We have tested the hypothesis that the antidiabetic biguanide metformin can be used to manipulate the threshold for stress-induced senescence (SIS), thus accelerating the onset of cancer-protective ...cellular senescence in response to oncogenic stimuli. Using senescence-prone murine embryonic fibroblasts (MEFs), we assessed whether metformin treatment modified the senescence phenotype that is activated in response to DNA damaging inducers. Metformin significantly enhanced the number of MEFs entering a senescent stage in response to doxorubicin, an anthracycline that induces cell senescence by activating DNA damage signaling pathways (e.g., ATM/ATR) in a reactive oxygen species (ROS)-dependent manner. Using WI-38 and BJ-1 human diploid fibroblasts (HDFs), we explored whether metformin supplementation throughout their entire replicative lifespan may promote the early appearance of the biomarkers of replicative senescence. Chronic metformin significantly reduced HDFs' lifespan by accelerating both the loss of replicative potential and the acquisition of replicative senescence-related biomarkers (e.g., enlarged and flattened cell shapes, loss of arrayed arrangement, accumulation of intracellular and extracellular debris and SA-β-gal-positive staining). Metformin functioned as a bona fide stressful agent, inducing monotonic, dose-dependent, SIS-like responses in BJ-1 HDFs, which are highly resistant to ROS-induced premature senescence. Metformin-induced SIS in BJ-1 fibroblasts was accompanied by the striking activation of several microRNAs belonging to the miR-200s family (miR-200a, miR-141 and miR429) and miR-205, thus mimicking a recently described ability of ROS to chemosensitize cancer cells by specifically upregulating anti-EMT (epithelial-to-mesenchymal transition) miR-200s. Because the unlimited proliferative potential of stem cells results from their metabolic refractoriness to SIS, we finally tested if metformin treatment could circumvent the stress (e.g., ROS)-resistant phenotype of induced pluripotent stem cells (iPSCs). Metformin treatment drastically reduced both the number and the size of iPSC colonies and notably diminished the staining of the pluripotency marker alkaline phosphatase. Our current findings, altogether, reveal for the first time that metformin can efficiently lower the threshold for SIS to generate an "stressed" cell phenotype that becomes pre-sensitized to oncogenic-like stimuli, including DNA damaging, proliferative and/or stemness inducers.
Abstract Ideal oncology drugs would be curative after a short treatment course if they could eliminate epithelium-originated carcinomas at their non-invasive, pre-malignant stages. Such ideal ...molecules, which are expected to molecularly abrogate all the instrumental mechanisms acquired by migrating cancer stem cells (CSCs) to by-pass tumour suppressor barriers, might already exist. We here illustrate how system biology strategies for repositioning existing FDA-approved drugs may accelerate our therapeutic capacity to eliminate CSC traits in pre-invasive intraepithelial neoplasias. First, we describe a signalling network signature that overrides bioenergetics stress- and oncogene-induced senescence (OIS) phenomena in CSCs residing at pre-invasive lesions. Second, we functionally map the anti-malarial chloroquine and the anti-diabetic metformin (“old drugs”) to their recently recognized CSC targets (“new uses”) within the network. By discussing the preclinical efficacy of chloroquine and metformin to inhibiting the genesis and self-renewal of CSCs we finally underscore the expected translational impact of the “old drugs–new uses” repurposing strategy to open a new CSC-targeted chemoprevention era.
Prompted by the ever-growing scientific rationale for examining the antidiabetic drug metformin as a potential antitumor agent in breast cancer disease, we recently tested the hypothesis that the ...assessment of metformin-induced global changes in gene expression -as identified using 44K (double density) Agilent's whole human genome arrays- could reveal gene-expression signatures that would allow proper selection of breast cancer patients who should be considered for metformin-based clinical trials. Using Database for Annotation, Visualization and Integrated Discovery bioinformatics (DAVID) resources we herein reveal that, at doses that lead to activation of the AMP-activated protein kinase (AMPK), metformin not only down-regulates genes coding for ribosomal proteins (i.e. protein and macromolecule biosynthesis) but unexpectedly suppresses numerous mitosis-related gene families including kinesins, tubulins, histones, auroras and polo-like kinases. This is, to our knowledge, the first genome-scale evidence of a mitotic core component in the transcriptional response of human breast cancer cells to metformin. These findings further support a tight relationship between the activation status of AMPK and the chromosomal and cytoskeletal checkpoints of cell mitosis at the transcriptional level.
Endogenous fatty acid metabolism is crucial to maintain the cancer cell malignant phenotype. Lipogenesis is regulated by the enzyme fatty acid synthase (FASN); and breakdown of fatty acids is ...regulated by carnitine palmitoyltransferase-1 (CPT-I). FASN is highly expressed in breast cancer and most common human carcinomas. Several compounds can inhibit FASN, although the degree of specificity of this inhibition has not been addressed. We have tested the effects of C75 and (-)-epigallocatechin-3-gallate (EGCG) on fatty acid metabolism pathways, cellular proliferation, induction of apoptosis and cell signalling in human breast cancer cells. Our results show that C75 and EGCG had comparable effects in blocking FASN activity. Treating cancer cells with EGCG or C75 induced apoptosis and caused a decrease in the active forms of oncoprotein HER2, AKT and ERK1/2 to a similar degree. We observed, in contrast, marked differential effects between C75 and EGCG on the fatty acid oxidation pathway. While EGCG had either no effect or a moderate reduction in CPT-I activity, C75 stimulated CPT-I activity (up to 129%), even in presence of inhibitory levels of malonyl-CoA, a potent inhibitor of the CPT-I enzyme. Taken together, these findings indicate that pharmacological inhibition of FASN occurs uncoupled from the stimulation of CPT-I with EGCG but not with C75, suggesting that EGCG might be free of the CPT-I related in vivo weight-loss that has been associated with C75. Our results establish EGCG as a potent and specific inhibitor of fatty acid synthesis (FASN), which may hold promise as a target-directed anti-cancer drug.
Circulating fatty acid synthase (FASN) is a biomarker of metabolically demanding human diseases. The aim of this study was to determine whether circulating FASN could be a biomarker of ...overnutrition-induced metabolic stress and insulin resistance in common metabolic disorders.
Circulating FASN was evaluated in two cross-sectional studies in association with insulin sensitivity and in four longitudinal studies investigating the effect of diet- and surgery-induced weight loss, physical training, and adipose tissue expansion using peroxisome proliferator-activated receptor agonist rosiglitazone on circulating FASN.
Age- and BMI-adjusted FASN concentrations were significantly increased in association with obesity-induced insulin resistance in two independent cohorts. Both visceral and subcutaneous FASN expression and protein levels correlated inversely with extracellular circulating FASN (P = -0.63; P < 0.0001), suggesting that circulating FASN is linked to depletion of intracellular FASN. Improved insulin sensitivity induced by therapeutic strategies that decreased fat mass (diet induced, surgery induced, or physical training) all led to decreased FASN levels in blood (P values between 0.02 and 0.04). To discriminate whether this was an effect related to insulin sensitization, we also investigated the effects of rosiglitazone. Rosiglitazone did not lead to significant changes in circulating FASN concentration.
Our results suggest that circulating FASN is a biomarker of overnutrition-induced insulin resistance that could provide diagnostic and prognostic advantages by providing insights on the individualized metabolic stress.
The AMP-activated protein kinase (AMPK) system, an evolutionary conserved low-energy checkpoint, functions as a canonical suppressor of cell proliferation. Proliferating cells, however, should also ...ensure a proper spatio-temporal bond between AMPK-sensed cell's metabolic status and cell division. A crucial linkage between cell proliferation and AMPK-interpreted cell bioenergetics appears to take place during the M-phase of the cell cycle. A recent description of a physical interplay between the active form the α-catalytic AMPK subunit with essential mitotic regulators in the centrosome and midbody has provided direct evidence that tumor-suppressive properties for AMPK closely relate to its ability to exquisitely coordinate sensing of energy resources and the fundamental biological process of genome division during mitosis and cytokinesis. Based on recent findings in our laboratory observing abortive cytokinesis followed by nuclear shape reorganization, mitotic catastrophe, polyploidization events, and cell giantism in p53-null cancer cells pharmacologically manipulated to exhibit sustained activation of AMPK, we now propose that AMPK is a novel and biologically significant participant with a tumor suppressive activity in the mitotic/cytokinetic phase of the cell cycle. In this scenario, molecular co-evolution of the energy-sensing cytokinetic tumor suppressor AMPK within the chronic biophysical constraints of the tumor microenvironment may inherently promote a continuous generation of structural and numerical changes in chromosomes favoring generation of nascent tumor cells and/or tumor-initiating cells over tumor cell death.
Raptor is the key scaffolding protein that recruits mTOR substrates to rapamycin-sensitive mTOR complex 1 (mTORC1), a molecular integrator of mitogenic and nutrient/energy environmental inputs into ...protein translation and cell growth. Although Raptor phosphorylation on various sites is pivotal in the regulation of mTORC1 activity, it remains to be elucidated whether site-specific phosphorylation differentially distributes Raptor to unique subcellular compartments. When exploring the spatiotemporal cell cycle dynamics of six different phospho (P)-Raptor isoforms (Thr
706
, Ser
722
, Ser
863
, Ser
792
and Ser
877
), a number of remarkable events differentially defined a topological resetting of P-RaptorThr706 on interphasic and mitotic chromosomes. In interphase nuclei, P-Raptor
Thr706
co-localized with fibrillarin, a component of the nucleolar small nuclear ribonucleoprotein particle, as well as with RNA polymerase I, the enzyme that transcribes nucleolar rRNA. Upon Actinomycin D-induced nucleolar segregation and disaggregation, P-RaptorThr706 was excluded from the nucleolus to accumulate at discrete nucleoplasmic bodies. During mitosis, CDK1 inhibition-induced premature assembly of nucleoli relocated fibrillarin to the surrounding regions of chromosomal-associated P-Raptor
Thr706
, suggesting that a subpopulation of mitotic P-Raptor
Thr706
remained targeted at chromosomal loops of rDNA or nuclear organizer regions (NORs). At the end of mitosis and cytokinesis, when reassembly of incipient nucleoli begins upon NORs activation of rDNA transcription, fibrillarin spatially reorganized with P-Raptor
Thr706
to give rise to daughter nucleoli. Treatment with IGF1 exclusively hyperactivated nuclear P-Raptor
Ser706
and concomitantly promoted Ser
2481
autophosphorylation of mTOR, which monitors mTORC1-associated catalytic activity. Nucleolar- and NOR-associated P-Raptor
Ser706
may physically link mTORC1 signaling to ever-growing nucleolus plurifunctionality including ribosome biogenesis, cell stress sensor and cell cycle/aging control.
AMP-activated protein kinase (AMPK) is being primarily studied as a central metabolic stress sensor, which regulates cell survival and growth-related metabolic pathways to preserve intracellular ATP ...levels in response to energy deprivation. Evidence is now accumulating that AMPK plays also an obligatory role to ensure proper cell division and faithful chromosomal segregation during mitosis. Increased phosphorylation in the AMPK catalytic subunit (AMPKα) was found in a proteomic study for kinases activated during G2/M and, more recently, activated AMPKα (PP-AMPKα
Thr172
) has been observed to transiently associate with several mitotic structures including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis. How AMPKα activation is controlled spatially and temporally during mitosis, however, remained undiscovered. Because Polo-like Kinases (PLKs) regulate many aspects of mitotic progression including centrosome maturation, bipolar spindle assembly, chromosome congression & segregation, and cytokinesis, we decided to combine an immunofluorescence microscopy analysis with a chemical biology approach employing a small-molecule PLK1 inhibitor to dissect a putative relationship between PLK1 and AMPKα during G2/M transition. PLK1 and PP-AMPKα
Thr172
were found to display a major spatio-temporal overlap early at centrosomes, from prophase until anaphase, and late at the midbody, during telophase and cytokinesis. Moreover, short-term treatment with the thiophene derivative GW843682X, a selective PLK inhibitor that has 400-fold greater potency for PLK1 than for PLK2 or PLK3, fully abolished mitotic activation of AMPKα. Upon long-term PLK1 inhibition, PP-AMPKα
Thr172
was barely detected surrounding the spindle poles of prometaphase-like arrested cells displaying the "polo" phenotype. Similarly, PP-AMPKα
Thr172
was largely inhibited in GW843682X-treated cells exhibiting cytokinesis failure and binucleate cell formation. Given that PLK1 is a well-recognized master regulatory kinase for the numerous protein substrates involved in mitosis, our current description of a causal link between PLK1 activity and mitotic phosphorylation of AMPKα may provide fundamental insights into how the energy sensor AMPK is directly coupled to mitotic cell division and cell cycle exist.
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