title: A high-content, multiplexed screen in human breast cancer cells identifies profilin-1 inducers with anti-migratory activities.
abstract: Profilin-1 (Pfn-1) is a ubiquitously expressed actin-binding protein that is essential for normal cell proliferation and migration. In breast cancer and several other adenocarcinomas, Pfn-1 expression is downregulated when compared to normal tissues. Previous studies from our laboratory have shown that genetically modulating Pfn-1 expression significantly impacts proliferation, migration, and invasion of breast cancer cells in vitro, and mammary tumor growth, dissemination, and metastatic colonization in vivo. Therefore, small molecules that can modulate Pfn-1 expression could have therapeutic potential in the treatment of metastatic breast cancer. The overall goal of this study was to perform a multiplexed phenotypic screen to identify compounds that inhibit cell motility through upregulation of Pfn-1. Screening of a test cassette of 1280 compounds with known biological activities on an Oris™ Pro 384 cell migration platform identified several agents that increased Pfn-1 expression greater than two-fold over vehicle controls and exerted anti-migratory effects in the absence of overt cytotoxicity in MDA-MB-231 human breast cancer cells. Concentration-response confirmation and orthogonal follow-up assays identified two bona fide inducers of Pfn-1, purvalanol and tyrphostin A9, that confirmed in single-cell motility assays and Western blot analyses. SiRNA-mediated knockdown of Pfn-1 abrogated the inhibitory effect of tyrphostin A9 on cell migration, suggesting Pfn-1 is mechanistically linked to tyrphostin A9's anti-migratory activity. The data illustrate the utility of the high-content cell motility assay to discover novel targeted anti-migratory agents by integrating functional phenotypic analyses with target-specific readouts in a single assay platform.
authors: Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors: Marion E
authors: ME
authors: Joy
authors: University of Pittsburgh Drug Discovery Institute, Pittsburgh, Pennsylvania, United States of America.
authors: Laura L
authors: LL
authors: Vollmer
authors: Platypus Technologies, LLC, Madison, Wisconsin, United States of America.
authors: Keren
authors: K
authors: Hulkower
authors: Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ; University of Pittsburgh Drug Discovery Institute, Pittsburgh, Pennsylvania, United States of America.
authors: Andrew M
authors: AM
authors: Stern
authors: University of Pittsburgh Drug Discovery Institute, Pittsburgh, Pennsylvania, United States of America.
authors: Cameron K
authors: CK
authors: Peterson
authors: Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ; University of Pittsburgh Drug Discovery Institute, Pittsburgh, Pennsylvania, United States of America.
authors: R C Dutch
authors: RC
authors: Boltz
authors: Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ; Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ; Magee Women's Research Institute, Pittsburgh, Pennsylvania, United States of America.
authors: Partha
authors: P
authors: Roy
authors: Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ; University of Pittsburgh Drug Discovery Institute, Pittsburgh, Pennsylvania, United States of America.
authors: Andreas
authors: A
authors: Vogt
mesh_terms: D000225:Adenine / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D054443:Cell Migration Assays; D002465:Cell Movement / Q000187:drug effects; D005260:Female; D055785:Gene Knockdown Techniques; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051304:Profilins / Q000378:metabolism*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: C495738:PFN1 protein, human; D051304:Profilins; D054852:Small Molecule Libraries; D020032:Tyrphostins; C406122:tyrphostin A9; D000225:Adenine