Mechanistic Role of miRNAs and Their Targets in Prostate Cancer Aggressiveness

描述(申请人提供)：前列腺癌(PCa)相关死亡是由去势抵抗型前列腺癌(CRPC)的出现和随后的转移引起的，这表明需要更好地从机制上了解肿瘤的侵袭性，以推动新疗法的发展。新的证据表明，上皮到间充质转化(EMT)的获得，这一类似于癌症干细胞样细胞发生的过程，有助于肿瘤的侵袭性，并由miR-200和let-7家族等microRNAs(MiRNAs)的非调控表达介导。MiR-200的缺失导致了在人类前列腺癌中普遍存在的Lin28b的过度表达。已知Lin28b还能阻断另一种miRNA(prelet-7和pri-let-7)的加工，导致成熟let-7减少，从而导致Suz12和EZH2表达增加，这是多梳抑制复合体2(PRC2)的重要组成部分。因此，Lin28b的过度表达以及miR-200和let-7的缺失似乎是导致PCa侵袭性的原因。我们发现，在具有EMT表型的PDGF-D过表达的PC3细胞(PC3 PDGF-D细胞)中，Lin28b的过度表达伴随着miR-200和let-7家族的表达降低，Suz12和EZH2的表达增加，这与从人PCA组织标本中获得的结果一致。此外，我们发现这些标记物在非裔美国人和高加索裔美国人患者中的表达存在差异。我们还发现，miR-200b、miR-200c和let-7的重新表达无论是通过遗传途径还是通过我们新开发的试剂(3，4-二氟苯并姜黄素或CDF)处理细胞，都下调了Lin28b和EZH2的表达。根据我们的初步结果，我们假设Lin28b的过表达导致PCa细胞(EMT表型细胞)通过下调miR-200b和miR-200c的表达而获得侵袭和转移特性，导致Suz12、ZEB1和ZEB2的表达增加。我们还假设Lin28b的过表达抑制了let-7家族的成熟，导致了EZH2的表达增加，并且这些过程可以被CDF处理的细胞所减弱。我们将通过三个具体目标来检验我们的假设。目的-1：建立Lin28b调节miRNAs和引起肿瘤细胞侵袭性(即增加细胞增殖、迁移、侵袭、克隆生长和自我更新能力)的机制(S)。目的：在体外和体内动物模型中，确定CDF介导的肿瘤细胞生长抑制是否是由于Lin28b、miR-200、LET-7、Suz12和EZH2表达的下调所致。目的-3：评估Lin28b、EZH2、miR-200和let-7在非裔美国人和高加索裔美国人患者的前列腺癌组织标本中的表达与肿瘤侵袭性的相关性。我们的临床前机制和基于PCa组织的研究将深入了解miRNAs及其靶点在表征PCa侵袭性方面的作用，并将提供一种新的方法(即使用CDF)来设计针对PCa患者的靶向治疗。

DESCRIPTION (provided by applicant): Prostate cancer (PCa)-related deaths are caused by the emergence of castrate-resistant prostate cancer (CRPC) and subsequent metastasis, suggesting the need for better mechanistic understanding of tumor aggressiveness in order to advance the development of novel therapies. Emerging evidence suggests that acquisition of the epithelial-to-mesenchymal transition (EMT), a process that resembles the genesis of cancer stem-like cells, contributes to tumor aggressiveness and is mediated by deregulated expression of microRNAs (miRNAs), such as miR-200 and let-7 family. Loss of miR-200 expression results in the over-expression of Lin28B, which is prevalent in human PCa. Lin28B is also known to block the processing of another miRNA (pre-let-7 and pri-let-7), resulting in decreased mature let-7, thereby leads to increased Suz12 and EZH2 expression, which are important components of the polycomb repressive complex 2 (PRC2). Thus, over- expression of Lin28B and loss of miR-200 and let-7 appear to be responsible for PCa aggressiveness. We found over-expression of Lin28B in PDGF-D-over-expressing PCa cells with the EMT phenotype (PC3 PDGF- D cells) concomitant with decreased expression of miR-200 and let-7 family and increased expression of Suz12 and EZH2, which is consistent with findings obtained from human PCa tissue specimens. Moreover, we found differential expression of these markers between African-American and Caucasian-American patients. We also found that the re-expression of miR-200b, miR-200c, and let-7 either by a genetic approach or by treating cells with our newly developed agent (3,4-difluorobenzo-curcumin or CDF) down-regulated the expression of Lin28B and EZH2. Based on our preliminary results, we hypothesize that over-expression of Lin28B leads to the acquisition of invasive and metastatic characteristics in PCa cells (EMT-phenotype cells) via down-regulation of miR-200b and miR-200c, resulting in increased expression of Suz12, ZEB1, and ZEB2. We also hypothesize that over-expression of Lin28B represses the maturation of let-7 family, leading to increased expression of EZH2, and these processes can be attenuated by treatment of cells with CDF. We will test our hypotheses by three specific aims. Aim-1: Establish the mechanism(s) by which Lin28B regulates miRNAs and causes tumor cell aggressiveness (i.e., increased cell proliferation, migration, invasion, clonogenic growth, and self-renewal capacity). Aim 2: Determine whether CDF-mediated inhibition of tumor cell growth is due to deregulation of Lin28B, miR-200, let-7, Suz12, and EZH2 expression both in vitro and in an animal model in vivo. Aim-3: Assess the relevance of Lin28B, EZH2, miR-200, and let-7 expression for characterizing tumor aggressiveness in human PCa tissue specimens in African-American compared to Caucasian-American patients. Our pre-clinical mechanistic and PCa-tissue-based research will provide insights into the roles of miRNAs and their targets in characterizing PCa aggressiveness, and will also provide a novel approach (i.e., use of CDF) toward designing targeted therapy for PCa patients.