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CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma
Regorafenib is used for hepatocellular carcinoma (HCC), but its response does not last long, partly due to chemoresistance acquisition. We performed a clustered regularly interspaced short palindromic repeats (CRISPR)-based loss-of-function genetic screen and aimed to discover molecules involved in...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770429/ https://www.ncbi.nlm.nih.gov/pubmed/31540262 http://dx.doi.org/10.3390/cancers11091362 |
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author | Suemura, Shigeki Kodama, Takahiro Myojin, Yuta Yamada, Ryoko Shigekawa, Minoru Hikita, Hayato Sakamori, Ryotaro Tatsumi, Tomohide Takehara, Tetsuo |
author_facet | Suemura, Shigeki Kodama, Takahiro Myojin, Yuta Yamada, Ryoko Shigekawa, Minoru Hikita, Hayato Sakamori, Ryotaro Tatsumi, Tomohide Takehara, Tetsuo |
author_sort | Suemura, Shigeki |
collection | PubMed |
description | Regorafenib is used for hepatocellular carcinoma (HCC), but its response does not last long, partly due to chemoresistance acquisition. We performed a clustered regularly interspaced short palindromic repeats (CRISPR)-based loss-of-function genetic screen and aimed to discover molecules involved in regorafenib resistance in HCC. Xenograft tumors established from Cas9-expressing HCC cells with pooled CRISPR kinome libraries were treated with regorafenib or a vehicle. Sequencing analysis identified 31 genes, with the abundance of multiple guide RNAs increased in regorafenib-treated tumors compared to that in vehicle-treated tumors, including 2 paralogues, LATS2 and LATS1, core components of the Hippo signaling pathway. Notably, all eight designed guide RNAs targeting LATS2 increased in regorafenib-treated tumors, suggesting that LATS2 deletion confers regorafenib resistance in HCC cells. LATS2 knockdown significantly mitigated the cytotoxic and proapoptotic effects of regorafenib on HCC cells. LATS2 inhibition stabilized the Hippo signaling mediator YAP, leading to the upregulation of antiapoptotic Bcl-xL and the multidrug resistance transporter ABCB1. Among 12 hepatoma cell lines, the half maximal inhibitory concentration (IC50) values of regorafenib were positively correlated with any of YAP, Bcl-xL and ABCB1 levels. The inhibition of YAP or Bcl-xL in regorafenib-insensitive HCC cells restored their susceptibility to regorafenib. In conclusion, our screen identified the Hippo signaling pathway as the mediator of regorafenib efficacy in HCC. |
format | Online Article Text |
id | pubmed-6770429 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-67704292019-10-30 CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma Suemura, Shigeki Kodama, Takahiro Myojin, Yuta Yamada, Ryoko Shigekawa, Minoru Hikita, Hayato Sakamori, Ryotaro Tatsumi, Tomohide Takehara, Tetsuo Cancers (Basel) Article Regorafenib is used for hepatocellular carcinoma (HCC), but its response does not last long, partly due to chemoresistance acquisition. We performed a clustered regularly interspaced short palindromic repeats (CRISPR)-based loss-of-function genetic screen and aimed to discover molecules involved in regorafenib resistance in HCC. Xenograft tumors established from Cas9-expressing HCC cells with pooled CRISPR kinome libraries were treated with regorafenib or a vehicle. Sequencing analysis identified 31 genes, with the abundance of multiple guide RNAs increased in regorafenib-treated tumors compared to that in vehicle-treated tumors, including 2 paralogues, LATS2 and LATS1, core components of the Hippo signaling pathway. Notably, all eight designed guide RNAs targeting LATS2 increased in regorafenib-treated tumors, suggesting that LATS2 deletion confers regorafenib resistance in HCC cells. LATS2 knockdown significantly mitigated the cytotoxic and proapoptotic effects of regorafenib on HCC cells. LATS2 inhibition stabilized the Hippo signaling mediator YAP, leading to the upregulation of antiapoptotic Bcl-xL and the multidrug resistance transporter ABCB1. Among 12 hepatoma cell lines, the half maximal inhibitory concentration (IC50) values of regorafenib were positively correlated with any of YAP, Bcl-xL and ABCB1 levels. The inhibition of YAP or Bcl-xL in regorafenib-insensitive HCC cells restored their susceptibility to regorafenib. In conclusion, our screen identified the Hippo signaling pathway as the mediator of regorafenib efficacy in HCC. MDPI 2019-09-13 /pmc/articles/PMC6770429/ /pubmed/31540262 http://dx.doi.org/10.3390/cancers11091362 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Suemura, Shigeki Kodama, Takahiro Myojin, Yuta Yamada, Ryoko Shigekawa, Minoru Hikita, Hayato Sakamori, Ryotaro Tatsumi, Tomohide Takehara, Tetsuo CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma |
title | CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma |
title_full | CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma |
title_fullStr | CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma |
title_full_unstemmed | CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma |
title_short | CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma |
title_sort | crispr loss-of-function screen identifies the hippo signaling pathway as the mediator of regorafenib efficacy in hepatocellular carcinoma |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770429/ https://www.ncbi.nlm.nih.gov/pubmed/31540262 http://dx.doi.org/10.3390/cancers11091362 |
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