Antitumorigenic mechanisms of the monoacylglycerol lipase inhibitor JZL184

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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.