Syndecan-1 (CD138) Modulates Triple-Negative Breast Cancer Stem Cell Properties via Regulation of LRP-6 and IL-6-Mediated STAT3 Signaling

<div><p>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.</p> </div

Syndecan−1 modulates activation of the STAT3 and NFκB signaling pathways and expression of LRP-6 in MDA−MB−231 breast cancer cells.

<p>Lysates of control and Syndecan−1 silenced cells were collected and 30-50µg protein/lane was immunoblotted and probed with the indicated antibodies. A) Western blot analysis reveals reduction of the phosphorylated form of STAT3 in Syndecan−1−silenced cells compared to controls. B) Syndecan−1 depletion leads to a significant reduced activation of NFκB. Immunoblot band intensities were normalized for tubulin expression and the data were analyzed using the paired Student's <i>t</i>-test. Data shown are triplicates from a single experiment representative of three independent experiments. **=p≤0.01, n≥3, error bars=SEM. C) Lysates of control and Syndecan−1 silenced cells were collected and 30-50µg protein/lane was immunoblotted and probed with an antibody recognizing LRP-6 (left panel). Immunoblot band intensities were normalized for tubulin expression and the data were analyzed using the paired Student's <i>t</i>-test (right panel). Data shown are triplicates from a single experiment representative of three independent experiments. **=p≤0.01, n≥3, error bars=SEM.</p

Syndecan-1 silencing impairs the formation of spheres and differentiation into cysts in MCF-7 cells.

<p>A) MCF-7 with sphere formation capacity were enriched for and transfected with a control siRNA or Syndecan-1 siRNA, followed by placing the cells in non-adherent culture conditions that promote sphere formation from single cells. After 4 days in suspension without siRNA it’s evident that Syndecan-1 knockdown is affecting proliferation. At this step it is already visible that the spheres are not only smaller but also more irregular compared to controls (upper panel). After 1 week, the spheres formed by Syndecan-1 transfected MCF-7 are less abundant, smaller and there are much more aggregates compared to controls (central panel). After 1 week in suspension culture, MCF-7 cells start to form cysts (bottom panel). The Syndecan-1-transfected cells show a drastically reduced cyst formation capability. The insert shows a Syndecan-1-depleted sphere that is generating a small “sphere/cyst hybrid”. Scale bar = 500µm (upper, central panels); 200µm (lower panel). B) Quantitative analysis of the sphere formation efficiency in control and Syndecan-1 siRNA-treated MCF-7 cells. Sphere number was determined 1 week after plating 3.000 transfected cells. Syndecan-1 siRNA-treatment results in a significant reduction of sphere formation efficiency (P<0.001, n=6).</p

siRNA-mediated knockdown of Syndecan-1 downregulates expression of IL-6R, IL-6 and CCL20 and dysregulates epithelial and mesenchymal marker protein expression in MDA-MB-231 breast cancer cells.

<p>A) left panel: RT-PCR analysis of IL-6R expression in MDA-MB-231 cells subjected to Syndecan-1 siRNA knockdown. Following total RNA isolation, mRNA was reverse transcribed and used as a template for PCR amplification of IL-6R. Right panel: PCR band intensities were normalized for actin expression and the data were analyzed using the paired Student's <i>t</i>-test. B) Left panel: Western blot analysis reveals reduction of IL-6R following Syndecan-1 silencing. Lysates of control and Syndecan-1 silenced cells were collected and 30-50µg protein/lane was immunoblotted and probed with sIL-6R antibody. Right panel: Immunoblot band intensities were normalized for tubulin expression and the data were analyzed using the paired Student's <i>t</i>-test. Data shown are triplicates from a single experiment representative of three independent experiments. C) left panel: RT-PCR analysis of IL-6 expression. Right panel: semiquantitative densitometric analysis (see panel A). D) left panel: RT-PCR analysis of CCL20 expression. Right panel: semiquantitative densitometric analysis (see panel A). *=p<0.05, ***=p<0.001, n≥3, error bars=SEM. E,F) The influence of IL-6 treatment on the expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (F) was studied by Western blotting. Cells were stimulated by 50ng/ml IL-6 24h after transfection with siRNA for 4h and 19h. In control cells, IL-6 treatment for 19h promoted EMT. Syndecan-1 depletion resulted in significant downregulation of E-cadherin expression. IL-6 treatment of Syndecan-1 depleted cells for 4h resulted in marker expression changes suggestive of enhanced mesenchymal-to-epithelial transition. (E,F) Upper panels = representative Western blots, lower panels = quantitative analysis. n≥3,*=P<0.05. G) Confocal immunofluorescence microscopy of phalloidin-labeled actin filaments reveals increased formation of actin stress fibers, filopodia (*) and lamellopodia (#) in Syndecan-1 siRNA-treated compared to control siRNA treated MCF-7 cells. </p

Characterization of SP cells concerning their CD44/CD24 expression.

<p>The combination of SP- and CD44/CD24 measurement revealed a proportion of 99.2% of SP cells with the CD44+/CD24(-/low) phenotype after control siRNA treatment while the Syndecan-1 knockdown approach showed that this proportion decreased only slightly to 98.7% (A). Influence of Syndecan-1 knockdown on CD24 expression. Compared to the control-siRNA treated cells, the Syndecan-1-siRNA transfected cells showed a 20% (±8.0%) increase in CD24 expression (*=P<0.05, n=3) (B).</p