SAHA treatment changes proteome profile in NPC cells
It has been reported that SAHA altered the global proteome in several kinds of cancers to deploy anti-tumor activity [23, 24]. In this study, 6391 proteins were quantified in 5-8F cell line upon SAHA treatment. Among the 6391 quantified proteins, 454 were up-regulated and 217 were down-regulated with 1.2 change folds.
To illuminate the functions of the differentially expressed proteins (DEPs), performed comprehensive bioinformatics analyses were performed via Gene Ontology (GO) analysis, protein domain analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 1A-1E). For the molecular function (MF) category, the DEPs were highly enriched in binding, catalytic activity and structural molecule activity (Fig. 1A). The analysis of cellular compartment (CC) displayed that proteins, which are involved in cell part, organelle and protein containing complex, were enriched toward SAHA treatment (Fig. 1B). Biological process (BP) analysis revealed that DEPs were mainly associated with cellular process, metabolic process biological process and cellular component organization (Fig. 1C).
Protein specific structural domain is one of its major functional characteristics. Consequently, we investigated the enriched domains of quantified proteins upon SAHA induction (Fig. 1D). We found that those domains related to ubiquitin-conjugating enzyme active site, nucleotide binding, histone H1/H5 domain and RNA recognition motif domain were highly enriched.
To further explore the relevant pathways regulated by SAHA treatment, KEGG pathway analysis was performed. As shown in Fig. 1E, spliceosome, endocytosis and ubiquitin-mediated proteolysis pathways were the three most robustly enriched ones in response to SAHA stimulation, indicating the function of SAHA in regulating the signallings.
Finally, we established the protein protein interaction network via STING database and visualized by Cytoscape software (Fig. 1F). The global network of PPI and the highly enriched MAPKinase signaling pathway were presented. MAPK signaling, which regulates gene expression, cellular growth and survival, plays a vital role in tumorigenesis. It has been demonstrated that dysregulation of it may lead to abnormal cell proliferation and resistance to apoptosis [25, 26]. This result indicated that MAPK signaling pathway might be a potential target of SAHA in NPC cells.
SAHA treatment changes acetylome profile in NPC cells
Given that SAHA is a pan HDAC inhibitor, we suspected that it will alter lysine acetylation in NPC cells. To this end, we performed quantitative acetylomics toward SAHA treatment in 5-8F cells by combination of TMT labeling, antibody enrichment of acetylation and LC-MS/MS analysis. Altogether, 441 lysine acetylation sites locating in 298 proteins were identified, of which 333 sites locating in 228 proteins were quantified. Within these quantified acetylation sites, 32 sites distributed on 26 proteins were upgraded and 47 sites distributed on 45 proteins were downward upon SAHA treatment (fold change > 1.2 or < -1.2). And top ten acetylated sites and corresponding proteins upon SAHA stimulation were concluded ( Table 1).
Table 1
Top ten acetylated sites and corresponding proteins with the highest change folds in acetylome upon SAHA treatment in 5-8F cells.
Symbol | Position | Fold change | Acetylated Probility |
PHIP | 1399 | 26.26 | AYTPSK(1)R |
PLXNB2 | 941 | 25.59 | VTK(1)FGAQLQCVTGPQATR |
SLC25A5 | 105 | 24.57 | QIFLGGVDK(1)R |
ALDOA | 147 | 23.78 | DGADFAK(1)WR |
CREBBP | 1595 | 23.2 | TNK(1)NKSSISR |
CREBBP | 1597 | 23.2 | TNKNK(1)SSISR |
NOLC1 | 251 | 23.15 | KQVVAK(1)APVK |
CTTN | 272 | 22.99 | TGFGGK(1)FGVQSER |
MACROH2A1 | 142 | 22.7 | SPSQK(0.964)KPVSK |
CTTN | 235 | 22.4 | GFGGK(1)FGVQTDR |
ACTN4 | 497 | -21.86 | CQK(1)ICDQWDALGSLTHSR |
AHNAK | 4239 | -21.9 | VDIDVPDVNIEGPDAK(1)LK |
KMT2A | 1130 | -21.98 | SSIAGSEDAEPLAPPIK(1)PIKPVTR |
AHNAK | 1305 | -22.07 | VDVEVPDVSLEGPEGK(1)LK |
SF3A1 | 486 | -22.32 | RTDIFGVEETAIGK(1)K |
MRPL47 | 144 | -22.36 | VVDSMDALDK(1)VVQER |
CLTA | 242 | -22.37 | SVLISLK(1)QAPLVH |
HSPD1 | 473 | -22.95 | TLK(1)IPAMTIAK |
SET | 7 | -23.9 | SAPAAK(1)VSKK |
EAF1 | 150 | -25.11 | APTK(1)PPVGPK |
To understand features of these acetylated proteins, integrated bioinformatics analyses by combination with motif analysis, GO category analysis, KEGG pathway and protein-protein interaction analysis were conducted. As shown in Fig. 2A, we studied the amino acids which located around acetylated sites via motif analysis approach. In total, 6 motifs were robustly enriched: KacL, EKac, Kac**R, KacF, KacL, and KacW (Kac refers to acetylated lysine, * refers to random amino acid site). These 6 motifs differed in abundance and KacL, EKac, Kac**R comprised approximately 66% in all quantified peptides (Fig. 2B). SAHA altered histone acetylation, which has a great impact on chromatin remodeling and epigenetics [27]. Accordingly, distribution of acetylated proteins across chromosome was analyzed. This result demonstrated that these proteins were mainly located at 11 and 14 chromatins (Fig. 2C).
When GO database was applied to analyze these acetylated proteins, we found that these proteins mainly participated in regulation of RNA splicing process, ribonucleotide triphosphate metabolic process and purine ribonucleotide triphosphate metabolic process with biological process (BP) category analysis (Fig. 3A, top panel). In terms of molecular function (MF) category, chromatin DNA binding, nuclear receptor transcription coactivator activity and nucleosome binding are the top three items (Fig. 3A, middle panel). And as shown in cellular component (CC) analysis, these acetylated DEPs mainly involved in spliceosome complex, catalytic step 2 spliceosome and mitochondrial protein complex (Fig. 3A, bottom panel). These results revealed that SAHA may regulate RNA splicing process, metabolic-related process and chromatin DNA binding activity to play anti-tumor activities in NPC cells.
Next, KEGG pathway was conducted to have a look into the associated cellular signaling toward SAHA treatment. Totally, based on the criteria: p < 0.05 and FDR < 0.05, the DEPs took part in 30 prevalent cellular signaling (Fig. 3B). Among them, MYC, EGFR, mRNA splicing, G2M checkpoint, oxidation phosphorylation and TCA cycle were mainly enriched pathways, which were closely associated with proliferation, cellular metabolism and cell cycle processes. It has been well characterized that these notable processes are hallmarks of cancer [28]. These results hinted that SAHA may modulate these tumor-associated signaling pathways via protein acetylation to display treatment function in NPC cells.
In Fig. 3C, we clustered the differentially expressed acetylated proteins via two-way hierarchical clustering, which clearly displayed the patterns of the acetylated and global proteins in response to SAHA treatment. Taking advantage of STING database PPI network was studied. Representative MYC and EGFR signaling pathway were showed in Fig. 3D. In the MYC signaling pathway, there were 14 sites exhibiting decrease at acetylation level upon SAHA treatment, while 4 sites exhibited increase. For EGFR signaling pathway, there were 15 proteins quantified to be acetylated, 6 of which were upregulated and 9 were downregulated (Fig. 3D).
Crosstalk between global proteome and acetylome in NPC cells
According to the whole proteome and acetylome data from 5-8F cells upon SAHA treatment, the crosstalk between these two modifications were analyzed. In total, 126 proteins were quantified by proteome and acetylome, including 14 DEPs (Fig. 4A and 4B). Subsequently, these 14 DEPs, showing the highest significance and change fold values, were selected for two-way hierarchical clustering analysis (Fig. 4C). Followed by, the protein-protein interaction analysis was studied to unveil the functional relationship between these two profiles and representative interacted proteins were displayed in Fig. 4D. Together, these analyses indicated a complex relationship between proteome and acetylome, which in turn determine the functions of SAHA in NPC cells.
SAHA treatment changes phosphorylome profile in NPC cells
Phosphorylation is one of the most important PTMs and is believed to take part in the process of cancer progression [12]. In previous study, we demonstrated that SAHA regulated the phosphorylation of p53 and Rb1 in NPC cells, hence protein phosphorylome towards SAHA stimulation was investigated here.
Classification and enrichment analysis based on GO revealed that the DEPs in phosphorylome were mainly associated with RNA related processes in biological process (BP) category, such as RNA localization process, nucleobase-containing compound transport process, establishment of RNA localization process and RNA/mRNA transport process. The cellular component (CC) category revealed that DEPs were primarily in chromosome, including chromosome, centromeric region, condensed chromosome and heterochromatin. For the molecular functional (MF) category, mRNA binding, histone binding, modification dependent protein binding and RNA polymerase binding were highly enriched (Fig. 5A).
To further study the related cellular signaling of phosphorylated DEPs, we conducted signaling analysis via KEGG database. In total, 30 significant cellular pathways were detected under the criteria p < 0.05 and FDR < 0.05 (Fig. 5B). It showed that cell cycle and mRNA splicing-related signaling pathways were robustly affected, which included G2M checkpoint pathway, M phase, mitotic spindle pathway and pre-mRNA/mRNA splicing pathway. Intriguingly, SUMOylation-related signaling was enriched in SAHA-induced phosphorylome profile, which consisted with the previous study that SAHA regulated protein Sumoylation to implement biological functions.
Then distribution of phosphorylated proteins across chromosome was also studied. The result presented that enhanced phosphorylated proteins were mainly gathered in No.16 and No.2 chromatins, while decreased phosphorylated proteins mostly located at No.11 and No.19 chromatins (Fig. 5C).
We next established the protein-protein interaction network about phosphorylated proteins. The overview network of PPI and the representative mRNA export process were presented in Fig. 5D. The result uncovered that SAHA boosted some proteins’ phosphorylation level (RANBP2, POM121, NUP214, SRSF10 and RNPS1), while reduced phosphorylation state of other proteins (SRSF5, NUP210, NUP188, NUP98, NUP107, NUP133 and SMG6) (Fig. 5D). Taken together, our results indicated that SAHA may manipulate the phosphorylation of some critical molecules to regulate pivotal signal pathways, leading to the therapeutic roles in NPC cells.
Crosstalk between the global proteome and phosphorylome in NPC cells
Based on the results of proteome and phosphorylome toward SAHA treatment in 5-8F cells, we found that 833 proteins in the global proteome were also phosphorylated, while 140 DEPs were detected to undergo the two modifications at the same time (Fig. 6A and 6B). We next assessed the correlation within the two modifications. The Pearson’s correlation coefficient ratio R2 was − 0.16, which suggested that global proteome and phosphorylome had weak negative relation in this situation (Fig. 6C). Following cellular signaling pathway analysis via KEGG database, we observed that several pathways were enriched in both two modifications, including cell cycle related and mRNA process related pathways (Fig. 6D). To evaluate the relevance between proteome and phosphorylome, we analyzed the protein-protein interaction network. The whole view of PPI between the global proteome and phosphorylome was showed in Fig. 6E. The complicated data hinted that there was closed crosstalk between global proteome and phosphorylome. Taken together, these results gave a hint that those important cellular processes and proteins underwent both proteome and phosphorylation might affect the functions of SAHA in NPC cells.
Crosstalk between phosphorylome and acetylome in NPC cells
It was reported that each protein modification can crosstalk with one or more other modifications to affect cellular functions [29]. As the most prominent two PTMs, phosphorylation and acetylation were demonstrated that linked closely with each other. To this point, we compared the acetylation and phosphorylation data from 5-8F cells treated with SAHA and 46 proteins were identified, which were both acetylated and phosphorylated (Fig. 7A). From the scatterplot, the connection ratio is 0.0319, which meant the two protein modifications were not directly linked in the overall (R2 = 0.0319, Fig. 7B).
To better understand the relationship about these two protein modifications, we conducted the interaction network of proteins. The overall diagram of protein interaction was presented in Fig. 7C. Then through clustering these proteins, we found out that both acetylated and phosphorylated proteins taking part in regulating those prevalent biological processes, such as gene expression, epigenetics, chromatin organization, chromatin assembly and disassembly processes (Fig. 7D). For molecular functional (MF) analysis, mRNA binding, histone binding, chromatin DNA binding and histone acetyltransferase activity were mostly clustered items. Subsequently, those proteins were detected that mainly located in chromosome, nuclear periphery, heterochromatin and acetyltransferase complex (Fig. 7D). The compressive analyses suggested that these cellular processes were mediated both by acetylation and phosphorylation, thus affecting SAHA’s activity.