Bioinformatics analysis based on computer calculations and the application of biological software is a very effective and essential supplement in proteomics research. It is a powerful tool that has been extensively used for MS/MS data analysis, such as PPI studies, functional clustering, pathway mapping, and prediction of protein function and structure [11, 28–34]. In this study, we examined the expression profiles of pTyr proteins in two HCC cell lines (MHCC97H and Hep3B) that have different metastatic potentials. For this purpose, we used an LC-MS/MS-based proteomics technique. We also compared and analyzed the interactions and functions of these proteins by PPI and functional clustering analyses based on their GO attributes that were determined by biological process clustering and signaling pathway analysis. The results revealed that some cell functions related to cancer metastasis were significantly upregulated in HCC metastasis, especially those involved in cell motility, migration, localization, communication, antiapoptosis processes, and protein autophosphorylation. These alterations have been partly reported in previous studies on cancer metastasis [35–38]. High motility and migration are prerequisites for the successful metastasis of tumor cells and their transfer to the target site, penetration into the basement membrane and blood vessels, and movement from poorly oxygenated sites to rich oxygenated sites [3, 39]. Antiapoptosis is a pivotal mechanism by which cancer cells are protected from elimination by means of various steps of a metastatic process . At present, there are large amounts of data that highlight the importance of this process in cancer metastasis (reviewed in [40–43]). Protein amino acid autophosphorylation is a specific attribute of tyrosine kinase, and its upregulation in the highly metastatic MHCC97H cells confirmed that alteration of tyrosine phosphorylation was in fact involved in HCC metastasis. Western blot analysis of the pTyr protein profile also confirmed these results. More importantly, we found that alteration in cell communication was the most obvious cellular event in HCC metastasis. Since cell communication driven by signal transduction controls all cell behavior and activity, aberrations in signal transduction could be a crucial factor leading to HCC metastasis and could also trigger changes in cancer cell behavior. Therefore, it is very important to explore the events that occur during the activation and regulation of the signaling pathway in HCC metastasis. For this purpose, a pathway clustering analysis was performed for the node proteins of the PPI networks in Hep3B and MHCC97H cell lines. The results revealed that EGFR signal transduction, cytokine- and chemokine-mediated signaling, and the PI3K and JAK-STAT cascades were closely related with HCC metastasis since these processes were significantly upregulated. These analytical results were similar to those obtained for liver metastasis of breast cancer [11, 29]. EGFR is an Erb-family transmembrane receptor tyrosine kinase. This receptor is shared by multiple ligands, including EGF, TGF, EGFF-like factor, and amphiregulin . EGFR-mediated signaling has been implicated in a variety of human cancers and is a key regulator of the cell proliferation, migration, metastasis, angiogenesis, and antiapoptosis processes [45, 46]. Recently, some studies have reported that EGFR inhibitors are effective first-line therapeutic agents in the treatment of some metastatic cancers [47, 48]. EGFR is a potential therapeutic target in HCC treatment [49, 50]. The JAK-STAT cascade, which is another example, is a critical signaling pathway that plays an important role in cell proliferation, differentiation, survival, motility, and apoptosis . Recently, it was reported that sustained activation of the JAK-STAT cascade was involved in hepatocarcinogenesis and metastasis [52, 53]. The data showed that increasing activity of the JAK-STAT cascade was associated with aberrant methylation silencing of the suppressor of cytokine signaling (SOCS), which is a negative regulator of the JAK-STAT pathway . However, another study also suggested that constitutive activation of STAT3 was closely related to HCC metastasis and was the main factor leading to the upregulation of the JAK-STAT cascade . Consistent with these results, our MS/MS results also indicated STAT3 activation. Unexpectedly, the JNK cascade was found to be negatively associated with HCC metastasis, thereby deviating from its canonical functions. This indicates that the role of this cascade in HCC metastasis should be re-evaluated. Negative regulation of the JNK cascade was recently reported in prostate and ovarian cancers [56, 57]. Researchers showed that JNKK1, an upstream kinase of JNK, could persistently phosphorylate JNK and activate the stress-activated protein kinase signaling pathway (SAPK) to induce cell apoptosis and decrease tumor metastasis. Moreover, the SAPK pathway also showed a negative correlation with HCC metastasis in this study. Therefore, these two pathways might play synergetic roles in HCC metastasis. In other words, cluster analysis of signaling pathways may provide some important information for predicting the functions of the pTyr proteins involved in HCC metastasis. These results have been obtained by bioinformatics studies and need to be verified experimentally prior to practical application.
In our study, FER, a nonreceptor tyrosine kinase, was shown to be significantly involved in the HCC metastatic process. FER was initially discovered in 1988 during studies on the proto-oncogene protein Fes/Fps . Since then, its involvement has been reported in growth factors/cytokine-mediated signaling as well as in the integrin/E-cadherin-mediated signaling pathways. It also plays a critical role in cytoskeletal regulation, cell adhesion, migration, and proliferation [59–61]. In this study, we examined the total and phosphorylated forms of FER; these were found to be overexpressed in the MHCC97H cell line but not in the Hep3B cell line. Similar expression patterns were observed for CTNND1 and CAV1, which are related to FER, and the results were also validated by western blotting and MS/MS analyses. FER and CTNND1 were shown to be involved in cadherin and integrin-mediated cell adhesion. Under normal circumstances, FER could indirectly sustain CTNNB1 dephosphorylation to ensure cadherin-mediated adhesion stability. When FER is overexpressed and phosphorylated, it can directly induce CTNNB1 and CTNND1 phosphorylation, resulting in the loss of cadherin-mediated adhesion [61, 62]. This suggests that increasing metastasis may be associated with CTNNB1 in some cancers. Moreover, CTNND1 had been implicated in the metastasis and pathogenesis of several human cancers . It was shown that overexpression and phosphorylation of CTNND1 in the cytoplasm could promote cadherin-deficient tumor metastasis by regulating the activity of the small GTPase [64, 65]. CAV1 is an important marker protein of caveolae and regulates signal transduction as a scaffolding protein. Recently, CAV1 was found to be an independent predictor of decreased survival in breast and rectal cancers and was significantly associated with the presence of distant metastasis in colon cancer patients . CAV1 could also sustain cadherin-mediated adhesion stability. It functioned by modulating the level and/or subcellular distribution of cadherin and CTNND1 by inhibiting Src kinase activity . Nevertheless, FER was identified as a Src substrate and is involved in tumor transformation . In this study, these three proteins displayed similar expression patterns in the two cell lines studied and also interacted directly in the MHHCC97H network. Therefore, we hypothesize that FER may be a key regulator in the adhesion event involving these three proteins and may participate in HCC metastasis. We also examined the influence of FER on cell invasion activity in vitro. The data illustrated that the knock-down of FER by RNAi significantly reduced MHCC97H cell invasion activity, suggesting that FER could positively contribute to HCC metastasis. To verify this, we further examined FER expression in HCC tumor tissues from patients with diverse distant metastasis outcomes after tumor excision. The results showed that FER overexpression frequently occurred in tumor cells with distant pulmonary metastasis, indicating that FER is strongly associated with HCC distant pulmonary metastasis.
Once it is confirmed that FER is a potential regulator in HCC metastasis, the next step is to understand the mechanism by which it regulates HCC cell metastasis. To date, there are few reports on the functional regulation of FER in cancer, and the main focus has been on cell adhesion. FER was shown to play a key role in the coordinate regulation of E-cadherin-mediated cell-cell adhesion and integrin-mediated focal adhesion. It could move between the two adhesion pathways to trigger adhesion transition [59, 61]. In a study on FER function using the Trojan peptide, researchers showed that FER that had dissociated from the cadherin complex could translocate to the integrin complex where it reduced p130CAS phosphorylation and interrupted integrin-mediated focal adhesion by affecting the phosphorylation of p130CAS binding partners, including PTP-PEST, PTP1B, FAK, and Crk . FER was also reported to be an upstream tyrosine kinase for CTTN (cortactin) as well as Src and Fyn [68, 69]. Phosphorylation of CTTN by FER is very critical for its recruitment to the cadherin and integrin complex . CTTN mainly participates in cytoskeletal regulation and plays a pivotal role in tumor metastasis [71, 72]. Recently, CTTN was shown to be an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation . In another study, it was shown that phosphorylation of tyrosine residues in CTTN was strongly associated with the potential to induce metastasis in nude mice . Whether CTTN phosphorylation by FER also has the same effects in HCC metastasis needs to be confirmed in future studies.
The results of this study suggested that FER is involved in HCC distant metastasis, and we have discussed the main mechanism and signaling pathways associated with FER functional regulation. The results from bioinformatics analysis also imply that FER is altered in MHCC97H cells in comparison with Hep3B cells. As a new functional molecule, the effect of FER on HCC metastasis remains unknown. It is possible that FER can alter the metastatic potential of HCC cells by one or several of the mechanisms mentioned here. Further studies are required to explore the exact role of FER in HCC cell invasion and metastasis. These would be both exciting and challenging.