IκB kinase-α coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy.

Activation of the IκB kinase (IKK) complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-κB has remained elusive. Here, analysis of IKK-dependent substrates in CRC cells after UV treatment revealed that phosphorylation of BRD4 by IKK-α is required for its chromatin-binding at target genes upon DNA damage. Moreover, IKK-α induces the NF-κB-dependent transcription of the cytokine LIF, leading to STAT3 activation, association with BRD4 and recruitment to specific target genes. IKK-α abrogation results in defective BRD4 and STAT3 functions and consequently irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK-α activity, BRD4, and the JAK/STAT pathway enhanced the therapeutic potential of 5-fluorouracil combined with irinotecan in CRC cells and is curative in a chemotherapy-resistant xenograft model. Finally, coordinated expression of LIF and IKK-α is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK-α, BRD4, and JAK/STAT signaling with clinical relevance.

Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas.

Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. Here, we demonstrate that haploinsufficient deletions of chromatin-bound tumor suppressor NFKB inhibitor alpha (NFKBIA) display distinct patterns of occurrence in relation to other genetic markers and are disproportionately present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation and induce a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wild-type tumors. An externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas confirms that NFKBIA deletions predict comparatively brief survival. Thus, NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into models predicting the disease fate of diffuse gliomas.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

Ethical Challenges of Early Identification of Advanced Chronic Patients in Need of Palliative Care: The Catalan Experience.

Palliative care must be early applied to all types of advanced chronic and life limited prognosis patients, present in all health and social services. Patients' early identification and registry allows introducing palliative care gradually concomitant with other measures. Patients undergo a systematic and integrated care process, meant to improve their life quality, which includes multidimensional assessment of their needs, recognition of their values and preferences for advance care planning purposes, treatments review, family care, and case management. Leaded by the National Department of Health, a program for the early identification of these patients has been implemented in Catalonia (Spain). Although the overall benefits expected, the program has raised some ethical issues. In order to address these challenges, diverse institutions, including bioethics and ethics committees, have elaborated a proposal for the program's advantages. This paper describes the process of evaluation, elaboration of recommendations, and actions done in Catalonia.

Novel phosphorylated TAK1 species with functional impact on NF-κB and ß-catenin signaling in human Cutaneous T-cell lymphoma.

Cutaneous T-cell lymphomas (CTCLs) represent different subtypes of lymphoproliferative disorders with no curative therapies for the advanced forms of the disease (namely mycosis fungoides and the leukemic variant, Sézary syndrome). Molecular events leading to CTCL progression are heterogeneous, however recent DNA and RNA sequencing studies highlighted the importance of NF-κB and ß-catenin pathways. We here show that the kinase TAK1, known as essential in B-cell lymphoma, is constitutively activated in CTCL cells, but tempered by the MYPT1/PP1 phosphatase complex. Blocking PP1 activity, both pharmacologically and genetically, resulted in TAK1 hyperphosphorylation at residues T344, S389, T444, and T511, which have functional impact on canonical NF-κB signaling. Inhibition of TAK1 precluded NF-κB and ß-catenin signaling and induced apoptosis of CTCL cell lines and primary Sézary syndrome cells both in vitro and in vivo. Detection of phosphorylated TAK1 at T444 and T344 is associated with the presence of lymphoma in a set of 60 primary human samples correlating with NF-κB and ß-catenin activation. These results identified TAK1 as a potential biomarker and therapeutic target for CTCL therapy.

Cytoplasmic accumulation of NCoR in malignant melanoma: consequences of altered gene repression and prognostic significance.

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy available in advanced stages. Nuclear corepressor (NCoR) is an essential regulator of gene transcription, and its function has been found deregulated in different types of cancer. In colorectal cancer cells, loss of nuclear NCoR is induced by Inhibitor of kappa B kinase (IKK) through the phosphorylation of specific serine residues. We here investigate whether NCoR function impacts in MM, which might have important diagnostic and prognostic significance. By IHC, we here determined the subcellular distribution of NCoR in a cohort of 63 primary invasive MM samples, and analyzed its possible correlation with specific clinical parameters. We therefore used a microarray-based strategy to determine global gene expression differences in samples with similar tumor stage, which differ in the presence of cytoplasmic or nuclear NCoR. We found that loss of nuclear NCoR results in upregulation of a specific cancer-related genetic signature, and is significantly associated with MM progression. Inhibition of IKK activity in melanoma cells reverts NCoR nuclear distribution and specific NCoR-regulated gene transcription. Analysis of public database demonstrated that inactivating NCoR mutations are highly prevalent in MM, showing features of driver oncogene.

Theoretical study on two-step mechanisms of peptide release in the ribosome.

A quantum mechanical study of different two-step mechanisms of peptide release in the ribosome has been carried out using the M06-2X density functional. Reoptimization with MP2 has also been carried out for the stationary points of some selected mechanisms. The uncatalyzed processes in solution have been treated with the SMD solvation model. From the results obtained in this paper for the peptide release process we can conclude that the energy barriers for the two-step mechanisms are lower than the ones for the concerted process, that the 2'OH plays also an important role in the catalytic process and that the side chain does not only accommodate a nucleophilic water molecule in the PTC, but it also contributes to activate this molecule through electron transfer to the water oxygen. We have also found that the second step is the rate-determining one, and that the two most favorable mechanisms, in which a water or a formamide molecule is added, follow a quite different strategy to catalyze the reaction. The main conclusion of our work is that the two-step mechanisms cannot be disregarded, since they can contribute to clarify the complex and yet unsolved problem of the mechanism of the peptide release process.

Quantum mechanical study on the mechanism of peptide release in the ribosome.

A quantum mechanical study of different concerted mechanisms of peptide release in the ribosome has been carried out using the M06-2X density functional. Reoptimization with MP2 has also been carried out for the stationary points of some selected mechanisms. The uncatalyzed processes in solution have been treated with the SMD solvation model. We conclude that the 2'-OH plays an important catalytic role and that it takes place via a zwitterionic transition state, this TS being stabilized by the presence of oxyanion holes or by the solvent. The comparison with our previous study on the peptide bond formation shows that both processes proceed via two different mechanisms, in such a way that the TS of the aminolysis has an ion-pair instead of a zwitterionic character. So, despite the limitations of the model we have used, we can conclude that there is catalytic promiscuity at the peptidyl transferase center (PTC) of the ribosome.

Quantum-mechanical study on the mechanism of peptide bond formation in the ribosome.

Ribosomes transform the genetic information encoded within genes into proteins. In recent years, there has been much progress in the study of this complex molecular machine, but the mechanism of peptide bond formation and the origin of the catalytic power of this ancient enzymatic system are still an unsolved puzzle. A quantum-mechanical study of different possible mechanisms of peptide synthesis in the ribosome has been carried out using the M06-2X density functional. The uncatalyzed processes in solution have been treated with the SMD solvation model. Concerted and two-step mechanisms have been explored. Three main points suggested in this work deserve to be deeply analyzed. First, no zwitterionic intermediates are found when the process takes place in the ribosome. Second, the proton shuttle mechanism is suggested to be efficient only through the participation of the A2451 2'-OH and two crystallographic water molecules. Finally, the mechanisms in solution and in the ribosome are very different, and this difference may help us to understand the origin of the efficient catalytic role played by the ribosome.

Effects of ionization on N-glycylglycine peptide: influence of intramolecular hydrogen bonds.

The ionization effects on 28 conformations of N-glycylglycine are analyzed by means of the hybrid B3LYP and the hybrid meta-MPWB1K density functionals and by single-point calculations at the CCSD(T) level of theory. The most favorable process observed corresponds to the ionization of the only neutral conformation that presents a OH...NH2 intramolecular hydrogen bond, which leads to CO2 elimination after a spontaneous proton transfer from -COOH to NH2. The remaining neutral structures evolve to 20 different conformations of N-glycylglycine radical cation, which lie about 25-40 kcal/mol higher than the decarboxylated [NH3CH2CONHCH2]+*...[CO2] complex. Structural changes induced by ionization depend on the intramolecular hydrogen bonds of the initial conformation, since they determine the nature of the electron hole formed. In most cases, ionization takes place at the terminal -NH2 and -CO of the amide bond, which produces a strengthening of the peptide bond and the formation of new -NH2...OC(amide) and -NH2...OCOH hydrogen bonds. However, if -NH2 and -CO(amide) simultaneously act as proton acceptor in the neutral conformation, ionization is mainly localized at the carboxylic group, which produces a strengthening of the -COOH...OC(amide) bond. Both functionals lead to similar trends and compare well with CCSD(T) results except for a few cases for which B3LYP provides a too delocalized picture of the electron hole and consequently leads to artificial geometry reorganization.

Arginine transport via cationic amino acid transporter 2 plays a critical regulatory role in classical or alternative activation of macrophages.

Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases L-arginine transport only through the y(+) system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in L-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages.

Macrophages require distinct arginine catabolism and transport systems for proliferation and for activation.

In murine macrophages, as a result of arginine catabolism during activation, citruline is produced under the effect of IFN-gamma and LPS, and ornithine and polyamines by IL-4 and IL-10. For proliferation, arginine is required from the extracellular medium and is used for protein synthesis. During activation, most arginine (>95% in 6 h) was metabolized, while under proliferation only half was incorporated into proteins. Under basal conditions, this amino acid was preferentially transported by y(+)L activity. During activation, arginine transport increased drastically (4-5-fold) through y(+) cationic amino acid transporter (CAT) activity. By contrast, M-CSF induced only a modest increase in uptake (0.5-fold). The increase in arginine transport during activation, but not proliferation, was mediated by the SLC7A2/Cat2 gene. SLC7A1/Cat1 is constitutively expressed, and is not modified by proliferating or activating agents. M-CSF-dependent proliferation was not affected in the macrophages of SLC7A2 knockout mice; however, these cells showed a drastic reduction in the production of citruline or ornithine and polyamines during activation. The data show that a large increase in a specific transport system (CAT2) is necessary for activation-induced arginine metabolism, while arginine is in excess for the requirements of proliferation and a modest increase in transport occurs.

Granulocyte-macrophage colony-stimulating factor increases L-arginine transport through the induction of CAT2 in bone marrow-derived macrophages.

L-arginine transport is crucial for macrophage activation because it supplies substrate for the key enzymes nitric oxide synthase 2 and arginase I. These enzymes participate in classic and alternative activation of macrophages, respectively. Classic activation of macrophages is induced by type I cytokines, and alternative activation is induced by type II cytokines. The granulocyte macrophage colony-stimulating factor (GM-CSF), in addition to inducing proliferation and differentiation of macrophages, activates arginase I, but its action on L-arginine transport is unknown. We studied the L-arginine transporters that are active in mouse primary bone marrow-derived macrophages (BMM) and examined the effect of GM-CSF treatment on transport activities. Under basal conditions, L-arginine entered mainly through system y(+)L (>75%). The remaining transport was explained by system y(+) (<10%) and a diffusion component (10-15%). In response to GM-CSF treatment, transport activity increased mostly through system y(+) (>10-fold), accounting for about 40% of the total L-arginine transport. The increase in y(+) activity correlated with a rise in cationic amino acid transporter (CAT)-2 mRNA and protein. Furthermore, GM-CSF induced an increase in arginase activity and in the conversion of L-arginine to ornithine, citrulline, glutamate, proline, and polyamines. BMM obtained from CAT2-knockout mice responded to GM-CSF by increasing arginase activity and the expression of CAT1 mRNA, which also encodes system y(+) activity. Nonetheless, the increase in CAT1 activity only partially compensated the lack of CAT2 and L-arginine metabolism was hardly stimulated. We conclude that BMM present mainly y(+)L activity and that, in response to GM-CSF, l-arginine transport augments through CAT2, thereby increasing the availability of this amino acid to the cell.

Lysinuric protein intolerance: mechanisms of pathophysiology.

Heteromeric amino acid transporters (HATs) are composed of two subunits, a polytopic membrane protein (the light subunit) and a disulfide-linked type II membrane glycoprotein (the heavy subunit). HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y(+)L isoforms, asc, xc-, and b(0,+)). The light subunits confer the amino acid transport specificity to the HAT. Two transporters of this family are relevant for inherited aminoacidurias. Mutations in any of the two genes coding for the subunits of system b(0,+) (rBAT and b(0,+)AT) lead to cystinuria (MIM 220100). Transport defects in a system y(+)L isoform, composed of 4F2hc and y(+)LAT-1, result in lysinuric protein intolerance (LPI) (MIM 222700). In this case, only mutations in the light subunit y(+)LAT-1, but not in the heavy chain 4F2hc, cause the disease. LPI, in addition to affecting intestinal and renal reabsorption of amino acids, is a multisystemic disease affecting the urea cycle and presents also with symptoms related to the immune system. The pathogenesis of these alterations is less well, or not understood at all.

Neuregulin signaling on glucose transport in muscle cells.

Neuregulin-1, a growth factor that potentiates myogenesis induces glucose transport through translocation of glucose transporters, in an additive manner to insulin, in muscle cells. In this study, we examined the signaling pathway required for a recombinant active neuregulin-1 isoform (rhHeregulin-beta(1), 177-244, HRG) to stimulate glucose uptake in L6E9 myotubes. The stimulatory effect of HRG required binding to ErbB3 in L6E9 myotubes. PI3K activity is required for HRG action in both muscle cells and tissue. In L6E9 myotubes, HRG stimulated PKBalpha, PKBgamma, and PKCzeta activities. TPCK, an inhibitor of PDK1, abolished both HRG- and insulin-induced glucose transport. To assess whether PKB was necessary for the effects of HRG on glucose uptake, cells were infected with adenoviruses encoding dominant negative mutants of PKBalpha. Dominant negative PKB reduced PKB activity and insulin-stimulated glucose transport but not HRG-induced glucose transport. In contrast, transduction of L6E9 myotubes with adenoviruses encoding a dominant negative kinase-inactive PKCzeta abolished both HRG- and insulin-stimulated glucose uptake. In soleus muscle, HRG induced PKCzeta, but not PKB phosphorylation. HRG also stimulated the activity of p70S6K, p38MAPK, and p42/p44MAPK and inhibition of p42/p44MAPK partially repressed HRG action on glucose uptake. HRG did not affect AMPKalpha(1) or AMPKalpha(2) activities. In all, HRG stimulated glucose transport in muscle cells by activation of a pathway that requires PI3K, PDK1, and PKCzeta, but not PKB, and that shows cross-talk with the MAPK pathway. The PI3K, PDK1, and PKCzeta pathway can be considered as an alternative mechanism, independent of insulin, to induce glucose uptake.

Gas phase dissociation energies of saturated AHn*+ radical cations and AHn neutrals (A = Li-F, Na-Cl): dehydrogenation, deprotonation, and formation of AHn-2*+ - H2 complexes.

The dissociation energies corresponding to the two possible A-H cleavages of A (A = Li-F and Na-Cl) radical cations (loss of a H(+) and loss of a H(.)) have been computed at the CCSD(T)/ 6-311++G(3df,2pd) level of theory and compared to those of their neutral precursors. Removing an electron from AH(n)() decreases dramatically its deprotonation energy, especially for the A molecules (C and ), which become one of the most acidic species of the row, their acid character being only exceeded by FH(.+) and ClH(.+), respectively. However, dehydrogenation energies only decrease for the systems on the left side of the row (up to C and SiH(4)(.+)) for which the electron is removed from a A-H bonding orbital. Nevertheless, the loss of hydrogen is the more favorable cleavage in all cases except FH(.+). Ionization of SiH(4) leads to a Jahn-Teller distorted structure that corresponds to a Si - H(2) complex. Other - eta(2)H(2) complexes in the doublet spin state have also been found to be stable for A = Be, Mg, Al, and P, the hydrogen molecule complexes being more stable than their corresponding radical cations, for Be, Mg, and Al.

The light subunit of system b(o,+) is fully functional in the absence of the heavy subunit.

The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System b(o,+) exchanges dibasic for neutral amino acids. It is composed of rBAT and b(o,+)AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. b(o,+)AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the b(o,+) substrates inside the liposomes. rBAT was essential for the cell surface expression of b(o,+)AT, but it was not required for reconstituted b(o,+)AT transport activity. No system b(o,+) transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted b(o,+)AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of b(o,+)AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted b(o,+)AT activity. Thus, subunit b(o,+)AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters.