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Targeting Prostate Cancer Subtype 1 by Forkhead box M1 Pathway Inhibition
Kirsi Ketola1, Ravi S.N Munuganti1, Alastair Davies1, Ka Mun Nip1, Jennifer L. Bishop1, Amina
Zoubeidi1,2
1: Vancouver Prostate Centre, Vancouver, BC, CAN 2: University of British Columbia, Faculty of Medicine, Department of Urology, Vancouver BC, CAN
Running title: Targeting PCS1 by FOXM1 Pathway Inhibition
Keywords: Cancer, Prostate, Subtypes, PCS1, Enzalutamide, Forkhead box M1
Financial support. This work (AZ) is supported by Prostate Cancer Canada and proudly funded by the Movember Foundation (T2013-01). AZ is supported by Michael Smith foundation for Health Research, KK is supported by Prostate Cancer Canada and US Department of Defense (PC141530). RSNM is supported by Prostate Cancer Canada and Michael Smith foundation for Health Research, AD is supported by Canadian Institute for Health Research and Prostate Cancer Foundation, JLB was supported by Prostate Cancer Foundation.
Corresponding author: Amina Zoubeidi, Ph.D Associate Professor Department of Urologic Sciences, UBC 2660 Oak Street Vancouver BC V6H 3Z6, Canada Phone: (604) 875-4111 # 68880 Fax: (604) 875-5654 Email: azoubeidi@prostatecentre.com
Conflict of Interest There are no conflicts of interest.
Word count: 4291 Total number of figures and tables: 5 Figures, 1 Table, 7 Supplementary Figures, 3 Supplementary Tables
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Abstract
Purpose: Prostate cancer was recently classified to three clinically relevant subtypes (PCS)
demarcated by unique pathway activation and clinical aggressiveness. In this preclinical study, we
investigated molecular targets and therapeutics for PCS1, the most aggressive and lethal subtype
with no treatment options available in the clinic.
Experimental Design: We utilized the PCS1 gene set and our model of enzalutamide (ENZR)
castration-resistant prostate cancer (CRPC) to identify targetable pathways and inhibitors for
PCS1. The findings were evaluated in vitro and ENZR CRPC xenograft model in vivo.
Results: The results revealed that ENZR CRPC cells are enriched with PCS1 signature and that
Forkhead box M1 (FOXM1) pathway is the central driver of this subtype. Notably, we identified
Monensin as a novel FOXM1 binding agent that selectively targets FOXM1 to reverse the PCS1
signature and its associated stem-like features and reduces the growth of ENZR CRPC cells and
xenograft tumors.
Conclusions: Our preclinical data indicate FOXM1 pathway as a master regulator of PCS1
tumours, namely in ENZR CRPC, and targeting FOXM1 reduces cell growth and stemness in ENZR
CRPC in vitro and in vivo. These preclinical results may guide clinical evaluation of targeting
FOXM1 to eradicate highly aggressive and lethal PCS1 prostate cancer tumours.
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Translational relevance
Androgen deprivation therapy including second-line treatment with enzalutamide is the mainstay of
therapy for metastatic and castration-resistant prostate cancer. Recently, three novel prostate
cancer subtypes were delineated from which PCS1 was the most aggressive and lethal subtype.
However, there are no specific therapeutic targets available for PCS1 tumours. Here, we report
Forkhead box M1 (FOXM1) pathway as a master regulator of the PCS1 subtype and ENZR CRPC
and identified Monensin as a novel FOXM1 inhibitor that selectively targets PCS1. These
preclinical results may guide clinical evaluation of targeting FOXM1 to target PCS1 in advanced
prostate cancer including ENZR CRPC.
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Introduction
Prostate cancer is the second leading cause of cancer death among Western men (1). Androgen
deprivation therapy targeting androgen receptor (AR) and blocking its signalling is the cornerstone
of therapies for prostate cancer patients with metastatic and castration-resistant disease (2, 3).
These include second-line therapies, namely enzalutamide (ENZ) and abiraterone acetate that
improve survival of patients with castration-resistant prostate cancer (CRPC) (4, 5). Nevertheless,
the effects are not curative and resistance to these therapies rapidly occurs (6, 7). Recent
advances in genotyping CRPC have underlined the role of heterogeneity in reactivation of AR
activity: the AR-driven resistance in CRPC remains dependent on AR signalling; for example, via
emergence of AR point mutations and splice variants (such as AR-V7) leading to acquired
resistance to androgen deprivation therapies (3, 6, 8-12). Moreover, cross-talk with other signalling
pathways that drive AR activity has been described (6, 8). In contrast, ‘AR-indifferent’ disease,
where the resistant cells lack AR expression and/or signalling activity, have recently been reported
to be associated with cellular plasticity and neuroendocrine molecular features (13).
Due to acquired resistance and the significant biological heterogeneity seen in prostate cancer
tumors, there has long been a clinical need to identify master regulators that could be targeted to
treat the most lethal, aggressive prostate cancer. In a recent study, prostate cancer was classified
to three prostate cancer subtypes (PCS), PCS1, PCS2 and PCS3, by utilizing and integrating
multiple publically available prostate cancer gene expression datasets (n>4,600) (14). The luminal-
like type 1 signature PCS1 was characterized as the most aggressive and lethal form of prostate
cancer (14). Interestingly, analysis of the circulating tumor cells from ENZ resistant (ENZR)
patient’s revealed that most of the ENZR patients belonged to the PCS1 subtype (14, 15).
However, there are no molecular targets or therapeutic options for patients with PCS1 tumors in
the clinic.
In this study, we sought to identify novel targets and therapeutics for the most lethal prostate
cancer subtype, PCS1. To achieve this goal, we utilized the PCS classifiers and our recently
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generated models of ENZR CRPC (14, 16) and found FOXM1 as a therapeutic target for tumors
with a PCS1 subtype.
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Materials and methods
Cells: The prostate carcinoma CRPC and ENZR CRPC cells were generated from LNCaP as
previously reported (16, 17), tested and authenticated by whole-genome and whole-transcriptome
sequencing (Illumina Genome Analyzer IIx, 2012), and tested as free of mycoplasma
contamination and grown in RPMI-1640 / 10% FBS / 1% glutamate / 1 % penicillin - streptomycin
(Hyclone) and 10 µM ENZ or DMSO.
Compounds. ENZ (Haoyuan Chemexpress, Shanghai, China) and Thiostrepton (Sigma-Aldrich)
were diluted in DMSO (Sigma-Aldrich) and Monensin (Sigma-Aldrich) was diluted in EtOH. Mon
concentration of 10 nM was used in all experiments unless otherwise noted.
Cell proliferation assay. Cell proliferation assay was performed on 96-/384-well plates (Greiner)
by plating 2,000/1,000cells/well in 100/35μl of media and left to attach overnight. Compound
dilutions were added and incubated for 72h. Cell viability was determined with CellTiter-Glo (CTG,
Promega, Inc.) and confluence with live-cell imaging (Incucyte, Essen Bioscience Inc. Ann Arbor
MI). The luminescence signal (700nm) from CTG was quantified using Tecan 200 plate reader
(Tecan, Männedorf, Switzerland).
Gene expression analysis using bead-arrays. ENZR CRPC cells were grown into approximately
70% confluence and total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). Integrity of
the RNA was monitored prior to hybridization using a Bioanalyzer 2100 (Agilent, Santa Clara, CA)
according to manufacturer’s instructions. 500ng of purified RNA was amplified with the TotalPrep
Kit (Ambion, Austin, TX) and the biotin labelled cDNA was hybridized to Agilent (Agilent, Santa
Clara, CA).
Analysis of gene expression data. Differentially expressed genes from microarray was set to a
minimum fold change of > 1.5. The functional gene ontology, pathway annotations and upstream
regulator pathway analyses (z-score) were analyzed for the sets of differentially expressed genes
using Ingenuity Pathway Analysis (IPA) Software (Ingenuity Systems Inc., Redwood City, CA,
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USA) and gene set enrichments were analyzed using MSigDB. In order to identify drugs with
similar or opposite effects on gene expression, Connectivity Map and LINCS Canvas database
was used (18).
In Silico Transcriptomics Analyses. Cancer Genome Browser, cBioPortal for Cancer Genomics
database and Genesapiens database were utilized in FOXM1 pathway gene expressions analyses
in prostate cancer patient samples and survival plots (19, 20).
Quantitative real-time PCR. Total RNA was extracted and 2µg of total RNA was reverse
transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science).
Real-time monitoring of PCR amplification of cDNA was performed using DNA primers on ABI
PRISM 7900 HT Sequence Detection System with SYBR PCR Master Mix (Applied Biosystems,
Foster City, CA). GAPDH levels were used as an internal standard and each assay was performed
in triplicate.
Molecular docking. EADock DSS engine based Swissdock web server was utilized to dock
molecular structures of FOXM1 DNA binding domain (pdb id: 3G73) and Mon (21). Mon chemical
structure was searched from Zinc database, dockings were performed five times for each
compound and the results were analysed using UCSF Chimera. Molecular dynamics (MD)
simulations of Mon was performed starting from their docking poses in DNA binding domain of the
FOXM1 protein as predicted by Glide SP program (22). Mutant forms of FOXM1 (R236A and
Y241A; K278A and H287A) were created using MOE (23). All MD simulations were performed with
the CUDA accelerated Amber 14 program. FOXM1 force field parameters were obtained from the
ff14SB force field and the ligand; Mon parameters came from generalized amber force field with
charges derived from a RESP fit using an HF/6-31G electrostatic potential calculated using the
Gaussian 09 program. MD simulations were carried out within AMBER 14 on WestGrid facilities
from Compute Calculation Canada (https.//www.westgrid.ca). The production MD simulation was
conducted for 10 ns without any restraints under the NPT ensemble condition at a temperature of
298 K and pressure of 1 atm.
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Mutagenesis of FOXM1. FOXM1 plasmid (DNASU) was double mutated on FOXM1-Mon binding
site (R236A_Y241A or K278A_H287A) using Q5® Site-Directed Mutagenesis Kit (New England
Biosciences) using following primers: R236A and Y241A: For:
TACTCTGCCATGGCCATGATACAATTC and Rev: GGGTGGCGCCTCAGACACAGAGTTCTG,
K278A and H287A: For: AACTCCATCCGCGCGAACCTTTCCCTGCACGAC and Rev:
CTTCCAGCCTGGCGCGGCAATGTGCTTAAAGTAGG.
Chromatin immunoprecipitation, ChIP. Cells treated with or without Mon (100 nM) for 6 hours
were cross-linked with PFA (Sigma-Aldrich) and sonicated to shear DNA. ChIP assay was
performed using the ChIP Assay Kit (Agarose Beads) according to the manufacturer’s protocol
(Millipore) and antibody against FOXM1. The binding or FOXM1 to its target genes’ promoters,
PLK1, CDC25B, AURKB and CCNB1, was addressed using qPCR. The primers for promoters
were PLK1 promoter: FOR CCAGAGGGAGAAGATGTCCA and REV
GTCGTTGTCCTCGAAAAAGC, CDC25B promoter: FOR AAGAGCCCATCAGTTCCGCTTG and
REV CCCATTTTACAGACCTGGACGC, AURKB promoter: FOR GGGGTCCAAGGCACTGCTAC
and REV GGGGCGGGAGATTTGAAAAG, CCNB1 promoter: FOR
CGCGATCGCCCTGGAAACGCA and REV CCCAGCAGAAACCAACAGCCG and Actin control:
FOR AGCGCGGCTACAGCTTCA and REV CGTAGCACAGCTTCTCCTTAATGT
Western blotting. Protein lysates (5–50μg) were run on SDS-PAGE and transferred to
nitrocellulose membranes, which were blocked in Odyssey Blocking Buffer (LI-COR Biosciences,
Lincoln, NE) at room temperature for 1h. Membranes were probed overnight at 4°C with primary
antibodies FOXM1 (1:1000), GAPDH (1:5000) and vinculin (1:5000) (Abcam, Cambridge, UK),
washed three times with PBS containing 0.1% Tween for 5 minutes and incubated for 1 hour with
1:5,000 diluted Alexa Fluor secondary antibodies (Invitrogen) at room temperature. Specific
proteins were detected using ODYSSEY IR imaging system (LI-COR Biosciences).
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DARTS assay. Cell lysates were incubated with vehicle or Mon and the proteins were degraded
with different concentrations of pronase according to the protocol as previously described (24).
FOXM1 protein level was obtained using western blot. GAPDH was used as a control.
Transfection and luciferase assay. 42DENZR and 42FENZR cells were reverse transfected on
96-well plates (20,000 cells/well) with FOXM1 promoter region cloned to pGL3-Basic Luciferase
reporter plasmid (Promega, Madison, WI) kindly provided by Dr. Pradip Raychaudhuri (University
of Chicago) (25) using Lipofectamin (0.5μL/well; Invitrogen). Renilla luciferase reporter construct
was used as a transfection control. After 24h, concentration series of Mon (10 to 100 nM) and
control dilutions were added onto the cells for 18h. FOXM1–luciferase activity was measured using
Dual-Luciferase Reporter Assay System (Promega) and microplate luminometer (Tecan) according
to the manufacturer’s instructions. All experiments were carried out in triplicate.
Cell transfections: 42DENZR and 42FENZR cells were plated on 10 cm plates (1 million cells / 10 ml
complete media, Corning Life Sciences, Corning, NY) for 18-24h prior to transfection with 10 nM
FOXM1 or control siRNA (Santa Cruz Biotechnology, Dallas TX) using Oligofectamine (Invitrogen)
and OPTI-MEM media (Gibco, Gaithersburg, MD). After 4h, OPTI-MEM media was replaced with
complete media and cells were incubated for 18h prior second transfection. After 48h, cells were
harvested. The same protocol as siRNA was used for shFOXM1 transfections using shFOXM1 or
control shRNA (Santa Cruz Biotechnology) and successfully transfected clones were selected for
and expanded in complete media containing 10 μg/mL puromycin. For transient FOXM1
overexpression, LNCaP and 16DCRPC cells were seeded on 6-well plates and FOXM1 plasmid (5
μg; DNASU) was transfected using Mirus T20/20 and OPTIMEM media (Invitrogen) according to
the manufacturer’s instructions. OPTI-MEM media were replaced after 24h with complete media ±
10 μmol/L ENZ, and cells were harvested after 48h.
Flow Cytometry. Cells were exposed to Mon for 24,48 and 72h (PI) or for 24h (ALDH activity and
CD24+/CD49b- staining), samples were stained with PI for 30min at 4°C (Sub-population), CD24
and CD49b antibodies (1:20) Biolegend, San Diego, CA) for 60min or ALDH reagent according to
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manufacturer’s instructions (Stem Cell Techonolgies, Vancouver, Canada). Live cells were gated
using staining with viability dye eFluor 506 (eBioscience, San Diego, CA). Data was acquired from
10,000 events on a Canto II (BD Biosciences). The results were analyzed using FlowJo (TreeStar,
Ashland, OR).
Xenograft experiments. Athymic nude male mice (Harlan Sprague-Dawley, Cumberland, IN), 5wk
of age, were injected s.c. in the both flanks with 1×106 42DENZR cells in 200μL of Matrigel without
growth factors (BD Biosciences, San Jose, CA). Mice were castrated and after two weeks of
castration, mice were given ENZ (10 mg/kg/d). When tumor size reached 200mm3, the mice were
divided into two groups: (a) vehicle only and (b) Mon (10 mg/kg/3 times a week). Mice were treated
for 3.5wk. Tumor volumes were calculated by caliper measurements twice a week to monitor tumor
growth (tumor volume = LW2×0.56). For ALDH activity experiments, Mon (10 mg/kg/3 times a
week) and ENZ (10mg/kg/d) treatments started the next day after injections of the cells and the
tumors were harvested at the size of 100-300mm3.
Statistical analyses. All in vitro and in vivo data were assessed using the Student's t test.
Disease-free survival was analysed using Kaplan–Meier curves. Levels of statistical significance
were set at *P<0.05, **P<0.01, ***P<0.001.
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Results
‘AR-indifferent’ ENZR CRPC cells are enriched with prostate cancer subtype 1 (PCS1)
signature. Our gene expression profiling results showed that ENZR CRPC 42DENZR and 42FENZR
cells are enriched with PCS1 signature (gene expression fold changes were compared to 16DCRPC
control cells, Fig. 1A and B) whereas PCS2 signature is significantly downregulated in both cells
(Fig. 1A). The results were confirmed by gene set enrichment analyses (Fig.1C) and
(Supplementary Fig. S1). Taken together, the analysis of 42DENZR and 42FENZR cells against the
novel subtypes indicate that these cells are enriched with the most aggressive and lethal prostate
cancer subtype, PCS1. Thus, these results reveal that our ENZR cells could be further utilized to
study novel targets and therapeutics for PCS1 patient subtype.
FOXM1 is a master regulator pathway in PCS1. To identify novel therapeutics for PCS1, we
utilized the PCS1 gene set signature as well as the gene expression profiling of 42DENZR and
42FENZR cells and performed Connectivity Map (cMap) combined with LINCS analyses (18, 26) to
identify compounds that could reverse all these three signatures (Fig. 2A). The results indicated
that Thiostrepton, a Forkhead box M1 (FOXM1) inhibitor, is the most enriched compound reversing
all three signatures (connectivity scores of -98, -92 and -90, in PCS1, 42DENZR and 42FENZR,
respectively). To confirm that FOXM1 pathway is a master regulator in PCS1; we first confirmed
that FOXM1 was up-regulated in 42DENZR and 42FENZR cells compared to parental LNCaP and
16DCRPC cells (Supplementary Fig. S2A). Second GSEA analysis were performed and the analysis
revealed that FOXM1 pathway is highly enriched and significantly activated in 42DENZR and 42FENZR
(Fig. 2B) (Enrichment scores of 0.52 and 0.55 in 42DENZR and 42FENZR cells respectively, p-values
< 0.01). Ingenuity upstream regulator pathway analysis also confirmed that FOXM1 pathway was
enriched in PCS1, 42DENZR and 42FENZR cells with z-scores of 4.5, 3.8 and 3.3 respectively
(Supplementary Fig. S2B). These data were further confirmed using qRT-PCR (Fig. 2C) showing
that FOXM1 pathway is upregulated in 42DENZR and 42FENZR. In contrast, we found that AR (16),
TP53 and RB1 pathways were downregulated in these cells with z-scores of -5.16, -5.02 and -4.15
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for AR, TP53 and RB1, respectively, p-values < 0.001 (Supplementary table 1) further suggesting
that these cells are ‘AR-indifferent’. Interestingly, FOXM1 pathway was found to represent 24% of
PCS1 signature (Fig. 2D) and PCS1 was induced when FOXM1 was overexpressed in LNCaP and
16DCRPC cells and downregulated when FOXM1 was silenced in 42DENZR and 42FENZR cells
(Fig. 2E). Together these data suggest that FOXM1 pathway is a major regulator for PCS1.
Since PCS1 has been linked to high-risk prostate cancer, we next addressed whether the
observed FOXM1 pathway activation in PCS1 subtype is also seen in high-risk prostate cancer
patients and whether the pathway expression has any correlation with disease free survival. We
found that high expression of FOXM1 and its target genes’ (AURKB, AURKA, BIRC5, PLK1,
CCNB1, CCNA2, GTSE1, CCNE2, CDK1, CDKN3 and CENPA) correlate with Gleason score
(TCGA, N=550, blue: downregulation, red: upregulation) (Fig. 2F). This was confirmed using Cox
regression analysis showing a significant relationship between FOXM1 and high Gleason
compared to normal prostate samples (correlation coefficient of 0.51, p-value < 0.0001) as well as
low PSA (correlation coefficient of -0.51, p-value < 0.0001) (Supplementary Table S2). In addition,
high FOXM1 pathway expression was also seen in metastatic prostate cancer patients compared
to primary patient samples analysed using in Genesapiens database (Supplementary Fig. S2C)
(27). Finally, FOXM1, AURKB, PLK1, CCNB1 and SKP2 mRNA expression correlate with poor
survival in prostate cancer patients (Fig. 2G) (analyzed from the data of Taylor et al. (28)). In
summary, these results from both unbiased compound and pathway analyses reveal FOXM1 as a
major pathway activated in patients with PCS1 tumors as well as 42DENZR and 42FENZR cells
enriched with PCS1. FOXM1 pathway activity correlated with high risk and poor survival, which is
in accordance with the previous findings that PCS1 is the most aggressive and lethal subtype of
prostate cancer and that FOXM1 activity is associated with poor survival, metastasis, and
resistance to therapy (29, 30).
Mon is a novel FOXM1 and PCS1 subtype targeting agent. As the Connectivity Map results
revealed the FOXM1 inhibitor Thiostrepton as a potential agent targeting PCS1 tumors, we first
explored its effect on 16DCRPC, 42DENZR and 42FENZR cells. The results revealed that Thiostrepton
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reduces cell proliferation, and although differential effect was seen in 42DENZR and 42FENZR
compared to 16DCRPC (p-value < 0.01) the EC50 values were similar between the cell lines
(Fig. 3A). The anti-proliferative effect of Thiostrepton was observed at lower concentration
compared to 40µM its IC50 for FOXM1 indicating possible toxicity, a concern that has been
reported previously (31, 32). Thiostrepton is a natural compound that belongs to a group of natural
antibiotics produced by Streptomyces species. Notably, other natural antibiotics were also among
the most enriched compounds for PCS1, 42DENZR and 42FENZR including bithionol, monensin,
manumycin, oligomycin, selamectin, idarubicin and CCCP (Table1). Thus, we tested the binding of
these compounds to the FOXM1 DNA binding domain (pdb id 3G73) using in silico Swissdock
molecular docking (21). Thiostrepton-FOXM1 binding affinity was used as a reference (31). We
found that monensin (Mon) has highest binding affinity to FOXM1 with ΔG of -12.94 kcal/mol and
FullFitness of -1556.22 kcal/mol compared to Thiostrepton (31) or other hit compounds (Table 1).
This effect was translated on the ability of Mon to exert a superior inhibitory effect on FOXM1
transcriptional activity compared to Thiostrepton (Supplementary Fig. 3A). Importantly, Mon
showed a dose dependent inhibition on FOXM1 transcriptional activity (Fig. 3B) and FOXM1
expression at protein levels (Supplementary Fig. 3B). Mon has predominant selectivity for high
FOXM1 expressing 42DENZR cells by reducing cell proliferation (Fig. 3C) and inducing apoptosis
(Supplementary Fig. S3C).
To further gain insight into the molecular interactions between Monensin-FOXM1, we conducted
explicit solvent molecular dynamics (MD) simulations. During 10ns-MD simulations, we observed
that Mon was tightly bound to the DNA binding region of FOXM1 throughout the simulation period
and Arg236, Tyr241, Lys278 and His287 residues of FOXM1 interact with Mon over 80% of the
total MD simulation time using a frequent contacts map (Fig. 3D). Importantly, the compound forms
strong H-bond interactions with His 287 and Tyr241 whereas Leu291, His287 and Trp281 residues
make strong hydrophobic contacts with the chemical core (Fig. 3D). Using drug affinity responsive
target stability assay (DARTS) (24), we confirmed that Mon binds to FOXM1 in cell assay
(Supplementary Fig. 3D). To further evaluate if Mon binds to FOXM1 DNA binding, in silico guided
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site-directed mutagenesis was performed. Based on the contact frequency map as shown in Fig.
3D, two double mutants (R236A and Y241A; K278A and H287A) were generated that contribute to
Mon binding to FOXM1 and were overexpressed LNCaP cells. We found that Mon exhibited
weaker effect on FOXM1 transcriptional activity compared to WT (p-values of < 0.01 and < 0.001
for R236A_Y241A or K278A_H287A, respectively) (Fig. 3E) as well as on cell proliferation (p-
values of < 0.001 and < 0.01 for R236A_Y241A or K278A_H287A, respectively) (Fig. 3F). We next
performed MD simulations on R236A and Y241A, K278A and H287A forms of FOXM1 mutants to
study the binding to Mon. We found that Monensin could not form critical H-bond interactions with
Tyr241 and His 287 as they were mutated to Ala (Fig.3G). These data support that Mon is binds to
FOXM1 DNA binding domain. Finally, we confirmed that Mon affects FOXM1 binding to DNA using
chromatin immunoprecipitation (ChIP) and confirmed that Mon reduced FOXM1 binding to
promoters of its target genes (PLK1, CDC25B, AURKB and CCNB1) (Fig. 3H).
A genome-wide analysis of 42DENZR cells treated with Mon (Supplementary Table S3) revealed
FOXM1 as the most significant master regulator pathway inhibited by Mon (z-score -3.8,
p-value < 0.001, Supplementary Fig. S3E) which was confirmed using GSEA (Fig. 3I, Enrichment
score -0.63, p-value < 0.001). Moreover, these data were further confirmed with qRT-PCR showing
that key FOXM1 target genes were downregulated after Mon treatment (Fig. 3J). To identify
whether FOXM1 inhibition by Mon affects prostate cancer subtype-specific genes and pathways,
we compared Mon gene expression data to PCS1, PCS2 and PCS3 signatures using GSEA. The
results showed that Mon significantly reduces the PCS1 signature (enrichment score -0.66, p-
value < 0.001) but does not have a significant effect on PCS2 or PCS3 indicating that Mon
selectively targets PCS1 (Fig. 3K). Taken together, these data confirm that Mon directly binds to
and targets FOXM1, resulting in blunting of the FOXM1 pathway and, in turn, PCS1 signature.
PCS1 and ENZR CRPC cells display cancer stem-like features that are targeted by FOXM1
inhibition. In particular, PCS1-specific gene signature was also reported to be enriched with stem-
like phenotype (14). We compared the subtype-specific genes of PCS1, PCS2 and PCS3 with
embryonic stem cell core (ESC) signatures and found that approximately one third of the PCS1
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genes belong to these signatures (33, 34) whereas PCS2 and PCS3 signatures did not display
these genes (<1% of the genes listed) (Fig. 4A). Our ENZR CRPC cells enriched with PCS1 were
also significantly enriched with ECS (ES score of 0.44, p-values < 0.01) whereas Mon significantly
downregulates ECS (ES score -0.40, p-value < 0.05) (Fig. 4B). Based on the above finding, we
explored the Mon effect on the population of CD24-/CD49b+ cells, ALDH activity and tumorspheres,
as readouts of stem-like phenotype. First, we found that 42DENZR and 42FENZR were enriched with
CD24-/CD49b+ population (Supplementary Fig. S4A) and exhibit high aldehyde dehydrogenase
(ALDH) (Supplementary Fig. S5A) compared to 16DCRPC. Targeting FOXM1 using Mon reduced or
siRNA reduces CD24-/CD49b+ population (Fig. 4C, Supplementary Fig. S4B) (Supplementary
Fig. S4C), ALDH activity (Fig. 4D, Supplementary Fig. S5B) (Supplementary Fig. S5C).
Interestingly, ALDHHigh cells which display higher expression of FOXM1 (cells sorted from 42DENZR
and 42FENZR cells using FACS) were more sensitive to Mon compared to ALDHLow cells (Fig. 4E)
(Supplementary Fig. S5D and E). In addition, Mon significantly reduced the number and size of
42DENZR and 42FENZR cell tumorspheres (Fig. 4F). These data suggest that Mon not only targets
FOXM1 but also stem cell-like phenotypes.
Mon reduces ENZR CRPC xenograft growth, FOXM1 pathway and ALDH activity in vivo.
Next, we investigated Mon effect on the growth of ENZ-resistant CRPC in vivo. Mice bearing
42DENZR xenograft tumors were treated with vehicle or Mon (10mg/kg) and monitored for tumor
sizes. The mice were observed for 30 days for signs of weight loss, toxicity, and behavioural
changes. No in vivo toxicity were seen in response to Mon (Supplementary Figure S6A). The
results indicated that Mon reduced tumor growth compared to vehicle treatment (Fig. 5A).
Moreover, Mon significantly reduced the expression of FOXM1 and its pathway members CCNB1
and SKP2 (Fig. 5B) as well as ALDH activity in the tumors (Fig. 5C, Supplementary Fig. S6B).
Together, these results convey that Mon reduces the growth, FOXM1 pathway activity and ALDH
activity in 42DENZR tumors in vivo.
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16
Discussion
This preclinical study addresses the major clinical challenge of targeting the most aggressive
subtype of prostate cancer PCS1 derived from 4,600 patient’s data (14). FOXM1 was found to be
the most enriched master regulator pathway in ‘AR-indifferent’ ENZR CRPC cells that we identified
as PCS1. Interestingly, we found that PCS1 signature is upregulated in ‘AR-indifferent’ CRPC-
NEPC patients in the 2016 Beltran cohort (compared to CRPC-Adeno) (Supplementary Figure
S7A) (13) and in NPp53 abiraterone-exceptional non responders mice (compared to NPp53
vehicle) that trans-differentiate to neuroendocrine (Supplementary Figure S7B) (38) as well as in
RB1, TP53 and PTEN double and triple knockout compared to wild type in the 2017 Ku dataset
(Supplementary Figure S7C) (39). These data suggest that PCS1 signature is found in a broad
phenotype of aggressive prostate cancer. In agreement, our ENZR cells exhibit a luminal B-like
subtype by PAM50 classification (Pearson corr. 0.47, p-values < 0.01), which is associated with
the poorest clinical prognoses in prostate cancer (40). In addition, FOXM1 and its target genes
were upregulated in high risk prostate cancer and correlated with poor survival and was found to
be increased in TP53Alt/RB1Alt phenotype characterized by aggressive NEPC features (39).
Importantly, mitotic kinase AURKA and N-Myc that previously were linked to NEPC phenotype (13)
are FOXM1 target genes (41, 42). Together these findings further link the PCS1 signature to
FOXM1 pathway, AR indifferent and/or neuroendocrine prostate cancer.
We discovered Mon as a novel FOXM1 binding agent with higher binding affinity to previously
established FOXM1 inhibitor Thiostrepton (43). Mon targets and reduces FOXM1 pathway activity
and reduces PCS1 signature. Mon selectively inhibits cell proliferation in high FOXM1 expressing
cells in vitro and in vivo without any toxicity, induces apoptosis and reduces self-renewal. These
data are in accordance with previous reports indicating that FOXM1 regulates major hallmarks of
cancer such as proliferation/cell cycle, metastasis, genomic instability, stem cell renewal, DNA
damage repair and drug resistance (29, 35-37). Together, these findings suggest that Mon as a
promising drug candidate to inhibit master regulator FOXM1 and PCS1 tumors.
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17
In conclusion, our results reveal FOXM1 as a major pathway activated in PCS1 and ENZR CRPC
and Mon as a novel FOXM1 and PCS1 subtype targeting agent that also reduces stem-like
phenotype in vitro and in vivo. High FOXM1 pathway expression also correlated with aggressive
prostate cancer phenotype in prostate cancer patients including high Gleason score and poor
survival. Since there is a lack of third-line treatment options for ENZR CRPC in the clinic and there
are no therapies available for PCS1, our results indicate that targeting FOXM1 pathway may
provide novel therapeutic strategy for this aggressive subset of prostate cancer associated with
treatment resistance.
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18
Acknowledgements
We thank Dr. Pradip Raychaudhuri, University of Chicago, for the FOXM1 luciferase plasmid.
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19
References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015 Jan-Feb;65(1):5-29. PubMed PMID: 25559415. 2. Huggins C, Hodges CV. Studies on prostatic cancer: I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. 1941. J Urol. 2002 Jul;168(1):9-12. PubMed PMID: 12050481. 3. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med. 2004 Jan;10(1):33-9. PubMed PMID: 14702632. 4. Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012 Sep 27;367(13):1187-97. PubMed PMID: 22894553. 5. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, et al. Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011 May 26;364(21):1995-2005. PubMed PMID: 21612468. Pubmed Central PMCID: 3471149. 6. Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer. 2015 Dec;15(12):701-11. PubMed PMID: 26563462. Pubmed Central PMCID: 4771416. 7. Scher HI, Beer TM, Higano CS, Anand A, Taplin ME, Efstathiou E, et al. Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study. Lancet. 2010 Apr 24;375(9724):1437-46. PubMed PMID: 20398925. Pubmed Central PMCID: 2948179. 8. Ferraldeschi R, Welti J, Luo J, Attard G, de Bono JS. Targeting the androgen receptor pathway in castration-resistant prostate cancer: progresses and prospects. Oncogene. 2015 Apr 2;34(14):1745-57. PubMed PMID: 24837363. Pubmed Central PMCID: 4333106. 9. Joseph JD, Lu N, Qian J, Sensintaffar J, Shao G, Brigham D, et al. A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509. Cancer Discov. 2013 Sep;3(9):1020-9. PubMed PMID: 23779130. 10. Korpal M, Korn JM, Gao X, Rakiec DP, Ruddy DA, Doshi S, et al. An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (enzalutamide). Cancer Discov. 2013 Sep;3(9):1030-43. PubMed PMID: 23842682. 11. Li Y, Chan SC, Brand LJ, Hwang TH, Silverstein KA, Dehm SM. Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines. Cancer Res. 2013 Jan 15;73(2):483-9. PubMed PMID: 23117885. Pubmed Central PMCID: 3549016. 12. Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med. 2014 Sep 11;371(11):1028-38. PubMed PMID: 25184630. Pubmed Central PMCID: 4201502. 13. Beltran H, Prandi D, Mosquera JM, Benelli M, Puca L, Cyrta J, et al. Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer. Nat Med. 2016 Mar;22(3):298-305. PubMed PMID: 26855148. Pubmed Central PMCID: 4777652. 14. You S, Knudsen BS, Erho N, Alshalalfa M, Takhar M, Al-Deen Ashab H, et al. Integrated Classification of Prostate Cancer Reveals a Novel Luminal Subtype with Poor Outcome. Cancer Res. 2016 Sep 1;76(17):4948-58. PubMed PMID: 27302169. 15. Miyamoto DT, Zheng Y, Wittner BS, Lee RJ, Zhu H, Broderick KT, et al. RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science. 2015 Sep 18;349(6254):1351-6. PubMed PMID: 26383955. Pubmed Central PMCID: 4872391. 16. Bishop JL, Thaper D, Vahid S, Davies A, Ketola K, Kuruma H, et al. The Master Neural Transcription Factor BRN2 Is an Androgen Receptor-Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer. Cancer Discov. 2017 Jan;7(1):54-71. PubMed PMID: 27784708. 17. Kuruma H, Matsumoto H, Shiota M, Bishop J, Lamoureux F, Thomas C, et al. A novel antiandrogen, Compound 30, suppresses castration-resistant and MDV3100-resistant prostate cancer growth in vitro and in vivo. Mol Cancer Ther. 2013 May;12(5):567-76. PubMed PMID: 23493310.
Research. on February 13, 2020. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 12, 2017; DOI: 10.1158/1078-0432.CCR-17-0901
20
18. Duan QN, Flynn C, Niepel M, Hafner M, Muhlich JL, Fernandez NF, et al. LINCS Canvas Browser: interactive web app to query, browse and interrogate LINCS L1000 gene expression signatures. Nucleic Acids Research. 2014 Jul 1;42(W1):W449-W60. PubMed PMID: WOS:000339715000074. English. 19. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Science signaling. 2013 Apr 2;6(269):pl1. PubMed PMID: 23550210. Pubmed Central PMCID: 4160307. 20. Kilpinen S, Autio R, Ojala K, Iljin K, Bucher E, Sara H, et al. Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues. Genome biology. 2008;9(9):R139. 21. Grosdidier A, Zoete V, Michielin O. SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res. 2011 Jul;39(Web Server issue):W270-7. PubMed PMID: 21624888. Pubmed Central PMCID: 3125772. 22. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem. 2004 Mar 25;47(7):1739-49. PubMed PMID: 15027865. 23. Chemical Computing Group ICMOEMCM, Quebec, Canada, 2008; . www. chemcomp.com. 24. Lomenick B, Jung G, Wohlschlegel JA, Huang J. Target identification using drug affinity responsive target stability (DARTS). Curr Protoc Chem Biol. 2011 Dec 1;3(4):163-80. PubMed PMID: 22229126. Pubmed Central PMCID: 3251962. Epub 2012/01/10. Eng. 25. Petrovic V, Costa RH, Lau LF, Raychaudhuri P, Tyner AL. Negative regulation of the oncogenic transcription factor FoxM1 by thiazolidinediones and mithramycin. Cancer Biol Ther. 2010 Jun 15;9(12):1008-16. PubMed PMID: 20372080. Pubmed Central PMCID: 3005150. Epub 2010/04/08. eng. 26. Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al. The connectivity map: Using gene-expression signatures to connect small molecules, genes, and disease. Science. 2006 Sep 29;313(5795):1929-35. PubMed PMID: WOS:000240832200043. English. 27. Kilpinen S, Autio R, Ojala K, Iljin K, Bucher E, Sara H, et al. Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues. Genome Biol. 2008;9(9):R139. PubMed PMID: 18803840. Pubmed Central PMCID: 2592717. 28. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell. 2010 Jul 13;18(1):11-22. PubMed PMID: 20579941. Pubmed Central PMCID: 3198787. 29. Aytes A, Mitrofanova A, Lefebvre C, Alvarez MJ, Castillo-Martin M, Zheng T, et al. Cross-species regulatory network analysis identifies a synergistic interaction between FOXM1 and CENPF that drives prostate cancer malignancy. Cancer cell. 2014 May 12;25(5):638-51. PubMed PMID: 24823640. Pubmed Central PMCID: 4051317. Epub 2014/05/16. eng. 30. Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV, et al. Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res. 2006 Feb 1;66(3):1712-20. PubMed PMID: 16452231. Pubmed Central PMCID: 1363687. Epub 2006/02/03. eng. 31. Chen Y, Ruben EA, Rajadas J, Teng NN. In silico investigation of FOXM1 binding and novel inhibitors in epithelial ovarian cancer. Bioorg Med Chem. 2015 Aug 1;23(15):4576-82. PubMed PMID: 26164623. 32. Gormally MV, Dexheimer TS, Marsico G, Sanders DA, Lowe C, Matak-Vinkovic D, et al. Suppression of the FOXM1 transcriptional programme via novel small molecule inhibition. Nat Commun. 2014 Nov 12;5:5165. PubMed PMID: 25387393. Pubmed Central PMCID: 4258842. 33. Wong JC, Jack MM, Li Y, O'Neill C. The epigenetic bivalency of core pancreatic beta-cell transcription factor genes within mouse pluripotent embryonic stem cells is not affected by knockdown of the polycomb repressive complex 2, SUZ12. PLoS One. 2014;9(5):e97820. PubMed PMID: 24845830. Pubmed Central PMCID: 4028244.
Research. on February 13, 2020. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 12, 2017; DOI: 10.1158/1078-0432.CCR-17-0901
21
34. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008 May;40(5):499-507. PubMed PMID: 18443585. Pubmed Central PMCID: 2912221. 35. Bella L, Zona S, Nestal de Moraes G, Lam EW. FOXM1: A key oncofoetal transcription factor in health and disease. Seminars in cancer biology. 2014 Dec;29:32-9. PubMed PMID: 25068996. 36. Halasi M, Gartel AL. FOX(M1) news--it is cancer. Mol Cancer Ther. 2013 Mar;12(3):245-54. PubMed PMID: 23443798. Pubmed Central PMCID: 3596487. 37. Koo CY, Muir KW, Lam EW. FOXM1: From cancer initiation to progression and treatment. Biochim Biophys Acta. 2012 Jan;1819(1):28-37. PubMed PMID: 21978825. 38. Zou M, Toivanen R, Mitrofanova A, Floch N, Hayati S, Sun Y, et al. Transdifferentiation as a Mechanism of Treatment Resistance in a Mouse Model of Castration-Resistant Prostate Cancer. Cancer Discov. 2017 Jul;7(7):736-49. PubMed PMID: 28411207. Pubmed Central PMCID: 5501744. 39. Ku SY, Rosario S, Wang Y, Mu P, Seshadri M, Goodrich ZW, et al. Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance. Science. 2017 Jan 06;355(6320):78-83. PubMed PMID: 28059767. Pubmed Central PMCID: 5367887. 40. Zhao SG, Chang SL, Erho N, Yu M, Lehrer J, Alshalalfa M, et al. Associations of Luminal and Basal Subtyping of Prostate Cancer With Prognosis and Response to Androgen Deprivation Therapy. JAMA oncology 2017 doi 10.1001/jamaoncol.2017.0751. 41. Lefebvre C, Rajbhandari P, Alvarez MJ, Bandaru P, Lim WK, Sato M, et al. A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers. Mol Syst Biol. 2010 Jun 08;6:377. PubMed PMID: 20531406. Pubmed Central PMCID: 2913282. 42. Wang IC, Ustiyan V, Zhang Y, Cai Y, Kalin TV, Kalinichenko VV. Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic Kras(G12D.). Oncogene. 2014 Nov 13;33(46):5391-6. PubMed PMID: 24213573. 43. Hegde NS, Sanders DA, Rodriguez R, Balasubramanian S. The transcription factor FOXM1 is a cellular target of the natural product thiostrepton. Nat Chem. 2011 Sep;3(9):725-31. PubMed PMID: 21860463. Epub 2011/08/24. eng.
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Figures
Figure 1. ‘AR-indifferent’ ENZR CRPC cells are enriched with the most aggressive, lethal
prostate cancer subtype PCS1. A and B) Expression of prostate cancer subtype-specific genes,
PCS1, PCS2 and PCS3 (14), in 42DENZR and 42FENZR cells (16). Fold changes were compared to
16DCRPC control cells C) Gene set enrichment analysis of PCS1 signature in 42DENZR and 42FENZR
cells.
Figure 2. FOXM1 is a master regulator pathway in PCS1. A) Common compounds reversing the
gene expression signature of prostate cancer (PCa) patient PCS1 subtype and 42DENZR and
42FENZR cells analysed using cMap and LINCS (18, 26). B) GSEA of FOXM1 pathway in 42DENZR
and 42FENZR cells. C) Expression of FOXM1, AURKB, PLK1, CCNB1 and SKP2 at mRNA levels by
qRT-PCR in 16DCRPC, 42DENZR and 42FENZR cells D) The percentage of PCS1, PCS2 and PCS3
subtype-specific genes in FOXM1 pathway. E) The effect of FOXM1 overexpression in LNCaP and
16DCRPC cells and silencing in 42DENZR and 42FENZR cells on PCS1 signature genes. F) The mRNA
expression of FOXM1, AURKB, AURKA, BIRC5, PLK1, CCNB1, CCNA2, GTSE1, CDK1, CDK3,
CENPA and KLK3 in prostate cancer patients (TCGA, n=550). G) Survival plots of FOXM1,
AURKB, PLK1, SKP2 and CCNB1 in prostate cancer patient data analysed using Taylor dataset in
cBioportal (28).
Figure 3. Mon is a novel FOXM1 and PCS1 subtype targeting agent. A) Relative cell
proliferation of 16DCRPC, 42DENZR and 42FENZR cells in response to various concentrations of
Thiostrepton using CTG cell proliferation assay. Graph represents pooled data from three
independent experiments. B) Effect of Mon (10 to 100 nM) on FOXM1 transcriptional activity
assessed by luciferase activity assay. C) Relative cell proliferation of 16DCRPC, 42DENZR and
42FENZR cells in response to various concentrations of Mon. Graph represents pooled data from
three independent experiments. D) Frequent contacts map of FOXM1-Mon binding reveal that
Arg236, Tyr241, Lys278 and His287 interact with Mon over 80% of the total MD simulation time. E)
Effect of Mon on mutated FOXM1 compared to WT as measured by transcriptional activity
(FOXM1 activity p-values of < 0.01 and < 0.001 for R236A_Y241A or K278A_H287A, respectively).
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F) Effect of Mon on mutated FOXM1 compared to WT as measured by cell proliferation (FOXM1
activity p-values -values of < 0.001 and < 0.01 for R236A_Y241A or K278A_H287A respectively).
G) MD stimulation showing thta Mon could not form critical H-bond interactions with Tyr241 and
His 287 as they were mutated to Ala. H) Cells treated with or without Mon (100 nM) for 6 hours
were cross-linked with PFA (Sigma-Aldrich) and sonicated to shear DNA. ChIP assay was
performed using the ChIP Assay Kit (Agarose Beads) according to the manufacturer’s protocol
(Millipore) and antibody against FOXM1. The FOXM1 binding to its target genes’ promoters, PLK1,
CDC25B, AURKB and CCNB1 was evaluated using qPCR. I) Effect of Mon on FOXM1 pathway in
42DENZR cells assessed by gene expression profiling and gene set enrichment analysis (GSEA) J)
mRNA expression of FOXM1 and its targets AURKB, PLK1, SKP2 and CCNB1 assessed by qRT-
PCR. Graph represents pooled data from three independent experiments. K) Effect of Mon on
prostate cancer subtype signatures PCS1, PCS2 and PCS3 in 42DENZR cells analysed by gene
expression profiling and GSEA.
Figure 4. PCS1 and ENZR CRPC cells display stem-like features that are targeted by FOXM1
inhibition. A) The percentage of PCS1, PCS2 and PCS3 subtype-specific genes in embryonic
stem cell signatures (33, 34). B) Mon effect on Wong et al. embryonic stem cell signature
assessed using GSEA on ctrl and Mon treated 42DENZR cells. C) CD49high population. Percentage
of high CD49b expressing cells in 42DENZR and 42FENZR cells in comparison to 16DENZR cells using
flow cytometry (left panel). Effect of Mon on the percentage CD49high population in 42DENZR and
42FENZR cells using flow cytometry (middle panel). Effect of FOXM1 siRNA on CD49high population
in 42DENZR and 42FENZR cells using flow cytometry (right panel). D) ALDH activity. Percentage of
ALDH activity in 42DENZR and 42FENZR cells in comparison to 16DENZR cells assessed by Aldefluor
ALDH activity using flow cytometry (left panel). Effect of Mon on ALDH activity in 42DENZR and
42FENZR cells in comparison to 16DENZR cells assessed by Aldefluor ALDH activity using flow
cytometry (middle panel). Effect of FOXM1 siRNA ALDH activity in 42DENZR and 42FENZR cells in
comparison to 16DENZR cells assessed by Aldefluor ALDH activity using flow cytometry (right
panel). E) Relative confluence of 42DENZR and 42FENZR cells with high and low ALDH activity in
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24
response to Mon treatment assessed by IncuCyte (Essen Biosciences). Graphs represent pooled
data from three independent experiments. F) Effect of Mon on the size and the number 42DENZR
and 42FENZR tumorspheres.
Figure 5. Mon reduces ENZR CRPC xenograft growth, FOXM1 pathway and ALDH activity in
vivo. A) Effect of vehicle or Mon on tumor growth of 42DENZR xenografts. Graph represents pooled
data from 6 vehicle and 6 Mon treated tumors. B) Relative mRNA expression assessed by qRT-
PCR of FOXM1, CCNB1 and SKP2 in vehicle vs. Mon treated xenografts. C) Percentage of cells
with high ALDH activity in 42DENZR tumor xenografts treated with vehicle or Mon. Graph represents
pooled data from 4 vehicle and 4 Mon treated tumors.
Table 1. In silico binding of antibiotic compounds reversing PCS1, 42DENZR and 42FENZR signatures
to FOXM1 DNA binding domain analysed using SwissDock.
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42D
42F
0.0
0.20.1
0.40.3
0.50.6
En
rich
me
nt
sco
re (
ES
)
p<0.001
42D : PCS1
42D 16D ENZR CRPC
PCS1 PCS2 PCS3
MCM4CCNB1CDC6CDKN3EZH2TPX2FOXM1KIF11HMMRMKI67KNTC1RAB3BANK3GJB1SLC12A2CFDPTGDSLTBP4SPEGGABRPACTC1ASPACOL4A6SGCASLC2A5PAGE4ACOX2C16orf45
PCS1
PCS2
PCS3
B
A
0.0
0.20.1
0.40.3
0.50.6
En
rich
me
nt
sco
re (
ES
)
42F : PCS1
42F 16D ENZR CRPC
ENZR
ENZR
ENZR
ENZR
p<0.001
Figure 1
10
-10
0
ES=0.64
ES=0.66
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A
42D
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ENZR
Dise
ase
Free
mRNA upregula�on no change
p<0.001 100
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50
0
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CancerNormalHigh Gleason
Months Disease Free Months Disease Free Months Disease Free Months Disease Free Months Disease Free 0 40 80 120 160 0 40 80 120 160 0 40 80 120 160 0 40 80 120
PCS1
1000-100
GTSE1CCNA2CCNB1
PLK1BIRC5
AURKAAURKBFOXM1
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CDK1CCNE2
FOXM1 AURKB PLK1 SKP2 CCNB1p<0.001 p<0.1p<0.1
Pa�ents (n=550), blue low, red high m
RNA expression
Figure 2
G
E
PCa pa�entsPCS1 signature
Compounds (connec�vity score)
Comparison
Common hits (incl. FOXM1 inhibitor
Thiostrepton)
PCa cell lines
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- - - - - - - -
1 13 0.3
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-0.5
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Figure 3
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Ctrl
Ctrl Ctrl Ctrl
Research. on February 13, 2020. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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Time (h)0 20 40 60 80
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A
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Figure 4
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C
0
2
4
6
% A
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pos
i�ve
cel
ls
Vehicle Mon
A B
mRN
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pres
sion
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�ve
to c
ontr
ol
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2
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0 0.5 1 1.5 2 2.5 3 3.5
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or g
row
th (f
old
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ge)
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Vehicle Mon
**
** *
Figure 5
VehicleMon
0 10 3 10 4 10 50
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100K
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0 10 3 10 4 10 50
50K
100K
150K
200K
250K
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MonVehicle
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ALDH (FITC) ALDH (FITC)
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Table 1.
Drug Target
FOXM1, Estimated ΔG
(kcal/mol)
FOXM1, FullFitness
(kcal/mol)
monensin antibiotic ‐12.94 ‐1556.22
idarubicin antibiotic, Topoisomerase II inhibitor ‐7.96 ‐807.24
bithionol antibacterial and anthelmintic ‐6.49 ‐788.62
manumycin antibiotic, Ras inhibitor ‐6.46 ‐601.04
CCCP antibiotic, inhibitor of oxidative phosphorylation ‐6.16 ‐776.52
thiostrepton FOXM1 inhibitor ‐9.4 kcal/mol (Chen et al., 2015)
oligomycin antibiotic, Mitochondrial ATP synthase Inhibitor 3D structure not available in the ZINC database
selamectin antihelminthic 3D structure not available in the ZINC database
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Published OnlineFirst September 12, 2017.Clin Cancer Res Kirsi Ketola, Ravi SN Munuganti, Alastair Davies, et al. Pathway InhibitionTargeting Prostate Cancer Subtype 1 by Forkhead box M1
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