Generation of c-FLIPshortshRNA was previously described.33Lentiviral vectors were co-transfected with the envelope vector pMD2.G (Addgene no. cell lines nevertheless exhibited that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIPLto be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIPLisoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours. Keywords:apoptosis, bladder cancer, cancer therapy resistance, death receptor Apoptosis is usually a controlled form of cell death, which is essential for normal development and the maintenance of tissue homeostasis in multicellular organisms. Deregulation of apoptosis disturbs the balance between the proliferation and death of cells, with too much proliferation leading to tumour formation and cancer.1Upregulation of anti-apoptotic cellular FLICE inhibitory proteins (c-FLIPs) have been demonstrated in different types of cancer.2Several mRNA isoforms of c-FLIP have been reported, from which three protein isoforms are expressed, namely c-FLIPlong(c-FLIPL), c-FLIPshort(c-FLIPS) and c-FLIPRaji(c-FLIPR).3,4,5c-FLIPLresembles procaspase-8 with two death effector domains (DEDs) and a catalytically inactive caspase-like domain name. The two shorter isoforms, c-FLIPSand c-FLIPR, mainly consist of the DEDs with a truncated C-terminal end. Interestingly, a functional single nucleotide polymorphism (SNP) regulates whether c-FLIPSor FLIPRis produced in humans.6The FLIP proteins can modulate apoptosis in close proximity to death receptors such as CD95 (Fas/Apo-1), tumour necrosis factor (TNF) receptor-1 (TNF-R1), TNF-related apoptosis inducing ligand (TRAIL) receptor-1 (TRAIL-R1; DR4) and TRAIL-R2 (DR5).7The so called death-inducing signalling complex (DISC) is formed upon triggering of death receptors and thereby activation of their intracellular domains, which allows GSK1265744 (GSK744) Sodium salt recruitment of an adaptor protein such as FADD (Fas-associated death domain-containing protein), which, in turn, can recruit the DED-containing proteins procaspase-8, procaspase-10 and the different c-FLIP isoforms.7The initiator caspases 8 and 10 become activated upon dimerisation and proteolytic cleavage at the DISC, initiating a signalling cascade resulting in apoptosis.8,9,10The DEDs of c-FLIP allow recruitment to the DISC,11where cleavage of initiator caspases can be prevented and caspase activation is consequently inhibited.3,5,12Although the short isoforms c-FLIPSand c-FLIPRhave been shown to solely possess anti-apoptotic functions,5,11,12the role of c-FLIPLhas been more controversially discussed.3,13c-FLIPLwas shown to exert either proapoptotic or anti-apoptotic functions depending on its expression level14,15and on the strength of CD95 receptor stimulation.16Moderate expression of c-FLIPLwas shown to promote apoptosis only upon strong receptor stimulation or in combination with overexpression of c-FLIPSor c-FLIPR. In contrast, high levels of c-FLIPLdiminished the sensitivity for CD95-induced apoptosis and caspase-8 activation.16 As numerous reports have demonstrated upregulation of c-FLIP in various cancers, prominently Hodgkin’s lymphoma17,18and melanoma,4,19may allow tumours avoiding endogenous mechanisms inducing apoptosis and especially escaping immune surveillance.2,20,21Moreover, upregulation of c-FLIP has been suggested as a possible mechanism for resistance to therapeutic triggering of death receptors and the extrinsic apoptotic pathway, which is studied as a promising approach to malignancy therapy.22In particular, the death receptor ligand TRAIL selectively induces apoptosis in various tumour cellsin vivowithout affecting cells in normal tissues.23,24However, more recent studies described resistance against TRAIL-induced apoptosis in many primary tumour cells.25 CD95 and TRAIL have also been implicated in the pathogenesis and response to therapy in bladder cancer.25,26In 2010, cancer of the urinary bladder was the fourth most common cancer in men in the United States as well as in the European Union Rabbit Polyclonal to PKC delta (phospho-Tyr313) (EU) and more than 90% of the cases were of the urothelial carcinoma histological subtype. Bladder cancer is primarily treated by surgery. Immunotherapy by BCG is commonly used to prevent recurrences and is thought to be mediated partly by effects of GSK1265744 (GSK744) Sodium salt neutrophil-derived TRAIL on residual tumour cells.27Cisplatin-based chemotherapy is used for the treatment of advanced stage cases, but is only moderately efficacious. In 2008, almost 30 000 patients died of bladder cancer in the EU. Because of the high morbidity and mortality of bladder cancers, there is an urgent need for improved treatment strategies and in particular, for understanding the mechanisms underlying resistance to immunotherapy and chemotherapy. The expression and function of c-FLIP in urothelial cancer are of obvious interest in that context, but few studies are available to date. One immunohistochemical study described an association of strong c-FLIP expression with GSK1265744 (GSK744) Sodium salt tumour progression in bladder cancer, but curiously a lack of expression in normal urothelium.28As many cancers retained CD95 expression, the authors suggested that c-FLIP might contribute to resistance against CD95-induced apoptosis. However, no functional experiments were performed. In contrast, another study provided evidence that c-FLIPLmight contribute to TRAIL resistance of some urothelial carcinoma cell lines.29Unfortunately, these studies have not been followed.