bims-ershed 2021-05-02 papers

bims-ershed

Biomed News

on ER Stress in Health and Diseases

Issue of 2021–05–02
nineteen papers selected by
Matías Eduardo González Quiroz, Worker’s Hospital

The Road of Solid Tumor Survival: From Drug-Induced Endoplasmic Reticulum Stress to Drug Resistance.
The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.
Expression of the Endoplasmic Reticulum Stress Marker GRP78 in the Normal Retina and Retinal Degeneration Induced by Blue LED Stimuli in Mice.
Combined Transcriptomic and Proteomic Analysis of Perk Toxicity Pathways.
Endoplasmic Reticulum Stress Contributes to Gefitinib-Induced Apoptosis in Glioma.
Functions and mechanisms of circular RNAs in regulating stem cell differentiation.
Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.
The molecular mechanism of nuclear signaling for degradation of cytoplasmic DNA: Importance in DNA damage response and cancer.
An in vivo Interaction Network of DNA-Repair Proteins: A Snapshot at Double Strand Break Repair in Deinococcus radiodurans.
The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.
Ultraviolet B irradiation decreases CXCL10 expression in keratinocytes through endoplasmic reticulum stress.
Daily briefing: How philosophy can make you a better scientist.
A protocol for in vivo analysis of liver tumorigenesis in mice using sleeping beauty transposon system.
Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model.
Establishment of a reporter system for monitoring activation of the ER stress transducer ATF6β.
PERK activation by CCT020312 chemosensitizes colorectal cancer through inducing apoptosis regulated by ER stress.
Critical DNA damaging pathways in tumorigenesis.
Nucleolin Rescues TDP-43 Toxicity in Yeast and Human Cell Models.
S-Nitrosylation in Tumor Microenvironment.

Front Mol Biosci. 2021 ;8 620514

The Road of Solid Tumor Survival: From Drug-Induced Endoplasmic Reticulum Stress to Drug Resistance.

Shulong Cao, Jingyi Tang, Yichun Huang, Gaofeng Li, Zhuoya Li, Wenqi Cai, Yuning Yuan, Junlong Liu, Xuqun Huang, Haiyuan Zhang.

  Endoplasmic reticulum stress (ERS), which refers to a series of adaptive responses to the disruption of endoplasmic reticulum (ER) homeostasis, occurs when cells are treated by drugs or undergo microenvironmental changes that cause the accumulation of unfolded/misfolded proteins. ERS is one of the key responses during the drug treatment of solid tumors. Drugs induce ERS by reactive oxygen species (ROS) accumulation and Ca2+ overload. The unfolded protein response (UPR) is one of ERS. Studies have indicated that the mechanism of ERS-mediated drug resistance is primarily associated with UPR, which has three main sensors (PERK, IRE1α, and ATF6). ERS-mediated drug resistance in solid tumor cells is both intrinsic and extrinsic. Intrinsic ERS in the solid tumor cells, the signal pathway of UPR-mediated drug resistance, includes apoptosis inhibition signal pathway, protective autophagy signal pathway, ABC transporter signal pathway, Wnt/β-Catenin signal pathway, and noncoding RNA. Among them, apoptosis inhibition is one of the major causes of drug resistance. Drugs activate ERS and its downstream antiapoptotic proteins, which leads to drug resistance. Protective autophagy promotes the survival of solid tumor cells by devouring the damaged organelles and other materials and providing new energy for the cells. ERS induces protective autophagy by promoting the expression of autophagy-related genes, such as Beclin-1 and ATG5-ATG12. ABC transporters pump drugs out of the cell, which reduces the drug-induced apoptosis effect and leads to drug resistance. In addition, the Wnt/β-catenin signal pathway is also involved in the drug resistance of solid tumor cells. Furthermore, noncoding RNA regulates the ERS-mediated survival and death of solid tumor cells. Extrinsic ERS in the solid tumor cells, such as ERS in immune cells of the tumor microenvironment (TME), also plays a crucial role in drug resistance by triggering immunosuppression. In immune system cells, ERS in dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) influences the antitumor function of normal T cells, which results in immunosuppression. Meanwhile, ERS in T cells can also cause impaired functioning and apoptosis, leading to immunosuppression. In this review, we highlight the core molecular mechanism of drug-induced ERS involved in drug resistance, thereby providing a new strategy for solid tumor treatment.

Keywords:  drug resistance; endoplasmic reticulum stress; immunosuppression; solid tumor; unfolded protein response

DOI:  https://doi.org/10.3389/fmolb.2021.620514
Int J Mol Sci. 2021 Apr 26. pii: 4494. [Epub ahead of print]22(9):

The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.

Wioletta Rozpędek-Kamińska, Grzegorz Galita, Natalia Siwecka, Steven L Carroll, John Alan Diehl, Ewa Kucharska, Dariusz Pytel, Ireneusz Majsterek.

  Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.

Keywords:  PERK; PERK inhibitor; apoptosis; eIF2α; endoplasmic reticulum stress; glaucoma; glaucoma treatment; unfolded protein response

DOI:  https://doi.org/10.3390/ijms22094494
Cells. 2021 Apr 23. pii: 995. [Epub ahead of print]10(5):

Expression of the Endoplasmic Reticulum Stress Marker GRP78 in the Normal Retina and Retinal Degeneration Induced by Blue LED Stimuli in Mice.

Yong Soo Park, Hong-Lim Kim, Seung Hee Lee, Yan Zhang, In-Beom Kim.

  Retinal degeneration is a leading cause of blindness. The unfolded protein response (UPR) is an endoplasmic reticulum (ER) stress response that affects cell survival and death and GRP78 forms a representative protective response. We aimed to determine the exact localization of GRP78 in an animal model of light-induced retinal degeneration. Dark-adapted mice were exposed to blue light-emitting diodes and retinas were obtained at 24 h and 72 h after exposure. In the normal retina, we found that GRP78 was rarely detected in the photoreceptor cells while it was expressed in the perinuclear space of the cell bodies in the inner nuclear and ganglion cell layers. After injury, the expression of GRP78 in the outer nuclear and inner plexiform layers increased in a time-dependent manner. However, an increased GRP78 expression was not observed in damaged photoreceptor cells in the outer nuclear layer. GRP78 was located in the perinuclear space and ER lumen of glial cells and the ER developed in glial cells during retinal degeneration. These findings suggest that GRP78 and the ER response are important for glial cell activation in the retina during photoreceptor degeneration.

Keywords:  GRP78; endoplasmic reticulum; retinal degeneration; retinal glial cell; stress response; unfolded protein response

DOI:  https://doi.org/10.3390/cells10050995
Int J Mol Sci. 2021 Apr 27. pii: 4598. [Epub ahead of print]22(9):

Combined Transcriptomic and Proteomic Analysis of Perk Toxicity Pathways.

Rebeka Popovic, Ivana Celardo, Yizhou Yu, Ana C Costa, Samantha H Y Loh, L Miguel Martins.

  In Drosophila, endoplasmic reticulum (ER) stress activates the protein kinase R-like endoplasmic reticulum kinase (dPerk). dPerk can also be activated by defective mitochondria in fly models of Parkinson's disease caused by mutations in pink1 or parkin. The Perk branch of the unfolded protein response (UPR) has emerged as a major toxic process in neurodegenerative disorders causing a chronic reduction in vital proteins and neuronal death. In this study, we combined microarray analysis and quantitative proteomics analysis in adult flies overexpressing dPerk to investigate the relationship between the transcriptional and translational response to dPerk activation. We identified tribbles and Heat shock protein 22 as two novel Drosophila activating transcription factor 4 (dAtf4) regulated transcripts. Using a combined bioinformatics tool kit, we demonstrated that the activation of dPerk leads to translational repression of mitochondrial proteins associated with glutathione and nucleotide metabolism, calcium signalling and iron-sulphur cluster biosynthesis. Further efforts to enhance these translationally repressed dPerk targets might offer protection against Perk toxicity.

Keywords:  Drosophila; Drosophila protein kinase RNA (PKR)-like ER kinase (dPerk); ER stress; activating transcription factor 4 (ATF4); unfolded protein response

DOI:  https://doi.org/10.3390/ijms22094598
Int J Mol Sci. 2021 Apr 11. pii: 3934. [Epub ahead of print]22(8):

Endoplasmic Reticulum Stress Contributes to Gefitinib-Induced Apoptosis in Glioma.

Cheng-Yi Chang, Ping-Ho Pan, Chih-Cheng Wu, Su-Lan Liao, Wen-Ying Chen, Yu-Hsiang Kuan, Wen-Yi Wang, Chun-Jung Chen.

  Adequate stress on the Endoplasmic Reticulum (ER) with the Unfolded Protein Response (UPR) could maintain glioma malignancy. Uncontrolled ER stress, on the other hand, predisposes an apoptosis-dominant UPR program. We studied here the proapoptotic actions of the Epidermal Growth Factor Receptor (EGFR) inhibitor gefitinib, with the focus on ER stress. The study models were human H4 and U87 glioma cell lines. We found that the glioma cell-killing effects of gefitinib involved caspase 3 apoptotic cascades. Three branches of ER stress, namely Activating Transcription Factor-6 (ATF6), Protein Kinase R (PKR)-Like ER Kinase (PERK), and Inositol-Requiring Enzyme 1 (IRE1), were activated by gefitinib, along with the elevation of intracellular free Ca2+, Reactive Oxygen Species (ROS), and NADPH Oxidase2/4 (NOX2/4). Specifically, elevated IRE1 phosphorylation, Tumor Necrosis Factor (TNF) Receptor-Associated Factor-2 (TRAF2) expression, Apoptosis Signal-Regulating Kinase-1 (Ask1) phosphorylation, c-Jun N-Terminal Kinase (JNK) phosphorylation, and Noxa expression appeared in gefitinib-treated glioma cells. Genetic, pharmacological, and biochemical studies further indicated an active ROS/ER stress/Ask1/JNK/Noxa axis causing the glioma apoptosis induced by gefitinib. The findings suggest that ER-stress-based therapeutic targeting could be a promising option in EGFR inhibitor glioma therapy, and may ultimately achieve a better patient response.

Keywords:  EGFR inhibitors; ER stress; Noxa; apoptosis; glioma

DOI:  https://doi.org/10.3390/ijms22083934
RNA Biol. 2021 Apr 26. 1-14

Functions and mechanisms of circular RNAs in regulating stem cell differentiation.

Zhengjun Lin, Xianzhe Tang, Jia Wan, Xianghong Zhang, Chunfeng Liu, Tang Liu.

  Stem cells are a class of undifferentiated cells with great self-renewal and differentiation capabilities that can differentiate into mature cells in specific tissue types. Stem cell differentiation plays critical roles in body homoeostasis, injury repair and tissue generation. The important functions of stem cell differentiation have resulted in numerous studies focusing on the complex molecular mechanisms and various signalling pathways controlling stem cell differentiation. Circular RNAs (circRNAs) are a novel class of noncoding RNAs with a covalently closed structure present in eukaryotes. Numerous studies have highlighted important biological functions of circRNAs, and they play multiple regulatory roles in various physiological and pathological processes. Importantly, multiple lines of evidence have shown the abnormal expression of numerous circRNAs during stem cell differentiation, and some play a role in regulating stem cell differentiation, highlighting the role of circRNAs as novel biomarkers of stem cell differentiation and novel targets for stem cell-based therapy. In this review, we systematically summarize and discuss recent advances in our understanding of the roles and underlying mechanisms of circRNAs in modulating stem cell differentiation, thus providing guidance for future studies to investigate stem cell differentiation and stem cell-based therapy.Abbreviations: CircRNAs: circular RNAs; ESCs: embryonic stem cells; ADSCs: adipose-derived mesenchymal stem cells; ecircRNAs: exonic circRNAs; EIciRNAs: exon-intron circRNAs; eiRNAs: circular intronic RNAs; tricRNAs: tRNA intronic circRNAs; pol II: polymerase II; snRNP: small nuclear ribonucleoprotein; m6A: N6-methyladenosine; AGO2: Argonaute 2; RBPs: RNA-binding proteins; MBNL: muscleblind-like protein 1; MSCs: mesenchymal stem cells; hiPSCs: human induced pluripotent stem cells; hiPSC-CMs: hiPSC-derived cardiomyocytes; hBMSCs: human bone marrow mesenchymal stem cells; hADSCs: human adipose-derived mesenchymal stem cells; hDPSCs: human dental pulp stem cells; RNA-seq: high-throughput RNA sequencing; HSCs: haematopoietic stem cells; NSCs: neural stem cells; EpSCs: epidermal stem cells; hESCs: human embryonic stem cells; mESCs: murine embryonic stem cells; MNs: motor neurons; SSUP: small subunit processome; BMSCs: bone marrow-derived mesenchymal stem cells; OGN: osteoglycin; GIOP: glucocorticoid‑induced osteoporosis; CDR1as: cerebellar degeneration-related protein 1 transcript; SONFH: steroid-induced osteogenesis of the femoral head; rBMSCs: rat bone marrow-derived mesenchymal stem cells; QUE: quercetin; AcvR1b: activin A receptor type 1B; BSP: bone sialoprotein; mADSCs: mouse ADSCs; PTBP1: polypyrimidine tract-binding protein; ER: endoplasmic reticulum; hUCMSCs: MSCs derived from human umbilical cord; MSMSCs: maxillary sinus membrane stem cells; SCAPs: stem cells from the apical papilla; MyoD: myogenic differentiation protein 1; MSTN: myostatin; MEF2C: myocyte enhancer factor 2C; BCLAF1: BCL2-associated transcription factor 1; EpSCs: epidermal stem cells; ISCs: intestinal stem cells; NSCs: neural stem cells; Lgr5+ ISCs: crypt base columnar cells; ILCs: innate lymphoid cells.

Keywords:  Circular RNAs; differentiation; miRNA sponges; stem cells

DOI:  https://doi.org/10.1080/15476286.2021.1913551
J Drug Target. 2021 Apr 29. 1-9

Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.

David Hernán Martínez-Puente, Rodolfo Garza-Morales, José Juan Pérez-Trujillo, Aracely García-García, Arnulfo Villanueva-Olivo, Humberto Rodríguez-Rocha, Laura Mireya Zavala-Flores, Odila Saucedo-Cárdenas, Roberto Montes de Oca-Luna, María de Jesús Loera-Arias.

  It has been previously reported that targeting and retaining antigens in the endoplasmic reticulum (ER) can induce an ER stress response. In this study, we evaluated the antitumor effect of E7 antigen fused to an ERresident protein, cyclooxygenase-2, which possesses a 19-aminoacid cassette that directs it to the endoplasmic reticulum-associated protein degradation (ERAD) pathway. The featured DNA constructs, COX2-E7 and COX2-E7ΔERAD, with a deletion in the 19-aminoacid cassette, were used to evaluate the importance of this sequence. In vitro analysis of protein expression and ER localisation were verified. We observed that both constructs induced an ER stress response. This finding correlated with the antitumor effect in mice injected with TC-1 cells and treated with different DNA constructs by biolistic vaccination. Immunisation with COX2-E7 and COX2-E7ΔERAD DNA constructs induced a significant antitumor effect in mice, without a significant difference between them, although the COX2-E7 construct induced a significant E7-specific immune response. These results demonstrate that targeting the E7 antigen to the ERAD pathway promotes a potent therapeutic antitumor effect. This strategy could be useful for the design of other antigen-specific therapies.

Keywords:  COX-2; DNA vaccine; E7; ER stress; GRP78/BiP; TC-1; endoplasmic reticulum; endoplasmic reticulum-associated protein degradation (ERAD); unfolded protein response (UPR)

DOI:  https://doi.org/10.1080/1061186X.2021.1919124
DNA Repair (Amst). 2021 Apr 26. pii: S1568-7864(21)00071-9. [Epub ahead of print]103 103115

The molecular mechanism of nuclear signaling for degradation of cytoplasmic DNA: Importance in DNA damage response and cancer.

Erfan Mohammadi, Fatemeh Sadoughi, Simin Younesi, Ansar Karimian, Zatollah Asemi, Nader Farsad-Akhtar, Fahime Jahanbakhshi, Hamidreza Jamilian, Bahman Yousefi.

  This review summarizes and addresses non-coding RNAs (rRNA, tRNA, Vault and Y RNA, snRNA, and miRNA) cytoplasmic decay pathways, the molecules, enzymes, and modifications such as uridylation, which play vital roles in the degradation processes in various eukaryotic organisms. Plus, SIRT1's role in fundamental cellular processes, including autophagy, DNA repair, DNA damage response (DDR), and the molecular mechanisms, is explored. Further, the HuR (an RNA-binding protein) impact on the expression of genes following DNA damage, and the pathways that regulate HuR function, which is through phosphorylation by Chk1/Cdk1 and Chk2, are specified. Finally, the role of DIF1/ Rnr2-Rnr4 in DDR has been discussed.

Keywords:  DNA damage response; DNA repair; Enzymes; Molecular mechanisms

DOI:  https://doi.org/10.1016/j.dnarep.2021.103115
J Proteome Res. 2021 Apr 30.

An in vivo Interaction Network of DNA-Repair Proteins: A Snapshot at Double Strand Break Repair in Deinococcus radiodurans.

Aman Kumar Ujaoney, Mahesh Kumar Padwal, Bhakti Basu.

  An extremophile Deinococcus radiodurans survives massive DNA damage by efficiently mending hundreds of double strand breaks through homology-dependent DNA repair pathways. Although DNA repair proteins that contribute to its impressive DNA repair capacity are fairly known, interactions among them or with proteins related to other relevant pathways remain unexplored. Here, we report in vivo cross-linking of the interactomes of key DNA repair proteins DdrA, DdrB, RecA, and Ssb (baits) in D. radiodurans cells recovering from gamma irradiation. The protein-protein interactions were systematically investigated through co-immunoprecipitation experiments coupled to mass spectrometry. From a total of 399 proteins co-eluted with the baits, we recovered interactions among diverse biological pathways such as DNA repair, transcription, translation, chromosome partitioning, cell division, antioxidation, protein folding/turnover, metabolism, cell wall architecture, membrane transporters, and uncharacterized proteins. Among these, about 80 proteins were relevant to the DNA damage resistance of the organism based on integration of data on inducible expression following DNA damage, radiation sensitive phenotype of deletion mutant, etc. Further, we cloned ORFs of 23 interactors in heterologous E. coli and expressed corresponding proteins with N-terminal His-tag, which were used for pull-down assays. A total of 95 interactions were assayed, in which we confirmed 25 previously unknown binary interactions between the proteins associated with radiation resistance, and 2 known interactions between DdrB and Ssb or DR_1245. Among these, five interactions were positive even under non-stress conditions. The confirmed interactions cover a wide range of biological processes such as DNA repair, negative regulation of cell division, chromosome partitioning, membrane anchorage, etc., and their functional relevance is discussed from the perspective of DNA repair. Overall, the study substantially advances our understanding on the cross-talk between different homology-dependent DNA repair pathways and other relevant biological processes that essentially contribute to the extraordinary DNA damage repair capability of D. radiodurans. The data sets generated and analyzed in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD021822.

Keywords:  DdrA; DdrB; RecA; Ssb; binary interactions; co-immunoprecipitation; interactome

DOI:  https://doi.org/10.1021/acs.jproteome.1c00078
Int J Mol Sci. 2021 Apr 14. pii: 4024. [Epub ahead of print]22(8):

The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.

Lee-Wen Chen, Shie-Shan Wang, Chien-Hui Hung, Ya-Hui Hung, Chun-Liang Lin, Pey-Jium Chang.

  The unfolded protein response (UPR) is an intracellular signaling pathway essential for alleviating the endoplasmic reticulum (ER) stress. To support the productive infection, many viruses are known to use different strategies to manipulate the UPR signaling network. However, it remains largely unclear whether the UPR signaling pathways are modulated in the lytic cycle of Epstein-Barr virus (EBV), a widely distributed human pathogen. Herein, we show that the expression of GRP78, a central UPR regulator, is up-regulated during the EBV lytic cycle. Our data further revealed that knockdown of GRP78 in EBV-infected cell lines did not substantially affect lytic gene expression; however, GRP78 knockdown in these cells markedly reduced the production of virus particles. Importantly, we identified that the early lytic protein BMLF1 is the key regulator critically contributing to the activation of the grp78 gene promoter. Mechanistically, we found that BMLF1 can trigger the proteolytic cleavage and activation of the UPR senor ATF6, which then transcriptionally activates the grp78 promoter through the ER stress response elements. Our findings therefore provide evidence for the connection between the EBV lytic cycle and the UPR, and implicate that the BMLF1-mediated ATF6 activation may play critical roles in EBV lytic replication.

Keywords:  ATF6; BMLF1; Epstein-Barr virus; GRP78; lytic replication; unfolded protein response

DOI:  https://doi.org/10.3390/ijms22084024
J Cell Biochem. 2021 Apr 28.

Ultraviolet B irradiation decreases CXCL10 expression in keratinocytes through endoplasmic reticulum stress.

Tomokazu Ohnishi, Mitsuhiro Hisadome, Kusuyama Joji, Norika Chiba, Muhammad Subhan Amir, Takuro Kanekura, Tetsuya Matsuguchi.

  Ultraviolet radiation is one of the standard treatment selections for psoriasis. interferon (IFN)-γ and IFN-γ-induced CXCL10, which are highly expressed by keratinocytes in psoriasis lesion, are therapeutic targets for psoriasis. In this study, we found that ultraviolet B (UVB) irradiation inhibited IFN-γ signaling events, including STAT1 phosphorylation and induction of CXCL10 messenger RNA (mRNA) expression in keratinocytes. IFN-γ-induced expression of CXCL10 mRNA in HaCaT cells, a human keratinocyte cell line, and human epithelial keratinocytes were also inhibited by H2 O2 or endoplasmic reticulum (ER) stress inducers. Conversely, a mixture of antioxidants, Trolox and ascorbic acid, and the ER stress inhibitor salubrinal partially counteracted the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA expression in HaCaT cells. We also found that UVB and ER stress reduced IFN-γ receptor 1 protein levels in the plasma membrane fraction of keratinocytes. These observations suggested that ER stress and the generation of reactive oxygen species are essential for the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA in keratinocytes.

Keywords:  CXCL10; ER stress; IFN-γ; STAT1; UVB; keratinocytes

DOI:  https://doi.org/10.1002/jcb.29936
Nature. 2021 Apr 27.

Daily briefing: How philosophy can make you a better scientist.

Flora Graham.

DOI: https://doi.org/10.1038/d41586-021-01150-4
STAR Protoc. 2021 Jun 18. 2(2): 100445

A protocol for in vivo analysis of liver tumorigenesis in mice using sleeping beauty transposon system.

Lingli He, Wentao Yu, Yi Lu, Wenxiang Zhang, Jinjin Xu, Lei Zhang.

  Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer death worldwide. However, the pathogenesis of HCC is complicated, and the drugs used for HCC treatment are limited. The following protocol combines a genetically engineered mouse model (GEMM) with a sleeping beauty system to establish an in vivo liver tumorigenesis model. By using this model, the impact of genes of interest in liver tumorigenesis and progression can be studied. This model can also be applied to develop new therapeutic drugs for HCC. For complete details on the use and execution of this protocol, please refer to He et al. (2020).

Keywords:  Cancer; Gene Expression; Genetics; Model Organisms; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2021.100445
Reproduction. 2021 Apr 01. pii: REP-20-0668.R1. [Epub ahead of print]

Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model.

Veronika Tandl, Denise Hoch, Julia Bandres-Meriz, Sanela Nikodijevic, Gernot Desoye, Alejandro Majali-Martinez.

Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1-10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24h, while eIF2α protein and CHOP expression were downregulated (-28%; P < 0.05 and -24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48h by low oxygen (-61%; P < 0.05), but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

DOI: https://doi.org/10.1530/REP-20-0668
Biochem Biophys Res Commun. 2021 Apr 21. pii: S0006-291X(21)00658-6. [Epub ahead of print]558 1-7

Establishment of a reporter system for monitoring activation of the ER stress transducer ATF6β.

Le Thi Hien, Sung Hoon Back.

  ATF6 has two isoforms, ATF6α and ATF6β, which are ubiquitously expressed type II transmembrane glycoproteins in the endoplasmic reticulum (ER). While the regulatory mechanisms and transcriptional roles of ATF6α in response to ER stress have been well-studied, those of its paralogue ATF6β are less understood. Moreover, there is no specific cell-based reporter assay to monitor ATF6β activation. Here, we developed a new cell-based reporter system that can monitor activation of endogenous ATF6β. This system expresses a chimeric protein containing a synthetic transcription factor followed by the transmembrane domain and C-terminal luminal domain of ATF6β. Under ER stress conditions, the chimeric protein was cleaved by regulated intramembrane proteolysis (RIP) to liberate the N-terminal synthetic transcription factor, which induced luciferase expression in the HeLa Luciferase Reporter cell line. This new stable reporter cell line will be an innovative tool to investigate RIP of ATF6β.

Keywords:  ATF6β; ER stress; GAL4 binding site; Luciferase; Regulated intramembrane proteolysis; Reporter cell line

DOI:  https://doi.org/10.1016/j.bbrc.2021.04.052
Biochem Biophys Res Commun. 2021 Apr 21. pii: S0006-291X(21)00423-X. [Epub ahead of print]557 316-322

PERK activation by CCT020312 chemosensitizes colorectal cancer through inducing apoptosis regulated by ER stress.

Yunpeng Lei, Lirui He, Chang Yan, Yuchen Wang, Guoqing Lv.

  Endoplasmic reticulum (ER) stress is a significant mechanism for chemoresistance to colorectal cancer (CRC) treatment. The RNA-like endoplasmic reticulum kinase (PERK) is critical for ER stress induction. In the present study, we attempted to explore whether PERK activator CCT020312 (CCT) could be effective for CRC treatment, and reveal the underlying mechanisms. We first found that CCT dose- and time-dependently reduced CRC cell proliferation. Importantly, it markedly improved the chemosensitivity of CRC cells that were drug-sensitive or -resistant to taxol treatment, as evidenced by the significantly decreased cell viability. Moreover, CCT at the non-toxic concentration exhibited obviously synergistic effects with taxol to induce apoptosis and cell cycle arrest in G2/M phase in vitro. In addition, we showed that CCT alone considerably induced ER stress in CRC cells through a dose- and time-dependent fashion. Meanwhile, CCT combined with taxol caused significant ER stress through improving phosphorylated PERK, eukaryotic translation initiation factor 2α (eIF2ɑ), C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78). More studies showed that the interaction between PERK and GRP78 was a potential target for CCT to perform its regulatory events. Intriguingly, PERK knockdown markedly abolished the regulatory role of CCT and taxol cotreatments in cell proliferation suppression and apoptosis induction, indicating the importance of PERK for CCT to perform its anti-cancer bioactivity. Our in vivo experiments confirmed that CCT plus taxol dramatically reduced tumor growth in CRC xenografts. Together, all these results suggested that promoting PERK activation by CCT may be an effective therapeutic strategy to improve CRC to taxol treatment.

Keywords:  Apoptosis; CCT020312; Colorectal cancer; ER stress; PERK

DOI:  https://doi.org/10.1016/j.bbrc.2021.03.041
Semin Cancer Biol. 2021 Apr 24. pii: S1044-579X(21)00113-9. [Epub ahead of print]

Critical DNA damaging pathways in tumorigenesis.

Jake A Kloeber, Zhenkun Lou.

  The acquisition of DNA damage is an early driving event in tumorigenesis. Premalignant lesions show activated DNA damage responses and inactivation of DNA damage checkpoints promotes malignant transformation. However, DNA damage is also a targetable vulnerability in cancer cells. This requires a detailed understanding of the cellular and molecular mechanisms governing DNA integrity. Here, we review current work on DNA damage in tumorigenesis. We discuss DNA double strand break repair, how repair pathways contribute to tumorigenesis, and how double strand breaks are linked to the tumor microenvironment. Next, we discuss the role of oncogenes in promoting DNA damage through replication stress. Finally, we discuss our current understanding on DNA damage in micronuclei and discuss therapies targeting these DNA damage pathways.

Keywords:  DNA damage; Genome instability; Tumorigenesis

DOI:  https://doi.org/10.1016/j.semcancer.2021.04.012
Front Cell Neurosci. 2021 ;15 625665

Nucleolin Rescues TDP-43 Toxicity in Yeast and Human Cell Models.

Caterina Peggion, Maria Lina Massimino, Roberto Stella, Raissa Bortolotto, Jessica Agostini, Arianna Maldi, Geppo Sartori, Fiorella Tonello, Alessandro Bertoli, Raffaele Lopreiato.

  TDP-43 is a nuclear protein involved in pivotal processes, extensively studied for its implication in neurodegenerative disorders. TDP-43 cytosolic inclusions are a common neuropathologic hallmark in amyotrophic lateral sclerosis (ALS) and related diseases, and it is now established that TDP-43 misfolding and aggregation play a key role in their etiopathology. TDP-43 neurotoxic mechanisms are not yet clarified, but the identification of proteins able to modulate TDP-43-mediated damage may be promising therapeutic targets for TDP-43 proteinopathies. Here we show by the use of refined yeast models that the nucleolar protein nucleolin (NCL) acts as a potent suppressor of TDP-43 toxicity, restoring cell viability. We provide evidence that NCL co-expression is able to alleviate TDP-43-induced damage also in human cells, further supporting its beneficial effects in a more consistent pathophysiological context. Presented data suggest that NCL could promote TDP-43 nuclear retention, reducing the formation of toxic cytosolic TDP-43 inclusions.

Keywords:  ALS; FTD; TDP-43 proteinopathies; misfolded proteins; neurodegenerative disorders; nucleo-cytoplasmic transport; nucleolin

DOI:  https://doi.org/10.3389/fncel.2021.625665
Int J Mol Sci. 2021 Apr 27. pii: 4600. [Epub ahead of print]22(9):

S-Nitrosylation in Tumor Microenvironment.

Vandana Sharma, Veani Fernando, Joshua Letson, Yashna Walia, Xunzhen Zheng, Daniel Fackelman, Saori Furuta.

  S-nitrosylation is a selective and reversible post-translational modification of protein thiols by nitric oxide (NO), which is a bioactive signaling molecule, to exert a variety of effects. These effects include the modulation of protein conformation, activity, stability, and protein-protein interactions. S-nitrosylation plays a central role in propagating NO signals within a cell, tissue, and tissue microenvironment, as the nitrosyl moiety can rapidly be transferred from one protein to another upon contact. This modification has also been reported to confer either tumor-suppressing or tumor-promoting effects and is portrayed as a process involved in every stage of cancer progression. In particular, S-nitrosylation has recently been found as an essential regulator of the tumor microenvironment (TME), the environment around a tumor governing the disease pathogenesis. This review aims to outline the effects of S-nitrosylation on different resident cells in the TME and the diverse outcomes in a context-dependent manner. Furthermore, we will discuss the therapeutic potentials of modulating S-nitrosylation levels in tumors.

Keywords:  ECM; NO; NOS; S-nitrosylation; cancer therapeutics; microbiome; microenvironment; tumor-associated immune cells

DOI:  https://doi.org/10.3390/ijms22094600