Regulation of drug disposition by a novel microRNA-mediated pathway

描述(由申请人提供)：我们建议调查和确定一种新的microRNA(MiRNA)介导的途径在药物处置和耐药性中的作用。ABCB1(Mdr1)等药物转运体的高表达参与了主动药物出口介导的药物耐药。这些转运蛋白的表达受两种主要的异源受体--构成性雄烷受体(CAR)和孕烷X受体(PXR)的转录调控，这两种受体都是与维甲酸X受体(RXR)的异源二聚体。PXR是配体激活的，而CAR在没有配体的情况下是结构性活性的，它的表达导致了其转录靶点的表达。最近的研究已经提供了miRNA介导的耐药性的证据。我们的初步研究发现，miR-137在阿霉素耐药细胞中下调，而CAR和MDR1上调。我们发现miR-137和CAR形成了一个不寻常的负反馈环，其中miR-137直接降低CAR水平，而CAR负调控miR-137的表达。我们还发现CAR反向激动剂降低MDR1，增加miR-137水平，并开发了一种对CAR有效和选择性的新型CAR反向激动剂(LTC27)。重要的是，异位表达的miR-137减少了CAR和MDR1，并在体外和体内使阿霉素耐药细胞对阿霉素重新敏感；这表明耐药性是可以逆转的。然而，人工合成的miRNAs的生物利用度有限，因为它们的稳定性和细胞摄取能力较低。通过筛选，我们鉴定出具有诱导miR-137表达和降低mdr1水平的抑涡剂(或SAHA)。SAHA是FDA批准的药物，是组蛋白脱乙酰酶(HDAC)的抑制剂；HDAC的抑制可以破坏转录抑制的CAR-HDAC1复合体。我们的初步数据使我们假设，增加miR-137水平或抑制CAR将减少耐药细胞中的mdr1，从而增加细胞内的药物积累并重新使细胞敏感；HDAC抑制剂诱导miR-137表达，至少部分是通过缓解 CAR的抑制作用，并能逆转耐药性。为了验证这些假设，我们将确定(1)miR-137的异位表达在多大程度上逆转了阿霉素的耐药性；(2)CAR-137的反向激动剂在多大程度上逆转了阿霉素的耐药性；(3)在我们独特的多柔比星耐药模型中，miR-137的表达被诱导的机制，以及HDAC抑制剂逆转耐药性的程度。我们还将研究HDAC1-CAR-miR-137启动子复合体，并确定HDAC抑制剂破坏该复合体的程度。这些研究将阐明耐药性是如何获得的，并确定逆转耐药性的潜在治疗方法。

DESCRIPTION (provided by applicant): We propose to investigate and determine the effect of a novel microRNA (miRNA)-mediated pathway in drug disposition and drug resistance. Elevated expression of drug transporters such as ABCB1 (MDR1) contributes to drug resistance mediated by active drug export. Expression of these transporters is transcriptionally controlled by two major xenobiotic receptors, constitutive androstane receptor (CAR) and pregnane X receptor (PXR), both of which function as heterodimers with retinoid X receptor (RXR). Whereas PXR is ligand- activated, CAR is constitutively active in the absence of ligand, and its expression leads to that of its transcriptional targets. Recent studies have provided evidence of miRNA-mediated drug resistance. Our preliminary studies found that miR-137 is down-regulated in doxorubicin-resistant cells, while CAR and MDR1 are up-regulated. We found that miR-137 and CAR form an unusual negative feedback loop wherein miR-137 directly reduces CAR levels, and CAR negatively regulates miR-137 expression. We also found that CAR inverse agonists reduce MDR1 and increase miR-137 levels, and have developed a novel CAR inverse agonist (LTC27) that is potent and selective for CAR. Importantly, ectopically expressed miR-137 reduces CAR and MDR1, and re-sensitizes doxorubicin-resistant cells to doxorubicin both in vitro and in vivo; suggesting that drug resistance can be reversed. However synthetic miRNAs have limited bioavailability because of their low stability and cellular uptake. By screening, we identified vorinostat (or SAHA) that induces miR-137 expression and reduce MDR1 level. SAHA, a drug approved by FDA, is an inhibitor of histone deacetylases (HDACs); HDAC inhibition can disrupt the transcriptionally repressive CAR-HDAC1 complex. Our preliminary data lead us to hypothesize that increasing miR-137 levels or inhibiting CAR will reduce MDR1 in drug-resistant cells, thereby increasing intracellular drug accumulation and re-sensitizing the cells; that HDAC inhibitors induce miR-137 expression, at least partly by relieving the repressive effect of CAR, and can reverse drug resistance. To test these hypotheses, we will determine (1) the extent to which doxorubicin resistance is reversed by ectopic expression of miR-137; (2) the extent to which doxorubicin resistance is reversed by CAR inverse agonists; and (3) the mechanism by which miR-137 expression is induced, and the extent to which drug resistance is reversed by HDAC inhibitors, in our unique doxorubicin- resistant models. We will also examine the HDAC1-CAR-miR-137 promoter complex and determine the extent to which the complex is disrupted by HDAC inhibitors. These studies will illustrate how drug resistance is acquired and identify potential treatments to reverse resistance.