Discussions
Mammalian target of rapamycin (mTOR) is frequently activated and
overexpressed in a variety of cancers, including colon cancer. Hence,
inhibition of mTOR is one of the crucial therapeutic steps in the course
of cancer treatment. PP242 is an ATP-competitive inhibitor of mTOR, and
the activity of PP242 in the inhibition of colon cancer growth in
vitro and in vivo has been previously reported (Blaser et al.,
2012; Gulhati et al., 2009; Roulin, Cerantola, Dormond-Meuwly,
Demartines, & Dormond, 2010; Y.J. Zhang et al., 2009). However, the
underlying metabolic mechanism behind the effects of PP242 is still not
clear. In the present study, using comprehensive metabolomics and
lipidomics approaches, we identified that the antitumor effects of PP242
are associated with the inhibition of energy metabolism pathways,
including glycolysis, the TCA cycle, fatty acid β-oxidation (β-FAO) and
glycerophospholipid metabolism, in an LS174T cell-induced colon cancer
xenograft mouse model.
According to the serum biochemistry and tissue histopathology test
results, no unusual toxicological changes were observed in the liver and
kidney when PP242 was administered at a daily dose of 60
mg.kg-1.day-1 for 21 days, and these
data were supported by previously published data (Tanaka et al., 2015).
Surprisingly, PP242 reduced LDL levels and increased the HDL levels in
mice with colon cancer. This means that the change in lipid metabolism
by PP242 treatment could also affect cholesterol metabolism.
In plasma, in order to disclose the therapeutic effects of PP242, a
comparison was carried out among the three experimental groups (NC, XC,
and PP242-treated). A total of 22 metabolites were identified in plasma,
which were significantly altered in either the XC or PP242-treated
group. However, no metabolites were significantly altered between the XC
and PP242-treated groups (Table 1). We observed that despite having the
visible effect of PP242 in reducing tumor size (Fig. 2), changes in the
plasma metabolome were not consequential to conclude the underlying
mechanism. Moreover, changes in the metabolites were inconsistent, where
the XC and PP242-treated groups displayed identical patterns of change
among the metabolites compared to the NC group. Therefore, we analyzed
the tumor tissues to further uncover PP242 activity at the metabolome
level.
The comprehensive metabolomics and lipidomics profiling of tumor tissues
provided a better understanding and delineation of the underlying
mechanism of PP242 compared to that provided by the plasma analysis, and
the major perturbed metabolic pathways were mainly related to energy
metabolism (glycolysis, the TCA cycle, and β-oxidation of mitochondrial
fatty acids) and glycerophospholipid metabolism. In tumors, due to the
daily dose of PP242 for three weeks, 59 metabolites, including a few
organic metabolites, fatty acids, glycerophospholipids, glycerolipids
and sphingolipids, were significantly altered (Table 2).
In order to maintain growth and survival, most cancer cells rely highly
on glycolysis to fulfill the elevated demand for nutrients and energy,
which finally leads to the elevation of lactic acid levels (Warburg,
1956; Hay, 2016; Mason & Rathmell, 2011). This elevation in lactic acid
levels causes acidosis in the extracellular tumor microenvironment to
maintain pH homeostasis and supports the migration and invasion of
cancer cells (de la Cruz-Lopez, Castro-Munoz, Reyes-Hernandez,
Garcia-Carranca, & Manzo-Merino, 2019; Alfarouk et al., 2014; Goetze,
Walenta, Ksiazkiewicz, Kunz-Schughart, & Muller-Klieser, 2011). In the
present study, after PP242 treatment, the lactic acid level was
significantly decreased in the tumors, indicating inhibition of
glycolysis and thereby inhibiting cancer growth and invasion. We also
observed a significant reduction in the level of the TCA cycle
intermediate aspartic acid after PP242 treatment. Aspartic acid is the
degradation metabolite of the TCA cycle, which is produced from the
glutaminolysis pathway. In the glutaminolysis pathway, the production of
α-ketoglutarate from glutamine via glutamic acid is another main
metabolic pathway for tumor growth and survival that replenishes TCA
cycle energy demand by acting as a carbon source (Jin, Alesi, & Kang,
2016; Tanaka et al., 2015). Thus, the decrease in the aspartic acid
level reflects the alteration of metabolites that may reduce the tumor
growth of colon cancer xenograft mice by inhibiting the energy supply to
the TCA cycle.
In addition to glycolysis, to meet the increased energy demand, cancer
cells carry out other metabolic strategies, such as β-FAO, to produce
more energy to support cancer cell growth and survival. Carnitine plays
a major role in transporting long-chain fatty acids inside the inner
membrane of mitochondria, which facilitates β-FAO to generate and supply
acetyl-CoA to the TCA cycle for energy production (Baci et al., 2018;
Melone et al., 2018; Qu, Zeng, Liu, Wang & Deng, 2016). Previous
studies have reported that higher blood carnitine levels denote higher
energy and functioning of cells, which is the main requirement of cancer
cells. Furthermore, increased levels of carnitines were also reported in
breast cancer and chronic lymphocytic leukemia (Armitage & Southam,
2016; Cala et al., 2018). According to our results, as the level of
carnitines, including L-carnitine, L-acetylcarnitine, and
L-palmitoylcarnitine, decreased after mTOR inhibition by PP242, the
transport of fatty acids also decreased, resulting in an increase in
fatty acid levels. Hence, it could be speculated that inhibition of mTOR
signaling by PP242 acts by blocking β-FAO in cancer cells by reducing
carnitine levels.
Treatment with PP242 also significantly reduced the glycerophospholipid
(PC, PE, and PI) levels, except lysophosphatidylcholine (LysoPC), where
only one LysoPC (LysoPC18:2) was significantly affected and whose level
increased. Phosphatidylcholine (PC) is a fundamental element of the cell
membrane and plays a pivotal role in the structure and function of cell
membranes (Furse & de Kroon, 2015; Gibellini & Smith, 2010).
Upregulation of PC has been observed in numerous cancers, including
colon cancer, and is considered one of the hallmarks of cancer growth
and progression (Cheng, Bhujwalla, & Glunde, 2016; Jones et al., 2019;
Kurabe et al., 2013). In this experiment, after PP242 treatment, the
level of PC was significantly downregulated, suggesting that PP242 is
able to inhibit cancer growth and progression. One LysoPC was
significantly increased when the mice were exposed to PP242. LysoPC is
normally generated from PC through the catalysis of phospholipase
A2 (PLA2), and few studies have reported
that the upregulation of LysoPC can be associated with the induction of
apoptosis (Law et al., 2019; M. Takahashi et al., 2002). Therefore,
according to our results, the increase in LysoPC levels could be due to
the apoptotic effects of PP242. Besides, LysoPC could also increase due
to the inhibition of its conversion from PC, which also decreases the
level of PC (Gao et al., 2016).
Phosphatidylethanolamine (PE) is another vital element of phospholipids,
and in mammalian cells, PE accounts for almost 15-25% of the total
lipid content and is also associated with a vast number of physiological
cellular processes (Lu et al., 2019; Patel & Witt, 2017; Tan et al.,
2017). In the normal cellular state, the existence of PE is only found
in the inner leaflet of the cell membrane. However, upregulation of PE
on the outer surface of cancer cells has been previously reported
(Stafford & Thorpe, 2011; Tan et al., 2017). PE was significantly
downregulated after PP242 treatment in our study, suggesting the
potential of PP242 in treating cancer by decreasing PE levels.
We also observed a significant reduction in all phosphatidylinositol
(PI) species after exposure to PP242 for three weeks. PI is another
important phospholipid class, accounting for approximately 5.6% of
total lipids, mainly exists in the inner leaflet of the cell membrane,
and plays a role in regulating cell survival, signaling and membrane
trafficking (Duan, 2016; Falkenburger, Jensen, Dickson, Suh, & Hille,
2010). PI overexpression is also associated with cancer progression, and
thus, a decrease in PI levels has shown cancer growth suppression
against various cancers (Baba et al., 2001; Kim, Jin, Bae, & Choi,
2019; Imoto et al., 1998). This evidence of PI level reduction supports
our results and indicates the potent antitumor activity of PP242.
Among the anionic phospholipids, phosphatidylserine (PS) is the most
abundant and is located in the plasma membrane’s inner leaflet of most
mammalian cells. It has also been reported that cancer cells possess
elevated levels of PS on their external surface (Davis et al., 2019;
Vallbhapurapu et al., 2015). Hence, the decrease in PS levels, could
suggest the suppression of cancer growth by the effects of PP242
treatment. Diacylglycerol (DG) and sphingomyelin (SM) were significantly
increased and decreased, respectively. In cellular signaling and lipid
metabolism, DG plays a very important role as an intermediate (Cala et
al., 2018). SM plays important roles in maintaining cell barrier
functions and fluidity as a structural component of the cell membrane
and regulate various cellular processes (Hannun & Obeid, 2008;
Ogretmen, 2018). Additionally, depending on the tumor biology, the
sphingolipid level could be increased or decreased (Knapp, Chomicz,
Swiderska, Chabowski, & Jach, 2019). Thus, the decrease in SM levels
could express the therapeutic effects of PP242. However, the association
of DG in this study is not clearly understood. A summary of the main
altered metabolic and lipidomic pathways due to three weeks of PP242
treatment in LS174T-induced colon cancer xenografts is shown in Fig. 8.
Herein, we investigated the antitumor effects of PP242 in LS174T
cell-induced colon cancer xenograft mouse model’s plasma and tumors
using comprehensive metabolomics and lipidomics approaches to discover
the metabolic mechanism of PP242 for the first time. Xenograft mice were
treated with PP242 for three weeks, and significant reductions in tumor
size and weight were observed. The plasma metabolic alterations were
identical in the XC and PP242-treated groups, and no metabolites were
significantly altered, suggesting that plasma samples were not enough to
disclose the metabolic mechanism of PP242. The metabolic and lipidomic
investigation of tumor tissues revealed that PP242 displayed its
antitumor activity by inhibiting energy and glycerophospholipid
metabolism, which are the major upregulated pathways in most cancers.
PP242 also exerts its anticancer effect through inhibiting the
β-oxidation of fatty acids. In addition, daily doses of PP242 over three
weeks did not induce any abnormal effects, indicating its safety level.
Together, this study provides valuable insights towards understanding
the underlying actions of PP242 on the basis of the metabolome and could
help to further implement PP242 in clinical analysis.