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Protein Kinase A-Induced Tamoxifen Resistance Is Mediated By Bentin Toaldo et al. BMC Cancer (2015) 15:588 DOI 10.1186/s12885-015-1591-4 RESEARCH ARTICLE Open Access Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13 Cristiane Bentin Toaldo1, Xanthippi Alexi1, Karin Beelen1, Marleen Kok1, Michael Hauptmann2, Maurice Jansen3, Els Berns3, Jacques Neefjes4, Sabine Linn1,5, Rob Michalides4 and Wilbert Zwart1* Abstract 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. Background treatment is tamoxifen. Tamoxifen competes with ERα’s Breast cancer is the most frequently diagnosed malig- natural ligand estradiol for binding to the ligand-binding nancy in women. Since 75 % of all breast tumors ex- pocket of the receptor. Tamoxifen forces the receptor in press Estrogen Receptor alpha (ERα), tumor growth is an alternative conformation as compared to estradiol, considered to be dependent on the activity of this thereby preventing recruitment of coregulators to the hormone-induced transcription factor. Thereby, treat- complex, which are essential for ERα-driven transcription ment is focused on inhibiting the function of ERα.One [1]. Although tamoxifen is considered a highly successful of the most frequently prescribed drugs in endocrine drug, resistance to treatment is common. Resistance to tamoxifen treatment can occur through a multitude of * Correspondence: [email protected] 1 mechanisms, including activation of the MAP kinase Division of Molecular Pathology, the Netherlands Cancer Institute, – Amsterdam, The Netherlands pathway [2 4] or overexpression of PAK1 [5], SRC1 [6], Full list of author information is available at the end of the article SRC3 [7] and ErbB2 [8]. © 2015 Bentin Toaldo et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bentin Toaldo et al. BMC Cancer (2015) 15:588 Page 2 of 12 An alternative mechanism of tamoxifen resistance is me- way, activated PKA can act locally and directly on its sub- diated by activation of the Protein Kinase A (PKA) path- strates, thereby orchestrating substrate specificity. AKAPs way [9]. Decreased expression of a regulatory component possess a PKA-anchoring domain composed of a 14–18 of the PKA complex, PKA-RIα, was found to correlate with residue long amphipathic helix [22], by which they interact a non-favorable prognosis in breast cancer patients treated withahydrophobicgrooveformedby4alpha-helicalstruc- with tamoxifen [10]. We could confirm these data [9], and tures on the PKA-RII subunit [23–26]. At least 50 AKAPs found in vitro that PKA-RIα knockdown enhances breast have been identified, with varying expression levels among cancer cell proliferation in the presence of tamoxifen. In different tissues and with their own unique intracellular addition, we showed in the same study that the major site localizations [27]. A number of these AKAPs have been PKA-responsive phosphorylation site on ERα is a serine reported to correlate with the occurrence of different can- residue found at position 305. This phosphorylation leads cer subtypes, including AKAP3 (ovarian cancer) [28, 29], to a conformational arrest within the receptor and results AKAP4 (multiple myeloma) [30], AKAP9 (breast cancer) in an agonistic response of the otherwise inhibitory com- [31], AKAP10 (breast cancer) [32] and AKAP13 (colorec- pound tamoxifen. In this altered conformation ERα re- tal cancer [33] and breast cancer [34]). Thus far, it remains orientates its C-terminus towards its coactivators, which unknown which AKAP family member is responsible for prevents a dissociation of RNA Polymerase II from the enabling the PKA-induced phosphorylation of Serine 305 complex that is normally observed in tamoxifen treated on ERα, resulting in tamoxifen resistance. cells, thereby resulting in transcriptional activation in the Here, we studied the enrichment of PKA-associated mo- presence of tamoxifen [11]. PKA-stimulated MCF-7 breast lecular pathways in a cohort of breast cancer patients, cancer cells express a unique repertoire of genes that are which received tamoxifen in a metastatic setting. Stratify- differentially expressed as compared to tamoxifen or PKA- ing tumors on ERαS305 phosphorylation status resulted in activating cAMP treatment alone. [12] This potentially un- the identification of a molecular pathway involving derlies a cell biological response for this pathway to tam- AKAP13, suggesting a functional link between AKAP13 oxifen resistance. In accordance, we recently reported S305 levels and PKA-induced ERα phosphorylation. AKAP13 phosphorylated ERα to bind a unique set of promoters expression levels correlated with a poor outcome after regulating transcriptional programs involved in tamoxifen tamoxifen treatment in breast cancer patients and corre- resistance [13]. lated with ERαS305 phosphorylation status. In vitro experi- In addition to in vitro data, in breast cancer patients ments could illustrate that AKAP13 interacts with ERα as treated with tamoxifen for metastatic disease, the phos- well as the regulatory subunit of PKA, and that AKAP13 phorylation status of ERαS305 was found to be indicative expression is essential for PKA-induced ERαS305 phos- for poor outcome [14]. Moreover, ERαS305 phosphoryl- phorylation. In summary, we demonstrate here that the ation was found to be a predictive marker for tamoxifen re- PKA-mediated phosphorylation of ERα at Serine 305, sistance in pre- [14, 15] and postmenopausal patients [16]. which leads to tamoxifen resistance, requires the PKA- However, it remains elusive what regulates the ERα/PKA anchoring protein AKAP13. interaction, which is an essential step in the ERαS305 phosphorylation pathway. Methods PKA is a multi-protein complex. The inactivated PKA Tissue culture, plasmids, antibodies, shRNA and siRNA complex is composed of a catalytic subunit (PKAcat) that MCF-7 cells were cultured in DMEM medium, supple- associates, in an inactive state, to its regulatory subunits mented with 8 % fetal-bovine serum and standard antibi- (PKA-RI and RII). After the regulatory subunits bind otics. The following antibodieswereused:AKAP13(Bethyl cAMP, the catalytic subunit can dissociate from the com- A301-404A-1), ERα (Santa Cruz sc-543), ERαS305P plex to phosphorylate its substrates. There are many known (Millipore, clone 124.9.4), PKA RII (Abcam ab57414), PKA PKA targets, and in order to achieve substrate-specificity, catalytic-α (Cell Signalling 4782), beta-actin (Abcam, PKA activity needs to be locally confined. Localized PKA ab8229) and AKAP95 (Bethyl, A301-061A). 3 days prior to activation can occur in multiple ways. Intracellularly, cAMP cell biological analyses, cells were switched to phenol-red levels are unevenly and dynamically distributed, [17, 18] free DMEM, supplemented with 5 % charcoal-treated which can partly be explained by the tethering of phospho- serum. For siRNA targeting of AKAP13 and AKAP95, a diesterases (PDEs) at distinct subcellular
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