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

The 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 2 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; ref. 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; refs. 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).

Glycogen synthase kinase-3 (GSK3), a ubiquitous serine/threonine kinase that is present in mammals in 2 isoforms: α and β (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 β-catenin undergo GSK3-dependent and β-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/β-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/β-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 β-catenin protein. Therefore, this study focused on mTOR inhibition-induced reduction of Snail and its underlying mechanisms.

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 DMSO at a concentration of 1 or 10 mmol/L, and aliquots were stored at −80°C. Stock solutions were diluted to the desired final concentrations with growth medium just before use. TGFβ1 was purchased from PeproTech. Rabbit monoclonal Snail (#3879), E-Cad (#3195), and β-TrCP (#4394) antibodies were purchased from Cell Signaling Technology. Other antibodies were the same as described previously (27, 28).

Human NSCLC cell lines used in this study were described previously (37, 38). MCF-7 and MDA-MB-453 were purchased from ATCC. HAP1, HAP1/β-TrCP-KO, and HAP1/rictor-KO cells were purchased from Horizon. 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. through analyzing short tandem repeat DNA profile, other cell lines have not been authenticated. 801BL is a metastatic large cell lung cancer cell line obtained by an in vivo selection from the parental 801D cells (39) and has been genetically authenticated. These cell lines were cultured in RPMI1640 or IMDM (HAP1 cells) medium containing 5% FBS at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Preparation of whole-cell protein lysates and Western blot analysis were performed as described previously (40).

Cells were collected in TRIzol (Sigma-Aldrich) for preparation of total RNA. Reverse transcription was then performed to generate cDNA template using OneScript cDNA Synthesis Kit from Abm Inc. qRT-PCR reaction was performed to amplify target genes using SYBR Green according to the manufacturer's instructions (Applied Biosystems). The primers used for Snail were 5′-GAGGCGGTGGCAGACTAG-3′ (forward) and 5′-GACACATCGGTCAGACCAG-3′ (reverse; ref. 41).

After drug treatment for a given time, the treated cells were exposed to 10 μg/mL CHX and then harvested at different times for Western blotting to detect the proteins of interest. Band intensities were quantified by NIH ImageJ software and levels of target protein were presented as a percentage of levels at 0 time post CHX treatment.

Rictor, raptor GSK3α/β, β-TrCP, Cul1, and SKP1 small interfering RNAs (siRNA) were the same as described previously (27–29, 42). Human Rictor (#2), raptor (#2), and murine raptor and rictor small hairpin RNAs (shRNA) in pLKO.1 lentiviral vector were purchased from Addgene, Inc. Human β-TrCP (1+2), β-TrCP1 #1, and β-TrCP1 #2 shRNAs (43) were generously provided by Dr. Wenyi Wei (Harvard Medical School, Boston, MA). Preparation of lentiviruses with a given shRNA, cell infection, and selection were the same as described previously (44, 45).

The tested cells seeded into chamber slides were fixed with formaldehyde for 15 minutes and washed with PBS for 3 times followed by blocking with 5% BSA in PBS for 1 hour at room temperature. The cells were then incubated with mouse anti-E-Cad antibody (Catalog no. 8426; Santa Cruz Biotechnology) at 1:50 dilution in PBS with 2% BSA at 4°C overnight followed by incubation with secondary Alexa Fluor 488 goat anti-mouse IgG antibody (Catalog no. A-11001; Thermo Fisher Scientific) at 1:100 dilution for 1 hour at room temperature in dark. After washing with PBS, cells were fixed with DAPI (Catalog no. P36941; Invitrogen) and examined under Olympus confocal microcopy.

Cell migration was evaluated with cell scratching (or wound healing) assay as follows: an incision was made with a tip in the central area of each well of 24-well plates to create an artificial wound after drug treatment. Images of the wound area were captured at 0, 24, and 48 hours after injury. The in vitro cell invasion assay was carried out in BD BioCoat Matrigel invasion chambers (Becton Dickinson) as described previously (46). Cell numbers in 96-well plates were determined with the SRB assay.

MMTV-PyMT spontaneous breast cancer with lung metastasis transgenic mice (stock no: 002374) were obtained from the Jackson Laboratory and housed in a room with constant temperature and humidity and a 12-hour/12-hour light/dark cycle. All experiments were performed according to protocols approved by the Center of Laboratory Animals Ethics Committee of Guangdong Pharmaceutical University. MMTV-PyMT mice (8 weeks old, female) were randomly divided into 3 groups and treatments initiated the following week with solvent, RAD001 and INK128, which were dissolved in solvent with 5% polyvinylpropyline, 15% N-meth-2-pyrrolidone, and 80% water. Mice were treated 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. INK128 was administered (oral gavage) to the mice at day 1, day 4, day 11, and day 23 at the doses of 0.5, 0.3, 0.1, and 0.3 mg/kg body weight, respectively. On the 29th day, the mice were sacrificed to collect tumors and lung tissues for measuring tumor weights and detecting lung metastatic foci and pulmonary nodules.

The statistical significance of differences between 2 experimental groups was analyzed with 2-sided unpaired Student t tests (for equal variances) or with Welch corrected t test (unequal variances) by use of Graphpad InStat 3 software. Results were considered to be statistically significant at P < 0.05.

To determine the involvement of mTOR in the regulation of Snail, we first examined the effects of different TORKinibs on the levels of Snail. We found that both INK128 and AZD8055 potently reduced the levels of Snail accompanied with suppressing the phosphorylation of Akt and S6 (Fig. 1A) in majority of the tested human lung cancer cell line in which basal levels of p-Akt and p-S6 were detectable. Similar results were also generated in MDA-MB-453 and MCF-7, 2 breast cancer cell lines (Fig. 1B). We 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 INK128 or AZD8055 (Fig. 1B). Hence, TORKinibs clearly decrease Snail levels in human cancer cells. Snail reduction occurred at 2-hour post-INK128 treatment and lasted for up to 24 hours in both A549 and HCC827 cells (Fig. 1C), indicating that Snail decrease is an early and sustained event. In agreement with our previous observations (28), we noted that INK128 quickly and effectively decreased p-Akt (S473) levels, with limited (A549) or no (HCC827) decrease in p-GSK3 levels (Fig. 1C). This result again shows that inhibition of Akt by TORKinibs is not necessarily accompanied with GSK3 activation.

We also examined the effects of rapamycin and RAD001, 2 widely-used conventional mTOR allosteric inhibitors, on modulation of Snail levels in A549 cells. As shown in Fig. 1D, both rapamycin and RAD001 at 1 to 100 nmol/L concentration ranges were more effective than INK128 in decreasing the levels of Snail as well as p-S6 and p-SGK1. As we reported previously (40, 45), both rapamycin and RAD001 increased p-Akt levels whereas INK128 decreased p-Akt levels at 100 nmol/L (Fig. 1D). In the 2 breast cancer cell lines, MCF-7 and MDA-MB-453, rapamycin also decreased Snail levels with elevated levels of p-Akt (Fig. 1B). Thus, suppression of Akt is not necessarily associated with Snail reduction induced by mTOR inhibitors.

In 7 lung cancer cell lines exposed to INK128 or AZD9291, Twist was detected only 3 cell lines (801BL, H23, and H1792) and its levels were not altered. Slug levels were not altered in HCC827, 801BL, Calu-1, H23, and H1792 cell lines, but reduced in PC-9 and EKVX cells (Supplementary Fig. S1). Therefore, TORKinibs have no or limited effects on altering Twist and Slug levels.

We next compared the effect of genetic inhibition of mTORC2 versus genetic inhibition of mTORC1 on modulation of Snail levels. To this end, we knocked down raptor and rictor, respectively, with 2 distinct siRNAs for each gene and then studied their impact on altering Snail protein levels. In A549 cells, transfection of the tested rictor and raptor siRNAs effectively knocked down raptor and rictor gene expression, respectively, accompanied with reduction of Snail, as detected by Western blotting. In HCC827 cells, raptor siRNA #2 effectively knocked down raptor gene expression and accordingly decreased Snail levels, whereas both rictor siRNAs effectively knocked down rictor gene expression accompanied with reduction of Snail (Fig. 2A). In the 801BL lung cancer cell line, rictor knockdown also decreased Snail levels (Fig. 2A, bottom). Similarly, knockdown of either raptor or rictor with a corresponding shRNA decreased Snail levels in A549, HCC827, 801BL, and even MEF cells (Fig. 2B). These data suggest that knockdown of both raptor and rictor causes Snail reduction.

In MEFs deficient in rictor or Sin1 and HAP1 cells deficient in rictor, Snail levels were clearly reduced (Fig. 2C–E). When rictor was re-introduced into rictor-KO MEFs, Snail reduction was not detected (Fig. 2E), indicating a specific event of rictor knockout. Given that both rictor and Sin1 are essential components of mTORC2 (1), it is clear that genetic inhibition of mTORC2 induces Snail reduction.

INK128 did not reduce Snail mRNA levels in both A549 and HCC827 cell lines as evaluated with qRT-PCR (Fig. 3A). The addition of MG132, a widely-used proteasome inhibitor, elevated basal levels of Snail and rescued Snail reduction induced by INK128 (Fig. 3B) or rapalogs (Supplementary Fig. S2A). In a CHX chase assay, Snail had a shorter half-life in INK128-treated cells (1–2 hours) than in DMSO-treated cells (3–5 hours) in both A549 and HCC827 cell lines (Fig. 3C). A similar effect was observed in rapamycin-treated cells (Supplementary Fig. S2B). Hence, it is clear that both TORKinibs and rapalogs enhance Snail protein degradation. Moreover, we found that knockdown of rictor, but not raptor, substantially facilitated the rate of Snail degradation in both A549 and HCC827 cell lines (Fig. 3D). Therefore, we suggest that inhibition of mTORC2, but not mTORC1, enhances Snail degradation.

It is well known that Snail undergoes GSK3-dependent degradation (25). We next determined whether GSK3 is involved in mediating Snail degradation induced by mTORC2 inhibition. The presence of either SB216763 or CHIR99021, 2 different GSK3 inhibitors, rescued Snail reduction induced by INK128 (Fig. 4A). Similarly, genetic inhibition of GSK3 by knocking down GSK3 (both α and β forms) also prevented Snail reduction induced by INK128 (Fig. 4B). As demonstrated above, INK128 clearly enhanced the rate of Snail degradation; however, the presence of SB216763 abolished this effect (Fig. 4C), indicating that GSK3 indeed mediates INK128-induced Snail degradation.

Given that SCF/β-TrCP mediates GSK3-dependent Snail degradation (25), we determined whether this E3 ligase is involved in mediating Snail degradation induced by mTORC2 inhibition. In both A549 and HCC827 cell lines, INK128 decreased β-TrCP levels while reducing Snail levels. Knockdown of β-TrCP with β-TrCP siRNA did not increase Snail levels in either cell line. Interestingly, treatment of β-TrCP siRNA-transfected cells with INK128 enhanced the reduction of both β-TrCP and Snail in comparison with the effect of INK128 or β-TrCP siRNA alone (Fig. 4D). Similar results were also generated with different shRNAs against β-TrCP1 or β-TrCP1+2 (Fig. 4E). β-TrCP deficiency in HAP1 cells neither elevated basal levels of Snail nor blocked Snail reduction induced by INK128. In this experiment, we also observed that INK128 decreased β-TrCP levels in HAP1 cells (Fig. 4F). Moreover, we knocked down SKP1, CUL1, or both, which are the essential components of the SCF complex, and then examined their impact on INK128-induced Snail reduction. Consistently, we failed to see any rescued effects of these gene knockdowns on Snail reduction induced by INK128 in both A549 and HCC827 cells (Fig. 4G). Hence, it is apparent that INK128 induces SCF/β-TrCP–independent Snail degradation.

We further examined the effect of TORKinibs on the levels of E-Cad, a well-known direct target of Snail (7). In 801BL cells with a high EMT phenotype, both INK128 and AZD8055 effectively decreased Snail levels accompanied with elevated levels of E-Cad through the tested time period (24–72 hours; Fig. 5A). The presence of GSK3 inhibitor, either SB216763 or CHIR99021, rescued Snail reduction induced by these TORKinibs and accordingly abolished the ability of the tested TORKinibs to increase E-Cad levels (Fig. 5B). These results clearly indicate that TORKinibs decrease Snail levels accompanied with E-Cad elevation in a GSK3-dependent fashion. Moreover, we detected much higher intensity of E-Cad staining in 801BL cells treated with INK128 or AZD8055 than in the control 801BL cells exposed to DMSO (Fig. 5C). Morphologically, we noted that 801BL cells changed from scattered and round cells into stretched and connected cells after treatment with INK128 or AZD9291 (Fig. 5D), suggesting a clear phenotypic suppression of EMT.

Considering the critical role of Snail in the regulation of cellular EMT and metastasis (7), we next determined the effects of TORKinibs on migration and invasion of cancer cells. The wound healing assay showed that both INK128 and AZD8055 slowed down the healing rates of the tested cell lines (Figs. 6A; Supplementary Fig. S3A). TGFβ facilitated cell healing rates (Figs. 6A; Supplementary Fig. S3A); this process was also slowed down when INK128 was present (Supplementary Fig. S3B). In agreement with the effect of GSK3 inhibitors on Snail reduction and E-Cad elevation induced by TORkinibs (Supplementary Fig. S4A), the presence of SB216763 or CHIR99021 compromised the effect of INK128 on suppressing cell migration (Supplementary Figs. S4B and S4C), indicating a GSK3-dependent event. Using Matrigel invasion chamber assay, we further demonstrated that INK128, at a concentration that apparently did not inhibit cell growth (Fig. 6B), significantly suppressed invasion of the tested cancer cells (Fig. 6C). Therefore, it is clear that these TORKinibs effectively inhibit cancer cell migration and invasion.

Finally, we used the MMTV-PyMT spontaneous breast cancer with lung metastasis transgenic mouse model (47) to demonstrate the effects of mTOR inhibition on cancer cell metastasis. In this model, primary breast tumors can metastasize to lung after about 8 weeks, thus allowing us to observe the effects of tested agents on suppression of lung metastasis. RAD001, but not INK128, significantly inhibited the growth of primary tumors (Fig. 7A). Both agents significantly suppressed lung metastasis assessed by counting tumor nodules on the lung surface (Fig. 7B and C) and on dissected lung tissue sections after hematoxylin and eosin (H&E) staining (Fig. 7D and E). In addition, metastatic nodules on lung surface or H&E-stained lung tissue sections in RAD001- and INK128-treated groups, in general, were smaller than those in solvent control groups. Hence, both RAD001 and INK128 effectively inhibit lung metastasis.

Lamouille and colleagues (14) previously demonstrated that TGFβ-induced elevation of Snail mRNA is in part mediated by mTORC2 through an undefined mechanism. However, this study did not show Snail protein elevation upon TGFβ stimulation. Our current study has demonstrated that inhibition of mTORC2 decreases Snail protein levels through facilitating its proteasomal degradation based on the following findings: (i) TORKinibs, dual inhibitors of mTORC1 and mTORC2, decreased Snail protein levels in multiple cancer cell lines; (ii) genetic inhibition of mTORC2 by knocking down or knocking out rictor or Sin 1, essential components of mTORC2, also decreased Snail levels; (iii) INK128 did not decrease Snail mRNA levels in the tested cell lines; (iv) proteasomal inhibition with MG132 rescued Snail reduction induced by INK128; and (v) both INK128 treatment and rictor knockdown promoted the rate of Snail degradation. Hence, it is clear that mTORC2 positively regulates Snail levels via a posttranslational mechanism, that is, through modulating its stability. In this study, inhibition of mTORC1 by knocking down raptor, an essential component of mTORC1, decreased Snail levels, but failed to affect the Snail degradation rate, suggesting that mTORC1 positively regulates Snail levels via a different mechanism, likely through positively modulating its translation as demonstrated previously (11). Therefore, it seems that there are 2 levels of Snail regulation by mTOR: mTORC1 primarily enhances protein translation and mTORC2 predominantly stabilizes Snail protein by slowing down its degradation. Our findings thus provide a biological basis that links mTORC2 to the positive regulation of EMT, cell invasion, and metastasis as reported previously (12–17).

In this study, we observed that both rapamycin and RAD001 effectively decreased Snail levels, as did INK128, in the tested A549 cells (Fig. 1C), facilitated Snail proteasomal degradation (Supplementary Fig. S2) and suppressed lung metastasis in vivo (Fig. 7). Rapalogs are generally thought to be ineffective against mTORC2. However, we recently have suggested that acute or short-term treatment of certain cancer cell lines (e.g., A549) with a rapalog disrupted the assembly of not only mTORC1, but also mTOCR2, despite increasing Akt phosphorylation, implying that rapalogs inhibit mTORC2 in addition to mTORC1 in some cancer cell lines (26). Therefore, it is reasonable to see Snail decrease in cells exposed to a rapalog. In this study, rapamycin and RAD001 effectively suppressed phosphorylation of SGK1, another well-known substrate of mTORC2 (48), as did INK128, although both agents increased p-Akt levels (Fig. 1D) as we previously reported (40, 45). This is consistent with our previous observation that disruption of mTORC2 assembly by rapamycin is tightly associated with suppression of SGK1 phosphorylation (26). Together with the fact that rapalogs enhanced Snail proteasomal degradation, we reasonably suggest that mTORC2 inhibition contributes to inhibition of cancer metastasis by RAD001 in addition to the involvement of mTORC1 inhibition.

It was previously suggested that Snail undergoes GSK3-dependent, SCF/β-TrCP-mediated proteasomal degradation (25). Indeed, TORKinib-induced Snail degradation is dependent on GSK3 because both chemical (e.g., small molecule inhibition) and genetic (e.g., gene knockdown) inhibition of GSK3 prevented Snail from reduction or degradation induced by mTOR inhibition (Fig. 4). However, we failed to demonstrate the involvement of SCF/β-TrCP in mediating this event based on the following findings: (i) TORKinibs decreased Snail levels accompanied with β-TrCP reduction; (ii) knockdown or deficiency of β-TrCP failed to rescue Snail reduction induced by INK128; and (iii) disruption of SCF complex by knocking down Cul1, SKP1, or both did not affect the ability of INK128 to decrease Snail (Fig. 4). Therefore, we suggest that mTORC2 inhibition induces GSK3-dependent degradation of Snail through a SCF/β-TrCP–independent mechanism (Fig. 7F). Beyond Snail, β-TrCP is also involved in degradation of Slug and Twist (49). In this study, we found that TORKinibs did not reduce the levels of Slug and Twist across the tested multiple cancer cell lines (Supplementary Fig. S1). These data again does not support the involvement of β-TrCP in mediating mTORC2 inhibition-induced Snail degradation. Several other SCF/F-box E3 ligases such as Fbxo45, Fbxo11, Fbxl14, and Fbxl5 are also involved in Snail ubiquitination and degradation (49). Because knockdown of SKP1, CUL1, or both, the essential components of the SCF complex, failed to rescue Snail reduction induced by INK128 (Fig. 4G), these E3 ligases are unlikely to be responsible for mTORC2 inhibition-induced Snail degradation either. A recent study has suggested that the SOCS box protein, SPSB3, function as a novel E3 ligase that ubiquitinates and degrades Snail in response to GSK-3β phosphorylation (50). Whether this E3 ubiquitin ligase is involved in mediating Snail degradation induced by mTORC2 inhibition is under investigation. Nonetheless, our findings warrant future study to identify a novel E3 ubiquitin ligase that mediates Snail ubiquitination and proteasomal degradation induced by mTORC2 inhibition (Fig. 7F).

In this study, INK128 decreased Snail levels accompanied with the elevation of E-Cad, a key marker of EMT and direct target gene of Snail (Fig. 5A and C); this effect is dependent on GSK3 because the presence of a GSK3 inhibitor abrogated the ability of INK128 not only to decrease Snail levels, but also to increase E-Cad expression (Fig. 5B). Consistently, INK128 inhibited cell migration including TGFβ-induced cell migration and cell invasion (Fig. 6A and C; Supplementary Fig. S3). Importantly, INK128 suppression of cell migration is also dependent on GSK3 since this effect was abolished by the presence of GSK3 inhibitor (Supplementary Fig. S4). These findings together support the notion that mTORC2 positively regulates cancer cell EMT, invasion, and metastasis. Therefore, it is clear that mTORC2 plays a critical role in positively regulating cancer cell EMT, invasion, and metastasis primarily by positively modulating Snail stability through preventing GSK3-dependent Snail degradation (Fig. 7F).

Our previous studies have suggested a critical role of GSK3 in maintaining the activity of mTOR inhibitors including rapalogs and TORKinibs against cancer cell growth largely due to GSK3-dependent degradation of cyclin D1, Mcl-1, and SREBP1 upon mTORC2 inhibition as an essential event contributing to the anticancer efficacy of mTOR inhibitors (26–29). This notion is further reinforced by the current finding of the critical role of GSK3-dependent Snail degradation induced by mTORC2 inhibition or mTOR inhibitors in mediating suppression of EMT, migration, and invasion of cancer cells. Clinically, our results suggest that it is critical to select cancers with activated GSK3 for mTOR-targeted cancer therapy in the clinic.

No potential conflicts of interest were disclosed.

Conception and design: Z.G. Chen, L.-J. Wang, S.-Y. Sun

Development of methodology: S. Zhang, D. Wang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zhang, G. Qian, Q.-Q. Zhang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Zhang, Q.-Q. Zhang, Y. Yao, M. Chen, S.-Y. Sun

Writing, review, and/or revision of the manuscript: S. Zhang, Z.G. Chen, S.-Y. Sun

Study supervision: L.-J. Wang, S.-Y. Sun

We are grateful to Dr. Wenyi Wei for providing β-TrCP shRNAs. We also thank Dr. Anthea Hammond in our department for editing the manuscript. Emory University Winship pilot funds (to S.-Y. Sun) and National Natural Science Foundation of China (No. 31771578 to Q.-Q. Zhang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.