Oncotarget

Research Papers:

Generation of lung cancer cell lines harboring EGFR T790M mutation by CRISPR/Cas9-mediated genome editing

Mi-Young Park, Min Hee Jung, Eun Young Eo, Seokjoong Kim, Sang Hoon Lee, Yeon Joo Lee, Jong Sun Park, Young Jae Cho, Jin Haeng Chung, Cheol Hyeon Kim, Ho Il Yoon, Jae Ho Lee and Choon-Taek Lee _

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Oncotarget. 2017; 8:36331-36338. https://doi.org/10.18632/oncotarget.16752

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Abstract

Mi-Young Park1,*, Min Hee Jung2,*, Eun Young Eo1, Seokjoong Kim2, Sang Hoon Lee1,3, Yeon Joo Lee1,3, Jong Sun Park1,3, Young Jae Cho1,3, Jin Haeng Chung4, Cheol Hyeon Kim5, Ho Il Yoon1,3, Jae Ho Lee1,3, Choon-Taek Lee1,3

1Division of Pulmonology and Critical Care Medicine, Department of Internal Medicine and Respiratory Center, Seoul National University Bundang Hospital, Seongnam, Republic of Korea

2Toolgen Inc., Seoul, Republic of Korea

3Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea

4Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Republic of Korea

5Department of Internal Medicine, Korea Cancer Center Hospital, Seoul, Republic of Korea

*These authors contributed equally to this work

Correspondence to:

Choon-Taek Lee, email: ctlee@snu.ac.kr

Keywords: lung cancer, resistance, EGFR T790M, CRISPR/Cas9

Received: August 15, 2016     Accepted: March 21, 2017     Published: March 31, 2017

ABSTRACT

Tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib are effective against lung adenocarcinomas harboring epidermal growth factor receptor (EGFR) mutations. However, cancer cells can develop resistance to these agents with prolonged exposure; in over 50% of cases, this is attributable to the EGFR T790M mutation. Moreover, additional resistance mutations can arise with the use of new drugs. Cancer cell lines with specific mutations can enable the study of resistance mechanisms. In this study, we introduced the EGFR T790M mutation into the PC9 human lung cancer cell line—which has a deletion in exon 19 of the EGFR gene—by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9-mediated genome editing. EGFR pyrosequencing and peptide nucleic acid clamping revealed that PC9 cells with EGFR T790M generated by CRISPR/Cas 9 had a higher T790M mutation rate than those with the same mutation generated by long-term exposure to gefitinib (PC9-G); moreover, resistance to gefitinib in these clones was higher than that in PC9-G cells. The clones were also highly sensitive to the 3rd-generation EGFR TKI AZD9291, which is cytotoxic to lung cancer cells with EGFR T790M. The CRISPR/Cas9 programmable nuclease system can be used to generate various cancer cell lines with specific mutations that can facilitate studies on resistance mechanisms and drug efficacy.


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