Constitutive Activation Of mTOR Research Articles

Immunophenotyping of peripheral blood mononuclear cells in tuberous sclerosis complex

Abstract Tuberous sclerosis complex (TSC) is a multisystem disorder caused by loss-of-function mutations in TSC1/2 proteins. This results in constitutive mTOR activation and predisposes to the development of benign tumours, with renal angiomyolipomas (AMLs) and tuber-related drug-resistant epilepsy (DRE) being major causes of morbidity and mortality in adults. Despite the central role of mTOR in leukocyte biology and the beneficial impact of mTOR inhibitors in TSC, the role of the immune system and inflammation in the development of tumours and DRE in people with TSC remains unknown. Using peripheral blood mononuclear cells (PBMCs) and serum from people with TSC, healthy controls (HC) and non-TSC DRE, we employed flow cytometry and multiplex assays to interrogate the immune profile associated with chronic loss of mTOR inhibition. We found that higher levels of the astroglial activation marker GFAP are elevated in TSC and show high intergenotype variability. We also observed differences in circulating immune cell populations, cytokines, chemokines and growth factors related to TSC. We found a higher proportion of B cells in parallel with increased total IgG but a reduction in IL-2 and IL-7 and a lower frequency of T cells. TSC with vs without AML showed differences in CD4:CD8 T cells ratio, monocytes and dendritic cells frequencies. Finally, loss of TSC1/2 affected T cell polarization, leading to an increased proportion of T c17CD8 +T cells, especially in subjects with DRE. Overall, our findings indicate TSC is associated with increased blood biomarkers for inflammation, glial activation, and distribution and profile of circulating PBMCs compared to controls.

Prospective phase II trial of the dual mTORC1/2 inhibitor vistusertib for progressive or symptomatic meningiomas in persons with neurofibromatosis 2.

Meningiomas occur in 80% of persons with neurofibromatosis 2 (NF2) and cause significant mortality and morbidity, yet there are no effective medical treatments. NF2-deficient tumors have constitutive activation of mammalian/mechanistic target of rapamycin (mTOR), and treatment with mTORC1 inhibitors results in growth arrest in a minority of tumors, with paradoxical activation of the mTORC2/AKT pathway. We studied the effect of vistusertib, a dual mTORC1/mTORC2 inhibitor, in NF2 patients with progressive or symptomatic meningiomas. Vistusertib was administered orally at 125 mg twice daily for 2 consecutive days each week. The primary endpoint was the imaging response in the target meningioma, defined as a volume decrease of 20% compared with the baseline. Secondary endpoints included toxicity, imaging response of nontarget tumors, quality of life, and genetic biomarkers. Eighteen participants (13 female), median age of 41 (range, 18-61) years, were enrolled. In target meningiomas, the best response was partial response (PR) in 1/18 tumors (6%) and stable disease (SD) in 17/18 tumors (94%). For all measured intracranial meningiomas and vestibular schwannomas, the best imaging response was PR in 6/59 tumors (10%) and SD in 53 (90%). Treatment-related grade 3/4 adverse events occurred in 14 (78%) participants, and 9 participants discontinued treatment due to side effects. Although the study did not meet the primary endpoint, vistusertib treatment was associated with high rates of SD in progressive NF2-related tumors. However, this dosing regimen for vistusertib was poorly tolerated. Future studies of dual mTORC inhibitors for NF2 should focus on optimizing tolerability and evaluating the relevance of tumor stability in participants.

Knockout of TSC2 in Nav1.8+ neurons predisposes to the onset of normal weight obesity

ObjectiveObesity and nutrient oversupply increase mammalian target of rapamycin (mTOR) signaling in multiple cell types and organs, contributing to the onset of insulin resistance and complications of metabolic disease. However, it remains unclear when and where mTOR activation mediates these effects, limiting options for therapeutic intervention. The objective of this study was to isolate the role of constitutive mTOR activation in Nav1.8-expressing peripheral neurons in the onset of diet-induced obesity, bone loss, and metabolic disease. MethodsIn humans, loss of function mutations in tuberous sclerosis complex 2 (TSC2) lead to maximal constitutive activation of mTOR. To mirror this in mice, we bred Nav1.8-Cre with TSC2fl/fl animals to conditionally delete TSC2 in Nav1.8-expressing neurons. Male and female mice were studied from 4- to 34-weeks of age and a subset of animals were fed a high-fat diet (HFD) for 24-weeks. Assays of metabolism, body composition, bone morphology, and behavior were performed. ResultsBy lineage tracing, Nav1.8-Cre targeted peripheral sensory neurons, a subpopulation of postganglionic sympathetics, and several regions of the brain. Conditional knockout of TSC2 in Nav1.8-expressing neurons (Nav1.8-TSC2KO) selectively upregulated neuronal mTORC1 signaling. Male, but not female, Nav1.8-TSC2KO mice had a 4–10% decrease in body size at baseline. When challenged with HFD, both male and female Nav1.8-TSC2KO mice resisted diet-induced gains in body mass. However, this did not protect against HFD-induced metabolic dysfunction and bone loss. In addition, despite not gaining weight, Nav1.8-TSC2KO mice fed HFD still developed high body fat, a unique phenotype previously referred to as ‘normal weight obesity’. Nav1.8-TSC2KO mice also had signs of chronic itch, mild increases in anxiety-like behavior, and sex-specific alterations in HFD-induced fat distribution that led to enhanced visceral obesity in males and preferential deposition of subcutaneous fat in females. ConclusionsKnockout of TSC2 in Nav1.8+ neurons increases itch- and anxiety-like behaviors and substantially modifies fat storage and metabolic responses to HFD. Though this prevents HFD-induced weight gain, it masks depot-specific fat expansion and persistent detrimental effects on metabolic health and peripheral organs such as bone, mimicking the ‘normal weight obesity’ phenotype that is of growing concern. This supports a mechanism by which increased neuronal mTOR signaling can predispose to altered adipose tissue distribution, adipose tissue expansion, impaired peripheral metabolism, and detrimental changes to skeletal health with HFD – despite resistance to weight gain.

MTOR signaling regulates central and peripheral circadian clock function.

The circadian clock coordinates physiology and metabolism. mTOR (mammalian/mechanistic target of rapamycin) is a major intracellular sensor that integrates nutrient and energy status to regulate protein synthesis, metabolism, and cell growth. Previous studies have identified a key role for mTOR in regulating photic entrainment and synchrony of the central circadian clock in the suprachiasmatic nucleus (SCN). Given that mTOR activities exhibit robust circadian oscillations in a variety of tissues and cells including the SCN, here we continued to investigate the role of mTOR in orchestrating autonomous clock functions in central and peripheral circadian oscillators. Using a combination of genetic and pharmacological approaches we show that mTOR regulates intrinsic clock properties including period and amplitude. In peripheral clock models of hepatocytes and adipocytes, mTOR inhibition lengthens period and dampens amplitude, whereas mTOR activation shortens period and augments amplitude. Constitutive activation of mTOR in Tsc2–/–fibroblasts elevates levels of core clock proteins, including CRY1, BMAL1 and CLOCK. Serum stimulation induces CRY1 upregulation in fibroblasts in an mTOR-dependent but Bmal1- and Period-independent manner. Consistent with results from cellular clock models, mTOR perturbation also regulates period and amplitude in the ex vivo SCN and liver clocks. Further, mTOR heterozygous mice show lengthened circadian period of locomotor activity in both constant darkness and constant light. Together, these results support a significant role for mTOR in circadian timekeeping and in linking metabolic states to circadian clock functions.

DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons

Mutations in DEPDC5 and NPRL3 subunits of GATOR1, a modulator of mechanistic target of rapamycin (mTOR), are linked to malformations of cortical development (MCD). Brain specimens from these individuals reveal abnormal cortical lamination, altered cell morphology, and hyperphosphorylation of ribosomal S6 protein (PS6), a marker for mTOR activation. While numerous studies have examined GATOR1 subunit function in non-neuronal cell lines, few have directly assessed loss of GATOR1 subunit function in neuronal cell types. We hypothesized that DEPDC5 or NPRL3 shRNA-mediated knockdown (DEPDC5/NPRL3 KD) leads to inappropriate functional activation of mTOR and mTOR-dependent alterations in neuronal morphology.Neuronal size was determined in human specimens harboring DEPDC5 or NPRL3 mutations resected for epilepsy treatment. DEPDC5/NPRL3 KD effects on cell size, filopodial extension, subcellular mTOR complex 1 (mTORC1) localization, and mTORC1 activation during nutrient deprivation were assayed in mouse neuroblastoma cells (N2aC) and mouse subventricular zone derived neural progenitor cells (mNPCs). mTORC1-dependent effects of DEPDC5/NPRL3 KD were determined using the mTOR inhibitor rapamycin. Changes in mTOR subcellular localization and mTORC1 pathway activation following DEPDC5/NPRL3 KD were determined by examining the proximity of mTOR to the lysosomal surface during amino acid starvation.Neurons exhibiting PS6 immunoreactivity (Ser 235/236) in human specimens were 1.5× larger than neurons in post-mortem control samples. DEPDC5/NPRL3 KD caused mTORC1, but not mTORC2, hyperactivation, soma enlargement, and increased filopodia in N2aC and mNPCs compared with wildtype cells. DEPDC5/NPRL3 KD led to inappropriate mTOR localization at the lysosome along with constitutive mTOR activation following amino acid deprivation. DEPDC5/NPRL3 KD effects on morphology and functional mTOR activation were reversed by rapamycin. mTOR-dependent effects of DEPDC5/NPRL3 KD on morphology and subcellular localization of mTOR in neurons suggests that loss-of-function in GATOR1 subunits may play a role in MCD formation during fetal brain development.

Inhibition of placental mTOR signaling provides a link between placental malaria and reduced birthweight

BackgroundPlacental Plasmodium falciparum malaria can trigger intervillositis, a local inflammatory response more strongly associated with low birthweight than placental malaria infection alone. Fetal growth (and therefore birthweight) is dependent on placental amino acid transport, which is impaired in placental malaria-associated intervillositis. Here, we tested the hypothesis that mechanistic target of rapamycin (mTOR) signaling, a pathway known to regulate amino acid transport, is inhibited in placental malaria-associated intervillositis, contributing to lower birthweight.MethodsWe determined the link between intervillositis, mTOR signaling activity, and amino acid uptake in tissue biopsies from both uninfected placentas and malaria-infected placentas with and without intervillositis, and in an in vitro model using primary human trophoblast (PHT) cells.ResultsWe demonstrated that (1) placental mTOR activity is lower in cases of placental malaria with intervillositis, (2) placental mTOR activity is negatively correlated with the degree of inflammation, and (3) inhibition of placental mTOR activity is associated with reduced placental amino acid uptake and lower birthweight. In PHT cells, we showed that (1) inhibition of mTOR signaling is a mechanistic link between placental malaria-associated intervillositis and decreased amino acid uptake and (2) constitutive mTOR activation partially restores amino acid uptake.ConclusionsOur data support the concept that inhibition of placental mTOR signaling constitutes a mechanistic link between placental malaria-associated intervillositis and decreased amino acid uptake, which may contribute to lower birthweight. Restoring placental mTOR signaling in placental malaria may increase birthweight and improve neonatal survival, representing a new potential therapeutic approach.

Curcumin targets the TFEB-lysosome pathway for induction of autophagy.

Curcumin is a hydrophobic polyphenol derived from the herb Curcumalonga and its wide spectrum of pharmacological activities has been widely studied. It has been reported that Curcumin can induce autophagy through inhibition of the Akt-mTOR pathway. However, the effect of Curcumin on lysosome remains largely elusive. In this study, we first found that Curcumin treatment enhances autophagic flux in both human colon cancer HCT116 cells and mouse embryonic fibroblasts (MEFs). Moreover, Curcumin treatment promotes lysosomal function, evidenced by the increased lysosomal acidification and enzyme activity. Second, Curcumin is capable of suppressing the mammalian target of rapamycin (mTOR). Interestingly, Curcumin fails to inhibit mTOR and to activate lysosomal function in Tsc2−/−MEFs with constitutive activation of mTOR, indicating that Curcumin-mediated lysosomal activation is achieved via suppression of mTOR. Third, Curcumin treatment activates transcription factor EB (TFEB), a key nuclear transcription factor in control of autophagy and lysosome biogenesis and function, based on the following observations: (i) Curcumin directly binds to TFEB, (ii) Curcumin promotes TFEB nuclear translocation; and (iii) Curcumin increases transcriptional activity of TFEB. Finally, inhibition of autophagy and lysosome leads to more cell death in Curcumin-treated HCT116 cells, suggesting that autophagy and lysosomal activation serves as a cell survival mechanism to protect against Curcumin-mediated cell death. Taken together, data from our study provide a novel insight into the regulatory mechanisms of Curcumin on autophagy and lysosome, which may facilitate the development of Curcumin as a potential cancer therapeutic agent.

The Mechanism of Adaptation of Breast Cancer Cells to Hypoxia: Role of AMPK/mTOR Signaling Pathway.

We studied the mechanisms of adaptation of human breast cancer cells MCF-7 to hypoxia and analyzed the role of AMPK/mTOR signaling pathway in the maintenance of cell proliferation under hypoxic conditions. It was found that long-term culturing (30 days or more) of MCF-7 cells under hypoxic conditions induced their partial adaptation to hypoxia. Cell adaptation to hypoxia was associated with attenuation of hypoxia-dependent AMPK induction with simultaneous constitutive activation of mTOR and Akt. These findings suggest that these proteins can be promising targets for targeted therapy of tumors developing under hypoxic conditions.

Wild type mesenchymal cells contribute to the lung pathology of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.

A carbazole alkaloid deactivates mTOR through the suppression of rictor and that induces apoptosis in lung cancer cells.

Non-small cell lung cancer (NSCLC) is known to be a difficult cancer to treat because of its poor prognosis, limited option for surgery, and resistance to chemo or radiotherapy. In this study, we have demonstrated that suppression of rictor expression in A549 and H1299 NSCLC cells by mahanine, a carbazole alkaloid, disrupted constitutive activation of mTOR and Akt. Mahanine suppression of rictor gene expression and consequent attenuation of its protein expression affected the inhibition of mTOR (Ser-2481) and Akt (Ser-473) phosphorylation. Since mahanine treatment revealed this new insight of rictor-mTOR relationship, we examined an association between mTOR activation with rictor expression. Interestingly, in rictor knockdown (KD) NSCLC cells, mTOR activation was significantly impaired. Transfection of rictor over-expression vector into the NSCLC cells reversed this situation. In fact, both rictor KD and mahanine treated cells showed considerably depleted phospho-mTOR level. These results indicate that rictor is required to maintain constitutive activation of mTOR in lung cancer cells. When mTOR kinase activity in rictor KD cells was examined with Akt as substrate, a significant reduction of Akt phosphorylation indicated impairment of mTOR kinase potentiality. Disruption of mTOR and Akt activation caused drastic mortality of NSCLC cancer cells through apoptosis. Hence, our study reveals a new dimension in mTOR-rictor relationship, where rictor stands to be a suitable therapeutic target for lung cancer.

Roles of mTORC1 in acute myeloid leukemia

Constitutive activation of mTOR is associated with acceleration of leukemia development. However, mTORC1 functions in established leukemia are unclear. We evaluated the role of mTORC1 in mouse acute myeloid leukemia (AML) cells using a murine model of conditional deletion of Raptor, an essential component of mTORC1, and an MLL-AF9-driven leukemia model. mTORC1 is essential for leukemia initiation, but a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. Our study demonstrated AML cells lacking mTORC1 activity to be capable of self-renewal as AML stem cells. This review will summarize our study on the roles of mTORC1 in AML, and discuss potential benefits and risks of mTORC2 inhibition and the potential use of mTOR inhibitors as AML therapy.

Melittin exerts antitumorigenic effects in human MM1.S multiple myeloma cells through the suppression of AKT/mTOR/S6K1/4E-BP1 signaling cascades

Although melittin, a water-soluble 26-amino acid peptide derived from bee venom of Apis mellifera, is known to exert anti-proliferative effects on various tumor cell lines, very little is known about its potential molecular mechanism(s) of action. In the present study, we investigated the effects of melittin on the AKT/mTOR/S6K1/4E-BP1 activation, associated gene products, cellular proliferation, and apoptosis in several tumor cells. We found that melittin inhibited both constitutive phosphorylation of AKT and mTOR and exerted a significant time-dependent anti-proliferative effect on MM1.S cells as compared to other types of tumor cells. Indeed, melittin clearly suppressed the constitutive activation of AKT/mTOR/S6K1/4E-BP1 signaling cascades, which correlated with the induction of apoptosis. Melittin can cause broad-spectrum inhibition of AKT/mTOR/S6K1/4E-BP1 axes in multiple myeloma cells when compared with various pharmacological AKT/mTOR inhibitors. Aberrant AKT activation by pcDNA3-myr-HA-AKT1 plasmid could not prevent the observed suppressive effect of melittin on constitutive mTOR, S6K1, and 4E-BP1 activation and overexpression of Bcl-2 also attenuated melittin-mediated apoptosis in the cells. Our results clearly indicate that melittin can interfere with multiple signaling cascades involved in carcinogenesis and thereby used as a potential drug candidate for both the prevention and treatment of cancer.

Abstract 4776: PDK1-dependent activation of AGC kinases is an absolute requirement for AKT oncogenic activity

Abstract The PI3K signaling cascade is frequently activated in human cancer, and AKT is commonly considered its major transforming conduit. Thus, PI3K and AKT represent relevant targets for therapeutic purposes. However, the many essential of roles played by this pathway in normal cells poses the problem of reaching therapeutic efficacy without harming healthy tissues. In principle, it would be ideal to target only molecules directly involved in the transformation process, thus reducing the toxicity associated with wide-range inhibitors. PDK1 plays a key role in the PI3K cascade, directly activating AKT. In addition, PDK1 possesses a substrate docking site named “PIF pocket” that is required to phosphorylate and activate members of the AGC kinase family, including S6K, PKC, SGK, and RSK. Mice in which the PI3K pathway is constitutively activated in the thyroid epithelium through loss of Pten are born with thyroid hyperplasia that progresses to invasive and metastatic follicular carcinoma, a process that is accelerated by the simultaneous loss of p27 or p53, and by Ras or Braf activation. Using in vivo as well as ex vivo and in vitro genetic and pharmacological approaches, we now show that although Akt activation is necessary, it is not sufficient to transform thyroid epithelial cells. Concomitant activation of Pdk1-dependent pathways is absolutely required to develop neoplastic lesions in vivo, and to induce cell proliferation ex vivo. Pten mutants develop adenomas and carcinomas by one year of age. Strikingly, complete rescue of this phenotype is observed in compound mutants in which, in addition to thyroid-specific Pten loss, the PIF-pocket of Pdk1 has been genetically disrupted, maintaining constitutive activation of Akt and mTor, but not of PIF-pocket dependent AGC kinases. We also show that the transcription factor STAT3 represents a key mediator of this essential Pdk1-dependent pathway: STAT3 phosphorylation is increased in Pten mutants, and abolished in compound mutants. Furthermore, genetic and pharmacological inhibition of STAT3 drastically impairs cell proliferation in vitro and rescues the hyperplastic phenotype of Pten mutant mice in vivo. Thus, we have identified an essential and druggable signaling cascade that critically cooperates with AKT activation to transform thyroid epithelial cells. Citation Format: Arturo Orlacchio, Valeria Antico Arciuch, Antonio Di Cristofano. PDK1-dependent activation of AGC kinases is an absolute requirement for AKT oncogenic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4776. doi:10.1158/1538-7445.AM2014-4776

Abstract A68: Activity of the TORC 1/2 kinase inhibitor, CC-223, in hormone receptor positive (HR+) breast cancer cell lines and patients (pts) with genetically characterized HR+ breast cancer in a Phase I clinical trial.

Abstract Introduction: PIK3CA and PTEN are among the top five most frequently mutated genes in breast cancer. Activating mutation of PIK3CA or loss of function mutation in PTEN leads to constitutive activation of AKT and mTOR driving multiple downstream metabolic and proliferative pathways important to oncogenesis. There is also cross-talk between the PI3K/AKT/mTOR pathway and ER signaling and activation of the PI3K/AKT/mTOR pathway is associated with resistance to endocrine therapy. CC-223 is a potent and selective ATP-competitive mTOR kinase inhibitor, targeting both TORC1 and TORC2 complexes. Results: Pre-clinically, CC-223 potently inhibits the growth of breast cancer cell lines with GI50 values below 1 uM in 38 of 43 lines. Luminal-derived cell lines are more sensitive to CC-223 than basal-derived lines. Within the luminal subset, ER+, HER2+, PIK3CA mutant, or wild-type TP53 cell lines are more sensitive to CC-223; PTEN loss is not associated with increased CC-223 sensitivity. During the dose escalation part of the phase I clinical trial, 3 pts with breast cancer were enrolled. One pt with HR+/Her2- breast cancer had a durable PR (31 weeks). An expansion cohort of HR+/Her2- breast cancer enrolled 17 pts at a CC-223 dose of 45 mg QD in 28-day cycles and 13 pts were evaluable for tumor response. Deep sequencing of tumors for multiple cancer-related genes was performed. The most common (> 20%) related adverse events (all grades) reported in the breast cancer cohort were nausea, stomatitis, fatigue, anorexia, diarrhea, vomiting, hyperglycemia, rash, and thrombocytopenia. Exposure-dependent TORC1 (p4EBP1) and TORC2 (pAKT) inhibition was observed in blood cells; analysis of paired tumor biopsies is ongoing. Reduction in glucose uptake (> 25% decrease in SUV) on PET imaging at day 15 was observed in 4 of 8 patients with PET imaging data currently available. Three pts demonstrated RECIST PR in target lesions (one categorized as PD due to a new bone lesion); PIK3CA mutations were present in all 3 subjects. Of the two additional pts with PIK3CA mutations, one had SD > 6 months. The one PIK3CA mutated subject with PD at first restaging had a concurrent p53 mutation. Additional genetic abnormalities in mTOR and related pathways in subjects with target lesion PR involved PTEN, Rictor, and IGFR1 genes. Six subjects had SD after 2 cycles, with minor target lesion regression (0 to -30%) in 5 of 6, and with SD > 24 weeks in 1 of 6. Conclusion: The safety profile of CC-223 is typical for drugs targeting the mTOR pathway. Preclinical and clinical data support the activity of CC-223 in HR+ positive breast cancer, particularly in tumors with PIK3CA mutations. Breast Cancer Accrual: Cedars-Sinai (Mita): 4; SCRIL (Arkenau): 4; IGR (Varga): 4; SCRI (Bendell): 3 (all Part A); Moffitt (Mahipal): 2; UCSF (Munster): 1; JSOM (Paz-Ares): 1; ICR (DeLord): 1. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A68. Citation Format: Monica M. Mita, Hendrik-Tobias Arkenau, Johanna C. Bendell, Pamela N. Munster, Amit Mahipal, Jean-Pierre Delord, Luis G. Paz-Ares, Jean-Charles Soria, Shuichan Xu, Tam Tran, Tao Shi, Xiaoling Wu, Rajesh Chopra, Kristen Hege, Andrea Varga. Activity of the TORC 1/2 kinase inhibitor, CC-223, in hormone receptor positive (HR+) breast cancer cell lines and patients (pts) with genetically characterized HR+ breast cancer in a Phase I clinical trial. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A68.

Brassinin Induces Apoptosis in PC‐3 Human Prostate Cancer Cells through the Suppression of PI3K/Akt/mTOR/S6K1 Signaling Cascades

The oncogenic PI3K/Akt/mammalian target of rapamycin (mTOR) signaling axis and its downstream effector, the ribosomal protein S6 kinase 1 (S6K1) play a key role in mediating cell survival in various tumor cells. Here, we investigated the effects of brassinin (BSN), a phytoalexin first identified as a constituent of cabbage, on the PI3K/Akt/mTOR/S6K1 activation, cellular proliferation, and apoptosis in PC-3 human prostate cancer. BSN exerted a significant dose-dependent cytotoxicity and reduced constitutive phosphorylation of Akt against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, knockdown of androgen receptor (AR) by small interfering RNA enhanced the potential effect of BSN on induction of apoptosis in LNCaP cells. BSN clearly suppressed the constitutive activation of PI3K/Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, TUNEL staining, loss of mitochondrial membrane potential, down-regulation of antiapoptotic and proliferative proteins, activation of caspase-3, and cleavage of PARP. Additionally, BSN could block broad-spectrum inhibition of PI3K/Akt/mTOR/S6K1 axes, and aberrant Akt activation by pcDNA3-myr-HA-Akt1 plasmid could not prevent the observed suppressive effect of BSN on constitutive mTOR activation. Finally, overexpression of Bcl-2 also attenuated BSN-mediated apoptosis in PC-3 cells. Taken together, our findings suggest that BSN can interfere with multiple signaling cascades involved in tumorigenesis and might be provided as a potential therapeutic candidate for both the prevention and treatment of prostate cancer.

Abstract 2326: S6K activation is essential for the development of follicular thyroid cancer.

Abstract The PI3K signaling cascade is frequently activated in follicular and anaplastic thyroid tumors. Mice in which the PI3K pathway is constitutively activated through loss of Pten (PtenL/L;TPO-Cre mice) develop thyroid hyperplasia that progresses to thyroid follicular carcinoma. The wide range of downstream targets of this pathway raises the question of the relative role and relevance of its multiple effectors. This issue has important therapeutic implications, since it would be ideal to only target molecules directly involved in the transformation process, thus reducing the toxicity associated with wide-range inhibitors. PDK1 plays a central role in the PI3K cascade, acting as a master regulator of both AKT and S6K. Notably, PDK1 possesses a substrate docking site named “PIF pocket” that is required to phosphorylate and activate S6K but not AKT. In order to define the relative contribution of Akt and S6k to the development of thyroid lesions and to their progression to follicular carcinoma, we have generated a compound mouse strain in which, in addition to thyroid-specific Pten loss, the PIF-pocket site of Pdk1 can be conditionally disrupted by the L155E mutation, resulting in constitutive activation of Akt and mTor, but not of S6k. We have found that young double mutant mice display a significant reduction in thyroid size, compared to PtenL/L;TPO-Cre mice, and that such rescue is associated with a marked decrease in the thyrocytes proliferation rate. More strikingly, while 60% of Pten mutants develop adenomas and carcinomas by one year of age, none of the age-matched double mutant mice displays neoplastic lesions, indicating that Akt activation, in the absence of S6k activation, is completely unable to drive transformation. Furthermore, we have identified STAT3 as a molecule that is selectively activated downstream of S6k, and that is inactive in double mutants. Pharmacological STAT3 inhibition drastically reduces the proliferation rate of thyroid cancer cell lines, suggesting that STAT3 may be one key conduit of S6k oncogenic activity. Citation Format: Valeria Gabriela Antico Arciuch, Antonio Di Cristofano. S6K activation is essential for the development of follicular thyroid cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2013-2326

Abstract 5247: Human melanoma cells resist oxidative stress due to overactive MEK/ERK and AKT/mTOR pathways and overproduction of melanin.

Abstract Melanoma is considered as one of the most aggressive and less treatable cancers. While various models of mechanisms have been proposed, the transformation from tranquil melanocytes to invasive melanoma cells remains an enigma. Alteration of cellular redox status has been suggested to be associated with cellular transformation. We compared primary human melanocytes and melanoma cells and their responses to oxidative stress. Both cells were treated with H2O2 at doses between 5 and 250μM. Microscopic data showed that melanoma cells are more resistant to H2O2 treatment. Western blot and confocal microscopic data showed that in melanoma cells, H2O2 does not induce CRT translocation which is considered as a mechanism through which melanocytes surrender to oxidative stress and induces immunogenic cell death. Furthermore, Western blot analysis showed that melanoma cells express EGFR, responsive to EGF treatment and have constitutive activation of AKT and mTOR, which are all related to better survival. In contrast, in the primary human melanocytes, EGFR expression is almost undetectable. And confocal microscopic data confirm this observation. Treatments of cells with MEK/ERK inhibitor U0126 and PI3K/AKT inhibitor LY294002 attenuates S6 phosphorylation. We also observed that in melanoma cells, but not in melanocytes, low dose (5 to 250μM) of H2O2 induces melanin production that is recognized to protect from further oxidative stress. Collectively, our data suggest that active MEK/ERK and AKT/mTOR pathways and up-regulation of melanin production contribute to the resistance of melanoma cells to oxidative stress and attribute to the aggressiveness of melanoma. Our data provides insights into the understanding of the molecular mechanisms of the transformation from melanocytes to melanoma and support the notion from the most recently published proteomic studies that EGFR remains one of the most important targets for better clinical management of melanoma. Citation Format: David Calianese, Garrett Cammarata, Gabriella Brum, Kevin Szulak, Charles Best, Katelyn Higgins, Yinsheng Wan. Human melanoma cells resist oxidative stress due to overactive MEK/ERK and AKT/mTOR pathways and overproduction of melanin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5247. doi:10.1158/1538-7445.AM2013-5247 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Erlotinib antagonizes ABC transporters in acute myeloid leukemia

Erlotinib was originally developed as an epidermal growth factor receptor (EGFR)-specific inhibitor for the treatment of solid malignancies, yet also exerts significant EGFR-independent antileukemic effects in vitro and in vivo. The molecular mechanisms underlying the clinical antileukemic activity of erlotinib as a standalone agent have not yet been precisely elucidated. Conversely, in preclinical settings, erlotinib has been shown to inhibit the constitutive activation of SRC kinases and mTOR, as well as to synergize with the DNA methyltransferase inhibitor azacytidine (a reference therapeutic for a subset of leukemia patients) by promoting its intracellular accumulation. Here, we show that both erlotinib and gefitinib (another EGFR inhibitor) inhibit transmembrane transporters of the ATP-binding cassette (ABC) family, including P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP), also in acute myeloid leukemia (AML) cells that do not overexpress these pumps. Thus, inhibition of drug efflux by erlotinib and gefitinib selectively exacerbated (in a synergistic or additive fashion) the cytotoxic response of KG-1 cells to chemotherapeutic agents that are normally extruded by ABC transporters (e.g., doxorubicin and etoposide). Erlotinib limited drug export via ABC transporters by multiple mechanisms, including the downregulation of surface-exposed pumps and the modulation of their ATPase activity. The effects of erlotinib on drug efflux and its chemosensitization profile persisted in patient-derived CD34+ cells, suggesting that erlotinib might be particularly efficient in antagonizing leukemic (stem cell) subpopulations, irrespective of whether they exhibit or not increased drug efflux via ABC transporters.

Inhibition of Mammalian target of rapamycin in human acute myeloid leukemia cells has diverse effects that depend on the environmental in vitro stress.

Effects of the mTOR inhibitor rapamycin were characterized on in vitro cultured primary human acute myeloid leukemia (AML) cells and five AML cell lines. Constitutive mTOR activation seemed to be a general characteristic of primary AML cells. Increased cellular stress induced by serum deprivation increased both mTOR signaling, lysosomal acidity, and in vitro apoptosis, where lysosomal acidity/apoptosis were independent of increased mTOR signaling. Rapamycin had antiproliferative and proapoptotic effects only for a subset of patients. Proapoptotic effect was detected for AML cell lines only in the presence of serum. Combination of rapamycin with valproic acid, all-trans retinoic acid (ATRA), and NF-κB inhibitors showed no interference with constitutive mTOR activation and mTOR inhibitory effect of rapamycin and no additional proapoptotic effect compared to rapamycin alone. In contrast, dual inhibition of the PI3K-Akt-mTOR pathway by rapamycin plus a PI3K inhibitor induced new functional effects that did not simply reflect a summary of single drug effects. To conclude, (i) pharmacological characterization of PI3K-Akt-mTOR inhibitors requires carefully standardized experimental models, (ii) rapamycin effects differ between patients, and (iii) combined targeting of different steps in this pathway should be further investigated whereas combination of rapamycin with valproic acid, ATRA, or NF-κB inhibitors seems less promising.

Everolimus with paclitaxel plus bevacizumab as first-line therapy for HER2-negative metastatic breast cancer (MBC): A randomized, double-blind, placebo-controlled phase II trial of the Sarah Cannon Research Institute (SCRI).

1018 Background: Constitutive activation of mTOR and amplified PI3K/Akt/mTOR signaling are common in MBC, and increase as treatment-resistance is acquired. Everolimus (E), an mTOR inhibitor, has single agent activity, combines well with paclitaxel (P) and bevacizumab (B), and prolonged PFS when added to AI therapy in BOLERO-2. In this randomized phase II trial, E was added to P/B as first-line treatment of HER2-negative MBC. Methods: Women with untreated HER2-negative MBC were randomized (1:1) to P 90mg/m2 IV (days 1, 8, and 15) and B10mg/kg IV (days 1 and 15) q28 days with E 10mg PO (Arm 1) or placebo PO (Arm 2) daily. Response assessment was performed q8 weeks until progression or intolerable toxicity. Primary endpoint was PFS. Secondary endpoints: safety, overall response rate, response duration, overall survival. 110 pts allowed detection of improvement in median PFS from 11 to 16 months with 70% power. Results: Between 8/2009 and 6/2011, 112 pts were randomized (Arm 1=55; Arm 2=57). Median age: 58 years (range: 25-79). 88% were ER+ or PR+. Pts received a median 5 treatment cycles (range: <1- 26+); 18 (16%) pts remain on treatment (Arm1, 9; Arm 2, 9). Median PFS were 8.8 months (Arm 1) and 7.1 months (Arm 2) (95% CIs 7.4-9.6; 5.5-9.1 months, p=0.79, HR 0.94). Complete responses were observed in 5% [Arm1, 4 (7%); Arm 2, 2 (4%)] with partial responses in 49% [Arm1, 29 (53%); Arm 2, 26 (46%)]. Responses rates in taxane pretreated pts Arm 1 22%, Arm 2 12%. Hematologic toxicity was similar in both arms; grade 3 mucositis occurred in 13% of E pts. Dose reductions (47% vs 25%) and interruptions (42% vs 26%) were more frequent with E due to mucositis and rash. Treatment discontinuation rates were similar. Conclusions: The addition of E did not result in a significant improvement in the efficacy of weekly paclitaxel/bevacizumab in the first-line treatment of HER2-negative MBC although response rates and median PFS were better with E. Possible explanation for these results may include lower dose intensity in the E arm, treatment of less resistant pts, or intrinsic differences in E activity when added to antiestrogen vs chemotherapy.