Regulation of tamoxifen sensitivity by a PAK1-EBP1 signalling pathway in breast cancer.
BACKGROUND
EBP1, an ErbB3-binding protein, sensitises breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21-regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2-overexpressing breast cancer cells.


METHODS
Effects of EBP1 on ErbB2 levels were measured by western blotting. Effects of EBP1 and IPA-3 on tamoxifen sensitivity were measured using a tetrazolium based cell viability assay.


RESULTS
Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1, tamoxifen sensitive. IPA-3, a specific small MW PAK1 inhibitor, sensitised cells to tamoxifen only when EBP1 was ectopically expressed. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels only in EBP1-overexpressing cells.


CONCLUSIONS
These studies suggest that phosphorylation of EBP1 may be one mechanism of PAK1-induced hormone resistance and that PAK1 inhibitors may be useful in cells in which EBP1 is overexpressed.
Greenebaum Cancer Center, University of Maryland School of Medicine, BRB 9-029, 655 W. Baltimore Street, Baltimore, MD 21201, USA.
A
A
Ghosh
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S
Awasthi
J R
JR
Peterson
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AW
Hamburger
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D010957:Plasmids; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013629:Tamoxifen / Q000494:pharmacology*; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D018931:Antineoplastic Agents, Hormonal; D016601:RNA-Binding Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D013629:Tamoxifen; D054462:p21-Activated Kinases; C101633:PA2G4 protein, human; C508053:ERBB2 protein, human; C515903:PAK1 protein, human