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1 Targeting Prostate Cancer Subtype 1 by Forkhead box M1 Pathway Inhibition Kirsi Ketola 1 , Ravi S.N Munuganti 1 , Alastair Davies 1 , Ka Mun Nip 1 , Jennifer L. Bishop 1 , Amina Zoubeidi 1,2 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: [email protected] 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 Research. on February 13, 2020. © 2017 American Association for Cancer clincancerres.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
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Targeting Prostate Cancer Subtype 1 by Forkhead …...1 Targeting Prostate Cancer Subtype 1 by Forkhead box M1 Pathway Inhibition Kirsi Ketola1, Ravi S.N Munuganti1, Alastair Davies1,

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Page 1: Targeting Prostate Cancer Subtype 1 by Forkhead …...1 Targeting Prostate Cancer Subtype 1 by Forkhead box M1 Pathway Inhibition Kirsi Ketola1, Ravi S.N Munuganti1, Alastair Davies1,

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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: [email protected]

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

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

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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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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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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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Acknowledgements

We thank Dr. Pradip Raychaudhuri, University of Chicago, for the FOXM1 luciferase plasmid.

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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

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A

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C

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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

  Updated version

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Access the most recent version of this article at:

  Material

Supplementary

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Access the most recent supplemental material at:

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