Compensatory Increase of Transglutaminase 2 Is Responsible for Resistance to mTOR Inhibitor Treatment

The mechanistic target of rapamycin complex 1 (mTORC1) plays a crucial role in controlling cell growth and homeostasis. Deregulation of mTOR signaling is frequently observed in some cancers, making it an attractive drug target for cancer therapy. Although mTORC1 inhibitor rapalog-based therapy has shown positive results in various pre-clinical animal cancer studies, tumors rebound upon treatment discontinuation. Moreover, several recent clinical trials showed that the mTORC1 inhibitors rapamycin and rapalog only reduce the capacity for cell proliferation without promoting cell death, consistent with the concept that rapamycin is cytostatic and reduces disease progression but is not cytotoxic. It is imperative that rapamycin-regulated events and additional targets for more effective drug combinations be identified. Here, we report that rapamycin treatment promotes a compensatory increase in transglutaminase 2 (TGM2) levels in mTORC1-driven tumors. TGM2 inhibition potently sensitizes mTORC1-hyperactive cancer cells to rapamycin treatment, and a rapamycin-induced autophagy blockade inhibits the compensatory TGM2 upregulation. More importantly, tumor regression was observed in MCF-7-xenograft tumor-bearing mice treated with both mTORC1 and TGM2 inhibitors compared with those treated with either a single inhibitor or the vehicle control. These results demonstrate a critical role for the compensatory increase in transglutaminase 2 levels in promoting mTORC1 inhibitor resistance and suggest that rational combination therapy may potentially suppress cancer therapy resistance.     

Targeting translation in acute myeloid leukemia: A new paradigm for therapy?

The mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway is commonly activated in cancer cells including acute myeloid leukemia (AML) and has been designed as a major target for cancer therapy. However, the efficacy of rapalogs (mTORC1 inhibitors) is limited in AML, due to the feedback activation of PI3K or ERK signaling pathways upon mTORC1 inhibition, which pathways should be simultaneously targeted to enhance the anti-leukemic activity of rapalogs. Moreover, the mRNA translation process is mTORC1-independent in AML, although markedly contributing to oncogenesis in this disease, and this also strongly participates to rapalogs resistance. Translation inhibition could be achieved by directly targeting the translation initiating complex using the 4EGI-1 compound, anti-eIF4E antisense oligonucleotides or the antiviral drug ribavirin or by second generation mTOR inhibitors (TORkinhibs). These new approaches represent promising perspectives for AML therapy that should have clinical development in the future.     

Targeting mTOR globally in cancer: Thinking beyond rapamycin

The mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for drug development. Until recently, we have relied largely on the use of rapamycin to study mTOR function and its anticancer potential. Recent insights now indicate that rapamycin is a partial inhibitor of mTOR through allosteric inhibition of mTOR complex-1 (mTORC1) but not mTOR complex-2 (mTORC2). Both the mechanism of action and the cellular response to mTORC1 inhibition by rapamycin and related drugs may limit the effectiveness of these compounds as antitumor agents. We and others have recently reported the discovery of second-generation ATP-competitive mTOR kinase inhibitors (TKIs) that bind to the active sites of mTORC1 and mTORC2, thereby targeting mTOR signaling function globally (see refs. 1-4). The discovery of specific, active-site mTOR inhibitors has opened a new chapter in the 40-plus year old odyssey that began with the discovery of rapamycin from a soil sample collected on Easter Island (see Vézina C, et al. J Antibiot 1975). Here, we discuss recent studies that highlight the emergence of rapamycin-resistant mTOR function in protein synthesis, cell growth, survival and metabolism. It is shown that these rapamycin-resistant mTOR functions are profoundly inhibited by TKIs. A more complete suppression of mTOR global signaling network by the new inhibitors is expected to yield a deeper and broader antitumor response in the clinic.     

Emerging strategies to overcome the resistance to current mTOR inhibitors in renal cell carcinoma

The mammalian target of rapamycin (mTOR) has emerged as an attractive cancer therapeutic target. Treatment of metastatic renal cell carcinoma (mRCC) has improved significantly with the advent of agents targeting the mTOR pathway, such as temsirolimus and everolimus. Unfortunately, a number of potential mechanisms that may lead to resistance to mTOR inhibitors have been proposed.In this paper, we discuss the mechanisms underlying resistance to mTOR inhibitors, which include the downstream effectors of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway, the activation of hypoxia-inducible factor (HIF), the PIM kinase family, PTEN expression, elevated superoxide levels, stimulation of autophagy, immune cell response and ERK/MAPK, Notch and Aurora signaling pathways. Moreover, we present an updated analysis of clinical trials available on PubMed Central and www.clinicaltrials.gov, which were pertinent to the resistance to rapalogs.The new frontier of inhibiting the mTOR pathway is to identify agents targeting the feedback loops and cross talks with other pathways involved in the acquired resistance to mTOR inhibitors. The true goal will be to identify biomarkers predictive of sensitivity or resistance to efficiently develop novel agents with the aim to avoid toxicities and to better choose the active drug for the right patient.     

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.     

Control of B lymphocyte development and functions by the mTOR signaling pathways

Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase originally discovered as the molecular target of the immunosuppressant rapamycin. mTOR forms two compositionally and functionally distinct complexes, mTORC1 and mTORC2, which are crucial for coordinating nutrient, energy, oxygen, and growth factor availability with cellular growth, proliferation, and survival. Recent studies have identified critical, non-redundant roles for mTORC1 and mTORC2 in controlling B cell development, differentiation, and functions, and have highlighted emerging roles of the Folliculin-Fnip protein complex in regulating mTOR and B cell development. In this review, we summarize the basic mechanisms of mTOR signaling; describe what is known about the roles of mTORC1, mTORC2, and the Folliculin/Fnip1 pathway in B cell development and functions; and briefly outline current clinical approaches for targeting mTOR in B cell neoplasms. We conclude by highlighting a few salient questions and future perspectives regarding mTOR in B lineage cells.     

A mechanism for synergy with combined mTOR and PI3 kinase inhibitors

Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.     

mTOR complex 2 signaling and functions

The mechanistic target of rapamycin (mTOR) plays a central role in cellular growth and metabolism. mTOR forms two distinct protein complexes, mTORC1 and mTORC2. Much is known about the regulation and functions of mTORC1 due to availability of a natural compound, rapamycin, that inhibits this complex. Studies that define mTORC2 cellular functions and signaling have lagged behind. The development of pharmacological inhibitors that block mTOR kinase activity, and thereby inhibit both mTOR complexes, along with availability of mice with genetic knockouts in mTOR complex components have now provided new insights on mTORC2 function and regulation. Since prolonged effects of rapamycin can also disrupt mTORC2, it is worth re-evaluating the contribution of this less-studied mTOR complex in cancer, metabolic disorders and aging. In this review, we focus on recent developments on mammalian mTORC2 signaling mechanisms and its cellular and tissue-specific functions.     

Crosstalks of GSK3 signaling with the mTOR network and effects on targeted therapy of cancer

The introduction of therapeutics targeting specific tumor-promoting oncogenic or non-oncogenic signaling pathways has revolutionized cancer treatment. Mechanistic (previously mammalian) target of rapamycin (mTOR), a highly conserved Ser/Thr kinase, is a central hub of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR network, one of the most frequently deregulated signaling pathways in cancer, that makes it an attractive target for therapy. Numerous mTOR inhibitors have progressed to clinical trials and two of them have been officially approved as anticancer therapeutics. However, mTOR-targeting drugs have met with a very limited success in cancer patients. Frequently, the primary impediment to a successful targeted therapy in cancer is drug-resistance, either from the very beginning of the therapy (innate resistance) or after an initial response and upon repeated drug treatment (evasive or acquired resistance). Drug-resistance leads to treatment failure and relapse/progression of the disease. Resistance to mTOR inhibitors depends, among other reasons, on activation/deactivation of several signaling pathways, included those regulated by glycogen synthase kinase-3 (GSK3), a protein that targets a vast number of substrates in its repertoire, thereby orchestrating many processes that include cell proliferation and survival, metabolism, differentiation, and stemness. A detailed knowledge of the rewiring of signaling pathways triggered by exposure to mTOR inhibitors is critical to our understanding of the consequences such perturbations cause in tumors, including the emergence of drug-resistant cells.Here, we provide the reader with an updated overview of intricate circuitries that connect mTOR and GSK3 and we relate them to the efficacy (or lack of efficacy) of mTOR inhibitors in cancer cells.     

mTORC1- and mTORC2-interacting proteins keep their multifunctional partners focused

The mammalian target of rapamycin, best known as mTOR, is a phylogenetically conserved serine/threonine kinase that controls life-defining cellular processes such as growth, metabolism, survival, and migration under the influence of multiple interacting proteins. Historically, the cellular activities blocked by rapamycin in mammalian cells were considered the only events controlled by mTOR. However, this paradigm changed with the discovery of two signaling complexes differentially sensitive to rapamycin, whose catalytic component is mTOR. The one sensitive to rapamycin, known as mTORC1, promotes protein synthesis in response to growth factors and nutrients via the phosphorylation of p70S6K and 4EBP1; while the other, known as mTORC2, promotes cell migration and survival via the activation of Rho GTPases and the phosphorylation of AKT, respectively. Although mTORC2 kinase activity is not inhibited by rapamycin, hours of incubation with this antibiotic can impede the assembly of this signaling complex. The direct mechanism by which mTORC2 leads to cell migration depends on its interaction with P-Rex1, a Rac-specific guanine nucleotide exchange factor, while additional indirect pathways involve the intervention of PKC or AKT, multifunctional ubiquitous serine/threonine kinases that activate effectors of cell migration upon being phosphorylated by mTORC2 in response to chemotactic signals. These mTORC2 effectors are altered in metastatic cancer. Numerous clinical trials are testing mTOR inhibitors as potential antineoplasic drugs. Here, we briefly review the actions of mTOR with emphasis on the controlling role of mTORC1 and mTORC2-interacting proteins and highlight the mechanisms linked to cell migration.     

mTOR complex 2 signaling and functions

The mechanistic target of rapamycin (mTOR) plays a central role in cellular growth and metabolism. mTOR forms two distinct protein complexes, mTORC1 and mTORC2. Much is known about the regulation and functions of mTORC1 due to availability of a natural compound, rapamycin, that inhibits this complex. Studies that define mTORC2 cellular functions and signaling have lagged behind. The development of pharmacological inhibitors that block mTOR kinase activity, and thereby inhibit both mTOR complexes, along with availability of mice with genetic knockouts in mTOR complex components have now provided new insights on mTORC2 function and regulation. Since prolonged effects of rapamycin can also disrupt mTORC2, it is worth re-evaluating the contribution of this less-studied mTOR complex in cancer, metabolic disorders and aging. In this review, we focus on recent developments on mammalian mTORC2 signaling mechanisms and its cellular and tissue-specific functions.Key words: mTOR, mTORC2, rictor, cancer, metabolism, ribosomes, protein synthesis, protein maturation, AGC kinases, growth factor signaling     

P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.     

PI3K/Akt/mTOR信号通路及临床相关肿瘤抑制剂

哺乳动物雷帕霉素靶（mTOR）和蛋白激酶B（Akt/PKB）与肿瘤发生的密切关系已被广泛地认可.mTOR是一种丝/苏氨酸激酶,可以通过影响mRNA转录、代谢、自噬等方式调控细胞的生长.它既是PI3K的效应分子,也可以是PI3K的反馈调控因子.mTORC1 和mTORC2是mTOR的两种不同复合物. 对雷帕霉素敏感的mTORC1受到营养、生长因子、能量和应激4种因素的影响.生长因子通过PI3K/Akt信号通路调控mTORC1是最具特征性调节路径.而mTORC2最为人熟知的是作为Akt473磷酸化位点的上游激酶. 同样,Akt/PKB在细胞增殖分化、迁移生长过程中发挥着重要作用. 随着Thr308和Ser473两个位点激活,Akt/PKB也得以全面活化.因此,mTORC2-Akt-mTORC1的信号通路在肿瘤形成和生长中是可以存在的.目前临床肿瘤治疗中,PI3K/Akt/mTOR是重要的靶向治疗信号通路.然而,仅抑制mTORC1活性,不是所有的肿瘤都能得到预期控制.雷帕霉素虽然能抑制mTORC1,但也能反馈性地增加PI3K信号活跃度,从而影响治疗预后.近来发现的第二代抑制剂可以同时抑制mTORC1/2和PI3K活性,这种抑制剂被认为在肿瘤治疗上颇具前景.本综述着重阐述了PI3K/Akt/mTOR信号通路的传导、各因子之间的相互调控以及相关抑制剂的发展.     

Induction of autophagy by dual mTORC1-mTORC2 inhibition in BCR-ABL-expressing leukemic cells

Over recent years, there have been substantial research advances on the mechanisms by which BCR-ABL transforms hematopoietic cells and promotes leukemic cell growth and survival. Among the diverse signaling cascades activated by BCR-ABL, the mTOR pathway plays a critical role in mRNA translation of genes that promote leukemogenesis and mitogenic responses. We have recently shown that dual targeting of mTORC1 and mTORC2 complexes using a catalytic mTOR inhibitor, OSI-027, results in generation of potent antileukemic effects against BCR-ABL transformed cells. Such effects were also seen in cells expressing the T315I mutation, which is resistant to all currently approved BCR-ABL kinase inhibitors. Our studies also demonstrate that such dual catalytic inhibition of mTORC2 and mTORC1 complexes in BCR-ABL-expressing K562 cells results in induction of autophagy, and that inhibition of the autophagic process using chloroquine promotes apoptosis of these cells. Altogether, our studies suggest that autophagy may be a limiting factor for the induction of apoptosis during dual mTORC2-mTORC1 targeting, in at least some types of BCR-ABL-expressing cells and have raised the potential of combinations of catalytic inhibitors of mTOR with autophagy inhibitors for the treatment of refractory Ph+ leukemias.     

第二代 mTOR 抑制剂的抗肿瘤研究进展

哺乳动物雷帕霉素靶蛋白(mTOR)是 PI3K/Akt/mTOR 等多种信号通路的下游分子,在细胞增殖、分化、转移和存活中发挥 重要作用,已成为癌症治疗的一个重要靶标。传统的 mTOR 抑制剂主要是雷帕霉素及其衍生物,能特异性抑制 mTORC1,但在部分癌 症临床治疗中未达到预期疗效,且易产生耐药性。第二代 mTOR 抑制剂即双重或多重 mTOR 抑制剂能与 mTOR 的催化位点竞争 ATP, 高度选择性地抑制 mTORC1 和 mTORC2,比单靶点 mTOR 抑制剂具有更大的治疗优势。此外,某些天然来源产物也具有对 mTOR 的 抑制作用,且毒性、副作用更小。综述近几年有关 mTOR 及其抑制剂在抗肿瘤方面的研究进展。     

Targeting LKB1 signaling in cancer

The serine/threonine kinase LKB1 is a master kinase involved in cellular responses such as energy metabolism, cell polarity and cell growth. LKB1 regulates these crucial cellular responses mainly via AMPK/mTOR signaling. Germ-line mutations in LKB1 are associated with the predisposition of the Peutz–Jeghers syndrome in which patients develop gastrointestinal hamartomas and have an enormously increased risk for developing gastrointestinal, breast and gynecological cancers. In addition, somatic inactivation of LKB1 has been associated with sporadic cancers such as lung cancer. The exact mechanisms of LKB1-mediated tumor suppression remain so far unidentified; however, the inability to activate AMPK and the resulting mTOR hyperactivation has been detected in PJS-associated lesions. Therefore, targeting LKB1 in cancer is now mainly focusing on the activation of AMPK and inactivation of mTOR. Preclinical in vitro and in vivo studies show encouraging results regarding these approaches, which have even progressed to the initiation of a few clinical trials. In this review, we describe the functions, regulation and downstream signaling of LKB1, and its role in hereditary and sporadic cancers. In addition, we provide an overview of several AMPK activators, mTOR inhibitors and additional mechanisms to target LKB1 signaling, and describe the effect of these compounds on cancer cells. Overall, we will explain the current strategies attempting to find a way of treating LKB1-associated cancer.     

The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.     

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

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.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.