RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells

ORIGINAL ARTICLE

See related commentary on pg 315

RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells Anja Berger1, Sandra-Annika Quast1, Michael Plo¨tz1, Nicholas-Frederik Kuhn1, Uwe Trefzer1 and Ju¨rgen Eberle1 Mutated BRAF represents a critical oncogene in melanoma, and selective inhibitors have been approved for melanoma therapy. However, the molecular consequences of RAF inhibition in melanoma cells remained largely elusive. Here, we investigated the effects of the pan-RAF inhibitor L-779,450, which inhibited cell proliferation both in BRAF-mutated and wild-type melanoma cell lines. It furthermore enhanced apoptosis in combination with the death ligand tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and overcame TRAIL resistance in melanoma cells. Enhanced apoptosis coincided with activation of mitochondrial pathways, seen by loss of mitochondrial membrane potential and release of cytochrome c, Smac (second mitochondria–derived activator of caspases), and apoptosis-inducing factor (AIF). Subsequently, caspase-9 and -3 were activated. Apoptosis induction by L-779,450/TRAIL was prevented by Bcl-2 overexpression and was dependent on Bax. Thus, activation of Bax by L-779,450 alone was demonstrated by Bax conformational changes, whereas Bak was not activated. Furthermore, the BH3-only protein Bim was upregulated in response to L-779,450. The significant roles of Smac, Bax, and Bim in this setting were proven by small interfering RNA (siRNA)-mediated knockdown experiments. L-779,450 also resulted in morphological changes indicating autophagy confirmed by the autophagy marker light chain 3-II (LC3-II). The pro-apoptotic effects of L-779,450 may explain the antitumor effects of RAF inhibition and may be considered when evaluating RAF inhibitors for melanoma therapy. Journal of Investigative Dermatology (2014) 134, 430–440; doi:10.1038/jid.2013.347; published online 19 September 2013

INTRODUCTION The high mortality of metastatic melanoma strongly relates to its pronounced resistance to chemotherapy as well as to an antitumor immune response (Garbe and Leiter, 2009). This is explained by defects in pro-apoptotic signaling, suggesting the overcoming of melanoma apoptosis deficiency as a predominant therapeutic target (Eberle et al., 2007). Mitogen-activated protein kinase (MAPK) pathways, initiated via Ras and RAF, have important roles in tumor development (Gray-Schopfer et al., 2005). The signaling cascade via MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) and ERK leads to phosphorylation of multiple target proteins, which drive the regulation of gene expression, cell differentiation, proliferation, and survival (Inamdar et al., 1

Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charite´, Berlin, Germany

Correspondence: Ju¨rgen Eberle, Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charite´, Charite´platz 1, 10117 Berlin, Germany. E-mail: [email protected] Abbreviations: AIF, apoptosis-inducing factor; FCS, fetal calf serum; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; pERK, phosphorylated extracellular signal-regulated kinase kinase; Smac, second mitochondria–derived activator of caspases; TRAIL, tumor necrosis factor– related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis; WT, wild type Received 17 October 2012; revised 14 May 2013; accepted 26 May 2013; accepted article preview online 16 August 2013; published online 19 September 2013

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2010). Three RAF isoforms (A, B, and C) have been reported, which exert distinct roles in different cells (Maurer et al., 2011). For melanoma, activating mutations in N-Ras (10–25%) and BRAF (40–60%) appear to be of particular interest (Davies et al., 2002; Sullivan and Flaherty, 2012). The high frequency of the BRAF(V600E) mutation resulted in the development of selective BRAF inhibitors, partially already approved for melanoma therapy (Flaherty et al., 2010; Chapman et al., 2011; Hauschild et al., 2012; Long et al., 2012; Sosman et al., 2012). However, the significance of RAF inhibitors in the regulation of apoptosis in melanoma cells has not been sufficiently explored. In apoptosis control, extrinsic proapoptotic pathways are initiated by binding of death ligands as tumor necrosis factor-a, CD95L/FasL, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) to their respective death receptors, leading to the formation of death-inducing signaling complexes and activation of initiator caspase-8 (Krammer et al., 2007). On the other hand, intrinsic apoptosis pathways may be initiated by cellular dysregulation and DNA damage and may result in pro-apoptotic mitochondrial activation. Key events are depolarization of the mitochondrial membrane potential (Dcm) and mitochondrial outer membrane permeabilization, leading to the release of cytochrome c, Smac (second mitochondria–derived activator of caspases), and AIF (apoptosis-inducing factor). Whereas cytochrome c activates initiator caspase-9, Smac functions as an antagonist & 2014 The Society for Investigative Dermatology

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Figure 1. Antiproliferative effects by L-779,450. (a) Levels of phosphorylated extracellular signal-regulated kinase (pERK) and total ERK were determined by western blotting in human melanoma cell lines treated for 24 hours with L-779,450 (0.1–50 mM): A-375, A-375-TS (V600E), and MeWo (BRAF-wild type (WT)). Two independent experiments revealed comparable results; equal loading was proven by glyceraldehyde dehydrogenase (GAPDH). (b) Apoptosis (% of sub-G1 cells) was determined in response to L-779,450 (5–40 mM; 24 hours) in A-375, Mel-HO-TS, Mel-2a, MeWo, and SK-Mel-13. Cytotoxicity (lactate dehydrogenase (LDH) release) was determined in parallel (below). Here, values of non-treated cells were set to 1. (c) Apoptosis was also determined at 48 and 72 hours (L-779,450, 10 mM). (b, c) Means and SDs of triplicates; at least two independent experiments. (d) Cell cycle analyses of human melanoma cell lines: open graphs, L-779,450-treated (10 mM, 24 hours); filled graphs, dimethyl sulfoxide (DMSO)-treated controls. (e) Growth curves determined in real time of melanoma cell lines treated with L-779,450 (5–20 mM) were compared with controls (0). Means and SDs (at 48 hours) of triplicate determinations are shown; two independent experiments were performed. Statistical significance is indicated (*Po0.05), when comparing L-779,450 and DMSO-treated cells.

of cellular inhibitor of apoptosis proteins (e.g. X-linked inhibitor of apoptosis (XIAP) and survivin), which themselves prevent effector caspase activity (Tait and Green, 2010). Mitochondrial apoptosis pathways are critically controlled by the family of Bcl-2 proteins, which encloses anti-apoptotic (e.g. Bcl-2 and Mcl-1), pro-apoptotic multidomain (Bax and Bak), and pro-apoptotic BH3-only proteins (e.g. Bid and Bim) (Chipuk et al., 2010). Initiator caspases activated in both

pathways may drive the processing and activation of the main effector caspase-3, which itself cleaves various death substrates to set apoptosis into work (Fischer et al., 2003). Mitochondrial pathways may also be activated by death ligands via caspase-8mediated processing and activation of Bid (Li et al., 1998). The death ligand TRAIL represents an additional therapeutic strategy and selectively induces apoptosis in cancer cells via TRAIL receptors DR4 and DR5 (Newsom-Davis et al., 2009). www.jidonline.org

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In melanoma cells and tissue samples, we have previously shown functional activity of TRAIL receptors (Kurbanov et al., 2005); however, some melanoma cell lines revealed perma-

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nent TRAIL resistance and others developed inducible resistance upon TRAIL treatment (Kurbanov et al., 2007). Inducible resistance was related to the downregulation of TRAIL

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receptors, initiator caspases, and pro-apoptotic Bcl-2 proteins (Zhang et al., 2006; Kurbanov et al., 2007), whereas the causes of permanent resistance in melanoma remained elusive. The correlation of MAPK activity and resistance to TRAILinduced apoptosis in melanoma cells had already been shown in a previous study when applying an MEK inhibitor (Zhang et al., 2003). Here, we prove the strong relation of RAF inhibition and apoptosis regulation in melanoma cells. As shown by small interfering RNA (siRNA)-mediated knockdown studies, the sensitization for TRAIL was particularly based on Bax activation and on upregulation of Bim in response to the inhibitor alone. This resulted in the activation of mitochondrial apoptosis pathways, particularly based on the release of Smac. RESULTS Direct effects of L-779,450

The pan-RAF inhibitor L-779,450 was applied for understanding the relations of RAF inhibition and apoptosis regulation in melanoma cells. Strong and dose-dependent phosphorylated ERK (pERK) downregulation was seen by L-779,450 in the BRAF(V600E)-mutated, TRAIL-sensitive melanoma cell line A-375, as well as in TRAIL-resistant A-375-TS. Reduced sensitivity of A-375-TS may be explained by its selection for TRAIL resistance. In contrast, only incomplete pERK downregulation was seen in the BRAF-wild-type (WT) melanoma cell line MeWo, even at high concentrations (Figure 1a). Downregulation of pERK largely remained without direct effect on apoptosis and cytotoxicity in melanoma cells up to 72 hours (Figure 1b and c). However, partial G1 arrest and depletion of S-phase cells were seen at 24 hours in four BRAF-mutated melanoma cell lines (A-375, Mel-HO, Mel-2a, and SK-Mel-28) as well as in their TRAIL-selected derivatives A-375-TS and Mel-HO-TS. G1 arrest was also seen in two N-Ras-mutated, BRAF-WT cell lines (SK-Mel-103 and SK-Mel-147), whereas another BRAFWT cell line (MeWo) and the BRAF-mutated cell line SK-Mel13 did not respond (Figure 1d). Treatment with L-779,450 resulted in dose-dependent decrease of cell numbers, as determined in real time (Figure 1e). Sensitization for death ligand–induced apoptosis

The sensitivity of melanoma cell lines for the death ligand TRAIL had been described in previous studies (Kurbanov et al., 2005, 2007; Berger et al., 2011). The effects of L-779,450 on TRAIL sensitivity were investigated here in melanoma cell lines with high TRAIL sensitivity (A-375 and SK-Mel-147),

moderate sensitivity (Mel-HO, SK-Mel-13, and SK-Mel-28), and permanent resistance (MeWo, Mel-2a, and SK-Mel-103), as well as in TRAIL-selected cell lines with acquired resistance (A-375-TS and Mel-HO-TS). Despite only moderate direct effects of L-779,450 on apoptosis, it strongly enhanced TRAILinduced apoptosis in sensitive melanoma cells and overruled TRAIL resistance in Mel-2a, SK-Mel-103, A-375-TS, and MelHO-TS. At 24 hours, 16–35% apoptosis induction was obtained, whereas cytotoxicity remained largely unaffected. BRAF-WT cells MeWo as well as BRAF-mutated cells SK-Mel-13 revealed no enhancement, not even at 72 hours after treatment (Figure 2a, b, and e). Morphologic changes characteristic for apoptosis became evident by bisbenzimide nuclear staining. Thus, nuclear fragmentation and chromatin condensation were seen at 24 hours in 18–30% of A-375 and A-375-TS cells (Figure 2c and d). The strong impact of induced apoptosis on cell proliferation was visualized by real-time cell analysis. Thus, complete detachment of cells was seen in A-375 and A-375-TS already at 36 hours in response to L-779,450/TRAIL (20 ng ml  1; Figure 2f). Treatment with the MEK inhibitor U0126 (20 mM) was applied for comparison and revealed a similar picture. Thus, it also enhanced TRAIL-induced apoptosis in A-375, A-375-TS, and Mel-HO but not in MeWo (Figure 2g–i). Also, treatment with the selective BRAF(V600E) inhibitor vemurafenib/PLX4032 resulted in comparable effects. Thus, PLX4032 similarly induced a G1 arrest and enhanced TRAIL-induced apoptosis in A-375, A-375-TS, and Mel-HO (Figure 2j and k). Activation of mitochondrial pro-apoptotic pathways

Involvement of caspases and Bid was investigated by monitoring activated cleavage products and loss of proforms, respectively. Processing of the extrinsic initiator caspase-8 and of Bid was already seen in A-375 and A-375-TS melanoma cells in response to TRAIL (16 hours), despite lacking TRAIL responsiveness of A-375-TS. This was evident by caspase-8 cleavage products (41 and 43 kDa) as well as by loss of caspase-8 and Bid proforms (57 and 22 kDa). This processing was, however, not enhanced by the combination with L-779,450 (Figure 3a). Decreased levels of the proform of the intrinsic initiator caspase-9 (47 kDa) were seen in A-375 in response to L-779,450 þ TRAIL but not in A-375-TS. Thus, initiator caspases could not (fully) explain enhanced apoptosis (Figure 3a). On the other hand, processing of the main effector caspase-3 was significantly enhanced by L-779,450/TRAIL, seen by its

Figure 2. Sensitization of melanoma cells for tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-induced apoptosis. (a) Percentages of apoptotic cells (sub-G1) were determined in human melanoma cell lines at 24 hours in response to L-779,450 (10–40 mM) and TRAIL. Examples are shown in insets. (b) Cytotoxicity (lactate dehydrogenase (LDH) release) was determined in parallel. Values of non-treated cells are set to 1. (c) Photographs (magnification 1:400) after bisbenzimide (Hoechst 33258) staining: TRAIL-sensitive A-375 and TRAIL-resistant A-375-TS cells treated for 24 hours with 10 mM L-779,450/TRAIL. White arrows show examples of fragmented or condensed nuclei. (d) Quantitative evaluation of cells with fragmented nuclei and/or condensed chromatin. (e) MeWo cells were treated also for 48 and 72 hours (percentages of sub-G1 cells). (f) Growth curves (real time) of A-375 and A-375-TS treated with TRAIL, L-779,450 (5 mM), or the combination were compared with non-treated controls. (g) Percentages of apoptotic cells (sub-G1) were determined in response to 20 mM U0126±TRAIL. (h) Cell cycle analyses of A-375 and MeWo treated with U0126 (20 mM, 24 hours, open graphs), as compared with dimethyl sulfoxide (DMSO) controls (filled graphs). (i) Growth curves of A-375 treated with U0126 (20 mM) and/or TRAIL (means and SDs of triplicate determinations). (j) Percentages of apoptotic cells (subG1) were determined in response to PLX4032 (3, 10, and 40 mM)±TRAIL. (k) Cell cycle analyses of A-375, A-375-TS, and Mel-HO treated with PLX4032 (10 mM, 2 hours, open graphs), as compared with DMSO controls (filled graphs). Always, two to three independent experiments revealed highly comparable results. Statistical significance (*Po0.05) is indicated, when comparing L-779,450/TRAIL treatment with TRAIL alone. HOECHST/bisbenzimide staining.

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cleavage products of 15, 17, and 20 kDa (Figure 3a). The decisive role of caspases was further demonstrated by the pancaspase inhibitor Q-VD-OPh, which completely abrogated apoptosis induction by L-779,450/TRAIL (Figure 3b). Lacking enhancement of caspase-8 and Bid processing suggested a role of Smac, which may antagonize cIAPs and thus activate caspase-3. Confirming the activation of mitochondrial apoptosis pathways, strong decrease of the mitochondrial membrane potential Dcm was seen at 24 hours in A-375 (490%) and in A-375-TS (50%) in response to L-779,450/TRAIL. In comparison, L-779,450 alone remained without effect on Dcm, and a response to TRAIL was seen in only 16% of A-375 (Figure 4a). Loss of Dcm appeared as in parallel to apoptosis induction, as not seen at 2 and 4 hours (data not shown). Finally proving the activation of mitochondrial apoptosis pathways, both A-375 and A-375-TS revealed strong release of mitochondrial apoptogenic factors (cytochrome c, AIF, and Smac) upon L-779,450/TRAIL treatment. This effect was already seen at 3 hours, before any apoptosis was induced (Figure 4b) and was still seen at 16 hours (data not shown). Mitochondrial release thus appeared as an early and initial effect. Smac represented a suitable candidate for overcoming the apoptosis blockade through cIAPs in melanoma cells. In agreement, siRNA-mediated Smac knockdown decreased apoptosis by L-779,450/TRAIL in A-375 by 60% (Figure 4c).

Induced Bim expression and autophagy by L-779,450

Decisive roles of Bcl-2 and Bax

DISCUSSION MAPK pathways initiated by Ras/RAF have decisive roles in melanoma (Davies et al., 2002; Jin et al., 2012; Mann et al., 2012). Thus, selective inhibitors for mutated BRAF as vemurafenib and dabrafenib have been approved or are in clinical trials (Flaherty et al., 2010; Chapman et al., 2011; Hauschild et al., 2012; Long et al., 2012; Sosman et al., 2012). The downstream kinase MEK represents an additional promising target, and combinations of both kinase inhibitors are evaluated (Eggermont and Robert, 2011; Flaherty et al., 2012). For targeting the MAPK pathway at an early step, we used here L-779,450, which was described as inhibitor for A-, B-, and CRAF (Shelton et al., 2003). As CRAF activation upon BRAF inhibition is discussed as a cause of resistance and melanoma relapse (Heidorn et al., 2010; Maurer et al., 2011; Sullivan and Flaherty, 2012), simultaneous inhibition of both RAF isoforms may be considered as a supportive strategy. Also other MAPKs, as p38 may be targeted by L-779,450, which may allow a comprehensive antitumor approach (Shelton et al., 2003). Efficient inhibition of the MAPK pathways by L-779,450 was seen in BRAF(V600E)-mutated melanoma cells by complete abrogation of ERK phosphorylation. Comparably high pERK levels were also seen in BRAF-WT cells, which were however less affected by L-779,450. Thus, ERK phosphorylation in these cells may depend on alternative pathways, e.g., as suggested for protein kinase Ca (Rucci et al., 2005). Reduced cell growth in response to L-779,450 was reported in hematopoietic cells, which appeared as related to ARAF and CRAF in these cells (Shelton et al., 2003). Similarly,

The significance of mitochondrial apoptosis pathways was further underscored by decisive roles of Bcl-2 and Bax. Thus, Bcl-2 stable overexpression in A-375 (A-375-Bcl-2) completely prevented apoptosis induction by L-779,450/TRAIL (20 ng ml  1; Figure 5a) and also abolished loss of Dcm (Figure 5b). Because of preferential binding of Bcl-2 to Bax, this suggested a particular role of the Bax pathway. The roles of Bax and Bak were distinguished in a BRAF-WT, K-Ras-mutated HCT-116 colon carcinoma cell model consisting of Bax þ Bak þ parental cells, Bax knockout, Bak knockdown, and double knockdown cells. Comparable apoptosis induction was seen at 24 hours in the parental and the Bak knockdown cells, whereas apoptosis was completely abrogated by Bax knockout (Figure 5c). Bax dependency was also proven in A-375 melanoma cells by siRNA strategies for Bax and Bak, respectively. Again, only the Bax knockdown resulted in resistance to TRAIL as well as to L-779,450/TRAIL, whereas Bak knockdown remained without effect (Figure 5d). The critical role of Bax was further investigated by N-terminus–specific antibodies indicative for Bax activation. Of note, significant Bax activation was seen in A-375 and A-375-TS already in response to L-779,450 alone, which itself did not induce apoptosis. This again appeared as an initial effect already at 2 hours. In contrast, TRAIL treatment remained without effect on Bax activation, and no activation was seen for Bak, as determined by a Bak-NT antibody (Figure 5e). Thus, Bax activation through L-779,450 did not trigger apoptosis by itself but enhanced TRAIL sensitivity of melanoma cells. 434

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For further understanding the enhancement of apoptosis, the expression of multiple apoptosis regulators was investigated in Mel-HO, A-375, and A-375-TS at 16 hours of treatment. No significant changes in response to L-779,450 were obtained by western blotting for Bcl-2, Mcl-1, Bax, Bak, survivin, or XIAP or by flow cytometry for TRAIL receptors DR4 and DR5 (data not shown). However, significant upregulation was found for the BH3-only protein BimEL, which may contribute to induction of mitochondrial, proapoptotic pathways (Figure 6a). TRAIL alone or in combination with L-779,450 did not affect Bim expression (Figure 6b). The significant role of Bim for L-779,450/TRAIL-mediated apoptosis was shown again by siRNA-mediated Bim knockdown in A-375, which abolished Bim upregulation and decreased apoptosis by 40% (Figure 6c). In course of L-779,450 treatment, responsive melanoma cells revealed consistent morphological changes, as large vacuoles strongly indicative of autophagy (Figure 6d). Again, these changes were not seen in MeWo and SK-Mel-13. Further substantiating autophagy, the marker light chain 3-II (LC3-II) was significantly upregulated upon treatment with L-779,450 (Figure 6e). Thus, apoptosis sensitization for TRAIL by activation of proapoptotic mitochondrial pathways via Bim and Bax as well as induced autophagy appeared as characteristic for the melanoma cell response to RAF inhibition.

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Figure 3. Caspase involvement. (a) Processing of caspase-8, -9, and -3 as well as of Bid was determined by western blotting in A-375- and A-375-TS-treated for 16 hours with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) (20 ng ml  1) and/or L-779,450 (10 mM). Cleavage products of caspase-3 and -8 are marked ( ). (b) Percentages of apoptotic cells (sub-G1) were determined after treatment (24 hours). In addition, the pancaspase inhibitor Q-VD-OPh (10 mM, 1 hour pretreatment, dark gray) was given. Statistical significance of abrogated apoptosis by Q-VD-OPh is indicated (*Po0.05). (a, b) At least two independent experiments revealed comparable results.

melanoma cells responded with reduced cell growth to PLX4720 (Tsai et al., 2008), and antiproliferative effects as well as G1 arrest were also seen here by L-779,450. These antiproliferative effects were not restricted to BRAF-mutated melanoma cells; however, there was a correlation between G1 arrest induced by L-779,450 and its ability to enhance TRAIL-induced apoptosis. TRAIL represents a promising antitumor strategy, due to selective apoptosis induction in tumor cells (Walczak et al., 1999; Ashkenazi, 2002), but the antitumor activity of TRAIL receptor agonists remained only limited in clinical trials (Herbst et al., 2010; Younes et al., 2010; Soria et al., 2011). Non-sufficient efficacy was attributed to inducible resistance, also seen in melanoma cells (Zhang et al., 2006; Kurbanov et al., 2007). Overcoming TRAIL resistance thus represents a promising therapeutic approach, and different strategies were investigated in melanoma cells as signaling inhibitors, chemotherapy, and UV irradiation (Ivanov et al., 2008; Berger et al., 2011; Hornle et al., 2011; Quast et al., 2012).

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Figure 4. Activation of mitochondrial (Mito) apoptosis pathways. (a) Mitochondrial membrane potential (Dcm) was determined in A-375 and A-375-TS treated for 24 hours with tumor necrosis factor–related apoptosisinducing ligand (TRAIL) (20 ng ml  1) and/or L-779,450 (10 mM). Treated cells (open graphs) were compared with dimethyl sulfoxide (DMSO) controls (gray). (b) Cytosolic extracts (Cyto) of TRAIL-sensitive A-375 and TRAIL-resistant A-375-TS were investigated by western blotting, after treatment for 3 hours with TRAIL±L-779,450 (10 mM). Mitochondrial extracts (Mito) served as controls, and analysis of the mitochondrial protein voltage-dependent anion channels (VDAC) ruled out contaminations of cytosolic extracts with mitochondria. Equal loading was proven by glyceraldehyde dehydrogenase (GAPDH). (c) Percentages of apoptotic cells (sub-G1) were determined in A-375 in response to 10 mM L-779,450±TRAIL (24 hours). Cells with small interfering (si)RNAmediated Smac knockdown (dark bars) were compared with scramble (Scr)-siRNA-transfected controls (light gray; means and SDs of triplicate values). Smac downregulation is shown by western blotting (inset; 24 hours, GAPDH as control). Statistical significance of abrogated apoptosis by Smac knockdown is indicated (*Po0.05). (a–c) At least two independent experiments revealed comparable results. AIF, apoptosis-inducing factor; Cyt c, cytochrome c; Dcm, mitochondrial membrane potential.

Although L-779,450 hardly induced apoptosis by itself, it efficiently enhanced TRAIL-induced apoptosis in eight of ten melanoma cell lines, suggesting a link between RAF activation and TRAIL resistance in melanoma cells. The relation between active MAPK pathways and TRAIL resistance had also been reported previously by using an inhibitor for the downstream MAPK MEK (Zhang et al., 2003). Activation of MAPK www.jidonline.org

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Figure 5. Dependency and activation of Bax. (a, c, d) Percentages of apoptotic cells (sub-G1) were determined in A-375 cells stably transfected with Bcl-2 (A-375-Bcl-2) or mock plasmid (A-375-mock), parental HCT-116 colon carcinoma cells (Bax þ , Bak þ ), and subclones with knockdown for Bax and/or Bak, as well as in A-375 cells with small interfering RNA (siRNA)-mediated knockdown for either Bax or Bak. Treatment was for 24 hours, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-treated cells (dark bars) were compared with cells without TRAIL (light gray). (a, c) L-779,450 was applied at 5 mM, but comparable results were also obtained for 10 mM (data not shown). (d) L-779,450 was applied at 10 mM. Means and SDs of representative experiments (triplicate values) are shown. Expression of Bcl-2, Bax, and Bak is shown in respective insets (24 hours, western blotting, glyceraldehyde dehydrogenase (GAPDH) as loading control). Statistical significance of differences is indicated (*Po0.05), when compared with parental/mock cells. (b) Mitochondrial membrane potential (Dcm) was determined in A-375-Bcl-2 and A-375-mock. L-779,450 (5 mM)/TRAIL treatment (open graphs) was compared with dimethyl sulfoxide (DMSO) controls (gray). (e) A-375 and A-375-TS cells were treated for 2 hours with L-779,450 (10 mM) and/or TRAIL and were analyzed for Bax/Bak conformational changes by flow cytometry (Bax-NT/Bak-NT antibodies). Treated cells (bold lines) were compared with non-treated controls (C, gray) and immunoglobulin G (IgG)–stained cells (thin lines). (a–e) At least two independent experiments revealed comparable results. Scr, scramble.

pathways and relations to TRAIL resistance were also seen in colon carcinoma (Oikonomou et al., 2011), and in agreement we found enhancement of TRAIL-induced apoptosis in BRAFWT- and K-Ras-mutated HCT-116 colon carcinoma cells. As also BRAF-WT and N-Ras-mutated melanoma cell lines were sensitized for TRAIL by L-779,450, pan-RAF inhibition also 436

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proved helpful when the pathway was activated at an upstream step. Enhanced activation of effector caspase-3, whereas activation of initiator caspase-8 and -9 was not further enhanced by L-779,450, indicated the role of mitochondrial apoptosis pathways and Smac release. Smac can activate caspase-3

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via preventing the blockage of XIAP (Chai et al., 2000). Thus, caspase-3 was processed to its intermediate 20 kDa cleavage product in response to TRAIL, whereas full caspase-3 processing to its mature 15 and 17 kDa cleavage products was particularly enabled upon combination with L-779,450. The significance of the Smac/XIAP rheostat in melanoma apoptosis resistance was also suggested in previous studies (Zhang et al., 2003; Hornle et al., 2011; Quast et al., 2012). Thus, mitochondrial apoptosis pathways were activated by

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L-779,450/TRAIL already after 3 hours, as seen by the release of apoptogenic mitochondrial factors. A role of mitochondrial pathways had also been seen in melanoma cells when combining other signaling inhibitors with TRAIL (Zhang et al., 2003; Berger et al., 2011; Quast et al., 2012). XIAP is considered as a major anti-apoptotic factor in cancer cells, which prevents caspase-3 activity (Mannhold et al., 2010; Fulda and Vucic, 2012), and its predominant role was demonstrated in melanoma cells (Hornle et al., 2011; Quast et al., 2012). It may therefore also explain enhanced apoptosis by L-779,450/TRAIL. In agreement, siRNA-mediated knockdown largely abolished induced apoptosis. Suppression of apoptosis by Smac knockdown was also seen in melanoma cells, when TRAIL was combined with UVB or an ion channel inhibitor (Hornle et al., 2011; Quast et al., 2012). Bcl-2 proteins, which control mitochondrial pathways, critically affected apoptosis induction by L-779,450/TRAIL. Thus, apoptosis was abrogated in melanoma cells by Bcl-2 overexpression or Bax knockdown. The decisive role of Bax activation was proven by Bax N-terminus-specific antibodies, whereas the alternative pathway via Bak was not affected. Interestingly, Bax was activated by L-779,450 treatment alone already after 2 hours. As L-779,450 did not induce apoptosis by itself, it only opened a gate for TRAIL sensitization, and the switch appeared to be the Bax activation. Apoptosis regulators may be controlled by MAPK pathways, as upregulation of Mcl-1 and survivin or downregulation of Bad and Bim (Ai et al., 2006; McCubrey et al., 2007; Cartlidge et al., 2008). Whereas no significant changes were obtained for other apoptosis regulators, the proapoptotic BH3-only protein BimEL was significantly upregulated in melanoma cells in response to L-779,450. Upregulation of Bim was also reported in response to the selective BRAF inhibitor PLX4720 (Wroblewski et al., 2013) and the MEK inhibitor U0126 (Wang et al., 2007). Bim isoforms represent efficient inductors of apoptosis through antagonizing all prosurvival Bcl-2 proteins (Chen et al., 2005). The relation of Bim and MAPKs is based on ERK-mediated Bim phosphorylation, which triggers its proteasomal degradation (Cartlidge et al., 2008). The critical role of Bim in sensitization of melanoma

Figure 6. Induction of Bim and autophagy. (a, b) Expression of BimEL was determined by western blotting in Mel-HO, A-375, and A-375-TS melanoma cells treated with 10 mM L-779,450 and in A-375 and A-375-TS treated with L-779,450±tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Glyceraldehyde dehydrogenase (GAPDH) served as loading control. (c) Percentages of apoptotic cells (sub-G1) were determined in A-375 cells with small interfering RNA (siRNA)-mediated knockdown for Bim (dark bars), as compared with scramble (Scr)-siRNA-transfected controls (light gray). Treatments: 10 mM L-779,450±TRAIL for 24 hours. Means and SDs of representative experiments (triplicate values) are shown. Statistical significance is indicated (*Po0.05), when comparing Bim and scramble siRNA. (Inset) Bim expression is shown by western blotting (24 hours), with GAPDH as loading control. (d) Cell photographs (magnification 1:200) of TRAIL-sensitive A-375 and TRAIL-resistant A-375-TS treated with L-779,450 (10 mM) are shown. (e) Expression of light chain 3-II (LC3-II) was determined by western blotting in A-375 and A-375-TS treated with TRAIL (20 ng ml  1) and/or L-779,450 (10 mM). (a–e) At least two independent experiments revealed comparable results.

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cells for TRAIL-induced apoptosis by L-779,450 was demonstrated here by its siRNA-mediated knockdown. Also, induced autophagy was seen upon L-779,450 treatment. Autophagy mainly represents a survival mechanism, which may enable tumor cells to outlive limiting growth conditions (Kondo et al., 2005; Levine, 2006). Thus, autophagy induced by MAPK inhibition may be seen as a response of the cells to survive the limitation caused by RAF inhibition. Enhanced autophagy in melanoma cells was recently also reported in response to selective BRAF inhibitors (Kim et al., 2012). Autophagy may thus limit an antitumor activity (Gump and Thorburn, 2011), but a possible autophagy-mediated survival was crossed here by apoptosis induction through TRAIL. In conclusion, inhibition of cell proliferation and sensitization for TRAIL-induced apoptosis are suggested here for explaining the antitumor activity of RAF inhibitors in melanoma. As TRAIL is expressed by cytotoxic T cells (Brincks et al., 2011), enhancement of TRAIL sensitivity may further support an antitumor immune response. Moreover, TRAIL receptor agonists, which have been seen in clinical trials, in combination with RAF inhibitors may be considered for melanoma therapy. MATERIALS AND METHODS Cell culture Experiments were performed in TRAIL-sensitive (A-375, Mel-HO, SK-Mel-28, SK-Mel-13, and SK-Mel-147) and in permanent resistant (Mel-2a, MeWo, and SK-Mel-103) human melanoma cell lines (Kurbanov et al., 2005). Mel-HO and A-375 were derived from primary tumors, whereas Mel-2a, SK-Mel-13, MeWo, SK-Mel-28, SK-Mel-147, and SK-Mel-103 were from metastasis. Sublines with acquired TRAIL resistance (Mel-HO-TS and A-375-TS) were derived from selection with 100 ng ml  1 TRAIL (Kurbanov et al., 2007). TRAIL resistance was regularly controlled in parallel to all experiments. Other A-375 subclones had been stably transfected with a pIRES-Bcl-2 plasmid (A-375-Bcl-2) or pIRES mock plasmid (A-375-mock), as described previously (Raisova et al., 2001). Parental BRAF-WT, K-Ras-mutated HCT-116 colon carcinoma cells were derived from ATCC (Maryland, MD). The HCT-116 Bax knockout, Bak knockdown, and Bax/Bak double knockdown cells were kindly provided by B Vogelstein (John Hopkins Cancer Center, Baltimore, MD) and had been described previously (Gillissen et al., 2010). Cells were cultivated at 37 1C, 5% CO2 in DMEM (4.5 g l  1 glucose; Gibco, Invitrogen, Karlsruhe, Germany) with 10% fetal calf serum (FCS) and antibiotics (Biochrom, Berlin, Germany). TRAILselected cells were continuously kept with 5 ng ml  1 TRAIL until 24 hours before treatment. Cells were plated in 6-, 24-, or 96-well plates with 2  105, 5  104, and 5  103 cells, respectively, and treatment started after 24 hours. For induction of apoptosis, TRAIL (Alexis, Gruenberg, Germany; ALX-201-073-C020; 20 ng ml  1), the pan-RAF inhibitor L-779,450 (Merck, Darmstadt, Germany; 0.1–50 mM), the MEK inhibitor U0126 (Sigma-Aldrich, Taufkirchen, Germany; 20 mM), and the selective BRAF(V600E) inhibitor vemurafenib/PLX4032 (Selleck Chemicals, Houston, TX) were used. For caspase inhibition, cells were preincubated for 1 hour with 10 mM of Q-VD-OPh (MP Biomedicals, Solon, OH). 438

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Cell proliferation, apoptosis, and cytotoxicity For continuous monitoring cell growth, the xCELLigence system (Roche Diagnostics, Penzberg, Germany) was applied. Relative cell indices correspond to attached cell numbers. Cell cycle analyses were performed for quantification of apoptosis and cell cycle arrest (Riccardi and Nicoletti, 2006). Cells harvested by trypsinization were stained for 1 hour with propidium iodide (Sigma-Aldrich; 200 mg ml  1), and sub-G1 fractions, corresponding to cells with fragmented DNA, were quantified by flow cytometry (FACS Calibur; BD Bioscience, Bedford, MA). Cytotoxicity was determined according to the release of lactate dehydrogenase by damaged cells into the culture supernatants. Values of non-treated cells were set to 1, and data were calculated as relative values of the non-treated controls (Cytotoxicity detection assay; Roche Diagnostics). Mitochondrial membrane potential was determined with the fluorescent dye tetramethylrhodamine methyl ester perchlorate Sigma-Aldrich; 1 mM (TMRM þ ; Sigma-Aldrich; 1 mM). Cells harvested by trypsinization were stained for 15 min at 37 1C and analyzed by flow cytometry. For identification of chromatin condensation and nuclear fragmentation in course of apoptosis, cells were harvested by trypsinization, centrifuged on cytospins, and fixed for 30 minutes in 4% formaldehyde. Cytospins were stained with bisbenzimide (Hoechst-33258; Sigma-Aldrich; 1 mg ml  1, 30 minutes) and examined by fluorescence microscopy. Apoptotic cells were identified by fragmented nuclei or by bright blue-stained nuclei with condensed chromatin.

Assays for Bax/Bak activation For analysis of Bax and Bak conformational changes related to their activation, primary antibodies specific for the N-terminal domains of Bax or Bak were applied in flow cytometry (Bax-NT, 1:100 (Upstate, Lake Placid, NY; no. 06-499); Bak-NT (Merck, Darmstadt, Germany), AM04, 1:10). The N-terminal domains become accessible in course of Bax/Bak activation (Gillissen et al., 2007; Martinou and Youle, 2011). Cells (105) were harvested by trypsinization and fixed for 30 minutes with 4% paraformaldehyde in phosphate-buffered saline. Cells were then incubated with the respective antibodies for 1 hour at 4 1C in phosphate-buffered saline/1% FCS, which also contained 0.1% saponin for cell permeabilization. Cells were subsequently incubated for 1 hour at 4 1C in the dark with the secondary antibody goat anti-rabbit IgG (H þ L)-FITC (Jackson Immuno Research, West Grove, FL). After washing and resuspension in phosphate-buffered saline/1% FCS, cells were immediately measured by flow cytometry.

Cell transfection with siRNA Transient cell transfection was performed in 6-well plates at 24 hours after seeding (70% confluence). Treatment with L-779,450/TRAIL followed after another 24 hours. Amounts of 20 pmol siRNA and 4 ml TurboFect (Fermentas, St Leon-Rot, Germany) were used per well. The siRNAs for Smac (sc-36505), Bax (sc-29212), Bak (sc-29786), Bim (sc-29802), and the scrambled control (sc-37007) were derived from Santa Cruz Biotechnology (Santa Cruz, CA).

Protein analysis For western blotting, total protein extracts were obtained by cell lysis in 150 mM NaCl, 1 mM EDTA, 2 mM phenylmethyl sulfonyl, 1 mM leupeptin, 1 mM pepstatin, 0.5% SDS, 0.5% NP-40, and 10 mM Tris-HCl (pH 7.5). Cytosolic and mitochondrial cell fractions were prepared by a

A Berger et al. RAF in TRAIL Resistance

kit of PromoKine (Heidelberg, Germany). Western blotting on nitrocellulose membranes was described previously (Eberle et al., 2003). Primary antibodies obtained were as follows: cleaved caspase-3 (3664, rabbit, 1:5,000), caspase-3 proform (9662, rabbit, 1:1,000), caspase-8 (9746, mouse, 1:1,000), caspase-9 (9505, rabbit, 1:1,000), ERK (4695, rabbit, 1:2,000), pERK (9101, rabbit, 1:2,000), and XIAP (2042, rabbit, 1:1,000) from Cell Signaling; Bcl-2 (sc-783, rabbit; 1:200), Bax (sc-493, rabbit, 1:200), Bak (sc-893, rabbit, 1:500), AIF (sc-9416, goat, 1:1,000), Smac (sc-56230, mouse, 1:1,000), Bim (sc11425, rabbit, 1:400), light chain 3 (sc-292354, rabbit, 1:500), Bid (sc-2002, rabbit, 1:1,000), glyceraldehyde dehydrogenase (GAPDH) (sc-3233, mouse, 1:1,000), and survivin (sc-17779, mouse, 1:500) from Santa Cruz Biotechnology. The antibodies used further were: cytochrome c (no. 556433, mouse, 1:1,000; BD Biosciences, Heidelberg, Germany) and VDAC (voltage-dependent anion channels; antiporin 31HL, 529536, mouse, 1:500; Calbiochem, Darmstadt, Germany). The secondary antibodies used were as follows: peroxidaselabeled goat anti-rabbit, goat anti-mouse, and rabbit anti-goat (Dako, Hamburg, Germany; 1:5,000).

Statistical analyses Assays consisted of triplicates, and at least two independent experiments were performed. Mean values and SDs were calculated either by enclosing all individual values of the independent experiments (at least six values) or a representative experiment is shown (triplicate values). Statistical significance was proven by Student’s t-test in case of normal distribution, and P-values of o0.05 were considered as statistically significant. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS The study was supported by the German Cancer Aid (Deutsche Krebshilfe), Melanomverbund, 10-8008, TP7.

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