Epigenetic Inactivation of EFEMP1 Is Associated with Tumor Suppressive Function in Endometrial Carcinoma

ObjectiveEFEMP1, the epidermal growth factor–containing fibulin-like extracellular matrix protein 1, functions as an oncogene or a tumor suppressor depending on the cancer types. In this study, we aim to determine whether EFEMP1 affects the tumorigenesis and progression of endometrial carcinoma.MethodsThe expression of EFEMP1 was investigated using immunohistochemistry in a panel of normal endometrium (n = 40), atypical hyperplasia (n = 10) and endometrial carcinoma tissues (n = 84). Methylation status of the EFEMP1 promoter was detected by methylation-specific PCR (MSP) and bisulphite genomic sequencing. Up- or down-regulation of EFEMP1 were achieved by stable or transient transfection with pCMV6/GFP/Neo-EFEMP1 or pGPU6/GFP/Neo-shEFEMP1 respectively. Effects of EFEMP1 on tumor proliferation, invasion and migration were evaluated by MTT, plate colony formation, Transwell and wound healing assay. The nude mouse tumor xenograft assay was used to investigate function of EFEMP1 in vivo.ResultsCompared with normal endometrium (32/40) and atypical hyperplasia (7/10), EFEMP1 expression was much lower in endometrial carcinoma tissues (16/84) (Pin vitro and suppressed tumorigenesis in nude mice. Besides, EFEMP1 increased expression of E-cadherin and suppressed expression of vimentin in endometrial carcinoma.ConclusionEFEMP1 is a new candidate tumor suppressor gene in endometrial carcinoma, and is frequently silenced by promoter hypermethylation. It could inhibit tumor growth and invasion both in vitro and in vivo. Our findings propose that targeting EFEMP1 might offer future clinical utility in endometrial carcinoma.

研究目的：EFEMP1（epidermal growth factor–containing fibulin-like extracellular matrix protein 1，含表皮生长因子域的纤连蛋白样细胞外基质蛋白1）的功能因癌症类型而异，既可作为癌基因，也可充当肿瘤抑制基因。本研究旨在明确EFEMP1对子宫内膜癌发生与进展的影响。
方法：采用免疫组化法检测40例正常子宫内膜组织、10例不典型增生组织及84例子宫内膜癌组织中EFEMP1的表达水平。通过甲基化特异性PCR（methylation-specific PCR, MSP）及亚硫酸氢盐基因组测序检测EFEMP1启动子的甲基化状态。分别通过稳定转染pCMV6/GFP/Neo-EFEMP1或瞬时转染pGPU6/GFP/Neo-shEFEMP1，实现EFEMP1的过表达或敲低。采用MTT实验、平板克隆形成实验、Transwell实验及划痕愈合实验评估EFEMP1对肿瘤增殖、侵袭及迁移能力的影响。通过裸鼠异种移植瘤实验探究EFEMP1在体内的功能。
结果：与正常子宫内膜组织（32/40）及不典型增生组织（7/10）相比，子宫内膜癌组织中EFEMP1的表达显著降低（16/84）。EFEMP1可在体外抑制子宫内膜癌细胞的增殖、侵袭与迁移，并在裸鼠体内抑制肿瘤发生。此外，EFEMP1可上调子宫内膜癌细胞中E-钙粘蛋白（E-cadherin）的表达，同时下调波形蛋白（vimentin）的表达。
结论：EFEMP1是子宫内膜癌中一种新型候选肿瘤抑制基因，其表达常因启动子高甲基化而发生沉默。该基因可在体内外抑制肿瘤的生长与侵袭。本研究结果提示，靶向EFEMP1有望为子宫内膜癌的临床治疗提供新的应用策略。