探討前列腺癌進程中hTop2β-DNA-PKcs複合體在EZH2造成的基因表現轉換所扮演的角色

Abstract

EZH2在生理上的角色一般被認知作為PRC2複合體的一部分，主要負責甲基轉移的酵素活性功能。PRC2複合體可在組蛋白三第27個離胺酸位點上進行三甲基修飾(H3K27me3)，抑制目標基因的表現。而在最近的研究指出EZH2在閹割抗性前列腺癌(CRPC)中第21個絲氨酸被磷酸化(pS21-EZH2)的情形較雄性激素依賴性前列腺癌(ADPC)來得高，且這樣的磷酸化會使得EZH2脫離PRC2複合體，從抑制基因表現的角色，轉化為促進基因表現，我們稱之為「EZH2基因表現轉換(EZH2-mediated gene expression switch)」。有鑑於過往研究指出，在雄性激素受體(androgen receptor, AR)所調控的基因表現中，需要人類拓樸異構酶hTop2β參與，由hTop2β造成DNA斷裂的訊號後誘發後續轉錄啟動的機制。故本篇我們想要探討的是在EZH2基因表現轉換中，hTop2β是否也會藉由形成DNA斷裂去誘導轉錄啟動。首先，我們進行細胞感受性的確認，並證明pS21-EZH2在CRPC中有較高的表現量。再者，我們發現在CRPC細胞中，solo genes的表現量明顯較高，且利用hTop2β的抑制劑ICRF-193，我們觀察到屬於solo genes的FKBP5的表現會受到ICRF-193明顯的抑制，顯示hTop2β可能參與在solo genes的表現中。下一步我們想看hTop2β所造成的DNA斷裂生成的情形，發現在雄性激素DHT的刺激下，會有明顯的γH2A.X的生成，且會被ICRF-193所抑制，代表雄性激素的刺激會活化hTop2β並造成DNA的斷裂，藉以活化基因表現。利用染色質免疫沉澱法，我們也發現了hTop2β確實可以結合至solo genes的啟動子上，進一步證明hTop2β參與在solo genes的表現中。最後，肇因於胞外小體(exosomes)相關重要性在近年被逐漸發現，並參與在細胞溝通與腫瘤微環境(tumor microenvironment)調控中，我們也發現CRPC細胞可以將pS21-EZH2分泌至胞外小體，說明pS21-EZH2可藉由胞外小體到達其他細胞或組織，可能在影響前列腺癌進展成CRPC的過程中有舉足輕重的角色。綜合以上，我們觀察到hTop2β參與在EZH2的基因表現轉換中，且EZH2的基因調控轉變在CRPC較為旺盛。現今對於前列腺癌的治療主要依賴賀爾蒙治療、手術與放射線治療，但以上療法並無法延緩前列腺的進程。根據我們的研究發現hTop2β在EZH2基因表現轉換中的參與，我們推測原本做為臨床上後線治療策略的hTop2β抑制劑，也許其早期運用，可以減緩或預防前列腺癌轉變為閹割抗性前列腺癌，藉以提高患者的預後，與癌症治癒的可能性。

Enhancer of zeste homolog 2 (EZH2) was first found as an enzymatic protein subunit responsible for methyltransferase activity in Polycomb repressive complex 2 (PRC2). PRC2 acts as repressor of transcription through generation of H3K27me3 marks. Interestingly, it was found that phosphorylated EZH2 at serine 21 residue (pS21-EZH2) was upregulated in CRPC, and EZH2 phosphorylation cause functional switch of EZH2, from transcriptional repressor to activator, which could activate solo genes expression, and it was known as EZH2 gene expression switch. We found that solo genes expression was higher in CRPC, and hTop2β inhibitor ICRF-193 could attenuate the expression of solo genes in CRPC cell lines. Furthermore, we found DNA breaks formation induced by androgen treatment could be attenuated by ICRF-193, which suggested that these breaks were generated by hTop2β. Moreover, we demonstrated the interaction between pS21-EZH2 and hTop2β, which indicated that EZH2-mediated gene expression may be conducted through cooperation with hTop2β. In addition, as exosomes became important targets for cancer research, we found pS21-EZH2 could be secreted to exosomes which indicated that pS21-EZH2 could be transported through exosomes, which may contribute to alter the tumor microenvironment. In conclusion, we found hTop2β could involve in EZH2-mediated gene expression switch, in this regard, hTop2β inhibitors may be able to prevent the prostate cancer progression from ADPC to CRPC. But now in clinical cancer management, first line treatments of prostate cancer were hormone therapy, surgery and radiation, hTop2β inhibitors were not the primary choices for cancer treatment. But those treatments were not able to prevent cancer progression from CRPC. Basing on our research, we speculate early usage of hTop2β inhibitors clinically may contribute to prevention of prostate cancer progression to CRPC.