In clinical settings, NPC is considered as a highly invasive and metastatic cancer. Clinical assays indicated that NPC patients with metastasis have a higher level of DNP in the serum than patients without metastasis [
13].Our previous work showed that DNP is known to mediate ezrin phosphorylation and promote NPC metastasis [
13]. Therefore, we speculated that DNP also induces the phosphorylation of other proteins when mediating metastasis. In the present study, we first confirmed DNP-mediated NPC metastasis, and subsequently used phosphoprotein proteomics to comprehensively identify DNP-regulated phosphoproteins and phosphosites. We found that DNP increased the expression of phosphorylated LYRIC, ferritin, Rho, and GTPase protein, and decreased the expression of phosphorylated ANKIB1 protein, dynein heavy chain 17, eukaryotic translation initiation factor 4B, triosephosphate isomerase, torsin-1A protein 1, membrane-associated progesterone receptor component 2, nuclear casein kinase, cyclin-dependent kinase substrate 1, and nucleolar RNA helicase, which are associated with protein synthesis, cellular movement, molecular transport, cell death, and signaling pathways related to cellular growth and proliferation.
Ferritins are important regulators of intracellular iron content. They are made of 24 subunits assembled to form the apoferritin shell. The protein subunits are of two types, light chain and heavy chain, which share extensive homology. Increased ferritin concentration in tumors has been recently reported in several malignancies, such as colon and breast cancers [
41‐
43], and seminoma. Patients with NPC have higher levels of ferritin in the serum [
44], and ferritin has been reported to be associated with NPC development [
45]. Furthermore, high ferritin expression can enhance celluler growth and improve resistance to oxidative stress in metastatic cancer cells by interfering with their cellular antioxidant system [
46]. Ferritins play important roles in chemokine receptor (CXCR)-mediated cell migration by forming a complex with CXCR4 [
47]. Phospho-ferritin at serine 178 increases binding to CXCR4 and nuclear translocation. We speculate that DNP might influence NPC metastasis by regulating ferritin phosphorylation.
Initiation of transcription is a complex biological process that critically determines gene expression. Enormous efforts over the decades have revealed a set of proteins essential for this initiation by each class of eukaryotic RNA polymerases. Some of them, Drosha and Dicer, are highly expressed in NPC tissue and predict NPC prognosis [
48]. For RNA polymerase I (Pol I), which is dedicated to transcription of the large rRNA precursor, 2 transcription factors have been defined in mammals. Phosphorylation at specific sites in Pol I is a prerequisite for proper transcription initiation, and phosphorylation/dephosphorylation of Pol I is one of the mechanisms by which cellular rDNA transcriptional activity could be modulated [
49]. In DNP-mediated NPC metastasis, DNP might induce Pol I phosphorylation, thereby regulating DNA translocation. Activating transcription factor 7-interacting protein 1 (ATF7IP) is highly expressed in human cancers of many different tissues, suggesting that it has an important role in tumorigenesis. ATF7IP is required for the Sp1-dependent maintenance of telomerase activity in cancer cells. Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine/growth factor that is capable of inducing a remarkable variety of biological activities, including cellular proliferation, scattering, tubulogenesis, and invasiveness. HGF/SF can induce cyclin D1 expression in mouse melanoma cells, and this up-regulation is mediated in part by activating transcription factor-2 [
50]. Rho family GTPases, including RhoA, B, C, Rac1, 2, and Cdc42, control cellular morphology and motility [
51]. Many of these effects are attributed directly or indirectly to their ability to regulate the cytoskeleton. The most distinct effects of these GTPases were noted in Swiss 3 T3 cells, in which RhoA induced stress fiber formation and Rac1 induced membrane ruffling and lamellipodia, whereas Cdc42 induced spike-like filopodia [
40]. In a previous study, Rho GTPase gene (
DLC1) is inactivated by DNA methylation, and was negatively regulated in NPC carcinogenesis, whereas DLC-1 expression was detected in NPC cells after 5-aza-dC treatment [
52]. In our previous work, we found that DNP activates Rho kinase and PKC, induces ezrin phosphorylation at threonine 567, increases fiber growth, and promotes motility and invasion of cells [
13]. In the present study, phospho-ezrin was not detected, probably because of the incompatibility of TiO
2 phosphopeptide enrichment or SILAC for ezrin phosphorylation at threonine. Nevertheless, DNP mediated the expression of Rho family GTPase and induced cancer metastasis. We believe that DNP also mediates NPC metastasis by increasing the expression of Rho family GTPases. LYRIC protein, also known as metastasis adhesion protein, is involved in cancer metastasis [
44]. Using the evidence from two independent experiments, we suggest that DNP mediates NPC metastasis by inducing LYRIC phosphorylation at serine 568. First, DNP enhanced cell motility and invasion following higher expression of phospho-LYRIC. When the LYRIC phosphorylation site serine 568 was mutated, DNP-mediated motility and invasion was dramatically decreased. Secondly, higher levels of phospho-LYRIC were detected in DNP-induced metastatic tumors. Additionally, higher phospho-LYRIC expression at serine 568 was also detected in the NPC tissues, and it was expressed at higher levels in the metastatic tissues than in primary site tissues (Additional file
3: Figure S1). The COOH terminal of LYRIC (amino acid 546–582) is the predominant regulator of nuclear localization, and its modification by ubiquitin regulates LYRIC nuclear localization [
40]. We speculate that DNP mediates the phosphorylation of LYRIC at serine 568, and regulates LYRIC nuclear localization, thereby enhancing its function and facilitate NPC metastasis. However, the function of phospho-LYRIC is not still clearly understood. Further studies are required to clarify whether LYRIC phosphorylation regulates LYRIC nuclear localization, and which kinase mediates LYRIC phosphorylation, in order to elucidate the mechanism that drives the enhanced metastasis of NPC.