Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.     

ZEB1‐mediated vasculogenic mimicry formation associates with epithelial–mesenchymal transition and cancer stem cell phenotypes in prostate cancer

The zinc finger E‐box‐binding homeobox 1 (ZEB1) induced the epithelial–mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid–Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1‐induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT‐related and CSC‐associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p‐Src⁵²⁷ level but not p‐Src⁴¹⁶ level, while ZEB1 knockdown also down‐regulated the level of p‐Src⁵²⁷ in PC3 and DU‐145 cells. PP2 treatment also significantly reduced the expression of VE‐cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.     

HDAC‐inhibitor (S)‐8 disrupts HDAC6‐PP1 complex prompting A375 melanoma cell growth arrest and apoptosis

Histone deacetylase inhibitors (HDACi) are agents capable of inducing growth arrest and apoptosis in different tumour cell types. Previously, we reported a series of novel HDACi obtained by hybridizing SAHA or oxamflatin with 1,4‐benzodiazepines. Some of these hybrids proved effective against haematological and solid cancer cells and, above all, compound (S)‐8 has emerged for its activities in various biological systems. Here, we describe the effectiveness of (S)‐8 against highly metastatic human A375 melanoma cells by using normal PIG1 melanocytes as control. (S)‐8 prompted: acetylation of histones H3/H4 and α‐tubulin; G₀/G₁ and G₂/M cell cycle arrest by rising p21 and hypophos‐phorylated RB levels; apoptosis involving the cleavage of PARP and caspase 9, BAD protein augmentation and cytochrome c release; decrease in cell motility, invasiveness and pro‐angiogenic potential as shown by results of wound‐healing assay, down‐regulation of MMP‐2 and VEGF‐A/VEGF‐R2, besides TIMP‐1/TIMP‐2 up‐regulation; and also intracellular accumulation of melanin and neutral lipids. The pan‐caspase inhibitor Z‐VAD‐fmk, but not the antioxidant N‐acetyl‐cysteine, contrasted these events. Mechanistically, (S)‐8 allows the disruption of cytoplasmic HDAC6‐protein phosphatase 1 (PP1) complex in A375 cells thus releasing the active PP1 that dephosphorylates AKT and blocks its downstream pro‐survival signalling. This view is consistent with results obtained by: inhibiting PP1 with Calyculin A; using PPP1R2‐transfected cells with impaired PP1 activity; monitoring drug‐induced HDAC6‐PP1 complex re‐shuffling; and, abrogating HDAC6 expression with specific siRNA. Altogether, (S)‐8 proved very effective against melanoma A375 cells, but not normal melanocytes, and safe to normal mice thus offering attractive clinical prospects for treating this aggressive malignancy.     

Suppressive effect of epigallocatechin‐3‐O‐gallate on endoglin molecular regulation in myocardial fibrosis in vitro and in vivo

Epigallocatechin‐3‐O‐gallate (EGCG), derived from green tea, has been studied extensively because of its diverse physiological and pharmacological properties. This study evaluates the protective effect of EGCG on angiotensin II (Ang II)‐induced endoglin expression in vitro and in vivo. Cardiac fibroblasts (CFs) from the thoracic aorta of adult Wistar rats were cultured and induced with Ang II. Western blotting, Northern blotting, real‐time PCR and promoter activity assay were performed. Ang II increased endoglin expression significantly as compared with control cells. The specific extracellular signal‐regulated kinase inhibitor SP600125 (JNK inhibitor), EGCG (100 μM) and c‐Jun N‐terminal kinase (JNK) siRNA attenuated endoglin proteins following Ang II induction. In addition, pre‐treated Ang II‐induced endoglin with EGCG diminished the binding activity of AP‐1 by electrophoretic mobility shift assay. Moreover, the luciferase assay results revealed that EGCG suppressed the endoglin promoter activity in Ang II‐induced CFs by AP‐1 binding. Finally, EGCG and the JNK inhibitor (SP600125) were found to have attenuated endoglin expression significantly in Ang II‐induced CFs, as determined through confocal microscopy. Following in vivo acute myocardial infarction (AMI)‐related myocardial fibrosis study, as well as immunohistochemical and confocal analyses, after treatment with endoglin siRNA and EGCG (50 mg/kg), the area of myocardial fibrosis reduced by 53.4% and 64.5% and attenuated the left ventricular end‐diastolic and systolic dimensions, and friction shortening in hemodynamic monitor. In conclusion, epigallocatechin‐3‐O‐gallate (EGCG) attenuated the endoglin expression and myocardial fibrosis by anti‐inflammatory effect in vitro and in vivo, the novel suppressive effect was mediated through JNK/AP‐1 pathway.     

Metabolic imaging using hyperpolarized 13C‐pyruvate to assess sensitivity to the B‐Raf inhibitor vemurafenib in melanoma cells and xenografts

Nearly all melanoma patients with a BRAF‐activating mutation will develop resistance after an initial clinical benefit from BRAF inhibition (BRAFi). The aim of this work is to evaluate whether metabolic imaging using hyperpolarized (HP) ¹³C pyruvate can serve as a metabolic marker of early response to BRAFi in melanoma, by exploiting the metabolic effects of BRAFi. Mice bearing human melanoma xenografts were treated with the BRAFi vemurafenib or vehicle. In vivo HP ¹³C magnetic resonance spectroscopy was performed at baseline and 24 hours after treatment to evaluate changes in pyruvate‐to‐lactate conversion. Oxygen partial pressure was measured via electron paramagnetic resonance oximetry. Ex vivo qRT‐PCR, immunohistochemistry and WB analysis were performed on tumour samples collected at the same time‐points selected for in vivo experiments. Similar approaches were applied to evaluate the effect of BRAFi on sensitive and resistant melanoma cells in vitro, excluding the role of tumour microenvironment. BRAF inhibition induced a significant increase in the HP pyruvate‐to‐lactate conversion in vivo, followed by a reduction of hypoxia. Conversely, the conversion was inhibited in vitro, which was consistent with BRAFi‐mediated impairment of glycolysis. The paradoxical increase of pyruvate‐to‐lactate conversion in vivo suggests that such conversion is highly influenced by the tumour microenvironment.     

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long‐term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK‐2206 in combination with paclitaxel and carboplatin. Although single agent MK‐2206 inhibited phospho‐AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK‐2206 with paclitaxel and carboplatin was cytotoxic in long‐term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase‐dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti‐oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.     

Thymus vulgaris alleviates UVB irradiation induced skin damage via inhibition of MAPK/AP‐1 and activation of Nrf2‐ARE antioxidant system

Solar ultraviolet (UV) radiation‐induced reactive oxidative species is mainly responsible for the development of photoageing. Rosmarinic acid was one of the main bioactive components detected in Thymus vulgaris (TV) we extracted. In this study, UVB‐induced skin damages have been shown to be ameliorated by treatment with TV in hairless mice (HR‐1) skin, demonstrated by decreased matrix metalloproteinases (MMPs) and increased collagen production. However, the underlying molecular mechanism on which TV acted was unclear. We examined the photoprotective effects of TV against UVB and elucidated the molecular mechanism in normal human dermal fibroblasts. Thymus vulgaris remarkably prevented the UVB‐induced reactive oxygen species and lactate dehydrogenase. Dose‐dependent increase in glutathione, NAD(P)H: quinone oxidoreductase1 and heme oxygenase‐1, by TV was confirmed by increased nuclear accumulation of Nrf2. Furthermore, 5‐Methoxyindole‐2‐carboxylic acid was introduced as a specific inhibitor of dihydrolipoyl dehydrogenase (DLD). We demonstrated that Nrf2 expression was regulated by DLD, which was a tricarboxylic acid cycle‐associated protein that decreased after UVB exposure. Besides, TV significantly diminished UVB induced phosphorylation of mitogen activated protein kinases pathway, containing extracellular signal‐regulated kinase, Jun N‐terminal kinase and p38, which consequently reduced phosphorylated c‐fos and c‐jun. Our results suggest that TV is a potential botanical agent for use against UV radiation‐induced oxidative stress mediated skin damages.     

Rictor regulates the vasculogenic mimicry of melanoma via the AKT‐MMP‐2/9 pathway

Vasculogenic mimicry (VM)‐positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin‐insensitive complex of mTOR (mTORC2), is up‐regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan–Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM‐2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down‐regulation. Western blotting assays indicated that down‐regulating Rictor significantly inhibited the phosphorylation of AKT at Ser⁴⁷³ and Thr³⁰⁸, which subsequently inhibited the expression and activity of downstream MMP‐2/9, as confirmed by real‐time PCR and gelatin Zymography. MK‐2206, a small‐molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP‐2/9, further supporting that Rictor down‐regulation inhibits the phosphorylation of AKT and activity of downstream MMP‐2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor—AKT—MMP‐2/9 signalling pathway.     

Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS‐mutant melanoma

The MEK inhibitor MEK162 is the first targeted therapy agent with clinical activity in patients whose melanomas harbor NRAS mutations; however, median PFS is 3.7 months, suggesting the rapid onset of resistance in the majority of patients. Here, we show that treatment of NRAS‐mutant melanoma cell lines with the MEK inhibitors AZD6244 or trametinib resulted in a rebound activation of phospho‐ERK (pERK). Functionally, the recovery of signaling was associated with the maintenance of cyclin‐D1 expression and therapeutic escape. The combination of a MEK inhibitor with an ERK inhibitor suppressed the recovery of cyclin‐D1 expression and was associated with a significant enhancement of apoptosis and the abrogation of clonal outgrowth. The MEK/ERK combination strategy induced greater levels of apoptosis compared with dual MEK/CDK4 or MEK/PI3K inhibition across a panel of cell lines. These data provide the rationale for further investigation of vertically co‐targeting the MAPK pathway as a potential treatment option for NRAS‐mutant melanoma patients.     

Mitochondria of a human multidrug‐resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P‐glycoprotein (P‐gp), breast cancer resistant protein and multiple resistance protein‐1. The MDR phenotype is associated with the constitutive expression of COX‐2 and iNOS, whereas celecoxib, a specific inhibitor of COX‐2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX‐2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1‐positive cells, whereas COX‐2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P‐gp in mitochondria of MDR cancer cells independently from inhibition of COX‐2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.     

PI3‐kinase/Akt pathway‐regulated membrane transportation of acid‐sensing ion channel 1a/Calcium ion influx/endoplasmic reticulum stress activation on PDGF‐induced HSC Activation

Acid‐sensing ion channel 1a (ASIC1a) allows Na⁺ and Ca²⁺ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non‐neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS‐related proteins were up‐regulated in carbon tetrachloride (CCl₄)‐induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS‐related biomarkers GRP78, Caspase12 and IREI‐XBP1. And, ERS inhibition by 4‐PBA down‐regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF‐induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF‐activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.     

Inhibition of P53/miR‐34a improves diabetic endothelial dysfunction via activation of SIRT1

Endothelial dysfunction contributes to diabetic macrovascular complications, resulting in high mortality. Recent findings demonstrate a pathogenic role of P53 in endothelial dysfunction, encouraging the investigation of the effect of P53 inhibition on diabetic endothelial dysfunction. Thus, high glucose (HG)‐treated endothelial cells (ECs) were subjected to pifithrin‐α (PFT‐α)—a specific inhibitor of P53, or P53‐small interfering RNA (siRNA), both of which attenuated the HG‐induced endothelial inflammation and oxidative stress. Moreover, inhibition of P53 by PFT‐α or P53‐siRNA prohibited P53 acetylation, decreased microRNA‐34a (miR‐34a) level, leading to a dramatic increase in sirtuin 1 (SIRT1) protein level. Interestingly, the miR‐34a inhibitor (miR‐34a‐I) and PFT‐α increased SIRT1 protein level and alleviated the HG‐induced endothelial inflammation and oxidative stress to a similar extent; however, these effects of PFT‐α were completely abrogated by the miR‐34a mimic. In addition, SIRT1 inhibition by EX‐527 or Sirt1‐siRNA completely abolished miR‐34a‐I's protection against HG‐induced endothelial inflammation and oxidative stress. Furthermore, in the aortas of streptozotocin‐induced diabetic mice, both PFT‐α and miR‐34a‐I rescued the inflammation, oxidative stress and endothelial dysfunction caused by hyperglycaemia. Hence, the present study has uncovered a P53/miR‐34a/SIRT1 pathway that leads to endothelial dysfunction, suggesting that P53/miR‐34a inhibition could be a viable strategy in the management of diabetic macrovascular diseases.     

Brain‐derived neurotrophic factor attenuates doxorubicin‐induced cardiac dysfunction through activating Akt signalling in rats

The clinical application of doxorubicin (Dox) is limited by its adverse effect of cardiotoxicity. Previous studies have suggested the cardioprotective effect of brain‐derived neurotrophic factor (BDNF). We hypothesize that BDNF could protect against Dox‐induced cardiotoxicity. Sprague Dawley rats were injected with Dox (2.5 mg/kg, 3 times/week, i.p.), in the presence or absence of recombinant BDNF (0.4 μg/kg, i.v.) for 2 weeks. H9c2 cells were treated with Dox (1 μM) and/or BDNF (400 ng/ml) for 24 hrs. Functional roles of BDNF against Dox‐induced cardiac injury were examined both in vivo and in vitro. Protein level of BDNF was reduced in Dox‐treated rat ventricles, whereas BDNF and its receptor tropomyosin‐related kinase B (TrkB) were markedly up‐regulated after BDNF administration. Brain‐derived neurotrophic factor significantly inhibited Dox‐induced cardiomyocyte apoptosis, oxidative stress and cardiac dysfunction in rats. Meanwhile, BDNF increased cell viability, inhibited apoptosis and DNA damage of Dox‐treated H9c2 cells. Investigations of the underlying mechanisms revealed that BDNF activated Akt and preserved phosphorylation of mammalian target of rapamycin and Bad without affecting p38 mitogen‐activated protein kinase and extracellular regulated protein kinase pathways. Furthermore, the beneficial effect of BDNF was abolished by BDNF scavenger TrkB‐Fc or Akt inhibitor. In conclusion, our findings reveal a potent protective role of BDNF against Dox‐induced cardiotoxicity by activating Akt signalling, which may facilitate the safe use of Dox in cancer treatment.     

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3‐nitropropionic acid (3‐NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum‐isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC), and that 3‐NPA increased DA, reactive oxygen species (ROS), DA‐quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3‐NPA‐induced increase in DA, ROS, lipid peroxidation, and DA‐quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α‐tocopherol. Results show that the increase in DA induced by 3‐NPA was inhibited by both 25 μM vinpocetine and 50 μM α‐tocopherol. Vinpocetine, as α‐tocopherol, also inhibited 3‐NPA‐induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA‐RS formation), and DA‐quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3‐NPA, 3‐NPA increases DA‐oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α‐tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA‐rich structures.     

Modification,Biological Evaluation and SAR Studies of Novel 1H‐Pyrazol Derivatives Containing N,N′‐Disubstituted Urea Moiety as Potential Anti‐melanoma Agents

Malignant melanomas are amongst the most aggressive cancers. BRAF Inhibitors have exhibited therapeutic effects against BRAF‐mutant melanoma. In continuation of our earlier studies on anti‐melanoma agents based on 1H‐pyrazole skeleton, two sets of novel compounds that include 1H‐pyrazole‐4‐amines FA 1 – FA13 and corresponding urea derivatives FN 1 – FN13 have been synthesized and evaluated for their BRAF^V600E inhibitory and antiproliferation activities. Compound FN 10 displayed the most potent biological activity against BRAF^V600E (IC₅₀ = 0.066 μm ) and the A375 human melanoma cell line (GI₅₀ = 0.81 μm ), which was comparable to the positive control vemurafenib, and more potent than our previously reported 1H‐pyrazole‐3‐amines and their urea derivatives. The results of SAR studies and molecular docking can guide further optimization and may help to improve potency of these pyrazole‐based anti‐melanoma agents.     

The role of S‐nitrosylation of kainate‐type of ionotropic glutamate receptor 2 in epilepsy induced by kainic acid

Epilepsy is a chronic brain disease affecting millions of individuals. Kainate receptors, especially kainate‐type of ionotropic glutamate receptor 2 (GluK2), play an important role in epileptogenesis. Recent data showed that GluK2 could undergo post‐translational modifications in terms of S‐nitrosylation (SNO ), and affect the signaling pathway of cell death in cerebral ischemia‐reperfusion. However, it is unclear whether S‐nitrosylation of GluK2 (SNO ‐GluK2) contributes to cell death induced by epilepsy. Here, we report that kainic acid‐induced SNO ‐GluK2 is mediated by GluK2 itself, regulated by neuronal nitric oxide synthase (nNOS ) and the level of cytoplasmic calcium in vivo and in vitro hippocampus neurons. The whole‐cell patch clamp recordings showed the influence of SNO ‐GluK2 on ion channel characterization of GluK2‐Kainate receptors. Moreover, immunohistochemistry staining results showed that inhibition of SNO ‐GluK2 by blocking nNOS or GluK2 or by reducing the level of cytoplasmic calcium‐protected hippocampal neurons from kainic acid‐induced injury. Finally, immunoprecipitation and western blotting data revealed the involvement of assembly of a GluK2‐PSD 95‐nNOS signaling complex in epilepsy. Taken together, our results showed that the SNO ‐GluK2 plays an important role in neuronal injury of epileptic rats by forming GluK2‐PSD 95‐nNOS signaling module in a cytoplasmic calcium‐dependent way, suggesting a potential therapeutic target site for epilepsy.

     

Near‐genomewide RNAi screening for regulators of BRAFV600E‐induced senescence identifies RASEF,a gene epigenetically silenced in melanoma

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene‐induced senescence (OIS), a highly potent in vivo tumor‐suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near‐genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF^V600E‐induced arrest. With genome‐wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence‐associated (SA)‐β‐galactosidase activity, interleukins, and tumor suppressor p15^INK4B. Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.