Differential Regulation of c-FLIP Isoforms Through Post-translational Modifications

Cells are constantly responding to signals from the surrounding tissues and the environment. To dispose of infected and potentially dangerous cells, to ensure the optimal execution of developmental processes and to maintain tissue homeostasis, a multicellular organism needs to tightly control both the number and the quality of its cells. Apoptosis is a form of active cellular self-destruction that enables an organism to regulate its cell number by deleting damaged or potentially dangerous cells. Apoptosis can be induced by death ligands, which bind to death receptors on the cell surface. Ligation of the receptors leads to the formation of an intracellular death inducing signaling complex (DISC). One of the DISC components is caspase-8, a protease that triggers the caspase cascade and is thereby a key initiator of programmed cell death. The activation of caspase-8 is controlled by the cellular FLICE-inhibitory proteins (c-FLIPs). Consequently, sensitivity towards receptor-mediated apoptosis is determined by the amount of c-FLIP, and the c-FLIP levels are actively regulated for example during erythroid differentiation of K562 erythroleukemia cells and by hyperthermia in Jurkat leukemia cells. The aim of my thesis was to investigate how c-FLIP is regulated during these processes. We found that c-FLIP isoforms are short-lived proteins, although c-FLIPS had an even shorter half-life than c-FLIPL. In both experimental models, increased death receptor sensitivity correlated with induced ubiquitylation and consequent proteasomal degradation of c-FLIP. Furthermore, we elucidated how phosphorylation regulates the biological functions and the turnover of c-FLIP, thereby contributing to death receptor sensitivity. We mapped the first phosphorylation sites on c-FLIP and dissected how their phosphorylation affects c-FLIP. Moreover, we demonstrated that phosphorylation of serine 193, a phosphorylated residue common to all c-FLIPs, is primarily mediated by the classical PKC. Furthermore, we discovered a novel connection between the phosphorylation and ubiquitylation of c-FLIP: phosphorylation of S193 protects c-FLIP from ubiquitylation. Surprisingly, although all c-FLIP isoforms are phosphorylated on this conserved residue, the biological outcome is different for the long and short isoforms, since S193 specifically prolongs the half-lives of the short c-FLIP isoforms, but not c-FLIPL. To summarize, we show that c-FLIP proteins are modified by ubiquitylation and phosphorylation, and that the biological outcomes of these modifications are isoform-specifically determined.Siirretty Doriast

NFAT promotes carcinoma invasive migration through glypican-6

Invasive migration of carcinoma cells is a prerequisite for the metastatic dissemination of solid tumours. Numerous mechanisms control the ability of cancer cells to acquire a motile and invasive phenotype, and subsequently degrade and invade the basement membrane. Several genes that are up-regulated in breast carcinoma are responsible for mediating the metastatic cascade. Recent studies have revealed that the NFAT (nuclear factor of activated T-cells) is a transcription factor that is highly expressed in aggressive breast cancer cells and tissues, and mediates invasion through transcriptional induction of pro-invasion and migration genes. In the present paper we demonstrate that NFAT promotes breast carcinoma invasion through induction of GPC (glypican) 6, a cell-surface glycoprotein. NFAT transcriptionally regulates GPC6 induction in breast cancer cells and binds to three regulatory elements in the GPC6 proximal promoter. Expression of GPC6 in response to NFAT signalling promotes invasive migration, whereas GPC6 silencing with shRNA (small-hairpin RNA) potently blocks this phenotype. The mechanism by which GPC6 promotes invasive migration involves inhibition of canonical β-catenin and Wnt signalling, and up-regulation of non-canonical Wnt5A signalling leading to the activation of JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Thus GPC6 is a novel NFAT target gene in breast cancer cells that promotes invasive migration through Wnt5A signalling

Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells

<p>Abstract</p> <p>Background</p> <p>cis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells.</p> <p>Methods</p> <p>5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone-associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Student's t-test (p < 0.05).</p> <p>Results</p> <p>Here we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases.</p> <p>Conclusions</p> <p>Our studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma.</p

Tumor endothelial cell up-regulation of IDO1 is an immunosuppressive feed-back mechanism that reduces the response to CD40-stimulating immunotherapy

CD40-stimulating immunotherapy can elicit potent anti-tumor responses by activating dendritic cells and enhancing T-cell priming. Tumor vessels orchestrate T-cell recruitment during immune response, but the effect of CD40-stimulating immunotherapy on tumor endothelial cells has not been evaluated. Here, we have investigated how tumor endothelial cells transcriptionally respond to CD40-stimulating immunotherapy by isolating tumor endothelial cells from agonistic CD40 mAb- or isotype-treated mice bearing B16-F10 melanoma, and performing RNA-sequencing. Gene set enrichment analysis revealed that agonistic CD40 mAb therapy increased interferon (IFN)-related responses in tumor endothelial cells, including up-regulation of the immunosuppressive enzyme Indoleamine 2, 3-Dioxygenase 1 (IDO1). IDO1 was predominantly expressed in endothelial cells within the tumor microenvironment, and its expression in tumor endothelium was positively correlated to T-cell infiltration and to increased intratumoral expression of IFN gamma. In vitro, endothelial cells up-regulated IDO1 in response to T-cell-derived IFN gamma, but not in response to CD40-stimulation. Combining agonistic CD40 mAb therapy with the IDO1 inhibitor epacadostat delayed tumor growth in B16-F10 melanoma, associated with increased activation of tumor-infiltrating T-cells. Hereby, we show that the tumor endothelial cells up-regulate IDO1 upon CD40-stimulating immunotherapy in response to increased IFN gamma-secretion by T-cells, revealing a novel immunosuppressive feedback mechanism whereby tumor vessels limit T-cell activation