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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 mRNA levels, survival and ERαS305P. a AKAP13 expression levels were analyzed and correlated with progression-free survival of a cohort of breast cancer patients treated for metastatic disease. b Tumors were stratified on the basis of ERαS305P positivity, the AKAP13 mRNA levels of each sample were analyzed. A Student’s T-test was performed; p = 0.06
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Fig3: AKAP13 mRNA levels, survival and ERαS305P. a AKAP13 expression levels were analyzed and correlated with progression-free survival of a cohort of breast cancer patients treated for metastatic disease. b Tumors were stratified on the basis of ERαS305P positivity, the AKAP13 mRNA levels of each sample were analyzed. A Student’s T-test was performed; p = 0.06

Mentions: Our findings indicated that expression of AKAP13 in tissue culture experiments is essential for the PKA-induced Serine 305 phosphorylation. Next, we investigated whether AKAP13 levels by themselves correlate with an unfavorable response to treatment in breast cancer patients. Therefore, tumor samples were analyzed, in which patients with metastatic disease were treated with tamoxifen. Time to tumor progression (TTP) was estimated according to the Kaplan–Meier method for AKAP13 expression, segmenting the continuous variable in two groups (low and high) with equal numbers of patients (Fig. 3a). Four different AKAP13 probes were available from the Agilent 44 K array data, and identical analyses were performed for all the remaining probes (Additional file 3: Figure S3). Of these four probes, 3 had significant log rank tests. In addition, we tested the four different AKAP13 probes for trend, using the significance of the coefficient for the continuous AKAP13 variable (Additional file 4: Table S1) and found a significant test for trend for probe 1 (p = 0.022). We then compared clinico-pathological characteristics for two subgroups (low and high) of this probe, Table 1. AKAP13 high tumours were more often lymph node positive (p = 0.033) and there was a trend towards negative progesterone receptor status (p = 0.071). Using a univariate Cox proportional hazard model we found a hazard ratio of 2.10 for patients with high AKAP13 levels (p = 0.007). Other clinico-pathological characteristics that were significant in the univariate analysis were ERα level (HR 0.20, p = 0.0002) and PR status (HR 0.45 p = 0.004) (Additional file 4: Table S2). Combining these variables in the multivariate analysis resulted in a hazard ratio for AKAP13 of 1.84 (p = 0.031) (Additional file 4: Table S3).Fig. 3


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 mRNA levels, survival and ERαS305P. a AKAP13 expression levels were analyzed and correlated with progression-free survival of a cohort of breast cancer patients treated for metastatic disease. b Tumors were stratified on the basis of ERαS305P positivity, the AKAP13 mRNA levels of each sample were analyzed. A Student’s T-test was performed; p = 0.06
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: AKAP13 mRNA levels, survival and ERαS305P. a AKAP13 expression levels were analyzed and correlated with progression-free survival of a cohort of breast cancer patients treated for metastatic disease. b Tumors were stratified on the basis of ERαS305P positivity, the AKAP13 mRNA levels of each sample were analyzed. A Student’s T-test was performed; p = 0.06
Mentions: Our findings indicated that expression of AKAP13 in tissue culture experiments is essential for the PKA-induced Serine 305 phosphorylation. Next, we investigated whether AKAP13 levels by themselves correlate with an unfavorable response to treatment in breast cancer patients. Therefore, tumor samples were analyzed, in which patients with metastatic disease were treated with tamoxifen. Time to tumor progression (TTP) was estimated according to the Kaplan–Meier method for AKAP13 expression, segmenting the continuous variable in two groups (low and high) with equal numbers of patients (Fig. 3a). Four different AKAP13 probes were available from the Agilent 44 K array data, and identical analyses were performed for all the remaining probes (Additional file 3: Figure S3). Of these four probes, 3 had significant log rank tests. In addition, we tested the four different AKAP13 probes for trend, using the significance of the coefficient for the continuous AKAP13 variable (Additional file 4: Table S1) and found a significant test for trend for probe 1 (p = 0.022). We then compared clinico-pathological characteristics for two subgroups (low and high) of this probe, Table 1. AKAP13 high tumours were more often lymph node positive (p = 0.033) and there was a trend towards negative progesterone receptor status (p = 0.071). Using a univariate Cox proportional hazard model we found a hazard ratio of 2.10 for patients with high AKAP13 levels (p = 0.007). Other clinico-pathological characteristics that were significant in the univariate analysis were ERα level (HR 0.20, p = 0.0002) and PR status (HR 0.45 p = 0.004) (Additional file 4: Table S2). Combining these variables in the multivariate analysis resulted in a hazard ratio for AKAP13 of 1.84 (p = 0.031) (Additional file 4: Table S3).Fig. 3

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