biomedicines

Article Anticancer Imidazoacridinone C-1311 is Effective in Androgen-Dependent and Androgen-Independent Prostate Cancer Cells

1, , 2 3 4 Magdalena Niemira * † , Barbara Borowa-Mazgaj , Samuel B. Bader , Adrianna Moszy ´nska , Marcin Ratajewski 5 , Kaja Kara´s 5 , Mirosław Kwa´sniewski 6, Adam Kr˛etowski 1,7 , 8 3 1, , Zofia Mazerska , Ester M. Hammond and Anna Skwarska * † 1 Clinical Research Centre, Medical University of Bialystok, 15-276 Bialystok, Poland; [email protected] 2 Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA; [email protected] 3 Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK; [email protected] (S.B.B.); [email protected] (E.M.H.) 4 Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, 80-416 Gdansk, Poland; [email protected] 5 Laboratory of Epigenetics, Institute of Medical Biology PAS, 93-232 Lodz, Poland; [email protected] (M.R.); [email protected] (K.K.) 6 Centre for Bioinformatics and Data Analysis, Medical University of Bialystok, 15-276 Bialystok, Poland; [email protected] 7 Department of Endocrinology, Diabetology and Internal Medicine, Medical University of Bialystok, 15-276 Bialystok, Poland 8 Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 80-233 Gdansk, Poland; zofi[email protected] * Correspondence: [email protected] (M.N.); [email protected] (A.S.); Tel.: +48-85-831-8893 (M.N.); +48-85-831-8893 (A.S.) These authors contributed equally to this work. †


Received: 3 July 2020; Accepted: 16 August 2020; Published: 19 August 2020


Abstract: The androgen receptor (AR) plays a critical role in prostate cancer (PCa) development and metastasis. Thus, blocking AR activity and its downstream signaling constitutes a major strategy for PCa treatment. Here, we report on the potent anti-PCa activity of a small-molecule imidazoacridinone, C-1311. In AR-positive PCa cells, C-1311 was found to inhibit the transcriptional activity of AR, uncovering a novel mechanism that may be relevant for its anticancer effect. Mechanistically, C-1311 decreased the AR binding to the prostate-specific antigen (PSA) promoter, reduced the PSA protein level, and, as shown by transcriptome sequencing, downregulated numerous AR target genes. Importantly, AR-negative PCa cells were also sensitive to C-1311, suggesting a promising efficacy in the androgen-independent PCa sub-type. Irrespective of AR status, C-1311 induced DNA damage, arrested cell cycle progression, and induced apoptosis. RNA sequencing indicated significant differences in the transcriptional response to C-1311 between the PCa cells. Gene ontology analysis showed that in AR-dependent PCa cells, C-1311 mainly affected the DNA damage response pathways. In contrast, in AR-independent PCa cells, C-1311 targeted the cellular metabolism and inhibited the genes regulating glycolysis and gluconeogenesis. Together, these results indicate that C-1311 warrants further development for the treatment of PCa.

Keywords: androgen receptor; prostate cancer; C-1311/Symadex™; transcriptomic profiling; next-generation sequencing

Biomedicines 2020, 8, 292; doi:10.3390/biomedicines8090292 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 292 2 of 19

1. Introduction Prostate cancer (PCa) remains one of the most common cancers in the male population and has a poor prognosis. Recent epidemiological data show that PCa is second only to lung cancer as a cause of cancer-related death among males in the United State [1]. Unfortunately, the 5-year survival rate of advanced PCa is 29% [2]. PCa is a highly heterogeneous disease, at both the molecular and cellular level, with a complex pattern of metastatic spread [3]. Such heterogeneity underlies the mixed response of PCa cells to treatment, highlighting the need for multi-targeted therapies. The androgen receptor (AR) is a key transcription factor that drives PCa tumorigenesis and progression. Accordingly, androgen deprivation therapy (ADT), which targets the AR via chronic administration of gonadotropin-releasing hormone analogs, anti-androgens, or their combination, is considered a standard therapy for patients with locally advanced or metastatic PCa [4]. However, despite initial response rates in excess of 80%, patients invariably develop resistance to this therapy within 2–3 years, transitioning to a more aggressive form of PCa due to adaptive mechanisms or clonal selections [5,6]. Advanced tumors, often termed metastatic castration-resistant PCa (mCRPC), may still have abnormally activated AR signaling pathways that fuel tumor survival and proliferation even in the presence of ADT [7]. In this context, novel therapeutic agents that target the AR are urgently required. The 1-nitroacridines are potent DNA-binding compounds that inhibit the growth of tumor cells [8], and have shown promising pre-clinical activity in colon [9] and prostate cancer cell lines [10]. The 1-nitroacridine, C-1748, has been previously reported to block AR activity in hormone-dependent PCa models in vitro and inhibit the growth of PCa xenografts in mice [8]. Despite those promising pre-clinical results, C-1748 has not progressed to clinical trial. In an effort to improve the efficacy and toxicity profile, a second generation of acridine derivatives, the imidazoacridinones, has been developed. The most active imidazoacridinone, C-1311 (Symadex), was well tolerated in Phase I clinical trials in patients with advanced solid tumors [11], and demonstrated promising efficacy in Phase II studies in patients with breast cancers refractory to anthracyclines and taxanes [12]. C-1311 is a DNA-damaging intercalator and inhibitor of topoisomerase II activity [13], which also blocks angiogenesis by targeting hypoxia-inducible factor 1α (HIF-1α)[14]. Additionally, C-1311 inhibits the oncogenic receptor tyrosine kinase FLT3 [15], and sensitizes p53-mutated cancer cells to radiation [16]. However, its efficacy in PCa cells never has been determined. Imidazoacridinones contain an imidazole ring attached to an acridinone core and diaminoalkyl side chain, similar to that found in topoisomerase II inhibitor, mitoxantrone. Given that mitoxantrone in combination with cabazitaxel has recently shown durable responses in patients with mCRPC [17], we investigated the anti-prostate cancer potential of the imidazoacridinones. Here, we show for the first time that imidazoacridinone C-1311, in addition to its known DNA-damaging properties, is a promising agent that blocks the transcriptional activity of the AR and effectively kills PCa cells, irrespective of their AR status.

2. Materials and Methods

2.1. Chemicals and Reagents The imidazo- and triazoloacridinones were synthesized at the Department of Pharmaceutical Technology and Biochemistry, at the Gdansk University of Technology (Poland), as described previously [18,19], and were a kind gift from the late Prof. Jerzy Konopa. Drugs were stored at 20 C as 50 mM stock solutions in 50% (v/v) ethanol, and freshly diluted in water before use. − ◦ All other chemicals, unless stated otherwise, were purchased from Sigma-Aldrich (Sigma-Aldrich, Darmstadt, Germany).

2.2. Cell Lines and 3D Spheroid Cultures The human prostate cancer LNCaP (ATCC® CRL-1740™) and DU-145 (ATCC® HTB-81D™) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The 22Rv1 cells were a kind gift from Prof. Valentine Macaulay (University of Oxford). LNCaP Biomedicines 2020, 8, 292 3 of 19 and 22Rv1 cells were maintained in RPMI 1640 medium. DU-145 cells were grown in DMEM. All media were supplemented with 10% FBS (Sigma-Aldrich, Darmstadt, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany). Cells were kept at 37 ◦C under a 5% CO2. Cells were routinely tested for mycoplasma and found to be negative. For spheroid formation, 5000 cells were seeded in 200 µL of complete growth medium in round bottom, ultra-low attachment 96-well plates (#7007, Corning, New York, NY, USA). Spheroids were grown for 5–10 days, after which the 22Rv1 and DU-145 cells produced spheroids of approx. 400 µM in diameter, while the LNCaP cells yielded much larger spheroids (approx. 800 µM in diameter). The medium was renewed, and the spheroids were subsequently treated with the drug for 96 h. Spheroid size was monitored using the GelCount imaging system (Oxford Optronix).

2.3. Plasmid Construction

AR reporter vector (AREIII)3-(AREII)3-(AREI)3-tk-Luc was generated by insertion of three copies of the chemically synthesized ARE sequence from the PSA gene and tk (tymidine kinase) promoter into a pGL4hygro vector (Promega, Madison, WI, USA). The three ARE sequences were as follows: AREIII (50-TCGACGAGGAACATATTGTATCGAG-30)[20], AREII (50-TCGACAGGGATCAGGGAGTCTCACCAGGGATCA-30)[21] and AREI (50-GATCCTTG CAGAACAGCAAGTGCTAGCTG-30)[22].

2.4. Generation of the LNCaP-ARE-Luc Reporter Cell Line

LNCaP cells were seeded on 6-well plates and transfected with 2 µg of the (AREIII)3-(AREII)3- (AREI)3-tk-Luc plasmid using the FugeneHD transfection reagent (Promega). Individual hygromycin B-resistant cell colonies were isolated, expanded, and screened for the DHT-inducible expression of luciferase. To assess the AR transactivation activity following 24 h drug treatment, cells were lysed, and the level of firefly luciferase was measured using the Luciferase Assay System (Promega), according to the manufacturer’s instructions.

2.5. Transient Transfection with the ARE-Luc Vector LNCaP cells were seeded on a 96-well plate (10,000 cells per well) overnight, co-transfected with the (AREIII)3-(AREII)3-(AREI)3-tk-Luc plasmid or the pG4.14luc/hygro (Promega) and pCMV-SEAP plasmid (a kind gift from Dr. S. Schlatter) using the FugeneHD transfection reagent. Cells were treated with an experimental compound 24 h after transfection, and reporter gene activity was assayed after a further 24 h. Luciferase activities were normalized to the corresponding SEAP activity, which was used as the transfection efficiency control.

2.6. Cell Viability Assays Cell viability was measured using an MTT (3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, cells (2 105) were seeded in 24-well plates, left to attach overnight, and × treated with the drugs for 72 h. After exposure, 0.5 mg/mL MTT was added into each well, and the plates were incubated for 4 h at 37 ◦C. The medium was removed, and the remaining formazan crystals were dissolved in DMSO. Absorbance was measured at 540 nm. The viability of the cells in the 3D spheroid cultures was measured using a resazurin assay. Briefly, single spheroids were grown in 200 µL of culture medium in 96-well plates. Following the drug treatment, 100 µL of the medium was removed, and replaced with a 20 µL resazurin solution (0.15 mg/mL) in PBS. After incubation for 4 h at 37 ◦C, the absorbance was measured at an excitation/emission wavelength of 544/590 nm. Cell viability was expressed as the percentage of the vehicle-treated control and the cytotoxic effect of the drug was assessed by the IC50 values. Biomedicines 2020, 8, 292 4 of 19

2.7. Western Blot Analysis Following the drug treatment, cells were lysed for 20 min on ice with buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA 1% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 50 mM β-glycerolophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Proteins (50 µg) were separated by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used: anti-PSA (#5877 Cell Signaling Technology, Danvers, MA, USA), anti-AR (#5153S Cell Signaling Technology), anti-GAPDH-HRP conjugate (#8884S Cell Signaling Technology), anti-γH2AX (#05-636-I, Millipore, Burlington, MA, USA), #anti-p53 (DO-I, Santa Cruz Biotechnology,Dallas, TX, USA), anti-p21 (#2947 Cell Signaling Technology), anti-PARP (#9542 Cell Signaling Technology), and anti-β-actin (C4, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoblots were developed using the Li-COR Odyssey imaging system, except for the anti-PSA, anti-AR, and anti-GAPDH-HRP conjugate, which were visualized using an enhanced chemiluminescence detection kit, the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

2.8. ChIP Assay for the Androgen Receptor LNCaP cells were cultured until an 80% confluence and exposed to C-1311. Proteins were cross-linked with formaldehyde, and then the cells were harvested, lysed, and the DNA sonicated with a VCX-130 sonicator (Sonics & Materials Inc., Newtown, CT, USA). Chromatin immunoprecipitation was performed using the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (EMD Millipore, Burlington, MA, USA) according to the manufacturer’s protocol. The following antibodies were used: control mouse IgG (EMD Millipore, Burlington, MA, USA) and anti-androgen receptor antibody—ChIP Grade (#ab74272, Abcam, City, UK). The relative enrichment of the PSA gene sequences was analyzed with real-time PCR using the SYBR Green I Master Mix on a LightCycler 96 (Roche, Basel, Switzerland). Primers P1 complementary to the PSA were as follows: forward 50-GAGTGCTGGTGTCTTAGGGC-30 and reverse 50-GCTAGCACTTGCTGTTCTGC-30; primers P2 complementary to the PSA intron 1 were as follows: forward 50-CCTCTTCCAGCAACTGAACC-30 and reverse 50-TCAGGGTTGACAGGAGGAAC-30. The AR negative-binding region in the first intron of the PSA was identified using Matinspector software [23]. The amplification of the soluble chromatin prior to immunoprecipitation was used as an input control. Quantification was performed using the dCt method with the Ct obtained for input DNA as a reference value: 1000 2-dCt, where dCt = Ct × sample Ct input DNA. − 2.9. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) Total RNA was purified from LNCaP and DU-145 cells with the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. The RNA concentration, purity, and integrity were assessed by Qubit (Invitrogen, Carslbad, CA, USA) and Tape Station 2200 (Agilent Technologies, Santa Clara, CA, USA). cDNA was synthesized using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol. The mRNA expression levels were measured using RT-qPCR performed in 20 µL volumes containing SYBR Green I Dye using the LightCycler® 480 SYBR Green I Master (Roche, Basel, Switzerland). The sequences of the primers were as follows: AR forward, 50-GGAATTCCTGTGCATGAAA-30; AR reverse, 50-CGAAGTTCATCAAAGAATT-30 [24]; GAPDH forward, 50-TGCACCACCAACTGCTTAGC-30; GAPDH reverse, 50-GGCATGGACTGTGGTCATGAG-30 [25]. The levels of AR were normalized to the GAPDH values. The quantitative PCR values were determined for each of the mRNAs levels using the comparative Ct (∆∆Ct) method. Biomedicines 2020, 8, 292 5 of 19

2.10. Flow Cytometry Analysis Following drug treatment, BrdU (20 µM) was added and the cells were incubated for 1 h in dark before fixation in ice-cold 70% ethanol. Samples were treated with 2 M HCl and blocked in 2% FBS in PBS solution before treatment with anti-BrdU primary antibody (BD Biosciences, San Jose, CA, USA) and secondary Alexa Fluoro 488 antibody (Invitrogen). Propidium iodide (Sigma Aldrich, Darmstadt, Germany) was added to the samples to determine the total DNA content. For the detection of apoptosis, cells, following drug treatment, were stained using the Annexin V-FITC Apoptosis Detection Kit (#556547, BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions. Samples were run on a BD FACSCalibur machine (BD Biosciences, San Jose, CA, USA) and plots were analyzed with FloJo software (v10, LLC, Ashland, OR, USA).

2.11. RNA-Seq Sample Preparation and Sequencing RNA-seq libraries were constructed from 1 µg of total RNA with an RNA integrity number (RIN) > 8, using the Illumina TruSeq® Stranded Total RNA Library Prep Kit (#20020598, Illumina, San Diego, CA, USA). Indexed libraries were pooled, clustered with use the cBot, and sequenced on the Illumina HiSeq 4000 platform generating 150 bp paired end reads (2 75 bp). Sequencing × data were processed to obtain fastq files with the bcl2fastq pipeline (Illumina, San Diego, CA, USA), including demultiplexing and adapter trimming steps. The quality of the reads was assessed using FastQC (Babraham Institute, Cambridge, UK). The BBduk tool (U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA, USA) was used to filter and soft trim all reads of high quality, as well as to filter out fragments originated from rRNAs. Cleaned reads were mapped to the human reference genome GRCh38 using the splice-aware aligner STAR [26]. Unique counts per gene were calculated with the in-built option in STAR and used for data visualization and further differential gene expression analysis. The quality of the mapping was assessed with the Flagstat module from Samtools [27] as well as RSeQC [28]. Mapped reads were also visually inspected with the Integrative Genomics Viewer [29]. Samples size factors were estimated, and counts were normalized using the trimmed mean of the M-values (TMM). Differential expression analysis was performed with EdgeR software [30], assuming a negative binomial distribution. After model fitting, the dispersion estimates were obtained, and a general linearized model was applied. The likelihood ratio test (LRT) was used to call differentially expressed genes (DEGs) under |log2FC| > 1 and a false discovery rate (FDR) corrected p-value < 0.05. For functional analysis, the gene ontology (GO) enrichment analysis—biological process (BP) by STRING (https://string-db.org) and the Ingenuity Pathway Analysis (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) were used. The significance of the association between the data set and the canonical pathway was determined based on two parameters: (a) the ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway; and (b) a p value calculated using Fischer’s exact test determining the probability that the association between the genes in the data set and the canonical pathways is due to chance alone. The RNA-seq data were deposited in the Gene Expression Omnibus (GEO) database (accession number: GSE151113).

2.12. Statistical Analysis Statistical differences were determined using GraphPad Prism 8 software (v8.4.3, GraphPad Software, Inc., San Diego, CA, USA). Unless otherwise indicated, a one-way ANOVA test was used, followed by a Dunnett test for each comparison. A p value less than 0.05 was considered significant (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Gene Set Enrichment Analysis (GSEA) was performed using the WebGestaltR R package with a ranked list of all the genes based on log10 (FDR-corrected p-value) and a change direction as up- (+) or downregulated ( ). Over-representation − analysis of the ‘AR targets upregulated by AR’ was performed with a hypergeometric test using the clusterProfiler R package and applying as the background the list of genes, which were analyzed in Biomedicines 2020, 8, 292 6 of 19

RNA-seq. Volcano plots were created in the R environment. Heat maps were generated with the “pheatmap” R package and GraphPad Prism 8. Venn diagrams were created with BioVinci software (v1.1.5, BioTuring, San Diego, CA, USA).

3. Results

3.1. Imidazoacridinones Are Potent Against AR-Dependent PCa Cells To begin, we examined the in vitro cytotoxic activity of the imidazoacridinones (Figure S1A) and their close analogs, the triazoloacridinones (Figure S1B), which contain a triazole ring condensed with the acridinone chromophore. The set of seven imidazoacridinones and triazoloacridinones, with diverseBiomedicines chemical 2020, 8, x FOR structures, PEER REVIEW were screened for their cytotoxic effect on AR-dependent6 of 18 LNCaP cells. Cells were incubated with the drugs for 72 h and the cell viability was determined using an MTT Cells were incubated with the drugs for 72 h and the cell viability was determined using an MTT assay. The resulting IC values indicated that the LNCaP cells were particularly sensitive to three of assay. The resulting50 IC50 values indicated that the LNCaP cells were particularly sensitive to three of the imidazoacridinones,the imidazoacridinones, C-1310, C-1310, C-1311, C-1311, and and C-1371 C-1371 (Figure (Figure 1A1A and and Figure Figure S2A). S2A).

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2 2 1 Cytotoxicactivity IC Cytotoxic activity IC activity Cytotoxic 0 0 2 0 1 0 1 5 2 6 1 3 7 9 98 05 33 3 4 29 303 531 -121 -1 -131 -13 -13 -141 -1 -1 -12 -1299 -1 -13 -1 -15 C C C C C C C C C C C C C C Figure 1.FigureCytotoxic 1. Cytotoxic activity activity of the of (A )the imidazoacridinones (A) imidazoacridinones and (andB) triazoloacridinones (B) triazoloacridinones in AR-dependentin AR- dependent prostate cancer LNCaP cells. IC50 values were determined after 72 h of treatment based on prostate cancer LNCaP cells. IC50 values were determined after 72 h of treatment based on the dose–responsethe dose–response curves andcurves presented and presented as bar as bar graphs. graphs. Data Data are the the mean mean ± SD, SD,n = 3.n Significance:= 3. Significance: One-way ANOVA with Tukey’s multiple comparison test; ** p< 0.01, * p< 0.05. ± One-way ANOVA with Tukey’s multiple comparison test; ** p < 0.01, * p < 0.05. These active compounds share structural features, including the presence of a diaminoethyl side Thesechain active and hydroxyl compounds group shareattached structural to an acridinone features, ring including (Figure S1A). the Interestingly, presence of among a diaminoethyl the side chaintriazoloacridinones, and hydroxyl only group the C-1303 attached derivative, to an which acridinone is a structural ring (Figure analogue S1A). of imidazoacridinone Interestingly, among the triazoloacridinones,C-1311, had a significantly only the lower C-1303 IC50 derivative,value (Figure which 1B and is Figure a structural S2B). Together, analogue these of data imidazoacridinone suggest that the selected imidazoacridinones may be effective against PCa cells. As C-1311 has already C-1311, had a significantly lower IC50 value (Figure1B and Figure S2B). Together, these data suggest that the selecteddemonstrated imidazoacridinones limited toxicity may and be high effective efficacy against in Phase PCa II clinical cells. trials, As C-1311 we focused has already further studies demonstrated on this compound. limited toxicity and high efficacy in Phase II clinical trials, we focused further studies on this compound. 3.2. C-1311 Induces Global Differences in the Transcriptome Profiles of PCa Subtypes 3.2. C-1311 Induces Global Differences in the Transcriptome Profiles of PCa Subtypes The AR acts as a ligand-regulated transcription factor [31]. Given the potent activity of the Theselected AR acts imidazoacridinones as a ligand-regulated in the transcription AR-positive factorLNCaP [31 cells,]. Given next thewe potent examined activity how of these the selected imidazoacridinonescompounds affect in theglobal AR-positive gene expression LNCaP in PCa cells, cells next with we a different examined AR how status. these To this compounds end, we affect global geneperformed expression transcriptomic in PCa cellsprofiling with using a di ffnext-genererent ARation status. sequencing To this (RNA-seq) end, we performed for one of the transcriptomic most active imidazoacridinones, C-1311. Differentially expressed genes (DEGs) were determined based on profiling using next-generation sequencing (RNA-seq) for one of the most active imidazoacridinones, the FDR < 0.05 and |log2FC|> 1. As shown by the volcano plots (Figure 2A) and Venn diagram C-1311.(Figure Differentially 2B), C-1311 expressed induced significant genes (DEGs) changes were in the determined whole transcriptome based on signature the FDR of the< 0.05 PCa andcells.| log2FC| > 1. AsInshown AR-positive by the LNCaP volcano cells, plots203 genes (Figure were2A) up andregulated Venn and diagram 533 genes (Figure were2 downregulated,B), C-1311 induced significantwhereas changes in AR-negative in the whole DU-145 transcriptome cells, 1035 signature genes were of the upregulated PCa cells. Inand AR-positive 588 genes LNCaPwere cells, 203 genesdownregulated. were upregulated Among andthe up- 533 and genes downregulated were downregulated, genes, only 23 whereas(1.9%) and in 25 AR-negative (2.2%), respectively, DU-145 cells, 1035 genesoverlapped were upregulated between LNCaP and and 588 DU-145, genes werewhich downregulated. was consistent with Among the different the up- AR-dependency and downregulated of these two PCa cell lines. genes, only 23 (1.9%) and 25 (2.2%), respectively, overlapped between LNCaP and DU-145, which was Next, we analyzed the functional categories and canonical pathways for the DEGs using consistentSTRING with the(https://string-db.org) different AR-dependency and the ofIngenu theseitytwo Pathway PCacell Analysis lines. (IPA; QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis), respectively. As shown by the GO BP enrichment analysis, the C-1311 treatment in AR-positive LNCaP cells induced significant changes in expression of genes involved in the DNA damage response, cell division, and cell cycle-related pathways (mitotic cell cycle, chromosome organization) (Figure S3A). A detailed IPA analysis of the altered canonical pathways showed the most significant gene transcript enrichment in the pathways was related to the G2/M DNA damage checkpoint regulation, ATM signaling, BRCA1-mediated DNA damage response, or cell cycle control of replication (Figure S3B Biomedicines 2020, 8, 292 7 of 19

Next, we analyzed the functional categories and canonical pathways for the DEGs using STRING (https://string-db.org) and the Ingenuity Pathway Analysis (IPA; QIAGEN Inc., https: //www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis), respectively. As shown by the GO BP enrichment analysis, the C-1311 treatment in AR-positive LNCaP cells induced significant changes in expression of genes involved in the DNA damage response, cell division, and cell cycle-related pathways (mitotic cell cycle, chromosome organization) (Figure S3A). A detailed IPA analysis of the altered canonical pathways showed the most significant gene transcript enrichment in the pathways was related to the G2/M DNA damage checkpoint regulation, ATM signaling, BRCA1-mediated DNA damage response, or cell cycle control of replication (Figure S3B and Table S1). Surprisingly, BP gene ontology enrichment revealed that in AR-negative DU-145 cells treated with C-1311, the DEGs were mainly involved in the regulation of metabolic processes (Figure S3A); in particular, glycolysis, gluconeogenesis, or glucocorticoid receptor signaling (Figure S3B and Table S2). Together, these results indicate that the transcriptional response of PCa cells to C-1311 is heterogeneous.

A LNCap/KLNCap LNCaP DU145/KDU145 Bioconductor packageA EnhancedVolcano Bioconductor package EnhancedVolcano DU-145

LNCaP NS Log2 FC P P & Log2 FC DU-145 NS Log2 FC P P & Log2 FC

6 6 CYLD OLR1 POMZP3

ZBTB21 MIR7107 RASEF C12orf50 ATF3 DTWD2 RAB30 PHF3 ZMYND10 WHAMM AC005264.2 SPC24 CDC7 DUSP9 ZNF184 LPXNCCNB1IP1 XIRP2−AS1 MXD3 FOXO3 BEST1 LOC101929435 LOC101927904 PGAM1 NFKB2 CCT6P1 RAET1L MICU3 DNAJC25 MMP19 ASF1B KNTC1 GPI PEX11A RRM2 RAB3A TTC19 ZNF267 LOC102723993 4 CCNA2 RTKN2 RRM2B LOC100132174 4 FBXO44 CEBPA CCL4L1 SNHG12 ROS1 EXO1 CENPU TIMELESS BTG2 SEMA4G RBL1 NADSYN1 SUOX ZNF79 SNHG17 VWF P HIST1H2AH FAM213B H2AFX LOC101929547 P RP4−553F4.6 FAM111B TRIP10 MDM2

CDKN1A LPCAT4 PTBP2 IFNB1 GPR19CENPE GINS1 FANCD2 LHX3 FAM13A−AS1 PROM2 ZFYUSP36 PMAIP1

10 HIST1H2BF SAPCD2 VCY CCDC157 PSG10P TGM4 HIST1H2BM GLS2 10 DHRS2 PLK1 MCM5 MIR210HG NDRG2 HSD17B8 KRTAP5−AS1 g SLC37A2 RPS27L AC004603.4 CDKN2C ORC1 LCE1B g IRF7 POLD1 TTC24 RHOH HERC3 RRP12 C10orf67 TXNIP o PTH FAS SLC1A1 CALCB SPIN4 o LSS VIPR1 PDGFC LINC00617 WDR63 LRRTM4 EDA LIF PHF21B GPR158 L CDC20 SYCE2 PPFIBP2 UCN VIP ABCC9 L PRDM4 IFI44 MNS1 LOC81691 LOC148696 RP11−167H9.6 FAM102B LHX3 GBP5 KLRC3 - BLNK OR4F13P APOBEC3H ALOXE3 PSG8 - CDT1 KIF1A PATL2 KIF26A HIST1H2BG C19orf57 BBC3 RNVU1−18 RAB6C LDHD DDX60 IFI44L HIST1H3C LOC101928248 ANKRD22 DIO2 LZTS1 ZNF519 FRMPD2 SULF2 JAKMIP1 LOC100506100 BIK TUBA4B XIRP2 HIST2H4B LOC642846 RINL ZNF334 VGF BHLHE41 HIST2H3A GPR137C TMEM217 DRAXIN MIR6827 MYT1 MANSC1 PIDD FAM157B IFNL1 GJB5 HPN NREP AP3B2 CCDC140 SPON1 2 LOC102724441 CDT1 LOC101929566 DCN TREH TNFRSF19LTA FGF9 PINLYP RASGEF1B EDN2 ZNF750 FAM196A 2 SORBS2 KCNIP2 PGPEP1 C5 DHRS2 NECAB1 HIST1H2BA LOC102723572 FAM57B FLJ35282 MUC19 LOC102723799 EPN3 CCDC64B AMHR2TTC29 IL1RL1 NEURL3 C14orf169 TERT ZNF492 TTBK1 LOC101929327 LOC101928738 PLEKHB1 PATE2FBN2 HIST1H2AA ARSI LOC100129083 STAB2 FAM87A RP3−326I13.1 METTL7A LCP1 COLEC12FGF19 SLC26A4−AS1 DNAJC5G LOC101927898 HSD17B6 GRHL3 LOC102724512 POU5F2 LOC101929177 DNAH10 F2 HIST1H3E TMPRSS2 TBX15 D

0 0 −3 0 3 6 −3 0 3 6 Log fold change 2 Log2 fold change Total = 29744 variables Total = 29744 variables

ANG ALOX12B BLACAT1 B ANKRD55 CCDC163P BBC3 CCNB1 BEST4 CCNF BTG2 CDCA3 CABP1 DUSP9 CDKN1A DEGs FAM64A CEACAM1

DEGs GTSE1 DRAXIN KIF20A FAM212B 23 508 25 563 KPNA2 180 1012 FCGBP LGI2 regulated

FLJ35282 - LRRC45

regulated GADD45A - LZTS1 GDNF

Up Down MALL KCNT1 MXD3 LHX3 NMU LIF PIF1 LINC00886 PLK1 LNCaP DU-145 PLK3 LNCaP DU-145 PLOD2 SLC12A4 PSRC1 SLC30A1 RNASE4 STAB2 SAPCD2 TRNAK13 SCARA3 SKP2

Figure 2. Global changes in the C-1311-regulated transcriptional profile in PCa cells. LNCaP and DU-145 cells were treated with 1 µM C-1311 for 24 h and subjected to RNA-seq analysis (n = 3). (A) Volcano plot of the differently expressed genes in PCa cells exposed to C-1311. The horizontal line at a false discovery rate (FDR) = 0.05; vertical line at |log2FC| = 1. (B) Venn diagram of differentially expressed genes (DEGs) in PCa cells exposed to C-1311.

3.3. C-1311 Significantly Blocks Androgen Receptor Transactivation Activity In AR-positive LNCaP cells treated with C-1311, the DEGs in BP were mainly enriched in processes regulating cell cycle progression. Several studies show that AR signaling is significantly implicated in DNA replication, checkpoint mechanisms, cell division, or DNA damage response, and inhibition of AR activity may lead to the induction of severe DNA damage and apoptosis of PCa cells [32,33]. Thus, next we focused on the effect of C-1311 on the AR-mediated transcriptional response. For this purpose, we designed the ‘AR targets upregulated by the AR’ gene signature (Table S3), using the Biomedicines 2020, 8, 292 8 of 19

TRRUST v2 database (Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining) (https://www.grnpedia.org/trrust/)[34] and recurrent AR target genes in the literature [32,35–37]. RNA-seq data were interrogated for the enrichment in the tested AR target gene signature. Gene Set Enrichment Analysis (GSEA) indicated the strong repression of the ‘AR targets upregulated by the AR’ gene signature (NES = 2.91, FDR = 1 10 10) (Figure3A). Detailed analysis showed that of the 409-gene ‘AR targets − × − upregulated by the AR’ signature, 117 genes identified as DEGs, were significantly repressed by C-1311 (over-representationBiomedicines 2020, analysis, 8, x FOR PEER Fisher’s REVIEW exact test p.adjust = 4.58 10 99). For example, C-13118 of 18 decreased × − the expression99). For of example, the AR C-1311 target decreased genes that the are expression known toof the regulate AR target DNA genes repair that (PARP1,are known RAD54L), to regulate control of the G2/MDNA phase repair progression (PARP1, RAD54L), (CDC6, CDC20,control of CDK1, the G2/M CCNA2, phase CCNB2,progression CDK2), (CDC6, mitotic CDC20, division, CDK1, spindle checkpoint,CCNA2, and cytokinesisCCNB2, CDK2), (MAD2L1, mitotic division, NUSAP1, spindle PRC1, checkpoint, KIF20A, and ZWINT) cytokinesis (Figure (MAD2L1,3B). NUSAP1, PRC1, KIF20A, ZWINT) (Figure 3B).

A AR Targets Upregulated by AR C

NES -2.91 FDR 1E-10

D 2.5

2.0

1.5 PRC1 TACC3 1.5 KNTC1 B 1 BUB1B 1.0 MAD2L1 0.5 * KIF20A ** ANLN 0 MELK 0.5 **** **** CCNA2 −0.5 Luciferase activity TOP2A Ctr) vs change (fold SPAG5 −1 UBE2T 0.0 PBK −1.5 CDKN3 POLA1 8 2 0 1 0 1 5 2 4 1 1 1 3 7 1 9 CCNB2 7 2 3 3 3 3 4 4 UBE2C -1 -1 -1 -1 -1 -1 -1 -1 CKS1B C C C C C C C C RNASEH2A RFC4 NUSAP1 NDC80 PRIM1 T T T FIGNL1 H H H SLC25A19 D D D T CDK2 E + + + RFC5 H D 2 2 0 0 1 1 NCAPD3 1 1 1 1 1 1 + RRM1 2 2 3 3 3 3 r r TRIP13 t t -1 -1 -1 -1 -1 -1 POLE2 C C C C C C C C RAD54B GINS3 AURKA FAM83D PSA NCAPD2 FANCD2 CDC7 β WEE1 -actin KIAA0101 FANCI IQGAP3 1.0 1.5 0.8 1.2 0.8 0.8 0.5 0.9 ESCO2 CDCA3 AURKB STIL POLQ TK1 POLA2 CDC20 F RFC3 CDC25C TIMELESS ANG HMMR PTTG1 CKS2 DNMT1 STMN1 DTYMK CCNF G CENPM TROAP 10000 TUBG1 IgG FOXM1 NMU anti-AR RNASE4 FAM64A 1000 HIST1H4C ATAD2 * ZWINT RAD51AP1 CDK1 100 FBXO5 FEN1 HIST1H3H H2AFX CHEK1 10 MCM7

DSN1 IgG) of (fold NCAPG ASF1B HMBG2 1 CDCA5 UGT8 PARP1 MCM5 associationRelativeARof CDCA7 0.10,1 MCM2 RAD54L XRCC3 Ctr C-1311 Ctr C-1311 TPRG1 TGFB3 MCM4 GINS2 PSA promoter PSA first intron UHRF1 CCNE2 1.5 DTL H MCM10 I CDC25A TM4SF1 UGT2A1 E2F2 11 HIST2H4B tr 3 ARL6IP6 1.0 C -1 CDCA4 C MCM3 FANCC XRCC2 AR XRCC4 CDC6 KLK3 0.5 CADPS2 SKP2

Relative AR mRNA ARmRNA Relative GAPDH RAB27A BCL2L1 (normalized to GAPDH) (normalized TUBB 1.0 0.96 EAF2 CXCR4 0.0 2 3 2 3 Ctr C-1311 Control C-1311

Figure 3.FigureC-1311 3. blocksC-1311 transcriptionalblocks transcriptional activity activity of the of androgen the androgen receptor receptor (AR). (AR). (A )( GeneA) Gene Set Set Enrichment AnalysisEnrichment (GSEA) plot Analysis showing (GSEA) repression plot showing of the repre ‘ARssion Targets of the Upregulated‘AR Targets Upregulated by the AR’ by gene the AR’ signature in gene signature in LNCaP cells treated with 1 µM C-1311 for 24 h. (B) Heat map of DEGs (|log2FC|> 1, FDR < 0.05) identified from the ‘AR Targets Upregulates by AR’ gene signature in LNCaP cells treated with 1 µM C-1311 for 24 h. (C) Schematic representation of the ARE-Luc reporter vector Biomedicines 2020, 8, 292 9 of 19

LNCaP cells treated with 1 µM C-1311 for 24 h. (B) Heat map of DEGs (|log2FC| > 1, FDR < 0.05) identified from the ‘AR Targets Upregulates by AR’ gene signature in LNCaP cells treated with 1 µM C-1311 for 24 h. (C) Schematic representation of the ARE-Luc reporter vector utilized for generation of the LNCaP-ARE-Luc reporter cell line. The ARE-Luc vector contains three copies of each of the three ARE sequences derived from the PSA gene and tk (thymidine kinase) basal promoter. Luc: luciferase gene. (D) AR transactivation assay. LNCaP-ARE-Luc cells were treated with imidazoacridinones at 1 µM for 24 h. Nitroacridine C-1748 (0.01 µM) was used as a reference AR inhibitor [8]. Luciferase activity (fold-change as compared to untreated cells) is shown. Data are the mean SD, n = 8. Significance: ± One-way ANOVA test; **** p < 0.0001, ** p < 0.01, * p < 0.05. (E)Effect of selected imidazoacridinones on the expression of prostate-specific antigen (PSA). LNCaP cells were exposed to 5 µM imidazoacridinone or pre-incubated with 10 nM 5-α-dihydrotestosterone (DHT) to potentiate the transcriptional activity of the AR. The level of PSA was determined 24 h later by Western blotting. Bands were analyzed by densitometry using ImageJ software and normalized to β-actin. Numbers represent a fold change in the PSA level relative to the untreated control cells not stimulated with DHT. (F–G) C-1311 blocks the recruitment of AR to ARE in the PSA promoter. LNCaP cells were treated with 5 µM C-1311 for 8 h followed by ChIP analyses with a control IgG and anti-AR antibody. (F) Schematic diagram showing the position of the AREs located in the proximal PSA promoter. P1 and P2: Locations of the PCR primers used in ChIP-qPCR. ATG: Start codon. (G) The effect of C-1311 on the recruitment of AR to the ARE located in the proximal promoter of the PSA and to the sequences of the first intron of the PSA, as indicated in (F). Binding of AR is shown as the fold enrichment over a normal IgG. Data are the mean SD of quadruplicates RT-PCR analyses from a representative ChIP experiment. Significance: ± Student’s t-test; * p < 0.05 in comparison to the control. (H) Real-time PCR analysis of the AR mRNA level in the LNCaP cells exposed to 1 µM C-1311 for 24 h. Data are the mean SD, n = 2. (I) Western ± blotting analysis of AR protein levels in LNCaP treated as in (H). Numbers represent the fold change in the AR level normalized to GAPDH and expressed relative to the control cells.

RNA-seq analysis suggested that C-1311 may block the function of the AR through the repression of its downstream transcriptional targets. To further test this, we generated a stable reporter cell line (LNCaP-ARE-Luc) expressing luciferase under the control of the tk promoter containing ARE (androgen response element) sequences derived from the prostate-specific antigen (PSA) promoter (Figure3C). LNCaP-ARE-Luc cells were treated and screened for AR-mediated luciferase activity. C-1311, as well as other imidazoacridinones like C-1310 and C-1371, at a 1 µM concentration significantly reduced the luciferase activity to a level lower than 50% of that observed in the untreated cells (Figure3D). C-1311 and C-1371 were also effective at ten-fold lower doses (0.1 µM, Figure S4A), indicating a dose-dependent inhibition of the transcriptional activity of the AR. Importantly, additional experiments using LNCaP cells transiently transfected with an empty vector or ARE-Luc reporter vector, confirmed that C-1311 significantly decreased the ARE-responsive luciferase activity, while it had no effect on the empty control vector (Figure S4B). The triazoloacridinones did not significantly inhibit AR transactivation activity (Figure S4C). Next, we confirmed the anti-AR activity of the selected imidazoacridinones by measuring the level of PSA. PSA expression is regulated by an upstream promoter and enhancer androgen response elements (ARE). Elevated PSA levels are a widely used marker to demonstrate activation of the AR signaling pathway [38]. Western blot analysis showed that imidazoacridinone C-1311 reduced the level of PSA in LNCaP cells. This inhibitory effect was still evident when AR was additionally stimulated using 5-alpha-dihydrotestosterone (DHT) (Figure3E). The C-1311-mediated decrease in AR transcriptional activity was further confirmed by a chromatin immunoprecipitation (ChIP) assay (Figure3G). C-1311 significantly reduced binding of AR to the AR response element (ARE) of the PSA promoter in LNCaP cells. Importantly, we did not observe the recruitment of AR to the AR-negative binding region located in the first intron of the PSA, in either the control or C-1311-treated cells (Figure3G). In addition, RT-PCR and Western blotting analyses showed no significant changes in the AR mRNA (Figure3H) and protein (Figure3I) level following C-1311 exposure. Together, these data indicate that the selected imidazoacridinones, Biomedicines 2020, 8, x FOR PEER REVIEW 10 of 18

Given that C‐1311 is a multi‐target therapeutic, we expanded our analysis of its effects on PCa cell lines to include those with a differing AR status. As expected, C‐1311 did not significantly change expressionBiomedicines of the2020 androgen, 8, 292 ‐regulated genes in AR‐negative DU‐145 cells (Figure S5). 10Surprisingly, of 19 however, C‐1311, as well as other imidazoacridinones, also inhibited the growth of DU‐145 cells (Figure S6Aincluding and theS6B), lead and compound this effect C-1311, was are promisingmore pronounced inhibitors of compared AR activity. Mechanistically,to the LNCaP C-1311 cells (Figure S5A vs. interferesFigure 1A). with The AR binding C‐1311 to IC the50 ARE value and for blocks the the DU AR-mediated‐145 cells was transcription. 0.15 μM, while for the LNCaP [Grab cells it was3.4. C-13110.47 μ IsM. Also To Potent further against confirm AR-Independent the efficacy PCa Cells of in C 2D‐1311 Monolayer in a more and 3D physiologically Spheroid Cultures relevant tumor model, we grew multicellular PCa spheroids in the presenceyour of C ‐1311 or DMSO. C‐1311 Given that C-1311 is a multi-target therapeutic, we expanded our analysis of its effects on PCa cell treatmentlines significantly to include those decreased with a di theffering diameter AR status. of Asthe expected, LNCaP C-1311 and DU did‐145 not significantly cell spheroids change in a time‐ dependentexpression manner of (Figure the androgen-regulated 4A–C). In addition, genes in C‐ AR-negative1311 effectively DU-145 blocked cells (Figure the growth S5). Surprisingly, of the spheroids formed fromhowever, 22Rv1 C-1311, cells. as These well as cells other have imidazoacridinones, partial androgen also sensitivity inhibited the due growth to the of DU-145 expression cells of both a full‐length(Figures AR S6A,B), and a constitutively and this effect was active more AR pronounced splice variant, compared AR‐ toV7, the which LNCaP lacks cells the (Figure ligand S5A‐binding µ domain vs.[39]. Figure As 1such,A). The the C-1311 22Rv1 IC 50cellsvalue serve for the as DU-145 a model cells for was a 0.15transitionM, while phenotype for the LNCaP between cells that of it was 0.47 µM. To further confirm the efficacy of C-1311 in a more physiologically relevant tumor androgenmodel,‐sensitive we grew LNCaP multicellular and AR PCa‐negative spheroids DU in‐ the145 presence cells [39]. of C-1311 or DMSO. C-1311 treatment Importantly,significantly as decreased shown theby diameterthe resazurin of the LNCaP assay and (Figure DU-145 4D), cell spheroidsC‐1311 also in a time-dependentsignificantly reduced viability mannerof the (FigurePCa cells4A–C). grown In addition, as spheroids. C-1311 e ff ectivelyAgain, blocked DU‐145 the cells growth were of the the spheroids most sensitive formed from to C‐1311 treatment.22Rv1 The cells. IC50 These value cells for have the partialDU‐145 androgen cells was sensitivity 0.3 μM, due whereas to the expression for 22Rv1 of both and a LNCaP full-length it was 1.2 and 4 μM,AR respectively. and a constitutively Numerous active AR intrinsic splice variant, differences AR-V7, which between lacks DU the ligand-binding‐145 and LNCaP domain or [22Rv139]. cells As such, the 22Rv1 cells serve as a model for a transition phenotype between that of androgen-sensitive may accountLNCaP for and this AR-negative difference DU-145 in sensitivity. cells [39]. Nonetheless, these results indicate that C‐1311 extends its activity also to the AR‐independent PCa cells.

A DMSO 1 µM 10 µM 1000 m)

 800 0 h 600 LNCaP 400 DMSO

96 h 200 1 µM p*

Spheroid diameter ( diameter Spheroid 10 µM p** 0 24 48 72 96 Time (h) B DMSO 1 µM 10 µM 800 m) 0 h  600

22Rv1 400 DMSO 96 h 200 1 µM p***

Spheroiddiameter ( 10 µM p**** 0 24 48 72 96 Time (h) C DMSO 1 µM 10 µM 600 m)  0 h 400

DU-145 200 DMSO 96 h 1 µM p****

Spheroid diameter ( 10 µM p**** 0 24 48 72 96 D Time (h) [Grab your reader’s D Figure 4. Cont.

100

DU-145 IC50 = 0.3 µM

22Rv1 IC50 = 1.2 µM **** * LNCaP IC50 = 4.0 µM 50 % of Control of %

0 0.00001 0.001 0.1 10 C-1311 [μM] Biomedicines 2020, 8, x FOR PEER REVIEW 10 of 18

Given that C‐1311 is a multi‐target therapeutic, we expanded our analysis of its effects on PCa cell lines to include those with a differing AR status. As expected, C‐1311 did not significantly change expression of the androgen‐regulated genes in AR‐negative DU‐145 cells (Figure S5). Surprisingly, however, C‐1311, as well as other imidazoacridinones, also inhibited the growth of DU‐145 cells (Figure S6A and S6B), and this effect was more pronounced compared to the LNCaP cells (Figure S5A vs. Figure 1A). The C‐1311 IC50 value for the DU‐145 cells was 0.15 μM, while for the LNCaP cells it was 0.47 μM. To further confirm the efficacy of C‐1311 in a more[Grab physiologically relevant tumor model, we grew multicellular PCa spheroids in the presenceyour of C ‐1311 or DMSO. C‐1311 treatment significantly decreased the diameter of the LNCaP and DU‐145 cell spheroids in a time‐ dependent manner (Figure 4A–C). In addition, C‐1311 effectively blocked the growth of the spheroids formed from 22Rv1 cells. These cells have partial androgen sensitivity due to the expression of both a full‐length AR and a constitutively active AR splice variant, AR‐V7, which lacks the ligand‐binding domain [39]. As such, the 22Rv1 cells serve as a model for a transition phenotype between that of androgen‐sensitive LNCaP and AR‐negative DU‐145 cells [39]. Importantly, as shown by the resazurin assay (Figure 4D), C‐1311 also significantly reduced viability of the PCa cells grown as spheroids. Again, DU‐145 cells were the most sensitive to C‐1311 treatment. The IC50 value for the DU‐145 cells was 0.3 μM, whereas for 22Rv1 and LNCaP it was 1.2 and 4 μM, respectively. Numerous intrinsic differences between DU‐145 and LNCaP or 22Rv1 cells may account for this difference in sensitivity. Nonetheless, these results indicate that C‐1311 extends its activity also to the AR‐independent PCa cells.

A DMSO 1 µM 10 µM 1000 m)

 800 0 h 600 LNCaP 400 DMSO

96 h 200 1 µM p*

Spheroid diameter ( diameter Spheroid 10 µM p** 0 24 48 72 96 Time (h) B DMSO 1 µM 10 µM 800 m) 0 h  600

22Rv1 400 DMSO 96 h 200 1 µM p***

Spheroiddiameter ( 10 µM p**** 0 24 48 72 96 Time (h) C DMSO 1 µM 10 µM 600 m)  0 h 400

DU-145 200 DMSO 96 h 1 µM p****

Spheroid diameter ( 10 µM p**** 0 Biomedicines 2020, 8, 292 24 48 72 96 11 of 19 D Time (h) [Grab your reader’s D

100

DU-145 IC50 = 0.3 µM

22Rv1 IC50 = 1.2 µM **** * LNCaP IC50 = 4.0 µM 50 % of Control of %

0 0.00001 0.001 0.1 10 C-1311 [μM] Figure 4. C-1311 is also potent against the AR-independent PCa cells in 3D spheroid cultures. (A–C) Representative images of the PCa cell spheroids treated with DMSO or C-1311 at the indicated doses for 96 h. Scale bars = 500 µm. Right panels show changes in spheroid diameter monitored over 96 h of C-1311 exposure. Results are the mean SD, n = 4. Significance: Two-way ANOVA; ± **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 in comparison to DMSO. For graph clarity, the corresponding p-values are presented next to the treatment conditions. (D)Effect of C-1311 on viability of the PCa cells grown as multicellular spheroids. Spheroids were treated with C-1311 for 96 h and the cell viability was determined by a resazurin assay. Results are the mean SD, n = 4. Significance: ± Two-way ANOVA; **** p < 0.0001, * p < 0.05.

Importantly, as shown by the resazurin assay (Figure4D), C-1311 also significantly reduced viability of the PCa cells grown as spheroids. Again, DU-145 cells were the most sensitive to C-1311 treatment. The IC50 value for the DU-145 cells was 0.3 µM, whereas for 22Rv1 and LNCaP it was 1.2 and 4 µM, respectively. Numerous intrinsic differences between DU-145 and LNCaP or 22Rv1 cells may account for this difference in sensitivity. Nonetheless, these results indicate that C-1311 extends its activity also to the AR-independent PCa cells.

3.5. C-1311 Induces DNA Damage and Arrests Cell Cycle Progression Next, we assessed the effect of C-1311 on the biological response of the PCa cells. C-1311, as a topoisomerase II inhibitor, has been previously demonstrated to induce DNA damage [16]. Correspondingly, we found that C-1311 treatment in LNCaP, 22Rv1, and DU-145 cells increased the phosphorylation of the histone H2AX (γH2AX) (Figure5A), a well-known marker of DNA damage [ 40]. In addition, as shown by the BrdU incorporation assay, C-1311, within 72 h of treatment, significantly reduced the percentage of cells actively replicating DNA (Figure5B). In LNCaP cells, a decrease in the BrdU-positive cell fraction was already evident within 24 h of treatment, while in 22Rv1 and DU-145 cells, a 72 h treatment was required to achieve a similar effect. Given that C-1311 triggered DNA damage and decreased the population of replicating cells, next we examined its effect on the cell cycle progression of the PCa cells. First, we determined the level of p53 and its transcriptional target, p21, which control the G1/S and G2/M phase cell cycle checkpoints. LNCaP cells contain the wild-type p53 gene. The 22Rv1 cells harbor one wild-type copy of p53 and one mutated copy of p53 (p53 wt/Q331R) and still retain p53 transcriptional activity. DU145 cells have inactivating p53 mutations (P223L and V274F mutations) [41]. Western blot analysis showed that in LNCaP and 22Rv1 cells, C-1311 treatment increased the level of p53 and p21 (Figure6A). In DU-145 cells, consistent with the non-functional p53, C-1311 did not increase the expression of p21. The cell cycle analysis showed that in response to C-1311, approximately 50% of the LNCaP cells remained in the G1 phase and failed to enter the S phase. In addition, C-1311 also induced arrest of the cell cycle in the G2/M phase (Figure6B and Figure S7). The 22Rv1 cells progressively accumulated in the G2/M phase, although approximately 20% of the cells remained arrested in the G1 phase after 72 h of treatment. In DU-145 cells, lacking a p53/p21 signaling axis, a population of cells in the G1 phase disappeared, and cells, after transient accumulation in the S phase, arrested in the G2/M phase. Biomedicines 2020, 8, x FOR PEER REVIEW 11 of 18

Figure 4. C-1311 is also potent against the AR-independent PCa cells in 3D spheroid cultures. (A–C) Representative images of the PCa cell spheroids treated with DMSO or C-1311 at the indicated doses for 96 h. Scale bars = 500 µm. Right panels show changes in spheroid diameter monitored over 96 h of C-1311 exposure. Results are the mean ± SD, n = 4. Significance: Two-way ANOVA; **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 in comparison to DMSO. For graph clarity, the corresponding p- values are presented next to the treatment conditions. (D) Effect of C-1311 on viability of the PCa cells grown as multicellular spheroids. Spheroids were treated with C-1311 for 96 h and the cell viability was determined by a resazurin assay. Results are the mean ± SD, n = 4. Significance: Two-way ANOVA; **** p < 0.0001, * p < 0.05.

3.5. C-1311 Induces DNA Damage and Arrests Cell Cycle Progression Next, we assessed the effect of C-1311 on the biological response of the PCa cells. C-1311, as a topoisomerase II inhibitor, has been previously demonstrated to induce DNA damage [16]. Biomedicines 2020, 8, 292 12 of 19 Correspondingly, we found that C-1311 treatment in LNCaP, 22Rv1, and DU-145 cells increased the phosphorylation of the histone H2AX (γH2AX) (Figure 5A), a well-known marker of DNA damage In contrast to LNCaP cells, in the 22Rv1 and DU-145 cells, the increase in the G2/M population was [40]. In addition, as shown by the BrdU incorporation assay, C-1311, within 72 h of treatment, also accompanied by the appearance of polyploid cells (>4N DNA). These results are consistent with significantlystudies reduced showing the that percentage in cancer cells of carrying cells activelyp53 mutations, replicating DNA damage DNA (Figure and a prolonged 5B). In G2LNCaP/M cells, a decrease phasein the arrest BrdU-positive can also lead to cell polyploidy fraction [42]. was Together, already these resultsevident indicate within that C-1311,24 h of in treatment, addition to while in 22Rv1 andtargeting DU-145 the cells, AR function, a 72 h inducestreatment DNA was damage required and eff ectivelyto achieve blocks a similar PCa cell cycleeffect. progression, irrespective of AR status.

A LNCaP 22Rv1 DU-145

0 24 48 72 0 24 48 72 0 24 48 72 (h) γH2AX β-actin

B DMSO 24 h 72 h LNCaP 31.3 % 8.4 % 0.4 %

22Rv1 29.7 % 24.6 % 1.7 %

58.9 % 23.1 % DU-145 39.3 % BrdU

PI

Figure 5. C-1311 induces DNA damage and decreases the percentage of replicating PCa cells Figure 5.irrespectively C-1311 induces of AR status. DNA (A) PCadamage cells were and treated decreases with 1 µM C-1311the percentage for the times indicated.of replicating Western PCa cells irrespectivelyblotting of was AR carried status. for γ(AH2AX.) PCaβ -actincells was were used treated as the loading with control,1 µM nC-1311= 3. (B )for Flow the cytometry times indicated. analysis of the BrdU incorporation (DNA synthesis) in PCa cells treated with C-1311 as in (A). Boxed Western blottingregion on the was dot plotscarried represents for γ theH2AX. BrdU-positive β-actin cells. was Results used are as representative the loading of three control, independent n = 3. (B) Flow cytometryexperiments analysis of and the are BrdU presented incorporation as FloJo dot-plot (DNA graphs, synthesis) which heat in maps PCa fluorescence cells treated intensities with ofC-1311 as in (A). Boxedgreatest region frequency on the in dot red. plots represents the BrdU-positive cells. Results are representative of three independent experiments and are presented as FloJo dot-plot graphs, which heat maps fluorescence intensities of greatest frequency in red. Biomedicines 2020, 8, x FOR PEER REVIEW 12 of 18

Given that C-1311 triggered DNA damage and decreased the population of replicating cells, next we examined its effect on the cell cycle progression of the PCa cells. First, we determined the level of p53 and its transcriptional target, p21, which control the G1/S and G2/M phase cell cycle checkpoints. LNCaP cells contain the wild-type p53 gene. The 22Rv1 cells harbor one wild-type copy of p53 and one mutated copy of p53 (p53 wt/Q331R) and still retain p53 transcriptional activity. DU145 cells have inactivating p53 mutations (P223L and V274F mutations) [41]. Western blot analysis showed that in LNCaP and 22Rv1 cells, C-1311 treatment increased the level of p53 and p21 (Figure 6A). In DU-145 cells, consistent with the non-functional p53, C-1311 did not increase the expression of p21. The cell cycle analysis showed that in response to C-1311, approximately 50% of the LNCaP cells remained in the G1 phase and failed to enter the S phase. In addition, C-1311 also induced arrest of the cell cycle in the G2/M phase (Figure 6B and Figure S7). The 22Rv1 cells progressively accumulated in the G2/M phase, although approximately 20% of the cells remained arrested in the G1 phase after 72 h of treatment. In DU-145 cells, lacking a p53/p21 signaling axis, a population of cells in the G1 phase disappeared, and cells, after transient accumulation in the S phase, arrested in the G2/M phase. In contrast to LNCaP cells, in the 22Rv1 and DU-145 cells, the increase in the G2/M population was also accompanied by the appearance of polyploid cells (>4N DNA). These results are consistent with studies showing that in cancer cells carrying p53 mutations, DNA damage and a prolonged G2/M phase arrest can also lead to polyploidy [42]. Together, these results indicate that C-1311, in addition to targeting the AR function, induces DNA damage and effectively blocks PCa cell cycle progression, Biomedicines 2020, 8, 292 13 of 19 irrespective of AR status.

A LNCaP 22Rv1 DU-145

0 24 48 72 0 24 48 72 0 24 48 72 (h) p53

p21

β-actin

B DMSO 24 h 72 h % % % LNCaP G1: 58 G1: 60 G1: 54 S: 24 S: 9 S: 9 G2/M : 17 G2/M : 31 G2/M : 37 >4N: 1 >4N: 0 >4N: 0

% % % 22Rv1 G1: 31 G1: 22 G1: 20 S: 38 S: 30 S: 15 G2/M : 20 G2/M : 35 G2/M : 47 >4N: 10 >4N: 13 >4N: 18

DU-145 % % % G1: 34 G1: 2 G1: 0 S: 42 S: 70 S: 63 G2/M : 20 G2/M : 21 G2/M : 23 >4N: 4 >4N: 7 >4N: 14 Counts PI

Figure 6. C-1311 blocks PCa cell cycle progression. (A) Western blotting analysis of p53 and p21 in PCa cells treated with 1 µM C-1311 for the times indicated. β-actin was used as the loading control, n = 3. (B) Cell cycle profiles of the PCa cells treated as in (A), stained with PI and analysed by flow cytometry. Histograms are representative of three independent experiments.

3.6. C-1311 Induces Apoptosis Irrespective of AR Status Next, we examined the cellular consequences of the C-1311 treatment in PCa cells. GO analysis revealed that several DEGs identified in the LNCaP and DU-145 cells exposed to C-1311 were also enriched in apoptosis-related pathways (Tables S4 and S5). In particular, we identified 93 DEGs in the LNCaP cells and 153 DEGs in the DU-145 cells, which were associated with positive and negative regulation of apoptosis. For example, C-1311 decreased the expression of classical anti-apoptosis genes, such as the IAP family member BIRC5 (survivin) or Bcl-2, but also BRCA1 and BRCA2, which connect DNA damage and stress response pathways to the execution of cell cycle arrest and apoptosis [43]. Western blot analysis confirmed that changes in the expression of the apoptosis-related genes were accompanied by those typical for apoptosis, cleavage of PARP (Figure7A). Similarly, flow cytometric analysis of Annexin V staining of cell surface phosphatidylserine, supported the observations that C-1311 was able to induce apoptosis in both LNCaP and DU-145 cells (Figure7B and Figure S8). Biomedicines 2020, 8, x FOR PEER REVIEW 13 of 18

Figure 6. C-1311 blocks PCa cell cycle progression. (A) Western blotting analysis of p53 and p21 in PCa cells treated with 1 µM C-1311 for the times indicated. β-actin was used as the loading control, n = 3. (B) Cell cycle profiles of the PCa cells treated as in (A), stained with PI and analysed by flow cytometry. Histograms are representative of three independent experiments.

3.6. C-1311 Induces Apoptosis Irrespective of AR Status Next, we examined the cellular consequences of the C-1311 treatment in PCa cells. GO analysis revealed that several DEGs identified in the LNCaP and DU-145 cells exposed to C-1311 were also enriched in apoptosis-related pathways (Table S4 and table S5). In particular, we identified 93 DEGs in the LNCaP cells and 153 DEGs in the DU-145 cells, which were associated with positive and negative regulation of apoptosis. For example, C-1311 decreased the expression of classical anti- apoptosis genes, such as the IAP family member BIRC5 (survivin) or Bcl-2, but also BRCA1 and BRCA2, which connect DNA damage and stress response pathways to the execution of cell cycle arrest and apoptosis [43]. Western blot analysis confirmed that changes in the expression of the apoptosis-related genes were accompanied by those typical for apoptosis, cleavage of PARP (Figure 7A). Similarly, flow cytometric analysis of Annexin V staining of cell surface phosphatidylserine, supported the

Biomedicinesobservations2020 ,that8, 292 C-1311 was able to induce apoptosis in both LNCaP and DU-145 cells (Figures14 of 7B 19 and S8).

AB 50 LNCaP DU-145 ** Ctr 40 * C-1311 0 24 48 72 0 24 48 72 time (h) PARP 30 cl. PARP 20

β-actin cells Apoptotic 10

0 (% ofAnnexin V-positivecells) LNCaP DU-145 Figure 7. C-1311 induces apoptosis irrespective of AR status. (A) Western blotting analysis of Figure 7. C-1311 induces apoptosis irrespective of AR status. (A) Western blotting analysis of apoptosis-related PARP cleavage. PCa cells were treated with DMSO or 1 µM C-1311 for the time apoptosis-related PARP cleavage. PCa cells were treated with DMSO or 1 µM C-1311 for the time indicated. β-actin was used as the loading control, n = 3. (B) The percentage of apoptotic cells indicated. β-actin was used as the loading control, n = 3. (B) The percentage of apoptotic cells determined by flow cytometry analysis of Annexin V/propidium iodide staining (PI). PCa cells were determined by flow cytometry analysis of Annexin V/propidium iodide staining (PI). PCa cells were treated with DMSO or 1 µM C-1311 for 72 h. The bar graph shows the percentage of total Annexin treated with DMSO or 1 µM C-1311 for 72 h. The bar graph shows the percentage of total Annexin V- V-positive cells, including Annexin V+/PI (early apoptosis) and Annexin V+/PI+ (late apoptotic cells). positive cells, including Annexin V+/PI-− (early apoptosis) and Annexin V+/PI+ (late apoptotic cells). Results are the mean SD, n = 3. Significance: Student’s t-test; * p < 0.05; ** p < 0.01. Results are the mean ±± SD, n = 3. Significance: Student’s t-test; * p< 0.05; ** p < 0.01.3. 4. Discussion 4. Discussion During PCa development, both AR-independent and AR-dependent signaling mechanisms contributeDuring to PCa the malignantdevelopment, transformation both AR-independe of epithelialnt and cells AR-dependent [44]. Here we signaling demonstrate mechanisms that the imidazoacridinonecontribute to the malignant C-1311 has transformation a potent anti-cancer of epithelial activity cells that[44]. extendsHere we to demonstrate AR-dependent that and the AR-independentimidazoacridinone PCa C-1311 cells. Regardlesshas a potent of anti-cancer AR status, C-1311activity caused that extends DNA damage, to AR-dependent effectively and arrested AR- cellindependent cycle progression PCa cells. in Regardless the G2/M phase, of AR status, and induced C-1311 apoptosis. caused DNA Importantly, damage, effectively in AR-dependent arrested PCa cell cells,cycle C-1311progression was found in the to G2/M inhibit phase, transcriptional and induce activityd apoptosis. of AR, uncoveringImportantly, a novelin AR-dependent mechanism thatPCa maycells, be C-1311 relevant was for found its anticancer to inhibit e fftranscriptionalect. Mechanistically, activity C-1311 of AR, decreased uncovering AR bindinga novel mechanism to the canonical that ARmay response be relevant element for (ARE)its anticancer sequence effect. in the MechPSAanistically,promoter andC-1311 reduced decreased the expression AR binding of the to PSA the protein.canonical RNA AR sequencingresponse element revealed (ARE) that C-1311sequence significantly in the PSA downregulated promoter and thereduced transcription the expression of several of otherthe PSA AR-dependent protein. RNA genes, sequencing confirming revealed that that C-1311 C-1311 targeted significantly AR transcriptional downregulated activity. the Thesetranscription results provideof several first other evidence AR-dependent that C-1311 genes, interferes confirming with the th ARat C-1311 binding targeted to DNA. AR We transcriptional have previously activity. found thatThese C-1311 results also provide inhibited first the evidence DNA-binding that C-1311 capacity interferes of transcriptional with the AR factor binding HIF1 toα DNA.to the We hypoxia have responsivepreviously elementfound that (HRE) C-1311 sequence also inhibited [14]. One the of theDNA-binding key mechanistic capacity characteristics of transcriptional of C-1311 factor is that HIF1 it isα ato DNA the hypoxia intercalator. responsive It is therefore element possible(HRE) sequence that through [14]. One intercalation, of the key C-1311mechanistic may interferecharacteristics with the binding of other transcription factors to their cognate sequences. In addition, given that C-1311 is also a topoisomerase II inhibitor, its non-specific effect on the global transcription cannot be ruled out. Topoisomerase II can bind within promoters of the actively transcribed genes [45], and topoisomerase II poisons, like adriamycin, were found to cause structural changes at or near the promoters and alter gene expression, although this effect depends on the arrangement of the promoter sites and the disposition of the topoisomerases [46]. While further studies are required to address these possible non-specific effects, the C-1311-mediated inhibition of the transcriptional activity of AR is a promising additional function of this drug, which may help to eliminate the androgen-dependent PCa cells. In PCa, mutations in the p53 tumor suppressor gene are associated with disease progression, increased metastasis, and androgen-independent growth [47]. Mechanism-of-action studies indicated that in AR-positive PCa cells expressing functional p53, C-1311 induced stable G1 and G2/M arrest. In contrast, AR-negative cells with mutated p53 underwent arrest in the G2/M phase of the cell cycle. These results are in agreement with our previous observations showing that C-1311 has the potential to kill cancer cells with an unfavorable p53 profile [16]. Strategies combining the standard anti-PCa therapy with cell cycle inhibitors targeting the G1 (palbociclib, ribociclib, AZD-5363, ipatasertib) or G2 (adavosertib) phases of the cell cycle are currently tested in several clinical trials in castration-resistant prostate cancer [48]. In this light, the potential of C-1311 to effectively block cell cycle progression of PCa cells in the G1 or G2/M phases is particularly promising. However, it should be noted that DNA Biomedicines 2020, 8, 292 15 of 19 damage and prolonged G2/M arrest can also carry the risk of abnormal mitosis and development of polyploid cells [42]. We found that in AR-independent PCa cells with mutated p53, C-1311-induced G2/M arrest was accompanied by the appearance of a population (~18%) of polyploid cells. While studies show that, following treatment with DNA-damaging agents, polyploid cells often undergo cell death or terminally arrest in senescence, polyploidy may also represent a survival mechanism [49,50]. Further studies are required to address the long-term fate of PCa polyploid cells arising following C-1311 exposure and their effect on the drug’s efficacy. Interestingly, using transcriptome sequencing, we identified differentially expressed genes (DEGs) and signaling pathways that may serve as additional targets for C-1311 and contribute to its anti-PCa activity. Multiple lines of evidence link AR signaling to the DNA damage response (DDR). AR regulates a transcriptional program of DNA repair, including homologous recombination (HR), activation of the poly(ADP-ribose) polymerase (PARP) function, and non-homologous end-joining recombination (NHEJ) [20]. Accordingly, inhibition of AR has been clinically shown to sensitize PCa to DNA-damaging chemo- and radiotherapy [51]. Employing RNA-seq, we found that in AR-positive cells, C-1311 significantly decreased the transcription of BRCA1 and BRCA2, two essential genes in the HR pathway. Moreover, C-1311 also decreased expression of direct AR target genes that play a functional role in HR, including CHEK1, RAD54L, RAD54B, POLE2, and XRCC2. Downregulation of HR in response to the inhibition of the AR by enzalutamide has been shown to sensitize PCa cells to PARP inhibitors [52]. Although it needs a separate study, this raises the possibility that C-1311, through the induction of HR deficiency, may be synthetically lethal with PARP inhibitors. Gene ontology and network analysis of the DEGs indicated that in AR-negative DU-145 cells, in contrast to AR-positive LNCaP cells, C-1311 treatment affected the transcriptional program of cellular metabolism. Studies show that PCa progression, aggressiveness, and poor prognosis are associated with an increased glycolytic phenotype. The highly metastatic PCa consume more glucose, with consequent lactate production, compared to the poorly metastatic PCa [53]. In AR-negative PCa cells, C-1311 significantly downregulated the expression of the key glycolytic enzymes PGK1, GPI, PPI1, PGAM1, ALDOA, and ALDOC. These results suggest that C-1311, by targeting glycolysis, may promote metabolic stress in androgen-independent PCa cells. Whether the C-1311-mediated modulation of the transcriptional program of PCa cell metabolism translates into a real cytotoxic effect warrants further studies.

5. Conclusions Here, we show that the anticancer imidazoacridinone C-1311 is highly effective in AR-dependent and AR-independent PCa cells. Previous studies identified C-1311 as a multitargeted compound, which blocks activity of topoisomerase II [13], HIF-1α/VEGF signalling, tumour angiogenesis [14], and FLT3 receptor tyrosine kinase FLT3 [15]. We demonstrated that, in addition to these functions, C-1311 blocks the transcriptional activity of the AR. Single drugs that affect multiple targets are considered as a valuable opportunity for drug discovery [54]. In this context, the newly identified inhibitory potential of C-1311 against the AR makes this drug a valuable candidate for further testing against prostate cancer.

Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9059/8/9/292/s1. Figure S1: Chemical structures of (A) imidazoacridinones and (B) triazoloacridinones. Figure S2: Cytotoxic activity of (A) imidazoacridinones and (B) triazoloacridinones in AR-dependent prostate cancer LNCaP cells. Figure S3: Top 10 GO: Biological process terms (A) and the most representative altered canonical pathways (B) in PCa cells treated with C-1311. Figure S4: Effect of imidazoacridinones and triazoloacridinones on AR transactivation activity measured by luciferase gene reporter assay. Figure S5: Effect of C-1311 on the expression of AR-regulated genes in AR-negative DU-145 cells. Figure S6: Cytotoxic activity of C-1311 and other imidazoacridinones in AR-negative DU-145 cells. Figure S7: Effect of C-1311 on the PCa cell cycle progression. Figure S8: C-1311 induces apoptosis in PCa cells irrespective of AR-status. Table S1: Top 10 deregulated canonical pathways with the list of up- and downregulated genes in LNCaP. Table S2: Top 10 deregulated canonical pathways with the list of up- and downregulated genes in DU-145. Table S3: ‘AR targets upregulated by AR’ gene signature. Table S4: Biomedicines 2020, 8, 292 16 of 19

The enriched GO terms in the biological processes related to apoptosis in LNCaP treated with 1 µM for 24 h. Table S5: The enriched GO terms in the biological processes related to apoptosis in DU-145 treated with 1 µM for 24 h. Author Contributions: Conceptualization, M.N. and A.S; methodology, A.S. and M.N.; formal analysis, M.K., M.N.; investigation, A.S., M.N., B.B.-M., A.M., S.B.B., M.R. and K.K.; writing—original draft preparation, A.S. and M.N.; writing—review and editing, E.M.H., S.B.B., Z.M., and A.K.; visualization, A.S., M.K., M.N.; supervision, A.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Centre, Poland; grant number 2013/09/D/NZ7/04185. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

AR androgen receptor ARE androgen response element ADT androgen deprivation therapy BRCA1 breast cancer type 1 susceptibility protein ChIP chromatin immunoprecipitation DDR DNA damage response DEG differentially expressed gene DHT dihydrotestosterone H2AX histone H2AX HR homologous recombination mCRPC metastatic castration-resistant prostate cancer MTT 3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PARP poly (ADP-ribose) polymerase PBS phosphate buffered saline PCa prostate cancer PSA prostate-specific antigen RIN RNA integrity number

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