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siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Waf1/Cip1) expression

Abstract

The transformation suppressor gene, programmed cell death gene 4 (Pdcd4), inhibits tumor-promoter-mediated transformation of mouse keratinocytes and has been implicated as a tumor suppressor gene in the development of human cancer. The Pdcd4 protein interacts with translation initiation factors eIF4A and eIF4G and binds to RNA, suggesting that it might be involved in regulating protein translation or other aspects of RNA metabolism. To study the function of Pdcd4 in more detail, we have downregulated Pdcd4 expression in HeLa cells by stable expression of shRNA. We have found that diminished Pdcd4 expression leads to increased expression of p21(Waf1/Cip1) and several other p53-regulated genes. Reporter gene studies demonstrate that Pdcd4 interferes with the activation of p53-responsive promoters genes by p53. Pdcd4 knockdown cells show decreased apoptosis and increased survival after UV irradiation. Taken together, our observations suggest a model in which low Pdcd4 expression after DNA damage favors the survival of cells, which would be eliminated by apoptosis under normal levels of Pdcd4 expression. Our results provide the first evidence that Pdcd4 is important role in the DNA-damage response and suggest that low levels of Pdcd4 expression observed in certain tumor cells contribute to tumorigenesis by affecting the fate of DNA-damaged cells.

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References

  • Afonja O, Juste D, Das S, Matsuhashi S, Samuels HH . (2004). Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene 23: 8135–8145.

    Article  CAS  PubMed  Google Scholar 

  • Bendjennat M, Boulaire J, Jascur T, Brickner H, Barbier V, Sarasin A et al. (2003). UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. Cell 114: 599–610.

    Article  CAS  PubMed  Google Scholar 

  • Biankin AV, Kench JG, Morey AL, Lee CS, Biankin SA, Head DR et al. (2001). Overexpression of p21(WAF1/CIP1) is an early event in the development of pancreatic intraepithelial neoplasia. Cancer Res 61: 8830–8837.

    CAS  PubMed  Google Scholar 

  • Bitomsky N, Böhm M, Klempnauer K-H . (2004). Transformation suppressor protein Pdcd4 interferes with JNK-mediated phosphorylation of c-Jun and recruitment of the coactivator p300 by c-Jun. Oncogene 23: 7484–7493.

    Article  CAS  PubMed  Google Scholar 

  • Bode AM, Dong Z . (2004). Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 4: 793–805.

    Article  CAS  PubMed  Google Scholar 

  • Böhm M, Sawicka K, Siebrasse JP, Brehmer-Fastnacht A, Peters R, Klempnauer K-H . (2003). The transformation suppressor protein Pdcd4 shuttles between nucleus and cytoplasm and binds RNA. Oncogene 22: 4905–4910.

    Article  PubMed  Google Scholar 

  • Burk O, Mink S, Ringwald M, Klempnauer K-H . (1993). Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors. EMBO J 12: 2027–2038.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen Y, Knosel T, Kristiansen G, Pietas A, Garber ME, Matsuhashi S et al. (2003). Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis. J Pathol 200: 640–646.

    Article  CAS  PubMed  Google Scholar 

  • Cmarik JL, Min H, Hegamyer G, Zhan S, Kulesz-Martin M, Yoshinaga H et al. (1999). Differentially expressed protein Pdcd4 inhibits tumor promoter-induced neoplastic transformation. Proc Natl Acad Sci USA 96: 14037–14042.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dorrello NV, Peschiaroli A, Guardavaccaro D, Colburn NH, Sherman NE, Pagano M . (2006). S6K1- and βTRCP-mediated degradation of Pdcd4 promotes protein translation and cell growth. Science 314: 467–471.

    Article  CAS  PubMed  Google Scholar 

  • Dupont J, Karas M, LeRoith D . (2003). The cyclin-dependent kinase inhibitor p21CIP/WAF is a positive regulator of insulin-like growth factor I-induced cell proliferation in MCF-7 human breast cancer cells. J Biol Chem 278: 37256–37264.

    Article  CAS  PubMed  Google Scholar 

  • El-Deiry WS, Tokino T, Velculesco VE, Levy DB, Parsons R, Trent JM et al. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 75: 817–825.

    Article  CAS  PubMed  Google Scholar 

  • Erber R, Klein W, Andl T, Enders C, Born AI, Conradt C et al. (1997). Aberrant p21(CIP1/WAF1) protein accumulation in head and neck cancer. Int J Cancer 74: 383–389.

    Article  CAS  PubMed  Google Scholar 

  • Fan Y, Borowsky AD, Weiss RH . (2003). An antisense oligodeoxynucleotide to p21(Waf1/Cip1) causes apoptosis in human breast cancer cells. Mol Cancer Ther 2: 773–782.

    CAS  PubMed  Google Scholar 

  • Fornace Jr AJ, Alamo Jr I, Hollander MC . (1988). DNA damage-inducible transcripts in mammalian cells. Proc Natl Acad Sci USA 85: 8800–8804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gao F, Zhang P, Zhou C, Li J, Wang Q, Zhu F et al. (2007). Frequent loss of PDCD4 expression in human glioma: possible role in the tumorigenesis of glioma. Oncol Rep 17: 123–128.

    CAS  PubMed  Google Scholar 

  • Gartel AL, Radhakrishnan SK . (2005). Lost in transcription: p21 repression, mechanisms, and consequences. Cancer Res 65: 3980–3985.

    Article  CAS  PubMed  Google Scholar 

  • Göke A, Göke R, Knolle A, Trusheim H, Schmidt H, Wilmen A et al. (2002). DUG is a novel homologue of translation initiation factor 4G that binds eIF4A. Biochem Biophys Res Commun 297: 78–82.

    Article  PubMed  Google Scholar 

  • Göke R, Barth P, Schmidt A, Samans B, Lankat-Buttgereit B . (2004). Programmed cell death protein 4 suppresses CDK1/cdc2 via induction of p21(Waf1/Cip1). Am J Physiol Cell Physiol 287: C1541–C1546.

    Article  PubMed  Google Scholar 

  • Gorospe M, Cirielli C, Wang X, Seth P, Capogrossi MC, Holbrook NJ . (1997). p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene 14: 929–935.

    Article  CAS  PubMed  Google Scholar 

  • Gu W, Roeder RG . (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90: 595–606.

    Article  CAS  PubMed  Google Scholar 

  • Harper JW, Adami GR, Wei N, Keymarsi K, Elledge S . (1993). The p21 Cdk interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75: 805–816.

    Article  CAS  PubMed  Google Scholar 

  • Herold S, Wanzel M, Beuger V, Frohme C, Beul D, Hillukkala T et al. (2002). Negative regulation of the mammalian UV response by Myc through association with Miz-1. Mol Cell 10: 509–521.

    Article  CAS  PubMed  Google Scholar 

  • Hershey JWB, Merrick WC . (2000). Translational control of gene expression: pathway and mechanism of initiation of protein synthesis. In: Sonenberg N, Hershey JWB, Mathews MB (eds). Translational Control. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, pp 33–88.

    Google Scholar 

  • Hilliard A, Hilliard B, Zheng SJ, Sun H, Miwa T, Song W et al. (2006). Translational regulation of autoimmune inflammation and lymphoma genesis by programmed cell death 4. J Immunol 177: 8095–8102.

    Article  CAS  PubMed  Google Scholar 

  • Jansen AP, Camalier CE, Colburn NH . (2005). Epidermal expression of the translation inhibitor programmed cell death 4 suppresses tumorigenesis. Cancer Res 65: 6034–6041.

    Article  CAS  PubMed  Google Scholar 

  • Jansen AP, Camalier CE, Stark C, Colburn NH . (2004). Characterization of programmed cell death 4 in multiple human cancers reveals a novel enhancer of drug sensitivity. Mol Cancer Ther 3: 103–110.

    CAS  PubMed  Google Scholar 

  • Jung JM, Bruner JM, Ruan S, Langford LA, Kyritsis AP, Kobayashi T et al. (1995). Increased levels of p21WAF1/Cip1 in human brain tumors. Oncogene 11: 2021–2028.

    CAS  PubMed  Google Scholar 

  • LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS et al. (1997). New functional activities for the p21 family of CDK inhibitors. Genes Dev 11: 847–862.

    Article  CAS  PubMed  Google Scholar 

  • Lankat-Buttgereit B, Göke R . (2003). Programmed cell death protein 4 (pdcd4): a novel target for antineoplastic therapy? Biol Cell 95: 515–519.

    Article  CAS  PubMed  Google Scholar 

  • LaRonde-LeBlanc N, Santhanam AN, Baker AR, Wlodawer A, Colburn NH . (2007). Structural basis for inhibition of translation by the tumor suppressor Pdcd4. Mol Cell Biol 27: 147–156.

    Article  CAS  PubMed  Google Scholar 

  • Linder P, Lasko P, Ashburner M, Leroy P, Nielsen P, Nishi K et al. (1989). Birth of the D-E-A-D box. Nature 337: 121–122.

    Article  CAS  PubMed  Google Scholar 

  • Martoriati A, Doumont G, Alcalay M, Bellefroid E, Pelicci PG, Marine JC . (2005). Dapk1, encoding an activator of a p19ARF-p53-mediated apoptotic checkpoint, is a transcription target of p53. Oncogene 24: 1461–1466.

    Article  CAS  PubMed  Google Scholar 

  • Moscovici C, Moscovici MG, Jimenez H, Lai MM, Hayman MJ, Vogt PK . (1977). Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell 11: 95–103.

    Article  CAS  PubMed  Google Scholar 

  • Mudduluru G, Medved F, Grobholz R, Jost C, Gruber A, Leupold JH et al. (2007). Loss of programmed cell death 4 expression marks adenoma-carcinoma transition, correlates inversely with phosphorylated protein kinase B, and is an independent prognostic factor in resected colorectal cancer. Cancer 110: 1697–1707.

    Article  CAS  PubMed  Google Scholar 

  • Ozanne BW, Spence HJ, McGarry LC, Hennigan RF . (2007). Transcription factors control invasion: AP-1 the first among equals. Oncogene 26: 1–10.

    Article  CAS  PubMed  Google Scholar 

  • Palamarchuk A, Efanov A, Maximov V, Aqeilan RI, Croce CM, Pekarsky Y . (2005). Akt phosphorylates and regulates Pdcd4 tumor suppressor protein. Cancer Res 65: 11282–11286.

    Article  CAS  PubMed  Google Scholar 

  • Prives C, Manley JL . (2001). Why is p53 acetylated? Cell 107: 815–818.

    Article  CAS  PubMed  Google Scholar 

  • Rozen F, Edery I, Meerovitch K, Dever T, Merrick W, Sonenberg N . (1990). Bidirectional RNA helicase activity of eukaryotic translation initiation factor 4A and 4F. Mol Cell Biol 10: 1134–1144.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seoane J, Le HV, Massague J . (2002). Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 419: 729–734.

    Article  CAS  PubMed  Google Scholar 

  • Schlichter U, Burk O, Worpenberg S, Klempnauer K-H . (2001). The chicken Pdcd4 gene is regulated by v-Myb. Oncogene 20: 231–239.

    Article  CAS  PubMed  Google Scholar 

  • Sherr CJ, Roberts JM . (1999). CDK inhibitors: positive and negative regulators of the G1-phase progression. Genes Dev 13: 1501–1512.

    Article  CAS  PubMed  Google Scholar 

  • Shibahara K, Asano M, Ishida Y, Aoki T, Koike T, Honjo T . (1995). Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death. Gene 166: 297–301.

    Article  CAS  PubMed  Google Scholar 

  • Sohn D, Essmann F, Schulze-Osthoff K, Jänicke RU . (2006). p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation. Cancer Res 66: 11254–11262.

    Article  CAS  PubMed  Google Scholar 

  • Toledo F, Wahl GM . (2006). Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer 6: 909–923.

    Article  CAS  PubMed  Google Scholar 

  • van de Wetering M, Oving I, Muncan V, Pon Fong MT, Brantjes H, van Leenen D et al. (2003). Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep 4: 609–615.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Q, Sun Z, Yang H-S . (2008). Downregulation of tumour suppressor Pdcd4 promotes invasion and activates both β-catenin/TCF and AP-1-dependent transcription in colon carvinoma cells. Oncogene 27: 1527–1535.

    Article  CAS  PubMed  Google Scholar 

  • Waters LC, Boehm M, Veverka V, Muskett FW, Frenkiel TA, Kelly GP et al. (2006). NMR assignment and secondary structure determination of the C-terminal MA-3 domain of the tumour suppressor protein Pdcd4. J Biomol NMR 36 (Suppl 1): 18.

    Article  PubMed  Google Scholar 

  • Waters LC, Veverka V, Boehm M, Schmedt T, Choong PT, Muskett FW et al. (2007). Structure of the C-terminal MA-3 domain of the tumour suppressor protein Pdcd4 and characterisation of its interaction with eIF4A. Oncogene 26: 4941–4950.

    Article  CAS  PubMed  Google Scholar 

  • Weiss RH . (2003). p21Waf1/Cip1 as a therapeutic target in breast and other cancers. Cancer Cell 4: 425–429.

    Article  CAS  PubMed  Google Scholar 

  • Weiss RH, Marshall D, Howard L, Corbacho AM, Cheung AT, Sawai ET . (2003). Suppression of breast cancer growth and angiogenesis by an antisense oligodeoxynucleotide to p21(Waf1/Cip1). Cancer Lett 189: 39–48.

    Article  CAS  PubMed  Google Scholar 

  • Wen YH, Shi X, Chiriboga L, Matsahashi S, Yee H, Afonja O . (2007). Alterations in the expression of PDCD4 in ductal carcinoma of the breast. Oncol Rep 18: 1387–1393.

    CAS  PubMed  Google Scholar 

  • Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D . (1993). p21 is a universal inhibitor of cyclin kinases. Nature 366: 701–704.

    Article  CAS  PubMed  Google Scholar 

  • Yang HS, Cho MH, Zacowicz H, Hegamyer G, Sonenberg N, Colburn N . (2004). A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A. Mol Cell Biol 24: 3894–3906.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang HS, Jansen AP, Komar AA, Zheng X, Merrick WC, Costes S et al. (2003a). The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol Cell Biol 23: 26–37.

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang HS, Jansen AP, Nair R, Shibahara K, Verma AK, Cmarik JL et al. (2001). A novel transformation suppressor, Pdcd4, inhibits AP-1 transactivation but not NF-kappaB or ODC transactivation. Oncogene 20: 669–676.

    Article  CAS  PubMed  Google Scholar 

  • Yang HS, Knies JL, Stark C, Colburn NH . (2003b). Pdcd4 suppresses tumor phenotype in JB6 cells by inhibiting AP-1 transactivation. Oncogene 22: 3712–3720.

    Article  CAS  PubMed  Google Scholar 

  • Yang H-S, Matthews CP, Clair T, Wang Q, Baker AR, Li C-CH et al. (2006). Tumorigenesis suppressor Pdcd4 down-regulates mitogen-activated protein kinase kinase kinase kinase 1 expression to suppress colon carcinoma cell invasion. Mol Cell Biol 26: 1297–1306.

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang W, Klos KS, Zhou X, Yao J, Yang Y, Smith TL et al. (2003c). ErbB2 overexpression in human breast carcinoma is correlated with p21Cip1 up-regulation and tyrosine-15 hyperphosphorylation of p34Cdc2: poor responsiveness to chemotherapy with cyclophoshamide methotrexate, and 5-fluorouracil is associated with Erb2 overexpression and with p21Cip1 overexpression. Cancer 98: 1123–1130.

    Article  CAS  PubMed  Google Scholar 

  • Young MR, Yang HS, Colburn NH . (2003). Promising molecular targets for cancer prevention: AP-1, NF-kappa B and Pdcd4. Trends Mol Med 9: 36–41.

    Article  CAS  PubMed  Google Scholar 

  • Zeng YX, Somasundaram K, el-Deiry WS . (1997). AP2 inhibits cancer cell growth and activates p21WAF1/CIP1 expression. Nat Genet 15: 78–82.

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Ozaki I, Mizuta T, Hamajima H, Yasutake T, Eguchi Y et al. (2006). Involvement of programmed cell death 4 in transforming growth factor-beta1-induced apoptosis in human hepatocellular carcinoma. Oncogene 25: 6101–6112.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank B Berkenfeld for excellent technical assistance, and W El-Deiry, R Eckner, M Gorospe, L-G Larsson, S Stein and T Hofmann for providing plasmids and M Königs for performing the caspase-3 assay. This work was supported by grants from the Deutsche Krebshilfe (10-1716) and the Wilhelm-Sander-Stiftung (2004.088.1) to K-HK. RM was supported by a fellowship from the Graduate School of Chemistry (GSC-MS) at the University of Münster.

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Correspondence to K-H Klempnauer.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Bitomsky, N., Wethkamp, N., Marikkannu, R. et al. siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Waf1/Cip1) expression. Oncogene 27, 4820–4829 (2008). https://doi.org/10.1038/onc.2008.115

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