14 research outputs found
The role of PPARβ/δ in skin SCCs
Switzerland is the country with the highest incidence rate of skin squamous cell carcinomas (SCCs) in Europe. However, in research, secs are mostly overlooked because they are well treatable in most cases. Yet, their high prevalence rate makes them a primary public concern. The alarmingly high number of sec induced deaths and the fact that researchers often neglected a search for molecular regulators makes it even more essential to find a cure of skin secs that goes beyond surgical intervention.
Our lab has previously shown that the llgand-activated transcription factors PPARβ/δ promoted UV induced skin sec progression. At the same time, PPARβ/δ loss-of-function prevented UV-induced skin response and skin secs in mice. This led us to our current hypothesis that ln human, PPARβ/δ supports the development of skin secs.
With thls project, 1 pursued to (i) lnvestigate if PPARβ/δ inhibition affects cancer hallmark in human skin sec cells in vitro, (ii) investigate the underlying mechanism and (iii) determine the therapeutic potential of PPARβ/δ inhibition in vivo.
I have found that (i) in some cancer cell lines, pharmacological PPARβ/δ inhibition decreases cancer hallmarks in human skin SCC cells in vitro, namely proliferation, migration, invasion and anoikis. ln other cell lines, PPARβ/δ inhibition had no effect compared to the control.
(ii) ln those cells where PPARβ/δ inhibition decreases cancer hallmarks, 1 analysed mRNA expression levels after PPARβ/δ inhibition compared to the control in an unbiased approach using mRNA sequencing. There, 1 observed decreased mRNA levels of genes associated with fatty acid oxidation. With the help of Dr Valentin Barqulssau, 1 could confirm a functional decrease in fatty acid oxidation after PPARl3/6 inhibition compared to the control. When I inhibited fatty acid oxidation in sec cells, it decreased prollferation, migration and anoikis compared to the contrai and therefore mimicked the effects of PPARβ/δ inhibition. This suggests that the in vitro decrease of cancer hallmarks after PPARβ/δ inhibition might be due to the decrease of fatty acid oxidation. Furthermore, PPARβ/δ inhibition increased cytotoxicity of human natural killer cells towards human sec cell lines compared to the contrai in vitro, suggesting that PPARj3/o inhibition affects tumour development on, both, a cell-autonomous level and regulates the tumour-microenvironment and its anti-tumour immunity.
(lii) ln an sec xenograft mouse model, 1 was surprised to observe an increase in tumour growth after treatment with the PPARβ/δ inhlbitor. These results are contrary to what I have expected based on the previous in vitro experiments because the in vitro results suggest that PPARβ/δ would decrease in tumour growth.
Overall, the role of PPARβ/δ in skin sec development is not yet clear, and it seems patient-specific whether PPARβ/δ inhibition has a beneficial outcome on tumour development or not.
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La Suisse est le pays qui présente le taux d'incidence le plus élevé de carcinomes épidermoïdes cutanés (CSC) en Europe. Cependant, dans la recherche, les CSC sont le plus souvent négligés parce qu'ils sont bien traitables dans la plupart des cas. Leur taux de prévalence élevé en fait toutefois une préoccupation publique majeure. Le nombre élevé de décès dus aux CSC et le fait que les chercheurs négligent souvent la recherche de régulateurs moléculaires rendent encore plus essentielle la recherche d'un traitement des CSC de la peau qui aille au-delà de l'intervention chirurgicale.
Notre laboratoire a précédemment montré que PPARl3/6, un facteur de transcription activé par des ligands, favorisaient la progression des CSC cutanés induits par les UV. Dans le même temps, la perte de fonction de PPARβ/δ a réduit la réponse cutanée induite par les UV et les CSC chez la souris. Cela nous a conduit à notre hypothèse actuelle selon laquelle, chez l'homme, PPARβ/δ favoriserait le développement des CSCs.
Dans le cadre de ce projet, j'ai cherché (i) à déterminer si l'inhibition de PPARβ/δ affecte les caractéristiques du cancer dans des cellules humaines de CSC in vitro, (ii) à étudier le mécanisme sous jacent et (iii) à déterminer le potentiel thérapeutique de l'inhibition de PPARβ/δ in vivo.
J'ai découvert que (i) dans certaines lignées de CSCs humaines, l'inhibition pharmacologique de PPARβ/δ diminue les caractéristiques du cancer in vitro par rapport au contrôle, à savoir la prolifération, la migration, l'invasion et l'anoikis. Dans d'autres lignées cellulaires, l'inhibition du PPARβ/δ n'a aucun effet par rapport au contrôle. (ii) Dans les cellules où l'inhibition du PPARβ/δ diminue les caractéristiques du cancer, j'ai analysé les niveaux d'expression de l'ARNm après l'inhibition du PPARβ/δ par rapport au contrôle dans une approche globale en utilisant le séquençage de l'ARNm. J'ai observé une diminution des niveaux d'ARNm des gènes associés à l'oxydation des acides gras. Nous avons confirmé une diminution fonctionnelle de l'oxydation des acides gras après l'inhibition du PPARβ/δ par rapport au contrôle. Lorsque j'ai inhibé l'oxydation des acides gras dans les cellules CSC, cela a diminué la prolifération, la migration et l'anoikis par rapport au contrôle et a imité les effets de l'inhibition du PPARβ/δ. Cela suggère que la diminution in vitro des signes cancéreux après l'inhibition de PPARβ/δ pourrait être due à la diminution de l'oxydation des acides gras. (iii) Dans un modèle de xénogreffe de
cellules de CSC humaines chez la souris, nous avons été surpris d'observer une augmentation de la
croissance tumorale après le traitement par l'inhibiteur de PPARβ/δ. Ces résultats sont contraires à ce que j'attendais sur la base des expériences in vitro précédentes, car les résultats in vitro suggèrent que l'inhibition de PPARβ/δ devrait ralentir la croissance tumorale.
Dans l'ensemble, le rôle de PPARβ/δ dans le développement des CSC de la peau n'est pas encore clair et il semble que l'inhibition de PPARβ/δ ait un effet bénéfique ou non sur le développement des tumeurs en fonction du patient
A CARD10-dependent tonic signalosome activates MALT1 paracaspase and regulates IL-17/TNF-a driven keratinocyte inflammation
The paracaspase MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) controls signaling downstream of several cell surface receptors, such as C-type lectin receptors on myeloid cells and antigen receptors on lymphocytes. Upon receptor engagement, MALT1, BCL10 (B-cell lymphoma/leukemia 10) and a CARD (Caspase recruitment domain) family member assemble into a ‘CBM’ complex, which is required to trigger MALT1 paracaspase activity and downstream transcriptional activation mechanisms (Meininger and Krappmann 2016; Rosebeck et al. 2011). Here, we found that CARD10 is highly expressed in proliferating keratinocytes and is responsible for a tonic level of paracaspase activity, driven by MALT1 isoform A. Furthermore, using the potent and selective MALT1 inhibitor MLT-827 (Bardet et al. 2018; Unterreiner et al. 2017), we reveal that MALT1 activity regulates pro-inflammatory responses downstream of IL-17/TNF-α
CARD10 cleavage by MALT1 restricts lung carcinoma growth in vivo
CARD-CC complexes involving BCL10 and MALT1 are major cellular signaling hubs. They govern NF-κB activation through their scaffolding properties as well as MALT1 paracaspase function, which cleaves substrates involved in NF-κB regulation. In human lymphocytes, gain-of-function defects in this pathway lead to lymphoproliferative disorders. CARD10, the prototypical CARD-CC protein in non-hematopoietic cells, is overexpressed in several cancers and has been associated with poor prognosis. However, regulation of CARD10 remains poorly understood. Here, we identified CARD10 as the first MALT1 substrate in non-hematopoietic cells and showed that protein-kinase-C-induced CARD10 cleavage by MALT1 at R587 dampens its capacity to activate NF-κB. Preventing CARD10 cleavage in the lung tumor A549 cell line increased basal levels of IL-6 and extracellular matrix components in vitro, and led to increased tumor growth in a mouse xenograft model, suggesting that CARD10 cleavage by MALT1 might be a built-in mechanism controlling tumorigenicity
Schematic representation of MALT1 isoforms and variants thereof.
<p>The two reported human MALT1 isoforms A (NP_006776.1) and B (NP_776216.1) are depicted. The drawings show the different domains, the auto-cleavage sites (red arrows), the catalytic cysteine residue (black), the reported ubiquitination site (green) and shows the regions required for binding BCL10 (grey) and TRAF6 (blue). The truncated variants of the two isoforms, used or discussed in the present work, are represented in the shaded boxes at the bottom, with predicted molecular weights. The 11-amino acid deletion of isoform B is depicted by a red triangle. Ig, Immunoglobulin-like domain.</p
Functional impact of C-terminal cleavage on MALT1 isoforms A and B.
<p><b>(A)</b> NF-κB luciferase reporter gene assay in HEK293 cells transfected with either, MALT1A WT, MALT1A-781, MALT1B WT, or MALT1B-770 in the absence or presence of CARD11-L244P. Luciferase activity was recorded after 24h. Data show the mean of triplicate determinations from 7 independent experiments. Statistical significance was calculated using the Student T-test. Western blot analysis, performed in parallel to control for protein expression, is shown below with samples from a representative experiment. An anti-tubulin immunoblot is provided as loading control. <b>(B)</b> CBM reconstitution was performed using MALT1A, MALT1B as well as their constitutively cleaved variants, together with the MALT1 protease substrate A20. Immunoblot analyses for MALT1 show the respective C-terminal auto-cleavage bands (white arrow heads). For A20, they show cleaved fragments as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref009" target="_blank">9</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref022" target="_blank">22</a>]. A20p37 is a proteasome sensitive degradation product of A20 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref009" target="_blank">9</a>]. Its weak detection when MALT1A WT is used is consistent with high proteolytic activity resulting in early processing of A20 and disappearance of A20p37 by the time of harvest. A non-specific (NS) band detected by the anti-A20 antibody is provided as loading control.</p
MALT1 auto-cleavage at R149 is induced by TRAF6.
<p><b>(A)</b> MALT1A was expressed in HEK293 cells, either alone, or together with CARD11-L244P, or as part of a CBM reconstitution assay (transfection ratio 1:1:1:1 using a control plasmid), or a TM reconstitution assay (TRAF6 + MALT1 (transfection ratio 1:1:2 with “2” referring to a control plasmid), or a CBMT reconstitution assay (transfection ratio 1:1:1:1). Western Blot analyses with anti-FLAG and anti-MALT1 (MT1/410) antibodies are shown, displaying MALT1 auto-cleaved fragments (C-terminal, white arrow head; N-terminal depicted as p16/p76, grey arrow heads). Loading controls with an anti-tubulin antibody are also provided. <b>(B)</b> The CBMT reconstitution assay was performed as in (A) using either TRAF6-WT, or TRAF6-C70A, or TRAF6 289–522. Anti-FLAG Western Blot analyses are shown, displaying MALT1 auto-cleaved fragments (C-terminal, white arrow heads; N-terminal p16, grey arrow heads) as well as MALT1 mono-ubiquitinated species (black arrow heads). An anti-cleaved BCL10 immunoblot providing evidence for proteolytic activity of MALT1 is also shown together with a non-specific (NS) band detected by the BCL10 antibody, as loading control.</p
MALT1 auto-proteolysis and mono-ubiquitination mechanisms.
<p><b>(A)</b> The TM reconstitution assay performed with MALT1A-C464A, MALT1A-R149A, MALT1A-R781A and combinations thereof. Immunoblotting with anti-FLAG antibody is shown. MALT1 C-terminal auto-cleavage is denoted with a white arrow head, N-terminal auto-cleavage with a grey arrow head (p16) and mono-ubiquitination of MALT1A-C464A with a black arrow head. * denotes a faint band that migrates above p16, which we observed in lanes loaded with MALT1A-C464A samples. <b>(B)</b> TM and CBM reconstitution assays were performed with MALT1A-C464A, MALT1A-K644R and the combination of the two. Immunoblots with anti-FLAG antibody are shown. MALT1 C-terminal auto-cleavage is denoted with white arrow heads, N-terminal auto-cleavage with a grey arrow head (p16) and mono-ubiquitination of MALT1A C464A with black arrow heads. The immunoblot with anti-BCL10 antibody (ep605y) shows reduced phosphorylated BCL10 when MALT1A-C464A and MALT1A-K644R are co-expressed, indicative of MALT1 protease activity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.g002" target="_blank">Fig 2A</a>).</p
TRAF6 is able to trigger auto-cleavage and mono-ubiquitination of MALT1.
<p><b>(A)</b> Comparison of CBM reconstitution assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.g001" target="_blank">Fig 1</a> (transfection ratio 1:1:1) and TM reconstitution assay (transfection ratio 1:1:1, using a control plasmid) in HEK293 cells. Immunoblot analyses are shown with anti-MALT1 antibodies (specified on the figure), anti-FLAG antibody to confirm the MALT1 p16 fragment, and anti-CARD11 as well as anti-TRAF6 antibodies. Both assays led to MALT1 C-terminal auto-cleavage (white arrow head) and mono-ubiquitination in the presence of z-VRPR-fmk (black arrow head). N-terminal MALT1 auto-cleavage was significantly detected only in the context of he TM assay, as shown by the generation of the two fragments p76 and p16 (both denoted with a grey arrow head). * and ** denote bands resulting from secondary auto-cleavage reactions, at the C- and N- terminus, respectively. The arrow in the TRAF6 immunoblot points to a possible minor cleavage product of TRAF6 which disappears in the presence of z-VRPR-fmk. <b>(B)</b> The TM reconstitution assay was performed using FLAG-TRAF6 WT or the FLAG-TRAF6-C70A mutant construct. Western Blot analysis with anti-FLAG antibody is shown. C-terminal auto-cleavage of MALT1 (white arrow head) and mono-ubiquitinated MALT1 (black arrow head) were observed with TRAF6 WT only. <b>(C)</b> Comparison of MALT1 WT and MALT1A-E4/A (TRAF6-binding deficient) in CBM and TM reconstitution assays. Immunoblotting with anti-FLAG antibody is shown. MALT1 C-terminal auto-cleavage is denoted with a white arrow head, N-terminal auto-cleavage (p16) is shown in the bottom panel, and mono-ubiquitination in the presence of z-VRPR-fmk is denoted with a black arrow head.</p
Post-translational modifications of MALT1 in human lymphocytes.
<p><b>(A)</b> OCI-Ly3 cells were grown for 4 days in the presence or absence of 50 μM z-VRPR-fmk. Cells were harvested for MALT1 immunoprecipitation and lysate analysis using an anti-MALT1 antibody (H-300), as described in the Methods section. The MALT1 faster and slower migrating species are indicated with white and black arrow heads, respectively. The BCL10 antibody recognizing more specifically the uncleaved form of BCL10 (ep606y) was reported already [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref038" target="_blank">38</a>]. Densitometry of MALT1and BCL10 signal intensities was measured in 3 independent experiments and is shown as Mean ± SD. Cells were counted at the end of each experiment and counts are represented as Mean ± SD (N = 3). Statistical significance was calculated using the Student T-test. <b>(B)</b> Jurkat cells were stimulated with PMA (10 ng/ml) anti-CD28 (1 μM) and anti-CD3 (1 μM) in the presence of 5 μM MG-132, for various times before full cell extraction and analysis of MALT1 (MT1/410 antibody) and BCL10 (antibodies specified on the figure) by immunoblotting. The MALT1 faster and slower migrating species are indicated with a white and a black arrow head, respectively. <b>(C)</b> Primary human CD3 T cells were stimulated with PMA (10 ng/ml) and Ionomycin (1 μM) in the presence of 5 μM MG-132, for various times before full cell extraction and analysis of MALT1 (MT1/410 antibody). The MALT1 faster and slower migrating species are indicated with a white and a black arrow head, respectively. Anti-tubulin immunoblots are provided as loading controls.</p
TRAF6 induces auto-proteolysis-dependent down-regulation of MALT1.
<p><b>(A)</b> The CBMT reconstitution assay in HEK293 was performed with either MALT1A-WT, or the TRAF6-binding deficient MALT1A-4E/A mutant construct, in the presence of TRAF6-WT, TRAF6-C70A or TRAF6 289–522. Immunoblotting with anti-FLAG antibody is shown. An anti-tubulin immunoblot is provided as loading control. <b>(B)</b> The TM reconstitution assay in HEK293 cells was performed using MALT1A, MALT1B as well as their C-terminal truncated variants. Anti-FLAG Western Blot analyses show the respective C-terminal auto-cleavage bands (white arrow head), the respective N-terminal auto-cleavage bands (grey arrow head, p16) as well as the respective mono-ubiquitinated species detected in the presence of z-VRPR-fmk (black arrow head). They also provide TRAF6 co-expression levels (bottom panels). <b>(C)</b> CBM and TM reconstitution assays in HEK293 cells were performed in the presence of co-expressed CYLD, using MALT1A, MALT1B, as well as their C-terminal truncated variants. Anti-FLAG Western Blot analyses show MALT1 C-terminal auto-cleavage bands (white arrow heads) as well as CYLD full length (<i>fl</i>) and cleaved fragment (<i>cl</i>) levels. Anti-CYLD immunoblots are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.s007" target="_blank">S7C Fig</a>. <b>(D)</b> CBM and TM reconstitution assays in HEK293 cells were performed using MALT1A-WT, several point mutant forms thereof: R149A, R781A, C464A, K644R, as well as the MALT1A-781 truncated form mimicking constitutive C-terminal auto-cleavage. Immunoblots with anti-FLAG antibody are shown. Densitometry analysis of MALT1 signals as percentage of the signal obtained with MALT1A 1–781 is provided underneath. MALT1 C-terminal auto-cleavage is denoted with a white arrow head and mono-ubiquitination with a black arrow head. An anti-tubulin immunoblot provides loading controls for the both assays.</p