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.

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine

“…Several in vivo and in vitro models support the idea that syndecan-1 promotes tumorigenesis by promoting Wnt signaling [203], tumor cell adhesion, spreading [230], angiogenesis [231], proliferation [232] and ECM signaling [233]. Recently, Ibrahim et al suggested that syndecan-1 promotes cancer stem cell properties in triple negative breast cancers [234], a factor that negatively impacts cancer therapies. The same study proposed that syndecan promotes stem cell properties via a pathway involving Wnt and IL-6/STAT3 signaling.…”

Secretome of tumor-associated leukocytes augment epithelial-mesenchymal transition in positive lymph node breast cancer patients via activation of EGFR/Tyr845 and NF-κB/p65 signaling pathway

“…Quantitative real-time PCR was conducted in duplicates for each gene of interest using SYBR Green dye and gene expression levels were measured in the step one plus detection System (Applied Biosystems, USA). We used the 2 −ΔΔCt method to analyze data after normalization to 18S ribosomal RNA (rRNA) as previously described [20]. Primer sequences are listed in Supplementary Table 1.…”

Targeting Cellular Signaling Pathways in Breast Cancer Stem Cells and its Implication for Cancer Treatment

“…Moreover, the classical co-receptor for growth and angiogenic factors in inflammatory response stimulus CD138 has been described as responsible for linking STAT3, Wnt and NF-ĸB signaling to modulate the BCSC phenotype (77). The self-renewal observed in BCSCs was referred to as being epigenetically regulated in the IL-6-STAT3-phosphatidylinositol 3-kinase (PI3K) pathway (78).…”