Table_1_CD44 Expression Predicts Prognosis of Ovarian Cancer Patients Through Promoting Epithelial-Mesenchymal Transition (EMT) by Regulating Snail, ZEB1, and Caveolin-1.DOC

Objectives: CD44, a transmembrane glycoprotein, is involved in the generation of a stem cell niche and maintaining stem cell quiescence. The aim of this study was to evaluate its contribution to ovarian cancer prognosis and progression, as well as explore the possible mechanisms.
Materials and Methods: The expression of CD44 in tissue microarray of 90 ovarian cancer patients was detected by immunohistochemistry. Kaplan-Meier method and Cox proportional hazard model were used to evaluate the factors associated with 5-year overall survival and disease-free survival. CD44 was knocked down by small interfering RNA, the expression of Snail, ZEB1, and Caveolin-1 in a stable Snail-expressing ovarian cancer cell line HO8910PM-Snail (HOPM-Snail) and its control cell line HO8910PM-vector (HOPM) was detected by western blotting analysis. Cell clone formation, migration, and invasion of HOPM-Snail and HOPM cells with CD44 silencing were examined by 3-D culture assay, wound healing assay, and transwell assay, respectively.
Results: Over-expression of CD44 was associated with advanced histological grade (p = 0.014) and FIGO stage (p = 0.001). Multivariate analysis showed that CD44 expression was an independent prognostic factor to predict both overall survival (p = 0.004) and disease-free survival (p = 0.025) of ovarian cancer patients. Down-regulation of CD44 expression by small silencing RNA abrogated both basal Snail expression and TGF-β1-induced Snail expression in HOPM and HOPM-Snail cells. In addition, CD44 knockdown caused a decrease in ZEB1 expression. RPPA data indicated that Caveolin-1 may be another regulative target of CD44, and western blotting analysis confirmed that CD44 knockdown caused an increase in Caveolin-1 expression. However, there was no noticeable reciprocal regulation among ZEB1, Caveolin-1, and Snail. Moreover, CD44 knockdown caused a decrease in cell clone formation, migration, and invasion of HOPM and HOPM-Snail cells.
Conclusions: As both Snail and ZEB1 are crucial inducers of epithelial-to-mesenchymal transition (EMT), our data suggested that CD44 may be crucial for the EMT process of ovarian cancer. Therefore, CD44 may be a potential prognostic marker as well as treatment target for ovarian cancer.

研究目的：CD44是一种跨膜糖蛋白（transmembrane glycoprotein），参与干细胞龛（stem cell niche）的构建并维持干细胞静息态（stem cell quiescence）。本研究旨在评估其对卵巢癌预后与进展的影响，并探讨潜在的作用机制。

材料与方法：采用免疫组织化学（immunohistochemistry）法检测90例卵巢癌患者组织芯片中CD44的表达水平。采用Kaplan-Meier法（Kaplan-Meier method）与Cox比例风险模型（Cox proportional hazard model）分析与5年总生存期及无病生存期相关的影响因素。通过小干扰RNA（small interfering RNA）敲低CD44的表达，采用蛋白质印迹（western blotting）分析分别检测稳定过表达Snail的卵巢癌细胞系HO8910PM-Snail（HOPM-Snail）及其对照细胞系HO8910PM-vector（HOPM）中Snail、ZEB1与Caveolin-1的表达水平。分别通过三维培养实验、划痕愈合实验与Transwell实验检测敲低CD44后的HOPM-Snail与HOPM细胞的克隆形成、迁移与侵袭能力。

结果：CD44高表达与较高的组织学分级（p = 0.014）及FIGO分期（FIGO）显著相关。多因素分析显示，CD44表达是预测卵巢癌患者总生存期（p = 0.004）与无病生存期（p = 0.025）的独立预后因素。在HOPM与HOPM-Snail细胞中，下调CD44的表达可同时抑制基础Snail表达与转化生长因子-β1（TGF-β1）诱导的Snail表达。此外，敲低CD44可降低ZEB1的表达水平。反向蛋白阵列分析（reverse phase protein array, RPPA）数据显示，Caveolin-1可能是CD44的另一调控靶点，蛋白质印迹实验证实敲低CD44可上调Caveolin-1的表达。但ZEB1、Caveolin-1与Snail三者之间未观察到明显的相互调控作用。进一步研究发现，敲低CD44可降低HOPM与HOPM-Snail细胞的克隆形成、迁移与侵袭能力。

结论：由于Snail与ZEB1均为上皮间质转化（epithelial-to-mesenchymal transition, EMT）的关键诱导因子，本研究数据表明CD44可能在卵巢癌的上皮间质转化过程中发挥关键作用。因此，CD44有望成为卵巢癌潜在的预后标志物与治疗靶点。