Breast Cancer Amplified Sequence 2, a Novel Negative Regulator of the P53 Tumor Suppressor

Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023 Published Online First on November 10, 2009 as 10.1158/0008-5472.CAN-09-2023

Cell, Tumor, and Stem Cell Biology

Breast Cancer Amplified Sequence 2, a Novel Negative Regulator of the p53 Tumor Suppressor

Ping-Chang Kuo,1 Yeou-Ping Tsao,2 Hung-Wei Chang,1 Po-Han Chen,1 Chu-Wei Huang,1 Shinn-Tsuen Lin,1 Yu-Tzu Weng,1 Tzung-Chieh Tsai,4 Sheau-Yann Shieh,3 and Show-Li Chen1

1Graduate Institute of Microbiology, College of Medicine, National Taiwan University; 2Department of Ophthalmology, Mackay Memorial Hospital; 3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan and 4Department of Microbiology & Immunology, National Chiayi University, Chiayi, Taiwan

Abstract human cancers (6, 7). The p53 protein has been shown to interact Breast cancer amplified sequence 2 (BCAS2) was reported with several cellular and viral proteins including SV40 large T, hu- previously as a transcriptional coactivator of estrogen recep- man papillomavirus E6 protein, adenovirus E1b, MDM2, E2F, p300/ tor. Here, we report that BCAS2 directly interacts with p53 to CBP, NUMB, and ASPP proteins (8, 9). These proteins are a mixture reduce p53 transcriptional activity by mildly but consistently of general and apoptosis-specific regulators of p53 and regulate decreasing p53 protein in the absence of DNA damage. How- how p53 protein determines the fate of cells (10). ever, in the presence of DNA damage, BCAS2 prominently In this study, BCAS2 interaction partners were identified and reduces p53 protein and provides protection against chemo- characterized using glutathione S-transferase (GST) pull-down therapeutic agent such as doxorubicin. Deprivation of BCAS2 assays combined with mass spectrometry. One such protein shown to bind BCAS2 was p53; its interaction was confirmed induces apoptosis in p53 wild-type cells but causes G2-M arrest in p53-null or p53 mutant cells. There are at least two by further GST pull-down in vitro and through reciprocal immu- apoptosis mechanisms induced by silencing BCAS2 in wild- noprecipitation in vivo. Because BCAS2 is a nuclear protein, we type p53-containing cells. Firstly, it increases p53 retention further examined whether BCAS2 could regulate p53 transactiva- in nucleus that triggers the expression of apoptosis-related tion and apoptosis. Here, we show for the first time that BCAS2 genes. Secondly, it increases p53 transcriptional activity by is a novel negative regulator of p53 that directly interacts with raising p53 phosphorylation at Ser46 and decreases p53 pro- p53 protein, decreasing p53 transcription activity. Deprivation of tein degradation by reducing p53 phosphorylation at Ser315. BCAS2 induces cell apoptosis in p53 wild-type cells but causes We show for the first time that BCAS2, a small nuclear protein G2-M arrest in p53-null or p53 mutant cells. It seems that, be- (26 kDa), is a novel negative regulator of p53 and hence a yond p53, other cellular factors exist that interact with BCAS2 potential molecular target for cancer therapy. [Cancer Res and are involved in the inhibition of cell growth. 2009;69(23):OF1–9] Materials and Methods Introduction In vitro GST pull-down analysis. The GST and GST fusion protein The sequence of breast cancer amplified sequence 2 (BCAS2) is plasmids were transformed into Escherichia coli BL21 (DE3) cells. the same as DAM1 (Mus musculus DNA amplified in mammary car- These fusion proteins were purified using glutathione-Sepharose 4B cinoma mRNA; GenBank accession no. AB020623) deposited at Na- beads (GE Life Sciences) according to the manufacturer’s procedures. tional Center for Biotechnology Information in July 2001 (1–3). The His-tagged p53 was synthesized by transformed pRSET-p53 plasmid BCAS2 gene maps to chromosome 1p13.3-21 region and contains into E. coli BL21 (DE3) cells and then purified by Ni-NTA agarose 678 bp encoding 225 amino acids, with a predicted molecular mass beads (Qiagen). The purified His-tagged p53 protein was incubated with of 26 kDa (2). BCAS2 was recently characterized as a transcription- the immobilized GST or GST-fused proteins beads and these bound al cofactor that enhances estrogen receptor–mediated gene ex- proteins were subjected to Western blotting with anti-His or anti-GST pression (4). In this article, we found that BCAS2 associates with antibodies. Preparation of cytoplasmic and nuclear extracts. Nuclear and cyto- the tumor suppressor p53 protein; we have also probed the cellular plasmic fractions were prepared from MCF-7 cells treated with the appro- and molecular effects of this interaction. priate anti-BCAS2 short hairpin RNAs (shRNA) for 48 h as described Tumor suppressor protein p53 is a multifunctional protein that previously (11). regulates cell growth and death by responding to various genotoxic Fluorescence microscopy assay. MCF-7 cells were fixed and then and oncogenic stresses (5). Tight regulation of p53 activity is essen- doubly stained with anti-p53 and anti-BCAS2 antibodies followed by incuba- tial for maintaining normal cell growth and for preventing tumor- tion with FITC-conjugated anti-rabbit (Abcam) and Alexa Fluor–conjugated igenesis. Mutations in p53 are a hallmark of at least 50% of all anti-mouse antibodies (Invitrogen). Nuclei were stained with 4′,6-diamidino- 2-phenylindole. Fluorescent images were monitored using a Carl Zeiss confocal microscope (LSM 510 META). Coimmunoprecipitation and Western blot analysis. To determine BCAS2-p53 binding, MCF-7 cells were transfected with full-length or Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). deletion p53 and BCAS2 plasmids. Forty-eight hours after transfection, cell P-C. Kuo and Y-P. Tsao contributed equally to this article. lysates were harvested and immunoprecipitated with the antibodies indi- Requests for reprints: Show-Li Chen, Graduate Institute of Microbiology, College cated in procedures described previously (11, 12). of Medicine, National Taiwan University, 7F, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan. Transient transfection and luciferase assays. Transient transfections Phone: 886-2-2312-3456, ext. 88289; Fax: 886-2-2391-5293; E-mail: [email protected]. ©2009 American Association for Cancer Research. were done by plasmid electroporation into MCF-7 or H1299 cells. Forty-eight doi:10.1158/0008-5472.CAN-09-2023 hours after transfection, the cells were assayed using a Dual-Glo Luciferase www.aacrjournals.org OF1 Cancer Res 2009; 69: (23). December 1, 2009

Assay System (Promega) according to the manufacturer’s instructions and Results detected using a Luminoskan Ascent luminometer (Thermo). The intensity of the firefly luciferase signals generated was normalized against an internal Protein-protein Interaction between BCAS2 and p53 tumor control (Renilla luciferase pRL-CMV). suppressor. To characterize BCAS2 function, we first examined Cell cycle analysis. Cells transfected with the indicated plasmids were which proteins could interact with BCAS2 by examining the nucle- collected and fixed and then stained with propidiumiodide and analyzed ar extracts of MCF-7 cells by GST pull-down assays followed by by flow cytometry (BD FACSCalibur). mass spectrometry (Fig. 1A, top). Tumor suppressor protein p53 Cell proliferation assay. MCF-7, H1299, C33A, and LNCaP cells were was just one of numerous potential BCAS2-interacting proteins transfected by electroporation. Twenty-four hours after transfection, identified (data not shown); immunoblotting confirmed that p53 MCF-7, C33A, and H1299 cells were replated at 1 × 105 per well in 12-well 4 was bound to GST-BCAS2 fusion protein (Fig. 1A, bottom). culture plates. LNCaP cells were reseeded at 2 × 10 per well in 24-well We then asked whether BCAS2 could directly interact with p53. culture plates. The total cell numbers were counted at 24, 48, 72, and 96 MDM2 protein is a well-studied p53 binding protein, and residues h time points by trypan blue staining. Real-time quantitative PCR. Total cellular RNA was extracted using 1 to 110 are required for p53 binding (13). GST-MDM2 (120) cov- Trizol reagents (Invitrogen) according to the manufacturer’s instructions. ering MDM2 amino acid sequence 1 to 120 was constructed and Real-time PCR was done with a Power SYBR Green PCR Master Mix (Ap- regarded as a positive control for p53 binding. The His-p53 protein plied Biosystems) according to the manufacturer’s protocol. synthesized by E. coli was incubated with the various purified GST- Chromatin immunoprecipitation-quantitative PCR assay. Chroma- fusion proteins in GST pull-down assay. The results showed that tin immunoprecipitation assays were done as described previously (11). the GST-MDM2 (120) and GST-BCAS2 proteins could be pulled For chromatin immunoprecipitation-quantitative PCR (qPCR), DNA was down with the purified His-p53 protein (Fig. 1B, top, lanes 2 and purified with a QIAquick PCR purification kit (Qiagen) and subjected 4). These data indicate that BCAS2 directly interacted with p53. to analysis by real-time qPCR. One percent of the preclear supernatant When investigating the interaction between endogenous BCAS2 fromthe reaction lacking primaryantibody was saved as total input of chromatin. and p53 in MCF-7 cells containing wild-type p53, p53 protein could Statistical analysis. Statistical procedures were carried out using SPSS be detected by anti-BCAS2 antibody (Fig. 1B, bottom). The detec- 16.0 software. ANOVA analysis was done. P values < 0.05 were considered tion of BCAS2 (molecular weight, 26 kDa) by precipitated p53 an- significant. tibody was hampered by the immunoglobulin light chain, which is

Figure 1. Interaction between BCAS2 and p53 in vitro and in vivo. A, BCAS2 associates with p53 in vitro. GST-BCAS2 and GST proteins were incubated with MCF-7 nuclear extracts and analyzed by silver staining (top). GST-pull-down proteins were analyzed by Western blot (WB) with p53 antibody (bottom). B, top, BCAS2 directly interacts with p53 in vitro. The His-p53 protein incubated with the GST, GST-BCAS2, or GST-MDM2 (120). The bound proteins were analyzed by Western blot using an anti-His or anti-GST antibody. Bottom, association of endogenous BCAS2 and p53 in vivo. Cell lysates of MCF-7 were subjected to immunoprecipitation (IP) with antibody against BCAS2 and then immunoblotted with p53 antibody (bottom). C, nuclear colocalization of BCAS2 and p53 by confocal laser microscopy. Bar, 10 μm.

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Figure 2. BCAS2-p53 complexes repress p53-mediated p21 promoter activity. MCF-7 cells were transiently transfected with p21-Luc promoter plasmid along with increasing doses of the FLAG-BCAS2 expression construct in the absence (A) or presence (B)of1μmol/L doxorubicin (Dox). C, H1299 cells were cotransfected with p53 expression plasmid in combination with increasing doses of FLAG-BCAS2 along with p21-Luc promoter plasmid. Top, all luciferase assays were done three times as independent experiments. Mean ± SD. Bottom, p53, p21, and BCAS2 protein expression. The expression levels of p53 and p21 protein quantified by UVP BioSpectrum-AC imaging software were normalized to β-actin. D, chromatin immunoprecipitation-qPCR assay to monitor the effect of BCAS2 on p53 binding to the p21 promoter.

25 kDa (data not shown). Instead, we conducted the confocal of immunoblot by increasing FLAG-BCAS2 plasmids was not de- microscopy assay that showed endogenous BCAS2 and p53 coloca- tectable accordingly can be explained by the transfection efficiency lized inside the MCF-7 nucleus (Fig. 1C). In summary, our data having reached its limits. Likewise, H1299 cells (lung cancer with showed that BCAS2 can directly interact with p53 in vitro and null p53 and weak endogenous p21) were cotransfected with a p53 in vivo. expression plasmid in combination with a p21-Luc promoter and Overexpression of BCAS2 reduces p53 protein and tran- increasing doses of FLAG-BCAS2. Similarly, the results showed the scriptional activity. The tumor suppressor p53 is a well-known significantly decreased p21 promoter activity (Fig. 2C, top) and the transcription factor that can activate downstreamtarget genes corresponding exogenous p53 protein and endogenous p21 protein (5, 10). Having identified a direct interaction between BCAS2 and were reduced proportionally to the increased amount of BCAS2 ex- p53, we investigated whether BCAS2 affected p53 transcriptional pression (Fig. 2C, bottom). Furthermore, we conducted chromatin activity. Transcription of the p21 gene is reportedly enhanced by immunoprecipitation-qPCR to measure the effect of BCAS2 on p53 p53 binding to its promoter region, a stretch that contains two binding to the p21 promoter. The results of Fig. 2D showed that copies of the p53 response element (14). Doxorubicin, a DNA-dam- BCAS2 decreased the p53 binding to its 5′ and 3′ binding site on aging agent, is a potent inducer of p53 protein and p53-dependent p21 promoter in both the absence and the presence of doxorubicin transcriptional activity (15). Firstly, we used the p21 promoter in MCF-7 cells compared with the mock and FLAG-vector control. (5 μg; kindly provided by Dr. Bert Vogelstein) along with Taken together, BCAS2 can reduce p53 protein expression to inhib- p3XFLAG-BCAS2 (15 or 20 μg) and internal control pRL-CMV it its transcriptional activity. (0.1 μg) as an assay model to determine p53 transcriptional activity Reciprocal binding domains between BCAS2 and p53. To (16). The total amount of plasmid per dish was equalized by adding verify our hypothesis that BCAS2 is an inhibitor of p53, we empty vectors. The results showed that the luciferase activity of mapped the domains within p53 and BCAS2 responsible for inter- the p21 promoter decreased (top), accompanied with increased acting with BCAS2 and p53, respectively. HA-tagged deletion BCAS2 protein expression and decreased levels of p53 and p21 mutants of p53 were constructed and named p53-FL, p53-Δ1, (bottom) in MCF-7 cells, regardless of doxorubicin treatment p53-Δ2, p53-Δ3, p53-Δ4, and p53-Δ5 (Fig. 3A, top). Figure 3A (Fig. 2B) or no treatment (Fig. 2A). However, that the quantitation showed that p53-Δ2, p53-Δ3, and p53-Δ4 complexed with BCAS2 www.aacrjournals.org OF3 Cancer Res 2009; 69: (23). December 1, 2009

Figure 3. Analysis of the mutual binding domains between BCAS2 and p53 proteins. A, schematic representation of the various p53 deletion mutants constructed (top). Specific domains are labeled as follows: TA, transactivation domain; DBD, specific DNA-binding domain; TET, tetramerization domain; NLS, nuclear localization signal; NES, nuclear export signal. Mapping of p53 domains used for binding with BCAS2. MCF-7 cells were transfected with HA-p53-FL or p53 deletion mutants. Cell extracts were immunoprecipitated with BCAS2 antibody. Bound proteins were subjected to Western blot analysis (bottom). B, map of BCAS2 domains. Two coiled-coil domains were identified and named CC1 (residues 139-173) and CC2 (residues 192-219), respectively (top). Mapping the BCAS2 domains required for interacting with p53. Cells were transfected with FLAG-BCAS2-FL or BCAS2 deletion mutants. Cell extracts were immunoprecipitated with p53 antibody. Bound proteins were subjected to Western blot analysis (bottom). C, effect of BCAS2 mutants on the expression of p53 and p21 protein in MCF-7 cells. MCF-7 cells were transfected with the indicated plasmids, and total cell lysates were subjected to Western blot analysis (top). The expression levels of p53 and p21 protein were measured as described in Fig. 2.

protein (bottom), indicating that p53 residues 132 to 324 are re- BCAS2 coiled-coil domains were responsible for binding to p53 sponsible for binding BCAS2, the same region required for DNA and regulating its activity. binding and nuclear signaling (17). Likewise, BCAS2 has two Depletion of BCAS2 affects p53-dependent transcription. To coiled-coil domains (Fig. 3B, top) that interact with other proteins evaluate the functions of BCAS2 gene by RNA interference (11, 12, (18, 19); we generated wild-type or single- or double-domain de- 20), four BCAS2 small interfering RNA sequences were designed letion mutants named BCAS2-FL, BCAS2-dCC1, BCAS2-dCC2, and and cloned into pSUPER plasmid and named according to the BCAS2-dCC1+2, respectively (Fig. 3B, top), to answer whether BCAS2 sequence to be disrupted (Supplementary Fig. S1). As coiled-coil domains of BCAS2 were involved in the interaction shown in Fig. 4A, BCAS2 protein expression was significantly di- with p53 protein. Figure 3B showed that those BCAS2 mutants minished by shBCAS2#434 and mildly diminished by shBCAS2#135 lacking either one or both domains were unable to bind p53 (bot- and shBCAS2#510 shRNAs in 293T cells (top). To further determine tom). When deletion mutants of BCAS2 were introduced into that the shBCAS2#434 construct could knock down BCAS2 gene MCF-7 cells, p53 and p21 protein expression was less disrupted expression is on-target effect, we generated the mutant plasmid than by full-length BCAS2 (Fig. 3C). This result indicated that the FLAG-BCAS2 (mutant), in which the sequence corresponding to

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023 BCAS2, a Negative Regulator of p53 shBCAS2#434-targeted sequence (position 434-452) was mutated cell types, LNCaP (prostate cancer cells containing wild-type p53), to 5′-AATCGAGCATGCTCAAAAA-3′. After cotransfection of wild- presented similar patterns of p53-induced gene expression (p21, type or mutant BCAS2 plasmid with shBCAS2#434 into 293T cells, PUMA, MDM2, and NOXA) after BCAS2 depletion as MCF-7 Western blot analysis showed that shBCAS2#434 only inhibited (Supplementary Fig. S2A) and A549 (lung cancer cells with wild- wild-type protein expression (Fig. 4A, bottom, lane 4) but not mu- type p53) showed the increased gene expression of PUMA, tant type expression (lane 6). In summary, the designed sequence MDM2, and NOXA but not p21 (Supplementary Fig. S2B). It can of shBCAS2#434 targeted the BCAS2 gene and diminished RNA be explained the different cell contexture influenced p53 binding expression (data not shown). efficiency to its targeted genes. To avoid off-target effects, we fur- We have shown that BCAS2 repressed the p53 transcriptional ther treated MCF-7 cells with the other targeting sequences, ability (Fig. 2). We therefore examined whether BCAS2 knockdown shBCAS2#135, which could decrease BCAS2 protein expression, could increase the p53 transcriptional activity of other p53- too (Fig. 4A, top). Similarly, qPCR of shBCAS2#135-treated cells targeted genes, such as p21, MDM2, PUMA, NOXA, and BAX showed the significantly increased gene expression of p21, PUMA, (10, 21). qPCR assays showed significantly increased gene expres- NOXA, and MDM2 but not BAX (Fig. 4B). To directly examine how sion of p21, PUMA, NOXA, and MDM2 but not BAX in MCF-7 cells BCAS2 depletion would affect p53-dependent transcription, RNA treated with shBCAS2#434 compared with the control (pSUPER) in interference studies showed that shBCAS2#434 could increase both doxorubicin treatment and no treatment (Fig. 4B). Two other p21 promoter activity in MCF-7 cells by ∼3.5-fold compared with

Figure 4. Depletion of BCAS2 expression increases the p53 transcriptional activity. A, top, identification of effective shRNA targets to BCAS2. Four BCAS2 shRNA clones were analyzed for inhibition of endogenous BCAS2 protein. Bottom, cells were transfected with shBCAS2#434 along with BCAS2 (wild-type) or mutant constructs and analyzed by Western blot. B, effect of BCAS2 knockdown on p53-targeted genes expression was analyzed by qPCR. C, silencing BCAS2 expression increases p21 promoter reporter activity. D, depletion of BCAS2 expression increased the protein level of p21 and PUMA by Western blot.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023 Cancer Research shBCAS2#331 and mock controls (Fig. 4C). Figure 4D shows that of BCAS2 would affect the relative distribution of p53 between p21 and PUMA protein levels were higher in shBCAS2#434-treated the cytosol and the nucleus. Our results show that p53 protein MCF-7 cells compared with mock, pSUPER, and shBCAS2#331. was increasingly nuclear in shBCAS2#434-treated MCF-7 cells Taken together, interfering with BCAS2 expression in wild-type (Fig. 5C, lane 6) post-transfection, indicating that BCAS2 deple- p53 cancer cells induces p21 and PUMA RNA and protein expres- tion increases nuclear p53 protein. Taken together, the knock- sion, indicating decreasing p53 transcriptional activity. down of BCAS2 caused the induction of p53 transcriptional Reducing BCAS2 expression affects p53 nuclear retention activity by nuclear p53 protein. and post-translational modifications. The expression of BCAS2 The transactivation function of p53 is believed to be regulated mildly reduced p53 protein expression (Fig. 2). We therefore inves- through protein accumulation and post-translational modifica- tigated whether the depletion of BCAS2 would increase the tions such as phosphorylation (25, 26). We therefore examined amount of p53 protein. As shown in Fig. 5A, the p53 protein levels changes in post-translational modifications to p53 on BCAS2 in- were mildly increased in MCF-7 cells treated with shBCAS2#434 duction and deprivation of BCAS2 (Fig. 5D). The effect on the ex- compared with mock, pSUPER, and shBCAS2#331 in normal content of p53 phosphorylation was analyzed after normalization to dition but in the presence of doxorubicin; the significant increase total p53 level. As shown in Fig. 5D, introduction BCAS2 in combi- of p53 protein was shown in shBCAS2#434 (Fig. 5A, lane 8). To fur- nation with doxorubicin treatment in MCF-7 cells caused a mea- ther assess whether BCAS2 regulates p53 protein turnover, Fig. 5B surable decrease at Ser46 phosphorylation status and increase at shows that there was more p53 protein in shBCAS2#434-treated Ser315 of p53 (left). In contrast, downregulation of BCAS2 by shRNA cells than in cells transfected with the control shRNAs. This indi- transfection increased the levels of Ser46 but reduced Ser315 phos- cates that the half-life of p53 protein in the shBCAS2#434-treated phorylation (right). The protein p53 phosphorylation at Ser315 is re- cells was longer than in cells treated with control shRNAs. ported to be required for its nuclear export and degradation by Besides being transcription-dependent, the p53-induced apo- Hdm2 (27), and phosphorylation at Ser46 is involved in p53-depen- ptotic programalso includes a transcription-independent path- dent transcription activity (26). It may explain BCAS2-degrading way (5, 22), probably mediated by cytosolic p53 protein (5, 10, p53 protein by increasing Ser315 phosphorylation and BCAS2-re- 22–24). In our studies, most p53 protein in MCF-7 cells is located ducing p53 transcriptional activity correlated with the effect of in the nucleus (Fig. 1C). Hence, we examined whether knockdown phosphorylation levels of Ser46.

Figure 5. Effect of BCAS2 on p53 protein stability, nuclear retention, and post-translational modifications. A, knockdown BCAS2 gene expression affects p53 protein expression in cells without and with doxorubicin treatment. B, effect of BCAS2 on p53 stability. As described in A, 48 h after transfection, the protein synthesis inhibitor cycloheximide (CHX) was added (100 μg/mL) to the cells at the different time points indicated. Band densities were quantitated using a UVP BioSpectrum-AC Imaging System. We set the p53 protein level in each control treatment as 100%, so the densitometer measured changes as a percentage of each control treatment. C, subcellular location of p53 by shBCAS2 treatment. Blots were probed for p53, BCAS2, poly (ADP-ribose) polymerase (PARP; for the nuclear fraction control), and tubulin (cytosolic fraction). D, left, BCAS2 suppresses the phosphorylation levels of p53 at Ser46 and induces phosphorylation at 315; right, deprivation of BCAS2 increases p53 phosphorylation at Ser46 but reduces at Ser315. The number presented the ratio of phosphorylated p53 to the corresponding total p53 protein in doxorubicin-treated experiments (control, set at 1).

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Figure 6. Biological significance of BCAS2 effects. Depletion of BCAS2 affects cell growth. A, knockdown of BCAS2 expression in cells containing wild-type p53 caused apoptosis in MCF-7 cells. Surviving cell numbers were counted by trypan blue staining (top). Bottom, Western blot. B, depletion of BCAS2 expression in H1299 cells (p53-null) results in growth retardation (top). Bottom, Western blot. C, silencing of BCAS2 in wild-type p53-cells induces cell apoptosis in a p53-dependent manner by flow cytometry (top). Bottom, Western blot. D, BCAS2 rescues doxorubicin-induced growth arrest. Left, cell proliferation assay of BCAS2 rescuing growth arrest induced by doxorubicin; top right, flow cytometry analysis; bottom, Western blot analysis of p53, p21, and BCAS2 protein expression levels from each treatment.

Reducing the expression of BCAS2 increases p53-dependent mock, pSUPER, and shBCAS2#331 control cells (Fig. 6A and apoptosis in cancer cells. Tumor suppressor p53 was reported Supplementary Fig. S3A, respectively). The dead cell number signifi- to play an important role in apoptosis and cell cycle arrest (28). cantly increased with time at 72 and 96 h in MCF-7 cells treated Cell proliferation assays were done to determine the biological with shBCAS2#434 (Supplementary Fig. S4A). The corresponding effects of knocking down BCAS2 gene expression on p53 activity. level of BCAS2 protein decreased in shBCAS2#434-treated cells at MCF-7 and LNCaP cells containing wild-type p53 (29, 30) were 48 h (Fig. 6A, bottom). The results fromthe trypan blue exclusion transfected with the pSUPER, shBCAS2#434, and shBCAS2#331 assay showed that knocking down BCAS2 expression in MCF-7 vectors separately to determine the cell growth rates. H1299 cells with shBCAS2#434 caused a dramatic increase (>40%) in cells containing null p53 and C33A cells harboring mutant p53 cell death after 72 h (Supplementary Fig. S4A). Similar results were also examined. The results showed that silencing BCAS2 were obtained by flow cytometry analysis, whereby the percent- expression by shBCAS2#434 caused dramatic decreases in the age of cells in sub-G1 phase increased at 72 h in MCF-7 cells cell growth rates of MCF-7 and LNCaP cells compared with treated with shBCAS2#434 compared with mock and pSUPER www.aacrjournals.org OF7 Cancer Res 2009; 69: (23). December 1, 2009

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023 Cancer Research vector controls (Supplementary Fig. S5; Supplementary Table S1). Discussion Likewise, growth rates for H1299 and C33A cells treated with We discovered that BCAS2 is a novel p53 binding protein. shBCAS2#434 were lower compared with the control groups Loss of p53 function is a characteristic of almost all human tu- (Fig. 6B and Supplementary Fig. S3B, respectively). The mors (32). We show that the binding of BCAS2 reduces p53 corresponding level of BCAS2 protein was reduced 48 h after function by BCAS2 mildly but consistently decreasing p53 pro- treatment (Fig. 6B and Supplementary S3B, bottom, respectively). tein in normal condition (Fig. 2A-C). However, in the presence Conversely, H1299 cells did not experience the apoptotic phenom- of DNA damage, BCAS2 prominently reduces p53 protein and enon seen in the MCF-7 cells following shBCAS2#434 treatment provides protection against chemotherapeutic agent such as (Supplementary Fig. S4B). The percentage of cells in sub-G1 doxorubicin (Fig. 6D). How BCAS2 is involved in p53 protein phase did not change significantly in H1299 cells (Supplemen- degradation will become interesting. Because BCAS2 is a small- tary Table S2) and C33A cells (Supplementary Table S3) treated sized protein (26 kDa) and only contains two coiled-coil domains with pSUPER, shBCAS2#331, and shBCAS2#434, respectively. The that are reportedly responsible for the other protein interaction, appearance of sub-G1 means that cells are undergoing apopto- it is less likely to function as E3 ligase activity for protein deg- sis (31), induced by shBCAS2#434 in wild-type p53-containing radation. On the other hand, MDM2 induces degradation and cells (MCF-7) but not in p53-null cells (H1299) or p53 mutant nuclear export of p53 by acting as a p53-specific E3 ubiquitin cells (C33A). Therefore, we can infer that apoptosis caused by ligase (6, 13, 33–35). It is interesting to examine whether BCAS2 silencing BCAS2 expression in MCF-7 (wild-type p53) cells can plays as a scaffold or adaptor molecule to mediate MDM2 or be mediated by p53. To further establish this, shp53 plasmid other E3 ligase-related proteins for p53 protein degradation. Var- was introduced to knock down p53 gene expression in MCF-7 ious negative apoptotic regulators of p53 were recently identified cells. Cell cycle analysis after the indicated treatments showed that that reduce p53 protein activity through different mechanisms the sub-G1 cell populations of the shp53 and shBCAS#434-treated (6, 8, 14, 15, 21, 36–42). For example, MDM2, PACT, aurora kinase cells were the same as control (Fig. 6C; Supplementary Table S4). A, and E6 function by causing p53 protein ubiquitination and The immunoblot presented p53 depletion in cells treated with degradation (6, 13, 33, 43). Transcriptional activation of p53 is shp53 (Fig. 6C, bottom, lane 2), BCAS2 depletion in cells with also repressed by p202 via binding to the PXXP motifs of p53 shBCAS2#434 (lanes 3-5), and ablation of both p53 and BCAS2 (39) and by LSD1 through demethylated p53 protein (31). Nota- in the combined shp53- and shBCAS2-treated cells (lane 5). In bly, HSP27 and PFTmfunction as small-sizedprotein inhibitors summary, silencing BCAS2-induced apoptosis is p53-dependent. of p53 by promoting the assembly of p53 into complexes (6, 15, 40). However, shBCAS2#434 caused cell cycle arrests at the G2-M Here, we describe the novel functioning of BCAS2 as a small- phase in p53-null H1299 cells and p53 mutant C33A cells (Sup- sized p53 binding protein, not previously known to be a p53 plementary Tables S2 and S3, respectively). This suggests inhibitor. that, beyond p53, BCAS2 may interact with other cellular factors Silencing BCAS2 for 72 h without stress treatment causes ∼40% to cause p53-independent cell growth arrest (Supplementary cells apoptotic in p53-containing cells (MCF-7; Fig. 6A; Supplemen- Fig. S6). tary Fig. S4A; Supplementary Table S1). There are several possible Ectopic overexpression of BCAS2 rescues p53-mediated explanations for this. Firstly, the increases of p53 Ser46 phosphor- growthinhibition. Because silencing BCAS2 expression caused ylation (Fig. 5D) prominently induce p53 activity that trigger apo- either cell apoptosis (wild-type p53) or growth arrest (p53-null ptosis. Secondly, in addition to p21, PUMA is also highly stimulated and mutant), we investigated whether ectopic expression of in both RNA and protein levels (Fig. 4B and D). PUMA is a key me- BCAS2 could rescue cells fromp53-induced growth inhibition. diator of p53-dependent apoptosis and targets to p53 that can re- Previous reports showed that low doses (50 nmol/L) of doxoru- lease p53 frominhibitory p53-BCL-XL complexesand then induce bicin caused cell growth arrest through the activation of the p53 mitochondrial outer membrane permeabilization (6). Thirdly, dep- pathway and p21 induction (15). Therefore, MCF-7 cells were rivation of BCAS2 only without stress in p53-null cells results in transiently transfected with FLAG-BCAS2 or FLAG-vector plas- growth arrest (Supplementary Tables S2 and S3), implying that mids and then treated with 50 nmol/L doxorubicin for 48 h. BCAS2 may also have direct effect in cell growth. This and our un- Using a cell proliferation assay, we showed that doxorubicin-in- published finding of accumulation of DNA damage after BCAS2 duced growth arrest in MCF-7 cells could be rescued by BCAS2 knockdown5 suggest that BCAS2 may be involved in DNA damage overexpression as shown in Fig. 6D (left). Similar results were repair and RNA splicing. Interestingly, BCAS2 is also known as also observed by flow cytometry analysis (Fig. 6D, right; Supple- spf27, a protein associated with the RNA spliceosome (3). spf27 mentary Table S5). In response to doxorubicin, the percentage of can forma pre-mRNAsplicing complexwith Pso4/Prp19, Cdc5L, the G1 population increased in MCF-7 control cells and in MCF-7 and Plrg1. The Pso4 complex was reported to be involved in cells treated with FLAG-vector at the expense of the respective DNA repair and RNA splicing (44–49). The function of BCAS2 in S-phase populations. By comparison, in doxorubicin-treated the cell cycle process remains unknown. It would be interesting MCF-7 cells overexpressing BCAS2, the percentage of the G1 pop- to identify the BCAS2-interacting proteins that induce p53-inde- ulation decreased, whereas the S-phase population increased pendent cell cycle arrest at G2-M. (Fig. 6D, right; Supplementary Table S5). The p53, p21, and BCAS2 In sum, our hypothesis model shows that the binding of BCAS2 protein expression levels derived from each treatment were com- reduces p53 function, whereby silencing the BCAS2 gene causes piled with the biological results (Fig. 6D, right and bottom). Taken p53 release to function as a transcription factor toward p21, NOXA, together, introducing BCAS2 in MCF-7 cells treated with low MDM2, and PUMA gene expression, resulting in an amplification doses of doxorubicin could decrease p21 protein expression, thereby accounting for the BCAS2-mediated protection from doxorubicin; this would imply that BCAS2 antagonizes p53-mediated growth inhibition. 5 Weng and Chen, unpublished results.

Cancer Res 2009; 69: (23). December 1, 2009 OF8 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023 BCAS2, a Negative Regulator of p53 of apoptotic signals leading to cell death in p53 wild-type cells. In Acknowledgments p53-null or p53 mutant cells, other cellular factors exist to interact Received 6/3/09; revised 9/22/09; accepted 9/24/09; published OnlineFirst 11/10/09. with BCAS2 to inhibit cell growth in a p53-independent manner Grant support: National Science Council grants NSC 95-2320-B-002-051-MY3 and (Supplementary Fig. S6). NSC 96-3112-B-002-030 and National Taiwan University grant 95R0066-BM 02-05. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Disclosure of Potential Conflicts of Interest with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. June-Tai Wu and Li-Po Wang for technical assistance and Drs. Shu- No potential conflicts of interest were disclosed. Chun Teng and Shin-Lian Doong for critical discussion.

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www.aacrjournals.org OF9 Cancer Res 2009; 69: (23). December 1, 2009

Breast Cancer Amplified Sequence 2, a Novel Negative Regulator of the p53 Tumor Suppressor

Ping-Chang Kuo, Yeou-Ping Tsao, Hung-Wei Chang, et al.

Cancer Res Published OnlineFirst November 10, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-2023

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