Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability
Identifieur interne : 000935 ( Pmc/Curation ); précédent : 000934; suivant : 000936Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability
Auteurs : E. Martín-Villar [Espagne, Royaume-Uni] ; B. Borda-D'Agua [Royaume-Uni] ; P. Carrasco-Ramirez [Espagne] ; J. Renart [Espagne] ; M. Parsons [Royaume-Uni] ; M. Quintanilla [Espagne] ; G E Jones [Royaume-Uni]Source :
- Oncogene [ 0950-9232 ] ; 2014.
Abstract
Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.
Url:
DOI: 10.1038/onc.2014.388
PubMed: 25486435
PubMed Central: 4430312
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability</title>
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<series><title level="j">Oncogene</title>
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<front><div type="abstract" xml:lang="en"><p>Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.</p>
</div>
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<front><journal-meta><journal-id journal-id-type="nlm-ta">Oncogene</journal-id>
<journal-id journal-id-type="iso-abbrev">Oncogene</journal-id>
<journal-title-group><journal-title>Oncogene</journal-title>
</journal-title-group>
<issn pub-type="ppub">0950-9232</issn>
<issn pub-type="epub">1476-5594</issn>
<publisher><publisher-name>Nature Publishing Group</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">25486435</article-id>
<article-id pub-id-type="pmc">4430312</article-id>
<article-id pub-id-type="pii">onc2014388</article-id>
<article-id pub-id-type="doi">10.1038/onc.2014.388</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability</article-title>
<alt-title alt-title-type="running">Podoplanin regulates invadopodia stability</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Martín-Villar</surname>
<given-names>E</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="aff" rid="aff2">2</xref>
<xref ref-type="corresp" rid="caf1">*</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Borda-d'Agua</surname>
<given-names>B</given-names>
</name>
<xref ref-type="aff" rid="aff2">2</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Carrasco-Ramirez</surname>
<given-names>P</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Renart</surname>
<given-names>J</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Parsons</surname>
<given-names>M</given-names>
</name>
<xref ref-type="aff" rid="aff2">2</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Quintanilla</surname>
<given-names>M</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Jones</surname>
<given-names>G E</given-names>
</name>
<xref ref-type="aff" rid="aff2">2</xref>
<xref ref-type="corresp" rid="caf2">*</xref>
</contrib>
<aff id="aff1"><label>1</label>
<institution>Instituto de Investigaciones Biomédicas ‘Alberto Sols' (CSIC-UAM)</institution>
, Madrid,<country>Spain</country>
</aff>
<aff id="aff2"><label>2</label>
<institution>Randall Division of Cell & Molecular Biophysics, King's College London</institution>
, London,<country>UK</country>
</aff>
</contrib-group>
<author-notes><corresp id="caf1"><label>*</label>
<institution>Instituto de Investigaciones Biomédicas ‘Alberto Sols' (CSIC-UAM), Arturo Duperier 4</institution>
, Madrid 28029, <country>Spain</country>
E-mail: <email>emvillar@iib.uam.es</email>
</corresp>
<corresp id="caf2"><label>*</label>
<institution>King's College London, Guy's Campus, Randall Division of Cell & Molecular Biophysics, Cell Motility & Cytoskeleton Section</institution>
, London SE1 1UL, <country>UK</country>
. E-mail: <email>gareth.jones@kcl.ac.uk</email>
</corresp>
</author-notes>
<pub-date pub-type="ppub"><day>20</day>
<month>08</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="epub"><day>08</day>
<month>12</month>
<year>2014</year>
</pub-date>
<volume>34</volume>
<issue>34</issue>
<fpage>4531</fpage>
<lpage>4544</lpage>
<history><date date-type="received"><day>19</day>
<month>05</month>
<year>2014</year>
</date>
<date date-type="rev-recd"><day>19</day>
<month>09</month>
<year>2014</year>
</date>
<date date-type="accepted"><day>11</day>
<month>10</month>
<year>2014</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2015 Macmillan Publishers Limited</copyright-statement>
<copyright-year>2015</copyright-year>
<copyright-holder>Macmillan Publishers Limited</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>
</license-p>
</license>
</permissions>
<abstract><p>Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.</p>
</abstract>
</article-meta>
</front>
<floats-group><fig id="fig1"><label>Figure 1</label>
<caption><p>Correlation between endogenous podoplanin expression and invadopodia formation in human SCC cell lines. (<bold>a</bold>
) Endogenous podoplanin expression in subconfluent SCC cell cultures by western blot. Two different exposure times are shown (upper panel 15 s; lower panel 40 s). The molecular mass variability observed between the different cell lines arises from the presence of O-linked carbohydrates on its extracellular domain as previously reported.<sup><xref ref-type="bibr" rid="bib21">21</xref>
, <xref ref-type="bibr" rid="bib26">26</xref>
, <xref ref-type="bibr" rid="bib54">54</xref>
</sup>
(<bold>b</bold>
) Determination of active invadopodia formation in SCC cells by gelatin-degradation assays. The different SCC cell lines were cultured on glass coverslips covered with crosslinked TRITC-gelatin for 18 h. Representative confocal images of SCC cells are shown. Insets show zoomed areas of individual invadopodia. Bar=10 μm. (<bold>c</bold>
) Quantification of active invadopodia formation in SCC cells (left and middle) and degradation area per cell (right) by gelatin-degradation assay. The results shown are the means±s.e.m. of <italic>n⩾</italic>
100 cells (left graph) or <italic>n⩾</italic>
10 cells-100 invadopodia (middle and right graphs) for each condition over three independent experiments.</p>
</caption>
<graphic xlink:href="onc2014388f1"></graphic>
</fig>
<fig id="fig2"><label>Figure 2</label>
<caption><p>Podoplanin defines adhesion rings at invadopodia of SCC cells. Confocal images showing specific localisation of podoplanin fused to GFP (PDPN-GFP) at invadopodia in HN5 cells cultured on glass coverslips covered with crosslinked gelatin (<bold>a, b</bold>
) or TRITC-gelatin (<bold>c</bold>
). Note that podoplanin is not present in all invadopodia (<bold>a</bold>
; left lower panels) but clusters to active invadopodia forming a ring structure around the actin or cortactin core (<bold>a, b</bold>
; lower panels and <bold>c</bold>
). Graphs indicate fluorescent intensity (in arbitrary units) of PDPN-GFP with respect to F-actin or cortactin over the indicated line scan. Data shown are representative from six invadopodia analysed per condition from two independent experiments. Bars=10 μm.</p>
</caption>
<graphic xlink:href="onc2014388f2"></graphic>
</fig>
<fig id="fig3"><label>Figure 3</label>
<caption><p>Podoplanin is required for efficient invadopodium-associated matrix degradation in SCC cells. (<bold>a</bold>
) Western blot of whole-cell lysates from HaCaT cells stably transfected with GFP (HaCaT-GFP) or GFP-tagged podoplanin (HaCaT PDPN-GFP) blotted for GFP and GAPDH (as loading control). Podoplanin undergoes proteolytic cleavage leading to the liberation of its intracellular domain into the cytosol (asterisk).<sup><xref ref-type="bibr" rid="bib55">55</xref>
</sup>
(<bold>b</bold>
) Gelatin-degradation assay using HaCaT cell transfectants (8h). Bar=25 μm. (<bold>c</bold>
) Quantification of the gelatin-degradation assay depicted in <bold>b</bold>
. Graphs represent the percentage of cells containing actin-rich protrusions that degrade the gelatin (left), number of active invadopodia per cell (middle) and degradation area per cell (right). Results shown are the means±s.e.m. of <italic>n⩾</italic>
100 cells (left graph) or <italic>n⩾</italic>
10 cells-100 invadopodia (middle and right graphs) over three independent experiments. (<bold>d</bold>
) Western blot showing podoplanin downregulation in HN5 cells. (<bold>e</bold>
) Gelatin-degradation assay using podoplanin-depleted HN5 cells (6 h). Podoplanin-specific silencing by shRNAs decreases active invadopodia formation in HN5 cells. Bar=25 μm. (<bold>f</bold>
) Quantification of the gelatin degradation assay represented in <bold>e</bold>
. Results shown are the means±s.e.m. of <italic>n</italic>
<italic>⩾</italic>
100 cells (left graph) or <italic>n</italic>
<italic>⩾</italic>
10 cells-100 invadopodia (middle and right graphs) for each condition over three independent experiments. ***<italic>P</italic>
<0.0001; **<italic>P</italic>
<0.001; *<italic>P</italic>
<0.01.</p>
</caption>
<graphic xlink:href="onc2014388f3"></graphic>
</fig>
<fig id="fig4"><label>Figure 4</label>
<caption><p>Dynamics of podoplanin during invadopodia assembly and matrix degradation. (<bold>a</bold>
) Podoplanin rings form following actin polymerisation. Live-cell imaging of HN5 cells expressing PDPN-GFP and Lifeact-Ruby cultured on 0.5% glutaraldehyde-crosslinked gelatin. Pictures represent zoomed areas of invadopodia dynamics over time. (<bold>b</bold>
) Panels 1, 2, 3 are kymographs showing the lifetime of representative invadopodia from time-lapse imaging in <bold>a</bold>
(boxes). White arrowheads indicate invadopodium formation time, and black arrowheads indicate the time of podoplanin ring assembly. (<bold>c</bold>
) Podoplanin ring assembly precedes matrix degradation. Time-lapse confocal microscopy of HN5 cells expressing PDPN-GFP cultured on 0.5% glutaraldehyde-crosslinked TRITC-gelatin. (<bold>d</bold>
) Panels 1 and 2 show Kymographs representing a subset of invadopodia from time-lapse in <bold>c</bold>
(boxes). White arrowheads indicate the time of podoplanin ring assembly, whereas black arrowheads indicate the time of gelatin degradation appearance. (<bold>e</bold>
) Graph represents the percentage of podoplanin-ringed invadopodia in the different populations of invadopodia. Invadopodia were classified into two main populations: precursors (newly formed invadopodia with no ability to degrade the matrix) and active (invadopodia actively degrading the matrix). Results shown are the means±s.e.m. of <italic>n⩾</italic>
70 invadopodia (five cells) for each condition over two independent experiments. ***<italic>P</italic>
<0.0001. Animate sequences of these data are shown in <xref ref-type="supplementary-material" rid="sup1">Supplementary Movies S1 and S2</xref>
. Bars=5 μm.</p>
</caption>
<graphic xlink:href="onc2014388f4"></graphic>
</fig>
<fig id="fig5"><label>Figure 5</label>
<caption><p>Podoplanin mediates invadopodia stabilisation. (<bold>a</bold>
–<bold>c</bold>
) HN5 control (Sc) and podoplanin-depleted (PDPN sh3 and sh4) cells expressing Lifeact-GFP were cultured on 0.5% glutaraldehyde-crosslinked gelatin for 3 h. Live-cell imaging was performed and analysed to calculate invadopodia lifetime (<bold>a</bold>
, <bold>b</bold>
) and total number of invadopodia per cell (<bold>c</bold>
). <italic>N⩾</italic>
90 invadopodia per condition from 10 cells over three independent experiments. Error bars represent s.e.m. An animate sequence of these data is shown in <xref ref-type="supplementary-material" rid="sup1">Supplementary Movie S3</xref>
. (<bold>d</bold>
) Invadopodia disassembly rates in control and podoplanin-depleted cells. Cells were grown on crosslinked unlabelled gelatin overnight. Invadopodia disassembly was induced by treatment with 10m<sc>M</sc>
methil-β-cyclodextrin (MβCD) for 20 min. Cells were fixed at the indicated time points, double stained for actin and cortactin and the number of cells with invadopodia was determined. (<bold>e</bold>
) Invadopodia reassembly rates were determined after washout of MβCD and incubation with normal medium. Rates of invadopodia disassembly and reassembly were calculated as the percentage of cells forming invadopodia on each time point. Values were normalised to <italic>t</italic>
=0 min (disassembly) or to <italic>t</italic>
=20 min after MβCD treatment (reassembly). The results shown are the means±s.e.m. of <italic>n⩾</italic>
200 cells for each condition and time point over three independent experiments. Bars=20 μm. ***<italic>P</italic>
<0.0005; * <italic>P</italic>
<0.05; NS=not significant.</p>
</caption>
<graphic xlink:href="onc2014388f5"></graphic>
</fig>
<fig id="fig6"><label>Figure 6</label>
<caption><p>Localisation of wild-type and mutant podoplanin proteins at invadopodia of HN5 cells. (<bold>a</bold>
) Schematic representation of podoplanin fusion constructs used for rescue experiments. SP, signal peptide; EC, ectodomain; TM, transmembrane domain; CT, cytoplasmic tail. Basic amino acids (bold) within the CT tail (RK…R) were substituted by uncharged polar residues (QN…<italic>N</italic>
; bold and underlined) disrupting the binding site for ERM proteins. The GXXXL motif within the TM domain was disrupted by substitution of the G in the position 137 by L. (<bold>b</bold>
) Graph represents the percentage of invadopodia showing recruitment of the indicated podoplanin constructs. The results shown are the means±s.e.m. of <italic>n⩾</italic>
250 invadopodia from two independent experiments. *<italic>P</italic>
<0.05. (<bold>c</bold>
) Confocal images of HN5 cells expressing the indicated podoplanin proteins. Bars=16 μm (upper panels) and 8 μm (lower panels). (<bold>d</bold>
) Graphs indicate fluorescent intensity (in arbitrary units) of each podoplanin mutant construct with respect to F-actin over the indicated line scan. Data shown are representative from 5–8 invadopodia analysed per condition from three independent experiments.</p>
</caption>
<graphic xlink:href="onc2014388f6"></graphic>
</fig>
<fig id="fig7"><label>Figure 7</label>
<caption><p>Podoplanin linkage to the cortical actin cytoskeleton and its association to lipid rafts are required for invadopodia-mediated ECM degradation. (<bold>a</bold>
) Western blot showing the re-expression of WT podoplanin and mutant constructs into podoplanin-depleted HN5 cells. (<bold>b</bold>
, <bold>c</bold>
) The ability of WT podoplanin or the indicated mutant constructs to rescue the invadopodia defects induced by depletion of podoplanin was evaluated by gelatin-degradation assays (6 h). Bar graph represents the percentage of cells forming active invadopodia on each condition. (<bold>b</bold>
) Representative images of the gelatin-degradation assay depicted in <bold>b</bold>
are shown in <bold>c</bold>
. Bars=20 μm. The results shown are the means±s.e.m. of <italic>n⩾</italic>
100 cells for each condition over three independent experiments. ***<italic>P</italic>
<0.0005.</p>
</caption>
<graphic xlink:href="onc2014388f7"></graphic>
</fig>
<fig id="fig8"><label>Figure 8</label>
<caption><p>ERM proteins localise at invadopodia adhesion rings and mediate podoplanin ring assembly. (<bold>a, b</bold>
) Specific localisation of moesin (<bold>a</bold>
) and ezrin (<bold>b</bold>
) at invadopodia of HN5 cells cultured on crosslinked gelatin. Note that ezrin and moesin cluster to invadopodia adhesion rings where they colocalise with podoplanin. Graphs indicate fluorescent intensity (in arbitrary units) of each marker over the indicated line scan. Data shown are representative from five invadopodia analysed per condition from three independent experiments. Bars=10 μm (upper panels) and 5 μm (lower panels). (<bold>c</bold>
) Western blot analysis of ezrin, moesin and podoplanin expression in HN5 cells upon specific siRNA treatment. (<bold>d</bold>
) Gelatin-degradation assay (6 h) of HN5 cells treated with ERM and podoplanin siRNAs. Bars=20 μm. (<bold>e</bold>
) Quantification of the gelatin-degradation assay depicted in <bold>d</bold>
. The results shown are the means±s.e.m. of <italic>n⩾</italic>
100 cells for each condition over three independent experiments. ***<italic>P</italic>
<0.0005; **<italic>P</italic>
<0.005; *<italic>P</italic>
<0.05.</p>
</caption>
<graphic xlink:href="onc2014388f8"></graphic>
</fig>
<fig id="fig9"><label>Figure 9</label>
<caption><p>Podoplanin regulates cofilin phosphorylation at Ser3 through the RhoC/ROCK/LIMK pathway. (<bold>a</bold>
) Determination of active RhoA and RhoC GTPase levels in control and podoplanin knockdown cells by GTP affinity pull-down assays (see also <xref ref-type="supplementary-material" rid="sup1">Supplementary Figure S7B</xref>
). (<bold>b</bold>
) The status of cofilin phosphorylation at Ser3 (pCofilinS3) was evaluated by western blot in podoplanin knockdown HN5 cells and HaCaT cells expressing PDPN-GFP. Quantification of pCofilinS3 levels (right panels) was performed relative to total cofilin levels and GAPDH loading control by densitometric analysis. Values were normalised to HN5 or HaCaT cells to which an arbitrary value of 1 was given. (<bold>c</bold>
,<bold>d</bold>
) Determination of pCofilinS3 levels during the stages of invadopodia formation in control and podoplanin-depleted HN5 cells. Invadopodia formation in HN5 cell transfectants was synchronised in order to evaluate invadopodia activity and the levels of pCofilinS3 during the stages of invadopodia formation (see Material and Methods section). Invadopodia activity was analysed by gelatin-degradation assay (<bold>c</bold>
), and pCofilinS3 changes were monitored by westen blot and quantified by densitometric analysis (<bold>d</bold>
and <xref ref-type="supplementary-material" rid="sup1">Supplementary Figure S7D</xref>
). pCofilinS3 levels were normalised to total cofilin levels and GAPDH-loading control. Graphs represent means±s.e.m. of two (<bold>c</bold>
) or three (<bold>d</bold>
) independent experiments. (<bold>e</bold>
) Western blot analysis of RhoC and RhoA expression in HN5 cells upon specific siRNA treatment (see also <xref ref-type="supplementary-material" rid="sup1">Supplementary Figure S7C</xref>
). (<bold>f</bold>
) Analysis of pCofilinS3 levels in RhoA- and RhoC-depleted cells. (<bold>g</bold>
) Effects of RhoA and RhoC knockdown in invadopodia-mediated degradation of HN5 cells (see also <xref ref-type="supplementary-material" rid="sup1">Supplementary Figure S7C</xref>
). (<bold>h</bold>
) Effects of ROCK inhibitor H-1152 in invadopodia-mediated degradation of control Sc and podoplanin-depleted HN5 cells. (<bold>i</bold>
) Western blot analysis pCofilinS3 levels after H-1152 treatment. Quantification of pCofilinS3 levels is shown in the right panel. (<bold>j</bold>
) Invadopodia-mediated gelatin degradation upon expression of the indicated LIMK1/2 mutant constructs in control Sc and podoplanin-depleted HN5 cells. (<bold>k</bold>
) Western blot analysis of pCofilinS3 and LIMK1/2 mutant expression (GFP). Quantification of pCofilinS3 levels is shown in the right panel. All Graphs represent means±s.e.m. of three or four (<bold>d</bold>
and <bold>e</bold>
) independent experiments. *<italic>P</italic>
<0.01; **<italic>P</italic>
<0.001; ***<italic>P</italic>
<0.0001; NS=not significant.</p>
</caption>
<graphic xlink:href="onc2014388f9"></graphic>
</fig>
</floats-group>
</pmc>
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