Abstract
Here, we used codon usage technology to generate two codon-modified human papillomavirus (HPV)16 E7 genes and, together with wild-type E7, to construct three HPV16 E7 gene plasmids: Wt-E7, HB1-E7, and HB2-E7. The three HPV 16 E7 plasmids were used to investigate how HPV16 E7 protein was expressed in different cells and how this oncoprotein deregulated cellular and molecular events in human keratinocytes to induce carcinogenesis. We discovered that codon usage of HPV16 E7 gene played a key role in determining expression of E7 oncoprotein in all tested cells. HPV16 E7 inhibited significantly expression of pRb to impair keratinocyte differentiation and disrupted development of skin epidermis in mice. HPV16 E7 increased substantially the number of G0/G1 cells associated with upregulation of cyclin D2 and downregulation of cyclin B1 in keratinocytes. HPV16 E7 not only inhibited expression of involucrin and α-spectrin but also disrupted the organization of involucrin filaments and spectrin cytoskeleton. Furthermore, HPV16 E7 inhibited expression of β-adducin, destroyed its cytoskeletal structure and induced phosphorylation of β-adducin(Ser662) in keratinocytes. Importantly, HPV16 E7 induced carcinogenesis in mice associated with expression of phosphorylated β-adducin(Ser662) and its nucleus-translocation. In conclusion, we provided evidence that HPV16 E7 oncoprotein inhibited keratinocyte differentiation in vitro and in vivo leading to carcinogenesis through cell cycle arrest and disruption of pRb/involucrin/spectrin/adducin cascade.
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References
Aylon Y, Oren M (2011) New plays in the p53 theater. Curr Opin Genet Dev 21(1):86–92. https://doi.org/10.1016/j.gde.2010.10.002
Blain SW (2008) Switching cyclin D-Cdk4 kinase activity on and off. Cell Cycle 7(7):892–898
Bodily JM, Hennigan C, Wrobel GA, Rodriguez CM (2013) Regulation of the human papillomavirus type 16 late promoter by E7 and the cell cycle. Virology 443(1):11–19. https://doi.org/10.1016/j.virol.2013.04.033
Boyer SN, Wazer DE, Band V (1996) E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res 56(20):4620–4624
Carvajal LA, Manfredi JJ (2013) Another fork in the road--life or death decisions by the tumour suppressor p53. EMBO Rep 14(5):414–421. https://doi.org/10.1038/embor.2013.25
Chen CL, Lin YP, Lai YC, Chen HC (2011) alpha-Adducin translocates to the nucleus upon loss of cell-cell adhesions. Traffic 12(10):1327–1340. https://doi.org/10.1111/j.1600-0854.2011.01245.x
Chen J, Shukla V, Farci P, Andricovich J, Jogunoori W, Kwong LN, Katz LH, Shetty K, Rashid A, Su X, White J, Li L, Wang AY, Blechacz B, Raju GS, Davila M, Nguyen BN, Stroehlein JR, Chen J, Kim SS, Levin H, Machida K, Tsukamoto H, Michaely P, Tzatsos A, Mishra B, Amdur R, Mishra L (2017) Loss of the transforming growth factor-beta effector beta2-Spectrin promotes genomic instability. Hepatology 65(2):678–693. https://doi.org/10.1002/hep.28927
Cid-Arregui A, Juarez V, zur Hausen H (2003) A synthetic E7 gene of human papillomavirus type 16 that yields enhanced expression of the protein in mammalian cells and is useful for DNA immunization studies. J Virol 77(8):4928–4937. https://doi.org/10.1128/jvi.77.8.4928-4937.2003
de Giorgi V, Sestini S, Massi D, Ghersetich I, Lotti T (2007) Keratinocyte growth factor receptors. Dermatol Clin 25(4):477–485, vii. https://doi.org/10.1016/j.det.2007.06.017
Deml L, Bojak A, Steck S, Graf M, Wild J, Schirmbeck R, Wolf H, Wagner R (2001) Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol 75(22):10991–11001. https://doi.org/10.1128/JVI.75.22.10991-11001.2001
Ding J, Doorbar J, Li B, Zhou F, Gu W, Zhao L, Saunders NA, Frazer IH, Zhao KN (2010) Expression of papillomavirus L1 proteins regulated by authentic gene codon usage is favoured in G2/M-like cells in differentiating keratinocytes. Virology 399(1):46–58. https://doi.org/10.1016/j.virol.2009.12.021
Doorbar J (2005) The papillomavirus life cycle. J Clin Virol 32(Suppl 1):S7–S15. https://doi.org/10.1016/j.jcv.2004.12.006
Doorbar J (2006) Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond) 110(5):525–541. https://doi.org/10.1042/CS20050369
Fang NX, Gu W, Ding J, Saunders NA, Frazer IH, Zhao KN (2007) Calcium enhances mouse keratinocyte differentiation in vitro to differentially regulate expression of papillomavirus authentic and codon modified L1 genes. Virology 365(1):187–197. https://doi.org/10.1016/j.virol.2007.03.038
Fernando GJ, Stewart TJ, Tindle RW, Frazer IH (1998) Th2-type CD4+ cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model. J Immunol 161(5):2421–2427
Fletcher GC, Elbediwy A, Khanal I, Ribeiro PS, Tapon N, Thompson BJ (2015) The Spectrin cytoskeleton regulates the Hippo signalling pathway. EMBO J 34(7):940–954. https://doi.org/10.15252/embj.201489642
Fowler L, Dong L, Bowes RC 3rd, van de Water B, Stevens JL, Jaken S (1998a) Transformation-sensitive changes in expression, localization, and phosphorylation of adducins in renal proximal tubule epithelial cells. Cell Growth Differ 9(2):177–184
Fowler L, Everitt J, Stevens JL, Jaken S (1998b) Redistribution and enhanced protein kinase C-mediated phosphorylation of alpha- and gamma-adducin during renal tumor progression. Cell Growth Differ 9(5):405–413
Freije A, Molinuevo R, Ceballos L, Cagigas M, Alonso-Lecue P, Rodriguez R, Menendez P, Aberdam D, De Diego E, Gandarillas A (2014) Inactivation of p53 in human keratinocytes leads to squamous differentiation and shedding via replication stress and mitotic slippage. Cell Rep 9(4):1349–1360. https://doi.org/10.1016/j.celrep.2014.10.012
Fu B, Quintero J, Baker CC (2003) Keratinocyte growth conditions modulate telomerase expression, senescence, and immortalization by human papillomavirus type 16 E6 and E7 oncogenes. Cancer Res 63(22):7815–7824
Fuchs E (2007) Scratching the surface of skin development. Nature 445(7130):834–842. https://doi.org/10.1038/nature05659
Gu W, Ding J, Wang X, de Kluyver RL, Saunders NA, Frazer IH, Zhao KN (2007) Generalized substitution of isoencoding codons shortens the duration of papillomavirus L1 protein expression in transiently gene-transfected keratinocytes due to cell differentiation. Nucleic Acids Res 35(14):4820–4832. https://doi.org/10.1093/nar/gkm496
Gu W, Li M, Zhao WM, Fang NX, Bu S, Frazer IH, Zhao KN (2004) tRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes. Nucleic Acids Res 32(15):4448–4461. https://doi.org/10.1093/nar/gkh748
Gyongyosi E, Szalmas A, Ferenczi A, Konya J, Gergely L, Veress G (2012) Effects of human papillomavirus (HPV) type 16 oncoproteins on the expression of involucrin in human keratinocytes. Virol J 9:36. https://doi.org/10.1186/1743-422X-9-36
Hatterschide J, Bohidar AE, Grace M, Nulton TJ, Kim HW, Windle B, Morgan IM, Munger K, White EA (2019) PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis. Proc Natl Acad Sci U S A 116(14):7033–7042. https://doi.org/10.1073/pnas.1819534116
Hebner CM, Wilson R, Rader J, Bidder M, Laimins LA (2006) Human papillomaviruses target the double-stranded RNA protein kinase pathway. J Gen Virol 87(Pt 11):3183–3193. https://doi.org/10.1099/vir.0.82098-0
Kalmodia S, Parameswaran S, Ganapathy K, Yang W, Barrow CJ, Kanwar JR, Roy K, Vasudevan M, Kulkarni K, Elchuri SV, Krishnakumar S (2017) Characterization and molecular mechanism of peptide-conjugated gold nanoparticle inhibiting p53-HDM2 interaction in retinoblastoma. Mol Ther Nucleic Acids 9:349–364. https://doi.org/10.1016/j.omtn.2017.10.012
Khanna MR, Mattie FJ, Browder KC, Radyk MD, Crilly SE, Bakerink KJ, Harper SL, Speicher DW, Thomas GH (2015) Spectrin tetramer formation is not required for viable development in Drosophila. J Biol Chem 290(2):706–715. https://doi.org/10.1074/jbc.M114.615427
Lan YJ, Chen H, Chen JQ, Lei QH, Zheng M, Shao ZR (2014) Immunolocalization of vimentin, keratin 17, Ki-67, involucrin, beta-catenin and E-cadherin in cutaneous squamous cell carcinoma. Pathol Oncol Res 20(2):263–266. https://doi.org/10.1007/s12253-013-9690-5
Leask A, Rosenberg M, Vassar R, Fuchs E (1990) Regulation of a human epidermal keratin gene: sequences and nuclear factors involved in keratinocyte-specific transcription. Genes Dev 4(11):1985–1998
Leder C, Kleinschmidt JA, Wiethe C, Muller M (2001) Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes. J Virol 75(19):9201–9209. https://doi.org/10.1128/JVI.75.19.9201-9209.2001
Leinonen PT, Hagg PM, Peltonen S, Jouhilahti EM, Melkko J, Korkiamaki T, Oikarinen A, Peltonen J (2009) Reevaluation of the normal epidermal calcium gradient, and analysis of calcium levels and ATP receptors in Hailey-Hailey and Darier epidermis. J Investigat Dermatol 129(6):1379–1387. https://doi.org/10.1038/jid.2008.381
Li B, Wang X, Zhou F, Saunders NA, Frazer IH, Zhao KN (2008) Up-regulated expression of Sp1 protein coincident with a viral protein in human and mouse differentiating keratinocytes may act as a cell differentiation marker. Differentiation 76(10):1068–1080. https://doi.org/10.1111/j.1432-0436.2008.00300.x
Lim JA, Baek HJ, Jang MS, Choi EK, Lee YM, Lee SJ, Lim SC, Kim JY, Kim TH, Kim HS, Mishra L, Kim SS (2014) Loss of beta2-spectrin prevents cardiomyocyte differentiation and heart development. Cardiovasc Res 101(1):39–47. https://doi.org/10.1093/cvr/cvt222
Liu W, Gao F, Zhao KN, Zhao W, Fernando GJ, Thomas R, Frazer IH (2002) Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity. Virology 301(1):43–52. https://doi.org/10.1006/viro.2002.1584
Liu WJ, Zhao KN, Gao FG, Leggatt GR, Fernando GJ, Frazer IH (2001) Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy. Vaccine 20(5–6):862–869. https://doi.org/10.1016/s0264-410x(01)00406-6
Macurek L, Lindqvist A, Medema RH (2009) Aurora-A and hBora join the game of Polo. Cancer Res 69(11):4555–4558 doi:69/11/4555 [pii]. https://doi.org/10.1158/0008-5472.CAN-09-0142
Matsuoka Y, Li X, Bennett V (2000) Adducin: structure, function and regulation. Cell Mol Life Sci 57(6):884–895. https://doi.org/10.1007/PL00000731
Metral S, Machnicka B, Bigot S, Colin Y, Dhermy D, Lecomte MC (2009) AlphaII-spectrin is critical for cell adhesion and cell cycle. J Biol Chem 284(4):2409–2418 doi:M801324200 [pii]. https://doi.org/10.1074/jbc.M801324200
Pickard A, Wong PP, McCance DJ (2010) Acetylation of Rb by PCAF is required for nuclear localization and keratinocyte differentiation. J Cell Sci 123(Pt 21):3718–3726. https://doi.org/10.1242/jcs.068924
Scoles DR, Huynh DP, Morcos PA, Coulsell ER, Robinson NG, Tamanoi F, Pulst SM (1998) Neurofibromatosis 2 tumour suppressor schwannomin interacts with betaII-spectrin. Nat Genet 18(4):354–359
Sevilla LM, Nachat R, Groot KR, Klement JF, Uitto J, Djian P, Maatta A, Watt FM (2007) Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier. J Cell Biol 179(7):1599–1612. https://doi.org/10.1083/jcb.200706187
Shen N, Liu C, Li J, Chen X, Yang Y, Zhu Y, Gong Y, Gong J, Zhong R, Cheng L, Miao X (2015) A phosphorylation-related variant ADD1-rs4963 modifies the risk of colorectal cancer. PLoS One 10(3):e0121485. https://doi.org/10.1371/journal.pone.0121485
Tindle RW, Croft S, Herd K, Malcolm K, Geczy AF, Stewart T, Fernando GJ (1995) A vaccine conjugate of ‘ISCAR’ immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants. Clin Exp Immunol 101(2):265–271. https://doi.org/10.1111/j.1365-2249.1995.tb08349.x
Tseng YH, Chang CS, Liu TY, Kao SY, Chang KW, Lin SC (2007) Areca nut extract treatment down-regulates involucrin in normal human oral keratinocyte through P13K/AKT activation. Oral Oncol 43(7):670–679. https://doi.org/10.1016/j.oraloncology.2006.08.003
Tuominen H, Sormunen R, Kallioinen M (1996) Non-erythroid spectrin (fodrin) in cutaneous tumours: diminished in cell membranes, increased in the cytoplasm. Br J Dermatol 135(4):576–580
von Harsdorf R, Hauck L, Mehrhof F, Wegenka U, Cardoso MC, Dietz R (1999) E2F-1 overexpression in cardiomyocytes induces downregulation of p21CIP1 and p27KIP1 and release of active cyclin-dependent kinases in the presence of insulin-like growth factor I. Circ Res 85(2):128–136. https://doi.org/10.1161/01.res.85.2.128
White EA, Munger K (2017) Crowd control: E7 conservation is the key to cancer. Cell 170(6):1057–1059. https://doi.org/10.1016/j.cell.2017.08.033
Woodworth CD, Wang H, Simpson S, Alvarez-Salas LM, Notario V (1993) Overexpression of wild-type p53 alters growth and differentiation of normal human keratinocytes but not human papillomavirus-expressing cell lines. Cell Growth Differ 4(5):367–376
Wu J, Masci PP, Chen C, Chen J, Lavin MF, Zhao KN (2015) beta-Adducin siRNA disruption of the spectrin-based cytoskeleton in differentiating keratinocytes prevented by calcium acting through calmodulin/epidermal growth factor receptor/cadherin pathway. Cell Signal 27(1):15–25. https://doi.org/10.1016/j.cellsig.2014.10.001
Xu XC, Mitchell MF, Silva E, Jetten A, Lotan R (1999) Decreased expression of retinoic acid receptors, transforming growth factor beta, involucrin, and cornifin in cervical intraepithelial neoplasia. Clin Cancer Res 5(6):1503–1508
Yun HY, Kim MW, Lee HS, Kim W, Shin JH, Kim H, Shin HC, Park H, Oh BH, Kim WK, Bae KH, Lee SC, Lee EW, Ku B, Kim SJ (2019) Structural basis for recognition of the tumor suppressor protein PTPN14 by the oncoprotein E7 of human papillomavirus. PLoS Biol 17(7):e3000367. https://doi.org/10.1371/journal.pbio.3000367
Zhao KN, Gu W, Fang NX, Saunders NA, Frazer IH (2005) Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo. Mol Cell Biol 25(19):8643–8655. https://doi.org/10.1128/MCB.25.19.8643-8655.2005
Zhao KN, Liu WJ, Frazer IH (2003) Codon usage bias and A+T content variation in human papillomavirus genomes. Virus Res 98(2):95–104
Zhao KN, Masci PP, Chen J, Lavin MF (2013) Calcium prevents retinoic acid-induced disruption of the spectrin-based cytoskeleton in keratinocytes through the Src/PI3K-p85alpha/AKT/PKCdelta/beta-adducin pathways. Cell Calcium 54(3):151–162. https://doi.org/10.1016/j.ceca.2013.05.009
Zhao KN, Masci PP, Lavin MF (2011) Disruption of spectrin-like cytoskeleton in differentiating keratinocytes by PKCdelta activation is associated with phosphorylated adducin. PLoS One 6(12):e28267. https://doi.org/10.1371/journal.pone.0028267
Zhi X, Lin L, Yang S, Bhuvaneshwar K, Wang H, Gusev Y, Lee MH, Kallakury B, Shivapurkar N, Cahn K, Tian X, Marshall JL, Byers SW, He AR (2015) betaII-Spectrin (SPTBN1) suppresses progression of hepatocellular carcinoma and Wnt signaling by regulation of Wnt inhibitor kallistatin. Hepatology 61(2):598–612. https://doi.org/10.1002/hep.27558
Zhou J, Liu WJ, Peng SW, Sun XY, Frazer I (1999) Papillomavirus capsid protein expression level depends on the match between codon usage and tRNA availability. J Virol 73(6):4972–4982
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This work was funded in part by grants from the National Nature Science Foundation of China (81772791, 81172463), from Cancer Council of Queensland, Australia (511210 to KNZ).
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Ci, X., Zhao, Y., Tang, W. et al. HPV16 E7-impaired keratinocyte differentiation leads to tumorigenesis via cell cycle/pRb/involucrin/spectrin/adducin cascade. Appl Microbiol Biotechnol 104, 4417–4433 (2020). https://doi.org/10.1007/s00253-020-10492-4
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DOI: https://doi.org/10.1007/s00253-020-10492-4