E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer

Oncogene, Journal Year: 2024, Volume and Issue: 43(28), P. 2184 - 2198

Published: May 24, 2024

Abstract Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ~5% all human cancers worldwide, including most cervical cancer cases and growing number anogenital oral cancers. The HPV viral oncogenes, E6 E7, manipulate many host cellular pathways that promote cell proliferation survival, predisposing infected cells malignant transformation. Despite the availability highly effective vaccines, there still no specific anti-viral therapies targeting or treatments for HPV-associated As such, better understanding viral-host interactions may allow identification novel therapeutic targets. Here, we demonstrate actin-binding protein LASP1 is upregulated in significantly correlates with poorer overall survival. In positive cancer, depletion inhibited oncogenic phenotype vitro, whilst having minimal effects negative cells. Furthermore, SH3 domain essential LASP1-mediated oncogenicity these Mechanistically, show E7 regulates at post-transcriptional level by repressing expression miR-203, which negatively mRNA levels binding its 3’UTR. Finally, required growth an vivo tumourigenicity model. Together, data induces survival thus identifying potential target

Language: Английский

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Human papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA

Journal of Virology, Journal Year: 2024, Volume and Issue: 98(9)

Published: Aug. 28, 2024

We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the human papillomavirus 16 (HPV16) life cycle. Here, we demonstrate in HPV-negative cervical cancer C33a cells foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required replication, viral replication promotes phosphorylation of SAMHD1, which converts it from dNTPase an HR independent E6/E7 expression. A phospho-mimic (SAMHD1 T592D) reduces E1-E2-mediated DNA has enhanced recruitment In HFK+HPV16 cells, T592D attenuates cellular growth, but does not attenuate growth isogenic HFK alone. also development foci following keratinocyte differentiation. The results indicated could be therapeutically beneficial with genomes. Protein phosphatase 2A (PP2A) can dephosphorylate PP2A function inhibited endothall. endothall DNA, mimicking phenotypes. Finally, head neck cell lines episomal genomes, their genome. suggest targeting phosphatases therapeutic potential treatment HPV infections cancers.

Language: Английский

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CRISPR-Cas9‒Based Genomic Engineering in Keratinocytes: From Technology to Application

JID Innovations, Journal Year: 2021, Volume and Issue: 2(2), P. 100082 - 100082

Published: Dec. 1, 2021

CRISPR-Cas9 is the most straightforward genome-editing tool to date. However, its implementation across disciplines hampered by variable efficiencies, reduced cell viability, and low success rates in obtaining clonal lines. This review aims recognize all CRISPR-Cas9‒related work within experimental dermatology field identify key factors for successful strategies different keratinocyte (KC) sources available. On basis of these findings, we conclude that groups use immortalized KCs generating knockout KCs. Our critical considerations future studies using CRISPR-Cas9, both fundamental clinical applications, may guide technologies (experimental) field. Introduction as a genomic editing toolCRISPRs were known bacterial genome hypervariable loci typically consisting direct repeats, separated sections sequences called spacers, proximity CRISPR-Cas genes. The mechanism system specifically target DNA was utilized successfully first time mammalian cells almost decade ago (Cong et al., 2013Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. al.Multiplex engineering CRISPR/Cas systems.Science. 2013; 339: 819-823Google Scholar; Jinek 2012Jinek M. Chylinski K. Fonfara I. Hauer Doudna J.A. Charpentier E. A programmable dual-RNA–guided endonuclease adaptive immunity.Science. 2012; 337: 816-821Google Mali 2013Mali P. Yang Esvelt K.M. Aach J. Guell DiCarlo J.E. al.RNA-guided human via Cas9.Science. 823-826Google Scholar), functions described extensively (Doudna Charpentier, 2014Doudna Genome editing. new frontier with CRISPR-Cas9.Science. 2014; 346: 1258096Google Scholar) schematically visualized Figure 1a. Many species have variants CRISPR Cas loci, investigated variant being (Makarova 2011Makarova K.S. Haft D.H. Barrangou Brouns S.J. Horvath al.Evolution classification CRISPR–Cas systems.Nat Rev Microbiol. 2011; 9: 467-477Google Scholar).CRISPR-Cas9‒mediated requires Cas9‒guide RNA (gRNA) complex containing Cas9, (crRNA), trans-activating (tracrRNA) (see Box 1: Terminology). can be introduced various methods, reviewed before (Lino 2018Lino C.A. Harper J.C. Carney J.P. Timlin Delivering CRISPR: challenges approaches.Drug Deliv. 2018; 25: 1234-1257Google Shi 2021Shi H. Smits J.P.H. van den Bogaard E.H. Brewer M.G. Research techniques made simple: delivery CRISPR/Cas9 components into epidermal cells.J Invest Dermatol. 2021; 141: 1375-1381.e1Google Scholar). By guidance crRNA, binds complement accompanied flanking protospacer adjacent motif 5′-NGG-3′ Streptococcus pyogenes Cas9 (Chylinski 2013Chylinski Le Rhun A. tracrRNA families type II immunity systems.RNA Biol. 10: 726-737Google Cas9‒gRNA induces double-stranded break at site (Deltcheva 2011Deltcheva Sharma C.M. Gonzales Chao Y. Pirzada Z.A. al.CRISPR maturation trans-encoded small host factor RNase III.Nature. 471: 602-607Google Shah 2013Shah S.A. Erdmann Mojica F.J. Garrett R.A. Protospacer recognition motifs: mixed identities functional diversity.RNA 891-899Google which repaired through either nonhomologous end joining (NHEJ) (Hefferin Tomkinson, 2005Hefferin M.L. Tomkinson A.E. Mechanism double-strand repair non-homologous joining.DNA Repair. 2005; 4: 639-648Google or homology-directed (HDR) (Liang 1998Liang F. Han Romanienko P.J. Jasin Homology-directed major pathway cells.Proc Natl Acad Sci USA. 1998; 95: 5172-5177Google In NHEJ, broken strands are religated, directly after random nucleotide insertions deletions (Takata 1998Takata Sasaki M.S. Sonoda Morrison C. Hashimoto Utsumi al.Homologous recombination end-joining pathways overlapping roles maintenance chromosomal integrity vertebrate cells.EMBO 17: 5497-5508Google Often, this leads frameshift mutations premature stop codons, therefore, readily used knock out protein expression interest. HDR, breaks sister chromatid homologous template strand. multiple crossovers, synthesis, ligation, damaged strand precisely Instead strand, an exogenous harboring desired mutation gene cassette single-strand DNA, arms on outsides (Chen 2011Chen Pruett-Miller S.M. Huang Gjoka Duda Taunton al.High-frequency ssDNA oligonucleotides zinc-finger nucleases.Nat Methods. 8: 753-755Google Radecke 2010Radecke Cathomen T. Schwarz Zinc-finger nuclease-induced oligodeoxynucleotides: wanted unwanted locus modifications.Mol Ther. 2010; 18: 743-753Google Rouet 1994Rouet Smih Expression site-specific stimulates 1994; 91: 6064-6068Google Scholar).Box 1CRISPR TerminologyCRISPRClustered Regularly Interspaced Short Palindromic RepeatsCas9CRISPR-associated 9Cas9nCas9 nickasedCas9Deactivated Cas9PAMProtospacer motifcrRNACRISPR RNAtracrRNATrans-activating RNA(s)gRNA(Single) RNARNPRibonucleoproteinHDRHomology-directed repairNHEJNonhomologous Open table tab Over years, increasing number harnessed toolbox, although current numbers limited but over past 5 years (Figure 1b c Table 1). performed keratinocytes (KCs) best practices determinants KC available, application.Table 1Characteristics Studies Utilize Human KeratinocytesCell sourcePublicationPMIDCell Types (All Human)Research GoalMethod IntroductionCarrierCas9 VersionRepairSelectionPrimary keratinocytesNöske 2016Nöske Stark H.J. Nevaril Berning Langbein Goyal al.Mitotic diversity homeostatic interfollicular epidermis.Int J Mol Sci. 2016; 167Google Scholar26828486Adult primary keratinocytesProtein knockoutElectroporationPlasmid vectorSpCas9NHEJFACSYue 2017Yue Gou X. Li Wicksteed B. Wu Engineered progenitor correct diet-induced obesity diabetes.Cell Stem Cell. 2017; 21: 256-263.e4Google Scholar28777946Foreskin keratinocytesGene activationElectroporationPlasmid vectorhCas9 D10AHDRPuromycinHainzl 2017Hainzl Peking Kocher Murauer E.M. Larcher Del Rio al.COL7A1 recessive dystrophic epidermolysis bullosa.Mol 2573-2584Google Scholar28800953RDEB correctionXfectCationic vectorSpCas9HDRPuromycin blasticidinKocher 2017Kocher Klausegger Hofbauer Wally V. al.Cut paste: efficient dominant negative KRT14 nickases.Mol 2585-2598Google Scholar28888469Adult vectorSpCas9NHEJBlasticidinFenini 2018aFenini G. Grossi Contassot Biedermann Reichmann French L.E. al.Genome reveals essential role NLRP1 inflammasome UVB sensing.J 138: 2644-2652Google Scholar29287762Adult knockoutlentiCRISPR v2LentivirusSpCas9NHEJPuromycinFenini 2018bFenini Gehrke Beer H.D. Satoh T.K. al.The p38 mitogen-activated kinase critically regulates activation.J 1380-1390Google Scholar30096351Adult v2LentivirusSpCas9NHEJPuromycinIzmiryan 2018Izmiryan Ganier Bovolenta Schmitt Mavilio Hovnanian Ex vivo COL7A1 correction bullosa repair.Mol Ther Nucleic Acids. 12: 554-567Google Scholar30195791RDEB correctionIDLVLentivirusSpCas9HDRNoneLiu 2018Liu Zhang Wang Liu Ruan Loss miR-143 miR-145 condyloma acuminatum promotes cellular proliferation inhibits apoptosis targeting NRAS.R Soc 5: 172376Google Scholar30225000Adult knockoutFuGene HDCationic vectorSpCas9NHEJPuromycinSlivka 2019Slivka P.F. Hsieh C.L. Lipovsky Pratt S.D. Locklear Namovic M.T. al.Small molecule pooled screens investigating IL17 signaling BRD2 novel contributor inflammatory responses.ACS Chem 2019; 14: 857-872Google Scholar30938974Adult keratinocytesCRISPR screenlentiCRISPR v2LentivirusSpCas9NHEJPuromycin blasticidinHerter 2019Herter E.K. Toma M.A. Vij Visscher al.WAKMAR2, long noncoding downregulated chronic wounds, modulates motility production chemokines.J 139: 1373-1384Google Scholar30594489Adult activationLipofectamine 2000Cationic vectordCas9n/aFACSJozic 2019Jozic Sawaya A.P. Pastar Head C.R. Wong L.L. Glinos G.D. al.Pharmacological genetic inhibition Caveolin-1 epithelialization wound closure.Mol 27: 1992-2004Google Scholar31409528Adult vectorSpCas9NHEJPuromycinGrossi 2020Grossi Fenini Hennig Di Filippo Generation CRISPR/Cas9.Methods 2020; 2109: 125-145Google Scholar31502220Adult v2LentivirusSpCas9NHEJPuromycinImmortalized keratinocytesLiu 2016Liu Y.C. Cai Z.M. X.J. Reprogrammed conserved regions HPV6/11 E7 genes E7-transformed keratinocytes.Asian Androl. 475-479Google Scholar26228041HPV16-transformed foreskin knockoutLipofectamine vectorSpCas9NHEJPuromycinDahlhoff 2017Dahlhoff Gaborit Bultmann Leonhardt Yarden Schneider M.R. CRISPR-assisted receptor deletion distinct ERBB2 ERBB3 skin keratinocytes.FEBS Journal. 284: 3339-3349Google Scholar28805349HaCaT 3000Cationic vectorSpCas9NHEJFACSGao 2017Gao Z. W. Hu Chen lysine methyltransferase SMYD2 methylates domain BMPR2 bone morphogenetic signaling.J Biol Chem. 292: 12702-12712Google Scholar28588028HaCaT knockoutpLKO.1-puroLentivirus adenovirusSpCas9NHEJPuromycinSarkar 2018Sarkar M.K. Hile G.A. Tsoi L.C. Xing Liang al.Photosensitivity I IFN responses cutaneous lupus driven epidermal-derived interferon kappa.Ann Rheum Dis. 77: 1653-1664Google Scholar30021804N/TERT knockoutTransfeXCationic vectorpSpCas9NHEJGeneticinSwindell 2018Swindell W.R. Beamer Sarkar Loftus Fullmer al.RNA-seq analysis IL-1b IL-36 identifies shared MyD88-dependent signature.Front Immunol. 80Google Scholar29434599N/TERT vectorpSpCas9NHEJFACSTrothe 2018Trothe Ritzmann Lang Scholz Ü Pul Kaufmann al.Hypotonic stress response involves LRRC8A component volume-regulated anion channels.Exp 1352-1360Google Scholar30252954HaCaT adult knockoutAd5-CMV-Cas9 Ad5-U6-sgRNAAdenovirusSpCas9NHEJNoneBenati 2018Benati Miselli Cocchiarella Patrizi Carretero Baldassarri al.CRISPR/Cas9-mediated situ LAMB3 derived from junctional patient.Mol 26: 2592-2603Google Scholar30122422Immortalized JEB knockoutIDLVLentivirusSpCas9NHEJNoneChiang 2018Chiang Pauli Biryukov Feister K.F. Meng White E.A. papillomavirus E6 oncoprotein targets USP15 TRIM25 suppress RIG-I-mediated innate immune Virol. 92 (e01737–17)Google Scholar29263274HaCaT knockoutpSicoR-CRISPR-PuroRLentivirusSpCas9NHEJPuromycinSawatsubashi 2018Sawatsubashi Joko Fukumoto Matsumoto Sugano S.S. Development versatile joining-based knock-in module editing.Sci Rep. 593Google Scholar29330493HaCaT knockin/knockoutElectroporationPlasmid vectorSpCas9NHEJPuromycinSun 2018Sun Y.Z. Ren Y.J. Gao Y.L. al.DNAJA4 deficiency enhances NF-kappa B-related growth arrest induced hyperthermia keratinocytes.J Dermatol 256-267Google Scholar29807809HaCaT knockoutDNAJA4-gRNA-EGFP Cas9-puroLentivirusSpCas9NHEJPuromycinZhong 2018Zhong Bai Pang He Du al.Advancing predictivity sensitization applying HMOX1 reporter system.Arch Toxicol. 92: 3103-3115Google Scholar30132045HaCaT knockinGenJetCationic vectorSpCas9HDRGeneticinBaida 2018Baida Bhalla Yemelyanov Stechschulte L.A. Shou Readhead al.Deletion glucocorticoid chaperone FKBP51 prevents glucocorticoid-induced atrophy.Oncotarget. 34772-34783Google Scholar30410676HaCaT knockoutRNAi-MaxRNP complexSpCas9NHEJNoneBonafont 2019Bonafont Mencía Á. García Torres Rodríguez al.Clinically relevant dual sgRNA CRISPR/Cas9-mediated editing.Mol 986-998Google Scholar30930113Immortalized knockoutElectroporationRNP complexSpCas9NHEJNoneJames 2019James C.D. Prabhakar A.T. Otoa Evans Bristol al.SAMHD1 16-induced viral replication during differentiation keratinocytes.mSphere. 4 (e00448-19)Google Scholar31391281N/TERT v2LentivirusSpCas9NHEJPuromycinHatterschide 2019Hatterschide Bohidar Grace Nulton T.J. Kim H.W. Windle al.PTPN14 degradation high-risk limits contributes HPV-mediated oncogenesis.Proc 116: 7033-7042Google Scholar32581101Foreskin N/TERT-1 v2 pXPR_011LentivirusSpCas9NHEJPuromycin blasticidinChoi 2019Choi Park Lee J.W. Choi W.J. Establishment Nrf2-deficient HaCaT characterization their ROS-induced cytotoxicity.Toxicol Vitro. 61: 104602Google Scholar31319135HaCaT v2LentivirusSpCas9NHEJPuromycinStump 2020Stump Feehan R.P. Jordan Shantz L.M. Nowotarski S.L. Knocking down raptor affects ornithine decarboxylase post-transcriptional Manner following ultraviolet B exposure.Amino 52: 141-149Google Scholar30972602HaCaT v2LentivirusSpCas9NHEJPuromycinMuraguchi 2019Muraguchi Nanba Nishimura Tashiro IGF-1R disrupts homeostasis stem maintenance.J 94: 298-305Google Scholar31122679HaCaT knockoutTransIT-LT1Cationic vectorSpCas9NHEJFACSWalter 2019Walter Vielmuth Wanuske Seifert Pollmann Eming al.Role Dsg1- Dsg3-mediated pemphigus autoantibody-induced loss cohesion.Front 1128Google Scholar31178865HaCaT vectorSpCas9NHEJFACSHatterschide 2020Hatterschide Brantly A.C. Munger amino acid C terminus mediates binding PTPN14 repression epithelial differentiation.J : 94Google blasticidinCasares 2020Casares Garrido-Rodríguez Millán Collado García-Martín al.Cannabidiol antioxidant BACH1.Redox 28: 101321Google Scholar31518892HaCaT v2LentivirusSpCas9NHEJPuromycinGálvez 2020Gálvez Chacón-Solano Bonafont W.L. Murillas al.Efficient CRISPR-Cas9-Mediated ablation recapitulate genodermatoses: modeling netherton syndrome.Mol Methods Clin Dev. 280-290Google Scholar32637457Immortalized complexSpCas9NHEJNoEnjalbert 2020Enjalbert Dewan Caley M.P. Jones Morse Kelsell D.P. al.3D model harlequin ichthyosis therapeutic targets.J Invest. 130: 4798-4810Google Scholar32544098N/TERT 6 HiperFectCationic vectorSpCas9NHEJFACSKocher 2020Kocher March O.P. Bischof Liemberger Hainzl al.Predictable reframing bullosa.J 140: 1985-1993.e5Google Scholar32142798Immortalized RDEB activation complexSpCas9NHEJNoneDabelsteen 2020Dabelsteen Pallesen E.M.H. Marinova I.N. Nielsen M.I. Adamopoulou Rømer T.B. al.Essential glycans epithelia dissected CRISPR-Cas9-Engineered organotypic model.Dev 54: 669-684.e7Google Scholar32710848N/TERT blasticidinImahorn 2020Imahorn Aushev Herms Hoffmann Cichon Reichelt al.Gene stable complete CIB1 model.Sci 14952Google Scholar32917957Immortalized epidermolytic knockoutXfectCationic vectorSpCas9NHEJFACSJames 2020James Das Morgan E.L. Macdonald I.M. Werner syndrome (WRN) 16 life cycle differentiation.mSphere. (e00858–20)Google Scholar32938703N/TERT knockoutCalcium phosphate transfectionPlasmid vectorSpCas9NHEJPuromycinSobiak Leśniak, 2020Sobiak Leśniak Effect SUV39H1 histone differentiation-associated keratinocytes.Cells. 9Google Scholar33297464HaCaT vectorSpCas9NHEJFACSBonafont 2021Bonafont Srifa Vaidyanathan Romano al.Correction Recessive Dystrophic repair-mediated 29: 2008-2018Google Scholar33609734Immortalized complexSpCas9NHEJNoneAbboodi 2021Abboodi Buckhaults Altomare Hosseinipour Banister C.E. al.HPV-inactive populations arise HPV16-transformed p53 knockout.Virology. 554: 9-16Google Scholar33321328HPV16-transformed vectorSpCas9NHEJFACSWanuske 2021Wanuske Brantschen Schinner Stüdle Walter Hiermaier al.Clustering desmosomal cadherins desmoplakin cell-cell adhesion.Acta Physiol (Oxf). 231e13609Google Scholar33354837HaCaT vectorSpCas9NHEJFACSO'Keeffe Ahern 2021O'Keeffe Lara-Sáez Zhou al.Non-viral complexes targeted polymer system[e-pub ahead print].Gene (accessed November 12, 2021)https://doi.org/10.1038/s41434-021-00282-6Google Scholar34363036Immortalized 3000RNP complexSpCas9NHEJFACSEvrard 2021Evrard Faway De Vuyst Svensek O. Glas Bergerat TNFAIP6 demonstrates TSG-6 retain hyaluronan inside epidermis.JID Innov. 1-19Google Scholarn/aN/TERT complexSpCas9NHEJNoneKocher 2021Kocher Haas al.A non-viral selection-free HDR approach improved safety profile 237-250Google Scholar34458008Immortalized fibroblastsGene correctionElectroporationRNP complexSpCas9 Cas9nHDRNoneiPSCSebastiano 2014Sebastiano Zhen H.H. Haddad Bashkirova Melo S.P. al.Human COL7A1-corrected pluripotent treatment bullosa.Sci Transl Med. 6: 264ra163Google Scholar25429056Induced cell‒derived correctionElectroporationPlasmid vectorSpCas9HDRGeneticin ganciclovirWebber 2016Webber B.R. Osborn M.J. McElroy A.N. Twaroski Lonetree DeFeo al.CRISPR/Cas9-based bullosa.NPJ Regen 1Google Scholar28250968Induced cellsGene vectorhCas9HDRPuromycinShinkuma 2016Shinkuma Guo Christiano A.M. Site-specific bullosa.Proc 113: 5676-5681Google Scholar27143720Induced vectorSpCas9NHEJFACSJacków 2019Jacków Hansen Abaci H.E. Doucet Y.S. Shin J.U. iPS Google Scholar31818947Induced complexSpCas9HDRFACSItoh 2020Itoh Kawagoe Tamai Nakagawa Asahina Okano Footprint-free (iPSC) (RDEB) piggyBac transposon system.J 98: 163-172Google Scholar32376152Induced complexSpCas9HDRPuromycinAbbreviations: Cas9n, nickase; hCas9, codon optimized Cas9; repair; IDLV, integrase-deficient lentiviral particles; iPSC, cell; JEB, bullosa; joining; PMID, PubMed identifier; RDEB, RNP, Ribonucleoprotein; SpCas9, Cas9. Delivery machinery KCCationic vectors, adenoviral vectors mostly transducing specific gRNA. Lentiviral especially designed purpose, such lentiCRISPR deposited Feng Zhang's laboratory (Sanjana 2014Sanjana N.E. Shalem Improved genome-wide libraries screening.Nat 11: 783-784Google available Addgene (Watertown, MA) (plasmid #52961) easily amendable encode gRNA sequence(s) infection often very incorporation encoded infected cell's genome, causing permanent transfer–and also induction–of sequence. Consequently, constitutive increases risk off-target cleavage potentially leading unforeseen changes. addition, result rearrangements transgene silencing adenovirus lentiviruses preferred, adenoviruses do not integrate (Stephen 2010Stephen Montini Sivanandam V.G. Al-Dhalimy Kestler H.A. Finegold al.Chromosomal integration vector vivo.J 84: 9987-9994Google Both lentivirus induce strong immunogenic (Nayak Herzog, 2010Nayak Herzog R.W. Progress prospects: vectors.Gene 295-304Google Zaiss Muruve, 2008Zaiss A.K. Muruve D.A. Immunity adeno-associated virus animals humans: continued challenge.Gene 2008; 15: 808-816Google complicating suitability use. Therefore, (AAV) particles, show immunogenicity compared (Zaiss 2002Zaiss Q. Bowen G.P. N.C. Bartlett J.S. Differential vectors.J 2002; 76: 4580-4590Google might more suitable. Nevertheless, drawback AAV particles smaller loading capacity than lentiviruses, limit relatively large plasmids encoding gRNAs Cas9.Electroporation transfection gRNAs, plasmids, mRNA, ribonucleoprotein (RNP) complexes, nowadays (Table These methods easy highly (especially electroporation RNP complexes), transient effects.CRISPR-Cas9 KCsTo study function, biological processes, disease mechanisms, tissue culture models include healthy individuals taken excess removed surgical procedures. Genetic predispositions pathogenesis many diseases, fr

Language: Английский

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Lactate drives cellular DNA repair capacity: Role of lactate and related short-chain fatty acids in cervical cancer chemoresistance and viral infection

Frontiers in Cell and Developmental Biology, Journal Year: 2022, Volume and Issue: 10

Published: Oct. 19, 2022

The characteristic feature of a cancer microenvironment is the presence highly elevated concentration L-lactate in tumor niche. lactate-rich environment also maintained by commensal mucosal microbiota, which has immense potential for affecting cells through its receptoric and epigenetic modes action. Some these lactate activities might be associated with failure anticancer therapy as consequence drug resistance acquired cells. Upregulation cellular DNA repair capacity enhanced efflux are most important mechanisms that account ineffective radiotherapy drug-based therapies. Here, we present recent scientific knowledge on role HCA1 receptor intrinsic activity an HDAC inhibitor development therapy-resistant phenotype, special focus cervical In addition, study highlighted viable interactions between mammalian microorganisms female reproductive tract demonstrated interesting mechanism regulating efficacy retroviral transduction lactate-driven modulation DNA-PKcs localization. To date, very few studies have focused enhancement upregulation particular multidrug-resistance proteins respect to their intracellular regulatory triggered lactate. This review presents main achievements field impact cell biology may promote undesirable alterations physiology mitigate infections.

Language: Английский

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Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Nov. 15, 2023

We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the HPV16 life cycle. Here we demonstrate in HPV negative cervical cancer C33a cells human foreskin keratinocytes immortalized by (HFK+HPV16), recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required replication viral promotes phosphorylation of SAMHD1, which converts it from dNTPase an HR independent E6/E7 expression. A phosphor-mimic (SAMHD1 T592D) reduces mediated DNA has enhanced recruitment In HFK+HPV16 T592D attenuates cellular growth, but does not attenuate growth isogenic HFK alone. also development foci following keratinocyte differentiation. The results indicated could be therapeutically beneficial with genomes. Protein phosphatase 2A (PP2A) can dephosphorylate PP2A function inhibited endothall. endothall DNA, mimicking phenotypes. Finally, head neck cell lines episomal genomes their genome. suggest targeting phosphatases therapeutic potential treatment infections cancers.

Language: Английский

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E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 8, 2024

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ~5% all human cancers worldwide, including most cervical cancer cases and growing number ano-genital oral cancers. The HPV viral oncogenes, E6 E7, manipulate many host cellular pathways that promote cell proliferation survival, predisposing infected cells malignant transformation. Despite the availability highly effective vaccines, there still no specific anti-viral therapies targeting or treatments for HPV-associated As such, better understanding viral-host interactions may allow identification novel therapeutic targets. Here, we demonstrate actin-binding protein LASP1 is upregulated in significantly correlates with poorer overall survival. In positive cancer, depletion inhibited vitro, whilst having minimal effects negative cells. Furthermore, show SH3 domain essential LASP1-mediated these Mechanistically, E7 regulates at post-transcriptional level by repressing expression miR-203, which negatively regulated mRNA levels binding its 3'UTR. Finally, demonstrated required growth an vivo tumourigenicity model. Together, data induces survival role thus identifying potential target

Language: Английский

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Applications of CRISPR-Cas System in Tumor Biology

ONCOLOGIE, Journal Year: 2021, Volume and Issue: 23(4), P. 463 - 492

Published: Jan. 1, 2021

Language: Английский

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CRISPR/Cas9-Mediated Generation of COL7A1-Deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa.

PubMed, Journal Year: 2023, Volume and Issue: 25(10), P. 665 - 673

Published: Oct. 9, 2023

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering disease caused by mutations in the COL7A1 gene which responsible for coding type VII collagen. Investigating pathological mechanisms novel candidate therapies RDEB could be fostered new cellular models. The aim of this study was to employ CRISPR/Cas9 technology development immortalized COL7A1-deficient keratinocyte cell lines intended application as model ex vivo studies.In experimental study, we used transient transfection express -targeting guide RNA (gRNA) Cas9 HEK001 line followed enrichment with fluorescent-activated sorting (FACS) via GFP expressing cells (GFP+ HEK001). Homogenous single-cell clones were then isolated, genotyped, evaluated collagen expression. We performed scratch assay confirm functional effect knockout.We achieved 46.1% (P<0.001) efficiency in/del induction enriched transfected population. Except 4% single nucleotide insertions, remaining in/dels deletions different sizes. Out nine expanded clones, two homozygous heterozygous obtained defined mutation sequences. No off-target detected knockout lines. Immunostaining western blot analysis showed lack (COL7A1) protein expression these also that had higher motility compared their wild-type counterparts.We reported first isogenic provide useful culture investigate aspects biology potential therapeutic options.

Language: Английский

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A critical role for E2-p53 interaction during the HPV16 life cycle

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2021, Volume and Issue: unknown

Published: Nov. 2, 2021

Abstract Human papillomaviruses (HPV) are causative agents in ano-genital and oral cancers; HPV16 is the most prevalent type detected human cancers. The E6 protein targets p53 for proteasomal degradation to facilitate proliferation of infected cell. However, immortalized cells predominantly spliced (E6*) unable degrade p53. Here we demonstrate that foreskin keratinocytes by (HFK+HPV16), positive oropharyngeal cancers, retain significant expression In addition, levels can be increased HPV16+ head neck cancer cell lines following treatment with cisplatin. Introduction full-length into HFK+HPV16 resulted attenuation cellular growth (in hTERT HFK, promoted enhanced proliferation). An understudied interaction between E2 investigated whether this was important viral life cycle. We generated mutant genomes interact resulting profound phenotypes primary HFK. induced hyper-proliferation, but an ultimate arrest growth; β-galactosidase staining demonstrated senescence, COMET assays showed DNA damage compared wild cells. There failure cycle organotypic rafts HFK premature differentiation reduced proliferation. results indicate E2-p53 critical during cycle, disruption has anti-viral potential. discuss potential mechanisms explain these phenotypes. Importance around 5% all currently no antivirals available combat infections therefore it remains a priority enhance our understanding HPV replication/transcription/segregation factor tumor suppressor p53, role demonstrates why case. If disrupted then fail proliferate, have there Results suggest targeting would therapeutic benefits, potentially attenuating spread HPV16.

Language: Английский

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