ABSTRACT
Metformin is a widely used and well-tolerated anti-diabetic drug that can reduce cancer risk and improve the prognosis of certain malignancies. However, the mechanism underlying its anti-cancer effect is still unclear. We studied the anti-cancer activity of metformin on colorectal cancer (CRC) by using the drug to treat HT29, HCT116 and HCT116 p53-/- CRC cells. Metformin reduced cell proliferation and migration by inducing cell cycle arrest in the G0/G1 phase. This was accompanied by a sharp decrease in the expression of c-Myc and down-regulation of IGF1R. The anti-proliferative action of metformin was mediated by two different mechanisms: AMPK activation and increase in the production of reactive oxygen species, which suppressed the mTOR pathway and its downstream targets S6 and 4EBP1. A reduction in CD44 and LGR5 expression suggested that the drug had an effect on tumour cells with stem characteristics. However, a colony formation assay showed that metformin slowed the cells' ability to form colonies without arresting cell growth, as confirmed by absence of apoptosis, autophagy or senescence. Our finding that metformin only transiently arrests CRC cell growth suggests that efforts should be made to identify compounds that combined with the biguanide can act synergistically to induce cell death.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Colorectal Neoplasms/metabolism , Metformin/pharmacology , Reactive Oxygen Species/metabolism , Antineoplastic Agents/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Female , HCT116 Cells , Humans , In Situ Hybridization , Membrane Potential, Mitochondrial/drug effects , Signal Transduction/drug effectsABSTRACT
BACKGROUND: Strategies aimed at obtaining a complete cytoreduction are needed to improve long-term survival for patients with colorectal cancer peritoneal carcinomatosis (CRC-pc). METHODS: We established organoid models from peritoneal metastases of two naïve CRC patients. A standard paraffin inclusion was conducted to compare their 3D structure and immunohistochemical profile with that of the corresponding surgical samples. RNA expression levels of the CRC stem cell marker LGR5 was measured by in situ hybridization. The secretome of organoids was profiled by mass spectrometry. Energy homeostasis of organoids was interfered with 4-IPP and metformin. Biochemical and metabolic changes after drug treatments were investigated by western blot and mass spectrometry. Mitochondria impairment was evaluated by electron microscopy and mitotraker staining. RESULTS: The two organoids recapitulated their corresponding clinical samples in terms of 3D structure and immmunoistochemical profile and were positive for the cancer stem cells marker LGR5. Proteomic analyses of organoids highlighted their strong dependence on energy producing pathways, which suggest that their targeting could be an effective therapeutic approach. To test this hypothesis, we treated organoids with two drugs that target metabolism acting on AMP-activated protein kinase (AMPK), the main regulator of cellular energy homeostasis, which may act as metabolic tumour suppressor in CRC. Organoids were treated with 4-IPP, an inhibitor of MIF/CD74 signalling axis which activates AMPK function, or metformin that inhibits mitochondrial respiratory chain complex I. As a new finding we observed that treatment with 4-IPP downregulated AMPK signalling activity, reduced AKT phosphorylation and activated a JNK-mediated stress-signalling response, thus generating mitochondrial impairment and cell death. Metformin treatment enhanced AMPK activation, decreasing the activity of the anabolic factors ribosomal protein S6 and p4EBP-1 and inducing mitochondrial depolarization. CONCLUSION: We provide evidence that the modulation of AMPK activity may be a strategy for targeting metabolism of CRC-pc organoids.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/metabolism , Colonic Neoplasms/metabolism , Histocompatibility Antigens Class II/metabolism , Intramolecular Oxidoreductases/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Metformin/pharmacology , Peritoneal Neoplasms/secondary , Pyrimidines/pharmacology , AMP-Activated Protein Kinases/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Energy Metabolism/drug effects , Humans , Molecular Targeted Therapy , Peritoneal Neoplasms/drug therapy , Peritoneal Neoplasms/genetics , Proteomics , Receptors, G-Protein-Coupled/genetics , Signal Transduction/drug effects , Tumor Cells, CulturedABSTRACT
BIM is a proapoptotic member of the Bcl-2 family. Here, we investigated the epigenetic status of the BIM locus in NPM/ALK+ anaplastic large cell lymphoma (ALCL) cell lines and in lymph node biopsies from NPM/ALK+ ALCL patients. We show that BIM is epigenetically silenced in cell lines and lymph node specimens and that treatment with the deacetylase inhibitor trichostatin A restores the histone acetylation, strongly upregulates BIM expression, and induces cell death. BIM silencing occurs through recruitment of MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2) corepressor complex. This event requires BIM CpG methylation/demethylation with 5-azacytidine that leads to detachment of the MeCP2 corepressor complex and reacetylation of the histone tails. Treatment with the ALK inhibitor PF2341066 or with an inducible shRNA targeting NPM/ALK does not restore BIM locus reacetylation; however, enforced expression of NPM/ALK in an NPM/ALK-negative cell line significantly increases the methylation at the BIM locus. This study demonstrates that BIM is epigenetically silenced in NPM/ALK-positive cells through recruitment of the SIN3a/HDAC1/2 corepressor complex and that NPM/ALK is dispensable to maintain BIM epigenetic silencing but is able to act as an inducer of BIM methylation.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Gene Silencing , Lymphoma, Large-Cell, Anaplastic/genetics , Membrane Proteins/genetics , Methyl-CpG-Binding Protein 2/metabolism , Proto-Oncogene Proteins/genetics , Repressor Proteins/metabolism , 5' Untranslated Regions , Acetylation , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Case-Control Studies , Cell Line, Tumor , Cell Survival , Chromatin/metabolism , CpG Islands , DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Histone Deacetylase Inhibitors/pharmacology , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Lymphoma, Large-Cell, Anaplastic/metabolism , Membrane Proteins/metabolism , Protein Processing, Post-Translational , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Sin3 Histone Deacetylase and Corepressor ComplexABSTRACT
Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor. Although a comprehensive assessment of BIM CpG island methylation revealed no consistent changes, glucocorticoid resistance in xenografts and patient biopsies significantly correlated with decreased histone H3 acetylation. Moreover, the histone deacetylase inhibitor vorinostat relieved BIM repression and exerted synergistic antileukemic efficacy with dexamethasone in vitro and in vivo. These findings provide a novel therapeutic strategy to reverse glucocorticoid resistance and improve outcome for high-risk pediatric ALL.