Abstract
Background
Protein arginine methyltransferases (PRMTs) regulate protein biological activity by modulating arginine methylation in cancer and are increasingly recognized as potential drug targets. Inhibitors targeting PRMTs are currently in the early phases of clinical trials and more candidate drugs are needed. Flavokawain A (FKA), extracted from kava plant, has been recognized as a potential chemotherapy drug in bladder cancer (BC), but its action mechanism remains unclear.
Methods
We first determined the role of a type II PRMT, PRMT5, in BC tissue samples and performed cytological experiments. We then utilized bioinformatics tools, including computational simulation, virtual screening, molecular docking, and energy analysis, to identify the potential use of PRMT5 inhibitors for BC treatment. In vitro and in vivo co-IP and mutation assays were performed to elucidate the molecular mechanism of PRMT5 inhibitor. Pharmacology experiments like bio-layer interferometry, CETSA, and pull-down assays were further used to provide direct evidence of the complex binding process.
Results
Among PRMTs, PRMT5 was identified as a therapeutic target for BC. PRMT5 expression in BC was correlated with poor prognosis and manipulating its expression could affect cancer cell growth. Through screening and extensive experimental validation, we recognized that a natural product, FKA, was a small new inhibitor molecule for PRMT5. We noticed that the product could inhibit the action of BC, in vitro and in vivo, by inhibiting PRMT5. We further demonstrated that FKA blocks the symmetric arginine dimethylation of histone H2A and H4 by binding to Y304 and F580 of PRMT5.
Conclusions
In summary, our research strongly suggests that PRMT5 is a potential epigenetic therapeutic target in bladder cancer, and that FKA can be used as a targeted inhibitor of PRMT5 for the treatment of bladder cancer.
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Background
Bladder cancer (BC) is the most common type of urinary cancer worldwide, with high recurrence and mortality [1]. Although numerous novel treatment regimens have been applied in the recent decade, no radical improvement in prognosis has been achieved in clinical practice. To date, local or systemic chemotherapy using widely accepted drugs is still the best therapeutic intervention to assist in surgery for BC treatment [2]. Although immune checkpoint inhibitors (ICIs) have emerged as an effective alternative for managing advanced disease and have shown durable responses in a subset of patients with BC [3], unfortunately, the overall response rate is only approximately 15–25% [4]. To improve the outcome of patients with BC, we need to urgently explore more therapeutic targets and identify appropriate drugs for treating BC [5].
Unbalanced methylation is gradually being accepted as a potential driver in human cancers [6]. Although lysine methyltransferases are widely known to regulate gene expression coupled with BC development, arginine methyltransferases and their roles in BC remain obscure [7]. Protein arginine methyltransferases (PRMTs) are enzymes that transfer a methyl group from S-adenosyl-L-methionine (SAM) to the substrate arginine side chain and can be categorized into different subtypes based on their catalytic routes [8]. Arginine methylation disorder has been reported to be prevalent in breast, lung, and colon cancers and leukemia [9]. Most PRMTs have been implicated in the regulation of cancer-associated epigenetics and chromatin transcription signaling, RNA metabolism, and DNA repair [10]. Recent reports have shown that some PRMTs play indispensable roles in regulating BC and are closely related to the proliferation, invasion, and poor prognosis of the disease [11]. However, no systematic analysis on PRMTs in BC has been performed to date, and the type of PRMT that specifically promotes the development of BC remains unknown.
The roles of PRMT enzymes in numerous diseases have spurred significant interest in targeting them pharmacologically [12]. Among these enzymes, including EPZ015866, EPZ015666, GSK3326595, and LY-283, the development of PRMT5 inhibitors is the most attractive approach [13,14,15,16]. Although some inhibitors have shown an inhibitory effect on PRMT5 enzyme activity and function, their toxicity and therapeutic effect are still being investigated [17].
Kava has been used for treating inflammatory bladder diseases for more than 100 years, and chalcones are the main classes of compounds identified from kava extracts, including flavokawain A, B, and C [18, 19]. These chalcones have also been found to have strong anti-tumor activity against various cancers, such as colon, lung, gastric, and breast cancers [20,21,22,23,24,25]. FKA, in particular, has a unique anti-cancer activity against urinary tract tumors [26]. Studies have demonstrated that FKA has the strongest anti-BC activity among the kava extracts discovered till date [27].
In previous studies, FKA could induce apoptosis in BC cells via the involvement of Bax protein-dependent and mitochondria-dependent apoptotic pathways [27]. In addition, FKA could induce a G2-M arrest in the bladder and prostate cancer cells [47, 48]. The combination of PRMT5 inhibitors with other regimens has also received great attention. NCT02783300 is evaluating PRMT5 inhibitors with immunotherapy like PD-1 antibody and NCT04794699 is testing the synthetic lethal effect of PRMT5 inhibitor with MTAP defect [49]. In our study, we also determined the combined effect of PRMT5 with some clinically used drugs. Inhibiting PRMT5 increased the chemotherapeutic effect of cisplatin and gemcitabine, and co-treatment of PRMT5 and EGFR inhibitor significantly inhibited cancer cell growth. The encouraging results of this work indicated that PRMT5 was a suitable target for develo** combined therapies with other drugs [50]. Further exploring its synergetic effect with PARP inhibitors, immunotherapeutic antibodies, or chemotherapeutic drugs is worthy for further research.
Conclusions
In conclusion, we systematically analyzed the role of the PRMT family in BC and confirmed that PRMT5 was highly correlated with the malignant properties of BC and could be an ideal epigenetic therapeutic target. As the first natural small molecule inhibitor of PRMT5, FKA had a strong inhibitory effect on BC and warrants further development to translate it into clinical applications.
Availability of data and materials
The data in the current study are available from the corresponding author upon request.
Abbreviations
- BC:
-
Bladder cancer
- CETSA:
-
Cellular thermal shift assay
- FKA:
-
flavokawain A
- GC:
-
Gemcitabine and cisplatin
- PRMT:
-
protein arginine methyltransferase
- SAM:
-
S-adenosyl methionine
- TGCA:
-
Cancer genome atlas program
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Acknowledgements
We would like to thank Editage for English language editing.
Funding
This work was supported by the National Nature Science Fund in China (Grants No. 81672525).
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SL: Conceptualization, writing, in vitro experiments, visualization. ZL: Bioinformatics analysis. CP: In vivo experiments. ZZ: Data curation, validation. CK: Funding acquisition. LY: Supervision. XL: Conceptualization, methodology, investigation, editing. All authors have read and approved the final version of the manuscript.
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The study design was approved (Approval No. [2022]102) by the Institutional Ethics Committees at the First Affiliate Hospital of China Medical University. All participants in the study provided informed consent before specimens were collected. All experiments on animals were conducted in accordance with the institutional guidelines approved (IACUC Issue No. KT2022608) by the Animal Care and Use Committee of China Medical University.
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Supplementary Information
Additional file 1: Supplementary Methods. Supplementary Table.
Correlation between expression of PRMT5 clinicopathological parameters in 60 cases of bladder cancer patients. Fig. S1. (a) PRMT5 expression correlated with BLCA gene signatures in the TCGA dataset. (b) PRMT5 expression correlated with BLCA marker genes in the TCGA dataset. (c) Kaplan–Meier analysis of survival prognosis based on PRMT5 expression in Imvigor datasets. (d) IHC analysis of PRMT5 in different stage BC. (e) Western blot analysis of PRMT5 expression and histone methylation level in BC tissues and adjacent normal bladder tissues. Statistical analysis of protein expression was shown on the right side. Fig. S2. (a) Cell viability assay performed by treating FKA in urothelial cell SV-HUC-1. (b) Cell viability assay performed after treatment with FKB, and FKB IC50 were calculated in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in three replications. (c) Cell viability assay performed after treatment with FKC, and FKC IC50 were calculated in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in three replications. (d) PRMT5 expression changed with different concentrations of FKB treatment times in T24 (upper) and UMUC3 (lower). (e) PRMT5 expression changed with different concentrations of FKC treatment times in T24 (upper) and UMUC3 (lower). (f) Different PRMT expression changed with different concentrations of FKA in T24 (left) and UMUC3 (right). Fig. S3. (a) Cell apoptosis measured by knocking down PRMT5 expression, FKA treatment, and supplied FKA in PRMT5 overexpressed UMUC3 using flow cytometry. (b) Apoptosis rates for replicated assays were counted. (c) Cell viability assay performed after treatment with FKA or PRMT5 shRNA combined with cetuximab in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in five replications. (d) Functional pathway enrichment of predicted FKA downstream targets. (e) Bladder cancer regulon genes changes after supplying FKA in UMUC3 measured by PCR, and the fold changes were standardized and normalized by log10. Fig. S4. (a) Bio-Layer Interferometry detecting the combination and dissociation constants of FKA and human recombinant PRMT5. (b) Fluorescent report CETSA assay confirmed that 25 μM FKA treatment could inhibit PRMT5 degradation when heated, thus enhancing PRMT5 stability in UMUC3. (c) Fluorescent report CETSA assay performed for PRMT5 expression when treated with FKA in wild-type and Y304A/ F580A mutated PRMT5 in UMUC3 cells. (d) Conservation of PRMT5 Y304 and F580 in different species.
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Liu, S., Liu, Z., Piao, C. et al. Flavokawain A is a natural inhibitor of PRMT5 in bladder cancer. J Exp Clin Cancer Res 41, 293 (2022). https://doi.org/10.1186/s13046-022-02500-4
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DOI: https://doi.org/10.1186/s13046-022-02500-4