title: Syndecan-1 (CD138) modulates triple-negative breast cancer stem cell properties via regulation of LRP-6 and IL-6-mediated STAT3 signaling.
abstract: Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis. Resistance of Syndecan-1-deficient mice to experimentally-induced tumorigenesis has been linked to altered Wnt-responsive precursor cell pools, suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. Here, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human triple-negative MDA-MB-231 and hormone receptor-positive MCF-7 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown in MDA-MB-231 cells significantly reduced putative cancer stem cell pools by 60% and 27%, respectively, compared to controls. In MCF-7 cells, Syndecan-1 depletion reduced the side population by 40% and Aldehyde dehydrogenase-1 by 50%, repectively. In MDA-MB-231 cells, the CD44(+)CD24(-/low) phenotype decreased significantly by 6% upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated by >40% in Syndecan-1-silenced MDA-MB-231 cells, which showed a dysregulated response to IL-6-induced shifts in E-cadherin and vimentin expression. Furthermore, activation of STAT-3 and NFkB transcription factors and expression of a coreceptor for Wnt signaling, LRP-6, were reduced by >45% in Syndecan-1-depleted cells compared to controls. At the functional level, Syndecan-1 siRNA reduced the formation of spheres and cysts in MCF-7 cells grown in suspension culture. Our study demonstrates the viability of flow cytometric approaches in analyzing cancer stem cell function. As Syndecan-1 modulates the cancer stem cell phenotype via regulation of the Wnt and IL-6/STAT3 signaling pathways, it emerges as a promising novel target for therapeutic approaches.
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital Münster, Münster, Germany ; Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt.
authors.forename: Sherif A
authors.initials: SA
authors.lastname: Ibrahim
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital Münster, Münster, Germany ; Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt.
authors.forename: Hebatallah
authors.initials: H
authors.lastname: Hassan
authors.affiliation: ITB-CNR, Segrate Milan, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Vilardo
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital Münster, Münster, Germany.
authors.forename: Sampath Katakam
authors.initials: SK
authors.lastname: Kumar
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital Münster, Münster, Germany.
authors.forename: Archana Vijaya
authors.initials: AV
authors.lastname: Kumar
authors.affiliation: Institute of Transfusion Medicine and Transplantation Immunology, University Hospital Münster, Münster, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Kelsch
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital Münster, Münster, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Schneider
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital Münster, Münster, Germany.
authors.forename: Ludwig
authors.initials: L
authors.lastname: Kiesel
authors.affiliation: Department of Radiotherapy - Radiooncology, University Hospital Münster, Münster, Germany.
authors.forename: Hans Theodor
authors.initials: HT
authors.lastname: Eich
authors.affiliation: ITB-CNR, Segrate Milan, Italy.
authors.forename: Ileana
authors.initials: I
authors.lastname: Zucchi
authors.affiliation: ITB-CNR, Segrate Milan, Italy.
authors.forename: Rolland
authors.initials: R
authors.lastname: Reinbold
authors.affiliation: Department of Radiotherapy - Radiooncology, University Hospital Münster, Münster, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Greve
authors.affiliation: Department of Gynecology and Obstetrics, University Hospital Münster, Münster, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Götte
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D002454:Cell Differentiation; D015536:Down-Regulation; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000378:metabolism*; D061986:MCF-7 Cells; D016328:NF-kappa B / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000473:pathology*; D034741:RNA, Small Interfering / Q000235:genetics; D050697:Retinal Dehydrogenase / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000473:pathology; D053668:Syndecan-1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D064726:Triple Negative Breast Neoplasms / Q000473:pathology*; D051153:Wnt Proteins / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D007527:Isoenzymes; C555475:LRP6 protein, human; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D016328:NF-kappa B; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor; D053668:Syndecan-1; D051153:Wnt Proteins; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse; D050697:Retinal Dehydrogenase