CDK19 belongs to the kinase module that also includes the subunits Mediator (MED) 12, MED13, cyclin C, and CDK8. The latter is the gene paralog of
CDK19; thus, CDK8 and CDK19 harbor a high degree of structural and functional similarities.
8Regulatory functions of the Mediator kinases CDK8 and CDK19.
Although CDK8 has been well investigated in numerous cancer entities including colon,
9- Firestein R.
- Bass A.J.
- Kim S.Y.
- Dunn I.F.
- Silver S.J.
- Guney I.
- Freed E.
- Ligon A.H.
- Vena N.
- Ogino S.
- Chheda M.G.
- Tamayo P.
- Finn S.
- Shrestha Y.
- Boehm J.S.
- Jain S.
- Bojarski E.
- Mermel C.
- Barretina J.
- Chan J.A.
- Baselga J.
- Tabernero J.
- Root D.E.
- Fuchs C.S.
- Loda M.
- Shivdasani R.A.
- Meyerson M.
- Hahn W.C.
CDK8 is a colorectal cancer oncogene that regulates β-catenin activity.
breast,
10- Xu D.
- Li C.-F.
- Zhang X.
- Gong Z.
- Chan C.-H.
- Lee S.-W.
- Jin G.
- Rezaeian A.-H.
- Han F.
- Wang J.
- Yang W.-L.
- Feng Z.-Z.
- Chen W.
- Wu C.-Y.
- Wang Y.-J.
- Chow L.-P.
- Zhu X.-F.
- Zeng Y.-X.
- Lin H.-K.
Skp2–MacroH2A1–CDK8 axis orchestrates G2/M transition and tumorigenesis.
and melanoma,
11- Kapoor A.
- Goldberg M.S.
- Cumberland L.K.
- Ratnakumar K.
- Segura M.F.
- Emanuel P.O.
- Menendez S.
- Vardabasso C.
- LeRoy G.
- Vidal C.I.
- Polsky D.
- Osman I.
- Garcia B.A.
- Hernando E.
- Bernstein E.
The histone variant macroH2A suppresses melanoma progression through regulation of CDK8.
and linked to many cancer-related pathways, such as WNT/ß-catenin-
9- Firestein R.
- Bass A.J.
- Kim S.Y.
- Dunn I.F.
- Silver S.J.
- Guney I.
- Freed E.
- Ligon A.H.
- Vena N.
- Ogino S.
- Chheda M.G.
- Tamayo P.
- Finn S.
- Shrestha Y.
- Boehm J.S.
- Jain S.
- Bojarski E.
- Mermel C.
- Barretina J.
- Chan J.A.
- Baselga J.
- Tabernero J.
- Root D.E.
- Fuchs C.S.
- Loda M.
- Shivdasani R.A.
- Meyerson M.
- Hahn W.C.
CDK8 is a colorectal cancer oncogene that regulates β-catenin activity.
and transforming growth factor-β/SMAD signaling,
12- Alarcón C.
- Zaromytidou A.-I.
- Xi Q.
- Gao S.
- Yu J.
- Fujisawa S.
- Barlas A.
- Miller A.N.
- Manova-Todorova K.
- Macias M.J.
- Sapkota G.
- Pan D.
- Massagué J.
Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-β pathways.
less is known about the role of CDK19 in carcinogenesis. As part of the kinase module, CDK8 and CDK19 reversibly associate with the Mediator complex as part of the transcription machinery.
13The Mediator complex: a central integrator of transcription.
Besides its function as transcriptional regulator within the Mediator, CDK8 and CDK19 influence cellular signaling by phosphorylation of numerous downstream substrates. Recently, Poss et al
14- Poss Z.C.
- Ebmeier C.C.
- Odell A.T.
- Tangpeerachaikul A.
- Lee T.
- Pelish H.E.
- Shair M.D.
- Dowell R.D.
- Old W.M.
- Taatjes D.J.
Identification of Mediator kinase substrates in human cells using cortistatin A and quantitative phosphoproteomics.
showed that blocking CDK8 and CDK19 by the selective inhibitor cortistatin A in colon cancer cells affects the phosphorylation of multiple proteins involved in inflammation, growth, and metabolic regulation. Of note, as seen after
CDK8/CDK19 gene knockdown, cellular changes occurred independent of these effects, highlighting the ability of
CDK8/CDK19 to modify cancer-related signaling by diverse mechanisms.
Materials and Methods
Cell Lines
Prostate cancer cell lines LNCaP, DU145, and PC3 cells were purchased from ATCC (Manassas, VA) and maintained according to provider's instructions. The cell line LNCaP Abl
18- Culig Z.
- Hoffmann J.
- Erdel M.
- Eder I.E.
- Hobisch A.
- Hittmair A.
- Bartsch G.
- Utermann G.
- Schneider M.R.
- Parczyk K.
- Klocker H.
Switch from antagonist to agonist of the androgen receptor blocker bicalutamide is associated with prostate tumour progression in a new model system.
was a kind gift of Dr. Zoran Culig (University of Innsbruck, Innsbruck, Austria) and was grown in full growth medium without steroids (charcoal-stripped fetal calf serum; ThermoFisher Scientific, Waltham, MA).
CDK8/CDK19 Inhibitors
CDK8/CDK19 inhibitor CCT251545 was a kind gift of Julian Blagg (Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, Sutton, UK).
15- Dale T.
- Clarke P.A.
- Esdar C.
- Waalboer D.
- Adeniji-Popoola O.
- Ortiz-Ruiz M.-J.
- Mallinger A.
- Samant R.S.
- Czodrowski P.
- Musil D.
- Schwarz D.
- Schneider K.
- Stubbs M.
- Ewan K.
- Fraser E.
- TePoele R.
- Court W.
- Box G.
- Valenti M.
- de Haven Brandon A.
- Gowan S.
- Rohdich F.
- Raynaud F.
- Schneider R.
- Poeschke O.
- Blaukat A.
- Workman P.
- Schiemann K.
- Eccles S.A.
- Wienke D.
- Blagg J.
A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.
CDK8/CDK19 inhibitor G02788177.93-1 was a kind gift of Michael F.T. Koehler (Discovery Chemistry, Genentech, South San Francisco, CA).
17- Koehler M.F.T.
- Bergeron P.
- Blackwood E.M.
- Bowman K.
- Clark K.R.
- Firestein R.
- Kiefer J.R.
- Maskos K.
- McCleland M.L.
- Orren L.
- Salphati L.
- Schmidt S.
- Schneider Ev
- Wu J.
- Beresini M.H.
Development of a potent, specific CDK8 kinase inhibitor which phenocopies CDK8/19 knockout cells.
Sensitivity of PCa cell lines toward CDK8/CDK19 inhibitors was assessed by dilution series in 96-well plates compared with dimethyl sulfoxide (DMSO) control. Viability was measured by MTT assay at different time points and was dose dependent.
CDK19 Overexpression
CDK19 plasmids (human-tagged true open reading frame, pCMV6-Entry vector) and human cytomegalovirus control plasmids were purchased from OriGene (OriGene Technologies, Rockville, MD) and used to perform CDK19 overexpression experiments. Transfection was performed using Screenfect A (Genaxxon Bioscience GmbH, Ulm, Germany). Cells were seeded in transfection medium containing the plasmid. Forty-eight hours later, medium was exchanged. CDK19 overexpression was determined by Western blot analysis.
Cell Viability Assay
Cell viability following various inhibitor treatment conditions or plasmid transfection was assessed using thiazolyl blue tetrazolium bromide (MTT). A total of 7000 LnCaP, 7000 LNCaP Abl, 5000 PC3, or 5000 DU145 cells per well were plated in 96-well plates (Corning, Glendale, AZ), and 500 μg/mL MTT (Sigma-Aldrich, Steinheim, Germany) dissolved in phosphate-buffered saline (PBS; Gibco Life Technologies, Waltham, MA) was added. Four hours later, 100 μL solubilization buffer (40% v/v dimethylformamide [Alfa Aesar, Thermo Fisher (Kandel) GmbH, Kandel, Germany], 2% glacial acetic acid [Merck, Darmstadt, Germany], 16% SDS [Applichem, Darmstadt, Germany], pH 4.7) was added; and absorbance was measured at 595 nm. For LNCaP Abl cells, the experiments were performed in medium containing charcoal-stripped fetal calf serum (ThermoFisher Scientific, Schwerte, Germany) without supplementation of dihydrotestosterone.
Cell Cycle Analysis
For cell cycle analysis, propidium iodide DNA staining followed by flow cytometric analysis was performed. In more detail, after 72 hours of specific cell treatments, cells were harvested and used for flow cytometric analysis. Cells were fixed in ice-cold 70% ethanol for 30 minutes at 4°C, washed three times with ice-cold PBS, and resuspended in 200 μL DNA staining solution [PBS containing 50 μg/mL propidium iodide (Sigma Aldrich, Steinheim, Germany) and 100 μg/mL Ribonuclease A (Sigma Aldrich, Steinheim, Germany)]. After 15 minutes of incubation in the dark, cell cycle was analyzed using an LSR II Cell Analyzer (BD Bioscience, Heidelberg, Germany). FCS Express 5 software (DeNovo Software, Glendale, CA) was used for analyzing flow cytometric data.
Wound Healing Assays
Wound healing assays were performed using IBIDI wound healing assays (ibidi GmbH, Graefelfing, Germany). Transfected or inhibitor treated cells were seeded in a concentration of 3 × 105 cells/mL in IBIDI chambers containing a 500-μm thick wall. After reaching a confluent cell layer, cells were treated with mitomycin to avoid overgrowth by proliferation. After 12 hours, cells were stained using crystal violet and migration was visually documented.
Cell Migration Assay
The 12-well cell inserts (ThermoFisher Scientific, Waltham, MA) were used to perform Boyden chamber migration assays. A total of 100 μL of serum-free medium was added to the upper chamber. Next, 200 μL serum-free medium containing 2.5 × 105 cells/mL was added to same chamber before adding 750 μL of 20% fetal calf serum–containing medium to the lower chamber. Cells were treated with DMSO or CDK8/CDK19 inhibitor at the time of plating. After 12 hours, the experiment was terminated by washing the inserts in Dulbecco’s PBS twice before using cotton swabs to remove noninvasive cells on the insert surface. Remaining cells were stained using crystal violet. Next, inserts were washed using Dulbecco’s PBS, and images were taken to document the amount of invasive cells.
Cell Adhesion Assay
A total of 25,000 cells were plated in 96-well plates, which were either nontreated or coated with 8 μg/cm2 collagen type I (Sigma Aldrich, St. Louis, MO). Cells were treated with 5 or 10 μmol/L CDK8/CDK19 inhibitor either at the beginning of the assay or 24 hours in advance. After various measuring time points, the medium was aspirated and wells were washed twice with Dulbecco’s PBS. Next, cells were stained using crystal violet for 20 minutes. After staining, remaining cells were washed four times with Dulbecco’s PBS to remove remaining dye. The amount of adherent cells was documented by taking images. Afterwards, methanol was added to dissolve crystal violet taken up by the adherent cells. Absorbance was measured at 595 nm.
Kinase Profiling Assay
Cells used for analysis were treated with DMSO or CDK8/CDK19 inhibitors for 4 hours before being harvested and lysed. Protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Schwerte, Germany). Protein tyrosine kinase (PTK) activity profiling was performed using a PamStation12 (PamGene International B.V., Hertogenbosch, the Netherlands) and PTK PamChip4 with four identical arrays. On each array, 142 tyrosine-containing peptides (13 amino acids long), derived from known human phosphorylation sites, are immobilized. An assay incubation mixture, containing 2 to 4 μg of protein (depending on total protein available), 100 μmol/L ATP, and fluorescein isothiocyanate–labeled antiphosphotyrosine antibody (PamGene International B.V.), was added in each array. The mixture was pumped up and down through the porous membrane. Peptide phosphorylation was monitored during the incubation by taking images with a charge-coupled device camera in combination with Evolve software version 1.2 (PamGene International B.V.), allowing real-time recording of the reaction kinetics. After washing of the arrays, fluorescence was detected at different exposure times (20, 50, 100, and 200 milliseconds). Analysis was performed using the Bionavigator software version 2.2 (PamGene International B.V.).
Discussion
Previous studies showed the Mediator complex subunit CDK19 to be specifically up-regulated in PCa and to be implicated in the pathogenesis of PCa progression.
5- Brägelmann J.
- Klümper N.
- Offermann A.
- von Mässenhausen A.
- Böhm D.
- Deng M.
- Queisser A.
- Sanders C.
- Syring I.
- Merseburger A.S.
- Vogel W.
- Sievers E.
- Vlasic I.
- Carlsson J.
- Andrén O.
- Brossart P.
- Duensing S.
- Svensson M.A.
- Shaikhibrahim Z.
- Kirfel J.
- Perner S.
Pan-cancer analysis of the Mediator complex transcriptome identifies CDK19 and CDK8 as therapeutic targets in advanced prostate cancer.
,6- Becker F.
- Joerg V.
- Hupe M.C.
- Roth D.
- Krupar R.
- Lubczyk V.
- Kuefer R.
- Sailer V.
- Duensing S.
- Kirfel J.
- Merseburger A.S.
- Brägelmann J.
- Perner S.
- Offermann A.
Increased mediator complex subunit CDK19 expression associates with aggressive prostate cancer.
Both findings, based on tissue-related data and the results of functional
in vitro studies, give strong evidence for an involvement of CDK19 in the growth, progression, and metastasis of PCa as well as the development of castration resistance. In this study, the aim was to explore underlying molecular mechanisms of pro-oncogenic effects of CDK19, and to further characterize recently published CDK8/CDK19 inhibitors in the context of PCa.
Senexin A and senexin B are widely used inhibitors to investigate the effects of CDK8/CDK19 inhibition.
19- Chen M.
- Liang J.
- Ji H.
- Yang Z.
- Altilia S.
- Hu B.
- Schronce A.
- McDermott M.S.J.
- Schools G.P.
- Lim C.
- Oliver D.
- Shtutman M.S.
- Lu T.
- Stark G.R.
- Porter D.C.
- Broude Ev
- Roninson I.B.
CDK8/19 Mediator kinases potentiate induction of transcription by NFκB.
,20- Menzl I.
- Zhang T.
- Berger-Becvar A.
- Grausenburger R.
- Heller G.
- Prchal-Murphy M.
- Edlinger L.
- Knab V.M.
- Uras I.Z.
- Grundschober E.
- Bauer K.
- Roth M.
- Skucha A.
- Liu Y.
- Hatcher J.M.
- Liang Y.
- Kwiatkowski N.P.
- Fux D.
- Hoelbl-Kovacic A.
- Kubicek S.
- Melo Jv
- Valent P.
- Weichhart T.
- Grebien F.
- Zuber J.
- Gray N.S.
- Sexl V.
A kinase-independent role for CDK8 in BCR-ABL1+ leukemia.
Subsequently, structurally different and potent CDK8/CDK19 inhibitors have been developed, aiming to optimize the potency and specificity of inhibitors.
15- Dale T.
- Clarke P.A.
- Esdar C.
- Waalboer D.
- Adeniji-Popoola O.
- Ortiz-Ruiz M.-J.
- Mallinger A.
- Samant R.S.
- Czodrowski P.
- Musil D.
- Schwarz D.
- Schneider K.
- Stubbs M.
- Ewan K.
- Fraser E.
- TePoele R.
- Court W.
- Box G.
- Valenti M.
- de Haven Brandon A.
- Gowan S.
- Rohdich F.
- Raynaud F.
- Schneider R.
- Poeschke O.
- Blaukat A.
- Workman P.
- Schiemann K.
- Eccles S.A.
- Wienke D.
- Blagg J.
A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.
,17- Koehler M.F.T.
- Bergeron P.
- Blackwood E.M.
- Bowman K.
- Clark K.R.
- Firestein R.
- Kiefer J.R.
- Maskos K.
- McCleland M.L.
- Orren L.
- Salphati L.
- Schmidt S.
- Schneider Ev
- Wu J.
- Beresini M.H.
Development of a potent, specific CDK8 kinase inhibitor which phenocopies CDK8/19 knockout cells.
In this study, the response of different PCa cell lines to CCT251545
15- Dale T.
- Clarke P.A.
- Esdar C.
- Waalboer D.
- Adeniji-Popoola O.
- Ortiz-Ruiz M.-J.
- Mallinger A.
- Samant R.S.
- Czodrowski P.
- Musil D.
- Schwarz D.
- Schneider K.
- Stubbs M.
- Ewan K.
- Fraser E.
- TePoele R.
- Court W.
- Box G.
- Valenti M.
- de Haven Brandon A.
- Gowan S.
- Rohdich F.
- Raynaud F.
- Schneider R.
- Poeschke O.
- Blaukat A.
- Workman P.
- Schiemann K.
- Eccles S.A.
- Wienke D.
- Blagg J.
A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.
and G02788177.93-1, and report their effects on cell proliferation, androgen-independent cell growth, migration, and the phosphorylation of CDK8/CDK19 substrates, was characterized. The results of this study support the efficiency of these inhibitors because doses of 5 to 10 μmol/L lead to CDK8/CDK19 inhibition confirmed by reduction of STAT1-S727 phosphorylation (data not shown), a substrate commonly used to measure CDK8/CDK19 kinase activity.
21- Nitulescu I.I.
- Meyer S.C.
- Wen Q.J.
- Crispino J.D.
- Lemieux M.E.
- Levine R.L.
- Pelish H.E.
- Shair M.D.
Mediator kinase phosphorylation of STAT1 S727 promotes growth of neoplasms with JAK-STAT activation.
In concordance with the recent observation that CDK/CDK19 inhibition does not significantly affect proliferation at doses that are sufficient to reduce migration and invasion of PCa cells,
5- Brägelmann J.
- Klümper N.
- Offermann A.
- von Mässenhausen A.
- Böhm D.
- Deng M.
- Queisser A.
- Sanders C.
- Syring I.
- Merseburger A.S.
- Vogel W.
- Sievers E.
- Vlasic I.
- Carlsson J.
- Andrén O.
- Brossart P.
- Duensing S.
- Svensson M.A.
- Shaikhibrahim Z.
- Kirfel J.
- Perner S.
Pan-cancer analysis of the Mediator complex transcriptome identifies CDK19 and CDK8 as therapeutic targets in advanced prostate cancer.
CCT251545 and G02788177.93-1 influenced the cell viability of PC3, DU145, and LNCaP cells at 10 μmol/L, whereas lower doses showed no significant effect (
Supplemental Figure S1). Interestingly, similar results have been reported in colon cancer cells (HCT116). Bergeron et al
22- Bergeron P.
- Koehler M.F.T.
- Blackwood E.M.
- Bowman K.
- Clark K.
- Firestein R.
- Kiefer J.R.
- Maskos K.
- McCleland M.L.
- Orren L.
- Ramaswamy S.
- Salphati L.
- Schmidt S.
- Schneider Ev
- Wu J.
- Beresini M.
Design and development of a series of potent and selective type II inhibitors of CDK8.
demonstrated a dramatically decreased proliferation at doses around 10 μmol/L, which was phenocopied by
CDK8 knockout. Of note, the effects observed herein were most pronounced after 24 hours of treatment, which most probably might be explained by pharmacokinetics, which was not a further focus of this study.
The progression of PCa is characterized by the development of castration resistance after initial response to androgen deprivation. Progressing or recurring disease in a hormone-naive status is commonly treated with luteinizing hormone-releasing hormone agonists, which remains the standard of care for androgen deprivation.
23- Cornford P.
- van den Bergh R.C.N.
- Briers E.
- Van den Broeck T.
- Cumberbatch M.G.
- De Santis M.
- Fanti S.
- Fossati N.
- Gandaglia G.
- Gillessen S.
- Grivas N.
- Grummet J.
- Henry A.M.
- der Kwast T.H.V.
- Lam T.B.
- Lardas M.
- Liew M.
- Mason M.D.
- Moris L.
- Oprea-Lager D.E.
- der Poel H.G.V.
- Rouvière O.
- Schoots I.G.
- Tilki D.
- Wiegel T.
- Willemse P.M.
- Mottet N.
EAU-EANM-ESTRO-ESUR-SIOG guidelines on prostate cancer: part II-2020 update: treatment of relapsing and metastatic prostate cancer.
For metastatic PCa, androgen deprivation is combined with novel antiandrogens, such as apalutamide, enzalutamide, and abiraterone, or with conventional chemotherapy (docetaxel).
23- Cornford P.
- van den Bergh R.C.N.
- Briers E.
- Van den Broeck T.
- Cumberbatch M.G.
- De Santis M.
- Fanti S.
- Fossati N.
- Gandaglia G.
- Gillessen S.
- Grivas N.
- Grummet J.
- Henry A.M.
- der Kwast T.H.V.
- Lam T.B.
- Lardas M.
- Liew M.
- Mason M.D.
- Moris L.
- Oprea-Lager D.E.
- der Poel H.G.V.
- Rouvière O.
- Schoots I.G.
- Tilki D.
- Wiegel T.
- Willemse P.M.
- Mottet N.
EAU-EANM-ESTRO-ESUR-SIOG guidelines on prostate cancer: part II-2020 update: treatment of relapsing and metastatic prostate cancer.
In the setting of metastatic CRPC, the use of enzalutamide rather than bicalutamide is recommended on the basis of clinical trials showing that enzalutamide has significantly improved progression-free survival in patients.
24- Shore N.D.
- Chowdhury S.
- Villers A.
- Klotz L.
- Siemens D.R.
- Phung D.
- van Os S.
- Hasabou N.
- Wang F.
- Bhattacharya S.
- Heidenreich A.
Efficacy and safety of enzalutamide versus bicalutamide for patients with metastatic prostate cancer (TERRAIN): a randomised, double-blind, phase 2 study.
Bicalutamide is currently recommended in combination with radiotherapy for patients with prostate-specific antigen relapse and high risk for cancer progression.
23- Cornford P.
- van den Bergh R.C.N.
- Briers E.
- Van den Broeck T.
- Cumberbatch M.G.
- De Santis M.
- Fanti S.
- Fossati N.
- Gandaglia G.
- Gillessen S.
- Grivas N.
- Grummet J.
- Henry A.M.
- der Kwast T.H.V.
- Lam T.B.
- Lardas M.
- Liew M.
- Mason M.D.
- Moris L.
- Oprea-Lager D.E.
- der Poel H.G.V.
- Rouvière O.
- Schoots I.G.
- Tilki D.
- Wiegel T.
- Willemse P.M.
- Mottet N.
EAU-EANM-ESTRO-ESUR-SIOG guidelines on prostate cancer: part II-2020 update: treatment of relapsing and metastatic prostate cancer.
Bicalutamide is a nonsteroidal AR antagonist that competitively inhibits the action of androgens by binding to the AR. Thereby, the androgen-induced expression of AR target genes and consequently subsequent oncogenic effects on AR-sensitive PCa cells are attenuated. Mechanistically, bicalutamide stimulates AR nuclear translocation and its binding to AR responsive genes, and ultimately induces a transcriptionally inactive state of the AR.
25- Masiello D.
- Cheng S.
- Bubley G.J.
- Lu M.L.
- Balk S.P.
Bicalutamide functions as an androgen receptor antagonist by assembly of a transcriptionally inactive receptor.
As a result, bicalutamide inhibits proliferation and the progression to S phase of the cell cycle in a dose-dependent manner, which is abrogated by adding dihydrotestosterone.
25- Masiello D.
- Cheng S.
- Bubley G.J.
- Lu M.L.
- Balk S.P.
Bicalutamide functions as an androgen receptor antagonist by assembly of a transcriptionally inactive receptor.
Interestingly, bicalutamide acquires agonistic properties during long-term androgen ablation of LNCaP cells,
18- Culig Z.
- Hoffmann J.
- Erdel M.
- Eder I.E.
- Hobisch A.
- Hittmair A.
- Bartsch G.
- Utermann G.
- Schneider M.R.
- Parczyk K.
- Klocker H.
Switch from antagonist to agonist of the androgen receptor blocker bicalutamide is associated with prostate tumour progression in a new model system.
whereas the new generation of anti-androgens, such as enzalutamide, prevent AR nuclear translocation and do not exhibit any agonistic properties.
Prior work showed CDK19 to be significantly up-regulated in CRPC
6- Becker F.
- Joerg V.
- Hupe M.C.
- Roth D.
- Krupar R.
- Lubczyk V.
- Kuefer R.
- Sailer V.
- Duensing S.
- Kirfel J.
- Merseburger A.S.
- Brägelmann J.
- Perner S.
- Offermann A.
Increased mediator complex subunit CDK19 expression associates with aggressive prostate cancer.
and the expression of CDK8 and CDK19 to be increased in androgen-sensitive LNCaP cells
5- Brägelmann J.
- Klümper N.
- Offermann A.
- von Mässenhausen A.
- Böhm D.
- Deng M.
- Queisser A.
- Sanders C.
- Syring I.
- Merseburger A.S.
- Vogel W.
- Sievers E.
- Vlasic I.
- Carlsson J.
- Andrén O.
- Brossart P.
- Duensing S.
- Svensson M.A.
- Shaikhibrahim Z.
- Kirfel J.
- Perner S.
Pan-cancer analysis of the Mediator complex transcriptome identifies CDK19 and CDK8 as therapeutic targets in advanced prostate cancer.
under androgen deprivation (versus androgen stimulation). On the basis of these results, we hypothesized that CDK8/CDK19 influences PCa growth in an androgen-dependent manner and therefore contributes to castration resistance. To verify this hypothesis, cell viability of androgen-sensitive LNCaP cells in response to dual blockade of CDK8/CDK19 and the AR was measured. Hereby, a synergistic effect of combined G02788177.93-1 and bicalutamide treatment was observed, which reduced LNCaP cell viability to 50% in a time-dependent manner (
Figure 1A). In contrast, G02788177.93-1 or bicalutamide treatment alone had only modest antiproliferative effects, which were stable over time. Therefore, these results might indicate that CDK8/CDK19 at least partially mediates its pro-oncogenic effects via the AR axis and that dual therapy combining AR and CDK8/CDK19 blockade might be a strategy to increase treatment efficiency.
On the basis of a recently published study suggesting that CDK8/CDK19 mediates antiproliferative activity by inducing a premature G
1/S-phase transition leading to DNA damage and subsequent cell death,
7- Nakamura A.
- Nakata D.
- Kakoi Y.
- Kunitomo M.
- Murai S.
- Ebara S.
- Hata A.
- Hara T.
CDK8/19 inhibition induces premature G1/S transition and ATR-dependent cell death in prostate cancer cells.
whether the synergistic effect of dual G02788177.93-1 and bicalutamide treatment can be explained by an altered cell cycle was explored further. Comparing the distribution of cell cycle phases in response to G02788177.93-1 alone or in combination with bicalutamide, there was no significant difference (
Figure 1B), suggesting a different mechanism conveying the synergistic effects on cell viability. However, previous results
7- Nakamura A.
- Nakata D.
- Kakoi Y.
- Kunitomo M.
- Murai S.
- Ebara S.
- Hata A.
- Hara T.
CDK8/19 inhibition induces premature G1/S transition and ATR-dependent cell death in prostate cancer cells.
in LNCaP cells, showing a significant reduction of cells in G
1 phase and an increased proportion of cells in the S phase in response to CDK8/CDK19 blockade, were confirmed (
Figure 1B). Strikingly, there was no effect of bicalutamide alone on the distribution of cell cycle phases (
Figure 1B). In previous studies, bicalutamide was shown to block LNCaP cells from entering S phase by regulating the expression of specific cell-cycle regulatory genes.
26- Bai V.U.
- Cifuentes E.
- Menon M.
- Barrack E.R.
- Reddy G.P.-V.
Androgen receptor regulates Cdc6 in synchronized LNCaP cells progressing from G1 to S phase.
Of note, in the current experiments, cell cycle phases were measured 72 hours after treatment with bicalutamide, whereas previous studies used an observation period of 24 hours after treatment, which might explain this disconcordance. But because there was an antiproliferative effect of bicalutamide 72 hours after treatment (
Figure 1A), it can be assumed that bicalutamide still has an inhibitory effect on LNCaP cells in this experimental setting.
To further explore the effects of CDK8/CDK19 on castration resistance, the effects of G02788177.93-1 alone or in combination with bicalutamide on LNCaP Abl cells were investigated. LNCaP Abl is an LNCaP subline that is long-term androgen deprived and characterized by androgen-independent growth. Interestingly, CDK8/CDK19 inhibition alone had moderate effects on cell viability in a time-dependent manner, highlighting that androgen-independent growth might be partially mediated by CDK8/CDK19 (
Figure 1C). AR blockade alone by bicalutamide had no antiproliferative effects in LNCaP Abl (
Figure 1D), as expected and described before. Intriguingly, combining bicalutamide with G02788177.93-1 dramatically reduced cell viability of LNCaP Abl in particular after long-term treatment (
Figure 1D), providing evidence that CDK8/CDK19 might support androgen-independent growth and its blockade rescues androgen dependency. The general course of cell viability over time indicates that the dual treatment leads to synergistic antiproliferative effects rather than conveying toxic effects (
Figure 1E). In conclusion, in the light of previous findings, these results provide evidence that CDK8/CDK19 inhibition might be a promising therapeutic approach to overcome resistance against anti-androgenic therapy, the main reason for PCa-specific death.
Next, the aim was to verify previous results showing that CDK8/CDK19 inhibition reduces metastatic capacity of PCa cells.
5- Brägelmann J.
- Klümper N.
- Offermann A.
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- Böhm D.
- Deng M.
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- Vogel W.
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Pan-cancer analysis of the Mediator complex transcriptome identifies CDK19 and CDK8 as therapeutic targets in advanced prostate cancer.
Two different assays clearly confirmed that CDK8/CDK19 inhibition by the two novel inhibitors reduces migration of PCa cells. Opposite effects were seen in CDK19 overexpressing cells, specifying our observations (
Figure 2). As described above, a previous study showed that CDK8/CDK19 inhibition has major impact on cellular features that mediate cell migration and invasion. In subsequent experiments, CDK8/CDK19-mediated effects observed on migration and invasion can be at least partially explained by modulation of cell adhesion dependence in the presence of collagen I (
Figure 3). Collagen I is part of the extracellular matrix of the bone, but also of prostatic periglandular stromal tissue, which was confirmed by immunohistochemistry (data not shown). On the basis of these results, we hypothesize that CDK8/CDK19 deregulation might reduce the capacity of PCa cells to bind collagen I and therefore affect cellular adhesion and specifically facilitate migration through collagen I–containing stromal tissue. Intracellular mediators of collagen I interaction include integrins, which were found to negatively correlate with high CDK19 levels using cBioportal
in silico analysis (data not shown). The observation that 24 hours pretreatment with G02788177.93-1 is required for subsequent increase in adhesion might indicate that CDK8/CDK19 act through changes in transcriptional regulation.
CDK8 and CDK19 are part of the Mediator complex and therefore exert major impact on gene transcription.
13The Mediator complex: a central integrator of transcription.
Apart from that, recent studies identified multiple peptides to be phosphorylated by CDK8/CDK19 kinase activity.
14- Poss Z.C.
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- Old W.M.
- Taatjes D.J.
Identification of Mediator kinase substrates in human cells using cortistatin A and quantitative phosphoproteomics.
Through phosphorylation of specific pathway molecules, CDK8/CDK19 is involved in immune-oncological processes
27- Putz E.M.
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CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance.
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SEL120-34A is a novel CDK8 inhibitor active in AML cells with high levels of serine phosphorylation of STAT1 and STAT5 transactivation domains.
and cancer-related signaling, such as NF-κB activity.
10- Xu D.
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Skp2–MacroH2A1–CDK8 axis orchestrates G2/M transition and tumorigenesis.
To identify downstream substrates of CDK8/CDK19 kinase function, a large-scale phosphoarray screen (PamGene International B.V.) was performed with three different PCa cell lines after treatment with the CDK8/CDK19 inhibitor G02788177.93-1. The phosphorylation of numerous substrates was found to be recurrently altered in more than one cell line, which are listed in
Figure 4D. These substrates were subsequently assigned to cell functions (
Figure 4D), providing evidence that CDK8/CDK19 affects the metastatic potential of PCa cells through their kinase activity. Of note, further studies are needed to experimentally explore the biological significance of these findings and to differentiate direct from indirect effects of CDK8/CDK19. Regarding the latter, it seems possible that the changed phosphorylation of some substrates results from altered upstream signaling due to CDK8/CDK19 inhibition but not by direct kinase interaction between CDK8/CDK10 and substrates. This seems particularly likely because the signaling molecules listed in
Figure 4 are closely connected. However, these results provide clues about the pathways affected by CDK8/CDK19 inhibition with regard to their kinase function and may serve as a result tool for further studies.
Additionally, these observations underline the importance of CDK8/CDK19 as kinases, apart from their function as transcriptional coregulators within the Mediator complex. The shift of focus to this specific property of CDK8/CDK19 unrelated to the Mediator complex has yielded promising results in the past because other groups were able to show direct CDK8/CDK19 kinase impact on essential oncogenic signaling pathways, such as the β-catenin/Wnt pathway.
29- Cai W.
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- Cao J.
Downregulation of CDK-8 inhibits colon cancer hepatic metastasis by regulating Wnt/β-catenin pathway.
The overall high similarity of affected substrates between different cell lines indicates relevance of CDK8/CDK19 kinase function in all stages of the disease, including hormone-sensitive PCa and CRPC phenotypes.
Collectively, the results of these functional experiments strengthen recent findings identifying CDK8/CDK19 as contributors to the progression of PCa. This study, for the first time, showed i) that CDK8/CDK19 inhibition sensitizes PCa cells to androgen blockade, ii) that reduced migratory capacity of PCa cells after CDK8/CDK19 inhibition is linked to increased adhesion in a collagen I–dependent manner, possibly via altered integrin signaling, and iii) that multiple phosphorylated substrates of CDK8/CDK19 highlight the significance of the CDK8/CDK19 kinase function.
Author Contributions
S.P., A.O., M.C.R., J.B., and V.J. conceptualized and designed the study; A.O., V.J., F.B., D.K., A.-L.L., and J.B. acquired data and performed experiments; A.O., V.J., M.C.R., F.B., S.P., J.B., A.S.M., V.S., L.T., J.K., and A.S.M. analyzed and interpretated data; Z.C. provided resources; S.P., A.O., V.J., J.B., M.C.R., V.S., L.T., and J.K. participated in data discussion; A.O. and M.C.R. performed statistical analysis; S.P., A.O., and V.J. drafted the manuscript; all coauthors revised the manuscript.
Article Info
Publication History
Published online: February 15, 2022
Accepted:
January 14,
2022
Footnotes
Supported by the Rudolf Becker-Foundation grant T0321/36080/2020/kg (S.P. and J.K.); the University of Luebeck fellowship (A.O. and M.C.H.); a University of Luebeck medical doctoral fellowship (V.J.); the German Cancer Aid (Deutsche Krebshilfe) through a Mildred Scheel Nachwuchszentrum grant (grant number 70113307 ) and the Else Kröner-Fresenius Stiftung Memorial grant 2018_EKMS.35 (J.B.); and the German Research Foundation (Deutsche Forschungsgesellschaft) grant (J.K.).
A.O. and V.J. contributed equally to this work.
Disclosures: None declared.
Copyright
© 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.