2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657633摘要：在台灣，大腸直腸癌是第三名國人常被診斷出的癌症且每年有逐漸增加的趨勢。此癌 症易於轉移的特性是造成高死亡率的主因。文獻指出，細胞周圍蛋白質水解作用的失 調在癌細胞的侵襲與轉移扮演著很重要的角色。在細胞表面的蛋白酶中，絲氨酸蛋白 酶是一群重要的成員，扮演樞紐的角色調節細胞周圍蛋白水解的起始與水解作用的疊 層。然而，目前鮮少有系統性的方法，有效地分析嵌膜絲氨酸蛋白酶在癌細胞侵襲、 腫瘤的生長、和轉移中所扮演的角色。在此整合型研究計晝中，我們將在大腸直腸癌 轉移模式中，利用活性基探針(由子計晝1設計與提供)去標記嵌膜絲氨酸蛋白酶且利用 質譜分析來鑑定這些被標記的蛋白。我們初步結果發現此活性基探針能夠標記上重組 嵌膜絲氨酸蛋白酶TMPRSS2。此外，TMPRSS2可以誘發matriptase的活化，而matriptase 的活化程度與大腸直腸癌細胞的侵襲能力有正相關，並且TMPRSS2和matriptase參與 了表皮生長因子(EGF)所誘導的大腸直腸癌細胞侵襲的過程中。同時，因有文獻報導指 出抗表皮生長因子(anti-EGFR)治療只能有效的治療帶有野生型K-Ras的大腸直腸癌病 人，但對其變異型無效。K-Ras是表皮生長因子接受者(EGFR)下游的調節者，當K-Ras 突變導致其獲得能力與大腸癌的轉移有很大的相關性並且有較差的預後。持續性地活 化的K-Ras是如何促進大腸直腸癌的惡化和轉移依然是非常不清楚。在此研究計晝 中，我們將利用活性基探針和質譜的分析來找出何種嵌膜絲氨酸蛋白酶在轉移性的大 腸直腸癌細胞扮演角色並描繪出在誘導的大腸直腸癌細胞轉移、腫瘤生成和轉移的過 程中可以被表皮生長因子或K-Ras訊息傳遞所活化的蛋白水解鏈。我們假設表皮生長 因子的訊息傳遞是經由活化K-Ras去誘導嵌膜絲氨酸蛋白酶的活化導致大腸直腸癌細 胞的侵襲和轉移。為回答這個假設，四個主要研究項目描述如下：1)在大腸直腸癌的 侵襲中，利用活化基探針和質譜分析找出參與大腸直腸癌轉移的嵌膜絲氨酸蛋白酶， 2)分析並描繪出這些被鑑定出的蛋白酶在大腸直腸癌的侵襲過程中的蛋白水解鏈，以 及在表皮生長因子或K-Ras誘導的癌細胞侵襲中所扮演的角色，3)利用異種移植的老 鼠來探討這些被鑑定出的蛋白酶在大腸直腸癌腫瘤生長和轉移中所扮演的角色，4)利 用大腸直腸癌的臨床檢體來分析這些被鑑定出來的蛋白酶、matriptase、表皮生長因子 接受者和K-Ras的蛋白表現量。此研究計晝我們將利用活化基探針及質譜分析建立一 個系統性的方法，以鑑定出在大腸直腸癌惡化過程中重要的細胞周圍絲氨酸蛋白酶。 此外，從此研究中所得到的資訊將提供更多的觀點，並且更進一步的探討在 EGFR/K-Ras所誘導的大腸直腸癌惡化中這些細胞周圍蛋白水解鏈的角色。並且可以提 供一些線索去發展全新的蛋白酶標靶治療(protease-targeting therapy)，進而去治療抗表 皮生長因子抗藥性(anti-EGFR-resistant)的大腸直腸癌。我們的長期目標為鑑定這些嵌膜 絲氨酸蛋白酶在癌細胞轉移中所扮演的重要角色，並利用這些蛋白酶做為生物標記物 (biomarkers)或藥物發展和癌症治療的標革巴<br> Abstract: Colorectal cancer is the third commonly diagnosed cancer and annually arising in Taiwan. The high mortality of colorectal cancers is due to metastasis. It has been strongly suggested that dysregulation of pericellular proteolysis plays important roles in tumor invasion and metastasis. Among cell-surface proteases, serine proteases are a major group with a pivotal role in initiating and modulating pericellular proteolytic cascades. However, there are few useful methods available to systematically identify membrane-anchored serine proteases that are involved in cancer cell invasion, tumor growth and metastasis. In this project of the integrated research program, we will employ activity-based probes (designed and provided by the Subproject 1) to label membrane-anchored serine proteases in a colorectal cancer metastatic model and identify the labeled proteins using LC/MS/MS analysis. Our preliminary data showed that the probe could label recombinant membrane-anchored serine protease TMPRSS2. Moreover, TMPRSS2 can activate matriptase and the activated levels of matriptase were correlated with the invasion capability of colorectal cancer cells, and TMPRSS2 and matriptase were involved in EGF-induced colorectal cancer cell invasion. Furthermore, it has been reported that anti-EGFR therapy is briefly effective for the colon cancer patients with wild-type K-Ras. K-Ras is a downstream mediator of EGFR and its gain-of-function mutants are associated with metastasis potentials of colon cancer and poor prognosis. How constitutively activated K-Ras promotes colorectal cancer progression and metastasis is still poorly understood. In this study, we will use activity-based probes and LC/MS/MS analysis to identify the membrane-anchored serine proteases in metastatic colorectal cancer cells and delineate the proteolytic cascades that can be activated by EGF or K-Ras signaling for inducing colorectal cancer cell invasion, tumorigenicity and metastasis. We hypothesize that EGF signaling is through activating K-Ras to induce the activation of membrane-anchored serine proteases, leading to colorectal cancer cell invasion and metastasis. To answer this hypothesis, three specific aims will be addressed: 1) To identify the membrane-anchored serine proteases involved in colorectal cancer cell invasion using activity-based probe labeling and LC/MS/MS analysis, 2) To examine and delineate the proteolytic cascade of the identified proteases in colorectal cancer cell invasion and their roles in EGF or K-Ras-induced cancer cell invasion, 3) To explore the role of the identified proteases in colorectal tumor growth and metastasis using xenograft mice, 4) To analyze the protein levels of the identified proteases and matriptase EGFR and K-Ras in archival specimens of colorectal cancer. The results of this study will establish a systematic approach to identify important pericellular serine proteases during the progression of colorectal cancer. Moreover, the information from this study will provide more insights to explore the roles of the pericellular proteolytic cascades in EGFR/K-Ras-promoted colorectal cancer progression, and give some clues to develop a novel protease-targeting therapy for anti-EGFR-resistant colon cancers. Our long-term goals are to identify those memebrane-anchored serine protease(s) which play key roles in cancer metastasis and to use them as biomarkers or targets for drug development and cancer treatment.