CTEN 於細胞核中累積對腫瘤形成特性的影響

Abstract

C-terminal tensin-like (CTEN) 蛋白質為 tensin 家族的成員之一，位在 focal adhesion 上，參與細胞的貼附、增生、遷移等行為，在許多癌症中皆發現 CTEN 高度表現。根據我們之前的研究顯示，相較於正常細胞中 CTEN 主要位在細胞質及 focal adhesion，大量的 CTEN 卻被發現聚集在癌細胞的細胞核中，顯示 CTEN 在細胞中的定位與腫瘤的發生具有高度的相關性。本論文假設 CTEN 在細胞核中累積會促進腫瘤發生，藉由在 CTEN 蛋白質加上幫助定位的胺基酸序列 nucleus localization signal (NLS) 或 Src myristoylation signal (myr)，進而改變 CTEN 在細胞內的分布，以了解 CTEN 分布的差異對細胞腫瘤形成特性的影響。實驗結果發現，在沒有內生性 CTEN 的 293A 細胞株中，穩定表現不同細胞定位的外源性 CTEN，細胞的生長與貼附能力並沒有因此而產生差異。而在只有少量內生性 CTEN 的大腸癌細胞株 HCT116 中，穩定表現於細胞核內累積的外源性 CTEN 不會影響 HCT116 細胞生長速率，但是會增加細胞移動與入侵能力。另一方面，我們先前發現在細胞核中的 CTEN 可能會與 DNA 結合，因此使用表現大量內生性 CTEN 的大腸癌細胞 HT29，以 chromatin enrichment proteomics (ChEP) assay 分析確認細胞核中的 CTEN 的確會與 DNA 結合，最後進行 chromatin immunoprecipitation sequencing (ChIP-seq) 找出 CTEN 結合的目標 DNA。

C-terminal tensin-like (CTEN) protein locates at focal adhesion and belongs to tensin family. It involve in cell adhesion, proliferation and migration. Elevated CTEN level has been detected in many cancers. On the basis of our previous studies, a high population of CTEN accumulates in the nucleus of cancer cells whereas it is predominantly associated with focal adhesions and localized in cytoplasm in normal cells. It suggests that the subcellular localization of CTEN is highly related to tumorigenesis. In this study, we hypothesize that the nuclear accumulation of CTEN contributes to tumorigenesis. By adding subcellular targeting sequences, such as nucleus localization signal (NLS) or Src myristoylation signal (myr), we manipulate the localization of CTEN to study the effect of different subcellular distributions of CTEN on cell tumorigenicity. The stably-expressed modified CTEN variants in 293A cells, which have no endogenous CTEN, did not affect cell adhesion and proliferation. However, when the stably-expressed CTEN accumulated in the nucleus in HCT116 colon cancer cell line, the cell migration and invasion ability were increased but the proliferation rate was not affected. On the other hand, our previous work found that nuclear CTEN may bind to DNA. Therefore, chromatin enrichment proteomics (ChEP) assay was first applied in HT29 colon cancer cell line to confirm the interaction of CTEN and DNA. Finally, chromatin immune-precipitation sequencing (ChIP-seq) was used to identify the target DNAs that bind to CTEN.