Sirtuin 5 regulates the proliferation, invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

Abstract Sirtuin 5 (SIRT5) is a NAD+‐dependent class III protein deacetylase, and its role in prostate cancer has not yet been reported. Therefore, to explore the diagnosis and treatment of prostate cancer, we investigated the effect of SIRT5 on prostate cancer. Sirtuin 5 was assessed by immunohistochemistry in 57 normal and cancerous prostate tissues. We found that the tissue expression levels of SIRT5 in patients with Gleason scores ≥7 were significantly different from those in patients with Gleason scores <7 (P < .05, R > 0). Further, mass spectrometry and pathway screening experiments showed that SIRT5 regulated the activity of the mitogen‐activated protein kinase (MAPK) pathway, which in turn modulated the expression of MMP9 and cyclin D1. Being a substrate of SIRT5, acetyl‐CoA acetyltransferase 1 (ACAT1) was regulated by SIRT5. SIRT5 also regulated MAPK pathway activity through ACAT1. These results revealed that SIRT5 promoted the activity of the MAPK pathway through ACAT1, increasing the ability of prostate cancer cells to proliferate, migrate and invade. Overall, these results indicate that SIRT5 expression is closely associated with prostate cancer progression. Understanding the underlying mechanism may provide new targets and methods for the diagnosis and treatment of the disease.

Sirtuins belong to a highly conserved family of proteins.
Mammals have seven sirtuins (SIRT1-7) that regulate different metabolic and stress response pathways. 14 SIRT5 plays a vital role in glycolysis, the tricarboxylic acid cycle, fatty acid oxidation, nitrogen metabolism, the pentose phosphate and antioxidant pathways, and apoptosis. [15][16][17] SIRT5 promotes the growth of human non-small cell lung cancer, 18 and patients with high SIRT5 expression have a poor prognosis. 19 In addition, SIRT5 is associated with liver, 20 ovarian 21 and breast 22 cancers. However, its role in prostate cancer has not been reported to date.
Acetyl-CoA acetyltransferase 1 (ACAT1) is a possible anticancer target. 23 Its expression can be a potential prognostic marker for prostate cancer, 24 and it may function as a promoter of this disease. 25 This study aimed to investigate the role of the SIRT5-ACAT1 axis in prostate cancer and identified new targets for the diagnosis and treatment of this disease.

| Patients and specimens
The prostate cancer specimens examined in this study were all

| Cell culture
All cell lines were obtained from the Shanghai Cell Bank (Shanghai, China) and cultured in RPMI 1640 medium with foetal bovine serum (FB15015; Clark Biosciences, Richmond, VA, USA). LNCaP and PC-3 cells were cultured according to the manufacturer's instructions in medium containing 10% foetal bovine serum (FBS) with no antibiotics and maintained in a 5% CO 2 incubator at 37°C.

| Immunohistochemistry and Gleason scores
Tissue sections were incubated with an SIRT5 rabbit polyclonal an-  Gene expression relative to β-actin was calculated using the 2 −ΔΔCt method.

| Colony formation assay
Cells with altered expression of SIRT5 protein were inoculated into 6-well culture plates at a density of 800 cells per well and cultured for 10-14 days. The cells were then washed with phosphate-buffered saline (PBS), fixed with pre-cooled methanol, washed three times with PBS and finally stained with crystal violet. After drying, the colonies were counted manually.

| Transwell invasion analysis
Prostate cancer cells (10 5 cells per 100 μL) with increased or decreased SIRT5 protein expression were inoculated into Transwell chambers (8 μm pore size, Corning, NY, USA) for 24 hours to observe their migration ability. Cells that crossed the membrane were stained with crystal violet, dried and counted manually.

| Immunofluorescence
An appropriate number of cells was seeded in a 10-cm Petri dish Tokyo, Japan) was used to obtain random cell images.

| Co-immunoprecipitation (Co-IP) assays
The desired cell line was plated in two 10-cm cell culture dishes.
After the cells grew to confluence, they were lysed and centrifuged at 12 000 rpm for 15 minutes at 4℃. Next, 60 μL of Protein A/G Sepharose (P2012; Beyotime Biosciences) was added to the supernatant and stirred for at least 2 hours. The mixture was then centrifuged at 4°C and 1000 rpm for 5 minutes, following which the supernatant was divided into two parts. To one part was added the target antibody (8 µg), and to the other part, anti-mouse/rabbit IgG (1:2000; ZSGB-BIO, Beijing, China). The mixture was shaken overnight at 4°C in a chromatography cabinet. The following day, 25 μL of agarose A/G magnetic beads was added to each tube and incubated at 4°C for 6 hours. Next, the cell lysate was washed, heated in boiling water for 10 minutes and immunoblotted.

| Active oxygen detection assays
The level of reactive oxygen species (ROS) in prostate cancer cells

| Statistical analyses
All data were analysed using SPSS version 24.0 (Beijing, China) to perform chi-square tests, or the Prism 5 (GraphPad, La Jolla, California, USA) software. All experiments were repeated at least three times independently under the same conditions. Values of P < .05 were considered statistically significant.

| High expression of SIRT5 in prostate cancer cells correlates with tumour Gleason score
To study the expression of SIRT5 in human prostate cancer tissues, we performed immunohistochemical experiments on 57 randomly selected prostate tissue sections. Expression of SIRT5 was absent in normal prostate tissues but high in tumour tissues ( Figure 1A). The 57 tissue sections were assigned and counted according to the Gleason score standard. When the Gleason score was equal to 7, the prostate cancer tissues could be divided into two groups: (3 + 4) and (4 + 3). The difference between the two groups was not statistically significant (P = .797). Similarly, the expression of SIRT5 in tissues with Gleason scores of 6 and 7 was not significantly different (P = .396); additionally, the expression was not significantly different in the two groups with scores of 8 and 9 (P = .262). However, when scores of 8 and 9 were combined as one group, SIRT5 protein expression was significantly different from that in the group with a score of 7 (P = .028) and the group with a score of 6 (P = .001) ( Table 1). Therefore, we concluded that SIRT5 expression was related to the prostate cancer Gleason score.
In the cBioPortal database, we found that the SIRT5 gene is mutated in prostate cancer and that most of these mutations are deep deletions ( Figure 1B) and missense mutations ( Figure 1C). These mutations may be related to the prognosis of prostate cancer patients.
However, there was no apparent statistical significance in our data ( Figure 1D); consequently, we ignored the effect of SIRT5 gene mutations on prostate cancer.

| SIRT5 promotes cell proliferation and migration
Based on the above findings, we next investigated the role of SIRT5

| SIRT5 regulates the MAPK signalling pathway
To understand how SIRT5 protein affects prostate cancer cells, we In order to highlight the role of SIRT5, we added a specific inhibitor of SIRT5 and found that the MAPK signalling pathway was inhibited and the expression level of related functional proteins was significantly decreased (Figure S2E,F).

| SIRT5 combines with ACAT1 and regulates its expression
To investigate how SIRT5 affected the MAPK signalling pathway, we performed mass spectrometry analyses ( Figure 4A). Results showed that ACAT1 combined with SIRT5. To further investigate this finding and determine whether SIRT5 regulated ACAT1 at the gene or protein level, qRT-PCR was performed. Irrespective of whether

SIRT5 expression was increased or decreased in LNCaP cells, ACAT1
mRNA level did not change. The same result was obtained in PC-3 cells ( Figure 4B), indicating that SIRT5 does not regulate ACAT1 mRNA.
Based on these findings, we shifted our focus to protein regulation and found that after interfering with the expression of SIRT5 in LNCaP cells, the protein level of ACAT1 decreased, whereas its levels increased when SIRT5 expression was increased ( Figure 4Ca). In contrast, after changing ACAT1 expression, SIRT5 level did not change ( Figure 4Cb); the same results were obtained in PC-3 cells (Figure 4Cc,D). These results indicate that SIRT5 can regulate ACAT1 at the protein level, but ACAT1 cannot regulate SIRT5 expression. However, after the expression of SIRT5 was decreased and MG-132 was added, the expression of ACAT1 protein did not increase significantly ( Figure S3A), indicating that SIRT5 does not regulate ACAT1 through the proteasome pathway.

Co-IP experiments performed in LNCaP and PC-3 cells showed
that SIRT5 and ACAT1 combined with each other in vitro ( Figure 4D).
The results were confirmed through confocal experiments, which revealed that the proteins SIRT5 and ACAT1 co-localized in the mitochondria in cells ( Figure 4E and Figure S3B).

| SIRT5 regulates the function of prostate cancer cells through ACAT1
In view of the above results, we understood the effect of ACAT1 protein on prostate cancer cells. Throughout the experiment, silencing of ACAT1 protein was effective ( Figure S4A,B). Under  Figure S4G,H). The colony formation assay shows that the ability of SIRT5 to promote colony formation is weakened in both LNCaP and PC-3 cells after co-transfection of the SIRT5 plasmid and ACAT1 siRNA. The right panel shows the number of colonies generated by these two cell lines. *P < .05,**P < .01. (E) The Transwell migration assay shows that the ability of SIRT5 to promote cell migration is weakened after co-transfection of the SIRT5 plasmid and ACAT1 siRNA in both LNCaP and PC-3 cells. *P < .05,**P < .01 Overall, these results demonstrate that SIRT5 regulates the MAPK signalling pathway through ACAT1 and has a tumour-promoting role in prostate cancer.

| D ISCUSS I ON
Prostate cancer is one of the most common malignancies in men. 26 The mortality rate in prostate cancer is related to obesity, physical exercise, smoking and antioxidants. 27 Presently, prostate cancer treatment imposes a serious economic burden. 28  We determined that ACAT1 can regulate the MAPK signalling pathway; therefore, we believe that SIRT5 regulates the MAPK signalling pathway through ACAT1, thereby promoting prostate cancer. MAPK protein is not only present in the nucleus and cytoplasm but may also be present in the mitochondria. 35 However, the mechanisms through which SIRT5 and ACAT1 affect the MAPK signalling pathway and by which the MAPK protein shuttles between the mitochondria, cytoplasm and nucleus remain to be studied further.
Nevertheless, we can confirm that the SIRT5-ACAT1 axis plays a very important role in prostate cancer. In addition, this axis may help develop new treatment strategies for prostate cancer, which may provide new hope for the treatment of this disease.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation Co., Ltd. for providing us with mass spectrometry services.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

This study was approved by the Ethics Committee of China Medical
University.