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志物。它的表达率与肿瘤的严重性代码，特别是其转移行为密切相关。然而，这种相关性背后的分子机制仍然几乎完全不清楚。PSMA的两种性质被怀疑是导致肿瘤生长和肿瘤细胞迁移增加的原因。(1)其羧肽酶的酶活性和（2）其内化到肿瘤细胞中。通过用RNA适体A9 g靶向PSMA来解决这两个特征，RNA适体A9 g抑制PSMA活性并被吸收到与PSMA结合的细胞中。最近，主办实验室发现A9 g在小鼠体内异种移植模型中也对人前列腺癌细胞表现出抗转移作用。本项目重点研究PSMA和RNA适体A9 g的亚细胞运输。从这项研究中获得的见解将阐明PSMA的细胞内作用，并旨在解开PSMA亚细胞途径报告的矛盾观察结果。申请人将检验以下假设，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 '，其主要存在于正常前列腺细胞中。大多数预期方法以前已由申请者或东道实验室确定。这些技术包括流式细胞术、RT-qPCR、共聚焦荧光显微镜和pulldown-assays。关于细胞内运输的推断信息也将用于开发优化的基于A9 g的治疗方法。在本文中，组合的第二（和第三）药物负载（例如化学治疗剂）将与A9 g缀合，其中药物的选择是关于它们在细胞内的作用位点。因此，通过将（1）A9 g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载相结合，旨在实现三模式治疗。

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.