Slit2是ㄧ個分泌型蛋白，在神經發育、腫瘤發展及血管新生方面扮演著重要的角色。 我們發現了 Slit2的兩種剪接變異型: Slit2-WT(有 Exon 15)及 Slit2-DE15(沒有 Exon 15)。Slit2-DE15具有抑制 CL1-5肺癌細胞的生長及侵犯能力，而 Slit2-WT只能抑制細 胞的侵犯能力。人類正常氣管上皮細胞 BEAS-2B及大部分的老鼠肺組織表現較高比例 的 Slit2-DE15。既然 Slit2是一個分泌型蛋白，我們好奇的問:如果正常的肺組織表現了 具有抑制細胞生長的 Slit2剪接變異型，Slit2-DE15，那麼肺腫瘤如何發展? 因此，我們 檢視了 18個肺癌病人之腫瘤鄰近的正常肺組織及 54個氣胸病人之肺組織中 Slit2的表 現。令人驚訝的是 100%的肺癌病人之”正常”肺組織表現極高的 Slit2-WT，而 Slit2-DE15 的表現極低或不表現(Slit2-WT>Slit2-DE15);然而 41%的氣胸病人則表現較高的 Slit2-DE15(Slit-WT Slit2≦ -DE15), P=0.001。這些結果暗示著 Slit2-Exon15的剪接變異型 可被不同的病理機制所調控。有趣的是，處理 gallic acid之後可以誘導 Slit2-DE15的表 現，這暗示著我們有機會將腫瘤微環境中 Slit2-WT型轉變成具有抑制腫瘤生長及侵犯 的 Slit2-DE15型。這些結果暗示著兩個可能性:(i)Slit2-WT的表現是可被誘導的;在腫瘤 發展過程，腫瘤微環境之 Slit2的表現由 Slit2-DE15轉為 Slit2-WT，使肺癌細胞逃脫生 長抑制的機制; (ii) Slit2-WT的表現是本來就存在的肺癌危險因子。為了進一步了解並 區分這兩個可能性我們設計了以下的研究目標: (1)利用動物模式和條件培養液之細胞 模式探討腫瘤的微環境是否會影響 Slit2剪接變異型的表現; (2)探討 gallic acid在 Slit2-Exon15所扮演的角色 (3)探討促進發炎反應的激素及香煙萃取物BaP是否會影響 Slit2剪接變異型的表現; (4)鑑定出在腫瘤微環境中具有影響Slit2剪接變異型的因子; (5) 鑑定出細胞內具有調控 Slit2剪接變異型的調控蛋白。本研究不但可以釐清 Slit2剪接 變異型的調控機制，並讓我們有機會尋找具有調控腫瘤微環境中 Slit2剪接變異型的天 然物，以達到預防肺癌的形成及有效的控制癌症的發展。 Slit2 is a secreted glycoprotein who plays important roles in neuronal development, tumorigenesis and angiogenesis. The expression of Slit2 is highly repressed in lung cancer. We identified two splicing form of Slit2, Slit2-WT (presence of exon 15) and Slit2-DE15 (absence of exon 15) in CL1-0 lung cancer cell line. Slit2-DE15 has ability to repress growth and invasion of CL1-5 lung cancer cells while Slit2-WT possesses invasion inhibitory activity only. Transformed normal human bronchial epithelium BEAS-2B and the normal lung tissue of BALB/c nude mice express higher level of Slit2-DE15 than Slit2-WT. Since Slit2 is a secreted protein and Slit2-DE15 is preferentially expressed in normal lung tissue of mice and human bronchial epithelial cells, we were wondering how lung cancer can ever progress if its microenvironment expresses this growth inhibitory splicing form of Slit2. We thus examined Slit2 splicing form in 18 adjacent normal lung tissues of lung cancer patients and 54 lung tissues of pneumothorax patients. Surprisingly, 100% of the “normal” lung tissue of lung cancer patients expressed predominantly Slit2-WT splicing form with little or undetectable Slit2-DE15 form (Slit2-WT>Slit2-DE15), while 41% of lung tissue from non-tumor specimen (from pneumothorax patients) expressed higher ratio of Slit2-DE15 (Slit-WT≦Slit2-DE15), P=0.001. These results suggested that Slit2-Exon15 splicing is regulated by different pathological mechanism. Interestingly, we found that gallic acid treatment induced the expression of Slit2-DE15, implicating that we may be able to reverse Slit2-WT splicing form to growth inhibitory Slit2-DE15 form in tumor microenvironment. These preliminary results raised two possible scenarios: (i) Slit2-WT splicing form expression is inducible; during tumor progression, the expression of Slit2 switches from Slit2-DE15 to Slit2-WT in tumor microenvironment that enable lung cancer cells to escape from growth inhibition; (ii) Slit2-WT splicing form preexists and is a risk factor for lung cancer. To discriminate these hypotheses we propose following goals in this study: (1) investigate whether tumor microenvironment has an impact on alternative splicing of slit2 expression by animal model and conditioned medium;(2) explore the role of gallic acid in regulating Slit2-Exon 15 splicing; (3) identify factors in microenvironment might affect alternative splicing of Slit2; (4) Investigate whether inflammatory cytokines/inducer affect alternative splicing of Slit2; (5) identify splicing factors that regulate alternative splicing of Slit2. This study not only allows us to study regulatory mechanisms involved in splicing of Slit2-Exon 15, but also to identify natural compounds which may regulate Slit2 splicing event for cancer prevention or control of cancer progression.
