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Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and inhibits ER-stress induced apoptosis
© Bruchmann et al; licensee BioMed Central Ltd. 2013
Received: 3 August 2012
Accepted: 18 February 2013
Published: 1 March 2013
The Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function.
Bag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni’s Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses.
Our results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death.
We have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel interaction partner of Bag5.
KeywordsUnfolded protein response Cell stress Endoplasmic reticulum Apoptosis Molecular chaperones Refolding
The Bag (Bcl-2 associated athanogene) protein family consists of 6 evolutionary conserved polypeptides (Bag1-Bag6) . They share a common, C-terminal, single-copied BAG domain consisting of three alpha helices that interact with and modulate the activity of the molecular chaperone Hsp70 . Structural biology and limited proteolysis studies identified the Bag domain as a 110-124 amino acid motif consisting of three antiparallel alpha helices of 30-40 amino acids each [2–4]. However the length of the Bag domain varies among the Bag family members, producing two distinct sub-groups: a ‘long’ Bag domain present in Bag-1 family of proteins and a ‘short’ Bag domain of Bag-3, Bag-4 and Bag-5 .
Several of the Bag proteins have been implicated in the control of apoptosis [6, 7]. Bag-1 and Bag-3 (Bis) interact with Bcl-2 to reduce apoptosis induced by several factors [6, 8]. Bag-4 (Sodd) associates with and blocks signaling of receptors of the tumor necrosis factor family [9, 10]. Bag-6 (Scythe) modulates the nuclear pathway that communicates with mitochondria and regulates the release of cytochrome c thereby controlling apoptosis [11, 12].
Other than the common Bag domain, Bag proteins do not share any homology in terms of sequence and encode for distinct domains: Bag-3 contains at its N-terminal region a WW domain  and a PXXP domain  while a ubiquitin-like domain is present in the central part of the Bag-1 proteins and in a double copy at the N-terminal part of Bag-6 . Bag5 is exceptional in this group of polypeptides since it consists solely of 5 BAG domains structured in two and a half helices .
Bag proteins enhance cell proliferation and survival [16, 17] and increase stress tolerance and therefore contribute to cancer development [18–20]. However the only function of Bag5 known so far is the inhibition of the activity of Hsp70 and the E3 ubiquitin ligase Parkin [21, 22] in Parkinson’s disease. A possibility exists that like the other Bag proteins it may also be involved in tumor progression although this has not been demonstrated.
In this communication we show that Bag5 is overexpressed in prostate cancer and exerts an anti-apoptotic function. We further demonstrate that Bag5 is a stress inducible gene that functions as a co-chaperone of GRP78/BiP and that its increased expression results in increased resistance to UPR-induced apoptosis.
Rabbit polyclonal anti-eIF2α FL315, goat monoclonal anti-GRP78 (N20), mouse monoclonal anti-Bag5 (18Z) and anti-tubulin (TU-02) antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit polyclonal anti-GRP78 (ab21685) and anti-Bag5 (ab97660) antibodies were purchased from Abcam (Cambridge, UK). Rabbit polyclonal anti-GRP78 (ET21) and anti-β-actin antibodies were purchased from Sigma (Steinheim, Germany). Mouse antibody against HA (HA.11 clone 16B12) was purchased from Covance (Munich, Germany). Antibodies against PARP, ATF4, IRE1α, CHOP, phospho-IRE1α, phospho-eIF2α and PERK were purchased from Cell Signaling Technology (Frankfurt am Main, Germany). Anti-ATF6 antibody was purchased from Imgenex (Hamburg, Germany).
All cell lines used in this work were purchased from ATCC.
22Rv.1, LNCaP and PC3 cells were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). HEK293 cells were cultivated in Dulbecco´s modified Eagle´s medium (DMEM) supplemented with 10% FCS. RWPE-1, WPE-NB14 and WPE-NB26 cells were cultivated in keratinocyte serum free medium. All the cell culture media were purchased from Invitrogen (Karlsruhe, Germany).
Unless otherwise stated, cells were treated for the indicated time points with a final concentration of 300 nM thapsigargin (Life Technology, Hamburg, Germany), 10 μg/ml tunicamycin (Sigma, Steinheim, Germany), 20 μM fenretinide (Enzo Life Sciences, Lörrach, Germany), 10 μM (-)-epigallocatechingallate (EGCG, Santa Cruz, Heidelberg, Germany) and 10 nM Taxol (Sigma, Steinheim, Germany). Glucose starvation was performed cultivating the cells in glucose-free medium supplemented with 10% dialyzed FCS. Serum starvation was performed cultivating the cells in serum-free medium.
Transfection experiments and siRNA
22Rv.1 and PC3 stably transfected with empty vector or Bag5 with FuGene6® (Promega, Mannheim, Germany). For the generation of stable pooled clones, cells were cultivated in RPMI supplemented with 10% FCS and 0.8 mg/ml G418 final concentration. HEK293 cells were transiently transfected with PromoFectin® (PromoKine, Heidelberg, Germany) according to the manufacturer’s recommendations. For siRNA experiment, cells were transfected with HiperFect (Qiagen, Hilden, Germany) with RNA antisense targeted against Bag5 or GFP as control. siRNA was purchased from Life Technologies (Darmstadt, Germany).
Immunofluorescence experiments were carried out on cells seeded in a 2-well glass slide (Lab-Tek® Chamber Slide System). After treatment with vehicle (ethanol 80%) or 300 nM thapsigargin, the medium was removed and the cells were stained with anti-Bag5 antibody and anti-PDI antibody (to track the ER). Samples were analyzed with a Leica TCS SPE confocal microscope. An IMARIS Coloc® (Bitplane, Zurich, Switzerland) module was used to calculate the co-localized voxels (volume unit, analogous to a pixel in two dimension images) between the two channels.
Quantification of protein extracts
Protein concentration was quantified with the Bio-Rad Protein Assay (Bio-Rad, Munich, Germany) according to manufacturer’s instructions.
For protein extraction, cells were washed once with PBS 1X and resuspended in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM PMSF). For protein extraction from patient material, 8 μm-thick frozen tissue sections were homogenized in lysis buffer with the TissueLyser (Qiagen, Hilden, Germany) and frozen at -80°C. Samples were centrifuged at 12000 × g for 10 min at 4°C, quantified, resuspended in sample buffer and boiled at 95°C for 5 minutes.
Endoplamsic reticulum fractionation
Endoplasmic Reticulum fractionation was performed with the ER enrichment kit from Imgenex (Hamburg, Germany). After thapsigargin treatment cells were washed with PBS 1X by centrifugation at 2000 × g for 5 min. For homogenization, the cell pellet was resuspended in 1.5 ml of 1X isosmotic homogenization buffer supplemented with protease inhibitor cocktail and transferred into a glass tubes for the homogenizer (Braun, Melsungen, Germany). The samples were homogenized with the TissueLyser (Qiagen, Hilden, Germany). The homogenate was transferred into a new tube and centrifuged for 10 minutes at 1000 × g at 4°C to eliminate nuclei and cell debris. The supernatant was transferred into a new tube and centrifuged for 15 minutes at 12000 × g at 4°C to eliminate the mitochondria and the cell debris. The resulting supernatant was ultracentrifuged 1 h at 90000 × g for 1 h at 4°C. The pellet containing the ER was resuspended in 1X suspension buffer supplemented with protease inhibitor cocktail and dissolved by pipetting and vigorous vortexing.
mRNA extraction and real time PCR
Total RNA was extracted with PeqGold (PeqLab, Erlangen, Germany) and first-strand cDNA synthesis was performed using the M-MLV reverse transcriptase (Promega, Mannehim, Germany) and random primers (Fermentas, St Leon-Rot, Germany). For q-RT-PCR analysis the Maxima SYBR Green/Rox qPCR Master Mix (Fermentas, St-Leon-Rot, Germany) and StepOne Plus Real-Time System apparatus (Applied Biosystems, Darmstadt, Germany) were used.
For Real Time PCR analysis the following primers were used: Bcl2, forward 5′-ATGTGTGTGGAGAGCGTCAACC-3′, reverse 5′-TGAGCAGAGTCTTCAGAGACAGCC-3′; Bax, forward 5′- CCTTTTCTACTTTGCCAGCAAAC-3′, reverse 5′- GAGGCCGTCCCAACCAC-3′; CHOP, forward 5′-TGGTCATTCCCCAGCCCGGG-3′, reverse 5′-TTCCCTGGTCAGGCGCTCGA-3′; Xbp1 spliced (Xbp1s) forward 5′-CCGGTCTGCTGAGTCCGCAGC-3′, reverse 5′-TGGCAGGCTCTGGGGAAGGG-3′; Bag5 forward 5′-TGTCCCCGGGTTTAGGGGTGTTC-3′, reverse 5′-TTCACAAGCACTGTCCCGCCC-3′; GRP78/BiP forward 5′-CGACCTGGGGACCACCTACT-3′, reverse 5′-TTGGAGGTGAGCTGGTTCTT-3′. Gene expression data analysis was normalized against the Ribosomal Protein 36 (Rib36). For Rib36 the forward 5′-GAAGGCTGTGGTGCTGATGG-3′ and reverse 5′-CCGGATATGAGGCAGCAGTT-3′ primers were used.
For Bag5 gene expression level in patient material, a set of 42 samples was used, including 15 benign prostatic hyperplasia (BPH) and 27 prostate cancer samples obtained from radical prostatectomy. The set of samples was obtained from the Tampere University Hospital (Tampere, Finland). The specimens were confirmed to contain >70% of malignant or non-malignant epithelial cells using hematoxylin and eosin-stained slides. Total RNA was extracted from the frozen sections with Trizol (Invitrogen, Hämeenlinna, Finland), and first-strand cDNA synthesis was performed using SuperScript III reverse transcriptase (Invitrogen, Hämeenlinna, Finland) and random primers (Fermentas, Glen Burnie, MA).
Bag5 gene expression was analyzed with the following primers: forward 5′-AGGTGTCCCCGGGTTTAG-3′ and reverse 5′-GATGTTGGTTTCCCATATCCA-3′. Values were normalized to β-actin using the primers: forward 5′-TGGGACGACATGGAGAAAAT-3′ and reverse 5′-AGAGGCGTACAGGGATAGCA-3′. For q-RT-PCR analysis the Maxima SYBR Green/Rox qPCR Master Mix (Fermentas, Helsinki, Finland) and CFX96 Real-Time System apparatus (Bio-Rad, Helsinki, Finland) were used.
Unless otherwise stated, calculations of statistical significance in this work were performed according to Student’s t test. For comparison of the mean values in Bag5 gene expression study in BPH and Prostate Cancer patients one-way ANOVA and Bonferroni’s Multiple Comparison test were used. For comparison of the mean values of Bag5 stained cells in the tissue array analysis the Mann-Whitney test was used.
Protein extraction from prostate tissue and tissue array
Radical prostatectomy specimens were obtained from patients undergoing surgery after prostate cancer diagnosis in the Tyrolean PSA Screening project for early detection of prostate cancer [23, 24] and were worked up according to the standard histopathology protocol. The use of archive tumor tissue samples was approved by the Ethics Committee of the Innsbruck Medical University.
A tissue microarray containing tissue cores of 91 cancer cases was prepared as described in  and double stained with a polyclonal rabbit Bag5 antiboby (Imgenex, Hamburg, Germany) diluted 1:500 in Ventana diluent and a monoclonal antibody directed against the basal cell marker p63 (Clone 4A4 + Y4A3, Neomarkers, MS Cat 1084-P0) diluted 1:100 in Ventana diluent using a Ventany Discovery-XT staining automate (Ventana, Roche, Mannheim, Germany). Immunoreactivity was scored by an uropathologist (G.S.) considering the number of positive cells and the intensity of immunostaining for Bag5. Each case included 3 cores of tumor and 1 core of benign tissue. According to the percentage of positive cells a score from 0 to 4 was assigned to the case (0 = no staining; 1 = 10-25%; 2 = 25-50%; 3 = 50-75%; 4 = 75-100%). For each case, the value assigned to the tumor is the average of the three tumor cores. Immunostaining for the basal cell marker p63 present only in benign tissue served as a control for accurately distinguishing benign and tumor tissue.
Colorimetric assay of ATP hydrolysis
ATP hydrolysis was measured using an ATPase assay kit from Innova Biosciences (Cambridge, UK). Briefly, 0.5 μg of purified GRP78 (StressMarq Biosciences, Victoria, Canada) was incubated in a buffer consisting of 0.5 M Tris pH 7.5 and 1 mM ATP in presence or absence of 0.17 μg of GST-Bag5 or GST-BagΔ 5 at 37°C. The experiment performed in presence only of GST-Bag5 was set as background control. After 30, 45 and 60 min, 50 μl PiColorLock™ Gold reagent and Accelerator were added to the solution. 2 minutes later 20 μl of stabilizer were added and the resulting green color was allowed to develop for 30 minutes at room temperature. Absorbance was measured at 595 nm. Enzymatic activity was calculated according to manufacturer’s instructions.
For in vivo protein-protein interaction studies, co-immunoprecipitation experiment was performed by continuous rotation of protein A sepharose beads in TE buffer (10 mM Tris pH8 and 0,1 mM EDTA pH 8) with 4 μg of anti-GRP78/BiP specific antibody for 2 h at 4°C. HEK293 cells were treated with 2 nM Dithiobis(succinimidyl propionate) (DSP) in 10 ml PBS 1X for 30 minutes at room temperature (RT) to crosslink endogenous proteins. Crosslinking was stopped by the addition of 20 mM Tris pH7.5 for 15 minutes at RT. Thereafter cells were centrifuged at 2000 rpm for 5 minutes and the pellet lysed in 1 ml lysis buffer (50 mM Tris pH7,4, 120 mM NaCl, 1 mM EDTA, 0.4% NP-40). Cell lysate was sheered by passage 10 times through a 23 G needle (Braun, Melsungen, Germany), sonified (Amp 60, 10 pulses) and centrifuged for 10 minutes at 12000 × g at 4°C. The cell lysate and the beads were then incubated over night at 4°C boiled at 95°C and finally subjected to SDS-PAGE and western blot analysis.
Caspase 3 cleavage measurement
Caspase 3 cleavage was measured with the CaspACE™ Assay System (Promega, Mannheim, Germany). 1·104 cells were seeded in a 96 well plate and treated for 24 hours as indicated in the figure legend. At the end of the treatment, cells were lysed and Caspase 3 activity was measured according to manufacturer’s instructions.
Bag5 is overexpressed in prostate cancer
The tumor-specific expression of Bag5 was not restricted to biopsies but could be reproduced in a cell culture model of prostate cancer progression where RWPE-1, WPE-NB14 and WPE-NB26 represent different stages of malignancy from benign to a more aggressive prostate tumor state (reviewed in ). Here again, increased Bag5 expression was found in the tumor compared to the benign cell lines (Figure 1E). In addition we could show in established prostate cell lines that Bag5 expression is high in the more aggressive PC3 cells compared to less aggressive 22Rv.1 and LNCaP cells and the benign-prostatic hyperplasia (BPH-1) derived cells (Figure 1F).
ER stress enhances Bag5 expression and alters its cytoplasmic localization
Stress induction did not only increase Bag5 expression, but it also modified its subcellular localization. In resting conditions Bag5 showed a diffuse staining in the cytoplasm. However when cells were treated for 12 hours with TG, Bag5 staining became perinuclear and a strong co-localization with the ER was observed as determined by the use of the ER tracker (Figure 2D). Quantification of the co-localization in three fields of three independent experiments making use of the software IMARIS Coloc® showed that indeed Bag5 was significantly enriched in the ER after stress induction (Figure 2E). This result was confirmed by a fractionation experiment in which it could be shown that already after 8 h of TG treatment the ratio of Bag5 in the ER compared to the cytoplasm was substantially increased (Figure 2F). A similar increase in the distribution of GRP78/BiP in the ER was also observed (Figure 2F).
Bag5 interacts with GRP78/BiP
As Bag5 is made up of five BAG domains, it was necessary to determine whether all the five domains bind equally to GRP78/BiP. A GST-pull down assay was therefore performed with 22Rv.1 cell lysate and GST-fused carboxy- and amino-terminal deletion mutants of Bag5 that sequentially deleted one BAG domain at a time (Figure 3C and E). Deletion of the fifth BAG-domain abrogated binding of GRP78/BiP to the full length Bag5 protein while sequential deletion of the N-terminal sequences up to the fifth BAG domain modulated but did not abolish binding to GRP78/BiP (Figures 3C and D). This confirmed that the fifth BAG domain is responsible for binding to GRP78/BiP.
Bag5 modulates GRP78/BiP activity
Bag5 expression levels modulate the unfolded protein response
Stressful conditions such as nutrient starvation, hypoxia or changes in pH to protect cells and promote cell survival activate a signaling pathway known as the UPR . However, when stressful conditions are prolonged, the UPR induces apoptosis by shutting down protein synthesis . Since the UPR is regulated by GRP78/BiP  and Bag5 binds this protein, we investigated if alteration of the level of Bag5 would affect the UPR. We overexpressed Bag5 by stable transfection in 22Rv.1 cell with an HA-Bag5 or an empty vector as control and induced the UPR by exposure of these cells to thapsigargin for 6 and 12 h.
To confirm these results, Bag5 expression was reduced in 22Rv.1 cells in a siRNA knock down experiment and the cells were treated for 6 and 12 h with thapsigargin (TG) to activate the UPR. Consistent with the overexpression results, a knock down of Bag5 expression is expected to produce the reverse results. Indeed the downregulation of Bag5 expression resulted in an increased eIF2α but a decreased IRE1α phosphorylation (Figure 5F). Taken together these results indicate that Bag5 induces the IRE1α pro-survival branch while it inhibits the PERK-eIF2α-ATF4 pro-apoptotic axis.
Bag5 overexpression increases stress tolerance in prostate cancer cells
In this work we showed that Bag5 interacts with the molecular chaperone GRP78/BiP demonstrating that in addition to the function of Bag proteins as interaction partners of Hsp70/Hsc70, a member of this family (Bag5) additionally interacts with the ER-resident chaperone GRP78/BiP. This expands the network of interaction partners of the Bag family of co-chaperones. In addition we showed for the first time at the RNA and protein levels that Bag5 is overexpressed in prostate cancer and that it plays a role as a pro-survival factor in UPR-induced apoptosis.
Bag5 is a co-chaperone of GRP78/BiP and promotes cancer cell survival
Bag proteins have been described to interact with the molecular chaperone Hsp/Hsc70  but recently it is reported that Bag-1 interacts with the ER chaperone GRP78/BiP  suggesting that other Bag proteins may share this property. In this study we could show that Bag5 also interacts with GRP78/BiP confirming that the Bag proteins may be more versatile in their interactions with molecular chaperones. However Bag5 does not interact with all ER-resident chaperones, it does not interact with the protein disulfide isomerase PDI or the Hsp90 homolog GRP94 showing selectivity in its interaction partners. The observation that Bag5 interacts with GRP78 and stimulates its ATPase activity expands the range of action of the Bag proteins in other cellular events such as ER-mediated stress response and the UPR.
From the analysis of the effect of Bag5 in the UPR, we could show that it preferentially stimulates IRE1α while it suppresses PERK/eIF2α pathways resulting in growth advantage for the cells. The action of Bag5 in regulating events in the ER is consistent with its increased association to this cellular compartment following thapsigargin treatment even if the mechanism by which it is recruited to the ER is not clear and needs further investigation. Intriguingly we could also observe that ectopic expression of Bag5 resulted already in untreated cells in increased Bcl-2 and decreased Bax expression independent from stress induction. It is possible therefore that Bag5 could contribute to cell survival both by interacting with GRP78 enhancing its enzymatic activity and by modulating Bcl-2/Bax ratio and that these two events could be independent from each other.
Bag5: a new tumor marker?
RNA and protein expression studies presented in this work show that the expression of Bag5 is increased in malignant compared to benign prostate tissue. Since other Bag family members are overexpressed in several types of cancers in addition to prostate cancer [35–37], such as breast [38, 39], colon  and pancreatic [41, 42] cancers, it is likely that increased Bag5 expression would be found in other type of tumors as well. If this is the case, it would be worth investigating the use of Bag5 as a novel tumor biomarker.
From the results of this work that Bag5 is a stress-inducible gene and it is anti-apoptotic, we would expect a tumor with high expression of Bag5 to be more aggressive and less responsive to stress-inducing chemotherapeutic agents. This hypothesis is supported by our observation that up- or downregulation of Bag5 levels modifies the ability of prostate cancer cells to respond to stress. This agrees with finds that Bag5 gene expression is increased in rat livers upon exposure to epatotoxants  and that it is induced in MCF7 breast cancer cells  and ovarian cancer spheroids  upon taxol treatment.
These finding together with the work described in this communication identify Bag5 as a gene whose expression is regulated by chemotherapeutic drugs and an anti-apoptotic gene. In addition we showed that Bag5 interacts with the molecular chaperone GRP78/BiP, often found overexpressed in chemoresistant tumors (reviewed in ). Downregulating Bag5 levels and/or interfering with Bag5-GRP78/BiP interaction could therefore represent a novel therapeutic approach to overcame chemoresistance and to treat late stage tumors.
Danilo Maddalo is a recipient of a Young Investigator Group (YIG) grant from the Karlsruhe Institute of Technology and the Deutsche Forschungsgemeinschaft (DFG). The experiments presented here were performed in part using laboratory material purchased with the Young Investigator Network Equipment Grant, from the Karlsruhe Institute of Technology and the Deutsche Forschungsgemeinschaft (DFG). We thank Nadine Leuchtner, Denise Kremer, Christof Seifarth and Irma Sottsas for their technical assistance.
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