Role of CD36 and free fatty acid uptake in epithelial-mesenchymal transition of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related death worldwide. The liver is the main site of free fatty acid (FFA) metabolism; epidemiological studies link HCC tumorigenesis and elevated mortality rates to obesity, which manifests as increased FFA. Previous studies investigated the cytotoxic and pro-inflammatory effects of FFA, but did not focus on its influence on HCC progression. In this study, we hypothesized that elevated FFAs induce the epithelial-mesenchymal transition (EMT) program, facilitating a metastatic, invasive HCC phenotype. We investigated the association between obesity, EMT progression and alterations in FFA-uptake proteins in TCGA HCC gene expression data and validated the results in protein samples from human HCC tumors. In order to scrutinize the phenotypic effects of elevated FFA, we assessed the migratory and invasive ability of HCC cell lines treated with various FFAs, and further verified the expression of EMT markers using qPCR, confocal microscopy and flow cytometry. Bioinformatic analysis of TCGA data revealed that obese patients have higher levels of CD36, a trans-membrane protein that facilitates FFA transport into the cell. CD36 expression levels were strongly correlated with an EMT gene signature. However, the degree of EMT itself was not associated with the body mass index (BMI) of the patients. These results were corroborated in protein measurements from human HCC tumor samples. Additionally, we observed that saturated and monounsaturated FFA-treated HCC cell lines exhibited increased migration, invasion, dissociation, and development of the characteristic EMT morphology. Next, we confirmed the expression of EMT markers using qPCR and confocal imaging, demonstrating that chemical inhibition of CD36 reversed the FFA-induced EMT phenotype. Given the known cytotoxic effects of elevated FFAs on hepatocytes, we tested a population-distribution hypothesis using flow cytometry. We found that although some cells succumbed to the cytotoxic effects of high FFA, a distinct population not only evaded cell death, but also acquired EMT. We further utilized PCR arrays to determine that Wnt and TGF-beta signaling pathways, potential drivers of the EMT program, were activated upon FFA treatment and showed that repression of these pathways prevented migration and invasion in FFA treated cells. Our research demonstrates the role of CD36 and FFAs in activating the EMT program via induction of Wnt and TGF-beta signaling, and provides the first direct evidence that elevated FFA uptake promotes progression of HCC through EMT. Further elucidation of this program may provide insights for management of advanced HC

Systems biology for identifying liver toxicity pathways

Drug-induced liver toxicity is one of the leading causes of acute liver failure in the United States, exceeding all other causes combined. The objective of this paper is to describe systems biology methods for identifying pathways involved in liver toxicity induced by free fatty acids (FFA) and tumor necrosis factor (TNF)-α in human hepatoblastoma cells (HepG2/C3A). Systems biology approaches were developed to integrate multi-level data, i.e., gene expression, metabolite profile, toxicity measurements and a priori knowledge to identify gene targets for modulating liver toxicity. Targets that modulate liver toxicity, in vitro, were computationally predicted and some targets were experimentally validated

Epigenetic modification of neural genes by the neuron restrictive silencer factor

The Neuron Restrictive Silencer Factor (NRSF) is the master transcriptional repressor of the neural phenotype and has been shown to regulate hundreds of neural genes. It is becoming very clear that NRSF maintains repression of gene expression by recruiting chromatin modifiers to target gene regions. Previous work of ours showed that the small molecules, forskolin and isobutylmethylxanthine (IBMX), could induce neural-like differentiation in mesenchymal stem cells by causing downregulation of NRSF and de-repression of neural gene expression1. We next set out to determine if there were epigenetic changes in the promoter regions of NRSF target genes. In our work, we look at changes in the methylation of DNA in the promoter regions through bisulfite conversion and sequencing, as well as the acetylation status of nearby histones through ChIP. NRSF is also dysregulated in several neurological diseases. In particular, repression of certain ion channels involved in the electrophysiological properties of neurons may underlie conditions such as neuropathic pain and epilepsy. Work has shown that in the disease state the genes for these ion channels show repressive epigenetic marks2. Using dCas9, we are able to bring chromatin modifiers to specific regions of the genome. Here, we use a dCas9-Tet1 fusion to demethylate NRSF regulated genes. As a proof of principle, we show that by reversing repressive epigenetic marks on genes that contribute to neurological disease, that the epigenetic activity of NRSF itself could be a therapeutic dimension. Please click Additional Files below to see the full abstract

Aquachlorido(2-{[6-(dimethylamino)pyrimidin-4- yl]sulfanyl}pyrimidine-4,6-diamine)copper(II) chloride hydrate

A copper(II) complex of the non-symmetric bidentate ligand 2-{[6-(di­methyl­amino)­pyrimidin-4-yl]sulfan­yl}pyrimidine-4,6-di­amine (L1) is reported. The single-crystal X-ray structure of aqua­[aqua/chlorido­(0.49/0.51)](2-{[6-(di­methyl­amino)­pyrimidin-4-yl]sulfan­yl}pyrimidine-4,6-di­amine)­copper(II) 0.49-chloride 1.51-hydrate, [CuCl1.51(C10H13N7S)(H2O)1.49]Cl0.49·1.51H2O or [(L1)Cl1.51(H2O)1.49Cu]0.49Cl·1.51H2O, exhibits distorted square-pyramidal geometry around the metal centre, with disorder in the axial position, occupied by chloride or water. The six-membered metal–chelate ring is in a boat conformation, and short inter­molecular S- - -S inter­actions are observed. In addition to its capacity for bidentate metal coordination, the ligand has the ability to engage in further supra­molecular inter­actions as both a hydrogen-bond donor and acceptor, and multiple inter­actions with lattice solvent water mol­ecules are present in the reported structure

A Dynamic Analysis of IRS-PKR Signaling in Liver Cells: A Discrete Modeling Approach

A major challenge in systems biology is to develop a detailed dynamic understanding of the functions and behaviors in a particular cellular system, which depends on the elements and their inter-relationships in a specific network. Computational modeling plays an integral part in the study of network dynamics and uncovering the underlying mechanisms. Here we proposed a systematic approach that incorporates discrete dynamic modeling and experimental data to reconstruct a phenotype-specific network of cell signaling. A dynamic analysis of the insulin signaling system in liver cells provides a proof-of-concept application of the proposed methodology. Our group recently identified that double-stranded RNA-dependent protein kinase (PKR) plays an important role in the insulin signaling network. The dynamic behavior of the insulin signaling network is tuned by a variety of feedback pathways, many of which have the potential to cross talk with PKR. Given the complexity of insulin signaling, it is inefficient to experimentally test all possible interactions in the network to determine which pathways are functioning in our cell system. Our discrete dynamic model provides an in silico model framework that integrates potential interactions and assesses the contributions of the various interactions on the dynamic behavior of the signaling network. Simulations with the model generated testable hypothesis on the response of the network upon perturbation, which were experimentally evaluated to identify the pathways that function in our particular liver cell system. The modeling in combination with the experimental results enhanced our understanding of the insulin signaling dynamics and aided in generating a context-specific signaling network

Lessons From the Field: Culturally Competent Support for Family, Friend and Neighbor Caregivers in Seattle

Refugee Women’s Alliance (ReWA) and Chinese Information and Service Center (CISC) both have many years of experience working with Seattle/King County\u27s immigrant communities. ReWA and CISC participate in an initiative to support family, friend and neighbor caregivers and promote the value of kith and kin care. They have learned valuable lessons about culturally respectful, empowering, and meaningful support and communication with caregivers. This paper highlights the nine most important factors they have found for creating a culturally inclusive support program for family, friend and neighbor caregivers