Regulation of drug disposition by a novel microRNA-mediated pathway

描述（由申请人提供）：我们建议研究和确定一种新的microRNA （miRNA）介导的途径在药物处置和耐药性中的作用。ABCB1 （MDR1）等药物转运体的表达升高有助于活性药物输出介导的耐药。这些转运体的表达受两种主要的异种受体的转录控制，即组成型雄甾烷受体（CAR）和孕烷X受体（PXR），两者都具有与类视黄酮X受体（RXR）的异源二聚体的功能。PXR是配体激活的，而CAR在没有配体的情况下具有组成性活性，其表达导致其转录靶标的表达。最近的研究提供了mirna介导的耐药证据。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回路，其中miR-137直接降低CAR水平，CAR负向调节miR-137的表达。我们还发现CAR逆激动剂可降低MDR1并增加miR-137水平，并开发了一种新型CAR逆激动剂（LTC27），它对CAR具有强效和选择性。重要的是，在体外和体内，异位表达的miR-137降低了CAR和MDR1，并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，合成mirna由于其低稳定性和细胞摄取而具有有限的生物利用度。通过筛选，我们发现伏立诺他（或SAHA）可诱导miR-137表达并降低MDR1水平。SAHA是一种获FDA批准的组蛋白去乙酰化酶（hdac）抑制剂；抑制HDAC可以破坏转录抑制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会降低耐药细胞中的MDR1，从而增加细胞内药物积累并使细胞重新敏感；HDAC抑制剂诱导miR-137表达，至少部分是通过缓解

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.