Regulation Of mTOR Pathway Research Articles

Regulation of mTOR Pathway in Exercise-induced Cardiac Hypertrophy.

This study was designed to examine whether the mTOR signaling pathway would respond to long-term different intensity exercises and to observe the impact of exercise upon possible cardiac damage. Male Sprague Dawley rats were randomly divided into control group, moderate-intensity exercise group and high-intensity exercise group, and each exercise group had 4 observation time points (1-24 h). Exercise training lasted 8 weeks with a 2-day break for each week. Serum cTnI was measured by ELSIA and myocardium histology was assessed by HE and HBFP. The expressions of Akt, mTOR, p70(S6K) and their phosphorylated forms were determined by western-blot. Both exercises were effective at inducing cardiac hypertrophy, wherein magnitude increased with exercise intensity. The significantly high level of serum cTnI in the high-intensity group was accompanied by obvious myocellular abnormalities and ischemia in the myocardium. Significant activation of Akt, mTOR and p70(S6K) were observed in the moderate exercise group but not in the high-intensity exercise group. Results indicate that long-term high-intensity exercise training would induce cardiac hypertrophy accompanied by damage to the heart, entailing a risk of pathological changes. There might be a pivotal regulatory role of the mTOR signaling pathway on cardiac hypertrophy after long-term moderate exercise, but not after high-intensity exercise.

Molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in bovine mammary epithelial cells

14-3-3γ, an isoform of the 14-3-3 protein family, was proved to be a positive regulator of mTOR pathway. Here, we analyzed the function of 14-3-3γ in protein synthesis using bovine mammary epithelial cells (BMECs). We found that 14-3-3γ interacted with eIF1AX and RPS7 by 14-3-3γ coimmunoprecipitation (CoIP) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) peptide mass fingerprinting analysis. These interactions of 14-3-3γ with eIF1AX and RPS7 were further confirmed by colocalization and fluorescence resonance energy transfer (FRET) analysis. We also found that methionine could promote protein synthesis and trigger the protein expression levels of 14-3-3γ, eIF1AX and RPS7. Analysis of overexpression and inhibition of 14-3-3γ confirmed that it positively affected the protein expression levels of eIF1AX, RPS7, Stat5 and mTOR pathway to promote protein synthesis and cell proliferation in BMECs. We further showed that overexpression of eIF1AX and RPS7 also triggered protein translation and cell proliferation. From these results, we conclude that molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in BMECs.

The cytoplasmic tail of FPC antagonizes the full-length protein in the regulation of mTOR pathway.

FPC (fibrocystin or polyductin) is a single transmembrane receptor-like protein, responsible for the human autosomal recessive polycystic kidney disease (ARPKD). It was recently proposed that FPC undergoes a Notch-like cleavage and subsequently the cleaved carboxy(C)-terminal fragment translocates to the nucleus. To study the functions of the isolated C-tail, we expressed the intracellular domain of human FPC (hICD) in renal epithelial cells. By 3-dimensional (3D) tubulogenesis assay, we found that in contrast to tubule-like structures formed from control cells, hICD-expressing cells exclusively formed cyst-like structures. By western blotting, we showed that the Akt/mTOR pathway, indicated by increased phosphorylation of Akt at serine 473 and S6 kinase 1 at threonine 389, was constitutively activated in hICD-expressing cells, similar to that in FPC knockdown cells and ARPKD kidneys. Moreover, application of mTOR inhibitor rapamycin reduced the size of the cyst-like structures formed by hICD-expressing cells. Application of either LY294002 or wortmannin inhibited the activation of both S6K1 and Akt. Expression of full-length FPC inhibited the activation of S6 and S6 kinase whereas co-expression of hICD with full-length FPC antagonized the inhibitory effect of full-length FPC on mTOR. Taken together, we propose that FPC modulates the PI3K/Akt/mTOR pathway and the cleaved C-tail regulates the function of the full-length protein.

Impaired lipid accumulation in the liver of Tsc2-heterozygous mice during liver regeneration

Tuberin is a negative regulator of mTOR pathway. To investigate the function of tuberin during liver regeneration, we performed 70% hepatectomy on wild-type and Tsc2+/− mice. We found the tuberin phosphorylation correlated with mTOR activation during early liver regeneration in wild-type mice. However, liver regeneration in the Tsc2+/− mice was not enhanced. Instead, the Tsc2+/− livers failed to accumulate lipid bodies, and this was accompanied by increased mortality.These findings suggest that tuberin plays a critical role in liver energy balance by regulating hepatocellular lipid accumulation during early liver regeneration. These effects may influence the role of mTORC1 on cell growth and proliferation.

Abstract 2498: Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer.

Abstract The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly dysregulated pathway in many human cancers, including non-small cell lung cancer (NSCLC). Therefore, targeting PI3K signaling has become a promising approach in cancer therapy. Although studies have indicated development of resistance to PI3K inhibitors, the molecular mechanism of drug resistance is largely unknown in NSCLC. Here, we sought to identify new potential biomarkers and pathways that confer resistance to PI3K inhibitors in NSCLC. To this end, we used reverse-phase protein array (RPPA) to assess the expression levels and activation status of 137 proteins involved in signaling pathways implicated in lung cancer. Seventy-four NSCLC cell lines were treated with the PI3K inhibitor BAY 80-6946 and lysates were collected for proteomic analysis. First, we evaluted the association between drug sensitivity (IC50) and RPPA expression levels at baseline in sixty NSCLC cell lines. Correlation test was applied to each marker vs drug and Wilcox Rank test was performed to compare the smallest and largest one third of all the cell lines. We found increased phosphorylation and inactivation of pro-apoptotic protein Bad and phospho-LKB1 in the resistant cell lines. An inverse correlation was found for phosphorylation and inactivation of c-Src. Interestingly, activation of EGFR and increased levels of PTCH and PKCα were found in the resistant cell lines. Next, paired t-test was applied to each marker comparing the difference between drug and control treated cells. As expected, treatment with BAY 80-6946, significantly downregulates components of PI3K/mTOR pathway, as illustrated by decreased levels of phospho-Akt (-4.28-fold relative expression) and phospho-S6 ribosomal protein (-9.64-fold), phospho-p70S6K (-3.47-fold) and phospho-mTOR (-1.42-fold). We observed elevated expression of negative regulators of mTOR pathway, LKB1 (1.11-fold), AMPKα (1.12-fold) and phospho-AMPKα (12-fold). On the other hand, our analysis identified new potential pathways of resistance that are activated upon PI3K inhibition. We found increased levels of phospho-EGFR (P<0.0001), PTCH (P<0.0001) and PKCα (P<0.0001). We observed activation of c-Src pathway upon treatment with BAY 80-6946, total and phospho-Src (P<0.0001 and P<0.0001). Accordingly, key downstream survival cascades regulated by c-Src, FAK (P<0.0001), Stat5 (P<0.0001), phospho-Stat5 (P<0.0001) and phospho-Stat3 (P<0.0001) were upregulated in treated cells. Our results suggest that PI3K inhibition leads to EGFR/MAPK and c-SRC/Stat signaling activation in vitro, suggesting a compensatory pathway that may allow NSCLC survival. Taken together, these results suggest that activation of Src/Stat pathway as an important mechanism of resistance to PI3K inhibitors in NSCLC and may be clinically relevant targets for combination therapy. Citation Format: Maria A. Cortez, Lauren Averett Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai Tran, Kevin Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung 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 2498. doi:10.1158/1538-7445.AM2013-2498

Targeting the mTOR-DEPTOR Pathway by CRL E3 Ubiquitin Ligases: Therapeutic Application

The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation, survival, and autophagy. The mTOR pathway is frequently activated in many human cancers, mainly resulting from alterations in the upstream regulators, such as phosphoinositide 3-kinase (PI3K)/AKT activation, PTEN loss or dysregulation of mTOR-negative regulators (e.g., TSC1/2), leading to uncontrolled proliferation. Thus, inhibiting the PI3K/AKT/mTOR pathways is widely considered as an effective approach for targeted cancer therapy. Recently, we and others found that DEPTOR, a naturally occurring inhibitor of both mTORC1 and mTORC2, was degraded by SCF (Skp1-Cullin-F box proteins) E3 ubiquitin ligase, the founding member of cullin-RING-ligases (CRLs), resulting in mTOR activation and cell proliferation. In addition to DEPTOR, previous studies have demonstrated that several other negative regulators of mTOR pathway are also substrates of CRL/SCF E3s. Thus, targeting CRL/SCF E3s is expected to cause the accumulation of these mTOR signal inhibitors to effectively block the mTOR pathway. In this review, we will discuss mTOR signaling pathway, how DEPTOR regulates mTOR/AKT axis, thus acting as a tumor suppressor or oncogene in some cases, how DEPTOR is ubiquitinated and degraded by SCFβ-TrCP E3, and how MLN4924, a small-molecule indirect inhibitor of CRL/SCF E3 ligases through blocking cullin neddylation, might be useful as a novel approach of mTOR pathway targeting for cancer therapy.

The role of mTOR inhibitors in the inhibition of growth and cortisol secretion in human adrenocortical carcinoma cells

Patients with adrenocortical carcinoma (ACC) need new treatment options. The aim of this study was to evaluate the effects of the mTOR inhibitors sirolimus and temsirolimus on human ACC cell growth and cortisol production. In H295, HAC15, and SW13 cells, we have evaluated mTOR, IGF2, and IGF1 receptor expressions; the effects of sirolimus and temsirolimus on cell growth; and the effects of sirolimus on apoptosis, cell cycle, and cortisol production. Moreover, the effects of sirolimus on basal and IGF2-stimulated H295 cell colony growth and on basal and IGF1-stimulated phospho-AKT, phospho-S6K1, and phospho-ERK in H295 and SW13 were studied. Finally, we have evaluated the effects of combination treatment of sirolimus with an IGF2-neutralizing antibody. We have found that H295 and HAC15 expressed IGF2 at a >1800-fold higher level than SW13. mTOR inhibitors suppressed cell growth in a dose-/time-dependent manner in all cell lines. SW13 were the most sensitive to these effects. Sirolimus inhibited H295 colony surviving fraction and size. These effects were not antagonized by IGF2, suggesting the involvement of other autocrine regulators of mTOR pathways. In H295, sirolimus activated escape pathways. The blocking of endogenously produced IGF2 increased the antiproliferative effects of sirolimus on H295. Cortisol production by H295 and HAC15 was inhibited by sirolimus. The current study demonstrates that mTOR inhibitors inhibit the proliferation and cortisol production in ACC cells. Different ACC cells have different sensitivity to the mTOR inhibitors. mTOR could be a target for the treatment of human ACCs, but variable responses might be expected. In selected cases of ACC, the combined targeting of mTOR and IGF2 could have greater effects than mTOR inhibitors alone.

MLN4924, An Investigational NAE Inhibitor, Suppresses AKT and mTOR Signaling Pathway Through up Regulating REDD1 in Human Myeloma Cells

Abstract 1870 Introduction:The development and survival of normal plasma cells as well as multiple myeloma cells depend on an elaborately regulated ubiquitin proteasome system (UPS). Proteasome inhibitors such as bortezomib have proved to be highly active in the treatment of multiple myeloma. MLN4924, a newly developed investigational NEDD8 activating enzyme (NAE) inhibitor, exhibits promising anti-tumor effect through both clinical and laboratory observation. We sought to evaluate the individual signaling effects of MLN4924 in multiple myeloma, with the intent of further understanding the mechanism of action and identifying potential combinations. Methods:Human myeloma cell lines (MM.1S, MM.1R and U266) were treated with increasing concentrations of MLN4924 for 24, 48 and 72 hrs respectively or concurrently with bortezomib. Cell viability (MTT), apoptosis, western blot, RT-qPCR and siRNA assays were used to identify the cellular and molecular sequelae of MLN4924 treatment. Results:Single agent studies demonstrate that MLN4924 induces cytotoxicity in all three MM cell lines. Cytotoxicity is associated with increased apoptosis and suppression of AKT and mTOR signaling pathway, as detected by FACS and western blot. MLN4924 suppresses protein turnover of Cullin-ring ligases substrates leading to stabilization of specific proteins, such as p27, CDT1, NRF2. We find that REDD1, a substrate of CUL4 A–DDB 1–ROC1–β-TRCP ubiquitin ligase and negative regulator of mTOR pathway, increases in as early as 2 hours when treated with MLN4924. Knock-down of REDD1 using siRNA alleviates MLN4924 induced AKT, mTOR signaling suppression as well as the growth inhibition, which suggests that MLN4924 inhibits AKT, mTOR signaling through upregulating REDD1 protein leading to cytotoxicity.Combining MLN4924 with bortezomib synergistically enhances the antitumor effect of MLN4924. Western blot confirms the development of significantly increased procaspase and PARP cleavage, as well as substantial down regulation of AKT and mTOR signaling. Neither IL-6 nor IGF-1 is able to abrogate these combination effects. When we silence REDD1 in the combination assay in MM.1R, we find less cell apoptosis and suppression of AKT, mTOR pathway, which further support that REDD1 is an important regulator for MLN4924 induced cytotoxicity in MMs. Conclusion:MLN4924 is a potent investigational antitumor compound suppressing mTOR signaling pathways in myeloma cell lines. Down-regulation of the mTOR signaling pathway is associated with increased expression of REDD1. Combination of MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MMs cell lines which can overcome the prosurvival effects of growth factor (IL-6 and IGF-1). These findings could positively impact clinical combination strategies. Disclosures:Kaufman:Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Onyx: Consultancy; Merck: Consultancy.

Abstract PR-11: Pharmacogenomics of mTOR inhibitors: A genome-wide association approach of biomarker identification.

Abstract Introduction: The mammalian target of rapamycin (mTOR), a downstream kinase of the PI3K/Akt signaling pathway, is a critical regulator of basic cellular functions, especially in tumor progression, and therefore has emerged as a key target for cancer chemotherapy. Rapamycin and its derivatives (e.g. Everolimus, Temsirolimus and Deferolimus) are mTOR inhibitors originally approved as immunosuppressants to prevent renal and cardiac allograft rejection. These mTOR inhibitors have recently been used in a large number of cancer clinical trials, including renal cell carcinoma, breast cancer, endometrial carcinoma, glioblastoma and mantle cell lymphoma. However, severe side effects such as toxicity and immune suppression limit the escalation of mTOR inhibitors' dosage, resulting in a low responsive rate. To maximize the efficacy and minimize side effects of mTOR inhibitors, there is a critical need to identify the genetic biomarkers for response to mTOR inhibitors and the mechanisms by which they manifest their therapeutic benefits in cancer. Purpose: This study aims to identify and validate novel genetic biomarkers that might influence the therapeutic responses of mTOR inhibitors. Methods: We used 273 human lymphoblastoid cell lines (LCLs), for which we have obtained genome-wide SNP data, Affymetric U133 plus 2 mRNA expression array data, and Illumina microRNA array data, to perform cytotoxicity assays with two mTOR inhibitors, Rapamycin and Everolimus. We then performed genome-wide association (GWA) analyses using SNPs, mRNA gene expression and microRNA data with cytotoxicity data (AUC values) collected on both mTOR inhibitors. Integrated analysis was then performed to identify the SNPs, and microRNAs that might be associated with genes, whose expressions in turn were significantly associated with the cytotoxicity. We then selected candidate genes to functionally validate their effects on sensitivity of mTOR inhibitors using an siRNA screening approach with 3 cell lines, a renal carcinoma (Caki2), a glioblastoma (U87) and a fibroblast (IMR90) cell line, followed by MTS cell proliferation assay and colony formation assay. Results: mRNA expression levels of 48 and 55 genes were associated with Rapamycin and Everolimus cytotoxicity (p<10−4) respectively, with 16 common genes between two drugs. One hundred and twenty seven and 100 SNPs were associated with Rapamycin and Everolimus cytotoxicity (p<10−4), respectively. We selected 23 candidate genes based on the GWA analyses (mRNA expression vs. cytotoxicity, SNPs vs. cytotoxicity and integrated analysis), and verified 13 genes that significantly altered cells' sensitivity to either one or both mTOR inhibitors in at least one cell line based on siRNA screening study. In addition, one microRNA, miR10a, was shown to be most significantly associated with cytotoxicity for both Rapamycin and Everolimus (p=3.3×10−4; p =1.4×10−4). Interestingly, this particular microRNA was also found to be strongly associated with gene expression levels of 9 out 23 selected candidate genes with p<10−4. Conclusions: This study identified a series of novel candidate genes and microRNA (miR10a) that might contribute to the response of mTOR inhibitors. These findings will enhance our understanding of the regulation of mTOR pathway and the mechanisms underlying the variation in response to mTOR inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-11.

Abstract 4009: Regulation of mTOR pathway by the orphan nuclear receptor NR4A1 (TR3/Nur77) in non-small cell lung cancer: Role of TR3 in AMPK activation

Abstract The orphan nuclear receptor NR4A1 (TR3/Nur77) is overexpressed as a nuclear protein in multiple human tumors and this receptor exhibits pro-oncogenic activity in many tumor types. A recent study in this laboratory has shown that deactivation of TR3 decreases proliferation and induces apoptosis through inhibition of Sp1-dependent anti-apoptotic genes. In this study, we show that TR3 is overexpressed in human non-small cell lung cancer (NSCLC) tissues compared with adjacent normal lung tissues and high TR3 expression levels are correlated with shorter survival for patients with NSCLC (P = 0.0112). Treatment of NSCLC cells with small interfering RNAs for TR3 or the TR3 deactivator, 1,1-bis(3’-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), decreased cell proliferation and induced apoptosis through dual-targeting of mTOR pathway and Sp1 transcription factor. Knockdown of TR3 inhibited mTOR and its two major downstream targets, p70S6K and 4E-BP1 through LKB1-independent activation of AMPK but not through inactivation of AKT nor Sp1. We also observed that AMPK activation by TR3 knockdown is primarily due to induction of sestrin2 by enhancing p53-dependent transactivation activity. Furthermore, DIM-C-pPhOH (30 mg/kg/day) induced apoptosis and inhibited tumor growth in an orthotopic mouse model of human NSCLC, and decreased tumor number and size in a lung metastatic tumor model using tail vein injection. These results demonstrate for the first time that endogenous nuclear TR3 regulates AMPK-mediated mTOR pathway and TR3 deactivation represents a novel approach for treatment of NSCLC. Grant support: National Institutes of Health (R01CA124998) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4009. doi:10.1158/1538-7445.AM2011-4009

Activation of mTOR in renal cell carcinoma is due to increased phosphorylation rather than protein overexpression

The altered expression and activation of the mammalian target of rapamycin (mTOR) promotes the invasiveness and metastatic potential in a variety of malignancies. The aim of the present pilot study was to determine mTOR expression in clear cell renal cell carcinoma (RCC) and to evaluate mTOR activation and phosphorylation at Ser2448. Tissue microarray immunohistochemistry and Western blot analysis of tumor and benign tissue from 10 patients subjected to tumor nephrectomy were investigated. Staining of mTOR and phosphorylated-mTOR (p-mTOR) was documented and determined as percentage of the maximum. Western blots were evaluated densitometrically. Ratios of tumor versus benign tissue were calculated and compared by the Wilcoxon/Kruskal-Wallis test. Immunohistochemical expressions of mTOR and p-mTOR were 49 and 40% in benign renal parenchyma, whereas it was 20 and 42% in tumor tissue. Ratios of tumor versus benign tissue revealed a reduction to 0.44 for mTOR and corresponding elevation to 1.29 for p-mTOR (p<0.05). The rate of p-mTOR to mTOR was 1.19 in benign, whereas it was 5.30 in tumor tissue. Western blot densitometry detected lower expressions of mTOR in tumor compared to benign tissues. Ratio of p-mTOR to mTOR were significantly different in benign versus tumor tissue (0.86 vs. 1.37; p<0.04). The observation that RCC specimens exhibit higher levels of p-mTOR in RCC compared to benign renal parenchyma indicate the role of mTOR phosphorylation in RCC tumor development and progression. This study found a concomitant reduction of the RCC mTOR protein expression, which suggests that elevated levels of activated p-mTOR result predominantly from an increased mTOR phosphorylation rather than from protein overexpression. These pilot study results may contribute to the clarification of mTOR-pathway regulation processes in RCC on the way to the protein profiling-predicted targeted therapy.

Bortezomib Inhibits mTOR Pathway in Multiple Myeloma Cell Lines Via Induced Expression of REDD1.

Pharmacogenomic profiles of genes involved in bortezomib - dexamethasone response may help to understand resistance and could provide new therapeutic targets as well as contributing to novel prognostic markers in multiple myeloma. We have used gene expression profiling to analyze the complex signaling pathways regulating the response to bortezomib - dexamethasone. Gene expression profiles were established in 9 cell lines, derived from 9 myeloma patients, incubated or not with a combination of bortezomib 10 nM and dexamethasone 1 μM. These concentrations correspond to the ones used for patients in the IFM 2005-01. Cells were collected after 6 hours of treatment. We focused our interest in early response genes, making the hypothesis that the comprehension of early effects would help to better understand the mechanisms of resistance that take place in at least two third of myeloma patients. Supervised analysis with permutations identified significantly up regulated genes involved in stress responses (heat shocks proteins, RTP801/dig2/REDD1/DDIT4), endoplasmic reticulum stress (HERP/HERPUD1, gadd145/CHOP/DDIT3), ubiquitin/proteasome pathway (proteasome 26S subunits PSMB7, PSMC4, PSMD3 and PSMD13), unfolded protein response (such as SQSTM1, ATF4) or redox equilibrium (PLRX, PRDX1). We assumed that these genes might represent a molecular signature of response to bortezomib and provide important insight into the complex mechanisms of action of these drugs. We focused on REDD1 a gene cloned in 2002 that is known to be rapidly induced by a wide variety of stress conditions (arsenic, hypoxia, dexamethasone, thapsigargin, tunimycin and heat shock) and DNA damages (ionizing radiation, ultraviolet radiation, DNA alkylant). We found that both REDD1 gene and protein expression were early and highly induced after bortezomib exposure alone or in combinaison with dexamethasone. This effect was dependent upon cell line: REDD1 was overexpressed within two hours in resistant cell lines in association with a cell size decrease while in sensitive cell lines, neither REDD1 induction nor morphological changes occured. REDD1 induction was associated with the dephosphorylation of S6K1, a key substrat of mTOR, a protein kinase which controls cell growth and cell size in response to various signals. SiRNA studies confirmed that bortezomib lead to a negative regulation of mRTor activity mediated by REDD1: disruption of REDD1 abrogates both S6K1 phosphorylation and early transitory cell size reduction. Our results are in accordance with data obtained in mouse showing an early regulation of mTOR pathway and cellular proliferation induced by REDD1 expression in response to stress. Our study suggests that mTOR regulation could be a resistance mechanism mediated by REDD1 expression. As we found that REDD1 was differentially induced in primary plasma cells from patients, this gene expression could help to predict response to bortezomib. Our objective is now to clarify the pathway that links bortezomib to REDD1 in multiple myeloma and to investigate REDD1 expression in patients enrolled in IFM 2005-01 clinical trial.

Generation and Characterization of Monoclonal Antibodies Against Tuberous Sclerosis Complex 2

TSC1 and TSC2 are two recently identified tumor suppressor genes encoding hamartin and tuberin, respectively. They have been implicated in the pathogenesis of tuberous sclerosis, a neurological disorder linked with the development of hamartomas in numerous organs, including the brain, kidneys, heart, and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase-activating (GAP) activity towards a small G protein Rheb (Ras homologue enriched in brain). Inhibition of Rheb is important for the positive regulation of mTOR pathway, while mutations of hamartin or tuberin result in uncontrolled cell cycle progression. Although the precise role for the TSC1/2 complex in tumor suppression is not clear, many studies have established a link with the regulation of transcription and protein biosynthesis, increasing susceptibility to apoptosis, cell differentiation, and cell cycle control. We describe the development of a monoclonal antibody specific towards TSC2/tuberin and characterize the suitability for Western blotting, immunoprecipitation, and immunofluorescent applications. The C-terminal region of TSC2 was expressed as a His-tag fusion protein in bacteria, affinity purified and used as an immunogen. Hybrid myelomas were produced from the spleenocytes of immunized mice and SP2/0 myeloma cells. Testing the specificity of cell culture supernatants from generated hybridomas towards recombinant His-TSC2C in ELISA assay allowed us to isolate a panel of positive clones. Further analysis of selected clones by Western blotting and immunoprecipitation revealed one clone, termed D6, which specifically recognized recombinant and endogenous TSC2. The specificity of generated antibody was also confirmed in TSC2(/) and TSC2(+/+) mouse embryo fibroblasts. In summary, the produced antibody is a useful tool in our research program and will be available for researchers investigating signal transduction pathways involving TSC1/2 signaling under physiological conditions and in human pathologies.

Hamartin and tuberin: Working together for tumour suppression

TSC1 and TSC2 are two recently identified tumour suppressor genes encoding hamartin and tuberin, respectively, and involved in pathogenesis of tuberous sclerosis, neurological disorder connected with the development of hamartomas in numerous organ systems, including the brain, kidneys, heart and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase-activating (GAP) activity towards a small G protein, Ras homologue enriched in brain (Rheb). Inhibition of Rheb is important for the regulation of mTOR pathway, while mutation of hamartin or tuberin results in uncontrolled cell cycle progression. Tuberin, possessing the Rheb-GAP domain, is phosphorylated by several kinases that confer the signals of growth factor stimulation or low cellular energy levels. Such a modification of tuberin influences its activity within the complex with hamartin and positively or negatively modulates mTOR-regulated protein translation and cellular proliferation. Current article describes biochemical properties of hamartin and tuberin, their known regulatory phosphorylation sites and binding partners.

The Stress-inducted Proteins RTP801 and RTP801L Are Negative Regulators of the Mammalian Target of Rapamycin Pathway

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an essential role in cell growth control. mTOR stimulates cell growth by phosphorylating p70 ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1 (4EBP1). The mTOR pathway is regulated by a wide variety of cellular signals, including mitogenic growth factors, nutrients, cellular energy levels, and stress conditions. Recent studies have proposed several mechanisms to explain how mTOR is regulated by growth factors and cellular energy levels. However, little is known as to how mTOR is regulated by stress conditions. We observed that two stress-induced proteins, RTP801/Redd1 and RTP801L/Redd2, potently inhibit signaling through mTOR. Our data support that RTP801 and RTP801L work downstream of AKT and upstream of TSC2 to inhibit mTOR functions. These results add a new dimension to mTOR pathway regulation and provide a possible molecular mechanism of how cellular stress conditions may regulate mTOR function.

Molecular Pathogenesis of Tuber Formation in Tuberous Sclerosis Complex

Tuberous sclerosis complex results from mutations in the TSC1 (hamartin) and TSC2 (tuberin) genes. Tubers are cortical developmental malformations in patients with tuberous sclerosis complex that are associated with intractable epilepsy and are composed of histologically distinct cell types, including giant cells and dysplastic neurons. We recently showed that tubers can be dynamic lesions characterized by populations of cells undergoing proliferation, migration, and death. We demonstrate that there is cell-specific activation of the mammalian target of rapamycin (mTOR)/p70S6 kinase/ribosomal S6 cascade in tubers and that giant cells express activated (phosphorylated) p70S6 kinase and ribosomal S6 protein. These findings support impaired hamartin- and tuberin-mediated mTOR pathway regulation. Tubers likely form by constitutive activation of the mTOR cascade during brain development as a consequence of impaired hamartin or tuberin function.