The mTOR positively regulates cell proliferation and survival through forming 2 complexes with raptor (mTOR complex 1; mTORC1) or rictor (mTOR complex 2; mTORC2). Compared with the mTORC1, relatively little is known about the biologic functions of mTORC2. This study focuses on addressing whether mTORC2 regulates apoptosis, particularly induced by TRAIL (TNFSF10). Using the mTOR kinase inhibitor, PP242, as a research tool, we found that it synergized with TRAIL to augment apoptosis of cancer cells. PP242 reduced the abundance of the short form of c-FLIP (FLIPS, CFLARS) and survivin (BIRC5). Enforced expression of ectopic FLIPS, but not survivin, attenuated augmented apoptosis induced by PP242 plus TRAIL. Thus, it is FLIPS downregulation that contributes to synergistic induction of apoptosis by PP242 plus TRAIL. PP242 decreased FLIPS stability, increased FLIPS ubiquitination, and facilitated FLIPS degradation. Moreover, knockdown of the E3 ligase Cbl (CBL) abolished PP242-induced FLIPS reduction. Thus, PP242 induces Cbl-dependent degradation of FLIPS, leading to FLIPS downregulation. Consistently, knockdown of rictor or mTOR, but not raptor, mimicked PP242 in decreasing FLIPS levels and sensitizing cells to TRAIL. Rictor knockdown decreased FLIPS stability, whereas enforced expression of rictor stabilized FLIPS. Moreover, silencing of Cbl abrogated FLIPS reduction induced by rictor knockdown. Collectively we conclude that it is mTORC2 inhibition that results in FLIPS downregulation and subsequent sensitization of TRAIL-induced apoptosis. Our findings provide the first evidence showing that mTORC2 stabilizes FLIPS, hence connecting mTORC2 signaling to the regulation of death receptor-mediated apoptosis. Cancer Res; 73(6); 1946–57. ©2012 AACR.
TRAIL (also called APO-2L) is a member of the tumor necrosis factor superfamily. Recombinant TRAIL is currently being tested in phase I clinical trials as a potential cancer therapeutic agent based on its unique ability to primarily trigger apoptosis in various types of cancer cells while sparing normal cells (1). However, cancer cells exhibit varied sensitivity to TRAIL and a substantial proportion of cancer cell lines are intrinsically insensitive to TRAIL (2). Thus, in these insensitive cancer cells, additional sensitization is needed to potentiate the killing effect of TRAIL.
TRAIL-initiated apoptosis involves the initial binding of TRAIL to death receptor 4 (DR4) or 5 (DR5) followed by oligomerization of the death receptor and formation of the death inducing-signaling complex (DISC). DISC assembly recruits the adaptor molecule FADD and procaspase-8, leading to autocleavage and activation of caspase-8, which further activates effector caspases (e.g., caspase-3) that eventually drive apoptotic death (1, 2). Cellular FLICE-inhibitory protein (c-FLIP) is a truncated form of caspase-8 that lacks enzymatic activity. It can also be recruited to the DISC, but suppresses apoptosis by blocking the activation of caspase-8 through competing with caspase-8 for binding to FADD (3). It has been well documented that elevated c-FLIP expression protects cells from death receptor–mediated apoptosis, whereas downregulation of c-FLIP by chemicals or siRNA sensitizes cells to death receptor–mediated apoptosis (4). Therefore, c-FLIP acts as a key inhibitor of TRAIL/death receptor-induced apoptosis. c-FLIP has multiple isoforms; however, only 2 forms have been well characterized at the protein level in human cells: short form (FLIPS) and long form (FLIPL). Elevated levels of c-FLIP have been found in a number of different cancers and are often correlated with a poor prognosis in certain types of cancers (5). Both FLIPL and FLIPS are rapidly turned over proteins regulated by ubiquitination/proteasome-mediated degradation (6, 7). However, the mechanisms underlying c-FLIP degradation are largely unclear even though the E3 ubiquitin ligases, Itch and Cbl, have been suggested to be involved in c-FLIP degradation (8, 9).
The mTOR is a serine-threonine protein kinase related tightly to the family of the phosphatidylinositol 3-kinase-related kinases. mTOR exerts a variety of different biologic functions primarily through forming 2 complexes characterized by the essential partner proteins raptor (mTOR complex 1; mTORC1) and rictor (mTOR complex 2; mTORC2; refs. 10, 11). mTORC1 is deeply involved in many cellular processes critical for the maintenance of cell metabolism and growth generally via regulating cap-dependent protein translation initiation; this involves phosphorylation of 2 key initiation factors S6K and 4E-BP1 followed by facilitating formation of the translation initiation complex (10). Although the mTORC1 is sensitive to rapamycin, the mTORC2 is generally thought to be rapamycin insensitive (12). Moreover, relatively little is known about the biologic functions of the mTORC2 other than its regulation of cytoskeleton and Akt-mediated cell survival (11). Several recent studies have suggested that mTORC2 exerts oncogenic functions in cancer development (13–15). Nonetheless, mTORC signaling is dysregulated in various types of human cancers and hence has emerged as an attractive cancer therapeutic target (16).
Rapamycin and its analogues (rapalogs) are conventional mTOR allosteric inhibitors with particular specificity to the mTORC1. Some rapalogs have shown encouraging results in improving overall survival among patients with metastatic renal cell carcinoma (17, 18) or advanced pancreatic neuroendocrine tumors (19). Consequently, these agents have been approved for clinical treatment of these indications. Despite this, the single agent activity of rapalogs in most other tumor types has been modest at best (20), likely due to the inaccessibility of the mTORC2 and their ability to activate AKT, MEK/ERK, and other survival pathways (16).
The discovery of mTORC2 as an Akt S473 kinase has spurred efforts to identify novel mTOR inhibitors that inhibit both mTORC1 and mTORC2 activity. As a result, several ATP-competitive inhibitors of mTOR kinase including PP242 and INK128 have been developed and tested in clinical trials (21, 22). These inhibitors have been shown to more dramatically inhibit protein synthesis, suppress Akt phosphorylation and induce G1 arrest and/or apoptosis in some cancer cells than rapamycin (23–26). A robust in vivo anticancer activity of these inhibitors against certain types of cancers was also observed (24, 27, 28). Therefore, these mTOR kinase inhibitors not only represent novel potential therapeutic agents, but are also valuable research tools for understanding the biology of mTORCs.
A previous study showed that rapamcyin sensitizes gliolastoma cells to TRAIL-induced apoptosis (29). However, others and we failed to show that rapalogs or mTOR knockdown can sensitize cancer cells including glioblastoma cells to TRAIL (30, 31). This study focuses on determining whether mTOR kinase inhibitors enhance TRAIL-induced apoptosis and if so, defining the underlying mechanisms.
Materials and Methods
Reagents and antibodies
PP242 and INK128 were purchased from Active Biochem. Rapamycin was purchased from LC Laboratories. BEZ235 was provided by Novartis Pharmaceuticals Corporation. The soluble recombinant human TRAIL was purchased from PeproTech, Inc.. The proteasome inhibitor MG132 and the protein synthesis inhibitor cycloheximide (CHX) were purchased from Sigma Chemical Co.. Monoclonal anti-FLIP antibody (NF6) was obtained Alexis Biochemicals. Mouse monoclonal caspase-8, survivin and polyclonal caspase-9, PARP, p-Akt (S473), p-Akt (T308), Akt, p-GSK3α/β (S21/9), p-S6 (S235/236), S6, p-PRAS40 (T246), and PRAS40 antibodies were purchased from Cell Signaling Technology, Inc. p-FOXO3a (T32) and GSK3α/β antibodies were purchased from Upstate/EMD Millipore. Mouse monoclonal caspase-3 antibody was purchased from Imgenex. Rabbit polyclonal DR5 antibody was obtained from ProSci Inc. Mouse monoclonal DR4 antibody (B-N28) was purchased from Diaclone. Polyclonal rictor and raptor antibodies were purchased from Bethyl Laboratories, Inc. Both polyclonal and monoclonal actin antibodies were purchased from Sigma Chemical Co.
Cell lines and cell culture
Human non–small cell lung carcinoma (NSCLC) cell lines used in this study were described in our previous work (32). Except for H157 and A549 cells, which were recently authenticated by Genetica DNA Laboratories, Inc. through analyzing short tandem repeat DNA profile, other cell lines have not been authenticated. The stable cell lines, H157-Lac Z-5 versus H157-FLIPS-1 and H157-Lac Z versus H157-survivin, were described previously (33). H157-scramble, H157-shRaptor, and H157-shRictor stable lines were described in our previous study (34). A549 stable lines with pLKO.1 (empty vector control), raptor short hairpin RNA (shRNA; shRaptor) or rictor shRNA (shRictor) were established as described previously (34). These cell lines were cultured in RPMI-1640 medium containing 5% FBS at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Cell survival and apoptosis assays
Cells were seeded in 96-well cell culture plates and treated the next day with the given agents. The viable cell number was determined using sulforhodamine B (SRB) assay as described previously (35). Combination index (CI) for drug interaction (e.g., synergy) was calculated using the CompuSyn software (ComboSyn, Inc.). Apoptosis was evaluated with Annexin V-PE Apoptosis Detection Kit purchased from BD Biosciences. The percentage of positive cells in the upper right and lower right quadrants represent the total apoptotic cell population. We also detected caspases and PARP cleavage by Western blot analysis as described below as additional indicators of apoptosis.
Western blot analysis
Preparation of whole-cell protein lysates and performance of the Western blot analysis were the same as described previously (32).
Immunoprecipitation for detection of ubiquitinated FLIPS
The given cells were cotransfected with HA-ubiquitin plus Flag-FLIPS plasmids using Lipofectamine 2000 transfection reagent (Invitrogen) based on the manufacturer's instructions. After 24 hours, the cells were treated with PP242, PP242 plus MG132 or MG132 alone for 4 hours. Cells were collected and lysed for immunoprecipitation (IP) using Flag M2 monoclonal antibody (Sigma) as previously described (33), followed by detection of ubiquitinated FLIPS with Western blot analysis using anti-HA antibody (Abgent).
Gene knockdown by siRNA or shRNA
Rictor #1 (5′-AAGCAGCCTTGAACTGTTTAA-3′), rictor #2 (5′-AAACTTGTGAAGAATCGTATC-3′), raptor #1 (5′-AAGGCTAGTCTGTTTCGAAAT-3′), raptor #2 (5′-AAGGACAACGGCCACAAGTAC-3′), and Cbl (5′-AACCTCTCTTCCAAGCACTGA-3′; ref. 36) siRNAs were synthesized by Qiagen. mTOR siRNA was purchased from Cell Signaling Technology, Inc. (Cat. # 6381). Transfection of these siRNA duplexes was conducted in 6-well plates using the HiPerFect transfection reagent (Qiagen) following the manufacturer's manual. The lentiviral Cbl shRNA set (Cat. # RHS4533), containing 7 different clones, was purchased from Thermo Scientific/Open Biosystems. Viral preparation, cell infection and subsequent cell selection with an antibiotic were carried out following the manufacturer's instructions and our previous description (34).
Detection of rictor and mTOR interaction
The given cells were transfected with myc-rictor expression plasmid (Addgene) and then lysed for detection of rictor and mTOR interaction with IP as described previously (34).
Adenoviral infection of cancer cells
Adenovirus harboring an empty vector (Ad-CMV) or a constitutively activated form of Akt (myristoylated Akt; Ad-myr-Akt) and cell infection were described previously (37).
PP242 cooperates with TRAIL to enhance apoptosis
We first determined whether PP242 has the ability to enhance TRAIL-induced apoptosis. As presented in Fig. 1A, the combination of PP242 and TRAIL was more effective than either agent alone in decreasing the survival of the tested NSCLC cell lines including H157, H226, and H358 cells. The CIs for these combinations were <1 (Supplementary Fig. S1), indicating that the combination of PP242 and TRAIL synergistically decreases the survival of NSCLC cells. In agreement, the combination of PP242 (e.g., 1 μmol/L) and TRAIL (e.g., 25 ng/mL) was also much more potent than either agent alone in increasing cleavage of caspase-8, caspase-9, caspase-3, and PARP in Western blot analysis (Fig. 1B) and in increasing the number of Annexin V-positive cells as shown by Annexin V assay (Fig. 1C) in 2 representative cell lines, H157 and H226. Taking the H226 cell line as an example, we detected approximately 16% and 3% of apoptotic cells in cells treated with TRAIL and PP242, respectively, but 46% of apoptotic cells after exposure to the combination of PP242 and TRAIL (Fig. 1C), which is greater than the sum of apoptosis induced by both single agents, further indicating that the combination of PP242 and TRAIL exerts synergistic apoptosis-inducing activity. Taken together, we conclude that the combination of PP242 and TRAIL synergistically induces apoptosis in NSCLC cells.
PP242 reduces the levels of FLIPS and survivin in NSCLC cells
To reveal the mechanism by which PP242 enhances TRAIL-initiated apoptosis, we analyzed alterations of several key proteins including c-FLIP, DR4, and DR5 in the TRAIL/death receptor-mediated apoptotic pathway in cells exposed to PP242. We also looked at the levels of survivin in cells exposed to PP242, because this is an mTOR-regulated protein (38) and is involved in regulation of TRAIL-induced apoptosis (39). Within the indicated concentration range (0.25–4 μmol/L), PP242 exerted a dose-dependent effect on reducing the levels of FLIPS and survivin, but not FLIPL (Fig. 2A and B). Even at 0.25 μmol/L, PP242 effectively reduced the levels of FLIPS in H226 cells. Time course analysis showed that both FLIPS and survivin reduction occurred at about 4 hours and was sustained up to 24 hours in H157 cells post PP242 treatment (Fig. 2C). PP242 did not increase DR5 expression in either of the tested cell lines. We noted that it increased DR4 expression in H157 cells, but not in H226 cells, suggesting a cell line-dependent effect on DR4 expression.
Enforced expression of ectopic FLIPs, but not survivin, abrogates synergistic induction of apoptosis by the PP242 and TRAIL combination
To explore the roles of FLIPS and survivin downregulation in the augmentation of apoptosis by PP242 plus TRAIL, we compared the effects of PP242 plus TRAIL on apoptosis induction in H157 cell lines that express ectopic Lac Z (as a control), FLIPS, or survivin. The combination of PP242 and TRAIL, as shown above, exerted augmented effects on induction of apoptosis and cleavage of caspase-8, caspase-3, and PARP in comparison with either single agent in H157-Lac Z control cells, but not in H157-FLIPS cells (Fig. 3A and B). In contrast, H157-Lac Z and H157-survivin cell lines were equally sensitive to PP242 plus TRAIL treatment evaluated both for caspase cleavage and for apoptosis induction (Fig. 3C and D). These data thus clearly indicate that downregulation of FLIPS, but not survivin, accounts for the enhancement of TRAIL-induced apoptosis by PP242.
Inhibition of mTORC2, but not mTORC1, decreases FLIPS expression
Given the important role of FLIPS downregulation in mediating the synergistic induction of apoptosis by PP242 and TRAIL, we focused our following studies on unraveling the mechanism by which PP242 reduces FLIPS levels. We asked whether PP242-mediated FLIPS downregulation is a consequence of mTORC inhibition. To this end, we evaluated the effects of PP242 on suppression of mTORC signaling pathways including Akt signaling. Two well-known events, phosphorylation of S6 and Akt, downstream of the mTORC1 and mTORC2, respectively, were effectively inhibited in cells exposed to PP242 in both dose- and time-dependent manners (Fig. 4A and Supplementary Fig. S2A), indicating that PP242 indeed effectively inhibits both mTORC1 and mTORC2 signaling. Considering recent finding on dual effects of mTOR kinase inhibitors on Akt (40), we further looked at effects of PP242 on Akt signaling in our cell systems or conditions. After treatment with PP242 for 16 hours, the levels of p-Akt (T308), p-GSK3 and p-FOXO3a were increased in H157 cells in a concentration-dependent manner accompanied with reduction of p-PRAS40 levels. In contrast, the levels of p-GSK3, p-FOXO3a and p-PRAS40 were concentration-dependently decreased in H226 cells (Fig. S2B). p-Akt (T308) levels in H226 cells were too low to be detected. Hence, PP242 exerts opposite effects on modulating the Akt signaling in these 2 cell lines.
Furthermore, we mimicked inhibition of mTORC signaling with mTOR siRNA, which is assumed to inhibit both mTORC1 and mTORC2, and then analyzed its effect on c-FLIP expression. Silencing of mTOR in both H157 and H226 cells reduced the levels of FLIPS as well as survivin (Fig. 4B), confirming that mTORC inhibition indeed causes the reduction of FLIPS and survivin. We noted that knockdown of mTOR also reduced FLIPL levels in H226 cells, but not in H157 cells (Fig. 4B).
We next addressed which of the mTORCs is involved in regulation of FLIPS levels. We knocked down rictor and raptor expression separately to mimic inhibition of the mTORC1 and mTORC2 and then examined their impact on FLIPS abundance. Our results showed that knockdown of rictor, but not raptor, reduced the levels of FLIPS in both H157 and H226 cell lines (Fig. 4C and D). Survivin levels were not reduced in either of the rictor siRNA-transfected cell lines (Fig. 4D). We also examined c-FLIP and survivin levels in our established A549-shRaptor and A549-shRictor stable cell lines and found that FLIPS levels were reduced in A549-shRictor, but not in A549-shRaptor cells. In contrast, survivin was reduced in A549-shRaptor cells, but not in A549-shRictor cells (Supplementary Fig. S3). In cells exposed to rapamycin, which is more selective for mTORC1 inhibition, we detected a dose-dependent reduction of survivin, but not of c-FLIP at all (Fig. 4E). Moreover, 2 additional mTOR kinase inhibitors, INK128 and BEZ235, also reduced FLIPs levels (Supplementary Fig. S4A and B) and synergized with TRAIL to kill cancer cells (Supplementary Fig. S4C and D). These data together clearly indicate that inhibition of the mTORC2 downregulates FLIPS levels, whereas inhibition of the mTORC1 causes survivin reduction. Accordingly, we suggest that mTORC2 is involved in the positive regulation of FLIPS levels.
Knockdown of rictor, but not raptor, sensitizes NSCLC cells to TRAIL-induced apoptosis
If knockdown of rictor downregulates c-FLIP expression, we reasonably speculated that it will also enhance TRAIL-induced apoptosis. Thus, we compared the impact of raptor and rictor knockdown on cell responses to TRAIL. As presented in Fig. 5A, H157-shRictor cells, in which rictor expression is stably knocked down, were much more sensitive to TRAIL than H157-scramble control cells and H157-shRaptor cells, in which raptor expression is stably silenced, as determined by measuring cell number changes (Fig. 5A). Similarly both H157 and H226 cell lines transfected with rictor siRNA were much more sensitive than those transfected with either control or raptor siRNA to TRAIL (Supplementary Fig. S5A and B). The Annexin V assay revealed that H157-shRictor cells exhibited higher sensitivity than both H157-scramble and H157-shRaptor cells to TRAIL, evidenced by 46% Annexin V-positive or apoptotic cells in H157-shRictor cells compared with 17% and 22% apoptotic cells in H157-scramble and H157-shRaptor cells, respectively (Fig. 5B). Consistently, TRAIL induced stronger cleavage of caspases (including caspase-8, caspase-9, and caspase-3) and PARP in H157-shRictor than in H157-scramble and H157-shRaptor cells although the cleavage of these proteins was slightly higher in H157-shRaptor than H157-scramble cells (Fig. 5C). Similar results were also generated in H226 cells transiently transfected with control, rictor or raptor siRNA. The strongest cleavage of caspase-8, caspase-3, and PARP was detected in rictor siRNA-transfected cells in comparison with those transfected with control or raptor siRNA (Supplementary Fig. S5C). Taken together, these data convincingly show that inhibition of rictor or mTORC2 sensitizes cancer cells to TRAIL-induced apoptosis.
PP242 and rictor knockdown facilitate FLIPS degradation, whereas enforced rictor expression stabilizes FLIPS
We explored whether a proteasome degradation-mediated mechanism is involved in the reduction of FLIPS levels by PP242 and rictor knockdown, because c-FLIP is known to be regulated by an ubiquitin/proteasome-dependent mechanism (6, 7). We first determined whether PP242 alters FLIPS stability. To this end, CHX was added to cells 16 hours after DMSO or PP242 treatment. The cells were then harvested at the indicated time post CHX for analysis of FLIPS degradation rates. Data in Fig. 6A revealed that the half-life of FLIPS in DMSO-treated cells was about 50 minutes; in contrast, its half-life in PP242-treated samples was <20 minutes. Therefore, it is apparent that PP242 reduces FLIPS protein stability. Next, we determined whether PP242 induces FLIPS degradation. Thus, we treated H157 and H1299 cells with PP242 in the absence and presence of the proteasome inhibitor MG132 followed by detection of FLIPS with Western blot analysis. In the absence of MG132, PP242 decreased FLIPS levels as we showed above. However, the presence of MG132 increased basal levels of FLIPS and prevented FLIPS from reduction by PP242 (Fig. 6B). This result suggests that PP242 induces FLIPS reduction through a proteasome-dependent mechanism. Furthermore, we determined whether PP242 increases FLIPS ubiquitination. As presented in Fig. 6C, the highest level of ubiquitinated FLIPS was detected in cells treated with PP242 plus MG132 compared with PP242 or MG132 alone, indicating that PP242 increases FLIPS ubiquitination. Our aforementioned results have shown that the mTORC2 is involved in positively regulating FLIPS levels. Therefore, we wondered whether the mTORC2 does this by regulating FLIPS degradation. To test this hypothesis, we knocked down rictor in H226 cells and then conducted a CHX chase assay to analyze FLIPS protein stability. Our results showed that rictor knockdown (Fig. 6D) greatly decreased FLIPS stability, because the half-life of FLIPS was shortened from about 50 minutes in control siRNA-transfected H226 control cells to approximately 15 minutes in H226 cells transfected with rictor siRNA (Fig. 6E). Moreover, rictor knockdown also increased ubiquitination of FLIPS (Fig 6F). Complementarily, co-expression of FLIPS and rictor resulted in elevated levels of FLIPS (Fig. 6G). In agreement, enforced expression of rictor substantially slowed down FLIPS degradation rate (Fig. 6H), indicating that rictor expression stabilizes FLIPS protein. Under the tested condition, expression of the ectopic rictor interacted with endogenous mTOR because endogenous mTOR could be pulled down with anti-myc (rictor) antibody in the IP experiment and increased p-Akt (S473) levels (Supplementary Fig. S6A), indicating that it does form and activate mTORC2. Besides, enforced expression of ectopic rictor protected cells from TRAIL-induced apoptosis because we detected less amounts of cleaved PARP and caspase-3 in cells transfected with rictor than in cells transfected with vector control (Supplementary Fig. S6B). Collectively, these data robustly indicate that mTORC2 indeed negatively regulates FLIPS stability. Hence we concluded that PP242 facilitates ubiquitin/proteasome-mediated FLIPS degradation through inhibition of the mTORC2, leading to downregulation of FLIPS.
Cbl is involved in mTORC2-mediated regulation of FLIPS degradation
Given that Cbl has been suggested to mediate FLIPS degradation (9), we then asked whether Cbl is involved in mediating FLIPS degradation induced by mTORC2 inhibition. Knockdown of Cbl with Cbl siRNA elevated basal levels of FLIPS and also prevented PP242-induced FLIPS reduction (Fig. 7A). A similar result was also generated in H1299 cells in which Cbl expression was stably silenced with 2 different Cbl shRNAs (Fig. 7B). Silencing of rictor decreased FLIPS levels in control siRNA-transfected H157 and H226 cells, but failed to do so in Cbl siRNA-transfected cells (Fig. 7C). These data together clearly indicate that Cbl is the E3 ubiquitin ligase that mediates FLIPS degradation induced by mTORC2 inhibition.
Expression of an active form of Akt does not impair the ability of PP242 to reduce FLIPS levels
Considering that Akt is a major substrate of mTORC2 and has been suggested to regulate c-FLIP expression (41–43), we further asked whether mTORC2 inhibition-induced FLIPS degradation is secondary to Akt inhibition. Thus, we expressed a constitutively active form of Akt (i.e., myr-Akt) through adenoviral infection and then examined its impact on PP2A-induced FLIPS reduction. As presented in Supplementary Fig. S7, expression of myr-Akt substantially increased the levels of p-GKS3, a well-known substrate of Akt, thus confirming Akt activation. However, PP242 was equally effective in reducing FLIPS levels in cells infected with either Ad-CMV or Ad-myr-Akt, indicating that enforced activation of Akt does not impair the ability of PP242 to induce FLIPS degradation.
In this study, we have shown that the newly developed mTOR kinase inhibitor, PP242, can augment TRAIL-induced apoptosis in NSCLC cells (Fig. 1). Although rapamcyin was previously shown to sensitize gliolastoma cells to TRAIL-induced apoptosis (29), we and others failed to show that rapalogs or mTOR knockdown could sensitize cancer cells, including glioblastoma cells, to TRAIL (30, 31). In the current study, knockdown of rictor rather than raptor sensitized cancer cells to TRAIL-induced apoptosis (Fig. 5 and Supplementary Fig. S5). Thus it is very likely that inhibition of mTORC2 signaling results in sensitization of TRAIL-induced apoptosis. To the best of our knowledge, this is the first report of the cooperative induction of apoptosis between an mTOR kinase inhibitor (e.g., PP242) or mTORC2 inhibition and TRAIL. Given that TRAIL is being tested as a cancer therapeutic agent in clinical trials (1), further study of the potential application of the PP242 and TRAIL combination in cancer therapy (e.g., NSCLC) is warranted.
DR4, DR5 and c-FLIP are key components in the regulation of TRAIL-induced apoptosis. DR4 and DR5 are receptors for TRAIL that initiate apoptosis upon binding with TRAIL and c-FLIP is the major inhibitor that suppresses TRAIL/death receptor-induced apoptosis (2). Modulation of the levels of these proteins (e.g., upregulation of DR4 and/or DR5 and/or downregulation of c-FLIP) generally results in sensitization of cancer cells to TRAIL-induced apoptosis (44). Survivin is a major inhibitor of the intrinsic apoptotic pathway and has also been suggested to regulate TRAIL-induced apoptosis (45). In this study, PP242 did not apparently increase DR5 expression, but reduced the levels of FLIPS and survivin in 2 tested NSCLC cell lines. However, enforced expression of ectopic FLIPS, but not survivin, conferred resistance of NSCLC cells to the combination of PP242 and TRAIL as evaluated with both the Annexin V assay and caspase cleavage (Fig. 3). These results suggest that downregulation of FLIPS, but not survivin, plays a critical role in mediating synergistic induction of apoptosis by PP242 and TRAIL. We also noted that DR4 expression was increased in H157 cells but not in H226 cells upon PP242 treatment, suggesting a cell line-dependent modulation. Whether DR4 upregulation also contributes to cooperative induction of apoptosis by the PP242 and TRAIL combination in some cell lines (e.g., H157) needs further investigation.
As an mTOR kinase inhibitor, PP242 efficiently inhibited both mTORC1 and mTORC2 signaling in our cell systems, evidenced by effective suppression of the phosphorylation of both S6 and Akt (Fig. 4), 2 well-known downstream markers of the mTORC1 and mTORC2. A previous study using glioblastoma cells suggested that mTORC1 signaling (i.e., mTOR/S6K signaling) positively regulates FLIPS translation and that inhibition of this pathway downregulates FLIPS through suppressing its translation and enhances TRAIL-induced apoptosis (29). In our study, mTOR knockdown reduced the levels of both FLIPS and survivin. However, we detected reduced levels of survivin, but not c-FLIP, in our cell systems exposed to rapamycin (Fig. 4E). Moreover, disruption of the mTORC2 (by rictor knockdown), but not the mTORC1 (by raptor knockdown), reduced FLIPS levels (Fig. 4 and Supplementary Fig. S3) although knockdown of raptor decreased survivin levels (Supplementary Fig. S3). Consistently, 2 other mTOR kinase inhibitors, INK128 and BEZ237, also reduced FLIPS levels effectively (Supplementary Fig. S4). Similar to our observations using PP242, knockdown of rictor, but not raptor, augmented TRAIL-induced apoptosis (Fig. 5 and Supplementary Fig. S5). These findings together clearly indicate that it is inhibition of the mTORC2 that positively regulates FLIPS levels, leading to enhancement of TRAIL-induced apoptosis. Our data also suggest that the mTORC1 positively regulates survivin expression, supporting the previous notion that mTORC1 signaling positively regulates survivin translation (38).
Our current results are consistent with our previous findings that inhibition of cap-dependent translation (e.g., with eIF4E siRNA or rapamycin) failed to decrease c-FLIP levels and augment TRAIL-induced apoptosis in other NSCLC cells (31). In this study, we found that knockdown of rictor did not decrease survivin levels although mTOR or raptor knockdown did reduce its expression (Fig. 4 and Supplementary Fig. S3). Thus, it is likely that the mTORC2-mediated regulation of c-FLIP may involve a novel mechanism independent of cap-dependent translation. We explored what this mechanism might be. It is known that c-FLIP including FLIPL and FLIPS are rapidly turned over proteins subjected to regulation through ubiquitin/proteasome-mediated protein degradation (6–8). Some small molecules negatively regulate c-FLIP levels through this mechanism as we showed previously (33, 46). In this study, we found that PP242 failed to decrease FLIPS levels in the presence of a proteasome inhibitor, increased c-FLIP ubiquitination and reduced the stability of FLIPS protein (Fig. 6). All of these results indicate that PP242 reduces FLIPS levels by facilitating its degradation through the ubiquitin/proteasome-dependent pathway. In agreement, knockdown of rictor substantially decreased FLIPS stability and increased FLIPS ubiquitination, whereas expression of ectopic rictor elevates FLIPS levels by enhancing its stability (Fig. 6). These data clearly indicate that modulation of the mTORC2 activity through genetic means also alters FLIPS levels by regulating its stability. Collectively, we suggest that mTORC2 signaling positively regulates FLIPS levels through negatively controlling its degradation. To the best of our knowledge, this is the first study suggesting mTORC2 regulation of c-FLIP degradation.
Currently, mechanisms underlying c-FLIP degradation have not been fully elucidated. Cbl is a pro-oncogene with E3 ubiquitin ligase activity and has been suggested to be involved in the development of TRAIL resistance in cancer cells through enhancing death receptor degradation and activating Akt (47–49). In our study, we found that inhibition of Cbl by knocking down its expression not only elevated basal levels of FLIPS, but also prevented FLIPS from reduction induced by either PP242 or rictor knockdown (Fig. 7). Thus, Cbl is clearly important for mTORC2 inhibition-induced FLIPS degradation. In other words, mTORC2 negatively controls or inhibits Cbl-dependent FLIPS degradation. Our ongoing studies seek to understand how the mTORC2 exerts this effect.
Akt is known to be a major substrate of mTORC2. Some studies have suggested that Akt is involved in positive regulation of c-FLIP expression, likely at transcriptional levels (41–43). However, Akt has also been shown to physically interact with and phosphorylate FLIPL, leading to its degradation (50). We recently have shown that the Akt inhibitor API-1 induces c-FLIP degradation independent of Akt inhibition (51). Moreover, celecoxib can induce c-FLIP degradation while increasing Akt phosphorylation (46). Nonetheless, the role of Akt in positive regulation of c-FLIP, particularly FLIPS degradation has not been clearly established. In our experimental conditions, PP242 activated Akt signaling in H157 cells whereas suppressing the Akt signaling in H226 cells evidenced by the findings that PP242 increased the levels of p-Akt (308), p-GSK3 and p-FOXO3a in H157 cells, but decreased them in H226 cells (Supplementary Fig. S2B). Regardless, PP242 decreased FLIPS levels in both cell lines (Fig. 2). Moreover, enforced activation of Akt (e.g., expression of myr-Akt) did not impair the ability of PP242 to decrease FLIPS levels (Supplementary Fig. S7). Hence we suggest that PP242- or mTORC2 inhibition-induced FLIPS degradation is unlikely to be the consequence of Akt inhibition or activation.
In summary, the current work has revealed a novel biologic function of the mTORC2, i.e., regulation of Cbl-dependent FLIPS degradation and TRAIL-induced apoptosis, hence providing the first evidence connecting mTORC2 signaling to the regulation of death receptor-mediated apoptosis. Our findings also highlight a novel strategy to use mTOR kinase inhibitors in combination with TRAIL for cancer therapy. Thus, further investigation in this direction is warranted.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: L. Zhao, S.-Y. Sun
Development of methodology: L. Zhao
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Zhao
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Zhao, S.-Y. Sun
Writing, review, and/or revision of the manuscript: L. Zhao, F. R. Khuri, S.-Y. Sun
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Zhao, P. Yue
Study supervision: S.-Y. Sun
We thank Dr. A. Hammond in our department for editing the manuscript.
Georgia Cancer Coalition Distinguished Cancer Scholar award and NIH R01 CA118450 (S-Y Sun), R01 CA160522 (S-Y Sun) and P01 CA116676 (Project 1 to FR Khuri and S-Y Sun).
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.