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.