Targeting CXCR4 and microRNA as Therapy for Chondrosarcoma

描述(申请人提供)：软骨肉瘤是唯一一种缺乏有效系统治疗的原发骨癌。它是成人最常见的原发骨肿瘤，存活率只有10%-25%，大多数患者死于肺转移。然而，转移的机制，一个强大的潜在的治疗靶点，是未知的。长期目标是确定治疗的分子靶点。CXCR4在软骨肉瘤中的内源性表达上调，其信号转导增加了促转移因子的表达。特定的microRNAs在软骨肉瘤中也过表达，特别是miR-181a通过增强CXCR4信号而发挥oncomir的作用。CXCR4信号和miR-181a的表达也因缺氧而增加。这个应用程序目标是同时使用蜂窝 和动物模型方法，以确定miR-181a的过度表达如何与软骨肉瘤的转移有关，并确定其作为治疗靶点的适宜性。CXCR4是一种G蛋白偶联受体。G蛋白信号蛋白16调节因子(RGS16)对CXCR4信号转导有抑制作用。特定的microRNAs在癌症中的过度表达可以破坏抑制通路，从而促进肿瘤的进展。中心假设是miR-181a过表达通过抑制RGS16和潜在的其他抑制通路来增强CXCR4信号转导。这一新的假设是基于申请人的实验室产生的初步数据。这个项目的基本原理是，目前针对CXCR4的拮抗剂只有部分有效，这表明基于靶向microRNA和受体的双管齐下的方法可能更有效。在本应用中，将系统地研究miR-181a作为CXCR4增强型oncomir的两个特定目标：1)确定缺氧与软骨肉瘤转移之间的机制联系；2)在软骨肉瘤小鼠模型中评价药理学、抗microRNA和联合治疗。在目标1中，将使用慢病毒为基础的抗miR-181a的表达构建体来抑制miR-181a在软骨肉瘤细胞中的过表达，该构建体在申请人手中已被证明是可行的。我们将分析这种抑制对RGS16和CXCR4信号表达的影响。3‘-UTR荧光素酶报告将用于验证RGS16是否为miR-181a的靶标。在第二个目标中，将在小鼠软骨肉瘤模型中评估抗miR-181a药物AMD3100和两者对肿瘤生长、血管生成和转移的影响。活体生物成像将用于监测肿瘤中基质金属蛋白酶的活性和血管生成。上述研究的贡献在于详细了解了microRNA和CXCR4信号如何调节MMPs、VEGF和软骨肉瘤转移的表达。该项目具有创新性，因为它利用抗microRNA来抑制CXCR4信号，这可能导致从细胞毒性化疗转向基于生物的治疗。这项拟议的研究意义重大，因为抑制CXCR4信号将为软骨肉瘤提供第一个生物靶向治疗。

DESCRIPTION (provided by applicant): Chondrosarcoma is the only primary bone cancer that lacks an effective systemic treatment. It is the most common primary bone tumor in adults and survival is only 10-25%, with most patients succumbing to lung metastases. However, the mechanisms of metastasis, a strong potential target for therapeutics, are unknown. The long-term goal is to identify molecular targets for treatment. CXCR4 expression is endogenously upregulated in chondrosarcoma and its signaling increases expression of pro-metastatic factors. Specific microRNAs are also overexpressed in chondrosarcoma, and miR-181a in particular functions as an oncomir by enhancing CXCR4 signaling. Both CXCR4 signaling and miR-181a expression are also increased by hypoxia. The objective of this application is to use both cellular and animal model approaches to determine how overexpression of miR-181a is linked to metastasis in chondrosarcoma, and determine its appropriateness as a therapeutic target. CXCR4 is a G-protein-coupled receptor. Regulator of G-protein signaling protein sixteen (RGS16) has an inhibitory effect on CXCR4 signaling. Overexpression of specific microRNAs in cancer can disrupt inhibitory pathways, thereby promoting tumor progression. The central hypothesis is that miR-181a overexpression enhances CXCR4 signaling by inhibiting RGS16 and potentially other inhibitory pathways. This novel hypothesis is based on preliminary data produced in the applicant's laboratory. The rationale for this project is that the current antagonists for CXCR4 are only partly effective, suggesting a two-pronged approach based on targeting microRNA as well as the receptor, may be more effective. In this application, miR-181a as a CXCR4- enhancing oncomiR will be systematically investigated by pursuing two specific aims: 1) Determine the mechanistic link between hypoxia and chondrosarcoma metastasis; 2) Evaluate pharmacologic, anti-microRNA, and combined therapy in a chondrosarcoma mouse model. In Aim #1, overexpression of miR-181a in chondro- sarcoma cells will be inhibited using a lentivirus based expression construct for anti-miR-181a which has been proven feasible in the applicant's hands. The effects of such inhibition on expression of RGS16 and CXCR4 signaling will be analyzed. 3'-UTR luciferase reporters will be used to validate RGS16 as a target of miR-181a. In Aim #2, CXCR4 inhibition with anti-miR-181a, the drug AMD3100, and both will be evaluated in a mouse chondrosarcoma model for their effects on tumor growth, angiogenesis, and metastasis. In vivo bioimaging will be used to monitor matrix metalproteinase activity and angiogenesis in the tumors. The contribution of the pro- posed research is expected to be a detailed understanding of how microRNA and CXCR4 signaling regulate expression of MMPs, VEGF, and chondrosarcoma metastasis. This project is innovative because it utilizes anti-microRNA to inhibit CXCR4 signaling which may cause a shift from cytotoxic chemotherapy to biologic based therapy. The proposed research is significant because inhibition of CXCR4 signaling will provide the first biologically targeted therapy for chondrosarcoma.