Mtorc2 Suppresses GSK3-Dependent Snail

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, inhibition 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 2019 American Association for Cancer Research. proteins (21–25).

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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-mediated 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 synthesis 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 80 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 (27, 28). 1:100 dilution for 1 hour at room temperature in dark. After washing with PBS, cells were fixed with DAPI (Catalog no. Cell lines and cell culture P36941; Invitrogen) and examined under Olympus confocal Human NSCLC cell lines used in this study were described microcopy. previously (37, 38). MCF-7 and MDA-MB-453 were purchased from ATCC. HAP1, HAP1/b-TrCP-KO, and HAP1/rictor-KO cells were purchased from Horizon. All MEFs used in this Cell migration, invasion, and growth assays study were described previously (27, 29). Except for H157 Cell migration was evaluated with cell scratching (or wound and A549 cells, which were authenticated by Genetica DNA healing) assay as follows: an incision was made with a tip in the fi Laboratories, Inc. through analyzing short tandem repeat central area of each well of 24-well plates to create an arti cial DNA profile, other cell lines have not been authenticated. wound after drug treatment. Images of the wound area were in vitro 801BL is a metastatic large cell lung cancer cell line obtained captured at 0, 24, and 48 hours after injury. The cell by an in vivo selection from the parental 801D cells (39) and invasion assay was carried out in BD BioCoat Matrigel invasion has been genetically authenticated. These cell lines were cul- chambers (Becton Dickinson) as described previously (46). Cell tured in RPMI1640 or IMDM (HAP1 cells) medium containing numbers in 96-well plates were determined with the SRB assay. 5% FBS at 37 Cinahumidified atmosphere of 5% CO2 and 95% air. Animals and treatments MMTV-PyMT spontaneous breast cancer with lung metastasis Western blot analysis transgenic mice (stock no: 002374) were obtained from the Preparation of whole-cell protein lysates and Western blot Jackson Laboratory and housed in a room with constant temper- analysis were performed as described previously (40). ature and humidity and a 12-hour/12-hour light/dark cycle. All

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experiments were performed according to protocols approved by In 7 lung cancer cell lines exposed to INK128 or AZD9291, the Center of Laboratory Animals Ethics Committee of Guang- Twist was detected only 3 cell lines (801BL, H23, and H1792) dong Pharmaceutical University. MMTV-PyMT mice (8 weeks old, and its levels were not altered. Slug levels were not altered in female) were randomly divided into 3 groups and treatments HCC827, 801BL, Calu-1, H23, and H1792 cell lines, but initiated the following week with solvent, RAD001 and INK128, reduced in PC-9 and EKVX cells (Supplementary Fig. S1). which were dissolved in solvent with 5% polyvinylpropyline, Therefore, TORKinibs have no or limited effects on altering 15% N-meth-2-pyrrolidone, and 80% water. Mice were treated Twist and Slug levels. with RAD001 at 2.5 mg/kg body weight (oral gavage, daily) for 8 days and then at 2 mg/kg body weight for an additional 20 days. Genetic inhibition of mTORC2 by knocking down or knocking INK128 was administered (oral gavage) to the mice at day 1, day 4, out rictor or Sin1 effectively induces Snail reduction day 11, and day 23 at the doses of 0.5, 0.3, 0.1, and 0.3 mg/kg We next compared the effect of genetic inhibition of mTORC2 body weight, respectively. On the 29th day, the mice were sacri- versus genetic inhibition of mTORC1 on modulation of Snail fi ced to collect tumors and lung tissues for measuring tumor levels. To this end, we knocked down raptor and rictor, respec- weights and detecting lung metastatic foci and pulmonary tively, with 2 distinct siRNAs for each gene and then studied their nodules. impact on altering Snail protein levels. In A549 cells, transfection of the tested rictor and raptor siRNAs effectively knocked Statistical analyses down raptor and rictor gene expression, respectively, accompa- fi The statistical signi cance of differences between 2 experimen- nied with reduction of Snail, as detected by Western blotting. t tal groups was analyzed with 2-sided unpaired Student tests (for In HCC827 cells, raptor siRNA #2 effectively knocked down t equal variances) or with Welch corrected test (unequal variances) raptor gene expression and accordingly decreased Snail levels, by use of Graphpad InStat 3 software. Results were considered to whereas both rictor siRNAs effectively knocked down rictor fi P < be statistically signi cant at 0.05. gene expression accompanied with reduction of Snail (Fig. 2A). In the 801BL lung cancer cell line, rictor knockdown also Results decreased Snail levels (Fig. 2A, bottom). Similarly, knockdown of either raptor or rictor with a corresponding shRNA decreased Chemical inhibition of mTOR with mTOR inhibitors decreases Snail levels in A549, HCC827, 801BL, and even MEF cells Snail levels in human cancer cells (Fig. 2B). These data suggest that knockdown of both raptor and To determine the involvement of mTOR in the regulation of rictor causes Snail reduction. fi Snail, we rst examined the effects of different TORKinibs on the In MEFs deficient in rictor or Sin1 and HAP1 cells deficient in levels of Snail. We found that both INK128 and AZD8055 rictor, Snail levels were clearly reduced (Fig. 2C–E). When rictor potently reduced the levels of Snail accompanied with suppres- was re-introduced into rictor-KO MEFs, Snail reduction was not sing the phosphorylation of Akt and S6 (Fig. 1A) in majority of the detected (Fig. 2E), indicating a specific event of rictor knockout. tested human lung cancer cell line in which basal levels of p-Akt Given that both rictor and Sin1 are essential components of and p-S6 were detectable. Similar results were also generated in mTORC2 (1), it is clear that genetic inhibition of mTORC2 MDA-MB-453 and MCF-7, 2 breast cancer cell lines (Fig. 1B). We induces Snail reduction. noted that both MCF-7 and T47D luminal breast cancer cell lines expressed very low or undetectable levels of Snail. After a very long exposure, we observed Snail reduction in MCF-7 cells treated with mTORC2 inhibition facilitates Snail degradation INK128 or AZD8055 (Fig. 1B). Hence, TORKinibs clearly decrease INK128 did not reduce Snail mRNA levels in both A549 and Snail levels in human cancer cells. Snail reduction occurred at HCC827 cell lines as evaluated with qRT-PCR (Fig. 3A). The 2-hour post-INK128 treatment and lasted for up to 24 hours in addition of MG132, a widely-used proteasome inhibitor, elevated both A549 and HCC827 cells (Fig. 1C), indicating that Snail basal levels of Snail and rescued Snail reduction induced by decrease is an early and sustained event. In agreement with our INK128 (Fig. 3B) or rapalogs (Supplementary Fig. S2A). In a previous observations (28), we noted that INK128 quickly and CHX chase assay, Snail had a shorter half-life in INK128-treated – – effectively decreased p-Akt (S473) levels, with limited (A549) or cells (1 2 hours) than in DMSO-treated cells (3 5 hours) in both no (HCC827) decrease in p-GSK3 levels (Fig. 1C). This result A549 and HCC827 cell lines (Fig. 3C). A similar effect was again shows that inhibition of Akt by TORKinibs is not necessarily observed in rapamycin-treated cells (Supplementary Fig. S2B). accompanied with GSK3 activation. Hence, it is clear that both TORKinibs and rapalogs enhance Snail We also examined the effects of rapamycin and RAD001, 2 protein degradation. Moreover, we found that knockdown of widely-used conventional mTOR allosteric inhibitors, on mod- rictor, but not raptor, substantially facilitated the rate of Snail ulation of Snail levels in A549 cells. As shown in Fig. 1D, both degradation in both A549 and HCC827 cell lines (Fig. 3D). rapamycin and RAD001 at 1 to 100 nmol/L concentration Therefore, we suggest that inhibition of mTORC2, but not ranges were more effective than INK128 in decreasing the levels mTORC1, enhances Snail degradation. of Snail as well as p-S6 and p-SGK1. As we reported previously (40, 45), both rapamycin and RAD001 increased p-Akt levels INK128 induces GSK3-dependent Snail degradation whereas INK128 decreased p-Akt levels at 100 nmol/L It is well known that Snail undergoes GSK3-dependent degra- (Fig.1D).Inthe2breastcancercelllines,MCF-7andMDA- dation (25). We next determined whether GSK3 is involved in MB-453, rapamycin also decreased Snail levels with elevated mediating Snail degradation induced by mTORC2 inhibition. The levels of p-Akt (Fig. 1B). Thus, suppression of Akt is not presence of either SB216763 or CHIR99021, 2 different GSK3 necessarily associated with Snail reduction induced by mTOR inhibitors, rescued Snail reduction induced by INK128 (Fig. 4A). inhibitors. Similarly, genetic inhibition of GSK3 by knocking down GSK3

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A PC-9 HCC827 H226 A549 H157 H522 801BL Calu-1 EKVX H23 H1792 AZD8055: −−+−−+− −+−−+ −− +−−+ −−+ −−+− −+ −− + − − INK128: −+−−+−− +−−+− −+ −−+− −+− −+−− +− −+ − − + Snail Fold reduction: 1 0 0 1 .4 .4 1 .2 .3 1 .2 .1 1 .1 .2 1 .2 .3 1 .04 .04 1 .8 1 1 .7 .7 1 .7 .6

p-Akt (S473)

Akt p-S6 (S235/236) S6 Actin

MCF-7 MDA-MB-453 BDT47D A549 INK128 Rap RAD001

Conc. (nmol/L): 0 1 10 100 1 10 100 1 10 100

Snail Snail Fold reduction: 1 .9 .5 .05 .2 .1 .2 .2 .2 .1 LE p-Akt (S473) Fold reduction: 1 .4 .1 .7 1 .8 .6 .8

p-Akt (S473) Akt p-GSK3 Akt (S21/9) GSK3 p-S6 (S235/236) p-SGK1 S6 (S422) SGK1 GAPDH p-S6 (S235/236) C A549 HCC827 Time (hours): 0 2 4 8 16 24 0 2 4 8 16 24 S6

Snail Tubulin Fold reduction: 1 .3 .04 .02 .1 .1 1 .6 .3 .2 .5 .4

p-Akt (S473)

Akt

p-S6 (S235/236)

S6 p-GSK3 (S21/9)

GSK3

GAPDH

Figure 1. mTOR inhibitors decrease Snail levels in different cancer cell lines. The indicated cell lines were exposed to 100 nmol/L INK128 or AZD8055 for 16 hours(A and B), 100 nmol/L INK128 for different times as indicated (C), or the indicated concentrations for 16 hours (D). Whole-cell protein lysates were then prepared from the aforementioned treatments and used for Western blotting to detect different proteins. LE, longer exposure

(both a and b forms) also prevented Snail reduction induced by INK128 induces SCF/b-TrCP–independent Snail degradation INK128 (Fig. 4B). As demonstrated above, INK128 clearly Given that SCF/b-TrCP mediates GSK3-dependent Snail deg- enhanced the rate of Snail degradation; however, the presence radation (25), we determined whether this E3 ligase is involved in of SB216763 abolished this effect (Fig. 4C), indicating that GSK3 mediating Snail degradation induced by mTORC2 inhibition. In indeed mediates INK128-induced Snail degradation. both A549 and HCC827 cell lines, INK128 decreased b-TrCP

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AB A549 801BL HCC827 MEFs

Raptor

Rictor Raptor Raptor

Snail A549 Rictor Rictor

Fold reduction: 1 .6 .4 .3 .2 Snail Snail Actin Fold reduction: 1 .6 .2 1 .4 .3 1 .5 .7 1 .2 .4

Tubulin Actin Raptor

Rictor HAP1 C MEFs D Rictor: WT KO Snail HCC827 Rictor: WT KO Fold reduction: 1 1 .4 .7 .4 Snail Snail E Actin Fold reduction: 1 0.4 Fold reduction: 1 0.3 MEFs Rictor Rictor 801BL Tubulin Actin

MEFs

Sin1: WT KO Snail Snail Snail Fold reduction: 1 .7 .4 Rictor Rictor Sin1 Tubulin Actin Actin

Figure 2. Impact of genetic knockdown (A and B) or knockout (C–E) of raptor, rictor, or Sin1 on modulating Snail levels. Whole-cell protein lysates were prepared from different cancer cell lines transiently transfected with the given siRNAs for 48 hours (A), from different cell lines stably expressing the indicated shRNAs (B), or from MEFs or HAP1 cells deﬁcient in a given gene (C–E) and then applied to Western blotting to detect proteins of interest.

levels while reducing Snail levels. Knockdown of b-TrCP with levels of E-Cad through the tested time period (24–72 b-TrCPsiRNAdidnotincreaseSnaillevelsineithercellline. hours; Fig. 5A). The presence of GSK3 inhibitor, either Interestingly, treatment of b-TrCP siRNA-transfected cells with SB216763 or CHIR99021, rescued Snail reduction induced by INK128 enhanced the reduction of both b-TrCP and Snail in these TORKinibs and accordingly abolished the ability of the comparison with the effect of INK128 or b-TrCP siRNA tested TORKinibs to increase E-Cad levels (Fig. 5B). These alone (Fig. 4D). Similar results were also generated with dif- results clearly indicate that TORKinibs decrease Snail levels ferent shRNAs against b-TrCP1 or b-TrCP1þ2(Fig.4E).b-TrCP accompanied with E-Cad elevation in a GSK3-dependent fash- deﬁciency in HAP1 cells neither elevated basal levels of Snail ion. Moreover, we detected much higher intensity of E-Cad nor blocked Snail reduction induced by INK128. In this exper- staining in 801BL cells treated with INK128 or AZD8055 than iment, we also observed that INK128 decreased b-TrCP levels in the control 801BL cells exposed to DMSO (Fig. 5C). Mor- in HAP1 cells (Fig. 4F). Moreover, we knocked down SKP1, phologically, we noted that 801BL cells changed from scattered CUL1, or both, which are the essential components of the SCF and round cells into stretched and connected cells after treat- complex, and then examined their impact on INK128-induced ment with INK128 or AZD9291 (Fig. 5D), suggesting a clear Snail reduction. Consistently, we failed to see any rescued phenotypic suppression of EMT. effects of these gene knockdowns on Snail reduction induced by INK128 in both A549 and HCC827 cells (Fig. 4G). Hence, it TORKinibs effectively inhibit cancer cell migration and is apparent that INK128 induces SCF/b-TrCP–independent invasion Snail degradation. Considering the critical role of Snail in the regulation of cellular EMT and metastasis (7), we next determined the effects TORKinibs decrease Snail levels accompanied with GSK3- of TORKinibs on migration and invasion of cancer cells. dependent E-Cad elevation The wound healing assay showed that both INK128 and We further examined the effect of TORKinibs on the levels of AZD8055 slowed down the healing rates of the tested cell lines E-Cad, a well-known direct target of Snail (7). In 801BL cells (Figs. 6A; Supplementary Fig. S3A). TGFb facilitated cell healing with a high EMT phenotype, both INK128 and AZD8055 rates (Figs. 6A; Supplementary Fig. S3A); this process was effectively decreased Snail levels accompanied with elevated also slowed down when INK128 was present (Supplementary

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A 1.2 B A549 HCC827 0.9 MG132: − − + + − − + + 0.6 INK128: − + − + − + − +

Snail mRNA 0.3 Snail 0 DMSO INK DMSO INK Actin Figure 3. HCC827 A549 INK128 does not decrease Snail HCC827 C A549 mRNA levels (A), but promotes DMSO INK128 Snail protein proteasomal DMSO INK128 degradation (B and C) as rictor CHX (hours): 0 1 2 4 6 0 1 2 4 6 CHX (hours): 0 1 2 4 6 0 1 2 4 6 knockdown does in facilitating Snail degradation (D). A, The Snail Snail indicated cell lines were exposed to different concentrations of GAPDH Actin INK128 for 3 hours and then harvested for preparation of total DMSO cellular RNA and subsequent 100 DMSO 100 INK128 detection of Snail mRNA with INK128 qRT-PCR. B, A549 or HCC827 75 75 cells were pretreated with 10 50 50 mmol/L MG132 for 1 hour and then cotreated with 100 nmol/L 25

25 Snail levels (% of 0 time) Snail levels

(% of 0 time) INK128 for 4 hours. The cells were 0 0 harvested for preparation of 0123456 0123456 whole-cell protein lysates and Time post CHX (hours) Time post CHX (hours) subsequent Western blot analysis. C, A549 or HCC827 cells A549 were treated with DMSO or D 100 nmol/L of INK128 for 8 hours pLKO.1 pLKO.1 shRaptor shRictor 100 and then exposed to 10 mg/mL shRaptor CHX. At the indicated times post CHX (min): 0 60 90 120 0 60 90 120 0 60 90 120 75 shRictor CHX, the cells were harvested for Snail preparation of whole-cell protein 50 lysates and subsequent Western blot analysis. Protein levels were Raptor 25 quantiﬁed with NIH ImageJ software and were normalized to Rictor 0 Snail levels (% of 0 time) actin or GAPDH. D, Whole-cell 0 30 60 90 120 protein lysates were prepared Actin Time post CHX (minutes) from the indicated transfectants exposed to 10 mg/mL CHX for 801BL pLKO.1 different times as indicated and pLKO.1 shRaptor shRictor 100 shRaptor then subjected to Western blot analysis. Protein levels were CHX (min): 0 60 90 120 0 60 90 120 0 60 90 120 shRictor 75 quantiﬁed with NIH ImageJ software and were normalized to Snail actin or tubulin. 50 Raptor 25 Rictor Snail level (% of 0 time) 0 0306090120 Tubulin Time post CHX (minutes)

Fig. S3B). In agreement with the effect of GSK3 inhibitors on Both INK128 and RAD001 effectively inhibit cancer metastasis Snail reduction and E-Cad elevation induced by TORkinibs in vivo (Supplementary Fig. S4A), the presence of SB216763 or Finally, we used the MMTV-PyMT spontaneous breast cancer CHIR99021 compromised the effect of INK128 on suppressing with lung metastasis transgenic mouse model (47) to demon- cell migration (Supplementary Figs. S4B and S4C), indicating a strate the effects of mTOR inhibition on cancer cell metastasis. In GSK3-dependent event. Using Matrigel invasion chamber this model, primary breast tumors can metastasize to lung after assay, we further demonstrated that INK128, at a concentration about 8 weeks, thus allowing us to observe the effects of tested that apparently did not inhibit cell growth (Fig. 6B), signifi- agents on suppression of lung metastasis. RAD001, but not cantly suppressed invasion of the tested cancer cells (Fig. 6C). INK128, significantly inhibited the growth of primary tumors Therefore, it is clear that these TORKinibs effectively inhibit (Fig. 7A). Both agents significantly suppressed lung metastasis cancer cell migration and invasion. assessed by counting tumor nodules on the lung surface (Fig. 7B

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A + GSK3i B C siRNA: Ctrl GSK3α/β CHIR99021: − − − − + + − − + + − − − + − + DMSO SB216763: INK128: 100 − + − + − + INK128 INK128: Snail 75 SB + INK Snail GSK3 50 A549 Actin Tubulin 25 0

Snail Snail levels (% of 0 time) 43210 HCC827 Time post CHX (hours) Actin DMSO INK128 SB + INK128 D CHX (hours): 0 1 2 4 0 1 2 4 0 1 2 4 A549 HCC827 Snail siRNA: Ctrl TrCP Ctrl TrCP INK128: − + − + − + − + GADPH Snail HAP1 β-TrCP F WT TrCP-KO Tubulin INK128: − + − + Snail

β E -TrCP Tubulin G INK128: − + − + − + − + siRNA: Ctrl Cul1 SKP1 Cul1+SKP1 INK128: − + − + − + − + Snail Snail β-TrCP A549 Cul1 Tubulin

SKP1 HCC827 Snail GAPDH β-TrCP HCC827 Tubulin Snail * Cul1

SKP1 A549

GAPDH

Figure 4. Inhibition of GSK3 with SB216763 or CHIR99021 (A and C) or siRNA (B), but not knockdown (D, E,andG) or knockout (F) of SCF/b-TrCP, rescues Snail degradation induced by INK128. A, The indicated cell lines were pretreated with 10 mmol/L SB216763 or CHIR99021 for 1 hour and then cotreated with 100 nmol/L INK128 for an additional 8 hours. B, HCC827 cells were transfected with the indicated siRNAs for 48 hours and then exposed to 100 nmol/L INK128 for an additional 8 hours. C, HCC827 cells were treated with DMSO, 100 nmol/L INK128, or INK128 plus 10 mmol/L SB216763 for 6 hours and then exposed to 10 mg/mL CHX for different times as indicated. D, The indicated cell lines were transfected with control (Ctrl) or b-TrCP siRNAs and after 48 hours were exposed to 100 nmol/L INK128 for an additional 8 hours. E, The indicated cell lines expressing the given shRNAs were exposed to 100 nmol/L INK128 for 8 hours. F, The indicated cell lines were exposed to 100 nmol/L INK128 for 8 hours. G, The given cell lines were transfected with the indicated siRNAs for 48 hours, followed with 100 nmol/L INK128 for additional 8 hours. Whole-cell protein lysates were then prepared from the above treatments and used for Western blot analysis. Protein levels in C were quantiﬁed with NIH ImageJ software and were normalized to GAPDH. The asterisk in G indicates a protein molecular weight marker band that showed up in ECL reaction.

and C) and on dissected lung tissue sections after hematoxylin RAD001- and INK128-treated groups, in general, were smaller and eosin (H&E) staining (Fig. 7D and E). In addition, metastatic than those in solvent control groups. Hence, both RAD001 and nodules on lung surface or H&E-stained lung tissue sections in INK128 effectively inhibit lung metastasis.

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A Time (hours): 24 48 72 AZD8055: − − + − − + − − + C DMSO INK128 AZD8055 INK128: − + − − + − − + −

Snail DAPI E-Cad

GAPDH

B SB216765: − − − + + + E-Cad AZD8055: − − + − − + INK128: − + − − + −

Snail

E-Cad Merge

Actin

− − − + + + CHIR99021: D AZD8055: − − + − − + DMSO INK128 AZD8055 INK128: − + − − + −

Snail

E-Cad

Actin

Figure 5. TORKinibs induce GSK3-dependent Snail reduction and E-Cad elevation (A and B) and drastic morphologic change with elevated staining of E-Cad (C and D). A and B, 801BL cells were exposed to 100 nmol/L INK128 or AZD8055 for different times as indicated (A) or were pretreated with 10 mmol/L SB216763 or CHIR99021 for 1 hour and then cotreated with 100 nmol/L INK128 or AZD8055 for an additional 48 hours (B). Different proteins were detected with Western blotting. C and D, 801BL cells were exposed to 100 nmol/L INK128 or AZD8055 for 48 hours, followed by immunocytoﬂuorescence staining of E-Cad (C)and picture of cell morphological changes (D).

Discussion mechanism, likely through positively modulating its translation Lamouille and colleagues (14) previously demonstrated that as demonstrated previously (11). Therefore, it seems that there are TGFb-induced elevation of Snail mRNA is in part mediated by 2 levels of Snail regulation by mTOR: mTORC1 primarily mTORC2 through an undefined mechanism. However, this study enhances protein translation and mTORC2 predominantly sta- did not show Snail protein elevation upon TGFb stimulation. Our bilizes Snail protein by slowing down its degradation. Our find- current study has demonstrated that inhibition of mTORC2 ings thus provide a biological basis that links mTORC2 to the decreases Snail protein levels through facilitating its proteasomal positive regulation of EMT, cell invasion, and metastasis as degradation based on the following findings: (i) TORKinibs, dual reported previously (12–17). inhibitors of mTORC1 and mTORC2, decreased Snail protein In this study, we observed that both rapamycin and RAD001 levels in multiple cancer cell lines; (ii) genetic inhibition of effectively decreased Snail levels, as did INK128, in the tested mTORC2 by knocking down or knocking out rictor or Sin 1, A549 cells (Fig. 1C), facilitated Snail proteasomal degradation essential components of mTORC2, also decreased Snail levels; (Supplementary Fig. S2) and suppressed lung metastasis in vivo (iii) INK128 did not decrease Snail mRNA levels in the tested cell (Fig. 7). Rapalogs are generally thought to be ineffective against lines; (iv) proteasomal inhibition with MG132 rescued Snail mTORC2. However, we recently have suggested that acute or reduction induced by INK128; and (v) both INK128 treatment short-term treatment of certain cancer cell lines (e.g., A549) with and rictor knockdown promoted the rate of Snail degradation. a rapalog disrupted the assembly of not only mTORC1, but also Hence, it is clear that mTORC2 positively regulates Snail levels via mTOCR2, despite increasing Akt phosphorylation, implying that a posttranslational mechanism, that is, through modulating its rapalogs inhibit mTORC2 in addition to mTORC1 in some cancer stability. In this study, inhibition of mTORC1 by knocking down cell lines (26). Therefore, it is reasonable to see Snail decrease in raptor, an essential component of mTORC1, decreased Snail cells exposed to a rapalog. In this study, rapamycin and RAD001 levels, but failed to affect the Snail degradation rate, suggesting effectively suppressed phosphorylation of SGK1, another well- that mTORC1 positively regulates Snail levels via a different known substrate of mTORC2 (48), as did INK128, although

3732 Cancer Res; 79(14) July 15, 2019 Cancer Research

mTORC2 Stabilization of Snail

A DMSO INK128 AZD8055 TGFβ1

0 hours

HCC827

24 hours

0 hours

801BL

24 hours

BCDMSO INK128 801BL HCC827 DMSO INK128 100 100 DMSO 75 75

50 50 Cell number (% of control) 25 25 INK128 0 0 801BL HCC827 Invasion (% of control) 801BL HCC827

Figure 6. TORKinibs inhibit cell migration (A) and invasion (B and C). A, The scratched cell lines as indicated were treated with 10 nmol/L INK128 or AZD8055 or 2 ng/mL TGFb for 24 hours and then photographed. B and C, Cells seeded in the upper chambers of the transwell assay for 2 hours were exposed to DMSO or INK128 (20 nmol/L for 801BL and 10 nmol/L for HCC827). After 30 hours, cells that migrated to the lower surface of the membrane were measured as invasive cells (C). Cells were seeded in 96-well plates and received the same treatments and cell numbers were determined by SRB assay (B). Columns are means SDs of triplicate determinations.

both agents increased p-Akt levels (Fig. 1D) as we previously SCF/b-TrCP in mediating this event based on the following reported (40, 45). This is consistent with our previous observation ﬁndings: (i) TORKinibs decreased Snail levels accompanied with that disruption of mTORC2 assembly by rapamycin is tightly b-TrCP reduction; (ii) knockdown or deﬁciency of b-TrCP failed to associated with suppression of SGK1 phosphorylation (26). rescue Snail reduction induced by INK128; and (iii) disruption of Together with the fact that rapalogs enhanced Snail proteasomal SCF complex by knocking down Cul1, SKP1, or both did not affect degradation, we reasonably suggest that mTORC2 inhibition the ability of INK128 to decrease Snail (Fig. 4). Therefore, we contributes to inhibition of cancer metastasis by RAD001 in suggest that mTORC2 inhibition induces GSK3-dependent deg- addition to the involvement of mTORC1 inhibition. radation of Snail through a SCF/b-TrCP–independent mechanism It was previously suggested that Snail undergoes GSK3-depen- (Fig. 7F). Beyond Snail, b-TrCP is also involved in degradation of dent, SCF/b-TrCP-mediated proteasomal degradation (25). Slug and Twist (49). In this study, we found that TORKinibs did Indeed, TORKinib-induced Snail degradation is dependent on not reduce the levels of Slug and Twist across the tested multiple GSK3 because both chemical (e.g., small molecule inhibition) cancer cell lines (Supplementary Fig. S1). These data again does and genetic (e.g., gene knockdown) inhibition of GSK3 prevented not support the involvement of b-TrCP in mediating mTORC2 Snail from reduction or degradation induced by mTOR inhibition inhibition-induced Snail degradation. Several other SCF/F-box E3 (Fig. 4). However, we failed to demonstrate the involvement of ligases such as Fbxo45, Fbxo11, Fbxl14, and Fbxl5 are also

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Zhang et al.

A B C (n = 14) 20 350 (n = 14) Ctrl P = 0.0031 P = 0.0817 300 P = 0.0363 P = 0.0437 15 250 200 10 (n = 8) 150 (n = 8) RAD001 100 (n = 8) 5 (n = 8) Tumor weight (g) 50

0 INK128

Lung surface nodules/mouse 0 Ctrl RAD001 INK128 Ctrl RAD001 INK128

D (n = 14) 80 70 P = 0.0418 P = 0.0169 E Ctrl RAD001 INK128 60 50 40 30 20 (n = 8) (n = 8) 10 0

Foci number/max lung section Ctrl RAD001 INK128

F mTOR mTORC2i mTORC2 Sin1 Rictor GSK3 ? U U U Dub3 U EMT Degradation Snail Snail invasion U metastasis U E3 ubiquitin ligase ? U U

Figure 7. Both INK128 and RAD001 inhibit lung metastasis in a transgenic mouse breast cancer model (A–E); this is likely to be in part due to suppression of mTORC2- mediated Snail stabilization as suggested (F). A–E, MMTV-PyMT mice at 9 weeks were treated with solvent control, RAD001, and INK128 as described in Materials and Methods. On day 29, mice were sacriﬁced to collect tumors and lungs for measuring tumor weights (A) and analyzing lung metastasis (B–E). F, The schema summarizes the possible working model, demonstrating the positive regulation of Snail stability, cell invasion, and metastasis by mTORC2. Red arrows indicate the possible effects caused by mTORC2 inhibition (mTORC2i).

involved in Snail ubiquitination and degradation (49). Because and proteasomal degradation induced by mTORC2 inhibition knockdown of SKP1, CUL1, or both, the essential components (Fig. 7F). of the SCF complex, failed to rescue Snail reduction induced by In this study, INK128 decreased Snail levels accompanied with INK128 (Fig. 4G), these E3 ligases are unlikely to be respon- the elevation of E-Cad, a key marker of EMT and direct target gene sible for mTORC2 inhibition-induced Snail degradation either. of Snail (Fig. 5A and C); this effect is dependent on GSK3 because A recent study has suggested that the SOCS box protein, SPSB3, the presence of a GSK3 inhibitor abrogated the ability of INK128 function as a novel E3 ligase that ubiquitinates and degrades not only to decrease Snail levels, but also to increase E-Cad Snail in response to GSK-3b phosphorylation (50). Whether expression (Fig. 5B). Consistently, INK128 inhibited cell migra- this E3 ubiquitin ligase is involved in mediating Snail degra- tion including TGFb-induced cell migration and cell invasion dation induced by mTORC2 inhibition is under investigation. (Fig. 6A and C; Supplementary Fig. S3). Importantly, INK128 Nonetheless, our ﬁndings warrant future study to identify a suppression of cell migration is also dependent on GSK3 since this novel E3 ubiquitin ligase that mediates Snail ubiquitination effect was abolished by the presence of GSK3 inhibitor

3734 Cancer Res; 79(14) July 15, 2019 Cancer Research

mTORC2 Stabilization of Snail

(Supplementary Fig. S4). These findings together support the Authors' Contributions notion that mTORC2 positively regulates cancer cell EMT, inva- Conception and design: Z.G. Chen, L.-J. Wang, S.-Y. Sun sion, and metastasis. Therefore, it is clear that mTORC2 plays a Development of methodology: S. Zhang, D. Wang critical role in positively regulating cancer cell EMT, invasion, and Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zhang, G. Qian, Q.-Q. Zhang metastasis primarily by positively modulating Snail stability Analysis and interpretation of data (e.g., statistical analysis, biostatistics, through preventing GSK3-dependent Snail degradation (Fig. 7F). computational analysis): S. Zhang, Q.-Q. Zhang, Y. Yao, M. Chen, S.-Y. Sun Our previous studies have suggested a critical role of GSK3 in Writing, review, and/or revision of the manuscript: S. Zhang, Z.G. Chen, maintaining the activity of mTOR inhibitors including rapalogs S.-Y. Sun and TORKinibs against cancer cell growth largely due to GSK3- Study supervision: L.-J. Wang, S.-Y. Sun dependent degradation of cyclin D1, Mcl-1, and SREBP1 upon mTORC2 inhibition as an essential event contributing to the Acknowledgments b anticancer efficacy of mTOR inhibitors (26–29). This notion is We are grateful to Dr. Wenyi Wei for providing -TrCP shRNAs. We also thank fi Dr. Anthea Hammond in our department for editing the manuscript. Emory further reinforced by the current nding of the critical role of University Winship pilot funds (to S.-Y. Sun) and National Natural Science GSK3-dependent Snail degradation induced by mTORC2 inhibi- Foundation of China (No. 31771578 to Q.-Q. Zhang). tion or mTOR inhibitors in mediating suppression of EMT, migration, and invasion of cancer cells. Clinically, our results The costs of publication of this article were defrayed in part by the payment of suggest that it is critical to select cancers with activated GSK3 for page charges. This article must therefore be hereby marked advertisement in mTOR-targeted cancer therapy in the clinic. accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conflicts of Interest Received January 15, 2019; revised April 23, 2019; accepted May 21, 2019; published first May 29, 2019. No potential conflicts of interest were disclosed.

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3736 Cancer Res; 79(14) July 15, 2019 Cancer Research

mTORC2 Suppresses GSK3-Dependent Snail Degradation to Positively Regulate Cancer Cell Invasion and Metastasis

Shuo Zhang, Guoqing Qian, Qian-Qian Zhang, et al.

Cancer Res 2019;79:3725-3736. Published OnlineFirst May 29, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0180

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