Epigenetic modifications by dietary PEITC in prostate cancer

背景和假设：前列腺癌（PCa）仍然是美国男性癌症死亡的第二大原因。在之前的资助期间，我们已经发现，苯乙基异硫氰酸酯（PEITC）和姜黄素（CUR）单独和联合抑制裸鼠PC-3异种移植和TRAMP小鼠中PCa的发展和进展，通过诱导促凋亡和抗增殖信号通路。本R 01延续申请的目的是研究PEITC对PCa的表观遗传效应。（i）随着TRAMP PCa的进展，抗氧化应激转录因子Nrf 2和Nrf 2-靶基因II期解毒酶和抗氧化酶逐渐丧失;（ii）用PEITC治疗导致TRAMP前列腺肿瘤中Nrf 2和Nrf 2靶基因UGT 1A 1的再表达，这与肿瘤抑制相关;（iii）Nrf 2基因在TRAMP肿瘤和TRAMP C1细胞系中通过启动子CpG超甲基化进行表观遗传学调节;（iv）ChIP测定揭示MBD 2和三甲基组蛋白H3（Lys 9）蛋白与这些CpG位点的结合增加;（vi）使用DNA甲基化PCR阵列，PEITC从一组96个基因启动子逆转LNCaP细胞中某些基因的甲基化状态;（vii）通过MSP测定和qPCR，PEITC恢复14-3-3基因的表达。尽管有这些有希望的结果，但我们对PEITC的表观遗传机制的理解存在重大差距。基于我们初步研究的结果，我们想检验我们的假设，即表观遗传事件将驱动TRAMP中PCa的发展和进展，PEITC修饰这些改变，导致其PCa化学预防作用。将在体内和体外详细研究TRAMP中Nrf 2和Nrf 2靶基因以及LNCaP中促凋亡/抗增殖基因的表观遗传事件。具体目标：本课题的具体目的是：（1）研究TRAMP小鼠前列腺肿瘤发生发展过程中的表观遗传学改变以及PEITC通过表观遗传学修饰的化学预防作用。(2)确定饮食PEITC引起的促凋亡/抗增殖基因的表观遗传修饰对SCID小鼠中LNCaP原位异种移植肿瘤的生长抑制作用。(3)阐明在TRAMP C1、C3和LNCaP细胞系中PEITC对从体内目的1和2获得的基因进行调控的体外表观遗传机制。建议研究的意义：通过饮食或化学干预对PCa进行癌症化学预防是合乎逻辑的，并将对美国数十万男性产生巨大的临床益处。拟议研究的成功将进一步推动PEITC的临床试验，以确定其化学预防表观遗传机制、生物标志物和针对人类前列腺癌的有效性。

DESCRIPTION (provided by applicant): Background and Hypothesis: Prostate cancer (PCa) remains the second leading cause of cancer death in men in the United States. During the previous funding period, we have found that phenethyl isothiocyanate (PEITC) and curcumin (CUR) alone and in combination inhibit the development and progression of PCa in nude mice PC-3 xenograft and TRAMP mice via induction of pro-apoptotic and anti-proliferative signaling pathways. The objective of this R01 continuation application is to investigate the epigenetic effects of PEITC against PCa. Rationale for the studies proposed in this application is derived from our published and unpublished preliminary studies showing that: (i) As TRAMP PCa progresses, there is a progressive loss of the anti-oxidative stress transcription factor Nrf2 and Nrf2-target genes phase II detoxifying and antioxidant enzymes; (ii) Treatment with PEITC resulted in the re-expression of Nrf2 and Nrf2-target gene UGT1A1 in the TRAMP prostate tumor correlating with tumor suppression; (iii) Nrf2 gene is epigenetically regulated in TRAMP tumor and TRAMP C1 cell line through promoter CpG hypermethylation; (iv) ChIP assays revealed increased binding of the MBD2 and trimethyl-histone H3 (Lys9) proteins to these CpG sites; (v) treatment with DNMT inhibitor 5-aza and HDACi TSA restores the expression of Nrf2; (vi) PEITC reverses the methylation status of certain genes in LNCaP cells from a panel of 96 gene promoters using DNA methylation PCR Array; (vii) PEITC restores expression of 14-3-3 gene by MSP assay and qPCR. Despite these promising results, significant gaps exist in our understanding of the epigenetic mechanism(s) of PEITC. Based on the results of our preliminary studies we would like to test our hypothesis that epigenetic events would drive the development and progression of PCa in TRAMP and PEITC modifies these alterations leading to its PCa chemopreventive effect. The epigenetic events of Nrf2 and Nrf2-target genes in TRAMP as well as pro- apoptotic/anti-proliferative genes in LNCaP will be investigated in detailed both in vivo and in vitro. Specific Aims: The specific aims of this project are to: (1) To investigate the epigenetic alterations during the development and progression of prostate tumorigenesis in TRAMP mice and the chemopreventive effects of PEITC through epigenetic modifications. (2) To determine the epigenetic modifications of pro-apoptotic/anti- proliferative genes elicited by dietary PEITC on the growth inhibition of LNCaP orthotopic xenograft tumors in SCID mice. (3) To elucidate the in vitro epigenetic mechanisms of regulation of the genes obtained from in vivo Aims 1 and 2 by PEITC in TRAMP C1, C3 and LNCaP cell lines. Significance of the Proposed Research: Cancer chemoprevention of PCa through dietary or chemical intervention would be logical and would be tremendous clinical benefit to hundreds of thousands of men in the United States. The success of the proposed study will further drive clinical trials of PEITC to determine its chemopreventive epigenetic mechanisms, biomarkers and effectiveness against human PCa.