Oncogene (2010) 29, 6543–6556 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis MD Lacher1,6, R Pincheira2, Z Zhu3, B Camoretti-Mercado4, M Matli3, RS Warren3 and AF Castro1,5,7 1Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA; 2Department of Biochemistry and Molecular Biology, University of Concepcion, Concepcion, Chile; 3Department of Surgery, University of California, San Francisco, CA, USA; 4Department of Medicine, University of Chicago, Chicago, IL, USA and 5Department of Dermatology, University of California, San Francisco, CA, USA Tuberous sclerosis complex (TSC) is an autosomally Introduction inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations Tuberous sclerosis complex (TSC) is an autosomally in the TSC2 tumor-suppressor gene. TSC2 negatively inherited disorder with benign tumor formation in the regulates the activity of the GTPase Rheb and thereby brain, kidney, heart, lung, skin and eye (Cheadle et al., inhibits mammalian target of rapamycin complex 1 2000; Dabora et al., 2001). The most frequent manifes- (mTORC1) signaling. Activation of mTORC1 as a result tation of TSC in the lung is known as lymphangioleio- of lack of TSC2 function is observed in TSC and sporadic myomatosis (LAM), a disease that also occurs lymphangioleiomyomatosis (LAM). TSC2 deficiency has sporadically (Carsillo et al., 2000). recently been associated with elevated AMP-activated TSC and LAM result from the inactivation of either protein kinase (AMPK) activity, which in turn correlated of the two tumor-suppressor genes, TSC1 that encodes with cytoplasmic localization of p27Kip1 (p27), a negative hamartin (TSC1), and TSC2 that encodes tuberin regulator of cyclin-dependent kinase 2 (Cdk2). How (TSC2) (van Slegtenhorst et al., 1997; Carsillo et al., AMPK in the absence of TSC2 is stimulated is not fully 2000; Sato et al., 2002). TSC1 and TSC2 form an active understood. In this study, we demonstrate that Rheb complex (TSC1/2) that negatively regulates mammalian activates AMPK and reduces p27 levels in Tsc2-null cells. target of rapamycin complex 1 (mTORC1) signaling, Importantly, both effects occur largely independent of which promotes cell growth (Huang and Manning, mTORC1. Furthermore, increased p27 levels following 2008). Along with others, we showed that the small Rheb depletion correlated with reduced Cdk2 activity and GTPase Rheb is the molecular link between TSC1/2 and cell proliferation in vitro, and with inhibition of tumor mTORC1. Rheb activates mTORC1, and TSC2 inhibits formation by Tsc2-null cells in vivo. Taken together, our Rheb by acting as a GTPase-activating protein (Castro data suggest that Rheb controls proliferation of TSC2- et al., 2003; Garami et al., 2003; Tee et al., 2003; Inoki deficient cells by a mechanism that involves regulation of et al., 2003a; Zhang et al., 2003b). AMPK and p27, and that Rheb is a potential target for The loss of TSC1/2 activity in TSC and LAM results TSC/LAM therapy. in aberrant mTORC1 activation, thereby contributing Oncogene (2010) 29, 6543–6556; doi:10.1038/onc.2010.393; to tumor growth (El-Hashemite et al., 2003; Choo and published online 6 September 2010 Blenis, 2006; Pollizzi et al., 2009). Thus, inhibition of mTORC1 with rapamycin may reduce the size of TSC Keywords: Rheb; p27; AMPK; mTORC1; TSC/LAM; tumors (Easton and Houghton, 2006; Paul and Thiele, cancer 2008). However, a clinical trial with rapamycin sug- gested that additional targets and therapeutic interven- tions are needed to efficiently treat TSC and LAM (Paul and Thiele, 2008). Elevated AMP-activated protein kinase (AMPK) activity has recently been reported in tumors and tumor-derived cells deficient in Tsc2 (Short et al., Correspondence: Dr AF Castro, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA. 2008). AMPK is a Ser/Thr kinase activated by changes E-mail: [email protected] in the AMP to ATP ratio that occur during metabolic 6Current address: BioTime, Inc., Alameda, CA 94502, USA. stress. Its primary role is to promote catabolic processes 7Current address: Department of Biochemistry and Molecular Biology, while inhibiting anabolic pathways, thus restoring University of Concepcion, Casilla 160-C, Correo 3, Ciudad energy levels. At the molecular level, AMPK is activated Universitaria, Concepcion, Chile. Received 31 August 2009; revised 6 July 2010; accepted 8 July 2010; by binding to AMP and phosphorylation by LKB1 published online 6 September 2010 (Viollet et al., 2009; Zhang et al., 2009). A consequence Rheb stimulates cell proliferation by regulating p27Kip1 MD Lacher et al 6544 of AMPK activation is the inhibition of cell growth. p70S6K substrate (Inoki and Guan, 2009) (Figure 1a). AMPK phosphorylates TSC2 and the mTOR partner We found that depletion of Rheb under serum-free Raptor to inhibit mTORC1 signaling (Inoki et al., conditions inhibited AMPK activity, as indicated by 2003b; Gwinn et al., 2008). However, AMPK and a reduction of the levels of both phospho-AMPK mTORC1 were both activated in Tsc2-null cells (p-T172), and phospho-acetyl-CoA carboxylase (Hahn-Windgassen et al., 2005), indicating that AMPK (p-ACC), an AMPK downstream target (Zhang et al., phosphorylation of Raptor is not sufficient to inhibit 2009) (Figure 1b). As inhibition of mTORC1 by mTORC1 signaling and cell growth when Tsc2 is depletion of Rheb may eliminate negative feedback absent. signaling to Akt and because it was shown that Akt can AMPK also phosphorylates the cyclin-dependent inhibit AMPK (Hahn-Windgassen et al., 2005; Short kinase (Cdk) inhibitor p27Kip1 (p27) (Liang et al., et al., 2008), we analyzed whether Rheb depletion can 2007; Short et al., 2008). Nuclear p27 inhibits cell cycle affect Akt activity, measured by the phosphorylation progression, whereas cytoplasmic p27 has been sug- levels at both S473 and T308. Phosphorylation of Akt gested to promote cell survival and motility (Assoian, was barely detected, and Rheb depletion only modestly 2004; Wu et al., 2006). Regulation of p27 localization is affected Akt phosphorylation at T308. Therefore, it is complex and may occur at multiple levels. For example, unlikely that Akt has a major role in the regulation of TSC2 increases p27 stability and nuclear localization by AMPK by Rheb (Figure 1b). To confirm this finding, we a mechanism that is independent of its effect on investigated the effect of Rheb depletion on AMPK mTORC1 (Rosner et al., 2006, 2007). Conversely, Akt activity in serum-starved Tsc2À/À MEFs. Similarly to has been shown to phosphorylate p27 at both T157 and the ELT3 cells, decreased Rheb protein levels correlated T198, and phosphorylation on these residues has been with inhibition of AMPK activity and reduction of the associated with reduced nuclear localization of p27 p-ACC levels in the absence of any effect on Akt activity (Fujita et al., 2002; Vervoorts and Luscher, 2008). (Figure 1c). These results highlight the role of Rheb in However, in TSC2-null cells, Akt activity is repressed by the activation of AMPK in Tsc2-null cells and indicate an inhibitory feedback loop by mTORC1-induced that Rheb is necessary for AMPK activation in Tsc2- activation of p70S6K (Manning, 2004), indicating that null cells under conditions of cellular stress caused by Akt does not have a critical role in the regulation of p27 serum deprivation. in the absence of TSC2. Instead, elevated AMPK To examine whether mTORC1 was involved in the activity controls p27 stability and subcellular localiza- regulation of AMPK activity, we used the mTORC1 tion in Tsc2-null cells, which affects Cdk2 activity and inhibitor rapamycin. As long exposure to rapamycin cell survival (Short et al., 2008, 2010). These observa- could affect mTORC2 activity (Sarbassov et al., 2006), tions suggest that AMPK may contribute to tumor and eliminate negative feedback signaling to Akt progression in TSC, and underscores the importance of (Manning, 2004), we inhibited mTORC1 for a short understanding how AMPK is activated in TSC cells. time in the absence of serum. Under this experimental As Rheb is constitutively activated in Tsc2-null cells condition, we found that while rapamycin was effective (Garami et al., 2003; Finlay et al., 2007), we sought to in inhibiting phosphorylation of S6, it failed to affect determine whether it regulates AMPK activity. Here, we AMPK activity (Figure 1d). Together, these data show that Rheb but not mTORC1 activates AMPK. We suggest that Rheb may affect AMPK function in an also demonstrate that Rheb regulates p27 and the mTORC1-independent manner. proliferation of TSC cells, including those derived from To confirm the effect of Rheb on AMPK obtained by lung specimens of patients with LAM. Our results Rheb depletion, we studied Rheb’s contribution to illustrate the importance of Rheb as a potential AMPK activation by overexpression of Rheb in Tsc2 þ / þ therapeutic target for TSC and LAM. MEFs. Rheb overexpression increased the phosphoryla- tion levels of AMPK, an effect that was blocked by the AMPK inhibitor compound C (Figure 2a). To corrobo- rate that Rheb activates AMPK independently of Results mTORC1, we treated vector and Rheb transfected Tsc2 þ / þ MEFs with rapamycin. Rapamycin was Rheb activates AMPK unable to inhibit the effect of Rheb overexpression on We explored whether Rheb regulates AMPK in Tsc2- AMPK activation as well as the phosphorylation of the null ELT3 cells, a tumor-derived rat cell line from the AMPK downstream targets acetyl-CoA carboxylase Eker model (Howe et al., 1995). As Rheb is constitu- (ACC) and Raptor (Figure 2b). As control, rapamycin tively activated in Tsc2-deficient cells (Garami et al., effectively blocked Rheb-induced phosphorylation of S6 2003; Finlay et al., 2007), we depleted Rheb using RNA (Figure 2b).