Arakawa H, Fukuda S, Kondoh H, Nakamura Y. p53RFP, a cyclin E/Cdk2 kinase involves induction of cyclin E gene transcription p53-inducible RING-finger protein, regulates the stability of and inhibition of p27(Kip1) binding to newly formed complexes. p21WAF1. Oncogene 2003;22:4449–58. Oncogene 1997;14:2373–81. 24. Kikuchi H, Uchida C, Hattori T, et al. ARA54 is involved in transcrip- 8. Prall OW, Sarcevic B, Musgrove EA, Watts CK, Sutherland RL. tional regulation of the cyclin D1 gene in human cancer cells. Carci-

Estrogen-induced activation of Cdk4 and Cdk2 during G1-S nogenesis 2007;28:1752–8. phase progression is accompanied by increased cyclin D1 ex- 25. Cong YS, Yao YL, Yang WM, Kuzhandaivelu N, Seto E. The hepatitis pression and decreased cyclin-dependent kinase inhibitor associ- B virus X-associated protein, XAP3, is a protein kinase C-binding ation with cyclin E-Cdk2. J Biol Chem 1997;272:10882–94. protein. J Biol Chem 1997;272:16482–9.

9. Nielsen NH, Loden M, Cajander J, Emdin SO, Landberg G. G1-S 26. Vallentin A, Mochly-Rosen D. RBCK1, a protein kinase CβI (PKCβI)- transition defects occur in most breast cancers and predict outcome. interacting protein, regulates PKCβ-dependent function. J Biol Chem Breast Cancer Res Treat 1999;56:105–12. 2007;282:1650–7. 10. Geradts J, Ingram CD. Abnormal expression of cell cycle regulatory 27. Tokunaga C, Kuroda S, Tatematsu K, Nakagawa N, Ono Y, Kikkawa proteins in ductal and lobular carcinomas of the breast. Mod Pathol U. Molecular cloning and characterization of a novel protein kinase 2000;13:945–53. C-interacting protein with structural motifs related to RBCC family 11. Toyoshima-Morimoto F, Taniguchi E, Shinya N, Iwamatsu A, Nishida E. proteins. Biochem Biophys Res Commun 1998;244:353–9. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus 28. Gordge PC, Hulme MJ, Clegg RA, Miller WR. Elevation of protein ki- during prophase. Nature 2001;410:215–20. nase A and protein kinase C activities in malignant as compared with 12. Lammer C, Wagerer S, Saffrich R, Mertens D, Ansorge W, Hoffmann normal human breast tissue. Eur J Cancer 1996;32A:2120–6. I. The cdc25B phosphatase is essential for the G2-M phase transition 29. Castoria G, Migliaccio A, Di Domenico M, et al. Role of atypical pro- – in human cells. J Cell Sci 1998;111:2445 53. tein kinase C in estradiol-triggered G1-S progression of MCF-7 cells. 13. Keyomarsi K, Pardee AB. Redundant cyclin overexpression and Mol Cell Biol 2004;24:7643–53. gene amplification in breast cancer cells. Proc Natl Acad Sci U S A 30. Li H, Weinstein IB. Protein kinase C β enhances growth and expres- 1993;90:1112–6. sion of cyclin D1 in human breast cancer cells. Cancer Res 2006;66: 14. Weichert W, Kristiansen G, Winzer KJ, et al. Polo-like kinase isoforms 11399–408. in breast cancer: expression patterns and prognostic implications. 31. Yi P, Feng Q, Amazit L, et al. Atypical protein kinase C regulates dual Virchows Arch 2005;446:442–50. pathways for degradation of the oncogenic coactivator SRC-3/AIB1. 15. Ito Y, Yoshida H, Uruno T, et al. Expression of cdc25A and cdc25B Mol Cell 2008;29:465–76. phosphatase in breast carcinoma. Breast Cancer 2004;11:295–300. 32. Fima E, Shtutman M, Libros P, et al. PKCeta enhances cell cycle

16. Burger A, Amemiya Y, Kitching R, Seth AK. Novel RING E3 ubiquitin progression, the expression of G1 cyclins and p21 in MCF-7 cells. ligases in breast cancer. Neoplasia 2006;8:689–95. Oncogene 2001;20:6794–804. 17. Garber K. Missing the target: ubiquitin ligase drugs stall. J Natl 33. Soule HD, Vazguez J, Long A, Albert S, Brennan M. A human cell line Cancer Inst 2005;97:166–7. from a pleural effusion derived from a breast carcinoma. J Natl 18. Tatematsu K, Yoshimoto N, Okajima T, Tanizawa K, Kuroda S. Iden- Cancer Inst 1973;51:1409–16. tification of ubiquitin ligase activity of RBCK1 and its inhibition by 34. Nilsson M, Dahlman I, Ryden M, et al. Oestrogen receptor α gene splice variant RBCK2 and protein kinase Cβ. J Biol Chem 2008; expression levels are reduced in obese compared to normal weight 283:11575–85. females. Int J Obes 2007;31:900–7.

www.aacrjournals.org Cancer Res; 70(3) February 1, 2010 1273

Gustafsson et al.

35. Matthews J, Wihlen B, Tujague M, Wan J, Strom A, Gustafsson JA. 43. Tatematsu K, Tokunaga C, Nakagawa N, Tanizawa K, Kuroda S, Estrogen receptor (ER) β modulates ERα-mediated transcriptional Kikkawa U. Transcriptional activity of RBCK1 protein (RBCC protein activation by altering the recruitment of c-Fos and c-Jun to estro- interacting with PKC 1): requirement of RING-finger and B-box mo- gen-responsive promoters. Mol Endocrinol 2006;20:534–43. tifs and regulation by protein kinases. Biochem Biophys Res Com- 36. Zhao C, Matthews J, Tujague M, et al. Estrogen receptor β2 nega- mun 1998;247:392–6. tively regulates the transactivation of estrogen receptor α in human 44. Ehrlund A, Anthonisen EH, Gustafsson N, et al. E3 ubiquitin ligase breast cancer cells. Cancer Res 2007;67:3955–62. RNF31 cooperates with DAX-1 in transcriptional repression of ste- 37. Stack G, Kumar V, Green S, et al. Structure and function of the pS2 roidogenesis. Mol Cell Biol 2009;29:2230–42. gene and estrogen receptor in human breast cancer cells. Cancer 45. Yuan J, Eckerdt F, Bereiter-Hahn J, Kurunci-Csacsko E, Kaufmann Treat Res 1988;40:185–206. M, Strebhardt K. Cooperative phosphorylation including the activity 38. Kos M, Reid G, Denger S, Gannon F. Minireview: genomic organiza- of polo-like kinase 1 regulates the subcellular localization of cyclin tion of the human ERα gene promoter region. Mol Endocrinol 2001; B1. Oncogene 2002;21:8282–92. 15:2057–63. 46. Mullenders J, Fabius AW, Madiredjo M, Bernards R, Beijersbergen 39. Tanimoto K, Eguchi H, Yoshida T, Hajiro-Nakanishi K, Hayashi S. RL. A large scale shRNA barcode screen identifies the circadian Regulation of estrogen receptor α gene mediated by promoter B clock component ARNTL as putative regulator of the p53 tumor sup- responsible for its enhanced expression in human breast cancer. pressor pathway. PLoS ONE 2009;4:e4798. Nucleic Acids Res 1999;27:903–9. 47. Gartel AL, Ye X, Goufman E, et al. Myc represses the p21(WAF1/ 40. Nigg EA. Mitotic kinases as regulators of cell division and its check- CIP1) promoter and interacts with Sp1/Sp3. Proc Natl Acad Sci points. Nat Rev Mol Cell Biol 2001;2:21–32. U S A 2001;98:4510–5. 41. Nikolaev AY, Gu W. PARC: a potential target for cancer therapy. Cell 48. Effects of chemotherapy and hormonal therapy for early breast Cycle 2003;2:169–71. cancer on recurrence and 15-year survival: an overview of the ran- 42. Tatematsu K, Yoshimoto N, Koyanagi T, et al. Nuclear-cytoplasmic domised trials. Lancet 2005;365:1687–717. shuttling of a RING-IBR protein RBCK1 and its functional interaction 49. Suzuki T, Urano T, Miki Y, et al. Nuclear cyclin B1 in human breast car- with nuclear body proteins. J Biol Chem 2005;280:22937–44. cinoma as a potent prognostic factor. Cancer Sci 2007;98:644–51.

1274 Cancer Res; 70(3) February 1, 2010 Cancer Research

RBCK1 Drives Breast Cancer Cell Proliferation by Promoting Transcription of Estrogen Receptor α and Cyclin B1

Nina Gustafsson, Chunyan Zhao, Jan-Åke Gustafsson, et al.

Cancer Res Published OnlineFirst January 26, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-2674

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2010/01/26/0008-5472.CAN-09-2674.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2010/01/26/0008-5472.CAN-09-2674. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.