No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Patients with the genetic disorder tuberous sclerosis complex (TSC) suffer from neoplastic growths in multiple organ systems. These growths are the result of inactivating mutations in either the TSC1 or TSC2 tumor suppressor genes, which negatively regulate the activity of mammalian target of rapamycin complex 1(mTORC1). There is currently no cure for this disease; however, my research has found that cells harboring TSC2-inactivating mutations derived from a rat model of TSC are sensitive to apoptosis induced by the clinically approved proteasome inhibitor, bortezomib, in a manner dependent on their high levels of mTORC1 activation. We see that bortezomib induces the unfolded protein response (UPR) in our cell model of TSC, resulting in cell death via apoptosis. The UPR is induced by accumulation of unfolded protein in the endoplasmic reticulum (ER) which activates the three branches of this pathway: Activating transcription factor 6 (ATF6) cleavage, phosphorylation of eukaryotic initiation factor 2α (eIF2α), and the splicing of X-box binding protein1 (XBP1) mRNA. Phosphorylation of eIF2α leads to global inhibition of protein synthesis, preventing more unfolded protein from accumulating in the ER. This phosphorylation also induces the transcription and translation of ATF4 and CCAAT-enhancer binding protein homologous protein (CHOP). Blocking mTORC1 activity in these cells using the mTORC1 inhibitor, rapamycin, prevented the expression of ATF4 and CHOP at both the mRNA and protein level during bortezomib treatment. Rapamycin treatment also reduced apoptosis induced by bortezomib; however, it did not affect bortezomib-induced eIF2α phosphorylation or ATF6 cleavage. These data indicate that rapamycin can repress the induction of UPR-dependent apoptosis by suppressing the transcription of ATF4 and CHOP mRNAs. In addition to these findings, we find that a TSC2-null angiomyolipoma cell line forms vacuoles when treated with the proteasome inhibitor MG-132. We found these vacuoles to be derived from the ER and that rapamycin blocked their formation. Rapamycin also enhanced expansion of the ER during MG-132 stress and restored its degradation by autophagy. Taken together these findings suggest that bortezomib might be used to treat neoplastic growths associated with TSC. However, they also caution against combining specific cell death inducing agents with rapamycin during chemotherapy.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Coronary heart disease (CHD) is one of the leading causes of death every year with nearly three-fourths of all deaths caused by the disease. The challenge scientists are facing today is discovering new drugs to protect the heart against cellular damage caused by ischemia-reperfusion injury (I-R injury). Rapamycin is one such drug that has been shown to protect the heart against ischemia-induced cellular injury. Rapamycin(sirolimus) inhibits protein synthesis through inhibition of the mammalian target ofrapamycin (mTOR). This property of rapamycin has led to its current clinical applications in drug-eluting stents and in immunosuppresive treatment to organ transplant patients. The mechanism by which this drug protects against I-R injury is currently unknown. The goal of this study is to elucidate rapamycin's cardioprotective signaling pathway. We hypothesized that upregulation of Akt occurs possibly as part of a positive feedback mechanism following the inhibition of mTOR by rapamycin. Adult male ICRmice were treated with rapamycin (0.25 mg/kg, i.p.), or volume-matched DMSO (solvent for rapamycin), or rapamycin (0.25mg/kg, i.p.) plus wortmannin (WTN, 15µg/kg, i.p.),an inhibitor of phosphatidylinositol 3-kinase, or wortmannin alone (15µg/kg, i.p.). After 30 min of stabilization, the hearts were subjected to 20 minutes of global ischemia and 30 minutes of reperfusion in Langendorff model. In a separate series of experiments mice were either injected with DMSO or rapamycin for 30 minutes, 1 hour, and 2 hours before harvesting the hearts for Western blot analysis of levels of total or phosphorylated Akt at Ser473. Our results showed that rapamycin protected the heart as observed by a reductionin infarct size from 33.8 ± 2.0% in DMSO-treated hearts to 19.3 ± 4.1% in rapamycin-treated hearts; a 43% reduction. This infarct-limiting effect was completely blocked by wortmannin (29.3 ± 4.8%). However, Western blot analysis showed no change in the level of Akt phosphorylation after administration of rapamycin. Our current resultsfurther confirmed rapamycin as a potential cardio-therapeutic drug to limit infarct size,potentially through the PI3K signaling pathway. However, the exact signaling pathway of this protection still remains elusive.

Acute myocardial infarction (AMI) is the leading cause of death worldwide. Currently, the best method of treating cardiac ischemia is early reperfusion which, itself, induces myocardial damage. The mTOR complex is a key regulator of cardioprotection against cell stressors. We hypothesized that reperfusion therapy with Rapamycin, a potent mTOR inhibitor, would reduce infarct size in adult mouse hearts. Rapamycin was administered at the onset of reperfusion following 30 min in situ LAD ligation. After 24 hours of reperfusion, myocardial infarct size and apoptosis were significantly reduced in rapamycin-treated mice compared to control. Rapamycin inhibited pro-apoptotic protein Bax and phosphorylation of ribosomal protein S6 (target of mTORC1), while it induced phosphorylation of AKT (target of mTORC2). Rapamycin also induced phosphorylation of ERK, while significantly reduced phosphorylation of p38. Thus, our study shows that reperfusion therapy with Rapamycin provides cardioprotection through induction of the phosphorylation of Akt and ERK.

An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and in vivo protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, Saccharomyces cerevisiae. Mutations in SAC7 and FAR11 suppressed lethality of both lst8 and tor2-21 mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. Far11, a component of a six-member complex, was found to interact with Tpd3 and Pph21, conserved components of Protein Phosphatase 2A (PP2A) via co-immunoprecipitation. Mutations in FAR11 and RTS1, which encodes a PP2A regulatory B subunit, restore phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. These data suggest that TORC2 signaling is antagonized by Far11-dependent PP2A activity. To characterize the assembly of the Far complex in vivo, intracellular localization of the Far complex was examined by fluorescence microscopy. It was found that the Far complex localizes to the endoplasmic reticulum (ER). The data show that Far9 and Far10 are tail-anchored proteins that localize to the ER first and recruit a Far8-Far7-Far3 pre-complex. Far11 is found at the ER only when all other Far proteins are assembled at the ER. Surprisingly, ER localization is required for the Far Complex’s role TORC2 signaling because deletion of the tail-anchor domain of Far9 results in partial bypass of the tor2-21 mutant growth defect at 37 ˚C.

The mammalian target of rapamycin (mTOR) signalling pathway takes part in both extracellular and intracellular signals. It is a major regulator of cell metabolism, growth, proliferation and survival. mTOR also regulates critical processes such as cytoskeletal organization, ribosomal biogenesis, transcription and protein synthesis. The mTOR pathway has been implicated in many diseases such as cancer, neurodegeneration and diabetes, which impact homeostasis and cellular functions. Moreover, mTOR has also been shown to play a critical role in immune cell regulation of T and B cells together with neutrophils and antigen presenting cells, as it integrates signals between them extending to the entire immune microenvironment. The aim of my study was to investigate the role of a component of the mTOR complex 1, Raptor, in myeloid cells. My findings show that the absence of Raptor knock out (KO) does not affect bone marrow derived macrophage (BMDM) differentiation and maturation. However, the absence of Raptor influences BMDM polarisation towards an inflammatory phenotype, at least at the level of transcription as observed by increases in mRNA expression of inflammatory cytokines such as TNFα, IL-12β, and IL-6. This finding was consolidated by an increase in NFκΒ pathway signalling in Raptor KO BMDMs. Downstream intracellular signalling in myeloid cells was affected by deletion of Raptor as I found reduced S6K phosphorylation in Raptor KO BMDMs compared to wild type (WT) BMDMs. As a consequence of Raptor absence in BMDMs, STAT3 phosphorylation was also reduced. Raptor deletion did not impact the PI3K/Akt signalling pathway, but decreased phosphorylation of ERK. BMDMs lacking Raptor had reduced phagocytic activity as they were also observed to migrate less towards a pancreatic cancer cell line. However preliminary experiments in pancreatic cancer models did not indicate a major role for Raptor in the activity of tumour associated myeloid cells. My results demonstrate that Raptor and by implication mTORC1, is involved in macrophage polarisation and function.

O câncer de mama representa um grave problema de saúde pública. Dentre os fármacos empregados, destaca-se o paclitaxel, um agente citotóxico eficaz, porém associado a severos efeitos colaterais. A metformina hidrocloreto tem obtido resultados promissores para o tratamento de neoplasias, porém é bastante hidrofílica, fator limitante da biodisponibilidade. A rapamicina tem demonstrado sinergismo com paclitaxel e potente atividade antitumoral. Todavia, é um fármaco lipofílico e possui desvantagens. Sistemas nanoestruturados de fármacos como lipossomas PEGlados são largamente empregados para a melhora da farmacocinética e potencialização da ação terapêutica. Ademais, a funcionalização de lipossomas com anticorpos monoclonais pode permitir a entrega seletiva do fármaco encapsulado à célula alvo. No presente trabalho objetivou-se desenvolver e caracterizar lipossomas e imunolipossomas funcionalizados com trastuzumabe, contendo paclitaxel, metformina hidrocloreto e/ou rapamicina, bem como avaliar as formulações através de estudos in vitro e in vivo. Os resultados mostraram que a metformina hidrocloreto foi encapsulada com baixa eficiência, menor que 20%, ao passo que paclitaxel e rapamicina puderam ser co-encapsulados com adequados valores de eficiência de encapsulação, equivalente a 56,32% para paclitaxel e 73,31% para rapamicina, e tamanho de partícula nanométrico, de 136,95 nm em composição biocompatível baseada em SPC:Col:DSPE-PEG(2000). Os dois fármacos apresentaram liberação lenta, e foram convertidos às formas molecular e amorfa, respectivamente para paclitaxel e rapamicina quando encapsulados. Os imunolipossomas foram funcionalizados com elevada eficiência com trastuzumabe e mantiveram o tamanho nanométrico, com adequados valores de encapsulação dos fármacos. Ainda, mostrou-se o sinergismo entre paclitaxel e rapamicina coencapsulados em lipossomas em células triplo negativas (4T1) e houve sinergismo entre os dois fármacos, mediado pelo anticorpo em imunolipossomas frente à linhagem celular HER2 positiva (SKBR3), em virtude do aumento do uptake celular mediado pelo trastuzumabe. Finalmente, os resultados obtidos in vitro foram confirmados in vivo, sendo que os lipossomas com paclitaxel e rapamicina coencapsulados foram capazes de controlar o crescimento tumoral em modelo de câncer de mama triplo negativo, ao passo que o imunolipossoma com os dois fármacos permitiu o controle do crescimento de tumores xenográficos HER2 positivos, cuja média de volume tumoral correspondeu a 25,27%, 44,38% e 47,78% das médias dos volumes tumorais de controle negativo, positivo e lipossoma, respectivamente. Portanto, a formulação desenvolvida nesse trabalho tem potencial para ser avaliada em estudos clínicos. / Breast cancer represents a severe public health problem. Among the drugs used in the treatment, paclitaxel is an effective cytotoxic drug, but associated with side effects. Hydrocloride metformin has shown promising results for cancer treatment, however it is very hydrophilic, a limiting factor for bioavailability. Rapamycin has demonstrated synergism with paclitaxel and potent anticancer activity, though it is a lipophilic drug with drawbacks that compromise its bioavailability. Nanostructured drug delivery systems, such as PEGylated liposomes are largely employed for pharmacokinetics improvement and enhancement of therapeutic effect. Furthermore, the functionalization of liposomes with monoclonal antibodies enables the selective delivery of the loaded drug to the target cell. In the present work, we aimed to develop and characterize liposomes and immunoliposomes functionalized with trastuzumab, containing paclitaxel, hydrocloride metformin and/or rapamycin, as well as to evaluate the formulations through in vitro and in vivo studies. The results showed that hydrocloride metformin was encapsulated with low efficiency, less than 20%, on the other hand paclitaxel and rapamycin could be co-loaded with suitable values of encapsulation efficiency, 56.32% for paclitaxel and 73.31% for rapamycin and nanometric particle size, 136.95 nm, based on a SPC:Chol:DSPE-PEG(2000) composition. The two drugs displayed slow release, and were converted to molecular and amorphous form, respectively for paclitaxel and rapamycin when encapsulated. The immunoliposomes were developed with high efficiency with trastuzumab and kept the nanometric size, with adequate encapsulation of drugs. Moreover, herein it was shown the synergism between paclitaxel and rapamycin co-loaded in liposomes in triple negative cells (4T1) and there was synergism between the two drugs mediated by the antibody in immunoliposomes in the HER2-positive cell line (SKBR3), due to the improved cell uptake mediated by trastuzumab. Finally, the results obtained in vitro were confirmed in vivo. Co-loaded paclitaxel and rapamycin were able to control tumor growth in a triple negative breast cancer animal model, while the immunoliposome containing the two drugs allowed for better control of tumor growth in a HER2-positive breast xenograft model, whose average tumor volume corresponded to 25.27%, 44.38% and 47.78% of the tumor volumes of positive control, negative control and liposome, respectively. Therefore, the formulation developed herein has potential to be evaluated in clinical trials.

No description available.

The Aryl Hydrocarbon Receptor (AhR) and the Aryl Hydrocarbon Nuclear Translocator (ARNT) play a role in mediating transcriptional responses to environmental pollutants, including the highly toxic compound 2,3,7,8-tetrachlorodibenzo -p-dioxin (TCDD) but also endogenous physiological ligands. More recent studies have also indicated that the AhR plays a role in the immune system notably in effector Th17 cells where it seems to be critical for the production of the IL-22 cytokine. It is known that AhR ligands such as dioxins can suppress CD8 T cell mediated antiviral immune responses but it is not known whether this reflects a direct role of the AhR in CD8 T cells.Accordingly, one objective of the present study was to explore AhR and ARNT expression in CD8 T cells. The initial strategy was to probe AhR and ARNT expression by western blot analysis. A second approach was to develop a mouse model that would fate mark single lymphocytes that have activated AhR signaling pathways. A third strategy was to examine the impact of deletion of AhR and ARNT on CD8 T-cell function.The data show that AhR and ARNT expression in CD8 T cells is limited to immune activated effector cells and these transcription factors are not expressed in naïve CD8 T cells. There are only low levels of AhR complexes in conventional CD8 positive cytotoxic T cells. To investigate AhR function at the single cell level we developed a mouse model to fate mark cells that have activated AhR signaling. In this model a mouse expressing Cre recombinase ‘knocked in’ to the CYP1A locus (CYP1A1Cre+/-) was backcrossed to the R26REYFP reporter mouse. In R26REYFP mice, a gene encoding EYFP is knocked into the ubiquitously expressed Rosa26 locus preceded by a loxP flanked stop sequence. CYP1A1 expression is controlled by AhR/ARNT complexes and the concept of our model was that cells that express AhR and ARNT complexes and are triggered with AhR ligands will express Cre recombinase and delete the loxP flanked stop sequence in the R26REYFP reporter locus and hence begin to express YFP.In vitro experiments demonstrated the validity of this AhR reporter model. The in vitro data reveal that expression of functional AhR/ARNT complexes occurs during Th17 and Tc17 cell differentiation but only a very low frequency of cytotoxic T cells activates the AhR. In vivo data found no evidence for AhR activation during T cell development in the thymus but show strong evidence for activation of AhR/ARNT signaling in innate lymphocytes in the gut. The ARNT transcription factor is highly expressed in cytotoxic T cells. These cells do not express functional AhR complexes, yet we considered that ARNT might play a role in CD8 T cell biology because of its ability to dimerise with the transcription factor Hif-1a. Our studies of T cells lacking ARNT expression revealed that in CD4 T cells the ARNT transcription factor regulates IL-17 and IL-22 production. In CD8 T cells we discovered that Hif-1a/ARNT signaling controls glycolysis in immune activated cells by sustaining expression of glucose transporters and multiple rate limiting glycolytic enzymes. ARNT was not required for CD8 T cell proliferation but was required for immune activated CD8 T cells to normally differentiate to express perforin and granzymes and to acquire the migratory program of effector T cells. Importantly, we discovered that Hif-1a/ARNT signaling is regulated by mTOR (mammalian target of rapamycin) thus revealing a fundamental mechanism linking nutrient sensing and transcriptional control of CD8 T-cell differentiation.