Cancer Cell Biology
BEX2 regulates mitochondrial apoptosis and G1 cell cycle in breast cancer
Article first published online: 26 AUG 2009
Copyright © 2009 UICC
International Journal of Cancer
Volume 126, Issue 7, pages 1596–1610, 1 April 2010
How to Cite
Naderi, A., Liu, J. and Bennett, I. C. (2010), BEX2 regulates mitochondrial apoptosis and G1 cell cycle in breast cancer. Int. J. Cancer, 126: 1596–1610. doi: 10.1002/ijc.24866
- Issue published online: 28 JAN 2010
- Article first published online: 26 AUG 2009
- Manuscript Accepted: 18 AUG 2009
- Manuscript Received: 3 MAY 2009
- The University of Queensland
- mitochondrial apoptosis;
- G1 arrest;
- breast cancer;
We have recently demonstrated that BEX2 is differentially expressed in primary breast tumors and BEX2 expression is required for the Nerve Growth factor inhibition of ceramide-induced apoptosis in breast cancer. In this study we investigate the functional role of BEX2 in the survival and growth of breast cancer cells. We demonstrate that BEX2 downregulation induces mitochondrial apoptosis and sensitizes breast cancer cells to the pro-apoptotic effects of ceramide, doxorubicin and staurosporine. In addition, BEX2 overexpression protects the breast cancer cells against mitochondrial apoptosis. We show that this effect of BEX2 is mediated through the modulation of Bcl-2 protein family, which involves the positive regulation of anti-apoptotic member Bcl-2 and the negative regulation of pro-apoptotic members BAD, BAK1 and PUMA. Moreover, our data suggests that BEX2 expression is required for the normal cell cycle progression during G1 in breast cancer cells through the regulation of cyclin D1 and p21. To further support the significance of BEX2 in the pathogenesis of breast cancer we demonstrate that BEX2 overexpression is associated with a higher activation of the Bcl-2/NF-κB pathway in primary breast tumors. Furthermore, we show that BEX2 downregulation results in a higher expression and activity of protein phosphatase 2A. The modulation of protein phosphatase 2A, which is also known to mediate the cellular response to ceramide, provides a possible mechanism to explain the BEX2-mediated cellular effects. This study demonstrates that BEX2 has a significant role in the regulation of mitochondrial apoptosis and G1 cell cycle in breast cancer.
Apoptosis is an important mechanism in maintaining cell homeostasis and an imbalance between the pro-apoptotic and anti-apoptotic pathways is a key feature of malignant transformation.1 Furthermore, since many anti-cancer therapies exert their function through the induction of apoptosis, the dysregulation of apoptotic signaling is commonly involved in drug resistance.2 Disruption of the balance between cell survival and cell death by alternation of regulatory mechanisms which function as mediators of survival or apoptotic signaling pathways can lead to enhanced cellular survival and cancer development.3 Ceramide is an endogenous signaling molecule, and its role in apoptosis has attracted much attention in recent years. Ceramide accumulation inside the cell is observed after numerous stress stimuli, such as chemotherapeutic agents and radiation.4, 5 Accumulated ceramide modulates a number of responses to stress, including apoptosis and cell cycle arrest.6 Previous studies have shown that ceramide-induced apoptosis involves a direct action on mitochondria.7–10 This effect is thought to be regulated by the Bcl-2 family of proteins.11–13 Furthermore, there is a known interaction between the ceramide and NF-κB pathways.14, 15 For example ceramide, in addition to inducing cell death, activates an opposing NF-κB-dependent survival pathway leading to 2 different cell fates.16, 17 Besides the effect on apoptosis, ceramide also induces a G1-cell cycle arrest in cancer cells.18, 19 In spite of these findings, the functional elements involved in the ceramide signaling pathway are poorly understood.
We have recently identified that BEX2, a member of brain-expressed-X-linked family, is differentially expressed in primary breast tumors.20 BEX2 expression, through the activation of NF-κB, is required for the nerve growth factor (NGF) inhibition of ceramide-induced apoptosis in breast cancer cells.20 Furthermore, BEX2 overexpression protects the breast cancer cells against the ceramide-mediated apoptosis.20 These findings led us to the hypothesis that BEX2 may have a key role in the regulation of survival and growth of breast cancer cells. In this study we show that BEX2 is a regulator of mitochondrial apoptosis and G1 cell cycle in breast cancer.
Material and Methods
All the culture media were obtained from Invitrogen. Breast cancer cell lines MCF-7, MDA-MB-231, T-47D and BT-474 were cultured in DMEM media, 10% fetal bovine serum (FBS). MDA-MB-453 and MDA-MB-361 cell lines were cultured in L15 media, 10% FBS. Sum-190 and SK-BR-3 cell lines were cultured in Ham's F12, 5% FBS and McCoy's 5a, 10% FBS media, respectively. Prostate cancer cell line LNCaP was cultured in RPMI, 10% FBS. U251MG and U87MG glioma cell lines were grown in E4 media, 10% FBS.
Cell line treatments
MCF-7, MDA-MB-231 and T-47D cell lines were treated with the following agents in serum free media overnight: (i) Ceramide analogue, C2 (Sigma) at 10 μM concentration; (ii) BAY11-7082 (BAY11), (Merck) which is an IκBα phosphorylation inhibitor, at 5 μM concentration. Treatments with staurosporine (STS), (Sigma) and doxorubicin (Sigma) were carried out in full media at 1 μM and 200 nM concentrations, respectively overnight. All experiments were performed in 4 biological replicates.
Real Time-PCR analysis in cell lines
Total RNA extraction was performed as described before.20 Real Time-PCR (RT-PCR) to assess the expression levels of BEX1 (assay ID: Hs00360592_g1), BEX2 (assay ID: Hs00607718_g1), cyclin D1 (assay ID: Hs00765553_m1), CDKN1A (assay ID: Hs00355782_m1), Bcl-2 (assay ID: Hs00153350_m1), BAK1 (assay ID: Hs00940249_m1), PUMA (assay ID: Hs00248075_m1), Bax (assay ID: Hs99999001_m1), Bcl-xL (assay ID: Hs99999146_m1) and PP2A B subunit (assay ID: Hs00270227_m1) were carried out using Taqman® Gene Expression Assays (Applied Biosystems) as instructed by the manufacturer. Housekeeping genes HPRT1 (part number: 4326321E) and RPLP0 (part number: 4326314E) were used as controls.
Relative gene expression = gene expression in the treated group/average gene expression in the control group. The amplification efficiencies of the above Taqman gene expression assayswere measured using the manufacturer's protocol (http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/ documents/generaldocuments/cms_040377.pdf) and found to be over 95% for all the assays. All cell line experiments were performed in 4 biological replicates.
Generating BEX2 knock-down in cell lines
BEX2-Knock Down (BEX2-KD) in MCF-7, MDA-MB-231 and T-47D cell lines was carried out using siRNA Oligos (duplex), (Sigma-Genosys). Three sets of siRNA duplexes for BEX2 were used. Duplex 1/2: (D1: 5′rCrArGUrAUrArGrAUrGrGrGrA rCrAUrArATT, D2: 5′UUrAUrGUrCrCrCrAUrCUrAUrArCUrGTT); Duplex 3/4: (D3: 5′rGrArGrCrGUUrArArArCrArAUrCUrCrAUTT, D4: 5′rAUrGrArGrAUUrGUUUrArArCrGrCUrCTT); Duplex 5/6: (D5: 5′UrCrArCrCrAUrGrArCrCrAUrCrArCrGrAUTT, D6:5′rAUrCrGUrGrAUrGrGUrCrAUrGrGUrGrATT). Transfection of siRNA oligos using Lipofectamine™ RNAiMAX (Invitrogen) was carried out using reverse transfection method as instructed by the manufacturer. In summary, 30 pmol of siRNA duplex (for a 6-well plate experiment) was diluted in Opti-MEM® I Medium (Invitrogen). Lipofectamine RNAiMAX was then added to each well containing the diluted siRNA molecules. The solution was gently mixed and incubated for 20 min at room temperature. Cells were diluted in complete medium without antibiotics so that each 500 μl contained 50,000 cells (cell densities were 40–50% confluent 24 hr after plating). Next, the diluted cells were added to each well with siRNA duplex-Lipofectamine RNAiMAX complex and transfected cells were then incubated at 37°C. The final siRNA duplex concentration was 10 nM for all the knock-down experiments. Cells transfected with siCONTROL™ Nontargeting siRNA, (Dharmacon) were used as controls. Treatments with ceramide, BAY11, STS and doxorubicin were performed 48 hr after the siRNA transfections followed by an overnight incubation. In all experiments the effects of BEX2-KD were assessed 72 hr after the siRNA transfections. BEX2-KD experiments were carried out separately with all 3 siRNA Oligos. The data presented for each knock-down experiment is the average result obtained from the 3 BEX2 siRNA-duplexes. All siRNA silencing experiments were performed in 4 replicates with each duplex.
Apoptosis assay using Hoechst 33258 staining was performed as described before.14 Apoptosis measurement with flow cytometry was carried out using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences). All experiments were performed in 4 biological replicates.
MCF-7 cells were grown to 60% confluence. BEX2 overexpression was performed using a BEX2 construct in pReciever expression vector (CMV promoter and a His-tag, GeneCopoeia, MD). A GFP construct in the same vector was used as the negative control. Transfection of MCF-7 cells was carried out using ExGen 500 reagent (Fermentas Life Sciences), as instructed by the manufacturer. All experiments were performed in 4 biological replicates.
Western blot analysis
Anti-phospho-Bcl2 (Ser70) rabbit antibody, anti-phospho-BAD (Ser112) rabbit antibody, anti-BAD rabbit antibody and anti-PP2A B subunit rabbit antibody were obtained from Cell Signalling, MA. Western blots were carried out at 1:1,000 dilution of each primary antibody using 20 μg of protein lysate. Western blots with anti-His mouse antibody (Sigma) and rabbit polyclonal BEX2 antibody were performed at 1:3,000 and 1:200 dilutions, respectively using 5 μg of protein lysate. This anti-BEX2 antibody was generated by us through Quality Controlled Biochemicals (MA) against the peptide; Ac-QENDEKDEKEQVANKGEPC-amide.
Immunofluorescence staining in cell lines
BEX2-KD experiments using BEX2 siRNA Oligos were carried out as described above in 6-well plates on cover-slips. Cells transfected with siCONTROL™ Nontargeting siRNA, (Dharmacon) were used as controls. Seventy-two hours after transfections, cells were washed with PBS and fixed/permeabilized with methanol for 10 min at −20°C. Cells were then washed 3 times with PBS and 2 times with PBS/5%BSA (Bovine Serum Albumin, Sigma). Immunofluorescence (IF) was carried out using BEX2 rabbit polyclonal antibody (see above). BEX2 antibody was diluted at 1:100 in PBS/5%BSA and incubated on the coverslips for 1 hr at room temperature (RT). Cells were then washed 3 times with PBS and 2 times with PBS/5%BSA and incubated with the secondary Alexa-594 anti-rabbit antibody (Invitrogen) at 1:500 dilutions for 1 hr at RT. After the secondary antibody incubation, coverslips were washed 3 times with PBS, counter-stained with DAPI and mounted to slides.
The amounts p21 in 10 μM of each lysate was measured using ELISA (PathScan Total p21 Sandwich ELISA kit, Cell Signaling, MA). The assays were performed in 4 biological replicates.
Mitochondrial apoptosis assays
- 1Mitochondrial permeability transition (MPT)-based apoptosis assay was carried out using MitoCapture Mitochondrial Apoptosis Assay Kit (Promokine). This assay utilizes a fluoresecent-based method using MitoCapture™ dye to distinguish between healthy and apoptotic cells by detecting the changes in the mitochondrial transmembrane potential.21 Treatment with STS (Sigma) at 1 μM overnight was used as a positive control for the MPT-based apoptosis assay.22 Transfections of BEX2-vector in MCF-7 cells and BEX2-siRNA in MDA-MB-231 cells were carried out as described above. Ceramide (10 μM) and STS (1 μM) treatments were performed 48 hr after the transfections for overnight. The fluorescent signals were measured using fluorescence microscopy and a total of 500 cells were assessed for each experiment. The ratio of cells staining with Rodamine/FITC was measured for each experimental group. All experiments were performed in 4 biological replicates.
- 2Cytosolic release of cytochrome c was measured using Cytochrome c Apoptosis Detection Kit (Promokine). In summary, 5 × 107 cells were collected by centrifugation at 600g for 5 min at 4°C. Cells were then washed with ice-cold PBS followed by another round of centrifugation as above. After removing the supernatant, cells were resuspended with 1 ml of 1× Cytosolic Extraction Buffer Mix (Promokine) containing DTT (1 mM) and Protease inhibitors followed by 10 min of incubation on ice. Next, cells were homogenized by 40 passes through a 27 gauge needle. The homogenized suspension was monitored under a microscope till 70–80% of the nuclei did not have a shiny ring. Homogenate was then centrifuged at 700g for 10 min at 4°C and the supernatant was collected into a fresh tube for the next step. After centrifugation at 10,000g for 30 min at 4°C, supernatant was collected as cytosolic fraction. The pellet was then resuspended in 0.1 ml of mitochondrial extraction buffer mix (Promokine) containing DTT (1 mM) and protease inhibitors, vortexed for 10 sec and saved as mitochondrial fraction. Protein concentrations from the isolates were measured using the BCA Protein Assay Kit (Thermo scientific). Western blots with anti-cytochrome c mouse monoclonal antibody (Promokine) were carried out at 1 μg ml−1 dilution using 10 and 20 μg from the isolated cytosolic and mitochondrial fractions, respectively. Beta-actin and COX IV (AbCam) were used as loading controls for the cytosolic and mitochondrial fractions, respectively. All experiments were performed in 4 biological replicates.
Flow cytometry analysis to assess the cell cycle
Seventy-two hours after the transfections, cells were harvested with trypsinization. A total of 1 × 106 cells were incubated in Hoechst 33342 (Sigma) at a concentration of 4 μg ml−1 in PBS for 30 min at 37°C. Fluorescence was then measured using flow cytometry for UV excitation. The fraction of cells in each phase of cell cycle was analyzed using Summit v4.3 software (Dako, CO). All experiments were performed in 4 biological replicates.
Cell lysis was carried out in lysis buffer deprived of phosphatase inhibitors as described before.23 PP2A assay was carried out using PP2A immunoprecipitation phosphatase assay kit (Millipore), and Pmoles of phosphate were measured for each group. Experiments were carried out in 4 replicates.
Primary breast tumors
The institutional research ethics committee approved this study and informed consent was obtained from each patient for the use of tissue samples. A total of 38 frozen tumor samples were obtained from the Princess Alexandra Hospital tissue bank. Total RNA extraction from the frozen breast tumor samples was performed as we previously described.24 RT-PCR to measure the expression of BEX2 and Bcl-2 was carried out as described above for the cell lines. IF staining was performed using 5-μm sections of each frozen tumor samples. Slides were fixed in acetone at −20°C for 10 min and blocked with 10% goat serum in PBS (Sigma) for 1 hr at RT. They were then incubated in anti-p65 rabbit primary antibody (AbCam) and Alexa-594 anti-rabbit secondary antibody (Invitrogen) at 1:200 and 1:500 dilutions, respectively. For each sample we examined 2 sections at high power field (100×). Images were obtained using a confocal microscope (Carl Zeiss) with ZEN 2008 imaging software. A total of 600 cells in each section and 1,200 cells per tumor sample were counted.
Biostatistical analysis was done using the Statistical Package SPSS® version 17.0 (Chicago, IL). Mann-Whitney U test was applied for the comparison of nonparametric data.
BEX2 is widely expressed in breast cancer cell lines
To study the function of BEX2 in breast cancer, we first assessed the expression of this gene in breast cancer cell lines using RT-PCR. In each cell line, BEX2 expression was measured relative to the expression of BEX2 in MCF-7 cells. We observed that all the tested breast cancer lines, with the exception of MDA-MB-453, had detectable levels of BEX2 and most of them had a higher BEX2 expression compared to the glioma cell line (Supporting Information Fig. S1a). We also assessed the expression of BEX1, an isoform of BEX2, in a number of cancer cell lines and only found detectable levels in the glioma lines (Supporting Information Fig. S1b). Furthermore, we observed that BEX2 expression was independent of estrogen receptor (ER) status since it was present in both ER+ (e.g., MCF-7) and ER- (e.g., MDA-MB-231) cell lines. To have a representative model of breast cancer, we used ER+ lines MCF-7 and T-47D as well as the ER- line MDA-MB-231 for our functional experiments.
Downregulation of BEX2 induces apoptosis and has synergy with proapoptotic agents
To study the effect of BEX2 expression on apoptosis, we first generated BEX2-KD in our studied cell lines using siRNA Oligos (duplex). Three sets of siRNA duplexes for BEX2 were tested in MCF-7, MDA-MB-231 and T-47D cell lines and nontargeting siRNA was used as a control. The level of BEX2-KD was assessed using RT-PCR. We observed more than 90% reduction of BEX2 transcript using the duplex 1/2 (D1/2) in all 3 cell lines (Fig. 1a). Furthermore, D3/4 and D5/6 also generated efficient BEX2-KD (Supporting Information Fig. S2). In addition, the downregulation of BEX2 protein after BEX2-KD was confirmed using IF with a rabbit polyclonal BEX2 antibody (Fig. 1b). All the knock-down experiments were carried out using each BEX2-siRNA duplex and the data presented for each knock-down experiment is the average result obtained from the 3 BEX2 siRNA-duplexes. We next assessed the effect of BEX2 downregulation on apoptosis and observed a significant increase at the baseline level of apoptosis following BEX2-KD in all 3 cell lines (p < 0.01, Fig. 1c). This effect was most prominent in MDA-MB-231 cells which showed a 5-fold increase in the level of apoptosis (Fig. 1c).
It is known that ceramide mediates a pro-apoptotic response in breast cancer cell lines and we have previously shown that BEX2 overexpression protects the cells against this effect of ceramide.14, 15, 20 Therefore, to further investigate the role of BEX2 in the cellular response to ceramide, we subsequently assessed the effect of BEX2-KD on ceramide-mediated apoptosis. The downregulation of BEX2 protein after BEX2-KD and ceramide treatment was confirmed using IF (Fig. 1b). The experiments were performed in each cell line with the dose escalation of ceramide at 3, 7, and 10 μM concentrations. We observed that BEX2-KD significantly sensitized all 3 cell lines to the ceramide-induced apoptosis (p < 0.01, Figs. 2a and 2b, Supporting Information Fig. S3). In addition, the synergy between BEX2-KD and ceramide-mediated apoptosis was independent of ceramide concentration (Supporting Information Fig. S3). It is notable that ceramide at 10 μM caused a significantly higher apoptotic response in MCF-7 (∼40%) and MDA-MB-231 (∼20%) cells compared to T-47D (<10%), (p < 0.03, Fig. 2a and Supporting Information Fig. S3). Importantly, this relative resistance of T-47D to ceramide was partially overcome by BEX2-KD (Fig. 2a and Supporting Information Fig. S3c).
It is known that ceramide, in addition to inducing apoptosis, activates an opposing NF-κB-dependent prosurvival pathway.16, 17 Since BEX2 expression has a protective function against the ceramide-mediated apoptosis, we postulated a prosurvival role for BEX2 in the axis between ceramide and NF-κB. To investigate this role we tested the effect of BEX2-KD on apoptosis mediated by BAY11 in breast cancer cell lines (BAY11 is an IκBα phosphorylation inhibitor). Breast cancer cells were treated with a low concentration of BAY11 at 5 μM. Although, this concentration of BAY11 led only to a slight induction of apoptosis, the addition of BEX2-KD resulted in a 3- to 5-fold increase in the BAY11-induction of apoptosis (p < 0.01, Fig. 2c). Moreover, we investigated the effect of BEX2 downregulation in sensitizing MDA-MB-231 and MCF-7 cells to the chemotherapy agent doxorubicin at 200 nM and mitochondrial proapoptotic agent STS at 1 μM. We observed that BEX2-KD markedly increased the level of apoptosis after doxorubicin treatment from ∼6 to 30% in MDA-MB-231 cells and from 7.5 to 18% in MCF-7 line (p < 0.03, Fig. 2d and Supporting Information Fig. S4). Furthermore, there was a significantly higher level of apoptosis with the combination of BEX2-KD and STS treatment compared to the STS treatment alone in both cell lines (p < 0.03, Fig. 2d). These data indicate that BEX2 downregulation induces apoptosis and sensitizes the breast cancer cells to the proapoptotic agents.
BEX2 protects the breast cancer cells against mitochondrial apoptosis
We next asked whether the effect of BEX2 on apoptosis is mediated through the mitochondrial intrinsic pathway, as it is known with the response to ceramide.7–10, 25 To investigate this we first carried out a mitochondrial permeability transition (MPT)-based apoptosis assay, which detects changes in the mitochondrial transmembrane potential using fluorescent staining (Fig. 3a). MCF-7 cells were studied in the following groups: (i) untreated control, (ii) ceramide treatment at 10 μM overnight, (iii) transfection with the control-vector followed by ceramide treatment, (iv) transfection with the BEX2-vector followed by ceramide treatment, (v) transfection with the control-vector followed by STS treatment, and (vi) transfection with the BEX2-vector followed by STS treatment. STS treatment was carried out at 1 μM overnight in the control group as a positive control for the MPT-based apoptosis. BEX2 overexpression was performed by the transfection of a BEX2-Histidine expression vector. A GFP-histidine expression vector was used as the negative control. Overexpression of BEX2 was confirmed using western blots both with an anti-Histidine (His) antibody and a rabbit polyclonal BEX2 antibody (Fig. 3b). To assess the transfection efficiency, co-transfection with Cyan Fluorescence Protein (CFP) was carried out and only the CFP labeled cells were assessed for the MPT-based apoptosis assay. The ratio of cells staining with Rodamine/FITC was measured for each experimental group and a decrease in this ratio was used as an indicator of mitochondrial apoptosis. We observed a significant reduction in the Rodamine/FITC ratio after ceramide and STS treatments (p < 0.03, Fig. 3c). Moreover, BEX2 overexpression restored the ceramide-induced reduction of Rodamine/FITC ratio to a level similar to the control group and partially reversed the reduction of Rodamine/FITC ratio observed after STS treatment in MCF-7 cells (p < 0.03, Fig. 3c). These findings indicate that BEX2 overexpression has a protective effect against the induction of mitochondrial apoptosis.
We next asked whether the apoptotic response observed after BEX2-KD is mediated through the mitochondrial pathway using MDA-MB-231 cells, which had a 5-fold increase in the level of apoptosis following BEX2-KD (Fig. 1c). MDA-MB-231 cells were studied in the following groups: (i) control-siRNA, (ii) BEX2-KD, (iii) control-siRNA + STS treatment at 1 μM, and (iv) BEX2-KD + STS treatment at 1 μM. STS treatment reduced the Rodamine/FITC ratio to 60% of baseline (Fig. 3c). Importantly, we observed that the Rodamine/FITC ratio decreased by about 50% after BEX2-KD in this cell line, indicating the induction of mitochondrial apoptosis (p < 0.03, Fig. 3c). Moreover, when STS treatment was added to BEX-KD there was a further decrease in the Rodamine/FITC ratio to ∼30% of control (p < 0.03, Fig. 3c), suggesting that BEX2-KD sensitized MDA-MB-231 cells to the mitochondrial apoptosis mediated by STS.
We further investigated the effect of BEX2 on the mitochondrial-mediated apoptosis by measuring the cytosolic release of cytochrome c from mitochondria. After extracting the cytosolic and mitochondrial cellular fractions, the amount of cytochrome c was determined by western blot. MDA-MB-231 cells were studied in the following groups: (i) control-siRNA, (ii) control-siRNA + ceramide treatment at 10 μM overnight, and (iii) BEX2-KD. We observed that cytochrome c, which was not present in cytosolic fraction of control-siRNA group, became detectable after the ceramide treatment (Fig. 3d, top panel). Importantly, cytochrome c was present in cytosolic fraction after BEX2-KD indicating a release from the mitochondria (Fig. 3d, top panel). Furthermore, we observed a corresponding reduction in mitochondrial cytochrome c level with both ceramide treatment and BEX2-KD (Fig. 3d, bottom panel). We next assessed MCF-7 cells in the following groups: (i) control-siRNA, (ii) control-siRNA + ceramide at 5 μM, (iii) BEX2-KD + ceramide at 5 μM. We observed that cytosolic cytochrome c, which was absent with ceramide at 5 μM, became detectable following the combination of BEX2-KD and ceramide (Fig. 3e, top panel). In addition, there was a corresponding reduction in mitochondrial cytochrome c level following the combination of BEX2-KD and ceramide (Fig. 3e, bottom panel). These findings indicate that BEX2 expression has a protective function against the mitochondrial apoptosis in breast cancer cells. In addition, BEX2 downregulation induces mitochondrial apoptosis and sensitizes breast cancer cells to the STS-mediated mitochondrial damage.
BEX2 regulates the phosphorylation and expression of Bcl-2 family
To investigate the underlying mechanism for BEX2 effect on mitochondrial apoptosis, we studied a potential role for BEX2 in the regulation of Bcl-2 family. It is known that Bcl-2 and BAD are regulated by ceramide through dephosphorylation.26, 27 It is also notable that phosphorylation results in the activation of anti-apoptotic protein Bcl-2 and inactivation of proapoptotic protein BAD.28, 29 Importantly, we observed a significant reduction in Bcl-2 expression by 20% (MDA-MB-231) to 40% (MCF-7 and T-47D) after BEX2-KD (p < 0.03, Fig. 4a). Furthermore, we found that the downregulation of BEX2 decreased Bcl-2 phosphorylation by 3- to 4-fold in T-47D and MCF-7 lines, respectively (Fig. 4b). This effect was not observed in MDA-MB-231 line. Moreover, we observed a reduction in the ratio of phospho-BAD/total-BAD to 0.4 (MDA-MB-231), 0.45 (T-47D) and 0.48 (MCF-7) after BEX2-KD compared to the controls (Fig. 4c).
To further study the effect of BEX2 on the regulation of Bcl-2 family, we measured the transcript levels of proapoptotic genes; BAK1 and PUMA using RT-PCR. Experiments were carried out in MCF-7 cell line using the following groups: (i) control, (ii) BEX2-KD, (iii) ceramide treatment at 10 μM and (iv) BEX2-KD + ceramide. We also assessed the effect of BEX2 downregulation on the expression of BAK1 and PUMA in MDA-MB-231 cells. We observed that BAK1 and PUMA expression significantly increased with both BEX2-KD and ceramide treatment in MCF-7 and MDA-MB-231 lines (p < 0.03, Fig. 4d). In addition, with the combined BEX2-KD and ceramide treatment there was a further increase in the expression of BAK1 to 9-fold and PUMA to 5-fold in MCF-7 cells (p < 0.03, Fig. 4d). We did not observe a significant change in the expression of Bax and Bcl-xL after BEX2-KD or ceramide treatment (data not shown). These findings suggest that BEX2 expression positively regulates the phosphorylation of Bcl-2 and BAD. Furthermore, BEX2 has a regulatory effect on the expression of mitochondrial pathway genes Bcl-2, BAK1 and PUMA. Moreover, in the absence of BEX2 expression the effect of ceramide on BAK1 and PUMA is significantly enhanced.
Downregulation of BEX2 results in G1-cell cycle arrest
It is known that ceramide induces G1-cell cycle arrest in cancer cells.18, 19 Since there is a close interaction between BEX2 and ceramide in apoptosis, we asked whether BEX2 expression has a role in cell cycle. To answer this question we studied the effect of BEX2 downregulation on cell cycle using flow cytometry. We observed a significant increase in the cell populations of G1 phase after BEX2-KD compared to the controls (ΔG1) in all 3 breast cancer cell lines, which indicated a G1 arrest (p < 0.03, Figs. 5a and 5b). To investigate the mechanism of this G1 arrest, we first measured cyclin D1 transcript level in breast cancer cell lines after BEX2-KD. Furthermore, we assessed cyclin D1 expression in MCF-7 cells after transfection with either BEX2-siRNA or control-siRNA followed by the treatment with ceramide at 10 μM overnight. We observed that BEX2-KD significantly decreased the expression of cyclin D1 between 3- and 5-fold in all 3 breast cancer lines (p < 0.03, Fig. 5c). In addition, ceramide treatment significantly reduced cyclin D1 expression and when combined with BEX2-KD the expression of cyclin D1 was further reduced to only 2% of the control (Fig. 5c). These findings suggest that BEX2 positively regulates cyclin D1 expression and protects the breast cancer cells against an excessive downregulation of cyclin D1 after ceramide treatment. We next assessed the p21 protein level using ELISA. We observed p21 level to increase by 3-fold in MCF-7 cells and by 23% in T-47D cells after BEX2-KD (p < 0.03, Fig. 5d). Interestingly, in MDA-MB-231 cells, BEX2-KD had an opposite effect compared to the other cell lines and resulted in a significant reduction of p21 expression (p < 0.03, Fig. 5d). We further validated this effect of BEX2 in MDA-MB-231 cells using ceramide treatment and BEX2 overexpression (Fig. 5d). To investigate whether the observed changes in p21 is regulated at the transcriptional or protein levels, we assessed the CDKN1A (p21 gene) transcript level in MCF-7 and MDA-MB-231 cells after BEX2-KD. CDKN1A expression decreased by 2-fold in MDA-MB-231, however, it did not significantly change in MCF-7 cells (Supporting Information Fig. S5). These findings suggest that in MCF-7 and T-47D cells, BEX2 expression has an inhibitory effect on the p21 level. Furthermore, the effect of BEX2 expression on p21 is mediated by posttranscriptional regulation in MCF-7 cells and at least partly by transcriptional regulation in MDA-MB-231 cells. Overall these data indicate that BEX2 expression is required for the normal cell cycle progression during G1 in breast cancer cells through the regulation of cyclin D1 and p21.
BEX2 regulates PP2A expression and activity
Ceramide is a known activator of protein phosphatase 2A (PP2A) and this activation mediates several functions of ceramide including G1 arrest and dephosphorylation of Bcl-2 and BAD proteins.26, 27, 30 Therefore, to find a mechanistic explanation for the similarities between the cellular effects of BEX2 downregulation and the response to ceramide, we investigated whether BEX2 expression has a regulatory effect on PP2A. We first studied the level of PP2A (B), subunit using western blot and observed this level to increase after BEX2-KD in T-47D and MDA-MB-231 cells by 1.5 and 1.8-fold, respectively (Fig. 6a). However, we did not observe a similar change for this subunit in MCF-7 cells. Interestingly, we noted a marked increase in a 45 KD PP2A (B) isoform after BEX2 downregulation in MDA-MB-231 cells compared to the control (Fig. 6a). We next measured the PP2A (B)-β isoform expression and found it to significantly increase by 2.4-fold after BEX2-KD in MDA-MB-231 cells (p < 0.03, Fig. 6b). As with the protein level, we did not find a significant change in the PP2A (B)-β isoform transcript in MCF-7 cells (Fig. 6b). To further investigate the effect of BEX2 expression on PP2A, we measured the PP2A phosphatase activity using the immunoprecipitation assay. MCF-7 cells were studied in the following groups: (i) control-siRNA, (ii) control-siRNA + ceramide at 10 μM, (iii) BEX2-KD, and (iv) BEX2-KD + ceramide at 10 μM. As expected, ceramide increased the PP2A phosphatase activity (Fig. 5c). Moreover, there was a significant increase in PP2A activity by about 1.5-fold after BEX2-KD, which was present in both untreated and ceramide-treated cells (p < 0.03, Fig. 6c). These data indicate that BEX2 expression regulates the level of PP2A regulatory subunit B and PP2A phosphatase activity.
Breast tumors with BEX2 overexpression have a higher Bcl-2 expression and p65 activation
To validate our in vitro findings regarding BEX2 interactions with the mitochondrial apoptosis and NF-κB pathways, we studied the association of BEX2 expression with Bcl-2 and p65 NF-κB levels in primary breast tumors. We first measured BEX2 expression using RT-PCR and normalized the expression results to the median expression of BEX2 across the cohort (Fig. 7a). To divide the cohort into 2 groups with either over- or under-expression of BEX2, we removed 7 samples with a borderline BEX2 expression so that the expression differences between the BEX2 over-expressed (BEX2 (+)) and BEX2 under-expressed (BEX2 (−)) samples were at least 3-fold (Fig. 7a). We next measured Bcl-2 expression in breast tumors using RT-PCR and normalized the data to the median expression of Bcl-2 across the cohort. Subsequently, we compared the level of Bcl-2 expression between BEX2 (+) and BEX2 (−) samples. We observed that Bcl-2 expression was significantly higher in the BEX2 (+) tumors by ∼3.7-fold compared to the BEX2 (−) samples (p < 0.01, Fig. 7b). In addition, we assessed the p65 nuclear staining in BEX2 (+) and BEX2 (−) tumors using p65-immunofluorescence (IF). Nuclear staining of p65 was used as a measurement of NF-κB activation. We first obtained the percentage of tumor cells with the p65 nuclear staining in each sample (Supporting Information table I) and then compared these measurements between the BEX2 (+) and BEX2 (−) tumors. We found that the p65-nuclear staining was ∼2-fold higher in the BEX2 (+) samples compared to the BEX2 (−) group (p < 0.01, Figs. 7c and 7d). These findings suggest that BEX2 (+) breast tumors have an overexpression of Bcl-2 and a higher activation of p65 NF-κB compared to the BEX2 (−) tumors.
Ceramide is a signaling molecule that regulates several cell processes, including apoptosis and cell growth. Although the existing data suggest that several inter-connecting pathways are involved in ceramide signaling, there is limited information available about the genes modulating this process. We have recently demonstrated that BEX2 is differentially expressed in primary breast tumors and BEX2 expression, through the activation of NF-κB, is required for the NGF-mediated inhibition of ceramide-induced apoptosis.20 In addition, BEX2 overexpression protects the breast cancer cells against ceramide-mediated apoptosis.20 Moreover, we observed that BEX2 downregulation increased the baseline level of apoptosis in breast cancer cells and sensitized them to the proapoptotic agents including ceramide, doxorubicin and STS (Figs. 1 and 2). Previous studies have shown that ceramide has a direct pro-apoptotic effect on the mitochondrial cascade.7, 8, 25 Overall, these findings led us to investigate the involvement of BEX2 in the mitochondrial apoptosis pathway.
In this study we demonstrated that BEX2 downregulation leads to the mitochondrial membrane damage and the cytosolic release of cytochrome c (Fig. 3). We also observed a clear interaction between BEX2 and the effect of ceramide on mitochondrial apoptosis. Interestingly, this effect of BEX2 is similar to the protective function of Bcl-2 overexpression against the ceramide-mediated mitochondrial apoptosis and cytochrome c release.31, 32 Moreover, BEX2 partially protected the breast cancer cells against STS-mediated mitochondrial apoptosis (Fig. 3c), which indicates that the anti-apoptotic function of BEX2 is not limited to the ceramide response. Bcl-2 family has an important function in the mitochondrial apoptosis pathway and ceramide is known to have a regulatory role in this process. This effect of ceramide includes the inactivation of Bcl-2 and activation of BAD by the way of dephosphorylation.26, 27 Therefore, to further investigate the role of BEX2 in mitochondrial apoptosis, we studied an interaction between BEX2 and the Bcl-2 family. We observed that BEX2 downregulation resulted in a significant reduction of Bcl-2 expression and phosphorylation as well as the reduction of phospho-BAD/total-BAD ratio in breast cancer cells (Figs. 4a–4c). Furthermore, we found an induction of proapoptotic genes BAK1 and PUMA with both BEX2 downregulation and ceramide treatment which was enhanced with their combination (Fig. 4d). These findings indicate that BEX2 has a protective function against the mitochondrial apoptosis in breast cancer, which is mediated through the regulation of Bcl-2 protein family. In this process BEX2 positively regulates the anti-apoptotic member Bcl-2 and negatively regulates the proapoptotic members BAD, BAK1 and PUMA.
It is notable that Bcl-2 and NF-κB mutually activate each other and there is an established Bcl-2/NF-κB pathway in breast tumors with significance in tumor progression.33–35 Here we demonstrate that BEX2 overexpression is associated with a higher activation of the NF-κB pathway in primary breast tumors (Fig. 7), which further supports our model of BEX2 interaction with both Bcl-2 and NF-κB pathways. Furthermore, these data suggest that BEX2 expression may potentially act as a marker for the activity of this Bcl-2/NF-κB pathway in breast cancer.
In addition to the regulation of cell survival, ceramide induces G1-cell cycle arrest in cancer cells.18, 19, 36 This effect of ceramide is mediated through the down-regulation of cyclin D1 and upregulation of p21.18, 19 This led us to investigate a functional role for BEX2 in cell cycle. We found that BEX2 down-regulation induced G1 phase arrest in breast cancer cell lines, which directly correlated with a decrease in the expression of cyclin D1 (Figs. 5a–5c). Furthermore, the combination of BEX2-KD and ceramide markedly reduced the expression of cyclin D1 to only 2% of the baseline. Moreover, we found that p21 protein level, as it has been shown with ceramide, significantly increased after BEX2 downregulation in MCF-7 and T-47D cells. Interestingly, in MDA-MB-231 cells we observed an opposite effect resulting in the reduction of p21 level after BEX2-KD. Although the reason for this opposite effect in MDA-MB-231 cells is not clear, this cell line has several biological differences with MCF-7 or T-47D which include a basal type, higher grade and p53 mutation.37 It is also notable that in MCF-7 cells the increase in p21 level after BEX2-KD was not associated with a significant transcriptional change, which is compatible with the known model of post-transcriptional regulation of p21 at the proteasome level.38, 39 Therefore, BEX2 expression is required for the normal cell cycle progression during G1 in breast cancer cells and prevents an excessive downregulation of cyclin D1 by ceramide.
The breast cancer cell lines used in this study were chosen to represent the heterogeneity of breast cancer. MCF-7 is a low grade ER positive cell line, which is sensitive to ceramide-mediated apoptosis and T-47D is an ER positive line with a relative resistance to ceramide. Moreover, MDA-MB-231 is an ER negative cell line with basal type and a high grade. Importantly, we observed that BEX2 function in the regulation of cell survival and growth showed a great degree of similarity across these cell lines. Furthermore, the majority of BEX2-regulated elements such as cyclin D1, Bcl-2 and BAD were also similar across these lines. The observed differences in some of the BEX2 regulatory functions between these cell lines, such as the effect on p21, are likely related to the underlying biological variations across breast cancer subtypes. Overall, our study suggests a significant role for BEX2 in the regulation of mitochondrial apoptosis and G1 cell cycle in breast cancer (Fig. 8).
The underlying mechanisms mediating the ceramide signaling are not well understood. However, there is evidence to suggest that PP2A activation is involved in several cellular responses to ceramide.26, 27, 30, 40–42 In view of the close interaction between BEX2 and the ceramide signaling pathway, we investigated a possible role for BEX2 in the regulation of PP2A. PP2A is implicated in a multitude of cellular functions and its core structure consists of a catalytic subunit (C), a scaffold protein (A), and a regulatory B subunit.43 There are 3 families of B subunit and each PP2A subunit has at least 2 isoforms. In this study we found that BEX2 down-regulation increased the PP2A B subunit protein levels in MDA-MB-231 and T-47D cell lines (Fig. 6a). This correlated with a rise in the transcriptional level of PP2A B subunit β-isoform in MDA-MB-231 cells (Fig. 6b). Interestingly, we also observed a marked increase in a 45 KD PP2A (B) isoform after BEX2 downregulation in MDA-MB-231 cells (Fig. 6a). Furthermore, we found a significant increase in the PP2A phosphatase activity after BEX2 down-regulation in MCF-7 line which was present in both untreated and ceramide treated cells (Fig. 6c). It is notable that the antibody used against the PP2A B subunit mainly detects the α-isoform, although it may also recognize other isoforms. Since in spite of a significant increase in the PP2A phosphatase activity after BEX2-KD we did not find a change at the level of PP2A B subunit in MCF-7 cells, we can conclude that other regulatory subunits/isofroms of PP2A may also be involved in this process. Overall, our findings suggest that the regulation of PP2A provides a possible mechanism to explain the BEX2-mediated cellular effects (Fig. 8). For example, we have shown that BEX2 protects the breast cancer cells against mitochondrial apoptosis by regulating some of the key steps in this process such as the phosphorylation of Bcl-2 and BAD, which are also known to be regulated by PP2A.26, 27 Therefore, the effect of BEX2 on the mitochondrial apoptosis pathway is upstream to the Bcl-2 protein family and can be resulted from the BEX2 regulation of PP2A.
It is notable that there are ongoing studies to investigate the potential clinical application of agents, which act by activating the sphingomyelinase pathway and generating ceramide in cancer models. One such agent is a semisynthetic vitamin E analogue, α-tocopheryl succinate (α-TOS), which has a high level of pro-apoptotic activity mediated through the generation of ceramide leading to the activation of PP2A and dephosphorylation of Bcl-2.44, 45 Furthermore, acid ceramidase inhibitors such as LCL204 are also being studied as new cancer therapies.46 Since BEX2 has a significant effect on the survival and growth of breast cancer cells, such as their sensitivity to proapoptotic agents and the regulation of PP2A activity in these cells, BEX2 is a potential therapeutic target in breast cancer.
In Summary, this study demonstrates that BEX2 has a significant regulatory function in the mitochondrial apoptosis pathway and G1 cell cycle. Furthermore, BEX2 over-expression in primary breast tumors is associated with a higher activation of the Bcl-2/NF-κB pathway. These findings indicate a key role for BEX2 in the biology of breast cancer with potential therapeutic implications.
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Additional Supporting Information may be found in the online version of this article.
|IJC_24866_sm_supfig1.tif||2632K||Supplementary Figure S1. BEX2 and BEX1 expression using Real-Time PCR in cell lines. (a) BEX2 expression folds in breast cancer cell lines relative to that of MCF-7 using RT-PCR. Error Bars: ± 2SEM. (b) BEX1 expression was assessed in breast cancer cell lines MCF-7, MDA-MB-231, T-47D, BT-474, Sum190, MDA-MB-361, SK-BR-3, and MDA-MB-453; prostate cancer cell line LNCap; and glioma cell lines U251MG and U87MG. Expression values are shown as: (40-CT) and a value of 0 means no detected expression.|
|IJC_24866_sm_supfig2.tif||2094K||Supplementary Figure S2. Real Time-PCR showing BEX2-knock down in breast cancer cell lines. BEX2-knock down efficiencies are shown for siRNA duplex 1/2 (Dup1/2), duplex 3/4 (Dup3/4), and duplex 5/6 (Dup 5/6) in MCF-7, MDA-MB-231, and T-47D cell lines. Expression of BEX2 after knock-down was assessed relative to non-targeting siRNA control and fold changes are shown for each duplex in each cell line.|
|IJC_24866_sm_supfig3.tif||319K||Supplementary Figure S3. The effect of BEX2 on ceramide pro-apoptotic response. (a) Percentage of apoptosis in MCF-7 cells after BEX2-knock down (KD) and treatment with ceramide. Ceramide treatment was carried out at 3 μM, 7 μM, and 10 μM concentrations overnight and apoptosis was measured using Hoechst assay. Non-targeting siRNA was used as a control. (b) Percentage of apoptosis in MDA-MB-231 cells as described in A. (c) Percentage of apoptosis in T-47D cells as described in A. (d) Hoechst staining to show the apoptotic cells in MDA-MB-231 cells after BEX2-KD and ceramide treatment at 3 μM. All error bars indicate: ± 2SEM.|
|IJC_24866_sm_supfig4.tif||208K||Supplementary Figure S4. BEX2-Knock Down and doxorubicin treatment. Histograms show the percentage of apoptosis using Annexin V-FITC assay inMDA-MB-231 cells. Cells were transfected with the control siRNA (a) or BEX2-siRNA (b) followed by doxorubicin treatment at 200 nM overnight.|
|IJC_24866_sm_supfig5.tif||1426K||Supplementary Figure S5. The effect of BEX2 down-regulation on p21. CDKN1A transcriptional change after BEX2 Knock-Down. RT-PCR was carried out in MCF-7 and MDA-MB-231 cells after transfections with either control-siRNA (control) or BEX2-siRNA. CDKN1A expression fold to control group is demonstrated for both cell lines. *, p < 0.03 is for the difference between knock-down and control groups in MDA-MB-231 cells. Error Bars: ± 2SEM.|
|IJC_24866_sm_supTable-1.doc||28K||Supplementary Table 1. Percentage of p65 nuclear staining.|
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