Mcl‐1 targeting strategies unlock the proapoptotic potential of TRAIL in melanoma cells

TNF‐related apoptosis‐inducing ligand (TRAIL) induces apoptosis selectively in cancer cells. For melanoma, the targeting of TRAIL signaling appears highly attractive, due to pronounced TRAIL receptor expression in tumor tissue. However, mechanisms of TRAIL resistance observed in melanoma cells may limit its clinical use. The Bcl‐2 family members are critical regulators of cell‐intrinsic apoptotic pathways. Thus, the antiapoptotic Bcl‐2 protein myeloid cell leukemia 1 (Mcl‐1) is overexpressed in many tumor types and was linked to chemotherapy resistance in melanoma. In this study, we evaluated the involvement of antiapoptotic Bcl‐2 proteins (Bcl‐2, Bcl‐xL, Bcl‐w, Mcl‐1, Bcl‐A1, and Bcl‐B) in TRAIL resistance. They were targeted by small interfering RNA‐mediated silencing in TRAIL‐sensitive (A‐375, Mel‐HO) and in TRAIL‐resistant melanoma cell lines (Mel‐2a, MeWo). This highlighted Mcl‐1 as the most efficient target to overcome TRAIL resistance. In this context, we investigated the effects of Mcl‐1‐targeting microRNAs as well as the Mcl‐1‐selective inhibitor S63845. Both miR‐193b and S63845 resulted in significant enhancement of TRAIL‐induced apoptosis, associated with decreased cell viability. Apoptosis induction was mediated by caspase‐3 processing as well as by Bax and Bak activation, indicating the critical involvement of intrinsic apoptosis pathways. These data may indicate a high relevance of Mcl‐1 targeting also in melanoma therapy. Furthermore, the data may suggest to consider the use of the tumor suppressor miR‐193b as a strategy for countering TRAIL resistance in melanoma.

targeting excessive tumor cell proliferation or targeting induction of apoptosis. Even immune-modulating therapies finally aim at the elimination of cancer cells by the induction of apoptosis. 4,5 Two major paths have been described for apoptosis induction, namely (a) extrinsic pathways through death ligands such as TNF-related apoptosis-inducing ligand (TRAIL) and (b) intrinsic, mitochondrial pathways in course of cellular stress situations, for example, DNA damage. 6,7 TRAIL triggers apoptosis via the two agonistic death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5. 8,9 Upon receptor binding, a membrane-bound, death-inducing signaling complex is formed, where initiator caspase-8 and -10 are activated. 10 In a caspase cascade, effector caspases as caspase-3 are further activated, which cleave a large number of death substrates to finally execute apoptosis' programs. 11 Caspase-3 is negatively regulated through the binding of XIAP (chromosome X-linked inhibitor of apoptosis protein). 12 A particular advantage of TRAIL is selective apoptosis induction in cancer cells, while normal tissue cells are largely spared. 13,14 Although melanoma cells have been shown to constitutively express DR5, this does not guarantee TRAIL sensitivity, as roughly half of melanoma cell lines reveal an intrinsic TRAIL resistance. 15,16 To address this limitation, different combination strategies have been identified, which can sensitize melanoma cells for TRAIL-induced apoptosis. In this context, the control of mitochondrial apoptosis pathways came into particular focus for explaining TRAIL resistance in melanoma. 17,18 Mitochondrial apoptosis pathways are decisively controlled by the family of Bcl-2 proteins, which share one to four distinct Bcl-2 homology (BH) domains. Bcl-2 proteins interact and control each other by heterodimerization. 19 In present models, proapoptotic, multidomain proteins with three BH domains (Bax and Bak) are  20 Activation of Bax and Bak is associated with permeabilization of the outer mitochondrial membrane and the release of mitochondrial proteins, such as cytochrome c and Smac (second mitochondrial activator of caspases). While cytochrome c promotes initiator caspase-9 activation via formation of the apoptosome, Smac inhibits the caspase-3 antagonist XIAP. [21][22][23] Expression of antiapoptotic Bcl-2 proteins provides a basic mechanism to allow survival of normal cells and on the other hand, may prevent apoptosis induction in malignant cells. 24 Mcl-1 appears of particular importance for the survival of different cell lineages during embryonic development, for example, hematopoietic and neuronal cells. 25,26 Its decisive role is also underlined by embryonic lethality of Mcl-1 knockout mice. 27 In recent years, the oncogenic activity of Mcl-1 received particular attention, 28   and MeWo) has been described previously. 35 Cells were cultivated at 37°C, 5% CO 2 in dulbecco's modified eagle's medium (DMEM) (4.5 g/L glucose; Gibco, Invitrogen, Karlsruhe, Germany), supplemented with 10% fetal calf serum (FCS) and antibiotics (Biochrom, Berlin, Germany).
For the different assays, melanoma cells were seeded in flatbottom 24-well plates at a density of 75.000 to 120.000 cells/well, according to the growth performance of the cell lines. Single treatments started at 48 hours after seeding (cell confluence at 50%-70%). Abmole Bioscience Inc, Houston, TX) were usually applied for 24 hours, when not stated differently.

| Western blotting
The activated pathways were generally based on the activation of Bax and Bak, underlining their particular roles in TRAIL-induced apoptosis in melanoma cells.

| DISCUSSION
Despite the development of new therapeutic approaches for metastatic melanoma, survival prognosis is still limited, particularly due to acquired therapy resistance. 3 New ideas and additional strategies may help to finally defeat this deadly disease. Apoptosis resistance represents a critical hallmark in cancer, 4 and the targeting of apoptosis pathways, for example, by the death ligand TRAIL, appears as a promising antitumor strategy. 18 The particular advantage of TRAIL is based on its capability to selectively induce apoptosis in cancer cells, while normal cells are largely spared. 13,14 TRAIL agonists have proven good tolerability and safety profiles in clinical trials, however, an additional clinical benefit, when TRAIL was applied in combination therapies, so far remained on a low level. Multiple strategies have been tested in melanoma cells for overcoming TRAIL resistance. 17 These have revealed an insufficient caspase cascade via caspase-8/caspase-3 as well as the requirement of the mitochondrial amplification loop. The level of Bcl-2 proteins thus represents a highly critical step in the control of TRAIL sensitivity. 17,41,49,50 Upregulation of antiapoptotic Bcl-2 proteins is a frequent issue in cancer, which was also associated with TRAIL resistance, for example, in pancreatic carcinoma and prostate cancer cells. [51][52][53] TRAIL-induced apoptosis in melanoma cells was particularly correlated with Bax activation and was abrogated by Bcl-2 overexpression. 17,54 Thus, the targeting of antiapoptotic Bcl-2 proteins represents a promising strategy to sensitize melanoma cells for TRAIL. This approach was further investigated here.
F I G U R E 2 Sensitization for TRAIL-induced apoptosis by Bcl-2 protein knockdown. Effects of Bcl-2 protein knockdown by siRNA on TRAIL-induced apoptosis were determined in TRAIL-sensitive cell lines A-375 and Mel-HO as well as in the TRAIL-resistant cell lines MeWo and Mel-2a. Assays were performed at 72 hours after the transfection of indicated siRNAs and at 24 hours after TRAIL treatment (100 ng/mL). Apoptosis was determined by PI staining and flow cytometry (cell cycle analysis). Cell cycle phases (G1, G2, and the S-phase) are indicated in overlays given below; apoptotic cells correspond to weakly PI-stained cells (sub-G1 cells). Indicated mean values and SDs correspond to all individual values of at least two independent experiments (each one with triplicates, at least six independent values). Statistical significance is indicated for the comparison of the combinations vs TRAIL treatment alone (*P < .05, ANOVA, two-way, multiple comparisons). ANOVA, analysis of variance; SD, standard deviation; siRNA, small interfering RNA; TRAIL, TNF-related apoptosis-inducing ligand The particular roles of six antiapoptotic Bcl-2 proteins (Bcl-2, Bcl-x L , Mcl-1, Bcl-w, Bcl-A1, Bcl-B) have been acknowledged in models explaining the mutual regulation of Bcl-2 proteins. 19  Whereas miR-339-3p was less effective in combination with TRAIL, the combination of miR-193b and TRAIL resulted in significant loss of cell viability and increased apoptosis. In parallel with the siMcl-1 approach, strong Bax and Bak activation was observed, indicating the activation of intrinsic apoptotic pathways.
In conclusion, tumor cells can be targeted by direct apoptosis agonists, for example, by TRAIL, as well as by inhibition of F I G U R E 5 Use of miRNAs and S63845 to overcome TRAIL resistance. A, MeWo cells were treated with 5 µM S63845 and TRAIL (100 ng/mL, light grey bars, following at 48 hours by determination of apoptosis (PI assay) and cell survival (calcein assay). Bax and Bak activation was quantified by NT antibodies at 12 hours posttreatment. B-E, MeWo cells were transfected with scrambled siRNA (OffT), siMcl-1, miR-193b, miR-339-3p, or were nontransfected (Ctr). In addition, they were treated with TRAIL at 48 hours posttransfection (100 ng/mL, light grey bars). TRAIL treatment was for 48 hours for determination of apoptosis (B) and cell survival (C), whereas for Bax (D) and Bak activation assays (E), TRAIL treatment was for only 12 hours. A-E, At least two independent experiments were performed, each one with at least duplicates; data are expressed as means ±SDs of all individual values (n = 4). Statistical significance of singletreated cells (w/o TRAIL, dark grey bars) is indicated vs nontreated controls, whereas the statistical significance of combination treatments (TRAIL + , light grey bars) is indicated vs TRAIL treatment alone (*P < .05). SD, standard deviation; siRNA, small interfering RNA; TRAIL, TNF-related apoptosis-inducing ligand SARIF ET AL. Of course, 2D cell cultures represent only a highly simplified model of cancer. The situation may improve when more complex models as 3D cultures or animals are used. Indeed, we have shown previously the antitumor effects of TRAIL in melanoma nude mouse models, when TRAIL was expressed by a replication-competent adenoviral vector. 68 As other drugs, BH3 mimetics may reveal varying efficacy in different tumor models as 2D, 3D, and in vivo, as previously shown. 56 Nevertheless, BH3 mimetics are presently promising enough for their clinical testing in patients with different tumors. [60][61][62] Thus, it appears conceivable that the good combination effects we saw here in a basic melanoma model may be finally translated into a clinical situation. Thus, after possibly further testing the particular effectors identified here in animal models, pharmacological use of TRAIL and S63845 or gene therapeutic use of miR-193b may be considered for melanoma therapy.