Vitamin D and androgen receptor-targeted therapy for triple-negative breast cancer
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Anti-estrogen and anti-HER2 treatments have been among the first and most successful examples of targeted therapy for breast cancer (BC). However, the treatment of triple-negative BC (TNBC) that lack estrogen receptor expression or HER2 amplification remains a major challenge. We previously discovered that approximately two-thirds of TNBCs express vitamin D receptor (VDR) and/or androgen receptor (AR) and hypothesized that TNBCs co-expressing AR and VDR (HR2-av TNBC) could be treated by targeting both of these hormone receptors. To evaluate the feasibility of VDR/AR-targeted therapy in TNBC, we characterized 15 different BC lines and identified 2 HR2-av TNBC lines and examined the changes in their phenotype, viability, and proliferation after VDR and AR-targeted treatment. Treatment of BC cell lines with VDR or AR agonists inhibited cell viability in a receptor-dependent manner, and their combination appeared to inhibit cell viability additively. Moreover, cell viability was further decreased when AR/VDR agonist hormones were combined with chemotherapeutic drugs. The mechanisms of inhibition by AR/VDR agonist hormones included cell cycle arrest and apoptosis in TNBC cell lines. In addition, AR/VDR agonist hormones induced differentiation and inhibited cancer stem cells (CSCs) measured by reduction in tumorsphere formation efficiency, high aldehyde dehydrogenase activity, and CSC markers. Surprisingly, we found that AR antagonists inhibited proliferation of most BC cell lines in an AR-independent manner, raising questions regarding their mechanism of action. In summary, AR/VDR-targeted agonist hormone therapy can inhibit HR2-av TNBC through multiple mechanisms in a receptor-dependent manner and can be combined with chemotherapy.
KeywordsHormone receptor Triple-negative breast cancer Androgen receptor Vitamin D receptor Cancer stem cells
Breast cancers (BCs) are categorized in the clinic into three subtypes, including, estrogen receptor positive (ER+), epidermal growth factor receptor 2 positive (HER2+), and triple-negative BCs (TNBCs) . Treatment of ER+ and HER2+ BCs has been successful through targeted therapy with anti-estrogen and anti-HER2 drugs. Due to the lack of these targets, neo-adjuvant chemotherapy is used for treatment of TNBC that are typically associated with poorer prognosis compared to other BC subtypes [2, 3]. In addition, up to half of the ER+ tumors eventually become resistant to anti-estrogens [4, 5, 6]. Therefore, there is an urgent need to identify and develop novel targeted therapy approaches for TNBC and hormone refractory ER+ BCs.
In an attempt to develop a normal cell lineage-based phylogenetic BC classification , we previously studied hormone receptors (HRs) in normal human breast tissues and compared them with human BCs . To do so, we used multiplex immunofluorescent staining and analyzed simultaneous co-expression of the 14 lineage markers in ~15,000 normal breast cells and ~3000 BCs, and found that both normal luminal breast cells and BCs conform to four hormonal states (HR3, HR2, HR1, and HR0) based on co-expression of ER, androgen receptor (AR) and vitamin-D (Vit-D) receptor (VDR) . We also found that there was 6.9-fold difference in overall survival between HR3 versus HR0 tumors . Compared to the 1.7–2.1-fold differences typically reported in overall survival between ER+/LumA versus TNBC/basal-like BC [8, 9], these results suggest that the HR0–3 classification reveals BC sub-groups with highly significant outcome differences.
Utilizing the HR0–3 classification, we discovered that approximately two-thirds of TNBCs co-express AR and VDR (HR2-av TNBC) or express VDR alone (HR1-v TNBC); the remaining one-third of the TNBCs are triple negative for ER, AR, and VDR (HR0 TNBC) . These findings raised the possibility of developing novel HR-targeted therapies for TNBC, for which the only existing option is chemotherapy at the moment. In the present study, we demonstrate that HR1-v and HR2-av TNBC cell lines can be targeted with AR and/or VDR agonist hormones alone and in combination. The combined effects of AR and VDR ligands not only reduce the viability of the cells, but also change their cancer stem cell (CSC) phenotype and differentiation. In addition, we found that AR and VDR agonist hormones can be combined with chemotherapeutic agents to successfully target TNBC cells.
Materials and methods
Cell culture and drug viability
All the BC and prostate cancer (PC) cell lines were purchased from ATCC and DSMZ and cultured in their respective media. The short tandem repeat profiling was used to validate the authenticity of the cell lines (Genetic Resources Core Facility, John Hopkins School of Medicine). All the drugs were prepared and used according to concentrations that were previously reported [10, 11, 12, 13, 14], detailed information for each drug is provided in Supplemental Table 2. Cell viability assays were carried out in 24 or 96 well plates with cell titer cell reagent as previously described . The LD50 values of all the AR antagonists were calculated as described previously . See supplemental methods for further details and analysis of additive or antagonistic activity determined by Bliss independent criterion.
Cell cycle and apoptosis assays
The cell cycle profile after AR or VDR agonist treatment was evaluated by bromodeoxy uridine (BrdU) pulse labeling followed by FACS analysis. Apoptosis was measured with FACS analysis of Annexin V and propidium iodide double staining following the manufacture’s protocol (Life Technologies). See supplemental methods for further details.
Tumorsphere and AldeFluor assays
Tumorsphere assays were performed as described previously . AldeFluor assays were performed as described  using ALDEFLUOR kit (StemCell Technologies). The cells were treated with agonist(s) for 8 days with media changes every 3 days and analyzed according to manufacturer’s protocol by FACS. See supplemental methods for further details.
PCR-based human stem cell array
Human stem cell RT2 Profiler™ PCR array was used and data were analyzed according to manufacturer’s protocol and software (SA Biosciences). The genes with a 2-fold difference compared to the vehicle treated control group were shortlisted and plotted as a Heatmap generated using Microsoft Excel.
The statistical significance of the data was evaluating by performing Student’s T test using a cut-off of P-value <0.05.
Majority of TNBC cell lines express AR and/or VDR
Inhibition of TNBC cell lines with calcitriol is VDR dependent
The role of VDR has been studied in cancers, showing that ligand bound VDR induces anti-proliferative, pro-apoptotic, and pro-differentiating effects both in vitro and in vivo [13, 18]. Here, we confirmed that natural VDR agonist 1α,25-dihyroxy vitamin D3 (calcitriol) inhibits cell viability in BC cell lines (Fig. 1b). No inhibition of cell viability was observed in VDR− breast cell line BT-549 demonstrating that the response to calcitriol is VDR dependent.
Inhibition of TNBC cell lines with dihydrotestosterone is AR dependent
While the notion of AR-targeted therapy for BC has been around since the early 1970s [19, 20, 21], whether AR agonists or AR antagonists should be used for this purpose has been contentious. Many studies show that AR agonists inhibit BC cell growth both in vivo and in vitro [22, 23, 24, 25, 26, 27, 28, 29, 30], and others indicated that AR antagonists can also inhibit breast tumor growth  and recently several clinical studies were initiated with AR antagonists in BC patients [32, 33]. Hence, based on the prior literature, it was not entirely clear whether AR agonists or antagonists should be used to treat AR+ TNBC. Thus, we started by testing the effects of both AR agonists and antagonists in a panel of AR+ and AR− BC cell lines including all three subtypes (ER+, HER2+, and TNBC). In addition, we used AR+ and AR− PCs as controls because PC cell lines have a well-established and specific response to AR ligands.
We found that AR agonists dihydrotestosterone (DHT) and R1881 stimulated proliferation of AR+ PC cell lines as expected. Importantly, there was no effect on AR− PC-3 cell line, which demonstrates that the effect of DHT and R1881 on cell proliferation is AR dependent in PC cell lines (Fig. 1c; Suppl. Fig. 1b).
Consistent with the opposing role of androgens in male versus female, DHT or R1881 treatment resulted in a decrease in cell proliferation and viability in AR+ BC cell lines (Fig. 1c; Suppl. Fig. 1b). The one exception was the ER− HER2+ BC cell lines in which AR agonists increase cell proliferation (data not shown), which was shown to be due to a cross-talk between HER2 and AR signaling pathways that is only observed in ER− background [10, 34, 35]. There was no response to DHT or R1881 in AR− BT-20, MDA-MB-468, and AR-low SUM-159PT cell lines indicating that at the effect of DHT and R1881 on cell proliferation is AR dependent in BC cell lines (Fig. 1c; Suppl. Fig. 1a, b).
In summary, AR agonists DHT and R1881 decrease proliferation of TNBC, ER+, and ER+/HER2+ BC cell lines and stimulate proliferation of ER−/HER2+/AR+ cell lines.
Inhibition of TNBC cell lines with AR antagonists is not dependent on AR
Next, we tested the effect of AR antagonists on BC cell line proliferation using three different drugs (flutamide, bicalutamide, and enzalutamide) on four AR+ and three AR−/low BC cell lines. In addition, two AR+ and two AR− PC cell lines were used as controls.
Surprisingly, in BC cell lines, we did not observe a similar difference in the LD50 values of AR antagonists in AR+ versus AR− lines (Fig. 2d; Suppl. Fig. 2c). There was decrease in cell proliferation in BC cell lines with AR antagonists regardless of their AR protein expression (Fig. 2c; Suppl. Fig. 2b). These results suggest that the effect of AR antagonists in BC cell lines can be AR-independent. Therefore, we concentrated on AR agonists as drug of choice for further experiments in TNBC lines (Fig. 7a).
Treatment of TNBC with AR and VDR agonists in combination with chemotherapy
We previously showed that nearly two-thirds of TNBC have HR1-v (45 %) or HR2-av (18 %) phenotype, i.e., these tumors are negative for ER, PR, and HER2, but positive for VDR (HR1-v) or both AR and VDR (HR2-av) . These TNBCs are currently treated with chemotherapeutic agents such as Taxol or cisplatin.
In HR2-av TNBC cell lines, we found that combination of AR and VDR agonists had an additive inhibitory effect in two different HR2-av TNBC cell lines (Fig. 3b). Next, we examined combining AR- and VDR-targeted therapy with chemotherapy, and found that combination of AR (DHT)- and VDR (calcitriol)-targeted therapy with Taxol or cisplatin has an additive effect in reducing cell viability (Fig. 3c, d).
It is worth pointing out that in order to demonstrate additivity in these experiments, we used doses lower than IC50 for each drug (Suppl. Fig. 4). Therefore, it is possible to achieve a greater reduction in cell numbers and viability, close to 100 % cell death, when these drugs are combined between IC50 and IC90 dose range.
The mechanism of tumor cell inhibition by AR- and VDR-targeted hormones
The VDR and AR agonists have been shown to have cell-context dependent pleiotropic effects on cell cycle, apoptosis, autophagy, or differentiation depending on the cell type and dose [13, 14, 25, 39, 40, 41, 42]. Therefore, we examined these potential mechanisms in HR2-av cell lines MFM-223 and CAL-148.
An increase in apoptosis was observed in CAL-148 with combination of DHT and calcitriol evaluated by Annexin V/PI co-staining (Fig. 4c) as well as PARP cleavage (Fig. 4d). Importantly, the apoptotic effects of DHT and calcitriol were minimal when they were used alone, consistent with an additive effect. In contrast, there was no change in apoptosis of MFM-223 cells with either hormone alone or in combination (Fig. 4c, d). Since many chemotherapeutic agents induce G2/M arrest followed by apoptosis [43, 44], we examined whether combining DHT and calcitriol with cisplatin in MFM-223 and CAL-148 would interfere with chemotherapy-induced G2/M arrest and apoptosis. We also found that co-treatment with AR/VDR agonists plus chemotherapy resulted in either no change or additive increase in G2/M arrest and apoptosis in MFM-223 and CAL-148 (Suppl. Fig. 5).
Lastly, we found that autophagy did not change with co-treatment of MFM-223 and CAL-148 cell lines with AR and VDR (DHT and Cal), measured by FACS using Cyto-ID® fluorescent dye or by western blots for autophagy marker LC3B (Suppl. Fig. 6).
Cumulatively, these results indicate that AR and VDR stimulation can additively inhibit proliferation of BC cells through cell cycle arrest or apoptosis depending on the cellular context, suggesting a rationale for their combined use. Furthermore, combining both hormones with chemotherapy can additively increase apoptosis in cancer cell lines.
AR- and VDR-targeted hormones can inhibit cancer stem cell phenotype
High aldehyde dehydrogenase (ALDH) activity is one of the features of CSCs, which is measured by AldeFluor assay [17, 50]. Consistent with the TFE results, we found that treatment with AR or VDR agonists decreased ALDH+ cells additively resulting in 5–100-fold decrease in the frequency of ALDH+ cells with co-treatment of HR2-av TNBC MFM-223 and CAL-148 cell lines (Fig. 5b; Suppl. Fig. 8).
Next, we examined other CSC-associated markers after treatment with AR or VDR agonists alone or in combination; we found that CD49f, Musashi, CD133, and CD326 are down-regulated with combination treatment (Fig. 5c). In addition, we observed an increase in differentiation markers Claudin-4 and cytokeratin 18 and down-regulation of cytokeratin 5 (MFM-223) as well as reciprocal down-regulation of vimentin and up-regulation of cytokeratin 18 (CAL-148) consistent with more differentiated epithelial phenotype (Fig. 5d; Suppl. Fig. 9).
Lastly, we used a PCR-based CSC pathway array that includes 84 common CSC-associated genes and found a 2–5-fold decrease in the mRNA expression of CD44, SOX2, MME (CD10), ALDH1A1, and PPAR-gamma with AR and VDR hormone co-treatment compared to control (Fig. 5e).
Cumulatively, these results indicate that in addition to increasing apoptosis and cell cycle arrest, AR and VDR agonists also inhibit CSC phenotype and induce differentiation in HR2-av TNBC cell lines.
Synthetic AR and VDR ligands can be used to treat HR2-av TNBC cell lines
The natural Vit-D ligands can cause hypercalcemia in patients, which prevents achieving clinically effective anti-tumor activity. However, over 1500 Vit-D analogs have been synthesized which may potentially have low calcemic effect [52, 53, 54]. We selected seocalcitol (EB1089), since its affinity to VDR is similar to natural ligand calcitriol, yet it has a 50–200-fold higher anti-proliferative activity and reduced hypercalcemic effect [55, 56]. We found that treatment of HR1-v and HR2-av cell lines with EB1089 resulted in a dose-dependent decrease in cell viability. Moreover, as reported before, EB1089 treatment was more potent than calcitriol at low doses (Fig. 6b; Suppl. Fig. 10a).
Next we examined combining synthetic VDR agonist EB1089 with chemotherapy in HR1-v BC cell lines, and found that there is an additive decrease in cell viability when EB1089 is combined with cisplatin or Taxol (Fig. 6c; Suppl. Fig. 10b). Furthermore, combining GTx-024 and EB1089 with or without Taxol produced additive inhibition of cell viability of the TNBC cell lines (Fig. 6d, e; Suppl. Fig. 10c, d). Cumulatively, these results suggest that AR- and VDR-targeted hormone therapy should be considered in the clinic using synthetic AR and VDR ligands with lower virilizing and calcemic side effects.
In brief, our study demonstrates that two different hormones that activate AR and VDR can be used alone, in combination with each other as well as with chemotherapy to inhibit proliferation of TNBC cell lines by increased apoptosis and G1/S arrest. In addition, these hormones inhibit TNBC CSC phenotype and induce differentiation. These observations raise the possibility of targeting approximately two-thirds of the TNBCs [ 1] with AR and/or VDR HR-targeted therapies.
Vitamin D receptor: its role in breast cancer treatment
VDR is expressed in around 90 % of the breast tumors . Of the six natural Vit-D compounds, calcitriol is the most active and stable form of Vit-D . Several clinical trials have been conducted with calcitriol as a single agent in breast tumors, with the conclusion that the therapeutic dose required to induce anti-tumor activity is difficult to achieve due to hypercalcemia, renal stones [52, 58, 59], and vascular calcification . Therefore, VDR agonists with low calcemic effects are needed to translate our findings to the clinic. Only a few of the more than 1500 Vit-D analogs have been tested in vivo so far [52, 53, 54]. Seocalcitol (EB1089) which we used in our study was tested in a Phase-I clinical trial and was found to be well tolerated and had some activity in BC patients .
The role of androgen agonists in breast cancer treatment
The role of androgen antagonists in breast cancer treatment
In our study, AR antagonists inhibited proliferation of all BC cell lines regardless of their high, low or negative AR expression status. This is very different than the observations in PC in which there was a >5-fold difference between in the LD50 of AR antagonists in AR+ versus AR− PC cell lines. Importantly, AR-independent inhibition of TNBC cell line proliferation was observed with all three antagonists indicating that this is not an isolated finding. Furthermore, the response of the same TNBC cell lines to the AR agonists DHT and R1881 was AR-specific, which indicates that these TNBC cell lines have a functional AR receptor. At the same time, the response of PC cell lines to AR antagonists was AR dependent ruling out methodological errors. These results indicate that the anti-proliferative effect of AR antagonists in BC cell lines may possibly be due to an off-target effect. Thus, while these drugs have some activity in the clinic, whether this is through AR or some other target remains to be determined. It is worth pointing out that it is already known that some AR antagonists can have non-AR activities; for example, enzalutamide inhibits GABA-A receptors , bicalutamide inhibits CYP27A1 , and both flutamide and bicalutamide bind to PR . Hence it is possible that these drugs may have activity in cells that are ostensibly negative for AR activity through other genes that are yet to be discovered. Therefore, genetic background of these individual cell lines may play a role in the AR antagonist response in BC cell lines. However, one might have expected the genetic background could affect agonist response equally, which was not the case. The seemingly off-target AR-antagonist effect appears to emerge only in breast cells and not in prostate cells, which may be due to tissue-specific regulation of signaling pathways. For example, at the gene expression level, it was found that AR binds to the PTEN promoter as a repressor, inhibiting its transcription in PC cells. In contrast, AR stimulates PTEN gene expression as an activator in BC cells . Therefore, while surprising, the tissue-specific drug response may not be completely unexpected. Many drugs have unexpected activities due to binding to unknown targets or unknown interactions between the known drug target and other biochemical pathways .
Role of hormones in inhibiting CSC population
There is growing evidence that BCs consist of a heterogeneous population of different subtypes of cells including non-CSCs and CSCs that possess the ability of self-renewal [80, 81] and thought to be associated with resistance to the standard therapies and metastasis [82, 83]. Hence, targeting CSCs may be important for complete tumor remission.
We found that hormonal co-stimulation of AR and VDR simultaneously leads to reduction of CSC population, evidenced by a decrease in TFE, decrease in ALDH activity and down-regulation of markers associated with the CSC phenotype. High expression of CD49f regulates pluripotency factors such as OCT4 and SOX2  and Musashi regulates Notch which is one of the key pathways that regulate self-renewal potency of the cells [85, 86]. Therefore, inactivation of CD49f, SOX2, and Notch signaling may provide a mechanism through which AR and VDR treatment can decrease CSC population.
In summary, we show that co-targeting AR and VDR with agonist hormones can be an effective strategy to target CSCs. Our results also suggest that the selection of AR agonists in the treatment of BC will depend on ER and HER2 status, and combination of AR and VDR agonists can be additive with chemotherapy.
Research for this article was supported in part by Funding to T. A. Ince from Breast Cancer Research Foundation and Play for P.I.N.K., NIEHS R01-ES024991 and NCI R01-CA146445 from NIH Roadmap Epigenomics Project, as well as Funds from Women’s Cancer Association of UM and Sylvester Comprehensive Cancer Center. We thank Dr. Aurea D. Sousa, Dr. Kerry Burnstein, Dr. Dorraya El-Ashry, and Dr. Joyce Slingerland for helpful discussion and their suggestions.
Compliance with ethical standards
Conflict of Interest
The authors declare no conflicts of interest.
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