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.
Highlights
Breast cancer is a complex disease and is the second leading cause of cancer mortality among women worldwide [1]
Flow cytometry-based ALDH isoform 1 (ALDH1) activity analysis revealed that Syndecan-1-silenced cells had a pool of 3.39% (±0.19%) ALDH1-positive cells compared to a pool of 4.63% (±0.31%) ALDH1-positive cells in controls, corresponding to a significant relative decrease by >25% upon Syndecan-1 depletion (P
We have combined flow cytometry analysis with a molecular biological approach to demonstrate a role for the heparan sulfate proteoglycan Syndecan-1 in human breast cancer stem cells (CSCs)
Summary
Breast cancer is a complex disease and is the second leading cause of cancer mortality among women worldwide [1]. Several lines of evidence suggest that, in contrast to the bulk of the tumor, a subset of cancer cells is characterized by the property of self-renewal, unlimited proliferative potential, expression of multidrug-resistance proteins, active DNA repair capacity, apoptosis resistance, and an enormous developmental plasticity [2,3,4,5,6,7] Due to these properties, these cancer stem cells (CSCs) display increased resistance to chemo- [8,9] and radiotherapy [10,11] and have the potential to reconstitute the bulk tumor after an otherwise successful therapy [9,12,13]. The development of therapies eliminating CD44(+)/CD24(-/low) CSCs or impeding activation of the signaling pathways these cells rely on may represent a promising approach for basal-like breast cancer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
