Sapanisertib

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

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.

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.

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).

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.

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. TGF1 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 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 RPMI 1640 or IMDM (HAP1 cells) medium containing 5% FBS at 37˚C in a humidified atmosphere of 5% CO2 and 95% air.Western blot analysis. Preparation of whole-cell protein lysates and Western blot analysis were performed as described previously (40). mRNA detection. Cells were collected in Trizol (Sigma-Aldrich co.) for preparation of total

RNA. Reverse transcription was then performed to generate cDNA template using OneScript® cDNA Synthesis Kit from Abm Inc (Richmond, BC). Quantitative real-time PCR (qPCR) 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) (41). CHX chase assay. 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 image J software and levels of target protein were presented as a percentage of levels at 0 time post CHX treatment. Small interfering RNA (siRNA) and small hairpin (shRNA)-mediated gene knockdown. Rictor, raptor GSK3, -TrCP, Cul1 and SKP1 siRNAs were the same as described previously (27- 29,42). Human Rictor (#2), raptor (#2) and murine raptor and rictor shRNAs in pLKO.1 lentiviral vector were purchased from Addgene, Inc. (Cambridge, MA). Human -TrCP (1+2), -TrCP1 #1 and -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).

Cell immunostaining. The tested cells seeded into chamber slides were fixed with formaldehyde for 15 min and washed with PBS for three times followed by blocking with 5% BSA in PBS for 1 h at room temperature. The cells were then incubated with mouse anti-E-Cad antibody (Cat#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 (Cat#A-11001; ThermoFisher .Scientific) at 1: 100 dilution for 1 h at room temperature in dark. After washing with PBS, cells were fixed with DAPI (Cat#P36941; Invitrogen) and examined under Olympus confocal microcopy.
Cell migration, invasion and growth assays. 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 h 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.
Animals and treatments. MMTV-PyMT spontaneous breast cancer with lung metastasis transgenic mice (stock no: 002374) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA) and housed in a room with constant temperature and humidity and a 12 h/12 h 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 three 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 mg/kg,
0.3 mg/kg, 0.1 mg/kg 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.

Statistical analyses. The statistical significance of differences between two experimental groups was analyzed with two-sided unpaired Student’s t tests (for equal variances) or with Welch’s corrected

Results

Chemical inhibition of mTOR with mTOR inhibitors decreases Snail levels in human cancer cells. 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, two 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 h post INK128 treatment and lasted for up to 24 h 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, two 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 nM 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 while INK128 decreased p-Akt levels at 100 nM (Fig. 1D). In the two 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 seven 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, 801 BL, Calu-1, H23 and H1792 cell lines, but reduced in PC-9 and EKVX cells (Fig S1). Therefore, TORKinibs have no or limited effects on altering Twist and Slug levels. Genetic inhibition of mTORC2 by knocking down or knocking out rictor or Sin1 effectively induces Snail reduction. 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 two 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 panel). 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 (Figs. 2C-2E). 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. mTORC2 inhibition facilitates Snail degradation. INK128 did not reduce Snail mRNA levels in both A549 and HCC827 cell lines as evaluated with qPCR (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 (Fig. S2A). In a CHX chase assay, Snail had a shorter half-life in INK128-treated cells (1-2 h) than in DMSO-treated cells (3-5 h) in both A549 and HCC827 cell lines (Fig. 3C). A similar effect was observed in rapamycin-treated cells (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.
INK128 induces GSK3-dependent 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, two different GSK3 inhibitors, rescued Snail reduction induced by INK128 (Fig. 4A). Similarly, genetic inhibition of GSK3 by knocking down GSK3 (both  and  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.

INK128 induces SCF/-TrCP-independent Snail degradation. Given that SCF/-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 -TrCP levels while reducing Snail levels. Knockdown of -TrCP with -TrCP siRNA did not increase Snail levels in either cell line. Interestingly, treatment of -TrCP siRNA- transfected cells with INK128 enhanced the reduction of both -TrCP and Snail in comparison with the effect of INK128 or -TrCP siRNA alone (Fig. 4D). Similar results were also generated with different shRNAs against -TrCP1 or -TrCP1+2 (Fig. 4E). -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 -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/-TrCP-independent Snail degradation. TORKinibs decrease Snail levels accompanied with GSK3-dependent E-Cad elevation. 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 h) (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. TORKinibs effectively inhibit cancer cell migration and invasion. 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 and S3A). TGF facilitated cell healing rates (Figs. 6A and S3A); this process was also slowed down when INK128 was present (Fig. S3B). In agreement with the effect of GSK3 inhibitors on Snail reduction and E-Cad elevation induced by TORkinibs (Fig. S4A), the presence of SB216763 or CHIR99021 compromised the effect of INK128 on suppressing cell migration (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.

Both INK128 and RAD001 effectively inhibit cancer metastasis in vivo. 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 (Figs. 7B and C) and on dissected lung tissue sections after HE staining (Fig. 7D and E). In addition, metastatic nodules on lung surface or HE-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.

Discussion

Lamouille et al (14) previously demonstrated that TGF-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 TGF- stimulation. Our current study has demonstrated that inhibition of mTORC2 decreases Snail protein levels through facilitating its proteasomal degradation based on the following findings: 1) TORKInibs, dual inhibitors of mTORC1 and mTORC2, decreased Snail protein levels in multiple cancer cell lines; 2) Genetic inhibition of mTORC2 by knocking down or knocking out rictor or Sin 1, essential components of mTORC2, also decreased Snail levels; 3) INK128 did not decrease Snail mRNA levels in the tested cell lines; 4) Proteasomal inhibition with MG132 rescued Snail reduction induced by INK128; and 5) 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, i.e., 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 two 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 the current 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 (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/-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/-TrCP in mediating this event based on the following findings: 1) TORKinibs decreased Snail levels accompanied with -TrCP reduction; 2) Knockdown or deficiency of -TrCP failed to rescue Snail reduction induced by INK128; and 3)

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/-TrCP-independent mechanism (Fig. 7F). Beyond Snail, -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 (Fig. S1). This data again does not support the involvement of -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). Since 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 (Figs. 5A and 5C); 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 TGF-induced cell migration and cell invasion (Figs. 6A, 6C and S3). Importantly, INK128 suppression of cell migration is also dependent on GSK3 since this effect was abolished by the presence of GSK3 inhibitor (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.

Acknowledgement

We are grateful to Dr. Wenyi Wei for providing -TrCP shRNAs. We also thank Dr. Anthea Hammond in our department for editing the manuscript.

References

1. Saxton RA, Sabatini DM. mTOR Signaling in Growth, Metabolism, and Disease. Cell
2017;168:960-76

2. Guertin DA, Stevens DM, Saitoh M, Kinkel S, Crosby K, Sheen JH, et al. mTOR complex 2 is required for the development of prostate cancer induced by Pten loss in mice. Cancer Cell 2009;15:148-59
3. Lee K, Nam KT, Cho SH, Gudapati P, Hwang Y, Park DS, et al. Vital roles of mTOR complex 2 in Notch-driven thymocyte differentiation and leukemia. J Exp Med 2012;209:713-28
4. Roulin D, Cerantola Y, Dormond-Meuwly A, Demartines N, Dormond O. Targeting mTORC2 inhibits colon cancer cell proliferation in vitro and tumor formation in vivo. Mol Cancer 2010;9:57
5. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-74

6. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 2014;15:178-96
7. Wang Y, Shi J, Chai K, Ying X, Zhou BP. The Role of Snail in EMT and Tumorigenesis. Curr Cancer Drug Targets 2013;13:963-72
8. Hsieh AC, Liu Y, Edlind MP, Ingolia NT, Janes MR, Sher A, et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 2012;485:55-61
9. Lamouille S, Derynck R. Cell size and invasion in TGF-beta-induced epithelial to mesenchymal transition is regulated by activation of the mTOR pathway. The Journal of cell biology 2007;178:437-51
10. Zhou H, Huang S. Role of mTOR signaling in tumor cell motility, invasion and metastasis.
Current protein & peptide science 2011;12:30-4218
11. Cai W, Ye Q, She QB. Loss of 4E-BP1 function induces EMT and promotes cancer cell migration and invasion via cap-dependent translational activation of snail. Oncotarget 2014;5:6015-27
12. Zong H, Yin B, Zhou H, Cai D, Ma B, Xiang Y. Inhibition of mTOR pathway attenuates migration and invasion of gallbladder cancer via EMT inhibition. Molecular biology reports 2014;41:4507-12
13. Gulhati P, Bowen KA, Liu J, Stevens PD, Rychahou PG, Chen M, et al. mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res 2011;71:3246-56
14. Lamouille S, Connolly E, Smyth JW, Akhurst RJ, Derynck R. TGF-beta-induced activation of mTOR complex 2 drives epithelial-mesenchymal transition and cell invasion. J Cell Sci 2012;125:1259-73
15. Kim EK, Yun SJ, Ha JM, Kim YW, Jin IH, Yun J, et al. Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis. Oncogene 2011;30:2954-63
16. Gupta S, Hau AM, Beach JR, Harwalker J, Mantuano E, Gonias SL, et al. Mammalian target of rapamycin complex 2 (mTORC2) is a critical determinant of bladder cancer invasion. PLoS One 2013;8:e81081
17. Gupta S, Hau AM, Al-Ahmadie HA, Harwalkar J, Shoskes AC, Elson P, et al. Transforming Growth Factor-beta Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2- Dependent Bladder Cancer Cell Migration and Invasion. Am J Pathol 2016;186:1351-60
18. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Biochem J19
19. Mills CN, Nowsheen S, Bonner JA, Yang ES. Emerging roles of glycogen synthase kinase 3 in the treatment of brain tumors. Frontiers in molecular neuroscience 2011;4:47
20. Mishra R. Glycogen synthase kinase 3 beta: can it be a target for oral cancer. Mol Cancer
2010;9:144
21. Welcker M, Clurman BE. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer 2008;8:83-93
22. Inuzuka H, Shaik S, Onoyama I, Gao D, Tseng A, Maser RS, et al. SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature 2011;471:104-9
23. Takahashi-Yanaga F, Sasaguri T. GSK-3beta regulates cyclin D1 expression: a new target for chemotherapy. Cell Signal 2008;20:581-9
24. Xu C, Kim NG, Gumbiner BM. Regulation of protein stability by GSK3 mediated phosphorylation. Cell Cycle 2009;8:4032-9
25. Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M, et al. Dual regulation of Snail by GSK- 3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol 2004;6:931-40
26. Koo J, Wang X, Owonikoko TK, Ramalingam SS, Khuri FR, Sun SY. GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth. Oncotarget 2015;6:8974-87
27. Koo J, Yue P, Deng X, Khuri FR, Sun SY. mTOR complex 2 stabilizes Mcl-1 protein by suppressing its GSK3-dependent and SCF-FBXW7-mediated degradation. Mol Cell Biol 2015;35:2344-55
28. Koo J, Yue P, Gal AA, Khuri FR, Sun SY. Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth. Cancer Res 2014;74:2555-6820
29. Li S, Oh YT, Yue P, Khuri FR, Sun SY. Inhibition of mTOR complex 2 induces GSK3/FBXW7- dependent degradation of sterol regulatory element-binding protein 1 (SREBP1) and suppresses lipogenesis in cancer cells. Oncogene 2016;35:642-50
30. Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol 2001;2:769-76
31. Nakayama KI, Nakayama K. Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer2006;6:369-81
32. Cuadrado A. Structural and functional characterization of NRF2 degradation by glycogen synthase kinase 3/beta-TrCP. Free radical biology & medicine 2015; 88:147-57.
33. Gao D, Wan L, Inuzuka H, Berg AH, Tseng A, Zhai B, et al. Rictor forms a complex with Cullin- 1 to promote SGK1 ubiquitination and destruction. Mol Cell 2010;39:797-808
34. Duan S, Skaar JR, Kuchay S, Toschi A, Kanarek N, Ben-Neriah Y, et al. mTOR generates an auto-amplification loop by triggering the betaTrCP- and CK1alpha-dependent degradation of DEPTOR. Mol Cell 2011;44:317-24
35. Gao D, Inuzuka H, Tan MK, Fukushima H, Locasale JW, Liu P, et al. mTOR drives its own activation via SCF(betaTrCP)-dependent degradation of the mTOR inhibitor DEPTOR. Mol Cell 2011;44:290-303
36. Zhao Y, Xiong X, Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy. Mol Cell 2011;44:304-16
37. Shi P, Oh YT, Zhang G, Yao W, Yue P, Li Y, et al. Met gene amplification and protein hyperactivation is a mechanism of resistance to both first and third generation EGFR inhibitors in lung cancer treatment. Cancer Lett 2016;380:494-504
38. Yao W, Yue P, Zhang G, Owonikoko TK, Khuri FR, Sun SY. Enhancing therapeutic efficacy of the MEK inhibitor, MEK162, by blocking autophagy or inhibiting PI3K/Akt signaling in human lung cancer cells. Cancer Lett 2015;364:70-8
39. Lu YL. [Spontaneous metastasis of clonal cell subpopulations of human lung giant cell carcinoma after subcutaneous inoculation in nude mice]. Zhonghua zhong liu za zhi [Chinese journal of oncology] 1989;11:1-7
40. Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, et al. Activation of Akt and eIF4E Survival Pathways by Rapamycin-Mediated Mammalian Target of Rapamycin Inhibition. Cancer Res 2005;65:7052-8
41. Hotz B, Arndt M, Dullat S, Bhargava S, Buhr HJ, Hotz HG. Epithelial to Sapanisertib mesenchymal transition: expression of the regulators snail, slug, and twist in pancreatic cancer. Clin Cancer Res 2007;13:4769-76
42. Ren H, Koo J, Guan B, Yue P, Deng X, Chen M, et al. The E3 ubiquitin ligases beta-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis. Mol Cancer 2013;12:146
43. Shimizu K, Fukushima H, Ogura K, Lien EC, Nihira NT, Zhang J, et al. The SCFbeta-TRCP E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to promote hepatic lipogenesis. Sci Signal 2017;10: eaah4117
44. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-101
45. Wang X, Yue P, Kim YA, Fu H, Khuri FR, Sun SY. Enhancing mammalian target of rapamycin (mTOR)-targeted cancer therapy by preventing mTOR/raptor inhibition-initiated, mTOR/rictor- independent Akt activation. Cancer Res 2008;68:7409-18
46. Oh YT, Yue P, Wang D, Tong JS, Chen ZG, Khuri FR, et al. Suppression of death receptor 5 enhances cancer cell invasion and metastasis through activation of caspase-8/TRAF2-mediated signaling. Oncotarget 2015;6:41324-38
47. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 2003;163:2113-26
48. Su B, Jacinto E. Mammalian TOR signaling to the AGC kinases. Crit Rev Biochem Mol Biol
2011;46:527-47
49. Yu Q, Zhou BP, Wu Y. The regulation of snail: on the ubiquitin edge. Cancer Cell Microenviron
2017;4: e1567
50. Liu Y, Zhou H, Zhu R, Ding F, Li Y, Cao X, et al. SPSB3 targets SNAIL for degradation in GSK-3beta phosphorylation-dependent manner and regulates metastasis. Oncogene 2018;37:768-76