Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Nodal enhances the activity of FoxO3a and its synergistic interaction with Smads to regulate cyclin G2 transcription in ovarian cancer cells

Oncogene volume 30, pages 3953–3966 (2011)Cite this article

Subjects

Abstract

Nodal, a member of the transforming growth factor-β superfamily, has been recently shown to suppress cell proliferation and to stimulate the expression of cyclin G2 (CCNG2) in human epithelial ovarian cancer cells. However, the precise mechanisms underlying these events are not fully understood. In this study, we investigated the transcriptional regulation of CCNG2 by the Nodal signaling pathway. In ovarian cancer cells, overexpression of Nodal or its receptors, activin receptor-like kinase 7 (ALK7) or ALK4, resulted in an increase in the CCNG2 promoter activity. Several putative Forkhead box class O (FoxO)3a-binding sites are present in the human CCNG2 promoter and overexpression of FoxO3a enhanced the CCNG2 promoter activity. The functional FoxO3a-binding element (FBE) was mapped to a proximal region located between −398 and −380 bp (FBE1) through deletion and mutation analyses, as well as chromatin immunoprecipitation (IP) assay. Interestingly, mutation of the FBE1 not only abolished the effect of FoxO3a, but also blocked Nodal-induced CCNG2 transcription. Nodal stimulated FoxO3a mRNA and protein expression through the canonical Smad pathway and suppressed FoxO3a inactivation by inhibiting AKT activity. Silencing of FoxO3a using small interfering RNA significantly reduced the effect of Nodal on the CCNG2 promoter activity. On the other hand, overexpression of Smad2 and Smad3 enhanced the FoxO3a-induced CCNG2 promoter activity whereas knockdown of Smad4 blocked the activity of FoxO3a. Furthermore, IP assays revealed that FoxO3a formed complexes with Smad proteins and that Nodal enhanced the binding of FoxO3a to the CCNG2 promoter. Finally, silencing of FoxO3a reversed the inhibitory effect of Nodal on cell proliferation. Taken together, these findings demonstrated that Nodal signaling promotes CCNG2 transcription by upregulating FoxO3a expression, inhibiting FoxO3a phosphorylation and enhancing its synergistic interaction with Smads. These results also suggest that FoxO3a is an important mediator of Nodal signaling in ovarian cancer cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10

Similar content being viewed by others

References

  • Accili D, Arden KC . (2004). FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117: 421–426.

    CAS  Google Scholar 

  • Adorno M, Cordenonsi M, Montagner M, Dupont S, Wong C, Hann B et al. (2009). A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis. Cell 137: 87–98.

    Article  CAS  Google Scholar 

  • Anderson MJ, Viars CS, Czekay S, Cavenee WK, Arden KC . (1998). Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 47: 187–199.

    Article  CAS  Google Scholar 

  • Baker K, Holtzman NG, Burdine RD . (2008). Direct and indirect roles for Nodal signaling in two axis conversions during asymmetric morphogenesis of the zebrafish heart. Proc Natl Acad Sci USA 105: 13924–13929.

    Article  CAS  Google Scholar 

  • Bates S, Rowan S, Vousden KH . (1996). Characterisation of human cyclin G1 and G2: DNA damage inducible genes. Oncogene 13: 1103–1109.

    CAS  Google Scholar 

  • Bennin DA, Don AS, Brake T, McKenzie JL, Rosenbaum H, Ortiz L et al. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B’ subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest. J Biol Chem 277: 27449–27467.

    Article  CAS  Google Scholar 

  • Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS et al. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857–868.

    Article  CAS  Google Scholar 

  • Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y et al. (2004). Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303: 2011–2015.

    Article  CAS  Google Scholar 

  • Chen J, Yusuf I, Andersen HM, Fruman DA . (2006). FOXO transcription factors cooperate with delta EF1 to activate growth suppressive genes in B lymphocytes. J Immunol 176: 2711–2721.

    Article  CAS  Google Scholar 

  • Cornforth AN, Davis JS, Khanifar E, Nastiuk KL, Krolewski JJ . (2008). FOXO3a mediates the androgen-dependent regulation of FLIP and contributes to TRAIL-induced apoptosis of LNCaP cells. Oncogene 27: 4422–4433.

    Article  CAS  Google Scholar 

  • Derynck R, Akhurst RJ, Balmain A . (2001). TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 29: 117–129.

    Article  CAS  Google Scholar 

  • Derynck R, Zhang YE . (2003). Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425: 577–584.

    Article  CAS  Google Scholar 

  • Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW et al. (2000). Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol Cell Biol 20: 9138–9148.

    Article  CAS  Google Scholar 

  • Fei M, Zhao Y, Wang Y, Lu M, Cheng C, Huang X et al. (2009). Low expression of Foxo3a is associated with poor prognosis in ovarian cancer patients. Cancer Invest 27: 52–59.

    Article  CAS  Google Scholar 

  • Fernandez de Mattos S, Essafi A, Soeiro I, Pietersen AM, Birkenkamp KU, Edwards CS et al. (2004). FoxO3a and BCR-ABL regulate cyclin D2 transcription through a STAT5/BCL6-dependent mechanism. Mol Cell Biol 24: 10058–10071.

    Article  CAS  Google Scholar 

  • Furuyama T, Nakazawa T, Nakano I, Mori N . (2000). Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues. Biochem J 349: 629–634.

    Article  CAS  Google Scholar 

  • Gomis RR, Alarcon C, He W, Wang Q, Seoane J, Lash A et al. (2006a). A FoxO-Smad synexpression group in human keratinocytes. Proc Natl Acad Sci USA 103: 12747–12752.

    Article  CAS  Google Scholar 

  • Gomis RR, Alarcon C, Nadal C, Van Poznak C, Massague J . (2006b). C/EBPbeta at the core of the TGFbeta cytostatic response and its evasion in metastatic breast cancer cells. Cancer Cell 10: 203–214.

    Article  CAS  Google Scholar 

  • Hauck L, Harms C, Grothe D, An J, Gertz K, Kronenberg G et al. (2007). Critical role for FoxO3a-dependent regulation of p21CIP1/WAF1 in response to statin signaling in cardiac myocytes. Circ Res 100: 50–60.

    Article  CAS  Google Scholar 

  • Horne MC, Donaldson KL, Goolsby GL, Tran D, Mulheisen M, Hell JW et al. (1997). Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest. J Biol Chem 272: 12650–12661.

    Article  CAS  Google Scholar 

  • Horne MC, Goolsby GL, Donaldson KL, Tran D, Neubauer M, Wahl AF . (1996). Cyclin G1 and cyclin G2 comprise a new family of cyclins with contrasting tissue-specific and cell cycle-regulated expression. J Biol Chem 271: 6050–6061.

    Article  CAS  Google Scholar 

  • Hu MC, Lee DF, Xia W, Golfman LS, Ou-Yang F, Yang JY et al. (2004). IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 117: 225–237.

    Article  CAS  Google Scholar 

  • Huang H, Tindall DJ . (2007). Dynamic FoxO transcription factors. J Cell Sci 120: 2479–2487.

    Article  CAS  Google Scholar 

  • Katayama K, Nakamura A, Sugimoto Y, Tsuruo T, Fujita N . (2008). FOXO transcription factor-dependent p15(INK4b) and p19(INK4d) expression. Oncogene 27: 1677–1686.

    Article  CAS  Google Scholar 

  • Kenney NJ, Adkins HB, Sanicola M . (2004). Nodal and Cripto-1: embryonic pattern formation genes involved in mammary gland development and tumorigenesis. J Mammary Gland Biol Neoplasia 9: 133–144.

    Article  Google Scholar 

  • Kim Y, Shintani S, Kohno Y, Zhang R, Wong DT . (2004). Cyclin G2 dysregulation in human oral cancer. Cancer Res 64: 8980–8986.

    Article  CAS  Google Scholar 

  • Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, Burgering BM . (1999). Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398: 630–634.

    Article  CAS  Google Scholar 

  • Kumar A, Novoselov V, Celeste AJ, Wolfman NM, Ten Dijke P, Kuehn MR . (2001). Nodal signaling uses activin and transforming growth factor-beta receptor-regulated Smads. J Biol Chem 276: 656–661.

    Article  CAS  Google Scholar 

  • Le XF, Arachchige-Don AS, Mao W, Horne MC, Bast Jr RC . (2007). Roles of human epidermal growth factor receptor 2, c-jun NH2-terminal kinase, phosphoinositide 3-kinase, and p70 S6 kinase pathways in regulation of cyclin G2 expression in human breast cancer cells. Mol Cancer Ther 6: 2843–2857.

    Article  CAS  Google Scholar 

  • Li Y, Wang Z, Kong D, Murthy S, Dou QP, Sheng S et al. (2007). Regulation of FOXO3a/beta-catenin/GSK-3beta signaling by 3,3′-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in prostate cancer cells. J Biol Chem 282: 21542–21550.

    Article  CAS  Google Scholar 

  • Lin K, Dorman JB, Rodan A, Kenyon C . (1997). daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278: 1319–1322.

    Article  CAS  Google Scholar 

  • Marshall GM, Gherardi S, Xu N, Neiron Z, Trahair T, Scarlett CJ et al. (2010). Transcriptional upregulation of histone deacetylase 2 promotes Myc-induced oncogenic effects. Oncogene 29: 5957–5968.

    Article  CAS  Google Scholar 

  • Martinez-Gac L, Marques M, Garcia Z, Campanero MR, Carrera AC . (2004). Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead. Mol Cell Biol 24: 2181–2189.

    Article  CAS  Google Scholar 

  • Massague J, Seoane J, Wotton D . (2005). Smad transcription factors. Genes Dev 19: 2783–2810.

    Article  CAS  Google Scholar 

  • Matsuyama S, Iwadate M, Kondo M, Saitoh M, Hanyu A, Shimizu K et al. (2003). SB-431542 and Gleevec inhibit transforming growth factor-beta-induced proliferation of human osteosarcoma cells. Cancer Res 63: 7791–7798.

    CAS  PubMed  Google Scholar 

  • Munir S, Xu G, Wu Y, Yang B, Lala PK, Peng C . (2004). Nodal and ALK7 inhibit proliferation and induce apoptosis in human trophoblast cells. J Biol Chem 279: 31277–31286.

    Article  CAS  Google Scholar 

  • Naora H, Montell DJ . (2005). Ovarian cancer metastasis: integrating insights from disparate model organisms. Nat Rev Cancer 5: 355–366.

    Article  CAS  Google Scholar 

  • Ogawa K, Saito A, Matsui H, Suzuki H, Ohtsuka S, Shimosato D et al. (2007). Activin-nodal signaling is involved in propagation of mouse embryonic stem cells. J Cell Sci 120: 55–65.

    Article  CAS  Google Scholar 

  • Papageorgiou I, Nicholls PK, Wang F, Lackmann M, Makanji Y, Salamonsen LA et al. (2009). Expression of nodal signalling components in cycling human endometrium and in endometrial cancer. Reprod Biol Endocrinol 7: 122.

    Article  Google Scholar 

  • Ramaswamy S, Nakamura N, Sansal I, Bergeron L, Sellers WR . (2002). A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2: 81–91.

    Article  CAS  Google Scholar 

  • Reissmann E, Jornvall H, Blokzijl A, Andersson O, Chang C, Minchiotti G et al. (2001). The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. Genes Dev 15: 2010–2022.

    Article  CAS  Google Scholar 

  • Schier AF . (2003). Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol 19: 589–621.

    Article  CAS  Google Scholar 

  • Schier AF, Shen MM . (2000). Nodal signalling in vertebrate development. Nature 403: 385–389.

    Article  CAS  Google Scholar 

  • Seoane J, Le HV, Shen L, Anderson SA, Massague J . (2004). Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117: 211–223.

    Article  CAS  Google Scholar 

  • Shen MM . (2007). Nodal signaling: developmental roles and regulation. Development 134: 1023–1034.

    Article  CAS  Google Scholar 

  • Stossi F, Likhite VS, Katzenellenbogen JA, Katzenellenbogen BS . (2006). Estrogen-occupied estrogen receptor represses cyclin G2 gene expression and recruits a repressor complex at the cyclin G2 promoter. J Biol Chem 281: 16272–16278.

    Article  CAS  Google Scholar 

  • Strizzi L, Postovit LM, Margaryan NV, Seftor EA, Abbott DE, Seftor RE et al. (2008). Emerging roles of nodal and Cripto-1: from embryogenesis to breast cancer progression. Breast Dis 29: 91–103.

    Article  Google Scholar 

  • Sunters A, Fernandez de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA et al. (2003). FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 278: 49795–49805.

    Article  CAS  Google Scholar 

  • Tian J, Andree B, Jones CM, Sampath K . (2008). The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signaling activities. Development 135: 2649–2658.

    Article  CAS  Google Scholar 

  • Topczewska JM, Postovit LM, Margaryan NV, Sam A, Hess AR, Wheaton WW et al. (2006). Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness. Nat Med 12: 925–932.

    Article  CAS  Google Scholar 

  • Tsai KL, Sun YJ, Huang CY, Yang JY, Hung MC, Hsiao CD . (2007). Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification. Nucleic Acids Res 35: 6984–6994.

    Article  CAS  Google Scholar 

  • Wang H, Jiang JY, Zhu C, Peng C, Tsang BK . (2006). Role and regulation of nodal/activin receptor-like kinase 7 signaling pathway in the control of ovarian follicular atresia. Mol Endocrinol 20: 2469–2482.

    Article  CAS  Google Scholar 

  • Weidinger C, Krause K, Klagge A, Karger S, Fuhrer D . (2008). Forkhead box-O transcription factor: critical conductors of cancer's fate. Endocr Relat Cancer 15: 917–929.

    Article  CAS  Google Scholar 

  • Xu G, Bernaudo S, Fu G, Lee DY, Yang BB, Peng C . (2008). Cyclin G2 is degraded through the ubiquitin-proteasome pathway and mediates the antiproliferative effect of activin receptor-like kinase 7. Mol Biol Cell 19: 4968–4979.

    Article  CAS  Google Scholar 

  • Xu G, Zhong Y, Munir S, Yang BB, Tsang BK, Peng C . (2004). Nodal induces apoptosis and inhibits proliferation in human epithelial ovarian cancer cells via activin receptor-like kinase 7. J Clin Endocrinol Metab 89: 5523–5534.

    Article  CAS  Google Scholar 

  • Xu G, Zhou H, Wang Q, Auersperg N, Peng C . (2006). Activin receptor-like kinase 7 induces apoptosis through up-regulation of Bax and down-regulation of Xiap in normal and malignant ovarian epithelial cell lines. Mol Cancer Res 4: 235–246.

    Article  CAS  Google Scholar 

  • Yang JY, Chang CJ, Xia W, Wang Y, Wong KK, Engelman JA et al. (2010). Activation of FOXO3a is sufficient to reverse mitogen-activated protein/extracellular signal-regulated kinase inhibitor chemoresistance in human cancer. Cancer Res 70: 4709–4718.

    Article  CAS  Google Scholar 

  • Yang JY, Zong CS, Xia W, Yamaguchi H, Ding Q, Xie X et al. (2008). ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation. Nat Cell Biol 10: 138–148.

    Article  CAS  Google Scholar 

  • Ye G, Fu G, Cui S, Zhao S, Bernaudo S, Bai Y et al. (2011). MicroRNA 376c enhances ovarian cancer cell survival by targeting activin receptor-like kinase 7: implications for chemoresistance. J Cell Sci 124: 359–368.

    Article  CAS  Google Scholar 

  • You H, Yamamoto K, Mak TW . (2006). Regulation of transactivation-independent proapoptotic activity of p53 by FOXO3a. Proc Natl Acad Sci USA 103: 9051–9056.

    Article  CAS  Google Scholar 

  • Zhang N, Kumar M, Xu G, Ju W, Yoon T, Xu E et al. (2006). Activin receptor-like kinase 7 induces apoptosis of pancreatic beta cells and beta cell lines. Diabetologia 49: 506–518.

    Article  CAS  Google Scholar 

  • Zhang YQ, Sterling L, Stotland A, Hua H, Kritzik M, Sarvetnick N . (2008). Nodal and lefty signaling regulates the growth of pancreatic cells. Dev Dyn 237: 1255–1267.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a CIHR grant (MOP-89931) and a CIHR/OWHC Mid-Career Award to CP.

Author information

Authors and Affiliations

  1. Department of Biology, York University, Toronto, Ontario, Canada

    G Fu & C Peng

Authors
  1. G Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C Peng.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fu, G., Peng, C. Nodal enhances the activity of FoxO3a and its synergistic interaction with Smads to regulate cyclin G2 transcription in ovarian cancer cells. Oncogene 30, 3953–3966 (2011). https://doi.org/10.1038/onc.2011.127

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2011.127

Keywords

This article is cited by

Search

Advanced search

Quick links