Development of an integrated CRISPRi targeting ΔNp63 for treatment of squamous cell carcinoma
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Masakazu Yoshida1, Etsuko Yokota1, Tetsushi Sakuma3, Tomoki Yamatsuji1, Nagio Takigawa2, Toshikazu Ushijima4, Takashi Yamamoto3, Takuya Fukazawa1 and Yoshio Naomoto1
1Department of General Surgery, Kawasaki Medical School, Okayama, 700-8505 Japan
2Department of General Internal Medicine 4, Kawasaki Medical School, Okayama, 700-8505 Japan
3Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526 Japan
4Division of Epigenomics, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, 104-0045 Japan
Takuya Fukazawa, email: [email protected]
Keywords: CRISPRi; ΔNp63; squamous cell carcinoma; molecular targeted therapy
Received: December 18, 2017 Accepted: May 23, 2018 Published: June 26, 2018
TP63 encodes TAp63, which is functionally similar to the tumor suppressor TP53, and ΔNp63, which lacks the transcription-activating domain of TAp63 and appears potently oncogenic in squamous cell carcinomas (SCCs). In this study, we developed an integrated CRISPR interference (CRISPRi) system to selectively suppress ΔNp63 (CRISPRiΔNp63). We engineered this CRISPRi using tandemized guide RNA expression cassettes that targeted the 50 to 100 bp downstream of the transcription start site of ΔNp63 in combination with inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain. The plasmid vector harboring CRISPRiΔNp63 repressed ΔNp63 transcription in lung and esophageal SCC cells. Likewise, Ad-CRISPRiΔNp63, an all-in-one adenoviral vector containing the tandemized gRNAs and dCas9/KRAB expression cassette suppressed ΔNp63 expression in SCC cells. Ad-CRISPRiΔNp63 also effectively decreased cell proliferation and colony formation and induced apoptosis in lung and esophageal SCC cells in vitro and significantly inhibited tumor growth in a mouse lung SCC xenograft model in vivo. These results indicate that ΔNp63 suppression using CRISPRiΔNp63 may be an effective strategy for treating lung and esophageal SCC.
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