MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer

Abstract Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR−, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Prostate cancer is driven by the androgen receptor (AR), an androgen-dependent transcription factor that promotes cell growth and proliferation (1).Castration-resistant prostate cancer (CRPC) is an advanced form of the disease in which cancer cells can survive despite androgen deprivation, frequently by restoring AR activity (2).Second-generation AR signaling inhibitors, such as enzalutamide, suppress AR activity by competitively inhibiting androgen binding, thereby significantly prolonging CRPC patient survival (3,4).However, cancer cells ultimately develop resistance to enzalutamide (5).One major mechanism of enzalutamide resistance is the enhanced expression of AR-V7, a constitutively active splice variant of AR that restores AR activity in patients treated with AR signaling inhibitors (6,7).Other mechanisms, such as the overexpression of AR coactivators, also contribute to the impairment of enzalutamide efficacy (5).Some subunits of the Mediator complex are coactivators of the AR in prostate cancer (8,9).The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale.It comprises the preinitiation complex on gene core promoters, where it mediates the interaction between RNA polymerase II and enhancer-bound transcription factors, thus promoting transcription initiation (10,11).The Mediator complex has a modular structure with the head, middle, and tail modules acting as the main core to which a kinase module can transiently associate (10).The kinase module is a key Mediator complex regulator that can disrupt its interaction with RNA polymerase II and phosphorylate transcription factors, consequently affecting their activity and stability (12)(13)(14).
MED12 is an essential subunit of the kinase module that plays a crucial role in preserving the structure of the entire module (15)(16)(17).Adler et al showed that MED12 is overexpressed in advanced CRPC compared to early-stage disease (18).They also related MED12 knockdown to decreased cell proliferation and reduced transition to the S stage of the cell cycle (18).Recent findings have shown that MED12 downregulation increased the proliferation of prostate cancer cells under androgen deprivation (19).
Cyclin-dependent kinase (CDK)8 and its alternative paralog, CDK19, are the enzymatic subunits of the kinase module of the Mediator complex (15).In prostate cancer cells, CDK8/19 inhibition downregulates cell proliferation and in vivo metastasis formation (20,21).Offermann et al demonstrated that combining CDK8/19 with bicalutamide, an AR inhibitor, additively decreased the proliferation of prostate cancer cells (22).
Taken together, these results suggest that MED12 and CDK8/19 play important roles in promoting tumorigenesis and resistance to therapy in prostate cancer cells.Our study investigated how the inhibition of MED12 and CDK8/19 subunits affects cancer signaling pathways and cell processes in prostate cancer, focusing on AR activity and cell responsiveness to enzalutamide.

Cell Proliferation Assay
The proliferation of adherent cells seeded in 6-well plates was evaluated 3 and 6 days after transfection.At both end time points, the cells were incubated with Hoechst 333423342 nucleic acid stain (Thermo Fisher Scientific, #H3570) at a 1:2000 dilution for 20 minutes.Cell nuclei were visualized using fluorescence imaging (CELENA ® S Digital Imaging System, Logos Biosystems) and counted using the Fiji ImageJ software.

Formation and Analysis of Spheroids
Three days after siRNA transfection, spheroids were generated in 96-well plates (Corning ® 96 Well Clear Round Bottom Ultra Low Attachment Microplate, Corning, #COR7007), and all downstream analyses were performed after 4 days.Spheroids were harvested and lysed with 25 µL 1× DNA/RNA Shield (Zymo Research, #R1100-50).Cell number estimation was performed by measuring the total DNA content of spheroids using the Qubit 1× dsDNA Assay Kit (Thermo Fisher Scientific, #Q33230) and comparing it with a standard known cell number.
cDNA was synthesized using the LunaScript ® RT SuperMix Kit (New England Biolabs, #M3010X), and gene expression was quantified by quantitative PCR (qPCR) using the Luna ® Universal qPCR Master Mix (New England Biolabs, #M3004X) and the CFX Connect Real-Time PCR Detection System (Bio-Rad, #1855200).qPCR was performed according to the manufacturer's protocol using the TaqMan gene expression assays listed in Supplementary Table 1 (23).

Prostate-Specific Antigen Quantification
Prostate-specific antigen (PSA) secreted by cells in the medium was quantified using chemiluminescent-based Elecsys total PSA assay (Roche, #04641655 190) and measured on a Cobas ® 8000 machine (Roche).

Enzalutamide Treatment
LNCaP and 22Rv1 cells were treated with 10 µM enzalutamide (MedChemExpress, #HY-70002) and the respective dimethylsulfoxide volumes immediately after siRNA transfection and medium change.After 3 days, the cells were trypsinized and reseeded.Downstream analyses were performed 6 days after enzalutamide treatment and siRNA transfection.

Dose-Response Assay
Cells were seeded in 96-well plates at a density of 8000 cells/ well.After 24 hours, the cells were treated with sequential 1:2 dilutions of a CDK8/19 inhibitor, SEL120-34A (Selleck Chemicals #S8840), at a concentration range of 0.47 to 15 µM.After 3 days, the cells were lysed in 1× Tris-acetate-EDTA buffer containing 1× TritonX-100 and 1% Proteinase K (Roth Industries, #7528.2).The total amount of DNA was measured by incubating the cell lysates with 1:1000 SYBR ® Green I nucleic acid gel stain (Sigma Aldrich, #S9430) for 10 minutes.The fluorescent signal was detected using a Cytation 5 Imaging Reader (BioTek Instruments).Cell number was calculated from the amount of DNA using a known standard.The IC50 values for cell proliferation for each cell line were calculated using GraphPad Prism 10.1.2(RRID: SCR_002798).

Population Doubling Level (Programmed Death Ligand) Assay
22Rv1 cells were seeded into T25 cell culture flasks and treated with single or combined treatments of 10 µM enzalutamide (MedChemExpress, #HY-70002) and 1.5 µM SEL120-34A (Selleck Chemicals, #S8840) for 18 days.The cells were passaged regularly into new flasks.At each passage, the cells were counted using the Schärfe Casy TT system.The cumulative programmed death ligand (PDL) was calculated using the formula: 3.32 × (log 10 (harvested cells) − log 10 (seeded cells)) + previous cumulative PDL.The experiments were performed in a single replicate.

Gene Correlation Analysis
Gene correlations were analyzed in publicly available prostate cancer tissue databases (TCGA_PRAD, GSE62872, GSE21034, GSE46691, and SU2C_PRAD) as previously described (24).The first 4 databases contain primary prostate cancer tissues, whereas the SU2C_PRAD database contains CRPC tissues, most of which are metastatic.

Gene Number Quantification
MED12 copy number aberration was extracted from the TCGA_PRAD and SU2C datasets.

Cell Line Dependency to MED12 Expression
The dependence of a benign prostatic hyperplasia cell line (BPH1) and prostate cancer cell lines (LNCaP, 22Rv1, VCaP, DuCaP, DU145, and PC3) on MED12 expression was analyzed using 2 independent publicly available screens from the DepMaP portal (RRID: SCR_017655).MED12 expression was downregulated using CRISPR or RNA interference (RNAi) and its effect on cell growth was measured.In the plot, complete cell dependence is located at '−1' on the x-axis, whereas complete cell independence is located at 0.

RNA Sequencing and Pathway Analysis
High-throughput 3′ RNA sequencing (RNA-seq) was performed using a BRB-seq Library Preparation Kit for Illumina (Alithea Genomics).Read alignment and counting were performed with STARsolo (SCR_021542) using Gencode Release 43 (GRCh38.p13)genome assembly.Strand-specific full transcriptome RNA-seq of the 22Rv1 samples was performed using NovoGene.Read alignment and counting were performed in the R software using the Rsubread package and the Gencode Release 43 (GRCh38.p13) genome assembly.Differential gene expression analysis was performed using the edgeR package in the R software.Pathway analysis (camera function) was performed using MSigDB hallmark gene sets version 7.5.1.

Statistics
Unpaired t-tests were used to analyze the differences between treated samples and their relative controls.A simple linear regression was applied to the PDL assay.Statistical significance was always considered as a P-value < .05(*P < .05,**P < .01,***P < .001).Data are presented as the mean ± SD.All experiments were performed in at least 3 biological replicates, unless otherwise noted.

MED12 Knockdown Inhibits Prostate Cancer Cell Proliferation
To investigate the role of MED12 in prostate cancer, we analyzed aberrations in the MED12 gene copy number status in 2 publicly available datasets: treatment-naïve (TCGA-PRAD, primary prostate cancer) and CRPC (SU2C-PRAD, predominantly metastatic prostate cancer) tissues.The MED12 copy number was increased in over 30% of CRPC samples, compared to less than 3% of treatment-naïve prostate cancer samples (Fig. 1A).Subsequently, we analyzed MED12 protein expression in a panel of prostate cancer cell lines (AR-positive: LNCaP, 22Rv1, VCaP, and DuCaP; AR-negative: DU145 and PC3) and in 1 benign prostatic hyperplasia cell line (BPH1) (Fig. 1B).MED12 protein expression was highly variable among the different prostate cancer cell lines.Consistent with the copy number data, we observed that 3 out of the 6 prostate cancer cell lines showed a strong increase in MED12 protein expression (>2× compared to benign BPH1 cells).LNCaP cells had the lowest MED12 protein levels.In addition, 2 independent datasets from CRISPR-and RNAi-based screens (DepMap portal) revealed that nearly all prostate cancer cell lines were highly sensitive to MED12 downregulation, suggesting its pivotal role in prostate cancer cell growth and survival.Of note, AR-positive cell lines (VCaP, LNCaP, MDA PCa 2b, and 22RV1) were more sensitive to MED12 downregulation than AR-negative cells (PC3 and DU145) in the RNAi-based screen (Fig. 1C).
Altogether, our results suggest that MED12 downregulates AR activity in 22Rv1 cells, whereas its role in LNCaP cells is less prominent.
Since AR-V7 promotes enzalutamide resistance (7), we investigated whether siMED12-mediated downregulation of AR-V7 protein influences 22Rv1 cell sensitivity to enzalutamide (2D models).The combination of MED12 knockdown and enzalutamide treatment after 6 days additively decreased PSA protein secretion and KLK3 mRNA expression compared with enzalutamide treatment alone (Fig. 4B).However, the combination treatment did not enhance the antiproliferative effects of the siMED12 single treatment at this relatively short time point (Fig. 4B).To clarify the inconsistency between AR activity and proliferation, we examined early cell responses [Supplementary Fig. S6 (25)].MED12 knockdown resulted in a rapid and sustained decrease in KLK3 and c-MYC mRNA expression levels.However, we did not observe To further explore the relationship between MED12 and enzalutamide, we performed the same experiments in LNCaP cells, which are sensitive to enzalutamide and do not express the AR-V7 protein.MED12 knockdown single treatment had weaker effects on AR activity in this cell line than 22Rv1.The combination of MED12 knockdown and enzalutamide treatment significantly decreased PSA protein secretion and KLK3 mRNA gene expression compared to enzalutamide treatment alone [Fig.4B, Supplementary Figure S7 (25)].This confirmed that MED12 knockdown and enzalutamide play an additive or synergistic role in AR inhibition, even in the absence of AR-V7 expression.Conversely, AR-V7 and c-Myc mRNA expression levels were not additively altered at 48 and 72 hours of treatment [Supplementary Fig. S7 (25)].After 6 days, the combination of siMED12 and enzalutamide resulted in a small but significant decrease in cell proliferation compared to enzalutamide alone (Fig. 4B).

CDK8/19 Inhibition Decreases Proliferation in Prostate Cancer Cell Lines
MED12 is a fundamental structural component of the kinase module of the Mediator complex (27).Its disruption impairs the enzymatic activity of the whole module exerted by CDK8 or its alternative paralog CDK19 (17).Thus, we hypothesized that MED12 knockdown affects prostate cancer cell lines mainly through the subsequent loss of the kinase  MED12 mRNA expression was positively correlated with CDK8 and CDK19 gene expression in multiple primary prostate cancer tissue datasets, as expected by their common presence in the kinase module.However, no significant correlation was detected in the CRPC tissue database [Fig.5A, Supplementary Fig. S8 (25)].High CDK8 expression significantly decreased relapse-free survival in treatment-naïve patients with prostate cancer (TCGA database), whereas CDK19 expression did not [Fig.5B, Supplementary Fig. S9  (25)].In prostate cancer cell lines, CDK8 protein expression was higher in PC3 cells than in LNCaP and 22Rv1 cells.Consistent with the alternative roles of CDK8 and CDK19 within the Mediator complex, CDK19 was expressed at lower levels in PC3 cells than in the other cell lines.All these expression trends were mirrored by their relative mRNA expression [Supplementary Fig. S10 (25)].SEL120-34A is a CDK8/19 subunit inhibitor that reduces tumor growth in acute myeloid leukemia (28).A phase 1B clinical trial (NCT04021368) is currently underway to assess its pharmaceutical potential.We treated LNCaP, 22Rv1, and PC3 cells with SEL120-34A to test their sensitivity to biochemical CDK8/19 inhibition.Consistent with observations for MED12, AR-positive cells were slightly more sensitive to CDK8/19 inhibition than the AR-negative PC3 cells (IC50: LNCaP-2.42 µM; 22Rv1-2.11µM; PC3-5.6 µM) (Fig. 5C).High-throughput 3′ RNA-sequencing and pathway analysis revealed that CDK8/19 inhibition downregulated the c-Myc pathway, E2F targets, and G2 M checkpoint hallmarks in at least 1 cell line (Fig. 5D).Conversely, it upregulated the tumor-suppressor p53 pathway in AR-positive cells (Fig. 5D).
Mirroring the effects of MED12 knockdown, CDK8/19 inhibition decreased PSA secretion in LNCaP (−64%; P = .001)and 22Rv1 cells (−64%; P = .0001)after 3 days of treatment (Fig. 5E).Notably, AR-V7 protein expression remained unchanged at this early stage (Fig. 5F), further underscoring the need for extended time points to assess potential synergy between CDK8/19 inhibition and AR.Consequently, we cotreated 22Rv1 cells with SEL120-34A and enzalutamide for 18 days.By day 8, the population doubling in the doubletreated cells began to decline, and the drug combination demonstrated a significant overall effect over the time course (P-value for CDK8/19 inhibition + ENZA vs CDK8/19 inhibition: 0.038) (Fig. 5G).Taken together, these findings suggest that CDK8/19 inhibition modulates the response to enzalutamide in 22Rv1 cells.

Discussion
Although elements of the Mediator complex have been implicated in multiple oncogenic processes, our knowledge of their specific functions in prostate cancer is limited.Our findings revealed that the downregulation of MED12 inhibits c-Myc signaling and cell proliferation.We showed that MED12 and CDK8/19 inhibition downregulate the AR response and that they both affect prostate cancer cell response to enzalutamide.
These results align with previous studies associating MED12 and CDK8/19 inhibition with reduced cell proliferation (18,20,29).Although our study is the first to relate MED12 to c-Myc in prostate cancer, MED12 depletion was already related to c-Myc expression in colon cancer (30).Our RNA-seq analysis revealed that CDK8/19 inhibition differentially modulates c-Myc signaling based on the in vitro    (20,29,31).While this manuscript was in preparation, Li and colleagues reported that inhibition of the Mediator complex kinase under castration conditions downregulated the Myc pathway in xenograft models of prostate cancer (29).Thus, the results of our study, along with those of Li et al, establish a basis for therapeutic intervention targeting the kinase module of the Mediator complex.c-Myc activation can modulate cell proliferation through various mechanisms, including the regulation of mitochondrial functions (32) and the metabolic changes resulting from AR inhibition (33).For example, c-Myc maintains the glycolytic activity (33) and increases the expression of key fatty acid synthesis genes, such as ATP citrate lyase, acetyl-CoA carboxylase alpha, and fatty acid synthase (34), thus promoting prostate cancer progression (35).
MED12 knockdown and CDK8/19 inhibition downregulated the AR activity in our 3D models.Other researchers have also recognized the value of 3D models for studying the androgenic regulation in vitro (36,37).MED12 affected the AR target genes FKBP5 and TMPRSS2 to a lesser extent than KLK3.This differential regulation may reflect the variable AR cofactors that are required for their expression.For example, the cofactors NCOA2 and NCOA7 are important modulators of KLK3 mRNA expression in VCaP cells (38), whereas TMPRSS2 expression can occur in an AR-independent manner (39).
Surprisingly, MED12 inhibition increased AR-FL expression in LNCaP cells.This could indicate a compensatory mechanism of the cells or suggest that MED12 might play a dual role depending on the cancer stage.However, it is noteworthy that combination with enzalutamide led to an additive effect.In 22Rv1 cells, MED12 knockdown consistently decreased AR-V7 protein expression, while AR-FL expression remained stable.AR-V7 is upregulated during antiandrogen resistance and modulates the proliferation and motility of prostate cancer cells (40), as well as cell sensitivity to cabazitaxel (40).Recent studies have also revealed that AR-V7 does not merely mimic AR-FL activity but can activate a unique set of target genes (41,42).
We demonstrated that CDK8/19 inhibition partially resensitized 22Rv1 cells to enzalutamide.This finding aligns with the study of Offermann et al, which showed that CDK8/19 inhibition combined with bicalutamide, an AR inhibitor, additively reduced cell proliferation (22).
To our knowledge, MED12 and CDK8/19 may influence the androgen and enzalutamide responses through both AR-V7-dependent and -independent mechanisms.Some Mediator complex subunits, such as MED1 and MED19, are cofactors of AR and enhance its transcriptional activity in prostate cancer (9,43).However, MED12 and CDK8/19 belong to the kinase module of the Mediator complex; therefore, their inhibition may disrupt the phosphorylation events that modulate the activity of AR and its variants (AR-Vs).Multiple kinases are already known to phosphorylate AR and AR-Vs, thus modulating their activity and protein stability (44).Considering the involvement of c-Myc in AR activity and enzalutamide resistance, MED12 may also influence these processes in a c-Myc-mediated manner (45).c-Myc overexpression contributes to suppression of AR activity, with low AR activity and high c-Myc being typical signatures of aggressive prostate cancer (46).According to our RNA-seq data, MED12 knockdown inhibits OXPHOS.Since enzalutamide-resistant cells primarily rely on OXPHOS for their growth, MED12-mediated regulation may increase the cell sensitivity to enzalutamide (47).
The MED12 and CDK8/19 subunits not only belong to the kinase module but are also essential for the kinase activity (16,17,48).Consistent with this knowledge, we observed that their inhibition partially altered the same cancer-promoting pathways (eg, the Myc pathway, E2F targets, and G2M checkpoints).However, these alterations did not completely overlap.An example is the P53 pathway, which is upregulated only following CDK8/19 inhibition.A potential explanation for this differential outcome may be the Mediator complexindependent functions of MED12.For instance, Huang et al showed that MED12 can translocate to the cytoplasm and regulate the TGF-β receptor (49).This phenomenon might be amplified by the overexpression of MED12 observed in advanced prostate cancer by Adler et al (18).
Our findings regarding the impact of CDK8/19 on prostate cancer suggest promising avenues for therapeutic interventions.SEL120-34A, the CDK8/19 inhibitor used in our study, is currently in a phase 1B clinical trial (NCT04021368) for acute myeloid leukemia.Although in vivo studies are beyond the scope of our research, our results may provide the basis for in vivo assessment of SEL120-34A feasibility for prostate cancer treatment.
Despite providing novel insights into the role MED12 and CDK8/19 in prostate cancer, our study has several limitations.Future research should aim to determine the effects of MED12 and CDK8/19 inhibition on AR signaling and enzalutamide responses over an extended period.Clarifying the roles of MED12 and CDK8/19 in modulating AR and AR-Vs is crucial for understanding their impact on prostate cancer.Addressing these questions requires further study on other AR-positive cell lines.
In conclusion, our study highlights the involvement of MED12 and CDK8/19 in c-Myc, AR activity, and cell response to enzalutamide in prostate cancer.These findings provide a valuable understanding of potential therapeutic targets for future research.

Figure 1 .
Figure 1.MED12 knockdown inhibits prostate cancer cell proliferation.(A) MED12 gene copy number in cohorts of primary prostate cancer (TCGA-PRAD) and castration-resistant prostate cancer (SU2C-PRAD) tissues.(B) MED12 protein quantification in a benign prostatic hyperplasia cell line (BPH1) and prostate cancer cell lines (LNCaP, 22Rv1, VCaP, DuCaP, Du145, and PC3).A representative Western blot is shown for each cell line.(C) RNA interference and CRISPR screens evaluating prostate cancer cell dependency on MED12 expression for their growth.Lower scores correspond to a higher dependency: a score of 0 is equivalent to nonessential genes and a score of −1 is equivalent to the median of essential genes.(D) MED12 protein quantification in LNCaP, 22Rv1, and PC3 cells transfected with siCtrl and siMED12.A representative Western blot is shown for each cell line.(E) Fluorescent-based cell number quantification of LNCaP, 22Rv1, and PC3 cells transfected with siCtrl and siMED12.(F) Fluorescent-based quantification of the total cell number in LNCaP, 22Rv1, and PC3 spheroids formed after pretransfection of siMED12 and siCtrl in 2D cells (total transfection time: 7 days).Data are represented as mean ± SD. *P < .05,**P < .01,***P < .001.

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Endocrinology, 2024, Vol.165, No. 10 module activity.This suggests that the direct inhibition of CDK8/19 may affect prostate cancer cells similarly to what was observed with MED12 loss.