Novel diagnosis and therapy for hepatoma targeting HBV-related carcinogenesis through alternative splicing of FIR (PUF60)/FIRΔexon2

Abstract

Aim: Disturbed alternative splicing of far upstream element-binding protein-interacting repressor (FIR) was found to be unable to repress c-Myc transcription and so it might be important for suppressing tumor development. FIR is a splicing variant of poly (U)-binding-splicing factor (PUF60), and forms complex with other splicing factors. FIR/PUF60 is a splicing factor of U2 small nuclear ribonucleoprotein auxiliary factor family, Thus FIR/PUF60 is a multifunctional protein. The expression of exon2-lacking splicing variant of FIR, FIRΔexon2, is elevated in many cancer tissues and promotes tumor development by disabling FIR-repression to sustain c-Myc activation. FIRΔexon2, as a dominant negative of FIR, opposed apoptosis in cancer cells. FIR/FIRΔexon2 interacts with degron pocket of F-box and W (Typ) D (Asp) repeat domain-containing 7 and inhibits proteolysis of substrates proteins. Recently, FIR/PUF60 was identified as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) pregenomic RNA (pgRNA) expression.

Methods: Small molecular chemical compounds against FIR and FIRΔexon2 were screened among 2,3275 chemicals by natural product depository array (RIKEN, Wako, Saitama, Japan).

Results: Nine chemicals against FIR and four chemicals against FIRΔexon2 were identified as candidates of interacting chemicals. Interestingly, BK697 contains WD -like structure. Among them, BK697 against FIRΔexon2 inhibited hepatoma cell growth.

Conclusion: Therefore, FIR (PUF60)/FIRΔexon2 is multifunctional and applicable for clinical use for HBV suppression and hepatoma treatment. Together, one clue to the development of hepatome diagnosis and therapies directed against FIR/FIRΔexon2/PUF60 with small molecular weight chemicals that inhibit HBV cccDNA replication.

Keywords

Hepatocellular carcinoma / hepatitis B virus / covalently closed circular DNA / far upstream element-binding protein-interacting repressor / poly (U)-binding-splicing factor / F-box and W (Typ) D (Asp) repeat domain-containing 7 / natural product depository array / U2AF homology motif / U2AF homology motif ligand motif

References

[1] Matsushita K,Kajiwara T,Itoga S,Kubo S,Matsubara H.c-Myc suppressor FBP-interacting repressor for cancer diagnosis and therapy.Front Biosci (Landmark Ed)2009;1:3401-8

[2] Pelengaris S,Evan GI.Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenicproperties of Myc and triggers carcinogenic progression.Cell2002;109:321-34

[3] Bazar L,Harris V,Levens D.Targeted melting and binding of a DNA regulatory element by a transactivator of c-Myc.J Biol Chem1995;270:8241-8

[4] Michelotti GA,Pullner A,Eick D.Multiple single-stranded cis elements are associated with activated chromatin of the human c-Myc gene in vivo.Mol Cell Biol1996;16:2656-69 PMCID:PMC231256

[5] Zubaidah RM,Tan SB,Lin Q.2-D DIGE profiling of hepatocellular carcinoma tissues identified isoforms of far upstream binding protein (FUBP) as novel candidates in liver carcinogenesis.Proteomics2008;8:5086-96

[6] Malz M,Singer S,Bissinger M,Longerich T,Vogel A,Schirmacher P.Overexpression of far upstream element binding proteins: a mechanism regulating proliferation and migration in liver cancer cells.Hepatology2009;50:1130-9

[7] Quinn LM.FUBP/KH domain proteins in transcription: back to the future.Transcription2017;8:185-92 PMCID:PMC5501386

[8] Liu J,Weber A,Chuikov S,Wang XW,Harris CC,Reinberg D.Defective interplay of activators and repressors with TFIH in xeroderma pigmentosum.Cell2001;104:353-63

[9] Kitamura A,Takiguchi Y,Tada Y,Hiroshima K,Tomonaga T,Inoue M,Sato Y,Tatsumi K.Synergistic effect of non-transmissible Sendai virus vector encoding the c-Myc suppressor FUSE-binding protein-interacting repressor plus cisplatin in the treatment of malignant pleural mesothelioma.Cancer Sci2011;102:1366-73

[10] Kano M,Rahmutulla B,Shimada H,Hiwasa T,Nomura F.Adenovirus-mediated FIR demonstrated TP53-independent cell-killing effect and enhanced antitumor activity of carbon-ion beams.Gene Ther2016;23:50-6

[11] Mizokami D,Tanaka N,Tomifuji M,Matsushita K,Shiotani A.Tacrolimus prevents laryngotracheal stenosis in an acute-injury rat model.Laryngoscope2015;125:E210-5

[12] Matsushita K,Ueda Y,Hasegawa M,Matsubara H.Non-transmissible Sendai virus vector encoding c-Myc suppressor FBP-interacting repressor for cancer therapy.World J of Gastroenterol2014;20:4316-28 PMCID:PMC3989966

[13] Mizokami D,Tanaka N,Tomifuji M,Ueda Y,Matsushita K.Gene therapy of c-Myc suppressor FUSE-binding protein-interacting repressor by Sendai virus delivery prevents tracheal stenosis.PloS One2015;

[14] Tanaka N,Mizokami D,Yamashita T,Ueda Y,Matsushita K,Shimada H.Sendai virus-mediated gene transfer of the c-Myc suppressor far-upstream element-binding protein-interacting repressor suppresses head and neck cancer.Gene Ther2015;22:297-304

[15] Black DL.Mechanisms of alternative pre-messenger RNA splicing.Annu Rev Biochem2003;72:291-336

[16] Matsushita K,Shimada H,Hayashi H,Komatsu A,Ochiai T.Strong HLA-DR antigen expression on cancer cells relates to better prognosis of colorectal cancer patients: possible involvement of c-Myc suppression by interferon-gamma in situ.Cancer Sci2006;97:57-63

[17] Duriez M,Lekbaby B,Schnuriger A,Guerrera CI,Fauveau V,Quetier I,Gourari S,Gill U,Debzi N,Maini MK,Soussan P.Alternative splicing of hepatitis B virus: a novel virus/host interaction altering liver immunity.J Hepatol2017;67:687-99

[18] Rahmutulla B,Satoh M,Tsuchida S,Shimada H,Miyazaki M.Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway.Oncotarget2014;5:2404-17 PMCID:PMC4058014

[19] Rahmutulla B,Nomura F.Alternative splicing of DNA damage response genes and gastrointestinal cancers.World J of Gastroenterol2014;20:17305-13 PMCID:PMC4265588

[20] Khageh Hosseini S,Steiner M,Gerlach K,Waidmann O,Schulze JO,Steinhilber D,Tampé R,Proschak E.Camptothecin and its analog SN-38, the active metabolite of irinotecan, inhibit binding of the transcriptional regulator and oncoprotein FUBP1 to its DNA target sequence FUSE.Biochem Pharmacol2017;146:53-62

[21] Brinkman BM.Splice variants as cancer biomarkers.Clin Biochem2004;37:584-94

[22] Matsushita K,Shimada H,Higashi M,Harigaya K,Libutti D,Ochiai T.An essential role of alternative splicing of c-Myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis.Cancer Res2006;66:1409-17

[23] Hastings ML,Duelli DM,Krainer AR.Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF(65).PLoS One2007;2:e538 PMCID:PMC1888729

[24] Page-McCaw PS,Sharp PA.PUF60: a novel U2AF65-related splicing activity.RNA1999;5:1548-60 PMCID:PMC1369877

[25] Zhang YM,Shi JL,Shu XM,Wang GC.The prevalence and clinical significance of anti-PUF60 antibodies in patients with idiopathic inflammatory myopathy.Clin Rheumatol2018;37:1573-80

[26] Fiorentino DF,Baer AN,Rieger KE,Rosen A,Christopher-Stine L.PUF60: a prominent new target of the autoimmune response in dermatomyositis and Sjögren's syndrome.Ann Rheum Dis2016;75:1145-51 PMCID:PMC4828328

[27] Kobayashi S,Hiwasa T,Rahmutulla B,Komukai Y,Matsubara H,Nomura F.Anti-FIRs (PUF60) auto-antibodies are detected in the sera of early-stage colon cancer patients.Oncotarget2016;7:82493-503 PMCID:PMC5347708

[28] Kobayashi S,Arasawa T,Ishii S,Ito M,Kano M,Kitamura K,Shin H,Nomura F,Matsushita K.Identification of specific and common diagnostic antibody markers for gastrointestinal cancers by SEREX screening using testis cDNA phage library.Oncotarget2018;9:18559-69 PMCID:PMC5915093

[29] Matsushita K,Tanaka N,Matsubara H,Levens D,Liu J,Nomura F.Interactions between SAP155 and FUSE-binding protein-interacting repressor bridges c-Myc and P27Kip1 expression.Mol Cancer Res2013;11:689-98

[30] Kajiwara T,Itoga S,Tanaka N,Matsubara H,Habara Y,Nomura F.SAP155-mediated c-Myc suppressor far-upstream element-binding protein-interacting repressor splicing variants are activated in colon cancer tissues.Cancer Sci2013;104:149-56

[31] Matsushita K,Tamura M,Tanaka N,Matsubara H,Yoshimoto R,Kubo S,Levens D,Nomura F.SAP155-mediated splicing of FUSE-binding protein-interacting repressor serves as a molecular switch for c-Myc gene expression.Mol Cancer Res2012;10:787-99

[32] Kotake Y,Owa T,Shimizu H,Ishihama Y,Mizui Y.Splicing factor SF3b as a target of the antitumor natural product pladienolide.Nat Chem Biol2007;3:570-5

[33] Kaida D,Tashiro E,Hagiwara M,Watanabe H,Yoshida T,Tani T,Yoshida M.Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA.Nat Chem Biol2007;3:576-83

[34] Sun S,Ito M,Chida T,Watashi K,Wakita T.Involvement of PUF60 in transcriptional and post-transcriptional regulation of hepatitis B virus pregenomic RNA expression.Sci Rep2017;7:12874 PMCID:PMC5634508

[35] Kanoh N,Simizu S,Hatakeyama S,Osada H.Immobilization of natural products on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions.Angew Chem Int Ed Engl2003;42:5584-7

[36] Osada H.Introduction of new tools for chemical biology research on microbial metabolites.Biosci Biotechnol Biochem2010;74:1135-40

[37] Kawatani M,Kondoh Y,Sekine T,Taniguchi N.Identification of matrix metalloproteinase inhibitors by chemical arrays.Biosci Biotechnol Biochem2015;79:1597-602

[38] Malz M,Samarin J,Sticht C,Roessler S,Renner M,Singer S,Weber A,Zörnig M,Breuhahn K.Overexpression of far upstream element (FUSE) binding protein (FBP)-interacting repressor (FIR) supports growth of hepatocellular carcinoma.Hepatology2014;60:1241-50

[39] Loerch S.Unmasking the U2AF homology motif family: a bona fide protein-protein interaction motif in disguise.RNA2016;22:1795-807 PMCID:PMC5113200

[40] Stepanyuk GA,Peralta E,Axelrod HL,Das D,Jaroszewski L,Lesley SA,Godzik A,Wüthrich K,Williamson JR.UHM-ULM interactions in the RBM39-U2AF65 splicing-factor complex.Acta Crystallogr D Struct Biol2016;72:497-511 PMCID:PMC4822562

[41] Loerch S,Manceau V,Kielkopf CL.Cancer-relevant splicing factor CAPERα engages the essential splicing factor SF3b155 in a specific ternary complex.J Biol Chem2014;289:17325-37 PMCID:PMC4067167

[42] Corsini L,Stier G,Scheffzek K,Sattler M.Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60.J Biol Chem2009;284:630-9

[43] Matsushita K,Rahmutulla B,Ishige T,Hoshino T,Mori T,Shimada H,Kito M,Kubo S,Hatano M,Matsuo M,Kaneda A,Nomura F.Haploinsufficiency of the c-Myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53- dependent T-cell acute lymphoblastic leukemia progression by activating Notch1.Oncotarget2015;6:5102-17 PMCID:PMC4467136

[44] Wang Z,Fukushima H,Gao D,Sarkar FH.Emerging roles of the FBW7 tumour suppressor in stem cell differentiation.EMBO Rep2011;13:36-43 PMCID:PMC3246256

[45] Wang Z,Zhong J,Fukushima H,Wei W.Tumor suppressor functions of FBW7 in cancer development and progression.FEBS Lett2012;586:1409-18 PMCID:PMC3372850

[46] Ogura Y,Tanaka N,Kobayashi S,Rahmutulla B,Murakami K,Nomura F,Matsubara H.Disturbed alternative splicing of FIR (PUF60) directed cyclin E overexpression in esophageal cancers.Oncotarget2018;9:22929-44 PMCID:PMC5955432

[47] Yanagita H,Matsumoto K,Takaesu Y,Murakami T,Chiba J,Hoshino T.Structural and biochemical study on the inhibitory activity of derivatives of 5-nitro-furan-2-carboxylic acid for RNase H function of HIV-1 reverse transcriptase.Bioorg Med Chem2011;19:816-25

[48] Yanagita H,Urano E,Ogata M,Murakami T,Chiba J,Hoshino T.Structural modulation study of inhibitory compounds for ribonuclease H activity of human immunodeficiency virus type 1 reverse transcriptase.Chem Pharm Bull (Tokyo)2012;60:764-71

[49] Fudo S,Nukaga M,Tashiro M,Hoshino T.Structural and computational study on inhibitory compounds for endonuclease activity of influenza virus polymerase.Bioorg Med Chem2015;23:5466-75

[50] Kimura A,Ailiken G,Minamoto T,Nomura F.FIR haplodeficiency promotes splicing to pyruvate kinase M2 in mice thymic lymphoma tissues revealed by six-plex tandem mass tag quantitative proteomic analysis.Oncotarget2017;8:67955-65 PMCID:PMC5620227

[51] Dayton TL,Miller KM,Bhutkar A,Davidson SM,Bronson RT,Vander Heiden MG.Germline loss of PKM2 promotes metabolic distress and hepatocellular carcinoma.Genes Dev2016;30:1020-33 PMCID:PMC4863734

[52] Gumireddy K,Yan J,Johannes GJ,Tchou J,Zhang L,Calin GA.Identification of a long non-coding RNA-associated RNP complex regulating metastasis at the translational step.EMBO J2013;32:2672-84 PMCID:PMC3801433

[53] Bowler E,Uzor S,Durand M,Rouschop KMA,Wilson I.Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells.BMC Cancer2018;18:355 PMCID:PMC5879922

[54] Lamontagne RJ,Bouchard MJ.Hepatitis B virus molecular biology and pathogenesis.Hepatoma Res2016;2:163-86 PMCID:PMC5198785

[55] Dixit U,Liu Z,Neiditch MB,Pandey VN.FUSE binding protein 1 facilitates persistent hepatitis C virus replication in hepatoma cells by regulating tumor suppressor p53.J Virol2015;89:7905-21 PMCID:PMC4505638

[56] Samarin J,Malz M,Stein I,Singer S,Cigliano A,Falk CS,Dooley S,Calvisi DF,Schirmacher P.PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells.Hepatology2016;63:813-26 PMCID:PMC5262441

[57] Rabenhorst U,Brezniceanu ML,Devens F,Rieker RJ,Chung HJ,Zörnig M.Overexpression of the far upstream element binding protein 1 in hepatocellular carcinoma is required for tumor growth.Hepatology2009;50:1121-9 PMCID:PMC3474328

[58] Moccia A,Skidmore JM,Wheeler M,Nickerson D,Hefner MA,Bielas SL.Genetic analysis of CHARGE syndrome identifies overlapping molecular biology.Genet Med2018;doi:10

[59] Zhao JJ,Zander CS,Georgii-Hemming P,Brandberg G,Frykholm C,Thuresson AC.Exome sequencing reveals NAA15 and PUF60 as candidate genes associated with intellectual disability.Am J Med Genet B Neuropsychiatr Genet2018;177:10-20 PMCID:PMC5765476

[60] Low KJ,Abou Jamra R,El Chehadeh S,Greenslade M,Hurst J,Kuentz P,Roessler F,Schneider MC,Tatton-Brown K,Vigeland MD,Willems M,Study DD.PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features.Eur J Hum Genet2017;25:552-9 PMCID:PMC5392357

[61] Santos-Simarro F,Del Pozo A,Silla JC,Nevado J,Monta-o VEF,Alba Valdivia LI,Lapunzina P.Eye coloboma and complex cardiac malformations belong to the clinical spectrum of PUF60 variants.Clin Genet2017;92:350-1

[62] Graziano C,Severi G,Wischmeijer A,Seri M.A de novo PUF60 mutation in a child with a syndromic form of coloboma and persistent fetal vasculature.Ophthalmic Genet2017;38:590-2

[63] Wells C,Malan V,Attie-Bitach T,Vekemans M,Romana S.First fetal case of the 8q24.3 contiguous genes syndrome. Am J Med Genet A2016;170A:239-42

[64] Dauber A,Guenot C,Kibaek M,Leheup B,Nowaczyk MJ,Zeesman S,Beckmann JS,Hastings ML,Katsanis N.SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant. Am J Hum Genet2013;93:798-811 PMCID:PMC3824129

[65] Královicová J,Stejskalová E,Hiller M,Vorechovsky I.PUF60-activated exons uncover altered 3′ splice-site selection by germline missense mutations in a single RRM.Nucleic Acids Res2018;46:6166-87 PMCID:PMC6093180