Intrabody targeting HIF-1α mediates transcriptional downregulation of target genes related to solid tumors

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Keywords :

HIF-1α; Nanobodies; Target genes; Transcriptional activation; HIF1A protein, human; Escherichia coli; HeLa cell line; Cell Hypoxia; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Protein Domains; Single-Domain Antibodies; Transcription, Genetic; Transfection; Uterine Cervical Neoplasms

Abstract :

[en] Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Disciplines :

Biochemistry, biophysics & molecular biology

Author, co-author :

Hu, Y.; Research Institute of Public Health, School of Medicine, Nankai University, Tianjin, 300071, China, Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

Romão, E.; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

Vincke, C.; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

Brys, L.; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

Elkrim, Y.; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

Vandevenne, Marylène ; Université de Liège - ULiège

Liu, C.; State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300301, China

Muyldermans, S.; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

Language :

English

Title :

Intrabody targeting HIF-1α mediates transcriptional downregulation of target genes related to solid tumors

Publication date :

2021

Journal title :

International Journal of Molecular Sciences

ISSN :

1661-6596

eISSN :

1422-0067

Publisher :

Multidisciplinary Digital Publishing Institute (MDPI), Switzerland

Volume :

Issue :

Pages :

12335

Peer reviewed :

Peer Reviewed verified by ORBi

Available on ORBi :

since 30 January 2022

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Bibliography

Botney, R.; Answine, J.F.; Cowles, C.E., Jr. Failures of the Oxygen Supply. Anesthesiol. Clin. 2020, 38, 901–921. [CrossRef]
Semenza, G.L. Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology. Annu. Rev. Pathol. 2014, 9, 47–71. [CrossRef] [PubMed]
Pouyssegur, J.; Dayan, F.; Mazure, N.M. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 2006, 441, 437–443. [CrossRef] [PubMed]
Ivan, M.; Fishel, M.L.; Tudoran, O.M.; Pollok, K.E.; Wu, X.; Smith, P.J. Hypoxia signaling: Challenges and opportunities for cancer therapy. Semin. Cancer Biol. 2021. [CrossRef]
Hockel, M.; Vaupel, P. Tumor hypoxia: Definitions and current clinical, biologic, and molecular aspects. J. Natl. Cancer Inst. 2001, 93, 266–276. [CrossRef]
Rankin, E.B.; Giaccia, A.J. Hypoxic control of metastasis. Science 2016, 352, 175–180. [CrossRef] [PubMed]
Pezzuto, A.; Carico, E. Role of HIF-1 in Cancer Progression: Novel Insights. A Review. Curr. Mol. Med. 2018, 18, 343–351. [CrossRef] [PubMed]
Wouters, A.; Pauwels, B.; Lardon, F.; Vermorken, J.B. Review: Implications of in vitro research on the effect of radiotherapy and chemotherapy under hypoxic conditions. Oncologist 2007, 12, 690–712. [CrossRef]
Williams, K.J.; Telfer, B.A.; Xenaki, D.; Sheridan, M.R.; Desbaillets, I.; Peters, H.J.; Honess, D.; Harris, A.L.; Dachs, G.U.; van der Kogel, A.; et al. Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1. Radiother. Oncol. 2005, 75, 89–98. [CrossRef]
Swartz, J.E.; Wegner, I.; Noorlag, R.; van Kempen, P.M.W.; van Es, R.J.J.; de Bree, R.; Willems, S.M.W. HIF-1a expression and differential effects on survival in patients with oral cavity, larynx, and oropharynx squamous cell carcinomas. Head Neck 2021, 43, 745–756. [CrossRef] [PubMed]
Vaupel, P.; Mayer, A. Hypoxia in cancer: Significance and impact on clinical outcome. Cancer Metastasis Rev. 2007, 26, 225–239. [CrossRef]
Multhoff, G.; Vaupel, P. Hypoxia Compromises Anti-Cancer Immune Responses. Adv. Exp. Med. Biol. 2020, 1232, 131–143. [CrossRef]
Masoud, G.N.; Li, W. HIF-1α pathway: Role, regulation and intervention for cancer therapy. Acta Pharm. Sin. B 2015, 5, 378–389. [CrossRef]
Greijer, A.E.; van der Groep, P.; Kemming, D.; Shvarts, A.; Semenza, G.L.; Meijer, G.A.; van de Wiel, M.A.; Belien, J.A.; van Diest, P.J.; van der Wall, E. Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1). J. Pathol. 2005, 206, 291–304. [CrossRef]
Semenza, G.L. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell. Dev. Biol. 1999, 15, 551–578. [CrossRef]
Koshiji, M.; To, K.K.; Hammer, S.; Kumamoto, K.; Harris, A.L.; Modrich, P.; Huang, L.E. HIF-1α induces genetic instability by transcriptionally downregulating MutSalpha expression. Mol. Cell 2005, 17, 793–803. [CrossRef] [PubMed]
Stebbins, C.E.; Kaelin, W.G., Jr.; Pavletich, N.P. Structure of the VHL-ElonginC-ElonginB complex: Implications for VHL tumor suppressor function. Science 1999, 284, 455–461. [CrossRef] [PubMed]
Chowdhury, R.; McDonough, M.A.; Mecinovic, J.; Loenarz, C.; Flashman, E.; Hewitson, K.S.; Domene, C.; Schofield, C.J. Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases. Structure 2009, 17, 981–989. [CrossRef]
Simon, M.C. Siah proteins, HIF prolyl hydroxylases, and the physiological response to hypoxia. Cell 2004, 117, 851–853. [CrossRef] [PubMed]
Semenza, G.L.; Nejfelt, M.K.; Chi, S.M.; Antonarakis, S.E. Hypoxia-inducible nuclear factors bind to an enhancer element located 3′ to the human erythropoietin gene. Proc. Natl. Acad. Sci. USA 1991, 88, 5680–5684. [CrossRef] [PubMed]
Mandriota, S.J.; Turner, K.J.; Davies, D.R.; Murray, P.G.; Morgan, N.V.; Sowter, H.M.; Wykoff, C.C.; Maher, E.R.; Harris, A.L.; Ratcliffe, P.J.; et al. HIF activation identifies early lesions in VHL kidneys: Evidence for site-specific tumor suppressor function in the nephron. Cancer Cell 2002, 1, 459–468. [CrossRef]
Maxwell, P.H.; Wiesener, M.S.; Chang, G.W.; Clifford, S.C.; Vaux, E.C.; Cockman, M.E.; Wykoff, C.C.; Pugh, C.W.; Maher, E.R.; Ratcliffe, P.J. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999, 399, 271–275. [CrossRef]
Scheuermann, T.H.; Yang, J.; Zhang, L.; Gardner, K.H.; Bruick, R.K. Hypoxia-inducible factors Per/ARNT/Sim domains: Structure and function. Methods Enzymol. 2007, 435, 3–24. [CrossRef]
Erbel, P.J.; Card, P.B.; Karakuzu, O.; Bruick, R.K.; Gardner, K.H. Structural basis for PAS domain heterodimerization in the basic helix–loop–helix-PAS transcription factor hypoxia-inducible factor. Proc. Natl. Acad. Sci. USA 2003, 100, 15504–15509. [CrossRef]
Yang, J.; Zhang, L.; Erbel, P.J.; Gardner, K.H.; Ding, K.; Garcia, J.A.; Bruick, R.K. Functions of the Per/ARNT/Sim domains of the hypoxia-inducible factor. J. Biol. Chem. 2005, 280, 36047–36054. [CrossRef] [PubMed]
Sharkey, R.M.; Goldenberg, D.M. Targeted therapy of cancer: New prospects for antibodies and immunoconjugates. CA Cancer J. Clin. 2006, 56, 226–243. [CrossRef] [PubMed]
Chiavenna, S.M.; Jaworski, J.P.; Vendrell, A. State of the art in anti-cancer mAbs. J. Biomed. Sci. 2017, 24, 15. [CrossRef] [PubMed]
Tariman, J.D. Changes in Cancer Treatment: Mabs, Mibs, Mids, Nabs, and Nibs. Nurs. Clin. N. Am. 2017, 52, 65–81. [CrossRef]
Hamers-Casterman, C.; Atarhouch, T.; Muyldermans, S.; Robinson, G.; Hamers, C.; Songa, E.B.; Bendahman, N.; Hamers, R. Naturally occurring antibodies devoid of light chains. Nature 1993, 363, 446–448. [CrossRef] [PubMed]
Muyldermans, S. Nanobodies: Natural single-domain antibodies. Annu. Rev. Biochem. 2013, 82, 775–797. [CrossRef]
Ackaert, C.; Smiejkowska, N.; Xavier, C.; Sterckx, Y.G.J.; Denies, S.; Stijlemans, B.; Elkrim, Y.; Devoogdt, N.; Caveliers, V.; Lahoutte, T.; et al. Immunogenicity Risk Profile of Nanobodies. Front. Immunol. 2021, 12, 632687. [CrossRef] [PubMed]
Wagner, T.R.; Rothbauer, U. Nanobodies—Little helpers unravelling intracellular signaling. Free Radic. Biol. Med. 2021, 176, 46–61. [CrossRef] [PubMed]
Zhao, L.; Liu, Z.; Yang, F.; Zhang, Y.; Xue, Y.; Miao, H.; Liao, X.; Huang, H.; Li, G. Intrabody against prolyl hydroxylase 2 promotes angiogenesis by stabilizing hypoxia-inducible factor-1α. Sci. Rep. 2019, 9, 11861. [CrossRef] [PubMed]
Groot, A.J.; Gort, E.H.; van der Wall, E.; van Diest, P.J.; Vooijs, M. Conditional inactivation of HIF-1 using intrabodies. Cell. Oncol. 2008, 30, 397–409. [CrossRef] [PubMed]
Hu, Y.; Romão, E.; Vertommen, D.; Vincke, C.; Morales-Yánez, F.; Gutiérrez, C.; Liu, C.; Muyldermans, S. Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins. Protein Expr. Purif. 2017, 137, 64–76. [CrossRef] [PubMed]
Cawez, F.; Duray, E.; Hu, Y.; Vandenameele, J.; Romao, E.; Vincke, C.; Dumoulin, M.; Galleni, M.; Muyldermans, S.; Vandevenne, M. Combinatorial Design of a Nanobody that Specifically Targets Structured RNAs. J. Mol. Biol. 2018, 430, 1652–1670. [CrossRef] [PubMed]
Wilson, W.R.; Hay, M.P. Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 2011, 11, 393–410. [CrossRef] [PubMed]
Rothbauer, U.; Zolghadr, K.; Tillib, S.; Nowak, D.; Schermelleh, L.; Gahl, A.; Backmann, N.; Conrath, K.; Muyldermans, S.; Cardoso, M.C.; et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 2006, 3, 887–889. [CrossRef] [PubMed]
Yuan, Y.; Hilliard, G.; Ferguson, T.; Millhorn, D.E. Cobalt inhibits the interaction between hypoxia-inducible factor-α and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-α. J. Biol. Chem. 2003, 278, 15911–15916. [CrossRef]
Arbabi Ghahroudi, M.; Desmyter, A.; Wyns, L.; Hamers, R.; Muyldermans, S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997, 414, 521–526. [CrossRef]
Vincke, C.; Gutierrez, C.; Wernery, U.; Devoogdt, N.; Hassanzadeh-Ghassabeh, G.; Muyldermans, S. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol. Biol. 2012, 907, 145–176. [CrossRef]
Pardon, E.; Laeremans, T.; Triest, S.; Rasmussen, S.G.; Wohlkonig, A.; Ruf, A.; Muyldermans, S.; Hol, W.G.; Kobilka, B.K.; Steyaert, J. A general protocol for the generation of Nanobodies for structural biology. Nat. Protoc. 2014, 9, 674–693. [CrossRef] [PubMed]
Vincke, C.; Loris, R.; Saerens, D.; Martinez-Rodriguez, S.; Muyldermans, S.; Conrath, K. General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold. J. Biol. Chem. 2008, 284, 3273–3284. [CrossRef]
Saerens, D.; Pellis, M.; Loris, R.; Pardon, E.; Dumoulin, M.; Matagne, A.; Wyns, L.; Muyldermans, S.; Conrath, K. Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J. Mol. Biol. 2005, 352, 597–607. [CrossRef] [PubMed]
Hu, Y.; Wu, S.; Wang, Y.; Lin, J.; Sun, Y.; Zhang, C.; Gu, J.; Yang, F.; Lv, H.; Ji, X.; et al. Unbiased Immunization Strategy Yielding Specific Nanobodies against Macadamia Allergen of Vicilin-like Protein for Immunoassay Development. J. Agric. Food Chem. 2021, 69, 5178–5188. [CrossRef] [PubMed]
Chen, R.; Xu, J.; She, Y.; Jiang, T.; Zhou, S.; Shi, H.; Li, C. Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia. Int. J. Mol. Med. 2018, 41, 2565–2572. [CrossRef] [PubMed]
Mellor, H.R.; Harris, A.L. The role of the hypoxia-inducible BH3-only proteins BNIP3 and BNIP3L in cancer. Cancer Metastasis Rev. 2007, 26, 553–566. [CrossRef] [PubMed]
Li, Z.; Jiang, L.; Chew, S.H.; Hirayama, T.; Sekido, Y.; Toyokuni, S. Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia. Redox Biol. 2019, 26, 101297. [CrossRef]
Zhang, J.Y.; Zhang, F.; Hong, C.Q.; Giuliano, A.E.; Cui, X.J.; Zhou, G.J.; Zhang, G.J.; Cui, Y.K. Critical protein GAPDH and its regulatory mechanisms in cancer cells. Cancer Biol. Med. 2015, 12, 10–22. [CrossRef]
Srivastava, C.; Irshad, K.; Dikshit, B.; Chattopadhyay, P.; Sarkar, C.; Gupta, D.K.; Sinha, S.; Chosdol, K. FAT1 modulates EMT and stemness genes expression in hypoxic glioblastoma. Int. J. Cancer 2018, 142, 805–812. [CrossRef]