Endoxifen downregulates AKT phosphorylation through protein kinase C beta 1 inhibition in ERα+ breast cancer

Abstract

Endoxifen, a secondary tamoxifen metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERα) binding at nanomolar concentrations. Phase I/II clinical trials identified clinical activity of Z-endoxifen (ENDX), in endocrine-refractory metastatic breast cancer as well as ERα+ solid tumors, raising the possibility that ENDX may have a second, ERα-independent, mechanism of action. An unbiased mass spectrometry approach revealed that ENDX concentrations achieved clinically with direct ENDX administration (5 µM), but not low concentrations observed during tamoxifen treatment (<0.1 µM), profoundly altered the phosphoproteome of the aromatase expressing MCF7AC1 cells with limited impact on the total proteome. Computational analysis revealed protein kinase C beta (PKCβ) and protein kinase B alpha or AKT1 as potential kinases responsible for mediating ENDX effects on protein phosphorylation. ENDX more potently inhibited PKCβ1 kinase activity compared to other PKC isoforms, and ENDX binding to PKCβ1 was confirmed using Surface Plasma Resonance. Under conditions that activated PKC/AKT signaling, ENDX induced PKCβ1 degradation, attenuated PKCβ1-activated AKT^Ser473 phosphorylation, diminished AKT substrate phosphorylation, and induced apoptosis. ENDX's effects on AKT were phenocopied by siRNA-mediated PKCβ1 knockdown or treatment with the pan-AKT inhibitor, MK-2206, while overexpression of constitutively active AKT diminished ENDX-induced apoptosis. These findings, which identify PKCβ1 as an ENDX target, indicate that PKCβ1/ENDX interactions suppress AKT signaling and induce apoptosis in breast cancer.

Fig. 1. Z-endoxifen (ENDX) effects on cell viability and apoptosis in estrogen deprived ERα+ /HER2- MCF7AC1 cells.

a Cells grown in CSS medium were treated with vehicle control or the indicated ENDX concentrations for 48 h. Cell viability was assessed by the crystal violet assay. b Cells were co-treated with vehicle control or the indicated ENDX concentrations, IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 h. The apoptosis (%) graphs are presented as the green object count (which correspond to cells that are stained with the IncuCyte green fluorescence Annexin V reagent) divided by the red object count (which correspond to the total number of cells in the culture that are stained with the IncuCyte red fluorescence Nuclight Rapid Red Cell Labeling reagent that labels the nucleus of all cells without perturbing cell function or biology) and displayed as percentage using the IncuCyte S3 analysis software. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 by one-way ANOVA.

Fig. 2. A schematic depicting the strategy used for quantitative proteomic and phosphoproteomic profiling of ENDX-treated MCF7AC1 cells.

All experiments were performed in triplicate. Cells maintained in CSS medium were treated with ETOH vehicle control or ENDX at specified dosages for 24 h. Following treatments, cells were harvested and lysed in 8 M urea buffer, followed by trypsin digestion, desalting and TMT labeling. The labeled peptides were fractionated, and phosphopeptides were enriched with immobilized metal affinity chromatography (IMAC) approach. Both fractionated peptides and IMAC-enriched phosphopeptides were analyzed by Orbitrap Lumos mass spectrometer.

Fig. 3. Effects of ENDX on the phosphoproteome of MCF7AC1 cells.

a A pie chart showing the distribution of identified phosphorylation sites. Volcano plots showing the total number of phosphosites, and the percentage that are upregulated (right side) and downregulated (left side) (Fold change (FC) |1.5|; p < 0.05) in cells treated for 24 h in CSS medium with 0.01 (b), 0.1 (c) or 5 μM (d) ENDX relative to vehicle treated cells, as detected by mass spectrometry analysis. e Venn diagram indicating the overlap of upregulated (pink) and downregulated (blue) phosphosites in ENDX-treated cells relative to vehicle treated cells. f Heatmap indicating relative abundance of the phosphosites analyzed in the ENDX-treated cells relative to vehicle treated cells. The hierarchical clustering of phosphosites is shown on the left.

Fig. 4. Kinase enrichment analysis predicts AKT signaling is regulated by high-dose ENDX.

210 phospho-regulated proteins from 0.01 μM (a), 224 phospho-regulated proteins from 0.1 μM (b), and 347 phospho-regulated proteins from 5 μM (c) ENDX treated cells were respectively used for KEA3 upstream kinase analysis. Left panels: Integrated rankings of most enriched kinases across libraries based on the MeanRank. The stacking bar chart shows the summation of the MeanRank derived from the libraries used for the KEA analysis. The libraries are color-coded. Right panels: Kinase co-regulatory networks constructed from kinase-kinase interactions between top-ranked kinase results for the integrated rankings. Directed edges indicate interactions supported by kinase-substrate evidence.

Fig. 5. Downregulated phosphosites following ENDX treatment are enriched for PKCβ, CDK1, and AKT target sequences.

a Fuzzy c-means clustering showing the classification of ENDX treatment effects on the phosphoproteome into three regulatory clusters. Cluster 1 represents phosphosites downregulated by ENDX in a dose-dependent manner. Cluster 2 represents phosphosites that are upregulated by ENDX at all concentrations examined. Cluster 3 represents phosphosites downregulated at 0.01 μM concentration but mostly unaffected at the 0.1 and 5 μM concentrations. b Molecular and cellular pathways potentially impacted by ENDX. Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis of the phosphosites altered by ENDX in cluster 1 showing the top biological pathways associated with these phosphosites. c Enriched phosphorylation motifs identified in cluster 1 phosphosites. d Graph showing the frequency of kinases known to phosphorylate cluster 1 phosphosites that were depleted by ENDX as assessed using NetworKIN and RoKAI prediction tools.

Fig. 6. ENDX specifically downregulates pAKT^Ser473 at 5 µM and inhibits PKCβ1 kinase activity.

a MCF7AC1 cells in CSS medium were treated for 24 h with vehicle control or 0.01, 0.1, and 5 µM ENDX. Immunoblot assays of pAKT^Ser473, pAKT^Thr308, AKT and p-AKT substrates are shown with β-actin as a loading control. b Serum starved MCF7AC1 cells were pretreated with vehicle control, 0.01, 0.1, 5 µM ENDX, 0.1 µM tamoxifen (TAM) or 0.1 µM ICI-182780 (ICI) followed by the addition of 100 nM insulin for 1 h as indicated. IB assays of pAKT^Ser473, pAKT^Thr308, AKT and β-actin are shown. c Serum starved MCF7AC1 cells were pretreated with vehicle control and 0.01, 0.1, and 5 µM ENDX for 2 h followed by the addition of 100 nM insulin for 1 h as indicated. IB assay of pAKT substrates and β-actin are shown. d, e In vitro kinase assay showing % PKCβ1 kinase activity in the presence of different concentrations of ENDX and TAM. The broad-spectrum kinase inhibitor staurosporine serves as a positive control. The IC₅₀ concentration of ENDX, TAM, and staurosporine are indicated.

Fig. 7. Role of PKCβ1 in mediating ENDX inhibition of AKT^Ser473 phosphorylation.

a Serum starved MCF7AC1 cells were treated with vehicle control or 20 and 200 nM PMA for 20 min. IB assays of pPKCβ1^Ser661, PKCβ1, pAKT^Ser473, AKT, p-AKT substrates and β-actin are shown. b Serum starved MCF7AC1 cells were pretreated with vehicle control or 0.01, 0.1, and 5 µM ENDX for 2 h followed by the addition of 200 nM PMA for 20 min as indicated. IB assays of pPKCβ1^Ser661, PKCβ1, pAKT^Ser473, AKT, p-AKT substrates and β-actin are shown. c Serum starved MCF7AC1 cells were pretreated with vehicle control or 1 µM ENZA for 2 h followed by the addition of 200 nM PMA for 30 min as indicated. IB assays of pPKCβ1^Ser661, PKCβ1, pAKT^Ser473, AKT and β-actin are shown. d Serum starved MCF7AC1 cells were pretreated with vehicle control, 0.01, 0.1, and 5 µM ENDX, 0.1 µM TAM or 0.1 µM ICI followed by the addition of 100 nM insulin for 1 h as indicated. IB assays of pPKCβ1^Ser661, PKCβ1 and β-actin are shown. e MCF7AC1 cells in CSS medium were transfected with non-targeting (siNT) or PKCβ-targeting (siPKCβ) siRNAs for 48 h. IB assays of PKCβ1, pAKT^Ser473 and β-actin are shown. The histogram indicates the percentage (%) of PKCβ1 and pAKT^Ser473 protein levels remaining upon PKCβ1 knockdown in siPKCβ-treated cells relative to siNT-treated cells from two biological replicates ± s.d. The vertical lines indicate that different lanes of the same blot were juxtaposed to remove intervening lanes. f MCF7AC1 cells were treated with siNT or siPKCβ1 in CSS medium for 6 days. Cell viability was assessed by crystal violet assays. Data represents the mean of six wells per treatment performed as biological triplicates ± s.d. *p ≤ 0.05; **p ≤ 0.01; ***p < 0.001 by one sample t-test.

Fig. 8. ENDX replicates the effects of the pan-AKT inhibitor, MK-2206, on apoptosis.

MCF7AC1 (a) and T47D-LTED (c) cells were co-treated with vehicle control or 0.01, 0.1, and 5 µM ENDX or MK-2206 in the presence of IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 h. The apoptosis (%) graph was generated as described in Fig. 1b. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ***p ≤ 0.001; ****p < 0.0001 by one-way ANOVA. MCF7AC1 (b) and T47D-LTED (d) cells in CSS medium were treated with vehicle control or 0.01, 0.1 and 5 µM ENDX for 24 h. IB assay of pAKT^Ser473, AKT, PARP, cleaved PARP, and β-actin are shown.

Fig. 9. Expression of catalytically active AKT diminishes ENDX ability to induce apoptosis.

a MCF7AC1^caAKT cells were grown in FBS medium in the absence (-) or presence (+) of cumate for 48 h. IB assay of C-terminally hemagglutinin tagged AKT (AKT-HA), endogenous AKT and β-actin. b MCF7AC1^caAKT cells grown in FBS medium in the (-) or (+) of cumate for 48 h. IB assay of pAKT-substrates and β-actin. c MCF7AC1^caAKT cells grown in CSS medium were treated for 48 h in the (-) or (+) of cumate, following which cells were co-treated with vehicle control or 5 µM ENDX and IncuCyte Annexin V green and NucLight red reagents for 48 h. The percentage (%) of cells undergoing apoptosis was calculated as indicated in Fig. 1b. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ns: nonsignificant. **p < 0.01 by one-way ANOVA.

Fig. 10. Summary of ENDX anticancer effects in ERα+ breast cancer cells.

a Activation of PKCβ1^Ser661 by the PKC agonist PMA and/or insulin phosphorylates AKT^Ser473 resulting in the activation of p-AKT downstream substrates, which mediates cell survival. b ENDX binds to PKCβ1 and facilitates PKCβ1 protein degradation, resulting in the attenuation of phosphorylation of AKT^Ser473 as well as downstream p-AKT substrates, leading to induction of apoptosis.