Pro-apoptotic effect of aurothiomalate in prostate cancer cells

[Cell Cycle 8:2, 306-313; 15 January 2009]; ©2009 Landes Bioscience

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Pro-apoptotic effect of aurothiomalate in prostate cancer cells 1Ludwig

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Marianna Trani,1 Alessandro Sorrentino,1,2 Christer Busch2 and Marene Landström1,2,*

Institute for Cancer Research; Uppsala University; Uppsala, Sweden; 2Department of Genetics and Pathology; Rudbeck Laboratory; Uppsala University; Uppsala,

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involves the activation of Rac1-Mek-Erk signalling.6 Recent findings suggested that this transformation can be prevented both in vitro and in vivo using a novel small molecule, aurothiomalate (ATM). ATM disrupts the interaction between the PB1 domain of PKCι and Par6,6,7 a protein involved in the polarity complex formation.8-10 Here, we report that ATM causes apoptosis of aggressive prostate cancer cells by a dose-dependent activation of ERK and p38 MAP kinases. We also showed that ATM leads to a release of cytochrome c from mitochondria to cytoplasm, suggesting that the mitochondrial pathway is also involved in this drug-mediated apoptotic program in prostate cancer cells. Our data suggest that targeting the PKCι and Par6 interaction is an effective and novel therapeutic strategy for treatment of aggressive prostate cancer.

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It has been recently demonstrated that small gold compounds could have a potential anti-tumoral activity. Here, we report that aurothiomalate (ATM), a gold compound already used in clinical therapy for the treatment of rheumatoid arthritis, has a pro-apoptotic effect in aggressive prostate cancer (PC3U) cells. In contrast, treatment of human primary epithelial prostate cells (PrEC) with ATM did not cause apoptosis. We demonstrated that ATM is able to disrupt the PKCι-Par6 complex in PC3U cells and that this disruption leads to the activation of ERK in a dose-dependent manner. Interestingly, we also showed that ERK acts upstream of the activation of caspase 3, leading to apoptosis. ATM treatment also causes activation of p38 and JNK MAP kinases. Moreover we could link ATM treatment to activation of the mitochondrial or so called intrinsic pathway, as we observed release of cytochrome c from mitochondria to cytoplasm, suggesting that the mitochondrial pathway is involved in the pro-apoptotic effect mediated by ATM. Taken together our data suggest that ATM could be a new promising drug for the treatment of advanced prostate cancer.

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Key words: aPKC, apoptosis, aurothiomalate, JNK, p38, Par6, prostate cancer

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Introduction

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Prostate cancer is the most common malignancy and it is the second leading cause of cancer death in males.1 Clinically, prostate cancer is diagnosed as local or advanced. Most early stage tumors are androgen-dependent and are often treated with surgery and chemotherapy, whereas advanced stage diseases are resistant to such treatments. Unfortunately, despite the early efficacy of androgen ablation, many advanced stage prostate tumors relapse into an androgen-independent disease with devastating results in mortality rates.2 Biochemically, prostate cancer progression is associated with the deregulation of specific growth factors with their respective signalling pathways.3 Recently, it has been demonstrated that in non-smallcell lung cancer cells, PKCι plays a pivotal role in growth and tumorigenicity.4,5 The molecular mechanism by which PKCι drives transformation of these cancer cells has been well elucidated and it

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*Correspondence to: Maréne Landström; Institution for Genetics and Pathology; Department of Genetics and Pathology; Husargatan 20; LICR, BOX 595, BMC; Uppsala SE-751 24 Sweden; Tel.: +46.070.231.6342; Email: Marene.Landstrom@ genpat.uu.se Submitted: 11/04/08; Accepted: 12/10/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/7596 306

Results

Effect of ATM on PC3U and PrEC cells. ATM has been shown to inhibit the growth of non-small-cell lung cancer (NSCLC) in vitro and tumorigenesis in vivo.7 In order to analyze the effects of ATM on prostate cancer cells we treated the PC3U cell line, as well as primary epithelial normal prostate cells (PrEC) as a control, with ATM. After ATM treatment, PC3U cells acquired a pro-apoptotic phenotype, distinguished by the formation of typical apoptotic bodies as determined by DAPI staining. The morphology of PrEC cells nuclei was, in contrast, not affected by the drug (data not shown). Moreover, we observed that after treating PC3U cells, but not PrEC cells, with ATM for 24 hours, there was a loss of the cells, suggesting that the drug specifically affect the viability of prostate cancer cells (Fig. 1A). Using the “Vybrant Apoptosis Assay” to quantify the apoptotic effect, almost 40% of the cells were found to be dead, when the highest concentration of the drug, 50 μM, was used. No increase in the amount of dead cells was observed when PrEC cells were treated with ATM for 24 hours (Fig. 1B). To elucidate the reason of the different response to ATM of PC3U and PrEC cells, the expression of proteins involved in formation of the polarity complex, was investigated. We found that PKCι was expressed at a higher level in PC3U cells than in PrEc cells, whereas the level of expression of other proteins investigated was equal in both cell lines (Fig. 1C). Since PKCι is a known target of ATM, its different expression levels could explain why PC3U cells are more sensible to the drug compared to PrEC cells. ATM disrupts the Par6-PKCι complex in PC3U cells. It has been previously reported that ATM inhibits growth of transformed

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NSCLC by targeting the PB1 domain of PKCι.13 In fact, ATM has been identified as a potent inhibitor of PB1-PB1 domain interaction between PKCι and the adaptor molecule Par6. To investigate whether ATM is able to disrupt the complex between these two proteins in PC3U cells, we used in situ proximity ligation assay (Fig. 2A and B). The interaction between Par6 and PKCι is represented by red dots. In control cells there was a strong interaction which was gradually disrupted after ATM treatment at both concentrations. The effect was even more evident when the highest concentration of the drug was used. To quantify the number of interacting complexes, red dots were counted using the Blob Finder software (Fig. 2C). In the control, there was an average of 7 red dots per cell suggesting a strong interaction. When PC3U cells were treated with 25 μM ATM for 24 hours, a decrease of the signal was observed, which decrease was even more evident when cells were treated with 50 μM ATM: at the highest concentration there was an average of less than one dot per cell. The interaction between Par6 and PKCι was confirmed by a co-immunoprecipitation experiment. PC3U cells were transiently transfected with a plasmid containing the wt FLAG-Par6 protein and then treated with ATM for 24 hours. Treatment with ATM decreased the amount of Flag-Par6 co-immunoprecipitated with PKCι in a dose-dependent manner (Fig. 2D) consistent with the PLA experiment. ATM treatment affects intracellular signal transduction pathways. It is known that the MEK-ERK pathway could be modulated by the polarity complex through the activation of Rac1.4,7 For this reason, we next investigated whether ATM treatment affected the activation downstream of Par6-PKCι effectors, such as ERK MAP kinase. Interestingly, whereas ERK levels were not affected by ATM treatment in neither PC3U nor PrEC cells, we found that ATM treatment selectively induced activation of ERK in a dose-dependent manner in PC3U cells (Fig. 3A). Stimulation of the cell lines with TGFβ for 2 hours did not cause any change on the expression of ERK. It is well known that ERK is mainly activated by MEK. ATM treatment did not affect the expression of MEK or the phosphorylation of MEK in either PC3U or PrEC cells, as shown by immunoblotting (Fig. 3B). Thus, most likely, it is not MEK that causes the direct activation of ERK after treatment with ATM. Recently, it has been demonstrated that ERK could be hyperactivated in some cancer cell lines and that this can be related to apoptosis through different pathways,14 e.g., AKT, a protein kinase mainly involved in pathways related to the cell survival and cell growth. To investigate whether the AKT pathway was modulated by active ERK in an ATM-dependent matter, we determined the expression of AKT and phospho-AKT, in both PC3U and PrEC cells (Fig. 3C). AKT was equally expressed in both cell lines and the level of expression was not affected by treatment with ATM or by TGFβ stimulation. Moreover, we found that ATM treatment in PC3U cells resulted in a downregulation of phospho-AKT, in a concentrationdependent manner. In contrast, the level or phosphorylation of AKT was not affected in PrEC cells after ATM treatment. TGFβ stimulation did not enhance this effect neither in PC3U nor in PrEC cells. ATM induces a pro-apoptotic effect in PC3U cells. As mentioned above, the hyperactivation of ERK in some cancer cell lines can be linked to apoptosis in several ways. In many cases, a member of the caspase family of proteins is involved. Caspase 3 is a key enzyme in

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Aurothiomalate specifically kills prostate cancer cells

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Figure 1. Effect of ATM in PC3U and PrEC cells. (A) DAPI staining visualizes the number of PC3U and PrEC cells after ATM treatment (25 μM or 50 μM) for 24 hours or treatment with vehicle (CTR) (objective 20X, Leica Microscope). (B) “Vybrant Apoptosis Assay” performed in PC3U and PrEC cells treated or treatment with vehicle (CTR) with ATM for 24 hours (25 μM or 50 μM). (C) PKCι is differently expressed in PC3U and PrEC cells. PC3U and PrEC cells were lysed and subjected to immuno-blotting using antisera against Par6, PKCζ and PKCι. Actin served as loading control.

the programmed cell death pathway. To investigate whether ATM treatment was able to induce apoptosis in prostate cancer cells through this mechanism, we performed two different types of experiments (Fig. 4A and B). As is shown in the blot in Figure 4A, ATM treatment caused an increase of the cleaved caspase 3, suggesting that

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Figure 2. ATM disrupts the Par6-PKCι complex. (A) In situ proximity ligation assay (PLA) for Par6 and PKCι in PC3U cells treated with ATM (25 μM or 50 μM) for 24 hours, or treatment with vehicle (CTR). (B) Red dots have been highlighted and isolated from the background to show the amount of interacting complexes. (C) Quantification of PLA experiment performed with Blob Finder Analysis Software (O-LINK technology) *p < 0.005. (D) Co-immunoprecipitation for FLAG-Par6 and endogenous PKCι in PC3U cells treated with ATM (25 μM or 50 μM) for 24 hours, or treatment with vehicle (CTR).

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this pathway is activated by the drug. The involvement of caspase 3 activation was demonstrated also by the use of PathScan ELISA caspase 3 kit. After treatment of PC3U cells with ATM (25 μM), we observed an increase of apoptotic cells from approximally 0.05% to about 5%, which increased to almost 20% after treatment with 50 μM ATM. It has recently been demonstrated that TGFβ-mediated apoptosis in PC3U cells, is induced by the activation of p38 by the TGFβ associated kinase-1; TAK1.15,16 TAK1 is known to activate also the JNK pathway.17 To understand if treatment with ATM could cause the activation of the p38 or JNK MAP kinases, we performed immunoblotting on cell lysates derived from PC3U and PrEC cells (Fig. 4C). After treatment with ATM, there was a clear drug-dependent activation of both p38 and JNK in PC3U cells, but not in PrEC cells, whereas the expression of p38 and JNK was not affected by the drug, neither in PC3U nor in PrEC cells. TGFβ stimulation did not affect activation or the level of expression of p38 or JNK in PC3U cells, but in PrEC cells it appeared to be responsible for the basal activation of p38 and JNK. These data suggest that both JNK and p38 MAP kinase pathways could be involved in the apoptotic effect induced by ATM in prostate cancer cells. To investigate whether p38 activation was required for the ATM-induced apoptotic program, we treated PC3U cells with 308

a specific p38 inhibitor (SB203580) and measured activation of caspase 3. After treating prostate cancer cells with 50 μM ATM with and without p38 inhibitor for 24 hours, there was a significant decrease of the number of apoptotic cells, from in 25% in ATM-treated PC3U cells to 3% in PC3U cells treated with 50 μM ATM in combination with SB203580 (Fig. 4D). This result suggests that p38 activation is important for activation of caspase 3 and is important for the apoptotic program induced by ATM. Programmed cell death is a highly regulated process and the release of the cytochrome c is one of the last and most important steps. It is a decisive event, also defined as “the point of no return”.18 Since ATM was able to activate different pathways involved in the apoptotic program, we wanted to investigate if ATM treatment could also lead to the release of cytochrome c from mitochondria to cytosol in our prostate cancer cell line. By a cell fractionation experiment we demonstrated that ATM treatment for 24 hours resulted in release of cytochrome c to the cytoplasm from the mitochondria in PC3U cells (Suppl. Fig. 1). Thus, most likely, the pro-apoptotic effect induced by the drug is mediated not only by the activation of caspase 3 and p38, but also by the activation of the mitochondrial pathway. ERK activation is required for the activation of caspase 3 in PC3U cells. We next wanted to clarify the role of ERK activation

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for the ATM-induced apoptotic effect. For this purpose, we silenced ERK using its specific siRNA, and then measured the activation of caspase 3, as previously described. We found a clear connection between the drug-dependent activation of ERK and the consequent activation of the caspase 3, in PC3U cells where ERK was silenced, when compared to control cells also treated with ATM. Also the decrease of apoptotic cells in cells where ERK was silenced, suggest that ERK activation is a requisite for the ATM-induced apoptotic program to occur (Fig. 5A and B).

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Prostate cancer is one the most common malignancies in the world. Unfortunately, to determine the prognosis of this tumor is complicated. As known from the literature, this is so for due to mainly two reasons: limited access to pretreatment biopsies that are representative for tumor grading and insufficient knowledge of which pathways are deregulated in malignancy grade progression of prostate cancer.2,19,20 Thus, understanding these mechanisms in detail could be useful for the identification and characterization of new potential drugs. In the last few years, small gold compounds have been studied for their potential anti-tumoral activity in different types of cancer.6 These compounds have been extensively used and are still used in clinical practice, for the treatment of the rheumatoid arthritis and other inflammatory diseases.21 One of these compound; ATM, has been shown to have an inhibitory effect on NSCLC transformation evoked by atypical PKCι.3 This inhibitory effect is mainly due to the ability of the drug to disrupt the interaction between aPKCι and the PB1 domain of Par6.7 In the present study, we investigated the potential role of ATM in advanced prostate cancer cells. In fact, preliminary results demonstrated that ATM had an inhibitory effect on the growth of prostate cancer cells in vitro (our unpublished data). Here, we report that ATM has a pro-apoptotic effect in prostate cancer cells without affecting the viability of normal epithelial prostate cells (Fig. 1A and B). This difference in response to the drug could be due to the different level of expression of ATM targets in our two cell lines. In fact, we noticed that PC3U cells express higher level of aPKCι than PrEC cells. Since aPKCι is the only known target of the drug, elevated expression of the protein only in PC3U cells, could explain why these cells are more sensible to ATM treatment compared to PrEC cells. We showed that ATM is able to disrupt Par6-PKCι interaction in PC3U cells in a dose-dependent manner and that the disruption of the polarity complex affects the expression of downstream molecules such as ERK and MEK. We expected to see a downregulation of both these proteins but, surprisingly, we proved that ATM is able to cause a dose-dependent activation of ERK in PC3U cells. Then, we showed that this activation is not due to the MEK kinase which expression and activation is not dependent on the drug. These results indicate that another kinase is involved in this pathway and that this molecule could be specifically activated by the drug. It would be interesting to further explore this mechanism to try to understand what leads to ERK activation after ATM treatment. It is known that the MEK-ERK pathway could be activated by reactive oxygen species (ROS). Moreover it has been already been demonstrated that ROS can induce activation of several forms of aPKCs.22 We were also able to demonstrate that this drug-dependent ERK activation is required for the activation of the caspase 3 and that this is important for the

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Discussion


Figure 3. ATM treatment affects intracellular signal transduction pathways. Total cell lysates from PC3U and PrEC cells were subjected to immuno-blotting using antisera against total or phosphorylated proteins. Tubulin (* indicates that the same immunoblot for tubulin was used as internal control, in A and B) or actin served as internal controls for immuno-blotting. Expression and activation of ERK (A), MEK (B) and AKT (C) in PC3U and PrEC cells, after treatment with ATM (25 μM or 50 μM) for 24 hours. Cells treated with vehicle served as control (CTR).

apoptotic effect. Recently, it has been reported that hyperactivation of ERK could be linked to an apoptotic programme although ERK is involved mainly in cell growth and survival processes. The programmed cell death could be achieved by different ways. Firstly,

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Figure 4. ATM induces a pro-apoptotic effect in PC3U cells. (A) Immuno-blotting on PC3U cell lysates to detect cleaved caspase 3 after treatment with ATM (25 μM or 50 μM) for 24 hours. Positive control (STS), PC3U cells treated with staurosporine (STS 5 μM) for 4 hours. Cells treated with vehicle served as negative control (CTR). (B) Activation of caspase 3 (PathScan ELISA Caspase3 Assay) in PC3U and PrEC cells after treatment with ATM (25 μM or 50 μM) for 24 hours or treated with vehicle (CTR). (C) Expression and activation of p38 and JNK in PC3U and PrEC cells after ATM treatment or with vehicle (CTR), for 24 hours investigated by immuno-blotting. Tubulin (*) indicates that the immunoblot for tubulin was used as internal control for equal loading of proteins for upper and lower panles (p-p38 and p-JNK). (D) Activation of caspase 3 (PathScan ELISA Caspase3 Assay) in PC3U cells after treatment with ATM (25 μM or 50 μM) or with vehicle (CTR), in the presence of the specific p38 inhibitor SB203580 (10 μM) for 24 hours. SB203580 was added to cells one hour before treatment with ATM. STS served as positive control for the assay.

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we proved that in PC3U cells there is a specific dose-dependent downregulation of AKT and that this pattern is specifically induced only in prostate cancer cells and not in normal epithelial prostate cells. It is known that AKT is a protein kinase which plays an important role in prostate cancer for survival. PC3U cells have PTEN mutations and express normally high levels of active AKT (our unpublished data). It is also known that there is a cross-talk between PI3K/AKT and MEK/ERK pathways even if it is not so clear how they influence each other. The downregulation of AKT specifically induced by the drug might be linked to the activation of ERK. This could be a mechanism through which the prostate cancer cell regulates its fate. Furthermore, PC3U cells do not express wt p53.23 Therefore it is likely that the cell must use alternative mechanisms to control its fate. A counterbalanced fine tuned regulation of ERK and AKT activation could be an option. The effect of ATM treatment could be considered by the cell as an extreme threat resulting 310

in activation of ERK, which in combination with activation of p38 and JNK MAP kinases (see further below), could push the cell to undergo apoptosis. Moreover, we have also shown that the ATM-induced activation of ERK is linked to apoptosis in PC3U cells via activation of p38, caspase 3 and cytochrome c release. Activation of p38 was shown to be essential for the activation of the caspase 3, consistent with role of p38α as a tumor suppressor.24 It would be interesting to further investigate the precise molecular mechanisms for ATM-induced apoptosis and if it specific for prostate cancer or not. It would also be of interest to achieve a better understanding of the putative role for TGFβ in the pro-apoptotic effect induced by ATM, as the activity of Par6 is known to be modulated by TGFβ in the EMT pathway.25,26 TGFβ is a key regulator in carcinogenesis, as it can act either as a shield against development of cancer,27 but also for promoting cancer initiation and progression, due to its capability to facilitate

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Figure 5. ERK activation is required for the activation of the caspase 3 in PC3U cells. Total cell lysates from PC3U cells, exposed to either control (CTR) or ERK1/2 specific siRNA and indicated treatment, were subjected to immuno-blotting for ERK1/2. (A) Note the efficiency of siRNA knock-down of ERK. Immuno-blotting for tubulin served as internal control for equal loading of proteins. (B) Activation of caspase 3 (PathScan ELISA Caspase 3 Assay) after ATM treatment (25 μM or 50 μM) or vehicle (CTR), in PC3U-cells transfected with ERK siRNA in the presence of TGFβ stimulation for 2 hours (lower) or in the presence of TGFβ stimulation (upper).

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EMT.28 However, in our current study, we did not observe any major effects by treatment of cells with TGFβ, on the biochemical response of ATM in PC3U cells. The apoptotic effect observed by ATM-treatment of aggressive PC-3U cells in this study, was achieved when cells were treated with 25 μM, and even more at 50 μM. Serum levels of ATM in patients treated with this compound has been reported to be 3–8 μg/mL.29,30 Interestingly, in a recent study by Regala et al.31 a potent antitumor effect of ATM on a small lung carcinoma cell line in vitro at IC50 46 μmol/L was observed, which is consistent with our data, presented here. Thus, it appears that the presumed cytotoxic effects exerted by ATM on prostate cancer cells in vivo, could be achieved without www.landesbioscience.com

having to increase serum concentrations of ATM to higher levels than in patients with rheumatoid arthritis, that receive treatment with ATM. In conclusion, we provide evidence that ATM induces death via apoptosis specifically in prostate cancer cells, as this pro-apoptotic effect is not observed in normal epithelial prostate cells. Taken together, our data presented here, suggest that ATM should be considered as a new promising drug for treatment of prostate cancer.

Materials and Methods Reagents. Aurothiomalate (ATM) was from Sanofi-Aventis (Myocrisin). FLAG-Par6 plasmid was obtained from Addgene.

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(10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 20 mM Hepes/Tris pH 7.4) supplemented with protease and phosphatase inhibitors. Samples were centrifuged at 12,500 g for 10 minutes at 4°C. After centrifugation, cells were further lysed and DNA was sheared, using a syringe with a medium needle (24G). The nuclear fraction was recovered as the pellet after another centrifugation for 5 min 3,000 g and was resuspended in 400 μl of nuclear buffer (20 mM Tris, 500 mM KCl, 2 mM MgCl2, 0.5% NP-40). EDTA was added to the supernatant to reach a final concentration of 0.5 mM and then centrifuged for 1 h at 1,00,000 g to obtain the cytosolic fraction.12 Protein concentration was determined by BCA method (Thermo). Path scan ELISA caspase 3 kit assay. Cells treated with ATM were collected by scraping in 0.5 ml (10 cm2 plate) of ice-cold cell lysis buffer (20 mM TrisHCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM ethylene diamintetraacetate (EDTA), 1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N’,N’-tetraacetic acid (EGTA), 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF)). The plate was incubated on ice for 5 minutes. Then lysates were sonicated for 5 minutes on ice at the highest power at cycles of 15’’ of sonication every 30” (Bioruptor, Diagenode). Lysates were treated in the assay following the producer’s recommendations. The spectrophotometric determination was performed using Wallac VICTOR2TM 1420 Multilabel Counter. The quantification was performed choosing the signal obtained from the control sample as the basal level. The basal level was subtracted to each sample and the obtained value has been divided again for the basal level to have the increase of apoptotic cells compared to the control. In situ proximity ligation assay (O-LINK technology). Cells were grown in a 6-well plate previously prepared with sterile glass microscopy slides. The slides were washed twice with phosphatebuffer saline (PBS), fixed in 3.7% paraformaldehyde for 20 minutes at room temperature, washed twice in PBS and subsequently permeabilized in 0.1% Triton X-100 in phosphate-buffered saline for 5 min, and washed again twice in PBS. Slides were incubated with Duolink Blocking Solution in a pre-heated humidity chamber for 30 minutes at 37°C. Then, primary antibodies (Ab anti-Par6 rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA; Ab anti-PKCι mouse, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were mixed and diluted at suitable concentrations in Antibody Diluent. After adding the primary solution to the slides, they were incubated for 20 minutes at room temperature. PLA probes were mixed and diluted in 1X Antibody Diluent. After washing slides with TBS-T, they were incubated with the PLA probes in a pre-heated humidity chamber for 2 hours at 37°C. Then, the PLA probe solution was tapped off from the slides, which then were incubated with Duolink Hybridization solution in a pre-heated humidity chamber for 15 minutes at 37°C. Thereafter, the ligation step was performed. The slides were washed once with TBS-T for 1 minute under gentle agitation. After diluting the Duolink Ligation Stock and adding the Duolink Ligase, the slides were incubated in a pre-heated humidity chamber for 15 minutes at 37°C. The amplification step was performed diluting the Duolink Amplification Stock and adding to this the Duolink Polymerase. Thereafter, the slides were incubated with this solution in a pre-heated humidity chamber for 90 minutes at 37°C. The last step was the detection. The slides were washed in TBS-T 2 x 2 minutes under gentle agitation. The detection solution was diluted

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Vybrant Apoptosis Assay Kit #13 (V35123) was from Molecular Probe Invitrogen. Path Scan ELISA Caspase 3 Assay was from Cell Signaling. In situ proximity ligation assay was from O-LINK company (Uppsala University). Cell cultures. Human advanced prostate cancer PC3U cells, a sub-line originated form PC3 cells,11 were routinely grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% α-glutamin. Primary epithelial prostate cells PrEC, were purchased form Cambrex Bio Science Walkersville, Inc., (CC-2555) and routinely grown in Clonetics PrEGMTM (CC-3166) prostate epithelial cell basal medium, supplemented with BPE, hydrocortisone, hEGF, epinephrine, transferring, insulin, retinoic acid, triidothyronine, GA-1000. One ReagentPackTM (CC-5034) contaning trypsin 0.025%/EDTA 0.01%, trypsin neutralizing solution and HEPES buffered saline solution was used to subculture PrEC cells. Both cell lines were treated with ATM at two different concentrations, 25 μM or 50 μM, in the same culturing conditions. Vybrant apoptotis assay kit. 5 x 104 cells/well of PC3U and PrEC cells were cultured in a 6-well plate in RPMI 1640, supplemented with 10% FBS, and in PrEGM medium respectively. Cells were treated with ATM at the fixed concentrations 25 μM or 50 μM for 24 hours, whereafter samples were treated according to the producer’s instructions. Western blot analysis. After ATM treatment, cells were collected by scraping in lysis buffer (RIPA buffer) containing protease and phosphatase inhibitors. The amount of proteins was determined using BCA assay (Thermo). After SDS-page and protein transfer, the membranes were blocked for 1 h with 5% BSA in TBS-T and then incubated with anti-Par6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PKCι, anti-PKCξ/λ, anti-p-PKCξ/λ, antiERK, anti-p-ERK, anti-MEK, anti-p-MEK, anti-p38, anti-p-p38, anti-JNK, anti-p-JNK (Cell Signaling Technologies, Danvers, MA), anti-FLAG (rabbit home made), overnight at 4°C. Afterwards, the membranes were washed and then incubated for 45 minutes with a secondary horseradish peroxidase-conjugated antibody and developed with Lumi-LightPLUS Western Blot Substrate (Roche Diagnostics, Germany). Transfection and immunoprecipitation. PC3U cells were transfected with a plasmid containing a wt FLAG-Par6. Twenty-four hours post-transfection, cells were treated with ATM (25 μM or 50 μM) for 24 hours, and were thencollected by scraping in lysis buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol). Cell lysates were centrifuged at 3,000 rpm for 5 minutes at 4°C and then incubated over-night at 4°C with 1 μg of anti-FLAG M2 monoclonal antibody (Sigma Aldrich, USA). Protein A was added to the samples and incubated for 45 minutes at 4°C. Beads were then washed four times with lysis buffer. Immunocomplexes were released by incubation for 3 min at 95°C in sample buffer containing SDS, and then subjected to SDS-polyacrylamide gel electrophoresis. siRNA transfection. PC3U cells were transfected with CTR siRNA and ERK1/2 siRNA (Dharmacon, CO, USA) using oligofectamineTM reagent (Invitrogen, CA, USA) following the producer’s instructions. One day post-transfection, PC3U cells were treated with ATM (25 μM or 50 μM) for 24 hours. Cytochrome c release. After ATM treatment cells were collected by scraping in 1 ml of phosphate-buffer saline. Cells were centrifuged at 540 g for 5 minutes and then treated with a hypotonic lysis buffer 312

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Marianna Trani is a Master Student from the University of Bologna. This work was supported by Ludwig Institute for Cancer Research, Swedish Cancer Foundation, Swedish Medical Research Council, Torsten and Ragnar Söderbergs Foundation and A.L.F. We thank Carl-Henrik Heldin and the members of the Apoptotic Signalling group for stimulating discussions. We thank also our colleagues at Ludwig Institute for Cancer Research, Uppsala Branch, for creating a beneficial scientific environment.

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Acknowledgements

21. Smith WE and Reglinski J. Distribution and reactivity of myocrisin. Met Based Drugs 1994; 1:497-507. 22. Wu WS. The signaling mechanism of ROS in tumor progression. Cancer Metastasis Rev 2006; 4:695-705. 23. Zhang S, Ekman M, Thakur N, Bu S, Davoodpour P, Grimsby S, et al. TGFβ1-induced activation of ATM and p53 is mediated by Smad7. Cell Cycle 2006; 23:2787-95. 24. Hui L, Bakiri L, Stepniak E, Wagner EF. p38alpha: a suppressor of cell proliferation and tumorigenesis. Cell Cycle 2007; 20:2429-33. 25. Bose R and Wrana J. Regulation of Par6 by extracellular signals. Curr Opin Cell Biol 2006; 18:206-12. 26. Ozdamar B, Bose R, Barrios-Rodiles M, Wang HR, Zhang Y, Wrana J. Regulation of the polarity protein Par6 by TGFβ receptors controls epithelial cell plasticity. Science 2005; 307:1603-9. 27. Honjo Y, Bian Y, Kawakami K, Molinolo A, Longenecker G, Boppana R, et al. TGFbeta receptor I conditional knockout mice develop spontaneous squamous cell carcinoma. Cell Cycle 2007; 11:1360-6. 28. Gregory PA, Bracken CP, Bert AG, Goodall GJ. MicroRNAs as regulators of epithelialmesenchymal transition. Cell Cycle 2008; 20:3112-8. 29. Blocka KL, Paulus HE, Furst DE. Clinical pharmacokinetics of oral and injectable gold compounds. Clin Pharmacokinet 1986; 11:133-43. 30. FASS.se 2008. www.FASS.se. 31. Regala RP, Thompson EA, Fields AP. Atypical protein kinase Ciota expression and aurothiomalate sensitivity in human lung cancer cells. Cancer Res 2008; 14:5888-95.

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and added to each sample. The slides were incubated in a pre-heated humidity chamber for 60 minutes at 37°C. Then, slides were washed using decreasing concentration of SSC solutions and finally 70% EtOH for 1 minute. After drying slides, samples were prepared for imaging using a minimal volume of Duolink Mounting Medium. The samples were analyzed in a fluorescence microscope (Zeiss Microscope 63X objective).

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Supplementary materials can be found at: www.landesbioscience.com/supplement/TraniCC8-2-Sup.pdf

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References 1. American Cancer Society. Surveillance research cancer statistics. Atlanta: American Cancer Society 2005. 2. Reynolds AR and Kyprianou N. Growth factor signalling in prostatic growth: significance in tumour development and therapeutic targeting. Br J Pharmacol 2006; 147:144-52. 3. Stangelberger A, Schally AV, Varga JL, et al. Antagonists of growth hormone releasing hormone (GHRH) and of bombesin/gastrin releasing peptide (BN/GRP) suppress the expression of VEGF, bFGF and receptors of the EGF/HER family in PC-3 and DU-145 human androgen-indipendent prostate cancers. Prostate 2005; 64:303-15. 4. Stallings-Mann M, Jamieson J, Regala RP, Weems C, Murray NR, Fields AP. A novel smallmolecule inhibitor of protein kinase Ciota blocks transformed growth of non-small-cell lung cancer cells. Cancer Res 2006; 66:1767-74. 5. Fields AP, Regala RP. Protein kinase Cι: Human oncogene, prognostic marker and therapeutic target. Pharmacol Res 2007; 55:487-97. 6. Regala RP, Weems C, Jamieson L, Copland JA, Thompson EA, Fields AP. Atypical protein kinase Ciota plays a critical role in human lung cancer growth and tumorigenicity. J Biol Chem 2005; 280:31109-15. 7. Fields AP, Frederick AP, Regala RP. Targeting the oncogenic protein kinase Cι signaling pathway for the treatment of cancer. Biochem Soc Trans 2007; 35:996-1000. 8. Etienne-Manneville S, Hall A. Cell polarity: Par6, aPKC and cytoskeletal crosstalk. Curr Opin Cell Biol 2003; 15:67-72. 9. Henrique D, Schweisguth F. Cell polarity: ups and downs of the Par6/aPKC complex. Curr Opin Genet Dev 2003; 13:341-50. 10. Hirano Y, Yoshinaga S, Takeya R, et al. Structure of a cell polarity regulator, a complex between atypical PKC and PB1 Par6 domains. J Biol Chem 2005; 280:9653-61. 11. Franzen P, Ichijo H, Miyazono K. Different signals mediate transforming growth factor-β1induced growth inhibition and extracellular matrix production in prostatic carcinoma cells. Exp Cell Res 1993; 207:1-7. 12. Marzano C, Gandin V, Folda A, Scutari G, Bindoli A, Rigobello MP. Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells. Free Radic Biol Med 2007; 42:872-81. 13. Erdogan E, Trond L, Stallings-Mann M, et al. Aurothiomalate inhibits transformed growth by targeting the PB1 domain of protein kinase Ciota. J Biol Chem 2006; 281:28450-9. 14. Zhuang S and Schnellmann RG. A death-promoting role for extracellular signal-regulated kinase. J Pharmacol Exp Ther 2006; 319:991-7. 15. Edlund S, Bu S, Schuster N, et al. Transforming growth factor-β1 (TGFβ)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGFβ-activated kinase 1 and mitogen-activated protein kinase kinase 3. Mol Biol Cell 2003; 14:529-44. 16. Sorrentino A, Thakur N, Grimsby S, et al. The type I TGFbeta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol 2008; 10:1199-207. 17. Takatsu Y, Nakamura M, Stapleton M, et al. TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development. Mol Cell Biol 2000; 20:3015-26. 18. Ow YP, Green DR, Hao Z, Mak TW. Cytochrome c: functions beyond the respiration. Nat Mol Cell Bio 2008; 9:532-42. 19. Morris DS, Tomlins SA, Rhodes DR, Mehra R, Shah RB, Chinnaiyan AM. Integrating biomedical knowledge to model pathways of prostate cancer progression. Cell Cycle 2007 10:1177-87. 20. Lee JT, Lehmann BD, Terrian DM, Chappell WH, Stivala F, Libra M, et al. Targeting prostate cancer based on signal transduction and cell cycle pathways. Cell Cycle 2008; 7:1745-62.


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