Faculty Bibliography

The long isoform of Fas apoptosis inhibitory molecule (FAIM-L) is a neuron-specific death receptor antagonist that modulates apoptotic cell death and mechanisms of neuronal plasticity. FAIM-L exerts its antiapoptotic action by binding to X-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases, which are the main effectors of apoptosis. XIAP levels are regulated by the ubiquitin-proteasome pathway. FAIM-L interaction with XIAP prevents the ubiquitination and degradation of the latter, thereby allowing it to inhibit caspase activation. This interaction also modulates non-apoptotic functions of caspases, such as the endocytosis of AMPA receptor (AMPAR) in hippocampal long-term depression (LTD). The molecular mechanism of action exerted by FAIM-L is unclear since the consensus binding motifs are still unknown. Here, we performed a two-hybrid screening to discover novel FAIM-L-interacting proteins. We found a functional interaction of SIVA-1 with FAIM-L. SIVA-1 is a proapoptotic protein that has the capacity to interact with XIAP. We describe how SIVA-1 regulates FAIM-L function by disrupting the interaction of FAIM-L with XIAP, thereby promoting XIAP ubiquitination, caspase-3 activation and neuronal death. Furthermore, we report that SIVA-1 plays a role in receptor internalization in synapses. SIVA-1 is upregulated upon chemical LTD induction, and it modulates AMPAR internalization via non-apoptotic activation of caspases. In summary, our findings uncover SIVA-1 as new functional partner of FAIM-L and demonstrate its role as a regulator of caspase activity in synaptic function.

Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover important tumor biology and/or yield promising therapeutic insights. Here, we investigated the role of circular RNAs (circRNA) in metastasis, using melanoma as a model aggressive tumor. We identified silencing of cerebellar degeneration-related 1 antisense (CDR1as), a regulator of miR-7, as a hallmark of melanoma progression. CDR1as depletion results from epigenetic silencing of LINC00632, its originating long non-coding RNA (lncRNA) and promotes invasion in vitro and metastasis in vivo through a miR-7-independent, IGF2BP3-mediated mechanism. Moreover, CDR1as levels reflect cellular states associated with distinct therapeutic responses. Our study reveals functional, prognostic, and predictive roles for CDR1as and expose circRNAs as key players in metastasis.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

Circular RNAs are a novel class of non-coding RNAs with functions that remain poorly characterized in normal and pathological conditions. CDR1as is a non-canonical circRNA observed to act as a sponge for miR-7 in brain tissues. Analysis of RNA-seq data of melanocytes and melanoma cell lines and short-term cultures revealed loss of CDR1as expression as a hallmark of melanoma cells. We confirmed silencing of CDR1as in melanoma cells and tissues by RT-qPCR using divergent primers. Clinically, we observed CDR1as loss associated with metastatic progression and poor patient outcomes in a cohort of fresh-frozen melanoma tissue samples. Depletion of CDR1as in melanoma cell lines enhanced invasion in vitro and lung metastasis in vivo, demonstrating functional significance of CDR1as silencing. Surprisingly, CDR1as depletion had no clear effect on miR-7 activity in melanoma cells, and miR-7 inhibition was insufficient to rescue CDR1as silencing-induced invasion. Moreover, GSEA analyses of proteomic profiling of melanoma cells depleted of CDR1as revealed reductions of proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial function, suggesting CDR1as loss may alter metabolism of melanoma cells. Mining of CLIP-Seq data sets and subsequent RIP-PCR revealed direct interactions of CDR1as with the IGF2BP family of proteins and TAR

Fas Apoptosis Inhibitory Molecule (FAIM) is an evolutionarily highly conserved death receptor antagonist, widely expressed and known to participate in physiological and pathological processes. Two FAIM transcript variants have been characterized to date, namely FAIM short (FAIM-S) and FAIM long (FAIM-L). FAIM-S is ubiquitously expressed and serves as an anti-apoptotic protein in the immune system. Furthermore, in neurons, this isoform promotes NGF-induced neurite outgrowth through NF-small ka, CyrillicB and ERK signaling. In contrast FAIM-L is found only in neurons, where it exerts anti-apoptotic activity against several stimuli. In addition to these two variants, in silico studies point to the existence of two additional isoforms, neither of which have been characterized to date. In this regard, here we confirm the presence of these two additional FAIM isoforms in human fetal brain, fetal and adult testes, and placenta tissues. We named them FAIM-S_2a and FAIM-L_2a since they have the same sequence as FAIM-S and FAIM-L, but include exon 2a. PCR and western blot revealed that FAIM-S_2a shows ubiquitous expression in all the tissues and cellular models tested, while FAIM-L_2a is expressed exclusively in tissues of the nervous system. In addition, we found that, when overexpressed in non-neuronal cells, the splicing factor nSR100 induces the expression of the neuronal isoforms, thus identifying it as responsible for the generation of FAIM-L and FAIM-L_2a. Functionally, FAIM-S_2a and FAIM-L_2a increased neurite outgrowth in response to NGF stimulation in a neuronal model. This observation thus, supports the notion that these two isoforms are involved in neuronal differentiation. Furthermore, subcellular fractionation experiments revealed that, in contrast to FAIM-S and FAIM-L, FAIM-S_2a and FAIM-L_2a are able to localize to the nucleus, where they may have additional functions. In summary, here we report on two novel FAIM isoforms that may have relevant roles in the physiology and pathology of the nervous system.

Caspases have recently emerged as key regulators of axonal pruning and degeneration and of long-term depression (LTD), a long-lasting form of synaptic plasticity. However, the mechanism underlying these functions remains unclear. In this context, XIAP has been shown to modulate these processes. The neuron-specific form of FAIM protein (FAIM-L) is a death receptor antagonist that stabilizes XIAP protein levels, thus preventing death receptor-induced neuronal apoptosis. Here we show that FAIM-L modulates synaptic transmission, prevents chemical-LTD induction in hippocampal neurons, and thwarts axon degeneration after nerve growth factor (NGF) withdrawal. Additionally, we demonstrate that the participation of FAIM-L in these two processes is dependent on its capacity to stabilize XIAP protein levels. Our data reveal FAIM-L as a regulator of axonal degeneration and synaptic plasticity.

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30-40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.