臺灣博碩士論文加值系統

食道癌是台灣男性十大癌症死因的第六名，每年將近有1500人死於食道癌。其五年存活率迄今仍小於20%。近年來，大家已經知道第二環氧化酶（cyclooxygenase-2, COX-2）在多種癌症之腫瘤進展中皆扮演重要的角色而PGE2與癌發生(carcinogenesis)的關係最密切。PGE2的直接作用對象是PGE2受體EP；截至目前為止，共發現EP1至EP4四種PGE2受體。在與食道癌有關的細胞株研究顯示，EP2的表現較其他三種EP強烈。
在本論文的第一部分，我們用免疫組織化學染色(immunohistochemical staining)分析了226位接受根除性食道癌切除手術(en bloc resection)的食道鱗狀上皮細胞癌(esophageal squamous cell carcinoma, ESCC)病患之檢體中，EP2的表現情形與臨床及病理因子的相關性。結果顯示，EP2過度表現(overexpression)的比率為43.4% (98/226)；而且與腫瘤侵犯深度T status呈正相關(p=0.016)，同時與較差的預後有關(p=0.047)。在較早期的食道癌病人(T1-3N0M0)共84人中，EP2過度表現不但會導致較差的預後(p=0.027)，而且在多變異分析中成為一個獨立因子(p=0.048)。
由於EP2過度表現與腫瘤侵犯深度T status呈正相關，因此與腫瘤侵犯(tumor invasion)有關的主題，自然成為研究重點。近年來，Snail被公認是上皮間質轉化(epithelial-mesenchymal transition, EMT)中一個關鍵的調節因子(key regulator)。在與Snail有關的研究中，曾有以頭頸部鱗狀上皮細胞癌(head and neck squamous cell carcinoma, HNSCC)為主的研究發現NBS1這個蛋白質的過度表現會引起Snail的表現增加，進而導致EMT的進行。由於頭頸部癌與食道癌主要皆為鱗狀上皮細胞癌，我們便想知道，上述關於NBS1與Snail的關係是否也存在於ESCC中呢?
在本論文的第二部分，我們先以免疫組織化學染色法，分析153位接受根除性食道癌切除手術的ESCC病患之檢體中，NBS1及Snail的表現與臨床及病理因子的相關性。結果顯示NBS1的過度表現與較佳的預後有關(p=0.002)；另一方面，Snail的過度表現與較差的預後有關(p=0.036)。至於NBS1與Snail之間則是有負相關趨勢(相關係數為-0.14、p=0.084)。由前述的結果可得知， Snail與EP2過度表現在ESCC皆會導致較差的預後。由於EP2是上游的受體，而Snail是一個轉錄因子，令人感興趣的是，EP2與 Snail之間是否有什麼相關性呢?
在本論文的第三部分，我們首先分析CE48T (簡稱48T)、CE81T (簡稱81T)、CE146T (簡稱146T) 與CETE2 (簡稱TE2) 四株食道癌細胞株EP2的表現；無論是在信使RNA (mRNA)或蛋白質層次，48T與146T的EP2表現皆較81T與TE2為強。同時，無論以PGE2或EP2的增效劑(agonist) Butaprost刺激，48T與146T移行性與侵犯性的增加皆較81T與TE2為強。以48T及Lentivirus作為轉染的媒介，我們成功地將48T-sh-EP2(144147)-0.5與48T-sh-EP2(144147)-1的EP2表現量降至約只剩五成與三成。接著我們發現無論有無Butaprost的存在，48T-sh-EP2(144147)-1的移行性與侵犯性皆有顯著的降低。為了瞭解Butaprost是否對細胞生長(cell proliferation)產生影響，我們對細胞株進行SRB(Sulforhodamine B)的測試，結果發現其細胞生長並未因加了Butaprost而有所改變。
在本論文的第四部分，利用即時聚合酶鏈鎖反應(real-time quantitative PCR, Q-PCR)、MMP陣列、反轉錄聚合酶鏈鎖反應與西方點墨法，發現經Butaprost刺激後，Snail與Slug為增加，而E-cadherin則減少；而MMP的變化則不明顯。最後，以不同的抑制物(inhibitor)包括adenylyl cyclase (cAMP)抑制物SQ22536、PKA抑制物H89與PI3K抑制物LY294002合併Butaprost刺激48T後，結果發現SQ22536與LY294002皆有效地抑制了經Butaprost刺激後的48T之移行性與侵犯性，但H89則增加了48T的移行性與侵犯性。由此可以得知，除了cAMP?袈KA?彪REB的訊息傳導路徑外，PI3K/Akt?都]-catenin的訊息傳導路徑，可能也在 EP2?蛀MT的過程中，扮演重要的角色。
根據本研究的結果顯示，PGE2受體EP2確實在食道鱗狀上皮細胞癌的侵犯性扮演重要的角色。EP2可能透過不同的訊息傳導路徑，調節EMT指標中的Snail與Slug而產生上皮間質轉化，進而增加食道癌細胞的侵犯性。

Esophageal cancer is ranking the sixth most lethal cause for male cancer patients in Taiwan and is responsible for nearly 1,500 deaths per year. Currently, its 5-year survival is still less than 20%. Recently, it has been well known that cyclooxygenase-2 (COX-2) plays an important role in tumor progression of several cancers and PGE2 is linked to carcinogenesis more closely than other prostaglandins. PGE2 elaborates its biological activities via PGE2 receptors, namely EPs, which are composed of four isoforms (EP1 to EP4). Regarding esophageal cancer, previous cell line studies have shown that the expression of EP2 was stronger than other EPs in esophageal cancer.
In the first part of this thesis, EP2 expression was evaluated by immunohistochemical staining in the surgical specimens in 226 patients of esophageal squamous cell carcinoma (ESCC) undergoing en bloc resection and the relationship between EP2 expression and clinicopathologic parameters was analyzed. The results showed that EP2 overexpression was found in 43.4% (98/226) of patients. Meanwhile, EP2 overexpression correlated positively with the T status (p=0.016) and was associated with a worse overall survival (p=0.047). EP2 overexpression also predicted a worse prognosis in 84 patients with earlier stages (T1-3N0M0, stage I and IIA) (p=0.027) and even became an independent factor under multivariate analysis among them (p=0.048).
Because EP2 overexpression was found to correlate positively with the depth of tumor invasion (T status) in ESCC, the topics regarding tumor invasion therefore became our interest of investigation. Recently, Snail is acknowledged as a key regulator of epithelial-mesenchymal transition (EMT). Regarding the studies investigating Snail, it has been reported that NBS1 overexpression induces EMT through up-regulation of Snail in head and neck squamous cell carcinoma (HNSCC). Because head and neck cancer and esophageal cancer are both composed of squamous cell carcinoma in majority, our question is whether the relationship between NBS1 and Snail in HNSCC also exists in ESCC.
In the second part of this thesis, NBS1 and Snail expression was evaluated in the surgical specimens in 153 patients of ESCC undergoing en bloc resection by immunohistochemical staining and the relationship between NBS1, Snail expression and clinicopathologic parameters was analyzed. NBS1 overexpression was found to associate with better overall survival (p=0.002). On the other hand, Snail overexpression was associated with worse overall survival (p=0.036). Interestingly, NBS1 overexpression correlated inversely with Snail overexpression marginally (correlation coefficient: -0.14, p=0.084). From the abovementioned results, we found that both EP2 and Snail were associated with worse prognosis in ESCC. Because EP2 is an upstream receptor and Snail is a transcription factor, it is interesting to know whether there exists some relationship between EP2 and Snail.
In the third part of this thesis, the expression of EP2 was evaluated in 4 ESCC cell lines including CE48T (or 48T), CE81T (or 81T), CE146T (or 146T) and CETE2 (or TE2). It was found that at both mRNA and protein level, the 48T and 146T cell lines exhibited stronger expression of EP2 than 81T and TE2. Subsequently, it was shown that treating with PGE2 or the selective EP2 agonist Butaprost caused significant increase of migration and invasion in 48T and 146T but not in 81T and TE2. Using 48T and viral transfection with Lentivirus, the protein expression of EP2 was successfully knocked down to 50% and 30% of original level in clone 48T-sh-EP2(144147)-0.5 and 48T-sh-EP2(144147)-1, respectively. Further studies revealed that irrespective of the treatment of Butaprost, migration and invasion of 48T-sh-EP2(144147)-1 decreased significantly. To elucidate whether Butaprost influences cell proliferation of ESCC cells, the SRB (Sulforhodamine B) test was also conducted on cell lines and it was found that cell proliferation was not significantly changed by the treatment of Butaprost.
In the fourth part of this thesis, using real-time quantitative polymerase chain reaction (Q-PCR), matrix metalloproteinase (MMP) array, reverse transcription PCR (RT-PCR) and Western blot analysis, it was found that Snail and Slug increased, whereas E-cadherin decreased, after treatment of Butaprost. The changes of MMPs were not remarkable. Finally, different inhibitors including SQ22536 (inhibitor of adenylyl cyclase), H89 (inhibitor of PKA) and LY294002 (inhibitor of PI3K) were applied in combination with Butaprost to treat 48T cells and it was found that SQ22536 and LY294002 both inhibited migration and invasion of 48T cells after treating by Butaprost but H89 increased migration and invasion. Based on the results, it was speculated that apart from the cAMPàPKAàCREB pathway, the PI3K/Aktàβ-catenin pathway also plays an important role in the process of EP2àEMT.
To sum up, our current study showed that the PGE2 receptor EP2 does play an important role in tumor invasion in ESCC cells. EP2 may mediate Snail, Slug and subsequent EMT through different signaling pathways to increase invasiveness of ESCC cells.

English Abstract …………………………………………………………… 4
Chinese Abstract …………………………………………………………… 8
List of Abbreviations …………………………………………………………… 11

Introduction ………………………………………… 13
? Specific Aims …………………………………………………………… 22
Materials and Methods …………………………………………………………… 23
? Patients
? Tissue Core Array
? Immunohistochemical Staining
? Evaluation of Immunostaining
? Clinicopathologic Variables and Statistical Analysis
? Western Blot Analysis
? Cell Lines and Cell Cultures
? RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
? Real-Time Quantitative Polymerase Chain Reaction (Q-PCR)
? Transwell Migration Assay
? Matrigel Invasion Assay
? Cyclic-AMP Assay
? shRNA and Viral Infection to Establish Transformed ESCC Clones
? Wound Healing Assay
? Sulforhodamine B (SRB) Cell Proliferation Assay
? MMP Array Evaluation
? Chemicals and Treatment
? Statistical Analysis
Results …………………………………………………… 41
? Results for the First Specific Aim …… 41 ? Clinical Data
? EP2 Immunostaining and Western Blot
? Relationship between EP2 Expression and Clinicopathologic Variables
? Survival Analysis
? Results for the Second Specific Aim …… 43
? Clinical Data
? Immunostaining of NBS1, Snail and Western Blot
? Relationship between NBS1, Snail Expression and Clinicopathologic Variables
? Survival Analysis
? Results for the Third Specific Aim …… 47
? Strength of EP2 Expression Correlated Positively with Migration and Invasion of ESCC Cells after PGE2 Stimulation
? Increase in cAMP Correlated Positively with Migration and Invasion of ESCC Cells after PGE2 Stimulation
? Knocking Down EP2 Decreased Migration and Invasion in CE48T Cells
? Butaprost Treatment Did Not Influence Proliferation of ESCC Cells
? Results for the Fourth Specific Aim…… 49
? Increased Expression of mRNA of MMP-1, MMP-2 and Slug after Butaprost Treatment
? MMP Assay Showed No Significant Increase of MMPs after Butaprost Treatment
? Expression of Slug and Snail Increased after Butaprost Treatment
? Inhibition of cAMP and PI3K Significantly Decreased Migration and Invasion Caused by EP2 Activation
Discussion …………………………………………… 53
Conclusion …………………………………………… 64
Perspectives ………………………………………… 65
References …………………………………………… 66
Figures and Tables ………………………………… 80
Publications ………………………………………… 115

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