BACKGROUND:
Breast cancer is a common cause of brain metastases occurring in at least 10-16% of patients and reaching 25-35% of TNBC patients (Hubalek, Hubalek et al., 2017; Yam, Mani et al., 2017; Yates, Knappskog et al., 2017; Omabe, Ezeani et al., 2014; Da Silva, Cardoso Nunes et al., 2020). Unfortunately, patients who are diagnosed with brain metastases often have poor prognosis with short overall survival times (Jézéquel, Kerdraon et al., 2019; Boire, Brastianos et al., 2020; Lehmann, Jovanović et al., 2016; Hu, Kang et al., 2009; Cacho-Díaz, García-Botello et al., 2020; Roma-Rodrigues, Mendes et al., 2019; Belli, Trapani et al., 2018; Nakamura & Smyth, 2017; Cruceriu, Baldasici et al., 2020).
Cancer cells endogenously synthesize 95% of free fatty acids (FFAs) de novo (Omabe, Ezeani et al., 2015) and incorporate and remodel exogenous palmitate into structural and oncogenic glycerophospholipids, sphingolipids and ether lipids (Omabe, Ezeani et al., 2015). Fatty acid synthesis (FAS) pathway confers a survival advantage to cancer cells and especially to tumor cells that are resistant to chemotherapy (Omabe, Ezeani et al., 2015). Fatty acid-binding protein 5 (FABP5) promoted lipolysis droplets, de-novo fatty acid synthesis, and coordinated lipid signaling that promoted prostate cancer metastasis (Senga, Kobayashi et al., 2018; Carbonetti, Wilpshaar et al., 2019). Increased FFA levels in tumors lead to enhanced tumor aggressiveness by increasing lipid synthesis mediated by lipolytic pathways (Omabe, Ezeani et al., 2015; Senga, Kobayashi et al., 2018; Carbonetti, Wilpshaar et al., 2019; Zhu, Zhao et al., 2016; Zhang, Guo et al., 2020; Li, Gao et al., 2019; Nomura, Long et al., 2010). Thus, targeting lipid metabolism may prevent the gain of survival advantage and improve treatment response in cancer cells.
MAGL is a serine hydrolase that regulates a fatty acid network that promotes cancer pathogenesis by enriching pro-tumorigenic signaling molecules. MAGL is a key hydrolytic enzyme in the FFA tumor network reported in colorectal cancer, neuroblastoma, nasopharyngeal carcinoma, and other cancers (Zhu, Zhao et al., 2016; Zhang, Guo et al., 2020; Li, Gao et al., 2019; Nomura, Long et al., 2010). MAGL primarily hydrolyzes endocannabinoid 2-AG and other monoacylglycerides and indirectly controls the levels of free fatty acids derived by their hydrolysis.
A new series of highly potent carbamate MAGL inhibitors were synthesized and characterized in our lab. In this study, we have investigated the inhibitor AM9928, which exhibited high potency against recombinant human MAGL with IC50 value of 8.9nM and lacked any affinity for the cannabinoid receptors CB1 and CB2 (Tyukhtenko, Ma et al., 2020; Tyukhtenko, Karageorgos et al., 2016; Tyukhtenko, Rajarshi et al., 2018). AM9928 demonstrated: (1) potency and selectivity for the target; (2) suitable physicochemical properties with low lipophilicity (ClogP 3-4); (3) good microsomal stability (>20 min) and plasma stability (>120 min); and (4) long target engagement by assessing the time required for MAGL reactivation (residence time) following the covalent inhibitory interaction between ligand and protein.
Since MAGL is important for lipid metabolism and lipid metabolism plays various roles in tumorigenesis, we investigated MAGL’s role in the process of tumor cell extravasation across the BBB and tumor colonization in brain. MAGL is highly expressed in TNBCs, which secrete high levels of inflammatory cytokines and chemokines. We hypothesized that tumor growth in the mammary fat pads and tumor cell infiltration across the BBB is facilitated via inflammatory chemokines/cytokines secreted from TNBC cells, leading to activation of HBMECs and resulting in TNBC spreading and colonization in brain. We found that TNBC adhesion to HBMECs and transmigration across HBMECs was inhibited by AM9928. In addition, AM9928 inhibited TNBC’s secretion of inflammatory cytokines and the angiogenic factor VEGF-A. Notably, AM9928 inhibited TNBC tumor growth in vivo, decreased in vivo BBB permeability and significantly reduced TNBC colonization in brain. Taken together, these results demonstrate novel mechanisms by which MAGL mediates its effects on brain metastasis by modulating BBB permeability.