In this study, we demonstrate a SMO-independent activation of Gli1 by mTOR/S6K1 pathway, in which activated S6K1 phosphorylates Gli1 at Ser84 resulting in its release from SuFu binding and translocation into the nucleus to activate its target genes (). We previously reported that the mTOR/S6K1 pathway facilitates progression from inflammation and tumorigenesis through upregulation of VEGF as well as the subsequent angiogenesis (Lee et al., 2007a
). In addition, TNFα/mTOR can be activated by chronic inflammation in esophagus (Yen et al., 2008
). Our work herein further implies that the mTOR/S6K1 pathway might also promote EAC through the activation of Gli1. Since Gli1 is known as an oncogene (Ng and Curran, 2011
), our results also provide further evidence to support the concept that chronic inflammation is an important stimulator for tumorigenesis of the esophagus (Lambert and Hainaut, 2007a
; Lambert and Hainaut, 2007b
The canonical HH pathway is well known to have a tight negative feedback regulation, which blocks the HH ligands and inhibits SMO activation through Gli1-promoted transcription of PTCH and hedgehog interacting protein (Katoh and Katoh, 2006
). When SMO is inactivated, SuFu binds to and inhibits Gli1 function (Katoh and Katoh, 2006
). Function-loss-mutation of SuFu has been shown to result in tumorigenesis due to the aberrant activation of the HH pathway (Cheng and Yue, 2008
; Lee et al., 2007b
). Therefore, SuFu is an important negative regulator for HH pathway and acts as tumor suppressor. In this study, we found that the phosphorylation of Gli1 by S6K1 blocked the interaction between SuFu and Gli1, allowing Gli1 to translocate into the nucleus to activate transcription of HH target genes. Thus, in contrast to the canonical HH pathway, SMO inhibitors seem not to affect S6K1-mediated Gli1 activation, suggesting that the S6K1-mediated release of SuFu from Gli1 occurs independently of SMO. In fact, SMO inhibitors, such as cyclopamine and GDC-0449, had little effects on the mTOR/S6K1-mediated Gli1 activation. These findings suggest that the mTOR/S6K1 pathway can act as a positive modulator to amplify and fuel Gli1 activation to promote tumorigenesis and disease progression.
The HH pathway has been considered as a therapeutic target for GI cancers, including esophageal cancers (Lee et al., 2009
; Wiedmann and Caca, 2005
). Several SMO inhibitors, including GDC-0449, are currently being tested in clinical trials, which are either structurally derived from or functionally similar to cyclopamine (Scales and de Sauvage, 2009
; Stanton and Peng, 2010
). Our data showed that the administration of GDC-0449 indeed decreased the EAC tumor size, supporting that GDC-0449 could also be used for treating EAC (). In this study, however, we disclose a SMO-independent activation of Gli1 by the mTOR/S6K1 pathway, which can not be inhibited by SMO inhibitors, but is sensitive to inhibitors of mTOR pathways. Co-treatment with mTOR/S6K1 and SMO inhibitors, RAD001 and GDC-0449, indeed showed better inhibitory effects on tumor growth in vivo
than single drug treatment. Therefore our results strongly suggest that a combination of inhibitors targeting the two pathways may be a more effective strategy to treat EAC.
In addition, through the immunostaining analysis of human EAC tissues, we found that in about 40% (28/70) patients, all of p-Gli1, Gli1, and p-S6K1 were positive, suggesting that these patients may bear both canonical HH pathway and mTOR/S6K1-mediated SMO-independent Gli1 activation. Based on the current study, we would predict that this population of patients may not have full response to GDC-0449 treatment alone, but could benefit from the proposed co-treatment of inhibitors targeting both mTOR and HH pathways. Therefore, a pre-selection procedure might be required for the patients before receiving the SMO inhibitors to determine whether the co-treatment strategy should be applied. It is worthwhile to mention that many inhibitors targeting these two pathways are being tested in clinical trials, such as GDC-0449 and IPI-926, targeting the hedgehog pathway (Stanton and Peng, 2010
), and RAD001 and AP23573, targeting the mTOR pathway (Konings et al., 2009
). Thus, exploring a vast array of possible therapeutic combinations will be useful to simultaneously target these pathways.
Although SMO inhibitors are known to inhibit several types of cancer and have shown hopeful tumor-inhibitory effects, the development of resistance has been reported due to the constitutive activation mutation of SMO or overactivation of PI3K/AKT pathway (Metcalfe and de Sauvage, 2011
). Buonamici et al. further showed that the resistance of medulloblastoma to SMO inhibitors could be decreased through a combination of SMO and PI3K/AKT inhibitors (Buonamici et al., 2010
). Interestingly, PI3K/AKT and RAS/MEK/ERK have also been found to activate Gli1 in a SMO-independent manner (Katoh and Katoh, 2009b
; Seto et al., 2009
; Stecca et al., 2007
) though the mechanisms are not well understood. Because AKT and ERK can activate the mTOR/S6K1 pathway (Ma et al., 2005
; Ozes et al., 2001
) and activation of Gli1 by AKT or ERK requires S6K1 (), our finding that S6K1 phosphorylates Gli1 and enhances its function provides a molecular mechanism not only for mTOR/S6K1-mediated but also AKT or ERK-induced SMO-independent Gli1 activation (). Thus, our results also provide a potential explanation for the resistance of tumor cells to SMO inhibitors. Similarly, our study offers a rationale for combining SMO with mTOR/S6K1 inhibitors to increase the effectiveness for treating EAC.
Taken together, our current study identifies Gli1 as a substrate for S6K1 and establishes a crosstalk between the mTOR/S6K1 and HH pathways, providing a mechanism for SMO-independent Gli1 activation. Our data also suggest that the combination of the inhibitors to these two pathways has a more potent inhibitory effect on the EAC cells than single agent alone. Moreover, we also found the correlation between p-S6K1 and Gli1 of multiple cancer types using tissue microarray, indicating that the combined targeted therapy, targeting both the mTOR/S6K1 and HH pathways, may be effective for treatment of EAC as well as other cancers.