The occurrence and development of tumors is modulated by the dual regulation of genetic instability and the tumor microenvironment. Several studies have shown that the tumor microenvironment is important in the development and progression of malignant tumors; however, its mechanisms remain to be fully elucidated. Metabolism is one of the basic biological life activities in the body; changes in the biological properties of normal cells when converted to malignant cells include metabolic reprogramming. In previous years, studies have revealed that the activation of certain oncogenes or tumor suppressor inactivation can regulate metabolic homeostasis, and are involved in promoting or inhibiting cancer progression (1,2). Certain metabolic enzymes can themselves be used as oncogenes or tumor suppressors involved in the process of cancer development.

Cardiolipin (CL) is a major mitochondrial membrane polyglycerophospholipid (3,4). It has been shown to be required for the activation of several pivotal mitochondrial membrane enzymes involved in ATP synthesis and mitochondrial respiratory complexes (5). CL may be involved in the pathway leading to cellular apoptosis (6,7). The final step for the de novo mitochondrial synthesis of CL is based on the substrates phosphatidylglycerol and cytidine diphosphate choline-diacylglycerol. This reaction is catalyzed by the enzyme cardiolipin synthase 1 (CRLS1) (8), which is ubiquitous in eukaryotes, archaebacteria and eubacteria. The human CRLS1 gene has previously been cloned and characterized (9,10).

Although CRLS1 has been demonstrated to be functional in lipid anyagcsere, the exact role in tumorigenesis remains to be fully elucidated, with the exception of investigations in patients with solid and non-solid malignancies, which showed that anti-cardiolipin antibodies were associated with an increased rate of thrombosis (11). The expression, prognostic effects and potential mechanism of CRLS1 in tumors remain to be elucidated. It is known that lung cancer is one of the malignant tumors with high morbidity and mortality rates in the world. Non-small cell lung cancer (NSCLC) accounts for almost 85% of all lung cancer cases and generally presents at an advanced stage at diagnosis with a 5-year survival rate of <5% (12,13). Although standard chemotherapy and molecular targeted therapy have improved overall survival (OS) and quality of life, the survival benefit is limited to those who lack a driver mutation or show resistance to drugs (14). Therefore, it is important to investigate the underlying and intertwined mechanisms of lung tumorigenesis and tumor progression to provide novel ideas and a theoretical basis for the treatment of NSCLC.

The present study focused on the association between metabolism-related genes and CRLS1 in addition to NSCLC, and comprehensively analyzed the mRNA expression of CRLS1 in tumors and patient prognosis. Furthermore, the mechanism in tumor development was examined using web-based and soft-based microarray databases. Together, the results suggested that CRLS1 is a promising candidate gene for prognostic and diagnostic approaches in NSCLC, and warrants further investigation.

Warburg first suggested that the prime cause of cancer was impaired energy metabolism, which involved irreversible injury to cellular respiration (20,21). The majority of normal mammalian cells achieve a constant delta G' of ATP hydrolysis of ~257 kJ/mol as energy required for normal life activities through respiration (22,23). By contrast, tumor cells maintain this level through a combination of respiration and glycolysis (24,25). Elevated glycolysis is a metabolic characteristic of almost all types of tumor, which can be observed through the use of labeled glucose analogs (26,27). Structural and biochemical abnormalities that have occurred in tumor cell mitochondria can compromise oxidative respiratory function. Several studies have assessed the lipid composition of tumor mitochondria (28,29), however, few have evaluated the content and the composition of CL in tumor mitochondria, which is a complex mitochondrial-specific phospholipid regulating numerous enzyme activities, particularly those associated with oxidative phosphorylation and coupled respiration (30,31). Kiebish et al investigated the content and composition of CL for the first time in purified mitochondria from mouse brain tumors. The results showed that the composition and content of CL in mouse brain tumor mitochondria differed distinctly from that derived from syngeneic host normal brain tissue. The mouse brain tumors contained CL abnormalities, which were unique to each tumor type. In addition, these CL abnormalities were associated with significant hypofunction in the electron transport chain (ETC). Taken together, the results suggested that abnormal CL underlies irreversible respiratory injury in tumors, which further supports the importance of CL in maintaining the structural integrity of the inner mitochondrial membrane and lined mitochondrial lipid defects to the Warburg theory of tumorigenesis (30).

CRLS1, also known as C20 or f155, is the human candidate gene for CL synthase, is localized in the mitochondria and is expressed at high levels in tissues, including the heart, skeletal muscle, lung and liver, belonging to the CDP-alcohol phosphatidyltransferase class-I family. It predominantly catalyzes the reversible phosphatidy l group transfer from one phosphatidylglycerol molecule to another to form CL and glycerol. As a result of these findings, the present study hypothesized that the reduction of CRLS1 caused by various factors contributes either directly or indirectly to abnormalities in CL synthesis or remodeling, and leads to damage to mitochondrial intima integrity and hypofunction in ETC activities. This suggests a reliance on glycolysis for cell survival, eventually resulting in the occurrence and development of tumors. In addition to the enzyme-mediated inhibition of CL synthesis, inherited mutations, a variety of epigenetic causes and environmental insults, including necrosis, hypoxia, ischemia, dietary imbalances and reactive oxygen species, can also produce mitochondrial defects to cause CL abnormalities (31,32), contributing to tumor initiation and progression. However, enzyme-mediated CL synthesis may be the most direct element and was investigated as one of the primary aims of the present study. In addition, based-wed DAVID GO term enrichment analysis showed that CRLS1-co-expressed genes were involved in phosphoprotein phosphatase activity, protein amino acid dephosphorylation, protein serine/threonine phosphatase activity and phosphate metabolic process. According to the co-expression analysis theory, which is based on hierarchical clustering in which groups of genes may have similar functions if they have similar expression patterns under a given pathological condition, it is possible to examine whether CRLS1 possesses putative and analogical functional activities with its co-expressed genes. These functional abnormalities caused by abnormalities in lipid metabolism may also be complementary contributors in tumorigenesis.

In previous years, studies have found that oncogene activation or tumor suppressor gene inactivation can regulate metabolic homeostasis in order to promote or inhibit cancer. Furthermore, certain metabolic enzymes can be used as oncogenes or tumor suppressors involved in the process of cancer progression. Members of the MAPK family, including the extracellular signal-regulated protein kinase (ERK) pathway, c-Jun N-terminal kinase (JNK) pathway, p38 pathway and ERK5 pathway, have been reported to be important in cell proliferation and apoptosis (33). From the extracellular stimulus to the cells to the corresponding biological effects, the MAPK signal transduction pathway involves a signaling cascade of multistage protein kinases, including three key kinases, MAPK, MAPKK and MAPKKK (34). The analysis of KEGG pathways via DAVID showed that CRLS1 may be involved in the MAPK signaling pathway as it is co-expressed with PPM1A, protein tyrosine phosphatase receptor type R protein (PTPRR) and DUSP7. PPM1A is a phosphatase belonging to the PP2C family, which dephosphorylates and inactivates a broad range of substrates, including MAPKs, p38 and JNK, and is involved in tumor suppression (35,36), Su et al (37) characterized the tumor suppressor function of PTPRR, particularly its role in metastasis via the MAPK signaling pathway, in a cervical cancer model. Taken together, it was suggested that PTPRR affects malignant phenotypes by dephosphorylating substrates, including ERK5 and the p38 pathway, which has also been observed in other species (38). However, the extent of kinases targeted by PTPRR in NSCLC has not been investigated. DUSPs show marked substrate preferences for specific MAPKs, which have been reported to limit the duration of MAPK phosphorylation and reduce microglial activation (39). Sharing a substrate preference for ERK1/2 may be categorized as inducible or constitutive according to their expression. Inducible DUSPs dephosphorylate JNK, p38 and ERK, whereas constitutive DUSPs are specific for ERK (40,41). Several studies of DUSPs in tumors have focused on constitutive and inducible DUSP7, which downregulates p38 and ERK in major inflammatory pathways (42,43). To date, few studies have reported on the roles of DUSP6 and other DUSPs in microglia.

The MAPK pathway consists of a three-tiered kinase cascade and the sequential phosphorylation of each kinase causes the activation of effector kinases JNK, p38, and ERK1/2. Three classes of protein phosphatases modulate the activities of upstream kinases: Type-2 Ser/Thr phosphatases (PP2A) including PPM1A, PTPs including PTPRR, and dual-specificity (Thr/Tyr) phosphatases (DUSPs), including DUSP7 (42). Using the DAVID functional annotation chart, several subclasses of MAPK signaling pathways may reveal the role of CRLS1 in tumorigenesis based on the co-expression analysis theory. The JNKs are members of the MAPK family and are activated by MAP2 kinases MKK-4 and MKK-7. This subsequently translocates to the nucleus where it can regulate the activity of multiple transcription factors. At the post-translational level, JNK enhances its transcriptional activity by double-phosphorylating Ser-63 and Ser-73 sites in the c-jun activation region, and eventually affects cell cycle, apoptosis, cell stress, and other physiological and pathological processes (44). According to the KEGG pathways and co-expression patterns identified in the present study, the putative CRLS1 JNK/c-Jun MAPK signaling pathway was demonstrated (Fig. 4A). CRLS1 prevents the phosphorylation of MKK-4 and MKK-7 by dephosphorylation and reducing its transcription by c-jun by suppressing the activation of JNK. The p38 signaling pathway (45) is activated through phosphorylation by the upstream MKK-3, which then translocates into the nucleus to interact with downstream protein kinases, chromosome remodeling and transcription factors, including mitogen-and stress-activated protein kinase 1/2 (MSK1/2), MYC associated factor X (MAX) and ETS transcription factor (ELK-1). Similarly, the putative CRLS1 p38 MAPK signaling pathway indicates the role of CRLS1 in tumors (Fig. 4B). CRLS1 inhibits the phosphorylation of MKK-3 and p38 by dephosphorylation, and reduces the transcription of MSK1/2, MAX/GADD153 and ELK-1 by suppressing the activation of p38. ERKs were the first subclass to be investigated and are also the most important members of the MAPK family (46). The ERK signaling pathway is activated by growth factors and mitotic stimuli, which are closely associated with cell proliferation, differentiation and transcription. ERK is activated continuously, and ultimately promotes cell proliferation and malignant transformation. The ERK cascade contains a typical three-level MAPK sequence activation process. Raf belongs to the MAPKKK family, which phosphorylates and activates MEK1/2 (MAPKK), and subsequently activates ERK1/2 by double phosphorylation. The same reaction model can also be applied to the CRLS1 ERK MAPK signaling pathway (Fig. 4C). CRLS1 prevents the phosphorylation of ERK by dephosphorylation, and reduces the transcription of MAP kinase-interacting kinase 1/2 and Elk-1 by suppressing the activation of ERK.

In terms of seleno-amino acid metabolism, KEGG pathways analysis via DAVID revealed that SEPHS1/2 is a CRLS1-co-expression gene involved in tumorigenesis. In the 1970s and 1980s, clinical data was collected examining the correlation between selenomethionine (Se-Met) and cancer incidence in various tumor types, including lung, skin and colon cancer (47). Several studies have identified selenium and Se-Met as potential chemopreventive agents against prostate cancer (48,49). SEPHS1/2 belongs to the selenophosphate synthetase 1 family, class II subfamily, which predominantly synthesizes selenophosphate from selenide and ATP (catalytic activity: ATP + selenide + H₂O=AMP + selenophosphate+phosphate). Studies have also shown that SEPHS affects cell viability upon ionization radiation through the modulation of p53, which further suggests that SEPHS and its reaction product selenophosphate may be involved in inhibiting cancer progression in a p53-dependent manner and offers potential as a novel anticancer measure (50,51). However, the antitumor mechanisms of selenophosphate and SEPHS described above have not been validated in the field of lung cancer. By contrast, the co-expression analysis theory indicates a similar role for CRLS1, which may be confirmed by further investigations of NSCLC-related seleno-amino acid metabolism and the p53 pathway.

In conclusion, using in silico approaches, the differential expression of CRLS1 in NSCLC and normal tissue was assessed in the present study, which revealed that the expression level of CRLS1 in lung cancer was lower, compared with that in normal lung tissues. In addition, a high expression of CRLS1 was found to be associated with improved OS in all patients with NSCLC and lung Ade. The meta-analysis for CRLS1-related co-expression analysis revealed that this molecule may be important for tumor-associated lipid metabolic processes, and its anti-oncogene position was also assessed, involving the MAPK signaling pathway. The above conclusions were obtained using various tools, software and large-scale databases, notably in silico rather than wet laboratory data. The results of the in silico experiments generally require confirmation in laboratory experiments. However, this remains an effective experimental approach for novel developments in understanding carcinogenesis and makes it convenient for further wet laboratory investigations.