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Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13.

Bentin Toaldo C, Alexi X, Beelen K, Kok M, Hauptmann M, Jansen M, Berns E, Neefjes J, Linn S, Michalides R, Zwart W - BMC Cancer (2015)

Bottom Line: Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. c.bentin.toaldo@nki.nl.

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.

No MeSH data available.


Related in: MedlinePlus

AKAP13 is required for PKA-induced ERαS305 phosphorylation and interacts with ERα and PKA-RII. a siRNA targeting AKAP13 prevents PKA-induced ERαS305 phosphorylation. MCF-7 breast cancer cells were transfected with an siRNA targeting AKAP13 or a control siRNA, after which the cells were treated for 1 h with 10 μM forskolin or left untreated. Samples were analysed by SDS-PAGE and Westernblotting, probing with antibodies detecting AKAP13, ERαS305P, ERα or actin as a loading control. b AKAP13 is required for a forskolin-enhanced ERα/PKA-cat interaction. Estrogen Receptor-negative U2OS cells were transfected with CFP-tagged ERα, YFP-tagged PKA catalytic subunit and a non-tagged PKA regulatory RII subunit. In addition, cells were transfected with an siRNA targeting AKAP13 or a control siRNA. Energy transfer from the CFP to the YFP fluorophore was measured in the same cell before and after 1 h of 10 μM forskolin treatment, and the average value prior to treatment was set on 1. N > 10. Bars indicate SEM. A student’s T-test was performed; p < 0.05. c, d, e AKAP13, ERα and PKA-RII form a complex. MCF-7 cells were hormone deprived for 3 days to deplete activated ERα transcriptional processes. Following that, cells were lysed for immunoprecipitations, directed at PKA-RII (c), AKAP13 (d), ERα (e) or a negative control protein. In addition, input and supernatant (sup) samples were taken. Western blots were probed with antibodies detecting AKAP13, ERα and PKA-RII
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Fig2: AKAP13 is required for PKA-induced ERαS305 phosphorylation and interacts with ERα and PKA-RII. a siRNA targeting AKAP13 prevents PKA-induced ERαS305 phosphorylation. MCF-7 breast cancer cells were transfected with an siRNA targeting AKAP13 or a control siRNA, after which the cells were treated for 1 h with 10 μM forskolin or left untreated. Samples were analysed by SDS-PAGE and Westernblotting, probing with antibodies detecting AKAP13, ERαS305P, ERα or actin as a loading control. b AKAP13 is required for a forskolin-enhanced ERα/PKA-cat interaction. Estrogen Receptor-negative U2OS cells were transfected with CFP-tagged ERα, YFP-tagged PKA catalytic subunit and a non-tagged PKA regulatory RII subunit. In addition, cells were transfected with an siRNA targeting AKAP13 or a control siRNA. Energy transfer from the CFP to the YFP fluorophore was measured in the same cell before and after 1 h of 10 μM forskolin treatment, and the average value prior to treatment was set on 1. N > 10. Bars indicate SEM. A student’s T-test was performed; p < 0.05. c, d, e AKAP13, ERα and PKA-RII form a complex. MCF-7 cells were hormone deprived for 3 days to deplete activated ERα transcriptional processes. Following that, cells were lysed for immunoprecipitations, directed at PKA-RII (c), AKAP13 (d), ERα (e) or a negative control protein. In addition, input and supernatant (sup) samples were taken. Western blots were probed with antibodies detecting AKAP13, ERα and PKA-RII

Mentions: Since AKAP13 and AKAP95 signaling cascades were significantly enriched in the ERαS305P positive patient subgroup, we tested in vitro whether this enrichment also implies a causal role for either of these two proteins in PKA-mediated ERα Serine 305 phosphorylation. siRNA-mediated knockdown of AKAP13 and AKAP95 was performed in MCF-7 breast cancer cells (Fig. 2a and Additional file 1: Figure S1, respectively). Cells were either treated or not treated with the PKA-activating compound forskolin for 1 h prior to cell lysis. As expected and previously observed [39], PKA activation by forskolin treatment sufficed to induce phosphorylation of ERα on Serine 305. Knocking down AKAP13 prevented the forskolin-induced increase in ERαS305P signal, implying that AKAP13 is required for the phosphorylation of ERα at this site. This effect was not observed after knock down AKAP95, and Serine 305 on ERα could still be phosphorylated upon PKA activation (Additional file 1: Figure S1). Furthermore, both AKAP13 as well as AKAP95 were not essential for MCF-7 cell proliferation arrest after tamoxifen treatment while proliferation under E2 conditions was slightly increased (Additional file 2: Figure S2). These data suggest that AKAP13 is not a crucial player in normal ERα biology and tamoxifen-response in absence of PKA activation.Fig. 2


Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13.

Bentin Toaldo C, Alexi X, Beelen K, Kok M, Hauptmann M, Jansen M, Berns E, Neefjes J, Linn S, Michalides R, Zwart W - BMC Cancer (2015)

AKAP13 is required for PKA-induced ERαS305 phosphorylation and interacts with ERα and PKA-RII. a siRNA targeting AKAP13 prevents PKA-induced ERαS305 phosphorylation. MCF-7 breast cancer cells were transfected with an siRNA targeting AKAP13 or a control siRNA, after which the cells were treated for 1 h with 10 μM forskolin or left untreated. Samples were analysed by SDS-PAGE and Westernblotting, probing with antibodies detecting AKAP13, ERαS305P, ERα or actin as a loading control. b AKAP13 is required for a forskolin-enhanced ERα/PKA-cat interaction. Estrogen Receptor-negative U2OS cells were transfected with CFP-tagged ERα, YFP-tagged PKA catalytic subunit and a non-tagged PKA regulatory RII subunit. In addition, cells were transfected with an siRNA targeting AKAP13 or a control siRNA. Energy transfer from the CFP to the YFP fluorophore was measured in the same cell before and after 1 h of 10 μM forskolin treatment, and the average value prior to treatment was set on 1. N > 10. Bars indicate SEM. A student’s T-test was performed; p < 0.05. c, d, e AKAP13, ERα and PKA-RII form a complex. MCF-7 cells were hormone deprived for 3 days to deplete activated ERα transcriptional processes. Following that, cells were lysed for immunoprecipitations, directed at PKA-RII (c), AKAP13 (d), ERα (e) or a negative control protein. In addition, input and supernatant (sup) samples were taken. Western blots were probed with antibodies detecting AKAP13, ERα and PKA-RII
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Fig2: AKAP13 is required for PKA-induced ERαS305 phosphorylation and interacts with ERα and PKA-RII. a siRNA targeting AKAP13 prevents PKA-induced ERαS305 phosphorylation. MCF-7 breast cancer cells were transfected with an siRNA targeting AKAP13 or a control siRNA, after which the cells were treated for 1 h with 10 μM forskolin or left untreated. Samples were analysed by SDS-PAGE and Westernblotting, probing with antibodies detecting AKAP13, ERαS305P, ERα or actin as a loading control. b AKAP13 is required for a forskolin-enhanced ERα/PKA-cat interaction. Estrogen Receptor-negative U2OS cells were transfected with CFP-tagged ERα, YFP-tagged PKA catalytic subunit and a non-tagged PKA regulatory RII subunit. In addition, cells were transfected with an siRNA targeting AKAP13 or a control siRNA. Energy transfer from the CFP to the YFP fluorophore was measured in the same cell before and after 1 h of 10 μM forskolin treatment, and the average value prior to treatment was set on 1. N > 10. Bars indicate SEM. A student’s T-test was performed; p < 0.05. c, d, e AKAP13, ERα and PKA-RII form a complex. MCF-7 cells were hormone deprived for 3 days to deplete activated ERα transcriptional processes. Following that, cells were lysed for immunoprecipitations, directed at PKA-RII (c), AKAP13 (d), ERα (e) or a negative control protein. In addition, input and supernatant (sup) samples were taken. Western blots were probed with antibodies detecting AKAP13, ERα and PKA-RII
Mentions: Since AKAP13 and AKAP95 signaling cascades were significantly enriched in the ERαS305P positive patient subgroup, we tested in vitro whether this enrichment also implies a causal role for either of these two proteins in PKA-mediated ERα Serine 305 phosphorylation. siRNA-mediated knockdown of AKAP13 and AKAP95 was performed in MCF-7 breast cancer cells (Fig. 2a and Additional file 1: Figure S1, respectively). Cells were either treated or not treated with the PKA-activating compound forskolin for 1 h prior to cell lysis. As expected and previously observed [39], PKA activation by forskolin treatment sufficed to induce phosphorylation of ERα on Serine 305. Knocking down AKAP13 prevented the forskolin-induced increase in ERαS305P signal, implying that AKAP13 is required for the phosphorylation of ERα at this site. This effect was not observed after knock down AKAP95, and Serine 305 on ERα could still be phosphorylated upon PKA activation (Additional file 1: Figure S1). Furthermore, both AKAP13 as well as AKAP95 were not essential for MCF-7 cell proliferation arrest after tamoxifen treatment while proliferation under E2 conditions was slightly increased (Additional file 2: Figure S2). These data suggest that AKAP13 is not a crucial player in normal ERα biology and tamoxifen-response in absence of PKA activation.Fig. 2

Bottom Line: Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. c.bentin.toaldo@nki.nl.

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.

No MeSH data available.


Related in: MedlinePlus