Targeting Tumor-Derived exRNA-Containing Microvesicles by High Throughput Screeni

描述（由申请人提供）：揭示肿瘤生长和转移的潜在机制对于开发针对去势抵抗性前列腺癌（CRPC）的治疗策略至关重要。最近的研究表明，肿瘤募集的脂肪干细胞（ASCs）可以促进肿瘤生长，特别是在肥胖患者中。与正常对应物不同，我们的初步数据表明，PC细胞分泌的因子诱导患者来源的ASC（pASC）的表型基因型变化，并破坏它们进行肿瘤转化。来自PC细胞（C4-2B和PC-3）而非正常前列腺上皮细胞（RWPE 1）的微泡（MV）（50-200 nm）引发肿瘤嗜性pASC以形成具有细胞遗传学畸变的肿瘤性病变，这让人联想到前列腺肿瘤和间充质-上皮转化（MET）的分子特征。pASC的致癌“重编程”与许多致癌miRNA（miR-125 b、miR-130 b、miR-155）和致癌mRNA（H-Ras和K-Ras）的转移相关，其进而下调pASC中的几种关键肿瘤抑制因子（TP 53、PDCD 4和Lats 2）。我们的数据揭示了肿瘤来源的细胞外RNA（exRNA）在通过受体pASC释放和摄取MV促进肿瘤生长中的先前未表征的作用。与正常受试者相比，癌症患者的血清含有高水平的循环MV;因此可以推测肿瘤来源的MV转移致癌exRNA货物使得癌症患者中的pASC能够发生肿瘤转化。因此，靶向肿瘤细胞的MV生物发生和释放和/或pASC的摄取，而不是单个exRNA，将更有效地消除pASC中多种致癌exRNA的转移和肿瘤发展。因此，我们假设临床批准的化合物的高通量筛选（HTS）将使我们能够选择抑制MV从肿瘤细胞的生物发生和释放和/或pASC对MV的摄取的先导剂。我们将通过利用细胞培养模型中的里程碑驱动实验和PC动物模型中的概念验证临床前研究来证实我们的假设。UH 2（I期）将鉴定在体外和体内有效消除MV生物合成、释放和/或摄取的化合物。UH 3（II期）将进一步验证体内抗肿瘤 这些药理学先导药物的有效性，并将支持在动物模型中进行更严格的里程碑驱动的临床前研究。以下具体目标（通过两个阶段执行）证实了我们的假设：第一阶段（UH 2）（1）证明从CRPC患者中获得的循环MV是否含有致癌exRNA并诱导pASC的肿瘤转化。(2)优化HTS检测，以鉴定抑制肿瘤细胞释放MV或受体pASC摄取MV或降低其致癌miRNA/RNA载量的先导化合物。(3)确定先导化合物是否在体内抑制肿瘤细胞释放和/或pASC对MV的摄取。II期（UH 3）（1）检查先导化合物在体内抑制ASC衍生肿瘤发展的功效。(2)NCATS库中先导化合物的临床适用性。尽管MV与癌症进展（新血管形成）有关，但尚未研究其在癌症患者干细胞肿瘤转化中的作用。我们证明了肿瘤细胞衍生的MV在细胞外癌RNA的转移和患者获得的ASC的转化中是新颖的，并证明了它们在癌症患者转移性疾病的生长和/或进展中的潜在作用。因此，所提出的工作是创新的，因为它不仅利用了MV介导的癌-miR和癌-mRNA的新功能作用的基础发现，而且还鉴定了新的先导治疗化合物以规避PC进展。通过建立预防性和/或治疗性干预策略，预计拟议研究的结果将对晚期PC的临床管理产生积极影响。

DESCRIPTION (provided by applicant): Unraveling the underlying mechanisms of tumor growth and metastasis is critical to developing curative strategies against castration-resistant prostate cancer (CRPC). Recent findings demonstrate that tumor-recruited adipose stem cells (ASCs) can promote tumor growth, especially in obese patients. Unlike normal counterparts, our preliminary data showed that factors secreted by PC cells induce phenotypic genotypic changes in patient-derived ASCs (pASCs) and subvert them to undergo neoplastic transformation. Microvesicles (MVs) (50-200 nm) from PC cells (C4-2B and PC-3), but not normal prostate epithelial cells (RWPE1), primed tumor-tropic pASCs to form neoplastic lesions with cytogenetic aberrations reminiscent of the molecular features of prostate tumors and mesenchymal-to-epithelial transition (MET). The oncogenic 'reprogramming' of pASCs was associated with the transfer of a number of oncogenic miRNAs (miR-125b, miR-130b, miR-155) and oncogenic mRNAs (H-Ras and K-Ras), which in turn down-regulated several key tumor suppressors (TP53, PDCD4 and Lats2) in pASCs. Our data deciphered previously uncharacterized roles for tumor-derived extracellular RNAs (exRNAs) in promoting tumor growth via the release and uptake of MVs by the recipient pASCs. Cancer patient's sera contain high levels of circulating MVs in comparison to normal subjects; thus it is possible to speculate the transfer of oncogenic exRNA cargo by the tumor-derived MVs enable neoplastic transformation of pASCs in cancer patients. Accordingly, targeting MVs biogenesis and release by tumor cells and/or uptake by pASCs, rather than individual exRNAs, would be more efficacious in abrogating the transfer and tumor development by multiple oncogenic exRNAs in pASCs. We therefore hypothesize that a high throughput screening (HTS) of clinically approved compounds will enable us to select lead agents that suppress the biogenesis and release of MVs from tumor cells and/or the uptake of MVs by pASCs. We will corroborate our hypothesis by utilizing milestone-driven experiments in cell culture models and by proof-of-concept pre-clinical studies in animal models of PC. The UH2 (phase-I) will identify compounds that potently abrogate the biogenesis, release and/or uptake of MVs in vitro and in vivo. The UH3 (phase-II) will further validate the in vivo anti-tumor efficacy of these pharmacological leads and will support more rigorous milestone-driven pre-clinical studies in animal models. The following specific aims, to be executes through two phases, corroborate our hypothesis: Phase-I (UH2) (1) Demonstrate if circulating MVs procured from CRPC patients harbor oncogenic exRNAs and induce neoplastic transformation of pASCs. (2) Optimize HTS assays to identify lead compounds which inhibit the release of MVs by tumor cells, or their uptake by recipient pASCs, or reduce their oncogenic miRNA/RNA load. (3) Establish if lead compounds inhibit tumor cell release and/or uptake of MVs by pASCs in vivo. Phase II (UH3) (1) Examine the efficacy of lead compounds in inhibiting ASC-derived tumor development in vivo. (2) Clinical applicability of the lead compounds from the NCATS library. Although MVs have been implicated in cancer progression (neovacularization), their role in neoplastic transformation of stem cells in cancer patients has not been investigated. Our demonstration of tumor cell derived MVs in transfer of extracellular onco-RNAs and transformation of patient procured ASCs is novel and lend credence to their potential role in outgrowth and/or progression of metastatic disease in cancer patients. Accordingly, the proposed work is innovative, because it capitalizes not only in underpinning discovery of new functional roles for MV-mediated onco-miRs and onco-mRNAs, but also in identifying new lead therapeutic compounds to circumvent PC progression. By establishing preventive and/or therapeutic intervention strategies, the outcome of the proposed studies is expected to exert a positive impact on the clinical management of advanced PC.