Genetic variants of kinase suppressors of Ras (KSR1) to predict survival in patients with ERα-positive advanced breast cancer

In patients with breast cancer (BC), deregulation of estrogen receptor (ERα) activity may account for most resistance to endocrine therapies. Our previous study used a whole-human kinome siRNA screen to identify functional actors in ERα modulation and showed the implication of proteins kinase suppressors of ras (KSR1). From those findings we evaluated the clinical impact of KSR1 variants in patients with ERα+ BC treated with TAM. DNA was obtained from 222 patients with advanced ERα+ BC treated with TAM who had undergone surgery from 1981 to 2003. We selected three potentially functional relevant KSR1 polymorphisms; two within the 3'UTR (rs224190, rs1075952) and one in the coding exon 7 (rs2293180). The primary end points were overall survival (OS) and disease-free survival (DFS). After a 6.4-year median follow-up, patients carrying the rs2241906 TT genotype showed shorter DFS (2.1 vs 7.1 years, P=0.005) and OS (2.6 vs 8.4 years P=0.002) than those with the TC or TT genotypes. Those associations remained significant in the multivariable analysis adjusting age, lymph node status, LMTK3 and IGFR variants and HER2 status. The polymorphisms rs2241906 and rs1075952 were in linkage disequilibrium. No association was shown between rs2293180 and survival. Among the actors of ERα signaling, KSR1 rs2241906 variants may predict survival in patients with advanced ERα+ BC treated with adjuvant TAM.

The dual EGFR/HER-2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN-38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER-2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN-38, the active metabolite of irinotecan (CPT-11), in colon and gastric cancer cell lines. Concentration-dependent antiproliferative effects of both lapatinib and SN-38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08-11.7 muM; SN-38 range 3.6-256 nM). Lapatinib potently inhibited the growth of a HER-2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER-2 expression. The combination of lapatinib and SN-38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN-38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN-38 when compared to SN-38 treatment alone. Finally, the combination of lapatinib and CPT-11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT-11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas.

Prognostic role of lemur tyrosine kinase-3 germline polymorphisms in adjuvant gastric cancer in Japan and the United States

Lemur tyrosine kinase-3 (LMTK3) was recently identified as an estrogen receptor (ER)-α modulator related to endocrine therapy resistance, and its polymorphisms rs9989661 (T>C) T/T genotype and rs8108419 (G>A) G/G or A/G genotype predicted improved outcomes in breast cancer. Because different predominant ER distributions link to breast and gastric cancer and little is known of the prognostic role of LMTK3 in gastric cancer, this study was carried out to clarify the prognostic role of these polymorphisms in gastric cancer. One-hundred and sixty-nine Japanese and 137 U.S. patients with localized gastric adenocarcinoma were enrolled. Genomic DNA was extracted from blood or tissue, and all samples were analyzed by PCR-based direct DNA sequencing. Overall, these polymorphisms were not associated with survival in both cohorts. When gender was considered, in multivariate analysis, harboring rs9989661 T/T genotype was associated with disease-free survival [HR, 4.37; 95% confidence interval (CI), 2.08-9.18; P < 0.0001] and overall survival (OS; HR, 3.69; 95% CI, 1.65-8.24; P = 0.0014) in the Japanese males and time to recurrence (HR, 7.29; 95% CI, 1.07-49.80; P = 0.043) in the U.S. females. Meanwhile, harboring rs8108419 G/G genotype was associated with OS in the Japanese females (HR, 3.04; 95% CI, 1.08-8.56; P = 0.035) and the U.S. males (HR, 3.39; 95% CI, 1.31-8.80; P = 0.012). The prognostic role of these polymorphisms may be negative in gastric cancer. These findings suggest that the estrogen pathway may play a prognostic role in patients with gastric cancer but this may be dependent on the regional differences both in physiology and genetic alterations of gastric cancer.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling.

METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided.

RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts.

CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440.

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. Δppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.

Developmental switch in the kinase dependency of long-term potentiation depends on expression of GluA4 subunit-containing AMPA receptors

The AMPA-receptor subunit GluA4 is expressed transiently in CA1 pyramidal neurons at the time synaptic connectivity is forming, but its physiological significance is unknown. Here we show that GluA4 expression is sufficient to alter the signaling requirements of long-term potentiation (LTP) and can fully explain the switch in the LTP kinase dependency from PKA to Ca2(+)/calmodulin-dependent protein kinase II during synapse maturation. At immature synapses, activation of PKA leads to a robust potentiation of AMPA-receptor function via the mobilization of GluA4. Analysis of GluA4-deficient mice indicates that this mechanism is critical for neonatal PKA-dependent LTP. Furthermore, lentiviral expression of GluA4 in CA1 neurons conferred a PKA-dependent synaptic potentiation and LTP regardless of the developmental stage. Thus, GluA4 defines the signaling requirements for LTP and silent synapse activation during a critical period of synapse development.

Synthesis, characterization, electrochemical behavior and in vitro protein tyrosine kinase inhibitory activity of the cymene-halogenobenzohydroxamato [Ru(eta(6)-cymene)(bha)Cl] complexes

The ruthenium(II)-cymene complexes [Ru(eta(6)-cymene)(bha)Cl] with substituted halogenobenzohydroxamato (bha) ligands (substituents = 4-F, 4-Cl, 4-Br, 2,4-F-2, 3,4-F-2, 2,5-F-2, 2,6-F-2) have been synthesized and characterized by elemental analysis, IR, H-1 NMR, C-13 NMR, cyclic voltammetry and controlled-potential electrolysis, and density functional theory (DFT) studies. The compositions of their frontier molecular orbitals (MOs) were established by DFT calculations, and the oxidation and reduction potentials are shown to follow the orders of the estimated vertical ionization potential and electron affinity, respectively. The electrochemical E-L Lever parameter is estimated for the first time for the various bha ligands, which can thus be ordered according to their electron-donor character. All complexes exhibit very strong protein tyrosine kinase (PTK) inhibitory activity, even much higher than that of genistein, the clinically used PTK inhibitory drug. The complex containing the 2,4-difluorobenzohydroxamato ligand is the most active one, and the dependences of the PTK activity of the complexes and of their redox potentials on the ring substituents are discussed. (C) 2012 Elsevier B.V. All rights reserved.

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas

Biophysical Chemistry 110 (2004) 83–92

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

PHYTOCHROME KINASE SUBSTRATE4 modulates phytochrome-mediated control of hypocotyl growth orientation

Gravity and light are major factors shaping plant growth. Light perceived by phytochromes leads to seedling deetiolation, which includes the deviation from vertical hypocotyl growth and promotes hypocotyl phototropism. These light responses enhance survival of young seedlings during their emergence from the soil. The PHYTOCHROME KINASE SUBSTRATE (PKS) family is composed of four members in Arabidopsis (Arabidopsis thaliana): PKS1 to PKS4. Here we show that PKS4 is a negative regulator of both phytochrome A- and B-mediated inhibition of hypocotyl growth and promotion of cotyledon unfolding. Most prominently, pks4 mutants show abnormal phytochrome-modulated hypocotyl growth orientation. In dark-grown seedlings hypocotyls change from the original orientation defined by seed position to the upright orientation defined by gravity and light reduces the magnitude of this shift. In older seedlings with the hypocotyls already oriented by gravity, light promotes the deviation from vertical orientation. Based on the characterization of pks4 mutants we propose that PKS4 inhibits changes in growth orientation under red or far-red light. Our data suggest that in these light conditions PKS4 acts as an inhibitor of asymmetric growth. This hypothesis is supported by the phenotype of PKS4 overexpressers. Together with previous findings, these results indicate that the PKS family plays important functions during light-regulated tropic growth responses

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

GLUT4, AMP kinase, but not the insulin receptor, are required for hepatoportal glucose sensor-stimulated muscle glucose utilization.

Recent evidence suggests the existence of a hepatoportal vein glucose sensor, whose activation leads to enhanced glucose use in skeletal muscle, heart, and brown adipose tissue. The mechanism leading to this increase in whole body glucose clearance is not known, but previous data suggest that it is insulin independent. Here, we sought to further determine the portal sensor signaling pathway by selectively evaluating its dependence on muscle GLUT4, insulin receptor, and the evolutionarily conserved sensor of metabolic stress, AMP-activated protein kinase (AMPK). We demonstrate that the increase in muscle glucose use was suppressed in mice lacking the expression of GLUT4 in the organ muscle. In contrast, glucose use was stimulated normally in mice with muscle-specific inactivation of the insulin receptor gene, confirming independence from insulin-signaling pathways. Most importantly, the muscle glucose use in response to activation of the hepatoportal vein glucose sensor was completely dependent on the activity of AMPK, because enhanced hexose disposal was prevented by expression of a dominant negative AMPK in muscle. These data demonstrate that the portal sensor induces glucose use and development of hypoglycemia independently of insulin action, but by a mechanism that requires activation of the AMPK and the presence of GLUT4.

PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism.

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.