Search tips
Search criteria

Results 1-25 (1485331)

Clipboard (0)

Related Articles

1.  Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2 
PLoS Biology  2009;7(2):e1000038.
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.
Author Summary
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.
PMCID: PMC2637922  PMID: 19209957
2.  Phosphatidylethanolamine-binding protein 4 promotes lung cancer cells proliferation and invasion via PI3K/Akt/mTOR axis 
Journal of Thoracic Disease  2015;7(10):1806-1816.
While phosphatidylethanolamine-binding protein 4 (PEBP4) is a key factor in the malignant proliferation and metastasis of tumor cells, the exact regulatory network governing its roles remains unclear. This study was designed to investigate the effect of PEBP4 on PI3K/Akt/mTOR pathway and explore its molecular network that governs the proliferation and metastasis of tumor cells.
After the recombinant plasmid pcDNA3.1-PEBP4 was constructed, the recombinant plasmid pcDNA3.1-PEBP4 and PEBP4-targeting siRNA were transfected into lung cancer HCC827 cell line. The expressions of PI3K/Akt/mTOR pathway components in HCC827 cells in each group were determined using Western blotting. In the HCC827 cells, the effect of PI3K pathway inhibitor LY294002 on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of LY294002 on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay. Furthermore, the effect of mTOR inhibitor rapamycin (RAPA) on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of RAPA on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay.
As shown by Western blotting, the protein expressions of p-Akt and phosphorylated mTOR (p-mTOR) were significantly higher in the pcDNA3.1-PEBP4-transfected group than in the normal control group and PEBP4 siRNA group (P<0.05); furthermore, the protein expressions of p-Akt and p-mTOR significantly decreased in the PEBP4 targeting siRNA-transfected group (P<0.05). Treatment with LY294002 significantly inhibited the protein expressions of p-Akt and p-mTOR in HCC827 cells (P<0.05). In contrast, treatment with RAPA only significantly inhibited the protein expression of p-mTOR (P<0.05). As shown by MTT, flow cytometry, and Transwell migration assay, both LY294002 and RAPA could significantly lower the viability of HCC827 cells and inhibit their proliferation and invasion (P<0.05); meanwhile, they could reverse the effect of PEBP4 in promoting the proliferation and migration of HCC827 cells (P<0.05).
The overexpression of PEBP4 increases the phosphorylation levels of Akt and mTOR in lung cancer cells. The PI3K/Akt/mTOR signaling axis may be a key molecular pathway via which PEBP4 promotes the proliferation and invasion of non-small cell lung cancer (NSCLC) cells; also, it may serve as a potential therapeutic target.
PMCID: PMC4635298  PMID: 26623104
Phosphatidylethanolamine-binding protein 4 (PEBP4); lung cancer; PI3K/Akt/mTOR; LY294002; rapamycin (RAPA)
3.  Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics 
PLoS ONE  2015;10(11):e0141253.
Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.
PMCID: PMC4646664  PMID: 26569621
4.  Gemcitabine resistance in breast cancer cells regulated by PI3K/AKT-mediated cellular proliferation exerts negative feedback via the MEK/MAPK and mTOR pathways 
OncoTargets and therapy  2014;7:1033-1042.
Chemoresistance is a major cause of cancer treatment failure and leads to a reduction in the survival rate of cancer patients. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen-activated protein kinase (MAPK) pathways are aberrantly activated in many malignant tumors, including breast cancer, which may indicate an association with breast cancer chemoresistance. In this study, we generated a chemoresistant human breast cancer cell line, MDA-MB-231/gemcitabine (simplified hereafter as “231/Gem”), from MDA-MB-231 human breast cancer cells. Flow cytometry studies revealed that with the same treatment concentration of gemcitabine, 231/Gem cells displayed more robust resistance to gemcitabine, which was reflected by fewer apoptotic cells and enhanced percentage of S-phase cells. Through the use of inverted microscopy, Cell Counting Kit-8, and Transwell assays, we found that compared with parental 231 cells, 231/Gem cells displayed more morphologic projections, enhanced cell proliferative ability, and improved cell migration and invasion. Mechanistic studies revealed that the PI3K/AKT/mTOR and mitogen-activated protein kinase kinase (MEK)/MAPK signaling pathways were activated through elevated expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-AKT, mTOR, p-mTOR, p-P70S6K, and reduced expression of p-P38 and LC3-II (the marker of autophagy) in 231/Gem in comparison to control cells. However, there was no change in the expression of Cyclin D1 and p-adenosine monophosphate-activated protein kinase (AMPK). In culture, inhibitors of PI3K/AKT and mTOR, but not of MEK/MAPK, could reverse the enhanced proliferative ability of 231/Gem cells. Western blot analysis showed that treatment with a PI3K/AKT inhibitor decreased the expression levels of p-AKT, p-MEK, p-mTOR, and p-P70S6K; however, treatments with either MEK/MAPK or mTOR inhibitor significantly increased p-AKT expression. Thus, our data suggest that gemcitabine resistance in breast cancer cells is mainly mediated by activation of the PI3K/AKT signaling pathway. This occurs through elevated expression of p-AKT protein to promote cell proliferation and is negatively regulated by the MEK/MAPK and mTOR pathways.
PMCID: PMC4063800  PMID: 24966685
chemoresistance; gemcitabine; breast cancer
5.  Targeted Therapy for Advanced Prostate Cancer: Inhibition of the PI3K/Akt/mTOR Pathway 
Current cancer drug targets  2009;9(2):237-249.
A large number of novel therapeutics is currently undergoing clinical evaluation for the treatment of prostate cancer, and small molecule signal transduction inhibitors are a promising class of agents. These inhibitors have recently become a standard therapy in renal cell carcinoma and offer significant promise in prostate cancer. Through an understanding of the key pathways involved in prostate cancer progression, a rational drug design can be aimed at the molecules critical to cellular signaling. This may enable administration of selective therapies based on the expression of molecular targets, more appropriately individualizing treatment for prostate cancer patients.
One pathway with a prominent role in prostate cancer is the PI3K/Akt/mTOR pathway. Current estimates suggest that PI3K/Akt/mTOR signaling is upregulated in 30-50% of prostate cancers, often through loss of PTEN. Molecular changes in the PI3K/Akt/mTOR signaling pathway have been demonstrated to differentiate benign from malignant prostatic epithelium and are associated with increasing tumor stage, grade, and risk of biochemical recurrence. Multiple inhibitors of this pathway have been developed and are being assessed in the laboratory and in clinical trials, with much attention focusing on mTOR inhibition. Current clinical trials in prostate cancer are assessing efficacy of mTOR inhibitors in combination with multiple targeted or traditional chemotherapies, including bevacizumab, gefitinib, and docetaxel. Completion of these trials will provide substantial information regarding the importance of this pathway in prostate cancer and the clinical implications of its targeted inhibition. In this article we review the data surrounding PI3K/Akt/mTOR inhibition in prostate cancer and their clinical implications.
PMCID: PMC2921605  PMID: 19275762
Prostate cancer; targeted therapy; PI3K; Akt; mTOR
6.  Potential targets for ovarian clear cell carcinoma: a review of updates and future perspectives 
Advances in surgical and medical treatments for ovarian cancer have improved prognoses. Platinum drugs in particular are pivotal for the medical treatment of ovarian cancer. However, previous studies have revealed that some histological subtypes, such as clear cell carcinoma, are resistant to medical treatment, including that with platinum drugs. Consequently, the clinical prognosis of advanced clear cell carcinoma is remarkably inferior, primarily because of its chemoresistant behavior. The prevalence of clear cell carcinoma is approximately 5 % in the West, but in Japan, its prevalence is particularly high, at approximately 25 %. Current medical treatments for advanced clear cell carcinoma are difficult to administer, and they have poor efficacy, warranting the development of novel target-based therapies. In this review, we describe medical treatments for clear cell carcinoma and discuss future prospects for therapy. In particular, we focus on the mechanism of platinum resistance in clear cell carcinoma, including the role of annexin A4, one of the most investigated factors of platinum resistance, as well as the mutant genes and overexpressed proteins such as VEGF, PI3K/AKT/mTOR signaling pathway, ARID1A, hepatocyte nuclear factor-1β, ZNF217. We also review targeted molecular therapeutics for epithelial ovarian cancer and discuss their role in clear cell carcinoma treatment. We review the drugs targeting angiogenesis (bevacizumab, sorafenib, and pazopanib), growth factors (gefitinib, erlotinib, lapatinib, trastuzumab, and AMG479), and signaling pathways (temsirolimus, dasatinib, and imatinib), and other drugs (oregovomab, volociximab, and iniparib). This current review summarizes and discusses the clinical significance of these factors in ovarian clear cell carcinoma as well as their potential mechanisms of action. It may provide new integrative understanding for future studies on their exact role in ovarian clear cell carcinoma.
PMCID: PMC4678619  PMID: 26675567
Clear cell carcinoma; Platinum resistance; Annexin A4; Target-based therapies; Ovarian cancer
7.  Targeting the PI3K/AKT/mTOR Signaling Axis in Children with Hematologic Malignancies 
Paediatric drugs  2012;14(5):299-316.
The phosphatidylinositiol 3-kinase (PI3K), AKT, mammalian target of rapamycin (mTOR) signaling pathway (PI3K/AKT/mTOR) is frequently dysregulated in disorders of cell growth and survival, including a number of pediatric hematologic malignancies. The pathway can be abnormally activated in childhood acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML), as well as in some pediatric lymphomas and lymphoproliferative disorders. Most commonly, this abnormal activation occurs as a consequence of constitutive activation of AKT, providing a compelling rationale to target this pathway in many of these conditions.
A variety of agents, beginning with the rapamycin analogue (rapalog) sirolimus, have been used successfully to target this pathway in a number of pediatric hematologic malignancies. Rapalogs demonstrate significant preclinical activity against ALL, which has led to a number of clinical trials. Moreover, rapalogs can synergize with a number of conventional cytotoxic agents and overcome pathways of chemotherapeutic resistance for drugs commonly used in ALL treatment, including methotrexate and corticosteroids. Based on preclinical data, rapalogs are also being studied in AML, CML, and non-Hodgkin’s lymphoma. Recently, significant progress has been made using rapalogs to treat pre-malignant lymphoproliferative disorders, including the autoimmune lymphoproliferative syndrome (ALPS); complete remissions in children with otherwise therapy-resistant disease have been seen.
Rapalogs only block one component of the pathway (mTORC1), and newer agents are under preclinical and clinical development that can target different and often multiple protein kinases in the PI3K/AKT/mTOR pathway. Most of these agents have been tolerated in early-phase clinical trials. A number of PI3K inhibitors are under investigation. Of note, most of these also target other protein kinases. Newer agents are under development that target both mTORC1 and mTORC2, mTORC1 and PI3K, and the triad of PI3K, mTORC1, and mTORC2. Preclinical data suggest these dual- and multi-kinase inhibitors are more potent than rapalogs against many of the aforementioned hematologic malignancies.
Two classes of AKT inhibitors are under development, the alkyl-lysophospholipids (APLs) and small molecule AKT inhibitors. Both classes have agents currently in clinical trials. A number of drugs are in development that target other components of the pathway, including eukaryotic translation initiation factor (eIF) 4E (eIF4E) and phosphoinositide-dependent protein kinase 1 (PDK1). Finally, a number of other key signaling pathways interact with PI3K/AKT/mTOR, including Notch, MNK, Syk, MAPK, and aurora kinase. These alternative pathways are being targeted alone and in combination with PI3K/AKT/mTOR inhibitors with promising preclinical results in pediatric hematologic malignancies. This review provides a comprehensive overview of the abnormalities in the PI3K/AKT/mTOR signaling pathway in pediatric hematologic malignancies, the agents that are used to target this pathway, and the results of preclinical and clinical trials, using those agents in childhood hematologic cancers.
PMCID: PMC4214862  PMID: 22845486
8.  Immunohistochemical expression of mTOR negatively correlates with PTEN expression in gastric carcinoma 
Oncology Letters  2012;4(6):1213-1218.
The phosphoinositide-3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway is a cellular pathway involved in cell growth, tumorigenesis and cell invasion which is frequently activated in various types of cancer. The downstream effector of the pathway is mTOR which is important in cellular growth and homeostasis and aberrant activation of mTOR has been reported in several types of cancer. The tumor suppressor gene phosphatase and tensin homolog (PTEN) is essential in this pathway for inhibiting tumor invasion and metastasis. However, the involvement of mTOR and PTEN in the progression of human gastric cancer remains to be identified. Immunohistochemical staining was performed to detect the expression of mTOR and PTEN in paraffin-embedded gastric tissue sections obtained from 33 patients with gastric cancer and 30 normal controls. The expressed mTOR was mainly distributed in the cytoplasm, while PTEN was mainly localized to the nucleus. By considering negative mTOR expression with positive PTEN expression as one group and negative PTEN expression with positive mTOR expression as the other, significant statistical differences were observed in various categories, including histological types and metastatic and clinical pathology stages, between the 2 groups (P<0.01 or 0.05). The results indicated that the expression levels of mTOR and PTEN were negatively correlated in the PI3K-AKT-mTOR signaling pathway. Combined detection of mTOR and PTEN expression may be used to evaluate the degree of malignancy in gastric cancer and may be a useful marker for the early diagnosis of gastric cancer.
PMCID: PMC3506752  PMID: 23205120
gastric carcinoma; mTOR; PTEN; immunohistochemistry
9.  Elevated phosphorylation and activation of PDK-1/AKT pathway in human breast cancer 
British Journal of Cancer  2005;93(12):1372-1381.
Activation of kinases signalling pathways contributes to various malignant phenotypes in human cancers, including breast tumour. To examine the possible activation of these signalling molecules, we examined the phosphorylation status in 12 protein kinases and transcription factors in normal primary human mammary epithelial cells, telomerase-immortalised human breast epithelial cell line, and two breast cancer lines, MDA-MB-468 and MCF-7, using Kinexus phosphorylated protein screening assays. The phosphorylation of FAK, mTOR, p70S6K, and PDK-1 were elevated in both breast cancer cell lines, whereas the phosphorylation of AKT, EGFR, ErbB2/Her2, PDGFR, Shc, and Stat3 were elevated in only one breast cancer line compared to normal primary mammary epithelial cells and telomerase-immortalised breast epithelial cells. The same findings were confirmed by Western blotting and by kinase assays. We further substantiated the phosphorylation status of these molecules in tissue microarray slides containing 89 invasive breast cancer tissues as well as six normal mammary tissues with immunohistochemistry staining using phospho-specific antibodies. Consistent findings were obtained as greater than 70% of invasive breast carcinomas expressed moderate to high levels of phosphorylated PDK-1, AKT, p70S6K, and EGFR. In sharp contrast, phosphorylation of the same proteins was nearly undetectable or was at low levels in normal mammary tissues under the same assay. Elevated phosphorylation of PDK-1, AKT, mTOR, p70S6K, S6, EGFR, and Stat3 were highly associated with invasive breast tumours (P<0.05). Taken together, our results suggest that activation of these kinase pathways by phosphorylation may in part account for molecular pathogenesis of human breast carcinoma. Particularly, moderate to high level of PDK-1 phosphorylation was found in 86% of high-grade metastasised breast tumours. This is the first report demonstrating phosphorylation of PDK-1 is frequently elevated in breast cancer with concomitantly increased phosphorylation of downstream kinases, including AKT, mTOR, p70S6K, S6, and Stat3. This finding thus suggested PDK-1 may promote oncogenesis in part through the activation of AKT and p70S6K and rationalised that PDK-1 as well as downstream components of PDK-1 signalling pathway may be promising therapeutic targets to treat breast cancer.
PMCID: PMC2361529  PMID: 16288304
PDK-1; AKT; mTOR; p70S6K; phosphorylation; breast cancer
10.  Preclinical Testing of PI3K/AKT/mTOR Signaling Inhibitors in a Mouse Model of Ovarian Endometrioid Adenocarcinoma 
Genetically engineered mouse (GEM) models of ovarian cancer that closely recapitulate their human tumor counterparts may be invaluable tools for preclinical testing of novel therapeutics. We studied murine ovarian endometrioid adenocarcinomas (OEAs) arising from conditional dysregulation of canonical WNT and PI3K/AKT/mTOR pathway signaling to investigate their response to conventional chemotherapeutic drugs and mTOR or AKT inhibitors.
Experimental Design
OEAs were induced by injection of adenovirus expressing Cre recombinase (AdCre) into the ovarian bursae of Apcflox/flox;Ptenflox/flox mice. Tumor-bearing mice or murine OEA-derived cell lines were treated with cisplatin and paclitaxel, mTOR inhibitor rapamycin, or AKT inhibitors API-2 or perifosine. Treatment effects were monitored in vivo by tumor volume and bioluminescence imaging, in vitro by WST-1 proliferation assays, and in OEA tissues and cells by immunoblotting and immunostaining for levels and phosphorylation status of PI3K/AKT/mTOR signaling pathway components.
Murine OEAs developed within 3 weeks of AdCre injection and were not preceded by endometriosis. OEAs responded to cisplatin + paclitaxel, rapamycin, and AKT inhibitors in vivo. In vitro studies showed that response to mTOR and AKT inhibitors, but not conventional cytotoxic drugs, was dependent on the status of PI3K/AKT/mTOR signaling. AKT inhibition in APC−/PTEN− tumor cells resulted in compensatory up-regulation of ERK signaling.
The studies demonstrate the utility of this GEM model of ovarian cancer for pre-clinical testing of novel PI3K/AKT/mTOR signaling inhibitors and provide evidence for compensatory signaling, suggesting that multiple rather than single agent targeted therapy will be more efficacious for treating ovarian cancers with activated PI3K/AKT/mTOR signaling.
PMCID: PMC3229658  PMID: 21903772
ovarian carcinoma; endometriosis; PI3K/AKT/mTOR signaling; mouse model; bioluminescence imaging
11.  mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis 
PLoS Genetics  2015;11(7):e1005291.
Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs) in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC)-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism.
Author Summary
The protein kinase mTOR is frequently activated in breast cancers, where it enhances cancer cell growth, survival, and metastastic spread to distant organs. Thus, mTOR is an attractive, clinically relevant molecular target for drugs designed to treat metastatic breast cancers. However, mTOR exists in two distinct complexes, mTORC1 and mTORC2, and the relative roles of each complex have not been elucidated. Moreover, as pathways that regulate normal tissue growth and development are often highjacked to promote cancer, understanding mTOR function in normal mammary epithelial development will likely provide insight into its role in tumor progression. In this study, we assessed the role of mTORC1 and mTORC2 complexes in normal mammary epithelial cell branching, survival, and invasion. Interestingly, while mTORC1 was not required for branching, survival and invasion of mammary epithelial cells, mTORC2 was necessary for these processes in both mouse and human models. Furthermore, we found that mTORC2 exerts its effects primarily through downstream activation of a PKC-alpha-Rac1 signaling axis rather than the more well-studied Akt signaling pathway. Our studies identify a novel role for the mTORC2 complex in mammary morphogenesis, including cell survival and motility, which are relevant to breast cancer progression.
PMCID: PMC4488502  PMID: 26132202
12.  Prognostic significance and therapeutic potential of the activation of anaplastic lymphoma kinase/protein kinase B/mammalian target of rapamycin signaling pathway in anaplastic large cell lymphoma 
BMC Cancer  2013;13:471.
Activation of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway has been demonstrated to be involved in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated tumorigenesis in anaplastic large cell lymphoma (ALCL) and correlated with unfavorable outcome in certain types of other cancers. However, the prognostic value of AKT/mTOR activation in ALCL remains to be fully elucidated. In the present study, we aim to address this question from a clinical perspective by comparing the expressions of the AKT/mTOR signaling molecules in ALCL patients and exploring the therapeutic significance of targeting the AKT/mTOR pathway in ALCL.
A cohort of 103 patients with ALCL was enrolled in the study. Expression of ALK fusion proteins and the AKT/mTOR signaling phosphoproteins was studied by immunohistochemical (IHC) staining. The pathogenic role of ALK fusion proteins and the therapeutic significance of targeting the ATK/mTOR signaling pathway were further investigated in vitro study with an ALK + ALCL cell line and the NPM-ALK transformed BaF3 cells.
ALK expression was detected in 60% of ALCLs, of which 79% exhibited the presence of NPM-ALK, whereas the remaining 21% expressed variant-ALK fusions. Phosphorylation of AKT, mTOR, 4E-binding protein-1 (4E-BP1), and 70 kDa ribosomal protein S6 kinase polypeptide 1 (p70S6K1) was detected in 76%, 80%, 91%, and 93% of ALCL patients, respectively. Both phospho-AKT (p-AKT) and p-mTOR were correlated to ALK expression, and p-mTOR was closely correlated to p-AKT. Both p-4E-BP1 and p-p70S6K1 were correlated to p-mTOR, but were not correlated to the expression of ALK and p-AKT. Clinically, ALK + ALCL occurred more commonly in younger patients, and ALK + ALCL patients had a much better prognosis than ALK-ALCL cases. However, expression of p-AKT, p-mTOR, p-4E-BP1, or p-p70S6K1 did not have an impact on the clinical outcome. Overexpression of NPM-ALK in a nonmalignant murine pro-B lymphoid cell line, BaF3, induced the cells to become cytokine-independent and resistant to glucocorticoids (GCs). Targeting AKT/mTOR inhibited growth and triggered the apoptotic cell death of ALK + ALCL cells and NPM-ALK transformed BaF3 cells, and also reversed GC resistance induced by overexpression of NPM-ALK.
Overexpression of ALK due to chromosomal translocations is seen in the majority of ALCL patients and endows them with a much better prognosis. The AKT/mTOR signaling pathway is highly activated in ALK + ALCL patients and targeting the AKT/mTOR signaling pathway might confer a great therapeutic potential in ALCL.
PMCID: PMC3852000  PMID: 24112608
Anaplastic large cell lymphoma (ALCL); Anaplastic lymphoma kinase (ALK); AKT; mTOR; 4E-BP1; p-70S6K1; Prognosis
13.  PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells 
AKT plays a pivotal role in the signal transduction of cancer cells. MK2206, an AKT inhibitor, has been shown to be an effective anti-cancer drug to a variety of cancer cell lines. However, some cancer cells acquire resistance to MK2206 and new strategies to suppress these cell lines remain to be developed.
Experimental design
Acquired MK-2206-resistant neuroblastoma (NB) cell sublines were induced by stepwise escalation of MK-2206 exposure (4–12 weeks). MTT assay was used to validate cell proliferation. Flow cytometry was performed for cell cycle analysis. Western blot assay was used for cell signaling study.
MK2206 (5–10 µmol) significantly suppressed cell growth of MK2206 non-resistant NB cells (LAN-1, KP-N-SIFA, NB-19 and SK-N-DZ), but is less efficient in inhibiting that of resistant sublines, even after 2-week MK2206-free incubation. MK2206 acted in mTOR-S6K dependent and independent methods. MK-2206 resistant sublines (LAN-1-MK, KP-N-SIFA-MK, and SK-N-DZ-MK) showed lower IC50 of GSK2334470 (PDK1 inhibitor). The cell growth of all sublines was prohibited by AZD8805 (mTOR inhibitor), with IC50 of AZD8805 3–10 times lower than MK2206 non-resistant cells. The signaling profiles of these resistant sublines were characterized by elevated PDK1-mTOR-S6K activity, accompanying by low phosphorylation of AKT compared with non-resistant counterparts. GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0–G1 ratio in LAN-1-MK than that in LAN-1 as well. PDK1 and mTOR inhibitors effected on phosphorylation of GSK3β in some of resistant sublines.
NB cells can acquire MK2206 resistance after exposure for 4–12 weeks. Resistant cells feature reliance on PDK1-mTOR-S6K pathway and are more sensitive to PDK1 and mTOR inhibitors than the non-resistant counterparts. Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.
PMCID: PMC4587771  PMID: 26421002
Neuroblastoma; MK2206; Akt; PDK1; mTOR; Cell growth
14.  Comprehensive analysis of microRNA-regulated protein interaction network reveals the tumor suppressive role of microRNA-149 in human hepatocellular carcinoma via targeting AKT-mTOR pathway 
Molecular Cancer  2014;13:253.
Our previous study identified AKT1, AKT2 and AKT3 as unfavorable prognostic factors for patients with hepatocellular carcinoma (HCC). However, limited data are available on their exact mechanisms in HCC. Since microRNAs (miRNAs) are implicated in various human cancers including HCC, we aimed to screen miRNAs targeting AKTs and investigate their underlying mechanisms in HCC by integrating bioinformatics prediction, network analysis, functional assay and clinical validation.
Five online programs of miRNA target prediction and RNAhybrid which calculate the minimum free energy (MFE) of the duplex miRNA:mRNA were used to screen optimized miRNA-AKT interactions. Then, miRNA-regulated protein interaction network was constructed and 5 topological features (‘Degree’, ‘Node-betweenness’, ‘Edge-betweenness’, ‘Closeness’ and ‘Modularity’) were analyzed to link candidate miRNA-AKT interactions to oncogenesis and cancer hallmarks. Further systematic experiments were performed to validate the prediction results.
Six optimized miRNA-AKT interactions (miR-149-AKT1, miR-302d-AKT1, miR-184-AKT2, miR-708-AKT2, miR-122-AKT3 and miR-124-AKT3) were obtained by combining the miRNA target prediction and MFE calculation. Then, 103 validated targets for the 6 candidate miRNAs were collected from miRTarBase. According to the enrichment analysis on GO items and KEGG pathways, these validated targets were significantly enriched in many known oncogenic pathways for HCC. In addition, miRNA-regulated protein interaction network were divided into 5 functional modules. Importantly, AKT1 and its interaction with mTOR respectively had the highest node-betweenness and edge-betweenness, implying their bottleneck roles in the network. Further experiments confirmed that miRNA-149 directly targeted AKT1 in HCC by a miRNA luciferase reporter approach. Then, re-expression of miR-149 significantly inhibited HCC cell proliferation and tumorigenicity by regulating AKT1/mTOR pathway. Notably, miR-149 down-regulation in clinical HCC tissues was correlated with tumor aggressiveness and poor prognosis of patients.
This comprehensive analysis identified a list of miRNAs targeting AKTs and revealed their critical roles in HCC malignant progression. Especially, miR-149 may function as a tumor suppressive miRNA and play an important role in inhibiting the HCC tumorigenesis by modulating the AKT/mTOR pathway. Our clinical evidence also highlight the prognostic potential of miR-149 in HCC. The newly identified miR-149/AKT/mTOR axis might be a promising therapeutic target in the prevention and treatment of HCC.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-253) contains supplementary material, which is available to authorized users.
PMCID: PMC4255446  PMID: 25424347
Hepatocellular carcinoma; microRNA-regulated protein interaction network; microRNA-149; AKT/mTOR pathway; Prognosis
15.  S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton 
PLoS ONE  2009;4(3):e4881.
Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of α-methylene-γ-lactone and 2-phenyl indole compound, possessed potent activity against this pathway.
Methodology/Principal Findings
Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or tubulin were analyzed by molecular modeling. Anti-tumor activity of S9 was evaluated in tumor cells and in nude mice bearing human cancer xenografts. S9 abrogated EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110α subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor xenografts.
Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development.
PMCID: PMC2654064  PMID: 19293927
16.  Morphoproteomic Confirmation of a Constitutively Activated mTOR Pathway in High Grade Prostatic Intraepithelial Neoplasia and Prostate Cancer 
Preclinical studies have implicated the mammalian target of rapamycin (mTOR) pathway in the cell cycle progression and growth of prostate cancer cells. Downstream signaling from PI3′-K/Akt leads to phosphorylation (p) of mTOR at serine 2448 and to activation of its substrate, p70S6Kinase (p70S6K), phosphorylated on threonine 389. This promotes translation and cell cycle progression. Morphoproteomic analysis, that combines both the application of phosphospecific probes directed against putative sites of activation on protein analytes and cellular compartmentalization [1] was carried out on tissue microarray (TMA) slides from 64 cases of primary, previously untreated adenocarcinomas of the prostate. Gleason scores ranged from 6 to 10. High grade prostatic intraepithelial neoplasia (HGPIN), which accompanied the invasive cancer in 20 cases, and 15 non-neoplastic controls from benign prostatic hypertrophy specimens in a separate TMA were also included. Ninety-three percent (93%) of tumors exhibited moderate to strong cytoplasmic/plasmalemmal expression of p-mTOR and eighty-five percent (85%) showed similar staining intensity for p-p70S6K. HGPIN demonstrated comparable and occasionally, stronger expression levels for these protein analytes. Quantitative digital imaging revealed an overall increase in the mean expression levels in HGPIN, reaching statistical significance for p-mTOR (Ser 2448) at p<0.05. Morphoproteomic analysis confirms the constitutive activation of the mTOR pathway in prostate cancer and HGPIN, with relative overexpression of p-mTOR in HGPIN. These findings coincide with preclinical studies in supporting a role for the mTOR pathway in the biology and development of prostate cancer through its putative precursor lesion, HGPIN and in suggesting a potential therapeutic target.
PMCID: PMC2480543  PMID: 18787612
mTOR pathway; prostate cancer; high grade PIN; morphoproteomics; tissue microarray
17.  Role of mTOR in anticancer drug resistance 
The mammalian target of rapamycin (mTOR) pathway plays a central role in regulating protein synthesis, ribosomal protein translation, and cap-dependent translation. Deregulations in mTOR signaling are frequently associated with tumorigenesis, angiogenesis, tumor growth and metastasis. This review highlights the role of the mTOR in anticancer drug resistance. We discuss the network of signaling pathways in which the mTOR kinase is involved, including the structure and activation of the mTOR complex and the pathways upstream and downstream of mTOR as well as other molecular interactions of mTOR. Major upstream signaling components in control of mTOR activity are PI3K/PTEN/AKT and Ras/Raf/MEK/ERK pathways. We discuss the central role of mTOR in mediating the translation of mRNAs of proteins related to cell cycle progression, those involved in cell survival such as c-myc, hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF), cyclin A, cyclin dependent kinases (cdk1/2), cdk inhibitors (p21Cip1 and p27Kip1), retinoblastoma (Rb) protein, and RNA polymerases I and III. We then discuss the potential therapeutic opportunities for using mTOR inhibitors rapamycin, CCI-779, RAD001, and AP-23573 in cancer therapy as single agents or in combinations to reverse drug resistance.
PMCID: PMC2519122  PMID: 18440854
mTOR; drug resistance; p70S6K1; PI3K; AKT; MAP kinase; VEGF; CCI-779; RAD001 (everolimus); AP-23573; neurofibromatosis 1
18.  Cooperative Effects of Akt-1 and Raf-1 on the Induction of Cellular Senescence in Doxorubicin or Tamoxifen Treated Breast Cancer Cells 
Oncotarget  2011;2(8):610-626.
Escape from cellular senescence induction is a potent mechanism for chemoresistance. Cellular senescence can be induced in breast cancer cell lines by the removal of estrogen signaling with tamoxifen or by the accumulation of DNA damage induced by the chemotherapeutic drug doxorubicin. Long term culturing of the hormone-sensitive breast cancer cell line MCF-7 in doxorubicin (MCF-7/DoxR) reduced the ability of doxorubicin, but not tamoxifen, to induce senescence. Two pathways that are often upregulated in chemo- and hormonal-resistance are the PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways. To determine if active Akt-1 and Raf-1 can influence drug-induced senescence, we stably introduced activated ΔAkt-1(CA) and ΔRaf-1(CA) into drug-sensitive and doxorubicin-resistant cells. Expression of a constitutively-active Raf-1 construct resulted in higher baseline senescence, indicating these cells possessed the ability to undergo oncogene-induced-senescence. Constitutive activation of the Akt pathway significantly decreased drug-induced senescence in response to doxorubicin but not tamoxifen in MCF-7 cells. However, constitutive Akt-1 activation in drug-resistant cells containing high levels of active ERK completely escaped cellular senescence induced by doxorubicin and tamoxifen. These results indicate that up regulation of the Ras/PI3K/PTEN/Akt/mTOR pathway in the presence of elevated Ras/Raf/MEK/ERK signaling together can contribute to drug-resistance by diminishing cell senescence in response to chemotherapy. Understanding how breast cancers containing certain oncogenic mutations escape cell senescence in response to chemotherapy and hormonal based therapies may provide insights into the design of more effective drug combinations for the treatment of breast cancer.
PMCID: PMC3248208  PMID: 21881167
Akt; ERK; mTOR; Senescence; Drug Resistance; Tamoxifen
19.  Antitumor Activity of Rapamycin in a Phase I Trial for Patients with Recurrent PTEN-Deficient Glioblastoma 
PLoS Medicine  2008;5(1):e8.
There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.
Methods and Findings
Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).
Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.
Trial registration: (#NCT00047073).
In a Phase I clinical trial Charles Sawyers and colleagues investigated the role of rapamycin in patients with PTEN-deficient glioblastoma.
Editors' Summary
Glioblastoma is a highly malignant tumor of the brain. As with other tumors, it can result from a number of different molecular changes. Traditional chemotherapy does little more than contain these tumors, and cannot cure it. An alternative approach to the treatment of such tumors is to target specific molecular changes in the tumor. Obviously such targeted treatment will work only in patients who have the specific molecular defect being targeted. Hence, traditional clinical trials, which include a large variety of different patients and tumors with different genetic changes, may be an inappropriate way to test how effective targeted treatments are.
One specific change that has been identified in around 40% of patients with glioblastoma is inactivation of a gene known as PTEN, which acts as a tumor suppressor gene. When PTEN is inactivated it has previously been shown to make cells more sensitive to a class of drugs known as mTOR inhibitors—one of which is rapamycin (trade name Sirolimus). mTOR is a protein that is involved in the regulation of a number of cellular processes including growth and proliferation. Drugs active against mTOR are currently being tested for effectiveness against other cancers and as immunosuppressive agents.
Why Was This Study Done?
This was a Phase I study—that is, the earliest type of a drug study that is done in humans—which aimed to look at the safety of rapamycin in a selected group of patients who were undergoing surgery after recurrence of glioblastoma, and whose tumors did not express PTEN. In addition, the authors also wanted to assess the feasibility of incorporating detailed molecular studies of the action of this drug into such a Phase I study and whether these molecular studies could predict whether patients were more or less likely to respond to rapamycin.
What Did the Researchers Do and Find?
A total of 15 patients were treated with rapamycin at differing doses for one week before surgery and then again after surgery until there was evidence that the tumors were progressing. There was no evidence of very severe toxicity in any of the patients, though there were some adverse effects that required treatment. When samples from the patients were tested after surgery, seven of them showed a reduction in how rapidly the tumor cells divided, and this reduction was associated with how much inhibition there was of mTOR. Two of these patients showed evidence on scans of a reduction in tumor mass. Cells from tumors that appeared resistant to rapamycin in patients were sensitive to rapamycin in tissue culture, suggesting that the lack of response was due to the drug not being able to penetrate the tumor. A second, unfortunate effect of rapamycin was to cause activation of another intracellular protein, Akt, in some patients; when this activation occurred, patients had a shorter time between surgery and a return of their disease.
What Do These Findings Mean?
The detailed molecular studies within this Phase I trial allow a better understanding of how this targeted drug works. These findings suggest that the rapamycin can reduce the proliferation rate of glioblastoma cells, and that this reduction appears to be related to how well the drug is able to penetrate the tumor and inhibit mTOR. However, in some patients the activation of a second pathway can speed up the course of the disease, so further trials should incorporate inhibitors of this second pathway.
Additional Information.
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides information on all aspects of cancer (in English and Spanish)
The UK charity Cancerbackup provides information on brain tumors
Wikipedia has a page on mTOR (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2211560  PMID: 18215105
20.  Two hits are better than one: targeting both phosphatidylinositol 3-kinase and mammalian target of rapamycin as a therapeutic strategy for acute leukemia treatment 
Oncotarget  2012;3(4):371-394.
Phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) are two key components of the PI3K/Akt/mTOR signaling pathway. This signal transduction cascade regulates a wide range of physiological cell processes, that include differentiation, proliferation, apoptosis, autophagy, metabolism, motility, and exocytosis. However, constitutively active PI3K/Akt/mTOR signaling characterizes many types of tumors where it negatively influences response to therapeutic treatments. Hence, targeting PI3K/Akt/mTOR signaling with small molecule inhibitors may improve cancer patient outcome. The PI3K/Akt/mTOR signaling cascade is overactive in acute leukemias, where it correlates with enhanced drug-resistance and poor prognosis. The catalytic sites of PI3K and mTOR share a high degree of sequence homology. This feature has allowed the synthesis of ATP-competitive compounds targeting the catalytic site of both kinases. In preclinical models, dual PI3K/mTOR inhibitors displayed a much stronger cytotoxicity against acute leukemia cells than either PI3K inhibitors or allosteric mTOR inhibitors, such as rapamycin. At variance with rapamycin, dual PI3K/mTOR inhibitors targeted both mTOR complex 1 and mTOR complex 2, and inhibited the rapamycin-resistant phosphorylation of eukaryotic initiation factor 4E-binding protein 1, resulting in a marked inhibition of oncogenic protein translation. Therefore, they strongly reduced cell proliferation and induced an important apoptotic response. Here, we reviewed the evidence documenting that dual PI3K/mTOR inhibitors may represent a promising option for future targeted therapies of acute leukemia patients.
PMCID: PMC3380573  PMID: 22564882
apoptosis; leukemia initiating cells; mRNA translation; PI3K/Akt/mTOR; targeted therapy
21.  Dysregulation of mammalian target of rapamycin pathway in plasmacytoid variant of urothelial carcinoma of the urinary bladder☆,☆☆ 
Human pathology  2012;44(4):612-622.
Plasmacytoid urothelial carcinoma is a rare but aggressive variant of bladder cancer with no clear therapeutic guidelines. Dysregulation of the mammalian target of rapamycin (mTOR) pathway has been linked to oncogenesis in conventional bladder cancer. Several antineoplastic agents targeting mTOR pathway are currently available. This study assesses mTOR pathway status as well as c-myc and p27 expression. We retrieved 19 archival cases of plasmacytoid urothelial carcinoma from two institutions. Whole tissue sections were evaluated for immunoexpression of phosphatase and tensin homolog (PTEN), phosphorylated mTOR, phosphorylated protein kinase B (AKT), phosphorylated S6, c-myc, and p27. We evaluated intensity (0 to 3+) and extent (0%–100%) of expression for all markers. An H score was calculated as the sum of products of intensity and extent for each marker and used during analysis. In addition, PTEN loss was defined as absence of expression in >10% of tumor cells. We encountered PTEN loss in 28%. Higher H score for nuclear phosphorylatedAKT and a lowerHscore for phosphorylated S6 was encountered in muscle invasive tumors compared to non-muscle invasive tumors (P = .007 and P = .009, respectively). Although a trend for negative prognostic impact on overall survival for higher phosphorylated mTOR expression was noted (P = .051), markers expression levels failed to predict survival in our cohort. We found dysregulation of mTOR pathway members in urinary bladder plasmacytoid urothelial carcinoma, suggesting that the use of mTOR pathway inhibitors might be beneficial for patients with this aggressive tumor.
PMCID: PMC3742093  PMID: 23084634
Mammalian target of rapamycin; PTEN; Plasmacytoid urothelial carcinoma; Bladder
22.  18F-FDG Is a Surrogate Marker of Therapy Response and Tumor Recovery after Drug Withdrawal during Treatment with a Dual PI3K/mTOR Inhibitor in a Preclinical Model of Cisplatin-Resistant Ovarian Cancer12 
Translational Oncology  2013;6(5):586-595.
AIM: Targeting the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming chemoresistance in ovarian cancer. We investigated the capability of 18F-fluororodeoxyglucose (18F-FDG) small-animal positron emission tomography (SA-PET) to predict the effects of a dual PI3K/mTOR inhibitor (BEZ-235) in a cisplatin-resistant ovarian cancer model. METHODS: In a first experiment, nude rats bearing subcutaneous SKOV3 tumors received BEZ-235 for 3 days given alone or after paclitaxel and were compared to controls (either untreated or that were given the excipients of paclitaxel and BEZ-235). SA-PET was performed at baseline, on day 3, and day 7. In a second experiment aiming at further exploring the kinetics of 18F-FDG tumor uptake during the first 48 hours following drug cessation, untreated controls were compared to rats receiving BEZ-235, which were imaged at baseline, on day 3, on day 4, and on day 5. SA-PET results were compared to cell proliferation assessment (Ki-67), PI3K/mTOR downstream target expression studies (pAKT and phospho-eukaryotic translation initiation factor 4E-binding protein 1), and apoptosis evaluation (cleaved caspase-3). RESULTS: In the first experiment, BEZ-235, compared to untreated controls, induced a marked decrease in 18F-FDG uptake on day 3, which was correlated to a significant decrease in cell proliferation and to a significant PI3K/mTOR pathway inhibition. No tumor necrosis or apoptosis occurred. Four days following treatment cessation, tumor recovery (in terms of PI3K/mTOR inhibition and cell proliferation) occurred and was identified by 18F-FDG SA-PET. Paclitaxel plus BEZ-235 showed results similar to BEZ-235 alone. In the second experiment, PI3K/mTOR pathways exhibited partial recovery as early as 24 hours following treatment cessation, but both 18F-FDG SA-PET and cell proliferation remained unchanged. CONCLUSIONS: 18F-FDG SA-PET is a surrogate marker of target inhibition during treatment with BEZ-235 and predicts tumor recovery 4 days after drug withdrawal, but not during the first 48 hours following drug cessation, when a lag between PI3K/mTOR pathway recovery and metabolic recovery is observed. 18F-FDG SA-PET could be used for therapy monitoring of PI3K/mTOR inhibitors, but our results also raise questions regarding the potential impact of the delay between PET imaging and the last drug intake on the accuracy of FDG imaging.
PMCID: PMC3799200  PMID: 24151539
23.  Regulation of FANCD2 by the mTOR pathway contributes to the resistance of cancer cells to DNA double strand breaks 
Cancer research  2013;73(11):3393-3401.
Deregulation of the mTOR pathway is closely associated with tumorigenesis. Accordingly mTOR inhibitors such as rapamycin and mTOR-selective kinase inhibitors have been tested as cancer therapeutic agents. Inhibition of mTOR results in sensitization to DNA damaging agents, however the molecular mechanism is not well understood. We found that an mTOR-selective kinase inhibitor, AZD8055, significantly enhanced sensitivity of a pediatric rhabdomyosarcoma xenograft toradiotherapy and sensitized rhabdomyosarcoma cells to interstrand crosslinker (ICL) melphalan. Sensitization correlated with drug-induced downregulation of a key component of the Fanconi anemia (FA) pathway, FANCD2 through mTOR regulation of FANCD2 gene transcripts via mTORC1-S6K1. Importantly, we show that FANCD2 is required for the proper activation of ATM-Chk2 checkpoint in response to ICL and that mTOR signaling promotes ICL-induced ATM-Chk2 checkpoint activation by sustaining FANCD2. In FANCD2 deficient lymphoblasts, FANCD2 is essential to suppress endogenous and induced DNA damage, and FANCD2-deficient cells demonstrated impaired ATM-Chk2 and ATR-Chk1 activation, which was rescued by re-introduction of wild type FANCD2. Pharmacological inhibition of PI3K-mTOR-AKT pathway in Rh30 rhabdomyosarcoma cells attenuated ICL-induced activation of ATM, accompanied with the decrease of FANCD2. These data suggest that the mTOR pathway may promote the repair of DNA double strand breaks by sustaining FANCD2 and provide a novel mechanism of how the FA pathway modulates DNA damage response and repair.
PMCID: PMC3674187  PMID: 23633493
mammalian target of rapamycin; DNA damage response; ATM; FANCD2; interstrand crosslink
Neuro-Oncology  2014;16(Suppl 5):v27.
BACKGROUND: Mammalian target of rapamycin (mTOR) signaling plays a critical role in tumor cell function and mTOR inhibitors have demonstrated promising activity in glioblastoma multiforme (GBM). Nevertheless, robust genetic predictors for sensitivity to mTOR inhibition remain elusive. As part of the biomarker discovery process, the North Central Cancer Treatment Group (NCCTG) incorporated functional imaging with 18FLT-PET/CT and focused exon sequencing into N057K, a Phase II clinical trial evaluating the mTORC1 inhibitor everolimus in combination with radiation(RT) and temozolomide(TMZ) in newly diagnosed GBM patients. METHODS: N057K began weekly everolimus one week prior to standard RT/TMZ. Patients who had >1cm3 of residual tumor were imaged with 18FLT-PET/CT before and after the initial two doses of everolimus, before initiating RT/TMZ. Imaged patients with a ΔSUVmax of >25% were classified as metabolic responders adapted from EORTC criteria for FDG-PET. Imaged patients with sufficient tumor samples also underwent immunohistochemical and focused exon sequencing analysis using the Seqwright 201 gene panel. Tertiary analysis focused on genes implicated in the PI3K/Akt/mTOR pathway (PTEN, PIK3CA, AKT, mTOR, Raptor/Rictor, S6, TSC1/2, NF1, CCND1, FGFR1/2). RESULTS: Nine patients had 18FLT-PET/CT imaging of which 4/9 had a metabolic response to everolimus alone. Six patients had sufficient tumor samples for further analysis, of which 3/6 had response. On IHC, 18FLT-PET responders had higher levels of PTEN expression and lower levels of pAKT and Ki-67 expression in comparison to non-responders. Focused exon sequencing found 1,458 coding alterations in 454 genes when compared to the reference genome. Only one 18FLT-PET responder had any genetic alterations within a PI3K/Akt/mTOR-implicated pathway, while all 18FLT-PET non-responders had multiple alterations within PI3K/Akt/mTOR-implicated pathways. CONCLUSIONS: 18FLT-PET successfully identified cohorts of patients who had robust, early metabolic responses to mTOR inhibition alone. These responders had robust PTEN expression on IHC and were less likely to have alterations within PI3K/Akt/mTOR-implicated pathways.
PMCID: PMC4217888
25.  PI3K/AKT/mTOR pathway activation in primary and corresponding metastatic breast tumors after adjuvant endocrine therapy 
Both preclinical and clinical data suggest that activation of the PI3K/AKT/mTOR pathway in response to hormonal therapy results in acquired endocrine therapy resistance. We evaluated differences in activation of the PI3K/AKT/mTOR pathway in estrogen receptor α (ERα) positive primary and corresponding metastatic breast cancer tissues using immunohistochemistry for downstream activated proteins, like phosphorylated mTOR (p-mTOR), phosphorylated 4E Binding Protein 1 (p-4EBP1) and phosphorylated p70S6K (p-p70S6K). For p-mTOR and p-4EBP1, the proportion of immunostained tumor cells (0–100%) was scored. Cytoplasmic intensity (0–3) was assessed for p-p70S6K. The difference between expression of these activated PI3K/AKT/mTOR proteins- in primary and metastatic tumor was calculated and tested for an association with adjuvant endocrine therapy. In patients who had received endocrine therapy (N = 34), p-mTOR expression increased in metastatic tumor lesions compared to the primary tumor (median difference 45%), while in patients who had not received adjuvant endocrine therapy (N = 37), no difference was found. Similar results were observed for p-4EBP1 and p-p70S6K expression. In multivariate analyses, adjuvant endocrine therapy was significantly associated with an increase in p-mTOR (p = 0.01), p-4EBP1 (p = 0.03) and p-p70S6K (p = 0.001), indicating that compensatory activation of the PI3K/AKT/mTOR pathway might indeed be a clinically relevant resistance mechanism resulting in acquired endocrine therapy resistance.
What's new?
Inhibitors of the PI3K/AKT/mTOR pathway can overcome the resistance to estrogen-depletion therapy that often develops in metastatic breast cancer. In this study, the authors compared primary and metastatic tumors; their results suggest that activation of the PI3K/AKT/mTOR pathway in patients who receive adjuvant endocrine therapy is a clinically relevant mechanism of acquired hormone resistance. For identification of companion diagnostics for PI3K/AKT/mTOR inhibitors, the authors conclude that analyzing primary tumor tissue may often fail to predict treatment response in metastatic breast cancer.
PMCID: PMC4277331  PMID: 24501006
PI3K/AKT/mTOR pathway; endocrine therapy, acquired hormone resistance

Results 1-25 (1485331)