Publications by authors named "Geertrui Denecker"

35 Publications

Kalirin-RAC controls nucleokinetic migration in ADRN-type neuroblastoma.

Life Sci Alliance 2021 May 3;4(5). Epub 2021 Mar 3.

Department of Neuroblastoma Genomics, Hopp-Children's Cancer Center at the (NCT) Nationales Centrum für Tumorerkrankungen Heidelberg (KiTZ), Heidelberg, Germany

The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin-GEF1-suppressed ADRN-type cells are a batch of AU-rich element-containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis-based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research.
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http://dx.doi.org/10.26508/lsa.201900332DOI Listing
May 2021

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Genes Chromosomes Cancer 2021 Apr 9;60(4):272-281. Epub 2021 Jan 9.

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.
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http://dx.doi.org/10.1002/gcc.22931DOI Listing
April 2021

The EMT Transcription Factor ZEB2 Promotes Proliferation of Primary and Metastatic Melanoma While Suppressing an Invasive, Mesenchymal-Like Phenotype.

Cancer Res 2020 07 5;80(14):2983-2995. Epub 2020 Jun 5.

Molecular and Cellular Oncology Laboratory, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of hampered outgrowth of primary melanomas , whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. SIGNIFICANCE: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/2983/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2373DOI Listing
July 2020

The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness.

Sci Rep 2020 01 14;10(1):218. Epub 2020 Jan 14.

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Neuroblastoma is an aggressive childhood cancer arising from sympatho-adrenergic neuronal progenitors. The low survival rates for high-risk disease point to an urgent need for novel targeted therapeutic approaches. Detailed molecular characterization of the neuroblastoma genomic landscape indicates that ALK-activating mutations are present in 10% of primary tumours. Together with other mutations causing RAS/MAPK pathway activation, ALK mutations are also enriched in relapsed cases and ALK activation was shown to accelerate MYCN-driven tumour formation through hitherto unknown ALK-driven target genes. To gain further insight into how ALK contributes to neuroblastoma aggressiveness, we searched for known oncogenes in our previously reported ALK-driven gene signature. We identified ETV5, a bona fide oncogene in prostate cancer, as robustly upregulated in neuroblastoma cells harbouring ALK mutations, and show high ETV5 levels downstream of the RAS/MAPK axis. Increased ETV5 expression significantly impacted migration, invasion and colony formation in vitro, and ETV5 knockdown reduced proliferation in a murine xenograft model. We also established a gene signature associated with ETV5 knockdown that correlates with poor patient survival. Taken together, our data highlight ETV5 as an intrinsic component of oncogenic ALK-driven signalling through the MAPK axis and propose that ETV5 upregulation in neuroblastoma may contribute to tumour aggressiveness.
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http://dx.doi.org/10.1038/s41598-019-57076-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959226PMC
January 2020

Author Correction: Integrative analysis identifies lincRNAs up- and downstream of neuroblastoma driver genes.

Sci Rep 2019 Jul 17;9(1):10536. Epub 2019 Jul 17.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-46785-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635357PMC
July 2019

Publisher Correction: In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors.

Sci Rep 2019 Jun 4;9(1):8360. Epub 2019 Jun 4.

Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-44435-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547841PMC
June 2019

Integrative analysis identifies lincRNAs up- and downstream of neuroblastoma driver genes.

Sci Rep 2019 04 5;9(1):5685. Epub 2019 Apr 5.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

Long intergenic non-coding RNAs (lincRNAs) are emerging as integral components of signaling pathways in various cancer types. In neuroblastoma, only a handful of lincRNAs are known as upstream regulators or downstream effectors of oncogenes. Here, we exploit RNA sequencing data of primary neuroblastoma tumors, neuroblast precursor cells, neuroblastoma cell lines and various cellular perturbation model systems to define the neuroblastoma lincRNome and map lincRNAs up- and downstream of neuroblastoma driver genes MYCN, ALK and PHOX2B. Each of these driver genes controls the expression of a particular subset of lincRNAs, several of which are associated with poor survival and are differentially expressed in neuroblastoma tumors compared to neuroblasts. By integrating RNA sequencing data from both primary tumor tissue and cancer cell lines, we demonstrate that several of these lincRNAs are expressed in stromal cells. Deconvolution of primary tumor gene expression data revealed a strong association between stromal cell composition and driver gene status, resulting in differential expression of these lincRNAs. We also explored lincRNAs that putatively act upstream of neuroblastoma driver genes, either as presumed modulators of driver gene activity, or as modulators of effectors regulating driver gene expression. This analysis revealed strong associations between the neuroblastoma lincRNAs MIAT and MEG3 and MYCN and PHOX2B activity or expression. Together, our results provide a comprehensive catalogue of the neuroblastoma lincRNome, highlighting lincRNAs up- and downstream of key neuroblastoma driver genes. This catalogue forms a solid basis for further functional validation of candidate neuroblastoma lincRNAs.
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http://dx.doi.org/10.1038/s41598-019-42107-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451017PMC
April 2019

ALK positively regulates MYCN activity through repression of HBP1 expression.

Oncogene 2019 04 11;38(15):2690-2705. Epub 2018 Dec 11.

Center for Medical Genetics Ghent (CMGG), Ghent University, Ghent, Belgium.

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PIK-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PIK antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.
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http://dx.doi.org/10.1038/s41388-018-0595-3DOI Listing
April 2019

In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors.

Sci Rep 2018 11 30;8(1):17468. Epub 2018 Nov 30.

Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium.

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway.
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http://dx.doi.org/10.1038/s41598-018-35868-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269481PMC
November 2018

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets.

Nat Commun 2018 11 19;9(1):4866. Epub 2018 Nov 19.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.
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http://dx.doi.org/10.1038/s41467-018-06699-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242972PMC
November 2018

Vehicle development, pharmacokinetics and toxicity of the anti-invasive agent 4-fluoro-3',4',5'-trimethoxychalcone in rodents.

PLoS One 2018 22;13(2):e0192548. Epub 2018 Feb 22.

Cancer Research Institute Ghent (CRIG), Ghent, Belgium.

Effective inhibitors of invasion and metastasis represent a serious unmet clinical need. We have recently identified 4-fluoro-3',4',5'-trimethoxychalcone or C16 as a potent anti-invasive molecule. In this paper, we report on the development of an optimized vehicle for oral administration of C16. We also explore its pharmacokinetic and toxicity profile in rodents as a prelude to a broad-scope evaluation as a pharmacological tool in animal models of disease. C16 showed suboptimal pharmacokinetics with limited oral bioavailability and whole blood stability. Rapid metabolism with elimination via glutathione conjugation was observed. An oral dosing routine using medicated gels was developed to overcome bioavailability issues and yielded sustained whole blood levels above the half maximal effective concentration (EC50) in a 7-day study. The compound proved well-tolerated in acute and chronic experiments at 300 mg/kg PO dosing. The medicated gel formulation is highly suitable for evaluation of C16 in animal models of disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192548PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823406PMC
April 2018

Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome.

Oncotarget 2017 Dec 6;8(63):106820-106832. Epub 2017 Nov 6.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Background: Neuroblastoma is an aggressive childhood malignancy of the sympathetic nervous system. Despite multi-modal therapy, survival of high-risk patients remains disappointingly low, underscoring the need for novel treatment strategies. The discovery of activating mutations opened the way to precision treatment in a subset of these patients. Previously, we investigated the transcriptional effects of pharmacological ALK inhibition on neuroblastoma cell lines, six hours after TAE684 administration, resulting in the 77-gene ALK signature, which was shown to gradually decrease from 120 minutes after TAE684 treatment, to gain deeper insight into the molecular effects of oncogenic ALK signaling.

Aim: Here, we further dissected the transcriptional dynamic profiles of neuroblastoma cells upon TAE684 treatment in a detailed timeframe of ten minutes up to six hours after inhibition, in order to identify additional early targets for combination treatment.

Results: We observed an unexpected initial upregulation of positively regulated MYCN target genes following subsequent downregulation of overall MYCN activity. In addition, we identified adrenomedullin (ADM), previously shown to be implicated in sunitinib resistance, as the earliest response gene upon ALK inhibition.

Conclusions: We describe the early and late effects of ALK inhibitor TAE684 treatment on the neuroblastoma transcriptome. The observed unexpected upregulation of ADM warrants further investigation in relation to putative ALK resistance in neuroblastoma patients currently undergoing ALK inhibitor treatment.
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http://dx.doi.org/10.18632/oncotarget.22423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739776PMC
December 2017

Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine-Nanobody Complex.

Front Immunol 2017 21;8:884. Epub 2017 Aug 21.

Ablynx N.V., Ghent, Belgium.

The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (V) domains (Nanobodies) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity and , as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn's disease, rheumatoid arthritis, and psoriasis.
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http://dx.doi.org/10.3389/fimmu.2017.00884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566574PMC
August 2017

Elevated ΔNp63α Levels Facilitate Epidermal and Biliary Oncogenic Transformation.

J Invest Dermatol 2017 02 7;137(2):494-505. Epub 2016 Oct 7.

Molecular Signaling and Cell Death Unit, Inflammation Research Center, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. Electronic address:

Unlike its family member p53, TP63 is rarely mutated in human cancer. However, ΔNp63α protein levels are often elevated in tumors of epithelial origin, such as squamous cell carcinoma and cholangiocarcinoma. To study the oncogenic properties of ΔNp63α in vivo, we generated transgenic mice overexpressing ΔNp63α from the Rosa26 locus promoter controlled by keratin 5-Cre. We found that these mice spontaneously develop epidermal cysts and ectopic ΔNp63α expression in the bile duct epithelium that leads to dilatation of the intrahepatic biliary ducts, to hepatic cyst formation and bile duct adenoma. Moreover, when subjected to models of 7,12-dimethylbenz[a]anthracene-based carcinogenesis, tumor initiation was increased in ΔNp63α transgenic mice in a gene dosage-dependent manner although ΔNp63α overexpression did not alter the sensitivity to 7,12-dimethylbenz[a]anthracene-induced cytotoxicity in vivo. However, keratinocytes isolated from ΔNp63α transgenic mice displayed increased survival and delayed cellular senescence compared with wild-type keratinocytes, marked by decreased p16 and p19 expression. Taken together, we show that increased ΔNp63α protein levels facilitate oncogenic transformation in the epidermis as well as in the bile duct.
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http://dx.doi.org/10.1016/j.jid.2016.09.026DOI Listing
February 2017

Deregulation of the replisome factor MCMBP prompts oncogenesis in colorectal carcinomas through chromosomal instability.

Neoplasia 2014 Sep;16(9):694-709

Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, 9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium. Electronic address:

Genetic instability has emerged as an important hallmark of human neoplasia. Although most types of cancers exhibit genetic instability to some extent, in colorectal cancers genetic instability is a distinctive characteristic. Recent studies have shown that deregulation of genes involved in sister chromatid cohesion can result in chromosomal instability in colorectal cancers. Here, we show that the replisome factor minichromosome maintenance complex-binding protein (MCMBP), which is directly involved in the dynamics of the minichromosome maintenance complex and contributes to maintaining sister chromatid cohesion, is transcriptionally misregulated in different types of carcinomas. Cellular studies revealed that both MCMBP knockdown and overexpression in different breast and colorectal cell lines is associated with the emergence of a subpopulation of cells with abnormal nuclear morphology that likely arise as a consequence of aberrant cohesion events. Association analysis integrating gene expression data with clinical information revealed that enhanced MCMBP transcript levels correlate with an increased probability of relapse risk in colorectal cancers and different types of carcinomas. Moreover, a detailed study of a cohort of colorectal tumors showed that the MCMBP protein accumulates to high levels in cancer cells, whereas in normal proliferating tissue its abundance is low, indicating that MCMBP could be exploited as a novel diagnostic marker for this type of carcinoma.
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http://dx.doi.org/10.1016/j.neo.2014.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4235010PMC
September 2014

Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation.

PLoS One 2013 30;8(7):e69115. Epub 2013 Jul 30.

Laboratory of Experimental Cancer Research (LECR), Department of Radiation Therapy & Experimental Cancer Research, Ghent University, Ghent, Belgium.

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA's anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069115PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728325PMC
April 2014

Caspase-14-deficient mice are more prone to the development of parakeratosis.

J Invest Dermatol 2013 Mar 27;133(3):742-750. Epub 2012 Sep 27.

Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. Electronic address:

Caspase-14 is an important protease in the proper formation of a fully functional skin barrier. Newborn mice that are deficient in caspase-14 exhibit increased transepidermal water loss and are highly sensitive to UVB-induced photodamage. Decreased caspase-14 expression and incomplete caspase-14 processing in lesional psoriatic parakeratotic stratum corneum has been reported previously. In this study, we show that caspase-14-deficient skin frequently displays incompletely cornified cells in the transitional zone between the granular and the cornified layers, pointing to a delay in cornification. We also demonstrate that after challenge of epidermal permeability barrier function by repetitive acetone treatment, a higher incidence of large parakeratotic plaques was observed in caspase-14-deficient skin. Furthermore, caspase-14-deficient mice are more prone than control mice to the development of parakeratosis upon induction of psoriasis-like dermatitis by imiquimod treatment. These results show that lack of caspase-14 expression predisposes to the development of parakeratosis and that caspase-14 has an important role in keratinocyte terminal differentiation and the maintenance of normal stratum corneum, especially in conditions causing epidermal hyperproliferation.
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http://dx.doi.org/10.1038/jid.2012.350DOI Listing
March 2013

Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.

J Invest Dermatol 2011 Nov 9;131(11):2233-41. Epub 2011 Jun 9.

Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium.

Caspase-14 is a protease that is mainly expressed in suprabasal epidermal layers and activated during keratinocyte cornification. Caspase-14-deficient mice display reduced epidermal barrier function and increased sensitivity to UVB radiation. In these mice, profilaggrin, a protein with a pivotal role in skin barrier function, is processed correctly to its functional filaggrin (FLG) repeat unit, but proteolytic FLG fragments accumulate in the epidermis. In wild-type stratum corneum, FLG is degraded into free amino acids, some of which contribute to generation of the natural moisturizing factors (NMFs) that maintain epidermal hydration. We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. These results indicate that accumulation of FLG fragments in caspase-14(-/-) mice is due to a defect in the terminal FLG degradation pathway. Consequently, we show that the defective FLG degradation in caspase-14-deficient skin results in substantial reduction in the amount of NMFs, such as urocanic acid and pyrrolidone carboxylic acid. Taken together, we identified caspase-14 as a crucial protease in FLG catabolism.
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http://dx.doi.org/10.1038/jid.2011.153DOI Listing
November 2011

Cop1 constitutively regulates c-Jun protein stability and functions as a tumor suppressor in mice.

J Clin Invest 2011 Apr 14;121(4):1329-43. Epub 2011 Mar 14.

Laboratory for Molecular Cancer Biology, Department of Molecular and Developmental Genetics, VIB-K.U.Leuven, Leuven, Belgium.

Biochemical studies have suggested conflicting roles for the E3 ubiquitin ligase constitutive photomorphogenesis protein 1 (Cop1; also known as Rfwd2) in tumorigenesis, providing evidence for both the oncoprotein c-Jun and the tumor suppressor p53 as its targets. Here we present what we believe to be the first in vivo investigation of the role of Cop1 in cancer etiology. Using an innovative genetic approach to generate an allelic series of Cop1, we found that Cop1 hypomorphic mice spontaneously developed malignancy at a high frequency in the first year of life and were highly susceptible to radiation-induced lymphomagenesis. Further analysis revealed that c-Jun was a key physiological target for Cop1 and that Cop1 constitutively kept c-Jun at low levels in vivo and thereby modulated c-Jun/AP-1 transcriptional activity. Importantly, Cop1 deficiency stimulated cell proliferation in a c-Jun-dependent manner. Focal deletions of COP1 were observed at significant frequency across several cancer types, and COP1 loss was determined to be one of the mechanisms leading to c-Jun upregulation in human cancer. We therefore conclude that Cop1 is a tumor suppressor that functions, at least in part, by antagonizing c-Jun oncogenic activity. In the absence of evidence for a genetic interaction between Cop1 and p53, our data strongly argue against the use of Cop1-inhibitory drugs for cancer therapy.
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http://dx.doi.org/10.1172/JCI45784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070608PMC
April 2011

Widespread overexpression of epitope-tagged Mdm4 does not accelerate tumor formation in vivo.

Mol Cell Biol 2010 Nov 20;30(22):5394-405. Epub 2010 Sep 20.

Laboratory for Molecular Cancer Biology, Department of Biomedical Molecular Biology, VIB-UGent, Technologiepark, B-9052 Ghent, Belgium.

Mdm2 and Mdm4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or Mdm4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and Mdm4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the Mdm4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of Mdm4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the Mdm4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or Eμ-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy.
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http://dx.doi.org/10.1128/MCB.00330-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976380PMC
November 2010

Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death.

Nat Cell Biol 2010 Jun 16;12(6):572-82. Epub 2010 May 16.

Interdisciplinary Research Institute (IRIBHM), Université Libre de Bruxelles (ULB), 808, route de Lennik, BatC, C6-130, 1070 Brussels, Belgium.

Adult stem cells (SCs) are at high risk of accumulating deleterious mutations because they reside and self-renew in adult tissues for extended periods. Little is known about how adult SCs sense and respond to DNA damage within their natural niche. Here, using mouse epidermis as a model, we define the functional consequences and the molecular mechanisms by which adult SCs respond to DNA damage. We show that multipotent hair-follicle-bulge SCs have two important mechanisms for increasing their resistance to DNA-damage-induced cell death: higher expression of the anti-apoptotic gene Bcl-2 and transient stabilization of p53 after DNA damage in bulge SCs. The attenuated p53 activation is the consequence of a faster DNA repair activity, mediated by a higher non-homologous end joining (NHEJ) activity, induced by the key protein DNA-PK. Because NHEJ is an error-prone mechanism, this novel characteristic of adult SCs may have important implications in cancer development and ageing.
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http://dx.doi.org/10.1038/ncb2059DOI Listing
June 2010

The "caveolae brake hypothesis" and the epidermal barrier.

J Invest Dermatol 2009 Apr 13;129(4):927-36. Epub 2008 Nov 13.

Department of Dermatology, Free University of Brussels, Brussels, Belgium.

Epidermal permeability barrier formation depends upon lamellar body (LB) secretion/fusion with the apical plasma membrane (APM) of outermost stratum granulosum (SG) cell, creating cholesterol/glycosphingolipid-enriched lipid rafts-like domains. We found that the dimensions of these domains are comparable to lipid raft in other cell types; and that acute barrier disruption regulates their size and dynamics. To assess the function of these LB-derived raft-like domains, we assessed APM dynamics and barrier recovery in methyl-beta-cyclodextrin (MbetaCD)-treated hairless mice and caveolin-1 knockouts (cav-1(-/-)). MbetaCD treatment impaired APM raft-like domain formation and barrier recovery. Accelerated barrier recovery is observed in cav-1(-/-) in parallel with expansion of raft-like domains. Barrier abrogation of normal epidermis resulted in translocation of cav-1 from the cytoplasm to raft-like membrane domains, restricting further raft-like domain formation and initiating terminal differentiation. Inhibition of LB secretion by monensin and absence of cav-1 delayed terminal differentiation. Furthermore, cav-1(-/-) mice exhibited an increased propensity to develop experimentally induced epidermal hyperplasia correlating with lipid raft persistence. Finally, the epidermal hyperplasia in psoriasis and Netherton syndrome is paralleled by increased lipid raft formation. These studies demonstrate that cav-1 delivery to the APM by LB trafficking to APM "brakes" further LB secretion, signals terminal differentiation, and regulates epidermal hyperproliferation.
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http://dx.doi.org/10.1038/jid.2008.328DOI Listing
April 2009

Caspase-14 reveals its secrets.

J Cell Biol 2008 Feb 4;180(3):451-8. Epub 2008 Feb 4.

Department for Molecular Biomedical Research, Flanders Institute for Biotechnology (VIB), 9052 Ghent, Belgium.

Caspase-14 is a unique member of the evolutionarily conserved family of cysteinyl aspartate-specific proteinases, which are mainly involved in inflammation and apoptosis. However, recent evidence also implicates these proteases in proliferation and differentiation. Although most caspases are ubiquitously expressed, caspase-14 expression is confined mainly to cornifying epithelia, such as the skin. Moreover, caspase-14 activation correlates with cornification, indicating that it plays a role in terminal keratinocyte differentiation. The determination of in vitro conditions for caspase-14 activity paved the way to identifying its substrates. The recent development of caspase-14-deficient mice underscored its importance in the correct degradation of (pro)filaggrin and in the formation of the epidermal barrier that protects against dehydration and UVB radiation. Here, we review the current knowledge on caspase-14 in skin homeostasis and disease.
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http://dx.doi.org/10.1083/jcb.200709098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234247PMC
February 2008

Acute modulations in permeability barrier function regulate epidermal cornification: role of caspase-14 and the protease-activated receptor type 2.

Am J Pathol 2008 Jan 21;172(1):86-97. Epub 2007 Dec 21.

Dermatology and Medical (Metabolism) Services, Veterans Administration Medical Center, 4510 Clement St., San Francisco, CA 94121, USA.

Stratum corneum comprises corneocytes, derived from outer stratum granulosum during terminal differentiation, embedded in a lipid-enriched extracellular matrix, secreted from epidermal lamellar bodies. Permeability barrier insults stimulate rapid secretion of preformed lamellar bodies from the outer stratum granulosum, regulated through modulations in ionic gradients and serine protease (SP)/protease-activated receptor type 2 (PAR2) signaling. Because corneocytes are also required for barrier function, we hypothesized that corneocyte formation could also be regulated by barrier function. Barrier abrogation by two unrelated methods initiated a wave of cornification, assessed as TdT-mediated dUTP nick end-labeling-positive cells in stratum granulosum and newly cornified cells by electron microscopy. Because cornification was blocked by occlusion, corneocytes formed specifically in response to barrier, rather than injury or cell replacement, requirements. SP inhibitors and hyperacidification (which decreases SP activity) blocked cornification after barrier disruption. Similarly, cornification was delayed in PAR2(-/-) mice. Although classical markers of apoptosis [poly(ADP-ribose)polymerase and caspase (Casp)-3] remained unchanged, barrier disruption activated Casp-14. Moreover, the pan-Casp inhibitor Z-VAD-FMK delayed cornification, and corneocytes were structurally aberrant in Casp14(-/-) mice. Thus, permeability barrier requirements coordinately drive both the generation of the stratum corneum lipid-enriched extracellular matrix and the transformation of granular cells into corneocytes, in an SP- and Casp-14-dependent manner, signaled by PAR2.
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http://dx.doi.org/10.2353/ajpath.2008.070161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2189608PMC
January 2008

Plakophilin-3-deficient mice develop hair coat abnormalities and are prone to cutaneous inflammation.

J Invest Dermatol 2008 Jun 13;128(6):1375-85. Epub 2007 Dec 13.

Department for Molecular Biomedical Research, VIB, Ghent, Belgium.

We generated mice deficient in plakophilin-3 (PKP3), a member of the Armadillo-repeat family and a component of desmosomes and stress granules in epithelial cells. In these mice, several subsets of hair follicles (HFs) had morphological abnormalities, and the majority of awl and auchene hair shafts had fewer medullar air columns. Desmosomes were absent from the basal layer of the outer root sheath of HFs and from the matrix cells that are in contact with dermal papillae. In the basal layer of PKP3-null epidermis, densities of desmosomes and adherens junctions were remarkably altered. Compensatory changes in several junctional proteins were observed. PKP3-null mice housed in conventional facilities were prone to dermatitis. Our animal model provides in vivo evidence that PKP3 plays a critical role in morphogenesis of HFs and shafts and in limiting inflammatory responses in the skin.
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http://dx.doi.org/10.1038/sj.jid.5701189DOI Listing
June 2008

Caspase-14 protects against epidermal UVB photodamage and water loss.

Nat Cell Biol 2007 Jun 21;9(6):666-74. Epub 2007 May 21.

Department for Molecular Biomedical Research, VIB, Technologie Park 927, B-9052, Ghent, Belgium.

Caspase-14 belongs to a conserved family of aspartate-specific proteinases. Its expression is restricted almost exclusively to the suprabasal layers of the epidermis and the hair follicles. Moreover, the proteolytic activation of caspase-14 is associated with stratum corneum formation, implicating caspase-14 in terminal keratinocyte differentiation and cornification. Here, we show that the skin of caspase-14-deficient mice was shiny and lichenified, indicating an altered stratum-corneum composition. Caspase-14-deficient epidermis contained significantly more alveolar keratohyalin F-granules, the profilaggrin stores. Accordingly, caspase-14-deficient epidermis is characterized by an altered profilaggrin processing pattern and we show that recombinant caspase-14 can directly cleave profilaggrin in vitro. Caspase-14-deficient epidermis is characterized by reduced skin-hydration levels and increased water loss. In view of the important role of filaggrin in the structure and moisturization of the skin, the knockout phenotype could be explained by an aberrant processing of filaggrin. Importantly, the skin of caspase-14-deficient mice was highly sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation, leading to increased levels of UVB-induced apoptosis. Removal of the stratum corneum indicate that caspase-14 controls the UVB scavenging capacity of the stratum corneum.
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http://dx.doi.org/10.1038/ncb1597DOI Listing
June 2007

Necrosis is associated with IL-6 production but apoptosis is not.

Cell Signal 2006 Mar 14;18(3):328-35. Epub 2005 Jul 14.

Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical Research, VIB and Ghent University, Technologiepark 927, B-9052, Ghent (Zwijnaarde), Belgium.

Due to loss of cell membrane integrity, necrotic cells passively release several cytosolic factors that can activate antigen presenting cells and other immune cells. In contrast, cells dying by apoptosis do not induce an inflammatory response. Here we show that necrotic cell death induced by several stimuli, such as TNF, anti-Fas or dsRNA, coincides with NF-kappaB-and p38MAPK-mediated upregulation and secretion of the pro-inflammatory cytokine IL-6. This event is greatly reduced or absent in conditions of apoptotic cell death induced by the same stimuli. This demonstrates that besides the capacity of necrotic cells to induce an inflammatory response due to leakage of cellular contents, necrotic dying cells themselves are involved in the expression and secretion of inflammatory cytokines. Moreover, inhibition of NF-kappaB and p38MAPK activation does not affect necrotic cell death in all conditions tested. This suggests that the activation of inflammatory pathways is distinct from the activation of necrotic cell death sensu strictu.
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http://dx.doi.org/10.1016/j.cellsig.2005.05.003DOI Listing
March 2006

A novel caspase-2 complex containing TRAF2 and RIP1.

J Biol Chem 2005 Feb 8;280(8):6923-32. Epub 2004 Dec 8.

Unit of Molecular Signalling and Cell Death, Department for Molecular Biomedical Research, Ghent University and Flemish Interuniversity Institute for Biotechnology, Technologiepark 927, Zwijnaarde B-9052, Belgium.

The enzymatic activity of caspases is implicated in the execution of apoptosis and inflammation. Here we demonstrate a novel nonenzymatic function for caspase-2 other than its reported proteolytic role in apoptosis. Caspase-2, unlike caspase-3, -6, -7, -9, -11, -12, and -14, is a potent inducer of NF-kappaB and p38 MAPK activation in a TRAF2-mediated way. Caspase-2 interacts with TRAF1, TRAF2, and RIP1. Furthermore, we demonstrate that endogenous caspase-2 is recruited into a large and inducible protein complex, together with TRAF2 and RIP1. Structure-function analysis shows that NF-kappaB activation occurs independent of enzymatic activity of the protease and that the caspase recruitment domain of caspase-2 is sufficient for the activation of NF-kappaB and p38 MAPK. These results demonstrate the inducible assembly of a novel protein complex consisting of caspase-2, TRAF2, and RIP1 that activates NF-kappaB and p38 MAPK through the caspase recruitment domain of caspase-2 independently of its proteolytic activity.
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http://dx.doi.org/10.1074/jbc.M411180200DOI Listing
February 2005

INCA, a novel human caspase recruitment domain protein that inhibits interleukin-1beta generation.

J Biol Chem 2004 Dec 21;279(50):51729-38. Epub 2004 Sep 21.

Unit of Molecular Signalling and Cell Death, Department for Molecular Biomedical Research, VIB-Ghent University, Technologiepark 927, Zwijnaarde B-9052, Belgium.

Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.
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http://dx.doi.org/10.1074/jbc.M407891200DOI Listing
December 2004

Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures.

Am J Pathol 2004 Sep;165(3):833-41

Department of Molecular Biomedical Research, Molecular Signaling and Cell Death Unit, Flanders Interuniversity Institute for Biotechnology (VIB) and Ghent University, Zwijnaarde, Belgium.

Caspase-14 is a nonapoptotic caspase family member whose expression in the epidermis is confined to the suprabasal layers, which consist of differentiating keratinocytes. Proteolytic activation of this caspase is observed in the later stages of epidermal differentiation. In psoriatic skin, a dramatic decrease in caspase-14 expression in the parakeratotic plugs was observed. Topical treatment of psoriatic lesions with a vitamin D3 analogue resulted in a decrease of the psoriatic phenotype and an increase in caspase-14 expression in the parakeratotic plugs. To investigate whether vitamin D3 directly affects caspase-14 expression levels, we used keratinocyte cell cultures. 1alpha,25-Dihydroxycholecalciferol, the biologically active form of vitamin D3, increased caspase-14 expression, whereas retinoic acid inhibited it. Moreover, retinoic acid repressed the vitamin D3-induced caspase-14 expression level. In addition, the use of organotypic skin cultures demonstrated that 1alpha,25-dihydroxycholecalciferol enhanced epidermal differentiation and caspase-14 activation, whereas retinoic acid completely blocked caspase-14 processing. Our data indicate that caspase-14 plays an important role in terminal epidermal differentiation, and its absence may contribute to the psoriatic phenotype.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618612PMC
http://dx.doi.org/10.1016/S0002-9440(10)63346-9DOI Listing
September 2004