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. 2017 Jul 1;77(13):3455-3466.
doi: 10.1158/0008-5472.CAN-16-3240. Epub 2017 May 16.

Androgen Receptor Supports an Anchorage-Independent, Cancer Stem Cell-like Population in Triple-Negative Breast Cancer

Valerie N Barton  1 , Jessica L Christenson  1 , Michael A Gordon  1 , Lisa I Greene  1 , Thomas J Rogers  1 , Kiel Butterfield  1 , Beatrice Babbs  1 , Nicole S Spoelstra  1 , Nicholas C D'Amato  1 , Anthony Elias  2 , Jennifer K Richer  3
Affiliations

Androgen Receptor Supports an Anchorage-Independent, Cancer Stem Cell-like Population in Triple-Negative Breast Cancer

Valerie N Barton et al. Cancer Res. .

Abstract

Preclinical and early clinical trials indicate that up to 50% of triple-negative breast cancers (TNBC) express androgen receptor (AR) and are potentially responsive to antiandrogens. However, the function of AR in TNBC and the mechanisms by which AR-targeted therapy reduces tumor burden are largely unknown. We hypothesized that AR maintains a cancer stem cell (CSC)-like tumor-initiating population and serves as an antiapoptotic factor, facilitating anchorage independence and metastasis. AR levels increased in TNBC cells grown in forced suspension culture compared with those in attached conditions, and cells that expressed AR resisted detachment-induced apoptosis. Culturing TNBC cells in suspension increased the CSC-like population, an effect reversed by AR inhibition. Pretreatment with enzalutamide (Enza) decreased the tumor-initiating capacity of TNBC cells and reduced tumor volume and viability when administered simultaneously or subsequent to the chemotherapeutic paclitaxel; simultaneous treatment more effectively suppressed tumor recurrence. Overall, our findings suggest that AR-targeted therapies may enhance the efficacy of chemotherapy even in TNBCs with low AR expression by targeting a CSC-like cell population with anchorage independence and invasive potential. Cancer Res; 77(13); 3455-66. ©2017 AACR.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no competing interests.

Figures

Figure 1
Figure 1
AR-regulated changes in gene expression in TNBC cells cultured in forced suspension. A, Heatmap of genes differentially regulated in BT549 cells grown in attached vs. suspended culture conditions for 24 hours. Biological quadruplicate samples of each treatment group are shown. Genes with > or < 2.0 fold-change difference between attached and suspended conditions (p < 0.05) are shown (yellow indicating increased gene expression and blue indicating decreased gene expression). Red stars indicate potentially AR-regulated genes in the Androgen Responsive Gene Database (http://argdb.fudan.edu.cn/index_info.php). B, MetaCore analysis of BT549 microarray data. Top, pathway analysis illustrating annotated connections between AR and genes altered in suspension culture. Bottom, summary of top transcriptional pathways predicted to be altered in suspension compared to attached culture conditions.
Figure 2
Figure 2
AR expression increases in forced suspension culture. A, Quantitative real-time PCR (qRT-PCR) for androgen receptor (AR) and glucocorticoid receptor (GR) gene expression in attached versus forced suspension culture conditions at 24 hours. B, Western blot for AR and GR in attached versus forced suspension culture conditions at 24 hours. α-Tubulin was used as a loading control. C, Immunohistochemistry for AR in attached versus forced suspension culture conditions at 48 hours. Representative images, magnification 400X. D, AR luciferase reporter assay in attached compared to suspended conditions in cells transduced with a non-targeting control (shNEG) or shRNAs targeting AR (shAR15, shAR17). Western blot for AR. α-Tubulin was used as a loading control. Mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
AR and cleaved-caspase 3 (CC3) increase in TNBC cells cultured in forced suspension and do not co-localize. A–B, TNBC cells were grown in attached and suspended conditions for 48 hours and stained for AR (green) and CC3 (red) using tyramide signal amplification. Representative images; A, magnification 400X, B, magnification 1000X to emphasize CC3 (red arrows) and AR (green arrows) localization. C, image analysis of BT549 suspended cells using inForm 2.2. Percentage of cells positive for Cy3.5 (CC3), FITC (AR), or double positive (CC3+AR) were calculated from the total number of positive cells for n = 3 random 200X fields; mean ± SEM.
Figure 4
Figure 4
A cancer stem cell-like population is increased in suspension. A, CD24 staining of MDA-MB-453 (MDA453) cells following 5 days in suspension culture. B, mammosphere formation efficiency (MFE) assay of MDA453 cells placed in attached or suspended conditions for 5 days prior to plating. C, ALDEFLUOR (ALDH+) assay of SUM159PT cells placed in suspended conditions for 3 days. DEAB was used as a control to set the gate. D, MFE assay of SUM159PT cells placed in attached or suspended conditions for 3 days prior to plating. E, SUM159PT cells cultured for 3 days in suspension were sorted for ALDH and AR gene expression was measured using quantitative real-time PCR (qRT-PCR). Shown is a representative image of sorted ALDH populations. Mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
AR inhibition decreases a cancer stem cell-like population. A, ALDEFLUOR assay of SUM159PT cells transduced with a non-targeting control (shNEG) or shRNAs targeting AR (shAR15 and shAR17). Western blot for AR. α-Tubulin is shown as a loading control. B, mammosphere formation efficiency (MFE) assays of SUM159PT cells transduced with shNEG, shAR15, or shAR17 or C, treated with enzalutamide (Enza). D, CD24 staining of MDA-MB-453 (MDA453) cells transduced with shNEG, shAR15, or shAR17. Western blot for AR. β-Actin is shown as a loading control. E, MFE assays of MDA453 cells transduced with shNEG, shAR15, or shAR17 or F, treated with Enza. Mean ± SD; * p < 0.05, # p = 0.06, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
Tumor initiation frequency of SUM159PT cells pre-treated with enzalutamide. A, schematic of experimental design. B, Tumor initiation frequency. Top, luciferase activity, measured as total flux, in mice injected with 102–104 cells pre-treated for 3 days with vehicle control (Veh) or enzalutamide (Enza). Bottom, summary of the number of mice per group with tumors as determined by palpation (left) and luciferase activity/total flux (right).
Figure 7
Figure 7
Enzalutamide and paclitaxel combination therapy is more effective than paclitaxel alone. SUM159PT-TGL cells were bilaterally injected into the 4th mammary fat pads of female, athymic nu/nu mice. Once tumors were established, mice were randomized and matched into three groups. Mice were administered 10 mg/kg/d paclitaxel (Pac) for five days. Group 1 started enzalutamide (Enza) treated measure simultaneously with Pac (SIM), while Group 2 started Enza subsequent to Pac (SEQ). A, tumor cell luciferase activity as measured by total flux over time. B, comparison of total flux on study day 49. C, tumor volume over time. D, comparison of tumor volume on study day 51. E, total flux per mouse between the final day of Pac treatment (Day 28) and Day 49. Mean ± SD.
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