Reaction Mechanisms of H2S Oxidation by Naphthoquinones.

1,4-naphthoquinones (NQs) catalytically oxidize H2S to per- and polysufides and sulfoxides, reduce oxygen to superoxide and hydrogen peroxide, and can form NQ-SH adducts through Michael addition. Here, we measured oxygen consumption and used sulfur-specific fluorophores, liquid chromatography tandem mass spectrometry (LC-MS/MS), and UV-Vis spectrometry to examine H2S oxidation by NQs with various substituent groups. In general, the order of H2S oxidization was DCNQ ~ juglone > 1,4-NQ > plumbagin >DMNQ ~ 2-MNQ > menadione, although this order varied somewhat depending on the experimental conditions. DMNQ does not form adducts with GSH or cysteine (Cys), yet it readily oxidizes H2S to polysulfides and sulfoxides. This suggests that H2S oxidation occurs at the carbonyl moiety and not at the quinoid 2 or 3 carbons, although the latter cannot be ruled out. We found little evidence from oxygen consumption studies or LC-MS/MS that NQs directly oxidize H2S2-4, and we propose that apparent reactions of NQs with inorganic polysulfides are due to H2S impurities in the polysulfides or an equilibrium between H2S and H2Sn. Collectively, NQ oxidation of H2S forms a variety of products that include hydropersulfides, hydropolysulfides, sulfenylpolysulfides, sulfite, and thiosulfate, and some of these reactions may proceed until an insoluble S8 colloid is formed.

Redox and Nucleophilic Reactions of Naphthoquinones with Small Thiols and Their Effects on Oxidization of H2S to Inorganic and Organic Hydropolysulfides and Thiosulfate.

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H2S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H2S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H2S to both inorganic and organic hydroper-/hydropolysulfides (R2Sn, R=H, Cys, GSH; n = 2-4) and organic sulfoxides (GSnOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H2S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H2S.

Characterizing the secretome of EGFR mutant lung adenocarcinoma.

Background: Lung cancer is the leading cause of cancer related death worldwide, mainly due to the late stage of disease at the time of diagnosis. Non-invasive biomarkers are needed to supplement existing screening methods to enable earlier detection and increased patient survival. This is critical to EGFR-driven lung adenocarcinoma as it commonly occurs in individuals who have never smoked and do not qualify for current screening protocols. Methods: In this study, we performed mass spectrometry analysis of the secretome of cultured lung cells representing different stages of mutant EGFR driven transformation, from normal to fully malignant. Identified secreted proteins specific to the malignant state were validated using orthogonal methods and their clinical activity assessed in lung adenocarcinoma patient cohorts. Results: We quantified 1020 secreted proteins, which were compared for differential expression between stages of transformation. We validated differentially expressed proteins at the transcriptional level in clinical tumor specimens, association with patient survival, and absolute concentration to yield three biomarker candidates: MDK, GDF15, and SPINT2. These candidates were validated using ELISA and increased levels were associated with poor patient survival specifically in EGFR mutant lung adenocarcinoma patients. Conclusions: Our study provides insight into changes in secreted proteins during EGFR driven lung adenocarcinoma transformation that may play a role in the processes that promote tumor progression. The specific candidates identified can harnessed for biomarker use to identify high risk individuals for early detection screening programs and disease management for this molecular subgroup of lung adenocarcinoma patients.

CCL5 production in lung cancer cells leads to an altered immune microenvironment and promotes tumor development.

Current immunotherapies for lung cancer are only effective in a subset of patients. Identifying tumor-derived factors that facilitate immunosuppression offers the opportunity to develop novel strategies to supplement and improve current therapeutics. We sought to determine whether expression of driver oncogenes in lung cancer cells affects cytokine secretion, alters the local immune environment, and influences lung tumor progression. We demonstrate that oncogenic EGFR and KRAS mutations, which are early events in lung tumourigenesis, can drive cytokine and chemokine production by cancer cells. One of the most prominent changes was in CCL5, which was rapidly induced by KRASG12V or EGFRL858R expression, through MAPK activation. Immunocompetent mice implanted with syngeneic KRAS-mutant lung cancer cells deficient in CCL5 have decreased regulatory T cells (Tregs), evidence of T cell exhaustion, and reduced lung tumor burden, indicating tumor-cell CCL5 production contributes to an immune suppressive environment in the lungs. Furthermore, high CCL5 expression correlates with poor prognosis, immunosuppressive regulatory T cells, and alteration to CD8 effector function in lung adenocarcinoma patients. Our data support targeting CCL5 or CCL5 receptors on immune suppressive cells to prevent formation of an immune suppressive tumor microenvironment that promotes lung cancer progression and immunotherapy insensitivity.

Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells.

Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.

Resistance to BET inhibitors in lung adenocarcinoma is mediated by casein kinase phosphorylation of BRD4.

Targeting the epigenome to modulate gene expression programs driving cancer development has emerged as an exciting avenue for therapeutic intervention. Pharmacological inhibition of the bromodomain and extraterminal (BET) family of chromatin adapter proteins has proven effective in this regard, suppressing growth of diverse cancer types mainly through downregulation of the c-MYC oncogene, and its downstream transcriptional program. While initially effective, resistance to BET inhibitors (BETi) typically occurs through mechanisms that reactivate MYC expression. We have previously shown that lung adenocarcinoma (LAC) is inhibited by JQ1 through suppression of FOSL1, suggesting that the epigenetic landscape of tumor cells from different origins and differentiation states influences BETi response. Here, we assessed how these differences affect mechanisms of BETi resistance through the establishment of isogenic pairs of JQ1 sensitive and resistant LAC cell lines. We found that resistance to JQ1 in LAC occurs independent of FOSL1 while MYC levels remain unchanged between resistant cells and their JQ1-treated parental counterparts. Furthermore, while epithelial-mesenchymal transition (EMT) is observed upon resistance, TGF-ß induced EMT did not confer resistance in JQ1 sensitive LAC lines, suggesting this is a consequence, rather than a driver of BETi resistance in our model systems. Importantly, siRNA knockdown demonstrated that JQ1 resistant cell lines are still dependent on BRD4 expression for survival and we found that phosphorylation of BRD4 is elevated in resistant LACs, identifying casein kinase 2 (CK2) as a candidate protein mediating this effect. Inhibition of CK2, as well as downstream transcriptional targets of phosphorylated BRD4-including AXL and activators of the PI3K pathway-synergize with JQ1 to inhibit BETi resistant LAC. Overall, this demonstrates that the mechanism of resistance to BETi varies depending on cancer type, with LAC cells developing JQ1 resistance independent of MYC regulation, and identifying CK2 phosphorylation of BRD4 as a potential target to overcome resistance in this cancer.

A new method for discovering EMAST sequences in animal models of cancer.

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) occur in up to 60% of colorectal cancers and may associate with aggressive and advanced disease in patients. Although EMAST occurs in many cancer types, current understanding is limited due to the lack of an animal model. Reported here is the design and implementation of an algorithm for detecting EMAST repeats in mice. This algorithm incorporates properties of known human EMAST sequences to identify repeat sequences in animal genomes and was able to identify EMAST-like sequences in the mouse. Seven of the identified repeats were analyzed further in a colon cancer mouse model and six of the seven displayed EMAST instability characteristic of that seen in human colorectal cancers. In conclusion, the algorithm developed successfully identified EMAST repeats in an animal genome and, for the first time, EMAST has been shown to occur in a mouse model of colon cancer.

Sensitivity to synchronicity of biological motion in normal and amblyopic vision.

Amblyopia is a developmental disorder of spatial vision that results from abnormal early visual experience usually due to the presence of strabismus, anisometropia, or both strabismus and anisometropia. Amblyopia results in a range of visual deficits that cannot be corrected by optics because the deficits reflect neural abnormalities. Biological motion refers to the motion patterns of living organisms, and is normally displayed as points of lights positioned at the major joints of the body. In this experiment, our goal was twofold. We wished to examine whether the human visual system in people with amblyopia retained the higher-level processing capabilities to extract visual information from the synchronized actions of others, therefore retaining the ability to detect biological motion. Specifically, we wanted to determine if the synchronized interaction of two agents performing a dancing routine allowed the amblyopic observer to use the actions of one agent to predict the expected actions of a second agent. We also wished to establish whether synchronicity sensitivity (detection of synchronized versus desynchronized interactions) is impaired in amblyopic observers relative to normal observers. The two aims are differentiated in that the first aim looks at whether synchronized actions result in improved expected action predictions while the second aim quantitatively compares synchronicity sensitivity, or the ratio of desynchronized to synchronized detection sensitivities, to determine if there is a difference between normal and amblyopic observers. Our results show that the ability to detect biological motion requires more samples in both eyes of amblyopes than in normal control observers. The increased sample threshold is not the result of low-level losses but may reflect losses in feature integration due to undersampling in the amblyopic visual system. However, like normal observers, amblyopes are more sensitive to synchronized versus desynchronized interactions, indicating that higher-level processing of biological motion remains intact. We also found no impairment in synchronicity sensitivity in the amblyopic visual system relative to the normal visual system. Since there is no impairment in synchronicity sensitivity in either the non-amblyopic or amblyopic eye of amblyopes, our results suggest that the higher order processing of biological motion is intact.

Sensitivity to biological motion drops by approximately 1/2 log-unit with inversion, and is unaffected by amblyopia.

The low-level deficits associated with amblyopia have been studied extensively, but very little is known about potential impairments to higher-level visual processing such as object recognition or structure-from-motion. Studies on biological motion, a complex form of structure-from-motion depicting human actions, have demonstrated that normal observers can analyze these patterns more effectively when they are shown in their original upright configuration as opposed to inverted upside-down (feet-up head-down). We measured this inversion effect quantitatively for both the dominant and amblyopic eyes of amblyopic observers. We found a modest ( approximately 30%) loss in sensitivity in the amblyopic eye for both upright and inverted actors, which we attribute to low-level deficits. However, we found no difference in the inversion effect between the two eyes, both showing an average 1/2 log-unit drop in sensitivity between upright and inverted displays. Our data provide a quantitative estimate of the inversion effect for biological motion, and demonstrate that higher-level processing in the motion hierarchy is not affected by amblyopia.

Meaningful interactions can enhance visual discrimination of human agents.

The ability to interpret and predict other people's actions is highly evolved in humans and is believed to play a central role in their cognitive behavior. However, there is no direct evidence that this ability confers a tangible benefit to sensory processing. Our quantitative behavioral experiments show that visual discrimination of a human agent is influenced by the presence of a second agent. This effect depended on whether the two agents interacted (by fighting or dancing) in a meaningful synchronized fashion that allowed the actions of one agent to serve as predictors for the expected actions of the other agent, even though synchronization was irrelevant to the visual discrimination task. Our results demonstrate that action understanding has a pervasive impact on the human ability to extract visual information from the actions of other humans, providing quantitative evidence of its significance for sensory performance.