Supplementary Table S7 from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations
In the last several years, accumulating evidence indicates that noncoding RNAs, especially long-noncoding RNAs (lncRNAs) and microRNAs, play essential roles in regulating angiogenesis. However, the contribution of lncRNA-mediated competing-endogenous RNA (ceRNA) activity in the control of capillary sprouting from the pre-existing ones has not been described so far. Here, by exploiting the transcriptomic profile of VEGF-A-activated endothelial cells in a consolidate three-dimensional culture system, we identified a list of lncRNAs whose expression was modified during the sprouting process. By crossing the lncRNAs with a higher expression level and the highest fold change value between unstimulated and VEGF-A-stimulated endothelial cells, we identified the unknown LINC02802 as the best candidate to take part in sprouting regulation. LINC02802 was upregulated after VEGF-A stimulation and its knockdown resulted in a significant reduction in sprouting activity. Mechanistically, we demonstrated that LINC02802 acts as a ceRNA in the post-transcriptional regulation of Mastermind-like-3 (MAML3) gene expression through a competitive binding with miR-486-5p. Taken together, these results suggest that LINC02802 plays a critical role in preventing the miR-486-5p anti-angiogenic effect and that this inhibitory effect results from the reduction in MAML3 expression.
Actinobacteria are potential producers of various secondary metabolites with diverse bioactivities. Streptomyces are one of the largest and valuable resource of bioactive and complex secondary metabolites, many of which have important clinical applications. Recent advancements in high-throughput sequencing technologies have made possible the mapping of Streptomyces genome which helps to elucidate the dynamic changes in gene expression in response to cellular status at both transcriptional and translation levels. In this chapter, we will provide a background on the approaches of the transcriptomic assembly along with the development of tools and algorithms that can be used for building prokaryotic transcriptomes.
Common fragile sites (cfs) are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Several investigations support the view that common fragile sites play a role in carcinogenesis. We discuss a genome-wide approach based on graph theory and Gene Ontology vocabulary for the functional characterization of common fragile sites and for the identification of genes that contribute to tumour cell biology. Common fragile sites were assembled in a network based on a simple measure of correlation among common fragile site patterns of expression. By applying robust measurements to capture in quantitative terms the non triviality of the network, we identified several topological features clearly indicating departure from the Erdos-Renyi random graph model. The most important outcome was the presence of an unexpected large connected component far below the percolation threshold. Most of the best characterized common fragile sites belonged to this connected component. By filtering this connected component with Gene Ontology, statistically significant shared functional features were detected. Common fragile sites were found to be enriched for genes associated to the immune response and to mechanisms involved in tumour progression such as extracellular space remodeling and angiogenesis. Moreover we showed how the internal organization of the graph in communities and even in very simple subgraphs can be a starting point for the identification of new factors of instability at common fragile sites. We developed a computational method addressing the fundamental issue of studying the functional content of common fragile sites. Our analysis integrated two different approaches. First, data on common fragile site expression were analyzed in a complex networks framework. Second, outcomes of the network statistical description served as sources for the functional annotation of genes at common fragile sites by means of the Gene Ontology vocabulary. Our results support the hypothesis that fragile sites serve a function; we propose that fragility is linked to a coordinated regulation of fragile genes expression.
# 17 LRP5 transcription and activation of the canonical Wnt signalling are protective signals in the myocardium after infarction {#article-title-2}
Background: LDL receptor-related protein 5 (LRP5) triggers the canonical Wnt pathway which participates in cell function regulation, including lipoprotein metabolism, macrophage mobility and phagocytosis, but its function in the heart is unknown.
Purpose: The aim of this study was to investigate LRP5 and the canonical Wnt signalling pathway in myocardial injury after acute-myocardial infarction (MI).
Methods: MI was induced in WT and LRP5-/- mice by coronary ligation. Infarct size, LRP5 and Wnt signalling proteins were measured. LRP5 and the different metabolic pathways involved in myocardial damage post-MI were analyzed in isolated cardiomyocytes, myofibroblasts and endothelial cells.
Results: LRP5-/- mice have significantly larger infarcts than WT mice (20.8 vs 9.9 p<0.5) suggesting a protective role of LRP5/Wnt in injured myocardium. Furthermore, administration of a GSK3 inhibitor that activates the Wnt pathway downstream LRP5, induced smaller infarcts in LRP5-/- mice indicating that an active Wnt pathway plays a protective role in the myocardium. Hypoxia induced LRP5 overexpression in isolated cardiomyocytes and endothelial cells indicating that a defensive and protective expression of LRP5 is triggered in both cell types. Induction of MI in WT and in LRP5-/- hypercholesterolemic animals, common risk factor in patients with ACS, induced larger infarcts in both genotypes. In isolated cardiomyocytes, LDL induced LRP5 overexpression and Wnt pathway activation whereas LRP5-silencing blocked the pathway.
Conclusions: LRP5 and the canonical Wnt pathway activation is a defensive pro-survival process triggered to protect the ischemic myocardium against different injury triggers, such as hypoxia and hypercholesterolemia to favour and restore cell viability.
# 18 FGF10 is required to promote cardiomyocyte proliferation after myocardial infarction {#article-title-3}
In mammals, cardiomyocyte proliferation decreases after birth resulting in severely limited regenerative capacities in the adult heart. Understanding the developmental processes controlling cardiomyocyte proliferation may thus identify new therapeutic targets to modulate the cell cycle activity of cardiomyocytes in the adult heart. We recently identified FGF10 as a regulator of regional cardiomyocyte-autonomous proliferation in the fetal heart and showed that overexpression of Fgf10 promotes cell cycle reentry of adult cardiomyocytes. These results identify FGF10 as a potential clinically relevant target for promoting adult cardiomyocyte cell cycle reentry after cardiac injury. Using an experimental mouse model of myocardial infarction (MI), together with Fgf10 loss of function mouse models, we investigated the role of FGF10 in pathological conditions. We first demonstrated that myocardial infarction leads to increased Fgf10 expression levels in the injured ventricle. Using an Fgf10-LacZ enhancer trap mouse line, we showed that Fgf10 is upregulated in cardiomyocytes, suggesting a role for FGF10 in pathological conditions. In order to investigate a potential protective role of FGF10 under pathological conditions, adult transgenic mice with reduced Fgf10 expression were subjected to myocardial infarction. Three weeks after myocardial infarction, cardiomyocyte hypertrophy, cell proliferation and heart function were evaluated. Immunofluorescence experiments revealed that while altered Fgf10 expression as no impact on cardiomyocyte hypertrophy, it significantly decreases post-MI cardiomyocyte proliferation. In addition, preliminary echocardiography experiments indicate that post-MI cardiac function is further impaired when Fgf10 levels are reduced. Together these results suggest that under pathological conditions FGF10 may play a protective role by promoting cardiomyocyte proliferation. FGF10 is thus as a potential target to improve the limited innate regenerative capacities of the myocardium after injury.
# 19 A new role for transcription factor EB (TFEB) in mouse epicardial development {#article-title-4}
Epicardium is the source of smooth muscle cells of coronary vessels (vSMCs) and intracardiac fibroblasts. Several pathways promoting epithelial to mesenchymal transition (EMT) of epicardial cells and their differentiation in vSMCs and fibrobalsts have been identified, but little is known about the factors limiting these processes. A member of MiTF-TFE family of transcription factors, TFEB is now extensively being studied due to its ability to increase lysosomal biogenesis and autophagy in response to the lack of nutrients. Although other MiTF family proteins are known to regulate differentiation of several lineages, TFEB involvement in embryo development has not yet been studied.
Our goal was to investigate the role of TFEB in mouse embryo development. Once we discovered Tfeb expression in epicardium, we aimed to understand its function during epicardial development.
To study Tfeb expression in the embryo, we used a transgenic mouse that expresses fusion protein TFEB-GFP. To evaluate the role of TFEB in epicardial development, we generated mice with epicardium-specific TFEB overexpression (Gata5-Cre+:Tfeb-3xflag) and knock-out (Gata5-Cre+:Tfeb flox/flox). Primary epicardial cells were cultured without or in the presence of vSMC differentiation-stimulating factor BMP4. Epicardial mesothelial cell line EMC was used for Tfeb silencing and overexpression experiments. Acquisition of myofibroblast (MF) phenotype was defined by the increased expression of vSMC (PDGFRβ, α-SMA, transgelin) and fibroblast (PDGFRα, fibronectin) markers evaluated by immunostaining, Real-Time PCR and Western blot and increased cell migration capacity measured in wound healing assay. ChIP-Seq analysis was performed in EMCs overexpressing constitutively active TFEB.
Tfeb is expressed in mouse epicardium at 11.5 and 13.5 dpc and is not detectable after 15.5 dpc. TFEB is present in epicardial cells, but not in epicardially derived cells (EPDCs), vSMCs and fibroblasts. The prolonged epicardial overexpression of Tfeb leads to embryonic lethality at dpc 15.5. Epicardium of transgenic embryos develops normally, but differentiation and migration of EDPCs are severely inhibited. Tfeb-overexpressing epicardiocytes proliferate in culture but fail to differentiate in vSMCs under BMP4 stimulation. Mice with epicardium-specific deletion of TFEB are vital. Mutant epicardiocytes in culture easily transform in MFs without BMP4 stimulus, when wild type cells maintain epithelial phenotype. Accordingly, in EMCs Tfeb silencing promotes transformation to MFs already in basic conditions, while Tfeb overexpression inhibits MF differentiation induced by TGFβ. ChIP-Seq analysis revealed about 2000 TFEB targets, among these functional groups of TGFβ and Notch pathways components are enriched.
Our results suggest a new role for Tfeb in restriction of epicardial EMT. More should be done to individuate TFEB targets crucial for this process and understand how Tfeb expression in epicardium is regulated.
Common fragile sites (cfs) are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Several investigations support the view that common fragile sites play a role in carcinogenesis. We discuss a genome-wide approach based on graph theory and Gene Ontology vocabulary for the functional characterization of common fragile sites and for the identification of genes that contribute to tumour cell biology. CFS were assembled in a network based on a simple measure of correlation among common fragile site patterns of expression. By applying robust measurements to capture in quantitative terms the non triviality of the network, we identified several topological features clearly indicating departure from the Erdos-Renyi random graph model. The most important outcome was the presence of an unexpected large connected component far below the percolation threshold. Most of the best characterized common fragile sites belonged to this connected component. By filtering this connected component with Gene Ontology, statistically significant shared functional features were detected. Common fragile sites were found to be enriched for genes associated to the immune response and to mechanisms involved in tumour progression such as extracellular space remodeling and angiogenesis. Our results support the hypothesis that fragile sites serve a function; we propose that fragility is linked to a coordinated regulation of fragile genes expression.
Supplementary Table S5 from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations
Background: MicroRNAs, post-transcriptional repressors of gene expression, play a pivotal role in gene regulatory networks. They are involved in core cellular processes and their dysregulation is associated to a broad range of human diseases. This paper focus on a minimal microRNA-mediated regulatory circuit, in which a protein-coding gene (host gene) is targeted by a microRNA located inside one of its introns. Results: Autoregulation via intronic microRNAs is widespread in the human regulatory network, as confirmed by our bioinformatic analysis, and can perform several regulatory tasks despite its simple topology. Our analysis, based on analytical calculations and simulations, indicates that this circuitry alters the dynamics of the host gene expression, can induce complex responses implementing adaptation and Weber's law, and efficiently filters fluctuations propagating from the upstream network to the host gene. A fine-tuning of the circuit parameters can optimize each of these functions. Interestingly, they are all related to gene expression homeostasis, in agreement with the increasing evidence suggesting a role of microRNA regulation in conferring robustness to biological processes. In addition to model analysis, we present a list of bioinformatically predicted candidate circuits in human for future experimental tests. Conclusions: The results presented here suggest a potentially relevant functional role for negative self-regulation via intronic microRNAs, in particular as a homeostatic control mechanism of gene expression. Moreover, the map of circuit functions in terms of experimentally measurable parameters, resulting from our analysis, can be a useful guideline for possible applications in synthetic biology.
The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients. Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use. Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells. Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level. Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution. At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance. However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution. Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process. However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis. Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.
'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='matrix(1 0 0 -1 0 549)'/%3e%3c/svg%3e)