Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.     

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8-3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.     

Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers

Purpose

We previously showed that 90% (47 of 52; 95% CI, 0.79 to 0.96) of lung adenocarcinomas from East Asian never-smokers harbored well-known oncogenic mutations in just four genes: EGFR, HER2, ALK, and KRAS. Here, we sought to extend these findings to more samples and identify driver alterations in tumors negative for these mutations.

Experimental Design

We have collected and analyzed 202 resected lung adenocarcinomas from never smokers seen at Fudan University Shanghai Cancer Center. Since mutations were mutually exclusive in the first 52 examined, we determined the status of EGFR, KRAS, HER2, ALK, and BRAF in stepwise fashion as previously described. Samples negative for mutations in these 5 genes were subsequently examined for known ROS1 fusions by RT-PCR and direct sequencing.

Results

152 tumors (75.3%) harbored EGFR mutations, 12 (6%) had HER2 mutations, 10 (5%) had ALK fusions all involving EML4 as the 5′ partner, 4 (2%) had KRAS mutations, and 2 (1%) harbored ROS1 fusions. No BRAF mutation were detected.

Conclusion

The vast majority (176 of 202; 87.1%, 95% CI: 0.82 to 0.91) of lung adenocarcinomas from never smokers harbor mutant kinases sensitive to available TKIs. Interestingly, patients with EGFR mutant patients tend to be older than those without EGFR mutations (58.3 Vs 54.3, P = 0.016) and patient without any known oncogenic driver tend to be diagnosed at a younger age (52.3 Vs 57.9, P = 0.013). Collectively, these data indicate that the majority of never smokers with lung adenocarcinoma could benefit from treatment with a specific tyrosine kinase inhibitor.     

Creatine kinase B is a target molecule of reactive oxygen species in cervical cancer.

Recently, a procedure for detecting ROS-sensitive proteins that contain active cysteine residues was devdoped. The method is based on the fact that biotin-conjugated iodoacetamide (BIAM) and ROS competitively and selectively react with the active cysteine residues in ROS-sensitive proteins. To investigate the role of ROS in cervical cancer, BIAM labeling on cytosolic proteins in normal and cancer tissues was performed, respectively. The BIAM labeling proteins are separated by 2-dimensional electrophoresis, and then identified by MALDI-TOF mass analysis. ROS-sensitive protein is identified as creatine kinase B containing cysteine residue in active center. Activity of creatine kinase B in normal tissue is higher than that of oxidized form in cervical cancer tissues. The result suggests that ROS play an important role in metabolic regulation in cervical cancer cells. However, molecular mechanisms that ROS and creatine kinase B are integrated into a physiological signal leading to the cellular transformation remain to be elucidated.     

The Natural Anticancer Agent Plumbagin Induces Potent Cytotoxicity in MCF-7 Human Breast Cancer Cells by Inhibiting a PI-5 Kinase for ROS Generation

Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI₅₀; 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.     

Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer

Tyrosine kinase (TK) fusions are attractive drug targets in cancers. However, rapid identification of these lesions has been hampered by experimental limitations. Our in silico analysis of known cancer-derived TK fusions revealed that most breakpoints occur within a defined region upstream of a conserved GXGXXG kinase motif. We therefore designed a novel DNA-based targeted sequencing approach to screen systematically for fusions within the 90 human TKs; it should detect 92% of known TK fusions. We deliberately paired ‘in-solution’ DNA capture with 454 sequencing to minimize starting material requirements, take advantage of long sequence reads, and facilitate mapping of fusions. To validate this platform, we analyzed genomic DNA from thyroid cancer cells (TPC-1) and leukemia cells (KG-1) with fusions known only at the mRNA level. We readily identified for the first time the genomic fusion sequences of CCDC6-RET in TPC-1 cells and FGFR1OP2-FGFR1 in KG-1 cells. These data demonstrate the feasibility of this approach to identify TK fusions across multiple human cancers in a high-throughput, unbiased manner. This method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 µg of DNA, and circumvents the need for complex computational sequence analysis.     

Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.     

Src-JNK-dependent pathway in cigarette smoke-induced mucous hypersecretion in airway epithelial cells

The aim of this work was to find out whether Src kinase family and c-Jun N-terminal kinase (JNK) are involved in the ROS signaling pathway that could induce mucin MUC5AC expression in cultured cells of airway epithelia (BEAS-2B). For this purpose, the impact of cigarette smoke extract (CSE) on ROS production and MUC5AC expression in BEAS-2B cells was studied. Effects of ROS scavenger dimethylthiourea (DMTU), JNK specific inhibitor SP600125, and Src specific inhibitor PP2 in the CSE-induced ROS generation and MUC5AC expression were also assessed. A dose-dependent increase of ROS production in cells exposed to different concentrations of CSE was detected. DMTU inhibited cigarette smoke-induced Src phosphorylation, suggesting the ROS involvement in activation of Src kinase. Furthermore, SP600125 reduced the expression of MUC5AC. The activation of JNK was suppressed by PP2 but not by TACE inhibitor TAPI-1 or EGFR inhibitor PD153035. These results suggest that Src kinase participate in JNK activation and MUC5AC synthesis, which is independent of the TACE/EGFR activation. We conclude that ROS-Src-JNK signal cascade plays a particular role in cigarette smoke-induced mucin MUC5AC expression in BEAS-2B cells.     

Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation

Two polyenylpyrroles from a soil ascomycete Gymnoascus reessii were previously identified as hit compounds in screening for cytotoxicity against lung cancer cells. These compounds and various analogs, which have been previously synthesized and tested for anti-lung cancer cell activity, were tested for anti-inflammatory activity. After preliminary screening for cytotoxicity for RAW 264.7 murine macrophage cells, the non-toxic compounds were tested for anti-inflammatory activity using lipopolysaccharide (LPS)-activated RAW 264.7 cells. Compounds 1h, 1i, and 1n reduced LPS-induced nitric oxide (NO) production, with respective ED₅₀ values of 15 ± 2, 16 ± 2, and 17 ± 2 µM. They also reduced expression of inducible NO synthase and interleukin-6 (IL-6) without affecting cyclooxygenase-2 expression. Compound 1h also reduced secretion of IL-6 and tumor necrosis factor-α by LPS-activated J774A.1 murine macrophage cells, primary mice peritoneal macrophages, and JAWSII murine bone marrow-derived dendritic cells and reduced NLRP3 inflammasome-mediated interleukin-1β (IL-1β) secretion by LPS + adenosine triphosphate-activated J774A.1 and JAWSII cells. The underlying mechanisms for the anti-inflammatory activity of compound 1h were found to be a decrease in LPS-induced reactive oxygen species (ROS) production, mitogen-activated protein kinase phosphorylation, and NF-κB activation and a decrease in ATP-induced ROS production and PKC-α phosphorylation. These results provide promising insights into the anti-inflammatory activity of these conjugated polyenes and a molecular rationale for future therapeutic intervention in inflammation-related diseases. They also show how compound 1h regulates inflammation and suggest it may be a new source for the development of anti-inflammatory agents to ameliorate inflammation- and NLRP3 inflammasome-related diseases.     

Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux

Macroautophagy (hereafter referred to as autophagy) has recently emerged as an attractive target for the treatment of various degenerative diseases and cancer. The discovery of effective pharmaceutical regulators of autophagy has, however, been hindered by a lack of feasible assay systems for autophagic flux. Here, we present a luciferase-based reporter assay that measures autophagic flux in real time in living cells and demonstrate that this assay system is apt for the detection of dose- and stimulus-dependent differences in autophagy kinetics. Furthermore, by screening a small molecule kinase inhibitor library containing 80 compounds we identified 12 compounds as inducers of autophagic flux. Importantly, six inhibitors of the class I phosphoinositide 3-kinase – protein kinase B – mammalian target of rapamycin complex 1 axis, the central signaling pathway repressing autophagy, scored as autophagy inducers adequately validating the screen. We conclude that the assay system presented here allows easy and rapid monitoring of autophagy kinetics and is suitable for screening of small molecule libraries.     

Cyclic GMP-dependent Stimulation of Serotonin Transport Does Not Involve Direct Transporter Phosphorylation by cGMP-dependent Protein Kinase

The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including “ecstasy” (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N⁶-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-³³P]ATP but not from similarly labeled N⁶-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.     

mt-Nd2a suppresses reactive oxygen species production by mitochondrial complexes I and III

Reactive oxygen species (ROS) play a critical role in the pathogenesis of human diseases. A cytosine to adenine transversion in the mitochondrially encoded NADH dehydrogenase subunit 2 (mt-ND2, human; mt-Nd2, mouse) gene results in resistance against type 1 diabetes and several additional ROS-associated conditions. Our previous studies have demonstrated that the adenine-containing allele (mt-Nd2(a)) is also strongly associated with resistance against type 1 diabetes in mice. In this report we have confirmed that the cytosine-containing allele (mt-Nd2(c)) results in elevated mitochondrial ROS production. Using inhibitors of the electron transport chain, we show that when in combination with nuclear genes from the alloxan-resistant (ALR) strain, mt-Nd2(c) increases ROS from complex III. Furthermore, by using alamethicin-permeabilized mitochondria, we measured a significant increase in electron transport chain-dependent ROS production from all mt-Nd2(c)-encoding strains including ALR.mt(NOD), non-obese diabetic (NOD), and C57BL/6 (B6). Studies employing alamethicin and inhibitors were able to again localize the heightened ROS production in ALR.mt(NOD) to complex III and identified complex I as the site of elevated ROS production from NOD and B6 mitochondria. Using submitochondrial particles, we confirmed that in the context of the NOD or B6 nuclear genomes, mt-Nd2(c) elevates complex I-specific ROS production. In all assays mitochondria from mt-Nd2(a)-encoding strains exhibited low ROS production. Our data suggest that lowering overall mitochondrial ROS production is a key mechanism of disease protection provided by mt-Nd2(a).     

Chemical screening identifies ROCK as a target for recovering mitochondrial function in Hutchinson‐Gilford progeria syndrome

Hutchinson‐Gilford progeria syndrome (HGPS) constitutes a genetic disease wherein an aging phenotype manifests in childhood. Recent studies indicate that reactive oxygen species (ROS) play important roles in HGPS phenotype progression. Thus, pharmacological reduction in ROS levels has been proposed as a potentially effective treatment for patient with this disorder. In this study, we performed high‐throughput screening to find compounds that could reduce ROS levels in HGPS fibroblasts and identified rho‐associated protein kinase (ROCK) inhibitor (Y‐27632) as an effective agent. To elucidate the underlying mechanism of ROCK in regulating ROS levels, we performed a yeast two‐hybrid screen and discovered that ROCK1 interacts with Rac1b. ROCK activation phosphorylated Rac1b at Ser71 and increased ROS levels by facilitating the interaction between Rac1b and cytochrome c. Conversely, ROCK inactivation with Y‐27632 abolished their interaction, concomitant with ROS reduction. Additionally, ROCK activation resulted in mitochondrial dysfunction, whereas ROCK inactivation with Y‐27632 induced the recovery of mitochondrial function. Furthermore, a reduction in the frequency of abnormal nuclear morphology and DNA double‐strand breaks was observed along with decreased ROS levels. Thus, our study reveals a novel mechanism through which alleviation of the HGPS phenotype is mediated by the recovery of mitochondrial function upon ROCK inactivation.     

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.     

Isoliquiritigenin isolated from licorice Glycyrrhiza uralensis prevents 6-hydroxydopamine-induced apoptosis in dopaminergic neurons

Licorice (Glycyrrhiza uralensis) is a medicinal herb containing various bioactive components implicated in antioxidative, anti-inflammatory, antiviral, and neuroprotective effects, but the effects of licorice against Parkinson's disease (PD)-related dopaminergic cell death have not been studied. In this study, we investigated the protective effects of isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a dopaminergic cell line, SN4741. ISL (1 μM) significantly attenuated 6-OHDA (50 μM)-induced reactive oxygen species (ROS) and nitric oxide (NO) generation and apoptotic cell death. ISL pretreatment effectively suppressed 6-OHDA-mediated upregulation of Bax, p-c-Jun N-terminal kinase (JNK), p-p38 mitogen-activated protein (MAP) kinase, cytochrome c release, and caspase 3 activation. In addition, ISL significantly attenuated 6-OHDA-induced Bcl-2, brain-derived neurotrophic factor (BDNF), and mitochondrial membrane potential (MMP) reduction. Pharmacological inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway reversed ISL-mediated neuroprotection against 6-OHDA toxicity in SN4741 cells. These results provide the first evidence that ISL can protect dopaminergic cells under oxidative stress conditions by regulating the apoptotic process.