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.
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.
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.
A 2-D-HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi-preparative HPLC (C18 column 250x10 mm, 5 ...μm) as a first dimension of separation to isolate phenolic fractions from commercial extra-virgin olive oils and CE coupled to TOF-MS (CE-TOF-MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF-MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2-D-HPLC-CE-MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra-virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti-carcinogenic capacity.
It has been recently proposed that AMP-activated protein kinase (AMPK) might indirectly promote the phosphorylation of MRLC (myosin II regulatory light chain) at Ser19 to regulate the transition from ...metaphase to anaphase and the completion of cytokinesis. Although these findings provide biochemical support for our earlier observations showing that the active form of the α catalytic AMPK subunit associates dynamically with essential mitotic regulators, several important issues remained unexplored. Does glucose starvation alter the ability of AMPK to bind to the mitotic apparatus and travel from centrosomes to the spindle midzone during mitosis and cytokinesis? Does AMPK activate MRLC exclusively at the cleavage furrow during cytokinesis? What is the mitosis-specific stimulus that activates the mito-cytokinetic AMPK/MRLC axis regardless of energy deprivation? First, we confirm that exogenous glucose deprivation fails to alter the previously described distribution of phospho-AMPKα
Thr172
in all of the mitotic phases and does not disrupt its apparent association with the mitotic spindle and other structures involved in cell division. Second, we establish for the first time that phospho-AMPKα
Thr172
colocalizes exclusively with Ser19-phosphorylated MRLC at the cleavage furrow of dividing cells, a previously unvisualized interaction between phospho-AMPKα
Thr172
and phospho-MRLC
Ser19
that occurs in cleavage furrows, intercellular bridges and the midbody during cell division that appears to occur irrespective of glucose availability. Third, we reveal for the first time that the inhibition of AMPK mitotic activity in response to PLK1 inhibition completely prevents the co-localization of phospho-AMPKα
Thr172
and phospho-MRLC
Ser19
during the final stages of cytokinesis and midbody ring formation. Because PLK1 inhibition efficiently suppresses the AMPK-mediated activation of MRLC at the cytokinetic cleavage furrow, we propose a previously unrecognized role for AMPK in ensuring that cytokinesis occurs at the proper place and time by establishing a molecular dialog between PLK1 and MRLC in an energy-independent manner.
Active avoidance by tumor cells from attack and elimination by immune cells is an emerging cancer hallmark that is achieved primarily through decreasing the levels of major histocompatibility complex ...class I (MHC-I) at the cancer cells' surface. Deficiencies in MHC-I antigen-restricted immunosurveillance may be intertwined with an altered, Warburg-like cancer cell-intrinsic metabolism, another emerging hallmark of cancer that involves a switch from mitochondrial respiration to glycolysis to efficiently support large-scale biosynthetic programs that are required for active cell proliferation. We recently envisioned that intervention strategies aimed at reversing the bioenergetic signature of cancer cells (e.g., the antidiabetic biguanide metformin) should correct oncogene (e.g., HER2)-driven MHC-I defects, thus preventing immune escape of oncogene transformants. First, we explored how metformin treatment impacted mitochondrial biogenesis in cultured breast cancer cells overexpressing the membrane tyrosine kinase receptor HER2, the best-characterized downregulator of MHC-I. Metformin exposure was found to dose-dependently increase the expression levels of cytochrome c oxidase I and mitochondrial succinate dehydrogenase, which are encoded by mitochondrial and nuclear DNA, respectively. Second, we explored whether metformin-enhanced mitochondrial biogenesis might significantly alter the MHC-I status in breast carcinoma cells. MHC-I expression, as assessed by flow cytometry using an anti-HLA-ABC monoclonal antibody, was fully restored (up to ~25-fold upregulation) in MHC-I-negative HER2 gene-amplified carcinoma cells. These findings may help delineate a previously unrecognized mechanism through which metformin (and metformin-like drugs) may enable a cancer patient's own immune system to mount an efficient anti-metastasis response that can prevent or delay disease recurrence. Restored antigenicity and immunogenicity of tumor cells may represent a previously unrecognized primary mode of action underlying the cancer-preventive effects of metformin.
Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the ...Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR
Ser2481
exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR
Ser2481
was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR
Ser2481
staining could be clearly observed among condensed chromosomes. Phospho-mTOR
Ser2481
then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR
Ser2481
displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR
Ser2481
largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR
Ser2481
was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR
Ser2481
to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR
Ser2448
with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR
Ser2481
to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR
Ser2481
to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.
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