sirolimus and Thymoma

The pathogenesis of thymic epithelial tumors remains poorly elucidated. The PIK3/Akt/mTOR pathway plays a key role in various cancers; interestingly, several phase I/II studies have reported a positive effect of mTOR inhibitors in disease control in thymoma patients. A major limit for deciphering cellular and molecular events leading to the transformation of thymic epithelial cells or for testing drug candidates is the lack of reliable in vitro cell system. We analyzed protein expression and activation of key players of the Akt/ mTOR pathway namely Akt, mTOR, and P70S6K in eleven A, B and AB thymomas as well as in normal thymuses. While only Akt and phospho-Akt were expressed in normal thymuses, both Akt and mTOR were activated in thymomas. Phospho-P70S6K was expressed in all thymic tumors whatever their subtypes, and absent in normal thymus. Interestingly, we report the activation of Akt, mTOR and P70S6 proteins in primary thymic epithelial cells maintained for short period of time after their derivation from seven AB and B thymomas. Finally, we showed that rapamycin (100 nM) significantly reduced proliferation of thymoma- derived epithelial cells without inducing cell death. Our results suggest that the activation of the Akt/ mTOR pathway might participate to the cell proliferation associated with tumor growth. Ultimately, our data enhance the potential role of thymic epithelial cells derived from tissue specimens for in vitro exploration of molecular abnormalities in rare thymic tumors.

Topics: Aged; Aged, 80 and over; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Female; Humans; Male; Middle Aged; Mutation; Neoplasms, Glandular and Epithelial; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Thymoma; Thymus Gland; Thymus Neoplasms; TOR Serine-Threonine Kinases; Transcription Factors, TFII; Tumor Cells, Cultured

Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4+ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8+ T cell effector and memory populations. Evaluation of mice with a T cell-specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8+ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8+ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8+ T cell effector responses, mTORC2 activity regulated CD8+ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8+ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8+ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity.

Topics: Adoptive Transfer; Animals; Carrier Proteins; CD4-CD8 Ratio; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Deoxyglucose; Female; Genes, Reporter; Glycolysis; Immunologic Memory; Interferon-gamma; Lymphocyte Activation; Lymphopoiesis; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Melanoma, Experimental; Mice; Mice, Congenic; Mice, Inbred C57BL; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Neuropeptides; Ovalbumin; Peptide Fragments; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Rapamycin-Insensitive Companion of mTOR Protein; Ras Homolog Enriched in Brain Protein; Recombinant Fusion Proteins; Sirolimus; Thymoma; TOR Serine-Threonine Kinases; Transduction, Genetic; Tumor Necrosis Factor-alpha

Mounting epidemiological evidence supports a role for insulin-signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. However, the underlying molecular mechanisms remain unknown. To study the oncogenic effect of chronically elevated insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (AKT/mTOR) pathway, a major downstream effector of insulin signaling, in this model of insulin-induced hepatocarcinogenesis. We found that activation of insulin signaling triggers a strong induction of the AKT/mTOR cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis, and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions, when compared with the healthy liver. AKT/mTOR metabolic effects on hepatocytes, after insulin stimulation, were found to be mTORC1 dependent and independent in human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint, despite insulin administration. Noticeably, metabolic abnormalities and proliferation driven by insulin were effectively reverted using the dual PI3K/mTOR inhibitor, NVP-BEZ235, both in vitro and in vivo.. The present results indicate that activation of the AKT/mTOR cascade by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. Thus, inhibition of AKT/mTOR and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin-induced hepatocarcinogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Diabetes Mellitus, Experimental; Disease Models, Animal; Fatty Acids; Hyperinsulinism; Immunoblotting; Lipogenesis; Liver Neoplasms; Male; Mice; Proto-Oncogene Proteins c-akt; Random Allocation; Rats; Rats, Inbred Lew; Sensitivity and Specificity; Sirolimus; Streptozocin; Thymoma; TOR Serine-Threonine Kinases; Transfection

Vaccine strategies aimed at generating CD8(+) T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8(+) T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8(+) T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8(+) T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8(+) T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8(+) T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Antigens, Neoplasm; Canarypox virus; Cancer Vaccines; CD8-Positive T-Lymphocytes; Clonal Selection, Antigen-Mediated; Immunologic Memory; Interleukin-15; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Multiprotein Complexes; Neoplasm Transplantation; Ovalbumin; Proteins; Sirolimus; T-Cell Antigen Receptor Specificity; Thymoma; Thymus Neoplasms; TOR Serine-Threonine Kinases; Vaccination

Rapamycin, a potent immunosuppressive drug that prevents rejection of organ transplants in many animals, caused profound growth inhibition in an immature B cell lymphoma, BKS-2, at very low concentrations (2 ng/ml). Similar growth inhibition was also observed in a series of B cell lymphomas (i.e., L1.2, NFS.1.1, and WEHI-279) as well as in thymoma cells. The cell death induced by rapamycin in BKS-2 lymphoma was found to be via programmed cell death, or apoptosis. In contrast to rapamycin, neither FK506 nor CsA affected the normal growth of these cells. FK506, but not CsA antagonized the effect of rapamycin and rescued the BKS-2 cells from undergoing apoptosis. Further, suboptimal concentrations of anti-IgM antibodies and rapamycin acted synergistically in causing the growth inhibition of BKS-2 cells and this inhibitory effect was also completely reversed by FK506. Thus, rapamycin appeared to inhibit lymphoma growth by binding to FK506 binding protein. These results indicate that rapamycin should be evaluated as an effective immunosuppressive therapeutic agent to prevent the incidence of lymphoma after transplantations.

Topics: Animals; Antibiotics, Antineoplastic; Antibodies, Anti-Idiotypic; Apoptosis; B-Lymphocytes; Cell Division; Drug Screening Assays, Antitumor; Immunosuppressive Agents; Lymphoma, B-Cell; Mice; Mice, Inbred CBA; Polyenes; Sirolimus; Tacrolimus; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured