Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.

Successful treatment of melanoma is still challenging, because metastasis remain chemoresistant and radioresistant. Accordingly, combinational treatments involving death ligands are mandatory. In a recent study from our lab, the majority out of 18 melanoma cell lines remained resistant against treatment with the death ligand TRAIL (tumor necrosis factor related apoptosis inducing ligand). Resistance was shown to be mainly due to incomplete processing of caspase-3 into catalytically inactive p21 by binding of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP). Coirradiation with sub-lethal ultraviolet (UV) B caused depletion of XIAP resulting in synergistic sensitization of all but two melanoma cell lines to TRAIL. We show here the XIAP depletion to essentially require initial caspasemediated cleavage, which promotes proteasomal degradation of XIAP. Utilizing specific caspase inhibitors and small interfering RNA-mediated knockdown, we further identified caspase-3 to be responsible for performing the initial cleavage of XIAP after UVB treatment. Additional evidence suggests an accelerated mitochondrial outer membrane permeabilization in response to co-treatment with TRAIL and UVB, which directs the release of XIAP antagonizing factors including Smac. Distraction of XIAP consequently liberates caspase-3 to autocatalytically process into active p17. Activated caspase-3 cleaves XIAP and further enhances its activation in a positive regulatory feedback loop. The molecular mechanism discovered here appears to have broader implications, because cleavage of XIAP was also shown to accompany cisplatin-induced sensitization of melanoma cells to TRAIL.

Regulatory systems often evolve by duplication of ancestral systems and subsequent specialization of the components of the novel signal transduction systems. In the Gram-positive soil bacterium Bacillus subtilis, four homologous antitermination systems control the expression of genes involved in the metabolism of glucose, sucrose and β-glucosides. Each of these systems is made up of a sensory sugar permease that does also act as phosphotransferase, an antitermination protein, and a RNA switch that is composed of two mutually exclusive structures, a RNA antiterminator (RAT) and a transcriptional terminator. We have studied the contributions of sugar specificity of the permeases, carbon catabolite repression, and protein–RAT recognition for the straightness of the signalling chains. We found that the β-glucoside permease BglP does also have a minor activity in glucose transport. However, this activity is irrelevant under physiological conditions since carbon catabolite repression in the presence of glucose prevents the synthesis of the β-glucoside permease. Reporter gene studies, in vitro RNA–protein interaction analyzes and northern blot transcript analyzes revealed that the interactions between the antiterminator proteins and their RNA targets are the major factor contributing to regulatory specificity. Both structural features in the RATs and individual bases are important specificity determinants. Our study revealed that the specificity of protein–RNA interactions, substrate specificity of the permeases as well as the general mechanism of carbon catabolite repression together allow to keep the signalling chains straight and to avoid excessive cross-talk between the systems.

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