Abstract
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor clinical outcomes and without effective targeted therapies. Numerous studies have suggested that HDAC inhibitors (TSA/SAHA) may be effective in TNBCs. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), is a key enzyme in the proline metabolism pathway and plays a vital role in tumorigenesis. In this study, we found that HDAC inhibitors (TSA/SAHA) significantly increased POX expression and autophagy through activating AMPK. Depletion of POX decreased autophagy and increased apoptosis induced by HDAC inhibitors in TNBC cells. These results suggest that POX contributes to cell survival under chemotherapeutic stresses and might serve as a potential target for treatment of TNBC.
Introduction
Among women, breast cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related deaths globally [1]. According to the expression of estrogen receptor (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), breast cancer is generally divided into four subtypes: HR+/HER2-, HR+/HER2+, HR-/HER2+, and triple-negative (HR-/HER2-) breast cancers [2]. Triple-negative breast cancer (TNBC) accounts for ~15% of breast cancers. Compared to other subtypes, TNBC not only is more aggressive, but also lacks targeted therapies. Thus, TNBC patients have low disease-free and overall survival rates. It is urgent to identify new and effective therapeutic targets [3–6].
Proline oxidase (POX), also known as proline dehydrogenase (PRODH), is often deleted in various human tumors [7]. POX is an enzyme involved in the first step of proline metabolism and plays an important role in tumor development. POX usually works as a mitochondrial tumor suppressor blocking cell cycle progression, initiating apoptosis, and inhibiting tumor growth by generating ROS [8]. The expression of POX is regulated by different tumor-associated factors, including p53, AMP-activated protein kinase (AMPK), inflammatory factor peroxisome proliferator-activated receptor gamma (PPARγ), oncogenic transcription factor c-Myc, and miR-23b, etc. [8–10]. The POX gene transcription was strongly induced by p53 [11] because there is a p53-response element at the POX gene promoter [12]. Additionally, PPARγ is a potent activator of the POX gene promoter [13]. In contrast, MYC suppresses POX expression primarily through upregulating miR-23b [9]. AMPK is a critical energy sensor for nutrient or oxygen deprivation [14]. In response to low-glucose stress, AMPK activation induces POX to maintain cellular energy levels [15].
Histone deacetylases (HDACs) contains 18 members, among of them, HDAC1–10 belongs to the Class I-II HDACs, which are frequently overexpressed in numerous tumors and are potential anti-cancer drug targets [16, 17]. HDAC inhibitors induce senescence, immunogenicity, differentiation, apoptosis, autophagy, necrosis, and cell growth arrest in different types of tumors [18–20]. Trichostatin A (TSA) and suberoylanili-dehydroxamic acid (SAHA) are two well-studied HDAC inhibitors [21], which inhibit class I/II HDAC’s activities [22]. TSA is a natural product, which inhibits HDAC1–7 and 9 at nanomolar levels but inhibits HDAC8 at a micromolar level [23]. SAHA, commercially known as vorinostat, is structurally similar to TSA. It is the first FDA-approved HDAC inhibitor and is admitted for the treatment of cutaneous T-cell lymphoma (CTCL) clinically [24–27]. It has been reported that SAHA in combination with BRD4 inhibitor [28], docetaxol [29], or epigallocatechin-3-gallate (EGCG) [30] synergistically inhibit breast cancers.
HDAC inhibitors induce apoptosis via multiple mechanisms, such as increasing the expression of Bcl-2 interacting mediator of cell death (Bim) [31, 32], increasing Fas-associated death domain protein (FADD) recruitment to DR4 in the death-inducing signaling complex [33, 34]. Recent studies showed that HDAC inhibitors induce autophagy in cancer cells [16, 35]. However, the role of autophagy in cell death remains controversial. Several studies reported that autophagy serves as a cell survival mechanism in HDAC inhibitors-mediated cancer cell death, while other studies suggest that autophagy acts as a cell death promoting mechanism [16, 35–39].
In this study, we demonstrated HDAC inhibitors (TSA/SAHA) induce POX and pro-survival autophagy through activating AMPK in TNBC cells. Depletion of POX increased HDAC inhibitors-induced apoptosis in TNBC cells. These results provided a novel insight into the functional mechanisms of HDAC inhibitors and POX in TNBC.
Materials and Methods
Cell lines and reagents
All cell lines used in this study, including HCC1806 and HCC1937, were purchased from American Type Culture Collection (ATCC, Manassas, USA) and validated by STR (short tandem repeat) analysis. HCC1806 and HCC1937 cells were cultured in RPMI 1640 medium (Thermo Fisher, Grand Island, USA) supplemented with 5% FBS, and were cultured at 37°C with 5% CO2. TSA (Cat#V900931) and SAHA (Cat#SML0061) were purchased from Sigma (St Louis, USA).
Cell viability assays
The cell viability was measured by SRB assays (Sulforhodamine B; Sigma). Briefly, HCC1806 and HCC1937 cells were seeded in 48-well plates at concentrations of 1.1 × 104 and 8 × 103 cells/well, respectively. After being treated with HDAC inhibitors or vehicle control, the cells were fixed with 10% trichloroacetic acid (TCA; Sigma) at room temperature for 1 h, followed by being washed with deionized water. Air dried cells were incubated with 0.4% SRB (W/V) solution in 1% acetic acid for 15 min, washed 5 times with 1% acetic acid and air dried completely. Finally, the SRB was dissolved with 10 mM unbuffered Trisbase, and the absorbance was measured using an Epoch microplate reader (BioTek, Shoreline, USA) at 530 nm. Each experiment was performed in triplicates for least three times independently.
Western blot analysis
HCC1806 and HCC1937 cells treated with TSA/SAHA at indicated concentrations were collected and lyzed using RIPA buffer containing protease inhibitors P8340 (Sigma). After quantification, around 40 μg protein were subject to 10% SDS-PAGE, then transferred to PVDF (polyvinylidene fluoride) membranes (0.45 μm; MerkMillipore, Darmstadt, Germany) for primary and secondary antibody incubation. The primary antibodies, including rabbit anti-AMPK antibody, anti-p-AMPK antibody, anti-p21 antibody, anti-Cyclin D1 antibody, anti-LC3 antibody, anti-P62 antibody, anti-PARP antibody, anti-P53 antibody, anti-PPARγ antibody and anti-cl-Caspase-3 antibody, and mouse anti-Myc antibody were all purchased from Cell signaling Technology (Danvers, USA) and diluted 1000 times. The mouse anti-POX antibody was purchased from Santa Cruz Biotech (Dallas, USA). The mouse anti-β-actin antibody (1:5000 dilution) were purchased from Sigma.
Real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA) and cDNA was synthesized using the Reverse Transcription Kit (Applied Biosystems, Austin, USA). For quantitative PCR, SYBR PCR Master Mix (Applied Biosystems) was used to quantify the expression level of target mRNA on a 7900HT Fast Real-Time PCR System (Applied Biosystems). The POX primer sequences are: Forward, 5′-AGGAGGCAGAGCACAAGGAGA-3′, and Reverse, 5′-CGGGCACTGATGACACCATTC-3′. GAPDH was used as the loading control. The GAPDH primer sequences are: Forward, 5′-GGTGAAGGTCGGAGTCAACG-3′, and Reverse, 5′-TGGGTGGAATCATATTGGAACA-3′.
RNA interference
HCC1806 and HCC1937 cells were plated in 6-well cell culture plates at a density of 2.0 × 105 cells/well. The day after plating, the cells were transfected with 50 nM of siRNA oligos using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. One day after transfection, the cells were treated with either TSA (1 μM)， SAHA (5 μM) or vehicle control for 24 h. Control siRNA (Cat. No. siN05815122147-1-5; RiboBio Company, Guangzhou, China) was used as the negative control and AMPK siRNA was purchased from Santa Cruz Biotech (Cat. No. sc-45312), and POX siRNA was purchased from RiboBio Company. The siRNA target sequences for the human POX gene are 5′-TAGAAGGTCATCTTCATGAGCTTGT-3′ (Poxsi#1), 5′-TATTCGTGCC ACTGCCATCCCTCTC-3′ (Poxsi#2), 5′-TTCGATGCAGCGCAAGAATGTCTCC-3′ (Poxsi#3). The siRNA target sequences for the human c-Myc gene are: 5′-AAGACCUUCAUCAAAAACAUTT-3′ (Mycsi#1), 5′-GAGCUAAAACGGAGCUUUUTT-3′ (Mycsi#2).
Apoptosis analyses
HCC1806 and HCC1937 cells were seeded in 6-well dishes at 2.0 × 105 cells/well. The day after plating, the cells were transfected with siRNAs followed by treatment with TSA (1 μM) or SAHA (5 μM), respectively for 24 h. The cells were collected and stained with Annexin V and Propidium lodide (PI) (BD Biosciences, San Jose, USA), then analyzed on an Accuri C6 Flow Cytometer (BD Biosciences).
Statistical analysis
All results were repeated at least three times and GraphPad Prism version 5.00 (GraphPad Software, San Diego, USA) was employed to analyze the data. All data are shown as the mean ± SD. Differences between groups were analyzed using Student’s t-test. P < 0.05 was considered statistically significant.
Results
HDAC inhibitors inhibit TNBC cell growth and induce POX expression
To test whether TSA/SAHA inhibits TNBC cell growth, we treated two TNBC cell lines, HCC1806 and HCC1937, with different concentrations of TSA (0–4 μM) or SAHA (0–40 μM) for 48 h and found that these inhibitors significantly decreased cell viability in a dose-dependent manner (Fig. 1A). It has been reported that HDAC inhibitors could induce TNBC cell cycle arrest, apoptosis, and autophagy [20]. In agreement with these, we found that HDAC inhibitors (TSA/SAHA) dramatically increased the CDK inhibitor P21, cleaved PARP, cleaved-Caspase-3, and LC3 II protein levels and decreased the Cyclin D1 and P62 protein levels (Fig. 1B). Interestingly, we found that both HDAC inhibitors induced the protein expression of POX in both TNBC cell lines (Fig. 1B).
HDAC inhibitors inhibit TNBC cell growth and induce POX expression (A) TSA (0–4 μM) and SAHA (0–40 μM) treatment for 48 h significantly decreased cell viability in HCC1806 and HCC1937 cells, as measured by the SRB assay. Data were presented as the mean ± SD. **P< 0.01. (B) TSA (0–2 μM) and SAHA (0–20 μM) treatment significantly increased POX protein expression in two cell lines in a dose-dependent manner, as detected by western blot analysis. Other apoptosis-, autophagy-, and cell cycle-related proteins were also examined by western blot analysis.
Depletion of POX decreases HDAC inhibitor-induced autophagy and HDAC inhibitor-increased apoptosis
To determine the function of POX in HDAC inhibitor-induced cellular process changes, we knocked down POX in HCC1806 and HCC1937 cells and treated the cells with TSA (1 μM) or SAHA (5 μM). As shown in Fig. 2A, HDAC inhibitor-induced LC3-II expression was dramatically decreased when POX was knocked down, but cleaved-PARP and cleaved-Caspase-3 expression was obviously increased. We simultaneously examined the expression of Cyclin D1 and P21 and found that POX knockdown did not rescue the downregulation for Cyclin D1 but partially rescued the induction of P21 by HDAC inhibitors.
Depletion of POX decreases HDAC inhibitor-induced autophagy and HDAC inhibitor-increased apoptosis (A) POX-depleted and control HCC1806 and HCC1937 cells were treated with TSA (1 μM) or SAHA(5 μM) for 24 h, and the expression levels of autophagy-, apoptosis-, cell cycle-related proteins were detected by western blot analysis. β-Actin was used as a loading control. POX was knocked down by an siRNA pool. (B) Depletion of POX suppressed HCC1806 and HCC1937 cell growth. The cells were transfected with POX siRNAs, and cell viability was measured by the SRB assay. Data were presented as the mean ± SD. (C) Depletion of POX increased HCC1806 and HCC1937 cell sensitivity to HDAC inhibits. The cell viability was detected by the SRB assay. Data were presented as the mean ± SD. (D) Depletion of POX increased HCC1806 and HCC1937 cell sensitivity to HDAC inhibits. HCC1806 and HCC1937 cells were transiently transfected with POX siRNA, followed by treatment with TSA (1 μM) or SAHA (5 μM) for another 24 h. The cells were collected for Annexin V staining and flow cytometric analysis. Data were presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Consistently, POX knockdown significantly suppressed TNBC cell growth, as determined by SRB assays in HCC1806 and HCC1937 cells (Fig. 2B). More importantly, POX knockdown significantly increased TNBC cell sensitivity to TSA and SAHA treatment (Fig. 2C). Finally, we detected apoptosis by staining the cells with Annexin V. By flow cytometry analysis, we found that POX knockdown significantly increased the proportion of AnnexinV-positive apoptotic cells in both cell lines (Fig. 2D). These data suggest that HDAC inhibitor-induced POX increases anti-apoptotic autophagy.
HDAC inhibitors induce POX mRNA expression in TNBC cell lines HCC1806 and HCC1937 cells were treated with either TSA (0–2 μM) or SAHA (0–20 μM) for 24 h. The total RNA was collected for RT-qPCR. Data were presented as the mean ± SD. **P< 0.01.
HDAC inhibitors induce POX expression through AMPK (A) HCC1806 and HCC1937 cells were treated with either TSA (0–2 μM) or SAHA (0–20 μM) for 24 h. The expressions of POX regulatory proteins, such as p-AMPK, AMPK, c-Myc, P53, and PPARγ, were detected by western blot analysis. β-Actin was used as a loading control. (B) POX induction by HDAC inhibits was abolished by AMPK depletion in HCC1806 and HCC1937 cells. The expression levels of POX, p-AMPK, and AMPK were detected by western blot analysis. (C) C-Myc knockdown did not affect the expression of POX in HCC1806 and HCC1937 cells.
HDAC inhibitors induce POX mRNA expression in TNBC cell lines
POX is expression in HCC1806 and HCC1937 cell lines that affiliated to triple negative breast cancer (Supplementary Fig. S1). In order to characterize the mechanism by which HDAC inhibitors induce POX expression in TNBC cell lines, we treated HCC1806 and HCC1937 cells with TSA or SAHA at different concentrations for 24 h. Subsequently, total RNA was collected to detect POX mRNA expression levels by RT-qPCR. As shown in Fig. 3A–D, TSA and SAHA significantly increased the POX mRNA expression in a dose-dependent manner. These results indicate that HDAC inhibitors may up-regulate POX expression at the transcriptional level.
HDAC inhibitors induce POX expression through AMPK
It has been reported that POX is induced by AMPK, p53, and PPARγ, but is suppressed by c-Myc through upregulating miR23b [8]. We treated HCC1806 and HCC1937 cells with TSA or SAHA at indicated concentrations and found that the expression of phosphorylated AMPK is increased and c-Myc is decreased at a dosage-dependent manner, which is consistent with previous studies (Fig. 4A). On the contrary, HDAC inhibitors (TSA/SAHA) decreased p53 and PPARγ protein expression levels (Fig. 4A). These results indicate that AMPK and c-Myc may be responsible for the POX induction by HDAC inhibitors.
To investigate whether TSA/SAHA induce POX expression via AMPK and c-Myc, we first knocked down AMPK in both HCC1806 and HCC1937 cells and examined the POX expression. As shown in Fig. 4B, AMPK knockdown suppressed TSA/SAHA-induced upregulation of POX expression. In contrast, after c-Myc was knocked down, the POX expression levels were not increased in either HCC1806 or HCC1937 cell lines (Fig. 4C). These results suggest that AMPK, but not c-Myc, is responsible for POX induction by HDAC inhibitors in TNBC cell lines.
Taken together, our findings suggest an important role of POX in HDAC inhibitors-mediated autophagy and apoptosis.
Discussion
Several studies suggested that POX plays a tumor suppressive role because POX induces apoptosis in a variety of cancer cells [13, 40–42]. The expression of POX in colon cancer tissues was downregulated compared with that in the normal tissues [43]. Both intrinsic and extrinsic apoptotic pathways are involved in POX-induced apoptosis [44]. On one hand, POX induces ROS and cytochrome C releasing, and activates Caspase-9 to initiate mitochondrial apoptosis (intrinsic pathway). On the other hand, POX stimulates the expressions of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and death receptor 5 (DR5), resulting in the cleavage of Caspase-8 (extrinsic pathway) [44].
However, POX was reported to protect cells from nutrient deprivation or hypoxic stress by producing either ATP or ROS for pro-survival autophagy [8]. We found that POX plays an anti-apoptotic role in HDAC inhibitors-induced TNBC cell death. Depletion of POX significantly increased apoptosis. Thus, POX may play a broader role than previously expected, which makes it a promising new therapeutic target for breast cancer as well as for other types of cancer, particularly in combinational therapy with HDAC inhibitors. It would be interesting to develop POX-specific inhibitors.
Autophagy is a cellular process whereby cytoplasm and cellular organelles are degraded in lysosomes for amino acid recovery and energy recycling [45]. Autophagy and apoptosis can be stimulated by the same stresses. Recent studies showed that a class of anti-cancer agents HDAC inhibitors emerged as novel therapeutic drugs by inducing either autophagy or apoptosis [46–48]. Currently, how HDAC inhibitors induce cell death is still controversial. The interplay between autophagy and apoptosis is a complex process whose outcome depends on cell type and environmental conditions. Autophagy usually suppresses apoptosis in response to chemotherapies [39]. Numerous reports have shown that stress-triggered autophagy depends on the activation of AMPK pathway [49, 50]. The well-known AMPK downstream pro-autophagy targets include mTOR, p27, and eukaryotic elongation factor-2 kinase (eEF-2 kinase), etc. [51–53]. We found that POX is an additional downstream target of AMPK to activate autophagy. As one of the downstream signalings of AMPK, mTOR may contribute to the upregulation of POX because rapamycin, an inhibitor of mTOR, also activates POX in response to glucose deprivation [14]. The exact transcriptional mechanism for POX by activated AMPK needs further investigation.
HDAC inhibitors can induce cell-cycle arrest by up-regulating cell cycle inhibitor p21 expression and down-regulating Cyclin D1 expression [54]. We confirmed that HDAC inhibitors indeed increased the expression of p21 and decreased the expression of Cyclin D1 in TNBC cells. The downregulation of Cyclin D1 by HDAC inhibitors could not be rescued by POX depletion, although the increase of p21 could be rescued in part.
In summary, we found that TSA and SAHA can induce autophagy and apoptosis at the same time. During this process, POX is induced to promote autophagy, which suppresses apoptosis. When induced POX is depleted, HDAC inhibitors induced more apoptotic cell death, indicating that POX is a potential therapeutic target for TNBC.
Funding
This work was supported by the grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA16010405 to C.C.), the National Natural Science Foundation of China (Nos. 81672624 to J.F., 81830087, 1602221, and 31771516 to C.C., and 81772847 to R.L.).
