Background
The understanding of the heterogeneity of cancer is of great importance for the development of effective therapeutic strategies in specific subgroups of patients. Indeed, genomic studies have classified breast cancer into different subtypes [
1]. At a clinical level, this heterogeneity corresponds to different prognosis, patterns of relapse, response to treatment and clinical behavior for each breast cancer subtype. For instance, tumors expressing estrogen receptors (ER) have a more benevolent behavior compared with those overexpressing HER2 [
2]. In addition, triple negative breast cancer (TNBC) shows a worse prognosis compared with tumors expressing ER, and the probability of early relapse during the first years after the diagnosed is higher compared with other subtypes [
2,
3].
The influence of sex hormones in the development of breast and prostate cancer is known. Estrogens and androgens act through their receptors by a direct transcriptional regulation of genes involved in cell growth and survival [
4]. In breast cancer, more than 70%–80% of tumors express the ER or progesterone receptor (PR), and therapies designed to neutralize their function have shown clinical benefit [
5]. In addition the androgen receptor (AR) is expressed in breast cancer in 60%–70% of tumors regardless of the ER status [
6] and has been linked with a good outcome in ER positive tumors [
7]. This good prognosis is associated with the impeding of the transcriptional activity mediated by the ER [
7]. By contrast, in the apocrine breast cancer subtype- an ER negative tumor- the AR acts by binding to the same transcription factors as the ER does, mainly through FOXA1, leading to a luminal gene expression phenotype [
8]. In this case the expression of AR is associated with worse prognosis [
8]. Recently, using gene expression analyses TNBC has been classified in subtypes including one termed luminal androgen that was enriched in genes related to this pathway [
9].
Testosterone and particularly dihydrotestosterone (DHT) are the main activators of the androgen receptor [
10]. Upon ligand binding AR translocate to the nucleus were it acts transcriptionally [
10,
11]. Androgens are the major sex hormones in males being produced at different levels but mainly in sex-related tissues [
11]. In addition, in females, androgens are also formed at the suprarenal gland, having a functional role after the deprivation of estrogens produced in the menopause [
11].
The control of AR is mediated at different levels, but signalling pathways play a key role by stabilizing or enhancing its transcriptional activity. In prostate cancer, membrane receptor tyrosine kinases (RTK) have been shown to modulate the AR expression. For instance, overexpression of HER2 in cell lines of prostate cancer results in increase AR activity and stability [
12]. Furthermore in breast cancer, a recent study has linked the activation of HER2 with the expression of AR [
13]. Of note, the PI3K pathway is the key node for the signal transmission of the stimuli from membrane RTKs in relation to the control of the AR in prostate cancer [
14,
15]. To this regard, in breast cancer, mutations at the kinase domain of the PI3KCA gene were linked with higher expression of the AR [
16].
In TNBC, expression of the epidermal growth factor receptor (EGFR) or Platelet-derived Growth Factor Receptor (PDGFRβ) has been described using immunohistochemical techniques [
17]. A frequent activation of the EGFR and other receptor (Met and Eph2) as well as non-receptor (Lyn, Src family kinases) tyrosine kinases has been confirmed using a profiling of tyrosine phosphorylated proteins [
18]. Moreover using shRNA library screening several kinases, including the EGFR and HER2, were identified as activated in TNBC [
19].
The mechanism of action of androgens in TNBC remains controversial and the relationship between activated RTKs and the expression of AR has not been fully explored, although some recent reports have suggested the AR as a potential therapeutic target [
13].
In order to contribute to a better understanding of the role of AR in TNBC, the aim of this study is to evaluate the expression of the AR in TNBC and its relationship to the activation status of RTKs and its downstream routes.
Methods
Reagents and antibodies
Cell culture media and supplements (fetal bovine serum (FBS), glutamine, penicillin/streptomycin) were purchased from Invitrogen (Gaithersburg, MD). The anti-pErk1/2, anti-Erk2, anti-PDGFR β, anti-pPDGFR β, anti-EGFR and anti-pEGFR antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Akt and anti-AR antibodies were from Cell Signalling Technologies (Beverly, MA). The anti-phosphorylated Akt (Serine 473) antibody was generated against the sequence RPHFPQFpS473YSAS (p = phosphorylated) [
20].
Cell culture
All cell lines were cultured at 37°C in a humidified atmosphere in the presence of 5% CO2–95% air. Cells were grown in DMEM or in RPMI medium containing a high glucose concentration (4,500 mg/liter) supplemented with antibiotics (penicillin at 100 U/ml, streptomycin at 100 μg/ml), glutamine 2 mM and 10% FBS. Cells were treated with different drugs: PD98059 (50 μM), BEZ235 (0.5 μM), BIC (20 μM), Imatinib (10 μM), Lapatinib (10 μM) and DHT (50nM).
Identification of human samples and studies
Human samples were obtained from the tumor bank of the Salamanca University Hospital and Albacete University Hospital following institutional and ethical guidelines. All patients signed the study consent form. TNBC samples were defined as those with HER2 negative by inmunohistochemistry (IHC), HER2 of 0 or 1+ or a negative fluorescence in situ hybridization (FISH). HER2 amplification (evaluated by FISH DAKO HER2 FISH pharmDxTM Kit, DakoCytomation, Glostrup, Denmark A/S) was defined as a HER2-chromosome 17 ratio of < .2.0, as required by guidelines. Hormone receptor (HR) negative was defined as follow <10% positive cells by IHC for both estrogen receptor (ER) and progesterone receptor (PR) following recommendations before 2011.
Frozen human samples were inspected by haematoxylin-eosin staining for epithelial tumor content by analysis of two slices at each end of the tumor. Only samples containing 70% epithelial tumoral cells were selected for Western analyses. The tumors were minced, washed with phosphate-buffered saline (PBS), and homogenized in ice-cold lysis buffer (140 mM NaCl; 10 mM EDTA; 10% glycerol; 2% Triton X-100; 20 mM Tris pH 7.0; pepstatin, 10 mM; aprotinin, 10 mg/ml; leupeptin, 10 mg/ml; PMSF, 1 mM; beta-glycerophosphate, 25 mM; sodium fluoride, 10 mM; and sodium orthovanadate, 10 mM) with a tight-fitting Dounce homogenizer. This homogenate was centrifuged at 10,000 g for 20 minutes at 4 uC, and the supernatants were transferred to new tubes.
Antibody arrays
Two commercial arrays were used for the studies; the human phospho-RTK array kit (R&D Systems, Abingdon, United Kingdom) and the PathScan RTK Signaling Antibody Array Kit (Cell Signaling). The Image J 1.44 software (National Institute of Health, Bethesda, MD, USA) was used for the quantification of the different RTKs in the human phospho-RTK array kit, and the Odyssey V3.0 program for the quantification of the cell signalling intermediates.
Western blotting
Cells were washed with phosphate-buffered saline and lysed in ice-cold lysis buffer (140 mM NaCl, 10 mM ethylenediaminetetraacetic acid, 10% glycerol, 1% Nonidet P-40, 20 mM Tris, pH 7.0, 1 μM pepstatin, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM phenylmethyl sulphonyl fluoride (PMSF), and 1 mM sodium orthovanadate) [
21]. Lysates were centrifuged at 10,000 × g at 4°C for 10 min, and supernatants were transferred to new tubes. Samples were then boiled in electrophoresis sample buffer and placed on 6%–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, depending on the molecular weight of the proteins to be analyzed. After electrophoresis, proteins in gels were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation). Membranes were blocked in Tris-buffered saline with Tween 20 (TBST) (100 mM Tris [pH 7.5], 150 mM NaCl, 0.05% Tween 20) containing 1% of bovine serum albumin for 1 hour and then incubated with the corresponding antibody for 2–16 hours. After washing with TBST, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies for 30 minutes and bands were visualized by using ECL Western Blotting Detection System (GE Healthcare, Buckinghamshire, United Kingdom).
Cell proliferation assays
Cells were plated in 24-well plates at 15,000–20,000 cells/well and cultured overnight in DMEM or RPMI + 10% FBS. The next day medium was replaced with DMEM or RPMI containing the different drugs. Cell proliferation was analyzed days later by an MTT-based assay as described [
22]. Unless otherwise indicated, the results are presented as the mean ± standard deviation (SD) of quadruplicates of a representative experiment that was repeated at least three times.
Revere transcription (RT)-PCR
Total RNA was extracted with RNeasy Mini Kit (Quiagen) as recommended by the supplier. cDNAs were synthesized from 5 μg of total RNA by using RevertAid H Minus First Stand cDNA Synthesis Kit (Fermentas) in a total volume of 20 μl. Reverse transcription was performed at 65° for 5 min followed by 60 min at 42°. PCR reactions were performed with 2 μl cDNA in a 50 μl volume and using Taq Polymerase (Biotools) and the following primer pairs (fragments size indicated in brackets): AR (247 bp) forward 5′-CTCACCAAGCTCCTGGACTC-3′ and reverse 5′-CAGGCAGAAGACATCTGAAAG -3′, β-actin (661 bp), forward 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ and reverse 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′. PCR cycle conditions were 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C for AR and β-actin. The number of cycles was 35 and the products were resolved on a 1% agarose gel and visualized with ethidium bromide.
The design of this study is approved by the Committee of Ethics in Research of the University hospital of Albacete (Reference PI-2010/017).
Discussion
In the present work, we describe the expression of the AR in TNBC and its relation to RTKs and their downstream signal routes. It is also interesting to remark that the expression of the AR was studied in cell lines not representative of a luminal androgen receptor gene expression subtype [
9].
In prostate cancer evidence support the link between the activation of RTKs, like the insulin-like growth factor, keratinocyte growth factor, EGFR or HER2, and the transactivation of the AR in the absence of androgens [
12,
23,
24]. Particularly signaling through ErbB receptors and HER2 augment the transcription of the AR; and activation of HER2 and HER3 through the HER3-ligand Neuregulin increases the amount of the AR [
12,
23]. Therefore inhibition of these kinases sensitizes cells to androgen withdrawal [
23,
25]. In estrogen negative breast cancer, the AR regulates the HER2 function suggesting that antiandrogens is a potential therapeutic approach [
13]. Indeed, studies in breast cancer using human samples confirm that those tumors that are HER2 positive express higher amounts of the AR [
26].
In our study we show a correlation between activated membrane tyrosine kinase receptors and the expression of the AR in TNBC. It is known that in triple negative tumors EGFR and PDGFRβ are phosphorylated and present in a significant number of tumors [
19,
27]. If extrapolating from mechanisms observed in prostate cancer, is not surprising to identify a correlation between activated RTKs and the expression of the AR. Interestingly this association was also observed in a panel of cell lines, and treatment with specific tyrosine kinase inhibitors against them reduced slightly the amount of the AR, particularly with lapatinib in BT549 when combined with bicalutamide. A slightly similar effect was observed for imatinib and bicalutamide in HS578T. As the PI3K/AKT pathway and the MAPK pathway are key intracellular signaling nodes from RTKs, we decided to evaluate their role in the activation of the AR. It is known that targeting key nodes has a more profound effect on the inhibition of deregulated functions that the inhibition of single receptors [
28]. In prostate cancer the PI3K/AKT pathway has been described as a key regulator in the transcription of the AR [
12], and in breast cancer mutations at the PIK3CA gene were associated with expression of the AR in patients [
16]. Our results show that the inhibition of the PI3K/mTOR pathway with BEZ235 decreased the amount of the AR in the absence of androgens.
It was interesting to find how the concomitant administration of an Erk1/2 inhibitor with bicalutamide reduced the amount of the AR in a more profound manner than when each agent was given alone. This effect was also observed when using inhibitors of EGFR and PDGFRβ.
We then studied the effect on proliferation of these combinations. Although an important effect was noted with BEZ235 given alone, no effect on proliferation was found when associated with an anti-androgen. Furthermore, the concomitant administration of PD98059 plus bicalutamide produced a profound arrest in proliferation compared to each agent given alone. This effect suggests that the inhibition of the MAPK pathway facilitates the anti-proliferative effect of anti-androgens. A synergistic interaction between anti-androgens and Erk1/2 inhibitors has been described in other models including apocrine breast cancer, but not in TNBC [
29]. A similar result was observed for the combination of lapatinib and bicalutamide in BT549 and for imatinib in HS578T.
Stimulation with DHT increased the amount of the AR, and this was not prevented by the administration of either an inhibitor of PI3K or Erk1/2; showing that the mechanism associated with the expression of the AR by androgens is independent of a kinase control. Furthermore, effects on the expression of AR after stimulation with DHT or when administering kinase inhibitors were independent of the expression of the AR gene, suggesting a posttranscriptional mechanism.
Our findings have some potential therapeutic translations. It has been suggested that the AR is a potential druggable target in TNBC and indeed, clinical studies are currently evaluating the role of androgen deprivation in the treatment of patients with breast cancer [
30]. In our work we identify a relationship between the expression of the AR and the activation of membrane kinases like EGFR and PDGFRβ. Furthermore, the concomitant inhibition EGFR and PDGFRβ with bicalutamide increased the antiproliferative effect compared with each agent given alone. A similar effect was observed for the administration of the Erk1/2 inhibitor with bicalutamide.
Conclusions
We observed a positive correlation between the activation of EGFR and PDGFRβ and the expression of the AR. Inhibition of PI3K pathway decreased the total amount of the AR. Inhibition of Erk1/2, PDGFRβ and EGFR decreased the expression of the AR when given concomitantly with bicalutamide and had an additive anti-proliferative effect, suggesting that administration of anti-androgens in TNBC should be given concomitantly with inhibitors of these kinases. Modification of AR expression by these kinase inhibitors was posttranscriptionally regulated. In conclusion, our results describe mechanisms associated with the control of the AR expression in TNBC, and demonstrate a potential therapeutic opportunity for the combination of anti-androgens with RTK inhibitors and its downstream routes.
Acknowledgements
This research is supported by: Fundación Sociosanitaria de Castilla La Mancha (FISCAM) Grant Number. PI/2010/017, Ministerio de Ciencia e Innovación, Grant Number BFU2009-07728/BMC, Fondo de Investigación Sanitaria (FIS), Grant Number PI09/02144, Cancer Center Network Program from the ISCIII (RD06/0020/0041). Fundación Científica AECC.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AO carried out the design and drafting the manuscript. AP participates in the design of the manuscript. MDC-L carried out the molecular studies, participated in the design and drafting manuscript. JC Montero, JC Morales and AP participate in the molecular studies. All authors read and approved the final manuscript.