A Molecular Analysis Provides Novel Insights into Androgen Receptor Signalling in Breast Cancer

<div><p>Background</p><p>Androgen Receptor (AR) is an essential transcription factor for the development of secondary sex characteristics, spermatogenesis and carcinogenesis. Recently AR has been implicated in the development and progression of breast and prostate cancers. Although some of the functions of the AR are known but the mechanistic details of these divergent processes are still not clear. Therefore understanding the regulatory mechanisms of the functioning of the AR in ER-/AR+ breast cancer will provide many novel targets for the purpose of therapeutic intervention.</p><p>Methods/Results</p><p>Using bioinformatics tools, we have identified 75 AR targets having prominent roles in cell cycle, apoptosis and metabolism. Herein, we validated 10 genes as AR targets by studying the regulation of these genes in MDA-MB-453 cell line on stimulation by androgens like 5α-dihydrotestosterone (DHT), using RT-qPCR and ChIP assay. It was observed that all the identified genes involved in cell cycle except MAD1L1 were found to be up regulated whereas expression of apoptosis related genes was decreased in response to DHT treatment. We performed an exhaustive, rigid-body docking between individual ARE and DNA binding domain (DBD) of the AR protein and it was found that novel residues K567, K588, K591 and R592 are involved in the process of DNA binding. To verify these specific DNA-protein interactions electrostatic energy term calculations for each residue was determined using the linearized Poisson–Boltzmann equation. Our experimental data showed that treatment of breast cancer cells with DHT promotes cell proliferation and decreases apoptosis. It was observed that bicalutamide treatment was able to reverse the effect of DHT.</p><p>Conclusion</p><p>Taken together, our results provide new insights into the mechanism by which AR promotes breast cancer progression. Moreover our work proposes to use bicalutamide along with taxanes as novel therapy for the treatment of TNBCs, which are positive for downstream AR signalling.</p></div

Drug resistance of MDA-MB-453.

<p>(A-C) MDA-MB-453 cells were treated with the indicated concentration of anti-cancer drugs paclitaxel, 5-fluorouracil and cyclophosphamide respectively and the cell survival was determined using MTT assay. (D) Table showing the IC₅₀ concentration of drugs determined using the survival curves. Error bars are means ± SD of three independent experiments; (*p < 0.05, **p < 0.01, ***p < 0.001).</p

DHT mediates cell proliferation and Bicalutamide (Bic) abrogates this effect.

<p>(A) MDA-MB-453 cells treated with indicated treatments for 48 hours and their morphology was observed at higher magnification and randomly selected microscopic fields were photographed. (B) 3 x 10⁵ cells were plated in 35mm dish and the cell were treated with IC₅₀ concentrations of the various drugs and harvested at 48 hours for DNA content analysis by flow cytometry. <b>(C)</b> AR binding to the promoter of CDT and KLF by ChIP-qPCR showing the binding of AR to CDT promoter in presence of Paclitaxel and various other treatments.(D) ChIP-qPCR showing the binding of AR to KLF promoter in presence of Paclitaxel and various other treatments. The gel panel below the picture shows the standard PCR validation of the result. All the means and standard deviation are obtained from three independent experiments. Pac: Paclitaxel; Bic: Bicalutamide; Cyclo: Cyclophosphamide; Fluoro: 5'-Fluorouracil, M: Marker, 1: Input, 2: Untreated control, 3: DHT +Pac,: 4: DHT+ Bic +Pac, 5: Bic + Pac, 6: Bic.</p

Strategy for the identification of novel androgen receptor regulated genes.

<p>(A) Flowchart of the methodology followed in finding novel AR targets. (B) Pie chart showing the distribution of the 75 putative AR targets in different cell processes. (C) Schematic diagram showing the various motifs and domains of the human androgen receptor; GLN RCH: Glutamine rich, GLY RCH: Glycine rich, DBD: DNA binding domain, NLS: nuclear localization signal, LBD: Ligand binding domain (D) The <i>in silico</i> model of AR containing both the LBD and DBD domain. The model is reproduced by applying the same protocol used by Helsen C. et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120622#pone.0120622.ref037" target="_blank">37</a>] The crystal coordinates from the model of PPARy-RXRα heterodiamer (PDB ID 3DZY), the rat AR-DBD (PDB-ID 1R4I) was used to aligned onto the RXRα-DBD and the human AR-LBD (1XQ3) was aligned onto the PPARy-LBD using VMD tool. The human AR-LBD (2AM9), and rat AR-DBD (1R4I) was used for protein-protein docking using the program HADDOCK.</p

Proposed model showing AR regulation of the breast tumour progression.

<p>Androgen receptor (AR) on DHT stimulation translocates into the nucleus and bind to its cognate androgen response elements (AREs). Inside the nucleus, AR up regulates the expression of ABL1, CDT1, KLF6 and SGOL2, while it represses MAD1L1 expression to induce cell proliferation. At the same it induces AATK expression and down regulates the expression of BOK, BIK and ENDOG to decrease apoptosis and promotes breast cancer progression. Bicalutamide (BIC) reverses the effect of AR on cell cycle and apoptosis by binding and preventing its activation. Due to its ability to negate the effects of AR, bicalutamide can be used to block breast cancer progression.</p

DHT stimulation decreases apoptosis and induces cell proliferation in MDA-MB-453 cells.

<p>(A) Effect of DHT stimulation on cell survival caused by paclitaxel, 5-fluorouracil and cyclophosphamide in presence of 10nM/L of DHT (+) or vehicle control (-) for 24 hrs. Error bars are means ± SD; (ns: not significant,*p < 0.05, **p < 0.01, ***p < 0.001). (B-D) Increased cell survival cause by concentration dependent increase in DHT (nM/L) in presence of paclitaxel, 5-fluorouracil and cyclophosphamide respectively. (E) Role of AR in regulating MDA-MB-453 cell proliferation on DHT stimulation. Monolayer growth rates of cells were determined by the MTT assay and 1.39 fold difference (P value = 0.0154) was observed between vehicle and DHT treated cells (F) Live cell counting was done with trypan blue done to estimate cell the survival. DHT treated cells showed 1.55 fold (P = 0.003) difference over vehicle treated cells. All the mean and standard deviation are obtained from three independent experiments. Error bars are means ± SD of three independent experiments; (*p < 0.05, **p < 0.01, ***p < 0.001).</p

Docking of Linear B-DNA fragments to AR-DBD.

<p>(A) AR-DBD showed two distinct DNA- binding sites. The DNA fragments docked at site I in the top ranked solutions (shown by their phosphate backbone, red), and the DNA fragments docked at site II (in green color) shows similar orientations. (B) Top 200 ranked solutions (first site in red and, second site in dark green spheres) found by DOT clusters at site I (representative docked DNA fragment, rank I, red DNA backbone), and site II (rank 3, green backbone). (C) Electrostatic surface view of the best conformer of DNA docked at AR binding site.</p

Interaction map showing the complexes formed by docking of ARE(s) to AR DBD.

<p>(A) Multiple sequence alignment of selected ARE genes. Novel conserved residues identified from literature are marked by deep red arrows, whereas the two purple arrows (at the bottom of the alignment) indicate the highly conserved contact points between ARE-DBD and cognate AREs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120622#pone.0120622.ref009" target="_blank">9</a>]. All ARE sequences shown were analyzed using Clustal W followed by glam2scan tools. All the small arrows mentioned at the top of panel A are showing the resemblance of the outcomes between sequence alignment and glam2scan. (B) The graphical representation of the consensus sequence generated through MEME software, where the height of the letters indicates the frequency of each base. In the consensus ARE sequence, dotted lines are used to highlight those nucleotide residues which actually involved in interactions is showing the ARE interaction site-1. (C-E) Docking results showing the ARE interaction site-1. (F-G) Docking results showing the ARE interaction site-2. The molecular basis for the interaction of B-DNA strands of AREs and the key residues of AR-DBD. The interacting protein residues are shown in green liquorice with transparent surface, while nucleotides are rendered in red liquorice with transparent surface.</p

DBD domain organization of androgen receptor.

<p>(A) Surface representation of the rat AR-DBD structure bound to the ARE (Androgen response element). (B) Cartoon representation of AR-DBD bound to ARE. The α helix in pink, beta sheets in cyan, loops and turns are in blue and yellow color, respectively. Zinc ions are presented as grey spheres.</p

Androgen regulation of previously uncharacterized genes in Her2 negative MDA-MB-453 cells.

<p>(A) Surface Her2 expression was checked using the IHC staining of (i) A Her2 positive axillary lymph node having breast cancer metastasis was taken as the positive control (ii) MDA-MB-231 cells were taken as negative control (iii) MDA-MB-453 cells showing negative staining for Her2. (B) MDA-MB-453 cell were steroid starved for 24 hrs and then they were treated with 10nM/L of DHT or vehicle control for the indicated time points. Total RNA was isolated and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. (C) ChIP assay were performed with AR antibody or control IgG antibody in MDA-MB-453 cells treated with 10nM/L of DHT or vehicle control for 24 hrs. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars are means ± SD of three independent experiments; (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Standard PCR for the AR, IgG and input DNA was performed for the indicated genes.</p