Proteins differently expressed in NSCLC patients
LC–MS-based proteomics has been proven to be a wide-ranging tool for plasma biomarker screening in recent years. All precursor ions within the selected m/z range could be fragmented in the process of DIA and analyzed in a single MS/MS scan. Thus, DIA allows for the measurement of peptides with low abundance and more accurate quantification. Multi-omics analysis is a powerful approach to jointly explore changes in the proteome and lipidome in vivo in this study. The protein–protein interactions as well as the relationship between lipids and proteins are clearly presented in Fig. 6. This strategy could be used to generate comprehensive testable hypotheses.
Dysregulated proteins highly correlated with the lipid response
The expression of many proteins was different between the NSCLC patient groups and the healthy group, including ALDOC, COL6A1, TUBA1B, DSG2, TXN, and CST3, and the differences were highly correlated with the lipid response, which might play crucial roles in NSCLC.
ALDOC is a member of the glycolysis enzyme family, which catalyzes the decomposition of β-D-fructose 1,6-bisphosphate into glycerone phosphate and d-glycraldehyde 3-phosphate, while its product is involved in glycerophospholipid metabolism. Meanwhile, ALDOC positively regulates the Wnt pathway, which is involved in tumor development, by blocking the GSK-3β-axin interaction and targeting axin to a Dvl-induced signalosome [11]. In addition, the glycolysis pathway in which ALDOC participates in could affect the energy metabolism of patients, and the differential expression of ALDOC may lead to different energy metabolism in patients with two syndromes, which was consistent with the influence of yin deficiency on energy metabolism [12]. In our study, ALDOC was highly correlated with LPCs and PCs in the lipidomics results, and affected the lipid metabolism of lung cancer, while the effects on QDLS and QDYD syndromes were discrepant. The role of ALDOC in the statistical correlation analysis with LPCs and PCs was consistent with that in lipid metabolism, which could contribute to syndrome differentiation as a key differential protein. Consequently, the discrepancies between QDLS and QDYD seemed to be related to the metabolic differences in glycerophospholipid metabolism and the glycolysis pathway involved in ALDOC.
COL6A1 is widely present in the extracellular matrix (ECM) and mediates the formation of microfibril networks. The ECM is emerging as an important component of the tumor microenvironment, providing structural support and regulating the activities of growth factors and cytokines. COL6A1 is reportedly a crucial regulator of lung cancer invasion and metastasis [13]. Its correlation with metastasis may be realized by changing the characteristics of ECM, promoting cell adhesion to ECM and supporting cell movement. The level of it was elevated in NSCLC patients with QDLS syndrome. As a subtype of α-tubulin, TUBA1B participates in the formation of microtubules and is generally involved in cell proliferation, adhesion, movement and division. Both G2/M cell cycle arrest and abnormal mitotic spindle formation, and subsequent apoptosis signal triggering could be caused by microtubule destruction [14]. Thus, the overexpression of TUBA1B in QDLS syndrome might be involved in the proliferation of cancer cells. DSG2, a protein of the cadherin superfamily, participates in cell adhesion and has been demonstrated to be overexpressed in NSCLC, which was consistent with the results in both syndrome groups [15]. TXN, a small molecule selenium-containing protein with a molecular weight of approximately 12 kDa, forms the thioredoxin system together with nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) and thioredoxin reductase (TRXR), which is one of the two redox regulatory systems supporting tumor growth. TXN is primarily responsible for defense against the oxidative stress burden caused by elevated reactive oxygen species (ROS) in lung cancer [16]. CST3 is a member of the cysteine protease inhibitor family, which mainly exists in extracellular fluid. The levels of cystatin C in lung cancer groups were elevated, consistent with the previous report [17]. “Qi” here was vital qi, and the interplay with evil qi determines the development of cancer. The deficiency of vital qi increases the ability of evil qi pathogenic factors to do harm and can aggravate the illness. The incidence and severity of qi deficiency in advanced cancer is higher than that in early stages [3]. Therefore, qi deficiency might be related to the progression of lung cancer by the metabolic pathways involving the above proteins.
Differential proteins involved in lipid metabolism
In addition, some differential proteins have been found to be involved in lipid metabolism based on the HMDB, KEGG, MetScape and LipidMaps databases and literature reports. These proteins, including ALDOC, PCSK9, ANGPTL3, and PRDX6, were correlated with certain lipids (Fig. 7). PCSK9 is emerging as a key regulator of plasma cholesterol homeostasis. PCSK9 in the circulation can bind to low-density lipoprotein receptor (LDLR), the receptor for LDL, which participates in cholesterol transport and clearance from the blood. PCSK9 promotes LDLR degradation and prevents its recirculation, leading to hypercholesteremia, and associated with a variety of malignant tumors [18, 19]. Consistent with the significant upregulation of cholesterol in our results, the overexpression of PCSK9 in QDLS lung cancer might become a key factor in the occurrence and development of lung cancer together with cholesterol synergistically. As one of the effective regulators of lipoprotein metabolism, ANGPTL3 inhibits the activity of lipoprotein lipase (LPL) through the N-terminal domain CCD fragment to prevent the clearance of plasma TGs [20]. The significantly elevated level of this protein in the QDYD lung cancer patients might be related to the abnormal TG levels in our results. Interestingly, ANGPTL3 had a positive correlation with certain TG species in the lipidomics results (|r| 0.5–0.8), which was consistent with the theory above. PRDX6 is a bifunctional enzyme with both peroxidase activity and phospholipase A2 activity. PRDX6 was enriched in the glycerophospholipid catabolic process by reducing the oxidized sn-2 fatty acyl group (peroxidase activity) and hydrolyzing the sn-2 ester bond (phospholipase activity) of phospholipids. LCAT could also be catalyzed by PRDX6. In summary, the membrane lipid peroxidation caused by oxidative stress could be prevented by PRDX6 to maintain the homeostasis of phospholipid metabolism. Recent studies have demonstrated that PRDX6 could activate Akt through the activation of phosphoinositide 3-kinase (PI3K) and p38 kinase, and further induce uPA (urokinase plasminogen activator) to promote the invasion of lung cancer cells [21]. In our study, PRDX6 was low expressed in QDLS lung cancer, and have a significant difference between the two syndromes, implying that PRDX6 focused on the influence of invasion ability and phospholipid metabolism homeostasis with “Yin deficiency” in the two groups of lung cancer.
Lipid changes in NSCLC patients
From the lipidomics screening, we observed elevated PC levels in the plasma of lung cancer patients with QDLS or QDYD syndrome. As a key component of the eukaryotic cell membrane, changes in PCs indicate variations in cell membrane function and affect the growth and proliferation of cancer cells [22]. Increased phosphatidylcholine metabolism has been confirmed in lung cancer as well as other cancer types. Hence, this effect could be interpreted as meeting the demands of the high proliferation rate of cancer cells [23]. In addition, the key enzyme choline kinase α, involved in the synthesis of PCs in the CDP-choline pathway, is overexpressed in lung cancer, breast cancer, and colorectal cancer [24], and its expression was correlated with poor prognosis of lung cancer [25], consistent with the increased PC levels in patient plasma in our study. Additionally, glycerol phosphodiesterase-mediated glycerophospholipid metabolism could also regulate signaling pathways through downstream products, as well as cell migration via protein kinase C signaling pathways [26]. Interestingly, based on the metabolic pathway analysis in our study, the glycerophospholipid metabolism pathway was significantly affected in both syndrome groups of lung cancer. Thus, the increase in PC levels suggested variation in cell membrane function in cancer cells, and cell migration might change in lung cancer. As the second most abundant phospholipid in the mammalian membrane, the levels of PEs also vary during cell growth and tumor progression [27]. PE binding proteins (PEBPs) increase secretion in A549 lung adenocarcinoma cells, and regulate tumor development, invasion and metastasis potential [28]. PEs may act in part as agonists for PEBP-mediated signal transduction. Most PCs and PEs were upregulated in cancer patients in our study, indicating that the occurrence of cancers with qi deficiency was closely associated to the metabolism of PCs and PEs.
LPCs, which contain a fatty acyl group bound to glycerol after hydrolysis of the ester bond of PC by phospholipase A2 (PLA2) [29], are also important intermediates in biosynthesis pathway of PCs. This biosynthesis pathway can be remodeled by lysophosphatidylcholine acyltransferase (LPCAT), a cytosolic enzyme catalyzing the transformation of LPCs to PCs [30], promoting the growth and metastasis of lung cancer cells and participating in the pathogenesis of lung cancer. LPCAT has been proved to be highly expressed in lung cancer patients [31]. Moreover, the low levels of most altered LPCs were consistent with plasma LPC levels in patients with advanced metastatic cancer, indicating that the balance of LPCs was disturbed by malignant tumors [32]. Notably, LPCs showed a more obvious decreasing trend in the QDLS group than in the QDYD group. LPEs can stimulate calcium signal transduction and induce the proliferation, migration and invasion of cancer cells [33]. The LPE plasma level in QDYD patients was elevated compared with that in healthy subjects and could also differentiate QDLS patients and QDYD patients. Notably, according to the consistent statistical and biological correlations, LPCs were selected as the key differential lipids and contributed to syndrome differentiation. Moreover, the increased trend in PCs and PEs was slightly more pronounced in the QDYD group than in the QDLS group, and LPCs and LPEs were prominently different between the QDLS and QDYD syndromes, indicating that the metabolism of glycerophospholipids was significantly associated with the difference between the two syndromes, which could be dominated by “Yin deficiency”.
Sphingolipid metabolism is also altered in lung cancer. Numerous SMs were dysregulated in both syndrome groups of lung cancer. As the main sphingolipids in mammalian cells, which are mainly transformed from sphingosine via sphingomyelin kinase, SMs play important roles in cellular signaling pathways and inhibit oxidative damage to tissues [34]. The downregulation of SMs indicates that the occurrence and progression of lung cancer inhibits the activity of sphingosine kinase and aggravates tissue oxidative damage. A similar trend was observed in both syndrome groups of lung cancer, indicating that SMs play similar roles in signaling pathways of lung cancer within the two syndromes.
TGs have been found to be upregulated in many types of cancer, and were correlated with a high risk of NSCLC [35]. TGs and cholesterol, jointly stored within lipid droplets, could serve as energy storage for cancer cells. Therefore, cancer cells can sustain the autonomy of growth, migration and proliferation as well as increased energy consumption [36]. Indeed, cancer cells are likely to contain more lipid droplets than normal cells [37]. TGs could also provide a fatty acid library to generate free fatty acids via hormone-sensitive lipase, adipose triglyceride lipase and monoacylglycerol lipase, which further undergo β-oxidation to release ATP as part of the energy source required by cancer patients [38]. Disrupted β-oxidation has been reported in various cancers, and its enhancement is related to tumor promotion [37]. Alternatively, chronic inflammation, an important factor in the development of cancer, is accompanied by an increase in TG levels [39]. As a well-known second messenger of lipids, DGs are intermediates of lipid metabolism and key elements of lipid-mediated signal transduction. They have been implicated in the maintenance of homeostasis during cell growth. A strong correlation between the disorder of DGs and human diseases, such as diabetes and malignant transformation, has been reported previously [40]. In our study, elevated DG and TG levels in both syndromes of NSCLC patients were observed.
Because cancer cells require excessive cholesterol and cholesterol intermediates to maintain additional proliferation, the synthesis of cholesterol is enhanced, leading to the accumulation of cholesterol [41, 42]. This accumulation allows cancer cells to evade apoptosis and support continuous cell division and proliferation [42]. TThe inhibitor of HMG-CoA-reductase (HMGCR), a rate-limiting enzyme in the mevalonate pathway where cholesterol is synthesized, has antiproliferative effects on LC cells [43]. Additionally, the enzymes involved in cholesterol synthesis could be regulated by sterol regulatory element binding protein (SREBP), the genes of which were overexpressed in cancer, suggesting the elevation of cholesterol synthesis in cancer [44]. The approximate manifestations of the cholesterol and neutral lipids above in both lung cancer syndrome groups imply similar growth rates and energy expenditure.
Dysregulated metabolism between NSCLC patients and healthy subjects
Among the proteins closely associated with the response or metabolism of PCs, PEs, LPCs, LPEs, SMs, Cer, DGs, TGs and cholesterol in NSCLC patients, ALDOC, PRDX6, COL6A1, TUBA1B, TXN, DSG2, CST3, PCSK9, and ANGPTL3 were involved in the regulation of glycerophospholipid metabolism, cell adhesion, proliferation and division, oxidative stress reaction, apoptosis, cholesterol homeostasis and the clearance of plasma TGs. These lipids were involved in glycerolipid metabolism, primary bile acid biosynthesis, sphingolipid metabolism and glycerophospholipid metabolism in both syndromes of lung cancer patients.
Discrepant metabolism between QDLS and QDYD syndromes in NSCLC
The discrepancies between QDLS and QDYD syndrome of lung cancer dominating boiled down by “Yin deficiency” were comprehensive and widespread. To name a few, TUBA1B in cell division and proliferation, PCSK9 in cholesterol homeostasis, ANGPTL3 and TG in triglyceride homeostasis and lipoprotein metabolism, ALDOC in glycolysis, and ALDOC, PRDX6, LPCs, and LPEs, in glycerophospholipid metabolism were all different between the syndromes. In addition, after the integration of statistical and biological analysis, ALDOC and LPCs were identified as differential proteins and lipids between the two syndromes of NSCLC patients and statistically and biologically correlated with AUC values greater than 0.8, which could contribute to syndrome differentiation in NSCLC. Importantly, glycerophospholipid metabolism they were both involved in was also the most significantly different pathway in patients with the two syndromes in lipidomics analysis. In addition, ALDOC participates in the glycolysis pathway and then affected the patient’s energy metabolism, which was consistent with the fact that “Yin deficiency” were related to energy metabolism. Therefore, there were different metabolic patterns in glycerophospholipid metabolism and glycolysis pathway in lung cancer patients with different syndromes, thus reflecting the differences of syndromes.