Introduction
Breast cancer is a disease with a number of diverse morphological subtypes. Invasive ductal carcinoma is the most common morphologic subtype representing 80% of invasive breast cancer cases [
1]. In addition, it can be subclassified into three major categories according to different expression levels of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) [
2]. In general, hormone receptor-positive breast cancer subtypes are less progressive and amenable to hormone therapy. Although HER2+ breast cancer subtype shows rapid progression, targeted therapy for treating breast cancers over-expressing HER2 has improved survival for HER2+ breast cancer patients [
3]. In contrast to these two subtypes, triple-negative breast cancer (TNBC) is resistant to various chemotherapy agents and targeted drugs because a widely available target for this subtype has not yet been discovered. Therefore, the development of new combined targeted therapy and identification of biomarkers that can help predict responses to treatment are still major challenges in TNBC.
Recent progress in the field of DNA repair has demonstrated that a synthetic lethal approach involving the use of poly (ADP-ribose) polymerase (PARP) inhibitors is a promising new therapeutic strategy for treating various cancers. DNA repair inhibitors have been shown to work as single agents in patients with DNA repair-defective tumors [
4,
5]. The most notable example so far is the use of PARP inhibitors to treat individuals with inherited breast and ovarian cancers lacking wild-type copies of the
BRCA1 and
BRCA2 genes [
6-
8]. PARP inhibitors have also produced promising results in TNBC patients harboring
BRCA-like genotypes or so-called BRCAness [
9]. Therefore, development of strategies for using PARP inhibitors and selecting populations within TNBC that will respond favorably to PARP inhibitor treatment based on predictive biomarkers represents both a challenge and an opportunity for breast cancer research. Additionally, enzyme-mediated DNA repair can cause resistance to DNA-damaging anticancer drugs and radiation, and inhibition of DNA repair may be therapeutically beneficial. In particular, it has been observed that combining chemotherapy or radiotherapy with PARP inhibitors kills human cancer cells more effectively than a genotoxic agent alone [
8,
10]. The development of new therapies including molecular targeting agents is eagerly awaited as well as treatment strategies to overcome chemoresistance using PARP inhibitors that are effective for ameliorating TNBC.
During the past few years, histone deacetylases (HDACs) have garnered great interest as anticancer therapeutic targets. Experimental data have suggested that HDACs are involved in mammary tumorigenesis at multiple levels [
11,
12]. HDACs participate in the negative regulation of genes such as ones encoding cell cycle inhibitors, differentiation factors, and pro-apoptotic factors. In addition, the expression of genes associated with angiogenesis along with cell invasion and migration are enhanced by HDACs. Thus, HDACs play important roles in cancer development by regulating the expression of numerous genes involved in both cancer initiation and progression. Based on the role of HDACs in cancer development, HDAC inhibition could have potent anti-tumor effects on various types of cancer by affecting tumor cells at multiple levels. More specifically, inhibition by HDACs could induce cell cycle arrest, apoptosis, and differentiation while inhibiting angiogenesis along with cell migration and invasion [
12].
HDACs also enable functional HRR by regulating the expression of homologous recombination repair (HRR)-related genes and promoting the accurate assembly of HRR-directed sub-nuclear foci [
13,
14]. There is evidence showing that dysfunctional HDACs lead to the downregulated expression of DNA repair genes including
RAD51 and
BRCA1/2, resulting in defective DNA repair which can result in the accumulation of DNA damage [
14,
15]. HDAC inhibitors have thus emerged recently as a class of anticancer therapeutic agents that prevent DNA repair. HDAC inhibition sustains DNA damage signaling and suppresses DNA repair gene expression, which can increase the sensitivity of cells to DNA damaging agents similar to BRCA deficiency in breast cancer. For this reason, HDAC inhibition could enhance the anti-tumor effect of PARP inhibitors in cases of TNBC by blocking the DNA repair pathway. Previous studies have shown that HDAC inhibition does enhance cellular sensitivity to DNA damaging agents; however, specific markers that can help predict the combinational effect have not yet been identified [
16-
18].
In the present investigation, we identified a determinant of the combined effects of a PARP inhibitor with an HDAC inhibitor in TNBC cell lines. We evaluated one possible combined strategy to treat the TNBC subtype. We discovered that suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, enhanced the growth inhibitory activities of olaparib, a PARP inhibitor, in TNBC cells. Additionally, the combination of olaparib plus SAHA induced the accumulation of DNA DSBs and downregulated signal transduction in TNBC cells that expressed phosphatase and tensin homolog (PTEN). Our results suggest that the expression of PTEN in TNBC cells significantly increased the anti-tumor effects of olaparib and SAHA through the induction of both apoptotic and autophagic cell death. Using a xenograft mouse model of TNBC cells expressing PTEN, we verified that co-treatment with olaparib and SAHA inhibited tumor growth. Taken together, these data suggest that the combination of olaparib with SAHA exerts a synergistic effect on TNBC cells that is associated with increased levels of both apoptosis and autophagy regulated by PTEN. More importantly, our results provide a rationale for conducting future clinical trials evaluating the effectiveness of using olaparib combined with SAHA to treat TNBC patients.
Methods
Reagents
Olaparib was provided by AstraZeneca (Macclesfield, UK) and SAHA was purchased from Selleck (Houston, TX, USA). Both reagents were dissolved in dimethyl sulfoxide (DMSO) as 10 mmol/L stock solutions.
Cell lines and culturing
Human breast cancer cells (MDA-MB-157, -231, -453, -468, BT-549, MCF7, T47D, SK-BR-3, HCC70, HCC1143, and Hs578T) whose identity was authenticated with a short tandem repeat analysis were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were banked and passaged for less than 6 months before use, and were maintained in a humidified atmosphere containing 5% CO2 at 37°C in RPMI-1640 (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% FBS; Welgene, Inc., Daegue, South Korea) and 10 μg/mL gentamicin (Cellgro, Manassas, VA, USA).
Cell growth inhibition assay
An MTT assay was used to determine cell viability as previously described [
19]. Cells were seeded at a density of 3 to 8 × 10
3 cells per well in 96-well plates and incubated overnight at 37°C. The cells were then treated with either olaparib or SAHA alone or with a combination of olaparib and SAHA at specific concentrations for 5 d. After treatment with the drugs, MTT solution was added to each well and the plates were incubated for 4 h at 37°C before the medium was removed. After dissolving the resulting formazan crystals with DMSO, cell viability was evaluated by measuring the absorbance of each well at 540 nm with a VersaMax™ microplate reader (Molecular Devices, Sunnyvale, CA, USA). The combined effect of olaparib and SAHA was assessed using Calcusyn software (Biosoft, Cambridge, UK). The combination index (CI), which is used to evaluate the effect of two-drug combinations, was calculated using the Chou-Talalay method [
20]. Drug synergism is defined by CI values <1 while antagonism is indicated by values >1.
Western blot analysis
Protein expression levels were measured by western blotting as previously described [
20]. Primary antibodies against MRE11, caspase3, PTEN, AKT, phosphorylated (p)-AKT, ERK, p-ERK, STAT3, p-STAT3, and LC3B were acquired from Cell Signaling Technology (Beverley, MA, USA). Anti-RAD51C (2H11/6) antibody was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies against p21 and Beclin-1 were obtained from Abcam (Cambridge, UK). Anti-p-histone H2A.X antibody (clone JBW301) was acquired from Millipore (Billerica, MA, USA) while anti-PARP antibody was purchased from BD Biosciences (Bedford, MA, USA). Anti-α-tubulin antibody (Sigma Aldrich, St Louis, MO, USA) was used as a control.
Cell cycle analysis
Cells treated with olaparib and/or SAHA were harvested, fixed in 70% ethanol, and then stored at -20°C. The cells were dissolved in 10 μg/mL RNase A (Sigma Aldrich) at 37°C for 20 minutes. Next, the cells were treated with 20 μg/mL propidium iodide (Sigma Aldrich) and the DNA contents of the cells (10,000 cells per experimental group) were measured using a fluorescence-activated cell sorting (FACS) Calibur flow cytometer (BD Biosciences).
Plasmid and siRNA transfection
The pcDNA3.1-PTEN expression plasmid was obtained from the Korea Human Gene Bank (Seoul, South Korea) and the GFP-LC3 construct was purchased from Cell Biolabs (San Diego, CA, USA). siRNA specific for PTEN and nonspecific controls were purchased from Qiagen (Hilden, Germany). Transfection was conducted using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The sequence of the PTEN-specific siRNA was 5′-AAGGCGTATACAGGAACAATA-3′. The sequence of the control (nonspecific) siRNA was 5′-AATTCTCCGAACGTGTCACG-3′.
Comet assays
An alkaline comet assay using a Trevigen Comet assay kit (Trevigen, Gaithersburg, MD, USA) was performed following the manufacturer’s protocol. Tail lengths were measured with the Comet assay IV program (Andor technology, Belfast, UK).
Immunofluorescence assay (GFP-LC3 localization)
Cells were plated on coverslips and transfected with the GFP-LC3 construct. After 2 d, the cells were fixed in 3.7% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS (PBS-T). The coverslips were mounted onto slides using Faramount aqueous mounting medium (Dako, Glostrup, Denmark). Immunofluorescence was visualized using a Zeiss LSM 510 laser scanning microscope.
Immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Immunohistochemistry and a TUNEL assay were performed as previously described [
19].
In vivo study
All animal experiments were carried out in the animal facility of Seoul National University (Seoul, South Korea) in accordance with institutional guidelines and prior approval from the Institutional Animal Care and Use Committee (IACUC) committee. To measure the
in vivo activity of olaparib and/or SAHA, 35 female Balb/c athymic nude 5-wk-old mice were purchased from Central Lab Animal Inc. (Seoul, South Korea). MDA-MB-231 cells (1 × 10
8) were subcutaneously injected into each mouse. After implantation of the tumor cells, the size of the resulting tumors and body weight of each mouse were measured. When the tumor volume reached 200 mm
3, the mice were randomly divided into different treatment groups (eight mice per group) and received vehicle, olaparib, SAHA, or a combination of olaparib and SAHA. All drugs were administered via oral gavages once daily at a concentration of 30 mg/kg for 28 consecutive days. Tumor volume was calculated using the following formula:
$$ \left({\left(\mathrm{width}\right)}^2\times \left(\mathrm{height}\right)\right)/2. $$
At the end of the measurement period, the mice were sacrificed with CO2 and the tumors were excised for further analysis.
Statistical analysis
Data were analyzed using SigmaPlot version 9.0 (Systat Software Inc., San Jose, CA, USA). All results are expressed as the mean ± standard error (SE). The two-sided Student’s t-test was used when appropriate. P-values <0.05 were considered statistically significant.
Discussion
Genomic instability is a key feature of cancer development, and DNA repair pathways have a significant impact on genomic stability. Defects in genome stability increase the sensitivity of cells to DNA damaging agents and provide an
Achilles heel for cancer therapeutics [
26,
27]. Olaparib, a PARP inhibitor that targets defects in the DNA repair pathway, has produced promising results in TNBC patients with BRCA deficiencies or BRCAness. However, the population of BRCAness in TNBC patients is reported to be limited, so many efforts have been made to extend the usage of PARP inhibitors [
19,
28-
30]. Various reports have demonstrated that compromised HRR activity sensitizes BRCA-proficient cancers to PARP inhibitors [
10,
19,
29]. Additionally, PARP inhibitors are a useful therapeutic strategy treating cases of cancer with a variety of HRR pathway deficiencies. Recent studies have suggested that the inhibition of HDAC activity impedes the HRR pathway, resulting in increased cellular sensitivity to DNA-damaging agents [
13,
31]. Thus, HDAC inhibition leads to the creation of cells that may mimic an HRR-deficient phenotype, resulting in increased PARP inhibitor sensitivity [
31,
32].
In the present study, we evaluated the synergistic effects of simultaneous PARP and HDAC inhibition on proliferation and cell cycle progression in sensitive TNBC cell lines. We also assessed the synergistic effects of PARP and HDAC co-targeting in TNBC cells. Our findings indicated that these effects are attributable to decreased DSB repair capacity due to HDAC inhibition, thereby resulting in DNA damage accumulation induced by PARP inhibition. We also discovered that TNBC cells showed different responses to the combination of PARP plus SAHA.
Interestingly, TNBC cells exhibiting synergistic responses to the olaparib-SAHA combination had a greater decrease of proliferative pathway activity observed as AKT and ERK phosphorylation. Our findings support the hypothesis that the synergistic effects on TNBC cells depend on PTEN expression.
PTEN is a well known target of HDACs. Not surprisingly, HDAC inhibition leads to the upregulation of PTEN expression [
13,
14]. Even though PTEN deficiency has been suggested as a marker that can help predict positive responses to PARP inhibitors [
33], the sensitivity of PARP inhibition is not associated with PTEN deficiency in at least two TNBC cell lines (HCC70 and MDA-MB-468) that we evaluated. Rather, PTEN deficiency appears to induce resistance to the combination effect of simultaneous inhibition of PARP and HDACs in TNBC cells. Activation of PTEN along with decreased AKT and ERK phosphorylation by treatment with olaparib plus SAHA in the present study suggested that proliferative signaling pathways are modulated by the combination of olaparib and SAHA. PTEN activation blocks cell cycle progression, thereby suppressing tumor formation and progression. In addition, PTEN is crucial for regulating and maintaining PI3K/AKT signaling [
34]. Loss of PTEN function mainly leads to over-activation of the PI3K/AKT pathway that is frequently observed in breast cancer. The PI3K/AKT pathway represents a mechanism of resistance to cancer therapeutic agents as well as PARP inhibitors [
34]. Therefore, upregulated PTEN expression induced by HDAC inhibition would enhance the cytotoxic effect of PARP inhibitors in PARP inhibitor-resistant breast cancer cells. This would be a rational argument for administering a combination regimen of olaparib plus SAHA for treating TNBC.
Another novel finding from the current investigation is that PTEN expression can determine the combined effects via the regulation of autophagic cell death. Induction of autophagy was clearly observed in TNBC cells expressing PTEN in which synergism between olaparib and SAHA was observed. Autophagy is a ubiquitous process of recycling cellular compartments and is mainly considered a cytoprotective response to metabolic stresses [
35-
38]. While autophagy is characterized as a mediator of cell death in the presence of chronic stress, it is unclear under which conditions autophagy promotes cell death or cell survival. Additionally, the interaction between autophagy and apoptosis is not well-established. Nevertheless, the effect of HDAC inhibition on autophagy has been studied in several types of cancers although many questions remain as to whether the induction of autophagy is cytoprotective or cytotoxic for cancer cells [
38]. In general, many studies in the field of cancer therapy have focused on a cell survival mechanism of autophagy in tumor cells [
37-
39]. Subsequently, autophagy suppression has been suggested to be a way to improve the therapeutic benefit of cancer treatments. It has also been hypothesized that autophagy induced by HDAC inhibition enhances the ability of cancer cells to escape cell death. However, we found that increased levels of autophagy correlated with increased cell death following olaparib and SAHA combination treatment. Based on data from the present study, we suggest a mechanism by which HDAC inhibition following SAHA treatment increases PTEN expression, leading to the downregulation of proliferative signaling pathways including the AKT/mTOR cascade and an associated increased sensitivity to PARP inhibitor-induced apoptosis. In addition, HDAC inhibition contributes to autophagy induction that also results in increased cancer cell death.
In summary, findings from the current investigation demonstrated that TNBC cells have different responses to olaparib and SAHA alone or in combination. Combination therapy with selective PARP and HDAC inhibitors may be an effective strategy for treating cases of TNBC with functional PTEN expression. The combination of PARP and HDAC inhibitors significantly promoted growth inhibition as a result of proliferative signaling pathway suppression, and also led the accumulation of DNA damage. Our data suggest that the combination of PARP and HDAC inhibitors also induces both apoptotic and autophagic cell death, which increases the cytotoxic effects of the inhibitors. These combined effects resulting in cell death are regulated by PTEN expression in TNBC cells. Results from our investigation indicate that olaparib plus SAHA could be a novel strategy for treating cases of TNBC with PTEN expression. In light of these findings, the combination of PARP and HDAC inhibitors may merit further clinical evaluation in patients suffering from TNBC.
Competing interests
Yung-Jue Bang: consultant/advisory board member of AstraZeneca, Inc. Seock-Ah Im: consultant board member and recipient of research funds from AstraZeneca Inc. Mark J O’Connor: employee of AstraZeneca Inc. None of the other authors have any potential conflicts of interest.
Authors’ contributions
MAR, ISA, and SSH designed the study, performed the experiments, interpreted the data, and wrote the manuscript. KDK assisted in the experiment, and wrote and reviewed the manuscript. KHJ, LKH, KT-Y, HSW, ODY, KT-Y, OMJ, BYJ, and ISA conceived of the study, and participated in its coordination. All authors of this paper participated in drafting the manuscript and approved the final version.