HDAC inhibitors調控ADAMTS1及EGFR表現之研究

Abstract

本論文探討組織蛋白去乙醯酶抑制劑(HDACi)影響血管新生抑制分子ADAMTS1及表皮生長因子受器(EGFR)表現之機轉。HDAC抑制劑(HDACi)是一種極具潛力的抗癌藥物且已臨床使用於治療皮膚T細胞淋巴瘤。抑制HDAC會使染色質結構呈現較鬆散之狀態，並啟動許多抑癌基因之表現，使細胞週期停滯或細胞凋亡。本論文發現廣效型HDAC抑制劑(TSA與SAHA)能誘發非小細胞肺癌細胞株(A549)表現血管新生抑制分子ADAMTS1。藉由專一性抑制劑及siRNA技術，證實係HDAC6壓制ADAMTS1之表現。更進一步證明，promoter近端之SP1 binding site為HDACi調控ADAMTS1表現之關鍵區域。HDACi能驅離promoter上之SP1及HDAC6，並吸引組織蛋白乙醯酶CBP結合至promoter上，重新促使ADAMTS1表現。 表皮生長因子受器(EGFR)之表現程度與大腸直腸癌是否轉移或復發有高度的關聯性，EGFR單株抗體亦應用於大腸直腸癌的臨床治療。然而EGFR單株抗體之療效，卻大幅受限於KRAS的突變狀態，突變之KRAS可不經EGFR而持續活化，並傳遞訊息促使癌細胞存活與增生。大腸直腸癌亦高度表現HDAC，其表現程度更與癌症之進程及預後有關。本論文發現，無論KRAS是否突變，HDACi皆顯著抑制大腸直腸癌細胞株之EGFR表現，並抑制EGF所活化之AKT及ERK。同時，HDACi亦降低SGLT1表現並減少細胞對葡萄糖之攝取。EGFR之降低亦貢獻於HDACi所造成之cell cycle arrest及apoptosis。利用overexpression與shRNA技術也證實Class I HDAC (HDAC1、2與3)與EGFR mRNA及蛋白質表現有正相關，於大腸直腸癌病人的檢體中，EGFR與HDAC3兩者之表現亦呈現高度相關。EGFR過度表現時，SP1、HDAC3與CBP皆被吸附至promoter，而HDACi會造成SP1、HDAC3及CBP與 promoter分離並降低histone乙醯化，因而減少EGFR表現。

The effects of histone deacetylase (HDAC) inhibitor on the expression of ADAMTS1 and EGFR were investigated in this study. In recent years, HDAC inhibitors are emerging as an exciting new class of potential anticancer agents for the treatment of solid and hematological malignancies. HDAC inhibition accumulates acetylated nuclear histones and loosens the compact chromatin structure to reactivate the silenced tumour-suppressor genes. HDAC inhibitors arrest cell cycle and trigger apoptosis by induction of p21 and death-receptor-mediated extrinsic apoptotic pathways. However, their effects on cancer progression, such as angiogenesis and metastasis, are largely unexplored. The first part of this study demonstrates that pan-HDAC inhibitors, TSA and SAHA, were capable to upregulate the angio-inhibitory ADAMTS1 which was originally silenced in non-small-cell lung cancer. We also demonstrated that specific inhibition or knockdown of HDAC6 led to higher expression of ADAMTS1. We further identify the proximal SP1 binding sites were essential for the TSA-induced ADAMTS1 expression and showed that TSA dispersed the HDAC6 and SP1 and recruited the CBP to the ADAMTS1 promoter. Expression of the epidermal growth factor receptor (EGFR) is highly correlated with metastasis and recurrence of colorectal cancer (CRC). The clinical strategy is suppressing EGF signaling by EGFR monoclonal antibodies. However, most patients are irresponsive to these antibodies due to the KRAS mutation, which constitutively activates the downstream survival signals bypassing EGFR. Histone deacetylase has also been reported to be overexpressed in colorectal cancer and correlates with poor prognosis. This study demonstrates that HDACi effectively reduced the viability of KRAS wild type and mutant cells in comparison with EGFR antibody, indicating their broad and effective antitumor effect. HDACi were capable to block the EGF signaling via silencing the EGFR expression and concurrent destabilized an active glucose transporter, SGLT1. Furthermore, we demonstrated that the class I HDACs was essential for EGFR gene transcription and a positive correlation between the expression of EGFR and HDAC3 in the specimens of colon cancer patients was also observed. More in-depth, we showed that the binding of SP1 to EGFR promoter and the accompanied recruitment of HDAC3 and CBP are essential for EGFR expression.