OPEN mTORC2 regulates hedgehog pathway activity by promoting stability to Gli2 protein and its nuclear translocation Samarpan Maiti 1 , Susmita Mondal 1 , Eswara M Satyavarapu 1 and Chitra Mandal* ,1 mTORC2 is aberrantly activated in cancer and therefore is considered to be an important therapeutic target. The hedgehog pathway, which is also often hyperactivated, regulates transcription of several genes associated with angiogenesis, metastasis, cellular proliferation and cancer stem cell (CSC) regeneration. However, the contribution of mTORC2 toward hedgehog pathway activity has not been explored yet. Here we have addressed the molecular cross talk between mTORC2 and hedgehog pathway activities in the context of glioblastoma multiforme, a malignant brain tumor using as a model system. We observed that higher mTORC2 activity enhanced the expression of a few hedgehog pathway molecules (Gli1, Gli2 and Ptch1) and amplified its target genes (Cyclin D1, Cyclin D2, Cyclin E, Snail, Slug and VEGF) both in mRNA and protein levels as corroborated by increased metastasis, angiogenesis, cellular proliferation and stem cell regeneration. Inhibition of mTORC2 formation decreased hedgehog pathway activity and attenuated all these above-mentioned events, suggesting their cross talk with each other. Further investigations revealed that mTORC2 inhibited ubiquitination of Gli2 by inactivating GSK3β, and thus it promotes stability to Gli2 and its nuclear translocation. Moreover, enhanced mTORC2 activity led to the increased clonogenic properties and CD133 + cells, indicating its role in CSC regeneration. mTORC2 inhibitor directed the reduction of hedgehog pathway proteins and also reduced CSCs. Thus, our observations support a role for elevated mTORC2 activity in regulating angiogenesis, metastasis, cellular proliferation and CSC regeneration via hedgehog pathway activity. Taken together, it provides a rationale for including the mTOR2 inhibitor as part of the therapeutic regimen for CSCs. Cell Death and Disease (2017) 8, e2926; doi:10.1038/cddis.2017.296; published online 13 July 2017 Glioblastoma multiforme (GBM) represents the most common form of malignant brain tumors in adults with extremely low survival rate. 1 Deregulation of mTORC2 signaling is one of the main mechanisms in GBM formation. 2 Hyperactivation of mTORC2 with Rictor overexpression was found to cause GBM. 3,4 mTORC2 has been linked with several metabolic reprogramming activities and is a major regulatory molecule. 5,6 The hedgehog (Hh) pathway is another major regulator of cellular differentiation, proliferation and stem cell mainte- nance. Aberrant activation of the Hh pathway is associated with GBM and leads to the activation of cancer stem-like cells (CSCs). 7 Transcriptional activation of Hh target genes occurs through the actions of Gli1, Gli2 and Gli3 (ref. 8). Gli1 acts as a transcriptional activator and is also regulated by Gli2 (ref. 9), whereas Gli2 and Gli3 act as both transcriptional activators and repressors based on post-translational modification. Evidence suggests that the full-length form of Gli2 proteins (Gli2 FL ) acts as a strong activator of Hh signaling. 10 Upon stabilization, Gli2 moves in the nucleus, which in turn promotes the transcription of both Gli1 and Gli2 and amplifies the transcription of other target genes. CSCs are considered the origin of tumorigenesis, drug resistance and relapse recurrence. 11,12 They display persistent activation of one or more highly conserved hedge- hog, Notch and Wnt signal transduction pathways. Therefore, CSCs are important targets for therapeutic intervention. Keeping in view the aberrant activation of both Hh and mTORC2 pathways and their role in GBM formation, there are needs to explore their contextual functioning in depth (Figure 1a). It will be useful to distinguish some specific molecules that are common to these pathways that can serve as potential drug targets. Here we have demonstrated a positive correlation between mTORC2 activity and Hh-pathway upregulation, leading to more invasiveness, angiogenesis and cellular proliferation of GBM cells. Further- more, we have observed that the interaction between mTORC2 and Hh-pathway molecules is associated with increased stemness properties. More importantly, we showed that inhibitors that target mTORC2 also target the Hh pathway and reduce the cell viabilities of CSCs. Results Differential mTORC2 activity leads to disparity in expres- sion of Hh pathway proteins in GBM cells. To understand the impact of mTORC2 activity in the regulation of the Hh pathway, we selected two GBM cell lines namely U87MG and 1 Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India *Corresponding author: C Mandal, Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India. Tel: +91 33 2429 8861; Fax: +91 33 2473 5197; E-mail: chitra_mandal@yahoo.com or cmandal@iicb.res.in Received 16.3.17; revised 22.4.17; accepted 03.5.17; Edited by A Oberst Citation: Cell Death and Disease (2017) 8, e2926; doi:10.1038/cddis.2017.296 Official journal of the Cell Death Differentiation Association www.nature.com/cddis