Combination of the novel histone deacetylase inhibitor YCW1 and radiation induces autophagic cell death through the downregulation of BNIP3 in triple-negative breast cancer cells in vitro and in an orthotopic mouse model
- Hui-Wen Chiu†1, 2,
- Ya-Ling Yeh3,
- Yi-Ching Wang4, 5,
- Wei-Jan Huang6,
- Sheng-Yow Ho†7, 8,
- Pinpin Lin9Email author and
- Ying-Jan Wang3, 10, 11Email author
© Chiu et al. 2016
Received: 28 August 2015
Accepted: 31 May 2016
Published: 10 June 2016
Triple-negative breast cancer (TNBC) is the most aggressive and invasive of the breast cancer subtypes. TNBC is a challenging disease that lacks targets for treatment. Histone deacetylase inhibitors (HDACi) are a group of targeted anticancer agents that enhance radiosensitivity. Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a member of the Bcl-2 subfamily. BNIP3 is not found in normal breast tissue but is up-regulated in breast cancer. In the present study, we investigated the anti-cancer effects of a newly developed HDACi, YCW1, combined with ionizing radiation (IR) in TNBC in vitro and in an orthotopic mouse model. Furthermore, we examined the relationship between autophagy and BNIP3.
Trypan blue exclusion was used to investigate the viability of 4 T1 (a mouse TNBC cell line) and MDA-MB-231 cells (a human TNBC cell line) following combined YCW1 and IR treatment. Flow cytometry was used to determine apoptosis and autophagy. The expression levels of BNIP3, endoplasmic reticulum (ER) stress- and autophagic-related proteins were measured using western blot analysis. An orthotopic mouse model was used to investigate the in vivo effects of YCW1 and IR alone and in combination. Tumor volumes were monitored using a bioluminescence-based IVIS Imaging System 200.
We found that YCW1 significantly enhanced toxicity in 4 T1 cells compared with suberoylanilide hydroxamic acid (SAHA), which was the first HDACi approved by the Food and Drug Administration for clinical use in cancer patients. The combined treatment of YCW1 and IR enhanced cytotoxicity by inducing ER stress and increasing autophagy induction. Additionally, the combined treatment caused autophagic flux and autophagic cell death. Furthermore, the expression level of BNIP3 was significantly decreased in cells following combined treatment. The downregulation of BNIP3 led to a significant increase in autophagy and cytotoxicity. The combined anti-tumor effects of YCW1 and IR were also observed in an orthotopic mouse model; combination therapy resulted in a significant increase in autophagy and decreased tumor tissue expression of BNIP3 in the tumor tissue.
These data support the possibility of using a combination of HDACi and IR in the treatment of TNBC. Moreover, BNIP3 may be a potential target protein for TNBC treatment.
Triple-negative breast cancer (TNBC) characterized by the absence of estrogen receptor alpha (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression, is a basal-like subgroup of breast cancers that accounts for 10–20 % of all breast cancers . Patients with this subtype are more likely to develop recurrence within the first 5 years, and survival following metastatic relapse is shorter for TNBC patients than those with other breast cancer subtypes . Currently, TNBC is one of the most attractive areas in cancer research. One reason for this scientific interest is the lack of therapeutic targets for TNBC. Therefore, identifying biological markers of TNBC progression could be helpful for the prevention of breast cancer metastasis and could provide novel therapeutic strategies for the disease. TNBC is typically treated with surgery, radiotherapy, and chemotherapy. Overcoming the deleterious consequences of radiotherapy and maximizing its anti-tumor effects to control tumor progression should be the goal of combined radio- and chemotherapy. Combination therapies aim to enhance radiosensitivity and prevent tumor recurrence. Numerous conventional cytotoxic drugs are used in conjunction with different radiation techniques . Recently, data accumulated by us and others have revealed that some compounds or drugs enhanced radiosensitivity through regulation of the cell cycle, induction of cell death and inhibition of DNA repair [1, 4–6].
Ionizing radiation (IR) induces important signal transduction pathways, such as the PI3K pathway, that are linked with radioprotective and growth-promoting events . The PI3K signaling pathway is associated with major radioresistance mechanisms, such as intrinsic radiosensitivity, tumor cell proliferation and hypoxia . Downstream molecular targets of PI3K up-regulate hypoxia-related proteins, stimulate mitogenic and pro-survival pathways and have anti-apoptotic effects via the induction of Bcl-XL, which is a member of the Bcl-2 family, and the inactivation of Bad and procaspase-9 . Positive Bcl-2 expression has been associated with poor survival and reduced sensitivity to chemotherapy in patients with TNBC . Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a member of the Bcl-2 subfamily of death-inducing mitochondrial proteins . Previous studies have demonstrated that BNIP3 provides a survival advantage in cancer cells by promoting autophagy and eliminating damaged mitochondria with low membrane potential that are a source of intracellular ROS [12, 13]. Additionally, BNIP3 expression is restricted to few normal tissues, including skeletal muscle and brain . In contrast to normal breast tissue in which BNIP3 was not expressed up-regulation of BNIP3 was observed in breast cancer . However, whether BNIP3 has an important role in TBNC remains unknown.
Many studies have implicated that HDAC enzymes have a role in the development of cancer and, therefore, are potential therapeutic targets [16, 17]. HDAC inhibitors (HDACi) block the deacetylation function of HDACs, causing cell cycle arrest, endoplasmic reticulum (ER) stress, differentiation, inhibition of angiogenesis, apoptosis and autophagy in many tumors [6, 17]. Normal cells are relatively resistant to HDACi-induced cell death . Moreover, HDACi can affect apoptosis and autophagy through regulation of the Bcl-2 family including inhibition of Bcl-2 and activation of Bax . However, several serious adverse events were reported in patients treated with suberoylanilide hydroxamic acid (SAHA), which was the first HDACi approved by the US Food and Drug Administration (FDA) for the clinical treatment of cutaneous T-cell lymphoma, and other HDACi . Therefore, the development of novel HDACi combination therapies or new HDACi with improved efficacies is urgently needed, particularly for solid tumors.
In this study, we used a newly developed HDACi, YCW1 (octanedioic acid [3-(2-(5-methoxy-1H-indol-1-yl)ethoxy)phenyl]-amide N-hydroxyamide), which was been optimized for HDAC inhibition using structure-based analyses [21, 22]. The murine TNBC cell line 4 T1 and human TNBC cell line MDA-MB-231 were used to investigate the anti-tumor effects of IR combined with this novel HDACi (YCW1) and the underlying mechanism of these effects, including the types of cell death and ER stress. Furthermore, we tested the inhibitory effect of IR combined with YCW1 in a 4 T1 orthotopic breast cancer model in mice. Our results suggest that the downregulation of BNIP3 in TNBC cells significantly increased the anti-tumor effects of IR and YCW1 through the induction of autophagic cell death. Using an orthotopic breast cancer mouse model of TNBC cells, we verified that co-treatment with IR and YCW1 inhibited tumor growth.
YCW1 inhibits HDAC activity and enhances cytotoxicity of IR in TNBC cells
Combined treatment induces ER stress and autophagy in 4 T1 cells
Autophagic cell death is induced by the combination of YCW1 and IR
BNIP3 expression influences the cytotoxic effects of YCW1 and IR in 4 T1 cells
Tumor growth in an orthotopic breast cancer model is suppressed by combined treatment with YCW1 and IR through the induction of autophagy and the inhibition of BNIP3
Biochemical tests including GOT, GPT, albumin and BUN
4 T1-Luc cells
YCW1 + IR
137.67 ± 42.17
147.33 ± 13.72
184.00 ± 32.32
181.67 ± 29.35
180.67 ± 18.22
48.00 ± 10.60
35.33 ± 3.18
33.67 ± 2.67
33.33 ± 2.19
40.33 ± 7.97
2.67 ± 0.13
2.37 ± 0.24
2.37 ± 0.12
2.37 ± 0.15
2.23 ± 0.13
21.60 ± 2.63
30.50 ± 10.42
26.73 ± 1.07
25.47 ± 1.60
26.53 ± 1.87
TNBC is the most invasive and aggressive among the breast cancer subtypes and there is no clinical therapy specific for patients with TNBC . In the present study, we found that the combination of a novel HDACi (YCW1) and IR may have anti-tumor potential against TNBC both in vitro and in vivo (Figs. 2 and 7). Moreover, our previous study demonstrated that YCW1 induced a broad spectrum of anticancer activities in lung cancer cell and animal models. It has been showed that HDACi induced cell death in many tumors and had less toxicity in normal cells [17, 18]. However, HDACi have demonstrated limited clinical benefit for patients with solid tumors . In our study, we used a novel HDACi, YCW1, and examined the combination effect of YCW1 and IR in TNBC cells. YCW1 showed a better inhibition of total HDAC activity and significantly enhanced toxicity compared with SAHA  (Fig. 1a). The combined treatment with YCW1 and IR enhanced the growth inhibition of 4 T1 and MDA-MB-231 cells compared with YCW1 or IR alone (Fig. 2). Furthermore, YCW1 caused no detectable toxicity as determined by either biochemical examination or in terms of the loss of body weight (Fig. 7b and Table 1). Therefore, YCW1 is a potential HDAC inhibitor and enhances radiosensitivity in TNBC cells.
HDACi cause ER stress, apoptosis and autophagy in many tumors [6, 17]. Previous research has shown that treatment of cancer cells with HDACi induced autophagy which promotes cancer cell survival when apoptosis induction is inhibited. Moreover, autophagy inhibition resulted in a higher level of apoptosis in response to SAHA treatment . Rao et al. indicated that combination of autophagy inhibitor and HDACi induced the accumulation of toxic polyubiquitylated proteins and caused inhibitory effects on TNBC cell growth . However, there are also numerous reports in the literature showing the pro-death function of autophagy. HDACi can induce caspase-independent autophagic cell death and have clear clinical implications in treating cancers with apoptotic defects [34, 35]. Therefore, the role of autophagy in regulating cancer cell death or survival remains controversial. Recent studies have investigated the induction of ER stress as a novel strategy for treating malignancies [36, 37]. ER stress triggers unfolded protein response (UPR) pathways, including the IRE1 pathway, the PKR-like ER-resistant kinase pathway and the activating transcription factor 6 pathway . Data accumulated by us and others have revealed that IR activates ER stress and UPR pathways through the induction of DNA damage [24, 39]. Here, IRE1α and phosphorylated eIF2α, which are both UPR-related proteins, increased in cells treated with combined YCW1 and IR (Fig. 3a). Using TEM, the ultrastructures of the 4 T1 cells indicated ER stress after the treatment with combined treatment (Fig. 4). Evidence indicating that ER stress can induce cell death, including apoptosis and autophagy, has been reported [40, 41]. We found that combined treatment mainly induced autophagy and a small amount of apoptosis in 4 T1 cells (Figs. 3, 4 and Additional file 1: Figure S1). In our in vivo study, tumor tissues from the mice treated with combined treatment showed higher autopahgic levels compared with mice treated with a single agent (Fig. 7d). However, the role of autophagy in regulating cancer cell death or survival remains controversial. Furthermore, an increase in autophagic flux may be the a key factor that modulates autophagy to cell death . The present study shows that Atg5 shRNA decreased combined treatment-induced cytotoxicity (Fig. 5c). Additionally, the combined treatment caused autophagic flux (Fig. 5d). Therefore, our results showed that the combination treatment with YCW1 and IR induced autophagic cell death in TNBC cells.
Members of the Bcl-2 family are contained in multiprotein complexes at the ER, where they regulate diverse cellular processes including autophagy, calcium homeostasis and the unfolded-protein response . The Bcl-2 family comprises three subfamilies, an anti-apoptotic family, a pro-apoptotic multi-domain family and a pro-apoptotic BH3-only protein family . Recent evidence shows that this family affects not only apoptosis but also autophagy [19, 44]. In the present study, Bcl-XL protein expression decreased in 4 T1 cells following combined treatment (Fig. 3a). Although BNIP3 is in the pro-apoptotic BH3-only protein family, it was recently found to increase resistance to apoptosis, and it was implicated that BNIP3 may also play an important role in autophagy [45, 46]. Moreover, growing evidence has shown that the overexpression of BNIP3 can be detected in many malignancies, such as glioma, breast cancer and prostate cancer [14, 47]. In addition, BNIP3 was not identified in normal breast but was up-regulated in breast cancer . Park et al. indicated that BNIP3 degradation was triggered by autophagy and could be regulated by mTORC1 and AMPK . In this study, we found that BNIP3 significantly decreased in cells or tumors following combined treatment compared with those that received YCW1 or IR alone (Figs. 6a and 7d). 4 T1 cells transfected with BNIP3 shRNA showed a significant increase the number of autophagic cells and reduced the viability compared with the control shRNA (Fig. 6c and d). Furthermore, the overexpression of BNIP3 decreased autophagy (Fig. 6f). Therefore, combined treatment induced autophagic cell death through the inhibition of BNIP3 in TNBC cells.
Taken together, these data suggest that the combination of YCW1 and IR exerts a cytotoxic effect on TNBC cells that is associated with increased levels of autophagy and ER stress. More importantly, BNIP3 was degraded by the combined treatment-induced autophagy. This combination benefit was also observed in vivo using an orthotopic breast cancer model. Our data show that combining YCW1 and IR and targeting BNIP3 are therapeutic options that should be further investigated for the treatment of TNBC.
Preparation of YCW1
The complete chemical name of YCW1 is octanedioic acid [3-(2-(5-methoxy-1H-indol-1-yl)ethoxy)phenyl]-amide N-hydroxyamide. Requests for this compound should be sent to firstname.lastname@example.org.
The murine breast cancer cell line 4 T1 (ATCC CRL-2539) and human breast cancer cell line MDA-MB-231 (ATCC HTB-26) were obtained from the American Type Culture Collection (ATCC). The luciferase-expressing murine breast cancer cell line 4 T1-Luc was obtained from Dr. M.L. Kuo (Institute of Toxicology, National Taiwan University, Taipei, Taiwan) . The cells were cultured in Dulbecco’s modified essential medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with an antibiotic containing 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco BRL, Grand Island, NY, USA) and 10 % fetal bovine serum (HyClone, South Logan, UT, USA). The cells were incubated in a humidified atmosphere containing 5 % CO2 at 37 °C. Exponentially growing cells were detached using 0.05 % trypsin-EDTA (Gibco BRL, Grand Island, NY) in DMEM.
Irradiation treatment and cell viability assay
IR was performed with 6 MV X-rays using a linear accelerator (Digital M Mevatron Accelerator, Siemens Medical Systems, CA, USA) at a dose rate of 5 Gy/min. An additional 2 cm of a tissue-equivalent bolus was placed on the top of the plastic tissue-culture flasks to ensure electronic equilibrium, and 10 cm of a tissue-equivalent material was placed under the flasks to achieve full backscatter. Cells were immediately treated with YCW1 following IR treatment. Next, the cells were centrifuged and resuspended in 0.1 ml PBS. Each cell suspension (0.02 ml) was mixed with 0.02 ml of a trypan blue solution (0.2 % in PBS). After 1 or 2 min, each solution was analyzed on a hemocytometer, with blue-stained cells counted as nonviable.
Determination of early apoptosis
Apoptosis was assessed by quantifying the translocation of phosphatidylserine to the cell surface, detected with Annexin V staining (Calbiochem, San Diego, CA, USA). Experiments were conducted according to our previous reports [5, 50].
The cells were cultured on coverslips. Cells were harvested and fixed in 4 % paraformaldehyde and blocked with 1 % BSA for 30 min. This was followed by incubation with a specific antibody against LC3 (MBL, Japan) for 1 h. After washing, the cells were labeled with a DyLight™ 488-conjugated affinipure goat anti-rabbit IgG (Jackson Immuno-Research Laboratories, PA, USA) for 1 h and with DAPI. Finally, the cells were washed in PBS, covered with a coverslip, and examined with a fluorescence microscope or confocal microscope (Carl Zeiess LSM780, Instrument Development Center, NCKU). To quantify the number of LC3 dots per cell, a minimum of 50 cells per sample was counted.
Transmission electron microscopy (TEM)
Cells were trypsinized and harvested, then fixed for 1 h in a solution containing 2.5 % glutaraldehyde and 2 % paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3. After fixation, the samples were postfixed with buffer containing 1 % OsO4 for 30 min. Ultra-thin sections were subsequently observed under a transmission electron microscope (JEOL JEM-1200EX, Japan) at 100 kV.
Western blot analysis
Total cellular protein lysate was prepared by harvesting cells in protein extraction buffer for 1 h at 4 °C, as described previously . GAPDH expression represented the protein loading control. Anti-GAPDH, anti-BNIP3, anti-IRE1α, phospho-eIF2α and anti-beclin 1 antibodies were obtained from Abcam (Cambridge, MA, USA); anti-LC3 and anti-eIF2α antibodies were obtained from Abgent (San Diego, CA, USA); anti-acetyl-histone H3 and acetyl-histone H4 antibodies were obtained from Millipore (Bedford, MA, USA); anti-acetyl-tubulin antibody was obtained from Sigma (St. Louis, Missouri, USA); anti-Bcl-XL antibody was obtained from Cell Signaling Technology (Ipswich, MA, USA); and anti-p62/SQSTM1 antibody was obtained from MBL (Nagoya, Japan).
Transfection of shRNA and plasmids
We used Arrest-In Transfection Reagent (Thermo, MA, USA) to transfect cells according to the manufacturer’s protocol. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, supported by the National Core Facility Program for Biotechnology Grants of NSC (NSC 100-2319-B-001-002). The mouse library is referred to as TRC-Mm 1.0. Individual clones are identified as shRNA TRCN0000072184, shRNA TRCN0000009691, TRCN0000099432, TRCN0000099433, TRCN0000375819 and shRNA TRCN0000229458. BNIP3 plasmid was obtained from OriGene Technologies Inc. (Rockville, MD, USA).
Orthotopic breast cancer model
All experiments on mice were performed according to the guidelines of our institute (the Guide for Care and Use of Laboratory Animals, Medical College, National Cheng Kung University). The animal use protocol listed below has been reviewed and approved by the Institutional Animal Care and Use Committee of National Cheng Kung University, Taiwan (Approval No: 104232). Six week-old female Balb/c mice were acquired from the National Laboratory Animal Center (Taiwan). The animals were housed five per cage at 24 ± 2 °C and 50 % ± 10 % relative humidity and subjected to a 12-h light/12-h dark cycle. 4 T1-Lluc cells (5 × 104 cells in 0.2 ml of PBS) were injected into the lactiferous ducts of the 4th mammary fat pads in the female Balb/c mice. The mice were randomized into four treatment groups (5 mice per group): (1) Control (DMSO), (2) 25 mg/kg YCW1 three times per week for three weeks, (3) a single dose of 4 Gy IR, or (4) a combination of treatments with 25 mg/kg YCW1 three times per week and a single dose of 4 Gy IR. Bioluminescence imaging was conducted using an IVIS 200 imaging system coupled to a data acquisition computer running Living Image Software (XENOGEN). Before imaging, the mice were anesthetized with isoflurane and injected i.p with 150 mg/kg body weight endotoxin-free luciferase substrate (VivoGlo™, Promega). Body weights were measured once per week and used as an indicator of the systemic toxicity of the treatment. There were no deaths in any group during the experimental period. Mice were sacrificed via CO2 exposure. After sacrificing, the tumor tissues were formalin fixed and paraffin embedded for immunohistochemistry.
Immunohistochemical (IHC) staining analysis
Paraffin-embedded tissue sections (4 μm) were dried, deparaffinized, and rehydrated. Following microwave pretreatment in citrate buffer (pH 6.0; for antigen retrieval), the slides were immersed in 3 % hydrogen peroxide for 20 min to block the activity of endogenous peroxidases. After extensive washing with PBS, the slides were incubated overnight at 4 °C with the anti-LC3 (MBL, Japan) or anti-BNIP3 (Abcam, MA, USA) antibody. The sections were then incubated with a secondary antibody for 1 h at room temperature, and the slides were developed using the STARR TREK Universal HRP detection kit (Biocare Medical, Concord, CA). Finally, the slides were counterstained using hematoxylin. Each slide was imaged at low magnification (×100).
Whole blood samples from the treated mice were collected by intracardiac puncture and centrifuged at 2000 × g for 20 min to separate the serum. The biochemistry evaluation included assessing the glutamate oxaloacetate transaminase (GOT) activity, glutamate pyruvate transaminase (GPT) activity, albumin levels and blood urea nitrogen (BUN) levels. All experiments and procedures were performed in accordance with the Institutional Care Use Committee guidelines.
Data are expressed as the mean ± SD. Statistical significance was determined using Student’s t-test for comparisons between the means or one-way analysis of variance with post-hoc Dunnett’s test . Differences were considered significant when p < 0.05.
3-MA, 3-methyladenine; AO, acridine orange; ATF6, activating transcription factor 6; AVOs, acidic vesicular organelles; BAF, bafilomycin A1; BNIP3, Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3; ER, endoplasmic reticulum; IR, ionizing radiation; IRE1α, inositol-requiring enzyme 1α; PERK, PKR-like ER-resistant kinase; UPR, unfolded protein response; UPS, ubiquitin-proteasome system.
This study was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2314-B-006-060-MY2, MOST 104-2320-B-038-065, NSC 100-2325-B-400-012, NSC 101-2325-B-400-010 and NSC 102-2325-B-400 -010), the Taipei Medical University, Taipei, Taiwan (TMU103-AE1-B28) and the Chi Mei Medical Center, Tainan, Taiwan (CMNCKU10422).
HWC, YLY, YHH, WJH and YFL participated in data collection and analysis. HWC, YCW, SYH, PL and YJW participated in the design of the study. HWC, SYH, PL and YJW participated in the writing of the manuscript and data interpretation. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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