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Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis.

Authors
  • Baker, Martin J1
  • Cooke, Mariana2, 3
  • Kreider-Letterman, Gabriel4
  • Garcia-Mata, Rafael4
  • Janmey, Paul A5
  • Kazanietz, Marcelo G1
  • 1 Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA [email protected] [email protected]
  • 2 Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
  • 3 Department of Medicine, Einstein Medical Center Philadelphia, Philadelphia, Pennsylvania, USA.
  • 4 Department of Biological Sciences, University of Toledo, Ohio, USA.
  • 5 Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Type
Published Article
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology
Publication Date
Oct 02, 2020
Volume
295
Issue
40
Pages
13698–13710
Identifiers
DOI: 10.1074/jbc.RA120.013919
PMID: 32817335
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

A large number of aggressive cancer cell lines display elevated levels of activated Rac1, a small GTPase widely implicated in cytoskeleton reorganization, cell motility, and metastatic dissemination. A commonly accepted methodological approach for detecting Rac1 activation in cancer cells involves the use of a conformation-sensitive antibody that detects the active (GTP-bound) Rac1 without interacting with the GDP-bound inactive form. This antibody has been extensively used in fixed cell immunofluorescence and immunohistochemistry. Taking advantage of prostate and pancreatic cancer cell models known to have high basal Rac1-GTP levels, here we have established that this antibody does not recognize Rac1 but rather detects the intermediate filament protein vimentin. Indeed, Rac1-null PC3 prostate cancer cells or cancer models with low levels of Rac1 activation still show a high signal with the anti-Rac1-GTP antibody, which is lost upon silencing of vimentin expression. Moreover, this antibody was unable to detect activated Rac1 in membrane ruffles induced by epidermal growth factor stimulation. These results have profound implications for the study of this key GTPase in cancer, particularly because a large number of cancer cell lines with characteristic mesenchymal features show simultaneous up-regulation of vimentin and high basal Rac1-GTP levels when measured biochemically. This misleading correlation can lead to assumptions about the validity of this antibody and inaccurate conclusions that may affect the development of appropriate therapeutic approaches for targeting the Rac1 pathway. © 2020 Baker et al.

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