Inducing ubiquitination and degradation of TrxR1 protein by LW-216 promotes apoptosis in non-small cell lung cancer via triggering ROS production.

Thioredoxin reductases are frequently overexpressed in various solid tumors as a protective mechanism against heightened oxidative stress. Inhibitors of this system, such as Auranofin, are effective in eradicating cancer cells. However, the clinical significance of thioredoxin reductase 1 (TrxR1) in lung cancer, as well as the potential for its antagonist as a treatment option, necessitated further experimental validation. In this study, we observed significant upregulation of TrxR1 specifically in non-small cell lung cancer (NSCLC), rather than small cell lung cancer. Moreover, TrxR1 expression exhibited associations with survival rate, tumor volume, and histological classification. We developed a novel TrxR1 inhibitor named LW-216 and assessed its antitumor efficacy in NSCLC. Our results revealed that LW-216 is effectively bound with intracellular TrxR1 at sites R371 and G442, facilitating TrxR1 ubiquitination and suppressing TrxR1 expression, while not affecting TrxR2 expression. Treatment of LW-216-induced DNA damage and cell apoptosis in NSCLC cells through the generation of reactive oxygen species (ROS). Importantly, supplementation with N-acetylcysteine (NAC) or ectopic TrxR1 expression reversed LW-216-induced apoptosis. Furthermore, LW-216 displayed potent tumor growth inhibition in NSCLC cell-implanted mice, reducing TrxR1 expression in xenografts. Remarkably, LW-216 exhibited superior antitumor activity compared to Auranofin in vivo. Collectively, our research provides compelling evidence supporting the potential of targeting TrxR1 by LW-216 as a promising therapeutic strategy for NSCLC.

TRAF4 Maintains Deubiquitination of Caveolin-1 to Drive Glioblastoma Stemness and Temozolomide Resistance.

Glioblastoma (GBM) is the most common type of primary adult brain tumor. Glioma stem cell (GSC) residence and temozolomide (TMZ) resistance in GBM both contribute to poor patient outcome. TRAF4 is a scaffold protein with E3 ubiquitin ligase activity that has recently been discovered to promote invasion and metastasis in several malignancies, but the effects and functions of TRAF4 in GBM remain to be determined. Here, we report that TRAF4 is preferentially overexpressed in GSCs and is required for stem-like properties as well as TMZ sensitivity in GBM cells. TRAF4 specifically interacted with the N-terminal tail of Caveolin-1 (CAV1), an important contributor to the tumorigenicity of GBM cells. TRAF4 regulated CAV1 stability by preventing ZNRF1-mediated ubiquitination and facilitating USP7-mediated deubiquitination independently of its E3 ubiquitin ligase catalytic activity. TRAF4-mediated stabilization of CAV1 activated protumorigenic AKT/ERK1/2 signaling, and disruption of this axis resulted in defects in stemness maintenance. In addition, expression of TRAF4 and CAV1 was positively correlated and predicted poor prognosis in human GBM samples. Screening of common nervous system drugs identified risperidone interaction with TRAF4, and risperidone treatment resulted in the dissociation of TRAF4 and CAV1. Importantly, pharmacologic inhibition of TRAF4 with risperidone potently inhibited self-renewal, abrogated tumorigenicity, and reversed TMZ resistance in GBM. Overall, TRAF4-mediated stabilization of CAV1 promotes stemness and TMZ resistance in GBM, providing a therapeutic strategy that could improve patient outcomes. SIGNIFICANCE: The identification of a TRAF4/Caveolin-1 axis that plays a crucial role in malignant progression of glioblastoma provides new insights into the function of TRAF4 in ubiquitin signaling and suggests TRAF4 as a potential therapeutic target.

Long noncoding RNA MAGI2-AS3 regulates the H2O2 level and cell senescence via HSPA8.

The redox homeostasis system regulates many biological processes, intracellular antioxidant production and redox signaling. However, long noncoding RNAs (lncRNAs) involved in redox regulation have rarely been reported. Herein, we reported that downregulation of MAGI2-AS3 decreased the superoxide level in Human fibroblasts (Fbs), a replicative aging model, as detected by the fluorescent probes dihydroethidium (DHE) and MitoSOX™ Red. RNA pulldown combined with mass spectrometry showed that HSPA8 is a novel interacting protein of MAGI2-AS3, which was further confirmed by photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Downregulation of MAGI2-AS3 decreased the hydrogen peroxide (H2O2) content by stabilizing the HSPA8 protein level via inhibiting the protesome degradation of HSPA8. Further evidence showed that MAGI2-AS3 interacted with the C-terminal domain (CTD) of HSPA8. Downregulation of MAGI2-AS3 delayed cell senescence, while this antiaging effect was abolished by HSPA8 knockdown. The underlying molecular mechanism by which MAGI2-AS3 knockdown inhibited cell senescence was mediated via suppression of the ROS/MAP2K6/p38 signaling pathway. Taken together, these findings revealed that downregulation of lncRNA MAGI2-AS3 decreased the H2O2 content and delayed cell senescence by stabilizing the HSPA8 protein level, identifying a potential antiaging application.

Differential Degradation of TRA2A and PYCR2 Mediated by Ubiquitin E3 Ligase E4B.

E4B belongs to the U-box E3 ligase family and functions as either an E3 or an E4 enzyme in protein ubiquitination. Transformer2A (TRA2A) and Pyrroline-5-carboxylate reductase 2 (PYCR2) are related to cancer development and are overexpressed in many cancer cells. The degradation of TRA2A and PYCR2 mediated by the ubiquitin-proteasome system (UPS) has not been reported. This study validated that E4B could ubiquitinate TRA2A and PYCR2 as an E3 ligase both in vitro and in the HEK293 cells. E4B mediated the degradation by forming K11- and K48- linked polyubiquitin chains on TRA2A and PYCR2, respectively. E4B regulated the alternative splicing function of TRA2A and affected RSRC2 transcription in the HEK293 cells. Although E4B is highly expressed, it hardly degrades TRA2A and PYCR2 in hepatocellular carcinoma (HCC) cells, suggesting other mechanisms exist for degradation of TRA2A and PYCR2 in the HCC cells. We finally reported that E4B interacted with substrates via its variable region.

CRMP2 is a therapeutic target that suppresses the aggressiveness of breast cancer cells by stabilizing RECK.

Metastatic breast cancer is characterized by high mortality and limited therapeutic target. During tumor metastasis, cytoskeletal reorganization is one of the key steps in the migration and invasion of breast cancer cells. Collapsin response mediator protein 2 (CRMP2) is a cytosolic phosphoprotein that plays an important role in regulating cytoskeletal dynamics. Previous researches have reported that altered CRMP2 expression is associated with breast cancer progression, but the underlying mechanism remains poorly understood. Here, we show that CRMP2 expression is reduced in various subtypes of breast cancers and negatively correlated with lymphatic metastasis. Overexpression of CRMP2 significantly inhibits invasion and stemness in breast cancer cells, while downregulation of CRMP2 promotes cell invasion, which is not required for tubulin polymerization. Mechanistic studies demonstrate that CRMP2 interacts with RECK, prevents RECK degradation, which, in turn, blocks NF-κB and Wnt signaling pathways. Furthermore, we find that phosphorylation of CRMP2 at T514 and S522 remarkably abolishes its functions to bind with RECK and to inhibit cell invasion. Pharmacologic rescue of CRMP2 expression suppressed breast cancer metastasis in vitro and in vivo and stimulated a synergetic effect with FN-1501 that induces CRMP2 dephosphorylation. Collectively, this study highlights the potential of CRMP2 as a therapeutic target in breast cancer metastasis and reveals a distinct mechanism of CRMP2.

Identification of a Vav2-dependent mechanism for GDNF/Ret control of mesolimbic DAT trafficking.

Dopamine (DA) homeostasis is essential for a variety of brain activities. Dopamine transporter (DAT)-mediated DA reuptake is one of the most critical mechanisms for normal DA homeostasis. However, the molecular mechanisms underlying the regulation of DAT activity in the brain remain poorly understood. Here we show that the Rho-family guanine nucleotide exchange factor protein Vav2 is required for DAT cell surface expression and transporter activity modulated by glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor Ret. Mice deficient in either Vav2 or Ret displayed elevated DAT activity, which was accompanied by an increase in intracellular DA selectively in the nucleus accumbens. Vav2(-/-) mice exposed to cocaine showed reduced DAT activity and diminished behavioral cocaine response. Our data demonstrate that Vav2 is a determinant of DAT trafficking in vivo and contributes to the maintenance of DA homeostasis in limbic DA neuron terminals.

Nitric oxide metabolism controlled by formaldehyde dehydrogenase (fdh, homolog of mammalian GSNOR) plays a crucial role in visual pattern memory in Drosophila.

Nitric oxide (NO) plays an important role in learning and memory which is essential for animals to adapt to the external environment. However, little is known about the role of NO metabolism in this process. S-nitrosoglutathione reductase (GSNOR) is a key protein in the control of NO metabolism and protein S-nitrosation. To study the relationship between NO metabolism and learning and memory, the expression of gene fdh which is homolog to mammalian GSNOR was modulated by the Gal4/UAS system in Drosophila. The over-expression of the fdh in the central nervous system significantly increased GSNOR activity and induced visual pattern memory defects of Drosophila. The role of fdh in learning and memory was independent of development and was neuron-specific: over-expression of the fdh in the fan-shaped body induced memory defect, while over-expression in the mushroom body did not. The visual pattern memory defect could be rescued by co-expression with exogenous cGMP-dependent protein kinase (PKG). Moreover, fdh over-expression resulted in denitrosation of multiple proteins functionally enriched in vesicle-mediated transport, which is important for learning and memory. These results showed that regulation of NO metabolism plays an important role in learning and memory, and the mechanism may involve both NO-cGMP-PKG signaling pathway and S-nitrosation modification.