Giuliana Papoff

Identification and Characterization of a Ligand-independent Oligomerization Domain in the Extracellular Region of the CD95 Death Receptor

The CD95 death receptor plays an important role in several physiological and pathological apoptotic processes involving in particular the immune system. CD95 ligation leads to clustering of the receptor cytoplasmic “death domains” and recruitment of the zymogen form of caspase-8 to the cell surface. Activation of this protease through self-cleavage, followed by activation of downstream effector caspases, culminates in cleavage of a set of cellular proteins resulting in apoptosis with disassembly of the cell. It is very well known that the extracellular region of the CD95 receptor is required for CD95L interaction and that the death domain is necessary for the induction of the apoptotic signaling. Here, we identified and characterized a novel CD95 ligand- and death domain-independent oligomerization domain mapping to the NH2-terminal extracellular region of the CD95 receptor. In vitro andin vivo studies indicated that this domain, conserved among all soluble CD95 variants, mediates homo-oligomerization of the CD95 receptor and of the soluble CD95 proteins, as well as hetero-oligomerization of the receptor with the soluble variants. These results offer new insight into the mechanism of apoptosis inhibition mediated by the soluble CD95 proteins and suggest a role of the extracellular oligomerization domain in the regulation of the non-signaling state of the CD95 receptor.

Protection of CD95-mediated apoptosis by activation of phosphatidylinositide 3-kinase and protein kinase B

Apoptosis may be triggered, in a variety of tissues, by interaction of the cell surface molecule CD95 with its specific ligand, CD95L. CD95 plays a physiological role in the regulation of the immune response; furthermore, alterations in CD95/CD95L function may contribute to the pathogenesis of a number of human diseases, including cancer, autoimmune diseases and viral infections. Many cells that express CD95, however, are not susceptible to CD95‐mediated apoptosis. It is therefore important to identify the mechanisms that counteract the CD95 apoptotic process that are still poorly understood. Growth factors and lymphokines such as interleukin (IL)‐4 that counteract CD95‐mediated apoptosis may activate phosphatidylinositide 3‐kinase (PI 3‐kinase). We therefore used two different approaches to investigate the role of PI 3‐kinase on CD95‐mediated apoptosis. First we tested the effect of two pharmacological PI 3‐kinase inhibitors, wortmannin and LY294002, on CD95 agonistic antibody‐induced apoptosis in three different cell lines. Second, we co‐expressed in COS7 cells CD95 with constitutively active PI 3‐kinase. Results of both approaches indicate that active PI 3‐kinase effectively protects against CD95‐mediated apoptosis. Furthermore we extended our studies on the CD95 downstream mediator, FADD, and on the PI 3‐kinase downstream mediator, the serine/threonine protein kinase PKB, using the co‐expression approach in COS7 cells. We provide evidence that apoptosis induced by triggering the CD95 cell death receptor is counteracted by PI 3‐kinase activation; moreover, PKB but not p70S6K represents the relevant downstream target of PI 3‐kinase signaling.

The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001–1 μg/ml, and VP16, at concentrations ranging from 0.05–1 μg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.

SOLUBLE FAS/APO-1 SPLICING VARIANTS AND APOPTOSIS

In addition to the full length mRNA activated human peripheral blood mononuclear cells (PBMC) and T cell tumor lines express several alternatively spliced Fas variants. At least five of these code for soluble Fas (CD95) molecules. In vitro studies suggest that these soluble Fas isoforms inhibit apoptosis induced by agonistic antibodies and, more importantly, by the natural Fas ligand in Fas-bearing sensitive cells. Interestingly, this functional property can be assigned to the first 49 aminoacids of the mature protein, the only region shared by the soluble Fas molecules.

Fas Splicing Variants and their Effect on Apoptosis

Higher vertebrates frequently contain multigene families of related ligands and their receptors, often with overlapping specificities. Presumably such an organization allows for greater flexibility in the timing and tissue distribution of these molecules. A further level of complexity is introduced by the fact that variants of the same growth factor or receptor can be encoded as alternative transcripts of the same gene. Such variants may remain membrane associated or may be efficiently secreted.

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.

FADD–calmodulin interaction: A novel player in cell cycle regulation

Analyses of knockout and mutant transgenic mice as well as in vitro studies demonstrated a complex role of FADD in the regulation of cell fate. FADD is involved in death receptor induced apoptosis, cell cycle progression and cell proliferation. In a search for mechanisms that might regulate FADD functions, we identified, upon the screening of a lambda-phage cDNA library, calmodulin (CaM) as a novel FADD interacting protein. CaM is a key mediator of signals by the secondary messenger calcium and it is an essential regulator of cell cycle progression and cell survival. Here, we describe the identification and characterization of two calcium dependent CaM binding sites in the alpha helices 8-9 and 10-11 of FADD. Phosphorylation of human FADD at the C-terminal serine 194, by casein kinase I alpha (CKIalpha), has been shown to regulate FADD-dependent non-apoptotic activities. Remarkably, we showed that both FADD and CaM are CKIalpha substrates and that in synchronized HeLa cells, FADD, CaM and CKIalpha co-localize at the mitotic spindle in metaphase and anaphase. Moreover, complementation experiments in Jurkat FADD-/- T cells indicated that: a) cells expressing FADD mutants in the CaM binding sites are protected from Taxol-induced G2/M cell cycle arrest; b) FADD/CaM interaction is not required for Fas receptor-mediated apoptosis although Fas and CaM might compete for binding to FADD. We suggest that the interplay of FADD, CaM and CKIalpha may have an important role in the regulation of cell fate.

Characterization of epithelial-mesenchymal transition intermediate/hybrid phenotypes associated to resistance to EGFR inhibitors in non-small cell lung cancer cell lines

Increasing evidence points to a key role played by epithelial-mesenchymal transition (EMT) in cancer progression and drug resistance. In this study, we used wet and in silico approaches to investigate whether EMT phenotypes are associated to resistance to target therapy in a non-small cell lung cancer model system harboring activating mutations of the epidermal growth factor receptor. The combination of different analysis techniques allowed us to describe intermediate/hybrid and complete EMT phenotypes respectively in HCC827- and HCC4006-derived drug-resistant human cancer cell lines. Interestingly, intermediate/hybrid EMT phenotypes, a collective cell migration and increased stem-like ability associate to resistance to the epidermal growth factor receptor inhibitor, erlotinib, in HCC827 derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small EMT-related gene list, which may have otherwise been overlooked by standard stand-alone methods for gene expression analysis.

N-Terminal and C-Terminal Domains of Calmodulin Mediate FADD and TRADD Interaction

FADD (Fas–associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1–2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding.

Tis21-gene therapy inhibits medulloblastoma growth in a murine allograft model

Medulloblastoma (MB), the tumor of the cerebellum, is the most frequent brain cancer in childhood and a major cause of pediatric mortality. Based on gene profiling, four MB subgroups have been identified, i.e., Wnt or Sonic Hedgehog (Shh) types, and subgroup 3 or 4. The Shh-type MB has been shown to arise from the cerebellar precursors of granule neurons (GCPs), where a hyperactivation of the Shh pathway leads to their neoplastic transformation. We have previously shown that the gene Tis21 (PC3/Btg2) inhibits the proliferation and promotes the differentiation and migration of GCPs. Moreover, the overexpression or the deletion of Tis21 in Patched1 heterozygous mice, a model of spontaneous Shh-type MB, highly reduces or increases, respectively, the frequency of MB. Here we tested whether Tis21 can inhibit MB allografts. Athymic nude mice were subcutaneously grafted with MB cells explanted from Patched1 heterozygous mice. MB allografts were then injected with adeno-associated viruses either carrying Tis21 (AAV-Tis21) or empty (AAV-CBA). We observed that the treatment with AAV-Tis21 significantly inhibited the growth of tumor nodules, as judged by their volume, and reduced the number of proliferating tumor cells (labeled with Ki67 or BrdU), relative to AAV-CBA-treated control mice. In parallel, AAV-Tis21 increased significantly tumor cells labeled with early and late neural differentiation markers. Overall the results suggest that Tis21-gene therapy slows down MB tumor growth through inhibition of proliferation and enhancement of neural differentiation. These results validate Tis21 as a relevant target for MB therapy.