胰臟癌具有高度致命性，它是由一群不同性質的癌細胞所組成的，其中有所謂的”胰臟癌幹細胞”。它是已經發生變異並具有對化療抗藥性及電療低敏感度之族群。最近癌幹細胞已變成是治癒癌症的新標的。在很多癌症包括胰臟癌、腦癌及乳癌，CD133被認為是癌幹細胞之重要的細胞膜表面分子標記(marker)。然而，與CD133相關的信號網絡(signal network)還未完全了解。我們初步的研究結果顯示將胰臟 癌細胞株高表達(overexpression) CD133時，會造成癌細胞生長速度加快，細胞群落形成(colony formation)變大，附著及移轉的能力增強。由我們的信號網絡研究資料發現:高表達CD133會促進EGFR的活化及PI3K/Akt路徑的磷酸化。但這些發現需要進一步確認。並需要在活體動物的異種移植(xenograft)研究中証實CD133的高表達較容易形成癌症腫瘤。而這些新鮮的檢體，我們可拿來進行共同免疫沉澱(co-immunoprecipitation)及蛋白質體學實驗。我們初步的證據更進一步的顯示: CD133可與EGFR呈物理上的接觸(physical association)。CD133的高表達可增加EGFR蛋白質的量及其磷酸化。若此重要發現可經過活體動物異種移植(xenograft)的新鮮癌症檢體再次證實，那CD133的信號網絡與EGFR的活化、蛋白質的磷酸化將確認有所連結，而這點可能與 CD133高表達時 (即所謂的”胰臟癌幹細胞”)，腫瘤會快速發展與其化療抗藥性有關。因此，本實驗的主要目的是利用免疫沉澱(immunoprecipitation LC MS/MS)等等的一些蛋白質體學方法來找尋與CD133 結合的蛋白質激酶(protein kinases ;task 2)，並了解高表達CD133 時相關的調控機制。最後我們想在活體動物實驗中證實，CD133 的高表達會產生對化療(Gemcitabine and 5-FU)的抗藥性。本實驗結果將可讓我們找到更有效的分子標的治療藥物用來對抗”胰臟癌幹細胞”；以避免胰臟癌的復發。

Most of tumor consists of a heterogeneous population of tumor cells among a tumor initiating and chemo or radiation resistant subpopulation, called cancer stem cells (CSCs), which have become increasingly important new anticancer targets. CD133 has been recently identified as a prominent marker for CSCs in pancreatic and other tumors; however, the signaling cascade of this cancer stem cell marker has not been fully explored. This study shows increased cell proliferation, colony formation, adhesion, and migration following CD133 overexpression in pancreatic ductal adenocarcinoma (PDAC) cells. Signaling studies have indicated that CD133 overexpression increases the epidermal growth factor receptor (EGFR) activation and phosphorylation of PI3K/Akt and MAPK/ ERK pathways. An in vivo xenograft study confirmed that overexpression of CD133 has higher tumorgentic ability than control mice. Molecular studies have found that CD133 physically associates with EGFR and promotes EGFR protein level and its phosphorlyation, which might be critical for PDAC tumor progression and chemoresistance. The data also showed that CD133 overexpression suppresses the EGF mRNA expression, which may imply that CD133 induces EGFR activation through an EGF ligand-independent process. The findings here point to an important mechanism of action for CD133 in PDAC. The EGFR inhibitor has potent anti-CD133 activity, and the current results have important implications for developing targeting CD133 activity as a novel cancer therapy strategy and the inhibitor approach presented here identifies the inhibition of CD133 activity by the EGFR inhibitor and sheds light on developing a new cancer therapeutic that functions by targeting CD133 activity in human cancer.

Contents

Abstract in Chinese ------------------------------------------- iii
Abstract in English -------------------------------------------- iv
Introduction ------------------------------------------------------ 1
Materials and Methods --------------------------------------- 5
Results ---------------------------------------------------------- 11
Discussion ------------------------------------------------------ 21
Figures and Tables ------------------------------------------- 27
References ------------------------------------------------------ 35

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