👤 Maikel P Peppelenbosch

Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 mutations contribute to HCC development remains unclear. In this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines. For each cell line we successfully obtained 2-4 correctly repaired clones, which all show reduced β-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored β-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive β-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling. Our study provides insights into the effects of repairing AXIN1 mutations on β-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations. Show less

AXIN1 is a major component of the β-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to β-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3β, β-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper β-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected β-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on β-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the β-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect β-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other β-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its β-catenin regulatory function, whereas the majority are passenger mutations. Show less

AXIN1 mutations are observed in 8-10% of hepatocellular carcinomas (HCCs) and originally were considered to support tumor growth by aberrantly enhancing β-catenin signaling. This view has however been challenged by reports showing neither a clear nuclear β-catenin accumulation nor clearly enhanced expression of β-catenin target genes. Here, using nine HCC lines, we show that AXIN1 mutation or siRNA mediated knockdown contributes to enhanced β-catenin signaling in all AXIN1-mutant and non-mutant lines, also confirmed by reduced signaling in AXIN1-repaired SNU449 cells. Both AXIN1 and AXIN2 work synergistically to control β-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels. The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of β-catenin-dependent cancers. At concentrations that inhibit tankyrase activity, some lines (e.g. HepG2, SNU398) were clearly affected in colony formation, but in most cases apparently independent from effects on β-catenin signaling. Overall, our analyses show that AXIN1 inactivation leads to enhanced β-catenin signaling in HCC cell lines, questioning the strong statements that have been made in this regard. Enhancing AXIN activity by tankyrase monotherapy provides however no effective treatment to affect their growth exclusively through reducing β-catenin signaling. Show less

The β-catenin signaling pathway is one of the most commonly deregulated pathways in cancer cells. Amino acid substitutions within armadillo repeats 5 and 6 (K335, W383, and N387) of β-catenin are found in several tumor types, including liver tumors. We investigated the mechanisms by which these substitutions increase signaling and the effects on liver carcinogenesis in mice. Plasmids encoding tagged full-length β-catenin (CTNNB1) or β-catenin with the K335I or N387K substitutions, along with MET, were injected into tails of FVB/N mice. Tumor growth was monitored, and livers were collected and analyzed by histology, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. Tagged full-length and mutant forms of β-catenin were expressed in HEK293, HCT116, and SNU449 cells, which were analyzed by immunoblots and immunoprecipitation. A panel of β-catenin variants and cell lines with knock-in mutations were analyzed for differences in N-terminal phosphorylation, half-life, and association with other proteins in the signaling pathway. Mice injected with plasmids encoding K335I or N387K β-catenin and MET developed larger, more advanced tumors than mice injected with plasmids encoding WT β-catenin and MET. K335I and N387K β-catenin bound APC with lower affinity than WT β-catenin but still interacted with scaffold protein AXIN1 and in the nucleus with TCF7L2. This interaction resulted in increased transcription of genes regulated by β-catenin. Studies of protein structures supported the observed changes in relative binding affinities. Expression of β-catenin with mutations in armadillo repeats 5 and 6, along with MET, promotes formation of liver tumors in mice. In contrast to N-terminal mutations in β-catenin that directly impair its phosphorylation by GSK3 or binding to BTRC, the K335I or N387K substitutions increase signaling via reduced binding to APC. However, these mutant forms of β-catenin still interact with the TCF family of transcription factors in the nucleus. These findings show how these amino acid substitutions increase β-catenin signaling in cancer cells. Show less

Aberrant activation of Wnt/β-catenin signaling plays a key role in the onset and development of hepatocellular carcinomas (HCC), with about half of them acquiring mutations in either CTNNB1 or AXIN1. However, it remains unclear whether these mutations impose sufficient β-catenin signaling or require upstream Wnt ligand activation for sustaining optimal growth, as previously suggested for colorectal cancers. Using a panel of nine HCC cell lines, we show that siRNA-mediated knockdown of β-catenin impairs growth of all these lines. Blocking Wnt secretion, by either treatment with the IWP12 porcupine inhibitor or knockdown of WLS, reduces growth of most of the lines. Unexpectedly, interfering with Wnt secretion does not clearly affect the level of β-catenin signaling in the majority of lines, suggesting that other mechanisms underlie the growth-suppressive effect. However, IWP12 treatment did not induce autophagy or endoplasmic reticulum (ER) stress, which may have resulted from the accumulation of Wnt ligands within the ER. Similar results were observed for colorectal cancer cell lines used for comparison in various assays. These results suggest that most colorectal and liver cancers with mutations in components of the β-catenin degradation complex do not strongly rely on extracellular Wnt ligand exposure to support optimal growth. In addition, our results also suggest that blocking Wnt secretion may aid in tumor suppression through alternative routes currently unappreciated. Show less

Apart from transporting lipids through the body, the human plasma lipoproteins very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are also thought to serve as a modality for intra-organismal protein transfer, shipping proteins with important roles in inflammation and thrombosis from the site of synthesis to effector locations. To better understand the role of VLDL and LDL in the transport of proteins, we applied a combination of LTQ ORBITRAP-XL (nLC-MS/MS) with both in-SDS-PAGE gel and in-solution tryptic digestion of pure and defined VLDL and LDL fractions. We identified the presence of 95 VLDL- and 51 LDL-associated proteins including all known apolipoproteins and lipid transport proteins, and intriguingly a set of coagulation proteins, complement system and anti- microbial proteins. Prothrombin, protein S, fibrinogen γ, PLTP, CETP, CD14 and LBP were present on VLDL but not on LDL. Prenylcysteine oxidase 1, dermcidin, cathelicidin antimicrobial peptide, TFPI-1 and fibrinogen α chain were associated with both VLDL and LDL. Apo A-V is only present on VLDL and not on LDL. Collectively, this study provides a wealth of knowledge on the protein constituents of the human plasma lipoprotein system and strongly supports the notion that protein shuttling through this system is involved in the regulation of biological processes. Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 - dyslipidaemia, 2 - atherosclerosis and vascular disease, and 3 - coagulation disorders. Show less

Adipocytes hypertrophy is the main cause of obesity and its affliction such as type 2 diabetes (T2D). Since superparamagnetic iron oxide nanoparticles (SPIONs) are used for a wide range of biomedical/medical applications, we aimed to study the effect of SPIONs on 22 and 29 risk genes (Based on gene wide association studies) for obesity and T2D in human adipocytes. The mRNA expression of lipid and glucose metabolism genes was changed upon the treatment of human primary adipocytes with SPIONs. mRNA of GULP1, SLC30A8, NEGR1, SEC16B, MTCH2, MAF, MC4R, and TMEM195 were severely induced, whereas INSIG2, NAMPT, MTMR9, PFKP, KCTD15, LPL and GNPDA2 were down-regulated upon SPIONs stimulation. Since SEC16B gene assist the phagocytosis of apoptotic cells and this gene were highly expressed upon SPIONs treatment in adipocytes, it is logic to assume that SPIONs may play a crucial role in this direction, which requires more consideration in the future. Show less