Therapeutic approaches that specifically target tumors and have few systemic side effects are important for cancer treatment. In recent years, many researchers have focused on therapeutic strategies targeting tumor-specific energy metabolism. However, there are few studies on cancer metabolism in childhood cancers, especially RMS. The present study yields novel findings concerning a therapeutic strategy for RMS, focusing on lipid metabolism.
It has been previously reported that the Warburg effect exists in RMS [10, 12]. The verification of intracellular metabolism using stable isotopes revealed that glucose uptake was significantly increased and lactate synthesis was enhanced in Rh30 compared to normal myocytes, demonstrating upregulated glycolytic function in RMS . Moreover, the mitochondrial TCA cycle was upregulated in Rh30 , indicating that energy substrates other than glucose, such as glutamic acid and fatty acids, were also essential for the TCA cycle. Thus, we hypothesized that suppressing lipid metabolism in RMS would upset the balance of tumor-specific energy metabolism, or the Warburg effect, leading to an antitumor effect.
Metabolomic analysis revealed that most intermediates in the mitochondrial TCA cycle were significantly reduced by the administration of MCDi, as would be expected if fatty acid oxidation was inhibited. Only 2-OG among the intermediate metabolites of the TCA cycle showed increased levels, likely owing to the anaplerotic pathway, which takes up glutamate from the cytoplasm into the mitochondria, . The metabolomic analysis further revealed that the PPP was down-regulated and nucleic acid synthesis was suppressed in RMS treated with the MCDi. AMPK is a key nutrient sensor that controls various intracellular signals by reflecting the nutritional status, not only in normal cells but also in tumor cells . Recently, AMPK has been reported to suppress the expression of G6PD, the rate-limiting enzyme in the first stage of PPP, by inactivating the transcriptional activities of cyclic AMP-response element-binding protein (CREB) and CREB-regulated transcriptional coactivator-1 (CRTC-1) [21, 22]. In our study, the inhibition of lipid metabolism in RMS cell lines increased AMPK phosphorylation, reflecting the intracellular low-energy state, and suppressed the expression of G6PD mRNA. This suggests that the inhibition of lipid metabolism suppressed the PPP in RMS cells via the AMPK-CRCT1-CREB-G6PD pathway. The inhibition of lipid metabolism induced cell cycle arrest in the G1 phase and increased the expression of p21 protein in all RMS cell lines. p21 is a major target of p53 activity and is thus associated with cell cycle arrest. In RMS cells, especially Rh30, Rh41, and RD cell lines, the tumor suppressor protein p53 is either mutated or deleted, and therefore its function is suppressed [23–25]. The inhibition of lipid metabolism suppressed the PPP in RMS cells, which might induce G1 arrest by promoting p21 expression via a p53-independent pathway [26, 27]. Overall, we proposed that the imbalanced tumor-specific energy metabolism due to the suppression of lipid metabolism led to an antitumor effect against RMS cells.
Autophagy facilitates the production of energy substrates required for cells to maintain their survival during starvation and breaks down the waste products accumulated inside cells, such as abnormal proteins and organelles with reduced function [28, 29]. While various signals control autophagy, AMPK is a major factor that induces autophagy, especially under nutrient deprivation . mTORC1 is a major suppressor of autophagy signals . As mentioned above, AMPK inhibits mTORC1 through the activation of TSC2 and also directly activates the unc-51 like autophagy activating kinase (Ulk1), followed by the induction of autophagy signals [32, 33]. Autophagy is present not only in normal cells but also in cancer cells [29, 34]. In this study, the suppression of lipid metabolism increased AMPK phosphorylation in RMS cell lines, resulting in the activation of the autophagy pathway. Additionally, western blotting and immunofluorescence staining confirmed the increased expression of LC3-II protein, which is a component of the autophagosome membrane and is therefore considered to be an autophagy marker.
Previous studies have shown that the role of autophagy, whether beneficial or harmful to tumors, is complex depending on the type of cancer. The advantage of autophagy in cancer cells is its protective role against various environmental stressors, and blocking this compensatory response increases cancer cell death [35, 36]. On the contrary, the activation of the autophagy pathway itself induces cancer cell death, which is called autophagic cell death . This type of programmed cell death is defined by the reduction of cancer cell death due to inhibition of autophagy. It has been reported that several therapeutic approaches induced autophagy in RMS. In addition, when the increased autophagy was suppressed, the apoptotic cell death was induced. [38, 39]. Consistent with these previous studies, we found that the suppression of lipid metabolism in RMS induced autophagy. Moreover, the inhibition of autophagy led to the promotion of apoptosis. Therefore, autophagy plays a tumor-protective role in RMS, and combination therapy by suppressing both lipid metabolism and autophagy may be promising as a therapeutic strategy.
Based on the results of in vitro experiments, we examined the lipid-dependence of RMS in orthotopic xenograft models by restricting dietary exogenous fats. Comparison between NCD-fed and LFD-fed mice showed no significant difference in body weight and total calorie intake during the observation period. However, bioluminescence imaging showed significantly suppressed tumor growth in LFD-fed mice compared to that in NCD-fed mice. Since calorie restriction was not involved, the difference in exogenous lipid uptake between the two groups was considered to be the cause of this difference. Moreover, CPT1A mRNA expression was significantly decreased in the tumors of LFD-fed mice, suggesting that not only the uptake of exogenous lipids but also the import of fatty acids into mitochondria were reduced. A significant decrease in the expression level of Ki67, a cell cycle-related protein, was observed in LFD-fed mice, indicating that the reduced lipid metabolism led to the suppression of the cell cycle. PPP might also have been affected, as the expression of G6PD mRNA tended to decrease. Thus, our in vivo experiments showed that the reduced exogenous lipid supply due to LFD affected lipid metabolism in RMS and exerted a tumor growth inhibitory effect.
Recently, there have been reports that HFD promotes the growth of several types of tumors. In colorectal cancer, increased level of HFD-derived palmitic acid induced the activation of hormone-sensitive lipase through the upregulation of cAMP/PKA signaling, which was promoted by increasing β2 adrenergic receptor expression, and enhancing the supply of fatty acids as an energy substrate in tumor cells; this affected the metabolic phenotype and made the tumor more malignant . HFD also increases the population of myeloid-derived suppressor cells, which have been reported as facilitators of tumor growth, resulting in the stimulation of inflammation in the microenvironment surrounding cancer cells and affecting the malignancy of the tumor [41, 42]. In other words, the availability of a large amount of fatty acids contributes to tumor growth in various ways. Like normal cells, cancer cells have lipid droplets in their cytoplasm, which enable them to adjust to adverse conditions such as starvation and oxidative stress . Furthermore, many lipid metabolism-related enzymes are associated with the undesirable characteristics of cancer cells, such as invasion and metastasis . High lipid availability is advantageous for tumor cells, whereas low lipid content may be disadvantageous for cancer growth; however, there have been few reports indicating that the suppression of lipid utilization exerts an antitumor effect.
The limitations of the present study are below: In the in vitro experiments, we only compared the effects of using the drug that suppresses intracellular lipid metabolism, and did not compare the effects on inhibiting the utilization of the exogeneous lipids, such as by reducing the fat content in the medium. Moreover, the biological experiments in this study were conducted in mice, and the effects of lipid metabolism inhibition on humans need to be fully investigated in the future.
In conclusion, the present study revealed that the inhibition of lipid metabolism suppressed the PPP in RMS. As a result, the synthesis of nucleic acids essential for the growth of cancer cells and antioxidants required for maintaining oxidative balance decreased, leading to a tumor growth inhibitory effect due to cell cycle arrest. It has been suggested that AMPK might play a key role in this antitumor effect. The autophagy signals induced by the AMPK-mTOR pathway were thought to be a tumor-protective reaction in RMS. This is because apoptotic cell death was induced via inhibition of the autophagy which was promoted by suppressing of the lipid metabolism. Including the experimental results in cancer-bearing mouse models, we reported for the first time that RMS relies on lipid metabolism to maintain its growth. Therefore, we believe that reducing lipid intake is a promising therapeutic approach for the treatment of RMS.