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

前列腺癌(PCa)是全球男性第二常见的恶性肿瘤。它通常对雄激素剥夺疗法反应良好，但最终会发展成仍然难以治疗的去势抵抗型前列腺癌(CRPC)。在CRPC的发生发展过程中，转录及其调控是关键过程。转录是由RNA聚合酶II执行的，在包括介体(MED)复合体的预起始复合体中发挥作用。然而，对MED的监管在很大程度上是未知的。在我们的项目中，我们将专注于MED的两个关键成分，MED12和MED15，最近被描述为与雄激素敏感的PCa或良性组织相比，它们在CRPC中强烈过度表达。MicroRNAs(MiRNAs)是重要的转录后调控因子，在我们的In Silico分析中，我们发现了在CRPC中低表达的候选miRNAs，并预测其靶向为MED12/MED15。因此，我们假设调节MED12/MED15表达的miRNA参与了CRPC的发生、去势抵抗和对抗雄激素治疗的抵抗。本项目的目的是从体外和体内肿瘤上皮细胞和间质细胞的角度分析miRNA对MED12/MED15的调控及其在CRPC中的功能相关性。该项目的主要目标：i)验证电子预测的miRNAs作为MED12或MED15的调节因子，并鉴定它们在体外治疗雄激素敏感以及苯扎鲁胺或阿比特龙耐药的前列腺癌细胞系中的功能相关性。Ii)以聚合物纳米粒形式配制的已识别miRNAs的治疗应用，以抑制肿瘤异种移植生长和/或影响体内CRPC异种移植瘤对抗雄激素治疗的敏感性。这也包括来自异种移植物的组织切片培养，以进行详细的分子体外分析。3)应用原位杂交和免疫组织化学的方法，评价MED12/15及其调控的miRNAs在PCa组织中的表达与CRPC患者疾病进展和生存的临床相关性。Iv)分析抗雄激素治疗下肿瘤旁基质细胞通过转化生长因子和miRNAs对MED12/15的调节。v)联合应用miRNA替代和转化生长因子？抑制在体外增强抗肿瘤效果的评估，以及在体内患者来源异种移植(PDX)中最佳组合的治疗评估。综上所述，我们将阐明在前列腺癌肿瘤细胞与基质细胞相互作用的背景下，miRNAs和转化生长因子ç对MED12/15的调节。我们期待miRNA替代的肿瘤抑制作用，以及它与抗雄激素治疗或体内/体外转化生长因子抑制联合使用时的相加/协同效应。

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.