Emilie A. Bard-Chapeau

Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness

Significance How cancer cells become invasive is key to understanding malignancy. Perturbations in O-glycosylation are strongly correlated with invasiveness. Here we report that tumor cells display relocation of O-glycosylation initiating glycosyltransferases from the Golgi apparatus to the endoplasmic reticulum (ER). ER-located O-glycosylation stimulates cell migration and invasiveness, which depend on cell surface O-glycoproteins. Inhibition of the glycosyltransferases in the ER reduces tissue invasion and metastasis formation in mice. Our study suggests that control of glycosylation via the subcellular localization of glycosyltransferases is a critical mechanism driving invasiveness in tumor cells. Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) drives high Tn levels in cancer cell lines and in 70% of malignant breast tumors. This process stimulates cell adhesion to the extracellular matrix, as well as migration and invasiveness. The GalNAc-Ts lectin domain, mediating high-density glycosylation, is critical for these effects. Interfering with the lectin domain function inhibited carcinoma cell migration in vitro and metastatic potential in mice. We also show that stimulation of cell migration is dependent on Tn-bearing proteins present in lamellipodia of migrating cells. Our findings suggest that relocation of GalNAc-Ts to the endoplasmic reticulum frequently occurs upon cancerous transformation to enhance tumor cell migration and invasiveness through modification of cell surface proteins.

Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model.

The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.

Ecotopic viral integration site 1 (EVI1) regulates multiple cellular processes important for cancer and is a synergistic partner for FOS protein in invasive tumors

Ecotropic viral integration site 1 (EVI1) is an oncogenic dual domain zinc finger transcription factor that plays an essential role in the regulation of hematopoietic stem cell renewal, and its overexpression in myeloid leukemia and epithelial cancers is associated with poor patient survival. Despite the discovery of EVI1 in 1988 and its emerging role as a dominant oncogene in various types of cancer, few EVI1 target genes are known. This lack of knowledge has precluded a clear understanding of exactly how EVI1 contributes to cancer. Using a combination of ChIP-Seq and microarray studies in human ovarian carcinoma cells, we show that the two zinc finger domains of EVI1 bind to DNA independently and regulate different sets of target genes. Strikingly, an enriched fraction of EVI1 target genes are cancer genes or genes associated with cancer. We also show that more than 25% of EVI1-occupied genes contain linked EVI1 and activator protein (AP)1 DNA binding sites, and this finding provides evidence for a synergistic cooperative interaction between EVI1 and the AP1 family member FOS in the regulation of cell adhesion, proliferation, and colony formation. An increased number of dual EVI1/AP1 target genes are also differentially regulated in late-stage ovarian carcinomas, further confirming the importance of the functional cooperation between EVI1 and FOS. Collectively, our data indicate that EVI1 is a multipurpose transcription factor that synergizes with FOS in invasive tumors.

Toll-Like Receptor 3 Expressing Tumor Parenchyma and Infiltrating Natural Killer Cells in Hepatocellular Carcinoma Patients

Background Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC. Methods HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3–9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan–Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided. Results TLR3 activation increased cell death in the TLR3+ SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002). Conclusions TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

EVI1 oncoprotein interacts with a large and complex network of proteins and integrates signals through protein phosphorylation

Significance Although ecotropic viral integration site 1 (EVI1) oncogenic transcription factor was discovered in 1988, its molecular functions and regulations are still underexplored. Through characterization of few EVI1-interacting proteins, EVI1 was identified as dynamic modulator of transcription and chromatin remodeling. We used proteomics approaches to define the EVI1 interactome. We found associations of EVI1 with not only transcriptional regulators, but also components of signaling pathways, DNA repair, DNA recombination, and mitosis complexes. We also identified functional EVI1 phosphorylation sites modified by casein-kinase II and protein phosphatase-1α that impact EVI1 activity. Thus, our study provides critical molecular insights on EVI1 action and regulation. Ecotropic viral integration site-1 (EVI1) is an oncogenic zinc finger transcription factor whose expression is frequently up-regulated in myeloid leukemia and epithelial cancers. To better understand the mechanisms underlying EVI1-associated disease, we sought to define the EVI1 interactome in cancer cells. By using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we could confidently assign 78 proteins as EVI1-interacting partners for FLAG-tagged EVI1. Subsequently, we showed that 22 of 27 tested interacting proteins could coimmunoprecipitate with endogenous EVI1 protein, which represented an 81.5% validation rate. Additionally, by comparing the stable isotope labeling by amino acids in cell culture (SILAC) data with high-throughput yeast two hybrid results, we showed that five of these proteins interacted directly with EVI1. Functional classification of EVI1-interacting proteins revealed associations with cellular transcription machinery; modulators of transcription; components of WNT, TGF-β, and RAS pathways; and proteins regulating DNA repair, recombination, and mitosis. We also identified EVI1 phosphorylation sites by MS analysis and showed that Ser538 and Ser858 can be phosphorylated and dephosphorylated by two EVI1 interactome proteins, casein kinase II and protein phosphatase-1α. Finally, mutations that impair EVI1 phosphorylation at these sites reduced EVI1 DNA binding through its C-terminal zinc finger domain and induced cancer cell proliferation. Collectively, these combinatorial proteomic approaches demonstrate that EVI1 interacts with large and complex networks of proteins, which integrate signals from various different signaling pathways important for oncogenesis. Comprehensive analysis of the EVI1 interactome has thus provided an important resource for dissecting the molecular mechanisms of EVI1-associated disease.

ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration

ER O-glycosylation can be induced through relocalisation GalNAc-Transferases from the Golgi. This process markedly stimulates cell migration and is constitutively activated in more than 60% of breast carcinomas. How this activation is achieved remains unclear. Here, we screened 948 signalling genes using RNAi and imaging. We identified 12 negative regulators of O-glycosylation that all control GalNAc-T sub-cellular localisation. ERK8, an atypical MAPK with high basal kinase activity, is a strong hit and is partially localised at the Golgi. Its inhibition induces the relocation of GalNAc-Ts, but not of KDEL receptors, revealing the existence of two separate COPI-dependent pathways. ERK8 down-regulation, in turn, activates cell motility. In human breast and lung carcinomas, ERK8 expression is reduced while ER O-glycosylation initiation is hyperactivated. In sum, ERK8 appears as a constitutive brake on GalNAc-T relocalisation, and the loss of its expression could drive cancer aggressivity through increased cell motility. DOI: http://dx.doi.org/10.7554/eLife.01828.001

Zip14 expression induced by lipopolysaccharides in macrophages attenuates inflammatory response

Objective and designWe investigated the role and regulation of zinc transporters in the activation of the inflammatory response in macrophages. Our exploratory computational study found that Zip14 (SLC39A14) was consistently up-regulated in activated macrophages; we therefore focused subsequently on that gene in the mechanistic study.MaterialThe expression and function of Zip14 was assessed in primary macrophages obtained by in-vitro differentiation of monocytes from human blood.MethodsPrimary macrophages were subjected to treatments with lipopolysaccharides, cytokines, chemicals, and pharmacological agents. SLC39A14 and inflammatory cytokine gene expressions were assessed by RT-qPCR. Zip14 siRNA knockdown was performed to explore the gene function.ResultsLipopolysaccharide’s inflammatory stimulus was a strong inducer of SLC39A14 mRNA expression in macrophages. This induction was dependent on calcium signaling, GC-rich DNA-binding, and NF-κB down-regulation. Impregnation of lipopolysaccharide-stimulated macrophages with the glucocorticoid dexamethasone further enhanced Zip14 expression while reducing interleukin-6 and tumor necrosis factor-α production. Zip14 knockdown in macrophages attenuated the expression and secretion of cytokines, indicating a buffering function for this zinc transporter.ConclusionsCollectively, our results identified the zinc transporter Zip14 as expressed downstream of lipopolysaccharide signals in macrophages. Zip14 induction had a regulatory function in cytokine production.

Mice Carrying a Hypomorphic Evi1 Allele Are Embryonic Viable but Exhibit Severe Congenital Heart Defects

The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1s critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1fl3) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1δex3/δex3 mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1fl3/fl3 mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1δex3/δex3 knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1δex3/δex3 mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.

Two-Step Forward Genetic Screen in Mice Identifies Ral GTPase-Activating Proteins as Suppressors of Hepatocellular Carcinoma

BACKGROUND & AIMS High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development. METHODS Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice. RESULTS Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice. CONCLUSIONS In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.

Functional features of EVI1 and EVI1Δ324 isoforms of MECOM gene in genome-wide transcription regulation and oncogenicity

The MDS1 and ecotropic viral integration site 1 (EVI1) complex locus (MECOM) gene encodes several transcription factor variants including MDS1-EVI1, EVI1 and EVI1Δ324. Although MDS1-EVI1 has been associated with tumor-suppressing activity, EVI1 is a known oncogene in various cancers, whose expression is associated with poor patient survival. Although EVI1Δ324 is co-transcribed with EVI1, its activity in cancer cells is not fully understood. Previous reports described that unlike EVI1, EVI1Δ324 protein cannot transform fibroblasts because of its disrupted N-terminal zinc finger (ZNF) domain. To better understand EVI1Δ324 biology and function, we obtained genome-wide binding occupancies and expression data in ovarian cancer cells. We characterized its DNA-binding sites, binding motif and target genes. Comparative analyses with previous study show that EVI1 and EVI1Δ324 share similar transcriptional activities linked to their common C-terminus ZNF domain. They bind to an E-twenty-six family (ETS)-like motif, target to a large extent the same genes and cooperate with AP1 transcription factor. EVI1Δ324-occupied genes were 70.7% similar to EVI1-bound genes. More strikingly, EVI1 and EVI1Δ324 differentially expressed genes were 99.87% identical, indicating comparable transcriptional regulatory functions. Consistently with gene ontologies linked to these target genes, EVI1Δ324 expression in HeLa cells could enhance anchorage-independent growth, such as EVI1, showing that EVI1Δ324 expression also lead to pro-oncogenic effects. The main specific feature of EVI1 variant is its N-terminus ZNF domain that binds DNA through GATA-like motif. We found that most GATA-like EVI1 chromatin immunoprecipitation sequencing peaks are far from genes and are not involved in transcriptional regulation. These genomic regions were enriched in simple sequence repeats and displayed high meiotic recombination rates. Overall, our genomics analyses uncovered common and specific features of two major MECOM isoforms. Their influence on transcription and downstream cell proliferation was comparable. However, EVI1-specific GATA-like binding sites, from its N-terminus ZNF domain, associated with high recombination rates, suggesting possible additional oncogenic potential for EVI1 in modulating genomic stability.