ACY1-mediated deacetylation of GSTP1 at lysine 30 suppresses cisplatin resistance in epithelial ovarian cancer

doi:10.21203/rs.3.rs-1949781/v1

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ACY1-mediated deacetylation of GSTP1 at lysine 30 suppresses cisplatin resistance in epithelial ovarian cancer

https://doi.org/10.21203/rs.3.rs-1949781/v1

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Resistance to platinum-based chemotherapy is the major limitation to the improvement of epithelial ovarian cancer (EOC) prognosis. Aminoacylase-1 (ACY1) hydrolyzes acylated l-amino acids and N-acetylated proteins; however, the roles of ACY1 in EOC and platinum resistance remain unknown. Accordingly, this study explored the biological functions and clinical significance of ACY1 in the resistance of EOC to cisplatin therapy. We found that lower levels of ACY1 were associated with worse prognosis and platinum-based chemoresistance in EOC. Moreover, ACY1 significantly sensitized EOC cells to cisplatin in vitro and in vivo. Mechanistically, ACY1 deacetylated glutathione S-transferase P1 (GSTP1) at lysine 30, which triggered its degradation and thereby reduced GSTP1 stability. Thus, ACY1 attenuated the intracellular reactive oxygen species (ROS) elimination and DNA damage repair. Finally, histone deacetylase 4 (HDAC4) downregulated ACY1 transcription by suppressing histone H3 lysine 27 acetylation on the ACY1 promoter, and inhibiting HDAC4 reversed cisplatin resistance in EOC cells. Collectively, these results revealed an HDAC4–ACY1–GSTP1 axis as vital signaling for cisplatin resistance in EOC. This suggested that ACY1 might be a novel mediator in EOC chemoresistance and HDAC4 inhibitors could be used to overcome platinum resistance in patients with low expression of ACY1.

epithelial ovarian cancer

platinum resistance

ACY1

GSTP1

HDAC4

reactive oxygen species

DNA damage repair

Ovarian cancer (OC) is the fifth leading cause of cancer death although not the common malignant tumor in women worldwide, and epithelial OC (EOC) accounts for over 95% of all cases of OC^1,2. Cytoreductive surgery combined with platinum-based chemotherapy remains the standard treatment for patients with advanced-stage EOC³. Approximately 80% of patients with EOC respond well to first-line platinum-based therapy; however, most relapse within 2 years from primary diagnosis and become acquire resistance to platinum-based chemotherapy⁴, which is the major obstacle to improve EOC prognosis. Therefore, understanding the mechanisms underlying resistance to platinum-based therapy is essential for developing better therapeutic biomarkers and strategies in EOC.

Aminoacylase-1 (ACY1), a homodimeric zinc-binding enzyme that catalyzes the hydrolysis of acylated l-amino acids and N-acetylated proteins^5,6. ACY1 is involved in the detoxification and bioactivation of xenobiotics^7,8. ACY1 was recently reported to play a role in the tumorigenesis of several cancers. However, controversies remain as to whether it is a pro- or anti-tumorigenic factor. Some found that low expression of ACY1 promoted angiogenesis, and cancer cell proliferation and invasion, in patients with hepatocellular carcinoma and renal carcinoma^9,10, whereas others found that increased expression of ACY1 activated the ERK/TGF-β pathway to promote colorectal cancer (CRC)¹¹. However, the biological role of ACY1 in EOC and its underlying mechanism remains unclear.

Cisplatin is a first-line chemotherapeutic for EOC; however, curing patients with EOC who have intrinsic or acquired resistance to cisplatin is challenging. Studies show that improving the ability of DNA repair and maintaining redox balance play crucial roles in cisplatin resistance^12–14. Glutathione S-transferase P1 (GSTP1), an isozyme in the glutathione S-transferase (GST) superfamily, is involved in detoxification and the regulation of oxidative stress¹⁵. GSTP1 regulates the metabolism of several chemotherapeutics, including cisplatin and carboplatin; prevents the excess accumulation of reactive oxygen species (ROS); and reduces DNA damage repair, which promotes resistance to chemotherapeutics^16–18. GSTP1 was found to play an essential role in EOC resistance to platinum-based chemotherapy^19,20. The expression of GSTP1 is regulated by multiple mechanisms, including promoter methylation and the action of transcription factors and microRNAs¹⁹. However, the mechanisms underlying the regulation of GSTP1 protein levels by post-translational modifications remain unclear.

In the present study, we found that ACY1 is closely correlated with favorable EOC prognosis and response to cisplatin chemotherapy. Mechanism investigations revealed that ACY1 deacetylates GSTP1 at lysine 30 (K30) and then induces its degradation, which destabilized GSTP1 to suppress cisplatin resistance. Furthermore, histone deacetylase 4 (HDAC4) inhibited ACY1 transcription by decreasing the level of histone H3 lysine-27 acetylation (H3K27Ac) on the ACY1 promoter. Thus, our study revealed a novel cisplatin-resistance mechanism mediated by the HDAC4–ACY1–GSTP1 axis which might be a new therapeutic target for overcoming platinum-based chemoresistance in EOC.

Reduced ACY1 expression correlates with poor prognosis and resistance to platinum-based chemotherapy in patients with EOC

Our previous study used gene expression microarray showed that ACY1 expression is higher in EOC tissue than in normal ovarian epithelial tissue (Figure S1A). The Gene Expression Profiling Interactive Analysis database (http://gepia.cancer-pku.cn/) also showed that the ACY1 mRNA levels were significantly higher in EOC tissue than in normal tissue (Figure S1B). Consistently, in samples from our institute (Sun Yat-sen University Cancer Center, Guangzhou, China), we found that ACY1 mRNA levels were significantly higher in EOC tissues (n = 19) than in normal tissues (n = 19) (Figure S1C). In an analysis performed using the online bioinformatics tool Kaplan-Meier Plotter (http://kmplot.com/analysis/), patients with EOC who had higher levels of ACY1 were found to have greater progression-free survival (PFS) and overall survival (OS) than those with EOC who had lower levels of ACY1 (Figure S1D).

We further determined the clinical significance of ACY1 expression by performing immunohistochemical staining of 120 archived EOC tissue specimens (Fig. 1A). As expected, patients with high levels of ACY1 (n = 59) had longer PFS (P = 0.003) and greater OS (P = 0.028) than those with low ACY1 levels (n = 61) (Fig. 1B and 1C). In addition, a multivariate analysis demonstrated that ACY1 expression was an independent prognostic factor for PFS and OS in patients with EOC (Fig. 1D and E). Furthermore, we found that elevated expression of ACY1 was inversely correlated with recurrence and a shorter progression-free interval (PFI; PFI ≤ 6 months or PFI > 6 months) (Table S1; Fig. 1F). Generally, platinum response is classified according to the PFI. The cut-off time is set at six months, defining platinum-sensitive as PFI since last line of platinum > 6 months and platinum-resistant as ≤ 6 months²¹. To explore the effects of ACY1 expression on response to platinum, we analyzed the correlation between ACY1 expression and platinum efficacy in 100 EOC patients treated with platinum-based chemotherapy (Figure S1E). The results showed that ACY1 levels were significantly higher in patients with EOC who were platinum-sensitive (n = 70) than in patients with EOC who were platinum-resistant (n = 30) (P < 0.01, Fig. 1G). Taken together, our results suggest that lower ACY1 levels predict resistance to platinum-based chemotherapy and poor clinical outcomes in patients with EOC.

ACY1 suppresses cisplatin resistance in EOC cells

Cisplatin is the mainstay of first-line chemotherapeutics for EOC. To further assess the effect of ACY1 on the efficacy of chemotherapy in EOC, stable ACY1-knockdown SKOV3 EOC cell lines using lentiviral short-hairpin RNA (shRNA) and ACY1-overexpressing A2780, TOV21G EOC cell lines were treated with cisplatin (Fig. 2A and S2A). A Cell Counting Kit-8 (CCK8) assay showed that ACY1 knockdown significantly increased the half-maximal inhibitory concentration (IC₅₀) of cisplatin required to inhibit cell proliferation, whereas ACY1 overexpression decreased this IC₅₀ (Fig. 2B–2E, S2B, and S2C). ACY1 knockdown consistently and substantially enhanced the colony-forming ability of cisplatin-treated EOC cells, whereas ACY1 overexpression inhibited this ability of cisplatin-treated EOC cells (Fig. 2F and 2G), indicating that ACY1 downregulation promoted cisplatin resistance in EOC cells. Moreover, annexin V/propidium iodide (PI) staining demonstrated that cisplatin-induced apoptosis was significantly attenuated in ACY1-knockdown cells and elevated in ACY1-overexpressing cells (Fig. 2H–2K, S2D, and S2E). Furthermore, we detected the levels of the DNA double-strand break marker gamma H2A histone family member X (γH2AX), anti-apoptotic marker B-cell leukemia/lymphoma 2 (Bcl-2), proapoptotic marker Bcl-2 associated X (Bax), and apoptotic marker cleaved poly (adenosine diphosphate-ribose) polymerase (PARP) via western blot analysis. The levels of γH2AX, Bax, and cleaved PARP were found to be reduced by ACY1 silencing but increased by ACY1 overexpression; by contrast, the level of Bcl-2 was increased by ACY1 silencing but reduced by ACY1 overexpression (Figure S2F). Taken together, the above findings indicated that ACY1 downregulation promoted cisplatin resistance in EOC cells.

To confirm the above findings in vivo, we subcutaneously inoculated three cohorts of female nude mice in the flank with ACY1-overexpressing, ACY1-knockdown, or control TOV21G cells, respectively. When the tumor volume of each cohort reached 100–150 cm³, the mice were treated with either cisplatin or saline solution. Cisplatin treatment reduced the tumor volume and weight in the ACY1-overexpressing cohort significantly more than in the ACY1-knockdown cohort (Fig. 2L–N), indicating that ACY1 upregulation attenuated cisplatin resistance in vivo. Overall, our results provide evidence that ACY1 depletion induces resistance to cisplatin in EOC cells.

ACY1 physically interacts with GSTP1

ACY1 is a cytosolic metalloenzyme that typically exerts its effects via direct interactions with proteins⁷. To characterize the molecular mechanisms underlying the roles of ACY1 in the resistance of EOC to cisplatin, we transfected an ACY1 plasmid into HEK-293T cells and performed mass spectrometry (MS) to identify ACY1-binding partners. There were several top potential ACY1-binding partners, such as RPS9, KRT1 and KRT9 (Fig. 3A and 3B; Table S2), with GSTP1 being chosen for further analysis owing to its known roles in drug metabolism and preventing oxidative damage. Moreover, GSTP1 upregulation markedly increased the resistance of patients with EOC to platinum-based chemotherapeutics.¹⁹. The results of the co-immunoprecipitation (co-IP) assay showed that endogenous ACY1 and GSTP1 co-precipitated in A2780 and TOV21G cells (Fig. 3C). Exogenous ACY1 was reciprocally co-immunoprecipitated with GSTP1 in HEK-293T cells transfected with Flag-ACY1 and HA-GSTP1 plasmids (Fig. 3D). Furthermore, GST pull-down assay identified that GSTP1 directly bound to ACY1 in vitro (Fig. 3E), and additional immunofluorescence assays revealed positive signals of ACY1 and GSTP1 colocalization in the cytoplasm of EOC cells (Fig. 3F). To determine which domain of ACY1 mediates the interactions between ACY1 with GSTP1, we constructed a series of deletion-mutant plasmids of Flag-ACY1 (Fig. 3G). Reciprocal co-IP assays revealed that the C-terminal M20 peptidase domain of ACY1 interacted with GSTP1 (Fig. 3H). Based on the above observations, we conclude that ACY1 physically interactes with GSTP1.

ACY1 decreases GSTP1 stability by facilitating FBX8-mediated ubiquitination and degradation of GSTP1

To explore whether physical interactions between GSTP1 and ACY1 regulate the expression of GSTP1, we quantified the levels of GSTP1 in the same cohort of EOC tissue samples used for analyzing the levels of ACY1 with IHC staining. This revealed that ACY1 levels were negatively correlated with GSTP1 levels (R = − 0.294, P = 0.0012) (Fig. 4A and 4B). Western blot analysis showed that ACY1 knockdown increased the levels of GSTP1 protein, whereas ACY1 overexpression decreased the levels of GSTP1 protein (Fig. 4C). Levels of ectopic HA-GSTP1 were also decreased in ACY1-overexpressing cells but increased in ACY1-silenced EOC cells (Fig. 4D and S3A). However, the expression of ACY1 did not significantly affect the levels of GSTP1 mRNA (Figure S3B and S3C), suggesting that ACY1 may regulate the translation of GSTP1 mRNA into protein or the stability of the protein.

To better understand the mechanisms via which ACY1 regulates GSTP1 protein levels, a cycloheximide (CHX) chase assay was conducted to assess GSTP1 protein half-life. GSTP1 protein half-life was found to be shortened by ectopic ACY1 (Fig. 4E and 4F) but lengthened by ACY1 silencing (Fig. 4G and 4H). Furthermore, GSTP1 protein levels were elevated in ACY1-overexpressing cells treated with the proteasome inhibitor MG132 (Fig. 4I and 4J). Similarly, the level of GSTP1 ubiquitination was enhanced by ACY1 overexpression (Fig. 4K) but reduced by ACY1 knockdown (Fig. 4L). Taken together, these results indicated that ACY1 attenuated the stability of GSTP1 via the ubiquitin-proteasome pathway.

F-box only protein 8 (FBX8), an E3 ubiquitin ligase, induces the degradation of GSTP1 by ubiquitin and to suppress CRC progression²². To determine whether ACY1 reduces the stability of GSTP1 via FBX8-mediated ubiquitination, we conducted a co-IP and western blot assay. This demonstrated that the ability of ACY1 to enhance GSTP1 ubiquitination was significantly inhibited in FBX8-silenced cells (Fig. 4M). Overall, these findings show that ACY1 promotes the ubiquitin–proteasome-dependent degradation of GSTP1 via the E3 ligase activity of FBX8.

ACY1 reduces GSTP1 stability and thereby increases intracellular ROS levels and inhibits DNA damage repair in cisplatin-treated EOC cells

To evaluate whether ACY1 suppresses cisplatin resistance through the action of GSTP1, we used small interfering RNA (siRNA) to downregulate the expression of GSTP1 in ACY1-knockdown cells. CCK8 and annexin V/PI assays revealed that GSTP1 knockdown significantly abolished the enhanced growth and survival capacities of cisplatin-treated EOC cells induced by ACY1-silencing (Fig. 5A–5D).

GSTP1 protects cancer cells from DNA damage and induced drug resistance by activating the ATM–CHK2–p53 signaling pathway¹⁷. Thus, to determine whether ACY1 regulates cell response to cisplatin via the anti-oxidation and DNA damage repair pathway activation effects of GSTP1, we measured the levels of intracellular ROS and related checkpoint kinases in the DNA damage repair signaling pathway, including ATM, phosphorylated ATM, CHK2, and phosphorylated CHK2. The results of flow cytometry assays revealed that ACY1 depletion decreased the intracellular levels of ROS in cisplatin-treated SKOV3 cells (Fig. 5E and 5F), whereas ACY1 overexpression increased the levels of ROS in cisplatin-treated A2780 and TOV21G cells (Fig. 5G and 5H). Moreover, the results of western blots revealed that the levels of phosphorylated ATM and CHK2 were increased upon ACY1 knockdown and decreased upon ACY1 overexpression (Fig. 5I). In sum, these findings show that ACY1 suppresses cisplatin resistance in EOC cells by blocking GSTP1-mediated regulation of intracellular ROS levels and activation of the ATM–CHK2 signaling pathway

ACY1 deacetylates GSTP1 at K30, which reduces GSTP1 stability

ACY1 deacetylates α-acetylated amino acids in the N-terminal peptide of intracellular proteins¹¹, and lysine (K) acetylation is the most common type of acetylation and can prevent ubiquitination and proteasome-dependent protein degradation²³. Thus, we realized it was important to assess whether ACY1 regulates GSTP1 acetylation. We therefore evaluated the effect of ACY1 expression on GSTP1 acetylation using a pan-acetylated K antibody. The K acetylation of ectopic HA-GSTP1 was increased in ACY1-knockdown SKOV3 cells (Fig. 6A, left panel) but decreased in ACY1-overexpressing A2780, HEK-293T, and TOV21G cells (Fig. 6A, right panel; S4A), implying that ACY1 may be responsible for GSTP1 deacetylation. To identify the specific acetylation sites of GSTP1, we isolated and purified GSTP1 from HEK-293T cells transfected with HA-GSTP1. MS analysis revealed that two K sites––K30 and K45––on the N-terminal GST domain of this purified GSTP1 were acetylated (Fig. 6B and 6C), which has not previously been reported. We therefore mutated both K30 and K45 to arginine (R) (K30R and K45R; acetylation-dead mutant), which resulted in significantly reduced the levels of total and acetylated ectopic GSTP1 protein in EOC cells (Fig. 6D). Moreover, a CHX chase assay showed that GSTP1-K30R had a shorter half-life than GSTP1-WT and GSTP1-K45R in EOC cells (Fig. 6E and 6F). Furthermore, the levels of ubiquitinated GSTP1-K30R were higher than the levels of ubiquitinated GSTP-WT and GSTP1-K45R (Fig. 6G), suggesting that K30 was the acetylation site that mediated GSTP1 ubiquitination. To confirm that ACY1 impaired GSTP1 stability via deacetylation at K30, we measured the levels of ubiquitinated ectopic HA-GSTP1 in HEK-293T cells transfected with GSTP1-acetylated site mutant plasmids and ACY1 siRNA. The results showed that ACY1 knockdown significantly reduced the levels of ubiquitinated GSTP1-WT and GSTP1-K45R but did not inhibit the K30 acetylation-mediated increase in GSTP1 ubiquitination (Fig. 6H). Moreover, CCK8 and annexin V/PI assays revealed that GSTP1-WT but not GSTP1-K30R significantly rescued the reduced growth and survival capacities of cisplatin-treated ACY1-overexpressing EOC (Fig. 6I–6L). Overall, our results demonstrate that ACY1 deacetylates GSTP1 at K30 to reduce GSTP1 stability in EOC cells, which leads to GSTP1 degradation via the ubiquitin–proteasome pathway.

HDAC4-mediated suppression of H3K27Ac inhibits ACY1 transcription in cisplatin-treated EOC cells

Owing to the lack of agonists that can bind ACY1, we explored the upstream mechanism underlying ACY1 expression in EOC cells to extend our study findings to a clinical setting. As histone acetylation plays an important role in chemoresistance mechanisms in EOC²⁴, we first evaluated modifications of the ACY1 promoter recorded on the UCSC Genome Browser (http://genome.ucsc.edu/). As shown in Fig. 7A, there was substantial H3K27Ac in the ACY1 promoter region, implying that ACY1 expression is regulated by chromatin acetylation. HDACs eliminate acetyl groups from histones, resulting in reduced gene expression. According to the LinkedOmics portal (http://linkedomics.org/), among all HDACs, only the mRNA levels of HDAC4 are negatively correlated with ACY1 mRNA levels in OC (R = − 0.229, P = 0.0390) (Fig. 7B and S5A). In addition, Stronach et al. reported that HDAC4 is significantly upregulated in clinical samples following acquired platinum resistant relapse and promotes platinum resistance in OC cells²⁵. Therefore, we speculated that HDAC4 regulates the expression of ACY1 via an epigenetic modification in EOC upon treatment of platinum-based chemotherapy.

Next, we measured the levels of ACY1 and HDAC4 in cisplatin-treated SKOV3, A2780 and TOV21G cells at various time points. This revealed that levels of ACY1 reduced gradually, whereas those of HDAC4 increased gradually upon cisplatin treatment at increasing time (Fig. 7C), implying that cisplatin induced HDAC4 expression but inhibited ACY1 expression. To confirm whether HDAC4 directly regulated ACY1 expression, we examined the levels of ACY1 mRNA and protein in EOC cell lines using two specific siRNAs targeting HDAC4. We found that HDAC4 knockdown significantly increased the levels of ACY1 mRNA and protein (Fig. 7D, 7E, and S5B). Moreover, a chromatin immunoprecipitation (ChIP) assay indicated that the ACY1 promoter region was enriched in H3K27Ac signals, and that this enrichment was increased by HDAC4 knockdown (Fig. 7F). Next, we performed CCK8 and annexin V/PI assays, which demonstrated that HDAC4 knockdown significantly attenuated the promotive effect of ACY1 silencing on the growth and survival capacities of cisplatin-treated EOC cells (Fig. 7G–7J). These data suggest that HDAC4-mediated suppression of H3K27Ac on the ACY1 promoter at least partly accounts for cisplatin treatment of EOC cells triggering the repression of ACY1 transcription, which leads to these cells exhibiting cisplatin resistance.

EOC is the most lethal gynecologic cancer, mainly because it is usually diagnosed at advanced stages and become chemoresistance following a frequent relapse–response pattern³. Platinum, particularly cisplatin and carboplatin, are the mainstay drug treatments for EOC^2,21,26. However, resistance to platinum occurs in most patients, and must be overcome to improve the survival rates of patients with EOC. Elucidating the molecular mechanisms underlying resistance to platinum is therefore crucial. In this study, we observed that low expression of ACY1 is significantly associated with resistance to platinum-based chemotherapeutics and poor prognosis in patients with EOC, implying that ACY1 should be targeted for overcoming this resistance.

ACY1 was recently shown to play complex roles in carcinogenesis. First, ACY1 was found to suppress the proliferation and induce the apoptosis of neuroblastoma cells by inhibiting the ERK/TGF-β pathway²⁷. By contrast, ACY1 was reported to promote the progression of non-small-cell lung cancer by activating the PTEN–PI3K–AKT signaling pathway²⁸. Extensive research is thus warranted to understand the functions of ACY1 in cancer. In this regard, our study demonstrates that ACY1 is an independent predictor of favorable EOC prognosis and that low ACY1 expression correlates with the resistance of EOC cells to platinum-based chemotherapeutic and thus tumor recurrence. Moreover, we found that ACY1 sensitizes EOC cells to cisplatin both in vitro and in vivo, indicating that ACY1 could be explored as a target to reverse cisplatin resistance.

There is currently no serum biomarker for detecting the response of EOC cells to platinum in the clinical setting. Nevertheless, ACY1 is known to be present in a stable form in the serum and is a potential biomarker of delayed graft function following kidney transplantation, and of hepatitis B virus-related liver cirrhosis, Parkinson’s disease, CRC, and small-cell lung cancer^6,11,29−31. Therefore, it is necessary to explore whether serum levels of ACY1 can help predict platinum resistance in patients with EOC, which is beneficial for EOC precision therapy.

Mechanistically, we found that ACY1 downregulated GSTP1 levels by reducing the stability of GSTP1 protein. As mentioned, there are multiple upstream regulators of GSTP1 expression. For example, hypermethylation of the GSTP1 promoter region silences GSTP1 transcription, inhibiting the progression of several types of cancers³². In contrast, GSTP1 expression is upregulated by miR-130b, which leads to chemoresistance of OC³³. Hasan et al. reported that the Nrf–antioxidant response element signaling pathway enhances the transcriptional activity of GSTP1³⁴. However, the mechanism of post-translational modification-mediated regulation of GSTP1 levels has thus far been unclear. Our results suggest there is a novel upstream mechanism responsible for the regulation of GSTP1 levels. Furthermore, we found that ACY1 reduces GSTP1 stability by deacetylating it at K30, thereby promoting its proteasome-independent degradation. ACY1 mainly catalyzes the hydrolysis of N-acylated amino acids to an acyl species and l-amino acids, and is involved in xenobiotic detoxification and bioactivation⁸. In addition, ACY1 interacts with sphingosine kinase type 1 and promotes its translocation from the cytosol to membranes, thereby potentiating its mitogenic and cytoprotective effects³⁵. In the current study, we found a novel function of ACY1 that it affects the K-30 acetylation of GSTP1 to regulate GSTP1 stability.

Protein acetylation is a type of post-translational modification that plays an essential role in regulating protein function. Non-histone acetylation regulates proteins’ stability, enzymatic activity, subcellular localization, and interactions with other macromolecules, which helps proteins participate in key cellular processes relevant to physiology and disease²³. For example, the acetylation of p53 at K370, K372, and K381 by p300 and CBP supports the open conformation of p53, which enhances its transcriptional activity³⁶. Similarly, p300/CBP-associated factor acetylates p38 at K152 and K53 to promote p38 phosphorylation, thereby sensitizing cells to apoptosis³⁷. Bhardwaj et al. showed that sirtuin 6-mediated pyruvate kinase M2 deacetylation at K433 results in its nuclear export, which suppresses its oncogenic functions³⁸. Cisplatin induces acetylation 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) at K472, which leads to PFKFB3 accumulation in the cytoplasm, thereby enhancing glycolysis and protecting cells from cisplatin-induced apoptosis³⁹. K acetylation is the most common form of protein acetylation and can affect protein stability by regulating both proteasome-dependent and -independent protein degradation.²³ The former comprises two mechanisms that affect acetylation-dependent protein stability: (1) sharing of the same K residues between the acetylated and ubiquitinylated modification sites; and (2) inhibition of the interactions of the E3 ubiquitin-protein ligase. By contrast, the mechanism via which ACY1 deacetylates GSTP1 to promote proteasome-independent GSTP1 degradation merits further study.

Finally, we observed that HDAC4, which was upregulated in the presence of cisplatin, inhibits the transcriptional activity of ACY1 by suppressing H3K27Ac, high levels of which occur on the ACY1 promoter in EOC cells. Similarly, Stronach et al. reported that compared with OC cells not treated with platinum-based chemotherapeutics, HDAC4 is upregulated in OC cells with acquired resistance to platinum-based chemotherapeutics, and that HDAC4 induces this resistance by inhibiting the activation of STAT1²⁵. Therefore, we proposed that the HDAC4–ACY1–GSTP1 axis may be a novel mechanism underlying acquired resistance to platinum-based chemotherapeutics in EOC. Based on all our above-mentioned findings, and given that a series of HDAC inhibitors are already approved for the treatment of malignancies⁴⁰, co-treatment of EOC with an HDAC4-selective inhibitor and a platinum-based chemotherapeutic may be effective. Further clinical research to explore this avenue is warranted.

In conclusion, our study revealed that ACY1 helps to reverse the resistance of EOC to platinum-based chemotherapeutics (Fig. 8). Specifically, ACY1 deacetylates GSTP1 at K30 and promotes FBX8-mediated ubiquitin–proteasome-dependent GSTP1 degradation, thereby reducing the capacity of EOC cells to eliminate intracellular ROS and activate DNA-damage repair. Moreover, HDAC4 decreases the transcriptional activity of ACY1 by suppressing H3K27Ac of the ACY1 promoter and blocking HDAC4 reverses cisplatin resistance in EOC cells. Finally, we determined the existence of an HDAC4–ACY1–GSTP1 axis as a novel pathway for EOC resistance to platinum-based chemotherapeutics, indicating that ACY1 is a potential biomarker of the response of EOC to such treatment. It also shows that combination therapy with a platinum-based chemotherapeutic and an HDAC4 inhibitor maybe a possible means to improve the prognosis of women with EOC.

Patient samples

A total of 120 archived formalin-fixed, paraffin-embedded EOC samples were obtained between February 2002 and December 2014 at the Sun Yat-sen University Cancer Center. Of the 120 samples, 113 belonged to patients who first underwent primary surgery and then completed platinum-based chemotherapy. Furthermore, 19 fresh EOC tissue samples and 19 paired normal ovarian epithelial tissue samples were collected and stored at − 80°C RNA later. RNA was isolated to detect the ACY1 mRNA levels. This study was approved by the Institutional Review Board of Sun Yat-sen University Cancer Center.

IHC

The paraffin-embedded EOC samples were stained via IHC following a standard protocol involving deparaffinization, rehydration, antigen retrieval (using a citrate buffer for ACY1 and an ethylenediaminetetraacetic acid buffer; pH 9.0 for GSTP1), antibody incubation, and diaminobenzidine staining. The primary antibodies used were anti-ACY1 (1:2000; HPA036174, Sigma, Germany) and anti-GSTP1 (1:800; 15902-1-AP, Proteintech, USA). The results of IHC staining were independently assessed by two experienced pathologists blinded to the patients’ clinical data. An H-score assessment was performed to obtain the staining score of each sample, as follows: staining intensity (negative: 0, wild: 1, moderate: 2, and strong: 3) × the staining extent score (0–100%). The median value was considered as the cutoff for ACY1 and GSTP1 expression to classify the low-and high-expression groups.

Cell culture and treatment

The human EOC cell lines A2780, SKOV3, and TOV21G were purchased from Shanghai Bioresource Collection Center (Shanghai, China), whereas HEK-293T was purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) in a humidified 5% CO₂ and incubator at 37°C.

A concentrated stock (5 mg/ml) of cisplatin (Hansoh Pharma, China) was dissolved in 0.9% sterile saline solution to give a working solution, which was then added to the cell growth media. The eukaryotic protein synthesis inhibitor cycloheximide (20 µg/ml; MCE, USA) and the proteasome inhibitor MG132 (10 µM; MCE, USA) were used to assess protein stability. The cells were harvested for subsequent experiments at the end of each time point.

Plasmid and RNA interference assays

pEZ-Lv242 was used as the vector of Flag-tagged ACY1 plasmid. His-tagged ubiquitin, HA-tagged GSTP1, and its mutant constructs were cloned into a pcDNA3.1 vector. ACY1-targeting shRNA oligonucleotides were cloned into a pLKO.1 vector. All plasmids were sequenced to confirm that the correct nucleotide sequence was inserted at the desired base position within the vector backbone. Flag-ACY1 or target-gene shRNA was co-transfected into HEK-293T cells with pLP1, pLP2, and pLP/VSVG to produce a virus. The target cells were infected with viral supernatants and selected with puromycin (2 µg/ml) to establish stable cell lines. siRNAs targeting GSTP1, FBX8, and HDAC4 were synthesized by GenePharma (Suzhou, China). The cells were transiently transfected with siRNA using Lipofectamine 3000 (Invitrogen, USA). The target sequences for shRNA and siRNA are shown in Table S3.

Protein extraction and western blot

After washing twice with phosphate-buffered saline (PBS), the cells were lysed using RNA immunoprecipitation buffer containing cocktail of protease and phosphatase inhibitors (Beyotime Biotechnology, China). A one-fourth volume of 5× sodium dodecyl sulfate (SDS) loading buffer was added to the supernatants from the cell lysis step, which were obtained by centrifugation of cell extracts at 16,000 ×g at 4°C for 10 min and then boiling of the supernatants at 95°C for 8 min. Protein samples were separated using SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin in PBS with 0.1% Tween-20 for 2 h at room temperature and then incubated successively with the corresponding primary antibodies at 4°C overnight and horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. A chemiluminescent reagent (Merck Millipore, Germany) was used to detect the signals. The primary antibodies used in this study are listed in Table S4.

RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was isolated from the patients’ samples and cell lines using TRIzol reagent (Invitrogen, USA), according to manufacturer’s instructions. cDNA was reverse transcribed using the GoScript™ Reverse Transcription System (Promega, USA). GoTaq qPCR Master Mix (Promega, USA) was used for real-time PCR, which was conducted using the LightCycler 480 Instrument (Roche, Switzerland). The primer sequences used are listed in Table S5.

Cell viability assay

EOC cells (8000 SKOV3 cells/well and 5000 A2780 and TOV21G cells/well) were seeded in 96-well plates and treated with various concentrations of cisplatin for 48 h. The CCK8 reagent (Bimake, USA) was added to the wells, according to the manufacturer’s instructions, and the plate was incubated for 2 h at 37°C. The absorbance of the contents of each well was measured using MD VersaMax (Molecular Devices, USA) at 450 nm.

Flow cytometry

Flow cytometry was performed for the cell apoptosis annexin V/PI assay and measurement of intracellular ROS levels. For the annexin V/PI assay, the cells were harvested, washed twice with 1× cold binding buffer and then stained with annexin V and PI (BestBio, China) at room temperature. For intracellular ROS level measurement, the cells were incubated with DCFH-DA (1:1000; Beyotime, China) in DMEM at 37°C for 30 min, washed with PBS, trypsinized, and resuspended in 200 µl PBS. All of the samples were assessed using CytoFLEX (Beckman Coulter, USA), and all data were analyzed using FlowJo version 10 software.

Colony-forming assay

The cells were seeded in 6-well plates (400 cells/well). Cisplatin was added to the cells after 12 h, and the plate was incubated for 24 h. After 14 days, the cells were fixed with methyl alcohol and stained with crystal violet. The colonies were then counted.

Co-IP and MS analyses

The cells were washed twice with PBS and lysed using NP-40 buffer (Beyotime, China). The cell lysates were then incubated with Anti-Flag Magnetic Beads or Anti-HA Magnetic Beads (MCE, USA) or a specific antibody and Protein A/G Magnetic Beads (MCE, USA) overnight at 4°C. The magnetic beads were then washed six times with NP-40 buffer, resuspended in 1× loading buffer, and then boiled to denature proteins. Finally, the samples were subjected to western blot analysis. For the MS analysis, the samples were first subjected to SDS-PAGE, and the gel was stained with Coomassie brilliant blue dye (Beyotime, China) for 3 h at room temperature and then eluted using de-staining solution for 20 h. The gel containing the separated bands was then cut and subjected to liquid chromatography–mass spectrometry analysis.

GST pull-down assay

GST–ACY1 fusion proteins, His-GSTP1, and GST proteins were expressed in Escherichia coli BL21 and purified using Glutathione Sepharose™ 4B beads or Ni beads (GE Healthcare, USA), respectively. Purified His-GSTP1 protein was incubated at 4°C overnight with GST or GST–ACY1 fusion proteins bound to glutathione Sepharose beads. The mixture was then boiled with 1× loading buffer for 5 min and subjected to western blotting.

Immunofluorescence assays

The cells were cultured on round glass coverslips and then washed with PBS twice, fixed with ice-cold methanol at room temperature for 10 min, permeabilized using 0.3% Triton X-100, and finally blocked with 5% bovine serum albumin in PBS for 1 h at room temperature. Next, the coverslips were incubated with the ACY1 and GSTP1 antibodies overnight at 4°C, followed by incubation with Alexa Flour 488-conjugated anti-rabbit immunoglobulin G (IgG; 1:500; A-21206; Thermo Fisher, USA) and Alexa Flour 555-conjugated anti-mouse IgG (1:500; A-31570; Thermo Fisher, USA) antibodies and 4′,6-diamidino-2-phenylindole (1:1000; 62248; Thermo Fisher, USA). Finally, the coverslips were mounted onto glass slides using a fluorescence mounting medium, and immunofluorescence was detected using confocal microscopy (Olympus FV1000, Japan).

ChIP assay

A ChIP assay kit (Cell Signaling Technology, USA) was used to examine protein–DNA interactions, according to the manufacturer’s instructions. For each reaction, 4 million EOC cells were crosslinked using formaldehyde (final concentration, 1%) at room temperature for 10 min; glycine was then added to the cell suspension to stop the reaction. The cell nuclei were lysed with membrane extraction buffer and isolated by centrifugation at 16,000 ×g at 4°C. Chromatin was digested using micrococcal nuclease and sonicated at 4°C to generate DNA fragments with a bulk size of 150–900 bp. After centrifugation, 2% of the supernatant was kept aside as the input, and the remaining supernatant was subjected immunoprecipitation using normal rabbit IgG control or anti-H3K27Ac antibody (1:100; 8173T; Cell Signaling Technology, USA) overnight at 4°C. The protein–DNA complex was mixed and incubated with 30-µl magnetic beads at 4°C for 2 h. The purified DNA was analyzed with qPCR using primers flanking the binding motifs on the ACY1 promoter region. The sequences of the ChIP–qPCR primers used are shown in Table S6.

Tumor xenograft assay

Four-week-old BALB/c-nude mice were provided by GemPharmatech (Guangdong, China). Stable ACY1-overexpressing or ACY1-knockdown or control TOV21G (5 × 10⁶ cells) cells suspended in 100 µl DMEM were subcutaneously injected into the flank of the nude mice. When the tumor volume reached 100–150 mm³, cisplatin (4 mg/kg body weight) was injected into the abdominal cavity twice a week for 3 weeks. The tumor size was measured with calipers every 4 days and calculated using the formula “volume = length × width² × 0.5.” After treatment with cisplatin for 3 weeks, the weights of mice and tumor tissues were measured, and the tumor tissues were collected for IHC staining. All animal experiments were approved by the Animal Care and Use Committee of Sun Yat-sen University Cancer center (approval no.: L025504202112004).

Statistical analysis

Data were analyzed using Statistical Package for the Social Sciences (SPSS) version 25.0 and GraphPad Prism version 7.0. Data were presented as mean ± standard deviation (SD). The Kaplan–Meier method was used to plot the survival curves, and the log-rank test was used for analyzing the survival rates. Multivariate analysis was performed using the Cox proportional hazards model to analyze the clinical prognostic factors. The association between ACY1 expression and the clinical prognostic factors was analyzed using the chi-square test. Between-group differences were evaluated using unpaired Student’s t-test. P < 0.05 was considered statistically significant.

ACKNOWLEDGMENTS

This work was supported by grant from the Natural Science Foundation of China (grant number 81872434), the National Natural Science Foundation of China (grant number 82103647), the Guangdong Basic and Applied Basic Research Foundation (grant number 2019A1515011191), Science and technology projects of GDPH (grant number 2018bq03), NSFC Incubation Program of GDPH (grant number KY012021158), Guangdong Medical Science and Technology Research Fund (grant number A2020016, A2020537) and the Project of Guangzhou Science and Technology Plan (grant number 202201010899). We thank Prof. Limin Zheng, from School of Life Sciences at Sun Yat-sen University for providing us with experimental platform, Prof. Jianhong Fang, from School of Life Sciences at Sun Yat-sen University for the essential technical assistance and suggestions for article conception, and Prof. Haiteng Deng and chongchong zhao in Proteinomics Facility at Technology Center for Protein Sciences, Tsinghua University, for protein MS analysis.

AUTHOR CONTRIBUTIONS

M.Z., X.Z. and Z.H. conceived the project and designed the study. X.Z., P.L., Y.J. and B.P. performed the experiments. H.X. and M.J. provided patients clinical information. Y.Z., S.H. and L.Z. analyzed and interpreted the data. X.Y., R.L. and W.J. provided intellectual contribution and technical assistance. X.Z. wrote the manuscript. M.Z. and Z.H. revised the manuscript. M.Z. provided study supervision. All authors approved the final content.

COMPETING INTERESTS

The authors declare no competing interests.

DATA AVAILABILITY

The data supporting the findings of this study are available in the article or available from the corresponding author upon reasonable request.

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ACY1-mediated deacetylation of GSTP1 at lysine 30 suppresses cisplatin resistance in epithelial ovarian cancer

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Abstract

Figures

Introduction

Results

ACY1 suppresses cisplatin resistance in EOC cells

ACY1 physically interacts with GSTP1

ACY1 decreases GSTP1 stability by facilitating FBX8-mediated ubiquitination and degradation of GSTP1

ACY1 deacetylates GSTP1 at K30, which reduces GSTP1 stability

Discussion

Materials And Methods

Patient samples

IHC

Cell culture and treatment

Plasmid and RNA interference assays

Protein extraction and western blot

RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Cell viability assay

Flow cytometry

Colony-forming assay

Co-IP and MS analyses

GST pull-down assay

Immunofluorescence assays

ChIP assay

Tumor xenograft assay

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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