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Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis
Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that g...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632093/ https://www.ncbi.nlm.nih.gov/pubmed/26479444 http://dx.doi.org/10.1038/oncsis.2015.32 |
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author | Miyoshi, N Wittner, B S Shioda, K Hitora, T Ito, T Ramaswamy, S Isselbacher, K J Sgroi, D C Shioda, T |
author_facet | Miyoshi, N Wittner, B S Shioda, K Hitora, T Ito, T Ramaswamy, S Isselbacher, K J Sgroi, D C Shioda, T |
author_sort | Miyoshi, N |
collection | PubMed |
description | Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction–validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan–Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields biologically meaningful outcomes by taking advantage of the existing knowledge of the physical and functional interactions between the predicted target genes. The resulting interaction maps provide useful information on signaling pathways cooperatively involved in clinically important features of the malignant cells, such as drug resistance. |
format | Online Article Text |
id | pubmed-4632093 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-46320932015-11-19 Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis Miyoshi, N Wittner, B S Shioda, K Hitora, T Ito, T Ramaswamy, S Isselbacher, K J Sgroi, D C Shioda, T Oncogenesis Original Article Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction–validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan–Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields biologically meaningful outcomes by taking advantage of the existing knowledge of the physical and functional interactions between the predicted target genes. The resulting interaction maps provide useful information on signaling pathways cooperatively involved in clinically important features of the malignant cells, such as drug resistance. Nature Publishing Group 2015-10 2015-10-19 /pmc/articles/PMC4632093/ /pubmed/26479444 http://dx.doi.org/10.1038/oncsis.2015.32 Text en Copyright © 2015 Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ Oncogenesis is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Original Article Miyoshi, N Wittner, B S Shioda, K Hitora, T Ito, T Ramaswamy, S Isselbacher, K J Sgroi, D C Shioda, T Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis |
title | Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis |
title_full | Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis |
title_fullStr | Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis |
title_full_unstemmed | Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis |
title_short | Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis |
title_sort | nodes-and-connections rnai knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632093/ https://www.ncbi.nlm.nih.gov/pubmed/26479444 http://dx.doi.org/10.1038/oncsis.2015.32 |
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