Study of Tumor Suppressor Functions of Contactin 4 in Colorectal Cancer

目前大腸直腸癌於全世界及臺灣皆是癌症死亡常見原因之一，大多數大腸直腸癌的發生為偶發性且多步驟染色體變異累積發展而成。於實驗室先前研究，利用179對大腸直腸癌腫瘤組織進行第3號染色體的刪除圖譜分析，定義出介於3p25.3-p26.3的最小刪除區域 (minimal deletion region, MDR)。接續以MDR內9個基因的RNA表現量進行篩選，發現細胞貼附分子Contactin 4 (CNTN4) 表現量在部分大腸直腸癌細胞株 (11/12, 91.6%) 及腫瘤檢體 (22/52, 42.3%) 都有顯著下降，顯示CNTN4可能為一個新的大腸直腸癌相關抑癌基因。本論文首先建構CNTN4表現質體 (pLAS3w-CNTN4) 並於大腸直腸癌細胞株HCT116挑選恢復表現CNTN4的單一穩定表現細胞株 ，隨後進行體外細胞實驗及活體動物實驗鑑定CNTN4的抑癌功能。結果顯示：於HCT116細胞表現CNTN4可以抑制細胞增生、固著依賴性及非固著依賴性胞落形成的能力，且會增加細胞自發性細胞凋亡，但是CNTN4的表現對於細胞的移動能力和侵襲性沒有一致性的影響。同時，在活體動物實驗則發現表現CNTN4可以減緩裸鼠皮下腫瘤的生成。初步探討CNTN4參與的訊息傳遞路徑，發現CNTN4會降低ERK1/2的磷酸化程度。綜合以上結果，推測CNTN4在大腸直腸癌的生成和發展具有抑癌的角色。另外，為了探討除了基因刪除外是否有其他機制造成CNTN4在大腸直腸癌表現量下降，將5株無CNTN4表現之大腸直腸癌細胞株以DNA去甲基化藥物5aza-CdR處理，發現DNA去甲基化無法使其CNTN4 mRNA恢復表現，因此推測DNA甲基化可能不是造成CNTN4於大腸直腸癌表現下降的主要機制之一。Colorectal cancer (CRC) is one of the most common causes of cancer death in the world and Taiwan. Most of CRC arise sporadically by the emergence of multiple chromosomal aberrations. In our previous study, a minimal deletion region (MDR) was delineated at 3p25.3-p26.3 by fine deletion mapping of chromosome 3 with 179 pairs of primary CRC tissues. By detecting the RNA transcripts of 9 genes in the MDR, the gene expression of Contactin 4 (CNTN4), a cell adhesion molecule, was remarkably down-regulated in 11 (91.6%) of 12 CRC cell lines and 22 (42.3%) of 52 primary tumors. The findings suggest that CNTN4 might be a novel CRC-associated tumor suppressor gene. In the present study, CNTN4-expression plasmid, pLAS3w-CNTN4, was constructed and then the single stable clones of CNTN4 re-expression in CRC cells, HCT116, were selected. Tumor suppressor activities of CNTN4 were identified by in vitro cell models and in vivo xenograft tumor mouse model. Ectopic expression of CNTN4 in HCT116 reduced cell proliferation, anchorage-dependent and anchorage-independent colony formation in vitro. CNTN4 expression also increased spontaneous apoptosis of HCT116 cells. However, ectopic expression of CNTN4 did not consistently affect the migration and invasion ability of HCT116 cells. Furthermore, we found that CNTN4 re-expression could suppress the tumorigenesis of subcutaneous xenograft in nude mice. The preliminary study of CNTN4 involved signaling pathway showed that CNTN4 could decrease the phosphorylated ERK1/2 in HCT116 cells. Taken together, the results support a role of CNTN4 as a novel tumor suppressor in colorectal tumorigenesis and progression. On the other hand, to identify other mechanisms of CNTN4 down-regulation expect genetic deletion in colorectal cancer, five colorectal cancer cell lines without CNTN4 expression were treated with DNA-demethylation agent, 5-aza-CdR. However, CNTN4 expression could not be restored after DNA demethylation. The result indicated that DNA type methylation might not the major mechanism of CNTN4 down-regulation in colorectal cancer