Epigenetic mechanisms underlie the crosstalk between growth factors and a steroid hormone.

Crosstalk between growth factors (GFs) and steroid hormones recurs in embryogenesis and is co-opted in pathology, but underlying mechanisms remain elusive. Our data from mammary cells imply that the crosstalk between the epidermal GF and glucocorticoids (GCs) involves transcription factors like p53 and NF-κB, along with reduced pausing and traveling of RNA polymerase II (RNAPII) at both promoters and bodies of GF-inducible genes. Essentially, GCs inhibit positive feedback loops activated by GFs and stimulate the reciprocal inhibitory loops. As expected, no alterations in DNA methylation accompany the transcriptional events instigated by either stimulus, but forced demethylation of regulatory regions broadened the repertoire of GF-inducible genes. We report that enhancers, like some promoters, are poised for activation by GFs and GCs. In addition, within the cooperative interface of the crosstalk, GFs enhance binding of the GC receptor to DNA and, in synergy with GCs, promote productive RNAPII elongation. Reciprocally, within the antagonistic interface GFs hyper-acetylate chromatin at unmethylated promoters and enhancers of genes involved in motility, but GCs hypoacetylate the corresponding regions. In conclusion, unmethylated genomic regions that encode feedback regulatory modules and differentially recruit RNAPII and acetylases/deacetylases underlie the crosstalk between GFs and a steroid hormone.

Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor.

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes.

Steering tumor progression through the transcriptional response to growth factors and stroma.

Tumor progression can be understood as a collaborative effort of mutations and growth factors, which propels cell proliferation and matrix invasion, and also enables evasion of drug-induced apoptosis. Concentrating on EGFR, we discuss downstream signaling and the initiation of transcriptional events in response to growth factors. Specifically, we portray a wave-like program, which initiates by rapid disappearance of two-dozen microRNAs, followed by an abrupt rise of immediate early genes (IEGs), relatively short transcripts encoding transcriptional regulators. Concurrent with the fall of IEGs, some 30-60 min after stimulation, a larger group, the delayed early genes, is up-regulated and its own fall overlaps the rise of the final wave of late response genes. This late wave persists and determines long-term phenotype acquisition, such as invasiveness. Key regulatory steps in the orderly response to growth factors provide a trove of potential oncogenes and tumor suppressors.

The translational landscape of mTOR signalling steers cancer initiation and metastasis.

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the 'cancerous' translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.

Combination of ATP-competitive mammalian target of rapamycin inhibitors with standard chemotherapy for colorectal cancer.

ATP-competitive mammalian target of rapamycin (mTOR) inhibitors are in early phase clinical trials. These novel targeted agents, including PP242, are mechanistically distinct from the allosteric, partial mTOR inhibitor, rapamycin. The goal of this study was to evaluate how PP242 best combines with standard chemotherapies for colorectal cancer (CRC), and which subsets of patients are most likely to benefit. The combination index for PP242 plus 5-fluorouracil, oxaliplatin, or irinotecan was determined in CRC cell lines with different mutational backgrounds. In KRAS mutant CRC cell lines, sensitivity to PP242 increases with co-mutation of PIK3CA. Mutation of p53 predicts resistance to chemotherapy, but not PP242. Efficacy of PP242 was comparable to that of standard chemotherapies over the dose range tested. Sensitivity or resistance to PP242 dictates relative synergy or antagonism, respectively, when PP242 is combined with 5-fluorouracil. The same trend exists for PP242 + oxaliplatin, but with a narrower dynamic range. Conversely potency of PP242 and the combination index for PP242 + irinotecan were unrelated, but synergy exists across all dose levels in PP242 and irinotecan sensitive, p53 wild-type cell lines. Overall, our in vitro analysis predicts that mutational status can be used to rank sensitivity to PP242 and standard chemotherapies. Single agent potency can in turn be used to predict the combination index in a drug-specific manner. Our data suggest a clinical trial to determine whether ATP-competitive mTOR inhibitors provide benefit in combination with standard chemotherapies for patients with PIK3CA mutant metastatic CRC, stratified by the presence or absence of KRAS co-mutation.

Constitutive mTORC1 activation by a herpesvirus Akt surrogate stimulates mRNA translation and viral replication.

All viruses require cellular ribosomes to translate their mRNAs. Viruses producing methyl-7 (m7) GTP-capped mRNAs, like Herpes Simplex Virus-1 (HSV-1), stimulate cap-dependent translation by activating mTORC1 to inhibit the translational repressor 4E-binding protein 1 (4E-BP1). Here, we establish that the HSV-1 kinase Us3 masquerades as Akt to activate mTORC1. Remarkably, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on the same sites as Akt. TSC2 depletion rescued Us3-deficient virus replication, establishing that Us3 enhances replication by phosphorylating TSC2 to constitutively activate mTORC1, effectively bypassing S6K-mediated feedback inhibition. Moreover, Us3 stimulated Akt substrate phosphorylation in infected cells, including FOXO1 and GSK3. Thus, HSV-1 encodes an Akt surrogate with overlapping substrate specificity to activate mTORC1, stimulating translation and virus replication. This establishes Us3 as a unique viral kinase with promising drug development potential.

New inhibitors of the PI3K-Akt-mTOR pathway: insights into mTOR signaling from a new generation of Tor Kinase Domain Inhibitors (TORKinibs).

mTOR (mammalian Target of Rapamycin) is the hub of the phosphoinositide 3-Kinase (PI3-K)→Akt→mTOR pathway, which is one of the most commonly mutated pathways in cancer. PI3-Ks and mTOR are related kinases which share an evolutionarily related kinase domain, although the former is a lipid kinase and the latter is a protein kinase. As a result of their similar ATP sites, the prototypical PI3-K inhibitors LY294002 and wortmannin inhibit both kinases, although the compounds have been primarily thought of as inhibitors of PI3-Ks. The widespread use of these reagents to understand PI3-K signaling and the likelihood that many of their effects are confounded by dual inhibition of PI3-K and mTOR make it essential to develop selective mTOR inhibitors in part to understand the unique cellular effects of inhibition of this key downstream component in the growth factor pathway. Rapamycin has historically provided a means for selective mTOR inhibition, yet it is not a typical ATP competitive inhibitor, making its effects difficult to reconcile with LY294002 and wortmannin. Several groups have recently reported pharmacological agents which inhibit mTOR but not PI3-K, providing a new pharmacological approach to selective mTOR inhibition. The TOR kinase domain inhibitors of mTOR have been termed TORKinibs to distinguish their mode of action from rapamycin and its analogs (rapalogs). These inhibitors bind to the ATP binding site of the kinase domain of mTOR and as a result inhibit both mTOR complexes, TORC1 (rapamycin sensitive) and TORC2 (rapamycin resistant). These molecules have allowed a reinvestigation of mTOR and in particular a reinvestigation of the mechanistic basis for incomplete proliferative arrest of cells by Rapamycin. A consensus has quickly emerged from the study of various TORKinibs that Rapamycin is ineffective at blocking cell proliferation because it only partially inhibits the activity of mTORC1. The profound anti-proliferative effect of TORKinibs suggests that as the molecules enter the clinic they may be successful in the treatment of cancers where rapamycin has failed.

Genetic dissection of the oncogenic mTOR pathway reveals druggable addiction to translational control via 4EBP-eIF4E.

We genetically dissect the contribution of the most prominent downstream translational components of mTOR signaling toward Akt-driven lymphomagenesis. While phosphorylation of rpS6 is dispensable for cancer formation, 4EBP-eIF4E exerts significant control over cap-dependent translation, cell growth, cancer initiation, and progression. This effect is mediated at least in part through 4EBP-dependent control of Mcl-1 expression, a key antiapoptotic protein. By using an active site inhibitor of mTOR, PP242, we show a marked therapeutic response in rapamycin-resistant tumors. The therapeutic benefit of PP242 is mediated through inhibition of mTORC1-dependent 4EBP-eIF4E hyperactivation. Thus, the 4EBP-eIF4E axis downstream of mTOR is a druggable mediator of translational control and Akt-mediated tumorigenesis that has important implications for the treatment of human cancers.

mTOR complex-2 activates ENaC by phosphorylating SGK1.

The serum- and glucocorticoid-induced kinase 1 (SGK1) plays a central role in hormone regulation of epithelial sodium (Na+) channel (ENaC)-dependent Na+ transport in the distal nephron. Phosphorylation within a carboxy-terminal domain, designated the hydrophobic motif (HM), determines the activity of SGK1, but the identity of the HM kinase is unknown. Here, we show that the highly conserved serine-threonine kinase mammalian target of rapamycin (mTOR) is essential for the phosphorylation of the HM of SGK1 and the activation of ENaC. We observed that mTOR, in conjunction with rictor (mTORC2), phosphorylated SGK1 and stimulated ENaC. In contrast, when mTOR assembled with raptor in the rapamycin-inhibited complex (mTORC1), it did not phosphorylate SGK1 or stimulate ENaC. Inhibition of mTOR blocked both SGK1 phosphorylation and ENaC-mediated Na+ transport, whereas specific inhibition of mTORC1 had no effect. Similarly, small hairpin RNA-mediated knockdown of rictor inhibited SGK1 phosphorylation and Na+ current, whereas knockdown of raptor had no effect. Finally, in co-immunoprecipitation experiments, SGK1 interacted selectively with rictor but not with raptor, suggesting selective recruitment of SGK1 to mTORC2. We conclude that mTOR, specifically mTORC2, is the HM kinase for SGK1 and is required for ENaC-mediated Na+ transport, thereby extending our understanding of the molecular mechanisms underlying Na+ balance.

Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2.

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)-->Akt-->mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.

SHIP prevents lipopolysaccharide from triggering an antiviral response in mice.

Gram-negative bacterial infections, unlike viral infections, do not typically protect against subsequent viral infections. This is puzzling given that lipopolysaccharide (LPS) and double-stranded (ds) RNA both activate the TIR domain-containing adaptor-inducing interferon beta (TRIF) pathway and, thus, are both capable of eliciting an antiviral response by stimulating type I interferon (IFN) production. We demonstrate herein that SH2-containing inositol-5'-phosphatase (SHIP) protein levels are dramatically increased in murine macrophages via the MyD88-dependent pathway, by up-regulating autocrine-acting transforming growth factor-beta (TGFbeta). The increased SHIP then mediates, via inhibition of the phosphatidylinositol-3-kinase (PI3K) pathway, cytosine-phosphate-guanosine (CPG)- and LPS-induced tolerance and cross-tolerance and restrains IFN-beta production induced by a subsequent exposure to LPS or dsRNA. Intriguingly, we found, using isoform-specific PI3K inhibitors, that LPS- or cytosine-phosphate-guanosine-induced interleukin-6 (IL-6) is positively regulated by p110alpha, -gamma, and -delta but negatively regulated by p110beta. This may explain some of the controversy concerning the role of PI3K in Toll-like receptor-induced cytokine production. Consistent with our in vitro findings, SHIP(-/-) mice overproduce IFN-beta in response to LPS, and this leads to antiviral hypothermia. Thus, up-regulation of SHIP in response to Gram-negative bacterial infections probably explains the inability of such infections to protect against subsequent viral infections.

Structure and properties of a re-engineered homeodomain protein-DNA interface.

The homeodomain (HD)-DNA interface has been conserved over 500 million years of evolution. Despite this conservation, we have successfully re-engineered the engrailed HD to specifically recognize an unnatural nucleotide using a phage display selection. Here we report the synthesis of novel nucleosides and the selection of mutant HDs that bind these nucleotides using phage display. The high-resolution crystal structure of one mutant in complex with modified and unmodified DNA demonstrates that, even with the substantial perturbation to the interface, this selected mutant retains a canonical HD structure. Dissection of the contributions due to each of the selected mutations reveals that the majority of the modification-specific binding is accomplished by a single mutation (I47G) but that the remaining mutations retune the stability of the HD. These results afford a detailed look at a re-engineered protein-DNA interaction and provide insight into the opportunities for re-engineering highly conserved interfaces.

RYR2 proteins contribute to the formation of Ca(2+) sparks in smooth muscle.

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.

Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle.

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.

A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells.

Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.

Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.

As a final step in endocytosis, clathrin-coated pits must separate from the plasma membrane and move into the cytosol as a coated vesicle. Because these events involve minute movements that conventional light microscopy cannot resolve, they have not been observed directly and their dynamics remain unexplored. Here, we used evanescent field (EF) microscopy to observe single clathrin-coated pits or vesicles as they draw inwards from the plasma membrane and finally lose their coats. This inward movement occurred immediately after a brief burst of dynamin recruitment and was accompanied by transient actin assembly. Therefore, dynamin may provide the trigger and actin may provide the force for movement into the cytosol.

Stretch-induced calcium release in smooth muscle.

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) release was not due to calcium-induced calcium release, as release was observed in Ca(2+)-free extracellular solution and when free Ca(2+) ions in the cytosol were strongly buffered to prevent increases in [Ca(2+)](i). Stretch-induced calcium release (SICR) was not affected by inhibition of InsP(3)R-mediated Ca(2+) release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca(2+) release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues.