Faculty Bibliography

BACKGROUND: Patients with high-risk neuroblastoma (NBL) tumors have a high mortality rate. Consequently, there is an urgent need for the development of new treatments for this condition. Targeting death receptor signaling has been proposed as an alternative to standard chemo- and radio-therapies in various tumors. In NBL, this therapeutic strategy has been largely disregarded, possibly because ~50-70% of all human NBLs are characterized by caspase-8 silencing. However, the expression of caspase-8 is detected in a significant group of NBL patients, and they could therefore benefit from treatments that induce cell death through death receptor activation. Given that cytokines, such as TNFalpha, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNFalpha and FasL in NBL. METHODS: For the purpose of the study we used a set of eight NBL cell lines. Here we explore the cell death induced by TNFalpha, FasL, cisplatin, and etoposide, or a combination thereof by Hoechst staining and calcein viability assay. Further assessment of the signaling pathways involved was performed by caspase activity assays and Western blot experiments. Characterization of Fas expression levels was achieved by qRT-PCR, cell surface biotinylation assays, and cytometry. RESULTS: We have found that TNFalpha is able to increase FasL-induced cell death by a mechanism that involves the NF-kappaB-mediated induction of the Fas receptor. Moreover, TNFalpha sensitized NBL cells to DNA-damaging agents (i.e. cisplatin and etoposide) that induce the expression of FasL. Priming to FasL-, cisplatin-, and etoposide-induced cell death could only be achieved in NBLs that display TNFalpha-induced upregulation of Fas. Further analysis denotes that the high degree of heterogeneity between NBLs is also manifested in Fas expression and modulation thereof by TNFalpha. CONCLUSIONS: In summary, our findings reveal that TNFalpha sensitizes NBL cells to FasL-induced cell death by NF-kappaB-mediated upregulation of Fas and unveil a new mechanism through which TNFalpha enhances the efficacy of currently used NBL treatments, cisplatin and etoposide.

The brains of patients with Alzheimer's disease (AD) present elevated levels of tumor necrosis factor-alpha (TNFalpha), a cytokine that has a dual function in neuronal cells. On one hand, TNFalpha can activate neuronal apoptosis, and on the other hand, it can protect these cells against amyloid-beta (Abeta) toxicity. Given the dual behavior of this molecule, there is some controversy regarding its contribution to the pathogenesis of AD. Here we examined the relevance of the long form of Fas apoptotic inhibitory molecule (FAIM) protein, FAIM-L, in regulating the dual function of TNFalpha. We detected that FAIM-L was reduced in the hippocampi of patients with AD. We also observed that the entorhinal and hippocampal cortex of a mouse model of AD (PS1(M146L)xAPP(751sl)) showed a reduction in this protein before the onset of neurodegeneration. Notably, cultured neurons treated with the cortical soluble fractions of these animals showed a decrease in endogenous FAIM-L, an effect that is mimicked by the treatment with Abeta-derived diffusible ligands (ADDLs). The reduction in the expression of FAIM-L is associated with the progression of the neurodegeneration by changing the inflammatory response mediated by TNFalpha in neurons. In this sense, we also demonstrate that the protection afforded by TNFalpha against Abeta toxicity ceases when endogenous FAIM-L is reduced by short hairpin RNA (shRNA) or by treatment with ADDLs. All together, these results support the notion that levels of FAIM-L contribute to determine the protective or deleterious effect of TNFalpha in neuronal cells.

Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for 15% of all pediatric cancer deaths. Several risk factors predict NBL outcome: age at the time of diagnosis, stage, chromosome alterations and MYCN (V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma-Derived Homolog) amplification, which characterizes the subset of the most aggressive NBLs with an overall survival below 30%. MYCN-amplified tumors develop exceptional chemoresistance and metastatic capacity. These properties have been linked to defects in the apoptotic machinery, either by silencing components of the extrinsic apoptotic pathway (e.g. caspase-8) or by overexpression of antiapoptotic regulators (e.g. Bcl-2, Mcl-1 or FLIP). Very little is known on the implication of death receptors and their antagonists in NBL. In this work, the expression levels of several death receptor antagonists were analyzed in multiple human NBL data sets. We report that Lifeguard (LFG/FAIM2 (Fas apoptosis inhibitory molecule 2)/NMP35) is downregulated in the most aggressive and undifferentiated tumors. Intringuingly, although LFG has been initially characterized as an antiapoptotic protein, we have found a new association with NBL differentiation. Moreover, LFG repression resulted in reduced cell adhesion, increased sphere growth and enhanced migration, thus conferring a higher metastatic capacity to NBL cells. Furthermore, LFG expression was found to be directly repressed by MYCN at the transcriptional level. Our data, which support a new functional role for a hitherto undiscovered MYCN target, provide a new link between MYCN overexpression and increased NBL metastatic properties.

Apoptosis is a major mechanism regulating immune tolerance by the elimination of autoreactive T lymphocytes. A failure of activation induced cell-death (AICD) has been described in T lymphocytes from patients with multiple sclerosis (MS). The aims of this study were to evaluate AICD in T lymphocytes from patients with MS and healthy controls, and to explore the molecular mechanisms underlying the deregulation observed in apoptosis induction. PHA-induced AICD was reduced in T lymphocytes from patients with relapsing-remitting MS compared with controls. This finding was associated with a diminished expression of Fas and a failure in caspase 3 activation.

TNFalpha can promote either cell survival or cell death. The activation of NF-kappaB plays a central role in cell survival while its inhibition makes TNFalpha-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-kappaB (super-repressor (SR)-IkappaBalpha) sensitizes HeLa cells towards TNFalpha-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFalpha-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-kappaB. Then, the activation of the NF-kappaB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFalpha-induced cell death, meaning that TNFalpha is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IkappaBalpha-overexpressing cells. This indicates that TNFalpha-induced apoptosis in SR-IkappaBalpha-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFalpha-induced apoptosis after Bcl-xL knockdown despite that NF-kappaB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFalpha-triggered NF-kappaB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-kappaB-dependent highly chemoresistant proliferative malignancies.

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells.

Polypyrimidine tract binding protein (PTB) regulates pre-mRNA splicing, having special relevance for determining gene expression in the differentiating muscle. We have previously shown that PTB protein abundance is progressively reduced during heart development without reduction of its own transcript. Simultaneous reduction of histone deacetylase (HDAC) expression prompted us to investigate the potential link between these events. HDAC5-deficient mice have reduced cardiac PTB protein abundance, and HDAC inhibition in myocytes causes a reduction in endogenous expression of cellular FLICE-like inhibitory protein (cFLIP) and caspase-dependent cleavage of PTB. In agreement with this, cardiac PTB expression is abnormally high in mice with cardiac-specific executioner caspase deficiency, and cFLIP overexpression prevents PTB cleavage in vitro. Caspase-dependent cleavage triggers further fragmentation of PTB, and these fragments accumulate in the presence of proteasome inhibitors. Experimental modification of the above processes in vivo and in vitro results in coherent changes in the alternative splicing of genes encoding tropomyosin-1 (TPM1), tropomyosin-2 (TPM2) and myocyte enhancer factor-2 (MEF2). Thus, we report a pathway connecting HDAC, cFLIP and caspases regulating the progressive disappearance of PTB, which enables the expression of the adult variants of proteins involved in the regulation of contraction and transcription during cardiac muscle development.

Upon activation, tumor necrosis factor alpha (TNF-alpha) receptor can engage apoptotic or survival pathways. Inhibition of macromolecular synthesis is known to sensitize cells to TNF-alpha-induced cell death. It is believed that this sensitization is due to the transcriptional blockade of genes regulated by NF-kappaB. Nevertheless, such evidence has remained elusive in the nervous system. Here, we show that TNF-alpha cannot normally induce apoptosis in PC12 cells or cortical neurons. However, cells treated with Actinomycin D (ActD) become susceptible to TNF-alpha-induced cell death through the activation of caspase-8, generation of tBid and activation of caspase-9 and -3. Analysis of several proteins involved in TNF-alpha receptor signaling showed no significant downregulation of NF-kappaB target genes, such as IAPs or FLIP, under such conditions. However, Bcl-x(L) protein levels, but not those of Bcl-2, Bax and Bak, are reduced by ActD or TNF-alpha/ActD treatments. Moreover, Bcl-x(L) overexpression fully protects cells against TNF-alpha/ActD-induced cell death. When endogenous levels of Bcl-x(L) are specifically downregulated by lentiviral-based RNAi, cells no longer require ActD to be sensitive to TNF-alpha-triggered apoptosis. Furthermore, Bcl-x(L) downregulation does not affect TNF-alpha-mediated NF-kappaB activation. Altogether, our results demonstrate that Bcl-x(L), and not Bcl-2, FLIP or IAPs, acts as the endogenous regulator of neuronal resistance/sensitivity to TNF-alpha-induced apoptosis in an NF-kappaB-independent manner.

Ligation of the cell surface molecule CD44 by anti-CD44 monoclonal antibodies (mAbs) has been shown to induce cell differentiation, cell growth inhibition and in some cases, apoptosis in myeloid leukemic cells. We report, herein, that exposure of human erythroleukemic HEL cells to the anti-CD44 mAb A3D8 resulted in cell growth inhibition followed by caspase-independent apoptosis-like cell death. This process was associated with the disruption of mitochondrial membrane potential (Delta Psi m), the mitochondrial release of apoptosis-inducing factor (AIF), but not of cytochrome c, and the nuclear translocation of AIF. All these effects including cell death, loss of mitochondrial Delta Psi m and AIF release were blocked by pretreatment with the poly (ADP-ribose) polymerase inhibitor isoquinoline. A significant protection against cell death was also observed by using small interfering RNA for AIF. Moreover, we show that calpain protease was activated before the appearance of apoptosis, and that calpain inhibitors or transfection of calpain-siRNA decrease A3D8-induced cell death, and block AIF release. These data suggest that CD44 ligation triggers a novel caspase-independent cell death pathway via calpain-dependent AIF release in erythroleukemic HEL cells.