ProNGF Is a Cell-Type-Specific Mitogen for Adult Hippocampal and for Induced Neural Stem Cells.

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.

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.

ATM kinase sustains breast cancer stem-like cells by promoting ATG4C expression and autophagy.

The efficacy of Ataxia-Telangiectasia Mutated (ATM) kinase signalling inhibition in cancer therapy is tempered by the identification of new emerging functions of ATM, which suggests that the role of this protein in cancer progression is complex. We recently demonstrated that this tumor suppressor gene could act as tumor promoting factor in HER2 (Human Epidermal Growth Factor Receptor 2) positive breast cancer. Herein we put in evidence that ATM expression sustains the proportion of cells with a stem-like phenotype, measured as the capability to form mammospheres, independently of HER2 expression levels. Transcriptomic analyses revealed that, in mammospheres, ATM modulates the expression of cell cycle-, DNA repair- and autophagy-related genes. Among these, the silencing of the autophagic gene, autophagy related 4C cysteine peptidase (ATG4C), impairs mammosphere formation similarly to ATM depletion. Conversely, ATG4C ectopic expression in cells silenced for ATM expression, rescues mammospheres growth. Finally, tumor array analyses, performed using public data, identify a significant correlation between ATM and ATG4C expression levels in all human breast cancer subtypes, except for the basal-like one.Overall, we uncover a new connection between ATM kinase and autophagy regulation in breast cancer. We demonstrate that, in breast cancer cells, ATM and ATG4C are essential drivers of mammosphere formation, suggesting that their targeting may improve current approaches to eradicate breast cancer cells with a stem-like phenotype.

CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response.

The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.

Targeting the MDM2/MDM4 interaction interface as a promising approach for p53 reactivation therapy.

Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function.

proNGF/NGF mixtures induce gene expression changes in PC12 cells that neither singly produces.

BACKGROUND: Growing evidence shows that, in vivo, the precursor of Nerve Growth Factor (NGF), proNGF, displays biological activities different from those of its mature NGF counterpart, mediated by distinct, and somewhat complementary, receptor binding properties. NGF and proNGF induce distinct transcriptional signatures in target cells, highlighting their different bioactivities. In vivo, proNGF and mature NGF coexist. It was proposed that the relative proNGF/NGF ratio is important for their biological outcomes, especially in pathological conditions, since proNGF, the principal form of NGF in Central Nervous System (CNS), is increased in Alzheimer's disease brains. These observations raise a relevant question: does proNGF, in the presence of NGF, influence the NGF transcriptional response and viceversa? In order to understand the specific proNGF effect on NGF activity, depending on the relative proNGF/NGF concentration, we investigated whether proNGF affects the pattern of well-known NGF-regulated mRNAs. RESULTS: To test any influence of proNGF on pure NGF expression fingerprinting, the expression level of a set of candidate genes was analysed by qReal-Time PCR in rat adrenal pheochromocytoma cell line PC12, treated with a mixture of NGF and proNGF recombinant proteins, in different stoichiometric ratios. These candidates were selected amongst a set of genes well-known as being rapidly induced by NGF treatment. We found that, when PC12 cells are treated with proNGF/NGF mixtures, a unique pattern of gene expression, which does not overlap with that deriving from treatment with either proNGF or NGF alone, is induced. The specific effect is also dependent on the stoichiometric composition of the mixture. The proNGF/NGF equimolar mixture seems to partially neutralize the specific effects of the proNGF or NGF individual treatments, showing a weaker overall response, compared to the individual contributions of NGF and proNGF alone. CONCLUSIONS: Using gene expression as a functional read-out, our data demonstrate that the relative availability of NGF and proNGF in vivo might modulate the biological outcome of these ligands.

LMNA knock-down affects differentiation and progression of human neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma. METHODOLOGY/PRINCIPAL FINDINGS: Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.

RACK1 is a ribosome scaffold protein for ß-actin mRNA/ZBP1 complex.

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including ß-actin mRNA. The release of ß-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the ß-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of ß-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the ß-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.

Nerve growth factor regulates axial rotation during early stages of chick embryo development.

Nerve growth factor (NGF) was discovered because of its neurotrophic actions on sympathetic and sensory neurons in the developing chicken embryo. NGF was subsequently found to influence and regulate the function of many neuronal and non neuronal cells in adult organisms. Little is known, however, about the possible actions of NGF during early embryonic stages. However, mRNAs encoding for NGF and its receptors TrkA and p75(NTR) are expressed at very early stages of avian embryo development, before the nervous system is formed. The question, therefore, arises as to what might be the functions of NGF in early chicken embryo development, before its well-established actions on the developing sympathetic and sensory neurons. To investigate possible roles of NGF in the earliest stages of development, stage HH 11-12 chicken embryos were injected with an anti-NGF antibody (mAb αD11) that binds mature NGF with high affinity. Treatment with anti-NGF, but not with a control antibody, led to a dose-dependent inversion of the direction of axial rotation. This effect of altered rotation after anti NGF injection was associated with an increased cell death in somites. Concurrently, a microarray mRNA expression analysis revealed that NGF neutralization affects the expression of genes linked to the regulation of development or cell proliferation. These results reveal a role for NGF in early chicken embryo development and, in particular, in the regulation of somite survival and axial rotation, a crucial developmental process linked to left-right asymmetry specification.

NGF and proNGF regulate functionally distinct mRNAs in PC12 cells: an early gene expression profiling.

The biological activities of NGF and of its precursor proNGF are quite distinct, due to different receptor binding profiles, but little is known about how proNGF regulates gene expression. Whether proNGF is a purely pro-apoptotic molecule and/or simply a "less potent NGF" is still a matter of debate. We performed experiments to address this question, by verifying whether a proNGF specific transcriptional signature, distinct from that of NGF, could be identified. To this aim, we studied gene expression regulation by proNGF and NGF in PC12 cells incubated for 1 and 4 hours with recombinant NGF and proNGF, in its wild-type or in a furin-cleavage resistant form. mRNA expression profiles were analyzed by whole genome microarrays at early time points, in order to identify specific profiles of NGF and proNGF. Clear differences between the mRNA profiles modulated by the three neurotrophin forms were identified. NGF and proNGF modulate remarkably distinct mRNA expression patterns, with the gene expression profile regulated by NGF being significantly more complex than that by proNGF, both in terms of the total number of differentially expressed mRNAs and of the gene families involved. Moreover, while the total number of genes modulated by NGF increases dramatically with time, that by proNGFs is unchanged or reduced. We identified a subset of regulated genes that could be ascribed to a "pure proNGF" signalling, distinct from the "pure NGF" one. We also conclude that the composition of mixed NGF and proNGF samples, when the two proteins coexist, influences the profile of gene expression. Based on this comparison of the gene expression profiles regulated by NGF and its proNGF precursor, we conclude that the two proteins activate largely distinct transcriptional programs and that the ratio of NGF to proNGF in vivo can profoundly influence the pattern of regulated mRNAs.

AIRAP, a new human heat shock gene regulated by heat shock factor 1.

Heat shock factor-1 (HSF1) is the central regulator of heat-induced transcriptional responses leading to rapid expression of molecular chaperones that protect mammalian cells against proteotoxic stress. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat shock genes encoding a variety of heat shock proteins, including HSP70, HSP90, HSP27, and other proteins of the network. Herein we report that the zinc finger AN1-type domain-2a gene, also known as AIRAP, behaves as a canonical heat shock gene, whose expression is temperature-dependent and strictly controlled by HSF1. Transcription is triggered at temperatures above 40 degrees C in different types of human cancer and primary cells, including peripheral blood monocytes. As shown by ChIP analysis, HSF1 is recruited to the AIRAP promoter rapidly after heat treatment, with a kinetics that parallels HSP70 promoter HSF1-recruitment. In transfection experiments HSF1-silencing abolished heat-induced AIRAP promoter-driven transcription, which could be rescued by exogenous Flag-HSF1 expression. The HSF1 binding HSE sequence in the AIRAP promoter critical for heat-induced transcription was identified. Because its expression is induced at febrile temperatures in human cells, AIRAP may represent a new potential component of the protective response during fever in humans.

Anti-cancer activity of plant-produced HPV16 E7 vaccine.

The E7 oncoprotein from Human Papilloma Virus (HPV) is an attractive candidate for anti-cancer vaccine development. In this study, we engineered HPV16 E7 coding sequence (wild type or mutagenized sequence, E7GGG) as fusions to beta-1,3-1,4-glucanase (LicKM) of Clostridium thermocellum and produced in Nicotiana benthamiana plants using a transient expression system. Target antigens were purified and evaluated in mice for their potential as prophylactic and therapeutic vaccine candidates. Both fusion proteins induced E7-specific IgG and cytotoxic T-cell responses and protected mice against challenge with E7-expressing tumor cells. Furthermore, when administered after challenge, these plant-produced antigens prevented tumor development.