RESUMEN
MYCN amplification (MNA) and disruption of tumor suppressor microRNA (TSmiR) function are key drivers of poor outcomes in neuroblastoma (NB). While MYCN and TSmiRs regulate glucose metabolism, their role in de novo fatty acid synthesis (FAS) and unsaturated FAS (UFAS) remains poorly understood. Here, we show that FAS and UFAS (U/FAS) genes FASN, ELOVL6, SCD, FADS2, and FADS1 are upregulated in high-risk (HR) NB and that their expression is associated with lower overall survival. RNA-Seq analysis of human NB cell lines revealed parallel U/FAS gene expression patterns. Consistent with this, we found that NB-related TSmiRs were predicted to target these genes extensively. We further observed that both MYC and MYCN upregulated U/FAS pathway genes while suppressing TSmiR host gene expression, suggesting a possible U/FAS regulatory network between MYCN and TSmiRs in NB. NB cells are high in de novo synthesized omega 9 (ω9) unsaturated fatty acids and low in both ω6 and ω3, suggesting a means for NB to limit cell-autonomous immune stimulation and reactive oxygen species (ROS)-driven apoptosis from ω6 and ω3 unsaturated fatty acid derivatives, respectively. We propose a model in which MYCN and TSmiRs regulate U/FAS and play an important role in NB pathology, with implications for other MYC family-driven cancers.
RESUMEN
Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (> 200 reprogrammed iPSC lines) using this protocol.
RESUMEN
A large number of Epstein Barr virus (EBV) immortalized lymphoblastoid cell lines (LCLs) have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into induced pluripotent stem cells (iPSCs) has paved the way to generate more relevant in vitro disease models using this existing bioresource. However, the latent EBV infection in the LCLs make them a unique cell type by altering expression of many cellular genes and miRNAs. These EBV induced changes in the LCL miRNome and transcriptome are reversed upon reprogramming into iPSCs, which allows a unique opportunity to better understand the miRNA and mRNA interactions that are EBV induced in LCLs and the changes that takes place during iPSC reprogramming. To identify the potential miRNA-mRNA interactions and better understand their role in regulating the cellular transitions in LCLs and their reprogrammed iPSCs, we performed a parallel genome-wide miRNA and mRNA expression analysis in six LCLs and their reprogrammed iPSCs. A total of 85 miRNAs and 5,228 mRNAs were significantly differentially expressed (DE). The target prediction of the DE miRNAs using TargetScan-Human, TarBase and miRecords databases identified 1,842 mRNA targets that were DE between LCLs and their reprogrammed iPSCs. The functional annotation, upstream regulator and gene expression analysis of the predicted DE mRNA targets suggest the role of DE miRNAs in regulating EBV induced changes in LCLs and self-renewal, pluripotency and differentiation in iPSCs.
RESUMEN
Macrophage activation/polarization to distinct functional states is critically supported by metabolic shifts. How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation, remains poorly understood. Here we show that IL-4 signaling co-opts the Akt-mTORC1 pathway to regulate Acly, a key enzyme in Ac-CoA synthesis, leading to increased histone acetylation and M2 gene induction. Only a subset of M2 genes is controlled in this way, including those regulating cellular proliferation and chemokine production. Moreover, metabolic signals impinge on the Akt-mTORC1 axis for such control of M2 activation. We propose that Akt-mTORC1 signaling calibrates metabolic state to energetically demanding aspects of M2 activation, which may define a new role for metabolism in supporting macrophage activation.