肝癌衍生生長因子 (hepatoma-derived growth factor, HDGF) 為一種存在細胞核之生長因子，會促進細胞生長及轉移。先前研究指出，在眾多癌症中，HDGF皆有大量表現的現象，進而造成癌細胞的增生及上皮細胞－間質轉移 (epithelial mesenchymal transition, EMT)。HDGF在癌症進程中扮演了重要的角色，但其訊息傳遞路徑仍為未知。本實驗室先前的研究發現，核仁素 (Nucleolin, NCL) 在肝癌細胞中會從細胞核移動到細胞膜與HDGF結合，並活化PI3K/AKT的訊息傳遞路徑，進而引發細胞的增生及移行。HDGF由PWWP及C140組成，而NCL則可分為三個區域 (domain I, II, III)。我們以螢光標記的HDGF觀察HDGF移動的過程，發現HDGF在四個小時內會進入細胞。接著，我們透過固相結合分析 (Solid phase binding assay)，提出HDGF是透過PWWP跟NCL第二個區域進行結合。從親和管柱分析及質譜分析中，我們發現除了NCL之外，核仁磷酸蛋白 (Nucleophosmin, B23)、膜聯蛋白A2 (Annexin A2) 及葡萄糖調節蛋白質78 (GRP78) 皆可能與HDGF有交互作用。因此，我們利用抗體中和方式將NCL、B23、Annexin A2及GRP78進行中和，發現當NCL、B23及GRP78被中和時，HDGF攝入細胞的情形會降低，顯示這幾個膜蛋白在HDGF與細胞結合扮演重要的角色。另外，先前研究也指出，HDGF為一個磷酸化蛋白，在103號位的絲胺酸 (Serine) 為一個重要的磷酸化位點，若將此為點突變為丙胺酸 (Alanine)，HDGF將喪失促進有絲分裂的功能。然而，HDGF於S103位點的突變是否會影響到與Nucleolin的結合及HDGF與Nucleolin結合後所誘發的下游訊息傳遞則尚未得知。我們透過固相結合分析及免疫螢光染色發現，當S103位點被突變為丙胺酸 (Alanine) 時，雖然HDGF S103A與NCL結合能力不會改變，但影響到HDGF攝入細胞中。我們推測，這可能是造成S103A 點突變造成HDGF喪失誘發細胞侵襲及喪失誘細胞增生能力的原因。綜合上述結果可了解NCL domain II 為HDGF與細胞結合的重要位置; HDGF S103A的突變將使HDGF喪失其生物功能。此發現可以做為HDGF未來癌症治療標靶開發及HDGF所誘發相關疾病之應用。

Hepatoma-derived growth factor (HDGF) is a nuclear targeted growth factor identified from human hepatocellular carcinoma (HCC) cell line, Huh7. Based on clinical studies, HDGF overexpression promotes tumor progression through stimulating proliferation and epithelial-mesenchymal transition (EMT). HDGF was considered as a poor prognostic factor for cancer patients in survival, however, the signaling pathway of HDGF is remains unclear. Our recent study indicates that HDGF may exerts its oncogenic function via binding to membrane Nucleolin (NCL) and activating the downstream PI3K/AKT signaling. HDGF is consist of PWWP and C140 domains; NCL is composed of three distinct domains (domain I, II, III). By using Alexa-488 fluorescent labeled HDGF, we have determined that the exogenous HDGF could be uptake within 4 hours. Subsequently, by using solid phase binding assay, we have demonstrated that HDGF bound to NCL domain II via its PWWP domain. By using affinity chromatography and MASS spectrum, we have characterized several membrane proteins such as B23, Annexin A2 and GRP78 that involved in HDGF-binding membrane complex. Using antibodies neutralization, we have confirmed not only NCL, but also B23 and GRP78 that play important roles in HDGF interaction and HDGF uptake. Furthermore, previous studies showed that S103 phosphorylation site playing a critical role in regulating HDGF mitogenic function. Our data has suggested HDGF S103A mutation lead to loss of HDGF-mediated cell invasion and cell proliferation. This study has revealed the importance of NCL domain II in HDGF binding; Besides, S103 position in HDGF is critical for the biological function in HDGF. This finding suggested that targeting HDGF membrane bound may provide a new strategy for cancer therapy and HDGF related diseases.

論文審定書 i
中文摘要 ii
Abstract iii
Contents iv
Figures and Legends index v
INTRODUCTION 1
MATERIALS AND METHODS 10
RESULTS 18
DISCUSSION 25
FIGURES AND LEGENDS 29
REFERENCES 51
Appendix 62

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