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Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis

Oncogene volume 29, pages 6543–6556 (2010)Cite this article

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Abstract

Tuberous sclerosis complex (TSC) is an autosomally inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations in the TSC2 tumor-suppressor gene. TSC2 negatively regulates the activity of the GTPase Rheb and thereby inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Activation of mTORC1 as a result of lack of TSC2 function is observed in TSC and sporadic lymphangioleiomyomatosis (LAM). TSC2 deficiency has recently been associated with elevated AMP-activated protein kinase (AMPK) activity, which in turn correlated with cytoplasmic localization of p27Kip1 (p27), a negative regulator of cyclin-dependent kinase 2 (Cdk2). How AMPK in the absence of TSC2 is stimulated is not fully understood. In this study, we demonstrate that Rheb activates AMPK and reduces p27 levels in Tsc2-null cells. Importantly, both effects occur largely independent of mTORC1. Furthermore, increased p27 levels following Rheb depletion correlated with reduced Cdk2 activity and cell proliferation in vitro, and with inhibition of tumor formation by Tsc2-null cells in vivo. Taken together, our data suggest that Rheb controls proliferation of TSC2-deficient cells by a mechanism that involves regulation of AMPK and p27, and that Rheb is a potential target for TSC/LAM therapy.

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References

  • Alkarain A, Slingerland J . (2004). Deregulation of p27 by oncogenic signaling and its prognostic significance in breast cancer. Breast Cancer Res 6: 13–21.

    Article  CAS  Google Scholar 

  • Assoian RK . (2004). Stopping and going with p27kip1. Dev Cell 6: 458–459.

    Article  CAS  Google Scholar 

  • Carsillo T, Astrinidis A, Henske EP . (2000). Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc Natl Acad Sci USA 97: 6085–6090.

    Article  CAS  Google Scholar 

  • Castro AF, Rebhun JF, Clark GG, Quilliam LA . (2003). Rheb binds TSC2 and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner. J Biol Chem 278: 32493–32496.

    Article  CAS  Google Scholar 

  • Cheadle JP, Reeve MP, Sampson JR, Kwiatkowski DJ . (2000). Molecular genetic advances in tuberous sclerosis. Hum Genet 107: 97–114.

    Article  CAS  Google Scholar 

  • Choo AY, Blenis J . (2006). TORgeting oncogene addiction for cancer therapy. Cancer Cell 9: 77–79.

    Article  CAS  Google Scholar 

  • Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J . (2008). Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci USA 105: 17414–17419.

    Article  CAS  Google Scholar 

  • Dabora SL, Jozwiak S, Franz DN, Roberts PS, Nieto A, Chung J et al. (2001). Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am J Hum Genet 68: 64–80.

    Article  CAS  Google Scholar 

  • Easton JB, Houghton PJ . (2006). mTOR and cancer therapy. Oncogene 25: 6436–6446.

    Article  CAS  Google Scholar 

  • El-Hashemite N, Zhang H, Henske EP, Kwiatkowski DJ . (2003). Mutation in TSC2 and activation of mammalian target of rapamycin signalling pathway in renal angiomyolipoma. Lancet 361: 1348–1349.

    Article  CAS  Google Scholar 

  • Finlay GA, Malhowski AJ, Liu Y, Fanburg BL, Kwiatkowski DJ, Toksoz D . (2007). Selective inhibition of growth of tuberous sclerosis complex 2 null cells by atorvastatin is associated with impaired Rheb and Rho GTPase function and reduced mTOR/S6 kinase activity. Cancer Res 67: 9878–9886.

    Article  CAS  Google Scholar 

  • Fujita N, Sato S, Katayama K, Tsuruo T . (2002). Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization. J Biol Chem 277: 28706–28713.

    Article  CAS  Google Scholar 

  • Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, Stocker H et al. (2003). Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell 11: 1457–1466.

    Article  CAS  Google Scholar 

  • Garcia-Martinez JM, Alessi DR . (2008). mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J 416: 375–385.

    Article  CAS  Google Scholar 

  • Gau CL, Kato-Stankiewicz J, Jiang C, Miyamoto S, Guo L, Tamanoi F . (2005). Farnesyltransferase inhibitors reverse altered growth and distribution of actin filaments in Tsc-deficient cells via inhibition of both rapamycin-sensitive and -insensitive pathways. Mol Cancer Ther 4: 918–926.

    Article  CAS  Google Scholar 

  • Gottlob K, Majewski N, Kennedy S, Kandel E, Robey RB, Hay N . (2001). Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev 15: 1406–1418.

    Article  CAS  Google Scholar 

  • Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS et al. (2008). AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30: 214–226.

    Article  CAS  Google Scholar 

  • Hahn-Windgassen A, Nogueira V, Chen CC, Skeen JE, Sonenberg N, Hay N . (2005). Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity. J Biol Chem 280: 32081–32089.

    Article  CAS  Google Scholar 

  • Halayko AJ, Camoretti-Mercado B, Forsythe SM, Vieira JE, Mitchell RW, Wylam ME et al. (1999). Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am J Physiol 276: L197–206.

    CAS  PubMed  Google Scholar 

  • Hong F, Larrea MD, Doughty C, Kwiatkowski DJ, Squillace R, Slingerland JM . (2008). mTOR-raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell 30: 701–711.

    Article  CAS  Google Scholar 

  • Howe SR, Gottardis MM, Everitt JI, Goldsworthy TL, Wolf DC, Walker C . (1995). Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am J Pathol 146: 1568–1579.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Huang J, Manning BD . (2008). The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem J 412: 179–190.

    Article  CAS  Google Scholar 

  • Huang J, Wu S, Wu CL, Manning BD . (2009). Signaling events downstream of mammalian target of rapamycin complex 2 are attenuated in cells and tumors deficient for the tuberous sclerosis complex tumor suppressors. Cancer Res 69: 6107–6114.

    Article  CAS  Google Scholar 

  • Inoki K, Guan KL . (2009). Tuberous sclerosis complex, implication from a rare genetic disease to common cancer treatment. Hum Mol Genet 18: R94–100.

    Article  CAS  Google Scholar 

  • Inoki K, Li Y, Xu T, Guan KL . (2003a). Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 17: 1829–1834.

    Article  CAS  Google Scholar 

  • Inoki K, Zhu T, Guan KL . (2003b). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115: 577–590.

    Article  CAS  Google Scholar 

  • Kim J, Jonasch E, Alexander A, Short JD, Cai S, Wen S et al. (2009). Cytoplasmic sequestration of p27 via AKT phosphorylation in renal cell carcinoma. Clin Cancer Res 15: 81–90.

    Article  CAS  Google Scholar 

  • Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR . (2003). Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422–39427.

    Article  CAS  Google Scholar 

  • Liang J, Shao SH, Xu ZX, Hennessy B, Ding Z, Larrea M et al. (2007). The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat Cell Biol 9: 218–224.

    Article  CAS  Google Scholar 

  • Ma L, Teruya-Feldstein J, Bonner P, Bernardi R, Franz DN, Witte D et al. (2007). Identification of S664 TSC2 phosphorylation as a marker for extracellular signal-regulated kinase mediated mTOR activation in tuberous sclerosis and human cancer. Cancer Res 67: 7106–7112.

    Article  CAS  Google Scholar 

  • Manning BD . (2004). Balancing Akt with S6K: implications for both metabolic diseases and tumorigenesis. J Cell Biol 167: 399–403.

    Article  CAS  Google Scholar 

  • Park HU, Suy S, Danner M, Dailey V, Zhang Y, Li H et al. (2009). AMP-activated protein kinase promotes human prostate cancer cell growth and survival. Mol Cancer Ther 8: 733–741.

    Article  CAS  Google Scholar 

  • Paul E, Thiele E . (2008). Efficacy of sirolimus in treating tuberous sclerosis and lymphangioleiomyomatosis. N Engl J Med 358: 190–192.

    Article  CAS  Google Scholar 

  • Planas-Silva MD, Weinberg RA . (1997). Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution. Mol Cell Biol 17: 4059–4069.

    Article  CAS  Google Scholar 

  • Pollizzi K, Malinowska-Kolodziej I, Stumm M, Lane H, Kwiatkowski D . (2009). Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis. Mol Cancer 8: 38.

    Article  Google Scholar 

  • Rosner M, Freilinger A, Hanneder M, Fujita N, Lubec G, Tsuruo T et al. (2007). p27Kip1 localization depends on the tumor suppressor protein tuberin. Hum Mol Genet 16: 1541–1556.

    Article  CAS  Google Scholar 

  • Rosner M, Freilinger A, Hengstschlager M . (2006). The tuberous sclerosis genes and regulation of the cyclin-dependent kinase inhibitor p27. Mutat Res 613: 10–16.

    Article  CAS  Google Scholar 

  • Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF et al. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 22: 159–168.

    Article  CAS  Google Scholar 

  • Sato T, Seyama K, Fujii H, Maruyama H, Setoguchi Y, Iwakami S et al. (2002). Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis. J Hum Genet 47: 20–28.

    Article  CAS  Google Scholar 

  • Shaw RJ, Cantley LC . (2006). Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441: 424–430.

    Article  CAS  Google Scholar 

  • Short JD, Dere R, Houston KD, Cai SL, Kim J, Bergeron JM et al. (2010). AMPK-mediated phosphorylation of murine p27 at T197 promotes binding of 14-3-3 proteins and increases p27 stability. Mol Carcinog 49: 429–439.

    CAS  PubMed  Google Scholar 

  • Short JD, Houston KD, Dere R, Cai SL, Kim J, Johnson CL et al. (2008). AMP-activated protein kinase signaling results in cytoplasmic sequestration of p27. Cancer Res 68: 6496–6506.

    Article  CAS  Google Scholar 

  • Tee AR, Manning BD, Roux PP, Cantley LC, Blenis J . (2003). Tuberous sclerosis complex gene products, tuberin and hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr Biol 13: 1259–1268.

    Article  CAS  Google Scholar 

  • Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y et al. (2009). An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem 284: 8023–8032.

    Article  CAS  Google Scholar 

  • van Slegtenhorst M, de Hoogt R, Hermans C, Nellist M, Janssen B, Verhoef S et al. (1997). Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277: 805–808.

    Article  CAS  Google Scholar 

  • Vervoorts J, Luscher B . (2008). Post-translational regulation of the tumor suppressor p27(KIP1). Cell Mol Life Sci 65: 3255–3264.

    Article  CAS  Google Scholar 

  • Viollet B, Athea Y, Mounier R, Guigas B, Zarrinpashneh E, Horman S et al. (2009). AMPK: lessons from transgenic and knockout animals. Front Biosci 14: 19–44.

    Article  CAS  Google Scholar 

  • Wu FY, Wang SE, Sanders ME, Shin I, Rojo F, Baselga J et al. (2006). Reduction of cytosolic p27(Kip1) inhibits cancer cell motility, survival, and tumorigenicity. Cancer Res 66: 2162–2172.

    Article  CAS  Google Scholar 

  • Yu JJ, Robb VA, Morrison TA, Ariazi EA, Karbowniczek M, Astrinidis A et al. (2009). Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells. Proc Natl Acad Sci USA 106: 2635–2640.

    Article  CAS  Google Scholar 

  • Zhang BB, Zhou G, Li C . (2009). AMPK: an emerging drug target for diabetes and the metabolic syndrome. Cell Metab 9: 407–416.

    Article  Google Scholar 

  • Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, Bajraszewski N et al. (2003a). Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

    Article  CAS  Google Scholar 

  • Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, Pan D . (2003b). Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol 5: 578–581.

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to the NIH-LAM registry, the LAM foundation and especially to the LAM patients for providing LAM specimens. We thank Prema S Idumalla and Xiaochen Yuan for technical assistance, and Drs David Donner and Martin McMahon for review of the paper and suggestions. Special thanks to Pamela Derish at UCSF for editing the paper. We also thank Dr Geoffrey Clark for providing the custom Rheb antibody, Dr Cheryl Walker for the ELT3 cells and Dr David Kwiatkowski for the MEFs Tsc2−/− and Tsc2+/+. This work was supported by grants from the Department of Defense (DOD TS043006) and Academic Senate (UCSF) (AFC), and LAM Foundation, ATS, ALA, and Blowitz-Ridgeway Foundation (BC-M). Zhe Zhu was supported by the DOD TS043006 grant to AFC.

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Author notes

  1. M D Lacher

    Present address: 6Current address: BioTime, Inc., Alameda, CA 94502, USA.,

  2. A F Castro

    Present address: 7Current address: Department of Biochemistry and Molecular Biology, University of Concepcion, Casilla 160-C, Correo 3, Ciudad Universitaria, Concepcion, Chile.,

Authors and Affiliations

  1. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA

    M D Lacher & A F Castro

  2. Department of Biochemistry and Molecular Biology, University of Concepcion, Concepcion, Chile

    R Pincheira

  3. Department of Surgery, University of California, San Francisco, CA, USA

    Z Zhu, M Matli & R S Warren

  4. Department of Medicine, University of Chicago, Chicago, IL, USA

    B Camoretti-Mercado

  5. Department of Dermatology, University of California, San Francisco, CA, USA

    A F Castro

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Correspondence to A F Castro.

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Lacher, M., Pincheira, R., Zhu, Z. et al. Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis. Oncogene 29, 6543–6556 (2010). https://doi.org/10.1038/onc.2010.393

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