表觀遺傳藥物之開發

Drugging the Epigenome

魏子堂(Tzu-Tang Wei)

指導教授 ： 陳青周

國立臺灣大學/醫學院/藥理學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.10069

摘要

c-Cbl是一種E3泛素接合酶，我們的研究發現，c-Cbl蛋白高度表現於正常肺組織，但不表現於非小細胞肺癌 (NSCLC) 病患之腫瘤組織，HDAC抑制劑WJ化合物或抑制HDAC1、HDAC2、HDAC3、HDAC6，於NSCLC細胞株皆可誘導c-Cbl蛋白之表現。外送c-Cbl蛋白可降低EGFR之蛋白表現並抑制NSCLC細胞之生長。EGFR蛋白受抑制亦可減少NSCLC細胞之生長，而EGFR 之Y1045位點突變，會降低WJ抑制腫瘤生長及降解EGFR蛋白之作用。共軛焦顯微鏡之分析顯示，WJ可促進c-Cbl蛋白及EGFR蛋白的交集，原位及尾靜脈注射之肺癌動物模式中，WJ可誘導c-Cbl之蛋白表現而抑制腫瘤之生長。因此，HDAC抑制劑誘導c-Cbl蛋白之表現為治療NSCLC之新穎策略。

Statins經由抑制HMG-CoA 還原酶 (HMGR) 之活性減少膽固醇之合成，HMGR藉由促進蛋白質prenylation而促進癌症之生長。我們先前研究發現，statins之 carboxylic acid結構類似hydroxamate，可抑制組蛋白去乙醯酶 (HDAC) 之活性，許多癌症都大量表現HDAC，降低抑癌基因之表現，因此HDAC為癌症治療之標的；為進一步增強statins抑制HDAC之活性，我們設計並合成小分子化合物(JMF3086, JMF3171 and JMF3173)，同時抑制HMGR及HDAC活性。最近亦有各種多重標靶HDAC抑制劑正在研究開發中。
我們發現大腸直腸癌 (CRC) 病患之HMGR活性會增加，而外加mevalonate可對抗JMF3086抑制CRC細胞生長之能力，顯示HMGR為治療CRC之新穎標靶。JMF3086可藉由caspase-dependent pathway促進CRC細胞凋亡。ChIP-on-chip分析顯示，JMF3086藉由活化NR3C1和NF-B促進細胞凋亡及調控發炎相關基因之表現，進一步發現，JMF3086調節acetyl-H3K27、CBP、HDAC1和HDAC3結合至不同基因之promoter，而調控不同基因之表現。口服JMF3086可治療azoxymethane (AOM)/dextran sulphate sodium (DSS)誘導之大腸直腸癌及ApcMin/+小鼠腫瘤之生長，並抑制大腸直腸癌之肝臟與肺臟轉移，抑制血管新生及人類大腸直腸癌幹細胞。此外，JMF3086可增強oxaliplatin在各種大腸直腸癌動物模式之治療效果，因此，此新穎之多重標靶HDAC抑制劑是具療效之先導藥物，可治療大腸直腸癌。

小鼠口服JMF3086亦可預防DSS誘導之腸炎及azoxymethane (AOM)/DSS誘導之大腸直腸癌，其預防效果更勝於lovastatin併用SAHA。JMF3086可降低發炎相關細胞激素、趨化素、COX-II及cyclin D1之表現，亦可抑制巨噬細胞和嗜中性球於大腸直腸癌腫瘤組織之浸潤，並預防癌幹細胞基因之表現，AOM/DSS鼠血漿中內毒素之增加亦受JMF3086抑制。因此，此新穎多重標靶HDAC抑制劑可預防大腸直腸癌。

關鍵字

表觀遺傳學 ； HMG-CoA還原? ； 去乙醯? ； Statin Hydroxamates

並列摘要

We found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, and inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth, and mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment.

Statins lower serum cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which could also be an oncotarget by regulating protein prenylation associated with cancer growth. We previously disclosed that statins containing carboxylic acid chain possess activity against histone deacetylases (HDACs) which are also attractive cancer targets. In this study, we strengthened the anti-HDAC activity of statins by designing three novel HDAC/HMGR dual inhibitors (JMF3086, JMF3171 and JMF3173). Recently, various polypharmacological molecules exhibiting dual inhibition on HDACs and other therapeutic targets have been developed.
We first found that HMGR activity is increased in colorectal cancer (CRC) patients, and that the addition of mevalonate reversed JMF3086-induced inhibition of CRC cell viability, suggesting that HMGR is a novel target for CRC treatment. JMF3086 induced apoptosis in colorectal cancer cells via a caspase-dependent pathway. A genome-wide ChIP-on-chip analysis in JMF3086-treated CRC cells using anti-acetyl-H3K27 antibody was performed. Apoptosis and inflammation categorized by Gene Ontology (GO) were the most significant biological functions regulated by JMF3086. Ingenuity Pathways Analysis (IPA) analysis predicted their respective regulation by NR3C1 and NF-B. JMF3086 down-regulated inflammatory and stemness genes and up-regulated tumor suppressor genes, in which differential bindings of acetyl-H3K27, CBP, HDAC1 and HDAC3 to their promoters explained this differential effect. Oral administration of JMF3086 exerted therapeutic efficacy against AOM/DSS-induced CRC in mice and inhibited tumor progression of ApcMin/+ mice. JMF3086 also inhibited CRC metastasis to lungs or livers and angiogenesis as well as the stemness in mouse models. Furthermore, JMF3086 potentiated the efficacy of oxaliplatin in different preclinical CRC models. Our data provide compelling evidence that novel polypharmacological HDAC inhibitors-statin hydroxamates exert significant therapeutic benefits against CRC in preclinical models and are promising new drugs for CRC treatment.

Oral administration of JMF3086 also prevented acute inflammation in the DSS-induced colitis mouse models. AOM/DSS-induced CRC was also attenuated by JMF3086, which showed superior protective effect than the combination of lovastatin and SAHA. It also decreased proinflammatory cytokines, chemokines, COX-II and cyclin D1 in tumor tissues as well as the infiltration of macrophages and neutrophils in tumor surrounding regions. Increased stemness factors in AOM/DSS mouse models was prevented by JMF3086. Elevated serum endotoxin level found in AOM/DSS mouse models was reduced by JMF3086. This work provides strong evidence that novel polypharmacological HDAC inhibitor JMF3086 has promising effect on the chemoprevention of CRC.

並列關鍵字

Epigenetics ； HMG-CoA Reductase ； Histone Deacetylase ； Statin Hydroxamates

參考文獻

1. Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA Cancer J Clin 63(1):11-30.

2. Herbst RS, Heymach JV, Lippman SM (2008) Lung cancer. N Engl J Med 359(13):1367-1380.

3. Detterbeck FC, Boffa DJ, Tanoue LT (2009) The new lung cancer staging system. Chest 136(1):260-271.

4. Collins LG, Haines C, Perkel R, Enck RE (2007) Lung cancer: diagnosis and management. Am Fam Physician 75(1):56-63.

5. Mountain CF (1997) Revisions in the International System for Staging Lung Cancer. Chest 111(6):1710-1717.