MYBBP1A suppresses breast cancer tumorigenesis by enhancing the p53 dependent anoikis

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1 kaogi et al. MC Cancer 213, 13:65 RESERCH RTICLE Open ccess suppresses breast cancer tumorigenesis by enhancing the dependent anoikis Kensuke kaogi 1, Wakana Ono 1, Yuki Hayashi 1, Hiroyuki Kishimoto 1,2 and Junn Yanagisawa 1,2* bstract ackground: Tumor suppressor is mutated in a wide variety of human cancers and plays a critical role in anoikis, which is essential for preventing tumorigenesis. Recently, we found that a nucleolar protein, Myb-binding protein 1a (), was involved in activation. However, the function of in cancer prevention has not been elucidated. Methods: Relationships between expression levels and breast cancer progression were examined using patient microarray databases and tissue microarrays. Colony formation, xenograft, and anoikis assays were conducted using cells in which was either knocked down or overexpressed. activation and interactions between and were assessed by immunoprecipitation and western blot. Results: expression was negatively correlated with breast cancer tumorigenesis. In vivo and in vitro experiments using the breast cancer cell lines MCF-7 and ZR-75-1, which expresses wild type, showed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by knockdown. We also found that binds to and enhances target gene transcription under anoikis conditions. Conclusions: These results suggest that is required for activation during anoikis; therefore, it is involved in suppressing colony formation and the tumorigenesis of breast cancer cells. Collectively, our results suggest that plays a role in tumor prevention in the context of activation. Keywords: reast cancer, Tumorigenesis, noikis,, ackground reast cancer is the most commonly occurring cancer in women worldwide. It has been estimated that more than 1.6 million new cases of breast cancer occurred in 21 [1]. Cancer cells develop features that are fundamentally different from those of normal cells. One hallmark of cancer cells is their ability to survive and proliferate in the absence of extracellular matrix (ECM)-derived signals [2]. The tumor suppressor plays a central role in coordinating the responses to stresses induced by a wide array of stimuli. Under normal conditions, cellular protein levels are maintained at basal levels. However, in response to genotoxic stresses, such as exposure to * Correspondence: junny@agbi.tsukuba.ac.jp Equal contributors 1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki , Japan 2 Life Science Center of Tsukuba dvanced Research lliance, University of Tsukuba, Tsukuba Science City, Ibaraki , Japan ultraviolet light or γ-irradiation, protein levels increase and trigger either cell cycle arrest or apoptosis [3]. plays a critical role in cancer prevention, because can suppress tumorigenesis by inducing cell cycle arrest and apoptosis through its transcriptional activity. is one of the tumor suppressor genes that is most frequently found to be inactivated in cancer [4]. also plays a critical role in anoikis. noikis, defined as detachment-induced apoptosis [5], reflects the essential requirement of most normal epithelial cells for ECM-derived survival signals [6]. When these signals are denied, for example, upon detachment and continued culture in suspension or in soft agar, cells will rapidly undergo cell cycle arrest and apoptosis. The capacity of cancer cells for anchorage-independent growth under conditions of detachment and suspension in soft agar correlates well with their tumorigenic potential [2]. is required for anoikis in many cell types [7], including epithelial cells [8-12]. Under conditions, 213 kaogi et al.; licensee iomed Central Ltd. This is an Open ccess article distributed under the terms of the Creative Commons ttribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 kaogi et al. MC Cancer 213, 13:65 Page 2 of 13 enhances p21, ax, and PUM transcription and induces cell cycle arrest and apoptosis [9,11]. However, the signaling pathways that regulate -dependent anoikis are largely unknown. Recently, we found that a nucleolar protein, Mybbinding protein 1a (), was involved in activation. When cells were exposed to cellular stresses, translocated from the nucleolus to the nucleoplasm. The translocated promoted acetylation and accumulation by facilitating the interaction between and histone acetyltransferase p3; thus, could enhance target gene transcription TCG reast Finak reast Richardson rest 2 5. P-value= 2.95E-25.5 P-value= 2.1E-6 4. P-value= 7.17E-4 log2 nedian-centerd ratio Normalized expression units Normalized expression units Normal reast (n=61) reast Carcinoma (n=392) Normal reast (n=6) reast Carcinoma (n=53) Normal reast (n=7) reast Carcinoma (n=4) C Normal reast 1 2 reast Carcinoma MCF-1 2 MCF-7 3 ZR Figure 1 expression decreases as breast cancer carcinogenesis progresses. () Decreased expression of mrn in breast carcinomas from three independent datasets for human breast cancers. Data and statistics were obtained from the Oncomine database [2-22,25]. () Decreased protein expression in carcinoma samples of breast cancer patients. IHC was used to examine expression in TMs and compare it with that of human normal breast tissues and human breast cancer tissues. Counterstaining used hematoxylin. Representatives of normal breast tissue (1 and 2) and breast carcinoma tissue (3 to 6) are shown. Sample 3 is an infiltrating duct carcinoma of T2N1aM, sample 4 is an infiltrating duct carcinoma of T3N2aM, sample 5 is an infiltrating duct carcinoma of T2NM, and sample 6 is an infiltrating duct carcinoma of T2NM. The areas within the black squares are enlarged and shown in the right-side panels. lack scale bars =.5 mm and white scale bars = 1 μm. (C) protein expression levels in MCF-1, MCF-7, and ZR-75-1 cells. protein levels in the cells were determined by immunoblottin.

3 kaogi et al. MC Cancer 213, 13:65 Page 3 of 13 [13]. Previous studies revealed that was involved in regulating intracellular energy status, inflammation, and myogenesis [14-16]. In addition, Sanhueza et al. recently reported that regulates the proliferation and migration of head and neck squamous cell carcinoma cells [17]. However, the role of in breast cancer prevention and the detailed mechanisms underlying these activities have not been determined. In this study, we show that expression is associated with breast cancer tumorigenesis through an extensive analysis of the Oncomine database. In vitro and in vivo experiments using the breast cancer cell lines, which expresses wild type, revealed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by knockdown. binds to under conditions and enhances target gene transcription, as evidenced mrn C Colony formation 5 shcontrol sh#1 sh# shcontrol sh#1 sh#2 shcontrol sh#1 sh#2 Number of colonies/9.4 cm D Tumor volume (mm3) Tumor volume day shcontrol sh#1 OEControl E sh#1 shcontrol OEControl F Tumor weight (g) Tumor weight shcontrol sh#1 shcontrol sh#1 Figure 2 suppresses colony formation and tumorigenesis in vitro and in vivo. () and () Efficiency of knockdown and overexpression in MCF-7 cells. () western blot with anti- antibody was used to detect protein in MCF-7 cells that stably expressed shrn for (sh #1 and sh #2), cells that expressed Luciferase shrn (shcontrol), cells that stably overexpressed (), and EGFP (). () mrn in these cells was quantified by RT qpcr. (C) Effects of expression on colony formation. Number of colonies per dish (area = 9.4 cm 2 ) are shown. (D) Tumor growth curves in nude mice that were inoculated with either shcontrol, sh #1,, or MCF 7 cells. Nude mice received bilateral subcutaneous injections of control or cells. Tumor volume is presented as the mean ± s.d. (n = 1) for 5 mice in each group. (E) and (F) Increased or decreased tumor weight in mice injected with sh #1 or cells. Photographs of mice (E) and tumors (F, right panels) are shown. Scale bars = 1 mm. Tumor weights after 49 days are shown in F, left panel. ars = mean + s.d. (n = 1).

4 kaogi et al. MC Cancer 213, 13:65 Page 4 of 13 noikis assay MCF-1 Living cells number /ml (1 5 ) OD (57 nm) noikis assay MCF-7 Living cells number /ml (1 5 ) OD (57 nm) OE OE noikis assay ZR-75-1 Living cells number /ml (1 5 ) OD (57 nm) OE OE OE OE C Figure 3 (See legend on next page.)

5 kaogi et al. MC Cancer 213, 13:65 Page 5 of 13 (See figure on previous page.) Figure 3 induces anoikis in normal breast and breast cancer cells. () induces anoikis in mammary epithelial cells. MCF-1 cells were transfected with sirn for or ; 48 h later, anoikis assay was performed. These cells were cultured under suspension conditions for 24 h, and viable cell numbers were determined by trypan blue dye exclusion or MTT assay. Protein levels of and in the cells were determined by immunoblotting. () and (C) MCF-7 and ZR-75-1 cells were transfected with sirn for 48 h, followed by anoikis assay, or expressed a plasmid for or for 24 h followed by anoikis assay. These cells were cultured under suspension conditions for 24 h, and viable cell numbers were counted or assessed by MTT assay. Protein levels of and in these cells were determined by immunoblotting. ars = mean + s.d. (n = 3). by co-immunoprecipitation experiments and RT-qPCR. These results suggest the physiological significance of in activation and cancer prevention. Methods Cell culture and transfection MCF-1, human mammary epithelial cells, were maintained in Dulbecco s modified Eagle s medium-f12 (DMEM-F12) (Invitrogen, Carlsbad, C) supplemented with.5 μg/ml hydrocortisone (Sigma-ldrich, St Louis, MO), 1 μg/ml insulin (Sigma-ldrich, St Louis, MO), and 2 ng/ml recombinant human EGF (Peprotech, Rocky Hill, NJ). MCF-7 human breast cancer cells were maintained in DMEM (Sigma-ldrich, St Louis, MO). ZR-75-1 human breast cancer cells were maintained in RPMI 164 (Nacalai Tesque, Kyoto, Japan). ll media were supplemented with 1% fetal bovine serum (FS) and 1% penicillin streptomycin solution (Nacalai Tesque, Kyoto, Japan). Transfection was performed using Lipofectamine LTX (Invitrogen, Carlsbad, C). Expression vectors and antibodies cdns encoding full-length and were amplified using PCR and subcloned into pcdn3 plasmids (Invitrogen, Carlsbad, C) and pqcxip (Clontech, Mountain View, C) containing sequences encoding FLG sequences. nti-β-ctin (Sigma-ldrich, St Louis, MO) and anti-human- (Santa Cruz, Santa Cruz, C) monoclonal antibodies and rabbit anti--k382c (Cell Signaling Technology, Danvers, M) polyclonal antibody were used according to the manufacturers instructions. Rabbit anti-human antibody was raised against a synthetic peptide corresponding to amino acids of human. Oncomine analysis The Oncomine database and gene microarray analysis tool, a repository for published complementary DN microarray data [18,19], were explored (July 212) for mrn expression in non-neoplastic and breast cancer tissues. Statistical analysis of the differences in expression between these tissues used Oncomine algorithms, which provided multiple comparisons among different studies [2-22]. Data sets obtained from TCG reast, Finak reast, and Richardson reast 2 included various stage, and all cancer samples were invasive. Immunohistochemistry (IHC) Human breast cancer tissue microarrays were purchased from SuperioChips Laboratories (Seoul, Korea) included various stages, and all cancer samples were invasive. Formalin-fixed tissues were dewaxed in xylene and rehydrated in alcohol. For antigen retrieval, the sections were subsequently heated in EDT buffer (1 mm, ph 8.) in a microwave oven for 5 min. Endogenous peroxidase activity was suppressed using a solution of 3% hydrogen peroxide in methanol for 6 min. The samples were stained with avidin biotin peroxidase complexes using a Histofine S-PO Immunohistochemical Staining Kit (Nichirei, Tokyo, Japan) according to the manufacturer s instructions. Rabbit anti- antibody was used at a dilution of 1:5. RN interference Methods for stable RN interference followed those described by Kajiro et al. [23]. To generate a shrn retroviral supernatant, GP2-293 cells (Clontech, Mountain View, C) were co-transfected with a p11 vector encoding an envelope protein and pretro-super (Oligo Engine, Seattle, W) vector containing either the or luciferase (control) target sequence. MCF-7 cells were incubated with the retroviral supernatant in the presence of 8 μg/ml polybrene. Infected cells were selected using 1 μg/ml of puromycin. The target sequences were 5 - TTCGGGC TTTTCTCTCGGCC-3 for MY1 #1, 5 -TTC GGGCTTTTCTCTCGGCC-3 for #2, and 5 -GGCTGCGCGGTGGTGTTGT-3 for luciferase. For sirn transfection, cells at 3% 5% confluency were transfected using Lipofectamine RNi MX (Invitrogen, Carlsbad, C) according to the manufacturer s protocol. ll sirns were purchased from Invitrogen. The sirn duplexes were, 5 -UCUUUCGUCGGU CGGCUGGUG-3 and, 5 -CCGUGGUUCU CUGGGCGG-3. Stealth RNi negative control was used as a negative control. Tumor xenograft models ll animal experiments were performed in accordance with institutional guidelines. The tumor xenograft

6 kaogi et al. MC Cancer 213, 13:65 Page 6 of 13 models have been described previously [23]. Each mouse was subcutaneously injected with 1 μl of cell suspension (5 1 6 ) in both flanks. t the time points indicated in the figures, the tumors were excised, weighed, and fixed or stored in liquid nitrogen. Soft agar colony-formation assay For soft agar assays, 22, cells were mixed with 1 ml of.35% top agar and plated onto a 35-mm six-well plate containing bottom plugs (.6% agar, 1% FS, 1 DMEM). fter the top agar had solidified (about 2 h at noikis assay MCF Living cells number /ml (1 5 ) OE Cont OD (57 nm) OE Cont OE Cont Living cells number /ml (1 5 ) OE Cont OD (57 nm) noikis assay ZR-75-1 OE Cont Figure 4 induces anoikis in a -dependent manner. () and () is required for -induced anoikis. s indicated, MCF-7 and ZR-75-1 cells were transfected with a plasmid for expression with or without sirn for. These cells were cultured under suspension conditions for 24 h, and viable cell numbers were counted using trypan blue dye exclusion or assessed by MTT assay. Protein levels of and in the cells were determined by immunoblotting. ars = mean + s.d. (n = 3). OE Cont

7 kaogi et al. MC Cancer 213, 13:65 Page 7 of C), 2 μl of DMEM was added into each well to prevent dehydration. This covering medium was changed every 2 or 3 days during culture. fter allowing growth for 2 weeks at 37 C, colonies with a diameter of >1 μm were counted. noikis assay MCF-1 cells were plated at a density of cells per well in six-well plates in an ultra-low attachment culture dish (Corning, Tewksbury, M) and maintained in DMEM-F12 (Invitrogen, Carlsbad, C) supplemented with 1% FS,.5 μg/ml hydrocortisone (Sigma-ldrich, St Louis, MO), 1 μg/ml insulin (Sigma-ldrich, St Louis, MO), and 2 ng/ml recombinant human EGF (Peprotech, Rocky Hill, NJ). MCF-7 cells were plated at a density of cells per well in DMEM with 5% FS. ZR-75-1 cells were plated at a density of cells per well in RPMI 164 with 1% FS. ll media were supplemented with 1% penicillin streptomycin solution (Nacalai Tesque, Kyoto, Japan). efore analysis, the samples were treated with trypsin to disperse the cells. Cell viability after detachment was determined by trypan blue dye exclusion. Viable cells were determined by MTT assay using an MTT cell counting kit (Nacalai Tesque, Kyoto, Japan). poptotic cell proportion was determined by fluorescence-activated cell sorting (FCS) analysis using PI and fluorescein isothiocyanate (FITC)-conjugated nnexin V (ML, Tokyo, Japan) staining. Real-time RT-PCR Real-time RT-PCR was performed as described previously [24]. Cells were homogenized in 1 ml Isogen (Nippon Gene, Tokyo, Japan), and the total RN was extracted according to the instruction manual. cdn was synthesized from total RN using Rever- Tracereversetranscriptase(Toyobo,Osaka,Japan) and oligo dt primers. Real-time PCR was used to amplify fragments representing the indicated mrn expressions. The primer sequences used were as follows: GPDH fw primer: 5 - GTTGCTCCCTCCGGC -3 GPDH rv primer: 5 - GGTCTCGCTCCTGGGTG-3 p21 fw primer: 5 -GGGCTCTCGGGTCG-3 p21 rv primer: 5 -TTGGGCTTCCTCTTGGG-3 ax fw primer: 5 -GCCTGGTGCTCGG -3 ax rw primer: 5 -CTTGGTCCGCC CCG -3 PUM fw primer: 5 - GGGCCCGCTGTGTCCT-3 PUM rv primer: 5 - CGTGCTCTCTCTCCTTGC-3.3% 2.%.2% FCS.4%.2%.4% FCS.2% 3.4%.5% 8.4% 68% 29% 75% 25% 74% 25% 68% 28% 55% 36% PI.3% 2.6%.5% OE 8.1%.5% 1% PI.3%.4%.1%.3% 68% 29% 56% 36% 54% 35% 74% 26% 74% 26% nnexin V nnexin V Figure 5 induces apoptosis during conditions in a -dependent manner. () MCF-7 cells were transfected with sirn or a plasmid for or expression for 48 h, followed by anoikis assay. These cells were cultured under suspension conditions for 24 h, and apoptotic cells were determined by nnexin V FITC/PI staining followed by flow cytometry analyses. () is required for induced anoikis. s indicated, MCF-7 cells were transfected with a plasmid for expression with or without sirn for. They were then cultured under suspension conditions for 24 h, after which apoptotic cells were determined by nnexin V FITC/PI staining and flow cytometry analyses.

8 kaogi et al. MC Cancer 213, 13:65 Page 8 of 13 Co-immunoprecipitation and immunoblotting Cells were lysed in TNE buffer [1 mm Tris HCl (ph 7.8), 1% Nonidet P-4 (NP-4),.15 M NaCl, 1 mm ethylenediaminetetraacetic acid (EDT), 1 M phenylmethylsulfonyl fluoride (PMSF), 1 g/ml aprotinin]. The extracted proteins were immunoprecipitated with antibody-coated protein G Sepharose beads (GE Healthcare Japan, Tokyo, Japan). ound proteins were separated by SDS PGE, transferred to polyvinylidene difluoride membranes (Millipore, Temecula, C), and detected with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence (ECL) western blot detection system (GE Healthcare Japan, Tokyo, Japan). Chromatin immunoprecipitation (ChIP) and real-time PCR detection ChIP assay was performed according to the published procedures [24]. The primers for real-time PCR were as follows: forward, TTCCCGCGCTTTGGG; reverse, TTGCTGTCCGGTCTCTGC for the upstream region of the ax gene. Immunofluorescence Cells were fixed in 3.7% formaldehyde in PS for 1 min. fter rinsing twice with PS, the cells were permeabilized in.1% Triton X-1 in PS and later blocked with TS-T buffer containing.5% bovine serum albumin and 1% goat serum for 1 h at room temperature. Subsequently, the cells were incubated with Real time-pcr MCF-7 ax mrn PUM mrn p21 mrn ax mrn PUM mrn p21 mrn Realtime-PCR ZR-75-1 Figure 6 enhances target genes expression during anoikis. () and () MCF-7 and ZR-75-1 cells were treated with sirns for 48 h prior to anoikis assay and cultured under or conditions for 2 h (ax mrn) or 4 h (PUM and p21 mrn). Total RNs were prepared and expressions of the indicated genes were analyzed by RT qpcr. ars = mean + s.d. (n = 3).

9 kaogi et al. MC Cancer 213, 13:65 Page 9 of 13 anti- and anti- antibodies for 1 h, stained with lexa Fluor-conjugated secondary antibodies (Invitrogen, Carlsbad, C) for 1 h, and mounted with Vectashield (Vector Laboratories, urlingame, C). Immunofluorescent images were obtained by iozero immunofluorescence microscopy (Keyence, Osaka, Japan). Results expression decreases as breast cancer carcinogenesis progresses We previously reported that MYP1 was involved in activating function. Therefore, we assumed that would have a cancer prevention function via. To examine the relationship between expression and breast cancer progression, we examined the expression profiles in breast carcinomas compared to those of normal tissue using the Oncomine database, which provides publicly available datasets of gene expression in cancer. Of the 12 datasets, 11 contained gene chip profiles classified as normal or breast carcinoma tissues, which indicated that mrn levels were significantly lower in breast carcinomas than in normal tissues. Three representative results from independent datasets characterized by large ChIP assay 12. IgG -K382c Relative DN bound (fold) α C DPI Marge D I.P. ttachedcondition Input αigg α I.. Detachedcondition Figure 7 enhances activation during anoikis. () cetylation at lysine 382 residues of induced under conditions requires. MCF-7 cells were treated with or for 48 h followed by culture under or conditions for 2 h. Cell lysates were analyzed by immunoblot using the indicated antibodies. () recruitment to the ax promoter under conditions requires. MCF-7 cells were treated as shown in (), and ChIP assay was performed using normal mouse IgG and anti- antibodies. The -binding region of the ax promoter was amplified and analyzed by RT qpcr. (C) translocates from the nucleolus to the nucleoplasm under conditions. MCF-7 cells were cultured under or conditions for 2 h, after which the localizations of and were visualized by immunofluorescence using anti- and anti- antibodies. (D) Endogenous associates with under conditions. MCF-7 cells were cultured under conditions for 2 h. The cell lysates were immunoprecipitated with normal rabbit IgG or anti- antibodies and analyzed by immunoblot using antibodies against and. ars = mean + s.d. (n = 3).

10 kaogi et al. MC Cancer 213, 13:65 Page 1 of 13 population sizes are shown in Figure 1 ( expression levels in normal vs. breast carcinoma; P = 2.95E-25, 2.1E-6, and 7.17E-4). Next, we used IHC to assess expression in non-neoplastic and breast cancer tissues using a human breast cancer tissue microarray. s shown in Figure 1, expression was significantly decreased in the breast cancer tumors. To confirm these results, we compared protein levels in MCF-1, MCF-7, and ZR-75-1 cells. The protein levels were much lower in MCF-7 and ZR-75-1 cells, the cancer cell lines derived from human breast cancer cells, than in MCF-1 cells, a normal breast tissue cell line (Figure 1C). These results suggested that had an inhibitory effect on carcinogenesis in these cancer patients. suppresses colony formation and tumorigenesis in vitro and in vivo To investigate a possible relationship between and breast cancer cell growth, we generated MCF-7 cells that had stably knocked down or overexpressed (Figure 2 and 2). MCF-7 is a breast cancer cell line that expresses wild type. s shown in Figure 2C, the colony numbers in soft agar were markedly increased when using knockdown cells (sh #1 and sh sh#2). Conversely, overexpression decreased the number of these colonies (Figure 2C: ). Next, we performed xenograft experiments to test the effect of expression on tumorigenicity in vivo. knockdown cells formed tumors that were significantly larger than those of control cells. In contrast, overexpressing cells formed smaller tumors than control cells (Figure 2D, 2E, and 2F). These results indicated that could suppress breast cancer tumor growth. induces anoikis in a -dependent manner To examine how could suppress tumor formation we focused on anoikis, because plays a critical role in anoikis [7-12]. Thus, we examined whether was involved in anoikis in the context of the pathway. MCF-1 mammary epithelial cells were transfected with or and cultured in non-adherent plates for 24 h. The number of viable cells under conditions increased with or knockdown (Figure 3). MCF-7 and ZR-75-1 breast cancer cells were also cultured under conditions. The number of viable cells increased with knockdown, while they decreased when was overexpressed. Similarly, knockdown increased and overexpression decreased the numbers of viable cells under conditions (Figure 3 and 3C). These results indicated that was involved in anoikis in breast tissues. To confirm that was involved in anoikis in context of activation, we tested the combination of knockdown and overexpression in anoikis assay using MCF-7 cells (Figure 4). ased on previous results (Figure 3), overexpression decreased the number of viable cells (compare lanes 1 and 2 in Figure 4). However, overexpression in knocked-down cells did not show any significant effects (compare lanes 3 and 4 in Figure 4). Similar results were obtained in ZR-75-1 cells (Figure 4). To further examine the role of in anoikis, we examined cellular apoptosis as determined by nnexin V FITC/PI staining, followed by flow cytometric analysis. In accordance with the results shown in nucleolus c c c 53 RE ax.etc Figure 8 Proposed model for the role of in dependent anoikis. See the text.

11 kaogi et al. MC Cancer 213, 13:65 Page 11 of 13 Figures 3 and 4, apoptosis decreased after and knockdown and increased when these genes were overexpressed (Figure 5). Moreover, overexpression in knocked-down cells did not result in any significant effects (Figure 5). These results indicate that regulates -dependent anoikis. enhances target genes expression during anoikis Next, the effects of knockdown on the induction of -target genes were examined. The mrn levels of ax, PUM, and p21 were increased under conditions, whereas the increases in these mrn levels were suppressed when was knocked down using sirn in MCF-7 cells (Figure 6). Similar results were obtained with ZR-75-1 cells (Figure 6). These results suggest that regulates -dependent anoikis by enhancing activation. enhances activation during anoikis The acetylation levels of are increased in response to stress and correlate well with activation and stabilization [26-28]. ccumulating evidence supports the conclusion that acetylation stabilizes and is indispensable for activation [29,3]. Therefore, to study the molecular mechanism by which induced anoikis in a -dependent manner, we examined whether was involved in the accumulation of protein and the acetylation of K382 under conditions. Immunoblotting revealed that conditions induced the accumulation and acetylation of (Figure 7 lane 3). However, accumulation and acetylation were not observed when was knocked down using sirn (Figure 7 lane 4). This suggested that was required for activation in anoikis. Consistent with these results, recruitment to the ax promoter was significantly enhanced under conditions, while recruitment was abrogated by knockdown (Figure 7). previous report showed that activates by facilitating its direct interaction with in response to stress when translocated from the nucleolus to the nucleoplasm [13]. Therefore, we examined the localization of and the interaction between and under conditions. Immunostaining revealed that translocated from the nucleolus to the nucleoplasm under conditions (Figure 7C). Moreover, co-immunoprecipitation showed that endogenous was bound to in MCF-7 cells under conditions (Figure 7D). These results indicated that, under conditions, translocates from the nucleolus to the nucleoplasm, and then binds to. Thus, MPP1 enhances target gene transcription. Discussion In this study, we revealed the physiological significance of in activation for prevention of cancer. was originally identified as a protein that interacted with the negative regulatory domain of c-myb [31]. However, in studies done by a number of different groups, was found to interact with and regulate several transcription factors. binds to Prep1 or PGC-1α and inhibits their activity. Prep1 is involved in development and organogenesis, and PGC-1α is a key regulator of metabolic processes such as mitochondrial biogenesis, respiration, and gluco neogenesis in the liver [32,33]. Correspondingly, MYP1 also interacts with NF κ and CRY1 and regulates their transcriptional activity [15,34]. We previously reported that interacts with and activates its transcriptional capacity. localizes predominantly in the nucleolus; however, it translocates from the nucleolus to the nucleoplasm in response to cellular stress and activates [13,14,35]. Similar to other kinds of stress, conditions induce acetylation and target gene transcription in an -dependent manner (Figures 6 and 7). plays an important role in activation in response to condition to induce anoikis. The regulation of localization under conditions is still to be elucidated. When cells are exposed to UV light, translocation is accompanied by nucleolar segregation [13]. ecause the nucleolus appears to be intact after 2 h under conditions, there may be another signal that releases from the nucleolus. Numerous reports have shown that other nucleolar proteins can activate similar to that by. RPL11 directly bind to HDM2 and inhibit HDM2- mediated ubiquitination [36-41]. Similarly, the nucleolar proteins NPM, NCL, NS, and RF can also directly bind to HDM2 and inhibit ubiquitination [42-46]. Unlike with these nucleolar factors, promotes activation by directly binding to without affecting HDM2 function. With regard to the pronounced effect of knockdown on accumulation and acetylation under conditions (Figure 7), may have a unique and essential function in response to conditions. In addition, Sanhueza et al. recently reported that regulates the proliferation and migration of head and neck squamous cell carcinoma cells [17]. However, the detailed mechanisms underlying these activities are unknown. In this study, we revealed a function for the nucleolar protein in breast cancer.

12 kaogi et al. MC Cancer 213, 13:65 Page 12 of 13 Therefore, our results provide a novel insight into the function of the nucleolar protein in the biology of cancer cells. Conclusion To determine the role of in cancer, we conducted an extensive analysis of the Oncomine database and IHC studies, and showed that expression was associated with breast cancer tumorigenesis. In vivo and in vitro experiments using the breast cancer cell lines revealed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by knockdown. Co-immunoprecipitation experiments revealed that binds to under conditions and enhances target gene transcription (Figure 8). These results revealed the physiological significance of in activation. Our results may lead to a novel strategy for breast cancer therapy. bbreviations : Protein 53; : Myb-binding protein 1a; ECM: Extracellular matrix; p21: Protein 21; ax: cl-2 associated X protein; PUM: upregulated modulator of apoptosis; p3: Protein 3; IHC: Immunohistochemistry; FCS: Fluorescence-activated cell sorting; FITC: Fluorescein isothiocyanate; RT-qPCR: Reverse transcription quantitative polymerase chain reaction; DMEM: Dulbecco s modified Eagle s medium; FS: Fetal bovine serum; PCR: Polymerase chain reaction; shrn: Short hairpin RN; sirn: Small interfering RN; RT-PCR: Reverse transcription polymerase chain reaction; PMSF: Phenylmethylsulfonyl fluoride; SDS-PGE: Sodium dodecyl sulfatepolyaclylamidegel electrophoresis; ECL: Enhanced chemiluminescence; ChIP: Chromatin immunoprecipitation; PS: Phosphate buffered saline; TS- T: Tris-buffered saline and tween 2; TM: Tissue microarray; OE: Overexpression; CP: CRE-binding protein; qpcr: Quantitativepolymerase chain reaction; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; NF-κ: nuclear factor kappa-light-chain-enhancer of activated cells; CRY1: Cryptochrome 1. Competing interests The authors declare that they have no conflicts of interests concerning this work. uthors contributions K participated at the design, execution and interpretation of the experiments, as well as writing up of the manuscript. WO participated at the immunoblotting experiments and the presentation of the manuscript. YH participated at the FCS analysis. HK participated at the interpretation of the data and the presentation of the manuscript. JY participated at the design and interpretation of the experiments, as well as writing up of the manuscript. ll authors read and approved the final manuscript. cknowledgements This work was supported by Grant-in-id for Japan Society for the Promotion of Science (JSPS). Received: 15 October 212 ccepted: 3 January 213 Published: 7 February 213 References 1. Forouzanfar MH, Foreman KJ, Delossantos M, Lozano R, Lopez D, Murray CJ, Naghavi M: reast and cervical cancer in 187 countries between 198 and 21: a systematic analysis. 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