Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability
Identifieur interne : 000464 ( Pmc/Curation ); précédent : 000463; suivant : 000465Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability
Auteurs : Andrea Díaz-Díaz ; Daniel Roca-Lema ; Alba Casas-Pais ; Gabriela Romay ; Giovanni Colombo ; Ángel Concha ; Bego A Gra A ; Angélica FigueroaSource :
- Cancers [ 2072-6694 ] ; 2020.
Abstract
The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.
Url:
DOI: 10.3390/cancers12010215
PubMed: 31952268
PubMed Central: 7017148
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability</title>
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<author><name sortKey="Romay, Gabriela" sort="Romay, Gabriela" uniqKey="Romay G" first="Gabriela" last="Romay">Gabriela Romay</name>
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<author><name sortKey="Colombo, Giovanni" sort="Colombo, Giovanni" uniqKey="Colombo G" first="Giovanni" last="Colombo">Giovanni Colombo</name>
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<author><name sortKey="Figueroa, Angelica" sort="Figueroa, Angelica" uniqKey="Figueroa A" first="Angélica" last="Figueroa">Angélica Figueroa</name>
<affiliation><nlm:aff id="af1-cancers-12-00215">Epithelial Plasticity and Metastasis Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), 15006 A Coruña, Spain;<email>andrea.diaz.diaz@sergas.es</email>
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<front><div type="abstract" xml:lang="en"><p>The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.</p>
</div>
</front>
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<biblStruct><analytic><author><name sortKey="Aparicio, L A" uniqKey="Aparicio L">L.A. Aparicio</name>
</author>
<author><name sortKey="Castosa, R" uniqKey="Castosa R">R. Castosa</name>
</author>
<author><name sortKey="Haz Conde, M" uniqKey="Haz Conde M">M. Haz-Conde</name>
</author>
<author><name sortKey="Rodriguez, M" uniqKey="Rodriguez M">M. Rodríguez</name>
</author>
<author><name sortKey="Blanco, M" uniqKey="Blanco M">M. Blanco</name>
</author>
<author><name sortKey="Valladares, M" uniqKey="Valladares M">M. Valladares</name>
</author>
<author><name sortKey="Figueroa, A" uniqKey="Figueroa A">A. Figueroa</name>
</author>
</analytic>
</biblStruct>
</listBibl>
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</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Cancers (Basel)</journal-id>
<journal-id journal-id-type="iso-abbrev">Cancers (Basel)</journal-id>
<journal-id journal-id-type="publisher-id">cancers</journal-id>
<journal-title-group><journal-title>Cancers</journal-title>
</journal-title-group>
<issn pub-type="epub">2072-6694</issn>
<publisher><publisher-name>MDPI</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">31952268</article-id>
<article-id pub-id-type="pmc">7017148</article-id>
<article-id pub-id-type="doi">10.3390/cancers12010215</article-id>
<article-id pub-id-type="publisher-id">cancers-12-00215</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Díaz-Díaz</surname>
<given-names>Andrea</given-names>
</name>
<xref ref-type="aff" rid="af1-cancers-12-00215">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Roca-Lema</surname>
<given-names>Daniel</given-names>
</name>
<xref ref-type="aff" rid="af1-cancers-12-00215">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Casas-Pais</surname>
<given-names>Alba</given-names>
</name>
<xref ref-type="aff" rid="af1-cancers-12-00215">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Romay</surname>
<given-names>Gabriela</given-names>
</name>
<xref ref-type="aff" rid="af1-cancers-12-00215">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Colombo</surname>
<given-names>Giovanni</given-names>
</name>
<xref ref-type="aff" rid="af2-cancers-12-00215">2</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Concha</surname>
<given-names>Ángel</given-names>
</name>
<xref ref-type="aff" rid="af3-cancers-12-00215">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Graña</surname>
<given-names>Begoña</given-names>
</name>
<xref ref-type="aff" rid="af4-cancers-12-00215">4</xref>
</contrib>
<contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0001-5530-5213</contrib-id>
<name><surname>Figueroa</surname>
<given-names>Angélica</given-names>
</name>
<xref ref-type="aff" rid="af1-cancers-12-00215">1</xref>
<xref rid="c1-cancers-12-00215" ref-type="corresp">*</xref>
</contrib>
</contrib-group>
<aff id="af1-cancers-12-00215"><label>1</label>
Epithelial Plasticity and Metastasis Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), 15006 A Coruña, Spain;<email>andrea.diaz.diaz@sergas.es</email>
(A.D.-D.);<email>Daniel.roca.lema@sergas.es</email>
(D.R.-L.);<email>alba.casas.pais@sergas.es</email>
(A.C.-P.);<email>Gabriela.Romay.Cousido@sergas.es</email>
(G.R.)</aff>
<aff id="af2-cancers-12-00215"><label>2</label>
Istituto per la Ricerca e l’Innovazione Biomedica (IRIB)—CNR di Palermo, Via Ugo La Malfa 153, 90146 Palermo, Italy;<email>giovanni.colombo@community.unipa.it</email>
</aff>
<aff id="af3-cancers-12-00215"><label>3</label>
Pathology Department and A Coruña Biobank from Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), 15006 A Coruña, Spain;<email>angel.concha.lopez@sergas.es</email>
</aff>
<aff id="af4-cancers-12-00215"><label>4</label>
Clinical Oncology Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), 15006 A Coruña, Spain;<email>Begona.Grana.Suarez@sergas.es</email>
</aff>
<author-notes><corresp id="c1-cancers-12-00215"><label>*</label>
Correspondence: <email>Angelica.Figueroa.Conde-Valvis@sergas.es</email>
</corresp>
</author-notes>
<pub-date pub-type="epub"><day>15</day>
<month>1</month>
<year>2020</year>
</pub-date>
<pub-date pub-type="collection"><month>1</month>
<year>2020</year>
</pub-date>
<volume>12</volume>
<issue>1</issue>
<elocation-id>215</elocation-id>
<history><date date-type="received"><day>17</day>
<month>12</month>
<year>2019</year>
</date>
<date date-type="accepted"><day>12</day>
<month>1</month>
<year>2020</year>
</date>
</history>
<permissions><copyright-statement>© 2020 by the authors.</copyright-statement>
<copyright-year>2020</copyright-year>
<license license-type="open-access"><license-p>Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (<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>The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.</p>
</abstract>
<kwd-group><kwd>Hsp90 chaperone</kwd>
<kwd>E3 ubiquitin-ligase Hakai</kwd>
<kwd>targeted therapy</kwd>
<kwd>colon cancer</kwd>
</kwd-group>
</article-meta>
</front>
<floats-group><fig id="cancers-12-00215-f001" orientation="portrait" position="float"><label>Figure 1</label>
<caption><p>Hakai interacts with heat shock protein 90 (Hsp90): Co-immunoprecipitation of Hakai and Hsp90 was performed in different cell lines: (<bold>a</bold>
) HEK293T. (<bold>b</bold>
) ACHN. (<bold>c</bold>
) HT29. (<bold>d</bold>
) HCT116. Hakai immunoprecipitation was performed by employing two different rabbit polyclonal Hakai antibodies indicated in material an methods: one from Bethyl (Bethyl Ab) and one kindly provided by Dr. Fujita (Hakai-2498). GAPDH signal was used as protein loading control.</p>
</caption>
<graphic xlink:href="cancers-12-00215-g001"></graphic>
</fig>
<fig id="cancers-12-00215-f002" orientation="portrait" position="float"><label>Figure 2</label>
<caption><p>Hakai, Annexin A2, and Hsp90 interact with other: The whole cell extracts of HCT116 cell line cells were prepared and subjected to a co-immunoprecipitation assay. Subsequent western-blotting analysis was performed by using Hakai, Annexin A2, or Hsp90 antibodies (<bold>a</bold>
) Hakai is specifically detected in anti-Hsp90 immunoprecipitates. (<bold>b</bold>
) Annexin A2 is specifically detected in anti-Hakai immunoprecipitates. (<bold>c</bold>
) Hsp90 is specifically detected in anti-Annexin A2 immunoprecipitates. GAPDH signal was used as protein loading control.</p>
</caption>
<graphic xlink:href="cancers-12-00215-g002"></graphic>
</fig>
<fig id="cancers-12-00215-f003" orientation="portrait" position="float"><label>Figure 3</label>
<caption><p>Hakai regulates Annexin A2 expression levels: (<bold>a</bold>
) HEK293T cells were transiently transfected with pcDNA-Flag-Hakai (4 µg), pBSSR-HA-ubiquitin (3 µg) and pSG-v-Src (3 µg) plasmids for 48 h. Cells were harvested and the levels of endogenous Annexin A2 levels and Hsp90 were determined by western blot analysis (left panel) and quantified by densitometry (right panel). The effect of the indicated transfected siRNA-Hakai on Annexin A2 and Hsp90 levels were tested in 293T cells (<bold>b</bold>
) and HCT116 (<bold>c</bold>
). Whole-cell lysates were subjected to western-blotting 72 h after transfection (top) and protein expression was quantified by densitometry (bottom) using GAPDH as loading control for normalization. Relative quantification of Annexin A2 expression levels was graphically represented as Mean ± SEM for two independent experiments for panel a and three for panel b and c (* <italic>p</italic>
< 0.05, ** <italic>p</italic>
< 0.01).</p>
</caption>
<graphic xlink:href="cancers-12-00215-g003"></graphic>
</fig>
<fig id="cancers-12-00215-f004" orientation="portrait" position="float"><label>Figure 4</label>
<caption><p>Geldanamycin treatment disrupts Hakai, Hsp90 and Annexin A2 interaction: HCT116 cell line was treated with 10 µM geldanamycin for 24 h. (<bold>a</bold>
) Immunoprecipitation was performed by using Hakai antibody. Subsequent western blotting-analysis was performed using Hakai, Annexin A2, or Hsp90 antibodies. (<bold>b</bold>
) Immunoprecipitation was performed by using Annexin A2 antibody and western blotting analysis was performed using Hakai and Hsp90 antibodies. GAPDH was used as protein loading control.</p>
</caption>
<graphic xlink:href="cancers-12-00215-g004"></graphic>
</fig>
<fig id="cancers-12-00215-f005" orientation="portrait" position="float"><label>Figure 5</label>
<caption><p>Geldanamycin reduces Hakai protein expression accompanied with an increase of Annexin A2: (<bold>a</bold>
) HEK293T and HCT116 cells were treated with geldanamycin at the indicated times and concentrations. Cell lysates were collected and subjected to western blot analysis with the indicated antibodies. (<bold>b</bold>
) HCT116 and HEK293T cell lines were treated with geldanamycin for 24 h at the indicated concentration. Cells were collected and subjected to mRNA extraction followed by RT-qPCR. Levels of Hakai mRNA normalized to control (HPRT) mRNA were measured and graphically represented as Mean ± SEM for three independent experiments (* <italic>p</italic>
< 0.05, ** <italic>p</italic>
< 0.01). (<bold>c</bold>
) HEK293T cells were transiently transfected with pcDNA 3.1 (10 µg) empty vector or pcDNA-Flag-Hakai (4 µg), pBSSR-HA-ubiquitin (3 µg) and pSG-v-Src (3 µg) plasmids for 48 h. Cells were treated with 10 µM geldanamycin 24 h after transfection. Cells were collected and protein levels were evaluated by western-blotting with the indicated antibodies. (<bold>d</bold>
) HCT116 cells were transfected with Hakai siRNA-1 and siRNA-2 oligos for 72 h and treated with 10 µM geldanamycin for the last 24 h of transfection. Cell lysates were collected and protein expression was evaluated by western-blotting with the indicated antibodies. Relative quantification of Annexin A2 expression levels was graphically represented as Mean ± SEM for three independent experiments (* <italic>p</italic>
< 0.05, ** <italic>p</italic>
< 0.01).</p>
</caption>
<graphic xlink:href="cancers-12-00215-g005"></graphic>
</fig>
<fig id="cancers-12-00215-f006" orientation="portrait" position="float"><label>Figure 6</label>
<caption><p>Geldanamycin induces an epithelial-like phenotype accompanied by an increase expression of E-cadherin and Annexin A2: HCT116 cell line was treated at the indicated concentrations of geldanamycin (GA) for 24 h. (<bold>a</bold>
) Images of HCT116 were taken by optical microscopy. (<bold>b</bold>
) Representative immunofluorescence images of Annexin A2 are shown (upper panel) and fluorescence intensity quantification is represented (lower panel). (<bold>c</bold>
) Representative immunofluorescence images of E-cadherin are shown (upper panel) and fluorescence intensity quantification is represented (lower panel). Images were taken with confocal microscope by employing 40× magnification objective. A zoom image of 80X magnification was included. Annexin A2 and E-cadherin were stained in green, and cell nuclei were counterstained with Hoechst. Quantification of intensity/area was represented as Mean ± SEM (**** <italic>p</italic>
< 0.0001).</p>
</caption>
<graphic xlink:href="cancers-12-00215-g006"></graphic>
</fig>
<fig id="cancers-12-00215-f007" orientation="portrait" position="float"><label>Figure 7</label>
<caption><p>Geldanamycin increases Hakai protein degradation in a lysosome-dependent manner: (<bold>a</bold>
) HEK293T cells were treated with chloroquine (left panel) and with MG132 (right panel) for 24 h at the indicated concentrations. Cell lysates were collected and evaluated by western blot analyses with Hakai and Annexin A2 antibodies. (<bold>b</bold>
) HEK293T cells were transiently transfected with pcDNA-Flag-Hakai (4 µg), pBSSR-HA-Ubiquitin (3 µg) and pSG-v-Src (3 µg) for 48 h. The day after transfection, cells were treated with chloroquine and geldanamycin at the indicated concentrations for 24 h. Cell lysates were collected and protein expression was evaluated by western blot analyses using the indicated antibodies. Chloroquine treatment-induced Hakai protein levels recovery during geldanamycin treatment. LC3 I/II levels was used as a positive control in chloroquine treatment and β-catenin as positive control in MG132 treatment.</p>
</caption>
<graphic xlink:href="cancers-12-00215-g007"></graphic>
</fig>
<fig id="cancers-12-00215-f008" orientation="portrait" position="float"><label>Figure 8</label>
<caption><p>Geldanamycin reduces Hakai-induced cell migration: HEK293T cells were transiently transfected with 8 µg of empty vector pcDNA 3.1 or pcDNA-Flag-Hakai for 54 h and motility was evaluated by transwell migration assay in presence or absence of 10 µM geldanamycin. Cells were seeded into migration transwells 48 h after transfection and let to migrate for 16 h in a gradient concentration of FBS (1–30%). Then cells were fixed and stained as indicated in methods. Images were obtained by optical microscopy with an objective of 10X magnification. Representative images taken using the 10× objective are shown (<bold>upper panel</bold>
) and quantification of migrating cells is shown (<bold>bottom panel</bold>
). Results are represented as as Mean ± SEM of triplicates (** <italic>p</italic>
< 0.01).</p>
</caption>
<graphic xlink:href="cancers-12-00215-g008"></graphic>
</fig>
<fig id="cancers-12-00215-f009" orientation="portrait" position="float"><label>Figure 9</label>
<caption><p>Hsp90 expression levels in colorectal cancer human samples: (<bold>a</bold>
) Representative images of Hsp90 immunoreactivity in normal colonic mucosa, adenoma and colorectal cancer (TNM stages I–IV). Images were obtained with 20× objective. Scale bar 125 µm. (<bold>b</bold>
) Statistical quantification of Hsp90 staining intensity in epithelial cancer cells at different colon cancer stages and in adenoma and normal colon tissue. A total of 30 human colon samples were analysed including: different TNM stages (I–IV) from colon adenocarcinomas, colon adenoma compared to human colon healthy tissues (normal colonic mucosa, n = 5; adenoma, n = 5; colorectal cancer, n = 5 of all stages) Scale bar, 200 µm. Black arrows show tumoral tissue and blue arrows show stromal tissue. Five photographs of each tissue were quantified and data are represented as scatter plot. Results are represented as Mean ± SEM for signal intensity scoring per area. Analysis was performed by employing Kruskal-Wallis with Tukey correction test (* <italic>p</italic>
< 0.05).</p>
</caption>
<graphic xlink:href="cancers-12-00215-g009"></graphic>
</fig>
</floats-group>
</pmc>
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