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Current Cancer Drug Targets

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ISSN (Print): 1568-0096
ISSN (Online): 1873-5576

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Research Article

Genome-Wide CRISPR-Cas9 Screening and Identification of Potential Genes Promoting Prostate Cancer Growth and Metastasis

Author(s): Wei Wang, Dongbo Yuan, Kehua Jiang, Ruidong Li, Han Qu, Fu-Neng Jiang, Wei-De Zhong, Fa Sun*, Zhenyu Jia* and Jianguo Zhu*

Volume 23, Issue 1, 2023

Published on: 20 August, 2022

Page: [71 - 86] Pages: 16

DOI: 10.2174/1568009622666220615154137

Price: $65

Abstract

Objective: Identification and validation of genes that functionally account for the growth and metastasis of prostate cancer.

Methods: DU145-KO cell line was constructed by transfecting DU145 cells with lentivirus packaged with the genome-wide knock-out library. The DU145-KO cells were transplanted into the armpits of immunocompromised Nu/Nu mice, followed by the tissue collection from the lung at week 3 (early lung tissue) or week 7 (late lung tissue with micro-metastasis), as well as from primary tumor site at week 7 (late primary tumor) after inoculation. Lung metastasis was retrieved at various time points for DNA sequencing analysis to identify enriched sgRNAs, thus candidate genes/miRNAs. Further bioinformatics analysis and limited functional validation studies were carried out.

Results: DU145-KO cells promoted the formation of transplanted tumors in mice and promoted the growth and metastasis of primary tumors, compared to the controls (DU145-NC cells). The analysis of sequence data showed that the abundance of sgRNAs significantly changed in the primary tumor and micro-metastasis site. Fifteen target genes (C1QTNF9B, FAM229A, hsa-mir-3929, KRT23, TARS2, CRADD, GRIK4, PLA2G15, LOXL1, SLITRK6, CDC42EP5, SLC2A4, PTGDS, MYL9 and ACOX2 for the enriched sgRNAs) have been selected for experimental validation, which showed that knock-out of any of these genes led to the enhanced potential of invasion and metastasis of DU145 cells.

Conclusion: Genome-wide CRISPR-Cas9 knock-out screening technology combined with highthroughput sequencing analysis identified genes that potentially relate to prostate tumor invasion and metastasis. Analysis of these genes provided insights into biological pathways relevant to the disease and disclosed innovative markers for diagnosis or prognosis as well as potential targets for therapy.

Keywords: Genome-wide, CRISPR-Cas9, DU145, prostate cancer, metastasis, screening.

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[1]
Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics. CA Cancer J. Clin., 2019, 69(1), 7-34.
[http://dx.doi.org/10.3322/caac.21551] [PMID: 30620402]
[2]
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin., 2018, 68(6), 394-424.
[http://dx.doi.org/10.3322/caac.21492] [PMID: 30207593]
[3]
Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell, 2011, 144(5), 646-674.
[http://dx.doi.org/10.1016/j.cell.2011.02.013] [PMID: 21376230]
[4]
Zugazagoitia, J.; Guedes, C.; Ponce, S.; Ferrer, I.; Molina-Pinelo, S.; Paz-Ares, L. Current challenges in cancer treatment. Clin. Ther., 2016, 38(7), 1551-1566.
[http://dx.doi.org/10.1016/j.clinthera.2016.03.026] [PMID: 27158009]
[5]
Gebler, C.; Lohoff, T.; Paszkowski-Rogacz, M.; Mircetic, J.; Chakraborty, D.; Camgoz, A.; Hamann, M.V.; Theis, M.; Thiede, C.; Buchholz, F. Inactivation of cancer mutations utilizing CRISPR/Cas9. J. Natl. Cancer Inst., 2016, 109(1)
[PMID: 27576906]
[6]
Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D.A.; Horvath, P. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007, 315(5819), 1709-1712.
[http://dx.doi.org/10.1126/science.1138140] [PMID: 17379808]
[7]
González, F.; Zhu, Z.; Shi, Z.D.; Lelli, K.; Verma, N.; Li, Q.V.; Huangfu, D. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell, 2014, 15(2), 215-226.
[http://dx.doi.org/10.1016/j.stem.2014.05.018] [PMID: 24931489]
[8]
Chira, S.; Gulei, D.; Hajitou, A.; Zimta, A.A.; Cordelier, P.; Berindan-Neagoe, I. CRISPR/Cas9: Transcending the reality of genome editing. Mol. Ther. Nucleic Acids, 2017, 7, 211-222.
[http://dx.doi.org/10.1016/j.omtn.2017.04.001] [PMID: 28624197]
[9]
Wiedenheft, B.; Sternberg, S.H.; Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature, 2012, 482(7385), 331-338.
[http://dx.doi.org/10.1038/nature10886] [PMID: 22337052]
[10]
Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.; Nakata, A. Nucleotide sequence of the iap gene, responsible for alkaline phospha-tase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol., 1987, 169(12), 5429-5433.
[http://dx.doi.org/10.1128/jb.169.12.5429-5433.1987] [PMID: 3316184]
[11]
Mojica, F.J.; Juez, G.; Rodríguez-Valera, F. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Mol. Microbiol., 1993, 9(3), 613-621.
[http://dx.doi.org/10.1111/j.1365-2958.1993.tb01721.x] [PMID: 8412707]
[12]
Mojica, F.J.; Díez-Villaseñor, C.; García-Martínez, J.; Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol., 2005, 60(2), 174-182.
[http://dx.doi.org/10.1007/s00239-004-0046-3] [PMID: 15791728]
[13]
Cong, L.; Ran, F.A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P.D.; Wu, X.; Jiang, W.; Marraffini, L.A.; Zhang, F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121), 819-823.
[http://dx.doi.org/10.1126/science.1231143] [PMID: 23287718]
[14]
Pourcel, C.; Salvignol, G.; Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology, 2005, 151(Pt 3), 653-663.
[http://dx.doi.org/10.1099/mic.0.27437-0] [PMID: 15758212]
[15]
Bolotin, A.; Quinquis, B.; Sorokin, A.; Ehrlich, S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology, 2005, 151(Pt 8), 2551-2561.
[http://dx.doi.org/10.1099/mic.0.28048-0] [PMID: 16079334]
[16]
Makarova, K.; Slesarev, A.; Wolf, Y.; Sorokin, A.; Mirkin, B.; Koonin, E.; Pavlov, A.; Pavlova, N.; Karamychev, V.; Polouchine, N.; Shakhova, V.; Grigoriev, I.; Lou, Y.; Rohksar, D.; Lucas, S.; Huang, K.; Goodstein, D.M.; Hawkins, T.; Plengvidhya, V.; Welker, D.; Hughes, J.; Goh, Y.; Benson, A.; Baldwin, K.; Lee, J.H.; Díaz-Muñiz, I.; Dosti, B.; Smeianov, V.; Wechter, W.; Barabote, R.; Lorca, G.; Altermann, E.; Barrangou, R.; Ganesan, B.; Xie, Y.; Rawsthorne, H.; Tamir, D.; Parker, C.; Breidt, F.; Broadbent, J.; Hutkins, R.; O’Sullivan, D.; Steele, J.; Unlu, G.; Saier, M.; Klaenhammer, T.; Richardson, P.; Kozyavkin, S.; Weimer, B.; Mills, D. Comparative genomics of the lactic acid bacteria. Proc. Natl. Acad. Sci. USA, 2006, 103(42), 15611-15616.
[http://dx.doi.org/10.1073/pnas.0607117103] [PMID: 17030793]
[17]
Makarova, K.S.; Grishin, N.V.; Shabalina, S.A.; Wolf, Y.I.; Koonin, E.V. A putative RNA-interference-based immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct, 2006, 1(1), 7.
[http://dx.doi.org/10.1186/1745-6150-1-7] [PMID: 16545108]
[18]
Brouns, S.J.; Jore, M.M.; Lundgren, M.; Westra, E.R.; Slijkhuis, R.J.; Snijders, A.P.; Dickman, M.J.; Makarova, K.S.; Koonin, E.V.; van der Oost, J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 2008, 321(5891), 960-964.
[http://dx.doi.org/10.1126/science.1159689] [PMID: 18703739]
[19]
Marraffini, L.A.; Sontheimer, E.J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science, 2008, 322(5909), 1843-1845.
[http://dx.doi.org/10.1126/science.1165771] [PMID: 19095942]
[20]
Wu, W.; Yang, Y.; Lei, H. Progress in the application of CRISPR: From gene to base editing. Med. Res. Rev., 2019, 39(2), 665-683.
[http://dx.doi.org/10.1002/med.21537] [PMID: 30171624]
[21]
Zhang, C.; Quan, R.; Wang, J. Development and application of CRISPR/Cas9 technologies in genomic editing. Hum. Mol. Genet., 2018, 27(R2), R79-R88.
[http://dx.doi.org/10.1093/hmg/ddy120] [PMID: 29659822]
[22]
Ma, H.; Dang, Y.; Wu, Y.; Jia, G.; Anaya, E.; Zhang, J.; Abraham, S.; Choi, J.G.; Shi, G.; Qi, L.; Manjunath, N.; Wu, H. A CRISPR-Based screen identifies genes essential for West-Nile-Virus-Induced cell death. Cell Rep., 2015, 12(4), 673-683.
[http://dx.doi.org/10.1016/j.celrep.2015.06.049] [PMID: 26190106]
[23]
Henser-Brownhill, T.; Monserrat, J.; Scaffidi, P. Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: From high-content screens to in vivo assays. Epigenetics, 2017, 12(12), 1065-1075.
[24]
Wang, T.; Wei, J.J.; Sabatini, D.M.; Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science, 2014, 343(6166), 80-84.
[http://dx.doi.org/10.1126/science.1246981] [PMID: 24336569]
[25]
Zhou, Y.; Zhu, S.; Cai, C.; Yuan, P.; Li, C.; Huang, Y.; Wei, W. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature, 2014, 509(7501), 487-491.
[http://dx.doi.org/10.1038/nature13166] [PMID: 24717434]
[26]
Parnas, O.; Jovanovic, M.; Eisenhaure, T.M.; Herbst, R.H.; Dixit, A.; Ye, C.J.; Przybylski, D.; Platt, R.J.; Tirosh, I.; Sanjana, N.E.; Shalem, O.; Satija, R.; Raychowdhury, R.; Mertins, P.; Carr, S.A.; Zhang, F.; Hacohen, N.; Regev, A. A Genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell, 2015, 162(3), 675-686.
[http://dx.doi.org/10.1016/j.cell.2015.06.059] [PMID: 26189680]
[27]
Alimirah, F.; Chen, J.; Basrawala, Z.; Xin, H.; Choubey, D. DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: Implications for the androgen receptor functions and regulation. FEBS Lett., 2006, 580(9), 2294-2300.
[http://dx.doi.org/10.1016/j.febslet.2006.03.041] [PMID: 16580667]
[28]
Litvinov, I.V.; Antony, L.; Dalrymple, S.L.; Becker, R.; Cheng, L.; Isaacs, J.T. PC3, but not DU145, human prostate cancer cells retain the coregulators required for tumor suppressor ability of androgen receptor. Prostate, 2006, 66(12), 1329-1338.
[http://dx.doi.org/10.1002/pros.20483] [PMID: 16835890]
[29]
Pulukuri, S.M.; Gondi, C.S.; Lakka, S.S.; Jutla, A.; Estes, N.; Gujrati, M.; Rao, J.S. Retraction: RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo. J. Biol. Chem., 2020, 295(37), 13136.
[http://dx.doi.org/10.1074/jbc.RX120.015588] [PMID: 32917829]
[30]
Stone, K.R.; Mickey, D.D.; Wunderli, H.; Mickey, G.H.; Paulson, D.F. Isolation of a human prostate carcinoma cell line (DU 145). Int. J. Cancer, 1978, 21(3), 274-281.
[http://dx.doi.org/10.1002/ijc.2910210305] [PMID: 631930]
[31]
Longo, P.A.; Kavran, J.M.; Kim, M.S.; Leahy, D.J. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol., 2013, 529, 227-240.
[http://dx.doi.org/10.1016/B978-0-12-418687-3.00018-5] [PMID: 24011049]
[32]
Faustino-Rocha, A.; Oliveira, P.A.; Pinho-Oliveira, J.; Teixeira-Guedes, C.; Soares-Maia, R.; da Costa, R.G.; Colaço, B.; Pires, M.J.; Colaço, J.; Ferreira, R.; Ginja, M. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Anim. (NY), 2013, 42(6), 217-224.
[http://dx.doi.org/10.1038/laban.254] [PMID: 23689461]
[33]
Chan, J.K. The wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology. Int. J. Surg. Pathol., 2014, 22(1), 12-32.
[http://dx.doi.org/10.1177/1066896913517939] [PMID: 24406626]
[34]
Chen, G.; Liang, Y.X.; Zhu, J.G.; Fu, X.; Chen, Y.F.; Mo, R.J.; Zhou, L.; Fu, H.; Bi, X.C.; He, H.C.; Yang, S.B.; Wu, Y.D.; Jiang, F.N.; Zhong, W.D. CC chemokine ligand 18 correlates with malignant progression of prostate cancer. BioMed Res. Int., 2014, 2014, 230183.
[http://dx.doi.org/10.1155/2014/230183] [PMID: 25197632]
[35]
Torres-Ruiz, R.; Rodriguez-Perales, S. CRISPR-Cas9: A revolutionary tool for cancer modelling. Int. J. Mol. Sci., 2015, 16(9), 22151-22168.
[http://dx.doi.org/10.3390/ijms160922151] [PMID: 26389881]
[36]
Mou, H.; Smith, J.L.; Peng, L.; Yin, H.; Moore, J.; Zhang, X.O.; Song, C.Q.; Sheel, A.; Wu, Q.; Ozata, D.M.; Li, Y.; Anderson, D.G.; Emer-son, C.P.; Sontheimer, E.J.; Moore, M.J.; Weng, Z.; Xue, W. CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion. Genome Biol., 2017, 18(1), 108.
[http://dx.doi.org/10.1186/s13059-017-1237-8] [PMID: 28615073]
[37]
Zuo, E.; Cai, Y.J.; Li, K.; Wei, Y.; Wang, B.A.; Sun, Y.; Liu, Z.; Liu, J.; Hu, X.; Wei, W.; Huo, X.; Shi, L.; Tang, C.; Liang, D.; Wang, Y.; Nie, Y.H.; Zhang, C.C.; Yao, X.; Wang, X.; Zhou, C.; Ying, W.; Wang, Q.; Chen, R.C.; Shen, Q.; Xu, G.L.; Li, J.; Sun, Q.; Xiong, Z.Q.; Yang, H. One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Res., 2017, 27(7), 933-945.
[http://dx.doi.org/10.1038/cr.2017.81] [PMID: 28585534]
[38]
Jiang, F.; Doudna, J.A. CRISPR-Cas9 structures and mechanisms. Annu. Rev. Biophys., 2017, 46(1), 505-529.
[http://dx.doi.org/10.1146/annurev-biophys-062215-010822] [PMID: 28375731]
[39]
Slaymaker, I.M.; Gao, L.; Zetsche, B.; Scott, D.A.; Yan, W.X.; Zhang, F. Rationally engineered Cas9 nucleases with improved specificity. Science, 2016, 351(6268), 84-88.
[http://dx.doi.org/10.1126/science.aad5227] [PMID: 26628643]
[40]
Hartenian, E.; Doench, J.G. Genetic screens and functional genomics using CRISPR/Cas9 technology. FEBS J., 2015, 282(8), 1383-1393.
[http://dx.doi.org/10.1111/febs.13248] [PMID: 25728500]
[41]
Chen, S.; Sanjana, N.E.; Zheng, K.; Shalem, O.; Lee, K.; Shi, X.; Scott, D.A.; Song, J.; Pan, J.Q.; Weissleder, R.; Lee, H.; Zhang, F.; Sharp, P.A. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell, 2015, 160(6), 1246-1260.
[http://dx.doi.org/10.1016/j.cell.2015.02.038] [PMID: 25748654]
[42]
Goodspeed, A.; Jean, A.; Costello, J.C. A whole-genome CRISPR screen identifies a role of MSH2 in cisplatin-mediated cell death in muscle-invasive bladder cancer. Eur. Urol., 2019, 75(2), 242-250.
[http://dx.doi.org/10.1016/j.eururo.2018.10.040] [PMID: 30414698]
[43]
Wang, C.; Jin, H.; Gao, D.; Wang, L.; Evers, B.; Xue, Z.; Jin, G.; Lieftink, C.; Beijersbergen, R.L.; Qin, W.; Bernards, R. A CRISPR screen identifies CDK7 as a therapeutic target in hepatocellular carcinoma. Cell Res., 2018, 28(6), 690-692.
[http://dx.doi.org/10.1038/s41422-018-0020-z] [PMID: 29507396]
[44]
Thiery, J.P.; Acloque, H.; Huang, R.Y.; Nieto, M.A. Epithelial-mesenchymal transitions in development and disease. Cell, 2009, 139(5), 871-890.
[http://dx.doi.org/10.1016/j.cell.2009.11.007] [PMID: 19945376]
[45]
Cho, E.S.; Kang, H.E.; Kim, N.H.; Yook, J.I. Therapeutic implications of cancer epithelial-mesenchymal transition (EMT). Arch. Pharm. Res., 2019, 42(1), 14-24.
[http://dx.doi.org/10.1007/s12272-018-01108-7] [PMID: 30649699]
[46]
Singh, M.; Yelle, N.; Venugopal, C.; Singh, S.K. EMT: Mechanisms and therapeutic implications. Pharmacol. Ther., 2018, 182, 80-94.
[http://dx.doi.org/10.1016/j.pharmthera.2017.08.009] [PMID: 28834698]
[47]
Huang, H. Matrix metalloproteinase-9 (MMP-9) as a cancer biomarker and MMP-9 biosensors: Recent advances. Sensors (Basel), 2018, 18(10), 3249.
[http://dx.doi.org/10.3390/s18103249] [PMID: 30262739]

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