Limits...
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

Molecular pathway enrichment in ERαS305P positive tumors. a Tissue microarrays, stained for ERαS305P were analysed and subgrouped into staining negative (left panel) or positive (right panel) for ERαS305P signal. Expression array data from these subgroups were clustered and pathway enrichement analyses were performed on the expression array data from each subgroup. b AKAP13 and AKAP95 were found to be the most significantly enriched pathways
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4536754&req=5

Fig1: Molecular pathway enrichment in ERαS305P positive tumors. a Tissue microarrays, stained for ERαS305P were analysed and subgrouped into staining negative (left panel) or positive (right panel) for ERαS305P signal. Expression array data from these subgroups were clustered and pathway enrichement analyses were performed on the expression array data from each subgroup. b AKAP13 and AKAP95 were found to be the most significantly enriched pathways

Mentions: Serine 305 phosphorylation on ERα was found to be a predictive marker for tamoxifen resistance in breast cancer patients [14, 15]. To define which cell biological factors may be causally involved in the PKA-induced phosphorylation of ERαS305, we performed pathway enrichment analyses from a cohort of breast cancer patients, which received tamoxifen for metastatic disease [14, 15]. The samples were stained for ERαS305P and scored for positivity by immunohistochemistry (Fig. 1a). Available expression array data [15] was reanalyzed using Biocarta pathway analysis. Among the 19 pathways differentially and significantly enriched in these two patient groups, five were found to be PKA-related. The top two differentially regulated pathways involved AKAP13 and AKAP95 signaling cascades. Since both of these proteins are members of the PKA-anchoring protein family, PKA-substrate specificity regulated by AKAP levels may be causally linked with ERαS305 phosphorylation status, and thus tamoxifen resistance.Fig. 1


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)

Molecular pathway enrichment in ERαS305P positive tumors. a Tissue microarrays, stained for ERαS305P were analysed and subgrouped into staining negative (left panel) or positive (right panel) for ERαS305P signal. Expression array data from these subgroups were clustered and pathway enrichement analyses were performed on the expression array data from each subgroup. b AKAP13 and AKAP95 were found to be the most significantly enriched pathways
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536754&req=5

Fig1: Molecular pathway enrichment in ERαS305P positive tumors. a Tissue microarrays, stained for ERαS305P were analysed and subgrouped into staining negative (left panel) or positive (right panel) for ERαS305P signal. Expression array data from these subgroups were clustered and pathway enrichement analyses were performed on the expression array data from each subgroup. b AKAP13 and AKAP95 were found to be the most significantly enriched pathways
Mentions: Serine 305 phosphorylation on ERα was found to be a predictive marker for tamoxifen resistance in breast cancer patients [14, 15]. To define which cell biological factors may be causally involved in the PKA-induced phosphorylation of ERαS305, we performed pathway enrichment analyses from a cohort of breast cancer patients, which received tamoxifen for metastatic disease [14, 15]. The samples were stained for ERαS305P and scored for positivity by immunohistochemistry (Fig. 1a). Available expression array data [15] was reanalyzed using Biocarta pathway analysis. Among the 19 pathways differentially and significantly enriched in these two patient groups, five were found to be PKA-related. The top two differentially regulated pathways involved AKAP13 and AKAP95 signaling cascades. Since both of these proteins are members of the PKA-anchoring protein family, PKA-substrate specificity regulated by AKAP levels may be causally linked with ERαS305 phosphorylation status, and thus tamoxifen resistance.Fig. 1

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