2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38718Slit2 was identified from a suppressive subtracted cDNA library generated by a set of lung cancer and its adjacent normal counterpart tissues from a female lung adenocarcinoma. Slit2 is a 200kD secreted glycoprotein. It was first identified as a repellent molecule for axon guidance in CNS via signaling through Robo1 receptor. Slit2 also participates in many normal biological functions including cell migration, axon branching, and initiate immune response, etc. Recently, slit2 has been implicated in pathological development of various cancers. Slit2 promoter was hypermethylated in many cancers that indicated slit2 possesses tumor suppressor activity. However, slit2 was also shown to induce angiogenesis in melanoma. We observed that expression of slit2 mRNA was repressed in 95% of female lung adenocarcinoma using microarray analyses. Interestingly, the expression of slit2 is inversely proportional to invasive ability of CL1 series cell line (established by Dr. Yang P.C.), i.e. the mRNA level of slit2 is high in low invasive cell lines CL1-0 and CL1-1, but the expression level of slit2 is low in high invasive cell lines CL1-5 and F4. To study the tumor suppressor role of Slit2 in lung cancer cell lines, RNA interference of slit2 in CL1-0 cells and overexpression of slit2 in CL1-5 cells were performed. Both experiments consistently showed that high level of slit2 inhibited cell growth. Moreover, overexperssion of slit2 in high invasive cell line, CL1-5, inhibited cell migration and invasion. Since Slit2 is a secreted protein and since it shows significant tumor suppressor activity in lung cancer cell lines, Slit2 has good potential to become an attractive therapeutic agent against lung cancers. To achieve the goal requires thorough study of slit2 signaling pathway. To study Slit2 signaling pathway involved in cell growth and invasion/migration in lung cancer, we will do following studies: (i) Slit2 can be cleaved in some cell types, the 140 kD N-terminal fragment is sufficient to affect cell migration in paracrine system, however the function of 60 kD C-terminal fragment is not identified yet. We will examine the function of each Slit2 cleavage product in lung cancer. (ii) To date, four Robo proteins were identified and all of them can associate with slit2. Robo1, 2, and 4 mRNAs were detected in lung cancer cell lines. We will clarify which Robo receptor(s) is/are involved in growth inhibition or invasion inhibition or both, by interfering robo expression. (iii) It is possible that not all tumor suppressor activities of Slit2 were mediated by Robo family. One way to identify other Slit2 interacting protein(s) is through yeast two-hybrid system. (iv) To investigate the signaling pathway of Slit2, we will perform microarray analyses to identify its target genes. Once target genes were identified and categorized, it is possible to unravel possible upstream receptor(s). Identified of target genes would also allow us to test Robo mediated Slit2 signaling pathway. (v) Finally, we would like to narrow down domain of Slit2 that might separate the effect of growth inhibition from invasion inhibition for therapeutic interests. All these experiments will be carefully performed in cell cultures and xenograft models.Slit2是一個分泌型蛋白，在中央神經系統之軸突遷徙中扮演一個排斥性的腳色。Slit2透過Robo1受體，使得軸突穿越中央線只有一次的機會。此外Slit2並扮演了許多生物功能，如細胞移動、神經軸突分支的形成、及調控免疫反應等。在許多癌症中，slit2基因的promoter有過度甲基化的現象，暗示著slit2扮演抑癌基因的角色。然而，slit2也會造成黑色素瘤的血管新生。 約95%的台灣女性肺腺癌，其slit2的表現被抑制。分析CL1系列之肺癌細胞株(楊绊池教授所建立)時發現，低侵犯性的CL1-0肺癌細胞株之slit2的表現很高;而在高侵犯性的CL1-5及F4細胞中，slit2的表現很低。欲了解slit2的抑癌功能，我們利用RNA干擾技術降低CL1-0細胞之slit2表現，並在CL1-5細胞中將slit2進行過度表現。這兩種實驗發現，高表現量的slit2會抑制癌細胞的生長。同時，高表現量的slit2抑制了CL1-5細胞的移動及侵犯的能力。由於Slit2是一個具有抑癌特質的分泌型蛋白，它可能成為一個重要的癌症治療標的基因。為了達成此一目標，必須徹底了解Slit2蛋白的訊息傳遞路徑。 欲了解slit2抑癌機制之訊息傳遞路徑，我們計劃進行如下的實驗: (i) 研究Slit2 蛋白被切割後，其N-terminal和C-terminal蛋白片段之抑癌功能。(ii) 探討已知之4種Robo受體中，那一種與Slit2在肺癌中抑制細胞生長與侵犯能力有關。 (iii) 如果Robo受體無法完全傳遞Slit2抑癌機制有關之訊息傳遞，我們將利用yeast two-hybrid系統尋找其他與Slit2有結合的蛋白。(iv) 利用微矩陣分析Slit2訊息傳遞之下游基因。(v) 鑑定能區分出抑制細胞生長或抑制細胞移動/侵犯之Slit2小片段蛋白。以上的實驗將會在細胞及動物體裡嚴謹的進行。Project