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CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro

Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two viral oncoproteins that promote cell growth: the...

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Autores principales: Temblador, Arturo, Topalis, Dimitrios, Andrei, Graciela, Snoeck, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770690/
https://www.ncbi.nlm.nih.gov/pubmed/31466237
http://dx.doi.org/10.3390/cancers11091260
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author Temblador, Arturo
Topalis, Dimitrios
Andrei, Graciela
Snoeck, Robert
author_facet Temblador, Arturo
Topalis, Dimitrios
Andrei, Graciela
Snoeck, Robert
author_sort Temblador, Arturo
collection PubMed
description Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two viral oncoproteins that promote cell growth: the small (sT) and the large (LT) tumor antigens (TAs). Despite the success of immunotherapies in patients with MCC, not all individuals respond to these treatments. Therefore, new therapeutic options continue to be investigated. Herein, we used CRISPR/Cas9 to target the viral oncogenes in two virus-positive MCC cell lines: MS-1 and WAGA. Frameshift mutations introduced in the target sequence upon repair of the Cas9-induced DNA break resulted in decreased LT protein levels, which subsequently impaired cell proliferation, caused cell cycle arrest, and led to increased apoptosis. Importantly, a virus-negative non-MCC cell line (HEK293T) remained unaffected, as well as those cells expressing a non-targeting single-guide RNA (sgRNA). Thus, we presumed that the noted effects were not due to the off-target activity of the TAs-targeting sgRNAs. Additionally, WAGA cells had altered levels of cellular proteins involved in cell cycle regulation, supporting the observed cell cycle. Taken together, our findings provide evidence for the development of a CRISPR/Cas9-based therapeutic option for virus-positive MCC.
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spelling pubmed-67706902019-10-30 CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro Temblador, Arturo Topalis, Dimitrios Andrei, Graciela Snoeck, Robert Cancers (Basel) Article Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two viral oncoproteins that promote cell growth: the small (sT) and the large (LT) tumor antigens (TAs). Despite the success of immunotherapies in patients with MCC, not all individuals respond to these treatments. Therefore, new therapeutic options continue to be investigated. Herein, we used CRISPR/Cas9 to target the viral oncogenes in two virus-positive MCC cell lines: MS-1 and WAGA. Frameshift mutations introduced in the target sequence upon repair of the Cas9-induced DNA break resulted in decreased LT protein levels, which subsequently impaired cell proliferation, caused cell cycle arrest, and led to increased apoptosis. Importantly, a virus-negative non-MCC cell line (HEK293T) remained unaffected, as well as those cells expressing a non-targeting single-guide RNA (sgRNA). Thus, we presumed that the noted effects were not due to the off-target activity of the TAs-targeting sgRNAs. Additionally, WAGA cells had altered levels of cellular proteins involved in cell cycle regulation, supporting the observed cell cycle. Taken together, our findings provide evidence for the development of a CRISPR/Cas9-based therapeutic option for virus-positive MCC. MDPI 2019-08-28 /pmc/articles/PMC6770690/ /pubmed/31466237 http://dx.doi.org/10.3390/cancers11091260 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
Temblador, Arturo
Topalis, Dimitrios
Andrei, Graciela
Snoeck, Robert
CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro
title CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro
title_full CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro
title_fullStr CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro
title_full_unstemmed CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro
title_short CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro
title_sort crispr/cas9 editing of the polyomavirus tumor antigens inhibits merkel cell carcinoma growth in vitro
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770690/
https://www.ncbi.nlm.nih.gov/pubmed/31466237
http://dx.doi.org/10.3390/cancers11091260
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