水解磷脂酸對人類卵巢癌SKOV3細胞分散及細胞連結分離之探討

Abstract

研究背景：水解磷脂酸(lysophosphatidic acid, LPA)是一種具有多樣生物功能的天然磷脂質，它能促進細胞增生、傷口癒合、以及神經細胞的突觸退縮等作用。此外，水解磷脂酸具有促進卵巢癌細胞生長的功能，在卵巢癌病患的腹水及血清中都含高濃度的脫脂磷脂酸。因此，水解磷脂酸被視為潛在性的卵巢癌生物標記。水解磷脂酸主要是藉由引發尿激素(urokinase)與基質金屬蛋白水解酶(MMP)的活性，同時促進應力纖維(stress fiber)及局部性黏著(focal adhesion)的形成，而達到促進卵巢癌細胞的移動。水解磷脂酸也會引發卵巢癌以外其他細胞株形態上的改變，包括：胞膜起皺褶(membrane ruffling)、板狀吸足生成(lamellipodia formation)、以及細胞與細胞相互分離。水解磷脂酸對卵巢癌細胞形態上的影響至今尚無定論。 研究目的：本研究針對水解磷脂酸在卵巢癌細胞形態上的影響，以及Src 家族蛋白質激酶與細胞黏連蛋白p120-catenin 的互動做探討，希望能藉由此研究，對水解磷脂酸促進卵巢癌細胞移動以及其分子機制，有更深入的了解。 研究題材及方法：本研究著重於水解磷脂酸對卵巢癌細胞間連結形態的探討，以及在卵巢癌細胞中，水解磷脂酸活化Src 家族蛋白質激酶對細胞間繫蛋白p120-catenin 的影響。並繼續探討水解磷脂酸活化上皮生長因子受體的機制，以及其對卵巢癌細胞間連結分離的影響。 研究結果：水解磷脂酸對具有混和上皮(epithelial)以及間質(mesenchymal)特性的卵巢癌細胞株SKOV3，引起細胞分散以及細胞黏連分離。本研究利用化學抑制劑PP2以及RNA干擾技術證明Src 家族蛋白質激酶為媒介水解磷脂酸此作用之重要蛋白。水解磷脂酸引發Src 家族蛋白質激酶活化，且促進其中之Fyn激酶與細胞連結蛋白p120-catenin的互動，進而調控與其他細胞黏連蛋白如β-catenin、α-catenin之穩定性。水解磷脂酸同時也活化其他的受體酪氨酸激酶(receptor tyrosine kinase)例如：上皮生長因子受體(EGFR)，本研究利用化學抑制劑AG 1478，證明上皮生長因子受體的活性對卵巢癌細胞間的連結分離扮演重要的角色。此外，藉由細胞間黏連蛋白質p120-catenin所提供之，被水解磷脂酸活化之Src 家族蛋白質激酶以及上皮生長因子受體，形成一個訊息傳遞整合的平台。 研究結論：水解磷脂酸對具有不同上皮特性的卵巢癌細胞株，造成不同的細胞分散以及細胞黏連分離的作用。水解磷脂酸經由活化Src 家族蛋白質激酶使卵巢癌細胞間的連結(cell-cell junction)分離，之後透過增加Src 家族蛋白質激酶之中的Fyn激酶與細胞間黏連蛋白質p120-catenin 的互動，影響p120-catenin 在細胞間連結的穩定性。水解磷脂酸與上皮生長因子受體之間的相互活化作用，也對於此一現象提供重要的調控機轉。

Abstract Background: Lysophosphatidic acid (LPA) is a natural phospholipid with various biological functions, such as cell proliferation, wound healing, and neurite retraction. LPA is also known as “ovarian cancer activating factor”, which was found to present at high concentrations in the plasma and ascites of ovarian cancer patients and is regarded as a potential biomarker for epithelial ovarian cancer. LPA has been known to promote invasion and migration of ovarian cancer cells via induction of urokinase secretion, MMP2 activation, stress fiber formation, and focal adhesion assembly. In addition, LPA has been found to cause colony dispersal effects by inducing morphological changes including membrane ruffling, lamellipodia formation, cell-cell dissociation and single cell migration in other carcinoma cell lines. However, the LPA effects on cell-cell dissociation of ovarian cancer cells are still inconclusive. Objectives: This study is proposed to investigate the LPA-induced morphological changes of cell-cell junctions and the signaling pathways of Src family kinases involved in the protein interactions with adherens junction protein p120-catenin in ovarian cancer cells and to clarify the effects of LPA on cell motility and cell-cell junctions and to give an in depth insight to the molecular mechanism of ovarian cancer cell migration. Materials and Methods: This study focuses on the morphological effects of LPA on cell-cell junctions and the Src-signaling pathway involved in ovarian cancer cells. In addition, this study also investigates the cross-talk of LPA and EGFR pathway and its significance on LPA-induced junction dispersal in ovarian cancer cells. Results: LPA induced cell dispersal and half junction formation in ovarian cancer cell line SKOV3, which coexpresses epithelial (E-cadherin, cytokeratin) and mesenchymal (N-cadherin, vimentin) markers. Src-family kinases were involved in both processes, since the effects were abolished by the selective tyrosine kinase inhibitor PP2. LPA induced rapid and transient activation of Src family kinases, which were recruited to cell-cell junctions by increasing the association with the adherens junction protein p120-catenin. Src family kinase, Fyn, was identified as the key component associated with p120-catenin after LPA stimulation in SKOV3 cells and was responsible for the cell dispersal. An EGFR-transactivation was also involved in LPA-induced cell dispersal, which could be abolished by the EGFR inhibitor, AG 1478. Conclusions: LPA induces differential cell dispersal responses in ovarian cancer cell lines harboring different epithelial and mesenchymal phenotypes. LPA-induced junction dispersal in ovarian cancer cells is caused by activating Src family kinases following by increasing p120-catenin and Src family kinase, Fyn, association to interfere with the stability of p120-catenin at cell-cell junction. The cross-talking to other receptor tyrosine kinase pathway, such as EGFR, is also required for the LPA effects on junction dispersal in ovarian cancer cells. Key words: ovarian cancer, lysophosphatidic acid, cell adhesion, Src family kinase, EGFR