Tuberous sclerosis complex (TSC) is a genetic autosomal dominant disorder characterized by benign tumor-like lesions, called hamartomas, in multiple organ systems, including the brain, skin, heart, ...kidneys, and lung. These hamartomas cause a diverse set of clinical problems based on their location and often result in epilepsy, learning difficulties, and behavioral problems. TSC is caused by mutations within the TSC1 or TSC2 genes that inactivate the genes' tumor-suppressive function and drive hamartomatous cell growth. In normal cells, TSC1 and TSC2 integrate growth signals and nutrient inputs to downregulate signaling to mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine kinase that controls cell growth and cell survival. The molecular connection between TSC and mTOR led to the clinical use of allosteric mTOR inhibitors (sirolimus and everolimus) for the treatment of TSC. Everolimus is approved for subependymal giant cell astrocytomas and renal angiomyolipomas in patients with TSC. Sirolimus, though not approved for TSC, has undergone considerable investigation to treat various aspects of the disease. Everolimus and sirolimus selectively inhibit mTOR signaling with similar molecular mechanisms, but with distinct clinical profiles. This review differentiates mTOR inhibitors in TSC while describing the molecular mechanisms, pathogenic mutations, and clinical trial outcomes for managing TSC.
Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 ...in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic "driver" mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mitochondria are dynamic organelles that undergo constant remodeling through the regulation of two opposing processes, mitochondrial fission and fusion. Although several key regulators and ...physiological stimuli have been identified to control mitochondrial fission and fusion, the role of mitochondrial morphology in the two processes remains to be determined. To address this knowledge gap, we investigated whether morphological features extracted from time-lapse live-cell images of mitochondria could be used to predict mitochondrial fate. That is, we asked if we could predict whether a mitochondrion is likely to participate in a fission or fusion event based on its current shape and local environment. Using live-cell microscopy, image analysis software, and supervised machine learning, we characterized mitochondrial dynamics with single-organelle resolution to identify features of mitochondria that are predictive of fission and fusion events. A random forest (RF) model was trained to correctly classify mitochondria poised for either fission or fusion based on a series of morphological and positional features for each organelle. Of the features we evaluated, mitochondrial perimeter positively correlated with mitochondria about to undergo a fission event. Similarly mitochondrial solidity (compact shape) positively correlated with mitochondria about to undergo a fusion event. Our results indicate that fission and fusion are positively correlated with mitochondrial morphological features; and therefore, mitochondrial fission and fusion may be influenced by the mechanical properties of mitochondrial membranes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA ...interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. ...However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.
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► Unbiased genomic and metabolomic approaches to reveal downstream functions of mTORC1 ► mTORC1 stimulates glucose uptake and glycolysis through HIF1 ► mTORC1 stimulates the pentose phosphate pathway and lipid biosynthesis through SREBP1 ► S6K1 regulates SREBP1 processing and transcriptional activation of target genes
Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. ...Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.
We constructed a mechanistic, computational model for regulation of (macro)autophagy and protein synthesis (at the level of translation). The model was formulated to study the system-level ...consequences of interactions among the following proteins: two key components of MTOR complex 1 (MTORC1), namely the protein kinase MTOR (mechanistic target of rapamycin) and the scaffold protein RPTOR; the autophagy-initiating protein kinase ULK1; and the multimeric energy-sensing AMP-activated protein kinase (AMPK). Inputs of the model include intrinsic AMPK kinase activity, which is taken as an adjustable surrogate parameter for cellular energy level or AMP:ATP ratio, and rapamycin dose, which controls MTORC1 activity. Outputs of the model include the phosphorylation level of the translational repressor EIF4EBP1, a substrate of MTORC1, and the phosphorylation level of AMBRA1 (activating molecule in BECN1-regulated autophagy), a substrate of ULK1 critical for autophagosome formation. The model incorporates reciprocal regulation of mTORC1 and ULK1 by AMPK, mutual inhibition of MTORC1 and ULK1, and ULK1-mediated negative feedback regulation of AMPK. Through analysis of the model, we find that these processes may be responsible, depending on conditions, for graded responses to stress inputs, for bistable switching between autophagy and protein synthesis, or relaxation oscillations, comprising alternating periods of autophagy and protein synthesis. A sensitivity analysis indicates that the prediction of oscillatory behavior is robust to changes of the parameter values of the model. The model provides testable predictions about the behavior of the AMPK-MTORC1-ULK1 network, which plays a central role in maintaining cellular energy and nutrient homeostasis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cancer cells have several hallmarks that define their neoplastic behavior. One is their unabated replicative potential that allows cells to continually proliferate, and thereby contribute to ...increasing tumor burden. The progression of a cell through the cell cycle is regulated by a series of checkpoints that ensures successful transmission of genetic information, as well as various cellular components, including organelles and protein complexes to the two resulting daughter cells. The mitochondrial reticulum undergoes coordinated changes in shape to correspond with specific stages of the cell cycle, the most dramatic being complete mitochondrial fragmentation prior to cytokinesis. To determine whether mitochondrial fission is a required step to ensure proper mitochondrial segregation into two daughter cells, we investigated the importance of mitochondrial dynamics to cell cycle progression. We found that mitochondrial hyperfusion promotes a defect in cell cycle progression characterized by an inability for cells to exit G2/M. Additionally, extended periods of persistent mitochondrial fusion led to robust caspase-dependent cell death. The cell death signals were coordinated through activation and cleavage of caspase-8, promoting a potent death response. These results demonstrate the importance of mitochondrial dynamics in cell cycle progression, and that inhibiting mitochondrial fission regulators may provide a therapeutic strategy to target the replicative potential of cancer cells.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CDC7-DBF4 kinase (DDK) initiates DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has diverse and apparently conflicting roles in the replication checkpoint response in ...various organisms, but the underlying mechanisms are far from settled. We show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1 and phosphorylates EXO1 in vitro. EXO1 is also required for nascent strand degradation following exposure to HU, so DDK might regulate EXO1 directly. Lastly, sublethal DDK inhibition causes various mitotic abnormalities, which is consistent with a checkpoint deficiency. In summary, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.