Ketone drink enhances therapeutic efficacy in prostate cancer by targeting EZH2

Abstract

It is well established that EZH2, a lysine methyltransferase, is upregulated in most aggressive cancers, highlighting the importance of EZH2 in cancer progression. Recent research has shown that metabolic reprogramming is pivotal in various biological processes, including cancer. Despite this, evidence of EZH2's role in regulating cancer metabolism remains limited. Our study reveals a negative correlation between EZH2 and HMGCS2, a gene belonging to the HMG-CoA synthase, in prostate and breast cancers. Interestingly, HMGCS2 is inversely related to cancer progression and prognosis in these cancers. Furthermore, HMGCS2 is epigenetically repressed by EZH2 both in vitro and in vivo. Notably, restored EZH2 reduces the elevated HMGCS2 levels observed upon EZH2 depletion. Overexpression of HMGCS2 decreases tumorigenesis in both prostate and breast cancers. Additionally, β-hydroxybutyrate (BHB), a downstream metabolite of HMGCS2, impedes prostate cancer progression by targeting EZH2 via direct protein-compound interaction-mediated protein degradation. More importantly, the ketone drink of BHB administration dramatically reduces tumor size and weight in a therapy-resistant, castration-resistant prostate cancer patient-derived xenograft model. Combining a ketone drink with FDA-approved drugs enzalutamide and Tazemetostat further suppresses tumor progression. Overall, the EZH2-HMGCS2-BHB regulatory network plays a critical role in the progression of prostate cancer, and a ketone drink is a novel therapeutic tool for patients with aggressive prostate cancer.

Fig. 1. HMGCS2 expression is lower in metastasis PCa and is negatively associated with EZH2 expression in PCa and BCa.

A, B HMGCS2 mRNA level in prostate tissues is shown based on sample types in normal prostate tissue (n = 52), primary (n = 500) and metastatic prostate tissues (n = 101) from TCGA-PRAD + SU2C dataset (A), and in normal prostate tissue (n = 29), primary (n = 131) and metastatic prostate tissues (n = 19) data from Taylor et al. (B). C, I HMGCS2 (C) and EZH2 (I) expression levels based on different groups of PCa by scRNA-seq. D, E Patients with PCa from Taylor et al. (total samples = 186) (D), TCGA-PRAD (n(high)=123, n(low)=123) (E) dataset were split into two groups based on mean expression of HMGCS2 and the resultant differences in disease free survival are shown by log-rank test. F Patients with BCa were split into two groups based on mean expression of HMGCS2 and the resultant differences in disease-free survival are shown by log-rank test (n(high) = 1014, n(low) = 1014). G, H HMGCS2 mRNA level in breast tissues is shown based on sample types (G) and individual cancer states (H) from TCGA data. J, K Inverse relationship between HMGCS2 and EZH2 in prostate adenocarcinoma (J) and BCa (K) using TCGA and METABRIC dataset. L Immunoblot of six different PCa cell lines for EZH2, HMGCS2, GAPDH, and H3 protein level. M Quantification of EZH2 and HMGCS2 protein levels in PCa cell lines. Protein levels were normalized to control values, with EZH2 in PC-3 cells set to 1.0 and HMGCS2 in LNCaP cells set to 1.0. Mean ± SE (n = 3). N Correlation of EZH2 and HMGCS2 Levels from PCa cell lines in Fig. 1M. PCa prostate cancer, BCa breast cancer.

Fig. 2. Depletion, inhibition, and degradation of EZH2 increased HMGCS2 level in PCa in vitro and in vivo.

A, C qPCR analysis showed HMGCS2 was increased in EZH2-depleted and inhibited C4-2 cells. B, D, E, F Immunoblot analysis confirmed increased-HMGCS2 protein level with depletion, inhibition, and degradation of EZH2. G ChIP-qPCR assay to monitor the enrichment of H3K27me3 at 2 kb upstream of the promoter regions of HMGCS2 in C4-2 cells with vehicle or 5uM of GSK126 treatment. F1(transcription start site (TSS)~−0.5 kb), F2 (−0.5~−1.0 kb), F3 (−1.0~−1.5 kb), F4 (−1.5~−2.0 kb). H, I Precastrated mice carrying LuCaP 35CR, an enzalutamide-resistant and abiraterone-resistant patient-derived xenograft model, received EPZ6438 (200 mg kg⁻¹ per day) for 28 days (five days per week). Tumor tissues were lysed and blotted for HMGCS2, EZH2, H3K27me3, H3, and β-actin. HMGCS2, EZH2, and H3K27me3 levels were normalized to the loading controls β-actin and H3, and the averages of the values normalized to β-actin and H3 were used. J, K C4-2 bearing NCG mice received MS8815 (25, 50, 100 mg kg⁻¹ per day) for 28 days (five days per week), and tumor tissues were used for immunoblotting analysis. Tumor tissues were lysed and blotted for HMGCS2, EZH2, H3K27me3, H3, and β-actin. HMGCS2, EZH2, and H3K27me3 levels were normalized to the loading controls β-actin and H3, and the averages of the values normalized to β-actin and H3 were used. MS: MS8815, Mean ± SE. Student t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3. Re-expression of EZH2 represses increased-HMGCS2 upon EZH2 depletion.

A, B qPCR analysis showed EZH2 and HMGCS2 mRNA level after depletion of EZH2 and reexpression of EZH2 in C4-2 cells. C, D Doxycycline-inducible EZH2-depletion cells were generated, treated with doxycycline for 4 days (1 ug/ml), and changed into fresh media. After 4 days of maintaining with fresh media (total 8 days from the start day of doxycycline treatment) and 26 days of maintaining of fresh media (total 30 days from the start day of doxycycline treatment), cell lysates were used for immunoblot analysis. The figure shows protein expression of EZH2 and HMGCS2 from immunoblot analysis with three different biological replicates. Mean ± SE. Student t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4. HMGCS2 overexpression decreases PCa progression.

A Immunoblot analysis confirmed HMGCS2 overexpression in C4-2 and PC-3 cell lines. B HMGCS2 overexpression decreased cell viability in C4-2 and PC-3 cell lines by cell titer glo. C Wound healing assay was conducted to evaluate the migration potential of HMGCS2 overexpression in C4-2 and PC-3 cell lines. D Boyden chamber migration assay was performed in C4-2 and PC-3 cell lines. Mean ± SE. Student t-test. *p < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001.

Fig. 5. BHB bound to EZH2 protein, and induced EZH2 degradation in a proteasome-dependent manner.

A After three days of transfection of siControl and siEZH2 in C4-2 cells, targeted metabolomics was conducted to measure BHB levels by HPLC-MS/MS. B Wound healing assay was performed after three days of treatment of BHB in C4-2. BHB was dissolved in RPMI medium, vehicle is RPMI medium. C Transwell migration assay was conducted with vehicle or BHB treatment for 3 days in C4-2. D EZH2 protein level was decreased with BHB treatment by western blot in C4-2, PC-3, and 22Rv1 cell lines. Vehicle (RPMI medium). E GSEA analysis of RNA-seq data in C4-2 cells after BHB treatment (left) and silencing EZH2 (right) revealed that genes of E2F targets, Myc targets, DNA repair, and G2M Checkpoint are enriched with both BHB treatment and EZH2 depletion. F Venn diagram with the shared signatures of BHB treatment and siEZH2. Number of positive enrichment (left) and negative enrichment (right) with BHB treatment and siEZH2. G C4-2 cells were treated with vehicle or BHB for three days. Cycloheximide was added to cells for the time indicated. The half-life of EZH2 was calculated. H MG132, a proteasome inhibitor, was used for 24 h. The cells were lysed and subjected to co-immunoprecipitation with anti-EZH2 antibody, followed by immunoblotting with anti-ubiquitin antibody. EZH2 levels were normalized to the loading controls β-actin. Ubiquitination levels were normalized to vehicle control. I Representative western blot obtained from pull-down assays using vehicle (RPMI medium) -conjugated or BHB-conjugated Sepharose beads. Binding proteins were eluted with BHB-containing buffer (0.1M Tris-HCl, BHB 0.5 mM). J GST and GST-EZH2 proteins were incubated with BHB-conjugated Sepharose beads for pull-down assays. Binding proteins were eluted with BHB-containing buffer (0.1 M Tris-HCl, BHB 0.5 mM). Student t-test. Mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. Ketone drink decreased the progression of PCa in vivo.

A–F Precastrated mice carrying LuCaP 35CR received vehicle or ketone drink (100 μl x twice per day) for 42 days (weekly schedule of five days on, two days off) (n = 5, each group). A The image of tumors was taken after sacrifice. B Tumor weight was measured after sacrifice. C Caliper measurements were taken twice a week to determine tumor volume. *P < 0.05, vehicle versus no dilution ketone drink. D Ketone drink does not change body wight in LuCap 35CR xenograft. E Tumor tissues were lysed and blotted for HMGCS2, EZH2, AR, H3 and β-actin. F HMGCS2, EZH2, and AR levels were normalized to the loading controls β-actin. Student t-test. Mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7. Ketone drink increased therapeutic efficacy of EZH2 inhibitor and enzaluatamide in PCa xenograft model.

A, D Combination index plot of BHB and EPZ-6438 (A), BHB and Enzalutamide (D) in C4-2 cells. B, C Immunoblot analysis showed decreased-AR protein level with BHB treatment in C4-2 (B) and 22Rv1 (C). E-J Precastrated mice injected C4-2 cells received vehicle, enzalutamide(10 mg/kg/day), ketone drink (100 μl x twice per day), EPZ6438 (200 mg/kg/day), and combination by gavage for 4 weeks (five days per week) (n = 6/group). E Serum was collected when the mice were sacrificed, and the serum level of BHB was measured. F Caliper measurements were taken every week to determine tumor volume. G Tumor weight was measured after sacrifice. H The image of the tumors was taken after sacrifice. I Representative images of IHC staining for Ki-67. J Quantification of IHC images by ImageJ. Mean ± SE. Student t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 8. The proposed model of this study.

EZH2 represses HMGCS2, a rate-limiting enzyme of ketogenesis, epigenetically. Downstream metabolite, BHB, binds to EZH2 and induces degradation of EZH2 as a negative feedback loop, leading to decrease PCa progression. Created with BioRender.com.