Division of Obstetrics and Gynecology, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, S-171 76 Stockholm, Sweden.
Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tartu, 51014 Tartu, Estonia.
Hum Reprod. 2020 Oct 1;35(10):2280-2293. doi: 10.1093/humrep/deaa164.
What is the physiological role of transforming growth factor-beta (TGF-β1) and syndecans (SDC1, SDC4) in endometriotic cells in women with endometriosis?
We observed an abnormal, pro-invasive phenotype in a subgroup of samples with ovarian endometriosis, which was reversed by combining gene silencing of SDC1 with the TGF-β1 treatment.
Women with endometriosis express high levels of TGF-β1 and the proteoglycan co-receptors SDC1 and SDC4 within endometriotic cysts. However, how SDC1 and SDC4 expression is regulated by TGF-β1 and the physiological significance of the high expression in endometriotic cysts remains unknown as does the potential role in disease severity.
STUDY DESIGN, SIZE, DURATION: We utilized a pre-validated panel of stem- and cancer cell-associated markers on endometriotic tissue (n = 15) to stratify subgroups of women with endometriosis. Furthermore, CD90+CD73+CD105+ (SC+) endometriotic stromal cells from these patient subgroups were explored for their invasive behaviour in vitro by transient gene inhibition of SDC1 or SDC4, both in the presence or absence of TGF-β1 treatment.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometriotic cyst biopsies (n = 15) were obtained from women diagnosed with ovarian endometriosis (ASRM Stage III-IV). Gene expression variability was assessed on tissue samples by applying gene clustering tools for the dataset generated from the pre-validated panel of markers. Three-dimensional (3D) spheroids from endometriotic SC+ were treated in vitro with increasing doses of TGF-β1 or the TGFBRI/II inhibitor Ly2109761 and assessed for SDC1, SDC4 expression and in vitro 3D-spheroid invasion. Transcriptomic signatures from the invaded 3D spheroids were evaluated upon combining transient gene silencing of SDC1 or SDC4, both in presence or absence of TGF-β1 treatment. Furthermore, nanoscale changes on the surface of endometriotic cells were analysed after treatment with TGF-β1 or TGFBRI/II inhibitor using atomic force microscopy.
Gene clustering analysis revealed that endometriotic tissues displayed variability in their gene expression patterns; a small subgroup of samples (2/15, Endo-hi) exhibited high levels of SDC1, SDC4 and molecules involved in TGF-β signalling (TGF-β1, ESR1, CTNNB1, SNAI1, BMI1). The remaining endometriotic samples (Endo-lo) showed a uniform, low gene expression profile. Three-dimensional spheroids derived from Endo-hi SC+ but not Endo-lo SC+ samples showed an aberrant expression of SDC1 and exhibited enhanced 3D-spheroid invasion in vitro, upon rhTGF-β1 treatment. However, this abnormal, pro-invasive response of Endo-hi SC+ was reversed upon gene silencing of SDC1 with the TGF-β1 treatment. Interestingly, transcriptomic signatures of 3D spheroids silenced for SDC1 and consecutively treated with TGF-β1, showed a down-regulation of cancer-associated pathways such as WNT and GPCR signalling.
Transcriptomic data were deposited in NCBI's Gene Expression Omnibus (GEO) and could be retrieved using GEO series accession number: GSE135122.
LIMITATIONS, REASONS FOR CAUTION: It is estimated that about 2.5% of endometriosis patients have a potential risk for developing ovarian cancer later in life. It is possible that the pro-oncogenic molecular changes observed in this cohort of endometriotic samples may not correlate with clinical occurrence of ovarian cancer later in life, thus a validation will be required.
This study emphasizes the importance of interactions between syndecans and TGF-β1 in the pathophysiology of endometriosis. We believe that this knowledge could be important in order to better understand endometriosis-associated complications such as ovarian cancer or infertility.
STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Cancerfonden (CAN 2016/696), Radiumhemmets Forskningsfonder (Project no. 154143 and 184033), EU MSCA-RISE-2015 project MOMENDO (691058), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695) and Karolinska Institute. Authors do not have any conflict of interest.
转化生长因子-β（TGF-β1）和连接蛋白（SDC1、SDC4）在子宫内膜异位症患者的子宫内膜异位症细胞中的生理作用是什么？
我们观察到卵巢子宫内膜异位症患者的亚组样本中存在异常的侵袭表型，这种表型通过联合基因沉默 SDC1 与 TGF-β1 处理而逆转。
子宫内膜异位症患者的子宫内膜异位囊肿中表达高水平的 TGF-β1 和蛋白聚糖共受体 SDC1 和 SDC4。然而，SDC1 和 SDC4 的表达如何受 TGF-β1 调节以及在子宫内膜异位囊肿中高表达的生理意义仍然未知，也不知道它们在疾病严重程度中的潜在作用。
研究设计、规模、持续时间：我们使用了经过验证的干细胞和癌症相关标志物的预验证面板，对子宫内膜异位症患者进行了分层（n=15）。此外，我们还探索了来自这些患者亚组的 CD90+CD73+CD105+（SC+）子宫内膜间质细胞的体外侵袭行为，通过瞬时基因抑制 SDC1 或 SDC4，以及在存在或不存在 TGF-β1 处理的情况下进行。
参与者/材料、设置、方法：从诊断为卵巢子宫内膜异位症（ASRM 分期 III-IV）的女性中获取子宫内膜异位症囊肿活检（n=15）。通过应用数据集的基因聚类工具，评估组织样本中的基因表达变异性，该数据集是由经过验证的干细胞和癌症相关标志物预验证面板生成的。将来自子宫内膜异位症 SC+的三维（3D）球体在体外用不同剂量的 TGF-β1 或 TGFBRI/II 抑制剂 Ly2109761 处理，并评估 SDC1、SDC4 的表达和体外 3D-球体侵袭。在存在或不存在 TGF-β1 处理的情况下，将瞬时基因沉默 SDC1 或 SDC4 结合在一起，评估入侵的 3D 球体的转录组特征。此外，使用原子力显微镜分析 TGF-β1 或 TGFBRI/II 抑制剂处理后子宫内膜异位症细胞表面的纳米级变化。
基因聚类分析显示，子宫内膜组织的基因表达模式存在变异性；一小部分样本（2/15，Endo-hi）表现出高水平的 SDC1、SDC4 和 TGF-β 信号转导中涉及的分子（TGF-β1、ESR1、CTNNB1、SNAI1、BMI1）。其余的子宫内膜异位症样本（Endo-lo）表现出均匀的、低水平的基因表达谱。来自 Endo-hi SC+的 3D 球体，但不是来自 Endo-lo SC+的 3D 球体，表现出 SDC1 的异常表达，并在 rhTGF-β1 处理后在体外表现出增强的 3D-球体侵袭。然而，这种 Endo-hi SC+的异常侵袭反应在 TGF-β1 处理时通过基因沉默 SDC1 而逆转。有趣的是，沉默 SDC1 并随后用 TGF-β1 处理的 3D 球体的转录组特征显示，WNT 和 GPCR 信号等癌症相关途径的下调。
转录组数据已被存入 NCBI 的基因表达综合数据库（GEO），并可使用 GEO 系列注册号：GSE135122 进行检索。
局限性、谨慎的原因：据估计，约有 2.5%的子宫内膜异位症患者有潜在的卵巢癌风险，在生命的后期发生。因此，在这个子宫内膜异位症样本的队列中观察到的促癌分子变化可能与生命后期卵巢癌的临床发生无关，因此需要进行验证。
这项研究强调了连接蛋白和 TGF-β1 在子宫内膜异位症发病机制中的相互作用的重要性。我们相信，这些知识对于更好地理解子宫内膜异位症相关并发症（如卵巢癌或不孕）可能很重要。
研究资金/利益冲突：本研究由 Cancerfonden（CAN 2016/696）、Radiumhemmets Forskningsfonder（项目编号 154143 和 184033）、欧盟 MSCA-RISE-2015 项目 MOMENDO（691058）、爱沙尼亚教育和研究部（IUT34-16）、Enterprise Estonia（EU48695）和卡罗林斯卡学院资助。作者没有任何利益冲突。
