Nasopharyngeal carcinoma (NPC) cell lines 5-8F and 6-10B are derived from the same human cell line SUNE1 that is derived from a female poorly differentiated nasopharyngeal squamous epithelial cell carcinoma. Those two cell sublines 5-8F and 6-10B with the same genetic background have tumorigenic ability that was confirmed by the colony formation in soft agar and in vivo mouse model, but are quite different in metastatic potential [1–3]. 5-8F cells are metastatic and 6-10B cells are nonmetastatic, which was confirmed by in vivo mouse model [2]. This different biological character shows that these two cell sublines are the useful experiment materials to study the molecular events of NPC metastasis. Based on those two subcelines, some research groups analyzed the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) using the whole-cell samples of those two cell lines. A total of 78 DEGs were identified with subtractive suppression hybridization [4] and microarray [5]. Of them, 11 DEGs including PTHLH formed a metastasis-related gene network [5]. A total of 28 DEPs were identified with two-dimensional gel electrophoresis (2DGE) coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) [6, 7]. Those DEPs were related to apoptosis, cell cycle, cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair [6, 7]. However, those DEGs and DEPs were not fully rationalized in the biological process of NPC metastasis, and there is no any overlap between those 78 DEGs and 28 DEPs [4–7]. It would be derived from the following limitation factors: (i) the range of the protein abundance of the whole-cell sample is too wide, not all proteins were extracted; (ii) 2DGE only can array very small partial components of a whole-cell proteome, and (iii) the process of translation of DEG to DEP is regulated by multiple factors, which results in the low consistence between the DEG and DEP profiles. The subcellular proteomics such as just focusing on one compartment of a cell would more effectively discover function-related proteins because the subcellular proteome is much simpler than the whole-cell proteome, it can maximally identify the protein components in a subcellular proteome.

Many evidences have demonstrated that mitochondria is the central to oxidative stress, and tumor cell self has anti-oxidant ability for its survival and metastasis [8–15]. Mitochondria are not just innocent bystander in cancer, but are related to tumorigenesis, apoptosis, cancer therapy and metastasis. The contribution of mitochondria to cancer involves three critical processes including alterations in glucose metabolism, production of reactive oxygen species (ROS), and compromise of intrinsic apoptotic function [8, 9]. According to the tumor-stroma co-evolution model opinion, cancer cells secrete hydrogen peroxide, induce oxidative stress in adjacent stromal cells, and result in an excess production of ROS in mitochondria. Excess stromal ROS induces cytoskeleton rearrangements, autophagy, and mitophagy in the tumor microenvironment to help cancer cells escape excess ROS in the primary tumor site, also induces the stromal over-production of recycled nutrients such as ketones and L-lactate that are fuel for tumor growth and metastasis [10–14]. This model provides fresh insight of how mitochondria involved in tumor metastasis. Therefore, a tight relationship exists between mitochondria and tumor metastasis. However, the causes, consequences, and other series of related questions regarding molecular mechanism of mictochondria in tumor metastasis still remain unclear. Thus, this current work focused on the mitochondrial proteome difference between metastatic 5-8F and 6-10B cells, and provided clues to clarification of the functional role of mitochondria in the process of NPC metastasis.

In order to screen mitochondrial proteins for the elucidation of the mechanism of NPC metastasis and the discovery of metastasis-related biomarkers, two-dimensional difference in-gel electrophoresis (2D-DIGE) was used to determine mitochondrial DEPs between metastatic 5-8F and nonmetastastic 6-10B cells, followed by protein characterization with peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS). siRNA transient transfection was used to suppress the expressions of up-regulated DEPs in metastatic 5-8F cells, followed by the observation of changes in cell mobility potential by Transwell Migration assay. Cytoscape BiNGO plugin was used for gene ontology (GO) analyses of DEPs to identify the enriched biological processes and molecular functions. A protein-protein interaction sub-network of DEPs was generated with literature data mining from HPRD and NCBI databases. Ten DEPs were rationalized in a tumor-stroma co-evolution model, and associated with tumor metastasis.

Nasopharyngeal carcinoma (NPC) is a malignant tumor with a high incidence of metastasis, has a great tendency to invade adjacent regions and metastasize to regional lymph node and distant organs. Though the molecular mechanisms of NPC metastasis remain poorly understood, the functions of mitochondria that involved in this process have been uncovered gradually: mitochondria are closely related to oxidative stress process in a cell. Different from the historical views, which assumed oxidative stress contributes to tumor initiation and progression solely by inducing genomic instability, the present views consider oxidative stress directly contributes to tumor metastasis. According to the tumor-stroma co-evolution model, cancer cells induce oxidative stress in adjacent stromal cells and result in an excess production of ROS in mitochondria. Excess stromal ROS induces cytoskeleton rearrangements, autophagy, and mitophagy in the tumor microenvironment to help cancer cells escape excess ROS in the primary tumor site, also induces the stromal over-production of recycled nutrients such as ketones and L-lactate that are fuel for tumor growth and metastasis [10–16]. Moreover, the enhanced cellular antioxidant capability and antioxidant function of redox-signaling in cancer cells result in an increase of cell resistance to oxidant, defend cancer cells themselves from oxidative damage, and favor their metastasis [14].

In general the balance between ROS generation and elimination is delicately maintained in cells. However, for many cancer cells, an increase in ROS production or decrease in ROS-scavenger capacity disturbs redox homeostasis which leads to an overall increase of intracellular ROS level. Over-expressed catalase to alleviate mitochondrial oxidative stress in a mouse animal model of breast cancer could reduce metastatic tumor burden by >12-fold [13]. Considered this catalase experimental result and our PRDX3 study, it is very likely that a molecular network based on redox-signaling pathways in cancer cells directly contributes to tumor metastasis, this process relates closely to mitochondrial oxidative stress and ROS produced by mitochondria might play an important role in tumorigenesis (its direct action on growth factors and transcription factors) and tumor metastasis as well.

Each mitochondrial DEP was rationalized in the tumor-stroma co-evolution model, and an experimental data-based diagram was proposed (Figure 9). In our comparative proteomic study of mitochondria between NPC metastatic 5-8F and nonmetastatic 6-10B cells, the first five enriched GO terms of DEPs were closely related to oxidative stress (Table 2). For PRDX3, our comparative proteomic data demonstrated the expression of PRDX3 in mitochondria of metastatic 5-8F cells is higher than in mitochondria of nonmetastatic 6-10B cells, it shows that the anti-oxidative stress capability in metastatic 5-8F cells is stronger than nonmetastatic 6-10B cells. This result is consistent with previous conclusion [14] that the enhanced anti-oxidant capabilities in cancer cells defend themselves from excess stromal oxidative damage and favors cell metastasis. Furthermore, among several DEPs (data not showed) selected to carry out siRNA transient transfections and then Transwell Migration assay, only suppression of PRDX3 expression resulted in the significantly increased number of cells migrated through Transwell membrane (P < 0.05). PRDX3, mitochondria thioredoxin-dependent peroxide reductase, is the main way by which cancer cells reduce their levels of H₂O₂ built up during active respiration [17, 18], suppression of this enzyme, a scavenger of peroxide, could increase oxidative stress in cancer cells to up-regulate the redox-signaling, promote the glycolysis, proliferation, and survival, and finally lead increased cells mobility potential [14] (Figure 8). To our knowledge, this was the first experimental evidence that PRDX3 was related to NPC metastasis.

PRDX6 increased about 2.5-fold in mitochondria of metastatic 5-8F cells than in mitochondria of nonmetastatic 6-10B cells. This result was consistent with the results of other research groups: PRDX6 expressions were increased in node-positive breast carcinoma samples compared with node-negative breast carcinoma samples [19]; enzymatic activities of PRDX6 in lung cancer cells metastasis [20]; over-expression of PRDX6 led to a more invasive phenotype and metastatic potential in human breast cancer [21]. Over-expression of PRDX6 is on the one hand to strengthen the peroxidase activity of a cell to reduce peroxidants such as H₂O₂ and hydroperoxides for the relief of oxidative stress. Besides of its peroxidase activity, PRDX6 has additional phospholipase A2 (PLA2) activity, this activity promotes accumulation of arochidonic acid, which, in turn, induces the invasive pathway involving p38 kinase, phosphoinositide 3-kinase, Akt and urokinase-type plasminogen activator. Over-expression of PRDX6 leads to a more invasive phenotype and metastasis potential, at least in part, through regulation of the levels of uPAR, Ets-1, MMP-9, RhoC, and TIMP-2 expression [20, 21]. Therefore, PRDX6 in NPC metastatic process demonstrates a double-side effect. To our knowledge this was the first report of the relationship between PRDX6 and NPC metastasis.

SOD2 increased in many tumor types as they progress from early stage non-invasive disease to late stage metastatic disease, and its over-expression in many instances enhance the metastasis phenotype because SOD2 is able to catalize toxic oxidant O₂ ^.- into another toxic oxidant H₂O₂ and hydroperoxides, and O₂ ^.- and H₂O₂ / hydroperoxides both directly contribute to oxidative stress. However, this metastatic phenotype can be reversed by efficient H₂O₂ scavenging [22–26]. Our proteomic results also showed that SOD2 increased about 3.3-fold in mitochondria of metastatic 5-8F than in nonmetastatic 6-10B cells. To our knowledge this was the first report of the relationship between SOD2 and NPC metastasis.

Over-expression of ECH1 can enhance the energy generation, and promote adhension and migration capability of cancer cell for cancer metastasis. The present experimental data showed that ECH1 increased in 4.1-fold in mitochondria of NPC metastatic 5-8F than nonmetastatic 6-10B cells. This result was consistent with the other studies [27], a higher expression level of ECH1 was confirmed in tissue from patients with gastric carcinoma with lymph node metastases, and down-regulation of ECH1 could inhibit tumor proliferation, increase the ratio in S phase to G1 phase and decrease the adhesion and migration capacity.

Studies [28] showed over-expression of each SERPINB gene could effectively suppress the invasiveness and motility of malignant cancer cells. Nuclear localization of SERPINB5 is required for its tumor metastasis suppressor function and it was enriched at the promoter of colony-stimulating factor (CSF-1) in chromatin immunoprecipitation and associated with diminished levels of CSF-1 mRNA. It suggests that its mechanism of action involves, in part, direct association of SERPINB5 with target genes to inhibit metastatic phenotype [29]. The present studies showed a down-regulation of SERPINB5 by 3.2-fold in mitochondria of NPC metastatic 5-8F than nonmetastatic 6-10B cells. Thus, the down-regulated SERPINB5 might enhance the metastatic capability of an NPC cancer cell.

COX subunit composition is regulated by COX5A and COX5B gene transcription in response to high and low O₂, respectively. This subunit switch maintains mitochondria redox homeostasis and relates to morphological and functional alteration of mitochondria [30]. PDIA5 also directly involves in the redox homeostasis. The present studies demonstrate that the down-regulation of COX5A and up-regulation of PDIA5 in NPC metastatic 5-8F cells relative to nonmetastatic 6-10B cells. The abnormal expression of COX5A and PDIA5 in NPC metastatic 5-8F cells might disturb the redox homeostasis to contribute to its metastatic process.

Studies [31] demonstrated over-expression of EIF5A enhances cell motility and promotes tumor metastasis in hepatocellular carcinoma. It was able to induce epithelial-mensenchymal transition (EMT), a key event in tumor invasion and metastasis, characterized by down-regulation of epithelial markers (E-cadherin and beta-catenin) and up-regulation of mesenchymal markers (fibronectin, N-cadherin, alpha-SMA and vimentin). It could also stimulate cytoskeleton rearrangement through activation of RhoA/Rac1 to contribute to the cancer cell metastasis. The present data showed EIF5A increased 2.5-fold in mitochondria of NPC metastatic 5-8F than nonmetastatic 6-10B cells. It suggests that over-expressed EIF5A contributes to NPC cancer cell metastasis.

IDH3B [32] involves in the mitochondrial tricarboxylic acid cycle that is initiated by acetyl-CoA. Acetyl-CoA is derived from the travel of pyruvic acid from cytoplasm into the interior of the mitochondrion. Pyruvic acid is generated from the glycolysis of glucose. The current study demonstrated the down-regulation of mitochondrial IDH3B in NPC metastatic 5-8F cells. Down-regulated IDH3B will obstacle the tricarboxylic acid cycle to reduce the generation of ATP, cause energy metabolism obstacle and mitochondrial dysfunction, and promote oxidative stress. Meanwhile, the tricarboxylic acid cycle obstacle will built up the pyruvic acid. Studies [33] demonstrated that pyruvic acid has strong angiogenic activity and plays an important role in angiogenesis for tumor growth and metastasis.

PSMC4 is a 26S protease that is involved in the ATP-dependent degradation of ubiquitinated proteins in the ubiquitin-proteasome system (UPS) that is extensively associated cancer pathogenesis [34]. The UPS plays a central role in fine-tuning the functions of core proangiogenic proteins including VEGF, VEGFR-2, angiogenic signaling proteins such as the PLCy1 and PI3 kinase/AKT pathways, and other non-VEGF angiogenic pathways to regulate the tumor angiogenesis [35]. The up-regulation of PSMC4 would contribute to this angiogenesis process for tumor metastasis.

Moreover, ETFA is a mitochondrial electron transfer flavoprotein [36]. The ETFA up-regulation in NPC metastatic cell could promote mitochondrial electron transport and oxidative phosphorylation, and enhance mitochondrial oxidative stress. Prohibitin (PHB) is a pro-oncogene, inhibits DNA synthesis, and regulates proliferation. However, PHB gene mutated in human cancers [37], PHB protein responds to mitochondrial stress and may play a role in regulating mitochondrial respiration activity [38]. MRPL12 may act as a scaffolding protein or stabilizer of respiratory chain supercomplexes, is involved in regulation of mitochondrial protein translation and respiration, and plays a role in mitochondrial-mediated death. The current studies found down-regulation of MRPL12, which would regulate mitochondria-mediated cell death and respiration [39, 40]. VDAC1 and VDAC2 forms a channel through the mitochondrial outer membrane with a weak anion selectivity at the open state and a cation-selectivity at the close state [41, 42], and GALU is involved in the binding of calcium ions [43]. The current studies showed the up-regulated expression of VDAC1, VDAC2, and GALU, they would disturb the mitochondrial ion homeostasis in the mitochondrial dysfunction and oxidative stress. The relationship of those mitochondrial DEPs and the tumor metastasis needs to be validated with experiments; however, these data are novel clues to insight into the mechanisms of NPC metastasis.

In summary, the present study provides new insights for exploring the significance of mitochondria and oxidative stress in NPC metastatic processes. Ten mitochondrial DEPs including PRDX3, PRDX6, SOD2, ECH1, SERPINB5, COX5A, PDIA5, EIF5A, IDH3B, and PSMC4 have been rationalized in the tumor-stroma co-evolution model that mitochondrial oxidative stress directly contributes to tumor metastasis (Figure 9). The expression suppression of mitochondrial PRDX3 protein in the anti-oxidative stress system enhanced the mobility potential of NPC metastatic cancer cells, which consolidates the roles of mitochondrial oxidative stress in NPC metastasis. Further investigation is required to determine the biological consequences of all identified mitochondrial DEPs and their relevance to the pathogenic mechanisms of NPC metastasis.