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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Y box binding protein 1 (YB1) has multiple functions associated with drug resistance, cell proliferation and metastasis through transcriptional and translational regulation. Increased expression of YB1 is closely related to tumor growth and aggressiveness. We showed that YB1 protein levels were decreased through replicative and premature senescence and were correlated with increased expression levels of p16INK4A tumor suppressor gene. Depletion of YB1 was associated with increased levels of p16 in human and murine primary cells. Forced expression of YB1 in mouse embryonic fibroblasts resulted in decreased expression of p16 and increased cell proliferation. Senescence-associated expression of β-galactosidase was repressed in YB1-over-expressing cells. Chromatin immunoprecipitation assays showed that YB1 directly associates with the p16 promoter. Taken together, all our findings indicate that YB1 directly binds to and represses p16 transcription, subsequently resulting in the promotion of cell growth and prevention of cellular senescence.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Y box binding protein 1 (YB1) is a member of the cold shock domain (CSD) protein family. This protein binds to DNA and/or RNA through the CSD and is involved in both transcriptional activation and repression by binding to the Y box elements of the target gene promoter (Kohno et al. 2003). YB1 is also known to play multiple roles in DNA repair, translation and RNA stabilization (Kohno et al. 2003). Clinical studies have shown that YB1 is expressed at high levels in a wide range of human cancers, including lung, breast, prostate and colon cancers (Bargou et al. 1997; Shibao et al. 1999; Gu et al. 2001; Shibahara et al. 2001; Gimenez-Bonafe et al. 2004). Increased YB1 levels are strongly associated with the expression of epidermal growth factor receptor (EGFR), HER-2 (Fujii et al. 2008; Lee et al. 2008) and CDC6 (Basaki et al. 2010) proteins in breast cancer.

Biochemical studies have showed that YB1 directly binds to the promoters of certain genes, including EGFR, HER-2 and CDC6, activating their expression (Wu et al. 2006; Basaki et al. 2010); this in turn leads to increased cell growth. YB1 has also been shown to be associated with drug resistance, by binding to and activating the multidrug resistance 1 (MDR1) protein in human cancer cells (Asakuno et al. 1994; Ohga et al. 1996). Studies in mice showed the role of YB1 in cell proliferation. Transgenic mice expressing human YB1 in mammary epithelia develop breast carcinomas with mitotic failure and centrosome amplification (Bergmann et al. 2005). For mice, the absence of YB1 can be embryonically lethal or lead to exencephaly (Lu et al. 2005; Uchiumi et al. 2006). YB1−/− mouse embryonic fibroblasts (MEFs) show retarded cell growth and are less responsive to oxidative, genotoxic and oncogenic stressors compared with their wild-type counterparts. Induction of cellular senescence by oxidative stress is enhanced in YB1−/− MEFs and is partly achieved by the increased mRNA expression of the cyclin-dependent kinase (CDK) inhibitors p16 and p21 (Lu et al. 2005). Basaki et al. have also showed that expression levels of p21 and p16 were increased when they were treated with YB-1 short interfering RNAs (siRNAs) in the MCF-7 human breast adenocarcinoma cell line (Basaki et al. 2010).

For cells entering the cell cycle, a mitogenic signal induces the expression of cyclin Ds, which bind to and activate CDK4/CDK6. This leads to inactivation of the pRB family of proteins through phosphorylation, causing pRB-E2F dissociation and promoting the cell cycle (Cobrinik 2005). Inhibition of CDK4/CDK6 activity by p16, which allows for the pRB family of proteins to be activated, contributes to cell cycle arrest (Serrano et al. 1993). The biochemical activity of p16 suggests that the protein functions as a tumor suppressor. The p16 gene is frequently mutated, or its expression silenced in human cancers (Ruas & Peters 1998; Sharpless 2005). Mice lacking p16 are prone to spontaneous and induced tumorigenesis (Krimpenfort et al. 2001; Sharpless et al. 2001). The expression of p16 is undetectable during embryogenesis and in young tissues. Levels of p16 protein increase with age and contribute to irreversible growth arrest known as replicative senescence (Gil & Peters 2006). Expression of p16 is also activated by a variety of oncogenes, leading to stable cell cycle arrest, termed ‘premature senescence’; this protects cells from hyperproliferative stimulation (Serrano et al. 1997). The p16 locus is histone H3 lysine 27 (H3K27)-trimethylated and epigenetically repressed by polycomb repression complex 1 (PRC1) and PRC2 (Bracken et al. 2007; Kotake et al. 2007, 2009). It was recently reported that a long noncoding RNA, ANRIL, is required for the recruitment of PRC1 and PRC2 to the p16 locus (Yap et al. 2010; Kotake et al. 2011). Ohtani et al. previously reported that the Ets2 transcription factor binds to and activates p16, leading to cellular senescence (Ohtani et al. 2001). Apart from Ets2, it is unclear which other transcription factors bind to and regulate p16 activity. In this study, we investigated the involvement of YB1 in repressing p16 transcription.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

YB1 protein levels were decreased during replicative and premature senescence

Previous studies in mice have shown that deletion of YB1 enhances premature senescence of MEFs via oxidative stress (Lu et al. 2005); therefore, we suggested that YB1 may have an important role in cellular senescence. We determined the levels of expression of YB1 in response to the oncogenic Ras signal as this is known to cause premature senescence by activating p16 transcription. MEFs ectopically expressing oncogenic RasG12V were established after retroviral transduction and used to examine the expression of YB1. Our Western blotting results indicated that p16 levels were increased by RasG12V transduction in MEFs. In contrast, YB1 levels were decreased by RasG12V transduction (Fig. 1A). Our qRT-PCR analysis showed that YB1 mRNA levels were unaffected by RasG12V transduction (Fig. 1B).

image

Figure 1. Inverse correlation between the levels of YB1 and p16 expression in mouse embryonic fibroblasts (MEFs). (A) MEFs were infected with control (Mock) or H-RasG12V-expressing retroviruses and selected for using puromycin. The levels of individual protein were determined by Western blotting. (B) YB1 mRNA levels were determined by qRT-PCR, and results were expressed relative to the corresponding values for MEFs that were mock-infected. Mean values and standard deviations were calculated from triplicates of a representative experiment. (C) Passage-dependent expression of YB1 and p16 in MEFs was determined by Western blotting. (D) YB1 mRNA levels were determined by qRT-PCR, and results were expressed relative to the corresponding values for passage 2 MEFs.

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We next examined the expression of YB1 in replicative senescence. Levels of p16 protein increased during in vitro passage of MEFs and contributed in part to replicative senescence. Concomitant with the increase in p16 levels, the amount of YB1 was decreased in a passage-dependent manner (Fig. 1C), whereas YB1 mRNA levels remained unchanged (Fig. 1D). Our data indicate that YB1 expression is post-transcriptionally down-regulated through both replicative and premature senescence and that reduction in YB1 levels is associated with an increase in p16 expression, suggesting that YB1 negatively regulates p16.

Silencing YB1 increases p16 gene expression in mice and humans

To examine whether YB1 is involved in the regulation of p16 expression, we knocked down mouse YB1 using specific siRNAs in MEFs. These siRNAs reduced YB1 levels such that they were almost undetectable (Fig. 2A). Associated with the reduction in YB1 levels was a substantial increase in mouse p16 (Fig. 2A). Our qRT-PCR analysis showed that silencing YB1 resulted in a twofold increase in p16 mRNA (Fig. 2B). Unexpectedly, the level of protein corresponding to p21 was actually decreased by YB1 silencing, although p21 protein expression was increased in MEFs where there was homozygous deletion of YB1 (Lu et al. 2005). The INK4 locus encodes three tumor suppressor genes, p16, ARF and p15 (a CDK inhibitor) (Gil & Peters 2006; Kim & Sharpless 2006; Sherr 2006). Transcription of the p16-ARF-p15 gene cluster is coordinately repressed by PRC1 and PRC2 (Bracken et al. 2007; Kotake et al. 2007). Therefore, we examined whether YB1 is also involved in the regulation of p15 and Arf. Our qRT-PCR and Western blotting analyses showed that p15 and Arf levels were unaffected by the silencing of YB1 (Fig. 2A and 2B). To confirm this, we transfected WI38 cells with human YB1-specific siRNAs and observed that depletion of YB1 correlated with increased expression of human p16, but not of p15, p21 and ARF (Fig. 2C and D). Our results suggest that YB1 is involved in p16 repression in both mouse and human cells.

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Figure 2. Silencing YB1 resulted in an increase in p16 mRNA levels, but not in p15 or ARF mRNA transcripts. (A) MEFs were transfected with siRNAs specific for YB1 or a control (ctr) siRNA. YB1 silencing efficiency and the effects of silencing on p16, p15 and p21 expression were determined by Western blotting. (B) Levels of p16 and Arf mRNA transcripts were determined by qRT-PCR. (C, D) WI38 cells were transfected with siRNAs specific for YB1 or a control (ctr) siRNA. YB1 silencing efficiency and the effects of silencing on p16, p15, p21 and ARF expression were determined by Western blotting (C) and qRT-PCR (D).

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YB1 represses p16 expression by binding to its promoter

We next examined conversely how forced expression of YB1 affects p16 expression. MEFs stably expressing YB1 were established with the aid of YB1-expressing retroviruses. Through the qRT-PCR assays, we showed that p16 mRNA levels in the virus-infected MEFs were substantially decreased to almost 35% of that in cells infected with control virus (Fig. 3A). Consistent with the decrease in mRNA levels, the amount of p16 protein was also decreased substantially (Fig. 3B). Associated with this, forced expression of YB1 increased the rate of proliferation for the MEFs (Fig. 3C) and corresponded to a decrease in the number of cells that stained positive for SA-β-Gal activity, an indicator of cellular senescence (Fig. 3D and E). Taken together, these data indicate that YB1 represses p16 transcription and prevents cellular senescence.

image

Figure 3. Ectopic expression of YB1 represses p16 expression in MEFs. (A, B) Early passage MEFs were infected with retroviruses expressing human YB1 or mock-infected as a control. Protein and mRNA quantification were determined by Western blotting and qRT-PCR, respectively. (C) The growth curves for MEFs infected with virus, or mock-infected, were determined by trypan blue staining. (D) MEFs stained for senescence-associated β-galactosidase (SA-β-gal) activity. (E) Proportion of SA-β-gal activity-positive cells.

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Given that YB1 is a transcription factor, we next examined whether YB1 binds to the p16 promoter. To clarify this issue, we carried out ChIP assay using oligonucleotide primers designed corresponding to a sequence within mouse p16 promoter region. Sixteen putative YB-1 responsive elements were identified within the first 2.5 kb of mouse p16 promoter (Fig. 4A). A ChIP assay showed that an anti-YB-1 antibody was able to precipitate DNA that was amplified with primer sets covering a region smaller than 1.5 kb of the mouse p16 promoter (Fig. 4B and Fig. S1 in Supporting Information). This would suggest that YB1 directly binds to the mouse p16 promoter.

image

Figure 4. YB1 directly associated with the p16 promoter region. (A) Schematic of potential YB1 binding sites and amplicons (a, b, c and d) used for ChIP assays targeting the mouse p16 locus. (B) The binding of YB1 to the mouse p16 promoter was determined by ChIP assay using IgG and an antibody against YB1. PCR was carried out using for each amplicon.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we have outlined that YB1 is involved in the repression of p16 transcription and cellular senescence. We observed that YB1 protein levels, but not the corresponding mRNA, were decreased during replicative and premature senescence, suggesting that YB1 is post-transcriptionally down-regulated through cellular senescence. Sorokin et al. previously reported that genotoxic stress induces a 20S proteasome-mediated cleavage of YB1 (Sorokin et al. 2005). However, we were unable to detect an increase in the occurrence of YB1 cleavage during cellular senescence (data not shown). It is possible that there are additional regulatory mechanisms involved with YB1 expression, such as ubiquitin–proteasome pathways and translational regulation.

We also found that p16 is a novel target of YB1 in both mouse and human cells. Reducing YB1 levels resulted in increased p16 mRNA levels, but not in increases in p15 and ARF mRNA levels. All these events occurred in both MEFs and WI38 cells, indicating that YB-1 specifically regulates the p16 gene at the INK4 locus. Additionally, our ChIP data show that YB1 binds to the p16 promoter, indicating direct regulation of p16 by YB1. Detailed biochemical mechanisms underlying the function of YB1 during p16 repression remain to be elucidated. PRC1 and PRC2 have been identified as repressors of p16 transcription through histone methylation (Bracken et al. 2007; Kotake et al. 2007, 2009). The binding of these to the p16 locus is limited during in vitro passage and through oncogenic signaling, which results in p16 activation. The functional relationship between YB1 and PRC1 and/or PRC2 during p16 repression is an important issue that requires further investigation.

YB1 is thought to be a key regulator of cell proliferation and an oncoprotein. Several genes targeted by YB1, such as p21, CDC6, EGFR and HER-2, are cell cycle regulators (Okamoto et al. 2000; Wu et al. 2006; Fujii et al. 2008; Lee et al. 2008; Basaki et al. 2010). Many studies have shown that increased expression of YB1 is related to tumor aggression in various human cancers (Kohno et al. 2003). We showed that ectopic expression of YB1 repressed p16 transcription and resulted in the repression of cellular senescence. Cellular senescence functions as a barrier to hyperproliferation by oncogenic signals, such as the activation of Ras, and is achieved by p16 induction (Serrano et al. 1997; Brookes et al. 2002; Braig et al. 2005; Collado et al. 2005). We postulate that YB1 up-regulation causes p16 repression, thus resulting in the repression of cellular senescence. The implication is that this might contribute to the progression of various cancers.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Cell culture and senescence-associated expression of β-galactosidase (SA-β-Gal) assay

Early passage WI38 (normal human fibroblasts) cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Primary MEFs were isolated on embryonic day 13.5 (E13.5) and cultured as previously described (Nakayama et al. 1996). The SA-β-Gal assays were conducted using a Senescence Detection Kit (BioVision), according to the manufacturer's protocol. Briefly, MEFs were seeded to a 60% confluency in 6-well plates. The next day, the cells were washed with PBS and fixed with 1 mL of fixative solution provided with the kit for 10 min at room temperature. After washing with PBS, the cells were treated with 1 mL of the staining solution mix provided with the kit for overnight at 37 °C and then observed under a microscope.

Retroviral transduction

Human YB1 and H-RasG12V (kindly provided by C J Der, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA) cDNAs were cloned into a pMX-puro retrovirus vector (kindly provided by T Kitamura, The University of Tokyo, Tokyo, Japan). Retroviral production was conducted as previously described (Kotake et al. 2007). Cells were infected with virus for 24 h and then treated again with virus-containing supernatant to increase infection efficiency. After infection, cells were selected using 2 μg/mL puromycin for 3 days.

Western blot

Western blot analysis was carried out as previously described (Kotake et al. 2007). Cells were lysed with RIPA buffer (50 mm Tris–HCl pH 8.0, 150 mm NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 1 mm Na3VO4, 1 mm DTT, 1 mm PMSF) supplemented with protease inhibitors (10 mg/L antipain, 10 mg/L leupeptin, 10 mg/L pepstatin, 10 mg/L trypsin inhibitor, 10 mg/L E64 and 2.5 mg/L chymostatin). The antibodies we used included anti-mouse p16 (M-156; Santa Cruz Biotechnology), anti-human p16 (ab50282; Abcam,), anti-mouse p21 (F-5; Santa Cruz Biotechnology), anti-p15 (Cell Signaling), anti-H-Ras (OP23; Calbiochem), anti-YB1 (ab12148; Abcam) and anti-α-tubulin (Sigma).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using an RNeasy Plus kit (Qiagen), with 1 μg of total RNA applied to RT reactions that contained oligo(dT)20 primers and SuperScript Reverse Transcriptase II (Invitrogen). The produced cDNA was added to a qRT-PCR mixture that contained 1 × SYBR Green PCR master mix (Qiagen) and 200 nm gene-specific primers. The expression level of each gene was normalized to a reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Assays were carried out in triplicate on an Mx3000P Real-Time Q-PCR System (Agilent Technologies) and Rotor-Gene 3000 system (Corbett Research). Primer sequences are available upon request.

RNA interference (RNAi)

The MEFs and WI38 cells were transfected with siRNA oligonucleotides using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer's protocol. The nucleotide sequences of siRNA for YB1 were mouse, 5′-CAA CGU CGG UAU CGC CGA AAC UUC A-3′; and human, 5′-GGU UCC CAC CUU ACU ACER U-3′ with 3′ dTdT overhangs.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were carried out as previous described (Kotake et al. 2007). Approximately 3 × 106 MEFs were fixed with 1% formaldehyde for 10 min before 125 mm glycine was added. Cells were lysed in a lysis buffer (10 mm HEPES pH 7.9, 0.5% NP-40, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT and protease inhibitor cocktail) on ice for 10 min and then centrifuged. Cell pellets were lysed in nuclear lysis buffer (20 mm HEPES pH 7.9, 25% glycerol, 0.5% NP-40, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA and protease inhibitor cocktail), sonicated and centrifuged. Lysates were diluted with an equal volume of dilution buffer (1% Triton X-100, 2 mm EDTA, 50 mm NaCl, 20 mm Tris–HCl pH 7.9, and protease inhibitor cocktail). Immunoprecipitation was conducted with a specific antibody against YB1 (RN015P; MBL). Normal rabbit IgG was used as a control. DNA fragments were purified with a PCR purification kit (Qiagen), and PCR was carried out using Platinum Taq polymerase (Invitrogen). The following primer pairs were used to amplify mouse p16 fragments: (i), 5′-ACG TGT GCA CTT CTT TGC TG-3′ and 5′-CAT AGG TGG CGC TAT TTG C-3′; (ii), 5′-CCC TCC AAA ATG AGT TGT TTG-3′ and 5′-CTG GTC ACC CTT TGA CAC G-3′; (iii), 5′-GAG GCA GAA GGG AGA CAG AG-3′ and 5′-AAG TCA TCG GAG GGC AAT C-3′; (iv), 5′-TCG TGG AGT TGG TAA ATG AGG-3′ and 5′-TCC TCA CCA GAA AGG CAA TG-3′; and (v), 5′-GGA AAG CCC TGC AAT TTA CTC-3′ and 5′-CCC TTA TGG AGT CGA TTT TCC-3′.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Yuuki Yasunaga and Mika Matsumoto for sample preparation and Dr. Kingo Chida and our laboratory members for helpful discussions. We also thank Professor Dr. Tetsuaki Nishida and Professor Dr. Masayuki Fujii, Kinki University, for their encouragement through this study and helpful discussions. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan 24700975 (Y. Kotake), 22300329 (M. Kitagawa), 24570151 (K. Kitagawa) and 23131504 (H. Niida) and from Iizuka City (Y. Kotake).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Asakuno, K., Kohno, K., Uchiumi, T., Kubo, T., Sato, S., Isono, M. & Kuwano, M. (1994) Involvement of a DNA binding protein, MDR-NF1/YB-1, in human MDR1 gene expression by actinomycin D. Biochem. Biophys. Res. Commun. 199, 14281435.
  • Bargou, R.C., Jurchott, K., Wagener, C., Bergmann, S., Metzner, S., Bommert, K., Mapara, M.Y., Winzer, K.J., Dietel, M., Dorken, B. & Royer, H.D. (1997) Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression. Nat. Med. 3, 447450.
  • Basaki, Y., Taguchi, K., Izumi, H., Murakami, Y., Kubo, T., Hosoi, F., Watari, K., Nakano, K., Kawaguchi, H., Ohno, S., Kohno, K., Ono, M. & Kuwano, M. (2010) Y-box binding protein-1 (YB-1) promotes cell cycle progression through CDC6-dependent pathway in human cancer cells. Eur. J. Cancer 46, 954965.
  • Bergmann, S., Royer-Pokora, B., Fietze, E., Jurchott, K., Hildebrandt, B., Trost, D., Leenders, F., Claude, J.C., Theuring, F., Bargou, R., Dietel, M. & Royer, H.D. (2005) YB-1 provokes breast cancer through the induction of chromosomal instability that emerges from mitotic failure and centrosome amplification. Cancer Res. 65, 40784087.
  • Bracken, A.P., Kleine-Kohlbrecher, D., Dietrich, N., Pasini, D., Gargiulo, G., Beekman, C., Theilgaard-Monch, K., Minucci, S., Porse, B.T., Marine, J.C., Hansen, K.H. & Helin, K. (2007) The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev. 21, 525530.
  • Braig, M., Lee, S., Loddenkemper, C., Rudolph, C., Peters, A.H., Schlegelberger, B., Stein, H., Dorken, B., Jenuwein, T. & Schmitt, C.A. (2005) Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436, 660665.
  • Brookes, S., Rowe, J., Ruas, M., Llanos, S., Clark, P.A., Lomax, M., James, M.C., Vatcheva, R., Bates, S., Vousden, K.H., Parry, D., Gruis, N., Smit, N., Bergman, W. & Peters, G. (2002) INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence. EMBO J. 21, 29362945.
  • Cobrinik, D. (2005) Pocket proteins and cell cycle control. Oncogene 24, 27962809.
  • Collado, M., Gil, J., Efeyan, A., Guerra, C., Schuhmacher, A.J., Barradas, M., Benguria, A., Zaballos, A., Flores, J.M., Barbacid, M., Beach, D. & Serrano, M. (2005) Tumour biology: senescence in premalignant tumours. Nature 436, 642.
  • Fujii, T., Kawahara, A., Basaki, Y., Hattori, S., Nakashima, K., Nakano, K., Shirouzu, K., Kohno, K., Yanagawa, T., Yamana, H., Nishio, K., Ono, M., Kuwano, M. & Kage, M. (2008) Expression of HER2 and estrogen receptor alpha depends upon nuclear localization of Y-box binding protein-1 in human breast cancers. Cancer Res. 68, 15041512.
  • Gil, J. & Peters, G. (2006) Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat. Rev. Mol. Cell Biol. 7, 667677.
  • Gimenez-Bonafe, P., Fedoruk, M.N., Whitmore, T.G., Akbari, M., Ralph, J.L., Ettinger, S., Gleave, M.E. & Nelson, C.C. (2004) YB-1 is upregulated during prostate cancer tumor progression and increases P-glycoprotein activity. Prostate 59, 337349.
  • Gu, C., Oyama, T., Osaki, T., Kohno, K. & Yasumoto, K. (2001) Expression of Y box-binding protein-1 correlates with DNA topoisomerase IIalpha and proliferating cell nuclear antigen expression in lung cancer. Anticancer Res. 21, 23572362.
  • Kim, W.Y. & Sharpless, N.E. (2006) The regulation of INK4/ARF in cancer and aging. Cell 127, 265275.
  • Kohno, K., Izumi, H., Uchiumi, T., Ashizuka, M. & Kuwano, M. (2003) The pleiotropic functions of the Y-box-binding protein, YB-1. BioEssays 25, 691698.
  • Kotake, Y., Cao, R., Viatour, P., Sage, J., Zhang, Y. & Xiong, Y. (2007) pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene. Genes Dev. 21, 4954.
  • Kotake, Y., Nakagawa, T., Kitagawa, K., Suzuki, S., Liu, N., Kitagawa, M. & Xiong, Y. (2011) Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene. Oncogene 30, 19561962.
  • Kotake, Y., Zeng, Y. & Xiong, Y. (2009) DDB1-CUL4 and MLL1 mediate oncogene-induced p16INK4a activation. Cancer Res. 69, 18091814.
  • Krimpenfort, P., Quon, K.C., Mooi, W.J., Loonstra, A. & Berns, A. (2001) Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 413, 8386.
  • Lee, C., Dhillon, J., Wang, M.Y., Gao, Y., Hu, K., Park, E., Astanehe, A., Hung, M.C., Eirew, P., Eaves, C.J. & Dunn, S.E. (2008) Targeting YB-1 in HER-2 overexpressing breast cancer cells induces apoptosis via the mTOR/STAT3 pathway and suppresses tumor growth in mice. Cancer Res. 68, 86618666.
  • Lu, Z.H., Books, J.T. & Ley, T.J. (2005) YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence. Mol. Cell. Biol. 25, 46254637.
  • Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N., Horii, I. & Loh, D.Y. (1996) Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85, 707720.
  • Ohga, T., Koike, K., Ono, M., Makino, Y., Itagaki, Y., Tanimoto, M., Kuwano, M. & Kohno, K. (1996) Role of the human Y box-binding protein YB-1 in cellular sensitivity to the DNA-damaging agents cisplatin, mitomycin C, and ultraviolet light. Cancer Res. 56, 42244228.
  • Ohtani, N., Zebedee, Z., Huot, T.J., Stinson, J.A., Sugimoto, M., Ohashi, Y., Sharrocks, A.D., Peters, G. & Hara, E. (2001) Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence. Nature 409, 10671070.
  • Okamoto, T., Izumi, H., Imamura, T., Takano, H., Ise, T., Uchiumi, T., Kuwano, M. & Kohno, K. (2000) Direct interaction of p53 with the Y-box binding protein, YB-1: a mechanism for regulation of human gene expression. Oncogene 19, 61946202.
  • Ruas, M. & Peters, G. (1998) The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim. Biophys. Acta 1378, F115F177.
  • Serrano, M., Hannon, G.J. & Beach, D. (1993) A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366, 704707.
  • Serrano, M., Lin, A.W., McCurrach, M.E., Beach, D. & Lowe, S.W. (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593602.
  • Sharpless, N.E. (2005) INK4a/ARF: a multifunctional tumor suppressor locus. Mutat. Res. 576, 2238.
  • Sharpless, N.E., Bardeesy, N., Lee, K.H., Carrasco, D., Castrillon, D.H., Aguirre, A.J., Wu, E.A., Horner, J.W. & DePinho, R.A. (2001) Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413, 8691.
  • Sherr, C.J. (2006) Divorcing ARF and p53: an unsettled case. Nat. Rev. Cancer 6, 663673.
  • Shibahara, K., Sugio, K., Osaki, T., Uchiumi, T., Maehara, Y., Kohno, K., Yasumoto, K., Sugimachi, K. & Kuwano, M. (2001) Nuclear expression of the Y-box binding protein, YB-1, as a novel marker of disease progression in non-small cell lung cancer. Clin. Cancer Res. 7, 31513155.
  • Shibao, K., Takano, H., Nakayama, Y., Okazaki, K., Nagata, N., Izumi, H., Uchiumi, T., Kuwano, M., Kohno, K. & Itoh, H. (1999) Enhanced coexpression of YB-1 and DNA topoisomerase II alpha genes in human colorectal carcinomas. Int. J. Cancer 83, 732737.
  • Sorokin, A.V., Selyutina, A.A., Skabkin, M.A., Guryanov, S.G., Nazimov, I.V., Richard, C., Th'ng, J., Yau, J., Sorensen, P.H., Ovchinnikov, L.P. & Evdokimova, V. (2005) Proteasome-mediated cleavage of the Y-box-binding protein 1 is linked to DNA-damage stress response. EMBO J. 24, 36023612.
  • Uchiumi, T., Fotovati, A., Sasaguri, T., Shibahara, K., Shimada, T., Fukuda, T., Nakamura, T., Izumi, H., Tsuzuki, T., Kuwano, M. & Kohno, K. (2006) YB-1 is important for an early stage embryonic development: neural tube formation and cell proliferation. J. Biol. Chem. 281, 4044040449.
  • Wu, J., Lee, C., Yokom, D., Jiang, H., Cheang, M.C., Yorida, E., Turbin, D., Berquin, I.M., Mertens, P.R., Iftner, T., Gilks, C.B. & Dunn, S.E. (2006) Disruption of the Y-box binding protein-1 results in suppression of the epidermal growth factor receptor and HER-2. Cancer Res. 66, 48724879.
  • Yap, K.L., Li, S., Munoz-Cabello, A.M., Raguz, S., Zeng, L., Mujtaba, S., Gil, J., Walsh, M.J. & Zhou, M.M. (2010) Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol. Cell 38, 662674.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
gtc12093-sup-0001-figureS1.pdfapplication/PDF47KFigure S1 YB1 binds to the mouse p16 promoter.

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