Antitumorigenic mechanisms of the monoacylglycerol lipase inhibitor JZL184

内源性大麻素系统作为治疗癌症的药理靶点已引起人们的极大兴趣。最近，我们报道了内源性大麻素降解酶脂肪酸酰胺水解酶(FAAH)的抑制剂可以深刻地抑制肺癌细胞的侵袭和转移，但不能抑制实体瘤的生长。FAAH底物具有抗侵袭和抗肿瘤转移的作用，包括内源性大麻素2-花生四烯基甘油(2-AG)。在初步实验中，主要的2-AG降解酶单酰甘油脂肪酶(MAGL)的抑制剂JZL184被发现可以抑制几种肺癌细胞系的侵袭，并抑制人内皮细胞(HUVEC)与肿瘤细胞相关的血管生成能力，但不改变肿瘤细胞的活力。在裸鼠中，JZL184被证明具有抑制转移的作用，并且与FAAH抑制剂相比，实体瘤的生长与抑制肿瘤血管生成有关。对JZL184的反应涉及多种侵袭和血管生成的调控，如诱导基质金属蛋白酶组织抑制因子-1(TIMP-1)、下调基质金属蛋白酶-2(MMP2)和胰岛素样生长因子-1(IGF-1)的表达。本提案阐述了MAGL抑制肺癌细胞的抗肿瘤机制。将使用肺癌细胞系以及肺癌患者活检组织的原代癌细胞进行改进的Boyden小室分析，以表征JZL184对肿瘤侵袭的影响。JZL184的抗血管生成作用将通过分析来自癌细胞的条件培养液对内皮细胞的迁移、活性、管状形成和三维萌芽形成的影响来测试。JZL184和2-AG的抗侵袭和抗血管生成作用的研究重点将分别放在TIMP-1、MMP-2和IGF-1的作用上，分别使用siRNA和加回方法。此外，大麻素受体和瞬时受体潜力香草素1(TRPV1)在抗肿瘤反应中的作用将被表征。此外，还将使用缺氧肺癌细胞的条件培养液来模拟启动血管生成途径的缺氧性癌症生长，以评估血管生成特征。为了进一步深入了解MAGL抑制所触发的转移和血管生成相关的细胞内通路，将进行TaqMan阵列分析。为了确定HUVEC内或其上的血管生成相关靶点，将分析条件培养液对HUVEC迁移参数的影响，如CD63、VE-钙粘附素、帕西林和粘着斑激酶。动物实验将最终揭示JZL184与2-AG联合给药的可能的协同作用，以及大麻素受体和TRPV1对JZL184和2-AG的抗转移和肿瘤消退作用的贡献。来自异种移植实验的样本将用于分析JZL184和2-AG对体内蛋白质的调节。

The endocannabinoid system has gained considerable interest as pharmacological target in cancer treatment. Recently, we reported inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) to profoundly suppress lung cancer cell invasion and metastasis, but not growth of solid tumors. Anti-invasive and antimetastatic effects were mimicked by FAAH substrates, including the endocannabinoid 2-arachidonoylglycerol (2-AG). In preliminary experiments, JZL184, an inhibitor of the major 2-AG-degrading enzyme monoacylglycerol lipase (MAGL), was found to inhibit invasion in several lung cancer cell lines and to suppress tumor cell-related angiogenic capacities of human endothelial cells (HUVEC), while not altering the viability of tumor cells. In athymic nude mice, JZL184 was demonstrated to confer suppression of metastasis and, in contrast to FAAH inhibitors, solid tumor growth associated with inhibition of tumor angiogenesis. Several invasion- and angiogenesis-related regulations have been found as response to JZL184 such as induction of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) and downregulation of matrix metalloproteinase-2 (MMP-2) and insulin-like growth factor-1 (IGF-1). The present proposal addresses the antitumorigenic mechanisms of MAGL inhibition in lung cancer cells. Modified Boyden chamber assays will be performed using lung cancer cell lines as well as primary cancer cells from lung cancer patients´ biopsies to characterize the impact of JZL184 on tumor invasion. The anti-angiogenic impact of JZL184 will be tested by analyzing the impact of conditioned media from cancer cells on migration, viability, tube formation and 3-dimensional sprout formation of endothelial cells. The particular focus in investigating anti-invasive and anti-angiogenic effects of JZL184 and 2-AG will be set on the role of TIMP-1, MMP-2 and IGF-1 by use of siRNA and add-back approaches, respectively. Moreover, the role of cannabinoid receptors and transient receptor potential vanilloid 1 (TRPV1) in antitumorigenic responses will be characterized. Angiogenic features will additionally be assessed using conditioned media of oxygen-deprived lung cancer cells to mimic hypoxical cancer growth that initiates angiogenic pathways. To provide further insight into metastasis- and angiogenesis-related intracellular pathways triggered by MAGL inhibition, TaqMan-Array analyses will be carried out. To identify angiogenesis-linked targets in or on HUVEC, the impact of conditioned media on migration parameters in HUVEC, such as CD63, VE-cadherin, paxilin and focal adhesion kinase, will be analyzed. Animal experiments will finally reveal a probable synergism of combinational administration of JZL184 with 2-AG and the contribution of cannabinoid receptors and TRPV1 to the antimetastatic and tumor-regressive effects of JZL184 and 2-AG. Samples from xenograft experiments will be used to analyze in vivo protein regulations by JZL184 and 2-AG.