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

描述(由申请人提供)：揭开肿瘤生长和转移的潜在机制对于开发抗去势前列腺癌(CRPC)的治疗策略至关重要。最近的发现表明，肿瘤募集的脂肪干细胞(ASCs)可以促进肿瘤的生长，特别是在肥胖患者中。与正常对照组不同，我们的初步数据显示，PC细胞分泌的因子诱导患者来源的ASCs(PASCs)发生表型变化，并颠覆它们经历肿瘤性转化。来自PC细胞(C4-2B和PC-3)的微泡(MVS)(50-200 nm)，而不是正常的前列腺上皮细胞(RWPE1)，启动了亲肿瘤的pASCs，形成具有细胞遗传学异常的肿瘤病变，使人想起前列腺癌的分子特征和间充质向上皮的转变(MET)。PASCs的致癌重编程与一些致癌的miRNAs(miR-125b、miR-130b、miR-155)和致癌的mRNAs(H-RAS和K-RAS)的转移有关，这反过来又下调了pASCs中的几个关键肿瘤抑制因子(TP53、PDCD4和Lats2)。我们的数据破译了先前未知的肿瘤来源的细胞外RNA(ExRNAs)通过受体pASCs释放和摄取MVS促进肿瘤生长的作用。与正常人相比，癌症患者血清中含有高水平的循环MVS；因此，可以推测肿瘤来源的MVS转移致癌的exRNA货物使癌症患者的pASCs发生肿瘤转化。因此，靶向MVS的生物发生、肿瘤细胞的释放和/或pASCs的摄取，而不是单个exRNAs，将更有效地消除pASCs中多个致癌exRNAs的转移和肿瘤的发展。因此，我们假设，对临床批准的化合物进行高通量筛选(HTS)将使我们能够选择能够抑制MVS的生物发生和从肿瘤细胞释放和/或pASCs摄取MVS的先导剂。我们将通过在细胞培养模型中利用里程碑驱动的实验以及在PC的动物模型中进行概念验证临床前研究来证实我们的假设。UH2(第一阶段)将确定在体外和体内有效地消除MVS的生物发生、释放和/或吸收的化合物。UH3(II期)将进一步验证体内抗肿瘤 这些药物的有效性领先，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。以下具体目标将分两个阶段执行，证实了我们的假设：阶段I(UH2)(1)证明从CRPC患者获得的循环MVS是否含有致癌外RNA并诱导pASCs的肿瘤性转化。(2)优化HTS检测，以确定能够抑制肿瘤细胞释放MVS的先导化合物，或受体pASCs摄取MVS的先导化合物，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pASCs摄取MVS。第二阶段(UH3)(1)检查铅化合物在体内抑制ASC来源的肿瘤发展的有效性。(2)NCATS文库中先导化合物的临床适用性。虽然MVS与癌症进展(新生血管形成)有关，但它们在癌症患者干细胞肿瘤性转化中的作用尚未被研究。我们证明了肿瘤细胞来源的微血管在细胞外肿瘤RNA的转移和患者获得的ASCs的转化中是新颖的，并证实了它们在癌症患者转移疾病的生长和/或进展中的潜在作用。因此，这项拟议的工作是创新的，因为它不仅有助于发现MV介导的onco-miRs和onco-mRNAs的新功能，而且还有助于识别新的先导治疗化合物来绕过PC进展。通过建立预防和/或治疗干预策略，拟议研究的结果有望对晚期PC的临床治疗产生积极影响。

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.