Biology subjects

Wangensteen, K. J.

Publications and source records attributed to Wangensteen, K. J..

3 recordsLinked to original sources

bioRxiv2019

In vivo screen identifies LXR agonism potentiates sorafenib killing of hepatocellular carcinoma

Existing drug therapies for hepatocellular carcinoma (HCC), including sorafenib, extend patient survival by only three months. We sought to identify novel druggable targets for use in combination with sorafenib to increase its efficacy. We implemented an in vivo genetic screening paradigm utilizing a library of 43 genes-of-interest expressed in the context of repopulation of the injured livers of Fumarylacetoacetate Hydrolase-deficient (Fah-/-) mice, which led to highly penetrant HCC. We then treated mice with vehicle or sorafenib to discover genetic determinants of sensitivity and resistance. Liver X Receptor alpha (LXR) emerged as a potential target. To examine LXR agonism in combination with sorafenib treatment, we added varying concentrations of sorafenib and LXR agonist drugs to HCC cell lines. We performed transcriptomic analysis to elucidate the mechanisms of HCC death. Fah-/- mice injected with the screening library developed HCC tumor clones containing Myc cDNA plus various other cDNAs. Treatment with sorafenib resulted in sorafenib-resistant HCCs that were significantly depleted in Nr1h3 cDNA, encoding LXR, suggesting that LXR activation is incompatible with tumor growth in the presence of sorafenib treatment in vivo. The combination of sorafenib and LXR agonism led to enhanced cell death as compared to monotherapy in multiple HCC cell lines, due to reduced expression of cell cycle regulators and increased expression of genes associated with apoptosis. Combination therapy also enhanced cell death in a sorafenib-resistant primary human HCC cell line. Our novel in vivo screen led to the discovery that LXR agonist drugs potentiate the efficacy of sorafenib in treating HCC.

cancer biology↗

bioRxiv2019

A high-content in vivo screen to identify microRNA epistasis in the repopulating mouse liver

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing; yet the roles of individual miRNAs or their combinatorial effects in this process are largely unknown. In this study, we sought to identify miRNAs that regulate hepatocyte repopulation following toxic liver injury in a high-throughput manner using the Fah-/- mouse. We constructed plasmid pools encoding over 30,000 tough decoy (TuD) miRNA inhibitors designed to target hepatocyte miRNAs in a pairwise manner. Plasmid libraries were delivered to hepatocytes of Fah-/- mice at the time of liver injury via hydrodynamic tail vein injection and integrated transgene-containing transposons were quantified following repopulation via high-throughput sequencing. Changes in polysome-bound transcripts following miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Analysis of TuD abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly altered following repopulation. We classified a subset of miRNA-binding sites (MBSs) as having strong effect on liver repopulation, thus implicating the targeted hepatocyte miRNAs as regulators of this process. Furthermore, we generated a high-content map of pairwise interactions between 171 MBSs and identified both synergistic and redundant effects. Our study highlights the power of higher-order screens to uncover miRNA functions that would go undetected by individual miRNA perturbations, and provides a new paradigm for the study of epistasis of miRNA activities.

systems biology↗

bioRxiv2019

The dynamic chromatin architecture of the regenerating liver

Background & AimsThe adult liver is the main detoxification organ and is routinely exposed to environmental insults but retains the ability to restore its mass and function upon tissue damage. However, massive injury can lead to liver failure, and chronic injury causes fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the transcriptional regulation of organ repair in the adult liver is incompletely understood.\n\nMethodsWe isolated nuclei from quiescent as well as repopulating hepatocytes in a mouse model of hereditary tyrosinemia, which recapitulates the injury and repopulation seen in toxic liver injury in humans. We then performed the assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) specifically in repopulating hepatocytes to identify differentially accessible chromatin regions and nucleosome positioning. Additionally, we employed motif analysis to predict differential transcription factor occupancy and validated the in silico results with chromatin immunoprecipitation followed by sequencing (ChIP-seq) for hepatocyte nuclear factor 4 (HNF4) and CCCTC-binding factor (CTCF).\n\nResultsChromatin accessibility in repopulating hepatocytes was increased in the regulatory regions of genes promoting proliferation and decreased in the regulatory regions of genes involved in metabolism. The epigenetic changes at promoters and liver enhancers correspond with regulation of gene expression, with enhancers of many liver function genes displaying a less accessible state during the regenerative process. Moreover, increased CTCF occupancy at promoters and decreased HNF4 binding at enhancers implicate these factors as key drivers of the transcriptomic changes in replicating hepatocytes that enable liver repopulation.\n\nConclusionsOur analysis of hepatocyte-specific epigenomic changes during liver repopulation identified CTCF and HNF4 as key regulators of hepatocyte proliferation and regulation of metabolic programs. Thus, liver repopulation in the setting of toxic injury makes use of both general transcription factors (CTCF) for promoter activation, and reduced binding by a hepatocyte-enriched factor (HNF4) to temporarily limit enhancer activity.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC=\"FIGDIR/small/664862v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@cefbc3org.highwire.dtl.DTLVardef@f2edc8org.highwire.dtl.DTLVardef@d90be9org.highwire.dtl.DTLVardef@19cf7a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗