PTENε suppresses tumor metastasis through regulation of filopodia formation

PTEN is one of the most frequently mutated genes in malignancies and acts as a powerful tumor suppressor. Tumorigenesis is involved in multiple and complex processes including initiation, invasion, and metastasis. The complexity of PTEN function is partially attributed to PTEN family members such as PTENα and PTENβ. Here, we report the identification of PTENε (also named as PTEN5), a novel N‐terminal‐extended PTEN isoform that suppresses tumor invasion and metastasis. We show that the translation of PTENε/PTEN5 is initiated from the CUG⁸¹⁶ codon within the 5′UTR region of PTEN mRNA. PTENε/PTEN5 mainly localizes in the cell membrane and physically associates with and dephosphorylates VASP and ACTR2, which govern filopodia formation and cell motility. We found that endogenous depletion of PTENε/PTEN5 promotes filopodia formation and enhances the metastasis capacity of tumor cells. Overall, we identify a new isoform of PTEN with distinct subcellular localization and molecular function compared to the known members of the PTEN family. These findings advance our current understanding of the importance and diversity of PTEN functions.     

PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation.     

Benchmarking AlphaFold‐enabled molecular docking predictions for antibiotic discovery

Efficient identification of drug mechanisms of action remains a challenge. Computational docking approaches have been widely used to predict drug binding targets; yet, such approaches depend on existing protein structures, and accurate structural predictions have only recently become available from AlphaFold2. Here, we combine AlphaFold2 with molecular docking simulations to predict protein‐ligand interactions between 296 proteins spanning Escherichia coli''s essential proteome, and 218 active antibacterial compounds and 100 inactive compounds, respectively, pointing to widespread compound and protein promiscuity. We benchmark model performance by measuring enzymatic activity for 12 essential proteins treated with each antibacterial compound. We confirm extensive promiscuity, but find that the average area under the receiver operating characteristic curve (auROC) is 0.48, indicating weak model performance. We demonstrate that rescoring of docking poses using machine learning‐based approaches improves model performance, resulting in average auROCs as large as 0.63, and that ensembles of rescoring functions improve prediction accuracy and the ratio of true‐positive rate to false‐positive rate. This work indicates that advances in modeling protein‐ligand interactions, particularly using machine learning‐based approaches, are needed to better harness AlphaFold2 for drug discovery.     

Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1‐CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1‐CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1‐CTDNEP1 downregulation also restores nuclear pore membrane fusion in post‐mitotic dTorsin^KO fat body cells. However, dTorsin‐associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel‐like structures in place of mature nuclear pores. We conclude that the NEP1R1‐CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid‐dependent and lipid‐independent mechanisms, explaining some of the pleiotropic effects of Torsins.     

Syncytiotrophoblast‐derived extracellular vesicles carry apolipoprotein‐E and affect lipid synthesis of liver cells in vitro

In normal pregnancy, hepatic metabolism adaptation occurs with an increase in lipid biosynthesis. Placental shedding of syncytiotrophoblast‐derived extracellular vesicles (STBEVs) into the maternal circulation constitutes a major signalling mechanism between foetus and mother. We investigated whether STBEVs from normal pregnant women might target liver cells in vitro and induce changes in lipid synthesis. This study was performed at the Nuffield Department of Women''s & Reproductive Health, Oxford, UK. STBEVs were obtained by dual‐lobe placental perfusion from 11 normal pregnancies at term. Medium/large and small STBEVs were collected by ultracentrifugation at 10,000g and 150,000g, respectively. STBEVs were analysed by Western blot analysis and flow cytometry for co‐expression of apolipoprotein‐E (apoE) and placental alkaline phosphatase (PLAP). The uptake of STBEVs by liver cells and the effect on lipid metabolism was evaluated using a hepatocarcinoma cell line (HepG2 cells). Data were analysed by one‐way ANOVA and Student''s t test. We demonstrated that: (a) STBEVs carry apoE; (b) HepG2 cells take up STBEVs through an apoE‐LDL receptor interaction; (c) STBEV incorporation into HepG2 cells resulted in (i) increased cholesterol release (ELISA); (ii) increased expression of the genes SQLE and FDPS (microarray) involved in cholesterol biosynthesis; (iii) downregulation of the CLOCK gene (microarray and PCR), involved in the circadian negative control of lipid synthesis in liver cells. In conclusion, the placenta may orchestrate the metabolic adaptation of the maternal liver through release of apoE‐positive STBEVs, by increasing lipid synthesis in a circadian‐independent fashion, meeting the nutritional needs of the growing foetus.     

Circular RNA VRK1 facilitates pre‐eclampsia progression via sponging miR‐221‐3P to regulate PTEN/Akt

Pre‐eclampsia (PE) is a worldwide pregnancy‐related disorder. It is mainly characterized by defect migration and invasion of trophoblast cells. Recently, circular RNAs (circRNAs) have been believed to play a vital role in PE. The expression patterns and the biological functions of circRNAs in PE remain elusive. Here, we performed a circRNA microarray to identify putative PE‐related circRNAs. Bioinformatics analyses were used to screen the circRNAs which have potential relationships with pre‐eclampsia, and we identified a novel circRNA (circVRK1) that was up‐regulated in PE placenta tissues. By using HTR‐8/SVneo cells, circVRK1 knockdown significantly enhanced cell migration and invasion abilities, as well as epithelial‐mesenchymal transition (EMT). Mechanistically, we found that circVRK1 and PTEN could function as the ceRNAs to miR‐221‐3p. Overexpression of miR‐221‐3p promoted cell migration, invasion and EMT via regulating PTEN. The cotransfection of miR‐221‐3p inhibitor or PTEN reversed the effect from circVRK1 knockdown. Moreover, the circVRK1/miR‐221‐3p/PTEN axis greatly regulated Akt phosphorylation. In general, circVRK1 suppresses trophoblast cell migration, invasion and EMT, by acting as a ceRNA to miR‐221‐3p to regulate PTEN, and further inhibit PI3K/Akt activation. The purpose of this paper is to open wide insights to investigate the onset of PE and provide new potential therapeutic targets in PE.     

Role of the Phosphatase PTEN in Early Vascular Remodeling

Background

The phosphatase PTEN represents an important physiological inhibitor of phosphatidylinositol-3 kinase (PI3-K)/protein kinase B (Akt) signalling, however, the functional role of PTEN in the initial phase of angioplasty-induced vascular injury remains elusive. In the present study we sought to determine PTEN''s effect on vascular smooth muscle cell (VSMC) apoptosis following acute injury in vivo and in vitro.

Methods and Results

Immunohistochemistry indicated a faint basal expression and equal distribution of PTEN in uninjured rat carotid arteries. 12 h following balloon-injury, PTEN expression was strongly increased in apoptotic (TUNEL+) VSMC. In vitro, stimulation with serum or different growth factors or subjecting VSMC to cyclic stretch had no effect on PTEN expression, whereas stimulation with H₂O₂ robustly increased PTEN expression in a time- and dose-dependent manner. To evaluate the functional role of PTEN expression, human VSMC were transduced with WT-PTEN. Overexpression of PTEN increased the number of apoptotic VSMC (19.8%±4.4 vs. 5.6%±2.3; P<0.001) as determined by TUNEL assay. In contrast, siRNA-mediated knock-down of PTEN attenuated the basal as well as H₂O₂-induced apoptosis of VSMC. Mechanistically, overexpression of PTEN prevented serum-induced Akt-phosphorylation, whereas siRNA-mediated knock down of PTEN augmented Akt-activation. Moreover, co-transfection of PTEN and a constitutive active Akt mutant prevented PTEN-dependent augmentation of VSMC apoptosis, indicating, that PTEN regulates VSMC apoptosis by inhibition of Akt phosphorylation/activation.

Conclusion

By interfering with the PI3-K/Akt-dependent survival signalling, the oxidative stress-induced up regulation of PTEN in VSMC of injured arteries augments the sensitivity of VSMC to apoptotic stimuli in the early phase following vascular injury, augmenting the initial injury and cell loss of the injured vessel wall. Thus, these data add to our understanding of PTEN''s role during vascular remodelling.     

Chemogenetic profiling reveals PP2A‐independent cytotoxicity of proposed PP2A activators iHAP1 and DT‐061

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT‐061 have recently been reported to selectively stabilize specific PP2A‐B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT‐061 on PP2A‐B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome‐wide CRISPR‐Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT‐061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT‐061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT‐061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A‐B56 biology.     

MicroRNA‐495‐3p diminishes doxorubicin‐induced cardiotoxicity through activating AKT

Doxorubicin (Dox) is a broad‐spectrum antitumour agent; however, its clinical application is impeded due to the cumulative cardiotoxicity. The present study aims to investigate the role and underlying mechanisms of microRNA‐495‐3p (miR‐495‐3p) in Dox‐induced cardiotoxicity. Herein, we found that cardiac miR‐495‐3p expression was significantly decreased in Dox‐treated hearts, and that the miR‐495‐3p agomir could prevent oxidative stress, cell apoptosis, cardiac mass loss, fibrosis and cardiac dysfunction upon Dox stimulation. In contrast, the miR‐495‐3p antagomir dramatically aggravated Dox‐induced cardiotoxicity in mice. Besides, we found that the miR‐495‐3p agomir attenuated, while the miR‐495‐3p antagomir exacerbated Dox‐induced oxidative stress and cellular injury in vitro. Mechanistically, we demonstrated that miR‐495‐3p directly bound to the 3′‐untranslational region of phosphate and tension homology deleted on chromosome ten (PTEN), downregulated PTEN expression and subsequently activated protein kinase B (PKB/AKT) pathway, and that PTEN overexpression or AKT inhibition completely abolished the cardioprotective effects of the miR‐495‐3p agomir. Our study for the first time identify miR‐495‐3p as an endogenous protectant against Dox‐induced cardiotoxicity through activating AKT pathway in vivo and in vitro.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

3,6'‐disinapoyl sucrose attenuates Aβ1‐42‐ induced neurotoxicity in Caenorhabditis elegans by enhancing antioxidation and regulating autophagy

The aggregation of β‐amyloid (Aβ) has the neurotoxicity, which is thought to play critical role in the pathogenesis of Alzheimer''s disease (AD). Inhibiting Aβ deposition and neurotoxicity has been considered as an important strategy for AD treatment. 3,6''‐Disinapoyl sucrose (DISS), one of the oligosaccharide esters derived from traditional Chinese medicine Polygalae Radix, possesses antioxidative activity, neuroprotective effect and anti‐depressive activity. This study was to explore whether DISS could attenuate the pathological changes of Aβ_1‐42 transgenic Caenorhabditis elegans (C. elegans). The results showed that DISS (5 and 50 μM) treatment significantly prolonged the life span, increased the number of egg‐laying, reduced paralysis rate, decreased the levels of lipofuscin and ROS and attenuated Aβ deposition in Aβ_1‐42 transgenic C. elegans. Gene analysis showed that DISS could up‐regulate the mRNA expression of sod‐3, gst‐4, daf‐16, bec‐1 and lgg‐1, while down‐regulate the mRNA expression of daf‐2 and daf‐15 in Aβ_1‐42 transgenic C. elegans. These results suggested that DISS has the protective effect against Aβ_1‐42‐induced pathological damages and prolongs the life span of C. elegans, which may be related to the reduction of Aβ deposition and neurotoxicity by regulating expression of genes related to antioxidation and autophagy.     

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline‐auxotroph yeast

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl‐CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl‐CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine‐tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

PTEN function in mammalian cell size regulation

The PTEN tumor suppressor gene is a lipid phosphatase that negatively regulates cell survival mediated by the phosphatidyl inositol 3' kinase-protein kinase B/Akt signaling pathway. Recent in vivo studies have revealed a novel role for PTEN in the size control of neurons. Dysregulation of cell growth control by PTEN is associated with the neurological disorder Lhermitte-Duclos disease. PTEN may regulate cell size through effects on protein translation.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

PTEN蛋白磷酸酶活性的作用

PTEN是一个具有磷酸酶活性的肿瘤抑制基因,是编码具有脂质磷酸酶活性和蛋白磷酸酶活性的双重特异性磷酸酶,其缺失或功能异常与人类恶性肿瘤的发生发展密切相关。PTEN的脂质磷酸酶活性和蛋白磷酸酶活性在调控肿瘤细胞的生物学行为、维持细胞正常的生理活动中均发挥了重要作用。但二者的作用重点及机制仍有不同,其蛋白磷酸酶活性主要侧重于调控细胞的黏附迁移及侵袭。为更好地认识PTEN蛋白磷酸酶活性的作用,该文对PTEN蛋白磷酸酶活性的作用及其机制作一简要综述。