VprBP drives prostate tumorigenesis by its kinase activity targeting histone H2A

Abstract

VprBP has been recently identified as an oncogenic kinase and a promising drug target in human malignant tumors. Although VprBP can phosphorylate histone H2A and some non-histone proteins, it seems to selectively target specific substrates in a cancer type-dependent manner by an unknown mechanism. Here we report that VprBP is highly expressed in prostate cancer cells and inactivates a group of genes encoding critical regulators of cell growth and proliferation in a manner dependent on its kinase activity toward H2AT120. As an extension of our previous finding of VprBP inhibitor B32B3, we also screened a series of small molecule compounds derived from B32B3 and identified B0045 as a second-generation VprBP inhibitor with much higher efficacy and potency. B0045 is far more effective in blocking VprBP-mediated H2AT120p and reactivating growth regulatory genes, resulting in a significantly lower proliferative capacity of prostate cancer cells. Similarly, B0045 treatment inhibits VprBP kinase activity, modulates H2AT120p-induced gene inactivation, and impairs prostate tumor growth in xenograft mouse models. Together, our findings establish a critical role for VprBP-mediated H2AT120p in oncogenic gene silencing and B0045 as a promising therapeutic strategy for prostate cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02956-3.

Introduction

Prostate cancer is the second most commonly diagnosed cancer and one of the leading causes of cancer death among men worldwide. Prostate cancer can be diagnosed as being confined only within the prostate gland or being in advanced stages spreading beyond the prostate, with several treatment options including surgery, radiation therapy, hormonal therapy, chemotherapy, immunotherapy, and combinations of these therapies [1–3]. Molecular features of prostate cancer have been the subject of active research for many years, and it is widely believed to arise from mutations and genetic alterations in genes encoding important regulators of cell growth and proliferation [4–7]. However, accumulating evidence indicates that epigenetic modifications are also involved in the development and progression of prostate cancer through perturbation of normal gene expression and cell growth states [8–11]. Adding support to this notion, it has been shown that aberrant DNA methylation contributes to the process of prostate carcinogenesis by inducing unintended inactivation of tumor suppressor genes [12, 13]. In studies focusing on histone modifications, EZH2, the catalytic subunit of PRC2 complex, was found to mediate H3K27me3, inactivate tumor suppressor genes, and drive prostate tumorigenesis [14, 15]. Similarly, p300 and CBP are often deregulated, and have oncogenic activities by disrupting normal histone acetylation status at genes associated with increased risk for prostate cancer [16, 17]. Related analyses also showed that HDACs are overexpressed in prostate cancer cells and induce an inactive state of specific genomic regions to enhance the cancer progression [18]. Despite these advances, however, it still remains elusive whether other epigenetic changes also drive transcriptional dysregulation and increase the risk of developing prostate cancer.

Of special relevance to the current report, the HIV-1 Vpr Binding Protein (VprBP), also known as DDB1 and CUL4 Associated Factor 1 (DCAF1), is a large protein that is highly expressed in cancer cells and whose function has been explored with respect to its role in regulating cell cycle, cell growth, and cell division [19–22]. Since VprBP was originally identified as a substrate recognition component of the DNA damage-binding protein 1 (DDB1)-Cullin 4 (Cul4)-RING finger protein (ROC1) E3 ubiquitin ligase complex, it has been believed that such regulatory function of VprBP is achieved by directing polyubiquitination and proteasomal degradation of specific protein targets. However, our systematic characterization of VprBP led to the unexpected observation that VprBP can bind nucleosomes and inhibit gene transcription in a manner dependent on its Lis homology motif [23]. More surprisingly, a further investigation of VprBP-induced transrepression process demonstrated that VprBP possesses an intrinsic kinase activity and phosphorylates histone H2A on threonine 120 (T120) for its action as a transcriptional modulator [24–26]. Our genome-wide analyses indicated a crucial role for H2AT120 phosphorylation (H2AT120p) in VprBP-induced silencing of growth regulatory genes in prostate cancer cells [24]. Additionally, VprBP has been shown to phosphorylate EZH2 at T367 (EZH2T367p) in colon cancer cells and p53 at S367(p53S367p) in DNA damage-response cascade [27, 28]. Because EZH2T367p and p53S367p are critical for VprBP function in these cases, distinct mechanisms seem to be employed by VprBP in different cell types and cellular processes. Such interpretation is further supported by our recent discovery of VprBP-mediated H2AT120p, not EZH2T367p, being a key epigenetic event that suppresses the expression of anti-osteoclastogenic genes and increases the rate of RANKL-induced osteoclast differentiation [29]. In view of these kinase activity-dependent functions of VprBP, we also have developed a small-molecule inhibitor, named B32B3, capable of specifically targeting VprBP, blocking H2AT120p/EZH2T367p, and reactivating growth regulatory genes in vitro and in vivo [24–27]. Considering that B32B3 treatment inhibits cell growth and bone resorption [24–27, 29], continuous identification and characterization of VprBP inhibitors have potential therapeutic implications for cancer and osteoporosis.

Here, in the major extension of previous studies, we first demonstrate a critical role of VprBP in driving gene silencing through H2AT120p rather than by EZH2T367p in prostate cancer cells. Consistent with these results, RNA-seq studies identified a group of genes whose expression is suppressed by VprBP in a manner that is strictly dependent on H2AT120p. We then screened a compound library derived from the first-generation VprBP inhibitor B32B3 and identified B0045 as the second-generation inhibitor with much higher potency at the lower, clinically relevant concentrations. More importantly, B0045 displays a highly potent anti-tumor activity through impairing VprBP-triggered H2AT120p and gene silencing in xenograft mouse models.

Results

VprBP promotes cancer cell growth via H2AT120p

In our earlier study, we demonstrated that VprBP is overexpressed and catalyzes H2AT120p to negatively regulate tumor suppressor genes in prostate cancer cells [24]. To understand the mode of oncogenic action of VprBP more precisely, we isolated nuclei from three prostate cancer cell lines (PC3, DU145, and LNcaP) and one normal prostate cell line (RWPE-1) and extracted chromatin fractions. Expectedly, our Western blot analysis detected VprBP expression at much higher levels in the prostate cancer cell lines PC3, DU145, and LNcaP compared with the normal prostate cell line RWPE-1 (Fig. 1A). The cancer cell lines PC3, DU145, and LNcaP exhibited high levels of H2AT120p, whereas H2AT120p was minimally detected in the normal cell line RWPE-1 (Fig. 1A). VprBP overexpression also correlated well with increased H2AT120p in human prostate tumor samples (Supplementary Fig. S1). VprBP is the kinase responsible for catalyzing H2AT120p in the prostate cancer cell lines, because specific knockdown of VprBP almost completely abrogated H2AT120p (Fig. 1B, Supplementary Fig. S2). Further supporting this idea, ectopic expression of VprBP wild-type (wt), but not VprBP kinase dead mutant (K194R), displayed almost complete rescue of H2AT120p defects caused by VprBP knockdown (Fig. 1B, Supplementary Fig. S2). As can be seen in Fig. 1C, immunofluorescence analysis also demonstrated that VprBPK194R mutant is incapable of restoring the original levels of H2AT120p in VprBP-depleted cells. Consistent with our previous reports [27], cell viability assays over a period of 3 days reproducibly showed that the prostate cancer cells grow much more slowly following the depletion of endogenous VprBP (Fig. 1D, Supplementary Figs. S3-5).

Fig. 1.

Cancer cell growth stimulated by VprBP-mediated H2AT120p. A Whole cell lysates and chromatin fractions were prepared from prostate cancer (PC3, DU145, and LNcaP) and normal prostate (RWPE-1) cells and subjected to Western blotting with indicated antibodies. Actin was used as loading control. The band intensities were quantified using Image J software (v1.53 k) after normalization to Actin. The corresponding optical densities are shown below. NS indicates no signal. Data are representative of three independent experiments. B VprBP-depleted PC3 cells were infected with lentiviruses expressing FLAG-tagged VprBP wild-type, K194R kinase-dead mutant, or S895D phospho-mimicking mutant as indicated on the top. Aliquots of cell lysates and chromatin fractions were analyzed by Western blotting, and data are representative of three independent experiments. The band intensities were quantified using Image J software (v1.53 k) after normalization to Actin. The corresponding optical densities are shown below. NS indicates no signal. C VprBP-depleted PC3 cells were complemented with lentivirus expressing VprBP wild-type, K194R mutant, or S895D mutant and immunostained with H2AT120p antibody. Scale bars correspond to 50 μm. Images are representative of three independent experiments. D Nucleosomes were reconstituted on the 601 nucleosome positioning sequence with recombinant histone octamers containing FLAG-tagged intact (1–135) or NT-cleaved (29–135) H3. In vitro binding assay was performed using those reconstituted nucleosomes with HA-tagged VprBP wild-type, K194R mutant, or S895D mutant. Nucleosome binding of VprBP was determined by Western blotting using anti-HA antibody. Data are representative of three independent experiments. E FLAG-tagged intact (1–135) or NT-cleaved (29–135) H3 was co-transfected with HA-tagged VprBP wild-type, K194R mutant, or S895D mutant in VprBP-depleted PC3 cells. After preparing nucleosomes, nucleosomes containing the ectopic H3 were selectively immunoprecipitated using anti-FLAG antibody. The binding of HA-tagged VprBP wild-type, K194R mutant, and S895D mutant was analyzed by Western blotting. Data are representative of three independent experiments. F Nucleosomes were reconstituted as described in (D) and incubated with VprBP wild-type or K194R mutant or S895D mutant in the presence of ATP for 30 min. The reactions were then resolved on 4–20% SDS-PAGE and analyzed by Western blotting with indicated antibodies. Data are representative of three independent experiments.

Related to the above observations, we recently showed that VprBP phosphorylates EZH2 at T367 (EZH2T367p) to augment its nuclear stabilization and enzymatic activity in colon cancer cells [27]. Thus, one would expect that VprBP also targets EZH2 for its oncogenic function in prostate cancer cells. However, the levels of EZH2T367p in the three cancer cell lines remained unchanged in our VprBP knockdown and rescue experiments (Fig. 1B, Supplementary Fig. S2), indicating that VprBP stimulates the growth of prostate cancer cells in an H2AT120p-dependent, but EZH2T367p-independent manner. In this regard, the role of VprBP-mediated H2AT120p in prostate cancer cells appears distinct from that of VprBP-mediated EZH2T367p in colon cancer cells, and such specialized functions of VprBP would predict that there might be additional activities and mechanisms to regulate VprBP substrate specificity and signaling networks.

VprBP Lis homology (LisH) motif serves as a high-affinity H3 N-terminal tail (H3NT) binding motif and is required for VprBP-nucleosome interaction [23]. Also, S895 phosphorylation almost completely abrogates the ability of VprBP to interact with nucleosomes [23]. Because kinase activity is often promoted by stably docking substrates to kinases, we reasoned that the physical association of VprBP with nucleosomes might also be critical for VprBP-mediated H2AT120p. To explore this possibility, we expressed VprBP mutant that harbors aspartic acid (D) substitution to mimic S895 phosphorylation in VprBP-depleted prostate cancer cells. It was apparent in our Western blotting and immunostaining that VprBPS895D mutant is defective in H3NT binding and thus unable to rescue H2AT120p in VprBP-depleted cells (Figs. 1B, C, Supplementary Fig. S2). Since VprBPS895D mutant still retains kinase activity comparable to VprBP wild-type (Supplementary Fig. S6), these results support the importance of VprBP-H3NT interaction in regulating VprBP-induced H2AT120p and cell growth.

As a more direct approach toward confirming our cellular data, we next reconstituted nucleosomes on 601 nucleosome positioning sequences and conducted in vitro binding assays. In accordance with our previous report [23, 30, 31], we detected a remarkable binding preference of VprBP for H3 intact nucleosome over H3NT deletion mutant nucleosome (Fig. 1D). On testing the nucleosome binding of VprBPK194R and VprBPS895D, VprBPK194R demonstrated a stable association with nucleosomes, but VprBPS895D failed to interact with nucleosomes (Fig. 1D). In further confirmation of this observation, ectopically expressed VprBP bound to H3 intact nucleosomes purified from FLAG-H3-transfected PC3 cells (Fig. 1E). This cellular interaction is dependent on H3NT, because H3NT deletion showed no VprBP-nucleosome interaction (Fig. 1E). Also, S895D phospho-mimicking mutation generated a complete abolishment of VprBP binding to nucleosomes in PC3 cells (Fig. 1E). When H2A phosphorylation assays were conducted with VprBPK194R and VprBPS895D, both mutants could not catalyze H2AT120p in our assays using H3 intact nucleosome substrates (Fig. 1F). In parallel immunostaining experiments, H2AT120p was minimally detected in VprBP-depleted cells expressing VprBPK194R and VprBPS895D (Fig. 1C), again indicating that VprBP kinase activity and nucleosome binding are critical for H2AT120p. We believe that these VprBP mutants represent unique tools to investigate the regulatory pathways governing H2AT120p and to unveil the contribution of VprBP kinase activity to specific epigenetic events in our future study.

VprBP-mediated H2AT120p inactivates growth regulatory genes

Since a major role for VprBP in mediating H2AT120p seems apparent in the above-described studies, we next wanted to examine the importance of H2AT120-targeted VprBP kinase activity in generating abnormal transcription state in prostate cancer cells. Toward this end, we performed RNA sequencing (RNA-seq) with total RNA isolated from control and VprBP-depleted PC3 prostate cancer cells. Our initial evaluation of RNA-seq data using principal component analysis (PCA) confirmed a clear separation among samples for each group, while close clustering of replicates from groups indicated minimal variability in the quality of analyzed replicates (Fig. 2A). Using a 1.5-fold cutoff, we identified a total of 1341 genes differentially expressed upon stable knockdown of VprBP in PC3 cells from transcriptome profiling (Figs. 2B, C, Supplementary Fig. S7). Among those genes, 717 genes were activated and 624 genes were repressed in response to VprBP knockdown (Figs. 2B, C, Supplementary Fig. S7, Table S1). In good agreement with our previous reports implicating VprBP in oncogenic gene silencing, gene ontology analysis of 717 upregulated targets also identified cell growth and proliferation as the most activated biological pathway in VprBP-depleted PC3 cells (Fig. 2D). Further supporting the role for VprBP in prostate cancer, our analysis of the leading-edge subset in the gene set detected a group of genes modulating interferon alpha response, interferon gamma response, and TNF-alpha signaling via NF-kB (Supplementary Fig. S8, Table S2).

Fig. 2.

Growth regulatory genes suppressed by VprBP-mediated H2AT120p. A Principal Component Analysis (PCA) was performed on RNA-seq datasets obtained from control and VprBP-depleted PC3 prostate cancer cells. The VprBP-knockdown group is shown in red, and the control group is represented in blue. Each group comprised three replicates. B The volcano plot illustrates the RNA-seq datasets with -log10 (P-value) displays on the Y-axis and the fold change of gene expression between VprBP knockdown and control groups on the X-axis. Genes exhibiting modulation following VprBP depletion are highlighted in red (upregulated) and blue (downregulated). Data show three biological replicates. C Venn diagram displays genes that were significantly upregulated or downregulated (> 1.5 fold; padj < 0.05) in VprBP-depleted PC3 cells compared to mock-depleted control cells. D Gene ontology analysis was conducted using the Ingenuity Pathway Analysis (IPA version 52912811, Qiagen) to examine the activated genes after VprBP knockdown. E Heatmap presents the expression levels (Z-scores) of the top 30 genes encoding positive regulators of cell growth and proliferation which are activated upon VprBP depletion. Upregulated and downregulated expressions are shown in red and blue, respectively. F RNA samples were isolated from mock-depleted control and VprBP-depleted PC3 cells and subjected to RT-qPCR analysis using primers listed in Supplementary Table S3. To check rescue effects, total RNA was also prepared from VprBP-depleted cells expressing VprBP wild-type, K194R mutant, or S958D mutant. All transcription levels were normalized to GAPDH. Data are represented as mean ± SD of three independent experiments; P values were calculated using two-way ANOVA. ***p < 0.001 versus ctrl sh; ^###p < 0.001 versus VprBP sh. G ChIP assays were performed on PC3 cells used in (F) using antibodies against VprBP, H2AT120p, and H2A antibodies as indicated. All ChIP DNAs were analyzed by real-time PCR with primer pairs amplifying the promoters and coding regions of the IRF1 (upper panel), CFB (middle panel), and TNFAIP3 (lower panel) genes. Primers used are listed in Supplementary Table S4. Error bars denote the mean ± SD obtained from triplicate real-time PCRs. All transcription levels were normalized to those of GAPDH. Data were expressed as mean ± SD (n = 3) of three independent experiments; *p < 0.05, ***p < 0.001 versus ctrl sh; ^#p < 0.05, ^###p < 0.001 versus VprBP sh.

As an experiment to validate our RNA-seq data, we conducted reverse transcription quantitative PCR (RT-qPCR) analysis of 8 representative target genes that are significantly upregulated upon VprBP knockdown and known to play critical roles in controlling cell growth in several types of cancers including prostate cancer. As summarized in Fig. 2F, our analysis clearly indicated the activation of the selected targets after VprBP knockdown in PC3 cells. The expression of VprBP wild-type, but not VprBPK194R mutant, in VprBP-depleted cells fully restored the inactive states of target genes (Fig. 2F). In view of the observed dependency of VprBP-mediated H2AT120p on H3NT, it was also reasonable to assume that VprBPS985D mutant defective in H3NT binding cannot reverse target gene activation in VprBP-depleted cells. Indeed, target genes were still expressed at very high levels after expressing VprBPS985D in VprBP-depleted PC3 cells, underscoring the significance of VprBP-H3NT interaction for VprBP-mediated H2AT120p and target gene inactivation (Fig. 2F). In additional experiments using DU145 and LNcaP prostate cancer cells, knockdown and rescue of VprBP generated similar effects on target gene transcription (Supplementary Fig. S9), again indicating a major role for VprBP-mediated H2AT120p in keeping the target genes in an inactive state in prostate cancer cells.

Although the above results established VprBP function in driving oncogenic gene silencing, we could not discriminate between direct and indirect effects of VprBP on target genes in this analysis. We therefore used chromatin immunoprecipitation (ChIP) assays to check the levels of VprBP and H2AT120p at IRF7, CFB, and TNFAIP3 genes that are highly ranked in RNA-seq data analysis and encode factors stimulating cell growth. In this study, crosslinked chromatin was isolated from control and VprBP-depleted PC3, DU145, and LNcaP cells, and the precipitated DNA was amplified by quantitative PCR (qPCR) with two primer sets specific for the promoters (P) and coding regions (C) of target genes. Whereas the precipitation levels were slightly different among the three target genes, we observed higher levels of VprBP and H2AT120p at the promoter region compared to the coding region. When ChIP-qPCR experiments were repeated in VprBP-depleted cells, almost complete loss of H2AT120p ChIP signals was detected at target genes (Fig. 2G). Expressing VprBP wild-type in VprBP-depleted cells could largely override VprBP ChIP signal defects caused by VprBP knockdown and recover H2AT120p ChIP signals at the target genes (Fig. 2G). However, similar rescue analysis after expressing VprBPK194R mutant in VprBP-depleted cells failed to show the recovery of H2AT120p ChIP signals at target genes (Fig. 2G), results indicative of the incapability of VprBPK194R mutant to generate H2AT120p. It was also apparent in our parallel ChIP assays that ectopic expression of VprBPS985D mutant failed to override H2AT120p defects caused by VprBP knockdown (Fig. 2G), again reinforcing the conclusion that VprBP-H3NT interaction is directly linked to the process of establishing H2AT120p at target genes.

B0045 is a second generation VprBP inhibitor exhibiting higher potency

Since VprBP-mediated H2AT120p induces oncogenic gene silencing in prostate cancer cells, targeting VprBP kinase activity is a promising strategy against prostate cancer. We previously reported B32B3 as a selective VprBP inhibitor that can suppress the growth of prostate cancer cells by specifically blocking H2AT120p. However, a high-molar IC₅₀ value (~ 0.5 μM) of this first-generation inhibitor has been a significant challenge in its use in a treatment condition in which inhibiting VprBP kinase activity is clinically useful [24]. To improve the potency of B32B3, we used the Chemically Advanced Template Search (CATS) distance metric for the prediction of their potential activities [32]. From those designs containing B32B3-like scaffold, 211 of new compounds were synthesized and screened for an ability to attenuate the kinase activity of recombinant VprBP. In initial assays using nucleosome substrates, eight compounds completely inhibited VprBP kinase activity toward H2AT120 at a final concentration of 5 μM, while the same concentration of other compounds failed to generate a complete abrogation of VprBP-mediated H2AT120p (Fig. 3A, Supplementary Fig. S10). Consistent with our previous report, 5 μM B32B3 was also effective in blocking VprBP kinase activity and thus H2AT120p under the same condition (Fig. 3A). To evaluate cellular effects of B32B3 derivatives, we next treated PC3, DU145, and LNcaP prostate cancer cells with those eight effective compounds over the concentration of 0–3 μM. Under these conditions and over a 24 h incubation period, B0045 most potently inhibited VprBP-mediated H2AT120p with half-maximal inhibitory concentration (IC₅₀) values ranging from 0.03 to 0.05 µM, as determined by Western blotting of chromatin fractions (Figs. 3B, C, Supplementary Fig. S11). Consistent with these results, our immunostaining of PC3, DU145, and LNcaP cells after 0.05 µM B0045 treatment also detected a significant reduction in H2AT120p—indicative of efficient blockage of VprBP kinase activity by B0045 (Figs. 3D, E, Supplementary Fig. S12). From our RT-qPCR, ChIP-qPCR, WST-1, and colony formation assays, it was also evident that changes in H2AT120p in response to B0045 treatment were paralleled by reactivation of VprBP target genes and suppression of cancer cell growth (Fig. 4, Supplementary Fig. S13). Moreover, that VprBP levels at target genes remained unchanged following B0045 treatment argues strongly that B0045 antagonizes VprBP kinase activity without affecting VprBP occupancy at target genes (Fig. 4F).

Fig. 3.

VprBP kinase activity toward H2AT120 inhibited by B0045. A Nucleosomes were reconstituted on the 601 nucleosome positioning sequence with recombinant histone octamers and subjected to in vitro kinase assays with VprBP in the presence of the indicated compounds (5 µM). The effects of the compounds were evaluated by Western blotting with H2A and H2AT120p antibodies. The band intensities were quantified using Image J software (v1.53 k) after normalization to H2A. The corresponding optical densities are shown below. NS indicates no signal. Data are representative of three independent experiments. B, C PC3 cells were grown in the presence of the indicated concentrations of compounds for 24 h. Then, chromatin fractions were prepared and analyzed by Western blotting with H2A and H2AT120p antibodies. The band intensities were quantified using Image J software (v1.53 k) after normalization to H2A. The corresponding optical densities are shown below. NS indicates no signal. Data are representative of three independent experiments. D PC3 cells were treated with DMSO, 0.05 µM B32B3, or 0.05 µM B0045 for 24 h and immunostained with H2AT120p antibody. Cell nuclei were also stained with DAPI. Images are representative of three independent experiments. Scale bar, 50 µm. E Molecular structure of B0045.

Fig. 4.

Cancer cell growth attenuated by B0045. A PC3, DU145, and LNcaP prostate cancer cells were treated with DMSO, B32B3, or B0045 at indicated concentrations, and their viability was assessed using the cell proliferation reagent WST-1. Data are represented as mean ± SD of three independent experiments. B The graph represents the half-maximal drug inhibitory concentration (IC₅₀) calculated by non-linear regression analysis using GraphPad Prism software. Data are represented as mean ± SD of three independent experiments. P values were calculated using one-way ANOVA with post-hoc Tukey’s test for multiple comparisons. ***p < 0.001 versus B32B3. C PC3, DU145, and LNcaP cells were treated with DMSO, 0.05 µM B32B3, or 0.05 µM B0045 over a period of 5 days, and their proliferation was assessed by the cell proliferation reagent WST-1. Data are represented as mean ± SD of three independent experiments. P values were calculated using one-way ANOVA with post-hoc Tukey’s test for multiple comparisons. ***p < 0.001 versus B32B3. D Colony formation assays were conducted with PC3, DU145, and LNcaP cells treated with DMSO, B32B3, or B0045 as in (C). Data represent the mean ± SD of three independent experiments in triplicate well; P values were calculated using on-way ANOVA with post-hoc Tukey’s test for multiple comparisons. ***p < 0.001 versus B32B3. E RNA samples were prepared from PC3 cells treated with DMSO, 0.05 µM B32B3, or 0.05 µM B0045 and analyzed by RT-qPCR using primers listed in Supplementary Table S3. All transcription levels were normalized to that of GAPDH. Data are expressed as mean ± SD three independent experiments. P values were calculated using one-way ANOVA with post-hot Tukey’s test for multiple comparisons. ***p < 0.001 versus B32B3. F ChIP assays were performed on PC3 cells used in (C) with antibodies against VprBP, H2AT120p, and H2A antibodies. All ChIP DNAs were analyzed by real-time PCR with primer pairs amplifying the promoters and coding regions of the IRF1 (left panel), CFB (middle panel), and TNFAIF3 (right panel) genes. IRF1, CFB, and TNFAIP3 primers used are listed in Supplementary Table S4. All transcription levels were normalized to those of GAPDH. Data were expressed as mean ± SD (n = 3); P values were calculated using one-way ANOVA with post-hoc Tukey’s test for multiple comparisons. *p < 0.05, ***p < 0.001 versus B32B3.

As an extension of the above-described studies using prostate cancer cell lines, it was important to check whether B0045 is also effective in vivo. For this objective, we generated xenograft models by subcutaneously injecting PC3 prostate cancer cells expressing enhanced green fluorescent protein (eGFP) into the right flank of nude mice. Because B32B3 exhibits antitumor efficacy at a dose of 5 mg kg^–1 in xenograft models [24], and because B0045 displays about 20-fold higher potency than B32B3 in our cellular assessments (Figs. 3B, C), it was reasonable to expect an antitumor efficacy of B0045 at much lower doses. To examine this possibility, B0045 was administered to PC3 xenograft mice at a dose of 0.3 mg kg^–1 twice a week over 28 days. Our fluorescence imaging at days 1, 14, and 28 demonstrated that B0045 treatment is highly effective in impairing the proliferative ability of PC3 xenograft tumors (Figs. 5A-D). However, the growth of PC3 xenograft tumors was only subtly impaired after injecting the same amount of B32B3 (Figs. 5A-D). As mice showed no significant changes in body weight after B0045 treatment, B0045 must have inhibitory effects on the growth of PC3 xenograft, specifically targeting PC3 cancer cells in mice (Fig. 5E). Mirroring the results in prostate cancer cell lines, H2AT120p levels were minimally changed after B32B3 treatment, as assessed by Western blotting with xenograft lysates (Fig. 5F). However, we detected a nearly 80% decrease in H2AT120p levels in B0045-treated xenograft tumors, strongly supporting that B0045 inhibits xenograft tumor growth in an H2AT120p-selective manner (Fig. 5F). Consistent with these findings, immunostaining of xenograft tumors confirmed lower levels of H2AT120p in B0045-treated mice, whereas H2AT120p levels were not much affected in B32B3-treated mice (Fig. 5G). As an experiment to elucidate the mechanistic basis of B0045 effects, our RT-qPCR analysis also showed that B0045 treatment resulted in, albeit to a varying extent, higher expression of VprBP target genes in PC3 xenograft tumors (Fig. 5H). These and the above noted data overall support the idea that B0045 treatment reactivates VprBP target genes and inhibits prostate tumor growth through controlling H2AT120p events.

Fig. 5.

Tumor xenograft progression reduced by B0045. A PC3 tumor xenografts were generated and treated with DMSO, B32B3 (0.3 mg/kg b.w.), or B0045 (0.3 mg/kg b.w.) for 28 days. Tumor growth was monitored by checking fluorescent signals detected at wavelengths of 581 nm with an in vivo imaging system (IVIS) after 1, 14, and 28 days of treatment. B Fluorescence intensities were quantified using regions of interest (ROIs) of equivalent-sized areas from lower abdominal regions at the indicated time points. Data are presented as mean ± SD (n = 6). P values were calculated using two-way ANOVA with post-hot Tukey’s test for multiple comparisons. ***p < 0.001 versus B32B3. C, D Mice were sacrificed 28 days after injection of PC3 cells and prostate tumor xenografts were surgically excised, and their volume (mm³) (C) and weights (mg) (D) were measured. Data are presented as the mean ± SD (n = 6). P values were calculated using two-way ANOVA with post-hot Tukey’s test for multiple comparisons. ***p < 0.001 versus B32B3. E Body weights of mice carrying xenografts were measured at indicated time points after inhibitor treatment. Data are presented as the mean ± SD (n = 6). P values were calculated using two-way ANOVA with post-hot Tukey’s test for multiple comparisons. F PC3 tumor xenografts were excised from mice after 28-day treatment with DMSO, B32B3 or B0045. Western blot analysis was performed on the excised PC3 tumor xenografts using the indicated antibodies. The band intensities were quantified using Image J software (v1.53 k) after normalization to Actin. The corresponding optical densities are shown below. NS indicates no signal. Data are representative of three independent experiments. G PC3 tumor xenografts were excised as in (F) and subjected to immunofluorescent staining with H2AT120p antibody. Shown are the representative results of three independent experiments. Scale bar, 50 μm. H RNA samples were prepared from PC3 tumor xenografts 28 days post-injection and analyzed by RT-qPCR using primers listed in Supplementary Table S3. Data represent the mean ± SD of three independent experiments. ***p < 0.001 versus B32B3.

Discussion

The role of VprBP kinase in transcriptional regulation has been studied in several distinct cellular contexts with different target substrates. In some cases, VprBP phosphorylates H2AT120, while in others it targets nonhistone substrates such as EZH2 and p53, for its oncogenic action. In this work, we have presented evidence indicating that VprBP is mainly responsible for catalyzing H2AT120p and inactivating a specific cohort of growth regulatory genes in prostate cancer cells. VprBP executes its function as a negative regulator of gene transcription in a H2AT120p-dependent manner, because shRNA-mediated knockdown of VprBP significantly decreased H2AT120p and impaired gene silencing. Given that no defects on EZH2T367p were observed in response to VprBP knockdown, EZH2T367p seems to be regulated by some other kinases and doesn’t appear to be the mechanism contributing to the oncogenic function of VprBP in prostate cancer cells. An earlier paper by our group established the importance of H3NT in driving the binding of VprBP to nucleosomes and the requirement of VprBP LisH motif for this binding process. We extended these findings by showing that VprBPS895p abolishes the ability of VprBP to bind nucleosomes through impeding VprBP LisH motif-H3NT interaction. Along with our later demonstration of S895p-induced attenuation of VprBP-mediated H2AT120p, these results strongly suggest that VprBPS895D phospho-mimicking mutant could be used as a tool to investigate the functional contribution of VprBP kinase activity targeting H2AT120 in the nucleosome. In support of this hypothesis, our biochemical and cellular analyses demonstrated (1) an impaired interaction of VprBPS895D with nucleosomes, (2) a clear inability of VprBPS895D to phosphorylate H2AT120 in nucleosomes, (3) a corresponding loss of transrespression potential of VprBPS895D, and (4) no apparent effects of VprBPS895D on cancer cell growth. These findings advance our previous studies describing the role of VprBP-mediated H2AT120p in oncogenic gene silencing and further highlight the importance of VprBP-H3NT interaction as a critical determinant of how VprBP recruitment and function are regulated in prostate cancer cells. Our data also raise the question of how VprBP-mediated H2AT120p establishes a stable inactivation of target genes in cancer cells. H2A is the only core histone containing a long carboxyl-terminal tail, and this tail domain protrudes in close proximity to the site where the DNA enters and exits the nucleosome. Therefore, it is likely that VprBP-mediated H2AT120p switches target genes from an active to inactive state by increasing the interaction affinity between H2A-H2B dimer and H3-H4 tetramer and inducing a conformational change in the nucleosome. Considering that modified histone tails could serve as an integrating platform for effector proteins, another possibility is that specific repressors are recruited to growth-regulatory genes by recognizing H2AT120p present on promoter nucleosomes. This stable docking of repressors across the promoter regions of VprBP target genes establishes the inactive state of transcription, especially impeding transcription initiation. Future studies exploring these two possibilities would be helpful for understanding to what extent VprBP-induced H2AT120p contributes to local chromatin folding and repressor recruitment, thereby achieving specific gene silencing.

In the current report, we also prepared and characterized a series of analogues of the first-generation VprBP inhibitor B32B3 to improve potency and efficacy. Our initial exploration of 211 analogues by in vitro kinase assays using recombinant VprBP and H2A identified eight analogues as fully inhibiting VprBP-mediated H2AT120p at the final concentration of 500 nM. In further characterization of those eight effective analogues using prostate cancer cells expressing high levels of VprBP, we identified B0045 being more potent than seven other compounds. B0045 can efficiently penetrate the cells and inhibit VprBP kinase activity with IC₅₀ values in the range of 30–50 nM, recapitulating the most relevant molecular phenotypes that are generated by VprBP knockdown. With VprBP as the only kinase impacting H2AT120p-induced gene silencing and cell growth, its selective inhibition results in decreasing nuclear levels of H2AT120p, reactivating a group of genes encoding growth-regulatory factors, and compromising the ability of prostate cancer cells to grow. Moreover, our observation of B0045 not influencing the growth of normal prostate cells is an important finding as it suggests that a beneficial therapeutic index might be achieved with B0045 inhibitor in clinical practice. One would expect B0045 to induce similar changes in intracellular oncogenic signaling and to display similar potent efficacy in all cell types highly expressing VprBP. However, we observed that some of the seven other analogues, showing substantial activity against VprBP in cellular assays (Fig. 3B), are more effective than B0024 at inducing growth inhibitory response in other cell types (data not shown). Although we do not have a clear explanation for this cell type-specific responsiveness to the analogues, it is likely a reflection of their differential cellular uptake, metabolic stability, or cellular adaptation, and we speculate that deeper understanding of these differences will improve our ability to use VprBP inhibitors in diseases. Further biological studies are underway to address this possibility and elucidate the complex and multiple cellular mechanisms that can underlie the differential inhibitory effects of these analogues observed in prostate and other types of cancer cells. In agreement with in vitro data, we also found that B0045-induced suppression of VprBP-mediated H2AT120p is highly effective at blocking oncogenic gene silencing in PC3 prostate tumor xenograft models. This result, together with its ability to impair tumor growth without affecting body weight of the animals, makes B0045 an attractive candidate for treatment of VprBP-driven prostate cancer. It is noteworthy that B0045 exhibited much higher potency against VprBP than the first generation VprBP inhibitor B32B3 which is different in the number and spatial arrangement of rings. Thus, we forecast that further optimization of specific ring structures and orientations in B0045 molecule may lead to the development of VprBP inhibitors with greater potency, possibly curative therapies for prostate cancer. In conclusion, we demonstrate that VprBP is overexpressed in prostate cancer cells and plays a key role in transcriptionally inactivating a group of growth-regulatory genes in a manner dependent on H2AT120p. For further support for such an H2AT120p-dependent function, S895D phosphomimetic mutation to specifically block H2AT120-targeted VprBP kinase activity almost completely abolishes the target gene silencing potential of VprBP in prostate cancer cells. More importantly, our systematic design, screening, and characterization of B32B3 analogues established B0045 as the second generation VprBP inhibitor that robustly disrupt VprBP-mediated H2AT120p and effectively attenuate cancer cell growth. These findings provide functional clues as to how VprBP establishes epigenetic gene silencing and establish a novel foundation to develop more anti-VprBP inhibitory agents as promising therapies with potential to treat a large portion of prostate cancer patients.

Materials and methods

Cell lines, constructs, and antibodies

All cell lines (RWPE-1, PC3, DU145, and LNcaP) were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO² at 37 °C. For mammalian expression of VprBP and H3, the corresponding cDNAs were amplified by PCR and ligated into the lentiviral expression vector pLenti-Hygro (Addgene, Cambridge, MA, USA) containing 5′ 3 × FLAG or hemagglutinin (HA) coding sequence. VprBP cDNA was amplified by PCR and inserted into the EcoRI and XhoI sites of pFASTBAC vector with an N-terminal 6 × His tag to generate VprBP baculovirus expression system. To generate VprBPS895D and K194R mutants, VprBP wild-type cDNA was mutated by using Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) after the construction. For bacterial expression of EZH2 and H3, their cDNAs were subcloned into the pET11d vector in frame with a 5’ FLAG tag. All constructs were verified by DNA sequencing. Antibodies used in this study are as follows: anti-FLAG antibody from Sigma-Aldrich (St. Louis, MO, USA); anti-HA; anti-actin, anti-His, anti-VprBP, and anti-EZH2 antibodies from Proteintech (Rosemont, IL, USA); anti-H3 and anti-H2A antibodies from Abcam (Cambridge, MA, USA); anti-H2AT120p from Active Motif (Carlsbad, CA, USA); and anti-rabbit and anti-mouse secondary antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

RNA interference

DNA oligonucleotides (5’-CGAGAAACTGAGTCAAATGAA-3’) encoding shRNA specific for VprBP coding region were annealed and ligated into the lentiviral expression vector pLKO.1 (Addgene, Berkeley, CA, USA). Lentiviral particles were generated in 293 T cells by transfecting plasmids encoding VSV-G, NL-BH, and the shRNA. Two days after transfection, the soups containing the viruses were collected and used to infect PC3 cells in the presence of polybrene (8 µg/mL). The cell lines were selected for two weeks in the presence of puromycin (4 µg/mL). For rescue experiments, VprBP-depleted cells were infected with lentiviruses expressing shRNA-resistant VprBP wild-type, K194R kinase-dead mutant, or S895D phospho-mimicking mutant and selected for two weeks in the presence of hygromycin (250 µg/mL).

Protein extraction and Western blotting

Whole cell lysates were prepared from RWPE-1, PC3, DU145, and LNcaP cells using M-PER™ Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For protein extraction from patient tissue samples, 10 prostate tumor specimens along with adjacent normal tissue counterparts were obtained from a commercial source. Tumor specimens were flash-frozen in liquid nitrogen and subsequently ground with a chilled mortar and pestle. Tissue lysates were prepared using T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA) following the manufacturer's instructions. The protein samples were separated using 4–20% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a 0.45 μM nitrocellulose membrane (GE Healthcare, Milwaukee, WI, USA). After blocking with 5% non-fat dry milk (Rockland, Limerick, PV, USA), the membrane was incubated at 4 °C with a primary antibody and washed with 0.05% Tween-20 in TBS (TBS-T). The membrane was washed with TBS-T, incubated at room temperature with a secondary antibody for 1 h, and visualized using an ECL reagent (Thermo Fisher Scientific, San Diego, CA, USA). All experiments were performed in triplicate.

Histone, nucleosome, and chromatin preparation

Histones were purified from RWPE-1, PC3, DU145, and LNcaP cells according to the acid extraction method as detailed previously [33]. Chromatin fraction was extracted from PC3, DU145, and LNcaP cells following the protocol described in our previous studies [30, 31, 34, 35]. To purify nucleosomes containing H3 wild-type, PC3 cells were infected with lentiviruses expressing H3 wild-type, FLAG-tagged intact (1–135), or NT-cleaved (29–135) H3 containing a N-terminal 3 × FLAG tag. The cells were selected for two weeks in the presence of hygromycin (250 μg/ml). Total nucleosomes were prepared using Nucleosome Preparation Kit (Active Motif, Carlsbad, CA, USA) and ectopic H3-containing mononucleosomes were isolated by immunoprecipitation using anti-FLAG M2 agarose beads in washing buffer (20 mM HEPES, pH 7.8, 300 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EGTA, 10% glycerol, 0.2% Triton X-100, and protease inhibitor cocktail). Levels of HA-tagged VprBP of bead-bound nucleosomes were analyzed by Western blotting. All experiments were performed in triplicates.

CATS molecular descriptor

Descriptor calculation was performed with a proprietary Java-based software tool. The software is provided at URL (http://modlab-cadd.ethz.ch/).

In vitro kinase assay

Nucleosomes were reconstituted using the 601 nucleosome positioning sequence and recombinant histones as recently described [36, 37]. For in vitro kinase assays, VprBP wild-type and mutants were incubated with reconstituted nucleosomes (2 µg) or FLAG-tagged EZH2 (1 µg) in kinase buffer (50 mM Tris–HCl [pH 7.5], 20 mM EGTA, 10 mM MgCl₂, 1 mM DTT, and 1 mM b-glycerophosphate) containing 4 mM ATP as recently described [24, 27, 28]. Following incubation for 30 min at 30 °C, VprBP and H2A or EZH2 proteins from each reaction were separated by SDS-PAGE, and phosphorylated H2A and EZH2 proteins were analyzed by Western blotting. All assays were conducted in triplicates.

RNA-seq

RNA was prepared from mock- or VprBP-depleted PC3 cells using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. After quality control, strand specific libraries were prepared using a KAPA Stranded mRNA-Seq Kit with KAPA mRNA Capture Beads (KAPA Biosystem, Wilmington, MA, USA), and validated on an Bioanalyzer with the DNA1000 kit (Agilent Technologies, Santa Clara, CA, USA). Pooled libraries were prepared, denatured, diluted to 15 pM, and then clonally clustered onto the sequencing flow cell using the cBOT Cluster Generation Station and TruSeq Paired-End Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA). The clustered flow cell was sequenced with 1X50 SE reads on the HiSeq HS (Illumina, San Diego, CA, USA) according to manufacturer’s protocol [25, 30, 35, 38]. Base conversion was made using OLB version 1.9, de-multiplexed, and converted to Fastq using CASAVA (ver. 1.8, Illumina, San Diego, CA, USA). The raw RNA-seq data were mapped to human genome assembly GRCh38/hg38 [39] using STAR (ver. 2.7.8a) aligner [40] and followed by quantification to GENECODE annotation release 46 using Partek E/M method. Aligned reads were applied a noise reduction filter to exclude low count alignment, and gene counts were normalized using the upper quantile normalization method. After principal components analysis with normalized gene counts, differentially expressed genes (DEGs) were selected by using the limma-voom from Partek® Flow® software (ver. 12.0.1, Partek Inc. St Louis, MO, USA). The absolute value of fold change > 1.5 and false discovery rate cut-off of 0.01 were used to identify genes that have a statistically significant difference in average expression across distinct biological groups. Gene ontology analysis of differentially expressed genes was performed using Ingenuity Pathway Analysis tool (IPA® ver. 111725566) (Qiagen Inc, Redwood City, CA, USA). The heat map was generated by calculating Z score of gene expression levels using the Generalized Minimum Distance R package heatmap.3 function [41].

RT-qPCR

Total RNA was isolated from PC3 cells by using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and converted to first-strand cDNA using the SuperScript III First-Strand System Kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time RT-qPCR was performed with SYBR Green Real-time PCR Master Mixes (Agilent Technologies, Santa Clara, CA, USA) using Agilent Aria software (ver. 2.1) according to the manufacturer’s instructions. The primers used for RT-qPCR are listed in Supplementary Table S3. All mRNA values were normalized to GAPDH mRNA levels, and all reactions were run in triplicate.

Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA)

The significantly upregulated genes (|log2FC|> 0.58, padj < 0.05) were subjected to pathway enrichment analysis utilizing Enrichr MSigDB hall mark 2020 pathway [42]. The data were filtered with the Benjamini–Hochberg method, and statistical significance was classified at a p-value and q-value threshold of < 0.05. To identify gene-signature-based difference in VprBP-depleted PC3 cells, we performed Gene Set Enrichment Analysis (GSEA) for Hallmark gene set from human MSigDB [43], based on a ranked gene list reflecting differentially expressed genes (p < 0.05) between control and VprBP-deficient PC3 cells. The number of permutations was set to 2000 for each analysis, and the enrichment score (ES) value was calculated for each gene set. The gene size smaller than 20 or larger than 500 was excluded.

ChIP-qPCR

Chromatin immunoprecipitation (ChIP) assays with PC3 cells were performed using the ChIP Assay Kit (Millipore Sigma, St. Louis, MO, USA) as recently described [26, 27]. After reversing the protein-DNA cross-links, immunoprecipitated DNA was purified and analyzed by quantitative real-time PCR (qPCR) using the primers that amplify the promoter region (P) and coding (C) region of IRF1, CFB and TNFAIP3 genes (Supplementary Table S4). Specificity of amplification was determined by melting curve analysis, and all samples were run in triplicate.

Cell viability and colony formation assays

Cell viability was quantified using the WST-1 Cell Proliferation Reagent (Roche Diagnostics, Switzerland) as recently described [26, 27] and according to the manufacturer’s protocol. In this assay, 10 μl of WST-1 assay solution was added to each well of 96-well plate containing 100 μl of PC3 cells in the culture. After 4 h incubation at 37 °C, the relative cell viabilities were given by determining the absorbance at 460 nm at each well using a microplate reader with CLARIOstar software (ver. 3.41). Each experiment was performed in triplicate. For colony formation assays, PC3 cells were seeded in a 6-well plate at a density of 1 × 10³ cells/well, and cells were allowed to form colonies for an additional 2 weeks. The colonies in each well were stained with 0.5% crystal violet and photographed. The colonies in each well were counted using ImageJ software (ver. 1.53 k, U.S. National Institutes of Health, Bethesda, MD, USA). All assays were run in triplicate, and the results presented are the average of three individual experiments.

Immunostaining

PC3, DU145, and LNcaP cells, the cells were cultured in 4-well chamber slides and fixed with 4% paraformaldehyde for 10 min. The cells were then permeabilized with 0.1% Triton X-100 for 10 min and treated with the blocking reagent (5% normal goat serum in TBS-T) for 60 min at room temperature. After incubating with H2AT120p antibody overnight and Alexa Fluor-conjugated secondary antibody for 60 min, the cells were then washed with PBS and imaged using a BZ-X fluorescence microscope (Keyence, San Diego, CA, USA).

Mice xenograft

8-week-old athymic male nude mice [(Crl:NU(NCr)-Foxn1nu] were obtained from Charles River Laboratories (Wilmington, MA, USA) and served with a general laboratory diet and water ad libitum in specific pathogen-free conditions. For experiment, mice were randomly divided into 3 xenograft groups (n = 6 mice in each group). EGFP expressing PC3 cells (1 × 10⁷) were subcutaneously injected into the right flank of each mouse and immediately treated with inhibitors for 28 days. To evaluate the effect of B0045 and B32B3 treatment, the first group was left untreated (Vehicle), the second and third group were intraperitoneally administrated with B32B3 (0.3 mg/kg b.w.) and B0045 (0.3 mg/kg b.w.), respectively, every three days. Fluorescent signals from the xenograft tumors were specifically detected at a wavelength of 581 nm with an in vivo imaging system (IVIS). Body weights were measured every 3 days over a period of 28 days. The treatment ended once Vehicle group reached the humane endpoint of 15 mm the maximum tumor size at largest diameter. At the end point of treatment, mice were euthanized by asphyxiation with CO₂, and tumors were excised, photographed, and weighed for further analysis as described previously [25, 27, 35, 38]. To determine the levels of VprBP, H2A, and H2AT120p, lysates were also prepared from the excised xenografts and analyzed by Western blotting. Sections (5 mm) of formalin-fixed and paraffin-embedded PC3 tumor xenografts were also subject to immunohistochemical analysis by treating with blocking reagent (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.3% Triton X-100, and 5% normal goat serum) for 30 min at room temperature. After incubating with VprBP and H2AT120p antibodies overnight and biotinylated secondary antibody for 60 min, immunodetection was performed using ABC reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. DAB was utilized for color development, and hematoxylin was used for counterstaining. All mouse experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee.

Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD). Statistical analyses of datasets were performed with one-way or two-way ANOVA followed by Tukey’s post-hoc test using GraphPad Prism software 9 (GraphPad Software Inc., San Diego, CA, USA) which was used for all analyses of the experiments. A p-value < 0.05 was considered statistically significant.

Accession numbers

The gene expression array data has been deposited in the NCBI Gene Expression Omnibus (GEO) database under the GEO accession number GSE308536.

Authors’ contributions

SK and WA conceived and designed the study. SK performed the experiments with contributions of WA. SK, YS, NG, and WA analyzed data. SK and WA wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Institutes of Health (AR073233). The study was also partly supported by award number P30CA014089 from the National Cancer Institute.

Data availability

The data supporting the findings of this study are available in the Gene Expression Omnibus (GEO) database under accession number GSE308536.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committee of University of Southern California, and were conducted in accordance with the committee’s institutional guidelines.

Consent for publication

All authors have read and approved the manuscript.

Competing interests

The authors declare no competing interests.