Ibrahim Sherif Abdelaziz, Gadalla Ramy, El-Ghonaimy Eslam A, Samir Omnia, Mohamed Hossam Taha, Hassan Hebatallah, Greve Burkhard, El-Shinawi Mohamed, Mohamed Mona Mostafa, Götte Martin
Department of Zoology, Faculty of Science, Cairo University, 12613, Giza, Egypt.
Department of Radiotherapy-Radiooncology, University Hospital Münster, Münster, Germany.
Mol Cancer. 2017 Mar 7;16(1):57. doi: 10.1186/s12943-017-0621-z.
Inflammatory breast cancer (IBC), a particularly aggressive form of breast cancer, is characterized by cancer stem cell (CSC) phenotype. Due to a lack of targeted therapies, the identification of molecular markers of IBC is of major importance. The heparan sulfate proteoglycan Syndecan-1 acts as a coreceptor for growth factors and chemokines, modulating inflammation, tumor progression, and cancer stemness, thus it may emerge as a molecular marker for IBC.
We characterized expression of Syndecan-1 and the CSC marker CD44, Notch-1 & -3 and EGFR in carcinoma tissues of triple negative IBC (n = 13) and non-IBC (n = 17) patients using qPCR and immunohistochemistry. Impact of siRNA-mediated Syndecan-1 knockdown on the CSC phenotype of the human triple negative IBC cell line SUM-149 and HER-2-overexpressing non-IBC SKBR3 cells employing qPCR, flow cytometry, Western blotting, secretome profiling and Notch pharmacological inhibition experiments. Data were statistically analyzed using Student's t-test/Mann-Whitney U-test or one-way ANOVA followed by Tukey's multiple comparison tests.
Our data indicate upregulation and a significant positive correlation of Syndecan-1 with CD44 protein, and Notch-1 & -3 and EGFR mRNA in IBC vs non-IBC. ALDH1 activity and the CD44CD24 subset as readout of a CSC phenotype were reduced upon Syndecan-1 knockdown. Functionally, Syndecan-1 silencing significantly reduced 3D spheroid and colony formation. Intriguingly, qPCR results indicate downregulation of the IL-6, IL-8, CCL20, gp130 and EGFR mRNA upon Syndecan-1 suppression in both cell lines. Moreover, Syndecan-1 silencing significantly downregulated Notch-1, -3, -4 and Hey-1 in SUM-149 cells, and downregulated only Notch-3 and Gli-1 mRNA in SKBR3 cells. Secretome profiling unveiled reduced IL-6, IL-8, GRO-alpha and GRO a/b/g cytokines in conditioned media of Syndecan-1 knockdown SUM-149 cells compared to controls. The constitutively activated STAT3 and NFκB, and expression of gp130, Notch-1 & -2, and EGFR proteins were suppressed upon Syndecan-1 ablation. Mechanistically, gamma-secretase inhibition experiments suggested that Syndecan-1 may regulate the expression of IL-6, IL-8, gp130, Hey-1, EGFR and p-Akt via Notch signaling.
Syndecan-1 acts as a novel tissue biomarker and a modulator of CSC phenotype of triple negative IBC via the IL-6/STAT3, Notch and EGFR signaling pathways, thus emerging as a promising therapeutic target for IBC.
炎性乳腺癌（IBC）是一种侵袭性特别强的乳腺癌，具有癌干细胞（CSC）表型。由于缺乏靶向治疗方法，鉴定IBC的分子标志物至关重要。硫酸乙酰肝素蛋白聚糖Syndecan-1作为生长因子和趋化因子的共受体，调节炎症、肿瘤进展和癌干性，因此它可能成为IBC的分子标志物。
我们使用qPCR和免疫组织化学方法，对三阴性IBC患者（n = 13）和非IBC患者（n = 17）癌组织中Syndecan-1、CSC标志物CD44、Notch-1和-3以及EGFR的表达进行了表征。采用qPCR、流式细胞术、蛋白质印迹、分泌组分析和Notch药理学抑制实验，研究了siRNA介导的Syndecan-1敲低对人三阴性IBC细胞系SUM-149和HER-2过表达的非IBC细胞系SKBR3细胞CSC表型的影响。数据采用Student's t检验/Mann-Whitney U检验或单因素方差分析，随后进行Tukey多重比较检验进行统计学分析。
我们的数据表明，与非IBC相比，IBC中Syndecan-1与CD44蛋白、Notch-1和-3以及EGFR mRNA上调且呈显著正相关。Syndecan-1敲低后，作为CSC表型读数的ALDH1活性和CD44CD24亚群减少。在功能上，Syndecan-1沉默显著减少了3D球体和集落形成。有趣的是，qPCR结果表明，在两种细胞系中，Syndecan-1抑制后IL-6、IL-8、CCL20、gp130和EGFR mRNA下调。此外，Syndecan-1沉默在SUM-149细胞中显著下调Notch-1、-3、-4和Hey-1，在SKBR3细胞中仅下调Notch-3和Gli-1 mRNA。分泌组分析显示，与对照相比，Syndecan-1敲低的SUM-149细胞条件培养基中IL-6、IL-8、GRO-α和GRO a/b/g细胞因子减少。Syndecan-1缺失后，组成型激活的STAT3和NFκB以及gp130、Notch-1和-2以及EGFR蛋白的表达受到抑制。机制上，γ-分泌酶抑制实验表明，Syndecan-1可能通过Notch信号调节IL-6、IL-8、gp130、Hey-1、EGFR和p-Akt的表达。
Syndecan-1通过IL-6/STAT3、Notch和EGFR信号通路，作为三阴性IBC的新型组织生物标志物和CSC表型的调节剂，因此成为IBC有前景的治疗靶点。
