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標題: | EphB3/ephrin-B分子訊息在人類大腸直腸癌之臨床和細胞分子生物學研究 Clinical, Cellular and Molecular Study of EphB3/ephrin-B Signaling in Human Colorectal Cancer |
作者: | Sou-Tyau Chiu 邱守苕 |
指導教授: | 謝豐舟,張金堅 |
關鍵字: | 大腸直腸癌,向上調控基因,臨床病理因子,cDNA微陣列實驗,微陣列實驗結果之再確認,定量RT-PCR,EphB3,ephrin-B,間質上皮轉換,腫瘤抑制基因, colorectal cancer,up-regulated gene,clinicopathological factors,cDNA microarray,post-array validation,real-time RT-PCR,EphB3,ephrin-B,mesenchymal-to-epithelial transition,tumor suppressor gene, |
出版年 : | 2009 |
學位: | 博士 |
摘要: | 在已開發國家,大腸直腸癌是造成癌症死亡的主因之一。近年來,某些標靶治療藥物已被證實能夠以有效率且少副作用的方式來治療外科手術無法切除的癌細胞,而瞭解癌症分子訊息傳遞路徑乃是發展和臨床應用標靶治療的先決條件。本研究有兩個部分。第一部分乃分子流行病學之研究,目地在尋找及確認人類大腸直腸癌中和臨床病理因子相關的向上調控基因。第二部分則專注於EphB3基因功能的剖析。已知EphB3是一種細胞膜上的酪氨酸磷酸激脢受體,在消化道中只有小腸絨毛隱窩以及大腸黏膜隱窩底部的始祖細胞(幹細胞)會表現EphB3。此外,EphB3也被證實是一個腫瘤抑制基因。這兩項引人注意的特點使我們決定要進一步探索EphB3的生物學功能。我們的研究不但揭露了EphB3在腸道和癌症生物學的一個嶄新的角色 (EphB3/ephrin-B分子訊息能夠促進細胞的極化以及上皮化,也就是所謂的間質上皮轉換),同時也顯示活化此一分子訊息極有潛力成為標靶治療的新標地。
在研究的第一部分,我們利用四位病人的四組樣本進行cDNA微陣列實驗。在這四個DNA晶片上都呈現向上調控的基因共有29個。我們挑選其中20個基因進行晶片實驗後之二次確認。確認的方法是定量RT-PCR實驗,樣本共有31組,取自於原來4位病人以及另外27位病人的外科檢體。20個基因中只有8個基因通過定量RT-PCR實驗的再確認。我們發現,經確認為向上調控的基因在31組樣本中,各有數組樣本沒有呈現向上調控,而這些樣本的腫瘤行為以及臨床病理特徵也不同於該基因呈現向上調控的其他腫瘤樣本。我們將8個基因在31組樣本中有或沒有呈現向上調控和31組樣本各自的10項臨床病理因子合或不合於於某條件,利用卡方檢定或是費雪氏精密檢定進行統計學的相關分析。結果發現其中6個基因的向上調控與否可以對應到是否出現某特定腫瘤行為或是臨床特徵: THY1以及 PHLAD1的向上調控和大腸直腸癌病人出現貧血狀態有統計相關 (P = 0.036 以及 P = 0.009);HNRPA1的向上調控和位於右側的大腸癌有統計相關 (P = 0.027);GPX2的向上調控和大腸直腸癌腫瘤細胞分化程度較高有統計相關 (P = 0.019);c-MYC的向上調控和男性大腸直腸癌腫瘤有統計相關 (P = 0.012);GRO1的向上調控和病人年齡小於65歲的大腸直腸癌有統計相關 (P = 0.010);此外,GRO1的基因表現愈強,大腸直腸腫瘤的侵襲性及惡性程度有愈低的傾向 (傾向乃指0.05 < P < 0.1)-即血中CEA level愈低、淋巴轉移愈少、Dukes’ stage愈早期 (P值分別為P = 0.058、P = 0.060、P = 0.075)。最後,我們也將20個基因在31組樣本中的mRNA levels利用統計學的廻歸分析兩兩互相進行統計檢定,期望找出20個基因之間那些基因在基因表現上相關。結果發現,HNPRA1和 c-MYC這兩個基因的基因轉錄強弱存在非常緊密的統計相關 (相關係數r = 0.948 )。本研究第一部分的結論如下:(1) 利用另外一種獨立的實驗方法來驗證DNA微陣列實驗的結果是必要的。(2) 利用統計學的相關分析可以找到和腫瘤生物行為以及臨床病理因子相對應的基因。 2005年Batlle等人提出基因剔除小鼠的活體證據證明Ephb3是腫瘤抑制基因。同一組研究學者於兩年後又提出證據證明EphB是藉由一種新的機轉-EphB媒介之腫瘤隔離作用 (EphB-mediated tumor compartmentalization) 來達到抑制腫瘤生長的效果。然而,是否還有其他因素能夠促成EphB媒介之腫瘤抑制作用則尚未有人報導。另一方面,Eph家族中的EphA4/ephrin-A以及EphB4/ephrin-B2分子訊息已經被證實能夠促進間質上皮轉換。因此,我們假設EphB3/ephrin-B分子訊息也會有類似的作用,並且臆測此一作用能夠抑制腫瘤生長。在研究的第二部分,我們首先檢視大腸直腸癌病人腫瘤樣本中EphB3的表現狀態。我們發現EphB3基因的表現在整體上是大腸直腸癌腫瘤高於正常大腸黏膜組織,但是如果將36組樣本依照腫瘤惡性程度分成Dukes’ stages B,C和D三組,就會發現:早期未擴散的大腸直腸癌 (即Dukes’ B) 腫瘤組織有很高的EphB3基因表現,而局部擴散的大腸直腸癌腫瘤 (Dukes’ C) 以及遠處轉移的大腸直腸癌腫瘤(Dukes’ D) 的EphB3基因表現都非常的微弱;這暗示EphB3在大腸直腸癌中扮演腫瘤抑制基因的角色;此部分的臨床證據呼應了Batlle等人在Ephb3-/- ; ApcMin/+基因剔除小鼠的實驗結果。接下來,我們將EphB3的表現質體轉染原本不會表現EphB3的HT-29人類大腸癌細胞株,並篩選出能夠持續表現EphB3的穩定細胞株。穩定細胞株可以同時表現EphB3 receptor以及其ligands (wildtype HT-29細胞本身即會表現ephrin-B1及ephrin-B2這兩種EphB3的ligands),所以EphB3/ephrin-B分子訊息在這些穩定細胞株是屬於同位交互作用 (interaction in cis)。分析這些穩定細胞株可以獲得和間質上皮轉換有關的細胞分子生物學證據。我們發現:持續表現EphB3能夠抑制HT-29細胞在培養皿平盤中的生長、在洋菜凝膠之中的懸浮生長,以及在裸鼠身上異種移植的腫瘤生長,並且誘發形態學、細胞行為以及分子傳遞訊息三個層面的變化,而這些變化都符合間質上皮轉換的條件。更專一的描述如下:持續表現EphB3能夠重組細胞骨架,包括將細胞的形態由伸展狀的外形變成上皮細胞的鵝卵石狀、構築細胞膜下層狀的肌動蛋白骨架 (cortical-actin-cytoskeleton),以及極化細胞膜上的E-cadherin和ZO-1分子。持續表現EphB3也會誘發細胞功能的改變,包括降低細胞的爬行能力、增加細胞的凋亡,以及增加依賴鈣離子的細胞-細胞間黏合力;顯然的,這些功能上的改變皆有利於間質上皮轉換。持續表現EphB3還可以減少間質細胞的分子標記 (降低fibronectin 以及 nuclear β-catenin),增加上皮細胞的分子標記 (包括ZO-1,E-cadherin 以及 plakoglobin),並且將能夠促進上皮間質轉換的已知分子路徑Crk-Rac1 pathway去活化。最後,我們發現:持續表現EphB3可以透過Wnt分子路徑增加ZO-1的表現,進而幫助建構細胞之間的tight junction。已知細胞之間tight junction的形成步驟是間質上皮轉換的最後一個環節,此一步驟和adherens junction、desmosome以及cortical-actin-cytoskeleton的形成代表了細胞極化程式的執行完畢,是上皮細胞分化終點的一項指標。 值得注意的另外一個事實就是,這些促進間質上皮轉換的因子同時也扮演著抑制腫瘤生長的角色,而這種角色的重疊也就是間質上皮轉換參與腫瘤抑制作用的證據。本研究第二部分的結論如下:(1) EphB3/ephrin-B分子訊息藉由重建上皮細胞與細胞間的連接來促進間質上皮轉換,而此種間質上皮轉換的促進作用同時也促成了EphB3媒介的腫瘤抑制作用。 (2) 活化EphB3/ephrin-B分子路徑能夠抑制大腸直腸癌的生長,EphB3 receptor有潛力成為大腸直腸癌標靶治療的分子標地。 Colorectal cancer is one of the leading causes of cancer death in developed countries. Recently, targeted therapy has raised hopes for treating unresectable cancers, including colorectal cancer, more effectively with fewer side effects. Understanding the details of signaling transduction is prerequisite for development and clinical application of targeted therapy. The first part of this study aimed to find individual up-regulated genes responsible for clinicopathological variations in human colorectal cancer. The second part of this study aimed to disclose EphB3 gene function. We provided evidence demonstrating that EphB3/ephrin-B signaling was able to promote apico-basal polarization and epithelialization (promote mesenchymal- to-epithelial transition) – a new role for EphB3 in the biology of the gut and cancer, and activation of such signaling has great potential for targeted therapy. PART I. Clinical Correlates of Up-regulated Genes in Colorectal Cancer (A Forward or Classical Genetics Study) We hypothesize that changes in the transcription of up-regulated genes are biologically meaningful and may be linked to variations in tumor behavior and clinical features. Genes up-regulated concurrently in four microarray experiments were taken as candidate genes. Twenty candidate genes were verified using real-time RT-PCR in these 4 and another 27 pairs of samples. The presence or absence of up-regulation of these genes was correlated with ten clinicopathological variables from 31 patients. The mRNA transcript levels of these 20 candidate genes in the 31 paired samples were also correlated with each other to disclose any expression relationship. 40% (8/20) of candidate genes were verified by real-time RT-PCR to have a tumor/normal expression ratio > 2. Up-regulation of THY1 and PHLAD1 was associated with the presence of anemia in colon cancer patients (p=0.036 and 0.009). Up-regulation of HNRPA1 was more significant in cancer growing in the right sided colon than the left side (p=0.027). Up-regulated GPX2 was related to a higher degree of tumor differentiation (p=0.019). c-MYC was significantly over-expressed in specimens from male rather than female colon cancer patients (p=0.012). GRO1 was significantly up-regulated in patients younger than 65 years old (p=0.010) and was found to be frequently over-expressed when cancers were less invasive. In addition, we found normalized transcript levels of HNPRA1 were tightly associated with that of c-MYC (r=0.948). We conclude that validation of microarray data using another independent laboratory approach is mandatory and statistical correlation between gene expression status and patient’s clinical features may reveal individual genes relevant to tumor behavior and clinicopathological variations in human colorectal cancer. PART II. EphB3 Promotes MET and Suppresses Tumor Growth (A Reverse Genetics Study) Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in ApcMin/+ mice and EphB-mediated compartment- alization was demonstrated to be a mechanism suppressing colorectal cancer progress- ion; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4/ephrin-A and EphB4/ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3/ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes’ stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar, and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression reorganized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical-actin- cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased Transwell migration, increased apoptosis and Ca2+- dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear β-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL–Rac1, a known epithelial-to-mesenchymal transition (EMT) signaling pathway. Additionally, crosstalk from Wnt signaling potentiated the restora- tion of epithelial cell-polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3/ephrinB interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect contributes to EphB3- mediated tumor suppression. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8796 |
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