Discussion
Breast cancer metastases in brain (BCM/B) show significant morphological
and genomic heterogeneity (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; Jézéquel, Kerdraon et al., 2019; 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). Patients with BCM/B have high mortality
resulted from the brain lesions and are resistant to chemotherapy
treatments (Hu, Kang et al., 2009; Cacho-Díaz, García-Botello et al.,
2020). Metastatic breast tumor cells transmigrate across the blood-brain
barrier (BBB) and form colonies of tumor cells in brain (Boire,
Brastianos et al., 2020; Hu, Kang et al., 2009; Cruceriu, Baldasici et
al., 2020). Under normal conditions, the BBB is a highly selective
barrier due to existence of tight junctions (TJs) between adjacent brain
microvascular endothelial cells (BMECs). However, in the process of
invasion of tumor cells to the brain, inflammatory cytokines and
chemokines secreted by these infiltrating tumor cells disrupt the BBB
integrity by causing damage to the continuous TJ structures formed by
endothelial cells of the brain parenchyma and astrocytes. Although the
BBB prevents the delivery of most therapeutic drugs into the brain, the
circulating metastatic tumor cells invade the damaged BBB to form
colonies in the brain (Hu, Kang et al., 2009; Cacho-Díaz, García-Botello
et al., 2020).
During tumor progression, cells undergo reprogramming of metabolic
pathways that regulate glycolysis and the production of lipids (Omabe,
Ezeani et al., 2014; Omabe, Ezeani et al., 2015; Zhang, Guo et al.,
2020; Li, Gao et al., 2019; Nomura, Long et al., 2010; Tyukhtenko, Ma et
al., 2020; Wang, Nag et al., 2015; Beloribi-Djefaflia, Vasseur et al.,
2016). Since MAGL is important in assigning the lipid stores in the
direction of pro-tumorigenic signaling lipids in cancer cells, we
studied the mechanisms by which MAGL contributes to promoting TNBC
metastases. The energy supply of lipids comes from de novosynthesis rather than circulating lipids during tumor development, which
are modulated and regulated by MAGL (Wang, Nag et al., 2015;
Beloribi-Djefaflia, Vasseur et al., 2016; Taïb, Aboussalah et al., 2019;
Yecies & Manning, 2010).
The assessment of MAGL inhibitors potency and selectivity was performed
to select compounds for the in vitro and in vivo studies
using the application of protein-based real-time 1H
NMR spectroscopy (Senga, Kobayashi et al., 2018; Carbonetti, Wilpshaar
et al., 2019; Zhu, Zhao et al., 2016), which allowed us to determine the
residence time on target for potent MAGL inhibitor AM9928 (46 h) (Figure
1). The longer inhibitory effect of AM9928 strongly suggest prolonged
pharmacodynamic effect of AM9928 which is required for our in
vivo studies.
Acute and chronic inflammation are regulated by interferons, the
interleukins, the chemokine family, mesenchymal growth factors, the
tumor necrosis factor family and adipokines (Tuo, Leleu-Chavain et al.,
2017; Karki, Man et al., 2017). Key pro-inflammatory cytokines involved
in inflammation include interleukin-1 (IL-1), IL-6, tumor necrosis
factor (TNFα), IL-8,
IL-12, IFN-γ
and IL-18.
Here, we analyzed the anti-inflammatory profile by targeting MAGL. Based
on our results, AM9928 (Figure 4) target tumor-inflammatory molecules
IL-6 and IL-8 and the angiogenic factor VEGF-A. These effects were like
the effects observed by siRNA-MAGL, but not with control siRNA (data not
shown).
Several genes were shown to be involved in the development of brain
metastases which include cyclooxygenase COX-2, EGFR ligand HBEGF and
α-2,6-sialyltransferase ST6GALNAC5 (Hu, Kang et al., 2009; Bos, Zhang et
al., 2009), all involved in facilitating cancer cell passage through the
BBB. We have previously reported the roles of the proinflammatory
peptide P in impairing the BBB integrity and the angiogenic factor
Angiopoietin-2 in mediating activation of brain microvascular
endothelial cells and BBB impairment, resulting in infiltration of TNBCs
into the brain (Rodriguez, Jiang et al., 2014; Avraham, Jiang et al.,
2014). The TME cells are the source of proinflammatory cytokines
(Cacho-Díaz, García-Botello et al., 2020; Omabe, Ezeani et al., 2015),
which are secreted by both autocrine and paracrine manner though
activation of STAT3 (Karki, Man et al., 2017; Bahiraee, Ebrahimi et al.,
2019). Indeed, the strong inhibition of IL-6 by AM9928 indicates the
potential of AM9928 to target inflammation associated with TNBC.
Therefore, MAGL may regulate lipid quality and/or quantity to promote
aggressiveness such as migration and inflammation in breast cancer
cells.
VEGF-A/VEGF-R signaling functions as an important survival pathway in
breast cancer cells 48-50 (Huang, Ouyang et al., 2018;
Mery, Rowinski et al., 2019; Banys-Paluchowski et al., 2018). VEGF-A
promoted tumor cell self-renewal through VEGF-A/VEGF-R/STAT3 signaling
resulting in activation of Myc, Sox2 and STAT3 (Huang, Ouyang et al.,
2018; Mery, Rowinski et al., 2019; Banys-Paluchowski, Witzel et al.,
2018). High VEGF-A levels strongly correlated with both STAT3 and Myc
expression as well as with tumor metastatic potential. We found
significant inhibition of VEGF-A expression by AM9928 (Figure 4c),
suggesting the effectiveness of targeting MAGL in inhibition of
angiogenesis.
Breast cancer-derived exosomes accumulate in the lung, spleen, and bone
marrow of naïve mice. At these sites, the exosomal content rich in IL-6
causes pro-metastatic alterations associated with reduction of T cell
proliferation, NK cell cytotoxicity and modulation of immune responses.
However, the role of TNBC-derived exosomes on HBMECs is not known. Here
we show that HBMECs were activated by TNBC-derived exosomes which
resulted in secretion of IL-8 and VEGF-A, thereby facilitating tumor
transmigration across HBMEC, and promoting tumor colonization. Further,
AM9929 treated TNBC-derived exosomes inhibited HBMECs activation as
indicated by the secretion levels of IL-8 and VEGF-A. Thus, TNBC-derived
exosomes may act as important mediators of intercellular communication
between TNBCs and brain endothelium facilitating the formation of
pre-metastatic niches and contributing to tumor dissemination in brain.
The BCM/B gene signature includes ANGLT4, MMP1, PTGS2 and
TNC16 . This gene signature facilitates the metastatic
process and changes in endothelial tight junction, cell migration and
invasion. In vivo studies demonstrated that AM9928 significantly
inhibited tumor growth in the mammary fat pads (Figure 5b), as compared
to untreated mice or mice treated with vehicle control. Further, AM9928
inhibited tumor cell colonization in the brain as detected by GFP
positive tumor cells ( Figure 5c + 5d). Indeed, quantitation of
GFP–4T1‐BrM5 tumor cell colonization in brain showed that the
expression of GFP-expressing Pan-CK positive tumor cells was
significantly lower in the mice group treated with AM9928, while the
presence of GFP–4T1‐BrM5 tumor cells in brain was significantly higher
in the untreated GFP-4T1BrM5 cells and the vehicle control treated mice
(Figure 5d). Since decrease in tumor colonization in the brain by AM9928
was observed, we then decided to determine BBB permeability. The
permeability of the BBB plays a crucial role in brain metastasis (Boire,
Brastianos et al., 2020; Bos, Zhang et al., 2009; Rodriguez, Jiang et
al., 2014; Avraham, Jiang et al., 2014). The changes in permeability of
the BBB‐BMEC‐TJs are a dynamic process dependent on the interaction
between TNBCs and BMECs. Our results showed that TNBC transmigration
across the BBB resulted in increased BBB permeability and these changes
in BBB permeability were reduced by AM9928 (Figure 6a) . Further, tumor
cells were shown to be in closed proximity to BMECs and the expression
of the tight junctions ZO-1 and Claudin 5 were impaired in the BBB from
mice untreated or treated with vehicle control (figure 6c), while
treatment of mice with AM9928, the expression of both ZO-1 and claudin-5
was observed as less damaged structures. Quantitation of TJs showed
there was lower expression levels of both ZO-1 and claudin-5 in mice
untreated or treated with vehicle control as compared to AM9928 treated
mice (Figure 6e-6f). However, these differences in TJ numbers were not
statistically significant between the untreated mice and the treated
AM9928 mice. Further, importantly, the number of alive mice at day 28
was higher in the AM9928 treated mice as compared to the vehicle control
group (Table 1).
Since AM9928 inhibited tumor growth in the mammary fat pads and
prevented changes in BBB integrity in vivo, resulting in reduced
tumor cell colonization in brain, we conclude that MAGL plays a role in
TNBC tumor growth and TNBC transmigration across the BBB. Taken
together, we suggest that targeting MAGL may provide a new approach to
reducing expression of proinflammatory factors as well as inhibiting
tumor growth and tumor cell colonization in brain.
Acknowledgments
We thank Shuxian Jiang for her help in the in vivo studies, Yigong Fu
for his technical support and help, Lili Wang for the quantitative
analysis of tight junctions in brain microvascular endothelial cells,
Lauren Graham and Elizabeth C. Ethier for text editing, compilation, and
figures preparations. We thank the following members at the CDD for
providing the MAGL inhibitors: Drs. Shakiru O. Alapafuja, Vidyanand
Shukla, Alexander Zvonok, and Kiran Vemuri.
The funders had no role in study design, data collection and analysis,
decision to publish or preparation of the manuscript. This study was
supported by departmental grants to Hava Karsenty Avraham and Shalom
Avraham.
Author
contributions
HKA and SA designed experiments, analyzed data, and wrote the
manuscript; OB performed in vitro experiments and analyzed data; SJ
helped with the in vivo experiments. ST helped with the NMR analysis of
MAGL inhibitors, AM for the synthesis of MAGL inhibitors, MKW, CY and AM
helped with the concept of MAGL as modulator of the BBB breaching.
* All in vivo studies were performed in the Division of
Experimental Medicine, BIDMC, Harvard Medical School, Boston and allin vitro studies were performed in CDD at Northeastern
University.
All in vivo studies were approved by BIDMC institutional Animal care and
use committee (IACUC) and the mice were handled in accordance with the
animal care policy of BIDMC, Harvard Medical School and in accordance
with the Declaration of Helsinki.