Research article | Open | Open Peer Review | Published:
MYBBP1A suppresses breast cancer tumorigenesis by enhancing the p53 dependent anoikis
BMC Cancervolume 13, Article number: 65 (2013)
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.
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.
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.
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.
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 . 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 .
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 . 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 .
p53 also plays a critical role in anoikis. Anoikis, defined as detachment-induced apoptosis , reflects the essential requirement of most normal epithelial cells for ECM-derived survival signals . 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 .
p53 is required for anoikis in many cell types , including epithelial cells [8–12]. Under detached conditions, p53 enhances p21, Bax, and PUMA transcription and induces cell cycle arrest and apoptosis [9, 11]. However, the signaling pathways that regulate p53-dependent anoikis are largely unknown.
Recently, we found that a nucleolar protein, Myb-binding 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 . Previous studies revealed that MYBBP1A was involved in regulating intracellular energy status, inflammation, and myogenesis [14–16]. In addition, Sanhueza et al. recently reported that MYBBP1A regulates the proliferation and migration of head and neck squamous cell carcinoma cells . 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 by co-immunoprecipitation experiments and RT-qPCR. These results suggest the physiological significance of MYBBP1A in p53 activation and cancer prevention.
Cell culture and transfection
MCF-10A, human mammary epithelial cells, were maintained in Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12) (Invitrogen, Carlsbad, CA) supplemented with 0.5 μg/ml hydrocortisone (Sigma-Aldrich, St Louis, MO), 10 μg/ml insulin (Sigma-Aldrich, St Louis, MO), and 20 ng/ml recombinant human EGF (Peprotech, Rocky Hill, NJ). MCF-7 human breast cancer cells were maintained in DMEM (Sigma-Aldrich, St Louis, MO). 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.
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 [20–22]. Data sets obtained from TCGA Breast, Finak Breast, and Richardson Breast 2 included various stage, and all cancer samples were invasive.
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.
Methods for stable RNA interference followed those described by Kajiro et al.. To generate a shRNA retroviral supernatant, GP2-293 cells (Clontech, Mountain View, CA) were co-transfected with a p10A1 vector encoding an envelope protein and pRETRO-SUPER (Oligo Engine, Seattle, WA) vector containing either the MYBBP1A 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′- TTCAGGGCATATTTCATCTCGGACC-3′ for MYBB1A #1, 5′-TTCAGGGCATATTTCATCTCGGACC-3′ for MYBBP1A #2, and 5′-GAAGCTGCGCGGTGGTGTTGT-3′ for luciferase. For siRNA transfection, cells at 30%–50% confluency were transfected using Lipofectamine RNAi MAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. All siRNAs were purchased from Invitrogen. The siRNA duplexes were MYBBP1A, 5′-UCUUUCAGUCAGGUCGGCUGGUGAA-3′ and p53, 5′-CCAGUGGUAAUCUACUGGGACGGAA-3′. Stealth RNAi negative control was used as a negative control.
Tumor xenograft models
All animal experiments were performed in accordance with institutional guidelines. The tumor xenograft models have been described previously . Each mouse was subcutaneously injected with 100 μl of cell suspension (5 × 106) 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, 22,000 cells were mixed with 1 ml of 0.35% top agar and plated onto a 35-mm six-well plate containing bottom plugs (0.6% agar, 10% FBS, 1× DMEM). After the top agar had solidified (about 2 h at 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.
MCF-10A cells were plated at a density of 3 × 105 cells per well in six-well plates in an ultra-low attachment culture dish (Corning, Tewksbury, MA) and maintained in DMEM-F12 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 0.5 μg/ml hydrocortisone (Sigma-Aldrich, St Louis, MO), 10 μg/ml insulin (Sigma-Aldrich, St Louis, MO), and 20 ng/ml recombinant human EGF (Peprotech, Rocky Hill, NJ). MCF-7 cells were plated at a density of 3 × 105 cells per well in DMEM with 5% FBS. ZR-75-1 cells were plated at a density of 3 × 105 cells per well in RPMI 1640 with 10% FBS. All media were supplemented with 1% penicillin–streptomycin solution (Nacalai Tesque, Kyoto, Japan). Before 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). Apoptotic cell proportion was determined by fluorescence-activated cell sorting (FACS) analysis using PI and fluorescein isothiocyanate (FITC)-conjugated Annexin V (MBL, Tokyo, Japan) staining.
Real-time RT-PCR was performed as described previously . 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 ReverTra 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:
GAPDH fw primer: 5′- GTATGACTCCACTCACGGCAAA -3′
GAPDH rv primer: 5′-GGTCTCGCTCCTGGAAGATG-3′
p21 fw primer: 5′-GGAGACTCTCAGGGTCGAAA-3′
p21 rv primer: 5′-TTAGGGCTTCCTCTTGGAGA-3′
Bax fw primer: 5′-AGCAAACTGGTGCTCAAGG -3′
Bax rw primer: 5′-CTTGGATCCAGCCCAACAG -3′
PUMA fw primer: 5′-GGGCCCAGACTGTGAATCCT-3′
PUMA rv primer: 5′-ACGTGCTCTCTCTAAACCTATGCA-3′
Co-immunoprecipitation and immunoblotting
Cells were lysed in TNE buffer [10 mM Tris–HCl (pH 7.8), 1% Nonidet P-40 (NP-40), 0.15 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 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). Bound proteins were separated by SDS–PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Temecula, CA), 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 . The primers for real-time PCR were as follows: forward, TAATCCCAGCGCTTTGGAAG; reverse, TTGCTAGATCCAGGTCTCTGCA for the upstream region of the Bax gene.
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 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 [7–12]. 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 [26–28]. 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 . 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.
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 . 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 . Because the nucleolus appears to be intact after 2 h under detached conditions, there may be another signal that releases MYBBP1A from the nucleolus.
Numerous reports have shown that other nucleolar proteins can activate p53 similar to that by MYBBP1A. RPL11 directly bind to HDM2 and inhibit HDM2-mediated p53 ubiquitination [36–41]. Similarly, the nucleolar proteins NPM, NCL, NS, and ARF can also directly bind to HDM2 and inhibit p53 ubiquitination [42–46]. Unlike with these nucleolar factors, MYBBP1A promotes p53 activation by directly binding to p53 without affecting HDM2 function. With regard to the pronounced effect of MYBBP1A knockdown on p53 accumulation and acetylation under detached conditions (Figure 7A), MYBBP1A may have a unique and essential function in response to detached conditions.
In addition, Sanhueza et al. recently reported that MYBBP1A regulates the proliferation and migration of head and neck squamous cell carcinoma cells . 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.
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.
Myb-binding protein 1a
Bcl-2–associated X protein
p53 upregulated modulator of apoptosis
Fluorescence-activated cell sorting
Reverse transcription quantitative polymerase chain reaction
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Polymerase chain reaction
Short hairpin RNA
Small interfering RNA
Reverse transcription polymerase chain reaction
Sodium dodecyl sulfate-polyaclylamidegel electrophoresis
Phosphate buffered saline
Tris-buffered saline and tween 20
Quantitativepolymerase chain reaction
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha
nuclear factor kappa-light-chain-enhancer of activated B cells
Forouzanfar MH, Foreman KJ, Delossantos AM, Lozano R, Lopez AD, Murray CJ, Naghavi M: Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis. Lancet. 2011, 378: 1461-1484. 10.1016/S0140-6736(11)61351-2.
Freedman VH, Shin SI: Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell. 1974, 3: 355-359. 10.1016/0092-8674(74)90050-6.
Vousden KH, Lu X: Live or let die: the cell’s response to p53. Nat Rev Cancer. 2002, 2: 594-604. 10.1038/nrc864.
Lacroix M, Toillon RA, Leclercq G: p53 and breast cancer, an update. Endocr Relat Cancer. 2006, 13: 293-325. 10.1677/erc.1.01172.
Frisch SM, Francis H: Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol. 1994, 124: 619-626. 10.1083/jcb.124.4.619.
Frisch SM, Screaton RA: Anoikis mechanisms. Curr Opin Cell Biol. 2001, 13: 555-562. 10.1016/S0955-0674(00)00251-9.
Grossmann J: Molecular mechanisms of “detachment-induced apoptosis–Anoikis”. Apoptosis: an international journal on programmed cell death. 2002, 7: 247-260. 10.1023/A:1015312119693.
Bachelder RE, Ribick MJ, Marchetti A, Falcioni R, Soddu S, Davis KR, Mercurio AM: p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB. J Cell Biol. 1999, 147: 1063-1072. 10.1083/jcb.147.5.1063.
Cheng H, Liu P, Wang ZC, Zou L, Santiago S, Garbitt V, Gjoerup OV, Iglehart JD, Miron A, Richardson AL, et al: SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis. Sci Signal. 2009, 2: ra35-10.1126/scisignal.2000369.
Ilic D, Almeida EA, Schlaepfer DD, Dazin P, Aizawa S, Damsky CH: Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol. 1998, 143: 547-560.
Ravid D, Maor S, Werner H, Liscovitch M: Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling. Oncogene. 2005, 24: 1338-1347. 10.1038/sj.onc.1208337.
Vitale M, Di Matola T, Bifulco M, Casamassima A, Fenzi G, Rossi G: Apoptosis induced by denied adhesion to extracellular matrix (anoikis) in thyroid epithelial cells is p53 dependent but fails to correlate with modulation of p53 expression. FEBS Lett. 1999, 462: 57-60. 10.1016/S0014-5793(99)01512-4.
Kuroda T, Murayama A, Katagiri N, Ohta YM, Fujita E, Masumoto H, Ema M, Takahashi S, Kimura K, Yanagisawa J: RNA content in the nucleolus alters p53 acetylation via MYBBP1A. EMBO J. 2011, 30: 1054-1066. 10.1038/emboj.2011.23.
Kumazawa T, Nishimura K, Kuroda T, Ono W, Yamaguchi C, Katagiri N, Tsuchiya M, Masumoto H, Nakajima Y, Murayama A, et al: Novel nucleolar pathway connecting intracellular energy status with p53 activation. J Biol Chem. 2011, 286: 20861-20869. 10.1074/jbc.M110.209916.
Owen HR, Elser M, Cheung E, Gersbach M, Kraus WL, Hottiger MO: MYBBP1a is a novel repressor of NF-kappaB. J Mol Biol. 2007, 366: 725-736. 10.1016/j.jmb.2006.11.099.
Yang CC, Liu H, Chen SL, Wang TH, Hsieh CL, Huang Y, Chen SJ, Chen HC, Yung BY, Chin-Ming Tan B: Epigenetic silencing of myogenic gene program by Myb-binding protein 1a suppresses myogenesis. EMBO J. 2012, 31: 1739-1751. 10.1038/emboj.2012.24.
Acuna Sanhueza GA, Faller L, George B, Koffler J, Misetic V, Flechtenmacher C, Dyckhoff G, Plinkert PP, Angel P, Simon C, Hess J: Opposing function of MYBBP1A in proliferation and migration of head and neck squamous cell carcinoma cells. BMC Cancer. 2012, 12: 72-10.1186/1471-2407-12-72.
Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey A, Chinnaiyan AM: ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia. 2004, 6: 1-6.
Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey A, Chinnaiyan AM: Large-scale meta-analysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression. Proc Natl Acad Sci USA. 2004, 101: 9309-9314. 10.1073/pnas.0401994101.
The Cancer Genome Atlas (TCGA) Data Portal. [https://tcga-data.nci.nih.gov/tcga/]
Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, Chen H, Omeroglu G, Meterissian S, Omeroglu A, et al: Stromal gene expression predicts clinical outcome in breast cancer. Nat Med. 2008, 14: 518-527. 10.1038/nm1764.
Richardson AL, Wang ZC, De Nicolo A, Lu X, Brown M, Miron A, Liao X, Iglehart JD, Livingston DM, Ganesan S: X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell. 2006, 9: 121-132. 10.1016/j.ccr.2006.01.013.
Kajiro M, Hirota R, Nakajima Y, Kawanowa K, So-ma K, Ito I, Yamaguchi Y, Ohie SH, Kobayashi Y, Seino Y, et al: The ubiquitin ligase CHIP acts as an upstream regulator of oncogenic pathways. Nat Cell Biol. 2009, 11: 312-319. 10.1038/ncb1839.
Murayama A, Ohmori K, Fujimura A, Minami H, Yasuzawa-Tanaka K, Kuroda T, Oie S, Daitoku H, Okuwaki M, Nagata K, et al: Epigenetic control of rDNA loci in response to intracellular energy status. Cell. 2008, 133: 627-639. 10.1016/j.cell.2008.03.030.
Alimonti A, Carracedo A, Clohessy JG, Trotman LC, Nardella C, Egia A, Salmena L, Sampieri K, Haveman WJ, Brogi E, et al: Subtle variations in Pten dose determine cancer susceptibility. Nat Genet. 2010, 42: 454-458. 10.1038/ng.556.
Kim JE, Chen J, Lou Z: DBC1 is a negative regulator of SIRT1. Nature. 2008, 451: 583-586. 10.1038/nature06500.
Knights CD, Catania J, Di Giovanni S, Muratoglu S, Perez R, Swartzbeck A, Quong AA, Zhang X, Beerman T, Pestell RG, Avantaggiati ML: Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate. J Cell Biol. 2006, 173: 533-544. 10.1083/jcb.200512059.
Zhao W, Kruse JP, Tang Y, Jung SY, Qin J, Gu W: Negative regulation of the deacetylase SIRT1 by DBC1. Nature. 2008, 451: 587-590. 10.1038/nature06515.
Li M, Luo J, Brooks CL, Gu W: Acetylation of p53 inhibits its ubiquitination by Mdm2. J Biol Chem. 2002, 277: 50607-50611. 10.1074/jbc.C200578200.
Tang Y, Zhao W, Chen Y, Zhao Y, Gu W: Acetylation is indispensable for p53 activation. Cell. 2008, 133: 612-626. 10.1016/j.cell.2008.03.025.
Keough R, Woollatt E, Crawford J, Sutherland GR, Plummer S, Casey G, Gonda TJ: Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3. Genomics. 1999, 62: 483-489. 10.1006/geno.1999.6035.
Fan M, Rhee J, St-Pierre J, Handschin C, Puigserver P, Lin J, Jaeger S, Erdjument-Bromage H, Tempst P, Spiegelman BM: Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1alpha: modulation by p38 MAPK. Genes Dev. 2004, 18: 278-289. 10.1101/gad.1152204.
Diaz VM, Mori S, Longobardi E, Menendez G, Ferrai C, Keough RA, Bachi A, Blasi F: p160 Myb-binding protein interacts with Prep1 and inhibits its transcriptional activity. Mol Cell Biol. 2007, 27: 7981-7990. 10.1128/MCB.01290-07.
Hara Y, Onishi Y, Oishi K, Miyazaki K, Fukamizu A, Ishida N: Molecular characterization of Mybbp1a as a co-repressor on the Period2 promoter. Nucleic Acids Res. 2009, 37: 1115-1126.
Tsuchiya M, Katagiri N, Kuroda T, Kishimoto H, Nishimura K, Kumazawa T, Iwasaki N, Kimura K, Yanagisawa J: Critical role of the nucleolus in activation of the p53-dependent postmitotic checkpoint. Biochem Biophys Res Commun. 2011, 407: 378-382. 10.1016/j.bbrc.2011.03.029.
Marechal V, Elenbaas B, Piette J, Nicolas JC, Levine AJ: The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes. Mol Cell Biol. 1994, 14: 7414-7420.
Lohrum MA, Ludwig RL, Kubbutat MH, Hanlon M, Vousden KH: Regulation of HDM2 activity by the ribosomal protein L11. Cancer Cell. 2003, 3: 577-587. 10.1016/S1535-6108(03)00134-X.
Zhang Y, Wolf GW, Bhat K, Jin A, Allio T, Burkhart WA, Xiong Y: Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway. Mol Cell Biol. 2003, 23: 8902-8912. 10.1128/MCB.23.23.8902-8912.2003.
Dai MS, Lu H: Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5. J Biol Chem. 2004, 279: 44475-44482. 10.1074/jbc.M403722200.
Dai MS, Zeng SX, Jin Y, Sun XX, David L, Lu H: Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition. Mol Cell Biol. 2004, 24: 7654-7668. 10.1128/MCB.24.17.7654-7668.2004.
Jin A, Itahana K, O’Keefe K, Zhang Y: Inhibition of HDM2 and activation of p53 by ribosomal protein L23. Mol Cell Biol. 2004, 24: 7669-7680. 10.1128/MCB.24.17.7669-7680.2004.
Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ: Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc Natl Acad Sci USA. 1998, 95: 8292-8297. 10.1073/pnas.95.14.8292.
Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L, Potes J, Chen K, Orlow I, Lee HW, et al: The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell. 1998, 92: 713-723. 10.1016/S0092-8674(00)81400-2.
Kurki S, Peltonen K, Latonen L, Kiviharju TM, Ojala PM, Meek D, Laiho M: Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation. Cancer Cell. 2004, 5: 465-475. 10.1016/S1535-6108(04)00110-2.
Saxena A, Rorie CJ, Dimitrova D, Daniely Y, Borowiec JA: Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization. Oncogene. 2006, 25: 7274-7288. 10.1038/sj.onc.1209714.
Dai MS, Sun XX, Lu H: Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2. Mol Cell Biol. 2008, 28: 4365-4376. 10.1128/MCB.01662-07.
The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/65/prepub
This work was supported by Grant-in-Aid for Japan Society for the Promotion of Science (JSPS).
The authors declare that they have no conflicts of interests concerning this work.
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.
Kensuke Akaogi, Wakana Ono contributed equally to this work.