TAK1 Phosphorylates RASSF9 and Inhibits Esophageal Squamous Tumor Cell Proliferation by Targeting the RAS/MEK/ERK Axis.

TGF-ß-activated kinase 1 (TAK1), a serine/threonine kinase, is a key intermediate in several signaling pathways. However, its role in tumorigenesis is still not understood well. In this study, it is found that TAK1 expression decreases in esophageal tumor tissues and cell lines. In vitro experiments demonstrate that proliferation of esophageal tumor cells is enhanced by knockdown of TAK1 expression and attenuated by elevated expression of TAK1. Using a subcutaneous tumor model, these observations are confirmed in vivo. Based on the results from co-immunoprecipitation coupled with mass spectrometry, Ras association domain family 9 (RASSF9) is identified as a downstream target of TAK1. TAK1 phosphorylates RASSF9 at S284, which leads to reduced RAS dimerization, thereby blocking RAF/MEK/ERK signal transduction. Clinical survey reveals that TAK1 expression is inversely correlated with survival in esophageal cancer patients. Taken together, the data reveal that TAK1-mediated phosphorylation of RASSF9 at Ser284 negatively regulates esophageal tumor cell proliferation via inhibition of the RAS/MEK/ERK axis.

Transcriptional regulator ArcA mediates expression of oligopeptide transport systems both directly and indirectly in Shewanella oneidensis.

In γ-proteobacterial species, such as Escherichia coli, the Arc (anoxic redox control) two-component system plays a major role in mediating the metabolic transition from aerobiosis to anaerobiosis, and thus is crucial for anaerobic growth but dispensable for aerobic growth. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, Arc (SoArc) primarily affects aerobic growth. To date, how this occurs has remained largely unknown although the growth defect resulting from the loss of DNA-binding response regulator SoArcA is tryptone-dependent. In this study, we demonstrated that the growth defect is in part linked to utilization of oligopeptides and di-tripeptides, and peptide uptake but not peptide degradation is significantly affected by the SoArcA loss. A systematic characterization of major small peptide uptake systems manifests that ABC peptide transporter Sap and four proton-dependent oligopeptide transporters (POTs) are responsible for transport of oligopeptides and di-tripeptides respectively. Among them, Sap and DtpA (one of POTs) are responsive to the SoarcA mutation but only dtpA is under the direct control of SoArcA. We further showed that both Sap and DtpA, when overproduced, improve growth of the SoarcA mutant. While the data firmly establish a link between transport of oligopeptides and di-tripeptides and the SoarcA mutation, other yet-unidentified factors are implicated in the growth defect resulting from the SoArcA loss.

EphA2 chimeric antigen receptor-modified T cells for the immunotherapy of esophageal squamous cell carcinoma.

BACKGROUND: It is urgent to explore an effective potential therapeutic strategy for ESCC. In recent years, cell-based cancer immunotherapy has become a potentially close for carcinoma therapy. Chimeric antigen receptor (CAR) T cell technology is a kind of adoptive cell therapy technique which has been developed rapidly. We sought to obtain EphA2.CAR-T cell and revealed the ability of EphA2.CAR-T cells to kill esophageal squamous cell carcinoma (ESCC) cells in vitro. METHODS: Firstly, the expression and location of EphA2 in ESCC tissues and cells was tested by immunohistochemistry staining and Western blot. Secondly, the second generation of EphA2.CAR was constructed via molecular biology technology, and transduced into T cells to obtain the EphA2.CAR-T cell. The transduction efficacies were assessed using flow cytometry (FCM). Thirdly, the effect of cell killing of EphA2.CAR-T cell on ESCC cells in vitro was detected by co-culture experiments. The productions of cytokines (TNF-α and IFN-γ) by EphA2.CAR-T cell after co-culture with ESCC cells were analyzed by ELISA assay. RESULTS: The expression of EphA2 was significantly upregulated in ESCC tissues and cells (P<0.05). EphA2 was expressed on the membrane of ESCC cells, so it could be served as tumor-associated surface antigens (TAA) of CAR for ESCC treatment. The EphA2.CAR-T cell was obtained successfully, and its' transduction efficacies was 61.4% by FCM. The ability of cell killing of EphA2.CAR-T cell was better than that of T cells (P<0.01), and demonstrated a dose-dependent cell killing. The results of ELISA assay showed that the levels of TNF-α and IFN-γ in EphA2.CAR-T cells were notably raised compared with T cells (P<0.05). CONCLUSIONS: We firstly constructed the second generation of EphA2.CAR and established EphA2.CAR-T cells. The EphA2.CAR-T cells showed a dose-dependent cell killing of ESCC cells, and promoted the production of cytokines in vitro. These findings open a new way for treatment of ESCC by immunotherapy in the future.

C-terminal binding protein­2 mediates cisplatin chemoresistance in esophageal cancer cells via the inhibition of apoptosis.

C-terminal binding protein­2 (CtBP2) is a transcriptional co-repressor that is associated with tumorigenesis and tumor progression. It has been reported to predict a poor prognosis in several human cancers, including esophageal squamous cell carcinoma (ESCC). The present study aimed to investigate the involvement of CtBP2 in the cisplatin (DDP) resistance of the ECA109 ESCC cell line and its effect on the expression of apoptosis-associated proteins. Constructed recombinant lentiviruses were used for the knockdown or overexpression of CtBP2 in ECA109 cells, and the expression of CtBP2 was measured using reverse transcription-quantitative polymerase chain reaction and western blotting following transfection. MTT assays, Hoechst 33342 staining and flow cytometry (FCM) were applied to detect the influence of CtBP2 on the DDP-induced viability and apoptosis of the transfected ECA109 cells. In addition, the levels of apoptosis-associated proteins, including p53, B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax) and activated caspase-3 were investigated in the transfected ECA109 cells. Stable ECA109 cells with CtBP2 overexpression or knockdown were successfully established. The results of the MTT, Hoechst 33342 and FCM assays demonstrated that overexpression of CtBP2 attenuated the reduction of cell viability and inhibited the cell apoptosis induced by DDP. Furthermore, the western blotting results indicated that CtBP2 overexpression inhibited the DDP-induced apoptosis of ECA109 cells via the reduction of p53, activated caspase-3 and Bax expression, and promotion of Bcl­2 expression. Therefore, the present study indicated that CtBP2 reduced the susceptibility of ECA109 cells to DDP by regulating the expression of apoptosis-related proteins, suggesting that it may be a promising therapeutic target in ESCC in the future.

Endogenous generation of hydrogen sulfide and its regulation in Shewanella oneidensis.

Hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. In this study, mechanisms of endogenous H2S generation in Shewanella oneidensis were investigated. As a research model with highly diverse anaerobic respiratory pathways, the microorganism is able to produce H2S by respiring on a variety of sulfur-containing compounds with SirACD and PsrABC enzymatic complexes, as well as through cysteine degradation with three enzymes, MdeA, SO_1095, and SseA. We showed that the SirACD and PsrABC complexes, which are predominantly, if not exclusively, responsible for H2S generation via respiration of sulfur species, do not interplay with each other. Strikingly, a screen for regulators controlling endogenous H2S generation by transposon mutagenesis identified global regulator Crp to be essential to all H2S-generating processes. In contrast, Fnr and Arc, two other global regulators that have a role in respiration, are dispensable in regulating H2S generation via respiration of sulfur species. Interestingly, Arc is involved in the H2S generation through cysteine degradation by repressing expression of the mdeA gene. We further showed that expression of the sirA and psrABC operons is subjected to direct regulation of Crp, but the mechanisms underlying the requirement of Crp for H2S generation through cysteine degradation remain elusive.

Impaired cell envelope resulting from arcA mutation largely accounts for enhanced sensitivity to hydrogen peroxide in Shewanella oneidensis.

Oxidative stress is one of the major challenges that Shewanella encounter routinely because they thrive in redox-stratified environments prone to reactive oxygen species (ROS) formation, letting alone that ROS can be generated endogenously. As respiration is the predominant process for endogenous ROS, regulators mediating respiration have been demonstrated and/or implicated to play a role in oxidative stress response. In our efforts to unveil the involvement of global regulators for respiration in the oxidative stress response, we found that loss of the Arc system increases S. oneidensis sensitivity to H2O2 whereas neither Fnr nor Crp has a significant role. A comparison of transcriptomic profiles of the wild-type and its isogenic arcA mutant revealed that the OxyR regulon is independent of the Arc system. We then provided evidence that the enhanced H2O2 sensitivity of the arcA mutant is due to an increased H2O2 uptake rate, a result of a cell envelope defect. Although one of three proteases of the ArcA regulon when in excess is partially accountable for the envelope defect, the major contributors remain elusive. Overall, our data indicate that the Arc system influences the bacterial cell envelope biosynthesis, a physiological aspect that has not been associated with the regulator before.

Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant.

UNLABELLED: Oxidative stresses triggered by reactive oxygen species (ROS) that damage various cellular components are unavoidable for virtually all living organisms. In defense, microorganisms have evolved sophisticated mechanisms to sense, respond to, and battle against ROS. Shewanella oneidensis, an important research model for applied and environmental microbes, employs OxyR to mediate the response to H2O2 by derepressing the production of the major H2O2 scavenger KatB as a major means toward these goals. Surprisingly, despite enhanced H2O2 degradation, the oxyR mutant carries a viability deficiency phenotype (plating defect), which can be suppressed by the addition of exogenous iron species. Experiments showed that the defect was not due to iron starvation. Rather, multiple lines of evidence suggested that H2O2 generated abiotically in lysogeny broth (LB) is responsible for the defect by quickly killing mutant cells. We then showed that the iron species suppressed the plating defect by two distinct mechanisms, either as an H2O2 scavenger without involving living cells or as an environmental cue to stimulate an OxyR-independent response to help cells cope with oxidative stress. Based on the suppression of the plating defect by overproduction of H2O2 scavengers in vivo, we propose that cellular components that are vulnerable to H2O2 and responsible for the defect may reside outside the cytoplasm. IMPORTANCE: In bacteria, OxyR is the major regulator controlling the cellular response to H2O2. The loss of OxyR results in reduced viability in many species, but the underlying mechanism is unknown. We showed in S. oneidensis that this defect was due to H2O2 generated abiotically in LB. We then showed that this defect could be corrected by the addition of Fe(2+) or catalase to the LB or increased intracellular production of catalase. Further analyses revealed that Fe(2+) was able not only to decompose H2O2 directly but also to stimulate the activity of OxyR-independent H2O2-scavenging enzymes. Our data indicate that iron species play a previously underappreciated role in protecting cells from H2O2 in environments.

PBP1a/LpoA but not PBP1b/LpoB are involved in regulation of the major ß-lactamase gene blaA in Shewanella oneidensis.

ß-Lactamase production is one of the most important strategies for Gram-negative bacteria to combat ß-lactam antibiotics. Studies of the regulation of ß-lactamase expression have largely been focused on the class C ß-lactamase AmpC, whose induction by ß-lactams requires LysR-type regulator AmpR and permease AmpG-dependent peptidoglycan recycling intermediates. In Shewanella, which is ubiquitous in aquatic environments and is a reservoir for antibiotic resistance, production of the class D ß-lactamase BlaA confers bacteria with natural resistance to many ß-lactams. Expression of the blaA gene in the genus representative Shewanella oneidensis is distinct from the AmpC paradigm because of the lack of an AmpR homologue and the presence of an additional AmpG-independent regulatory pathway. In this study, using transposon mutagenesis, we identify proteins that are involved in blaA regulation. Inactivation of mrcA and lpoA, which encode penicillin binding protein 1a (PBP1a) and its lipoprotein cofactor, LpoA, respectively, drastically enhances blaA expression in the absence of ß-lactams. Although PBP1b and its cognate, LpoB, also exist in S. oneidensis, their roles in blaA induction are dispensable. We further show that the mrcA-mediated blaA expression is independent of AmpG.

Distinct roles of major peptidoglycan recycling enzymes in ß-Lactamase production in Shewanella oneidensis.

ß-Lactam antibiotics were the earliest discovered and are the most widely used group of antibiotics that work by inactivating penicillin-binding proteins to inhibit peptidoglycan biosynthesis. As one of the most efficient defense strategies, many bacteria produce ß-lactam-degrading enzymes, ß-lactamases, whose biochemical functions and regulation have been extensively studied. A signal transduction pathway for ß-lactamase induction by ß-lactam antibiotics, consisting of the major peptidoglycan recycling enzymes and the LysR-type transcriptional regulator, AmpR, has been recently unveiled in some bacteria. Because inactivation of some of these proteins, especially the permease AmpG and the ß-hexosaminidase NagZ, results in substantially elevated susceptibility to the antibiotics, these have been recognized as potential therapeutic targets. Here, we show a contrasting scenario in Shewanella oneidensis, in which the homologue of AmpR is absent. Loss of AmpG or NagZ enhances ß-lactam resistance drastically, whereas other identified major peptidoglycan recycling enzymes are dispensable. Moreover, our data indicate that there exists a parallel signal transduction pathway for ß-lactamase induction, which is independent of either AmpG or NagZ.

Evidence for function overlapping of CymA and the cytochrome bc1 complex in the Shewanella oneidensis nitrate and nitrite respiration.

Shewanella oneidensis is an important model organism for its versatility of anaerobic respiration. CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome, plays a central role in anaerobic respiration by transferring electrons from the quinone pool to a variety of terminal reductases. Although loss of CymA results in defect in respiration of many electron acceptors (EAs), a significant share of the capacity remains in general. In this study, we adopted a transposon random mutagenesis method in a cymA null mutant to identify substituent(s) of CymA with respect to nitrite and nitrate respiration. A total of 87 insertion mutants, whose ability to reduce nitrite was further impaired, were obtained. Among the interrupted genes, the petABC operon appeared to be the most likely candidate given the involvement of the cytochrome bc1 complex that it encodes in electron transport. Subsequent analyses not only confirmed that the complex and CymA were indeed functionally overlapping in nitrate/nitrite respiration but also revealed that both proteins were able to draw electrons from ubiquinone and menaquinone. Furthermore, we found that expression of the bc1 complex was affected by oxygen but not nitrate or nitrite and by global regulators ArcA and Crp in an indirect manner.