N‐α‐Acetyltransferase 10 inhibits invasion and metastasis of oral squamous cell carcinoma via regulating Pirh2‐p53 signalling pathway

Abstract N‐α‐Acetyltransferase 10 (NAA10) was reported to be involved in tumour invasion and metastasis in several of tumours. However, the role and mechanism of NAA10‐mediated invasion and metastasis in oral squamous cell carcinoma (OSCC) remains undetermined. Herein, our study showed that NAA10 inhibits cell migration and invasion in vitro and attenuates the xenograft tumorigenesis in nude mice. Mechanistically, we demonstrated that there is a physical interaction between NAA10 and RelA/p65 in OSCC cells, thereby preventing RelA/p65‐mediated transcriptional activation of Pirh2. Consequently, inhibition of Pirh2 increased p53 level and suppressed the expression of p53 downstream targets, matrix metalloprotein‐2 (MMP‐2) and MMP‐9. Therefore, NAA10 may function as a tumour metastasis suppressor in the progression of OSCC by targeting Pirh2‐p53 axis and might be a prognostic marker as well as a therapeutic target for OSCC.

Nα-Acetyltransferase 10 (NAA10/ARD1), the catalytic subunit of N-terminal acetyltransferase complex (NatA), has both Nα and Nε acetylation activities. [4][5][6] NAA10 is involved in regulating cell proliferation, [6][7][8] apoptosis, 9,10 autophagy, 11,12 tumour metastasis 13,14 and cell cycle arrest. 15 NAA10 overexpression has been documented in breast cancer, 16 colorectal cancer, 17 hepatocellular cancer 18 and lung cancer. 19 While downregulation of NAA10 in thyroid neoplasm, 20 non-small cell lung cancer (NSCLC) 11 was also reported. Although the efforts to elucidate the biological function of NAA10, it remains disputed regarding its roles in cancer. NAA10 was shown to physically interact with and acetylated the androgen receptor (AR) and form a positive feedback loop for AR-dependent prostate tumorigenesis. 8 Moreover, in lung cancer cells, NAA10 potentiates DNMT1's affinities with promoter regions of tumour suppressor gene E-cadherin and LATS, thereby suppressing their transcription and facilitating tumorigenesis. 21 NAA10 was also reported to interact with β-catenin and promote transcription of cyclin D1 in lung cancer cells. 6 By binding to p65 subunit of nuclear factor-κB (NF-κB), NAA10 may increase MCL1 transcription and resistance to stimuli-induced apoptosis. 10 In osteosarcoma, NAA10 was directly associated with MMP-2 protein through its acetyltransferase domain and maintained MMP-2 protein stability via NatA complex activity. 14 However, other studies found that NAA10 may serve as a tumour suppressor. By binding to PIX proteins, NAA10 inhibited Cdc42/Rac1 activity and, therefore, suppressed tumour metastasis. 19 Besides, NAA10 inhibits the migration and invasion of breast cancer cells by binding to STAT5a and decreases STAT5astimulated ID1 expression. 13 TSC2 was found to be acetylated and stabilized by NAA10, through which NAA10 inhibits mammalian target of rapamycin signalling pathway and suppressed tumorigenesis. 11 NAA10 may play diverse roles in different types of cancer cells or different stages during cancer tumorigenesis, and thus, identifying cancer-type-specific targets will help to understand the role of NAA10 in a certain cancer type. 14 p53 has been shown to involved in the regulation of cell cycle arrest, DNA damage repair and apoptosis. 22,23 Of particular interest, the emerging evidence demonstrated that p53 plays a critical role in inhibiting cancer invasion and metastasis. 24,25 Matrix metalloproteinases (MMPs) play an important role in tumour invasion, metastasis and tumour-induced angiogenesis by degrading basement membrane and extracellular matrix (ECM). 26,27 It has been shown that p53 can potently attenuate the expression of MMP-1, 28 MMP-2, 29 MMP-9 30 and MMP-13. 31 Pirh2 is a p53-induced protein, and has been shown to ubiquitylate p53 in vivo and in vitro. 32 Indeed, Pirh2, as an oncoprotein, was found to be stabilized and upregulated in several of tumour tissues, including head and neck cancer. 33 The overexpression of Pirh2 was concomitant with decreased p53 levels in malignant tissues, suggesting a role for Pirh2 in tumorigenesis through regulation of p53 stability and expression. 34 In previous study, we revealed that the expression of NAA10 was negatively correlated with that of Pirh2 in OSCC tissues.
Besides, positive NAA10 and negative Pirh2 might be independent biomarkers for better prognosis in OSCC patients. 35 However, the precise mechanism that NAA10 downregulated Pirh2 remains unknown. Here, we demonstrated that NAA10 plays a role in the invasion and metastasis of OSCC. Mechanistically, we elucidated that NAA10 interacts with RelA/p65 in the cytoplasm of OSCC cells, and subsequently inhibits RelA/p65-mediated transcriptional activation of Pirh2 by preventing the nuclear translocation of RelA/ p65. Consequently, inhibition of Pirh2 increased p53 level and suppressed the expression of p53 downstream targets, MMP-2 and MMP-9. These data revealed that NAA10 may function as a tumour metastasis suppressor in the progression of OSCC by targeting Pirh2-p53 axis and might be a prognostic marker as well as a therapeutic target for OSCC.

| Western blot
Protein was lysed by using RIPA lysis buffer containing 1% PMSF to extract the total cellular protein. Cell lysates were incubated on ice for 30 min and then centrifuged for 15min at 13,000 g to remove debris. Aliquots of proteins were boiled in 1× loading buffer for 10 min, samples containing 30 µg of total proteins were resolved by SDS-PAGE, and proteins were transferred to PVDF membrane (Millipore Corporation). Membranes were incubated with primary antibodies overnight at 4°C and appropriate HRP-secondary antibodies for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence detection (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Scientific). The antibody information was listed in Table S1. Real-time RT-PCR (qRT-PCR) assays were performed following reported procedures.

| Transwell migration and invasion assays
We performed cell migration and invasion assays according to a previous study. 19 Briefly, 8 ×

| In vivo tumorigenesis in nude mice
For nude mice tumorigenicity assays, 5 ×

| GST-Pull Down assays and Immunoprecipitation
Human full-length NAA10 cDNA was cloned into pGEX-5X-3vector, and the recombinant Glutathione-S-transferases (GST)-  CAL 27 and SCC-15 Cells in the logarithmic growth period were harvested and the fractionation of nuclear and cytoplasmic proteins was performed as described previously, 10 and the qualities of cytoplasmic and nuclear extracts were, respectively, verified by Western blot with antibodies against β-actin and Histone H 3 .

| Immunofluorescence
CAL 27 and SCC-15 cells were grown on coverslips and fixed in 4% paraformaldehyde for 30 min at 4°C, followed by permeabilization with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 5 min and blocked with 3% bovine serum albumin at room temperature for 1 h. Anti-NAA10 antibody and anti-p65 antibody were then applied to the cells overnight at 4°C, followed by washing with PBS/0.1% Triton X-100 and probing with tetramethylrhodamine isothiocyanate-conjugated anti-rabbit secondary antibody and fluorescein isothiocyanate-conjugated anti-mouse antibody for 45 min at room temperature. After washing, cells were stained with 4',6-diamidino-2-phenylindole and mounted on 50% glycerol/PBS. A Leica SP2 confocal system (Leica Microsystems) was used to observe the localization of NAA10 and RelA/p65.

| Luciferase reporter assay
CAL 27 cells cultured in 24-well plates were cotransfected indicated plasmids by using lipofectamine 2000. After 48 h, the luciferase activity was measured using a dual-luciferase reporter assay kit (E1910, Promega) according to the manufacturer's protocol. The firefly luciferase intensity was normalized based on transfection efficiency measured by Renilla luciferase activity.

| Chromatin immunoprecipitation assay
Quantitative chromatin immunoprecipitation (qCHIP) was performed as described previously. 10 The sequences of specific primers were listed in Table S3.

| Gene expression microarray data analysis and statistics
The raw data files from microarray profiling were imported into the Partek Genomics Suite (v. 6.6; Partek) for analysis, and two-way analysis of variance (2-way ANOVA) was applied with a fold change of 1.5 for the selection of differentially expressed genes at a significance level of p < 0.05.
The differentially expressed gene lists were further correlated for their relevant biological function and reaction pathway by analysing the GSEA (Gene Set Enrichment Analysis) and KEGG (Kyoto Encyclopedia of Genes and Genomes) using the Partek Genomic Suite. A significance level of p < 0.05 in the GSEA analysis to identify the significant biological process involved was observed, whereas an enrichment score of p < 0.05 in the KEGG pathway to identify the significant pathway was observed.

| Statistical analysis
All data for each group derived from three independent experiments were presented as the means ± SD. Statistically significant differences using a Student's t-test method that was evaluated using SPSS 20.0 (SPSS). A p values < 0.05 was considered statistically significant.

| NAA10 has tumour-suppressive function in vitro and in vivo
Our previous study revealed that NAA10 is overexpressed in OSCC tissues, and NAA10 expression correlates to TNM stage and lymph node status. Moreover, our data confirmed that NAA10 functions as an independent prognostic factor for OSCC patients. 35 To further explore the role of NAA10 in OSCC cells, CAL 27 and SCC-15 cells were transfected with three siRNAs targeting NAA10, and the NAA10 expression had a better-reduced trend in OSCC cells which were transfected with siNAA10-2 in the level of mRNA and protein, and cell migration was significantly elevated in CAL 27 cells with siNAA10 compared with control cells ( Figure S1). Next, CAL 27 and SCC-15 cells with stable interference of NAA10 expression were generated by lentiviral infection of shRNA targeting NAA10, whose target was identical to that of siNAA10-2. As illustrated in Figure 1A, silencing of NAA10 was confirmed by Western blot analysis in the OSCC cell lines (Left panel, Figure 1A). Subsequently, we found that knockdown of NAA10 significantly elevated the cell proliferation capacity (Right panel, Figure 1A), migration ( Figure 1B) and invasion ( Figure 1C The acetyltransferase activity of NAA10 plays an essential role in some NAA10-regulated biological events. 4 To uncover whether the acetylation activity of NAA10 was involved in regulating cellular migration and invasion. Plasmids encoding R82A mutant NAA10, NAA10-MT, which had lost its ability to associate with acetyl-CoA, and, therefore, exhibited low acetyltransferase activity 37 and wild-type were transfected into CAL 27 and SCC-15 cells. We found that the two constructs exhibited similar inhibitory effects on cellular migration and invasion ( Figure S2B,C).
Taken together, these results indicated that NAA10 regulates cell migration and invasion through a mechanism-independent intrinsic acetyltransferase activity.

| NAA10 knockdown inhibits P53 signalling pathway
Next, we sought to gain insight into the mechanism by which NAA10 regulates invasion and metastasis phenotype in OSCC. We performed cellular Gene Expression Profile with NAA10 stably silenced CAL 27 cells, and the differentially regulated genes were selected for KEGG pathway analysis. The analysis result showed that the P53 signalling pathway was the most relevant downstream signalling pathway of NAA10 (Figure 2A). Next, we performed Gene Set Enrichment Analysis (GSEA) and found that P53 signalling pathway was enriched in this dataset ( Figure 2B). Subsequently, we performed the genetic variations of P53 signalling pathways of clustering analysis ( Figure 2C). Consequently, some genes in P53 signalling pathway were determined by qRT-PCR, and Pirh2 was upregulated and p53 downregulated after knocking down NAA10 ( Figure 2D).
p53 is a major substrate of Pirh2, and Pirh2 promotes p53 degradation. 32 Furthermore, we previously demonstrated that the expression of NAA10 was negatively correlated with that of Pirh2 in OSCC. 35 Therefore, the Pirh2-p53 signalling pathway is selected for verification and study in the next step.

Pirh2-p53 signalling pathway
To uncover whether NAA10 was involved in regulating P53 signalling pathway. Firstly, human OSCC tissue microarrays (TMAs) and tumour xenograft tissues of nude mice were performed HE staining and staining images were viewed and evaluated independently by two experienced pathologists to ensure that the selected tissues were squamous cell carcinoma. Next, we verified the expression correlation of NAA10, Pirh2 and p53 by immunohistochemical staining, which suggested that NAA10 abundance was negatively associated with that of Pirh2 but positively associated with that of p53 ( Figure 3A). Moreover, tumour invasion is often associated with the enhanced synthesis of matrix metalloproteinases (MMPs), among which MMP-2 and MMP-9 are of central importance. 38

| NAA10 interacts with RelA/p65 and attenuates phosphorylation of p65 in OSCC
In the previous study, there was a significant inverse correlation between the expression of NAA10 and Pirh2 in OSCC patient tissues. 35 Furthermore, the effect of NAA10 on the expression of Pirh2 was determined by qRT-PCR in OSCC cells. The results indicated that NAA10 down-regulates the mRNA expression of Pirh2 ( Figure S3). These data raised a possibility that NAA10 regulates Pirh2 expression at the transcriptional level. Accumulating evidence demonstrated that NAA10 interacts with various transcription factors to regulate the expression of tumour-related target genes. 10 And, densitometric data showed a considerably higher ratio of p-p65 to p65 in OSCC cells with knockdown of NAA10 in comparison with control cells (p < 0.01, Figure S4).

| NAA10 suppresses RelA/p65-activated Pirh2 transcription
Our results raised the possibility that NAA10 could modulate the signalling events upstream of Pirh2 transcription, thereby promoting Pirh2 mRNA expression. Thus, we speculated whether RelA/p65 has a transcriptional activation effect on Pirh2, and NAA10 suppresses RelA/p65-mediated transcription activity of Pirh2 by interacting with the RelA/p65. Next, we explored whether RelA/p65 can affect the transcription of Pirh2, the human Pirh2 promoter-luciferase plasmid pGL3-Pirh2 and the RelA/p65 overexpression plasmid were cotransfected to CAL 27 cells, and the transcriptional activation was detected. The luciferase activity F I G U R E 2 NAA10 expression level is closely associated with Pirh2-p53 signalling pathway. (A) NAA10 gene expression profile was analysed after the stable interference of CAL 27 cells, and genes with differential expression of more than two folds were selected for KEGG analysis. The top 10 KEGG pathways were displayed. (B) GSEA result showed that high NAA10 expression was positively correlated with p53 signalling in OSCC. NES, normalized enrichment score. (C) Differentially expressed genes of P53 signalling affected by NAA10 silencing in CAL 27 cells. The colour intensity was proportional to the log 2 of expression ratio (blue, downregulated; red, upregulated). (D) Expression levels of selected genes in P53 pathway were examined by qRT-PCR in CAL 27 cells. Each bar represents the mean ± SD from three independent experiments was significantly higher than in the control group after RelA/p65 overexpression (Lanes 1 and 3 in Left and Right panels; Figure 5A).
The result elucidated that RelA/p65 has a transcriptional activation effect on Pirh2. Furthermore, we showed that overexpression of RelA/p65 augmented Pirh2's promoter activity, which was enhanced by silencing of NAA10 but was compromised by co-expression of NAA10 (Lane 3 and 4 in Left and Right panels; Figure 5A), indicating NAA10 could alleviate RelA/p65-regulated Pirh2 transcription.
To elucidate the RelA/p65 and Pirh2 promoter-specific binding sites, the truncated luciferase reporter plasmid: S1: pGL3-Pirh2-S1 with RelA/p65 proteins in vitro. V5-NAA10 and His-p65 were cotransfected in 293T cells, and reciprocal immunoprecipitation and immunoblotting were carried out with the indicated antibodies. (C) Cytoplasmic and nuclear interactions between NAA10 and RelA/p65. Indicated cells were subjected to subcellular fractionation, and the qualities of cytoplasmic and nuclear extracts were, respectively, verified by Western blot with antibodies against β-actin and H 3 . Cytoplasmic protein (500 µg) and nuclear protein (200 µg) were immunoprecipitated with an anti-p65 antibody, followed by Western blot with anti-NAA10 and anti-RelA/p65 antibodies. (D) Binding assay of His-p65 with GST or GST-NAA10. (E) Colocalization of NAA10 and RelA/p65. CAL 27 and SCC-15 cells were subjected to immunofluorescence staining with anti-NAA10 (green) and anti-RelA/p65 (red) antibodies. Colocalization was shown by the merge (yellow). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). (F) shCon and shNAA10 were transfected into CAL 27 and SCC-15 cells, respectively. The endogenous level of p65 and p-p65 (Ser536) was detected by Western blot. All experiments were carried out in triplicate promoter (length 1907 bp) was further constructed based on identified three potential RelA/p65 binding sites on the promoter region of Pirh2 by using Promo software ( Figure 5B). Then, the truncated luciferase reporter plasmids were cotransfected to CAL 27 cells with p65 overexpression plasmid and NAA10 knockdown or overexpression plasmid, respectively. The results showed that the luciferase activity of the truncated Pirh2 promoter region S1 was increased after NAA10 knockdown and is comparable with the full-length Pirh2 promoter. However, the luciferase activity of the truncated S2 had no significant difference with that of the control group ( Figure 5C). Similar data could be observed following stable expression of NAA10 (Right panel, Figure 5C).
We performed chromatin immunoprecipitation (ChIP)-qPCR assays in CAL 27 cells, and the results revealed that p65 bound to S1 site, but not to S2 site in qCHIP assays (Left panel, Figure 5D).
Once NAA10 was knockdown, binding of RelA/p65 to S1 site was dramatically increased (Right panel, Figure 5D), suggesting that binding of RelA/p65 to S1 site was negatively regulated by the cellular levels of NAA10. Collectively, these data indicated that RelA/p65 binds to the promoter region S1 to regulate Pirh2 transcription and NAA10 suppresses RelA/p65 induced Pirh2 transcription.

| Pirh-p53 signalling pathway is essential for NAA10 medicated OSCC tumour suppressor function
To further confirm that NAA10 affects the migration and invasion of OSCC through Pirh2-p53 signalling pathway, CAL 27 cells were transfected with NAA10 siRNA or Pirh2 siRNA, respectively, or in combination. Notably, silencing of NAA10 elevated cellular migration and invasion, and while silencing of Pirh2-inhibited cellular migration, invasion in CAL 27 cell. Pirh2 silencing indeed relieved the cell migration induced by NAA10 knockdown ( Figure 6A). Similar results were also observed in cell invasion ( Figure 6B). These data suggested that NAA10 plays a role in the migration and invasion of OSCC cells in part via regulating Pirh2-p53 signalling pathway.

| DISCUSS ION
In this study, we demonstrated the interaction between RelA/ p65 subunit of NF-κB with NAA10, and NAA10 suppressed Pirh2 expression via RelA/p65-dependent transcription in OSCC cells.
Pirh2 is a Ring-H2 domain containing E3 ubiquitin ligase, which targets several suppressors genes including p53. 32 Emerging evidence further confirmed that Pirh2 might be a novel critical oncoprotein in the development and progression of tumour. Overexpression of Pirh2 had been found in various cancers, including head and neck squamous cell carcinoma (HNSCC). And, Pirh2 expression was correlated with poor prognosis, at least partially through degradation of p27 in HNSCC. 33 The emerging evidence demonstrated that p53 plays an important role in inhibiting tumour invasion and metastasis by regulating the expression and activity of Matrix metalloproteinases (MMPs). 24,25,28,31 Recently, Lee et al. found that p53 is implicated in the regulation of EMT process, and p53 inhibits the expression of Ecadherin and increases that of Snail in OSCC cells. 39  In the present study, we found that NAA10 inhibits the migration, invasion of OSCC cells, and attenuates xenograft tumorigenesis in vivo ( Figure 1). Mechanistically, NAA10 decreases Pirh2 expression, and thus, it rescues p53 expression and decreases the expression of MMP-2 and MMP-9 to block migration and invasion of OSCC cells (Figure 7). We previously found that the expression of NAA10 was negatively correlated with that of Pirh2 in OSCC tissues.
Besides, positive NAA10 and negative Pirh2 might be independent biomarkers for better prognosis in OSCC patients. 35  The bars represent the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.005 F I G U R E 6 Pirh2-p53 signalling pathway is essential for NAA10 medicated OSCC invasion and metastasis. (A and B) Transwell assays were used to evaluate whether Pirh2 knockdown blocked the promoting effects of silencing NAA10 on the migration (A) and invasion (B) of CAL 27 cells. Values are presented as the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 F I G U R E 7 Schematic representation depicting the effects of NAA10 on OSCC invasion and metastasis. NAA10, by interacting with p65 and inhibiting the phosphorylation of p65, inhibits p65 goes into the cell nucleus and binds to Pirh2 promoter, and further attenuates the transcription expression of Pirh2 gene. Thereby the inhibition of Pirh2 increased the expression of the down-stream tumour suppressor genes p53, which leads to the down-regulation of the expression of the downstream MMP-2 and MMP-9 genes and plays the role of inhibiting the invasion and metastasis of OSCC activation, and this nuclear translocation is not regulated by IκBα. 42 Here, we hypothesized that NAA10 interacts with p65 in the cytoplasm to inhibit p65 translocation from cytoplasm into the nucleus, resulting in suppressed activation of NF-κB, and thereby inhibiting Pirh2 transcription in OSCC cells. However, our results did not elucidate the mechanism that NAA10 regulates the phosphorylation of p65. Phosphorylation of p65 is an important active form, which is regulated by various kinases and phosphatases. Activation of IKK kinase leads to phosphorylation of IκBα, which is separated from the p65-p50 complex, and then p65 enters the nucleus and functions as a transcription factor. 43 Whether the inhibition of p65 phosphorylation by NAA10 in OSCC cells is dependent on the IKKs deserves further studies.
Based on the above results, we speculated whether RelA/ p65 has a transcriptional activation effect on Pirh2, and NAA10 suppresses RelA/p65-mediated transcription activity of Pirh2. Therefore, we constructed the luciferase plasmid of the Pirh2 promoter region and detected the transcriptional activation of Pirh2 by RelA/p65 by using luciferase reporter assay. The results demonstrated that RelA/p65 binds to Pirh2 promoter to regulate its transcription, and NAA10 suppresses RelA/p65 binding to Pirh2 promoter, thus inhibiting Pirh2 expression. Hua et al.'s study reported NAA10 decreases GIT-assisted localization of PIX on membrane protrusions, thus alleviating CDC42/ RAC1-dependent cell metastasis. 19 In addition, Lee et al. 21 found silencing of NAA10 resulted in diminished recruitment of DNMT1 to E-cadherin promoter in qCHIP assay, but silencing DNMT1 had no effects on NAA10's binding to the same site. They also showed that NAA10 could stabilize DNMT1-DNA association by interaction with both non-methylated and hemimethylated DNA.
Taken together, this study elucidated that NAA10, as a tumour suppressor, inhibited tumorigenesis, migration and invasion in OSCC. Mechanically, we demonstrated that NAA10-suppressed RelA/p65 mediated the transcription of Pirh2, and decreased its expression. Therefore, NAA10 elevated p53 protein expression and stability via impairing the effect of Pirh2 on p53 protein degradation, and thus, inhibited p53 downstream genes expression involved in migration and invasion of tumour, such as MMP-2 and MMP-9 (as illustrated in Figure 7). Our study suggested that NAA10 may serve as a therapeutic target for the prevention of metastasis in OSCC.

ACK N OWLED G EM ENTS
We appreciate Professor Chengchao Shou (Peking University Cancer Hospital & Institute) for providing the plasmids and antibody. This study was supported by grants 81560473 from National Natural Science Foundation of China, grants 2021BC004 and 2018CB002 from Xinjiang Production and Construction Corps Science and Technology Cooperation Project, and grants GJHZ201901, CGZH202001 from Shihezi University.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published paper.