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.