Author Manuscript Published OnlineFirst on May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0180 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. mTORC2 suppresses GSK3-dependent Snail degradation to positively regulate cancer cell invasion and metastasis Shuo Zhang,1,3 Guoqing Qian,3 Qian-Qian Zhang,2 Yuying Yao,2, Dongsheng Wang,3 Zhuo G. Chen,3 Li-Jing Wang,2 Mingwei Chen,1 and Shi-Yong Sun3 1First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, P. R. China; 2Vascular Biology Research Institute, School of Basic Science, Guangdong Pharmaceutical University, Guangzhou, Guangdong, P. R. China; 3Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, Georgia, USA Running title: mTORC2 stabilization of Snail Key words: mTOR, mTORC2, Snail, degradation, GSK3, -TrCP, invasion, metastasis Abbreviations: mTOR, mammalian target of rapamycin; mTORC2, mTOR complex 2; CHX, cycloheximide; siRNA, small-interfering RNA; shRNA, short-hairpin RNA. Grant Support: Emory University Winship pilot funds (to SYS) and National Natural Science Foundation of China (No. 31771578 to QQZ). Note: SYS is a Georgia Research Alliance Distinguished Cancer Scientist. Request for reprints: Shi-Yong Sun, Ph.D., Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, 1365-C Clifton Road, C3088, Atlanta, GA 30322. Phone: (404) 778-2170; Fax: (404) 778-5520; E-mail: [email protected] 1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0180 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) positively regulates cell invasion and metastasis by enhancing translation of Snail. A connection between mTOR complex 2 (mTORC2) and cell invasion and metastasis has also been suggested, yet the underlying biology or mechanism is largely unknown and thus is the focus of this study. Inhibition of mTOR with both mTOR inhibitors and knockdown of key components of mTORC, including rictor, Sin1 and raptor, decreased Snail protein levels. Inhibition of mTOR enhanced the rate of Snail degradation, which could be rescued by inhibition of the proteasome. Critically, inhibition of mTORC2 (by knocking down rictor) but not mTORC1 (by knocking down raptor) enhanced Snail degradation. Therefore, only mTORC2 inhibition induces Snail proteasomal degradation, resulting in eventual Snail reduction. Interestingly, inhibition of GSK3 but not SCF/β-TrCP rescued the Snail reduction induced by mTOR inhibitors, suggesting GSK3-dependent, but SCF/β-TrCP-independent proteasomal degradation of Snail. Accordingly, mTOR inhibitors elevated E-cadherin levels and suppressed cancer cell migration and invasion in vitro and metastasis in vivo. Collectively, this study reveals that mTORC2 positively regulates Snail stability to control cell invasion and metastasis. Significance Findings delineate a new regulation mechanism of Snail, an important master regulator of EMT and invasion in cancers. 2 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0180 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction The mammalian target of rapamycin (mTOR) is critical for the regulation of cell growth, metabolism, survival and other biological functions. It mediates these functions primarily through interacting with other proteins to form two distinct complexes: mTOR complex 1 (mTORC1), which is composed of mTOR, raptor, mLST8, PRAS40 and DEPTOR, and mTOR complex 2 (mTORC2), which contains mTOR, rictor, mLST8, DEPTOR, mSin1 and protor (1). mTORC1 signaling is crucial for regulating cap-dependent translation initiation, an essential process for synthesizing many oncogenic proteins such as cyclin D1, c-Myc, Mcl-1 and VEGF, through phosphorylating S6 kinase (S6K) and eIF4E-binding protein 1 (4E-BP1), whereas mTORC2 may positively regulate cell survival and proliferation, primarily by phosphorylating Akt and serum and glucocorticoid-inducible kinase (SGK) (1). In comparison with mTORC1 signaling, relatively little is known about the biological functions of mTORC2, particularly those related to the regulation of oncogenesis, although mTORC2 is involved in promoting cancer development (2-4). Invasion and metastasis is a cancer hallmark and the leading cause of cancer death (5). Epithelial- mesenchymal transition (EMT) is a key step toward cancer metastasis; this process is in part mediated by Snail, a major transcription factor for repression of E-cadherin (E-Cad) (6,7). The role of mTORC1 in the positive regulation of the EMT process and metastasis through translational control of gene expression has long been recognized (8-10). It has been shown that mTORC1/4EBP1/eIF4E-mediated Snail translation and subsequent repression of E-Cad plays a critical role in EMT induction, tumor cell migration and invasion (11). Although some studies suggest that mTORC2 is also involved in mediating EMT, invasion and metastasis of cancer cells (12-17), the underlying biology or mechanisms are largely unknown (10). 3 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0180 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Glycogen synthase kinase-3 (GSK3), a ubiquitous serine/threonine kinase that is present in mammals in two isoforms: and (18), plays a key role in regulating a diverse range of cellular functions including glycogen metabolism, cell survival and death (18). However, GSK3 has complex roles in the regulation of oncogenesis: it can function as a tumor suppressor in some cancer types while potentiating the growth of cancer cells in others (19,20). It is well known that GSK3 enhances proteasomal degradation of several oncogenic proteins, including Snail, c-Myc, Mcl-1, sterol regulatory element-binding proteins (SREBPs) and cyclin D, through phosphorylating these proteins (21-25). In the past few years, we have demonstrated that mTORC2 is tightly associated with the negative regulation of GSK3-dependent, SCF E3 ligase (FBX4 or FBXW7)-mediated degradation of cyclin D1, Mcl-1 and SREBP1; inhibition of mTORC2 (e.g., with rictor knockdown or mTOR inhibitors) accordingly induces the degradation of these proteins (26-29). These findings have suggested a novel biological function of mTORC2 in the positive regulation of cancer cell metabolism, growth and survival via the direct negative regulation of protein degradation. In addition to the FBXW7- or FBX4- mediated degradation mechanism, several other proteins such as Snail and -catenin undergo GSK3- dependent and -TrCP (another SCF E3 ligase)-mediated degradation (25,30-32). Rictor, a key component of mTORC2, interacts with a core component of the SCF E3 complex, Cul1 (33). Moreover, SCF/-TrCP interacts with DEPTOR, another key component of both mTORC1 and mTORC2, to promote its degradation (34-36). Hence, we were interested in determining whether mTORC2 also regulates GSK3-dependent and SCF/-TrCP-mediated degradation of these proteins. Using chemical approaches, we found that inhibition of mTOR with mTOR kinase inhibitors (TORKinibs) effectively decreased the levels of Snail, but not -catenin protein. Therefore, the current study focused on mTOR inhibition-induced reduction of Snail and its underlying mechanisms. 4 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0180 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Material and Methods Reagents. The mTOR inhibitors, rapamycin, RAD001, INK128 and AZD8055, the proteasome inhibitor, MG132, the protein synthesis inhibitor, cycloheximide (CHX), and the GSK3 inhibitors, SB216763 and CHIR99021, were the same as described previously (28). These agents were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mM or 10 mM, and aliquots were stored at -80˚C. Stock solutions were diluted to the desired final concentrations with growth medium just before use. TGF1 was purchased from PeproTech (Rocky Hill, NJ). Rabbit monoclonal Snail (#3879), E-Cad (#3195) and -TrCP (#4394) antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA). Other antibodies were the same as described previously (27,28). Cell lines and cell culture. Human NSCLC cell lines used in this study were described previously (37,38). MCF-7 and MDA-MB-453 were purchased from ATCC (Manassas, VA). HAP1, HAP1/-TrCP-KO and HAP1/rictor-KO cells were purchased from Horizon (Cambridge, CB). All MEFs used in this study were described previously (27,29). Except for H157 and A549 cells, which were authenticated by Genetica DNA Laboratories, Inc. (Cincinnati, OH) through analyzing short tandem repeat DNA profile, other cell lines have not been authenticated. 801BL is a metastatic large cell lung