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Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression

Abstract

Recurrence is frequently associated with the acquisition of radioresistance by tumors and resulting failures in radiotherapy. We report, in this study, that long-term fractionated radiation (FR) exposures conferred radioresistance to the human tumor cells, HepG2 and HeLa with cyclin D1 overexpression. A positive feedback loop was responsible for the cyclin D1 overexpression in which constitutively active AKT was involved. AKT is known to inactivate glycogen synthase kinase-3β (GSK3β), which is essential for the proteasomal degradation of cyclin D1. The resulting cyclin D1 overexpression led to the forced progression of S-phase with the induction of DNA double strand breaks. Cyclin D1-dependent DNA damage activated DNA-dependent protein kinase (DNA-PK), which in turn activated AKT and inactivated GSK3β, thus completing a positive feedback loop of cyclin D1 overproduction. Cyclin D1 overexpression led to the activation of DNA damage response (DDR) consisted of ataxia telangiectasia mutated (ATM)- and Chk1-dependent DNA damage checkpoint and homologous recombination repair (HRR). Long-term FR cells repaired radiation-induced DNA damage faster than non-FR cells. Thus, acquired radioresistance of long-term FR cells was the result of alterations in DDR mediated by cyclin D1 overexpression. Inhibition of the AKT/GSK3β/cyclin D1/Cdk4 pathway by the AKT inhibitor, Cdk4 inhibitor or cyclin D1 targeting small interfering RNA (siRNA) suppressed the radioresistance. Present observations give a mechanistic insight for acquired radioresistance of tumor cells by cyclin D1 overexpression, and provide novel therapeutic targets for recurrent radioresistant tumors.

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Abbreviations

API-2:

AKT/protein kinase B signaling inhibitor-2

APC:

anaphase promoting factor

BSA:

bovine serum albumin

BrdU:

bromodeoxyuridine

CSCs:

cancer stem cells

CDKs:

cyclin-dependent kinases

CHX:

cycloheximide

DDR:

DNA damage response

DNA-PK:

DNA-dependent protein kinase

DSBs:

double strand breaks

FACS:

fluorescence-activated cell sorting

FR:

fractionated radiation

GSK3β:

glycogen synthase kinase-3β

HE:

hematoxylin-eosin

IHC:

immunohistochemistry

PBS:

phosphate-buffered saline

PI:

propidium iodide

PKB:

protein kinase B

SR:

single radiation

SDS–PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  • Agami R, Bernards R . (2000). Distinct initiation and maintenance mechanisms cooperate to induce G1 cell cycle arrest in response to DNA damage. Cell 102: 55–66.

    Article  CAS  PubMed  Google Scholar 

  • Alao JP . (2007). The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol Cancer 6: 24.

    Article  PubMed  PubMed Central  Google Scholar 

  • Alt JR, Cleveland JL, Hannink M, Diehl JA . (2000). Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev 14: 3102–3114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arnaudeau C, Lundin C, Helleday T . (2001). DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J Mol Biol 307: 1235–1245.

    Article  CAS  PubMed  Google Scholar 

  • Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G . (1993). Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 7: 812–821.

    Article  CAS  PubMed  Google Scholar 

  • Bani-Hani K, Martin IG, Hardie LJ, Mapstone N, Briggs JA, Forman D et al. (2000). Prospective study of cyclin D1 overexpression in Barrett's esophagus: association with increased risk of adenocarcinoma. J Natl Cancer Inst 92: 1316–1321.

    Article  CAS  PubMed  Google Scholar 

  • Bartkova J, Lukas J, Muller H, Lutzhoft D, Strauss M, Bartek J . (1994a). Cyclin D1 protein expression and function in human breast cancer. Int J Cancer 57: 353–361.

    Article  CAS  PubMed  Google Scholar 

  • Bartkova J, Lukas J, Strauss M, Bartek J . (1994b). The PRAD-1/cyclin D1 oncogene product accumulates aberrantly in a subset of colorectal carcinomas. Int J Cancer 58: 568–573.

    Article  CAS  PubMed  Google Scholar 

  • Baumann M, Krause M, Hill R . (2008). Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 8: 545–554.

    Article  CAS  PubMed  Google Scholar 

  • Benzeno S, Lu F, Guo M, Barbash O, Zhang F, Herman JG et al. (2006). Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1. Oncogene 25: 6291–6303.

    Article  CAS  PubMed  Google Scholar 

  • Bommi-Reddy A, Almeciga I, Sawyer J, Geisen C, Li W, Harlow E et al. (2008). Kinase requirements in human cells: III. Altered kinase requirements in VHL-/- cancer cells detected in a pilot synthetic lethal screen. Proc Natl Acad Sci USA 105: 16484–16489.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bozulic L, Surucu B, Hynx D, Hemmings BA . (2008). PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell 30: 203–213.

    Article  CAS  PubMed  Google Scholar 

  • Cann KL, Hicks GG . (2007). Regulation of the cellular DNA double-strand break response. Biochem Cell Biol 85: 663–674.

    Article  CAS  PubMed  Google Scholar 

  • Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G . (2004). Cell death by mitotic catastrophe: a molecular definition. Oncogene 23: 2825–2837.

    Article  CAS  PubMed  Google Scholar 

  • Chang AR, Wu HG, Park CI, Jun YK, Kim CW . (2008). Expression of epidermal growth factor receptor and cyclin D1 in pretreatment biopsies as a predictive factor of radiotherapy efficacy in early glottic cancer. Head Neck 30: 852–857.

    Article  PubMed  Google Scholar 

  • Connell-Crowley L, Harper JW, Goodrich DW . (1997). Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. Mol Biol Cell 8: 287–301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Diehl JA, Zindy F, Sherr CJ . (1997). Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev 11: 957–972.

    Article  CAS  PubMed  Google Scholar 

  • Duronio V . (2008). The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem J 415: 333–344.

    Article  CAS  PubMed  Google Scholar 

  • Filmus J, Robles AI, Shi W, Wong MJ, Colombo LL, Conti CJ . (1994). Induction of cyclin D1 overexpression by activated ras. Oncogene 9: 3627–3633.

    CAS  PubMed  Google Scholar 

  • Giacinti C, Giordano A . (2006). RB and cell cycle progression. Oncogene 25: 5220–5227.

    Article  CAS  PubMed  Google Scholar 

  • Gillett C, Fantl V, Smith R, Fisher C, Bartek J, Dickson C et al. (1994). Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res 54: 1812–1817.

    CAS  PubMed  Google Scholar 

  • Gladden AB, Diehl JA . (2005). Location, location, location: the role of cyclin D1 nuclear localization in cancer. J Cell Biochem 96: 906–913.

    Article  CAS  PubMed  Google Scholar 

  • Harper JW, Elledge SJ . (2007). The DNA damage response: ten years after. Mol Cell 28: 739–745.

    Article  CAS  PubMed  Google Scholar 

  • Hartwell LH, Weinert TA . (1989). Checkpoints: controls that ensure the order of cell cycle events. Science 246: 629–634.

    Article  CAS  PubMed  Google Scholar 

  • Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, Orr AI et al. (2004). Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res 64: 9152–9159.

    Article  CAS  PubMed  Google Scholar 

  • Higuchi E, Oridate N, Homma A, Suzuki F, Atago Y, Nagahashi T et al. (2007). Prognostic significance of cyclin D1 and p16 in patients with intermediate-risk head and neck squamous cell carcinoma treated with docetaxel and concurrent radiotherapy. Head Neck 29: 940–947.

    Article  PubMed  Google Scholar 

  • Hitomi M, Stacey DW . (1999). Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner. Curr Biol 9: 1075–1084.

    Article  CAS  PubMed  Google Scholar 

  • Kang T, Wei Y, Honaker Y, Yamaguchi H, Appella E, Hung MC et al. (2008). GSK-3 beta targets Cdc25A for ubiquitin-mediated proteolysis, and GSK-3 beta inactivation correlates with Cdc25A overproduction in human cancers. Cancer Cell 13: 36–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim JJ, Tannock IF . (2005). Repopulation of cancer cells during therapy: an important cause of treatment failure. Nat Rev Cancer 5: 516–525.

    Article  CAS  PubMed  Google Scholar 

  • Lakin ND, Jackson SP . (1999). Regulation of p53 in response to DNA damage. Oncogene 18: 7644–7655.

    Article  CAS  PubMed  Google Scholar 

  • Lin DI, Lessie MD, Gladden AB, Bassing CH, Wagner KU, Diehl JA . (2008). Disruption of cyclin D1 nuclear export and proteolysis accelerates mammary carcinogenesis. Oncogene 27: 1231–1242.

    Article  CAS  PubMed  Google Scholar 

  • Massague J . (2004). G1 cell-cycle control and cancer. Nature 432: 298–306.

    Article  CAS  PubMed  Google Scholar 

  • Nicholson KM, Anderson NG . (2002). The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 14: 381–395.

    Article  CAS  PubMed  Google Scholar 

  • Pagano M, Theodoras AM, Tam SW, Draetta GF . (1994). Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts. Genes Dev 8: 1627–1639.

    Article  CAS  PubMed  Google Scholar 

  • Pierce AJ, Johnson RD, Thompson LH, Jasin M . (1999). XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13: 2633–2638.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pilch DR, Sedelnikova OA, Redon C, Celeste A, Nussenzweig A, Bonner WM . (2003). Characteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem Cell Biol 81: 123–129.

    Article  CAS  PubMed  Google Scholar 

  • Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Roussel MF et al. (1993). Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 7: 1559–1571.

    Article  CAS  PubMed  Google Scholar 

  • Rich JN . (2007). Cancer stem cells in radiation resistance. Cancer Res 67: 8980–8984.

    Article  CAS  PubMed  Google Scholar 

  • Russell A, Thompson MA, Hendley J, Trute L, Armes J, Germain D . (1999). Cyclin D1 and D3 associate with the SCF complex and are coordinately elevated in breast cancer. Oncogene 18: 1983–1991.

    Article  CAS  PubMed  Google Scholar 

  • Sedelnikova OA, Pilch DR, Redon C, Bonner WM . (2003). Histone H2AX in DNA damage and repair. Cancer Biol Ther 2: 233–235.

    Article  CAS  PubMed  Google Scholar 

  • Sherr CJ, McCormick F . (2002). The RB and p53 pathways in cancer. Cancer Cell 2: 103–112.

    Article  CAS  PubMed  Google Scholar 

  • Sherr CJ, Roberts JM . (1995). Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9: 1149–1163.

    Article  CAS  PubMed  Google Scholar 

  • Shimura T, Martin MM, Torres MJ, Gu C, Pluth JM, DeBernardi MA et al. (2007). DNA-PK is involved in repairing a transient surge of DNA breaks induced by deceleration of DNA replication. J Mol Biol 367: 665–680.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shimura T, Torres MJ, Martin MM, Rao VA, Pommier Y, Katsura M et al. (2008). Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress. J Mol Biol 375: 1152–1164.

    Article  CAS  PubMed  Google Scholar 

  • Takahashi-Yanaga F, Sasaguri T . (2008). GSK-3beta regulates cyclin D1 expression: a new target for chemotherapy. Cell Signal 20: 581–589.

    Article  CAS  PubMed  Google Scholar 

  • Toda Y, Kono K, Abiru H, Kokuryo K, Endo M, Yaegashi H et al. (1999). Application of tyramide signal amplification system to immunohistochemistry: a potent method to localize antigens that are not detectable by ordinary method. Pathol Int 49: 479–483.

    Article  CAS  PubMed  Google Scholar 

  • Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, Durkacz BW . (2003). Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. Cancer Res 63: 6008–6015.

    CAS  PubMed  Google Scholar 

  • Yang L, Dan HC, Sun M, Liu Q, Sun XM, Feldman RI et al. (2004). Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res 64: 4394–4399.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr Ohtsura Niwa and Dr Keiko Nakayama for their critical reading of the paper. We thank Dr J Alan Diehl for providing pFleX-cyclin D1 expression vectors. We thank Dr Shuntaro Ikawa for providing some chemicals. This study was in part supported by the Grant-in-Aid for young scientists (Start-up-20810003) and (Wakate-B-21710054), by the Grant-in-Aid from Ministry of Education, Culture, Sports, Science and Technology, by grants from the Ministry of Health, Labour and Welfare of Japan, and by the ‘Frontier Science Research Program’ in the Center for Interdisciplinary Research, Tohoku University.

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Correspondence to M Fukumoto.

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Shimura, T., Kakuda, S., Ochiai, Y. et al. Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. Oncogene 29, 4826–4837 (2010). https://doi.org/10.1038/onc.2010.238

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