探討新穎Cullin4泛素接合酶調控PML泛素化降解影響肺癌進程之機制

Abstract

PML蛋白在細胞當中具有多效性的抑癌作用，並且在許多人類癌症當中都被發現PML蛋白表現降低的情形。目前許多研究指出泛素-蛋白酶體系統參與了腫瘤細胞中PML蛋白的降解，然而，當中PML泛素化的機制尚未被透徹地了解。在此篇研究中，我們找到一個以Cullin4為基底的新穎PML泛素化接合酶複合物，其包含了受質辨識器WDR4以及兩普遍之子單元DDB1和Roc1。我們發現透過與DDB1的結合，WDR4能促進PML的泛素化程度以及蛋白酶體降解，並且縮短了PML的半衰期。此外，臨床上的證據也顯示出在肺癌當中WDR4/PML的作用路徑有高度活化的現象，因此我們接著想研究此路徑在肺癌進程中的生物意義。為了找出WDR4/PML作用路徑的下游基因，我們將WDR4表現質體或PML siRNA送入肺癌細胞株A549，並做基因微陣列的分析。經過確認後，我們發現CD73、uPAR以及SAA2均會被WDR4/PML路徑所調控。重要的是，這些基因都被報導能調控腫瘤微環境而促進腫瘤進程，進而促進腫瘤細胞增生、遷移、入侵以及轉移。我們也利用實驗證實WDR4可能藉由增加數個腫瘤促進因子和胞外間質重組基因的表現而促進肺癌細胞的增生、遷移以及入侵。總而言之，我們發現一個新穎的PML泛素化接合酶，並利用細胞實驗指出此PML泛素化路徑在肺癌進程中的功能。未來，我們會利用動物實驗來確立這條路徑在肺癌進程中扮演的角色。

The PML (promyelocytic leukemia) protein plays pleiotropic roles in tumor suppression and its downregulation has been observed in multiple types of human cancers. While previous studies revealed the contribution of ubiquitin-proteasome system to PML degradation in tumors, the precise mechanism for PML ubiquitination has not been completely understood. Here, we discovered that a Cullin4-based complex containing WDR4 substrate adaptor and two common subunits DDB1 and Roc1 as a novel PML ubiquitin ligase. WDR4 potentiated PML ubiquitination and proteasomal degradation and decreased PML half-life through a mechanism depending on its interaction with DDB1. Since clinical evidence implicated a hyperactivation of WDR4/PML axis in lung cancer, we sought to investigate the biological significance of this pathway in lung cancer progression. To this end, lung cancer cell line A549 transfected with WDR4 or siPML was subjected to microarray analysis to identify genes that were coregulated under both treatments. CD73, uPAR, and SAA2 were identified and verified as target genes of WDR4/PML axis. Importantly, they are all reported to potentiate tumor progression by regulating tumor microenvironment, thereby potentiating tumor proliferation, migration, invasion, and metastasis. Consistent with the discovery of these target genes, we showed that WDR4/PML axis simulated lung cancer proliferation, migration, and invasion, which is likely mediated by upregulation of several tumor-promoting factors and ECM remodeling genes. Together, we identified a novel PML ubiquitin ligase and demonstrated the functions of this PML ubiquitination pathway in lung cancer progression using cell-based analyses. In the future, we will test the role of this pathway in lung cancer progression by animal models.