Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13.
BACKGROUND
Estrogen Receptor alpha (ERα)-positive breast cancer patients receive endocrine therapy, often in the form of tamoxifen. However, resistance to tamoxifen is frequently observed. A signalling cascade that leads to tamoxifen resistance is dictated by activation of the Protein Kinase A (PKA) pathway, which leads to phosphorylation of ERα on Serine 305 and receptor activation, following tamoxifen binding. Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.


METHODS
We performed immunohistochemistry to detect ERαSerine 305 phosphorylation in a cohort of breast cancer patients who received tamoxifen treatment in the metastatic setting. From the same tumor specimens, Agilent 44 K gene expression analyses were performed and integrated with clinicopathological data and survival information. In vitro analyses were performed using MCF7 breast cancer cells, which included immunoprecipitations and Fluorescence Resonance Energy Transfer (FRET) analyses to illustrate ERα complex formation. siRNA mediated knockdown experiments were performed to assess effects on ERαSerine 305 phosphorylation status, ERα/PKA interactions and downstream responsive gene activity.


RESULTS
Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13. AKAP13 mRNA expression levels correlate with poor outcome in patients who received tamoxifen treatment in the metastatic setting. In addition, AKAP13 mRNA levels correlate with ERαSerine 305 phosphorylation in breast tumor samples, suggesting a functional connection between these two events. In a luminal breast cancer cell line, AKAP13 was found to interact with ERα as well as with a regulatory subunit of PKA. Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process.


CONCLUSIONS
We show that the PKA-anchoring protein AKAP13 is essential for the phosphorylation of ERαS305, which leads to tamoxifen resistance both in cell lines and tamoxifen-treated breast cancer patients.
Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. c.bentin.toaldo@nki.nl.
Cristiane
C
Bentin Toaldo
Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. x.alexi@nki.nl.
Xanthippi
X
Alexi
Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. k.beelen@nki.nl.
Karin
K
Beelen
Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. m.kok@nki.nl.
Marleen
M
Kok
Division of Psychosocial Research and Epidemiology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. m.hauptmann@nki.nl.
Michael
M
Hauptmann
Department of Medical Oncology, Josephine Nefkens Institute and Cancer Genomics Center, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands. m.p.h.m.jansen@erasmusmc.nl.
Maurice
M
Jansen
Department of Medical Oncology, Josephine Nefkens Institute and Cancer Genomics Center, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands. p.berns@erasmusmc.nl.
Els
E
Berns
Division of Cell Biology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. j.neefjes@nki.nl.
Jacques
J
Neefjes
Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. s.linn@nki.nl.|Department of Medical Oncology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. s.linn@nki.nl.
Sabine
S
Linn
Division of Cell Biology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. michalides10@gmail.com.
Rob
R
Michalides
Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. w.zwart@nki.nl.
Wilbert
W
Zwart
D054758:A Kinase Anchor Proteins / Q000378:metabolism*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D015778:Minor Histocompatibility Antigens; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013629:Tamoxifen / Q000494:pharmacology*
D054758:A Kinase Anchor Proteins; C085467:AKAP13 protein, human; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D015778:Minor Histocompatibility Antigens; D011518:Proto-Oncogene Proteins; D013629:Tamoxifen; D017868:Cyclic AMP-Dependent Protein Kinases