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The cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as a cytosolic double stranded DNA sensor that plays a pivotal role in the type I interferon and inflammation responses via the STING-dependent signaling pathway. In the past several years, a growing body of evidence has revealed that cGAS is also localized in the nucleus where it is associated with distinct nuclear substructures such as nucleosomes, DNA replication forks, the double-stranded breaks, and centromeres, suggesting that cGAS may have other functions in addition to its role in DNA sensing. However, while the innate immune function of cGAS is well established, the non-canonical nuclear function of cGAS remains poorly understood. Here, we review our current understanding of the complex nature of nuclear cGAS and point to open questions on the novel roles and the mechanisms of action of this protein as a key regulator of cell nuclear function, beyond its well-established role in dsDNA sensing and innate immune response.

Background: The most common preparation of tocotrienols is the Tocotrienol-Rich Fraction (TRF). This study aimed to investigate whether TRF induced liver Nrf2 nuclear translocation and influenced the expression of Nrf2-regulated genes. Methods: In the Nrf2 induction study, mice were divided into control, 2000 mg/kg TRF and diethyl maleate treated groups. After acute treatment, mice were sacrificed at specific time points. Liver nuclear extracts were prepared and Nrf2 nuclear translocation was detected through Western blotting. To determine the effect of increasing doses of TRF on the extent of liver nuclear Nrf2 translocation and its implication on the expression levels of several Nrf2-regulated genes, mice were divided into 5 groups (control, 200, 500 and 1000 mg/kg TRF, and butylated hydroxyanisole-treated groups). After 14 days, mice were sacrificed and liver RNA was extracted for qPCR assay. Results: 2000 mg/kg TRF administration initiated Nrf2 nuclear translocation within 30 min, reached a maximum level of around 1 h and dropped to half-maximal levels by 24 h. Incremental doses of TRF resulted in dose-dependent increases in liver Nrf2 nuclear levels, along with concomitant dosedependent increases in the expressions of Nrf2-regulated genes. Conclusion: TRF activated the liver Nrf2 pathway resulting in increased expression of Nrf2-regulated cytoprotective genes.

Blood–brain barrier (BBB) damage after ischemia significantly influences stroke outcome. Compound LFHP-1c was previously discovered with neuroprotective role in stroke model, but its mechanism of action on protection of BBB disruption after stroke remains unknown. Here, we show that LFHP-1c, as a direct PGAM5 inhibitor, prevented BBB disruption after transient middle cerebral artery occlusion (tMCAO) in rats. Mechanistically, LFHP-1c binding with endothelial PGAM5 not only inhibited the PGAM5 phosphatase activity, but also reduced the interaction of PGAM5 with NRF2, which facilitated nuclear translocation of NRF2 to prevent BBB disruption from ischemia. Furthermore, LFHP-1c administration by targeting PGAM5 shows a trend toward reduced infarct volume, brain edema and neurological deficits in nonhuman primate Macaca fascicularis model with tMCAO. Thus, our study identifies compound LFHP-1c as a firstly direct PGAM5 inhibitor showing amelioration of ischemia-induced BBB disruption in vitro and in vivo, and provides a potentially therapeutics for brain ischemic stroke., Graphical abstract LFHP-1c directly binding to PGAM5 inhibits PGAM5 enzyme activity and impairs PGAM5 interaction with NRF2, promoting NRF2 transcription activity to protect against blood–brain barrier disruption and ischemic brain injury.Image 1

Mifepristone (Mif), an effective synthetic steroidal antiprogesterone drug, is widely used for medical abortion and pregnancy prevention. Due to its anti-glucocorticoid effect, high-dose Mif is also used to treat Cushing’s syndrome. Mif was reported to active pregnane X receptor (PXR) in vitro and PXR can induce hepatomegaly via activation and interaction with yes-associated protein (YAP) pathway. High-dose Mif was reported to induce hepatomegaly in rats and mice, but the underlying mechanism remains unclear. Here, the role of PXR was studied in Mif-induced hepatomegaly in C57BL/6 mice and Pxr-knockout mice. The results demonstrated that high-dose Mif (100 mg · kg(−1) · d(−1), i.p.) treatment for 5 days significantly induced hepatomegaly with enlarged hepatocytes and promoted proliferation, but low dose of Mif (5 mg · kg(−1) · d(−1), i.p.) cannot induce hepatomegaly. The dual-luciferase reporter gene assays showed that Mif can activate human PXR in a concentration-dependent manner. In addition, Mif could promote nuclear translocation of PXR and YAP, and significantly induced the expression of PXR, YAP, and their target proteins such as CYP3A11, CYP2B10, UGT1A1, ANKRD, and CTGF. However, Mif (100 mg · kg(−1) · d(−1), i.p.) failed to induce hepatomegaly in Pxr-knockout mice, as well as hepatocyte enlargement and proliferation, further indicating that Mif-induced hepatomegaly is PXR-dependent. In summary, this study demonstrated that PXR-mediated Mif-induced hepatomegaly in mice probably via activation of YAP pathway. This study provides new insights in Mif-induced hepatomegaly, and provides novel evidence on the crucial function of PXR in liver enlargement and regeneration.

Early and accurate diagnosis of prostate cancer (PCa) is extremely important, as metastatic PCa remains hard to treat. EWI‐2, a member of the Ig protein subfamily, is known to inhibit PCa cell migration. In this study, we found that EWI‐2 localized on both the cell membrane and exosomes regulates the distribution of miR‐3934‐5p between cells and exosomes. Interestingly, we observed that EWI‐2 is localized not only on the plasma membrane but also on the nuclear envelope (nuclear membrane), where it regulates the nuclear translocation of signaling molecules and miRNA. Collectively, these functions of EWI‐2 found in lipid bilayers appear to regulate PCa cell metastasis through the epidermal growth factor receptor‐mitogen‐activated protein kinase‐extracellular‐signal‐regulated kinase (EGFR‐MAPK‐ERK) pathway. Our research provides new insights into the molecular function of EWI‐2 on PCa metastasis, and highlights EWI‐2 as a potential PCa biomarker., Our findings identify EWI‐2 as a potential prostate cancer (PCa) biomarker. EWI‐2 is involved in PCa metastasis through its localization on lipid bilayers where it facilitates nucleocytoplasmic shuttling of EGFR signaling molecules and also regulates microRNA sorting in exosomes. These molecular functions of EWI‐2 result in downstream activation of the epidermal growth factor receptor‐mitogen‐activated protein kinase‐extracellular‐signal‐regulated kinase (EGFR‐MAPK‐ERK) signaling pathway and promote PCa cell migration.

Through integration of multiple large database, two oncogenic signatures (YAP conserved and TGFβ‐up signatures) were commonly upregulated in the cancer cell lines with the mesenchymal properties. AXL, the expression of which is highly induced in EMT, contributes the doxorubicin resistance in EMT. Doxorubicin treatment induced YAP‐dependent gene response and promoted AXL expression coordinated with TGFβ‐SMAD4., The acquisition of chemoresistance remains a major cause of cancer mortality due to the limited accessibility of targeted or immune therapies. However, given that severe alterations of molecular features during epithelial‐to‐mesenchymal transition (EMT) lead to acquired chemoresistance, emerging studies have focused on identifying targetable drivers associated with acquired chemoresistance. Particularly, AXL, a key receptor tyrosine kinase that confers resistance against targets and chemotherapeutics, is highly expressed in mesenchymal cancer cells. However, the underlying mechanism of AXL induction in mesenchymal cancer cells is poorly understood. Our study revealed that the YAP signature, which was highly enriched in mesenchymal‐type lung cancer, was closely correlated to AXL expression in 181 lung cancer cell lines. Moreover, using isogenic lung cancer cell pairs, we also found that doxorubicin treatment induced YAP nuclear translocation in mesenchymal‐type lung cancer cells to induce AXL expression. Additionally, the concurrent activation of TGFβ signaling coordinated YAP‐dependent AXL expression through SMAD4. These data suggest that crosstalk between YAP and the TGFβ/SMAD axis upon treatment with chemotherapeutics might be a promising target to improve chemosensitivity in mesenchymal‐type lung cancer.

Si Chen,* Yingling Liu,* Kaiguang Zhang, Lele Chen Department of Gastroenterology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230001, Anhui, People’s Republic of China*These authors contributed equally to this workCorrespondence: Si ChenDepartment of Gastroenterology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, #17 Lvjiang Road, Hefei, 230001, Anhui, People’s Republic of ChinaEmail css81729@sina.comIntroduction: Gastric cancer is one of the main reasons of cancer-induced death, exploring the molecular mechanisms of gastric cancer progression is critical for gastric cancer therapy. Here, we studied the role of cysteine protease inhibitor CST1 in gastric cancer progression.Methods: Matrigel-coated or -uncoated transwell assay was used to determine the effect of CST1 on gastric cancer invasion and migration, luciferase reporter system was used to determine the effect of CST1 on Wnt pathway activity.Results: CST1 had high expression levels in gastric cancer tissues and cells, patients who had high CST1 expression had poor outcome. Overexpression of CST1 increased gastric cancer migration and invasion, while knockdown of CST1 suppressed gastric cancer migration invasion. Mechanism analysis showed CST1 promoted WNT signaling pathway activity, promoted the nuclear translocation of β-catenin and the expression of Wnt signaling targets. Inhibition of Wnt pathway in CST1 overexpression cells inhibited migration and invasion, suggesting CST1 promoted gastric cancer cell migration and invasion through activating the Wnt pathway.Conclusion: In summary, we found CST1 promoted gastric cancer migration and invasion through activating Wnt signaling, providing a novel target for gastric cancer therapy.Keywords: CST1, gastric cancer, invasion, migration, Wnt pathway

Rationale: Annexin-A1 (ANXA1) has previously been proposed to play a crucial role in neuronal apoptosis during ischemic stroke injury. Our recent study demonstrated that ANXA1 was modified by SUMOylation, and that this modification was greatly weakened after cerebral ischemia, but its effect on neuronal death and the underlying mechanism have not been fully elucidated. Methods: Mice subjected to middle cerebral artery occlusion were established as the animal model and primary cultured neurons treated with oxygen-glucose deprivation and reperfusion was established as the cell model of ischemic stroke. The Ni2+-NTA agarose affinity pull-down assay was carried out to determine the SUMOylation level of ANXA1. Co-immunoprecipitation assays was utilized to explore the protein interaction. Immunoblot analysis, quantitative real-time PCR, Luciferase reporter assay were performed to identify the regulatory mechanism. LDH release and TUNEL staining was performed to investigate the neuronal cytotoxicity and apoptosis, respectively. Results: In this study, we identified the deSUMOylating enzyme sentrin/SUMO-specific protease 6 (SENP6) as a negative regulator of ANXA1 SUMOylation. Notably, we found that SENP6-mediated deSUMOylation of ANXA1 induced its nuclear translocation and triggered neuronal apoptosis during cerebral ischemic injury. A mechanistic study demonstrated that SENP6-mediated deSUMOylation of ANXA1 promoted TRPM7- and PKC-dependent phosphorylation of ANXA1. Furthermore, blocking the deSUMOylation of ANXA1 mediated by SENP6 inhibited the transcriptional activity of p53, decreased Bid expression, suppressed caspase-3 pathway activation and reduced the apoptosis of primary neurons subjected to oxygen-glucose deprivation and reperfusion. More importantly, SENP6 inhibition by overexpression of a SENP6 catalytic mutant in neurons resulted in significant improvement in neurological function in the mouse model of ischemic stroke. Conclusions: Taken together, the results of this study identified a previously unidentified function of SENP6 in neuronal apoptosis and strongly indicated that SENP6 inhibition may provide therapeutic benefits for cerebral ischemia.

Atherosclerosis is a persistent inflammatory state, while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development. Although ligustilide (Lig) was shown to exert obvious antiatherogenic effects in previous studies, its precise mechanism has not been deeply discussed. In this paper, we designed a Lig-derived photoaffinity labelling (PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach. Mothers against decapentaplegic homologue 3 (SMAD3), a signal transmitter of transforming growth factor-β (TGF-β) which promotes the development of vascular fibrosis, was identified as a potential target of Lig. Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1, thereby inhibiting the collagen synthesis process. Hence, developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.

Our study examined for the inhibitory effect of ethanolic extract of Eleagnus Conferta Roxb leaves on TNF –α induced NF- kB nuclear translocation in lung cancer A549 cell line using flow cytometry. Apoptosis also studied to know about the antiproliferative and anticancer effects. However, our results revealed in apoptosis, Elaeagnus conferta Roxb leaves showed (33.54%) increased in proportion of cells. In the study of pre-treatment of A549 cells with Elaeagnus conferta Roxb leaves followed by TNF- α caused the increased proportion of cells (20.78%) at apoptosis induced cell death, which was statistically significant (p< 0.001). The untreated A549 cells had minimal NF-kB expression (0.25± 0.01%). However, the approach of A549 cells with Elaeagnus conferta Roxb had induced NF-kB production many fold (11.50± 1.05%). Therefore, we conclude our study was proved the impact of Elaeagnus conferta Roxb leaves inhibit the cellular growth of NSCLC-A549 cell line and induces apoptosis. Hence, from our findings, we proved this plant has anticancer activity, further feasibly taken for drug formulation.

Anthracyclines are a class of conventional and commonly used frontline chemotherapy drugs to treat breast cancer. However, the anthracycline-based regimens can only reduce breast cancer mortality by 20–30%. Furthermore, there is no appropriate biomarker for predicting responses to this kind of chemotherapy currently. Here we report our findings that may fill this gap by showing the AQP1 (Aquaporin1) protein as a potential response predictor in the anthracycline chemotherapy. We showed that breast cancer patients with a high level of AQP1 expression who underwent the anthracycline treatment had a better clinical outcome relative to those with a low level of AQP1 expression. In the exploration of the underlying mechanisms, we found that the AQP1 and glycogen synthase kinase-3β (GSK3β) competitively interacted with the 12 armadillo repeats of β-catenin, followed by the inhibition of the β-catenin degradation that led to β-catenin’s accumulation in the cytoplasm and nuclear translocation. The nuclear β-catenin interacted with TopoIIα and enhanced TopoIIα’s activity, which resulted in a high sensitivity of breast cancer cells to anthracyclines. We also found, the miR-320a-3p can attenuate the anthracycline’s chemosensitivity by inhibiting the AQP1 expression. Taken together, our findings suggest the efficacy of AQP1 as a response predictor in the anthracycline chemotherapy. The application of our study includes, but is not limited to, facilitating screening of the most appropriate breast cancer patients (who have a high AQP1 expression) for better anthracycline chemotherapy and improved prognosis purposes.

Mycobacterium avium (Mav) causes chronic infections in immunocompromised patients that require long-term antibiotic treatment. We have previously shown that Mav takes residence in host Mϕs and establishes a compartment (MavC) in which it is hidden from host defenses. Failure to establish the MavC traps Mav in Lamp1+ phagolysosomes where growth is prevented, and inflammatory signaling activated through TLRs 7/8. To elucidate how antibiotic treatment affects mycobacterial trafficking and host defenses, we infected human primary Mϕs with Mav for 4 days prior to treatment with a macrolide, aminoglycoside, and ethambutol. We show that Mav is killed and the MavC fuses with Lamp1+ lysosomes following antibiotic treatment. However, this does not result in nuclear translocation of NF-κB or production of inflammatory cytokines, suggesting different Lamp1+ lysosomal compartments can form that differ in their innate signaling capabilities. Thus, we show that upon antibiotic treatment of a chronic infection, Mav is quietly disposed of by Mϕs.

Hypoxia-inducible factors (HIFs) are key mediators expressed under hypoxic condition and involved in many kinds of disease such as cancer and abnormal angiogenesis. Thus, development of their inhibitor has been extensively explored. Here, we describe a finding that Remodelin, a specific inhibitor of NAT10, could also inhibit the expression of HIFs. The presence of Remodelin could suppress the elevated level of HIF-1α protein and its nuclear translocation induced by either treatment of cobalt chloride (CoCl2) or hypoxia in dose or time-dependent way. More importantly, Remodelin could also inhibit the constitutional expression of HIF-1α and HIF-2α in VHL mutant 786-0 cells. With using of cells with depletion of NAT10 by shRNA or Crispr-Cas9 edited, we further demonstrated that inhibition of HIFs by Remodelin should need NAT10 activity. In biological analysis, the treatment of cultured HUVECs with Remodelin could inhibit in vitro cell migration and invasion and tube-formation. Our investigation implied that Remodelin could be a new potential inhibitor of HIFs for using in angiogenesis targeting therapy in either cancers or inflammatory diseases.

DNA damage signals transducer RING finger protein 8 (RNF8) is involved in maintaining genomic stability by facilitating the repair of DNA double-strand breaks (DSB) via ubiquitin signaling. By analyzing the TCGA database and colon cancer tissue microarrays, we found that the expression level of RNF8 was positively correlated with that of c-Myc in colon cancer, which were closely associated with poor survival of colon cancer patients. Furthermore, overexpressing and knocking down RNF8 increased and decreased the expression of c-Myc in colon cancer cells, respectively. In addition, RNF8 interacted with β-catenin and facilitated its nuclear translocation by conjugating K63 polyubiquitination on it. These observations suggested a de novo role of RNF8 in promoting the progression of colon cancer by inducing β-catenin-mediated c-Myc expression.

An aberrant elongated tract of glutamine residues (polyQ) in proteins induces multiple diseases treated in the clinic. In our previous study of progressive myoclonic epilepsy (PME), using whole‑exome sequencing, a mutant Cav2.1 protein with an aberrant elongated polyQ tract was identified in PME patients. To investigate the molecular mechanism and cell biology of this aberrant elongated polyQ tract, wild‑type Cav2.1 with 13 polyQ repeats (Cav2.1 wt‑Q13) and mutant‑type Cav2.1 with 26 polyQ repeats (Cav2.1 mt‑Q26) were prepared and introduced into human SH‑SY5Y neuroblastoma cells. Using a WST‑1 assay, it was revealed that Cav2.1 mt‑Q26 markedly suppressed the proliferation of the SH‑SY5Y cells, a result not observed for the Cav2.1 wt‑Q13‑transfected cells. It was also revealed that Cav2.1 mt and its truncated molecules suppressed cell proliferation by inducing apoptosis rather than arresting the cell cycle. Further investigations indicated a nuclear translocation phenomenon associated with the Cav2.1 mt molecules. Mechanistically, it was revealed that the Cav2.1 mt molecules activated the Bcl‑2/Bax, caspase‑3 and poly ADP‑ribose polymerase (PARP) apoptotic pathways. The present study may provide new insights for interpreting the pathogenesis of PME and the relationship among polyQ, CACNA1A gene mutations and PME.

Asthma is a chronic disorder characterized by inflammation, mucus metaplasia, airway remodeling, and hyperresponsiveness. We recently showed that IL-1–induced glycolytic reprogramming contributes to allergic airway disease using a murine house dust mite model. Moreover, levels of pyruvate kinase M2 (PKM2) were increased in this model as well as in nasal epithelial cells from asthmatics as compared with healthy controls. Although the tetramer form of PKM2 converts phosphoenolpyruvate to pyruvate, the dimeric form of PKM2 has alternative, nonglycolysis functions as a transcriptional coactivator to enhance the transcription of several proinflammatory cytokines. In the current study, we examined the impact of PKM2 on the pathogenesis of house dust mite–induced allergic airways disease in C57BL/6NJ mice. We report, in this study, that activation of PKM2, using the small molecule activator, TEPP46, augmented PKM activity in lung tissues and attenuated airway eosinophils, mucus metaplasia, and subepithelial collagen. TEPP46 attenuated IL-1β–mediated airway inflammation and expression of proinflammatory mediators. Exposure to TEPP46 strongly decreased the IL-1β–mediated increases in thymic stromal lymphopoietin (TSLP) and GM-CSF in primary tracheal epithelial cells isolated from C57BL/6NJ mice. We also demonstrate that IL-1β–mediated increases in nuclear phospho-STAT3 were decreased by TEPP46. Finally, STAT3 inhibition attenuated the IL-1β–induced release of TSLP and GM-CSF, suggesting that the ability of PKM2 to phosphorylate STAT3 contributes to its proinflammatory function. Collectively, these results demonstrate that the glycolysis-inactive form of PKM2 plays a crucial role in the pathogenesis of allergic airways disease by increasing IL-1β–induced proinflammatory signaling, in part, through phosphorylation of STAT3.

International audience; The impact on human health of long-term exposure to low doses of silver nanoparticles (AgNPs) remainsunderstudied. Cellular studies have shown the intracellular dissolution of AgNPs within endolysosomesfollowed by Ag(I) binding to biomolecular thiolate-containing molecules. However, the precise subcellulardistribution of Ag(I) and the nature of the disrupted physiological pathways remained unknown. Novelimaging approaches enabled us to visualize the trafficking of AgNP-containing lysosomes towards aperinuclear location and a nuclear transfer of Ag(I) species with accumulation in the nucleoli. These Ag(I)species impaired nuclear receptor activity, disrupting critical mechanisms of liver physiology in low doseexposure scenarios, thus justifying further research into defining a framework for the safe use of AgNPs.

Non-coding RNAs represent a class of important regulators in immune response. Previously, LINC02605 was identified as a candidate regulator in innate immune response by lncRNA microarray assays. In this study, we systematically analyzed the functions and the acting mechanisms of LINC02605 in antiviral innate immune response. LINC02605 was up-regulated by RNA virus, DNA virus, and type I IFNs in NF-κB and Jak-stat dependent manner. Overexpression of LINC02605 promotes RNA virus-induced type I interferon production and inhibited viral replication. Consistently, knockdown of LINC02605 resulted in reduced antiviral immune response and increased viral replication. Mechanistically, LINC02605 released the inhibition of hsa-miR-107 on the expression of phosphatase and tensin homolog (PTEN). By microRNA mimics and inhibitors, hsa-miR-107 was demonstrated to not only inhibit PTEN’s expression but also negatively regulate the antiviral immune response. Knockdown of LINC02605 led to the reduction of PTEN expression both in mRNA and protein levels. Overexpression of LINC02605 had an opposite impact. Moreover, LINC02605 attenuated the serine 97 phosphorylation level of interferon regulatory factor 3 (IRF3) by promoting PTEN expression. Nucleoplasmic fragmentation assay showed that knocking down LINC02605 inhibited the nuclear translocation of IRF3, rendering the host cells more susceptible to viral invasion, while overexpression showed opposite effects. Therefore, LINC02605 is an induced lncRNA by viral infection and plays a positive feedback in antiviral immune response through modulating the nuclear translocation of IRF3.

Background: CXCR7 is an atypical chemokine receptor that transmits biased signal independent of G-protein activation. However, whether CXCL12/CXCR7 biased signal activation plays an essential role in colorectal cancer (CRC) progression and metastasis remains obscure. Methods: The functional role of CXCL12/CXCR7 biased signal in CRC was investigated by RNA-sequencing, Transwell assay and in vivo tumor xenografts. YAP1 nuclear translocation and molecular mechanisms were determined by cell transfection, luciferase activity assay, immunofluorescence, coimmunoprecipitation and immunohistochemistry and RT-qPCR analysis.Results: In this study, CXCR7 CXCL12/overexpression promotes Epithelial-to-mesenchymal transition (EMT) and upregulates the expression of stem marker doublecortin-like kinase 1 (DCLK1) in CRC cells with concurrent repression of miR-124-3p and miR-188-5p. Further luciferase assay prove that these miRNAs could regulate EMT by direct targeting vimentin and DCLK1. More importantly, CXCL12/CXCR7/β-arrestin1-mediated biased signal induces YAP1 nuclear translocation, which functions as a transcriptional repressor by interacting with Yin Yang 1 (YY1) and recruiting YY1 to the promoter of miR-124-3p and miR-188-5p. Pharmacological inhibitor of YAP1 recapitulates the anti-tumorigenesis and anti-metastasis effects of YAP1 depletion upon CXCR7 activation in tumor xenografts. Clinically, the expression of CXCR7 was positively correlated with nuclear YAP1 levels and EMT markers. Conclusions: Our findings revealed the novel role of YAP1 nuclear translocation in promoting EMT of CRC by repressing miR-124-3p and miR-188-5p through CXCL12/CXCR7/β-arrestin1 biased signal activation. These findings highlight the potential of targeting YAP1 nuclear translocation in hampering CXCL12/CXCR7 biased signal-induced metastasis of CRC.

The extracellular signal-regulated kinases (ERKs) are mitogen-activated protein kinases (MAPKs) that are utilized downstream of Ras to Raf to MEK signaling to control activation of a wide array of targets. Activation of ERKs is elevated in Ras-driven tumors and RASopathies, and thus is a target for pharmacological inhibition. Regulatory mechanisms of ERK activation have been studied extensively in vitro and in cultured cells, but little in living animals. In this study, we tagged the Caenorhabditis elegans ERK-encoding gene, mpk-1. MPK-1 is ubiquitously expressed with elevated expression in certain contexts. We detected cytosol-to-nuclear translocation of MPK-1 in maturing oocytes and hence validated nuclear translocation as a reporter of some activation events. During patterning of vulval precursor cells (VPCs), MPK-1 is necessary and sufficient for the central cell, P6.p, to assume the primary fate. Yet MPK-1 translocates to the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK nuclear kinase translocation reporter of substrate activation, raising questions about mechanisms and indicators of MPK-1 activation. This system and reagent promise to provide critical insights into the regulation of MPK-1 activation within a complex intercellular signaling network., Summary: Tagged endogenous C. elegans MPK-1 shows activation-dependent cytosol-to-nuclear translocation. This tool provides novel insights into MPK-1 localization compared with other markers of in vivo ERK activation.

BackgroundDenosumab is a game-changing drug for giant cell tumor of bone (GCTB); however, its clinical biomarker in terms of post-administration ossification of the tumors has not been elucidated. The aims of this study were to investigate the relationship between Wnt/β-catenin signaling and the ossification of GCTB and to evaluate whether endogenous nuclear β-catenin expression predicted denosumab-induced bone formation in GCTB. MethodsWe established cultures of patient-derived primary GCTB stromal cells (pGCTB-SCs) and assessed the osteoblastic characteristics of pGCTB-SCs using cytochemical staining, optical density method (405 nm), and immunocytochemistry. Several mRNA expressions of osteoblastic markers (ALP, collagen type Ⅰ alpha 1 (COL1A1), integrin-binding sialoprotein (IBSP), runt-related transcription factor2 (RUNX2), and bone gamma-carboxyglutamate protein (BGLAP) was also investigated using quantitative real-time PCR (qRT-PCR) analysis. The impact on nuclear β-catenin translocation (NBT) by differentiation-induction was evaluated using Western blotting and immunocytochemistry. Wnt signaling inhibitor, GGTI-286, and selective Rac1 inhibitor, NSC23766, were added with OGM to evaluate the essential role of NBT. Furthermore, the distribution of endogenous NBT and the relationship with the tumoral ossification were assessed. Its predictability for the ossification in GCTB was also evaluated using a nuclear β-catenin labeling index (NBLI) of clinical samples (n = 86) and quantitative analyses of CT images.ResultspGCTB-SCs significantly expressed the osteoblastic markers with osteogenic medium (OGM) in a time-dependent manner (P < 0.0001, vs. vehicle). NBT was upregulated within 12 hours after OGM treatment. In addition, decreasing NBT by treatment with 40μM GGTI-286 significantly abolished the osteoblastic differentiation of pGCTB-SCs (P < 0.0001, vs. OGM). The selective Rac1 inhibitor, NSC23766, also suppressed the OGM-induced osteoblastic differentiation and NBT in pGCTB-SCs. Furthermore, in GCTB clinical samples, the NBTs were significantly associated with the tumor ossification following denosumab treatment (P = 0.003 for entire and peripheral tumor, and P = 0.02 for intra-tumor of GCTB) as well as endogenous intra-tumoral ossification. ConclusionsIn summary, our findings suggest a close relationship between the NBT and the osteoblastic differentiation of GCTB. Investigations of NBTs in naïve GCTB samples will provide a promising biomarker for predicting the ossification of GCTB following denosumab treatment.

Background: HPV16 is the most common infection subtype, among which E6 and E7 proteins are the most common carcinogenic proteins. Our previous studies found that E6 and E7 proteins regulated the expression of GLUT1 through multiple molecular signaling pathways in lung cancer. However, whether they can regulate the glucose uptake of GLUT1 and the underlying molecular mechanism has not been identified. Methods: The modulating effects of E6 or E7, NDRG2, β-catenin, and GLUT1 were detected by double directional genetic manipulations in lung cancer cell lines; The immunofluorescence was used to detect the effect of NDRG2 on the nuclear translocation of β-catenin; The glucose uptake level of GLUT1 was observed under the confocal microscope.Results: We demonstrated for the first time that E6 and E7 had inhibitory effects of NDRG2 which further resulted in increased β-catenin expression and promoted β-catenin nuclear translocation, furthermore promoted the expression and glucose uptake of GLUT1. Therefore, we hypothesized both E6 and E7 in HPV16 promoted the expression and glucose uptake of GLUT1 through HPV-NDRG2- β-catenin-GLUT1 axis. Conclusion: Our findings confirmed the regulatory role of tumor suppressor NDRG2 in the pathogenesis of lung cancer, and we further demonstrate the detail relationships among E6 and E7, NDRG2, β-catenin, and GLUT1; which provided a novel therapeutic target for tumor treatment.

Myocardial dysfunction accompanied by severe sepsis could significantly increase the mortality rate of septic patients. This study investigated the effects and the potential mechanisms of sevoflurane preconditioning on septic myocardial dysfunction, which was induced by lipopolysaccharide (LPS; from Escherichia coli O55:B5; 18 mg/kg) in mice. Results indicated that 1 hour after the administration, LPS induced a significant increase in cell-surface Toll-like receptor 4 (TLR4), cytoplasmic IKKα protein expression, and nuclear translocation of nuclear factor kappa-B (NF-κB) protein (P0.05), which was attenuated by preconditioning with sevoflurane. Two hours after the administration, inhalation of sevoflurane significantly reduced the serum levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-10 (P0.05). Twelve hours after administration, LPS caused pathological damage to the heart and elevated the serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (P0.05). Echocardiography indicated that sevoflurane preconditioning significantly improved systolic and diastolic function. The inhalation of sevoflurane inhibited increases in myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2), TNF-α, and IL-1β levels (P0.05) induced by endotoxemia, whereas IL-6 release was facilitated. Sevoflurane attenuated the myocardial levels of nitric oxide (P0.05) without an apparent influence on malondialdehyde (MDA) or superoxide dismutase (P0.05). In conclusion, our study indicates that exposure to 2% sevoflurane before LPS challenge is protective against myocardial dysfunction. Sevoflurane preconditioning may attenuate neutrophil infiltration and the release of inflammatory mediators during endotoxemia.

Aberrantly elevated expression levels of histone deacetylase 1 (HDAC1) and vimentin are closely associated with disease progression in hepatocellular carcinoma (HCC). It was previously demonstrated that knocking down expression of HDAC1 resulted in a concurrent decrease in the expression levels of vimentin. However, a causal link between these two proteins has not yet been demonstrated, to the best of our knowledge. In the present study, the association between HDAC1 and vimentin was investigated using an HDAC1 overexpression platform. HDAC1 and vimentin were significantly increased in HCC cells, and HDAC1 overexpression enhanced vimentin mRNA and protein expression levels in an HDAC1 dose-dependent manner. Subsequently, truncation and mutation of a vimentin promoter demonstrated that HDAC1-induced vimentin expression was dependent on a nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding site in the vimentin promoter sequence. Furthermore, HDAC1 induced vimentin expression by promoting NF-κB translocation between the cytoplasm and the nucleus, as opposed to modulating the total expression level of vimentin directly. The data in the present study demonstrated that HDAC1 is overexpressed in HCC and that HDAC1 may upregulate vimentin expression through the NF-κB signaling pathway, thus demonstrating a causal link between HDAC1 and vimentin in HCC, and may provide valuable information in understanding the pathogenesis of HCC.

The human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. Research reports on the mechanism of HEF1 in gastric cancer (GC) differentiation are limited. In this study, we reveal that HEF1 plays an essential role in regulating of differentiation in human GC. HEF1 was found to be highly expressed in GC tissues. Besides, In GC tissues or cells, cellular level of HEF1 negatively correlated with tumor differentiation. In addition, we showed that upregulation of HEF1 increased Wnt5a expression and the nuclear translocation of β-catenin, thereby resulting in poor differentiation in GC. Notably, GC patients with a higher expression of HEF1 showed significantly poorer disease-free and overall survival. Thus, our findings suggest that HEF1 reduces differentiation through the Wnt5a/β-catenin signaling pathway and that HEF1 is an independent unfavorable prognostic death factor in GC.

Dorsal-ventral patterning of the Drosophila embryo depends on the NFκB superfamily transcription factor Dorsal (Dl). Toll receptor activation signals for degradation of the IκB inhibitor Cactus (Cact), leading to a ventral-to-dorsal nuclear Dl gradient. Cact is critical for Dl nuclear import, as it binds to and prevents Dl from entering the nuclei. Quantitative analysis of cact mutants revealed an additional Cact function to promote Dl nuclear translocation in ventral regions of the embryo. To investigate this dual Cact role, we developed a predictive model based on a reaction-diffusion regulatory network. This network distinguishes non-uniform Toll-dependent Dl nuclear import and Cact degradation, from the Toll-independent processes of Cact degradation and reversible nuclear-cytoplasmic Dl flow. In addition, it incorporates translational control of Cact levels by Dl. Our model successfully reproduces wild-type data and emulates the Dl nuclear gradient in mutant dl and cact allelic combinations. Our results indicate that the dual role of Cact depends on the dynamics of Dl-Cact trimers along the dorsal-ventral axis: In the absence of Toll activation, free Dl-Cact trimers retain Dl in the cytoplasm, limiting the flow of Dl into the nucleus; in ventral-lateral regions, Dl-Cact trimers are recruited by Toll activation into predominant signaling complexes and promote Dl nuclear translocation. Simulations suggest that the balance between Toll-dependent and Toll-independent processes are key to this dynamics and reproduce the full assortment of Cact effects. Considering the high evolutionary conservation of these pathways, our analysis should contribute to understanding NFκB/c-Rel activation in other contexts such as in the vertebrate immune system and disease., Author summary In Drosophila, Toll pathway establishes spatially distinct gene expression territories that define the embryonic dorsal-ventral axis. Toll activation leads to degradation of the IκB inhibitor Cactus, releasing the NFκB superfamily transcription factor Dorsal for nuclear entry. Recently, quantitative analysis of cact mutants revealed that Cact displays an additional function to promote Dl nuclear translocation in ventral regions of the embryo. To understand this novel activity, we developed a predictive theoretical model that shows that the kinetics of Dorsal-Cactus complex formation prior to their recruitment to Toll-signaling complexes is an essential regulatory hub. Cactus controls the balance between the recruitment of these complexes by active Toll receptor and association-dissociation events that generate free Dorsal for direct nuclear import.

PI3Ks consist of p110 catalytic subunits and p85 regulatory subunits. PIK3CA, encoding p110α, is frequently mutated in human cancers. Most PIK3CA mutations are clustered in the helical domain or the kinase domain. Here, we report that p85β disassociates from p110α helical domain mutant protein and translocates into the nucleus through a nuclear localization sequence (NLS). Nuclear p85β recruits deubiquitinase USP7 to stabilize EZH1 and EZH2 and enhances H3K27 trimethylation. Knockout of p85β or p85β NLS mutant reduces the growth of tumors harboring a PIK3CA helical domain mutation. Our studies illuminate a novel mechanism by which PIK3CA helical domain mutations exert their oncogenic function. Finally, a combination of Alpelisib, a p110α-specific inhibitor, and an EZH inhibitor, Tazemetostat, induces regression of xenograft tumors harboring a PIK3CA helical domain mutation, but not tumors with either a WT PIK3CA or a PIK3CA kinase domain mutation, suggesting that the drug combination could be an effective therapeutic approach for PIK3CA helical domain mutant tumors.

In the progression of castration-resistant prostate cancer (CRPC), the androgen receptor (AR) that serves as a transcription factor becomes the most remarkable molecule. The transcriptional activity of AR is regulated by various coregulators. As a result, altered expression levels, an aberrant location or activities of coregulators promote the development of prostate cancer. We describe herein results showing that compared with androgen-dependent prostate cancer (ADPC) cells, AR nuclear translocation capability is enhanced in androgen-independent prostate cancer (AIPC) cells. To gain insight into whether AR coregulators are responsible for AR translocation capability, we performed coimmunoprecipitation (CO-IP) coupled with LC-MS/MS to screen 27 previously reported AR cofactors and 46 candidate AR cofactors. Furthermore, one candidate, myosin heavy chain 9 (MYH9), was identified and verified as a novel AR cofactor. Interestingly, the distribution of MYH9 was in both the cytoplasmic and nuclear compartments yet was enriched in the nucleus when AR was knocked down by AR shRNA, suggesting that the nuclear translocation of MYH9 was negatively regulated by AR. In addition, we found that blebbistatin, an inhibitor of MYH9, not only promoted AR nuclear translocation but also enhanced the expression of the AR target gene PSA, which indicates that MYH9 represses nuclear AR signaling. Taken together, our findings reveal that MYH9 appears to be a novel corepressor of AR plays a pivotal role in the progression of CRPC.

Lychee is a favorite fruit of the Cantonese and native to Southeast Asia. In this study, the anti-neuroinflammatory bioactive compounds of lychee seeds have been carried out. Five new jasmonates (1, 2, 6-8) and seventeen known compounds were isolated using a series of chemical and chromatographic methods. Their chemical structures were identified through comprehensive spectroscopic analysis. Anti-neuroinflammatory activities were assayed and evaluated for the purified compounds. Most of the compounds exhibited pronounced anti-neuroinflammatory activities on nitric oxide (NO) induced by lipopolysaccharide (LPS) in BV-2 microglia cells. Moreover, compounds 1, 2 and 20 could reduce the expression of LPS-induced pro-inflammatory factors (iNOS and COX-2), inhibit the expression of mRNA levels of iNOS, COX-2, IL-6 and block NF-κB nuclear translocation in dose-dependent manners. This study suggested that lychee phytochemicals could be benefit to some neuroinflammatory-associated diseases, such as Alzheimer's disease.

We recently established a new plasma peptidomic technique and comprehensively identified a large number of low-molecular weight and low-abundance native peptides using a single drop of human plasma. To discover a novel polypeptide that potently modulates the cardiovascular system, we performed a bioinformatics analysis of the large-scale identification results, sequentially synthesized the selected peptide sequences, tested their biological activities, and identified a 30-amino-acid proatherogenic peptide, GIP_HUMAN[22–51], as a potent proatherosclerotic peptide hormone. GIP_HUMAN[22–51] has a common precursor with the glucose-dependent insulinotropic polypeptide (GIP) and is located immediately N-terminal to GIP. Chronic infusion of GIP_HUMAN[22–51] into ApoE−/− mice accelerated the development of aortic atherosclerotic lesions, which were inhibited by co-infusions with an anti-GIP_HUMAN[22–51] antibody. GIP_HUMAN[22–51] increased the serum concentrations of many inflammatory and proatherogenic proteins, whereas neutralising antibodies reduced their levels. GIP_HUMAN[22–51] induced IκB-α degradation and nuclear translocation of NF-κB in human vascular endothelial cells and macrophages. Immunoreactive GIP_HUMAN[22–51] was detected in human tissues but there was no colocalization with the GIP. The plasma GIP_HUMAN[22–51] concentration in healthy humans determined using a stable-isotope tagged peptide was approximately 0.6 nM. This study discovered a novel endogenous proatherogenic peptide by using a human plasma native peptidomic resource.

Background and aim: Patients with end stage lung disease rely on mechanical ventilation (MV) to be able to breathe. However, MV often induces substantial lung injury known as ventilator-induced-lung-injury (VILI). Current in-vitro models of VILI fail to recapitulate the complex dynamic lung condition such as air-liquid interface (ALI) and 3D structures. To provide ALI conditions and customizable scaffolds for modelling VILI, we aimed to develop a lung-on-a-chip (LOC) device which allows for controllable stretch and is easily manufactured. Methods and results: We successfully fabricated a LOC using 3D printable molds and a commercially available porous membrane mimicking the lung ECM. This LOC was used to expose murine lung epithelial cells to stretch seen in MV (i.e. 25% cyclic strain). We observed higher nuclear translocation of the mechanosensors YAP/TAZ under stretched conditions (dynamic) compared to non-stretched conditions (static). Furthermore, confocal and scanning electron microscopy (SEM) images show higher amounts of cell loss in the dynamic condition compared to the static one. Cell death was measured using lactate dehydrogenase (LDH) levels in the supernatants where we confirmed higher levels of cell death under dynamic conditions compared to the static ones. Conclusion: Our study supports the potential use of 3D-printing as a fabrication technique for LOCs. Using the manufactured device, we were successfully able to accurately model lung injury on chip.

RNA-binding protein 24 (RBM24) has been shown to play tumor-suppressive functions in various types of cancer. The present study aimed to investigate the role of RBM24 in liver cancers and its downstream mechanisms. The present study demonstrated that RBM24 functioned as a tumor suppressor in liver cancer cells, and inhibited nuclear translocation of β-catenin and tumor protein 63 expression by immunocytochemistry. In addition, RBM24 could suppress sphere formation in a multicellular tumor spheroid model of liver cancer cells. In conclusion, it is hypothesized that RBM24 is a tumor suppressor of liver cancer cells, which could be a potential novel therapeutic target for treatment of patients with liver cancer.

Burgeoning evidence has indicated that normal autophagy is required for nuclear factor erythroid 2-related factor (Nrf2)-mediated cardiac protection whereas autophagy inhibition turns on Nrf2-mediated myocardial damage and dysfunction in a setting of pressure overload (PO). However, such a concept remains to be fully established by a careful genetic interrogation in vivo. This study was designed to validate the hypothesis using a mouse model of PO-induced cardiomyopathy and heart failure, in which cardiac autophagy and/or Nrf2 activity are genetically inhibited. Myocardial autophagy inhibition was induced by cardiomyocyte-restricted (CR) knockout (KO) of autophagy related (Atg) 5 (CR-Atg5KO) in adult mice. CR-Atg5KO impaired cardiac adaptations while exacerbating cardiac maladaptive responses in the setting of PO. Notably, it also turned off Nrf2-mediated defense while switching on Nrf2-operated tissue damage in PO hearts. In addition, cardiac autophagy inhibition selectively inactivated extracellular signal regulated kinase (ERK), which coincided with increased nuclear accumulation of Nrf2 and decreased nuclear translocation of activated ERK in cardiomyocytes in PO hearts. Mechanistic investigation revealed that autophagy is required for the activation of ERK, which suppresses Nrf2-driven expression of angiotensinogen in cardiomyocytes. Taken together, these results provide direct evidence consolidating the notion that normal autophagy enables Nrf2-operated adaptation while switching off Nrf2-mediated maladaptive responses in PO hearts partly through suppressing Nrf2-driven angiotensinogen expression in cardiomyocytes.

Introduction Signal transducer and activator of transcription 3 (STAT3) is involved in cancer initiation and resistance to chemo-radiation therapies and targeted agents. The role of STAT3 in inflammation and immunity together with its involvement in a variety of diseases including genitourinary, gastrointestinal, lung, ovarian and brain tumors makes STAT3 an ideal candidate for therapeutic strategies. Areas covered The authors provided an overview on STAT3 inhibitors and examined the most recent results obtained by these agents in cancer patients. The authors discussed the results published since 2015 and the ongoing clinical trials on anti-STAT3 agents in cancer patients. The authors also provide our opinion on the future perspectives of this therapeutic approach in this context. The manuscript includes information from trial databases and scientific literature. Expert opinion Future challenges include the development of non-peptide small-molecule inhibitors of STAT3 designed to directly inhibit STAT3 activity. In addition, inhibitors of STAT3/STAT3 nuclear translocation or DNA binding activity are also emerging as novel promising therapeutic approaches A better comprehension of the role of STAT3 in modulating immune response together with advances in understanding the mechanisms of STAT3-induced chemo and/or radio-resistance will also help the design of combined strategies in cancer patients.

In a genome-wide DNA methylation analysis of glioblastoma we identified aberrant methylation of the HIV-1 Tat interactive protein 2 (HTATIP2) gene promotor. This was strongly correlated with downregulation of HTATIP2 gene expression, suggesting a potential tumor suppressor function in glioblastoma. HTATIP2 has been shown to inhibit nuclear translocation through interaction with β-importins. We hypothesize that deregulation of HTATIP2 expression inhibits nuclear entry of cancer-relevant proteins, thereby disturbing their specific function in the nucleus. We identified N-Methylpurine-DNA Glycosylase (MPG) as a potential cancer relevant candidate with the observation of nuclear and/or cytoplasmic localization in glioblastoma. MPG is a DNA repair protein that recognizes DNA lesions (N7-meG, N3-meA) and initiates base excision repair. We have shown that nuclear MPG expression contributes to resistance of glioblastoma to treatment with the alkylating agent temozolomide. Here we investigated the effect of HTATIP2 on cellular localization of MPG using (i) an inducible system (TET-ON) to express HTATIP2 in non-expressing glioblastoma cells, and (ii) HTATIP2-targeting siRNAs to knock down HTATIP2 in cells with endogenous expression. Results from confocal microscopy or Imagestream analyses showed a significant cytoplasmic retention of MPG in presence of HTATIP2, while knock-down of HTATIP2 resulted in nuclear MPG localization. Cytoplasmic retention of MPG in the presence of HTATIP2 was associated with a significant increase in γ-H2Ax signal after treatment with the alkylating agent: methyl methanesulfonate (MMS), suggesting increase in DNA damage. Mechanistically, we found that HTATIP2 co-localizes with importin β1, and excludes MPG localization. Furthermore, HTATIP2 displayed a similar effect on cytoplasmic retention of MPG as pharmacologic inhibition of Importin β 1. Taken together, these results suggest that epigenetic silencing of HTATIP2 may increase nuclear localization of MPG, thereby increasing the capacity of the tumor cells to repair treatment related lesions and thereby contributing to treatment resistance.

OBJECTIVES Tumor Treating Fields (TTFields) are approved in combination with temozolomide for newly diagnosed glioblastoma (GBM). TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly. The addition of TTFields resulted in a significant improvement in overall survival. However, most GBM patients eventually develop resistance to TTFields and the mechanism remains unexplored. METHODS Multiple GBM cell lines were treated continuously at clinically approved frequency of 200 kHz using an in vitro TTFields system until cells with relative resistance to the cytotoxic effects of TTFields. A systems approach aided by innovative network ranking computational algorithms were utilized to analyze global gene expression profiles and identify resistance pathways, which were subsequently validated experimentally. RESULTS TTFields-induced chromosomal instability is preserved in resistant cells, indicating that TTFields resistance is mediated through a non-biophysical mechanism. This acquired TTFields resistance phenotype is associated with a transition of GBM cells to a stem-like state as determined by a neurosphere assay, stemness markers such as CD44 and increased tumorigenesis when implanted into mouse brain. Using an innovative computational platform-NETZEN, we methodically dissected this stemness program in resistant cells. 3 networks were found disrupted and all play critical roles in GBM stemness. Mechanistically, Prostaglandin E Receptor 3 (PTGER3) is the top ranked regulator responsible for resistance. PTGER3 is rapidly upregulated both in vitro and in vivo upon exposure to TTFields and further increases with prolonged treatment as resistance sets in. Immunofluorescence staining shows PTGER3’s nuclear translocation along with Lamin A/C disruption in response to TTFields. Pharmacological inhibition of PTGER3 using aspirin or PTGER3-specific inhibitors resensitized or prevent cells becoming resistance to TTFields. CONCLUSIONS We have identified a novel regulator PTGER3 at the apex that plays a critical role in TTFields resistance. This is a potential therapeutic target to reduce resistance to TTFields therapy in GBM.