The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation. Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma. Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy. Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent. We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis. Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts. These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.
Penta-O-galloyl-β-D-glucose (PGG) suppresses the in vivo growth of human DU145 and PC-3 prostate cancer (PCa) xenografts in nude mice, suggesting potential utility as a PCa chemotherapeutic or chemopreventive agent. Our earlier work implicates caspase-mediated apoptosis in DU145 and LNCaP PCa cells as one mechanism for the anti-cancer activity. We show here that in the more aggressive PC-3 PCa cell line, PGG induced programmed cell deaths (PCD) lacking the typical caspase-mediated apoptotic morphology and biochemical changes. In contrast, PGG induced patent features of autophagy, including formation of autophagosomes and lipid modification of LC-3 after 48 h of PGG exposure. The autophagic responses were also observed in the murine TRAMP-C2 cells. Caspase inhibition exacerbated PGG-induced overall death. As for molecular changes, we observed a rapid inhibition of the phosphorylation of mTOR-downstream targets S6K and 4EBP1 by PGG in PC-3 and TRAMP C2 cells, but not that of mTOR itself, along with increased AKT phosphorylation. Whereas inhibition of PI3K increased both PGG-induced apoptosis and autophagy, experiments with pharmacologic inducer or inhibitor of autophagy or by knocking down autophagy mediator Beclin-1 showed that autophagy provided survival signaling that suppressed caspase-mediated apoptosis. Knocking down of RIP-1 kinase increased overall death, without changing LC-3 II or caspase activation, thus not supporting RIP1-necroptosis for PGG-induction of autophagy or other PCD. Furthermore, PGG-treated PC-3 cells lost clonogenic ability. The induction by PGG of caspase-independent PCD in aggressive PCa cell lines supports testing its merit as a potential drug candidate for therapy of caspase-resistant recurrent PCa.
autophagy; programmed cell death; polyphenols
The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.
Growth factor pathways are required for normal development but are often inappropriately activated in many cancers. One growth-factor–sensitive pathway of increasing interest to cancer researchers relies on the mammalian target of rapamycin (mTOR), a kinase that (like all kinases) delivers phosphate groups from ATP to amino acid residues of downstream proteins. TOR proteins were first discovered in yeast as the cellular targets of rapamycin, a small, naturally occurring molecule derived from bacteria that is widely used as an immunosuppressant and more recently in some cancer therapies. The study of TOR proteins has relied heavily on the use of rapamycin, but rapamycin does not directly inhibit TOR kinase activity; rather, rapamycin influences TOR's enzymatic activities by binding to a domain far from the kinase's active site. Some mTOR functions are resistant to rapamycin, as a result of the kinase activity of one kind of multiprotein complex, the mTOR complex 2 (mTORC2), whereas rapamycin-sensitive functions of mTOR are due to the mTOR complex 1 (mTORC1). We have developed new inhibitors of mTOR that bind to the ATP-binding site of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2 without inhibiting other kinases. Unexpectedly, these inhibitors had profound effects on protein synthesis and cell proliferation due to their inhibition of mTORC1 rather than mTORC2. We found that the phosphorylation of a protein that controls protein synthesis, the mTORC1 substrate 4E binding protein (4EBP) is partially resistant to rapamycin but fully inhibited by our new inhibitors. The finding that 4EBP phosphorylation is resistant to rapamycin suggests that active-site inhibitors may be more effective than rapamycin in the treatment of cancer and may explain why rapamycin is so well tolerated when taken for immunosuppression.
Cells rely on the mammalian target of rapamycin kinase (mTOR) to sense growth factors. Inhibition of all forms of mTOR using newly developed inhibitors of its active site reveals new insights into the function of two mTOR-containing protein complexes and their potential as therapeutic targets.
Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1–RIPK3–pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3β, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1–RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1–RIPK3–pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.
necroptosis; neuron; Akt; mTOR; RIPK1; RIPK3
Signaling through phosphatidylinositol 3-kinase (PtdIns3K)-Akt-mTOR is frequently activated in cancers including glioblastoma multiforme (GBM), where this kinase network regulates survival. It is thus surprising that inhibitors of these pathways induce minimal cell death in glioma. We showed that the dual PtdIns3K-mTOR inhibitor PI-103 induces autophagy in therapy-resistant, PTEN-mutant glioma, with blockade of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) contributing independently to autophagy. Inhibition of autophagosome maturation synergizes with PI-103 to induce apoptosis through the Bax-dependent intrinsic mitochondrial pathway, indicating that PI-103 induces autophagy as a survival pathway in this setting. Not all inhibitors of PtdIns3K-Akt-mTOR signaling synergize with inhibitors of autophagy. The allosteric mTORC1 inhibitor rapamycin fails to induce apoptosis in conjunction with blockade of autophagy, due to feedback-activation of Akt. Apoptosis in the setting of rapamycin therapy requires concurrent inhibition of both autophagy and of PtdIns3K-Akt. Moreover, the clinical PtdIns3K-mTOR inhibitor NVP-BEZ235 cooperates with the clinical lysosomotropic autophagy inhibitor chloroquine to induce apoptosis in PTEN-mutant glioma xenografts in vivo, offering a therapeutic approach translatable to patients.
glioma; signal transduction; PtdIns3K; kinase inhibitors; apoptosis; autophagy; combination therapy
Liver cancer is one of the most frequent cancers in the world. Targeted therapy of cancer with specific inhibitors is developing and has shown promising antitumor efficacy. CCI-779 (temsirolimus), a specific inhibitor of mTOR (mammalian target of rapamycin), can block the mTOR signaling pathway. Here, we systematically examined the expression of mTOR and its downstream targets in liver cancer cells and normal liver cells, then investigated inhibitory effects of CCI-779 on mTOR signaling pathway and its role in regulating liver cancer cell growth.
The expression of mTOR and its downstream targets in Bel-7402 liver cancer cells and HL-7702 normal liver cells were examined by western blot. The mTOR specific inhibitor (CCI-779) was used to treat Bel-7402 cells to identify its effects on Bel-7402 cell growth and activity of mTOR signaling pathway in vitro. Cell viability tests were performed after the treatment of CCI-779. Western blot was applied to assess the changes of mTOR pathway and flow cytometry was used to analyze cell cycle of Bel-7402 cells after the treatment of CCI-779.
mTOR, p70S6K, S6, and 4EBP1 were overexpressed in Bel-7402 cells compared with HL-7702 cells. Bel-7402 cells were sensitive to CCI-779. The survival rate of the cells treated with CCI-779 over 0.312 μM was significantly different compared with that of control (P < 0.05). CCI-779 inhibited the phosphorylation of mTOR (Ser2448), p70S6K (Thr389), S6 (Ser240/244), and 4EBP1 (Thr37/46) in different grades and the expressions of p70S6K, S6, and 4EBP1. As a result, CCI-779 induced a dose-dependent decrease in cell proliferation, G1/S arrest and damage of cell shape.
Taken together, these data showed that CCI-779 can inhibit mTOR signaling and proliferation in Bel-7402 liver cancer cells in vitro. It offers a therapeutic intervention through inhibition of mTOR as a potential strategy for liver cancer.
CCI-779 (temsirolimus); mTOR signaling; Cell growth; Liver cancer cell
Triple-negative breast cancer (TNBC) is aggressive and lacks targeted therapies. Phosphatidylinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are frequently activated in TNBC patient tumors at the genome, gene expression and protein levels, and mTOR inhibitors have been shown to inhibit growth in TNBC cell lines. We describe a panel of patient-derived xenografts representing multiple TNBC subtypes and use them to test preclinical drug efficacy of two mTOR inhibitors, sirolimus (rapamycin) and temsirolimus (CCI-779).
We generated a panel of seven patient-derived orthotopic xenografts from six primary TNBC tumors and one metastasis. Patient tumors and corresponding xenografts were compared by histology, immunohistochemistry, array comparative genomic hybridization (aCGH) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) sequencing; TNBC subtypes were determined. Using a previously published logistic regression approach, we generated a rapamycin response signature from Connectivity Map gene expression data and used it to predict rapamycin sensitivity in 1,401 human breast cancers of different intrinsic subtypes, prompting in vivo testing of mTOR inhibitors and doxorubicin in our TNBC xenografts.
Patient-derived xenografts recapitulated histology, biomarker expression and global genomic features of patient tumors. Two primary tumors had PIK3CA coding mutations, and five of six primary tumors showed flanking intron single nucleotide polymorphisms (SNPs) with conservation of sequence variations between primary tumors and xenografts, even on subsequent xenograft passages. Gene expression profiling showed that our models represent at least four of six TNBC subtypes. The rapamycin response signature predicted sensitivity for 94% of basal-like breast cancers in a large dataset. Drug testing of mTOR inhibitors in our xenografts showed 77 to 99% growth inhibition, significantly more than doxorubicin; protein phosphorylation studies indicated constitutive activation of the mTOR pathway that decreased with treatment. However, no tumor was completely eradicated.
A panel of patient-derived xenograft models covering a spectrum of TNBC subtypes was generated that histologically and genomically matched original patient tumors. Consistent with in silico predictions, mTOR inhibitor testing in our TNBC xenografts showed significant tumor growth inhibition in all, suggesting that mTOR inhibitors can be effective in TNBC, but will require use with additional therapies, warranting investigation of optimal drug combinations.
Autophagy has emerged as an important antimicrobial host defense mechanism that not only orchestrates the systemic immune response, but also functions in a cell autonomous manner to directly eliminate invading pathogens. Pathogenic bacteria such as Salmonella have evolved adaptations to protect themselves from autophagic elimination. Here we show that signaling through the non-receptor tyrosine kinase focal adhesion kinase (FAK) is actively manipulated by the Salmonella SPI-2 system in macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface of the Salmonella-containing vacuole (SCV), leading to amplified signaling through the Akt-mTOR axis and inhibition of the autophagic response. In FAK-deficient macrophages, Akt/mTOR signaling is attenuated and autophagic capture of intracellular bacteria is enhanced, resulting in reduced bacterial survival. We further demonstrate that enhanced autophagy in FAK−/− macrophages requires the activity of Atg5 and ULK1 in a process that is distinct from LC3-assisted phagocytosis (LAP). In vivo, selective knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral infection with S. typhimurium. Clearance was correlated with reduced infiltration of inflammatory cell types into infected tissues and reduced tissue damage. Together, these data demonstrate that FAK is specifically targeted by S. typhimurium as a novel means of suppressing autophagy in macrophages, thereby enhancing their intracellular survival.
Salmonella enterica is a food- and water-borne pathogen that has evolved closely with vertebrate hosts. Two medically relevant serovars include S. typhimurium, which causes gastroenteritis and S. typhi, which is the causative agent of typhoid fever. Host cells can utilize a process called autophagy, normally involved in the elimination of defective proteins and organelles, to capture and degrade intracellular pathogens. Enteric Salmonella express numerous virulence factors that enable the bacterium to subvert host defense mechanisms. Here we report that Salmonella specifically activates the host molecule focal adhesion kinase (FAK) in macrophages, triggering a signaling cascade that suppresses the autophagic elimination of intracellular bacteria. A key regulator of autophagy in mammalian cells is the target of rapamycin, mTOR, which transmits inhibitory signals that downregulate the autophagic response. We show that Salmonella-induced FAK activation leads to the Akt-dependent activation of mTOR, thereby repressing autophagic signaling. Inhibition of autophagy results in increased bacterial survival, while in FAK-deficient cells, autophagy is enhanced and intracellular Salmonella are eliminated. We also show that in mice lacking macrophage-specific FAK, animals were less susceptible to oral Salmonella infection. Together, these data identify FAK as a novel regulator of autophagy in macrophages with broad implications for host survival.
Although the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway promotes survival signaling, inhibitors of PI3K and mTOR induce minimal cell death in PTEN (phosphatase and tensin homolog deleted from chromosome 10 ) mutant glioma. Here, we show that the dual PI3K-mTOR inhibitor PI-103 induces autophagy in a form of glioma that is resistant to therapy. Inhibitors of autophagosome maturation cooperated with PI-103 to induce apoptosis through the mitochondrial pathway, indicating that the cellular self-digestion process of autophagy acted as a survival signal in this setting. Not all inhibitors of mTOR synergized with inhibitors of autophagy. Rapamycin delivered alone induced autophagy, yet cells survived inhibition of autophagosome maturation because of rapamycin-mediated activation of Akt. In contrast, adenosine 5′-triphosphate–competitive inhibitors of mTOR stimulated autophagy more potently than did rapamycin, with inhibition of mTOR complexes 1 and 2 contributing independently to induction of autophagy. We show that combined inhibition of PI3K and mTOR, which activates autophagy without activating Akt, cooperated with inhibition of autophagy to cause glioma cells to undergo apoptosis. Moreover, the PI3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, synergized with the lysosomotropic inhibitor of autophagy, chloroquine, another agent in clinical use, to induce apoptosis in glioma xenografts in vivo, providing a therapeutic approach potentially translatable to humans.
Targeting signaling pathways is an attractive approach in many malignancies. The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and/or disease free survival. mTOR kinase inhibitors have been introduced in the therapy of renal cell carcinoma and mantle cell lymphoma, and several trials are currently underway. However, the pathological characterization of mTOR activity in lymphomas is still incomplete.
mTOR activity and the elements of mTOR complexes were investigated by immunohistochemistry on tissue microarrays representing different human non-Hodgkin-lymphomas (81 cases) and Hodgkin-lymphomas (87 cases). The expression of phospho-mTOR, phospho-4EBP1, phospho-p70S6K, phospho-S6, Rictor, Raptor and Bcl-2, Bcl-xL, Survivin and NF-kappaB-p50 were evaluated, and mTOR activity was statistically analyzed along with 5-year survival data. The in vitro and in vivo effect of the mTOR inhibitor rapamycin was also examined in human Hodgkin-lymphoma cell lines.
The majority (>50%) of mantle cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma and Hodgkin-lymphoma cases showed higher mTOR activity compared to normal lymphoid tissues. Hodgkin-lymphoma was characterized by high mTOR activity in 93% of the cases, and Bcl-xL and NF-kappaB expression correlated with this mTOR activity. High mTOR activity was observed in the case of both favorable and unfavorable clinical response. Low mTOR activity was accompanied by complete remission and at least 5-year disease free survival in Hodgkin-lymphoma patients. However, statistical analysis did not identify correlation beetween mTOR activity and different clinical data of HL patients, such as survival. We also found that Rictor (mTORC2) was not overexpressed in Hodgkin-lymphoma biopsies and cell lines. Rapamycin inhibited proliferation and induced apoptosis in Hodgkin-lymphoma cells both in vitro and in vivo, moreover, it increased the apoptotic effect of chemotherapeutic agents.
Targeting mTOR activity may be a potential therapeutic tool in lymphomas. The presence of mTOR activity probably indicates that the inclusion of mTOR inhibition in the therapy of Hodgkin-lymphomas may be feasible and beneficial, especially when standard protocols are ineffective, and it may also allow dose reduction in order to decrease late treatment toxicity. Most likely, the combination of mTOR inhibitors with other agents will offer the highest efficiency for achieving the best clinical response.
mTOR activity; Hodgkin-lymphoma; Rapalogs; TMA; Hodgkin-lymphoma xenograft
The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors’ effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycin's ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.
The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway promotes the initiation of autophagy, and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) is well known to induce autophagy. Autophagy is a self-defense mechanism of cancer cells that are subjected to antitumor agents, and blocking autophagy can trigger apoptosis. In the present study, we demonstrate that an mTOR inhibitor, rapamycin, induces autophagy in the Nara-H malignant fibrous histiocytoma (MFH) cell line through the activation of ERK1/2. Rapamycin-induced apoptosis was enhanced following the inhibition of the MEK/ERK pathway. In the Nara-H cells, we examined the effects of rapamycin treatment on cell proliferation and on the phosphorylation of the mTOR pathway components and autophagy by western blot analysis. Furthermore, we examined the effects of rapamycin with or without the MEK inhibitor, U0126, on the induction of apoptosis by using fluorescence microscopy. Rapamycin inhibited Nara-H cell proliferation and decreased the phosphorylation of the mTOR pathway in the Nara-H cells. Rapamycin induced the apoptosis of Nara-H cells, and this apoptosis was enhanced by U0126. Simultaneously, phospho-ERK1/2 was activated by rapamycin. The present study demonstrates that rapamycin induces autophagy in Nara-H cells by activating the MEK/ERK signaling pathway, and the rapamycin-induced apoptosis can be enhanced by the MEK inhibitor, U0126. These results suggest that self-protective mechanisms involving mTOR inhibitors in Nara-H cells are prevented by the inhibition of the MEK/ERK pathway. The combination of an mTOR inhibitor (e.g., rapamycin) and an MEK inhibitor (e.g., U0126) may offer effective treatment for MFH, as this combination effectively activates apoptotic pathways.
autophagy; apoptosis; mammalian target of rapamycin; mitogen-activated protein kinase
Perifosine is an alkylphospholipid exhibiting antitumor activity as demonstrated in both preclinical studies and clinical trials. This activity is partly associated with its ability to inhibit Akt activity. It has been shown that the mTOR axis plays a critical role in regulation of cell proliferation and survival, primarily through functioning both downstream and upstream of Akt. The current study reveals a novel mechanism by which perifosine inhibits Akt and the mTOR axis. In addition to inhibition of Akt, perifosine inhibited the assembly of both mTOR/raptor and mTOR/rictor complexes. Strikingly, perifosine reduced the levels of Akt and other major components including mTOR, raptor, rictor, p70S6K, and 4E-BP-1 in the mTOR axis by promoting their degradation through a GSK3/FBXW7-dependent mechanism. These results thus suggest that perifosine inhibits the mTOR axis through a different mechanism from inhibition of mTOR signaling by classical mTOR inhibitors such as rapamycin. Moreover, perifosine substantially increased the levels of type II LC3, a hallmark of autophagy, in addition to increasing PARP cleavage, suggesting that perifosine induces both apoptosis and autophagy. The combination of perifosine with a lysosomal inhibitor enhanced apoptosis and inhibited the growth of xenografts in nude mice, suggesting that perifosine-induced autophagy protects cells from undergoing apoptosis. Collectively, we conclude that perifosine inhibits mTOR signaling and induces autophagy, highlighting a novel mechanism accounting for perifosine’s anticancer activity and a potential strategy to enhance perifosine’s anticancer efficacy by preventing autophagy.
Perifosine; Akt; mTOR; degradation; autophagy; lung cancer cells
Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.
mTOR pathway hyperactivation occurs in nearly 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacological strategy to overcome it.
We performed in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin resistant signaling and blocking GBM growth and a novel single cell technology, DNA Encoded Antibody Libraries, was used to identify mechanisms of resistance.
Here we demonstrate that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling; block mTORC2 signaling and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes GBM cells and orthotopic xenografts to CC214-1 and CC214-2 induced cell death.
These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in GBM and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. This study also demonstrates a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacological strategy for overcoming it.
The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase. It plays an evolutionarily conserved role in regulating cell growth, proliferation, survival, and metabolism via different cellular processes. The purpose of this study was to explore the inhibitory effects of CCI-779 (temsirolimus), a specific mTOR inhibitor, on mTOR signaling, and examine the mechanism of cell growth suppression by CCI-779 in Cashmere goat fetal fibroblasts (GFb cells). GFb cells were sensitive to CCI-779 and the survival rate of cells treated with >3.0 μM of CCI-779 was significantly reduced compared with the control (p<0.01). CCI-779 inhibited the phosphorylation of mTOR (at Ser2448) and S6 (at Ser240/244), and the expression of mTOR, p70S6K, and S6. Thus, CCI-779 was toxic to GFb cells, and it induced a dose-dependent decrease in cell proliferation and caused G1/S cell cycle arrest. Taken together, these data show that CCI-779 can inhibit mTOR signaling and proliferation in GFb cells in vitro. Therefore, mTOR is an important regulator for GFb cell growth and proliferation.
Metastatic colorectal cancer (CRC) is incurable for most patients. Since mammalian target of rapamycin (mTOR) has been suggested as a crucial modulator of tumor biology, we aimed at evaluating the effectiveness of mTOR targeting for CRC therapy. To this purpose, we analyzed mTOR expression and the effect of mTOR inhibition in cancer stem-like cells isolated from three human metastatic CRCs (CoCSCs).
CoCSCs exhibited a strong mTOR complex 2 (mTORC2) expression, and a rare expression of mTOR complex 1 (mTORC1). This latter correlated with differentiation, being expressed in CoCSC-derived xenografts. We indicate Serum/glucocorticoid-regulated kinase 1 (SGK1) as the possible main mTORC2 effector in CoCSCs, as highlighted by the negative effect on cancer properties following its knockdown. mTOR inhibitors affected CoCSCs differently, resulting in proliferation, autophagy as well as apoptosis induction. The apoptosis-inducing mTOR inhibitor Torin-1 hindered growth, motility, invasion, and survival of CoCSCs in vitro, and suppressed tumor growth in vivo with a concomitant reduction in vessel formation. Torin-1 also affected the expression of markers for cell proliferation, angio-/lympho-genesis, and stemness in vivo, including Ki67, DLL1, DLL4, Notch, Lgr5, and CD44. Importantly, Torin-1 did not affect the survival of normal colon stem cells in vivo, suggesting its selectivity towards cancer cells. Thus, we propose Torin-1 as a powerful drug candidate for metastatic CRC therapy.
colorectal cancer; cancer stem cells; mTOR; apoptosis
BACKGROUND & AIMS
Aspirin reduces the incidence of and mortality from colorectal cancer (CRC) by unknown mechanisms. Cancer cells have defects in signaling via the mechanistic target of rapamycin (mTOR), which regulates proliferation. We investigated whether aspirin affects adenosine monophosphate–activated protein kinase (AMPK) and mTOR signaling in CRC cells.
The effects of aspirin on mTOR signaling, the ribosomal protein S6, S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) were examined in CRC cells by immunoblotting. Phosphorylation of AMPK was measured; the effects of loss of AMPKα on the aspirin-induced effects of mTOR were determined using small interfering RNA (siRNA) in CRC cells and in AMPKα1/α2−/− mouse embryonic fibroblasts. LC3 and ULK1 were used as markers of autophagy. We analyzed rectal mucosa samples from patients given 600 mg aspirin, once daily for 1 week.
Aspirin reduced mTOR signaling in CRC cells by inhibiting the mTOR effectors S6K1 and 4E-BP1. Aspirin changed nucleotide ratios and activated AMPK in CRC cells. mTOR was still inhibited by aspirin in CRC cells after siRNA knockdown of AMPKα, indicating AMPK-dependent and AMPK-independent mechanisms of aspirin-induced inhibition of mTOR. Aspirin induced autophagy, a feature of mTOR inhibition. Aspirin and metformin (an activator of AMPK) increased inhibition of mTOR and Akt, as well as autophagy in CRC cells. Rectal mucosal samples from patients given aspirin had reduced phosphorylation of S6K1 and S6.
Aspirin is an inhibitor of mTOR and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism. These could contribute to its protective effects against development of CRC.
Chemoprevention; Colon Cancer; Oncogene; Tumor Suppressor
The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. Several mTOR inhibitors are currently being tested in cancer clinical trials. Both PI3K/Akt and MEK/ERK signaling regulate mTOR axis. However, inhibition of mTOR activates Akt survival signaling, which in turn attenuates mTOR inhibitors’ anticancer efficacy. We are interested in developing strategies for enhancing mTOR-targeted cancer therapy. In this study, we report that mTOR inhibition also induced activations of the MEK/ERK signaling pathway in some cancer cell lines after a prolonged treatment. The combination of rapamycin with the MEK inhibitor U0126 significantly enhanced growth inhibitory effects of cancer cells, suggesting that MEK/ERK activation may counteract mTOR inhibitors’ anticancer efficacy. Similarly, the combination of an mTOR inhibitor with the EGF receptor inhibitor erlotinib synergistically inhibited the growth of both human cancer cells in cell cultures and xenografts in nude mice. Moreover, the presence of erlotinib suppressed rapamycin-induced phosphorylation of Akt, ERK and eIF4E as well, implying that erlotinib can suppress mTOR inhibition-induced feedback activation of several survival signaling pathways including Akt, ERK and eIF4E. Thus, we suggest a therapeutic strategy for enhancing mTOR-targeted cancer therapy by preventing mTOR inhibition-induced feedback activation of several survival mechanisms.
mTOR inhibitors; erlotinib; survival signaling; Akt; ERK; eIF4E
Autophagy is a cellular response to intracellular pathogens including mycobacteria and is induced by the direct inhibitors of mammalian target of Rapamycin (mTOR), a major negative regulator of autophagy. Autophagy induction by mTOR inhibition (mTOR dependent autophagy), through chemical means or starvation, leads to mycobacterial killing in infected cells. However, previous work by our group has shown that mycobacterial infection of macrophages naturally induces both autophagy and mammalian target of Rapamycin (mTOR) activity (mTOR independent autophagy). In the current work, we further explore the relationship between mTOR activity and mycobacterial killing in macrophages.
While low concentrations of the mTOR inhibitors, Rapamycin, Torin 1, and Torin 2, can effectively reduce or block mTOR activity in response to lipopolysaccharides (LPS) or mycobacteria, higher concentrations (10 uM) are required to observe Mycobacterium smegmatis killing. The growth of M. smegmatis was also inhibited by high concentrations of Rapamycin in LC3B and ATG5 deficient bone marrow derived macrophages, suggesting that non-autophagic mechanisms might contribute to killing at high doses. Since mycobacterial killing could be observed only at fairly high concentrations of the mTOR inhibitors, exceeding doses necessary to inhibit mTOR, we hypothesized that high doses of Rapamycin, the most commonly utilized mTOR inhibitor for inducing autophagic killing, may exert a direct bactericidal effect on the mycobacteria. Although a short-term treatment of mycobacteria with Rapamycin did not substantially affect mycobacterial growth, a long-term exposure to Rapamycin could impact mycobacterial growth in vitro in select species.
This data, coupled with previous work from our laboratory, further indicates that autophagy induction by mTOR inhibition is an artificial means to increase mycobacterial killing and masks more relevant endogenous autophagic biochemistry that needs to be understood.
Autophagy; Mycobacteria; mTOR; Inhibitors; Bacille Calmette-Guérin (BCG); M. tuberculosis
A growing body of evidence suggests that microRNAs (miRNAs) play an important role in cancer diagnosis and therapy. MicroRNA-99a (miR-99a), a potential tumor suppressor, is downregulated in several human malignancies. The expression and function of miR-99a, however, have not been investigated in human renal cell carcinoma (RCC) so far. We therefore examined the expression of miR-99a in RCC cell lines and tissues, and assessed the impact of miR-99a on the tumorigenesis of RCC.
MiR-99a levels in 40 pairs of RCC and matched adjacent non-tumor tissues were assessed by real-time quantitative Reverse Transcription PCR (qRT-PCR). The RCC cell lines 786-O and OS-RC-2 were transfected with miR-99a mimics to restore the expression of miR-99a. The effects of miR-99a were then assessed by cell proliferation, cell cycle, transwell, and colony formation assay. A murine xenograft model of RCC was used to confirm the effect of miR-99a on tumorigenicity in vivo. Potential target genes were identified by western blotting and luciferase reporter assay.
We found that miR-99a was remarkably downregulated in RCC and low expression level of miR-99a was correlated with poor survival of RCC patients. Restoration of miR-99a dramatically suppressed RCC cells growth, clonability, migration and invasion as well as induced G1-phase cell cycle arrest in vitro. Moreover, intratumoral delivery of miR-99a could inhibit tumor growth in murine xenograft models of human RCC. In addition, we also fond that mammalian target of rapamycin (mTOR) was a direct target of miR-99a in RCC cells. Furthermore, siRNA-mediated knockdown of mTOR partially phenocopied the effect of miR-99a overexpression, suggesting that the tumor suppressive role of miR-99a may be mediated primarily through mTOR regulation.
Collectively, these results demonstrate for the first time, to our knowledge, that deregulation of miR-99a is involved in the etiology of RCC partially via direct targeting mTOR pathway, which suggests that miR-99a may offer an attractive new target for diagnostic and therapeutic intervention in RCC.
MicroRNA-99a; mTOR; Renal cell carcinoma
Inhibitors of TORC1 have been shown to be active in patients with metastatic renal cell carcinoma (RCC). As the PI3-K pathway activates numerous other kinases, transcription factors and proteins associated with cell growth and survival besides mTOR, disruption of this pathway upstream of mTOR may be more effective than inhibition of TORC1 alone.
To investigate this possibility, the dual PI3-K/mTOR inhibitor NVP-BEZ235 was compared with rapamycin in RCC cell lines and xenografts generated from 786-O and A498 cells.
Treatment of RCC cell lines with NVP-BEZ235 in vitro resulted in the nuclear translocation of p27, greater reduction in tumor cell proliferation, and more complete suppression of Akt, Mnk-1, eIF4E, and 4EBP-1 phosphorylation and Cyclin D1 and HIF2α expression than that achieved with rapamycin. The reduction of HIF2α levels correlated with reduced HIF activity as determined by luciferase assay. NVP-BEZ235 induced growth arrest in both the 786-O and A498 xenografts that was associated with inhibition of Akt and S6 phosphorylation as well as the induction of apoptosis and reduction in markers of tumor cell proliferation. In contrast, rapamycin induced only minimal growth retardation.
Dual inhibition of PI3-K/mTOR with NVP-BEZ235 induced growth arrest in RCC cell lines both in vitro and in vivo more effectively than inhibition of TORC1 alone. These results provide the rationale for the clinical assessment of agents such as NVP-BEZ235 in patients with advanced RCC.
Renal Cancer; PI3-Kinase; Akt; mTOR; HIF
PI3K and mTOR are key components of signal transduction pathways critical for cell survival. Numerous PI3K inhibitors have entered clinical trials, while mTOR is the target of approved drugs for metastatic renal cell carcinoma (RCC). We characterized expression of p85 and p110α PI3K subunits and mTOR in RCC specimens and assessed pharmacologic co-targeting of these molecules in vitro.
We employed tissue microarrays containing 330 nephrectomy cases using a novel immunofluorescence-based method of Automated Quantitative Analysis (AQUA) of in situ protein expression. In RCC cell lines we assessed synergism between PI3K and mTOR inhibitors and activity of NVP-BEZ235, which co-targets PI3K and mTOR.
p85 expression was associated with high stage and grade (P < 0.0001 for both). High p85 and high mTOR expression were strongly associated with decreased survival, and high p85 was independently prognostic on multi-variable analysis. Strong co-expression of both PI3K subunits and mTOR was found in the human specimens. The PI3K inhibitor LY294002 and rapamycin were highly synergistic in all six RCC cell lines studied. Similar synergism was seen with all rapamycin concentrations used. NVP-BEZ235 inhibited RCC cell growth in vitro with IC50s in the low ηM range and resultant PARP cleavage.
High PI3K and mTOR expression in RCC defines populations with decreased survival, suggesting that they are good drug targets in RCC. These targets tend to be co-expressed, and co-targeting these molecules is synergistic. NVP-BEZ235 is active in RCC cells in vitro; suggesting that concurrent PI3K and mTOR targeting in RCC warrants further investigation.
•We examine the interplay of mTOR and unfolded protein response in the control of cell survival and cell death.•There is a mutual exclusion between the survival and self-killing mechanisms during ER stress.•Metyrapone has similar effect to rapamycin in promoting autophagy.•An increase in autophagy by mTOR inhibition can delay cell death via feedback loops.•The main role of mTOR is to suppress autophagy-dependent survival during intolerable ER stress.
Unfolded or misfolded proteins in the endoplasmic reticulum (ER) trigger an adaptive ER stress response known as unfolded protein response (UPR). Depending on the severity of ER stress, either autophagy-controlled survival or apoptotic cell death can be induced. The molecular mechanisms by which UPR controls multiple fate decisions have started to emerge. One such molecular mechanism involves a master regulator of cell growth, mammalian target of rapamycin (mTOR), which paradoxically is shown to have pro-apoptotic role by mutually interacting with ER stress response. How the interconnections between UPR and mTOR influence the dynamics of autophagy and apoptosis activation is still unclear. Here we make an attempt to explore this problem by using experiments and mathematical modeling. The effect of perturbed mTOR activity in ER stressed cells was studied on autophagy and cell viability by using agents causing mTOR pathway inhibition (such as rapamycin or metyrapone). We observed that mTOR inhibition led to an increase in cell viability and was accompanied by an increase in autophagic activity. It was also shown that autophagy was activated under conditions of severe ER stress but that in the latter phase of stress it was inhibited at the time of apoptosis activation. Our mathematical model shows that both the activation threshold and temporal dynamics of autophagy and apoptosis inducers are sensitive to variation in mTOR activity. These results confirm that autophagy has cytoprotective role and is activated in mutually exclusive manner with respect to ER stress levels.
ER, endoplasmic reticulum; mTOR, mammalian target of rapamycin; UPR, unfolded protein response; Autophagy; Apoptosis; Endoplasmic reticulum stress; Metyrapone; Unfolded protein response; mTOR
The mammalian target of rapamycin (mTOR) kinase is an important component of PTEN/PI3K/Akt signaling pathway, which is frequently deregulated in prostate cancer (CaP). Recent studies suggest that targeting PTEN/PI3K/Akt and mTOR signaling pathway could be an effective strategy for the treatment of hormone refractory CaP. Here, we show that the treatment of androgen-independent and PTEN-negative human CaP PC3 cells with fisetin, a dietary flavonoid, resulted in inhibition of mTOR kinase signaling pathway. Treatment of cells with fisetin inhibited mTOR activity and downregulated Raptor, Rictor, PRAS40 and GβL that resulted in loss of mTOR complexes (mTORC)1/2 formation. Fisetin also activated the mTOR repressor TSC2 through inhibition of Akt and activation of AMPK. Fisetin-mediated inhibition of mTOR resulted in hypophosphorylation of 4EBP1 and suppression of Cap-dependent translation. We also found that fisetin treatment leads to induction of autophagic-programmed cell death rather than cytoprotective autophagy as shown by small interfering RNA Beclin1-knockdown and autophagy inhibitor. Taken together, we provide evidence that fisetin functions as a dual inhibitor of mTORC1/2 signaling leading to inhibition of Cap-dependent translation and induction of autophagic cell death in PC3 cells. These results suggest that fisetin could be a useful chemotherapeutic agent in treatment of hormone refractory CaP.