MYBBP1A suppresses breast cancer tumorigenesis by enhancing the p53 dependent anoikis

Background Tumor suppressor p53 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 (MYBBP1A), was involved in p53 activation. However, the function of MYBBP1A in cancer prevention has not been elucidated. Methods Relationships between MYBBP1A 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 MYBBP1A was either knocked down or overexpressed. p53 activation and interactions between p53 and MYBBP1A were assessed by immunoprecipitation and western blot. Results MYBBP1A 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 p53, showed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by MYBBP1A knockdown. We also found that MYBBP1A binds to p53 and enhances p53 target gene transcription under anoikis conditions. Conclusions These results suggest that MYBBP1A is required for p53 activation during anoikis; therefore, it is involved in suppressing colony formation and the tumorigenesis of breast cancer cells. Collectively, our results suggest that MYBBP1A plays a role in tumor prevention in the context of p53 activation.


Background
Breast 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 2010 [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 p53 plays a central role in coordinating the responses to stresses induced by a wide array of stimuli. Under normal conditions, cellular p53 protein levels are maintained at basal levels. However, in response to genotoxic stresses, such as exposure to ultraviolet light or γ-irradiation, p53 protein levels increase and trigger either cell cycle arrest or apoptosis [3]. p53 plays a critical role in cancer prevention, because p53 can suppress tumorigenesis by inducing cell cycle arrest and apoptosis through its transcriptional activity. p53 is one of the tumor suppressor genes that is most frequently found to be inactivated in cancer [4].
p53 also plays a critical role in anoikis. Anoikis, 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].
Recently, we found that a nucleolar protein, Mybbinding protein 1a (MYBBP1A), was involved in p53 activation. When cells were exposed to cellular stresses, MYBBP1A translocated from the nucleolus to the nucleoplasm. The translocated MYBBP1A promoted p53 acetylation and accumulation by facilitating the interaction between p53 and histone acetyltransferase p300; thus, MYBBP1A could enhance p53 target gene transcription   [13]. Previous studies revealed that MYBBP1A was involved in regulating intracellular energy status, inflammation, and myogenesis [14][15][16]. In addition, Sanhueza et al. recently reported that MYBBP1A regulates the proliferation and migration of head and neck squamous cell carcinoma cells [17]. However, the role of MYBBP1A in breast cancer prevention and the detailed mechanisms underlying these activities have not been determined.
In this study, we show that MYBBP1A 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 p53, revealed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by MYBBP1A knockdown. MYBBP1A binds to p53 under detached conditions and enhances p53 target gene transcription, as evidenced   ZR-75-1 human breast cancer cells were maintained in RPMI 1640 (Nacalai Tesque, Kyoto, Japan). All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Nacalai Tesque, Kyoto, Japan). Transfection was performed using Lipofectamine LTX (Invitrogen, Carlsbad, CA).

Expression vectors and antibodies
cDNAs encoding full-length p53 and MYBBP1A were amplified using PCR and subcloned into pcDNA3 plasmids (Invitrogen, Carlsbad, CA) and pQCXIP (Clontech, Mountain View, CA) containing sequences encoding FLAG sequences. Anti-β-Actin (Sigma-Aldrich, St Louis, MO) and anti-human-p53 (Santa Cruz, Santa Cruz, CA) monoclonal antibodies and rabbit anti-p53-K382Ac (Cell Signaling Technology, Danvers, MA) polyclonal antibody were used according to the manufacturers' instructions. Rabbit anti-human MYBBP1A antibody was raised against a synthetic peptide corresponding to amino acids 1265-1328 of human MYBBP1A.

Oncomine analysis
The Oncomine database and gene microarray analysis tool, a repository for published complementary DNA microarray data [18,19], were explored (July 2012) for MYBBP1A mRNA expression in non-neoplastic and breast cancer tissues. Statistical analysis of the differences in MYBBP1A expression between these tissues used Oncomine algorithms, which provided multiple comparisons among different studies [20][21][22]. Data sets obtained from TCGA Breast, Finak Breast, and Richardson Breast 2 included various stage, and all cancer samples were invasive.

Immunohistochemistry (IHC)
Human breast cancer tissue microarrays were purchased from SuperBioChips 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 EDTA buffer (1 mM, pH 8.0) 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 SAB-PO Immunohistochemical Staining Kit (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. Rabbit anti-MYBBP1A antibody was used at a dilution of 1:50.

Tumor xenograft models
All animal experiments were performed in accordance with institutional guidelines. The tumor xenograft MCF-10A cells were transfected with siRNA for p53 or MYBBP1A; 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 MYBBP1A and p53 in the cells were determined by immunoblotting. (B) and (C) MCF-7 and ZR-75-1 cells were transfected with siRNA for 48 h, followed by anoikis assay, or expressed a plasmid for p53 or MYBBP1A 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 MYBBP1A and p53 in these cells were determined by immunoblotting. Bars = mean + s.d. (n = 3). models have been described previously [23]. Each mouse was subcutaneously injected with 100 μl of cell suspension (5 × 10 6 ) in both flanks. At 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  37°C), 200 μl of DMEM was added into each well to prevent dehydration. This covering medium was changed every 2 or 3 days during culture. After allowing growth for 2 weeks at 37°C, colonies with a diameter of >100 μm were counted.

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 RNA was extracted according to the instruction manual. cDNA was synthesized from total RNA using Rever-Tra Ace reverse transcriptase (Toyobo, Osaka, Japan) and oligo dT primers. Real-time PCR was used to amplify fragments representing the indicated mRNA expressions. The primer sequences used were as follows:

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, TAATCCCAGCGCTTTGGAAG; reverse, TTGCTAGATCCAGGTCTCTGCA for the upstream region of the Bax gene.

Immunofluorescence
Cells were fixed in 3.7% formaldehyde in PBS for 10 min. After rinsing twice with PBS, the cells were permeabilized in 0.1% Triton X-100 in PBS and later blocked with TBS-T buffer containing 0.5% bovine serum albumin and 10% goat serum for 1 h at room temperature. Subsequently, the cells were incubated with anti-MYBBP1A and anti-p53 antibodies for 1 h, stained with Alexa Fluor-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) for 1 h, and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Immunofluorescent images were obtained by Biozero immunofluorescence microscopy (Keyence, Osaka, Japan).

MYBBP1A expression decreases as breast cancer carcinogenesis progresses
We previously reported that MYBBBP1A was involved in activating p53 function. Therefore, we assumed that MYBBP1A would have a cancer prevention function via p53. To examine the relationship between MYBBP1A expression and breast cancer progression, we examined the MYBBP1A 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 MYBBP1A mRNA levels were significantly lower in breast carcinomas than in normal tissues. Three representative results from independent datasets characterized by large I.P. population sizes are shown in Figure 1 (MYBBP1A expression levels in normal vs. breast carcinoma; P = 2.95E-25, 2.10E-6, and 7.17E-4). Next, we used IHC to assess MYBBP1A expression in non-neoplastic and breast cancer tissues using a human breast cancer tissue microarray. As shown in Figure 1B, MYBBP1A expression was significantly decreased in the breast cancer tumors. To confirm these results, we compared MYBBP1A protein levels in MCF-10A, MCF-7, and ZR-75-1 cells. The MYBBP1A 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-10A cells, a normal breast tissue cell line ( Figure 1C). These results suggested that MYBBP1A had an inhibitory effect on carcinogenesis in these cancer patients.

MYBBP1A suppresses colony formation and tumorigenesis in vitro and in vivo
To investigate a possible relationship between MYBBP1A and breast cancer cell growth, we generated MCF-7 cells that had stably knocked down or overexpressed MYBBP1A (Figure 2A and 2B). MCF-7 is a breast cancer cell line that expresses wild type p53. As shown in Figure 2C, the colony numbers in soft agar were markedly increased when using MYBBP1A knockdown cells (shMYBBP1A #1 and shMYBBP1A sh#2). Conversely, MYBBP1A overexpression decreased the number of these colonies ( Figure 2C: OE MYBBP1A). Next, we performed xenograft experiments to test the effect of MYBBP1A expression on tumorigenicity in vivo. MYBBP1A knockdown cells formed tumors that were significantly larger than those of control cells. In contrast, MYBBP1A overexpressing cells formed smaller tumors than control cells ( Figure 2D, 2E, and 2F). These results indicated that MYBBP1A could suppress breast cancer tumor growth.

MYBBP1A induces anoikis in a p53-dependent manner
To examine how MYBBP1A could suppress tumor formation we focused on anoikis, because p53 plays a critical role in anoikis [7][8][9][10][11][12]. Thus, we examined whether MYBBP1A was involved in anoikis in the context of the p53 pathway. MCF-10A mammary epithelial cells were transfected with sip53 or siMYBBP1A and cultured in non-adherent plates for 24 h. The number of viable cells under detached conditions increased with p53 or MYBBP1A knockdown ( Figure 3A). MCF-7 and ZR-75-1 breast cancer cells were also cultured under detached conditions. The number of viable cells increased with p53 knockdown, while they decreased when p53 was overexpressed. Similarly, MYBBP1A knockdown increased and MYBBP1A overexpression decreased the numbers of viable cells under detached conditions ( Figure 3B and 3C). These results indicated that MYBBP1A was involved in anoikis in breast tissues.
To confirm that MYBBP1A was involved in anoikis in context of p53 activation, we tested the combination of p53 knockdown and MYBBP1A overexpression in anoikis assay using MCF-7 cells ( Figure 4A). Based on previous results ( Figure 3B), MYBBP1A overexpression decreased the number of viable cells (compare lanes 1 and 2 in Figure 4A). However, MYBBP1A overexpression in p53 knocked-down cells did not show any significant effects (compare lanes 3 and 4 in Figure 4A). Similar results were obtained in ZR-75-1 cells ( Figure 4B).
To further examine the role of MYBBP1A in anoikis, we examined cellular apoptosis as determined by Annexin V-FITC/PI staining, followed by flow cytometric analysis. In accordance with the results shown in

Figures 3 and 4, apoptosis decreased after p53 and
MYBBP1A knockdown and increased when these genes were overexpressed ( Figure 5A). Moreover, MYBBP1A overexpression in p53 knocked-down cells did not result in any significant effects ( Figure 5B). These results indicate that MYBBP1A regulates p53-dependent anoikis.
MYBBP1A enhances p53 target genes expression during anoikis Next, the effects of MYBBP1A knockdown on the induction of p53-target genes were examined. The mRNA levels of Bax, PUMA, and p21 were increased under detached conditions, whereas the increases in these mRNA levels were suppressed when MYBBP1A was knocked down using siRNA in MCF-7 cells ( Figure 6A). Similar results were obtained with ZR-75-1 cells ( Figure 6B). These results suggest that MYBBP1A regulates p53-dependent anoikis by enhancing p53 activation.

MYBBP1A enhances p53 activation during anoikis
The acetylation levels of p53 are increased in response to stress and correlate well with p53 activation and stabilization [26][27][28]. Accumulating evidence supports the conclusion that acetylation stabilizes p53 and is indispensable for p53 activation [29,30]. Therefore, to study the molecular mechanism by which MYBBP1A induced anoikis in a p53-dependent manner, we examined whether MYBBP1A was involved in the accumulation of p53 protein and the acetylation of p53 K382 under detached conditions. Immunoblotting revealed that detached conditions induced the accumulation and acetylation of p53 ( Figure 7A lane 3). However, p53 accumulation and acetylation were not observed when MYBBP1A was knocked down using siRNA ( Figure 7A lane 4). This suggested that MYBBP1A was required for p53 activation in anoikis. Consistent with these results, p53 recruitment to the Bax promoter was significantly enhanced under detached conditions, while p53 recruitment was abrogated by MYBBP1A knockdown (Figure 7B).
A previous report showed that MYBBP1A activates p53 by facilitating its direct interaction with p53 in response to stress when MYBBP1A translocated from the nucleolus to the nucleoplasm [13]. Therefore, we examined the localization of MYBBP1A and the interaction between MYBBP1A and p53 under detached conditions. Immunostaining revealed that MYBBP1A translocated from the nucleolus to the nucleoplasm under detached conditions ( Figure 7C). Moreover, co-immunoprecipitation showed that endogenous MYBBP1A was bound to p53 in MCF-7 cells under detached conditions ( Figure 7D). These results indicated that, under detached conditions, MYBBP1A translocates from the nucleolus to the nucleoplasm, and then binds to p53. Thus, MPBBP1A enhances p53 target gene transcription.

Discussion
In this study, we revealed the physiological significance of MYBBP1A in p53 activation for prevention of cancer. MYBBP1A 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, MYBBP1A was found to interact with and regulate several transcription factors. MYBBP1A 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, MYBBBP1A also interacts with NF-κB and CRY1 and regulates their transcriptional activity [15,34].
We previously reported that MYBBP1A interacts with p53 and activates its transcriptional capacity. MYBBP1A localizes predominantly in the nucleolus; however, it translocates from the nucleolus to the nucleoplasm in response to cellular stress and activates p53 [13,14,35]. Similar to other kinds of stress, detached conditions induce p53 acetylation and target gene transcription in an MYBBP1A-dependent manner (Figures 6 and 7A). MYBBP1A plays an important role in p53 activation in response to detached condition to induce anoikis.
The regulation of MYBBP1A localization under detached conditions is still to be elucidated. When cells are exposed to UV light, MYBBP1A translocation is accompanied by nucleolar segregation [13]. Because the nucleolus appears to be intact after 2 h under detached conditions, there may be another signal that releases MYBBP1A from the nucleolus.
In addition, Sanhueza et al. recently reported that MYBBP1A 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 MYBBP1A in breast cancer. Therefore, our results provide a novel insight into the function of the nucleolar protein MYBBP1A in the biology of cancer cells.

Conclusion
To determine the role of MYBBP1A in cancer, we conducted an extensive analysis of the Oncomine database and IHC studies, and showed that MYBBP1A 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 MYBBP1A knockdown. Co-immunoprecipitation experiments revealed that MYBBP1A binds to p53 under detached conditions and enhances p53 target gene transcription ( Figure 8). These results revealed the physiological significance of MYBBP1A in p53 activation. Our results may lead to a novel strategy for breast cancer therapy.

Competing interests
The authors declare that they have no conflicts of interests concerning this work.
Authors' contributions KA 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 FACS 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. All authors read and approved the final manuscript.