Invasion and metastasis of carcinomas is promoted by the activation of the embryonic 'epithelial to mesenchymal transition' (EMT) program, which triggers cellular mobility and subsequent dissemination of tumour cells. We recently showed that the EMT-activator ZEB1 (zinc finger E-box binding homeobox 1) is a crucial promoter of metastasis and demonstrated that ZEB1 inhibits expression of the microRNA-200 (miR-200) family, whose members are strong inducers of epithelial differentiation. Here, we report that ZEB1 not only promotes tumour cell dissemination, but is also necessary for the tumour-initiating capacity of pancreatic and colorectal cancer cells. We show that ZEB1 represses expression of stemness-inhibiting miR-203 and that candidate targets of miR-200 family members are also stem cell factors, such as Sox2 and Klf4. Moreover, miR-200c, miR-203 and miR-183 cooperate to suppress expression of stem cell factors in cancer cells and mouse embryonic stem (ES) cells, as demonstrated for the polycomb repressor Bmi1. We propose that ZEB1 links EMT-activation and stemness-maintenance by suppressing stemness-inhibiting microRNAs (miRNAs) and thereby is a promoter of mobile, migrating cancer stem cells. Thus, targeting the ZEB1-miR-200 feedback loop might form the basis of a promising treatment for fatal tumours, such as pancreatic cancer.

Mammalian target of rapamycin complex 1 (mTORC1) controls growth and survival in response to metabolic cues. Oxidative stress affects mTORC1 via inhibitory and stimulatory inputs. Whereas downregulation of TSC1-TSC2 activates mTORC1 upon oxidative stress, the molecular mechanism of mTORC1 inhibition remains unknown. Here, we identify astrin as an essential negative mTORC1 regulator in the cellular stress response. Upon stress, astrin inhibits mTORC1 association and recruits the mTORC1 component raptor to stress granules (SGs), thereby preventing mTORC1-hyperactivation-induced apoptosis. In turn, balanced mTORC1 activity enables expression of stress factors. By identifying astrin as a direct molecular link between mTORC1, SG assembly, and the stress response, we establish a unifying model of mTORC1 inhibition and activation upon stress. Importantly, we show that in cancer cells, apoptosis suppression during stress depends on astrin. Being frequently upregulated in tumors, astrin is a potential clinically relevant target to sensitize tumors to apoptosis.

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational–experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

Modeling and testing network structures reveal a distinct mechanism of mTORC2 activation by insulin.

Background and Aims Prior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant. Methods We examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features. Key Results The water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development. Conclusions Cuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

Permeability is a crucial trait that affects seed longevity and is regulated by different polymers including proanthocyanidins, suberin, cutin and lignin located in the seed coat. By testing mutants in suberin transport and biosynthesis, we demonstrate the importance of this biopolymer to cope with seed deterioration. Transcriptomic analysis of cog1‐2D, a gain‐of‐function mutant with increased seed longevity, revealed the upregulation of several peroxidase genes. Reverse genetics analysing seed longevity uncovered redundancy within the seed coat peroxidase gene family; however, after controlled deterioration treatment, seeds from the prx2 prx25 double and prx2 prx25 prx71 triple mutant plants presented lower germination than wild‐type plants. Transmission electron microscopy analysis of the seed coat of these mutants showed a thinner palisade layer, but no changes were observed in proanthocyanidin accumulation or in the cuticle layer. Spectrophotometric quantification of acetyl bromide‐soluble lignin components indicated changes in the amount of total polyphenolics derived from suberin and/or lignin in the mutant seeds. Finally, the increased seed coat permeability to tetrazolium salts observed in the prx2 prx25 and prx2 prx25 prx71 mutant lines suggested that the lower permeability of the seed coats caused by altered polyphenolics is likely to be the main reason explaining their reduced seed longevity.

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Mammalian target of rapamycin (mTOR) kinase responds to growth factors, nutrients and cellular energy status and is a central controller of cellular growth. mTOR exists in two multiprotein complexes that are embedded into a complex signalling network. Adenosine monophosphate-dependent kinase (AMPK) is activated by energy deprivation and shuts off adenosine 5¢-triphosphate (ATP)-consuming anabolic processes, in part via the inactivation of mTORC1. Surprisingly, we observed that AMPK not only responds to energy deprivation but can also be activated by insulin, and is further induced in mTORC1-deficient cells. We have recently modelled the mTOR network, covering both mTOR complexes and their insulin and nutrient inputs. In the present study we extended the network by an AMPK module to generate the to date most comprehensive data-driven dynamic AMPK-mTOR network model. In order to define the intersection via which AMPK is activated by the insulin network, we compared simulations for six different hypothetical model structures to our observed AMPK dynamics. Hypotheses ranking suggested that the most probable intersection between insulin and AMPK was the insulin receptor substrate (IRS) and that the effects of canonical IRS downstream cues on AMPK would be mediated via an mTORC1-driven negative-feedback loop. We tested these predictions experimentally in multiple set-ups, where we inhibited or induced players along the insulin-mTORC1 signalling axis and observed AMPK induction or inhibition. We confirmed the identified model and therefore report a novel connection within the insulin-mTOR-AMPK network: we conclude that AMPK is positively regulated by IRS and can be inhibited via the negative-feedback loop.Abbreviations AIC, akaike information criterion; AMP, adenosine monophosphate; AMPK, adenosine monophosphate-dependent kinase; ATM, ataxiatelangiectasia mutated; ATP, adenosine 5¢-triphosphate; BIC, Bayesian Information Criterion; CaMKK, Ca 2+ -sensitive calmodulin-dependent protein kinase kinase; CV, coefficient of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA-myr-Akt, myristoylated version of Akt; HRP, horseradish peroxidase; IR, insulin receptor; IRE1, inositol-requiring enzyme 1; IRS, insulin receptor substrate; LKB1, liver kinase B1; MOTA, Mean Optimal Transformation Approach; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of rapamycin complex 2; NFL, negative-feedback loop; PDK1, phosphoinositidedependent protein kinase 1; p.i., postinduction; PI3K, phosphatidylinositol 3-kinase; PRAS40, proline-rich Akt substrate of 40 kDa; PTEN, phosphatase and tensin homologue; p70-S6K, S6-kinase; shRaptor, inducible short hairpin Raptor knockdown;TAK1 ⁄ MAP3K7, transforming growth factor-beta-activated kinase-1; TSC1-TSC2, tuberous sclerosis complex 1 ⁄ 2 dimer; 4E-BP, 4E-binding protein.