Published OnlineFirst May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0180 Cancer Tumor Biology and Immunology Research mTORC2 Suppresses GSK3-Dependent Snail Degradation to Positively Regulate Cancer Cell Invasion and Metastasis Shuo Zhang1,2, Guoqing Qian2, Qian-Qian Zhang3, Yuying Yao3, Dongsheng Wang2, Zhuo G. Chen2, Li-Jing Wang3, Mingwei Chen1, and Shi-Yong Sun2 Abstract mTOR complex 1 (mTORC1) positively regulates cell Snail proteasomal degradation, resulting in eventual Snail invasion and metastasis by enhancing translation of Snail. reduction. Interestingly, inhibition of GSK3 but not SCF/ A connection between mTOR complex 2 (mTORC2) b-TrCP rescued the Snail reduction induced by mTOR and cell invasion and metastasis has also been suggested, inhibitors, suggesting GSK3-dependent, but SCF/b-TrCP– yet the underlying biology or mechanism is largely independent proteasomal degradation of Snail. According- unknown and thus is the focus of this study. Inhibition ly, mTOR inhibitors elevated E-cadherin levels and sup- of mTOR with both mTOR inhibitors and knockdown of pressed cancer cell migration and invasion in vitro and key components of mTORC, including rictor, Sin1, and metastasis in vivo. Collectively, this study reveals that raptor, decreased Snail protein levels. Inhibition of mTOR mTORC2 positively regulates Snail stability to control cell enhanced the rate of Snail degradation, which could be invasion and metastasis. rescued by inhibition of the proteasome. Critically, inhi- bition of mTORC2 (by knocking down rictor) but not Significance: These findings delineate a new regulation mTORC1 (by knocking down raptor) enhanced Snail deg- mechanism of Snail, an important master regulator of radation. Therefore, only mTORC2 inhibition induces epithelial–mesenchymal transition and invasion in cancers. Introduction inducible kinase (SGK; ref. 1). In comparison with mTORC1 signaling, relatively little is known about the biological functions The mTOR is critical for the regulation of cell growth, metab- of mTORC2, particularly those related to the regulation of onco- olism, survival, and other biological functions. It mediates these genesis, although mTORC2 is involved in promoting cancer functions primarily through interacting with other proteins to development (2–4). form 2 distinct complexes: mTOR complex 1 (mTORC1), which is Invasion and metastasis is a cancer hallmark and the leading composed of mTOR, raptor, mLST8, PRAS40, and DEPTOR; and cause of cancer death (5). Epithelial–mesenchymal transition mTOR complex 2 (mTORC2), which contains mTOR, rictor, (EMT) is a key step toward cancer metastasis; this process mLST8, DEPTOR, mSin1, and protor (1). mTORC1 signaling is is in part mediated by Snail, a major transcription factor for crucial for regulating cap-dependent translation initiation, an repression of E-cadherin (E-Cad; refs. 6, 7). The role of mTORC1 essential process for synthesizing many oncogenic proteins such in the positive regulation of the EMT process and metastasis as cyclin D1, c-Myc, Mcl-1, and VEGF, through phosphorylating through translational control of gene expression has long been S6 kinase (S6K) and eIF4E-binding protein 1 (4E-BP1), whereas recognized (8–10). It has been shown that mTORC1/4EBP1/ mTORC2 may positively regulate cell survival and proliferation, eIF4E-mediated Snail translation and subsequent repression of primarily by phosphorylating Akt and serum and glucocorticoid- 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 1 fi First Af liated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, cancer cells (12–17), the underlying biology or mechanisms are Shaanxi, P.R. China. 2Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, Georgia. largely unknown (10). 3Vascular Biology Research Institute, School of Basic Science, Guangdong Glycogen synthase kinase-3 (GSK3), a ubiquitous serine/ Pharmaceutical University, Guangzhou, Guangdong, P.R. China. threonine kinase that is present in mammals in 2 isoforms: a b Note: Supplementary data for this article are available at Cancer Research and (18), plays a key role in regulating a diverse range of Online (http://cancerres.aacrjournals.org/). cellular functions including glycogen metabolism, cell survival, and death (18). However, GSK3 has complex roles in the S.-Y Sun is a Georgia Research Alliance Distinguished Cancer Scientist. regulation of oncogenesis: it can function as a tumor suppressor Corresponding Author: Shi-Yong Sun, Ph.D., Emory University School of Med- in some cancer types while potentiating the growth of cancer icine and Winship Cancer Institute, 1365 Clifton Road NE, C-3088, Atlanta, GA 30322. Phone: 404-778-2170; Fax: 404-778-5520; E-mail: [email protected] cells in others (19, 20). It is well known that GSK3 enhances proteasomal degradation of several oncogenic proteins, includ- – Cancer Res 2019;79:3725 36 ing Snail, c-Myc, Mcl-1, sterol regulatory element-binding pro- doi: 10.1158/0008-5472.CAN-19-0180 teins (SREBPs), and cyclin D, through phosphorylating these Ó2019 American Association for Cancer Research. proteins (21–25). www.aacrjournals.org 3725 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0180 Zhang et al. In the past few years, we have demonstrated that mTORC2 mRNA detection is tightly associated with the negative regulation of GSK3- Cells were collected in TRIzol (Sigma-Aldrich) for prepara- dependent, SCF E3 ligase (FBX4 or FBXW7)–mediated degra- tion of total RNA. Reverse transcription was then performed to dation of cyclin D1, Mcl-1, and SREBP1; inhibition of generate cDNA template using OneScript cDNA Synthesis mTORC2 (e.g., with rictor knockdown or mTOR inhibitors) Kit from Abm Inc. qRT-PCR reaction was performed to amplify accordingly induces the degradation of these proteins (26–29). target genes using SYBR Green according to the manufacturer's These findings have suggested a novel biological function of instructions (Applied Biosystems). The primers used for mTORC2 in the positive regulation of cancer cell metabolism, Snail were 50-GAGGCGGTGGCAGACTAG-30 (forward) and growth, and survival via the direct negative regulation of 50-GACACATCGGTCAGACCAG-30 (reverse; ref. 41). protein degradation. In addition to the FBXW7- or FBX4-medi- ated degradation mechanism, several other proteins such as CHX chase assay Snail and b-catenin undergo GSK3-dependent and b-TrCP After drug treatment for a given time, the treated cells were (another SCF E3 ligase)–mediated degradation (25, 30–32). exposed to 10 mg/mL CHX and then harvested at different times Rictor, a key component of mTORC2, interacts with a core for Western blotting to detect the proteins of interest. Band component of the SCF E3 complex, Cul1 (33). Moreover, SCF/ intensities were quantified by NIH ImageJ software and levels of b-TrCP interacts with DEPTOR, another key component of both target protein were presented as a percentage of levels at 0 time mTORC1 and mTORC2, to promote its degradation (34–36). post CHX treatment. Hence, we were interested in determining whether mTORC2 b – also regulates GSK3-dependent and SCF/ -TrCP mediated deg- Small interfering RNA and small hairpin RNA–mediated gene radation of these proteins. Using chemical approaches, we knockdown found that inhibition of mTOR with mTOR kinase inhibitors Rictor, raptor GSK3a/b, b-TrCP, Cul1, and SKP1 small (TORKinibs) effectively decreased the levels of Snail, but not interfering RNAs (siRNA) were the same as described b -catenin protein. Therefore, this study focused on mTOR previously (27–29, 42). Human Rictor (#2), raptor (#2), and inhibition-induced reduction of Snail and its underlying murine raptor and rictor small hairpin RNAs (shRNA) in mechanisms. pLKO.1 lentiviral vector were purchased from Addgene, Inc. Human b-TrCP (1þ2), b-TrCP1 #1, and b-TrCP1 #2 shRNAs (43) Materials and Methods were generously provided by Dr. Wenyi Wei (Harvard Medical School, Boston, MA). Preparation of lentiviruses with a given Reagents shRNA, cell infection, and selection were the same as described The mTOR inhibitors, rapamycin, RAD001, INK128, and previously (44, 45). AZD8055, the proteasome inhibitor, MG132, the protein syn- thesis inhibitor, cycloheximide (CHX), and the GSK3 inhibitors, Cell immunostaining SB216763 and CHIR99021, were the same as described previ- The tested cells seeded into chamber slides were fixed with ously (28). These agents were dissolved in DMSO at a concen- formaldehyde for 15 minutes and washed with PBS for 3 times tration of 1 or 10 mmol/L, and aliquots were stored at À80 C. followed by blocking with 5% BSA in PBS for 1 hour at room Stock solutions were diluted to the desired final concentrations temperature. The cells were then incubated with mouse anti-E- with growth medium just before use. TGFb1 was purchased from Cad antibody (Catalog no. 8426; Santa Cruz Biotechnology) at PeproTech. Rabbit monoclonal Snail (#3879), E-Cad (#3195), 1:50 dilution in PBS with 2% BSA at 4C overnight followed by and b-TrCP (#4394) antibodies were purchased from Cell Sig- incubation with secondary Alexa Fluor 488 goat anti-mouse IgG naling Technology. Other antibodies were the same as described antibody (Catalog no. A-11001; Thermo Fisher Scientific) at previously