前列腺細胞中 ΔNp63α 調控 CTEN 基因進而參與細胞貼附與遷移機制

Abstract

p63 轉錄因子屬於 p53 蛋白質家族的一員,包含六個主要的異構體,在上皮組織 的發育與平衡中扮演重要的角色。p63 的其中一種異構物 ΔNp63α 和 Cten 在前列 腺基底細胞 (basal cells) 中皆大量表現,並且在前列腺癌中表現量大幅降低,其他 研究顯示,knockdown ΔNp63α 或是 Cten 時皆會造成細胞失去貼附能力。而我們 發現 knockdown ΔNp63α 會使得 CTEN 的 mRNA 表現量降低,這樣的現象促使我 們欲研究 ΔNp63α 對於 CTEN 啟動子是否具有 trans-activation 的能力,且細胞生理 是否因此受到影響。因此我們假設 ΔNp63α 能夠 trans-activate CTEN 啟動子,並影 響細胞的貼附與遷移能力。本論文的研究發現,在人類正常前列腺細胞株 RWPE- 1 中, knockdown ΔNp63α 會造成 Cten 的 mRNA 與蛋白質表現量下降,並且證明 了 ΔNp63α 會直接結合在 CTEN 啟動子上,且可能結合於 CTEN 啟動子上的 p53/p63 預測結合序列,而活化 CTEN 的基因表現。此外,knockdown ΔNp63α 會 使得 RWPE-1 細胞株的貼附與移動能力下降,並且在過量表現 Cten 之後貼附與移 動能力回升。除了 in vitro 的實驗,我們也分析了線上資料庫 Oncomine 與 Gene Expression Omnibus (GEO) 所開放下載的前列腺癌症病患之 microarray 資料,我們 發現 TP63 與 CTEN 除了在前列腺癌症中的表現量會下降之外,轉移型癌症的 TP63 與 CTEN 表現量會比原位癌症更進一步地降低,另外,免疫染色的結果顯示, 人類前列腺中的 Cten 與 ΔNp63 之蛋白質表現量與表現位置具有相關性。本論文 證明 ΔNp63α 會結合在 CTEN 的啟動子以調控其基因表現,並透過 Cten 調控細胞 的貼附與移動能力,且可能參與前列腺癌細胞的轉移過程。

p63, a transcription factor of p53 family which contains six major isoforms, is a linchpin of epithelial development and homeostasis. ΔNp63α and Cten are both highly expressed in normal prostate basal cells and down-regulated in prostate cancer. previous evidence has indicated that knockdown of either Cten or ΔNp63α would cause decrease in cell adhesion. Moreover, we discovered that ΔNp63α depletion by siRNA results in reduced CTEN mRNA level. It prompted us to investigate whether CTEN promoter is trans-activated by ΔNp63α and the resulting cell morphological changes. Therefore, we hypothesized that ΔNp63α would trans-activate CTEN expression and subsequently lead to affects cell adhesion and mobility, which could possibly have connection with cancer metastasis. In our study, siRNA-mediated knockdown of ΔNp63α caused decrease of Cten expression in normal prostate cell line RWPE-1. Moreover, we showed that ΔNp63α physically binds to CTEN promoter and the putative p53/p63 consensus binding sites are critical for ΔNp63α binding and activation. In addition, we demonstrated that knockdown of p63 results in decrease of cellular adhesion and migration, which could be rescued by over-expression of Cten. Consistent with our in vitro findings, we confirmed a positive correlation between TP63 and CTEN expression by analyzing the microarray data set from online database Oncomine and Gene Expression Omnibus (GEO). Based on these downloaded data set, we also discovered that the expressions of TP63 and CTEN in metastatic cancers are both lower than those in primary cancers. Additionally, immunohistochemical staining analysis showed a correlation between Cten and ΔNp63 expression and localization in human prostate. Altogether, our findings revealed that ΔNp63α regulates the expression of CTEN by trans-activating its promoter, and CTEN up-regulation results in increased capacity of cell adhesion and migration ability.