Non-canonical AR activity facilitates endocrine resistance in breast cancer

in Endocrine-Related Cancer
Correspondence should be addressed to T E Hickey or E Lim: theresa.hickey@adelaide.edu.au or e.lim@garvan.org.au
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The role of androgen receptor (AR) in endocrine-resistant breast cancer is controversial and clinical trials targeting AR with an AR antagonist (e.g., enzalutamide) have been initiated. Here, we investigated the consequence of AR antagonism using in vitro and in vivo models of endocrine resistance. AR antagonism in MCF7-derived tamoxifen-resistant (TamR) and long-term estrogen-deprived breast cancer cell lines were achieved using siRNA-mediated knockdown or pharmacological inhibition with enzalutamide. The efficacy of enzalutamide was further assessed in vivo in an estrogen-independent endocrine-resistant patient-derived xenograft (PDX) model. Knockdown of AR inhibited the growth of the endocrine-resistant cell line models. Microarray gene expression profiling of the TamR cells following AR knockdown revealed perturbations in proliferative signaling pathways upregulated in endocrine resistance. AR loss also increased some canonical ER signaling events and restored sensitivity of TamR cells to tamoxifen. In contrast, enzalutamide did not recapitulate the effect of AR knockdown in vitro, even though it inhibited canonical AR signaling, which suggests that it is the non-canonical AR activity that facilitated endocrine resistance. Enzalutamide had demonstrable efficacy in inhibiting AR activity in vivo but did not affect the growth of the endocrine-resistant PDX model. Our findings implicate non-canonical AR activity in facilitating an endocrine-resistant phenotype in breast cancer. Unlike canonical AR signaling which is inhibited by enzalutamide, non-canonical AR activity is not effectively antagonized by enzalutamide, and this has important implications in the design of future AR-targeted clinical trials in endocrine-resistant breast cancer.

Downloadable materials

  • Supplementary Figure 1: AR siRNAs reduce AR mRNA levels. The efficacy of AR siRNA A and B was assessed using qRT-PCR. RNA extracted from MCF7 TamR (A) and MCF7 LTED (B) cells, which were transfected with nonsense (NS), AR siRNA A (siA) or B (siB) for 2 days, was converted into cDNA. RT-qPCR was performed using the cDNA and AR mRNA level was normalized to GAPDH. Data is represented as the expression of normalized AR in AR siRNA-transfected cells relative to that of NS-transfected cells. The experiment was performed in 3 biological replicates.
  • Supplementary Figure 2: AR knockdown increases nuclear ER in MCF7 TamR cells. The effect of AR knockdown on nuclear ER was assessed using immunofluorescence. (A) Representative images of MCF7 TamR cells stained with 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI), anti-AR (green) or anti-ER (red) antibodies post 2-day transfection with NS, AR si A or AR siB. (B) Quantification of nuclear ER intensity in 500 cells using ImageJ. *** p<0.001 using Student’s t test for NS vs AR siA or siB.
  • Supplementary Figure 3: AR is predominantly cytoplasmic in MCF7 cells. (A) The subcellular localization of AR and ER was evaluated in endocrine-sensitive (ES), TamR and LTED MCF7 cells in their basal growth media. Subcellular fractionation was performed to enrich for cytoplasmic and nuclear fractions, as represented alpha-tubulin (α-tub) and lamin A respectively, prior to immunoblotting for AR and ER.
  • Supplementary Figure 4: Heatmap of the top 50 differentially expressed genes post AR knockdown in MCF7 TamR cells. Most significant up- (red) and down-(blue) regulated genes induced by transient knockdown of AR.
  • Supplementary Figure 5: AR knockdown enhances ER signaling in MCF7 LTED cells. The effect of AR knockdown on the expression of ER and ER-regulated PGR in the MCF7 LTED cells was assessed. MCF7 LTED cells transfected with either nonsense (NS) or AR siRNA A (siA) were harvested 2 days post-transfection. (A) Protein lysates extracted from these cells were immunoblotted for AR, ER and GAPDH. (B) RNA extracted from these cells was used to determine the effect of AR knockdown on the expression levels of ESR1 and PGR using RT-qPCR. Data is represented as log2 ratio and the experiment was repeated in triplicates. * p<0.05 using Student’s t test when comparing AR siRNA A versus NS. Error bars = SEM.
  • Supplementary Figure 6: Effect of enzalutamide treatment on Gar15-13 PDX. Endpoint tumors treated with vehicle or 20 mg/kg/day enzalutamide were subjected to immunohistochemical staining with antibodies against ER, AR, SEC14L2, ER and PR. Four biological replicates from each treatment condition are presented. Representative images at 80x are presented. Scale bar = 25 µm.
  • Supplementary Figure 7: Enzalutamide reduces viability of AR-null T47D cells at high doses. Knock out of AR in T47D cells was achieved by transfecting these cells with AR Double Nickase Plasmid (#SCZSC-400026-NIC-2, Santa Cruz) and control cells were transfected with the Control Double Nickase Plasmid (#SCZSC-437281, Santa Cruz). Transfection of these plasmids was carried out using the LipofectamineTM 3000 Transfection Reagent (Thermo fisher). (A) The loss of AR protein in the AR-knockout T47D cells was confirmed by immunoblotting with an anti-AR antibody in response to 24 h treatment with vehicle (Veh) or 10 nM 5 alpha-dihydrotestosterone (DHT) relative to control plasmid-transfected T47D cells. Alpha-tubulin (αtub) was immunoblotted for as a loading control. (B) AlamarBlue assay assessing the dose response of control and AR-knockout T47D cells to 4 days of enzalutamide (Enz) treatment. ** p<0.01 using Student’s t test comparing Enz versus Veh-treatment. Error bars = SEM.
  • Supplementary Figure 8: Loss of AR abrogates AR-agonist induced growth inhibition in MCF7 TamR cells. (A) Summary of experiment where MCF7 TamR cells transfected with NS, ARsiA or ARsiB for 2 days were treated with either Veh or 1 nM DHT for 5 days. (B) Cell counting was performed to determine the effect of DHT on the proliferation of these cells. * p<0.05 and n.s. = not significant using Student’s t test. Error bars = SEM.

 

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    AR knockdown reduces proliferation of ER+AR+ endocrine-resistant cells. (A) Expression of steroid hormone receptors including AR, ER, PR and FOXA1 in endocrine-sensitive (ES), tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cell lines by immunoblotting. (B) The efficiency of AR siRNA-A (siA) and siRNA B (siB) on AR protein in the endocrine-resistant cell lines was compared to nonsense (NS) siRNA 4 days post transfection by immunoblotting for AR and GAPDH (loading control). The effect of AR loss on proliferation was assessed using cell counting and AlamarBlue assay 3 and 6 days post transfection in TamR (C and E) and LTED (D and F) cells. Cell numbers in log2 scale are presented in (C) and (D). *P < 0.05, **P < 0.01 using Student’s t-test. Error bars = s.e.m. from three biological replicates.

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    AR knockdown restores characteristics of classical ER activity. The effect of AR knockdown on gene expression using microarray profiling and on ER chromatin binding at selected sites in MCF7 TamR cells was assessed. The efficiency of AR siA vs that of NS 2 days post transfection was assessed using RT-qPCR (A) and immunoblotting (B). Each reaction was performed in three technical replicates and error bars = s.d. (C) Differentially expressed genes identified from microarray profiling were subjected to MSigDB hallmark GSEA. The top nine enrichment groups for the up- and downregulated genes, with q values of <0.001 are presented. (D) The differential gene set induced by the transient knockdown of AR was overlapped with that of the MSigDB hallmark Estrogen Response Early signature. (E) RT-qPCR was used to validate changes in the selected genes ESR1, PGR, CXCL8 and CTGF identified in the expression profiling. Changes in these genes post AR knockdown are presented as log2-fold change of the expression of each gene in AR siRNA-transfected cells relative to NS siRNA-transfected cells. (F) ER binding at distal and proximal CXCL8 promoters post AR knockdown was assessed using chromatin immunoprecipitation (ChIP). Data is represented as fold change relative to NS-transfected cells. *P < 0.05, **P < 0.01 using Student’s t-test. Error bars = s.e.m. from three biological replicates.

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    AR knockdown re-sensitizes MCF7 TamR cells to tamoxifen. The effect of AR knockdown on the response of MCF7 TamR cells to tamoxifen was evaluated using AlamarBlue and colony formation assays. (A) The viability of MCF7 TamR cells was assessed using AlamarBlue assay in response to 3 and 5 days treatment with vehicle (Veh) or 5 µM tamoxifen (Tam) after transfection with NS, AR siA or AR siB RNA for 2 days. Data is presented as the fold change of Tam-treated cells to Veh-treated cells in log2 ratio. (B) 6-day colony-forming assay assessing the response of MCF7 TamR to Tam as per the treatment and transfection conditions in (A). (C) Quantification of the colony area coverage using ImageJ with data presented as the fold change of tamoxifen to Veh-treated cells in log2 ratio. (D) Cell lysates extracted from MCF7 TamR cells transfected with NS, AR siA or AR siB for 2 days, were immunoblotted for AR, phospho-ER at serine-118 (pER s118), ER and GAPDH. (E) Cell lysates in (D) were immunoblotted for phospho-Akt at serine 473 (pAKT s473), total Akt, FOXO3a and GAPDH. Densitometry was performed using ImageJ and data is represented relative to control cells (RR; relative ratio). *P < 0.05, **P < 0.01 using Student’s t-test. Error bars = s.e.m. from three biological replicates.

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    AR pharmacological inhibition does not recapitulate effects of AR knockdown in MCF7 TamR cells. The effect of enzalutamide (Enz) treatment on MCF7 TamR cells in relation to growth and ER signaling was assessed. The effect of 10 µM Enz on AR expression and subcellular localization was evaluated using immunoblotting and immunofluorescence. Cells were treated with enzalutamide for 48 h in the presence or absence of 10 nM DHT prior to harvest for immunoblotting for AR, ER and GAPDH (A) and immunofluorescence with DAPI (blue) and antibodies against cytoplasmic marker alpha-tubulin (αtub; red) and AR (green) (B). Densitometric analysis was performed using ImageJ and data is represented relative to vehicle (Veh)-treated cells in the absence of DHT (RR; relative ratio). (C) Colony-forming assays evaluating the effect of 9-day Veh or Enz treatment on MCF7 TamR cells. (D) Quantification of the colony area coverage using ImageJ and data is presented as the fold change of Enz to Veh treatment. (E) AlamarBlue assessing the effect of 10 µM Enz on the viability of MCF7 TamR cells at 3 and 6 days post treatment in the absence or presence of 5 µM tamoxifen (Tam). Data is presented as relative to day 0. (F) RT-qPCR was performed to determine the effect of 48 h 10 µM Enz treatment on ESR1, PGR, CXCL8 and CTGF mRNA levels in MCF7 TamR cells relative to Veh-treated cells. Data is presented as log2 ratio. (G) Cell lysates from MCF7 TamR cells treated with Veh or 10 µM Enz for 48 h were immunoblotted for phospho-Akt at serine 473 (pAKT s473), total Akt, FOXO3a and GAPDH. (H) AlamarBlue assessing the effect of 10 µM Enz on the viability of MCF7 LTED cells at 3 and 6 days post treatment. *P < 0.05, n.s. = not significant, using Student’s t-test. Error bars = s.e.m. from three biological replicates.

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    Enzalutamide does not inhibit the growth of an endocrine-resistant PDX. The effect of antagonizing AR using enzalutamide (Enz) on an ER+AR+ endocrine-resistant PDX was assessed. (A) This PDX model was established from the liver metastasis of a patient with ER+PR+HER2− breast cancer who relapsed after 1 year of adjuvant aromatase inhibitor treatment. (B) PDX-bearing mice were treated with either vehicle (Veh) or 20 mg/kg Enz when tumors reached ~150–200 mm3. Tumors were harvested when they reached the ethical endpoint of 1000 mm3 and data is presented as fold change of tumor volumes at harvest from baseline tumor volumes. (C) IHC staining was performed to determine the effect of treatment on Ki-67, AR, SEC14L2 and ER. Representative images at 80× are presented. Scale bar = 25 µm (D) Proliferation index of tumors treated with Veh or Enz were determined. This was based on the quantification of proportion of cells positive for Ki-67 in >1000 cells from at least three random high-magnification fields. (E) Protein lysates extracted from Veh- or Enz-treated tumors were immunoblotted for AR, cyclin A and GAPDH. (F) Intensity of nuclear AR and total SECL14L2 was performed using ImageJ and data is represented as intensity of Enz (n = 6) relative to Veh (n = 4). (G) RT-qPCR was performed to determine the effect of Enz treatment (n = 3) on SECL14L2 and FKBP5 relative to Veh treatment (n = 3). *P < 0.05, **P < 0.01 and n.s. = not significant using Student’s t-test. Error bars = s.e.m.

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