Slug轉錄因子的轉譯後修飾與其在肺腺癌上癌侵襲及轉移之探討

Abstract

轉譯後修飾在細胞生理調節上扮演很重要的角色，主要是透過對蛋白質穩定及定位，進而影響他們的功能。在本論文裡，我們會探討鋅指結構的轉錄抑制因子Slug被glycogen synthase kinase 3 beta (GSK3β)磷酸化及之後被C terminus of HSC70-Interacting Protein (CHIP)泛素化的蛋白質降解。我們利用體外磷酸化試驗與液相層析質譜儀等方式發現GSK3β磷酸的位置主要在Ser100及Ser104，其次為Ser92及Ser96。磷酸過後的Slug會使其蛋白穩定度下降。接著，我們利用蛋白質分析法找到E3泛素蛋白連接酶CHIP，並驗證了減少CHIP的表現則可以提高Slug蛋白的表現並降低Slug的泛素與降解，且不影響Slug信使核糖核酸的多寡。相反地，不被磷酸的Slug-4SA變異體，則不易被CHIP降解，也因此這些不被降解的Slug促進了E-cadherin的轉錄抑制，使得肺腺癌細胞更容易發生上皮- 間質細胞的轉換(EMT)而造成癌侵襲，另外我們也在動物活體試驗中證明不降解的Slug更促進癌轉移的發生。最後，我們檢驗了肺腺癌檢體與癌細胞株，發現GSK3β的活性高低與Slug蛋白表現有關，並且在癌細胞中也是如此。進一步在病人檢體的[GSK3β具活性，Slug蛋白低表現量]的子群中，我們發現60%病人的CHIP表現量也相對較高，意味著CHIP可能在GSK3β調控的Slug蛋白降解裡扮演著重要的角色。總括而言，我們的研究發現一條新的GSK3β-CHIP-Slug機制，透過Slug蛋白的累積或降解，調控肺腺癌的轉移。

Post-translational modification plays an important role in regulating the stability, localization, and the functions of many proteins. In this thesis, we report that the zinc-finger-containing transcriptional repressor, Slug, can be phosphorylated by glycogen synthase kinase 3 beta (GSK3β) and later ubiquitinated by the C terminus of HSC70-Interacting Protein (CHIP) for proteasomal degradation. We identified S100 and S104 as the major phosphorylation sites for GSK3β, and S92 and S96, the minor ones. Further analysis shows that phosphorylation of Slug facilitates its protein turnover. To characterize which E3 ligase is involved in GSK3β-mediated Slug degradation, we conducted the proteomic analysis which reveals that CHIP interacts with wild-type Slug (wtSlug). Knockdown of CHIP stabilizes Slug-WT protein without a change in the Slug mRNA level and reduces Slug ubiquitination and degradation. In contrast, nonphosphorylatable Slug-4SA is not degraded by CHIP. The accumulation of nondegradable Slug further leads to the repression of E-cadherin expression and enhances epithelial-mesenchymal transition (EMT), therefore promoting cancer cell migration, invasion, and metastasis. Furthermore, we show that the GSK3β-pSer9 level correlates with the expression of Slug in patients with non-small cell lung cancer (NSCLC). Within the group of [GSK3β active, Slug low], 60% of the cases have an overexpression of CHIP, implicating the significant role CHIP may play in the GSK3β-mediated Slug degradation. Collectively, our findings provide evidence of a de novo GSK3β-CHIP-Slug pathway that may be involved in the progression of metastasis in lung cancer.