Geranylgeranylacetone promotes human osteosarcoma cell apoptosis by inducing the degradation of PRMT1 through the E3 ubiquitin ligase CHIP

Abstract Geranylgeranylacetone (GGA), an inducer of heat shock proteins, exerts anticancer activity in some tumours. However, the effect of GGA on human osteosarcoma (OS) has not been reported. This work is designed to evaluate the effect of GGA on the proliferation and apoptosis of human OS cells and to explore the underlying mechanisms. It was found that GGA markedly inhibited the proliferation and induced apoptosis of U‐2 OS cells in a dose‐dependent manner and also up‐regulated the expression of heat shock protein 70 (Hsp70). The degradation and ubiquitination of protein arginine N‐methyltransferase 1 (PRMT1) were obviously enhanced in U‐2 OS cells with CHIP overexpression and GGA treatment. The expression of PRMT1 was reversed in GGA‐treated cell after CHIP knockdown. The turnover of PRMT1 was obviously faster in cells overexpressing CHIP than that in control cells. The methylation and activity of STAT3 were induced by PRMT1, resulting in the inhibition of FAS transcription. Overexpression of PRMT1 reversed the effect of GGA on activation of apoptosis‐related proteins and U‐2 OS cell apoptosis. The expressions of PRMT1 were significantly up‐regulated in OS tissues compared with the adjacent normal tissues and benign bone tumours. In conclusion, GGA promotes the degradation of PRMT1 through the Hsp70‐CHIP‐mediated proteasome pathway, thereby inducing the FAS‐triggered cell apoptosis. Inhibition of PRMT1 may be a potential therapeutic strategy for OS patients.

as an inducer of heat shock protein 70 (Hsp70), which rapidly accumulates in cells after heat stress. 5 At the same time, GGA inhibits cell survival and induces apoptosis in multiple types of cancer, such as leukaemia, colon cancer and melanoma. [6][7][8] GGA also exerts antiinvasion effects on ovarian cancer and breast carcinoma cells. 9,10 Although pro-apoptotic effects of GGA have been reported, the underlying mechanisms have not been well defined.
The carboxyl terminus of Hsp70-interacting protein (CHIP), also named STIP1 homology and U-box-containing protein 1 (STUB1), is both an Hsp70/Hsp90 co-chaperone and an E3 ubiquitin ligase. 11 CHIP performs cellular protein quality control and regulates some important signalling pathways by inducing ubiquitin-mediated degradation of its substrate proteins via the proteasomal pathway. 12 The activity of CHIP is initiated through interacting with Hsp70, subsequently promotes protein degradation by polyubiquitination. 13 Recently, our preliminary data demonstrated that CHIP could promote ubiquitination and degradation of protein arginine N-methyltransferase 1 (PRMT1) in HEK293 cells. 14 Other studies have reported that PRMT1 is one of the major protein arginine methyltransferases that catalyses mono-methylation and asymmetric demethylation of arginine-bearing substrates. 15 Thus, PRMT1 is involved in regulating a wide range of cellular processes, including RNA processing, DNA damage response and signal transduction.
Aberrant expression of PRMT1 has been found in several malignancies. 16 Therefore, we speculate that the suppressive effects of GGA on cancer cells are associated with Hsp70-CHIP-induced ubiquitination and degradation of PRMT1.
Although there are several reports about the anticancer activity of GGA in tumours, the effect of GGA on human OS has not been evaluated. Therefore, in the present study, the role of GGA in the proliferation and apoptosis of U-2 OS cell, a human osteosarcoma cell line, was investigated. We also aimed to determine whether the effects of GGA are mediated by CHIP-induced ubiquitination and degradation of PRMT1.

| Cell proliferation assay
Cell proliferation was detected by Cell Counting Kit-8 (CCK-8; CK04-11, Dojindo, Kumamoto, Japan) assay. U-2 OS cells were seeded into 96-well plates at a density of 1 × 10 4 cells/mL and treated with GGA at various doses for 24 hours. After washing, 10 µl of CCK-8 was added to each well and incubated for 2 hours at 37℃. The optical density (OD) at 450 nm was detected by Epoch 2 Microplate Spectrophotometer.

| EdU staining
Detection of EdU incorporation into the DNA was performed with the Click-iT EdU Alexa Fluor-647 Cell Proliferation Kit (C10340, Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, cultured U-2 OS cells were plated in 24-well plates at a density of 2x10 5 cells/well. The cells were treated with GGA for 24 hours before exposure to EdU. After incubation for 2 hours, cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. The cells were washed three times and then incubated with the EdU staining cocktail for 30 minutes protected from light. Images were taken by using fluorescence microscope.

| Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay
Cell apoptosis was determined by TUNEL assay (Roche), as previously described. 17 Briefly, U-2 OS cells were treated with GGA for 24 hours. After washing with PBS, U-2 OS cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100. The fixed cells were subjected to the TUNEL assay according to the manufacturer's instructions.
Finally, the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 15 minutes. Fluorescent images were taken in 10 random fields for each sample using fluorescence microscope.

| Western blotting analysis
Western blot experiments were performed as previously described. 17 Cells were lysed in RIPA buffer. Proteins were separated and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with primary and secondary antibodies. The immunoblotted proteins were visualized with enhanced chemiluminescence (ECL) reagents. ImageJ software, an open-source image processing program, was used to quantify blots.

| Co-immunoprecipitation
The co-immunoprecipitation assay was performed as described previously. 14 In brief, cells were treated with 10 μM MG132 for 2 hours and then lysed in RIPA buffer on ice for 30 minutes. Then, the lysates were incubated with anti-PRMT1 primary antibody and protein A/Gagarose beads (sc-2003, Santa Cruz, CA, USA) overnight at 4℃. For dimethyl-arginine immunoprecipitations, without MG132 treatment, cell lysates were incubated with mono/dimethyl-arginine antibody and protein A/G-agarose beads overnight at 4℃. After the final wash, the beads were resuspended in 40 μL 1 × Laemmli buffer.
The samples were boiled for 5 minutes and analysed by Western blot probed with the antibody specific for ubiquitin.

| Quantitative real-time polymerase chain reaction (qPCR) analysis
Total RNA was isolated from cells or tissues using TRIzol reagent according to the manufacturer's instructions. RNA samples were reverse-transcribed using random hexamer primers in the presence of RNase inhibitor (Takara Bio, Shiga, Japan). The cDNA was amplified using the ChemoHS qPCR mix  ing to the percentage of positive cells as follows: 1, <5%; 2, 5%-25%;

| Statistical analysis
Mean densitometry values and all other quantitative data are presented as the mean ± standard error of the mean (SEM). Comparisons among groups were made using Student's t test or one-way ANOVA followed by the Student-Newman-Keuls test. Differences with P values <0.05 were considered statistically significant. SPSS software was used to analyse the data.

| GGA dose-dependently inhibited proliferation of U-2 OS cells and promoted cell apoptosis
We investigated whether GGA influenced the viability and proliferation of U-2 OS cells in vitro using the CCK-8 assay and EdU staining. As shown in Figure 1A, Therefore, we selected 20 µM of GGA for subsequent experiments.

| GGA increased tumour necrosis factor receptor superfamily member 6 (FAS) expression and activation of caspase-3, caspase-8 and caspase-9, but did not affect p53 level in U-2 OS cell
FAS-and p53-mediated signalling pathways play important roles in the process of apoptosis. 18 We detected the levels of FAS and p53 in U-2 OS cells treated with 20 µM GGA at different time points. As shown in

| The level of PRMT1 was decreased in GGAtreated U-2 OS cells with concomitantly enhanced PRMT1 polyubiquitination that was regulated by Hsp70
Previous studies have reported that GGA acts as an inducer of Hsp70, which interacts with CHIP to promote protein degradation by polyubiquitination. 5, 13 We have confirmed that CHIP could decrease PRMT1 level in HEK293 cells via the ubiquitinationproteasome pathway. 14

| The effect of GGA on PRMT1 degradation was achieved by CHIP-mediated ubiquitination modification
To investigate whether GGA-induced ubiquitination and degradation of PRMT1 are dependent on CHIP, we transfected U-2 OS cells with short hairpin RNA against CHIP (shCHIP) and pLenti-CHIP plasmids to knockdown or overexpress CHIP, respectively.
The results showed that the expression of PRMT1 in GGA-treated OS cells with shRNA stabilized PRMT1 level ( Figure 4G and H).
These results confirm that the effect of GGA on PRMT1 degradation and ubiquitination is dependent on CHIP expression in U-2 OS cells. were collected to detect methylation of STAT3 by immunoprecipitation using anti-mono and dimethyl-arginine antibody. The isolated proteins were then analysed for the presence of STAT3 by immunoblotting. As shown in Figure 5A and B, the higher expression of PRMT1 was observed in U-2 OS cells with pLenti-PRMT1 transfection. The STAT3 was methylated even in untreated cells. However, the level of STAT3 immunoprecipitated was Overexpression of PRMT1 promoted phosphorylated STAT3 entry into nucleus of U-2 OS cells ( Figure 5C). To confirm whether the transcriptional level of FAS was affected by PRMT1, we performed qPCR analysis for level of FAS mRNA. The data showed that DB75 increased the mRNA level of FAS, which was reversed by exogenous PRMT1 ( Figure 5D). These data indicated that methylation and activity of STAT3 are induced by PRMT1, resulting in the inhibition of FAS transcription.

| Overexpression of PRMT1 suppressed GGAinduced U-2 OS cell apoptosis by inhibiting the FAStriggered apoptotic pathway
The role of PRMT1 in translation regulation has been recognized. 21 This study next sought to determine whether high expression of PRMT1 inhibits GGA-induced U-2 OS cell apoptosis. We overexpressed PRMT1 in U-2 OS cells by pLenti-PRMT1 transfection and then measured cell apoptosis and apoptotic protein levels after GGA stimulation. As shown in Figure 6A-C, Western blot of the cell lysates with an anti-PRMT1 antibody revealed higher PRMT1 expression in transfected U-2 OS cells.
In parallel, overexpression of PRMT1 reduced apoptotic cell proportion after GGA treatment for 24 hours. We further investigated the effect of These observations indicate that PRMT1 restrains GGA-mediated U-2 OS cell apoptosis by inhibiting the FAS-triggered apoptotic pathway.

| PRMT1 was up-regulated at the protein level in OS tissues of patients
Aberrant expression of PRMT1 has been reported in several malignancies. 16 We detected the expression of PRMT1 via  Figure S1). GGA has been identified as a non-invasive Hsp70 inducer, and the cooperative activity of Hsp70 and CHIP is involved in the quality control of multiple client proteins. 23 In our previous study, we found that CHIP can promote PRMT1 ubiquitination and degradation in HEK293 cells. PRMT1 is a histone arginine methyltransferase that mainly mono-and demethylates histone H4 arginine 3, which is an activation site for gene expression. 24,25 In colon cancer, PRMT1 is involved in epidermal growth factor receptor methylation during the resistance to cetuximab treatment. In addition, the down-regulation proteasome pathway. The degradation of PRMT1 in U-2 OS cells induces the inhibition of STAT3 methylation and activation of the FAStriggered apoptotic pathway, resulting in cell apoptosis ( Figure 7E).

| D ISCUSS I ON
In addition, high expression of PRMT1 is observed in the three major subtypes of OS but not in osteoid osteoma and chondroblastoma.
Our study found a novel effect of GGA on human OS cells. However, more studies are needed to dissect further the role of GGA in OS animal model for future clinical trials.

ACK N OWLED G EM ENT
This research was supported by the Fundamental Research Funds for the Central Universities.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available on request from the corresponding author.