Superoxide induced inhibition of death receptor signaling is mediated via induced expression of apoptosis inhibitory protein cFLIP.

The death inhibitory proteins, cFLIP and Bcl-2, canonically act at different steps to regulate receptor-mediated apoptosis in cancer cells. Here we report that pharmacological or genetic means to effect an increase in intracellular superoxide result in cFLIP upregulation. Interestingly, Bcl-2 overexpression is associated with a concomitant increase in cFLIP, and reducing superoxide sensitizes Bcl-2 overexpressing cancer cells to receptor-mediated apoptosis via downregulation of cFLIP. Moreover, inhibiting glycolytic flux overcomes apoptosis resistance by superoxide-dependent downregulation of cFLIP. Superoxide-induced upregulation of cFLIP is a function of enhanced transcription, as evidenced by increases in cFLIP promoter activity and mRNA abundance. The positive effect of superoxide on cFLIP is mediated through its reaction with nitric oxide to generate peroxynitrite. Corroborating these findings in cell lines, subjecting primary cells derived from lymphoma patients to glucose deprivation ex vivo, as a means to decrease superoxide, not only reduced cFLIP expression but also significantly enhanced death receptor sensitivity. Based on this novel mechanistic insight into the redox regulation of cancer cell fate, modulation of intracellular superoxide could have potential therapeutic implications in cancers in which these two death inhibitory proteins present a therapeutic challenge.

FLIP: a flop for execution signals.

Resistance to apoptosis is one of the established hallmarks of cancer cells. This is a function of an imbalance between the proteins that facilitate death execution and those that inhibit apoptosis or promote cell proliferation. The anti-apoptotic protein, FLICE inhibitory protein (FLIP), first identified as a viral protein, is over-expressed in a variety of human pathologies. Initial observations linked FLIP expression to inhibition of death receptor induced apoptosis, due to its structural homology to the cysteine protease, caspase-8. FLIP impedes full processing of pro-caspase-8 to its active form and its release to the cytosol, and by doing so blocks apoptotic signaling downstream of the membrane death initiating signaling complex (DISC). Recent observations have highlighted the complex regulation of this protein and its cross talk with diverse signaling networks and metabolic processes. As FLIP expression is directly associated with chemotherapy resistance, a better understanding of its genomic organization, gene transcription, as well as post-transcriptional regulation could yield novel targets with potential therapeutic implications against drug refractory cancers. In this short review, we provide a brief overview of the structural and functional biology of this somewhat complex protein with direct relevance to carcinogenesis.

The 3'-untranslated region length and AU-rich RNA location modulate RNA-protein interaction and translational control of ß2-adrenergic receptor mRNA.

Posttranscriptional controls play a major role in ß(2)-adrenergic receptor (ß(2)-AR) expression. We recently reported that ß(2)-AR mRNA translation is suppressed by elements in its 3'-untranslated region (UTR). We also identified T-cell-restricted intracellular antigen-related protein (TIAR) and HuR as prominent AU-rich (ARE) RNA-binding proteins that associate with ß(2)-AR mRNA 3'-UTR. In this study, we identified a poly(U) region at the distal end of the 3'-UTR as critical for TIAR binding to ß(2)-AR mRNA and for translational suppression. Here, we also report that the locations of the poly(U) and ARE sequences within the 3'-UTR are important determinants that control the translation of ß(2)-AR mRNA. Consistent with this finding, a 20-nucleotide ARE RNA from the proximal 3'-UTR that did not inhibit mRNA translation in its native position was able to suppress translation when re-located to the distal 3'-UTR of the receptor mRNA. Immunoprecipitation and polysome profile analysis demonstrated the importance of 3'-UTR length and the ARE RNA location within the 3'-UTR, as key determinants of RNA/protein interactions and translational control of ß(2)-AR mRNA. Further, the importance of 3'-UTR length and ARE location in TIAR and HuR association with mRNA and translational suppression was demonstrated using a chimeric luciferase reporter gene.

Transcriptional down-regulation of IGFBP-3 in human hepatocellular carcinoma cells is mediated by the binding of TIA-1 to its AT-rich element in the 3'-untranslated region.

Insulin-like growth factor binding protein-3 (IGFBP-3) plays key roles in regulating cell growth, differentiation, and apoptosis in a variety of cellular systems. We have observed significant down-regulation of IGFBP-3 expression in primary human hepatocellular carcinoma (HCC) tissues when compared to adjacent histologically normal tissues. In this study, we functionally mapped the entire 3'-UTR of the IGFBP-3 mRNA, spanning 1471 nt and identified a 210 bp fragment consisting of AT-rich elements at the distal downstream region preceding the consensus pre-mRNA polyadenylation signal that provide high affinity binding for TIA-1 to mediate the specific suppression of IGFBP-3 expression in human HCC cells.

Translational control of beta2-adrenergic receptor mRNA by T-cell-restricted intracellular antigen-related protein.

Cellular expression of the beta(2)-adrenergic receptor (beta(2)-AR) is suppressed at the translational level by 3'-untranslated region (UTR) sequences. To test the possible role of 3'-UTR-binding proteins in translational suppression of beta(2)-AR mRNA, we expressed the full-length 3'-UTR or the adenylate/uridylate-rich (A+U-rich element (ARE)) RNA from the 3'-UTR sequences of beta(2)-AR in cell lines that endogenously express this receptor. Reversal of beta(2)-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of beta(2)-adrenergic receptor expression. Using a 20-nucleotide ARE RNA from the receptor 3'-UTR as an affinity ligand, we purified the proteins that bind to these sequences. T-cell-restricted intracellular antigen-related protein (TIAR) was one of the strongly bound proteins identified by this method. UV-catalyzed cross-linking experiments using in vitro transcribed 3'-UTR RNA and glutathione S-transferase-TIAR demonstrated multiple binding sites for this protein on beta(2)-AR 3'-UTR sequences. The distal 340-nucleotide region of the 3'-UTR was identified as a target RNA motif for TIAR binding by both RNA gel shift analysis and immunoprecipitation experiments. Overexpression of TIAR resulted in suppression of receptor protein synthesis and a significant shift in endogenously expressed beta(2)-AR mRNA toward low molecular weight fractions in sucrose gradient polysome fractionation. Taken together, our results provide the first evidence for translational control of beta(2)-AR mRNA by TIAR.

The 3'-untranslated region of the beta2-adrenergic receptor mRNA regulates receptor synthesis.

beta(2)-Adrenergic receptors (beta(2)-ARs) are low abundance integral membrane proteins that mediate the effects of catecholamines at the cell surface. Post-transcriptional regulation of beta(2)-AR is dependent, in part, on sequences within the 5'- and 3'-untranslated regions (UTRs) of the receptor mRNA. In this work, we demonstrate that 3'-UTR sequences regulate the translation of the receptor mRNA. Deletion of the 3'-UTR sequences resulted in 2-2.5-fold increases in receptor expression. The steadystate levels of beta(2)-AR mRNA did not change significantly in the presence or absence of the 3'-UTR, suggesting that the translation of the receptor mRNA is suppressed by 3'-UTR sequences. Introduction of the receptor 3'-UTR sequences into the 3'-UTR of a heterologous reporter gene (luciferase) resulted in a 70% decrease in reporter gene expression without significant changes in luciferase mRNA levels. Sucrose density gradient fractionation of cytoplasmic extracts from Chinese hamster ovary cells transfected with full-length receptor cDNA demonstrated that the receptor transcripts were distributed between polysomal and non-polysomal fractions. Deletion of 3'-UTR sequences from the receptor cDNA resulted in a clear shift in the distribution of receptor mRNA toward the polysomal fractions, favoring increased translation. The 3'-UTR sequences of the receptor mRNA were sufficient to shift the distribution of luciferase mRNA from predominantly polysomal fractions toward non-polysomal fractions in cells transfected with the chimeric luciferase construct. Taken together, our results provide the first evidence for translational control of beta(2)-AR expression by 3'-UTR sequences. Presumably, this occurs by affecting the receptor mRNA localization.