Abstract
Purpose: Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach.
Experimental Design: We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1.
Results: We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell–like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor–directed therapies, without toxic effects on normal tissues.
Conclusions: Our data offer a paradigm for targeting TICs and support the development of BMI-1–targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176–91. ©2016 AACR.
BMI-1 is associated with tumor initiation, progression, and resistance to therapy. We identified a class of small-molecule post-transcriptional inhibitors of BMI-1. BMI-1 inhibitors that target self-renewal of tumor initiating cells may be developed to treat specific cancers.
Introduction
Prostate cancer is the most common cancer affecting men in the developed world (1). Current therapies including androgen deprivation (ADT) and androgen receptor (AR)-directed therapies are only temporarily effective, and therapy resistance and relapse are commonly inevitable (2). We and others have shown that primary prostate cancer contain cells endowed with self-renewal and tumorigenic potential (3–8), known as tumor-initiating cells (TIC; ref. 9). By virtue of their resistance to therapy, TICs could be the prime cause of tumor relapse. Thus, to accomplish tumor eradication, efforts are made to design TIC-tailored therapy that would selectively target these highly aggressive tumorigenic cells.
An attractive treatment strategy is to use agents capable of impeding the self-renewal abilities of TICs, therefore targeting heterogeneous cells in all tumor clone(s) within a given patient. BMI-1 (B-cell–specific MMLV insertion site-1), a member of the polycomb family of the chromatin-remodeling complex, was shown to regulate stem cell self-renewal (10) and play a key role in prostate cancer initiation and progression (11). In clinical specimens, BMI-1 expression correlates with high rates of prostate cancer recurrence (12), and downstream targets of BMI-1 are associated with therapy-resistant prostate cancer (13). These data closely associate BMI-1 with the presence of tumor-initiating stem-like cells in clinical prostate cancer samples, making it reasonable to assume that small-molecule inhibitors targeting BMI-1 could be the first in a new class of antitumor therapy directed against self-renewing and chemoresistant TICs.
Previously, we developed a surrogate self-renewal assay that allowed us to isolate TICs from prostate cancer tissue based on α2β1-integrin (also called CD49b/CD29) and CD44 protein expression (6). Herein, we demonstrate that in prostate TICs, BMI-1 is overexpressed and functionally regulates their survival and maintenance. Targeting of BMI-1 with a novel inhibitor impaired self-renewal and migratory potential in vitro. Consistently, BMI-1 inhibition in vivo decreased tumor growth and significantly reduced TICs in patient-derived samples and tumor xenografts, as evaluated by CD49b/CD29/CD44 staining, serial transplantation in vivo, and clonogenic prostasphere assays ex vivo. Remarkably, these outcomes were not observed following conventional chemotherapy treatments, while targeting BMI-1 also inhibited prostate cancer cells resistant to AR-directed therapies. Given the role of TICs in therapy resistance, these observations support the evaluation of BMI-1 inhibitors for a more effective prostate cancer management.
Materials and Methods
Materials
Initial small-molecule inhibitors were from PTC Therapeutics. C-209 was latter synthetized and purified by the Department of Chemistry at Rutgers University (New Brunswick, NJ). Docetaxel (also called taxotere), doxorubicin, and methotrexate were from Rutgers Cancer Institute of New Jersey (CINJ) pharmacy. Cycloheximide was purchased from Cell Signaling Technology. Collagen-I was bought from BD Biosciences, and NOD/SCID/IlRγmice were from the The Jackson Laboratory.
Collagen adherence assay
Putative cancer stem–like cells, or TICs, were isolated by combining phenotypic analyses (3) with collagen adherence as described previously (6). Briefly, tissue culture dishes were coated with 70 μg/mL of collagen-I for 1 hour at room temperature or overnight at 4°C. Subsequently, plates were washed with PBS and blocked in 0.3% BSA for 30 minutes. Cells were plated on collagen plates for 5 or 20 minutes. Next, cells adhering in 5 minutes and not adhering after 20 minutes were collected and used for further experiments.
Identification of BMI-1 post-transcriptional inhibitors
We have previously examined a small-molecule library (PTC Therapeutics) for post-transcriptional inhibitors of BMI-1 utilizing luciferase reporters encompassing the 5′UTR and 3′UTR of human BMI-1 (14). Anti-BMI-1 antibody (Millipore, clone F6) was used for ELISA assays and Western blotting. The principal BMI-1′s downstream target, mono-ubiquitinated (γ) histone H2A, was examined using a mouse monoclonal anti-ubiquityl-histone H2A antibody (clone E6C5; Millipore). The selectivity of C-209 was further investigated by profiling it against both a library of purified protein kinase targets using the Z'-LYTE SelectScreen profiling activity assay (Invitrogen) against 245 kinases at [ATP] Km and C-209 (3 μmol/L), and a phosphatase profiler assay with an IC50 profiler (Millipore). Both assays yielded <10% activity for C-209.
Electrostatic potential and docking of C-209 to the human BMI-1 RNA
All quantum mechanics calculations were performed using Gaussian 09. C-209 was geometry optimized at the PM6 level using tight convergence. A single‐point energy calculation at the B3LYP/6‐31G(d) level was performed and Merz‐-Kollman partial atomic charges were estimated from the electrostatic potential. The reported energy is gas phase. The surface and contour plot was prepared using the GaussView program. The electrostatic potential allowed us to build a model for docking (15) of C-209 to the human BMI RNA. We used the UCSF DOCK program (v6.7). The small-molecule C-209 was built using the Spartan (Wavefunction, Inc) quantum mechanics package and geometry optimized at the PM6 semiempirical level. The Amber99SB partial atomic charges were used on the RNA and AM1-BCC partial atomic charges were calculated for C-209 within the UCSF Chimera molecular graphics package (15). The interaction energy scores (Eint) estimate the binding energy in the DOCK scoring of C-209 with BMI-1 RNA, using guanine as a reference. DOCK scores, Evdw (kcal/mol), Eelec (kcal/mol), and Eint (kcal/mol), were generated using the following equation: Eint = Evdw + Eelec. C-209 DOCK scores were Evdw (kcal/mol) −60.6, Eelec (kcal/mol) −5.2, and Eint (kcal/mol) −65.8, as compared with guanine scores of Evdw (kcal/mol) −36.4, Eelec (kcal/mol) −4.1, and Eint (kcal/mol) −40.5. The lower the energy score, the more stable the complex contacts with the RNA due to complete fitting into the binding pocket.
Patient-derived cell culture
Primary normal and prostate cancer cells were isolated from specimens obtained at Rutgers CINJ in accordance with an Institutional Review Board (IRB)-approved protocol and upon informed consent from patients undergoing surgical resection. Normal and tumor samples were each extracted from the same patient as adjacent tissues examined and verified following pathologic examination. For cell isolation from surgical specimen, tissue was minced into small pieces and incubated with 1× collagenase (Sigma Aldrich) for 2 to 4 hours depending on the size of the tissue. After incubation, the dissociated pieces were strained with a 70-μm filter to remove debris and the dissociated cells were washed with PBS at 250 × g for 2 minutes to eliminate fibroblasts. The recovered cells were cultured in prostate epithelial basal media (PrEBM, Lonza) for at least 14 days before being used for experiments at low passage numbers. Cells from immortalized prostate cancer lines were maintained at low passage numbers in RPMI media (Gibco), 10% FBS, and 1% penicillin–streptomycin. TICs, obtained from DU145 cells after selection (6), were maintained in keratinocyte serum–free medium (KSFM) supplemented with EGF and bovine pituitary extract (KSFM media; all from Invitrogen). Protein analyses, flow cytometry, cell sorting, cell viability and survival assays, and cell migration assays are described in further detail in the Supplementary Methods.
Spheroid forming assay
We have previously characterized spheroid forming (prostasphere) abilities from multiple prostate cancer cell lines and primary cells (6). To measure prostasphere-forming abilities, 2 × 103 cells/well were suspended in KSFM media and plated on 1% agarose-coated plates. Every 3 days, half of the media were replaced and prostaspheres of >50 μm in diameter and consisting of >50 cells were counted on day 14. Single cells from day 7 spheroids were used in secondary and tertiary spheroid assays. Colony-forming abilities of prostate cancer cells plated at 1 × 103 cells/well in 6-well dishes coated with 1% agar were performed as described previously (16). Methotrexate (10 nmol/L), doxorubicin (50 nmol/L), and cycloheximide (10 μg/mL) were employed. The prostasphere and colony-forming assays are described in further details in the Supplementary Methods.
Luciferase target assay
Cells were transfected with Lipofectamine 2000 (Invitrogen) using the following plasmids: pCDNA3.1+Luc Vector UTR, pCDNA3.1+Luc BMI-1 3′UTR, pCDNA3.1+ Luc BMI-1 5′UTR, and pCDNA3.1+Luc BMI-1 5′ and 3′UTR as described previously (14). To normalize the transfection efficiency, cells were transfected with pCMV-AcGFP1 at a ratio of 1/3 along with the UTR plasmids. Cells were then visually counted for GFP+ cells. After 19 hours, 5,000 cells/well were seeded in a 96-well plate and 6 hours later, C-209 was added. At 24 hours post-treatment, cells were assayed for luciferase activity using Steady-Glo system (Promega). For qPCR, total RNA was extracted using TRIzol Reagent (Life Technologies) and purification was assessed with RNeasy plus Mini kit (Qiagen). cDNA was synthesized from 100 ng of total RNA using SuperScript VILO cDNA Synthesis Kit (Life Technologies) according to the manufacturer's instruction. Synthesized cDNAs (10 ng) were used as templates for real-time PCR using EXPRESS SYBR GreenER qPCR supermix. qPCR was performed in the StepOnePlus real-time PCR system (Applied Biosystems).
Labeling, transplantation, and drug treatment of prostate cancer grafts in zebrafish
Wild-type EKK, Casper, and *AB zebrafish (Danio rerio) were maintained following an approved aquatic animal protocol. Adult fish were spawned and reared in conditioned water at 28.5°C on a 14-hour-light 10-hour-dark cycles. Embryos were staged as described (http://zfin.org). Quantum dots (QD) labeled human prostate cancer cells were tracked in embryos and juvenile Casper fish as described previously (6). After initial imaging, transplanted embryos were maintained at 33°C for up to 12 days. Juvenile zebrafish at 6–8 weeks of age were immune-suppressed with 10 μg/mL dexamethazone for 2 days as described previously (17). Xenografts were examined for QD fluorescence upon tumor formation and treatment, and equal numbers of QD-positive cells from pooled primary grafts were used for injection into secondary recipients. Sections were examined for histologic and IHC analyses and compared with primary tissues as described previously (6). Further details are provided in the Supplementary Methods.
Small molecular translation assay
Transcription and translation of BMI-1 in vitro was done utilizing the human BMI-1 cDNA. Briefly, the full-length 3.2 Kb fragment of the human BMI-1 cDNA (containing 5′UTR and 3′UTR) was subcloned into the BamHI site of pSK+ downstream of the T7 promoter. The resulting pSK+-hBMI-1-cDNA vector was linearized with SacI, purified, and utilized for TNT coupled transcription/translation systems (Promega) following the manufacturer's instructions. T7-mediated translation of mRNA (133 nmol/L), after preincubation with or without 2 μmol/L C-209 for 60 minutes at 30°C was performed in cell-free reticulocyte lysates. Aliquots of the transcribed products were run on an agarose gel to confirm equal transcription. The newly synthesized proteins were analyzed on SDS-PAGE and probed for BMI-1 expression using the rabbit monoclonal anti-BMI-1 (D20B7) antibody (Cell Signaling Technology).
Treatment of mouse xenografts
Animal studies were performed according to Robert Wood Johnson Medical School IACUC protocol #I12-024-5. To differentiate tumor from contaminating nontumor mouse cells, prior to mice injection, DU145 cells were infected with lentiviral vector encoding luciferase2/enhanced GFP (Luc2/EGFP) that was generated as described previously (18). Luc2/EGFP cells were sorted (or enriched with the adherence assay) and CD49hi/CD29hi/CD44hi cells were isolated, suspended in 100 μL mixed 1:1 with Matrigel (BD Biosciences), and injected subcutaneously into the left flank of 6-week-old NOD SCID IL-2Rnull (NSG) mice. After tumor formation, mice were randomized and subcutaneously administered with docetaxel 6 mg/kg once per week for 2 weeks, or C-209 at a dose of 60 mg/kg daily for 12 days. Tumor growth was evaluated with an electronic caliper before every administration, and measured every 3 days until day 30 and tumors were subsequently removed. Paraffin sections (5 μm) of day 30 mouse xenografts were H&E stained and incubated with anti-Ki67 (Upstate-Millipore), -BMI-1 (Cell Signaling Technology), and -CD44 (R&D Systems). To calculate the retention of tumor-seeding capacity, tumor xenografts were dissociated after treatments, and recovered cells were sorted for EGFP. Equal numbers of EGFP-positive cells were next reinjected into secondary recipients.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc). Data are presented as mean ± SD. Statistical significance was determined by Student t test or ANOVA (one-way or two-way) with Bonferroni post hoc test. Mann–Whitney U test was used to compare the differences in xenograft tumor volumes between two groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Results
BMI-1 is a potential target for human prostate TICs
BMI-1 is a key player in prostate cancer initiation, recurrence, and progression (11, 12). Accordingly, we found that BMI-1 is differentially expressed in prostate cancer cell lines, but low in normal prostate epithelial cells (Supplementary Fig. S1A). To assess the functional role(s) of BMI-1 in prostate cancer, we performed BMI-1 loss-of-function analyses in DU145 prostate cancer cells (Supplementary Fig. S1B). Downregulation of BMI-1 was associated with decreased cell motility and clonogenic capability, as well as decreased cell survival, alone and when combined with chemotherapy (Supplementary Fig. S1C–S1E). Notably, resistance to drug-induced apoptosis, motility, invasiveness, and clonogenicity have been traced to TICs (19).
We recently found that in prostate cancer, clonogenic, migratory, and in vivo tumorigenic potentials are enriched in the collagen-I rapidly adherent (5 minutes) CD49bhiCD29hiCD44hi cell population (6), identified therefore as TICs. Thus, we utilized this functional adherence assay to analyze BMI-1 expression. Indeed, the rapidly adherent DU145 cells were enriched in the CD49bhiCD29hiCD44hi phenotype (Fig. 1A and B), and significantly overexpressed BMI-1, both at the RNA (Fig. 1C) and protein levels (Fig. 1D and E and Supplementary Fig. S2A). Furthermore, as expected, the rapidly adherent CD49bhiCD29hiCD44hi TICs were enriched for the other prostate TIC markers integrin-α6 (CD49f) and TROP (refs. 4, 8; Supplementary Fig. S2B), suggesting that BMI-1 is enhanced in the more tumorigenic cell compartment (6) of prostate cancer. To confirm our results and model, we also assessed BMI1 expression upon sorting of the high and low CD49CD29CD44 cells and found the same outcomes (Supplementary Fig. S2C).
Assessment of BMI-1 in prostate cancer TICs isolated by combined adherence and phenotypic assays. A, Flow cytometric analyses. B, Percentage of cells identified by flow cytometry to express the TIC phenotype. C, Relative mRNA BMI-1 level in total, CD49bhiCD29hiCD44hi (high) and CD49blowCD29lowCD44low (low) DU145 cells. D, Western blot analysis showing BMI-1 expression levels between total, CD49bhiCD29hiCD44hi and CD49blowCD29lowCD44low DU145 cells. E, BMI-1 expression quantitation from six independent experiments. Anti-actin was used as a loading control. F, Fold adhesion of rapidly adherent CD49bhiCD29hiCD44hi cells assessed over total DU145 control (Sh-Scr) and DU145 BMI-1–depleted (Sh-BMI-1) cells. Results are shown as mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001.
Assessment of BMI-1 in prostate cancer TICs isolated by combined adherence and phenotypic assays. A, Flow cytometric analyses. B, Percentage of cells identified by flow cytometry to express the TIC phenotype. C, Relative mRNA BMI-1 level in total, CD49bhiCD29hiCD44hi (high) and CD49blowCD29lowCD44low (low) DU145 cells. D, Western blot analysis showing BMI-1 expression levels between total, CD49bhiCD29hiCD44hi and CD49blowCD29lowCD44low DU145 cells. E, BMI-1 expression quantitation from six independent experiments. Anti-actin was used as a loading control. F, Fold adhesion of rapidly adherent CD49bhiCD29hiCD44hi cells assessed over total DU145 control (Sh-Scr) and DU145 BMI-1–depleted (Sh-BMI-1) cells. Results are shown as mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001.
Thus, to study whether BMI-1 expression modulates the levels of TICs, we employed the collagen adherence assay after lentiviral-mediated knockdown. Loss of BMI-1 in sh-BMI-1 DU145 cells resulted in a significant (∼40%) decrease in the numbers of rapidly adherent CD49bhiCD29hiCD44hi cells (Fig. 1F), suggesting an impact of BMI-1 on the TIC population.
Identification of pharmacologic BMI-1 inhibitors
We previously demonstrated that BMI-1 expression is tightly controlled by posttranscriptional processes mapping to the 5′- and 3′-untranslated regions (UTRs) (14). In reporter cells containing the luciferase open reading frame flanked with the human BMI-1 5′- and 3′-UTRs, the BMI1 3′-UTR enhanced BMI-1 expression, while the internal ribosome entry site (IRES)-containing 5′-UTR impaired BMI-1 expression and controlled the effect of the 3′-UTR (14).
These observations allowed the construction of a platform to identify compounds impacting the regulatory mechanisms within the BMI-1 5′UTR and 3′UTR to modulate BMI-1 protein expression (14, 20).
A high-throughput screen against a library of >200,000 small molecules (PTC Therapeutics) identified seven compounds targeting the posttranscriptional control mechanisms described above (14, 20). To examine their antitumor activity, we determined their IC50 concentrations in DU145 cells (Supplementary Fig. S3A). Among them, three compounds: C-209, C-210, and C-211 significantly decreased the number of rapidly adherent and highly BMI-1 expressing CD49bhiCD29hiCD44hi cells by an average of 30%–50% (Fig. 2A). Accordingly, inhibition of proliferation was also observed following C-209 treatment in sorted CD49bCD29CD44hi versus CD49bCD29CD44low cells (Supplementary Fig. S3B).
BMI-1 inhibition reduces TIC number and interferes with self-renewal capacity in vitro. A, Fold adhesion of rapidly adherent CD49bhiCD29hiCD44hi cells evaluated upon treatment of total DU145 with inhibitors targeting BMI-1 for 72 hours. B, IC50s of compounds C-209-211 assessed in DU145 cells through an ELISA assay. C, Effects of BMI-1 post-transcriptional inhibitors versus the nonspecific protein translation inhibitor cycloheximide (CHX), or the chemotherapeutics methotrexate (MTX) and doxorubicin (Doxo) on secondary (left) and tertiary (right) prostate spheroids formation. Treatments that were statistically significant were indicated as *, P < 0.05 and **, P < 0.01, compared with untreated.
BMI-1 inhibition reduces TIC number and interferes with self-renewal capacity in vitro. A, Fold adhesion of rapidly adherent CD49bhiCD29hiCD44hi cells evaluated upon treatment of total DU145 with inhibitors targeting BMI-1 for 72 hours. B, IC50s of compounds C-209-211 assessed in DU145 cells through an ELISA assay. C, Effects of BMI-1 post-transcriptional inhibitors versus the nonspecific protein translation inhibitor cycloheximide (CHX), or the chemotherapeutics methotrexate (MTX) and doxorubicin (Doxo) on secondary (left) and tertiary (right) prostate spheroids formation. Treatments that were statistically significant were indicated as *, P < 0.05 and **, P < 0.01, compared with untreated.
BMI-1 enables transcriptional repression of >1,600 target genes through the PRC1 complex (21); therefore, targeting BMI-1 would evoke complex cellular responses depending on the cell type and/or activated pathways. We evaluated BMI-1 protein inhibition by ELISA and Western blotting in comparison with EZH2; a closely related PRC2 protein with a similarly short half-life (22). Indeed, all three compounds reduced BMI-1 expression in a dose-dependent manner (Fig. 2B and Supplementary Fig. S3C), but had no effects on EZH2 levels (Supplementary Fig. S3C).
Furthermore, BMI-1 knockdown induces senescence (10). Treatment of mouse embryonic fibroblasts (MEF), that are either Bmi-1+/+ or Bmi-1−/−, with C-209, C-210, and C-211, respectively, elicited a significant dose-dependent increase in senescence in Bmi-1+/+ MEFs, and sh-BMI-1 DU145 cells, but not in the highly senescent Bmi-1−/− MEFs (Supplementary Fig. S3D and S3E). Accordingly, when we assessed β-Gal staining in CD49bhiCD29hiCD44hi in comparison with CD49blowCD29lowCD44low cells, we observed in TICs a greater senescence following C-209 treatment, as expected in regard to a much higher BMI-1 expression in CD49bhiCD29hiCD44hi cells (Supplementary Fig. S3F and S3G), thus suggesting a potential functional specificity of the selected inhibitors in targeting BMI-1′s effects on cellular senescence.
Pharmacologic targeting of BMI-1 in human prostate TICs
To establish that BMI-1 inhibitors have activity against the putative TICs in prostate cancer, we treated different prostate cancer cells with C-209. This treatment significantly impaired the percentage of rapidly adherent CD49bhiCD29hiCD44hi (TICs; Supplementary Fig. S4A and S4B), induced a cell-cycle accumulation (Supplementary Fig. S4C), which was anticipated from loss of BMI-1′s cell-cycle–regulatory function(s) (23), reduced the number of cells in S-phase (Supplementary Fig. S4C) and, critically, evoked a dose-dependent reduction in both BMI-1 and C-terminal lysine-119 mono-ubiquitinated form of γ-H2A, a specific product of the BMI-1 PRC1 complex activity (ref. 23; Supplementary Fig. S4D and S4E).
Self-renewal capacity is a distinguishing property of stem cells. Serial clonogenic spheroid assays estimate the frequency of TICs, especially after treatments (24). To evaluate the ability of these compounds to target serial clonogenic capacity, single cells collected from primary spheroids were plated in the presence of C-209, C-211, and two commonly used chemotherapies, methotrexate and doxorubicin. Unlike the initial slight inhibitory effects of methotrexate and doxorubicin, treatment with C-209 and C-211 considerably diminished the number of single cell–derived secondary and, more importantly, tertiary spheroids (Fig. 2C). While methotrexate had no effect, the outcomes of C-209 and C-211 treatment were remarkable, as nearly a 10-fold reduction was observed (Fig. 2C). We therefore hypothesized that inhibiting BMI-1 might eliminate self-renewing, hence more tumorigenic cells, while chemotherapies only affect the proliferating transit-amplifying cells sparing the ones endowed with clonogenic capacities, and characterized by a lower proliferative kinetic. In line with this notion, serial spheroid formation in presence of the general protein biosynthesis inhibitor cycloheximide (CHX; ref. 25), similar to chemotherapies, did not impact cells capable of self-renewal (Fig. 2C), thus strengthening our hypothesis.
Effects of BMI-1 inhibition on normal cells
When targeting TICs, an important concern is the effect on “normal” cell compartments. To assess the effect of BMI-1 inhibition on normal tissues, we initially used zebrafish embryos, valuable models for in vivo drug toxicity screening studies (26).
In toxicologic assays, C-209, C210, and C-211 had no notable effects on zebrafish development at their respective IC50s (Supplementary Fig. S5A). However, unlike C-209, higher doses of C-210 and C-211 impeded embryo hatching and caused embryo curling, thus suggesting narrow safety margins (Supplementary Fig. S5A and S5B). These data prompted us to dismiss C-210 and 211 inhibitors and focus on establishing the safety profile of C-209. To provide evidence that the effects of C-209 were not related to a general disruption of mRNA translation, zebrafish embryos were exposed in parallel to the universal protein inhibitor CHX (Supplementary Fig. S6A). CHX treatment resulted in developmental arrest and profound embryonic toxicity at low concentrations, while embryos treated with C-209 at 3 μmol/L survived and progressed normally throughout development (Supplementary Fig. S6A). Likewise, treatment of adult zebrafish with C-209, but not CHX, yielded no apparent impact on survival (Supplementary Fig. S6B).
To further assess C-209 adverse effects on normal mammalian tissues, BMI-1 inhibition was also tested on normal prostate epithelial RWPE1 cells by examining clonogenic potential pre- and post-C-209 treatment. While C-209 drastically reduced DU145 prostate cancer colony formation, RWPE1 cells, showing an expected lower clonogenic proclivity than DU145 cancer cells, BMI-1 inhibition was mostly ineffective (Supplementary Fig. S7A).
Furthermore, because self-renewal of hematopoietic stem and progenitor cells (HSPC) is vital for the sustained production of blood cells (10), we evaluated the clonogenic capacity of primary human CD34+ HSPCs upon treatment with C-209 and observed no significant effect (Supplementary Fig. S7B). In addition, treatment of mice with C-209 did not induce any anemia and/or thrombocytopenia, nor were histologic changes observed in the bone marrow of treated versus untreated mice (Supplementary Fig. S7C), suggesting that dosage limited targeting of BMI-1 would be selective in inhibiting the clonogenic potential of tumor versus normal stem cells.
C-209 could target post-transcriptional regulation of BMI-1
To gain insight into the mechanism of action of C-209 (Fig. 3A) against prostate cancer, we assessed the electrostatic potential of C-209 (Fig. 3B), and then used the UCSF DOCK program to model the docking of C-209 to the human BMI 5′UTR RNA. The docking model suggests that C-209 could bind to pockets formed in the BMI-1 RNA fold structures (Fig. 3C). We next utilized the BMI-1 UTR luciferase reporter constructs previously used to demonstrate the regulatory roles of BMI-1 UTRs (14), to examine the effects of C-209 on cells harboring these BMI-1 UTR–regulatory reporters. Indeed, C-209 treatment significantly reduced normalized luciferase expression in cells harboring the BMI-1 3′UTR and reversed the BMI-1 expression–inducing effects of the BMI-1 3′UTR (14) in cells harboring both the BMI-1 5′ and 3′UTR (Fig. 3D). Thus, in prostate cancer cells, C-209 engages the BMI-1–regulatory mechanisms embedded within the UTRs.
Modulation of BMI-1 post-transcriptional regulation by C-209. A, Chemical structure of C-209. B, The electrostatic potential of C-209 mapped to electron density surface. At an IC50 of 2 μmol/L, the electrostatic potential E(RB3LYP) = −1423.42386733 au and dipole moment = 9.4906 Debye. C, Docking of C-209 to the human BMI-1 RNA. View of C-209 (space filling model colored magenta) within the binding pocket of the BMI-1 5′UTR model (ribbon). Illustration was created using the Pymol software package. D, Left, schematic diagram of the luciferase (Luc) constructs used. The diagrams display the IRES containing 5′UTR and the micro-RNA (miR)-binding sites within the 3′UTR. The base pair (bp) length of the human BMI-1 5′ and 3′UTRs are displayed (from full-length cDNA # L.13689.1). Boxes and sites are drawn neither to scale nor to exact locations. Right, DU145 cells containing Luc flanked by control UTRs or BMI-1 5′ or 3′ UTR regions were treated for 24 hrs with 2 μmol/L C-209 and compared against untreated and DMSO controls. IRES-containing BMI-1 5′UTR and 3′UTR were shown to either inhibit or upregulate Luc expression, respectively (14). The 5′UTR reduced Luc expression, while when combined with the 3′UTR reversed the Luc expression enhancing effects of the 3′UTR. Treatment with 2 μmol/L C-209 resulted in reduced Luc expression opposing the effects of the 3′UTR. Data plotted represent six independent experiments. E, Percentage of inhibition of Luc reporter cells in control (Cont.) versus BMI-1 5′ and 3′UTR cells following C-209 (0.0195–20 μmol/L) treatments for 72 hours. F, Selective effects of C-209 on BMI-1 mRNA translation in cell-free extracts. Top, Western blot analysis of translated full-length BMI-1 RNA (complete cDNA including BMI-1 5′ and 3′UTRs) in in vitro transcription/translation (TNT) assays in eukaryotic cell-free rabbit reticulocytes. A cellular lysate in the most right lane was used as a positive control to determine the BMI-1 migrated band on the polyacrylamide gel at a position of approximately 37 KDa. Bottom, quantitation of normalized translated BMI-1 from BMI-1 cDNA pretreated or not with 2 μmol/L C-209 for one hour compared with control. G, BMI-1 expression in vector-transduced (Sh-Scr), BMI-1-overexpressing (EGFP-BMI-1), and BMI-1–depleted (shBMI-1) DU145 cells. H, Cell viability evaluated in vector-transduced (Sh-Scr), BMI-1–overexpressing (EGFP-BMI-1) and BMI-1–depleted (shBMI-1) DU145 cells following C-209 (0.0195–20 μmol/L) treatments for 72 hours.
Modulation of BMI-1 post-transcriptional regulation by C-209. A, Chemical structure of C-209. B, The electrostatic potential of C-209 mapped to electron density surface. At an IC50 of 2 μmol/L, the electrostatic potential E(RB3LYP) = −1423.42386733 au and dipole moment = 9.4906 Debye. C, Docking of C-209 to the human BMI-1 RNA. View of C-209 (space filling model colored magenta) within the binding pocket of the BMI-1 5′UTR model (ribbon). Illustration was created using the Pymol software package. D, Left, schematic diagram of the luciferase (Luc) constructs used. The diagrams display the IRES containing 5′UTR and the micro-RNA (miR)-binding sites within the 3′UTR. The base pair (bp) length of the human BMI-1 5′ and 3′UTRs are displayed (from full-length cDNA # L.13689.1). Boxes and sites are drawn neither to scale nor to exact locations. Right, DU145 cells containing Luc flanked by control UTRs or BMI-1 5′ or 3′ UTR regions were treated for 24 hrs with 2 μmol/L C-209 and compared against untreated and DMSO controls. IRES-containing BMI-1 5′UTR and 3′UTR were shown to either inhibit or upregulate Luc expression, respectively (14). The 5′UTR reduced Luc expression, while when combined with the 3′UTR reversed the Luc expression enhancing effects of the 3′UTR. Treatment with 2 μmol/L C-209 resulted in reduced Luc expression opposing the effects of the 3′UTR. Data plotted represent six independent experiments. E, Percentage of inhibition of Luc reporter cells in control (Cont.) versus BMI-1 5′ and 3′UTR cells following C-209 (0.0195–20 μmol/L) treatments for 72 hours. F, Selective effects of C-209 on BMI-1 mRNA translation in cell-free extracts. Top, Western blot analysis of translated full-length BMI-1 RNA (complete cDNA including BMI-1 5′ and 3′UTRs) in in vitro transcription/translation (TNT) assays in eukaryotic cell-free rabbit reticulocytes. A cellular lysate in the most right lane was used as a positive control to determine the BMI-1 migrated band on the polyacrylamide gel at a position of approximately 37 KDa. Bottom, quantitation of normalized translated BMI-1 from BMI-1 cDNA pretreated or not with 2 μmol/L C-209 for one hour compared with control. G, BMI-1 expression in vector-transduced (Sh-Scr), BMI-1-overexpressing (EGFP-BMI-1), and BMI-1–depleted (shBMI-1) DU145 cells. H, Cell viability evaluated in vector-transduced (Sh-Scr), BMI-1–overexpressing (EGFP-BMI-1) and BMI-1–depleted (shBMI-1) DU145 cells following C-209 (0.0195–20 μmol/L) treatments for 72 hours.
To examine whether C-209 is selective for BMI-1 posttranscriptional inhibition, we utilized control and BMI-1 UTRs reporter cells and found that C-209 preferentially inhibit expression of a reporter, the translation of which is under the BMI-1 5′- and 3′UTR control, rather than alternate control UTRs (Fig. 3E). We also treated DU145 cells concomitantly with C-209 2 μmol/L and CHX, a general translational inhibitor, and analyzed mRNA expression of BMI-1 and the CHX target epithelial sodium channel (αENaC; ref. 27). While treatment with CHX mutually lowered mRNA levels of both BMI-1 and αENaC, C-209 exposure did not exert a lowering effect on the levels of transcribed BMI-1 mRNA, explained by a reported cancer cellular addiction to BMI-1 (11, 12, 28). Thus, C-209 reduces the production of BMI-1 protein likely by modulating its posttranscriptional regulation.
Nonetheless, to specifically link C-209 effects to targeting posttranscriptional regulation of BMI-1 transcripts, we analyzed BMI-1 translation directed by full-length (UTR-containing) human BMI-1 cDNA (29) in eukaryotic cell–free expression system. Incubation of the BMI-1 cDNA with C-209 for just one hour before translation decreased BMI-1 protein synthesis by 27% (Fig. 3F).
Moreover, we performed a dose–response cell viability assay in BMI-1–deficient (sh-BMI-1) and BMI-1–overexpressing (EGFP BMI-1) DU145 cells, and compared the results with control transduced DU145 Sh-Scr cells expressing endogenous level of the protein (Fig. 3G). Unlike DU145 Sh-Scr cells, whose survival was impaired even at low C-209 doses (IC50 1.25–2.5 μmol/L), both DU145 BMI-1–deficient (lacking the target) and -overexpressing (with excess target) cells showed a lower sensitivity, being affected only at very high concentrations of C-209 (DU145 EGFP-BMI-1 IC50 ∼10 μmol/L and DU145 sh-BMI-1 IC50 ∼15 μmol/L). Survival analyses comparison between DU145 Sh-Scr and DU145 sh-BMI-1 cells revealed a highly significant response of DU145 Sh-Scr cells to C-209 even starting at 0.3125 μmol/L (Fig. 3H). Accordingly, to experience the growth-inhibitory effect of C-209, BMI-1–overexpressing cells required a higher dose than DU145 Sh-Scr (Fig. 3H), therefore providing additional evidence that C-209 effects could be selective towards BMI-1 targeting.
Also, to examine other potential mechanisms of action, C-209 inhibitory activity was examined against a panel of 245 kinases and 21 phosphatases. These assays elucidated a lack of significant inhibition (<10%; data not shown).
BMI-1 inhibition in patient-derived TICs
To assess the value of a new treatment, primary patient-derived cells represent a much more relevant model compared with cell lines. Despite the known difficulties in culturing primary prostate cancer cells in vitro, even if for brief periods, we have recently successfully maintained primary prostate cancer cells endowed with self-renewal and in vivo tumorigenic potential in short-term culture (6). Therefore, we examined C-209 treatment in a panel of short-term cultures from primary prostate cancer cells differentially expressing BMI-1 (Table 1; Supplementary Fig. S8). Exposure of patient-derived prostate cancer cells to C-209 resulted in a significant BMI-1 downregulation (Fig. 4A) followed by antitumor activity at an IC50 similar to that found in androgen-responsive LNCaP cells and lower, but not significantly different, from the IC50 found in the androgen-insensitive DU145 cells (Fig. 4B). Notably, only a slight nonsignificant downregulation of BMI-1 was observed in patient-derived normal counterparts treated with C-209 (Fig. 4A). Remarkably, as observed with tumor cell lines, treatment with C-209 caused a critical reduction in the rapidly adherent CD49bhiCD29hiCD44hi (TIC) population in primary prostate cancer cultures (Fig. 4C, left and right panels). In contrast, treatment with docetaxel, a treatment for advanced prostate cancer (2), resulted in enrichment of the highly aggressive CD49bhiCD29hiCD44hi TICs.
Primary prostate cancer patient characteristics
. | . | . | . | . | . | BMI-1 expression by IHC . |
---|---|---|---|---|---|---|
Primary prostate cancer . | Age . | Type . | Grade . | pTNM . | Gleason . | H-score . |
15728 | 62 | Adc | 3 | pT2c | 3 + 3 | 120 |
17148 | 52 | Adc | 3 | pT2c | 3 + 3 | 150 |
17761 | 57 | Adc | 3 | pT2c | 3 + 3 | ND |
19803 | 55 | Adc | 3 | pT2c | 3 + 3 | 70 |
24126 | 53 | Adc | 4 | pT3a | 3 + 3 | 190 |
40181 | 60 | Adc | 4 | pT3a | 3 + 4 | ND |
25185 | 67 | Adc | 3 | pT2c | 3 + 3 | 200 |
25315 | 66 | Adc | 4 | pT3a | 3 + 4 | 150 |
26136 | 67 | Adc | 3 | pT3b | 4 + 5 | 220 |
25854 | 55 | Adc | 4 | pT3b | 4 + 5 | 190 |
28838 | 65 | Adc | 4 | pT3b | 4 + 4 | 160 |
28864 | 68 | Adc | 3 | pT3b | 3 + 4 | 210 |
28869 | 67 | Adc | 4 | pT2c | 4 + 4 | 200 |
29032 | 68 | Adc | 4 | pT3b | 4 + 5 | 210 |
29084 | 58 | Adc | 3 | pT2c | 3 + 3 | ND |
29092 | 71 | Adc | 4 | pT3b | 4 + 4 | 210 |
29110 | 69 | Adc | 4 | pT3c | 4 + 5 | 130 |
29663 | 50 | mAdc | 4 | pT3b | 4 + 5 | 250 |
29834 | 44 | Adc | 3 | pT2c | 3 + 4 | 160 |
29990 | 63 | Adc | 4 | pT3a | 4 + 3 | 200 |
33020 | 45 | Adc | 3 | pT2c | 3 + 4 | 165 |
33106 | 64 | mAdc | 5 | pT3b | 5 + 4 | 180 |
33072 | 48 | Adc | 4 | pT3a | 4 + 3 | 200 |
33120 | 47 | Adc | 3 | pT2c | 3 + 4 | 200 |
. | . | . | . | . | . | BMI-1 expression by IHC . |
---|---|---|---|---|---|---|
Primary prostate cancer . | Age . | Type . | Grade . | pTNM . | Gleason . | H-score . |
15728 | 62 | Adc | 3 | pT2c | 3 + 3 | 120 |
17148 | 52 | Adc | 3 | pT2c | 3 + 3 | 150 |
17761 | 57 | Adc | 3 | pT2c | 3 + 3 | ND |
19803 | 55 | Adc | 3 | pT2c | 3 + 3 | 70 |
24126 | 53 | Adc | 4 | pT3a | 3 + 3 | 190 |
40181 | 60 | Adc | 4 | pT3a | 3 + 4 | ND |
25185 | 67 | Adc | 3 | pT2c | 3 + 3 | 200 |
25315 | 66 | Adc | 4 | pT3a | 3 + 4 | 150 |
26136 | 67 | Adc | 3 | pT3b | 4 + 5 | 220 |
25854 | 55 | Adc | 4 | pT3b | 4 + 5 | 190 |
28838 | 65 | Adc | 4 | pT3b | 4 + 4 | 160 |
28864 | 68 | Adc | 3 | pT3b | 3 + 4 | 210 |
28869 | 67 | Adc | 4 | pT2c | 4 + 4 | 200 |
29032 | 68 | Adc | 4 | pT3b | 4 + 5 | 210 |
29084 | 58 | Adc | 3 | pT2c | 3 + 3 | ND |
29092 | 71 | Adc | 4 | pT3b | 4 + 4 | 210 |
29110 | 69 | Adc | 4 | pT3c | 4 + 5 | 130 |
29663 | 50 | mAdc | 4 | pT3b | 4 + 5 | 250 |
29834 | 44 | Adc | 3 | pT2c | 3 + 4 | 160 |
29990 | 63 | Adc | 4 | pT3a | 4 + 3 | 200 |
33020 | 45 | Adc | 3 | pT2c | 3 + 4 | 165 |
33106 | 64 | mAdc | 5 | pT3b | 5 + 4 | 180 |
33072 | 48 | Adc | 4 | pT3a | 4 + 3 | 200 |
33120 | 47 | Adc | 3 | pT2c | 3 + 4 | 200 |
NOTE: Table shows the deidentified number of each patient, age, prostate cancer type (Adc, adenocarcinoma, mAdc, metastatic adenocarcinoma), histologic grade, pathologic staging based on the pTNM classification, where pT2c indicates bilateral prostate disease, and total Gleason scores. BMI-1 expression is assessed as the extent of nuclear immunoreactivity by IHC and indicated as an H-score. The H-score is obtained using the following formula: H-Score = (% at 0) × 0 + (% at 1+) × 1 + (% at 2+) × 2 + (% at 3+) × 3. Scoring was determined as (3X percentage of BMI-1 strongly staining nuclei + 2X percentage of BMI-1 moderately staining nuclei + 1X percentage of BMI-1 weakly staining nuclei), giving a range of 0 to 300. Weak cytoplasmic and/or stromal staining was seen in a few sections and was not considered in the score. BMI-1 staining in multiple sections from the same patient's tumor displayed marked heterogeneity. ND, not determined due to insufficient tissue material.
Antitumor activities of C-209 against patient-derived TICs. A, BMI-1 expression levels assessed in normal (N) and tumoral (T) patient-derived samples before and after C-209 (2 μmol/L) treatment for 72 hours. Right, quantitation of BMI-1 expression levels in normal (N) and tumoral (T) patient-derived cells after C-209 (2 μmol/L) treatment for 72 hours. B, Antitumor activity of C-209 in androgen-responsive LNCaP cells (orange), androgen-insensitive DU145 cells (red), and primary prostate cancer cells (blue; patient #25854). Percentage of survival was evaluated by MTS assay. C, Representative cytofluorimetric analysis (left) and graphical plotting (right) of TIC modulation in primary patient-derived cells untreated and treated with C-209 (2 μmol/L) and docetaxel (2.5 nmol/L) for 72 hours (patient #28869, #33020, #33072, #33120, #33106). D–F, Cell survival, clonogenicity, and motility assessed on unselected primary prostate cancer (#29084, #29092, #29110, #29663, #29834, #29990, #28869, #28864). Following treatments with DMSO, C-209 (2 μmol/L) or docetaxel (2.5 nmol/L) for 96 hours, cells were washed, counted, and replated in fresh media to assess cell survival (assessed at 96 hours after cell wash), colony formation (assessed at 2–3 weeks after cell wash), and migration (24 hours after cell wash). Data are displayed as mean percentage ± SD. Single independent experiments were performed with four to eight distinct patient-derived cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Antitumor activities of C-209 against patient-derived TICs. A, BMI-1 expression levels assessed in normal (N) and tumoral (T) patient-derived samples before and after C-209 (2 μmol/L) treatment for 72 hours. Right, quantitation of BMI-1 expression levels in normal (N) and tumoral (T) patient-derived cells after C-209 (2 μmol/L) treatment for 72 hours. B, Antitumor activity of C-209 in androgen-responsive LNCaP cells (orange), androgen-insensitive DU145 cells (red), and primary prostate cancer cells (blue; patient #25854). Percentage of survival was evaluated by MTS assay. C, Representative cytofluorimetric analysis (left) and graphical plotting (right) of TIC modulation in primary patient-derived cells untreated and treated with C-209 (2 μmol/L) and docetaxel (2.5 nmol/L) for 72 hours (patient #28869, #33020, #33072, #33120, #33106). D–F, Cell survival, clonogenicity, and motility assessed on unselected primary prostate cancer (#29084, #29092, #29110, #29663, #29834, #29990, #28869, #28864). Following treatments with DMSO, C-209 (2 μmol/L) or docetaxel (2.5 nmol/L) for 96 hours, cells were washed, counted, and replated in fresh media to assess cell survival (assessed at 96 hours after cell wash), colony formation (assessed at 2–3 weeks after cell wash), and migration (24 hours after cell wash). Data are displayed as mean percentage ± SD. Single independent experiments were performed with four to eight distinct patient-derived cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
The effectiveness of any targeted therapy is based on the absence of relapse and/or secondary clonal lesions (30). As TICs account for tumor progression by the virtue of their treatment resistance, self-renewal, and tumor-seeding capacity (19), it is reasonable to deduce that the efficacy of a TIC-tailored strategy relies on a diminished clonogenic and tumorigenic capacity.
To evaluate C-209 efficiency in targeting patient-derived TICs, we pretreated distinct unselected primary prostate cancer cells for several days with either C-209 or docetaxel. Subsequently, to investigate the long-term impact of treatments, particularly in a post-therapy discontinuation setting, cells were washed and replated. Cell rescue and soft agar assays were assessed to evaluate differences in cell survival and colony-forming repopulation abilities. Interestingly, both docetaxel and C-209 treatments impaired short-term survival of primary prostate cancer, although C-209 to a more significant extent (Fig. 4D). Critically, patient-derived prostate cancer cells maintained the ability to form colonies after single treatments with docetaxel but significantly less with C-209 (Fig. 4E), indicating that BMI-1 inhibition impairs survival and clonogenic activity of primary prostate cancer TICs.
BMI-1 has been implicated in contributing to prostate cancer metastasis (31). As loss of function of BMI-1 impaired prostate cancer cell migration (Supplementary Fig. S1C), we assessed the posttreatment propensity of patient-derived prostate cancer cells to migrate in modified Boyden chambers. We found that, while docetaxel-treated cell-migratory potential was almost unchanged, C-209 exposure significantly diminished their motility (Fig. 4F), thus suggesting a notable role for BMI-1 in cancer dissemination.
Evaluation of C-209 in vivo
Successful murine xenografting of primary human prostate cancer, in the absence of inducing murine urogenital mesenchyme (32), has rarely been achieved. We have shown that embryonic and juvenile zebrafish could be successfully used as prostate cancer xenograft models (6). Here, we utilized these xenografts to identify small-molecule inhibitors that functionally target BMI-1 and self-renewal activities (Fig. 5A). We isolated prostate cancer cells from 24 patients undergoing surgical prostatectomy (Table 1), and examined their tumor initiation potential in zebrafish xenografts (Supplementary Fig. S9A). Prostate cancers were diagnosed on the basis of histologic examination (Supplementary Fig. S9B–S9D). The expression of the prostate cancer–specific alpha-methylacyl coenzyme-A racemase (AMACR), when combined with overexpression of Erg (33), provide excellent dual prostate cancer–specific biomarkers (ref. 6; Supplementary Figs. S9E–S9G and S10A and S10B). We detected Erg overexpression associated with AMACR in the mirror sections of sampled prostate cancer tissue (Supplementary Fig. S10E–S10G) and within zebrafish xenografts (Supplementary Fig. S10C–S10E), in cells that expressed the human isoform of CD44 and BMI-1 (Supplementary Fig. S10F–S10H). TICs isolated from primary prostate cancer engrafted robustly in the preimmune zebrafish embryos (Supplementary Fig. S10H), forming xenografts (Supplementary Fig. S9H–S9K) with cells morphologically similar to the patient's biopsy cells (compare cells in Supplementary Fig. S9D to those in Supplementary Fig. S9K) and were positive for prostate-specific antigen (PSA) staining (Supplementary Fig. S9L–S9O).
Treatment effects of C-209 on zebrafish xenografts. A, Schematic illustration of the experimental procedure for the use of zebrafish prostate cancer xenografts to identify small molecules targeting BMI-1 in vivo. B, Antitumor activity of C-209. Reduction in tumor size monitored with reduced QD fluorescence (blue arrows). C, Antitumor activity of C-209 (2 μmol/L) against xenografts derived from either parental cells (yellow) or the TIC fraction (red) from three primary samples. The graph demonstrates responses to C-209 as a percentage of total treated xenografts (n = 25 xenograft per cell fraction per patient). Zebrafish with established grafts were sorted before treatment. D, Evaluation of tumor area variation calculated as fluorescence intensity of untreated and treated xenografts with C-209 (2 μmol/L). Data are presented as mean value ± SD. E, Tumorigenic capacity of primary prostate cancer TICs in zebrafish xenografts. Cells, pretreated with C-209 (2 μmol/L) or docetaxel (2.5 nmol/L), were washed after 4 days, plated in fresh media for 3 days and subsequently injected in equal number into adult zebrafish. Data are displayed as mean percentage ± SD. from four distinct patients (#29663, #29834, #29084 and #29990). F, The graph displays the percentage ± SD. of Ki67-positive cells in DMSO (control), C-209 (2 μmol/L), or docetaxel (2.5 nmol/L)-treated cells. G, Strategy employed to determine inhibition of tumor initiation potential of remaining treated cells in secondary xenografts. TICs of patient samples #40181, #26136, and #25854 were transplanted to generate primary xenografts (1°). Diagram on the right demonstrates primary graft take rates. Xenografts were treated (TRT) with either DMSO or C-209 at 2 μmol/L for 72 hours, tumor areas were dissected, pooled, and TICs were sorted and injected into secondary recipients. Treatment with C-209 significantly reduced the rates of secondary xenografts (2°). Scale bars are 250 μm in B.
Treatment effects of C-209 on zebrafish xenografts. A, Schematic illustration of the experimental procedure for the use of zebrafish prostate cancer xenografts to identify small molecules targeting BMI-1 in vivo. B, Antitumor activity of C-209. Reduction in tumor size monitored with reduced QD fluorescence (blue arrows). C, Antitumor activity of C-209 (2 μmol/L) against xenografts derived from either parental cells (yellow) or the TIC fraction (red) from three primary samples. The graph demonstrates responses to C-209 as a percentage of total treated xenografts (n = 25 xenograft per cell fraction per patient). Zebrafish with established grafts were sorted before treatment. D, Evaluation of tumor area variation calculated as fluorescence intensity of untreated and treated xenografts with C-209 (2 μmol/L). Data are presented as mean value ± SD. E, Tumorigenic capacity of primary prostate cancer TICs in zebrafish xenografts. Cells, pretreated with C-209 (2 μmol/L) or docetaxel (2.5 nmol/L), were washed after 4 days, plated in fresh media for 3 days and subsequently injected in equal number into adult zebrafish. Data are displayed as mean percentage ± SD. from four distinct patients (#29663, #29834, #29084 and #29990). F, The graph displays the percentage ± SD. of Ki67-positive cells in DMSO (control), C-209 (2 μmol/L), or docetaxel (2.5 nmol/L)-treated cells. G, Strategy employed to determine inhibition of tumor initiation potential of remaining treated cells in secondary xenografts. TICs of patient samples #40181, #26136, and #25854 were transplanted to generate primary xenografts (1°). Diagram on the right demonstrates primary graft take rates. Xenografts were treated (TRT) with either DMSO or C-209 at 2 μmol/L for 72 hours, tumor areas were dissected, pooled, and TICs were sorted and injected into secondary recipients. Treatment with C-209 significantly reduced the rates of secondary xenografts (2°). Scale bars are 250 μm in B.
Again, we employed the zebrafish toxicity assay to demonstrate that the compounds under investigation have no notable toxicities when used at their corresponding IC50s (Supplementary Fig. S11A and S11B). We next treated zebrafish embryos that were engrafted with QD-labeled TIC-derived prostate cancer (Fig. 5A and Supplementary Fig. S12A). Treatment of prostate cancer xenografts from multiple patient samples (Fig. 5B and C) with C-209 at 2 μmol/L led to tumor shrinkage (Fig. 5B and D and Supplementary Fig. S12B, left and right). Likewise, treatment of juvenile xenograft fish with C-209 led to tumor reduction (Supplementary Fig. S12C, left and right), suggesting that although governing the cell fate of TICs, BMI-1 likely regulates the viability of prostate cancer cells in general.
To determine C-209 efficacy in targeting TICs, hence tumor reinitiation, primary prostate cancer were treated with either docetaxel or C-209 for 3 days before being washed and injected into zebrafish embryos. After 10 days, C-209–treated cells gave rise to significantly less tumors than control- or docetaxel-treated cells (Fig. 5E). Notably, these effects were associated with a significant reduction in Ki67 staining (Fig. 5F).
Xenotransplantation, followed by serial repopulation, is considered an essential criterion to assess serial maintenance of stemness in defining TICs. Thus, we sorted labeled, pooled primary tumor cells from primary zebrafish xenografts treated either with DMSO or C-209, and used them for secondary xenografts (Fig. 5G). TICs from DMSO-treated embryos were able to initiate secondary grafts in 81.8% of cases (n = 54/66 secondary xenograft embryos from three patient samples), while C-209–treated cells had significantly less tumor initiation potential in only 29.3% of cases (n = 22/75 from three patient samples; P < 0.001; Fig. 5G), suggesting that C-209 treatment is effectively impairing the frequency of TICs in zebrafish xenografts.
Zebrafish provide a powerful organism to study the cancer self-renewal population (34). To examine whether C-209 treatment affects tumor response and TIC survival in a murine model, we employed a strategy aimed at unraveling the targeting of self-renewing TICs (Fig. 6A). We injected rapidly adherent CD49bhiCD29hiCD44hi DU145 TICs (6), that were previously infected with a lentiviral vector encoding luciferase2/enhanced GFP (Luc2/EGFP), into NOD-SCID-IL-2R null (NSG) mice. Tumors, allowed to grow until the size of approximately 100 mm3, were treated with C-209 or the chemotherapeutic agent docetaxel for approximately 2 weeks (Fig. 6B). At the end of treatments, while vehicle-treated tumors grew exponentially and docetaxel exerted a minimal effect on xenografts growth, C-209–treated tumors were significantly inhibited (Fig. 6B). In addition, as tumor relapse is frequently observed following treatment discontinuation, tumor volume was monitored with an electronic caliper for additional 15 days. Noticeably, at the end of this period, the results in Fig. 6B, while revealing a significant difference in tumor growth between docetaxel-treated and untreated mice, show an even higher disparity in C-209–treated xenografts. In line with this, Ki67+ cells in grafts with reduced nuclear BMI-1 and surface CD44 expression from C-209–treated mice were significantly lower when compared with controls (Fig. 6C and D). Also, severe tumor damage, indicated by large necrotic areas and scarce cellularity, was present two weeks after the last delivery of chemotherapy and BMI-1 inhibitors (Supplementary Fig. S13) thus corroborating the efficiency of anti-BMI-1–based therapy. Furthermore, the same outcomes were observed when treatments were performed on unsorted androgen-independent DU145 cells or androgen-dependent 22rv1 cell xenografts (Supplementary Fig. S14). In both mouse xenografts, C-209 showed tumor growth inhibition and lastly regression, although sooner and to a higher extent in androgen-independent DU145 cell (with relatively higher BMI-1 expression) xenografts (Supplementary Fig. S14).
In vivo pharmacologic targeting of BMI-1 in mouse prostate cancer xenografts. A, Strategy for examining the antitumor activity of C-209 in serial mouse xenografts and clonogenic repopulation assays of treated cells. B, Growth rate of mouse xenografts generated after subcutaneous injection of CD49bhiCD29hiCD44hi Luc2EGFP cells. Mice were randomized and administered daily with 60 mg/kg/day of C-209 for 12 days and docetaxel 6 mg/kg once a week for two consecutive weeks. Results are mean ± SD of six independent experiments. Comparison of tumor volumes between the three groups was determined by two-way ANOVA with Bonferroni post hoc test. Graph indicates significance of docetaxel versus control at day 30 (**P < 0.01) and C-209 versus control at day 30 (****P < 0.0001). There was a trend towards significance (P = 0.08) when comparing tumor volumes in xenograft treated with C-209 versus docetaxel at day 30 using Mann–Whitney U test. At the earlier days 20 and 25, docetaxel was not significantly different than control, while C-209 was; C-209 versus control at day 20 (*P < 0.05); C-209 versus control at day 25 (**P < 0.01). Red arrow indicates treatment discontinuation. In each experiment, n = 8/group. C, intratumor IHC revealed reduced nuclear BMI-1 (brown) and surface CD44 (red) staining upon treatment with C-209. Staining was performed on tumor xenografts taken at day 30. D, Quantitation of Ki67-positive cells in sections from treated xenografts. (***, P < 0.001; *, P < 0.01). E, Colony-forming ability assay performed on freshly dissociated and EGFP sorted xenograft-cells. Average number of colonies/plate for each treatment mean ± SD of two independent experiments with 12 wells/condition is reported. *, P < 0.05; ***, P < 0.001. F, Tumor initiation potential in serial grafting in secondary mouse xenografts of cells dissociated from treated primary mouse xenografts (n = 8 mice/group, *, P < 0.01). G, Cell proliferation of LNCaP and DU145 cells treated with C-209 (2 μmol/L), abiraterone (5 μmol/L), enzalutamide (5 μmol/L), and docetaxel (2.5 nmol/L) for 72 hours. H, Cell survival of therapy-resistant cells. LNCaP and DU145 cells pretreated with abiraterone (5 μmol/L), enzalutamide (5 μmol/L), and docetaxel (2.5 nmol/L) for 72 hours were washed and remaining cells that survived treatments (therapy-resistant cells) were treated with C-209 (2 μmol/L) for another 3 days. Results are indicated as ± SD of two independent experiments with *, P < 0.05; **, P < 0.01.
In vivo pharmacologic targeting of BMI-1 in mouse prostate cancer xenografts. A, Strategy for examining the antitumor activity of C-209 in serial mouse xenografts and clonogenic repopulation assays of treated cells. B, Growth rate of mouse xenografts generated after subcutaneous injection of CD49bhiCD29hiCD44hi Luc2EGFP cells. Mice were randomized and administered daily with 60 mg/kg/day of C-209 for 12 days and docetaxel 6 mg/kg once a week for two consecutive weeks. Results are mean ± SD of six independent experiments. Comparison of tumor volumes between the three groups was determined by two-way ANOVA with Bonferroni post hoc test. Graph indicates significance of docetaxel versus control at day 30 (**P < 0.01) and C-209 versus control at day 30 (****P < 0.0001). There was a trend towards significance (P = 0.08) when comparing tumor volumes in xenograft treated with C-209 versus docetaxel at day 30 using Mann–Whitney U test. At the earlier days 20 and 25, docetaxel was not significantly different than control, while C-209 was; C-209 versus control at day 20 (*P < 0.05); C-209 versus control at day 25 (**P < 0.01). Red arrow indicates treatment discontinuation. In each experiment, n = 8/group. C, intratumor IHC revealed reduced nuclear BMI-1 (brown) and surface CD44 (red) staining upon treatment with C-209. Staining was performed on tumor xenografts taken at day 30. D, Quantitation of Ki67-positive cells in sections from treated xenografts. (***, P < 0.001; *, P < 0.01). E, Colony-forming ability assay performed on freshly dissociated and EGFP sorted xenograft-cells. Average number of colonies/plate for each treatment mean ± SD of two independent experiments with 12 wells/condition is reported. *, P < 0.05; ***, P < 0.001. F, Tumor initiation potential in serial grafting in secondary mouse xenografts of cells dissociated from treated primary mouse xenografts (n = 8 mice/group, *, P < 0.01). G, Cell proliferation of LNCaP and DU145 cells treated with C-209 (2 μmol/L), abiraterone (5 μmol/L), enzalutamide (5 μmol/L), and docetaxel (2.5 nmol/L) for 72 hours. H, Cell survival of therapy-resistant cells. LNCaP and DU145 cells pretreated with abiraterone (5 μmol/L), enzalutamide (5 μmol/L), and docetaxel (2.5 nmol/L) for 72 hours were washed and remaining cells that survived treatments (therapy-resistant cells) were treated with C-209 (2 μmol/L) for another 3 days. Results are indicated as ± SD of two independent experiments with *, P < 0.05; **, P < 0.01.
To investigate whether C-209 treatment was able to target TICs in vivo, clonogenic assays ex vivo and serial transplantations were assessed from treated and untreated xenografts (Fig. 6A). Interestingly, the clonogenic potential of cells derived from C-209–treated tumors was significantly reduced (Fig. 6E). To determine the frequency of cells having clonogenic, hence tumorigenic function, in the mixed tumor bulk population, we performed ex vivo a limiting dilution assay between C-209–treated and untreated xenograft-derived cells. While control-derived cells were highly clonogenic, C-209–treated xenograft-derived cells generated colonies at a lower frequency (Supplementary Fig. S15). Importantly, while both control- and docetaxel xenograft–derived cells could be serially transplanted in secondary recipients, the graft repopulation capacity of C-209–treated tumors was significantly reduced (Fig. 6F), hence demonstrating that BMI-1 targeting is effective against tumor-propagating cells.
Clinical management of advanced prostate cancer remains a challenge. To gain insights into the clinical relevance of BMI-1 inhibition, we treated androgen-responsive LNCaP and 22rv1 cells (Fig. 6G and Supplementary Fig. S16) and androgen-insensitive DU145 cells with C-209 (Fig. 6G). We observed that, compared with the current standard-of-care treatments (docetaxel, enzalutamide, and abiraterone), C-209 was more significantly effective in inhibiting cell growth, in both androgen-dependent and -independent cells (Fig. 6G and Supplementary Fig. S16A, left). Furthermore, when cells resistant to these therapies were subsequently treated with C-209, not only cell survival was strongly impaired, but more importantly, clonogenic activity was completely abrogated (Fig. 6H and Supplementary Fig. S16A, right and S16B).
Collectively, our data demonstrate that C-209, a novel small molecule that targets posttranscriptional regulation of BMI-1, displays anti-TICs and antitumor activities in both zebrafish and mouse prostate cancer xenografts.
Discussion
Mounting evidence support the notion that distinct tumor subpopulations termed cancer stem cells (CSC) or TICs are responsible for tumor generation and treatment failure (35, 36). Accordingly, to achieve tumor eradication, new approaches capable of targeting the tumorigenic core of cancers are needed. TICs possess indefinite replicative ability due to an inherent or acquired self-renewal capacity. Consequently, targeting self-renewal potential of a given tumor may be the key toward developing more effective treatments.
Here, we initially demonstrated that knockdown of BMI1 impairs stem cell–like traits in prostate cancer, likely by reducing TIC frequency. Next, through high-throughput followed by selective approaches, our group was first to identify small molecules that target BMI-1 posttranscriptional regulation (37), and here, we investigated the ability of the prototype molecule C-209 to interfere with prostate cancer cell survival and self-renewal, similar to the effects in colon cancer (38).
Human prostate spheroids have increased BMI-1 (39). Herein, we showed that in the 5-minute adherent CD49bhiCD29hiCD44hi tumorigenic stem-like cells, BMI-1, Integrin-α6 (CD49f), and TROP2 levels are higher than in the nontumorigenic counterpart. Moreover, BMI-1 protein is highly enriched in these 5-minute adherent CD49bhiCD29hiCD44hi cells. Colony, serial spheroid formation and tumor xenograft studies showed that BMI-1 controls self-renewal, hence tumor-seeding capacity of prostate TICs. Importantly, the same outcomes were not observed with conventional chemotherapies or general translational inhibitor treatments. These data suggest that, unlike chemotherapies, which largely spare cells endowed with tumorigenic capacities (35), exposure to C-209 efficiently reduces the survival and clonogenic potential of TICs in prostate cancer.
Multiple molecular pathways regulate the self-renewal potential of stem cells and are therefore potential targets in TICs (40). Among these, BMI-1, the key component of PRC1 transcriptional repressor complex that plays important roles in cell-cycle regulation and cellular senescence, represents a critical target. BMI-1 is in fact also necessary for Hh- (41), β-catenin- (42), and Akt-mediated self-renewal (28, 43), thus is a key stem cell self-renewal regulator (43). In prostate cancer, BMI-1 activation occurs in primary tumors (31), transgenic mice (11, 28), in stem cells from metastatic prostate cancer with poor prognosis (13), and is highly predictive of PSA recurrence (12).
A possible drawback for the development of agents targeting stem cell self-renewal may be the potential toxicity deriving from inhibition of normal differentiation, particularly in HSPCs (44). Importantly, C-209 had less of an effect on CD34+ HSPCs. The in vivo administration of C-209 did not alter bone marrow integrity, nor did it induce anemia and/or thrombocytopenia in treated mice. In addition, no notable toxicity was observed in zebrafish at the tumor IC50 employed. These findings suggest that it might be possible to target TICs overexpressing BMI-1 with potent targeted therapy, and without notable toxic effects on normal cells. Indeed, we have recently demonstrated that lower (more tolerated) doses of AKT-targeted therapy could impair and radiosensitize glioblastoma TICs overexpressing BMI-1 (43).
C-209 was identified in a screen utilizing reporter cells that harbor BMI-1 5′UTR and 3′UTR (37). Treatments of tumor cells with C-209 reduced BMI-1 protein levels but not EZH2 nor a panel of 245 kinases and 21 phosphatases, increased cellular senescence, reduced the specific BMI-1 product of activity lysine-119 monoubiquitinated form of γ-H2A (23), induced a cell-cycle accumulation, and impaired TICs by abolishing serial spheroid formation in vitro and graft repopulation potential in vivo. Altogether, these outcomes are functional effects suggesting that C-209 may directly or indirectly target BMI-1 to evoke a complex cellular response.
Structure–activity relationship in UTR reporters and modeling studies suggest that C-209 is relatively selective towards targeting the BMI-1 transcript, harboring the BMI-1 UTR–regulatory elements. Thus, the anti-proliferative effects of C-209 could be due to direct modulation of BMI-1 post-transcriptional control mechanisms regulating BMI-1 translation and embedded within the UTRs, such as control of translation initiation either directly in a cap-independent fashion or possibly through riboswitches, which not only control gene expression through the 5′UTR, but also control splicing in the 3′UTR by coupling metabolite binding to mRNA processing, or indirectly by regulating mRNA stability (45). Few small molecules elicit their effects by modulating RNA function(s) outside of the bacterial ribosome. As the exact site(s) on the BMI-1 5′UTR and/or 3′UTR targeted by C-209 remains to be determined, detailed biophysical and RNA-binding affinity studies are necessary to delineate the exact selectivity of C-209 towards BMI-1 RNA.
ChIP-Seq and global mapping revealed that BMI-1 transcriptionally represses approximately 1,600 targets through the PRC1 complex, with many targets involved in apoptotic and cell survival pathways, therefore driving the proliferation-promoting function of BMI-1 (21). We observed that C-209 treatment interruption led to a rapid tumor growth rebound. Notably, BMI-1 inhibition affected both androgen-dependent and -independent prostate cancer cell proliferation and inhibited in vivo tumor growth.
ADT has been the standard care for patients with advanced prostate cancer. Although ADT shows clear benefits for many patients, castration-resistant prostate cancer (CRPC) inevitably occurs. Moreover, despite the initial effectiveness of second-generation AR-directed therapy with abiraterone and enzalutamide, resistance to these agents develops in patients with CRPC. ADT, abiraterone and enzalutamide stimulate TIC expansion by multiple pathways that drive resistance to these therapies (2). Notably, BMI-1 targets are associated with lethal therapy-resistant prostate cancer (13). We demonstrate that, following C-209 exposure, both cell survival and clonogenic capacities are strongly impaired in chemo- or AR directed therapy-treated prostate cancer cells. These effects were observed regardless of their hormonal dependence. Although, and in line with correlation of BMI-1 overexpression with prostate cancer progression (12), targeting BMI-1 with C-209 was more effective in hormone-independent cells. Notably the efficacy of BMI-1 inhibition in androgen-independent cells did not change with the presence of previous treatments. Accordingly, while targeting of TICs should complement the current androgen- and proliferation-based approaches, BMI-1 inhibition could possibly be attempted as a mono-therapeutic approach to target therapy-resistant prostate cancer in the clinic.
We found BMI-1 to be overexpressed in secondary tumor lesions, also BMI-1 inhibition was accompanied by reduced cell motility in prostate cancer; therefore, a link may exist between BMI-1-expressing self-renewing TICs and cancer dissemination. Genetic modulation of BMI-1 in prostate cancer cell lines revealed a linear correlation between protein expression and the antiproliferative as well as anti-TIC response to C-209. Instead, tumors from different patients exhibit variable responses, due to their extensive heterogeneity (46) that, unlike immortalized cell lines (47), are derived from distinct genetic alterations and cell proliferation kinetics. Mutations, such as those affecting AR signaling and PTEN/PI3K/AKT activation, could render prostate cancer cells addicted to the activity of BMI-1 signaling (28). The defects in Bmi-1–null mice are caused by inappropriate Ink4a/Arf expression (11). The proteins p16Ink4a and p19Arf, which induce cell-cycle arrest and apoptosis through activation of Rb and p53, respectively, are not expressed in normal tissue but induced upon oncogenic signaling (48). Addiction of prostate cancer cells to BMI-1 likely activates cellular signals in tumor cells, which are absent in normal tissue, to elicit aberrant proliferative and antiapoptotic effects. This could explain the distinct cellular responses to BMI-1 inactivation among normal and tumor cells. The robust expression of BMI-1 in most prostate cancer samples analyzed, the extreme sensitivity of prostate cancer cells to BMI-1 inactivation versus normal cells, upregulation of BMI-1 mRNA as an initial response to C-209 treatment, all suggest that prostate cancer cells might have an oncogenic dependence over BMI-1 activity, possibly through increased transcriptional self-regulation, and similarly to the oncogenic addiction to Myc (49). This dependence distinguishes TICs from normal cells and can be viewed as a survival mechanism, to maintain cancer cell viability, which could be exploited for targeted therapy. Overall, our data suggest that prostate cancer cells are more sensitive to BMI-1 inhibition, corroborating similar preferential sensitivity of prostate tumors versus normal tissues to Bromodomain and Extra-Terminal motif (BET) domain inhibitors (49). We conclude that the identification of molecules targeting BMI-1, a self-renewal target involved in oncogenic addiction, may open new avenues to directly target TICs for prostate cancer treatment while preserving normal stem cell populations.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: N. Bansal, M. Bartucci, S. Davis, L. Cao, D. Medina, I.Y. Kim, T.W. Davis, R.S. DiPaola, J. Bertino, H.E. Sabaawy
Development of methodology: N. Bansal, S. Yusuff, S. Davis, L. Cao, T.W. Davis, R.S. DiPaola, H.E. Sabaawy
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Bansal, M. Bartucci, S. Davis, K. Flaherty, E. Huselid, M. Patrizii, D. Jones, L. Cao, M.N. Stein, I.Y. Kim, T.W. Davis, R.S. DiPaola, J. Bertino, H.E. Sabaawy
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Bansal, M. Bartucci, S. Davis, M. Patrizii, L. Cao, H. Zhong, J. Kerrigan, M.N. Stein, I.Y. Kim, T.W. Davis, J. Bertino, H.E. Sabaawy
Writing, review, and/or revision of the manuscript: M. Bartucci, Y.-C. Moon, H. Zhong, D. Medina, J. Kerrigan, M.N. Stein, I.Y. Kim, R.S. DiPaola, J. Bertino, H.E. Sabaawy
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H.E. Sabaawy
Study supervision: J. Bertino, H.E. Sabaawy
Other (designed and synthesized BMI-1 inhibitors C-206–C-212): N. Sydorenko
Other (pathology data analysis and interpretation and review of the manuscript): H. Zhong
Acknowledgments
We thank Drs. Maarten van Lohuizen (The Netherlands Cancer Institute) for the BMI-1 knockout mouse embryonic fibroblasts (MEFs), and Leonard Zon (Harvard University, Cambridge, MA) for the Casper zebrafish. We thank Dr. David Augeri and members of his laboratory at the Department of Chemistry at Rutgers University for the synthesis and purification of the small molecules utilized in this study.
Grant Support
This project was supported by the Department of Defense Grant (W81XWH-12-1-0249 to H.E. Sabaawy), National Cancer Institute (P30 CA072720; to R.S. DiPaola), the New Jersey Commission on Cancer Research (NJCCR) Grant (09-1137-CCR-EO to H.E. Sabaawy), Rutgers Cancer Institute of New Jersey (pilot grant to J. Bertino and H.E. Sabaawy), Wellcome Trust grant (SDDI award # 092687; to PTC Therapeutics) and New Jersey Health Foundation award (research grant; to H.E. Sabaawy).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.