MicroRNA mediated regulation of key components of the Mediator Complex (MED) and its functional role in CRPC

前列腺癌（PCa）是全球男性中第二常见的恶性肿瘤。雄激素阻断治疗对前列腺癌的治疗效果良好，但最终仍会发展为去势抵抗性前列腺癌（CRPC），其治疗仍然困难。转录由RNA聚合酶II进行，其在包括介体（MED）复合物的前起始复合物中起作用。然而，MED的调节在很大程度上是未知的。在我们的项目中，我们将专注于MED的两个关键组成部分，MED 12和MED 15，最近被描述为与雄激素敏感的PCa或良性组织相比，在CRPC中强烈过表达。microRNA（miRNAs）是重要的转录后调节因子，在我们的计算机模拟分析中，我们已经鉴定了在CRPC中表达不足并预测靶向MED 12/MED 15的候选miRNAs。因此，我们推测调控MED 12/MED 15表达的miRNAs与CRPC的发生、去势抵抗和抗雄激素治疗抵抗有关，本课题的目的是从肿瘤上皮细胞和间质细胞的角度分析miRNAs对MED 12/MED 15表达的调控及其在CRPC中的作用。这也包括通过miRNA替代和与TGF β拮抗剂的联合治疗，调节候选miRNA和MED组分来寻求CRPC的抗雄激素治疗的再敏感化。该项目的关键目标：i）验证计算机预测的miRNA作为MED 12或MED 15的调节剂，并表征其在体外治疗雄激素敏感性以及恩杂鲁胺或阿比特龙抗性PCa细胞系的功能相关性。ii）配制在聚合物纳米颗粒中的鉴定的miRNA的治疗应用，以抑制肿瘤异种移植物生长和/或影响CRPC异种移植物对体内抗雄激素疗法的敏感性。这还包括来源于异种移植物的组织切片培养物，用于详细的离体分子分析。iii）通过PCa组织中的同时miRNA原位杂交和免疫组织化学评估MED 12/15及其调节miRNA的表达对于CRPC患者的疾病进展和存活的临床相关性。iv）在抗雄激素治疗下通过TGF β 1和miRNA通过肿瘤邻近基质细胞的MED 12/15调节的分析。v）评估组合的miRNA替代和TGF β 1抑制在体外增强的抗肿瘤作用，以及在患者来源的异种移植物（PDX）中在体内对最佳组合的治疗评价。总之，我们将阐明在PCa肿瘤细胞中miRNA和TGF β 1与基质细胞相互作用的背景下对MED 12/15的调节。我们预期miRNA替代的肿瘤抑制作用以及其与抗雄激素治疗或TGF β抑制剂组合在体外/体内的相加/协同作用。

Prostate cancer (PCa) is the second most frequent malignant tumor in men worldwide. It usually responds well to androgen deprivation therapy, but eventually castration resistant prostate cancer (CRPC) will develop that is still difficult to treat. In CRPC development and progression, transcription and its regulation are key processes. Transcription is performed by RNA polymerase II, functioning in a pre-initiation complex that includes the Mediator (MED) complex. However, the regulation of MED is largely unknown. In our project, we will focus on two key components of MED, MED12 and MED15, which have recently been described as strongly overexpressed in CRPC compared to androgen-sensitive PCa or benign tissue. MicroRNAs (miRNAs) are important post-transcriptional regulators, and in our in silico analyses we have identified candidate miRNAs that are underexpressed in CRPC and predicted to target MED12/MED15. Therefore, we hypothesize that miRNAs regulating the expression of MED12/MED15 contribute to the development of CRPC, castration resistance and resistance against anti-androgen therapy.The aims of our project are to analyze the miRNA-mediated regulation of MED12/MED15, and its functional relevance in CRPC from the side of the tumor epithelial cells and the stromal cells in vitro and in vivo. This also includes pursuing a resensitization of CRPC for anti-androgen treatment by miRNA replacement and combined treatment with an antagonist of TGFß, regulating candidate miRNAs and MED components.Key objectives of the project:i) Validation of in silico predicted miRNAs as regulators of MED12 or MED15, and characterization of their functional relevance for the treatment of androgen-sensitive as well as enzalutamide or abiraterone resistant PCa cell lines in vitro. ii) Therapeutic application of the identified miRNAs, formulated in polymeric nanoparticles, to inhibit tumor xenograft growth and/or to affect sensitivity of CRPC xenografts towards anti-androgen therapy in vivo. This also includes tissue slice cultures derived from the xenografts for detailed molecular ex vivo analyses. iii) Assessing the clinical relevance of the expression of MED12/15 and their regulating miRNAs for disease progression and survival of CRPC patients by simultaneous miRNA in situ hybridization and immunohistochemistry in PCa tissues. iv) Analysis of MED12/15 regulation by tumor adjacent stromal cells through TGFß and miRNAs under anti-androgen therapy.v) Assessment of combined miRNA replacement and TGFß inhibition for enhanced antitumor effects in vitro, and the therapeutic evaluation of optimal combinations in patient-derived xenografts (PDX) in vivo. In summary, we will elucidate the regulation of MED12/15 by miRNAs and TGFß in PCa tumor cells in context of their interaction with stromal cells. We expect tumor suppressive effects of miRNA replacement and additive/synergistic effects upon its combination with anti-androgen therapy or TGFß inhibition in vitro/in vivo.