sirolimus and Carcinoma--Lewis-Lung

Immune cell activation occurs concurrently with metabolic reprogramming. As important components of the tumor microenvironment, monocytic myeloid-derived suppressor cells (M-MDSCs) are featured by their potent immunosuppressive abilities on anti-tumor effector cells. However, little is known about the contribution of metabolic adaptations to their suppressive roles. In this study, we found that tumor-infiltrating M-MDSCs had the same phenotype with splenic M-MDSCs. Compared with splenic M-MDSCs, tumor-infiltrating M-MDSCs exhibited stronger suppressive activities which was accompanied by higher glycolysis. Inhibition of glycolysis impaired the suppressive function of tumor M-MDSCs. Meanwhile, the results demonstrated that mTOR was responsible for this function regulation. mTOR inhibition by rapamycin decreased the glycolysis and reduced the suppressive activities of these cells. Furthermore, rapamycin treatment inhibited the tumor growth and reduced the percentage of M-MDSCs in 3LL tumor bearing mice. These results demonstrated that modulation of metabolism in immune cells can be an effective way to enhance anti-tumor effects.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Lewis Lung; Glucose; Glycolysis; Mice; Mice, Inbred C57BL; Myeloid-Derived Suppressor Cells; Phosphorylation; Sirolimus; Spleen; TOR Serine-Threonine Kinases

We found that conditioned medium derived from Lewis Lung Carcinoma cells down-regulated Semaphorin3a (Sema3a) mRNA expression and increased the activity of mammalian target of rapamycin complex 1 (mTORC1) in osteoblast-like MC3T3-E1 cells. Furthermore, mTORC1 inhibition with rapamycin counteracted the effect of conditioned media on Sema3a mRNA expression. These results suggest that tumor cells decrease Sema3a mRNA expression in osteoblast in an mTORC1-dependent manner.

Topics: Animals; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Culture Media, Conditioned; Gene Expression Regulation; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Osteoblasts; RNA, Messenger; Semaphorin-3A; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Aberration of signaling pathways by genetic mutations or alterations in the surrounding tissue environments can result in tumor development or metastasis. However, signaling molecules responsible for these processes have not been completely elucidated. Here, we used mouse Lewis lung carcinoma cells (LLC) to explore the mechanism by which the oncogenic activity of Semaphorin3A (Sema3A) signaling is regulated. Sema3A knockdown by shRNA did not affect apoptosis, but decreased cell proliferation in LLCs; both the mammalian target of rapamycin complex 1 (mTORC1) level and glycolytic activity were also decreased. In addition, Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation by oligomycin, but conferred resistance to decreased cell viability induced by glucose starvation. Furthermore, recombinant SEMA3A rescued the attenuation of cell proliferation and glycolytic activity in LLCs after Sema3A knockdown, whereas mTORC1 inhibition by rapamycin completely counteracted this effect. These results demonstrate that Sema3A signaling exerts its oncogenic effect by promoting an mTORC1-mediated metabolic shift from oxidative phosphorylation to aerobic glycolysis.

Topics: Animals; Apoptosis; Carcinogenesis; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Proliferation; Gene Knockdown Techniques; Glucose; Glycolysis; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Oxidative Phosphorylation; Semaphorin-3A; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3'-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.

Topics: Androstadienes; Animals; Blotting, Western; Carcinoma, Lewis Lung; Cell Line, Tumor; Chloramphenicol O-Acetyltransferase; Chromones; Enzyme Activation; Enzyme Inhibitors; Extracellular Matrix; Flavonoids; Gene Expression Regulation, Enzymologic; Genes, Dominant; Genes, Reporter; Insulin-Like Growth Factor I; Matrix Metalloproteinase 2; Mice; Mitogen-Activated Protein Kinases; Models, Biological; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Plasmids; Proto-Oncogene Proteins c-raf; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sirolimus; Time Factors; Tumor Suppressor Proteins; Wortmannin

The membrane type 1 matrix metalloproteinase (MT1-MMP) has been identified as a major activator of MMP-2 - a process involving the formation of a trimolecular complex with TIMP-2. We previously identified the IGF-I receptor as a positive regulator of MMP-2 synthesis. Here, we investigated the role of IGF-IR in the regulation of MT1-MMP. Highly invasive Lewis lung carcinoma subline H-59 cells express MT1-MMP and utilize it to activate their major extracellular matrix degrading proteinase-MMP-2. These cells were transiently transfected with a plasmid vector expressing a luciferase reporter gene downstream of the mouse MT1-MMP promoter. IGF-I treatment increased luciferase activity in the transfected cells by up to 10-fold and augmented endogenous MT1-MMP mRNA and protein synthesis by up to 2-3-fold, relative to controls. MT1-MMP induction and invasion were blocked by the PI 3-kinase inhibitors LY294002 and wortmannin and by rapamycin, but not by the MEK inhibitor PD98059. Overexpression of a dominant negative Akt mutant or of the tumor suppressor phosphatase and tensin homologue, PTEN, in these cells also caused a significant reduction in MT1-MMP expression and invasion. The results demonstrate that IGF-IR controls tumor cell invasion by coordinately regulating MMP-2 expression and its MT1-MMP-mediated activation and identify PI 3-kinase/Akt/mTOR signaling as critical to this regulation.

Topics: Amino Acid Substitution; Androstadienes; Animals; Carcinoma, Lewis Lung; Chromones; Collagen; Drug Combinations; Enzyme Induction; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Neoplastic; Insulin-Like Growth Factor I; Laminin; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Mice; Morpholines; Neoplasm Invasiveness; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoric Monoester Hydrolases; Phosphorylation; Point Mutation; Promoter Regions, Genetic; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteoglycans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptor, IGF Type 1; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wortmannin