Investigation of subcellular trafficking of prostate specific membrane antigen (PSMA) and development of optimised aptamer therapeutics targeting PSMA.

前列腺特异性膜抗原（PSMA）是前列腺癌细胞的重要细胞表面标记物。其表达率与肿瘤的严重程度密切相关，特别是与肿瘤的转移行为密切相关。然而，这种相关性背后的分子机制仍然几乎完全不清楚。PSMA的两个特性被怀疑可以解释肿瘤生长和肿瘤细胞迁移的增加。(1)其羧肽酶活性和(2)其内化到肿瘤细胞。这两个特征都是通过RNA适配体A9g靶向PSMA来解决的，A9g抑制PSMA活性，并被吸收到与PSMA结合的细胞中。最近，宿主实验室在小鼠体内异种移植模型中发现A9g对人前列腺癌细胞也表现出抗转移作用。目前的项目重点是PSMA和RNA适配体A9g的亚细胞运输。从这项研究中获得的见解将揭示PSMA的细胞内作用，并旨在解开PSMA亚细胞途径的矛盾观察报告。申请人将检查假设，即内化的PSMA部分转移到细胞质中并被蛋白水解以产生非膜形式的PSM'，这主要存在于正常前列腺细胞中。大多数预期的方法以前是由申请人或主办实验室建立的。这些包括流式细胞术、RT-qPCR、共聚焦荧光显微镜和拉下试验。关于细胞内交通的推断信息也将用于开发优化的基于A9g的治疗方法。在此，组合的二级（和三级）药物负荷（例如，化疗药物）将与A9g结合，药物的选择取决于它们在细胞内的作用部位。因此，三模式治疗是通过结合(1)A9g的抗转移能力与(2)针对关键肿瘤基因的sirna和(3)上述位点特异性药物负荷来实现的。

Prostate specific membrane antigen (PSMA) is a prominent cell surface marker of prostate cancer cells. Its expression rate is strongly correlated to the severity code of the tumor, in particular to its metastatic behaviour. However the molecular mechanisms behind this correlation remain almost totally unclear. Two properties of PSMA are suspected to account for increased tumor growth and tumor cell migration. (1) Its enzymatic activity of a carboxypeptidase and (2) its internalisation into the tumor cell. Both features are addressed by targeting PSMA with the RNA aptamer A9g, which inhibits PSMA activity and is taken up into the cell bound to PSMA. Recently, the hosting laboratory found that A9g also exhibits an anti-metastatic effect on human prostate cancer cells in an in vivo xenograft model in mice.The present project focusses on the subcellular trafficking of PSMA and the RNA aptamer A9g. The insights derived from this study will shed light on the intracellular actions of PSMA and are intended to unravel the contradictory observations reported for PSMAs subcellular pathway. The applicant will examine the hypothesis, that internalised PSMA is in part transferred into the cytoplasm and proteolysed to yield the non-membrane form PSM', which is predominantly found in normal prostate cells. The majority of the intended methods has formerly been established by the applicant or by the hosting laboratory. These include flow cytometry, RT-qPCR, confocal fluorescence microscopy and pulldown-assays.The deduced information about the intracellular traffic will also be used for the development of optimised A9g based therapeutics. Herein combinatorial secondary (and tertiary) drug loads (e.g. chemotherapeutics) will be conjugated to A9g with the choice of drug being in respect to their site of action within the cell. As a result a trimodal therapeutic is intended by combining (1) A9g's anti-metastatic ability with (2) siRNAs against crucial tumor genes and (3) the aforementioned site-specific drug load.