2018-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657064摘要：卵巢上皮細胞癌是女性最致命的婦科癌症，通常在診斷時已進入第三期，以標準的减積手術治療及包含鉑類和紫杉醇的化學治療，預後仍然很差。目前應用較新的抗血管形成（angiogenesis）的鏢靶治療，可以有效延長病人的存活期，但很快的多數病人會產生對抗血管形成治療藥物的抗藥性。目前認為，產生抗藥性原理有：血管形成轉換(angiogenesisswitch)、周邊血管形成（vascular co-option）、血管生成拟態(vasculogenic mimicry)、腫瘤改變成不依賴血管的表態型（phenotype）等。為了了解卵巢上皮細胞癌的侵襲及轉移機制，我們建立並發表了一卵巢癌侵襲模式。經由基因晶片分析，我們發現IGFBP-3 在抑制癌細胞的侵襲能力上扮演重要角色，在臨床上有顯著的意義。我們進一步發現IGFBP-3 是由於其啟動子上p53 的調控區域被甲基化的超基因調控而不表現。在初步的研究中，我們進行微陣列基因分析，找到與IGFBP-3 有相同表現模式的一個抑制血管形成功能的基因Thrombospondin-1(THBS-1)。在動物研究中，我們證明IGFBP-3 可誘導腫瘤缺氧的狀態，並通過其抑制血管形成作用使癌細胞瘤進入休眠。然而，持續表現IGFBP-3 及THBS-1 一段時間之後，新的血管再度形成同時腫瘤又再生長及轉移。此動物模式，可作為細胞在缺氧下血管形成原理及抗血管形成藥物機轉的研究平台。在本研究中，我們將研究重點放在IGFBP-3在抑制卵巢癌的腫瘤血管形成的調節機制，及在IGFBP-3 如何與THBS-1 和p53 緊密合作達到抑制腫瘤血管形成的功能。為了證明這個假說，將首先驗證IGFBP-3 與THBS-1 的關係，並確定IGFBP-3 具有直接調節THBS-1 啟動子的功能。同時我們也將以染色質免疫沉澱（CHIP）辨別參與IGFBP-3 對THBS-1 啟動子調控的其他可能因子。由於缺氧是造成血管形成的啟動條件，因此會在缺氧環境下分析癌細胞的血管形成調控。藉由HUVEC 和CAM 分析法分析，當不同的IGFBP-3 與THBS-1 調控子被抑制時，細胞在缺氧的環境中對血管形成的作用。我們還將回顧過去的微陣列基因分析結果，並參考細胞生物訊息傳遞路徑、皮間質轉型、超基因調控、小分子核糖核酸等資料庫，尋找可能參與這些血管增生及抑制機制和血管形成開關的調節因子。最後，我們將整合這些確定的基因和蛋白質，以對腫瘤血管形成過程有更好的理解。因此，本計畫的研究結果，在學術上可以理解癌血管形成、惡化及轉移的分子機轉，並可提供為判斷癌惡化的分子標靶，在臨床上可以為卵巢癌患者提供更準確的診斷以及更好的治療。<br> Abstract: Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer in women worldwide.The majority of EOC were at advanced stage at diagnosis and the overall survival rate was dismaldespite debulking surgery and standard platinum-taxane chemotherapy. New target therapy such asanti-angiogenesis therapy was recently proved to be effective to prolong progression free survival inEOC patients. However, anti-angiogenesis therapy was found to have frequent subsequent treatmentresistance. Several processes could have happened to cause anti-angiogenesis therapy resistance,such as the development of angiogenic switch, vascular co-option, vasculogenic mimicry, andshifting of angiogenic to non-angiogenic phenotype tumor during treatment. To study the molecularsignature and mechanism of ovarian cancer invasion and metastasis, we have established andpublished an EOC invasion model containing sublines with increasing order of invasive activities.Through microarray, series of invasion-related genes were identified and IGFBP-3 was found as akey negative regulator of cancer invasion. IGFBP-3 was silenced through epigenetic regulation,specifically at p53 trans-activation sites. Lower IGFBP-3 expression and higher methylation weresignificantly related to poor patient survival. In our preliminary study, using further microarray, wefound IGFBP-3 co-expressed with thrombospondin-1 (THBS-1), an endogenous inhibitor ofangiogenesis. And by animal study, we demonstrated that IGFBP-3 could induce tumor dormancythrough its anti-angiogenesis effect in tumors with invasive phenotype. Under persisting hypoxia,re-vascularization in associated with tumor re-growth and metastasis occurred, despite persistingTHBS-1 activation. This in vivo model is an ideal platform to study angiogenesis at hypoxiacondition and the mechanism of how anti-angiogenesis resistances occur. In this study, we willfocus on how IGFBP-3 acts as an angiogenesis modulator in the cooperation of THBS-1 and p53.We first verify the correlation of IGBP-3 with THBS-1 on angiogenesis inhibition and analyze thebiological events during the reversion of anti-angiogenesis. We then study promoter regulation ofIGFBP-3 on THBS-1. By Chromatin Immunoprecipitation assays (CHIP), promoter regulationcomplexes and associated factors acting on IGFBP-3 or THBS-1 promoters will be identified.Selected genes obtained from our previous microarray data will be used for further study. And basedon bioinformation on signal transduction pathways, epithelial-mesenchymal transition, epigeneticgene regulation and micro-RNA, mechanism participated in such microenvironment could bedisclosed. Angiogenesis functional assays and hypoxia conditions simulating tumormicroenvironment will be used to study events that might occur with angiogenesis and the reverseof anti-angiogenesis. We aim to provide insight to a better understanding of caner angiogenesis,growth, invasion and metastases in EOC. And most importantly, to a better treatment of EOC.卵巢癌血管形成IGFBP-3THBS-1p53Epithelial ovarian cancerangiogenesisIGFBP-3THBS-1p53