Haploinsufficiency of Cyfip2 Causes Lithium-Responsive Prefrontal Dysfunction.

OBJECTIVE: Genetic variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) encoding an actin-regulatory protein are associated with brain disorders, including intellectual disability and epilepsy. However, specific in vivo neuronal defects and potential treatments for CYFIP2-associated brain disorders remain largely unknown. Here, we characterized Cyfip2 heterozygous (Cyfip2+/- ) mice to understand their neurobehavioral phenotypes and the underlying pathological mechanisms. Furthermore, we examined a potential treatment for such phenotypes of the Cyfip2+/- mice and specified a neuronal function mediating its efficacy. METHODS: We performed behavioral analyses of Cyfip2+/- mice. We combined molecular, ultrastructural, and in vitro and in vivo electrophysiological analyses of Cyfip2+/- prefrontal neurons. We also selectively reduced CYFIP2 in the prefrontal cortex (PFC) of mice with virus injections. RESULTS: Adult Cyfip2+/- mice exhibited lithium-responsive abnormal behaviors. We found increased filamentous actin, enlarged dendritic spines, and enhanced excitatory synaptic transmission and excitability in the adult Cyfip2+/- PFC that was restricted to layer 5 (L5) neurons. Consistently, adult Cyfip2+/- mice showed increased seizure susceptibility and auditory steady-state responses from the cortical electroencephalographic recordings. Among the identified prefrontal defects, lithium selectively normalized the hyperexcitability of Cyfip2+/- L5 neurons. RNA sequencing revealed reduced expression of potassium channel genes in the adult Cyfip2+/- PFC. Virus-mediated reduction of CYFIP2 in the PFC was sufficient to induce L5 hyperexcitability and lithium-responsive abnormal behavior. INTERPRETATION: These results suggest that L5-specific prefrontal dysfunction, especially hyperexcitability, underlies both the pathophysiology and the lithium-mediated amelioration of neurobehavioral phenotypes in adult Cyfip2+/- mice, which can be implicated in CYFIP2-associated brain disorders. ANN NEUROL 2020;88:526-543.

Association of TIM-3 expression with glucose metabolism in Jurkat T cells.

BACKGROUND: T cell activation is associated with increase in glycolysis and glutaminolysis. T cell immunoglobulin and mucin domain containing protein-3 (TIM-3), a T cell surface molecule, downregulates T cell activation and leads to insufficient immunity in cancer and chronic infection. TIM-3 regulates T cell activation possibly through alterations in metabolism; however, the relationship between TIM-3 expression and T cell metabolic changes has not been well studied. RESULTS: We investigated the association between TIM-3 expression and metabolic changes by analyzing glucose metabolism, glutamine metabolism, and mitochondrial function in TIM-3 overexpressing or knockout Jurkat T cell lines relative to their control cell lines. Glucose uptake and consumption, and lactate release were downregulated by TIM-3 expression but upregulated by TIM-3 knockout. Concomitantly, the expression of the glucose transporter, Glut1, but not Glut2, 3, or 4 was altered by TIM-3 expression. However, TIM-3 expression alone could not account for the change in glutamine consumption, glutamate release, and mitochondrial mass, ROS production or membrane potential in these cell lines. CONCLUSION: Our results show the association of TIM-3 expression with T cell glucose metabolism. These results are significant in chronic infections and cancers where it is necessary to control TIM-3 expressing T cells.

Raf kinase inhibitory protein is required for cerebellar long-term synaptic depression by mediating PKC-dependent MAPK activation.

It was demonstrated previously that a positive feedback loop, including protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), is required for the gradual expression of cerebellar long-term depression (LTD). PKC and MAPK are mutually activated in this loop. MAPK-dependent PKC activation is likely to be mediated by phospholipase A2. On the other hand, it is not clear how PKC activates MAPK. Therefore, the entire picture of this loop was not fully understood. We here test the hypothesis that this loop is completed by the PKC substrate, Raf kinase inhibitory protein (RKIP). To test this hypothesis, we used a mutant form of RKIP that is not phosphorylated by PKC and thus constitutively inhibits Raf-1 and MEK, upstream kinases of MAPK. When this RKIP mutant was introduced into Purkinje cells of mouse cerebellar slices through patch-clamp electrodes, LTD was blocked, while wild-type (WT) RKIP had no effect on LTD. Physiological epistasis experiments demonstrated that RKIP works downstream of PKC and upstream of MAPK during LTD induction. Furthermore, biochemical analyses demonstrated that endogenous RKIP dissociates from Raf-1 and MEK during LTD induction in a PKC-dependent manner, suggesting that RKIP binding-dependent inhibition of Raf-1 and MEK is removed upon LTD induction. We therefore conclude that PKC-dependent regulation of RKIP leads to MAPK activation, with RKIP completing the positive feedback loop that is required for LTD.

Modulation of the Gut Microbiota Alters the Tumour-Suppressive Efficacy of Tim-3 Pathway Blockade in a Bacterial Species- and Host Factor-Dependent Manner.

T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) is an immune checkpoint molecule and a target for anti-cancer therapy. In this study, we examined whether gut microbiota manipulation altered the anti-tumour efficacy of Tim-3 blockade. The gut microbiota of mice was manipulated through the administration of antibiotics and oral gavage of bacteria. Alterations in the gut microbiome were analysed by 16S rRNA gene sequencing. Gut dysbiosis triggered by antibiotics attenuated the anti-tumour efficacy of Tim-3 blockade in both C57BL/6 and BALB/c mice. Anti-tumour efficacy was restored following oral gavage of faecal bacteria even as antibiotic administration continued. In the case of oral gavage of Enterococcus hirae or Lactobacillus johnsonii, transferred bacterial species and host mouse strain were critical determinants of the anti-tumour efficacy of Tim-3 blockade. Bacterial gavage did not increase the alpha diversity of gut microbiota in antibiotic-treated mice but did alter the microbiome composition, which was associated with the restoration of the anti-tumour efficacy of Tim-3 blockade. Conclusively, our results indicate that gut microbiota modulation may improve the therapeutic efficacy of Tim-3 blockade during concomitant antibiotic treatment. The administered bacterial species and host factors should be considered in order to achieve therapeutically beneficial modulation of the microbiota.

Free-standing nanocomposite multilayers with various length scales, adjustable internal structures, and functionalities.

We introduce an innovative and robust method for the preparation of nanocomposite multilayers, which allows accurate control over the placement of functional groups as well as the composition and dimensions of individual layers/internal structure. By employing the photocross-linkable polystyrene (PS-N(3), M(n) = 28.0 kg/mol) with 10 wt % azide groups (-N(3)) for host polymer and/or the PS-N(3)-SH (M(n) = 6.5 kg/mol) with azide and thiol (-SH) groups for capping ligands of inorganic nanoparticles, nanocomposite multilayers were prepared by an efficient photocross-linking layer-by-layer process, without perturbing underlying layers and nanostructures. The thickness of individual layers could be controlled from a few to hundreds of nanometers producing highly ordered internal structure, and the resulting nanocomposite multilayers, consisting of polymer and inorganic nanoparticles (CdSe@ZnS, Au, and Pt), exhibit a variety of interesting physical properties. These include prolonged photoluminescent durability, facile color tuning, and the ability to prepare functional free-standing films that can have the one-dimensional photonic band gap and furthermore be patterned by photolithography. This robust and tailored method opens a new route for the design of functional film devices based on nanocomposite multilayers.

Determination of low molecular weight monocarboxylic Acid gases in the atmosphere by parallel plate diffusion scrubber-ion chromatography.

A method has been developed for the determination of the concentration of low molecular weight monocarboxylic acid gases in the atmosphere. The method involves quantitative collection of analyte gases using a parallel plate diffusion scrubber and subsequent ion analysis by ion chromatography. Among the gases are formic acid, acetic acid, propionic acid, n-butyric acid, and n-valeric acid. Method detection limits are on the order of sub to low parts-per-trillion by volume. The method has been successfully applied to ambient air analysis.

Regulation of TIM-3 expression in a human T cell line by tumor-conditioned media and cyclic AMP-dependent signaling.

T cell immunoglobulin and mucin domain-3 (TIM-3) expression increases in exhausted T cells, which inhibits T cell function. TIM-3 expression is supposedly up-regulated in tumor-bearing individuals via chronic antigenic stimulation of T cells. Considering the immunosuppressive nature of the tumor microenvironment, we investigated whether tumor-secreted molecules might enhance TIM-3 expression in Jurkat T cells. We observed that TIM-3 expression was increased by the activation of prostaglandin (PG) E2 and cyclic AMP (cAMP) signaling pathways. Adenylate cyclase activation led to protein kinase A (PKA)-dependent upregulation of the TIM-3 minimal promoter region and of upstream conserved non-coding sequences. TIM-3 expression in Jurkat T cells was increased by the exposure to breast tumor cell-conditioned media partially through the interaction between PGE2 and its receptor, EP4. Our results propose that tumor-secreted molecules such as PGE2, which activates PKA and EPAC, may regulate TIM-3 expression in T cells.

Silver nanoparticles induce reactive oxygen species-mediated cell cycle delay and synergistic cytotoxicity with 3-bromopyruvate in Candida albicans, but not in Saccharomyces cerevisiae.

Background: Silver nanoparticles (AgNPs) inhibit the proliferation of various fungi; however, their mechanisms of action remain poorly understood. To better understand the inhibitory mechanisms, we focused on the early events elicited by 5 nm AgNPs in pathogenic Candida albicans and non-pathogenic Saccharomyces cerevisiae. Methods: The effect of 5 nm and 100 nm AgNPs on fungus cell proliferation was analyzed by growth kinetics monitoring and spot assay. We examined cell cycle progression, reactive oxygen species (ROS) production, and cell death using flow cytometry. Glucose uptake was assessed using tritium-labeled 2-deoxyglucose. Results: The growth of both C. albicans and S. cerevisiae was suppressed by treatment with 5 nm AgNPs but not with 100 nm AgNPs. In addition, 5 nm AgNPs induced cell cycle arrest and a reduction in glucose uptake in both fungi after 30 minutes of culture in a dose-dependent manner (P<0.05). However, in C. albicans only, an increase in ROS production was detected after exposure to 5 nm AgNPs. Concordantly, an ROS scavenger blocked the effect of 5 nm AgNPs on the cell cycle and glucose uptake in C. albicans only. Furthermore, the growth-inhibition effect of 5 nm AgNPs was not greater in S. cerevisiae mutant strains deficient in oxidative stress response genes than it was in wild type. Finally, 5 nm AgNPs together with a glycolysis inhibitor, 3-bromopyruvate, synergistically enhanced cell death in C. albicans (P<0.05) but not in S. cerevisiae. Conclusion: AgNPs exhibit antifungal activity in a manner that may or may not be ROS dependent, according to the fungal species. The combination of AgNPs with 3-bromopyruvate may be more useful against infection with C. albicans.

Smaller Body Size, Early Postnatal Lethality, and Cortical Extracellular Matrix-Related Gene Expression Changes of Cyfip2-Null Embryonic Mice.

Cytoplasmic FMR1-interacting protein 2 (CYFIP2) is a key component of the WAVE regulatory complex (WRC) which regulates actin polymerization and branching in diverse cellular compartments. Recent whole exome sequencing studies identified de novo hotspot variants in CYFIP2 from patients with early-onset epileptic encephalopathy and microcephaly, suggesting that CYFIP2 may have some functions in embryonic brain development. Although perinatal lethality of Cyfip2-null (Cyfip2 -/-) mice was reported, the exact developmental time point and cause of lethality, and whether Cyfip2 -/- embryonic mice have brain abnormalities remain unknown. We found that endogenous Cyfip2 is mainly expressed in the brain, spinal cord, and thymus of mice at late embryonic stages. Cyfip2 -/- embryos did not show lethality at embryonic day 18.5 (E18.5), but their body size was smaller than that of wild-type (WT) or Cyfip2 +/- littermates. Meanwhile, at postnatal day 0, all identified Cyfip2 -/- mice were found dead, suggesting early postnatal lethality of the mice. Nevertheless, the brain size and cortical cytoarchitecture were comparable among WT, Cyfip2 +/-, and Cyfip2 -/- mice at E18.5. Using RNA-sequencing analyses, we identified 98 and 72 differentially expressed genes (DEGs) from the E18.5 cortex of Cyfip2 +/- and Cyfip2 -/- mice, respectively. Further bioinformatic analyses suggested that extracellular matrix (ECM)-related gene expression changes in Cyfip2 -/- embryonic cortex. Together, our results suggest that CYFIP2 is critical for embryonic body growth and for early postnatal survival, and that loss of its expression leads to ECM-related gene expression changes in the embryonic cortex without severe gross morphological defects.

Age-dependent decrease of GAD65/67 mRNAs but normal densities of GABAergic interneurons in the brain regions of Shank3-overexpressing manic mouse model.

Dysfunction of inhibitory GABAergic interneurons is considered a major pathophysiological feature of various neurodevelopmental and neuropsychiatric disorders. The variants of SHANK3 gene, encoding a core scaffold protein of the excitatory postsynapse, have been associated with numerous brain disorders. It has been suggested that abnormalities of GABAergic interneurons could contribute to the SHANK3-related disorders, but the limitation of these studies is that they used mainly Shank3 knock-out mice. Notably, Shank3-overexpressing transgenic mice, modeling human hyperkinetic disorders, also show reduced inhibitory synaptic transmission, abnormal electroencephalography, and spontaneous seizures. However, it has not been investigated whether these phenotypes of Shank3 transgenic mice are associated with GABAergic interneuron dysfunction, or solely due to the cell-autonomous postsynaptic changes of principal neurons. To address this issue, we investigated the densities of parvalbumin- and somatostatin-positive interneurons, and the mRNA and protein levels of GAD65/67 GABA-synthesizing enzymes in the medial prefrontal cortex, striatum, and hippocampus of adult Shank3 transgenic mice. We found no significant difference in the measurements performed on wild-type versus Shank3 transgenic mice, except for the decreased GAD65 or GAD67 mRNAs in these brain regions. Interestingly, only GAD65 mRNA was decreased in the hippocampus, but not mPFC and striatum, of juvenile Shank3 transgenic mice which, unlike the adult mice, did not show behavioral hyperactivity. Together, our results suggest age-dependent decrease of GAD65/67 mRNAs but normal densities of certain GABAergic interneurons in the Shank3 transgenic mice.

Integrative Analysis of Brain Region-specific Shank3 Interactomes for Understanding the Heterogeneity of Neuronal Pathophysiology Related to SHANK3 Mutations.

Recent molecular genetic studies have identified 100s of risk genes for various neurodevelopmental and neuropsychiatric disorders. As the number of risk genes increases, it is becoming clear that different mutations of a single gene could cause different types of disorders. One of the best examples of such a gene is SHANK3, which encodes a core scaffold protein of the neuronal excitatory post-synapse. Deletions, duplications, and point mutations of SHANK3 are associated with autism spectrum disorders, intellectual disability, schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Nevertheless, how the different mutations of SHANK3 can lead to such phenotypic diversity remains largely unknown. In this study, we investigated whether Shank3 could form protein complexes in a brain region-specific manner, which might contribute to the heterogeneity of neuronal pathophysiology caused by SHANK3 mutations. To test this, we generated a medial prefrontal cortex (mPFC) Shank3 in vivo interactome consisting of 211 proteins, and compared this protein list with a Shank3 interactome previously generated from mixed hippocampal and striatal (HP+STR) tissues. Unexpectedly, we found that only 47 proteins (about 20%) were common between the two interactomes, while 164 and 208 proteins were specifically identified in the mPFC and HP+STR interactomes, respectively. Each of the mPFC- and HP+STR-specific Shank3 interactomes represents a highly interconnected network. Upon comparing the brain region-enriched proteomes, we found that the large difference between the mPFC and HP+STR Shank3 interactomes could not be explained by differential protein expression profiles among the brain regions. Importantly, bioinformatic pathway analysis revealed that the representative biological functions of the mPFC- and HP+STR-specific Shank3 interactomes were different, suggesting that these interactors could mediate the brain region-specific functions of Shank3. Meanwhile, the same analysis on the common Shank3 interactors, including Homer and GKAP/SAPAP proteins, suggested that they could mainly function as scaffolding proteins at the post-synaptic density. Lastly, we found that the mPFC- and HP+STR-specific Shank3 interactomes contained a significant number of proteins associated with neurodevelopmental and neuropsychiatric disorders. These results suggest that Shank3 can form protein complexes in a brain region-specific manner, which might contribute to the pathophysiological and phenotypic diversity of disorders related to SHANK3 mutations.

Phosphorylation of CYFIP2, a component of the WAVE-regulatory complex, regulates dendritic spine density and neurite outgrowth in cultured hippocampal neurons potentially by affecting the complex assembly.

Actin dynamics is a critical mechanism underlying many cellular processes in neurons. The heteropentameric WAVE-regulatory complex (WRC), consisting of WAVE, CYFIP1/2, Nap, Abi, and HSPC300, is a key regulator of actin dynamics that activates the Arp2/3 complex to initiate actin polymerization and branching. The WRC is basally inactive because of intermolecular interactions among the components, which can be modulated by bindings of phospholipids and Rac1, and phosphorylations of WAVE and Abi. However, the phosphorylation of other components of WRC and their functional significance remain largely unknown. To address this issue, we focused on CYFIP1/2, in which we found two brain-specific phosphorylation sites (S582 of CYFIP2 and T1068/T1067 of CYFIP1/2) from a publicly available phosphoproteome database. To understand their functional effects, we overexpressed wild-type, phospho-blocking, or phospho-mimetic mutants of CYFIP2 in cultured hippocampal neurons, and found that only T1067A CYFIP2 decreased the density of stubby spines. Moreover, overexpression of wild-type CYFIP2 increased neurite length, but T1067A did not exert this effect. To understand the mechanism, we modeled CYFIP2 phosphorylation in the crystal structure of WRC and found that T1067 phosphorylation could weaken the interaction between CYFIP2 and Nap1 by inducing conformational changes of CYFIP2 α-helical bundles. In the co-immunoprecipitation assay, however, wild-type, T1067A, and T1067E CYFIP2 showed similar interaction levels to Nap1, suggesting that T1067 phosphorylation alone is not sufficient to disrupt the interaction. Considering that the activation of WRC requires disassembly of the complex, our results suggest that T1067 phosphorylation, together with other factors, could contribute toward the activation process.

Striatal Transcriptome and Interactome Analysis of Shank3-overexpressing Mice Reveals the Connectivity between Shank3 and mTORC1 Signaling.

Mania causes symptoms of hyperactivity, impulsivity, elevated mood, reduced anxiety and decreased need for sleep, which suggests that the dysfunction of the striatum, a critical component of the brain motor and reward system, can be causally associated with mania. However, detailed molecular pathophysiology underlying the striatal dysfunction in mania remains largely unknown. In this study, we aimed to identify the molecular pathways showing alterations in the striatum of SH3 and multiple ankyrin repeat domains 3 (Shank3)-overexpressing transgenic (TG) mice that display manic-like behaviors. The results of transcriptome analysis suggested that mammalian target of rapamycin complex 1 (mTORC1) signaling may be the primary molecular signature altered in the Shank3 TG striatum. Indeed, we found that striatal mTORC1 activity, as measured by mTOR S2448 phosphorylation, was significantly decreased in the Shank3 TG mice compared to wild-type (WT) mice. To elucidate the potential underlying mechanism, we re-analyzed previously reported protein interactomes, and detected a high connectivity between Shank3 and several upstream regulators of mTORC1, such as tuberous sclerosis 1 (TSC1), TSC2 and Ras homolog enriched in striatum (Rhes), via 94 common interactors that we denominated "Shank3-mTORC1 interactome". We noticed that, among the 94 common interactors, 11 proteins were related to actin filaments, the level of which was increased in the dorsal striatum of Shank3 TG mice. Furthermore, we could co-immunoprecipitate Shank3, Rhes and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) proteins from the striatal lysate of Shank3 TG mice. By comparing with the gene sets of psychiatric disorders, we also observed that the 94 proteins of Shank3-mTORC1 interactome were significantly associated with bipolar disorder (BD). Altogether, our results suggest a protein interaction-mediated connectivity between Shank3 and certain upstream regulators of mTORC1 that might contribute to the abnormal striatal mTORC1 activity and to the manic-like behaviors of Shank3 TG mice.

Bipolar Disorder Associated microRNA, miR-1908-5p, Regulates the Expression of Genes Functioning in Neuronal Glutamatergic Synapses.

Bipolar disorder (BD), characterized by recurrent mood swings between depression and mania, is a highly heritable and devastating mental illness with poorly defined pathophysiology. Recent genome-wide molecular genetic studies have identified several protein-coding genes and microRNAs (miRNAs) significantly associated with BD. Notably, some of the proteins expressed from BD-associated genes function in neuronal synapses, suggesting that abnormalities in synaptic function could be one of the key pathogenic mechanisms of BD. In contrast, however, the role of BD-associated miRNAs in disease pathogenesis remains largely unknown, mainly because of a lack of understanding about their target mRNAs and pathways in neurons. To address this problem, in this study, we focused on a recently identified BD-associated but uncharacterized miRNA, miR-1908-5p. We identified and validated its novel target genes including DLGAP4, GRIN1, STX1A, CLSTN1 and GRM4, which all function in neuronal glutamatergic synapses. Moreover, bioinformatic analyses of human brain expression profiles revealed that the expression levels of miR-1908-5p and its synaptic target genes show an inverse-correlation in many brain regions. In our preliminary experiments, the expression of miR-1908-5p was increased after chronic treatment with valproate but not lithium in control human neural progenitor cells. In contrast, it was decreased by valproate in neural progenitor cells derived from dermal fibroblasts of a BD subject. Together, our results provide new insights into the potential role of miR-1908-5p in the pathogenesis of BD and also propose a hypothesis that neuronal synapses could be a key converging pathway of some BD-associated protein-coding genes and miRNAs.

Using mammalian GFP reconstitution across synaptic partners (mGRASP) to map synaptic connectivity in the mouse brain.

Many types of questions in neuroscience require the detection and mapping of synapses in the complex mammalian brain. A tool, mammalian GFP reconstitution across synaptic partners (mGRASP), offers a relatively easy, quick and economical approach to this technically challenging task. Here we describe in step-by-step detail the protocols for virus production, gene delivery, brain specimen preparation, fluorescence imaging and image analysis, calibrated substantially and specifically to make mGRASP-assisted circuit mapping (mGRASPing) practical in the mouse brain. The protocol includes troubleshooting suggestions and solutions to common problems. The mGRASP method is suitable for mapping mammalian synaptic connectivity at multiple scales: microscale for synapse-by-synapse or neuron-by-neuron analysis, and mesoscale for revealing local and long-range circuits. The entire protocol takes 5-6 weeks, including time for incubation and virus expression.

Structured synaptic connectivity between hippocampal regions.

The organization of synaptic connectivity within a neuronal circuit is a prime determinant of circuit function. We performed a comprehensive fine-scale circuit mapping of hippocampal regions (CA3-CA1) using the newly developed synapse labeling method, mGRASP. This mapping revealed spatially nonuniform and clustered synaptic connectivity patterns. Furthermore, synaptic clustering was enhanced between groups of neurons that shared a similar developmental/migration time window, suggesting a mechanism for establishing the spatial structure of synaptic connectivity. Such connectivity patterns are thought to effectively engage active dendritic processing and storage mechanisms, thereby potentially enhancing neuronal feature selectivity.

Prx I suppresses K-ras-driven lung tumorigenesis by opposing redox-sensitive ERK/cyclin D1 pathway.

AIMS: Coupled responses of mutated K-ras and oxidative stress are often an important etiological factor in non-small-cell lung cancer (NSCLC). However, relatively few studies have examined the control mechanism of oxidative stress in oncogenic K-ras-driven NSCLC progression. Here, we studied whether the redox signaling pathway governed by peroxiredoxin I (Prx I) is involved in K-ras(G12D)-mediated lung adenocarcinogenesis. RESULTS: Using human-lung adenocarcinoma tissues and lung-specific K-ras(G12D)-transgenic mice, we found that Prx I was significantly up-regulated in the tumor regions via activation of nuclear erythroid 2-related factor 2 (Nrf2) transcription. Interestingly, the increased reactive oxygen species (ROS) by null mutation of Prx I greatly promoted K-ras(G12D)-driven lung tumorigenesis in number and size, which appeared to require the activation of the ROS-dependent extracellular signal-regulated kinase (ERK)/cyclin D1 pathway. INNOVATION: Taken together, these results suggest that Prx I functions as an Nrf2-dependently inducible tumor suppressant in K-ras-driven lung adenocarcinogenesis by opposing ROS/ERK/cyclin D1 pathway activation. CONCLUSION: These findings provide a better understanding of oxidative stress-mediated lung tumorigenesis.