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.
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.