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APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing

Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehe...

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Autores principales: Shah, Fenil, Goossens, Emery, Atallah, Nadia M., Grimard, Michelle, Kelley, Mark R., Fishel, Melissa L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709621/
https://www.ncbi.nlm.nih.gov/pubmed/28922540
http://dx.doi.org/10.1002/1878-0261.12138
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author Shah, Fenil
Goossens, Emery
Atallah, Nadia M.
Grimard, Michelle
Kelley, Mark R.
Fishel, Melissa L.
author_facet Shah, Fenil
Goossens, Emery
Atallah, Nadia M.
Grimard, Michelle
Kelley, Mark R.
Fishel, Melissa L.
author_sort Shah, Fenil
collection PubMed
description Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT‐PCR. Testing additional patient‐derived pancreatic cancer cells reveals particular genes (ITGA1,TNFAIP2,COMMD7,RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox‐specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.
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spelling pubmed-57096212017-12-04 APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing Shah, Fenil Goossens, Emery Atallah, Nadia M. Grimard, Michelle Kelley, Mark R. Fishel, Melissa L. Mol Oncol Research Articles Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT‐PCR. Testing additional patient‐derived pancreatic cancer cells reveals particular genes (ITGA1,TNFAIP2,COMMD7,RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox‐specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments. John Wiley and Sons Inc. 2017-10-19 2017-12 /pmc/articles/PMC5709621/ /pubmed/28922540 http://dx.doi.org/10.1002/1878-0261.12138 Text en © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Shah, Fenil
Goossens, Emery
Atallah, Nadia M.
Grimard, Michelle
Kelley, Mark R.
Fishel, Melissa L.
APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing
title APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing
title_full APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing
title_fullStr APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing
title_full_unstemmed APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing
title_short APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing
title_sort ape1/ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell rna sequencing
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709621/
https://www.ncbi.nlm.nih.gov/pubmed/28922540
http://dx.doi.org/10.1002/1878-0261.12138
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