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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 tamoxifen-driven gene expression in PKA-activated cells. a Activation of the PKA pathway by shRNA-PKA-RIα. Western blot analysis of ERαS305P, ERα, phospho-CREB and PKA-RIα. Actin was used as loading control. b PKA-RIα knockdown gives rise to tamoxifen resistance. Control (white) or PKA-RIα knockdown (black) cells were used. An MTT assay was performed after cells were seeded in hormone-deprived medium (t = 0) and cultured for a week in the absence or presence of tamoxifen. Error bars indicate SD values from six independent measurements. c RT-QPCR analyses for Control (left) or shPKA-RIα (right) cells. Cells were hormone-deprived and transfected with siCntrl (white) or siAKAP13 (black). Three days after transfection, cells were incubated with estradiol (E2), tamoxifen (Tam), Fulvestrant (ICI) or DMSO control (veh). Expression levels of XBP-1 (top) and TFF1 (bottom) were analysed. Error bars indicate SD values from 3 independent measurements
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Fig4: AKAP13 is required for tamoxifen-driven gene expression in PKA-activated cells. a Activation of the PKA pathway by shRNA-PKA-RIα. Western blot analysis of ERαS305P, ERα, phospho-CREB and PKA-RIα. Actin was used as loading control. b PKA-RIα knockdown gives rise to tamoxifen resistance. Control (white) or PKA-RIα knockdown (black) cells were used. An MTT assay was performed after cells were seeded in hormone-deprived medium (t = 0) and cultured for a week in the absence or presence of tamoxifen. Error bars indicate SD values from six independent measurements. c RT-QPCR analyses for Control (left) or shPKA-RIα (right) cells. Cells were hormone-deprived and transfected with siCntrl (white) or siAKAP13 (black). Three days after transfection, cells were incubated with estradiol (E2), tamoxifen (Tam), Fulvestrant (ICI) or DMSO control (veh). Expression levels of XBP-1 (top) and TFF1 (bottom) were analysed. Error bars indicate SD values from 3 independent measurements

Mentions: AKAP13 interacts with ERα and is required for ERαS305 phosphorylation. Does AKAP13 knockdown also result in a decrease of ERα-driven gene expression in tamoxifen-resistant cells? For this, we generated an MCF-7 derivative cell line that is tamoxifen-resistant through enhanced PKA activity. An shRNA was used targeting the regulatory subunit of PKA, PKA-RIα, as was performed before [9]. Knocking down PKA-RIα activated the PKA pathway, as shown by a phosphorylation of CREB, and increased ERαS305P levels (Fig. 4a). Furthermore, while control cells could be effectively blocked in their cell proliferation using tamoxifen, this was not the case when PKA-RIα was knocked down (Fig. 4b). Now that we have established a PKA-driven tamoxifen resistant cell line, the next question was whether the agonistic features of tamoxifen could be blocked by targeting AKAP13. Since PKA activation stimulates cell proliferation both in an ERα-dependent as well as an ERα-independent fashion [9], interpretation of cell proliferation in shPKA-RIα cells following siAKAP13 may be challenging. To focus the analysis on ERα-functioning in the context of PKA-driven tamoxifen resistance, we therefore decided to perform RT-QPCR for two well-annotated ERα-responsive genes: TFF1 and XBP1 (Fig. 4c). For both these genes, shPKA-RIα knockdown greatly increased ERα action. Importantly, siAKAP13 did not affect TFF1 and XBP1 expression in control tamoxifen-treated cells, where expression levels were comparable to those found for the full ERα antagonist Fulvestrant (ICI). In cells with PKA-RIα knockdown, XBP1 and TFF1 levels were considerably higher under tamoxifen conditions as compared to Fulvestrant conditions. Targeting AKAP13 however decreased TFF1 and XBP1 levels in tamoxifen-treated shPKA-RIα cells, now comparable to those levels found under Fulvestrant conditions. These data indicate that activation of PKA increases ERα-driven gene expression under tamoxifen conditions, which can be reverted by knocking down AKAP13.Fig. 4


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 tamoxifen-driven gene expression in PKA-activated cells. a Activation of the PKA pathway by shRNA-PKA-RIα. Western blot analysis of ERαS305P, ERα, phospho-CREB and PKA-RIα. Actin was used as loading control. b PKA-RIα knockdown gives rise to tamoxifen resistance. Control (white) or PKA-RIα knockdown (black) cells were used. An MTT assay was performed after cells were seeded in hormone-deprived medium (t = 0) and cultured for a week in the absence or presence of tamoxifen. Error bars indicate SD values from six independent measurements. c RT-QPCR analyses for Control (left) or shPKA-RIα (right) cells. Cells were hormone-deprived and transfected with siCntrl (white) or siAKAP13 (black). Three days after transfection, cells were incubated with estradiol (E2), tamoxifen (Tam), Fulvestrant (ICI) or DMSO control (veh). Expression levels of XBP-1 (top) and TFF1 (bottom) were analysed. Error bars indicate SD values from 3 independent measurements
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4536754&req=5

Fig4: AKAP13 is required for tamoxifen-driven gene expression in PKA-activated cells. a Activation of the PKA pathway by shRNA-PKA-RIα. Western blot analysis of ERαS305P, ERα, phospho-CREB and PKA-RIα. Actin was used as loading control. b PKA-RIα knockdown gives rise to tamoxifen resistance. Control (white) or PKA-RIα knockdown (black) cells were used. An MTT assay was performed after cells were seeded in hormone-deprived medium (t = 0) and cultured for a week in the absence or presence of tamoxifen. Error bars indicate SD values from six independent measurements. c RT-QPCR analyses for Control (left) or shPKA-RIα (right) cells. Cells were hormone-deprived and transfected with siCntrl (white) or siAKAP13 (black). Three days after transfection, cells were incubated with estradiol (E2), tamoxifen (Tam), Fulvestrant (ICI) or DMSO control (veh). Expression levels of XBP-1 (top) and TFF1 (bottom) were analysed. Error bars indicate SD values from 3 independent measurements
Mentions: AKAP13 interacts with ERα and is required for ERαS305 phosphorylation. Does AKAP13 knockdown also result in a decrease of ERα-driven gene expression in tamoxifen-resistant cells? For this, we generated an MCF-7 derivative cell line that is tamoxifen-resistant through enhanced PKA activity. An shRNA was used targeting the regulatory subunit of PKA, PKA-RIα, as was performed before [9]. Knocking down PKA-RIα activated the PKA pathway, as shown by a phosphorylation of CREB, and increased ERαS305P levels (Fig. 4a). Furthermore, while control cells could be effectively blocked in their cell proliferation using tamoxifen, this was not the case when PKA-RIα was knocked down (Fig. 4b). Now that we have established a PKA-driven tamoxifen resistant cell line, the next question was whether the agonistic features of tamoxifen could be blocked by targeting AKAP13. Since PKA activation stimulates cell proliferation both in an ERα-dependent as well as an ERα-independent fashion [9], interpretation of cell proliferation in shPKA-RIα cells following siAKAP13 may be challenging. To focus the analysis on ERα-functioning in the context of PKA-driven tamoxifen resistance, we therefore decided to perform RT-QPCR for two well-annotated ERα-responsive genes: TFF1 and XBP1 (Fig. 4c). For both these genes, shPKA-RIα knockdown greatly increased ERα action. Importantly, siAKAP13 did not affect TFF1 and XBP1 expression in control tamoxifen-treated cells, where expression levels were comparable to those found for the full ERα antagonist Fulvestrant (ICI). In cells with PKA-RIα knockdown, XBP1 and TFF1 levels were considerably higher under tamoxifen conditions as compared to Fulvestrant conditions. Targeting AKAP13 however decreased TFF1 and XBP1 levels in tamoxifen-treated shPKA-RIα cells, now comparable to those levels found under Fulvestrant conditions. These data indicate that activation of PKA increases ERα-driven gene expression under tamoxifen conditions, which can be reverted by knocking down AKAP13.Fig. 4

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