{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T23:06:24Z","timestamp":1790723184249,"version":"4.1.0"},"reference-count":42,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T00:00:00Z","timestamp":1770681600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"MUR PRIN 2017","award":["2017T8CMCY"],"award-info":[{"award-number":["2017T8CMCY"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Metastatic breast cancer (BC) remains a major clinical challenge, and identifying molecular mechanisms driving tumor cell migration and invasion is critical to develop effective therapeutic strategies. Clusterin (CLU), a secreted chaperone-like protein, is upregulated in BC and metastatic tissue; however, its functional contribution to tumor aggressiveness remains unclear. Here, we silenced CLU by siRNA in two BC cell lines with distinct aggressiveness and examined its impact on migration, invasion, and associated signaling pathways. Following CLU silencing, cell migration and invasion were assessed using transwell assays. Cytoskeletal organization was evaluated by F-actin staining, while downstream signaling pathways were analyzed by RT-PCR, Western blotting, and Rho GTPase pull-down. A comparative proteomic analysis was performed in CLU-expressing and CLU-silenced MDA-MB-231 cells. CLU knockdown significantly reduced migration and invasion in MDA-MB-231, concomitantly with loss of F-actin-rich membrane protrusions, reduced expression of MMP9, COL1A1, and COL4A1, and decreased activation of Akt, NF-\u03baB, and RhoA. Proteomic profiling revealed extensive remodeling of pathways involved in cell adhesion, cytoskeletal dynamics, and extracellular matrix interactions. Differently, no or very mild effects were observed in CLU-silenced MCF-7 cells. These findings identify CLU as an upstream regulator of a pro-metastatic adhesion\u2013cytoskeleton signaling in BC, selectively operative in EMT-engaged, basal-like cells, highlighting the importance of patient stratification for CLU-targeted therapeutic strategies.<\/jats:p>","DOI":"10.3390\/ijms27041721","type":"journal-article","created":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T17:10:52Z","timestamp":1770743452000},"page":"1721","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Clusterin Promotes the Migration and Invasion of Highly Aggressive Breast Cancer Cells Through Molecular Mechanisms That Affect the Cell Cytoskeleton and Extracellular Matrix Dynamics"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4109-310X","authenticated-orcid":false,"given":"Alessia","family":"Ciringione","sequence":"first","affiliation":[{"name":"Laboratory of Biochemistry, Molecular Biology and Oncometabolism, Department of Medicine and Surgery, University of Parma, Via Volturno 39, 43125 Parma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Marozzi","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Molecular Biology and Oncometabolism, Department of Medicine and Surgery, University of Parma, Via Volturno 39, 43125 Parma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0262-4485","authenticated-orcid":false,"given":"Silvana","family":"Belletti","sequence":"additional","affiliation":[{"name":"Histology and Embryology Laboratory, Department of Medicine and Surgery, University of Parma, Via Volturno 39, 43126 Parma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-0394-9414","authenticated-orcid":false,"given":"Margot","family":"Lo Pinto","sequence":"additional","affiliation":[{"name":"Proteomics Group of Ri.MED Foundation, Research Department, IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Via E. Tricomi 5, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7300-6153","authenticated-orcid":false,"given":"Simone Dario","family":"Scilabra","sequence":"additional","affiliation":[{"name":"Proteomics Group of Ri.MED Foundation, Research Department, IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Via E. Tricomi 5, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3951-7213","authenticated-orcid":false,"given":"Patrizia","family":"Cancemi","sequence":"additional","affiliation":[{"name":"Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale Delle Scienze, Ed. 16, 90128 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6670-2588","authenticated-orcid":false,"given":"Federica","family":"Rizzi","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Molecular Biology and Oncometabolism, Department of Medicine and Surgery, University of Parma, Via Volturno 39, 43125 Parma, Italy"},{"name":"National Institute of Biostructure and Biosystems (INBB), 00136 Rome, Italy"},{"name":"COMT-Center for Molecular and Translational Oncology, 43124 Parma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,10]]},"reference":[{"key":"ref_1","first-page":"229","article-title":"Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries","volume":"74","author":"Bray","year":"2024","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"R65","DOI":"10.1186\/bcr3679","article-title":"Proposed new clinicopathological surrogate definitions of luminal A and luminal B (HER2-negative) intrinsic breast cancer subtypes","volume":"16","author":"Maisonneuve","year":"2014","journal-title":"Breast Cancer Res."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"\u0141ukasiewicz, S., Czeczelewski, M., Forma, A., Baj, J., Sitarz, R., and Stanis\u0142awek, A. (2021). Breast cancer-epidemiology, risk factors, classification, prognostic markers, and current treatment strategies-an updated review. Cancers, 13.","DOI":"10.3390\/cancers13174287"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"S26","DOI":"10.1016\/j.breast.2015.07.008","article-title":"Clinical implications of the intrinsic molecular subtypes of breast cancer","volume":"24","author":"Prat","year":"2015","journal-title":"Breast"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"7714","DOI":"10.1016\/S0021-9258(18)32238-5","article-title":"Purification and characterization of a cell-aggregating factor (clusterin), the major glycoprotein in ram rete testis fluid","volume":"258","author":"Blaschuk","year":"1983","journal-title":"J. Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1080\/14728222.2017.1267142","article-title":"Clusterin as a therapeutic target","volume":"21","author":"Wilson","year":"2017","journal-title":"Expert Opin. Ther. Targets"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.bbagrm.2014.11.003","article-title":"Distinct promoters, subjected to epigenetic regulation, drive the expression of two clusterin mRNAs in prostate cancer cells","volume":"1849","author":"Bonacini","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"111174","DOI":"10.1016\/j.biopha.2020.111174","article-title":"Clusterin: Always protecting. Synthesis, function and potential issues","volume":"134","author":"Garcia","year":"2021","journal-title":"Biomed. Pharmacother."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1016\/j.lungcan.2012.11.024","article-title":"Prognostic role of clusterin in resected adenocarcinomas of the lung","volume":"79","author":"Panico","year":"2013","journal-title":"Lung Cancer"},{"key":"ref_10","first-page":"1","article-title":"Clusterin (CLU) and prostate cancer","volume":"105","author":"Rizzi","year":"2009","journal-title":"Adv. Cancer Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"514","DOI":"10.1159\/000351207","article-title":"The molecular chaperone apolipoprotein J\/clusterin as a sensor of oxidative stress: Implications in therapeutic approaches\u2014A mini-review","volume":"59","author":"Trougakos","year":"2013","journal-title":"Gerontology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1375","DOI":"10.1007\/s10238-022-00885-2","article-title":"The role and function of CLU in cancer biology and therapy","volume":"23","author":"Zhang","year":"2023","journal-title":"Clin. Exp. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"R1","DOI":"10.1677\/ERC-09-0140","article-title":"The clusterin paradigm in prostate and breast carcinogenesis","volume":"17","author":"Rizzi","year":"2010","journal-title":"Endocr. Relat. Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4081624","DOI":"10.1155\/2019\/4081624","article-title":"Clusterin Silencing in Prostate Cancer Induces Matrix Metalloproteinases by an NF-\u03baB-Dependent Mechanism","volume":"2019","author":"Bonacini","year":"2019","journal-title":"J. Oncol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4344","DOI":"10.1038\/onc.2009.286","article-title":"Genetic inactivation of ApoJ\/clusterin: Effects on prostate tumourigenesis and metastatic spread","volume":"28","author":"Bettuzzi","year":"2009","journal-title":"Oncogene"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"R86","DOI":"10.1186\/bcr1835","article-title":"Anticlusterin treatment of breast cancer cells increases the sensitivities of chemotherapy and tamoxifen and counteracts the inhibitory action of dexamethasone on chemotherapy-induced cytotoxicity","volume":"9","author":"Redondo","year":"2007","journal-title":"Breast Cancer Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1016\/S0002-9440(10)64552-X","article-title":"Overexpression of clusterin in human breast carcinoma","volume":"157","author":"Redondo","year":"2000","journal-title":"Am. J. Pathol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e373","DOI":"10.1016\/j.clbc.2017.08.007","article-title":"Stromal clusterin expression predicts therapeutic response to neoadjuvant chemotherapy in triple negative breast cancer","volume":"18","author":"Wang","year":"2018","journal-title":"Clin. Breast Cancer"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1837","DOI":"10.1158\/1535-7163.MCT-05-0178","article-title":"Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells both in vitro and in vivo","volume":"4","author":"So","year":"2005","journal-title":"Mol. Cancer Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"708","DOI":"10.1158\/1078-0432.CCR-08-1159","article-title":"Phase II trial of OGX-011 in combination with docetaxel in metastatic breast cancer","volume":"15","author":"Chia","year":"2009","journal-title":"Clin. Cancer Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e108647","DOI":"10.15252\/embj.2021108647","article-title":"Dynamic EMT: A multi-tool for tumor progression","volume":"40","author":"Brabletz","year":"2021","journal-title":"EMBO J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1038\/s41580-018-0067-1","article-title":"Actin-microtubule crosstalk in cell biology","volume":"20","author":"Dogterom","year":"2019","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5507","DOI":"10.1111\/bph.12704","article-title":"Movers and shakers: Cell cytoskeleton in cancer metastasis: Cytoskeleton and cancer metastasis","volume":"171","author":"Fife","year":"2014","journal-title":"Br. J. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"170","DOI":"10.4161\/cam.5.2.14403","article-title":"Dynamics of the Rho-family small GTPases in actin regulation and motility","volume":"5","author":"Spiering","year":"2011","journal-title":"Cell Adh Migr."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1038\/s41392-020-0134-x","article-title":"Molecular principles of metastasis: A hallmark of cancer revisited","volume":"5","author":"Fares","year":"2020","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1093\/abbs\/gms091","article-title":"Small interfering RNA targeted to secretory clusterin blocks tumor growth, motility, and invasion in breast cancer","volume":"44","author":"Niu","year":"2012","journal-title":"Acta Biochim. Biophys. Sin."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3131","DOI":"10.7150\/jca.18457","article-title":"Breast cancer cell line classification and its relevance with breast tumor subtyping","volume":"8","author":"Dai","year":"2017","journal-title":"J. Cancer"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Ciringione, A., and Rizzi, F. (2025). Facing the Challenge to Mimic Breast Cancer Heterogeneity: Established and Emerging Experimental Preclinical Models Integrated with Omics Technologies. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26104572"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1007\/s10549-014-3159-4","article-title":"Generation of MCF-7 cells with aggressive metastatic potential in vitro and in vivo","volume":"148","author":"Ziegler","year":"2014","journal-title":"Breast Cancer Res. Treat."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Aseervatham, J. (2020). Cytoskeletal remodeling in cancer. Biology, 9.","DOI":"10.3390\/biology9110385"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1042\/EBC20190015","article-title":"Actin dynamics in cell migration","volume":"63","author":"Schaks","year":"2019","journal-title":"Essays Biochem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"150","DOI":"10.3892\/or.2023.8587","article-title":"Downregulation of fascin induces collective cell migration in triple negative breast cancer","volume":"50","author":"Yamamoto","year":"2023","journal-title":"Oncol. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/j.ymthe.2004.08.029","article-title":"Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo","volume":"11","author":"Denoyelle","year":"2005","journal-title":"Mol. Ther."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"988","DOI":"10.1038\/nrd1902","article-title":"Exploiting the PI3K\/AKT pathway for cancer drug discovery","volume":"4","author":"Hennessy","year":"2005","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1080\/21541248.2017.1304855","article-title":"Cross-talk between Rho GTPases and PI3K in the neutrophil","volume":"10","author":"McCormick","year":"2019","journal-title":"Small GTPases"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"113978","DOI":"10.1016\/j.yexcr.2024.113978","article-title":"RANK\/RANKL axis promotes migration, invasion, and metastasis of osteosarcoma via activating NF-\u03baB pathway","volume":"436","author":"Takeda","year":"2024","journal-title":"Exp. Cell Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"104969","DOI":"10.1016\/j.yexmp.2025.104969","article-title":"The multifunctional protein CCN1\/CYR61: Bridging physiology and disease","volume":"142","author":"Kerek","year":"2025","journal-title":"Exp. Mol. Pathol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1124\/mi.8.4.8","article-title":"G Protein-Coupled Receptors Go Extracellular: RhoA Integrates the Integrins","volume":"8","author":"Walsh","year":"2008","journal-title":"Mol. Interv."},{"key":"ref_39","first-page":"59","article-title":"Functional domains of CCN1 (Cyr61) regulate breast cancer progression","volume":"33","author":"Chung","year":"2008","journal-title":"Int. J. Oncol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"60","DOI":"10.3892\/ijmm.2022.5116","article-title":"Talin-1 interaction network in cellular mechanotransduction (Review)","volume":"49","author":"Zhao","year":"2022","journal-title":"Int. J. Mol. Med."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3776","DOI":"10.1083\/jcb.201808061","article-title":"Talin as a mechanosensitive signaling hub","volume":"217","author":"Goult","year":"2018","journal-title":"J. Cell Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2435","DOI":"10.1242\/jcs.190991","article-title":"Talin\u2014The master of integrin adhesions","volume":"130","author":"Klapholz","year":"2017","journal-title":"J. Cell Sci."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1721\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T17:11:44Z","timestamp":1770743504000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1721"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,10]]},"references-count":42,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["ijms27041721"],"URL":"https:\/\/doi.org\/10.3390\/ijms27041721","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,10]]}}}