YB-1致癌角色之探討：YB-1誘發基因體不穩定性之分子機制

Abstract

致癌蛋白Y-box binding protein-1 (YB-1)為一多功能性蛋白，並與轉錄、DNA 錯誤修補(DNA mismatch repair)及化療藥劑抗藥性(chemoresistance)之引發等過程相關。YB-1 已被報導可藉由誘發基因體不穩定性(genomic instability)而導致腫瘤生成，然而其相關之分子機制迄今仍未明瞭。為了確保基因體之完整性，真核細胞中之錯配位修補複合物(MMR complex)可修補錯配位核.酸並引發MMR-dependent DNA checkpoint pathway。其中PCNA 可與MMR complex 結合並有助於DNA錯誤修補之進行。值得注意的是，錯配位修補功能之失活與基因體不穩定性、腫瘤生成及化療藥劑抗藥性之引發直接相關。本實驗室近來初步結果顯示：YB-1 可藉由干擾錯配位修補複合物之組成及影響錯配位修補系統相關因子，如MSH2、MSH6、ATR、ATRIP 及 Chk1 之表現，而得以抑制錯配位修補之進行。此一觀察暗示著YB-1 可經由不同層面影響MMR 活性及MMR-dependent checkpoint signaling 而導致細胞之染色體不穩定性，最終造成腫瘤形成與抗藥性引發。因此，闡明YB-1 與錯配位修補系統之功能性交互作用將可提供一嶄新之分子機制以解釋YB-1 致癌與抗藥性引發之機轉。為達此目的，本四年研究計劃將探討YB-1 影響錯配位修補系統之分子機制及此一YB-1 與錯配位修補系統之功能性交互作用於基因體不穩定性、腫瘤生成及抗藥性引發中扮演的角色為何。本計劃之研究方向分述如下：為了解YB-1 蛋白造成MMR失活之分子機制，我們將分別探討：(1) YB-1 是否藉由與PCNA之交互作用干擾MMR complex 之形成；(2) YB-1 是否影響錯配位DNA 上MMR complex 之形成；(3) YB-1 是否透過干擾MMR complex 之形成而抑制細胞內MMR之活性。同時，我們亦將分析YB-1 與PCNA 之交互作用對於MMR complex 形成所扮演之角色。此外，我們也將尋找無法與PCNA 進行交互作用之YB-1 突變蛋白，以利後續研究之進行。 另一方面，我們亦將探討YB-1 對於因染色體受損害而誘發MMR-dependent checkpoint signaling 及細胞凋亡所扮演之調控角色。此部分將進一步釐清YB-1 對於MMR-dependent ATRChk1 pathway 所扮演之功能角色，並觀察YB-1 是否藉由抑制MMR 活性而誘發細胞之抗藥性。同時，我們將全面性尋找基因表現受YB-1 影響之damage response signaling 的相關因子，以釐清YB-1 於DNA 受損時對MMR 相關之訊息傳遞鏈與細胞週期進行的影響。此外，我們也將分別測試YB-1 誘發染色體不穩定性及微衛星序列不穩定性之能力，以期進一步釐清YB-1 所導致MMR失活對於誘發此二種基因體不穩定性表型所扮演之角色。 此外，YB-1 已被報導可藉由被PI3K/AKT 磷酸.磷酸化而促進腫瘤生長；本實驗室近來亦發現YB-1 可與SUMO 蛋白產生直接交互作用並經由SUMO 修飾系統進一步進行SUMO 修飾。由於轉譯後修飾作用為一種可以快速改變蛋白活性及提供與其他蛋白交互作用並完成下游反應之方式，因此轉譯後修飾作用是否參與YB-1 蛋白所導致MMR 之失活為另一值得探討的主題。是故本計畫亦將釐清轉譯後修飾作用，如SUMO 修飾或磷酸化修飾，是否參與YB-1 蛋白所導致MMR失活、damage response signaling、染色體不穩定及抗藥性引發之過程。 綜合本計劃之研究方向，將有助於進一步釐清YB-1 促進腫瘤生成及藥物抗藥性之可能機轉，期能提供治療腫瘤所需之新穎治療策略。

The oncoprotein, Y-box binding protein-1 (YB-1), is a multifunctional protein involved in transcription, chemoresistance and DNA repair. Notably, YB-1 has been reported to provoke carcinomas through the induction of genetic instability. However, the underlying mechanism remains largely unknown. In eukaryotic cells, the mismatch repair (MMR) complex could repair the mispaired DNA and trigger MMR-dependent DNA checkpoint pathway to ensure the integrity of the genomes, whereas PCNA interacts with the mismatch complex and assists the DNA repair. Most remarkably, MMR deficiency is directly linked to genetic instability, tumorigenesis and chemoresistance. Recently, our preliminary results indicated that YB-1 could suppress MMR activity through interfering MMR complex assembly and modulating the expression levels of several MMR related factors, including MSH2, MSH6, ATR, ATRIP and Chk1. Therefore, it is likely that YB-1 could impart pleiotropic effects on MMR activity and MMR-dependent checkpoint signaling, ultimately leading to malignancy and drug resistance. Thus, knowledge of the functional interaction between YB-1 and MMR machinery may provide a novel underlying mechanism accounting for the oncogenic and chemoresistance-inducing potential of YB-1. In view of these, the objective of this project in the next four years will decipher the detailed molecular mechanisms by which YB-1 modulates MMR machinery and whether the interactions between YB-1 and MMR machinery is associated with genomic instability, tumorigenesis and chemoresistance. The specific items which we intend to accomplish are outlined below. In the aspects of the molecular mechanism of YB-1-induced MMR deficiency, efforts will focus on (1)whether YB-1 interferes with the MMR complex assembly via interacting with PCNA; (2) Whether YB-1 interferes with the MMR complex assembly on mismatch sites; (3) Whether YB-1 reduces the MMR activity via preventing functional MMR assembly. Along these lines, the roles of YB-1/PCNA interaction in MMR complex assembly would be addressed. Additionally, the YB-1 mutant deficient in PCNA binding will also be identified for the following investigation. Another line of this project will focus on the regulatory role of YB-1 in MMR-dependent cell cycle checkpoint and apoptosis in the response of DNA damage. Briefly, this includes the delineation of the functional role of YB-1 in MMR dependent ATR-Chk1 pathway. Meanwhile, the investigation on whether YB-1 induces the chemoresistance via modulating the MMR activity and a global search of damage response signaling components affected by YB-1 will be carried out. Furthermore, to demonstrate the functional role and molecular mechanism of YB-1 in genome instability, we will examine the abilities of YB-1 to induce microsatellite instability or chromosomes instability phenotype. Additionally, efforts will also be made to elucidate the contribution of YB-1- mediated MMR deficiency in the induction of genomic instability. More interestingly, YB-1 has been shown to be phosphorylated by PI3K/AKT and our recent results also indicated that YB-1 not only serves as a substrate for sumoylation pathway but also directly interacts with SUMO paralogs. Given that post-translational modification (PTM) provides an efficient and rapid way to change the activities of protein and supplies extra interacting surfaces for effector recognition to induce downstream events, these observations raise an interesting question as to whether PTM plays any role in modulation of YB-1-mediated MMR deficiency. Therefore, efforts will specifically direct to the investigation of whether PTM of YB-1, such as sumoylation and phosphorylation, participates in the process of YB-1-mediated MMR deficiency, DNA damage response signaling, genome instability and chemoresistance induction. The approaches outlined above may lead a better view of the YB-1-induced malignancy and drug resistance, which may be informative for the future basic research on tumorigenesis and ultimately development of more effective means for cancer treatment.