【作者】 陈慧君；

【导师】 吴绪峰；

【作者基本信息】 武汉大学 ， 临床医学肿瘤学， 2010， 博士

【摘要】 目的：上皮性卵巢癌以种植转移为主要转移方式,癌细胞常随腹腔液或腹水的流动播散至盆、腹腔,并种植生长于各脏器表面。卵巢癌患者腹水中常有溶血凝脂酸(Lysophosphatidic acid, LPA)水平的显著增高,LPA的生物功能之一即诱导细胞迁移,而细胞迁移正是肿瘤转移的关键步骤。故我们推测由LPA刺激导致的细胞迁移在卵巢癌的临床转移中具有关键性作用,然而目前尚无有关LPA诱导肿瘤细胞迁移的具体机制的报道。本研究探讨了LPA在卵巢癌转移中的作用、信号转导机制以及决定癌细胞转移能力的关键因子。方法：采用小室细胞迁移分析及裸鼠体内转移分析检测卵巢癌细胞株对LPA的反应性与细胞体内侵袭转移能力之间的关系。GST融合蛋白下拉分析检测比较侵袭性与非侵袭性卵巢癌细胞中Rho GTPases (Rac、Cdc42、RhoA)的活化情况。采用慢病毒介导的RNA干扰技术沉默相关基因的表达,腺病毒技术导入呈显性活性或失活的基因,慢病毒技术外源性导入缺失基因,以研究LPA诱导Rac活化的信号转导机制。免疫组化S-P法检测64例上皮性卵巢癌手术标本石蜡切片中SOS1、EPS8及ABIl的表达,并分析其与患者临床病例参数及预后的关系。结果：卵巢癌细胞对LPA诱导细胞迁移的反应性与其体内转移能力呈正相关。参与细胞迁移调控的Rho GTPases中,Rac的活化是LPA诱导细胞迁移的关键步骤：Rac(-)可阻断侵袭性细胞对LPA诱导迁移的反应性及体内转移瘤的形成(P<0.05),而Rac(+)可恢复非侵袭性细胞的LPA反应性及转移能力(P<0.05)。信号通路的研究表明：LPA诱导卵巢癌细胞迁移的信号转导机制为LPAR1-Ras-SOS1/EPS8/ABI1-Rac。LPA主要通过其跨膜受体LPAR1将诱导迁移的信号传至细胞内,封闭LPAR1而非其他受体的表达可显著阻断LPA诱导的卵巢癌细胞迁移(P<0.05)及Rac活化；随后,再通过Ras激活Rac的特异性鸟甘酸交换因子SOS1,SOS1与另两种蛋白EPS8及ABI1形成三重复合物,并在后二者的协同下激活Rac,进而诱导卵巢癌细胞的迁移。三重复合物SOS1/EPS8/ABI1的完整性对卵巢癌细胞的转移至关重要：沉默任一成员的表达均可阻断侵袭性细胞中LPA诱导的Rac活化、细胞迁移及体内转移(P<0.05)；所检测的非侵袭性细胞均有复合物某一成员的缺失,而外源性导入相应的缺失基因可恢复细胞的LPA反应性及转移能力(P<O.05)。临床检测显示,SOS 1/EPS8/ABI1的联合表达与卵巢癌患者的手术病理分期有关,期别越高则阳性率越高(P=0.01)。生存分析显示SOS1/EPS8/ABI1的联合表达与患者的预后有关：联合表达阳性患者的生存时间显著低于阴性患者(P=0.025)。结论：卵巢癌细胞的侵袭转移能力与其对LPA的反应性呈正相关,LPA诱导的Rac活化为卵巢癌细胞转移的关键步骤,引起该生物效应的信号转导途径为LPAR1-Ras-SOS1/EPS8/ABI1-Rac,其中三重复合物SOS1/EPS8/ABI1的完整性对决定卵巢癌细胞的转移能力具关键作用。SOS1/EPS8/ABI1的联合表达可成为指示卵巢癌患者预后的可靠指标,该三重复合物可成为阻断卵巢癌转移的有效靶点。更多还原

【Abstract】 Objective:Ovarian cancer is mainly confined in peritoneal cavity and its spread is aided by peritoneal ascites. Lysophosphatidic acid (LPA) is present at high levels in ovarian cancer patients’ ascites and potently stimulates cell migration, which faciliate the procession of cancer metastasis. Thus, we hypothesize that LPA-induced cell migration might be a key step in ovarian cancer metastasis, in which the mechanisam in detail is still unkown. Our purpose is to determine the role of LPA in ovarian cancer metastasis, the underling signaling mechanisms and the key factors responsible for the migratory capacity of cancer cells.Methods:Transwell migration and peritoneal metastatic colonization assays were performed to determine the correlation between LPA-stimulated cell migration and metastatic potentials. We compared LPA-induced Rho GTpases activation between metastatic and non-metastatic ovarian cancer cell lines and characterized the involved signaling mechanisms with genetic mutants and shRNAs. Finally, we analyzed the correlation between SOS1/EPS8/ABI1 co-expression and clinical status of ovarian cancer patients by immunohistochemistry.Results:Ovarian cancer cell lines with LPA migratory response were metastatic and vice versa. Among Rho GTPases, Rac activation is essential for LPA-induced cell migration. Because Rac(-) could blocked the LPA response and metastasis of invasive cells (P<0.05); Rac(+) could confer the non-invasive cells with LPA response and metastatic capability (P<0.05).The signaling mechanism study demonstrated that: the pathway responsible for LPA-induced cell migration was LPAR1-Ras-SOS1/EPS8/ABI1-Rac. LPA transduce the migratory signal into the cells through LPAR1 but the other LPA receptors, because knock down the expression of LPAR1 could block the LPA-indcuced cell migration (P<0.05) and Rac activation. Then signal from LPA will active Rac-specific guanine nucleotide exchange factor SOS1. SOS1 forms a tri-complex with EPS8 and ABI1, and active Rac with the coordination of these two proteins. The integrity of SOS1/EPS8/ABI1 tri-complex was necessary for ovarian cancer metastasis, becasue silence any member of the complex could block the LPA-induced Rac activation, cell migration and metastatic colonization (P<0.05). Members of SOS1/EPS8/ABI1 tri-complex were absent in non-metastatic ovarian cancer cells and re-expressing the missing one conferred them with LPA responsiveness and metastatic capability (P<0.05).Importantly, clinical samples detection showed that SOS1/EPS8/ABI1 co-expression correlated to advanced clinic stages (P=0.00l) and shorter survival of ovarian cancer patients (P=0.025).Conclusions:The magritory capability of the ovarian cancer cells is closely correlated to the LPA responsiveness of the cells. The LPA-induced Rac activation is essential for ovarian cancer metastasis, and LPAR1-Ras-SOS1/EPS8/ABIl-Rac signaling pathway is responsible for this biologic effect. The integrity of SOS1/EPS8/ABI1 tri-complex is a determinant of metastatic capability of ovarian cancer cells. Co-expression of SOS1/EPS8/ABI1 might be an reliable indicator of patients’survival. This tri-complex could be a target for anti-cancer treatment.更多还原