2017-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/650505摘要：肺癌是在台灣以及世界各地癌症死亡最常見的原因之一，初次診斷時大約只有15-20%的非小細胞肺癌病患有機會接受手術治療。如何早期診斷肺癌且給予有效的治療，並找出轉移率高的危險群施以有效的治療，將有助於減少肺癌的死亡率。我們先前的研究發現SERPIN D1（serine proteinase inhibitor，clade D，member 1，又稱為Heparin cofactor II, HCII）為非小細胞肺癌新的轉移促進因子，PI3K、Rac1及 Cdc42 為SERPIN D1 誘發肺癌細胞絲狀偽足形成和移動的下游作用因子。SERPIN D1 的表現，與癌症復發增加及治療結果不佳有關聯性。由於我們已知SERPIN D1 是透過heparin binding site 與癌細胞作用，因此可與heparan sulfate proteoglycans（HSPGs）上共價鍵結的heparan sulfate 交互作用。而HSPGs 與receptor tyrosine kinase（RTK）經常形成coreceptor，使得RTK 與生長素結合時較易被活化。因此我們利用RayBio Human RTK Phosphorylation Antibody Array 大規模篩檢RTK 磷酸化的增減，發現CL1-0 細胞其anaplastic lymphoma kinase (ALK) 的磷酸化在SERPIN D1 刺激後顯著增加，而且我們發現SERPIN D1 與ALK 在細胞膜表面有colocalization。另外我們發現SERPIN D1 與Syndecan 1和Syndecan 4 也有colocalization，而且最近的研究顯示ALK 的ectodomain 有heparin binding site，因此我們假設SERPIN D1 與ALK 的交互作用可能是透過第三者，如syndecans 等HSPG，提供heparansulfate 鏈與ALK 形成coreceptor，進而與SERPIN D1 連結將訊息傳遞到細胞內。本研究計畫希望透過證實SERPIN D1 與ALK 以及syndecan 的交互作用找到ALK 這個orphan receptor 可能的ligand，另外也可更加了解SERPIN D1 與native ALK 在肺癌致病機轉的角色以及未來ALK inhibitor 更多的臨床應用。<br> Abstract: Although there has been progress in the diagnosis and treatment of lung cancer, NSCLC carries a 5-yearsurvival rate of only 17%. Identifying early stage NSCLC with metastatic potential and providingindividualized treatment strategies will have a higher probability of truly curing the disease. Our previouswork demonstrated that SERPIN D1 is a novel metastatic enhancer, which increases cancer cell motility andpromotes cancer metastasis in vitro and in vivo. Rac1 and Cdc42 were found to be the downstream effectorsof SERPIN D1 through a PI3K-dependent manner and these effects can be blocked by heparin. Wehypothesized that SERPIN D1 interacts with cancer cells through cell surface receptors as well as heparansulfate proteoglycans (HSPG) to activate downstream signal pathways. Among transmembrane HSPG family,syndecans have prominent roles in regulating cell adhesion and motility, processes which are central tocancer progression. Syndecan may serve as coreceptor for various receptor tyrosine kinases (RTKs) and theprocess is facilitated through heparan sulfate (HS) chains. To explore the candidate RTK that interacts withSERPIN D1, we used RayBio Human RTK Phosphorylation Antibody Array to capture the levels of RTKtyrosine phosphorylation. The result showed increased phosphorylation of anaplastic lymphoma kinase (ALK)about 2-fold after SERPIN D1 stimulation and overexpression. To validate the result of RTKPhosphorylation Antibody Array, we determined the level of ALK phosphorylation and the activity ofdownstream AKT by western blot analysis of CL1-0 mock transfactant and SERPIN D1overexpression cells.The result showed that ALK phosphorylation at Tyr1604 as well as AKT phosphorylation at Ser473increased after SERPIN D1 overexpression. We further demonstrated that SERPIN D1 colocalizes with ALKby confocal micrograph. Although previous studies have identified pleiotrophin and midkine as putativeALK ligands, a detailed understanding of ALK receptor activation and function remains to be determined.Recently, it has been reported that heparin binds specifically to a highly basic region at the N terminus of theALK extracellular domain, therefore, it is possible that the ectodomain regions of ALK are involved in thebinding of a common protein ligand or closely related ligands, which, in concert with an HSPG, activateALK. Our preliminary result demonstrated the colocalization of SERPIN D1 with Syndecan 1 and 4 as wellas ALK with Sndecan 1. For this study, we hypothesized that the promigratory effect of SERPIN D1 on lungcancer cells is through activation of ALK and facilitated by interacting with SDC1 and/or SDC4 ascoreceptors, which provide heparan sulfate chains for binding of SERPIN D1 and AKL ectodomain. Ourstudy has potential to discover novel ligand for ALK, as well as its biological function and its role on cancerprogression in its native form. Furthermore, the interaction of SDCs with ALK through heparan sulfatechains may play a role in ALK activation. Similar to other RTKs, ligand-mediated increases in ALKsignaling may contribute to tumorigenesis. Thus ALK inhibition by use of tyrosine kinase inhibitor, such ascrizotinib or alectinib, may be beneficial in patients with excessive ALK ligands in tumor microenvironmentSERPIN D1癌症轉移Anaplastic lymphoma kinase (ALK)SyndecanSERPIN D1MetastasisAnaplastic lymphoma kinase (ALK)Syndecan