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Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells

Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demo...

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Autores principales: Matossian, Margarite D., Elliott, Steven, Rhodes, Lyndsay V., Martin, Elizabeth C., Hoang, Van T., Burks, Hope E., Zuercher, William J., Drewry, David H., Collins-Burow, Bridgette M., Burow, Matthew E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: D.A. Spandidos 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988660/
https://www.ncbi.nlm.nih.gov/pubmed/33777204
http://dx.doi.org/10.3892/ol.2021.12641
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author Matossian, Margarite D.
Elliott, Steven
Rhodes, Lyndsay V.
Martin, Elizabeth C.
Hoang, Van T.
Burks, Hope E.
Zuercher, William J.
Drewry, David H.
Collins-Burow, Bridgette M.
Burow, Matthew E.
author_facet Matossian, Margarite D.
Elliott, Steven
Rhodes, Lyndsay V.
Martin, Elizabeth C.
Hoang, Van T.
Burks, Hope E.
Zuercher, William J.
Drewry, David H.
Collins-Burow, Bridgette M.
Burow, Matthew E.
author_sort Matossian, Margarite D.
collection PubMed
description Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. ‘Hits’ or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-ΔCTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the ‘active’ inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-ΔCTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers.
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spelling pubmed-79886602021-03-26 Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells Matossian, Margarite D. Elliott, Steven Rhodes, Lyndsay V. Martin, Elizabeth C. Hoang, Van T. Burks, Hope E. Zuercher, William J. Drewry, David H. Collins-Burow, Bridgette M. Burow, Matthew E. Oncol Lett Articles Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. ‘Hits’ or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-ΔCTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the ‘active’ inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-ΔCTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers. D.A. Spandidos 2021-05 2021-03-16 /pmc/articles/PMC7988660/ /pubmed/33777204 http://dx.doi.org/10.3892/ol.2021.12641 Text en Copyright: © Matossian et al. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
spellingShingle Articles
Matossian, Margarite D.
Elliott, Steven
Rhodes, Lyndsay V.
Martin, Elizabeth C.
Hoang, Van T.
Burks, Hope E.
Zuercher, William J.
Drewry, David H.
Collins-Burow, Bridgette M.
Burow, Matthew E.
Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells
title Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells
title_full Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells
title_fullStr Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells
title_full_unstemmed Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells
title_short Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells
title_sort application of a small molecule inhibitor screen approach to identify cxcr4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988660/
https://www.ncbi.nlm.nih.gov/pubmed/33777204
http://dx.doi.org/10.3892/ol.2021.12641
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