PHRF1促進肺癌轉移藉由ZEB1的轉錄調控

Abstract

PHRF1 是一個E3泛素連接酶，並且在C端包含一個SRI功能區塊，被預測能與RNA 聚合複合體中最大的次單元RPB1的 C-terminal domain (CTD) 進行結合。當DNA遭受雙股斷裂，PHRF1能促進非同源性末端接合 (non-homologous end joining, NHEJ)。另一方面，PHRF1也能促進細胞增生與癌症的發生。然而，PHRF1促使細胞進行入侵與轉移的機制，目前仍處於未知的狀態。從分子機制的層面切入，我們發現PHRF1能夠增加一個和癌症轉移相關的因子ZEB1的mRNA的表現量。從肺癌病人解體的組織切片染色結果中，PHRF1 具有相對較高的表現量。另外， PHRF1也能促進上皮變間質型的轉換 (Epithelial-mesenchymal transition)，在整個癌症轉移中，扮演早期一個重要的步驟。我們的實驗結果指出，PHRF1可以和RPB1-CTD中的磷酸化Ser-2與Ser-2/5進行結合。Ser-2與Ser-2/5的磷酸化是一個代表調控轉錄延長階段 (transcription elongation stage)的重要特徵。藉由反轉錄-定量聚合酶連鎖反應 (RT-qPCR)，我們發現PHRF1也能促進ZEB1其mRNA的表現量。此外，PHRF1能對pVHL進行K48型的聚泛素化的蛋白質修飾，造成pVHL被降解，pVHL是一個腫瘤抑制子與轉錄延長階段抑制子。因此PHRF1能調控ZEB1的RNA表現量，是藉由與RPB1進行結合，減少轉錄延長階段抑制子pVHL，進而促進其轉錄延長階段的效率。總結上述，我們的研究發現PHRF1能促進ZEB1表現，造成癌症轉移的發生，並了解其背後的分子機制。

PHRF1 is an E3 ligase and contains an SRI domain which is a putative binding domain with the C-terminal domain (CTD) of RPB1, the biggest subunit on RNA polymerase II complex. PHRF1 promotes non-homologous end joining (NHEJ) when DNA suffers double-strand breaks, and also increases cell proliferation and cancer progression. However, the mechanism that PHRF1 facilitates cancer invasion and metastasis remains unknown. On molecular aspect, we report that PHRF1 can increase mRNA levels of ZEB1 which contributes to metastasis development in lung cancer. PHRF1 has relatively higher expression in human lung cancer specimens on immunohistochemistry and enhances epithelial-mesenchymal transition (EMT), which is a crucial step in early metastasis. Our results indicated that PHRF1 could bind phospho-Ser2 and phospho-Ser2/Ser5 of the CTD of RPB1, a prominent signature of transcription elongation, and elevated the level of ZEB1 transcription on RT-qPCR assay. Besides, PHRF1 ubiquitinates pVHL, which is a tumor suppressor and a transcription elongation inhibitor, with K48-linked polyUb chains and makes pVHL degraded. Therefore, promotion of ZEB1 by PHRF1 may be explained by the PHRF1- RPB1 interaction which regulates transcription elongation. Collectively, our studies indicate that PHRF1 promotes metasatasis by ZEB1 transcription regulation and realize the mechanism behind it.