articles

Self-renewal as a therapeutic target in human colorectal cancer

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© 2014 Nature America, Inc. All rights reserved.

Antonija Kreso1,2, Peter van Galen1, Nicholas M Pedley1,3, Evelyne Lima-Fernandes4, Catherine Frelin1,5, Thomas Davis6, Liangxian Cao6, Ramil Baiazitov6, Wu Du6, Nadiya Sydorenko6, Young-Choon Moon6, Lianne Gibson1,3, Yadong Wang1,3, Cherry Leung1,3, Norman N Iscove1,5,7, Cheryl H Arrowsmith1,4,5, Eva Szentgyorgyi8, Steven Gallinger9,10, John E Dick1,2,11 & Catherine A O’Brien1,3,9,11 Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichment of cancer-initiating cells (CICs) in tumors that survive therapy. Together with recent reports showing that CIC gene signatures influence patient survival, these studies predict that targeting self-renewal, the key ‘stemness’ property unique to CICs, may represent a new paradigm in cancer therapy. Here we demonstrate that tumor formation and, more specifically, human colorectal CIC function are dependent on the canonical self-renewal regulator BMI-1. Downregulation of BMI-1 inhibits the ability of colorectal CICs to self-renew, resulting in the abrogation of their tumorigenic potential. Treatment of primary colorectal cancer xenografts with a small-molecule BMI-1 inhibitor resulted in colorectal CIC loss with long-term and irreversible impairment of tumor growth. Targeting the BMI-1–related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Evidence suggests that colorectal cancer usually follows the cancer stem cell model where a subset of cancer cells possess transcriptional and epigenetic programs that endow them with stemness properties, including the capacity for self-renewal1–4. Such cells, functionally defined as CICs, are uniquely capable of initiating and sustaining tumor growth in serial transplantation assays. CICs have a number of other biological properties that distinguish them from the remainder of tumor cells, including resistance to treatment5–7, evasion of cell death8,9 and dormancy10. These properties, together with variation in the ability of CICs to self-renew10, suggest that they may have a central role in tumor recurrence. Although CICs have been characterized in several human cancers2–4,11–13, their clinical relevance remains uncertain. One unattained piece of evidence that would render strong support for the concept that stemness and CICs play a part in human cancer would be to control tumor growth using a clinically relevant method that targets CIC self-renewal. Bmi-1 represents the first regulator that was strongly linked to self-renewal and has been implicated in the maintenance of stem cells in several tissues14–16. Bmi-1 is part of the polycomb repressive complex 1 (PRC1), where it functions as an epigenetic chromatin modifier targeting many genes17,18. In the intestinal epithelium, Bmi1 expression marks mouse intestinal stem cells and is important for crypt maintenance19,20. Bmi-1 has also been shown to have a role in the propagation of several types of cancer21–25, although only a few

s­ tudies have directly assessed its impact on CIC function26,27. In the context of colorectal cancer, BMI-1 can be overexpressed in tumors, and its expression has been associated with increased risk of metastasis and high-grade tumors28–31; however, studies on the role of BMI-1 in colorectal CICs and self-renewal have not been reported. We describe here that BMI-1 is required for CIC function in colorectal tumors and that small-molecule–mediated BMI-1 inhibition reduces tumor burden in primary human colorectal tumor xenograft models. Thus, inhibiting a recognized regulator of self-renewal is an effective approach to control tumor growth, providing strong evidence for the clinical relevance of self-renewal as a biological process for therapeutic targeting. RESULTS BMI-1 knockdown reduces human colorectal cancer growth We detected BMI-1 expression in most cells of a well-characterized human colon adenocarcinoma cell line, LS174T, and five patientderived colorectal tumor samples under conditions that permitted expansion in vitro (Fig. 1a,b). When we sorted these samples into phenotypic fractions enriched for colorectal CICs and non-CICs, BMI-1 was expressed in both fractions, similar to other markers used to isolate normal intestinal stem cells and colorectal CICs (Supplementary Figs. 1–3). To determine whether reduction of BMI-1 levels affected the growth of human colorectal cancer cells, we stably

1Princess

Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada. 2Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. 4Structural Genomics Consortium, Toronto, Ontario, Canada. 5Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. 6PTC Therapeutics, South Plainfield, New Jersey, USA. 7Department of Immunology, University of Toronto, Toronto, Ontario, Canada. 8Department of Pathology, Toronto General Hospital, Toronto, Ontario, Canada. 9Department of Surgery, Toronto General Hospital, Toronto, Ontario, Canada. 10Fred Litwin Centre for Cancer Genetics, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. 11These authors contributed equally to this work. Correspondence should be addressed to J.E.D. ([email protected]). Received 26 January; accepted 1 November; published online 1 December 2013; doi:10.1038/nm.3418

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knocked down BMI-1 transcript levels using a total of four different lentivirus encoded shRNAs. The different shRNAs yielded similar results, and we employed one shRNA for most studies, as it reliably yielded cells with >60% BMI-1 knockdown at the mRNA and protein levels (BMI-1-KD cells) (Fig. 1a,b). In addition to an shRNA to BMI1 (shBMI-1), the lentivirus also encoded eGFP. BMI-1-KD cells were progressively outcompeted and lost their ability to grow in vitro, as evidenced by a decline in the fraction of shBMI-1–expressing eGFP+ cells over time in both cell lines and primary patient-derived cell cultures (Fig. 1c). To evaluate effects on in vivo tumor-forming potential, we injected BMI-1-KD cells subcutaneously into nonobese diabetic– severe combined immunodeficiency (NOD-SCID) and interleukin-2 receptor γc–deficient (NSG) mice. Four weeks following transplantation, BMI-1-KD LS174T cells displayed a 12-fold reduction in tumor growth as compared to control cells expressing an shRNA to luciferase (shLUC) (P < 0.0001, Fig. 1d). Likewise, the mass of tumors from patient-derived colorectal cell cultures was significantly reduced in the BMI-1-KD group of sample 02 (P < 0.0001, Fig. 1d) and sample 04 (P = 0.02, Fig. 1d); no tumors were detected in mice given BMI-1-KD sample 01 cells (Fig. 1d). We obtained similar results using additional shRNAs targeting BMI-1 (Supplementary Fig. 4a). To ensure that the dependence on BMI-1 for tumor cell growth was not due to short-term culturing, we generated single-cell suspensions from five additional colorectal cancer specimens immediately following surgical resection and directly transduced them without expansion in vitro. We observed considerably reduced tumor growth following injection under the renal capsule in the BMI-1-KD group compared to the control transduced cells expressing shLUC for three of the five tested samples (Fig. 1e). For sample 05 and LS174T cells, BMI-1-KD cells generated smaller tumors upon re-injection into secondary recipients,

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5,000 2,000 Figure 1  BMI-1 knockdown impairs human colorectal cancer cell growth. 4,000 (a) BMI-1 transcript levels in LS174T cells transduced with the shBMI-1 knockdown vector 1,500 * 3,000 (normalized to GAPDH). (b) Immunoblots showing BMI-1 and GAPDH protein in lysates of LS174T 1,000 2,000 colon adenocarcinoma cell line and five patient colorectal tumor samples transduced with the shLUC 500 or shBMI-1 vectors. (c) The percentage of cells expressing eGFP among human colorectal cancer cells 1,000 transduced with the shBMI-1 or shLUC virus and passaged every 4–7 d. For LS174T cells, data are 0 0 expressed as mean ± s.e.m. of triplicate cultures. (d) eGFP+ tumor weight of control and BMI-1-KD colorectal cancer cells that were grown subcutaneously in immunocompromised mice. Tumor weight was calculated by multiplying tumor mass by percentage eGFP expression, which was similar between shLUC and shBMI-1 cells before injection. Each circle represents one mouse, and horizontal lines indicate the mean tumor weight. (e) eGFP+ tumor growth in immunodeficient mice injected in the renal capsule with primary human colorectal cancer cells that were not expanded in vitro and that were transduced with control or shBMI-1 lentiviruses. Circles represent the weight of one tumor, and horizontal lines show the mean (*P < 0.05, **P < 0.01 and ***P < 0.001). Tumor weight (mg)

© 2014 Nature America, Inc. All rights reserved.

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indicating that BMI-1-KD cells exhibit a sustained reduction in tumor growth (Fig. 1e and Supplementary Fig. 4b). Collectively, these data indicate that human colorectal cancer cells require BMI-1 to generate and maintain tumor growth in vivo. The observed decrease in tumor mass upon BMI-1 knockdown suggested a defect in cell proliferation. Consistent with this, BMI-1-KD LS174T cells, when grown as nonadherent spheres in vitro, had a smaller sphere diameter compared to controls (Supplementary Fig. 5a). BMI-1 knockdown did not lead to a statistically significant difference in BrdU uptake (P > 0.05, Supplementary Fig. 5b) in LS174T cells; however, there was a marked increase in the proportion of cells in G0 (Ki67−) (Supplementary Fig. 5c). In accordance with these findings, BMI-1 exerted its effects on sphere initiation through its repressive function of the CDKN2A locus, which encodes p14ARF and p16INK4a (Supplementary Fig. 6a–c and Supplementary Results). We analyzed three primary human colorectal cancer cell cultures and observed a modest 1.7-fold reduction in S phase cells upon BMI-1 knockdown (P = 0.036, Fig. 2a,b and Supplementary Fig. 7a,b). There were 1.2-fold more cells in G0 after BMI-1 knockdown (P = 0.050, Fig. 2c,d and Supplementary Fig. 7c,d). In addition, in LS174T cells expressing shBMI-1, there was a marked increase in the proportion of cells expressing cleaved caspase 3 (Supplementary Fig. 5d), indicative of apoptosis induction. In primary cell cultures, we found only a twofold increase (P = 0.04) in cleaved caspase 3 in the BMI-1-KD group (Fig. 2e,f and Supplementary Fig. 7e). Consistent with these small changes in proliferation and apoptosis, inhibiting expression of the CDKN2A locus did not rescue the BMI-1-KD phenotype in primary patient-derived cell cultures (Supplementary Fig. 6d,e and Supplementary Results). Taken together, these data indicate that the reduced tumor mass of BMI-1-KD cells is due in part to the

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Figure 2  Attenuated proliferation and increased apoptosis upon BMI-1-KD. (a) Representative flow cytometric profile of BrdU (marker of cell cycle progression) and 7-AAD (marker of DNA content) staining of control and BMI-1-KD primary colorectal cancer cells expanded in vitro. (b) Quantification of BrdU staining in three biological samples with two replicate experiments each. (c) Flow cytometric profile of Ki67 expression in control and BMI-1-KD cells (n = 4 biological samples with two to three replicates each); the data are quantified in d. (e) Flow cytometric profiles of control and BMI-1-KD cells stained for cleaved caspase-3. Blue boxes indicate the percentage of cells that stain positive for cleaved caspase-3. (f) Quantification and normalization to control of cleaved caspase-3 detection (n = 4 biological samples with two repeats each). Measurements were taken 10 d after transduction. All values are normalized to the shLUC groups, and data are expressed as mean ± s.e.m. (*P < 0.05). Data for individual experiments are displayed in Supplementary Figure 7.

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combination of a reduced fraction of cells going through S phase, a smaller proportion of actively cycling cells and a modest increase in cells undergoing cell death.

a single colorectal CIC into secondary recipients using limiting dilution (Fig. 3e). There was a 60-fold decrease in the frequency of selfrenewing CICs in the BMI-1-KD group in LS174T cells (P < 0.0001, Fig. 3f). For sample 01, the frequency of CICs in the BMI-1-KD group was reduced eightfold (P = 0.005), and for sample 02, we observed a 15-fold reduction in CICs (P < 0.0001, Fig. 3f and Supplementary Table 3). Taken together, these findings indicate that BMI-1 silencing leads to a considerable decrease in the number of colorectal CICs in primary human tumors.

BMI-1-KD impairs colorectal CIC self-renewal In addition to the reduction in tumor mass of BMI-1-KD cells, there was also a decrease in the total number of tumors initiated, suggesting that BMI-1 may regulate colorectal CICs (Fig. 3a). To enumerate the frequency of CICs directly, we employed clonal tumor initiation assays performed with limiting-dilution analyses (LDAs) (Fig. 3b). BMI-1 knockdown decreased the frequency of tumor-initiating cells 12-fold in LS174T cells (P < 0.0001, Fig. 3c and Supplementary Table 1). In four primary samples, the frequency was 11-, 65-, 19- and 54-fold lower in the BMI-1-KD groups as compared to controls (Fig. 3c and Supplementary Table 1). These quantitative studies establish that BMI-1 knockdown prevents tumor initiation, thereby functionally lowering the frequency of CICs. To directly determine whether BMI-1 was affecting CIC selfrenewal, we took two additional approaches. We employed the widely used in vitro assay of sphere initiation but used LDA to ensure the accuracy of our measurements. We determined that there was a 283to 661-fold decrease in the sphere re-initiating cell frequency in the BMI-1-KD group compared to controls (Fig. 3d and Supplementary Table 2). We observed similar results with two additional shRNAs (Supplementary Fig. 8). Therefore, BMI-1 knockdown severely abrogates the self-renewal capacity of colon cancer cells in vitro. The definitive method to assess the self-renewal potential of CICs is to carry out secondary tumor formation assays in vivo using tumors initiated by a single CIC (see details in Supplementary Methods). We therefore injected shLUC and shBMI-1 cells from three samples at limiting doses into mice; we transplanted tumors generated from

Targeting BMI-1 by small-molecule inhibition We identified a low-molecular-weight compound, PTC-209 (Fig. 4a), by high-throughput screening of compounds using gene expression modulation by small molecules (GEMS) technology, where reduced luciferase BMI-1 reporter activity was measured to identify compounds that lower BMI-1 transcript levels. The GEMS reporter vector contains the luciferase open reading frame flanked by and under post-transcriptional control of the BMI-1 5′ and 3′ untranslated regions (UTRs). Subsequent characterization demonstrated that PTC-209 inhibited not only the UTR-mediated reporter expression (Fig. 4b) but also endogenous BMI-1 expression in human colorectal HCT116 (Fig. 4c) and human fibrosarcoma HT1080 tumor cells (Fig. 4d). The inhibition was dose dependent, with half-maximum inhibitory concentration (IC50) at about 0.5 µM in both reporter and ELISA assays (Fig. 4b,d). PTC-209 caused no inhibition of cell growth or viability in HEK293 human embryonic kidney cells after overnight treatment (Fig. 4b) and had limited effects on cell proliferation in HT1080 cells after a 48-h treatment (Fig. 4d), indicating that the activity of PTC-209 is not due to cytotoxicity. BMI-1 facilitates transcriptional silencing through the PRC1 complex by acting as a cofactor for RING1A, a monoubiquitination E3 ligase with activity specific for Lys119 on histone H2A, a mark associated with repressive chromatin structures. PTC-209 selectively reduced global ubiquitinated histone H2A (uH2A) but not total H2A and RING1A levels (Fig. 4c), indicating its ability to specifically reduce PRC1 activity. Compound PTC-209 preferentially inhibits the proliferation of human lymphoma U937 and HT1080 tumor cells, whereas it is less

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effective in primary human peripheral blood 10 0.1 1 mononuclear cells and human hematopoietic stem cells (Fig. 4e). 1 0.01 0.1 *** To more directly establish PTC-209 spe0.1 0.001 0.01 *** cificity, we took two genetic approaches *** using BMI-1-KD and BMI-1 overexpression 0.01 0.0001 0.001 shLUC shBMI-1 shLUC shBMI-1 shLUC shBMI-1 (BMI-1-OE) lentiviral vectors. In BMI-1-KD colon cancer cells from patient tumor tis100 e sue and the LS174T cell line, we found a 80 shBMI-1 60 lower sensitivity to the growth-inhibitory 100 Lentiviral shRNA 37 shLUC 80 transduction effects of PTC-209 over a range of PTC-209 shBMI-1 20 60 doses (Fig. 4f–i, Supplementary Figs. 9 Inject Inject 0 Calculate Calculate dilution dilution 37 shLUC CIC CIC Cell and 10 and Supplementary Results), indiseries of series of frequency 20 frequency dose viable cells viable cells cating that PTC-209 is specific for BMI-1. Isolate cells from tumours Human 0 generated at clonal cell dose cancer cells Cell dose Likewise, BMI-1-OE colon cancer cells Cell number Cell number injected were relatively less sensitive to the action of injected PTC-209 than cells expressing endogenous LS174T Sample 01 Sample 02 f 1 BMI-1 levels (Fig. 4j–l, Supplementary 0.1 1 Fig. 11 and Supplementary Results), further 0.1 0.1 0.01 supporting the specificity of PTC-209 for *** ** 0.01 BMI-1. In vitro treatment of human color*** 0.01 0.001 ectal cancer cells with doses between 0.1 and 0.001 10 µM reduced BMI-1 protein levels in a dose0.001 0.0001 0.0001 dependent manner (Fig. 5a) with a concomishLUC shBMI-1 shLUC shBMI-1 shLUC shBMI-1 tant reduction in cell growth (Fig. 5b). These data indicate that PTC-209 selectively lowers BMI-1 levels and decreases We obtained similar results when cells were assayed 5 d following colorectal tumor cell growth. The ability to control the length of BMI-1 inhibitor removal (Supplementary Fig. 12 and Supplementary inhibition through the addition or removal of the inhibitor provides Results), indicating that the effects on sphere formation are stable a critical tool to further investigate the effects of BMI-1 function in and result from the viability changes induced by drug treatment at the colorectal CICs. start of the experiment. Therefore, the frequency of sphere-initiating cells was affected after transient downregulation of BMI-1, suggesting BMI-1 inhibitor irreversibly impairs colorectal CICs that BMI-1 inhibition results in irreversible reduction of CICs. To determine whether BMI-1 reduction–mediated impairment To examine how PTC-209 affected CICs, we analyzed the response of colorectal CICs was transient or irreversible, we exposed three of CICs and non-CICs to PTC-209. The proportion of cells with high colorectal cancer samples to PTC-209 for 4 d, removed the inhibitor Wnt activity, as measured by quantifying expression of the T cell and seeded viable cells at limiting doses in medium lacking inhibitor. factor/lymphoid enhancer factor (TCF/LEF)-eGFP reporter, which For all samples, treatment of cells with the inhibitor at 0.1 µM was previously used to mark colorectal CICs32, was reduced following yielded a statistically significant decrease in the frequency of sphere- PTC-209 treatment (Supplementary Fig. 13a–d and Supplementary ­initiating cells, ranging from a 2- to 11-fold reduction as compared to Results). PTC-209 treatment also resulted in decreased Lgr5 mRNA vehicle-treated cells (P < 0.01, Fig. 5c and Supplementary Table 4). levels; Lgr5 is a Wnt target gene and its expression marks mouse CIC frequency (%)

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Figure 3  BMI-1 knockdown reduces the frequency of self-renewing colorectal CICs. (a) Representative image of tumors generated in mice that were injected with cells described in Figure 1d. (b) Schematic illustrating the experimental approach taken to assay the frequency of CICs after BMI-1 knockdown (see Online Methods). (c) CIC frequency of shBMI-1– or shLUC-transduced cells as measured by LDA in vivo. Data are expressed as mean and 95% confidence interval (CI). (d) The number of secondary sphere-forming units for the shLUC and shBMI-1 groups. Data are expressed as mean and 95% CI. (e) Schematic representation of the experimental approach taken to quantify the self-renewal ability of a single colorectal CIC in vivo. (f) Frequency of secondary CICs in vivo. Data are expressed as mean and 95% CI. Frequency and probability estimates were computed using the ELDA software (**P < 0.01 and ***P < 0.001).

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Figure 4  BMI-1 inhibitor reduces BMI-1 levels and is not overtly toxic. (a) Chemical structure of PTC-209, C17H13Br2N5OS (molecular weight 495.2 g/mol). (b) Dose-dependent inhibition of UTR-mediated luciferase BMI-1 reporter expression and cytotoxicity measurements in HEK293 cells. The assays were run in triplicate for each point, and the percentage of inhibition was calculated against vehicle control. Data are expressed as mean ± s.d. of n = 3 replicates. (c) Western blot analysis of HCT116 cell lysates following overnight PTC-209 treatment. (d) BMI-1 inhibition (as measured by ELISA) and cytotoxicity in HT1080 cells following PTC-209 treatment. Data are expressed as mean ± s.d. of n = 3 replicates. (e) Cytotoxicity following 72-h PTC-209 treatment in U937 and HT1090 tumor cells. 50% cytotoxic concentration (CC 50) for human peripheral blood mononuclear cells (hPBMCs) is shown as mean ± s.d., n = 4 separate assays; CC50 for human hematopoietic stem cells (hHSCs) was determined by CFU assays. (f–i) shBMI-1–expressing colon cancer cell sensitivity to PTC-209 compared to shLUC-expressing cells. Data are shown as pools of three, four and two experiments for samples 01 and 03 and LS174T cells, respectively. Data are expressed as mean ± s.e.m. of 11–23 replicates at each dose. Norm., normalized. (j–l), BMI-1-OE cell sensitivity to PTC-209. For both the LUC-OE and BMI-1-OE groups, the number of viable eGFP + cells in the PTC-209 treatments was normalized to the average number of cells in the 0-nM PTC-209–treated group. Data are expressed as pooled mean ± s.e.m. of two to five independent experiments for samples 01 and 03 and LS174T cells and one experiment with sample 11; all experiments have three to eight replicates at each dose (*P < 0.05, **P < 0.01 and ***P < 0.001).

intestinal stem cells (Supplementary Fig. 13e). These data support the conclusion that BMI-1 downregulation specifically affects CICs. CD133 and/or CD44 expression was not changed following BMI-1 downregulation (Supplementary Fig. 14), and induction of differentiation markers was variable upon PTC-209 treatment (Supplementary Fig. 15). Analysis of cell cycle and apoptosis markers in CD133+ and CD44+ cells showed an induction of cells in G0 and increased expression of cleaved caspase 3 following PTC-209 treatment (Supplementary Figs. 16,17 and Supplementary Results), indicating that CICs undergo cell cycle exit and apoptosis upon BMI-1 downregulation. Taken together, these data indicate that the effects of PTC-209 are mediated through irreversible growth inhibition of CICs. To assess the effects of the BMI-1 inhibitor on CICs activity in vivo, we treated colorectal cancer samples ex vivo, followed by evaluation in functional CIC xenograft assays in vivo. Pretreatment of cells with 1 or 10 µM inhibitor reduced tumor growth in recipient animals both for sample 01 (P = 0.0056, Fig. 5d) and for sample 03 (P = 0.0006, Fig. 5e).

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When viable cells from sample 11 were injected following ex vivo treatment with 10 µM PTC-209, no tumors formed, whereas equal numbers of viable cells generated tumors in the control group (Fig. 5f). Therefore, transient depletion of BMI-1 levels irreversibly reduces the potential of colorectal cancer cells to initiate tumors. Therapeutic targeting of colorectal CICs Having established that BMI-1 inhibition has an irreversible effect on colorectal CIC function, we evaluated the ability of PTC-209 to inhibit the growth of an established primary human colon cancer xenograft in nude mice. Pharmacokinetic analysis demonstrated that PTC-209 effectively inhibited BMI-1 production in tumor tissue in vivo (Supplementary Table 5). The general health, blood indices and numbers of hematopoietic stem cells and peripheral blood leukocytes of control and BMI-1 inhibitor treatment groups was not altered (Supplementary Table 6, Supplementary Fig. 18 and Supplementary Results). For mice transplanted with cells from sample 01, 9 d following the initial drug administration, at which point control animals

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increase in size. Likewise, for mice given cells from sample 03, 9 d after PTC-209 initiation, the drug-treated tumors displayed a significantly smaller tumor volume as compared to the control tumors (P = 0.002, Fig. 6b). LS174T cell–derived tumors displayed significantly

reached endpoint and were killed, the tumor volume of the PTC209–treated group was significantly reduced (P = 0.0005, Fig. 6a). Continued treatment of animals with PTC-209 for an additional week did not affect the overall health of the mice, and tumors did not

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34

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Figure 6  Therapeutic targeting of the BMI-1–related self-renewal 1.0 2.0 Control Control machinery. (a–e) Tumor growth curves of colorectal tumors that were –1 –1 PTC-209 (60 mg kg ) PTC-209 (60 mg kg ) 0.8 treated with PTC-209, where each point represents the mean tumor 1.5 0.6 volume ± s.e.m. of at least eight tumor measurements. mg kg −1, 1.0 mg per kg body weight. (f,g) Tumor growth curve of recipients bearing 0.4 0.5 LIM1215 cell–derived (f) and HCT1116 cell–derived (g) tumors that 0.2 *** * were previously treated with control or PTC-209 (data shown in d for 0 0 LIM1215 and e for HCT116); treatment was terminated and tumor 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 10111213141516 size monitored for an additional 30 d without any additional treatment. Time (d) Time (d) (h) Frequency of colorectal CICs following PTC-209 treatment in vivo. At the end of in vivo dosing experiments (as shown in b and c), tumor cells were isolated and viable cells were injected into secondary animals in a limiting-dilution series; the recipients did not receive any PTC-209 treatment during these secondary assays. Frequency and probability estimates were computed using the ELDA software. The mean and 95% CI are shown. (i,j) Tumor growth curves of previously PTC-209–treated tumors. Colorectal tumors, which were treated in the experiments in d and retransplanted for the experiments in i or treated in the experiments in e and retransplanted for the experiments in j, were transplanted to assess possible development of resistance. Once transplanted cells formed tumors, recipients received daily PTC-209 for the indicated number of days. For a–g and i–j, P values were derived by comparing values from the control- or PTC-209–treated groups that were normalized to the tumor size on day 0 of each experiment (*P < 0.05, **P < 0.01 and ***P < 0.001). Squares represent mean tumor volume, and data are expressed as mean ± s.e.m. Tumor volume (cm3)

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Tumor weight (mg)

Sample 03 Sample 11 Sample 01 Figure 5  BMI-1 inhibitor permanently reduces colorectal 2.5 2.0 4 CICs. (a) Western blot of vehicle (DMSO)- or PTC-209–treated 2.0 colorectal cancer cells. (b) Cell viability of PTC-209–treated 3 1.5 cells 6 d following inhibitor addition. Each point represents 1.5 1.0 2 the mean of three technical replicates; data are expressed as ** 1.0 *** *** mean ± s.e.m. (c) Frequency of sphere-initiating cells after a *** 0.5 1 No 0.5 4-d pretreatment with the BMI-1 inhibitor. Data are expressed as tumors 0 0 0 mean and 95% CI. (d–f) Tumor mass recorded after injection of 0 0.01 0.1 1 10 0 0.1 1 10 0 0.01 0.1 1 10 colorectal cancer cells that were pretreated with PTC-209 for BMI-1 inhibitor (µM) BMI-1 inhibitor (µM) BMI-1 inhibitor (µM) 4 d ex vivo. For each condition, a fixed number of viable cells were injected into recipient mice (d, sample 01, 50,000 viable cells; e, sample 03, 10,000 viable cells and f, sample 11, 30,000 viable cells). Each circle represents the weight of one tumor and horizontal bars indicate the mean (*P < 0.05, **P < 0.01 and ***P < 0.001).

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articles reduced tumor volumes in the BMI-1 inhibitor group (P < 0.0001, Fig. 6c). We saw similar results using tumors derived from human colon cancer cell lines LIM1215 and HCT116 cells (Fig. 6d,e). These data indicate that inhibition of BMI-1 with PTC-209 halts growth of preestablished tumors in vivo. To test whether BMI-1 inhibitor–treated recipients should receive continuous BMI-1 inhibitor to control tumor size, we discontinued PTC-209 treatment and observed xenografts derived from two independent samples for the following 30 d. In recipients bearing LIM1215 cell–derived tumors, the previously PTC-209–treated and the controltreated recipients displayed similar tumor volumes when examined 30 d following PTC-209 termination (Fig. 6f). In recipients bearing HCT116 cell–derived tumors (from Fig. 6e), significantly smaller tumors were present 30 d following PTC-209 termination (P = 0.008; Fig. 6g). Although there is heterogeneity between these two models, for the HCT116 group, tumors from inhibitor-treated animals did not grow back when inhibitor treatment was stopped. To confirm whether the tumors harvested at the end of BMI-1 inhibitor treatment exhibited an irreversible decrease in CICs, we transplanted tumor cells of two samples at the end of the in vivo dosing regimen into secondary recipients at limiting doses. PTC-209 treatment in primary tumors reduced the frequency of functional tumor-initiating cells by 16-fold for LS174T cell–derived tumors (P < 0.0001) and fourfold for sample 03 tumors (P = 0.01, Fig. 6h and Supplementary Table 7). These data demonstrate that in vivo treatment with a BMI-1 inhibitor reduces the frequency of functional colorectal CICs. Finally, to assess whether tumors growing in secondary recipients were generated by cells with acquired resistance to PTC-209, we isolated and re-injected previously treated LIM1215 cell– and HCT116 cell–derived tumor cells into new recipients. Following tumor establishment, we treated recipients with PTC-209. Compared to the control, there was a reduction in tumor size following PTC-209 treatment for both samples (Fig. 6i,j), indicating that tumors remained sensitive to the BMI-1 inhibitor. Taken together, our data indicate that colorectal CICs are irreversibly impaired upon BMI-1 inhibition and that a small molecule targeting BMI-1 can control tumor growth in a preclinical model of primary human colorectal cancer. DISCUSSION Our results establish that BMI-1 regulates primary human colorectal cancer cells, including functionally defined CICs. The identification of a molecular regulator of CICs enabled us to successfully test the relevance of the cancer stem cell model in colorectal cancer; if CICs are clinically relevant, then therapeutically targeting them should result in effective and durable therapies. Self-renewal is largely measured in functional assays that require proliferation, making it difficult to investigate molecules that affect both processes. BMI-1 does not function solely as a self-renewal regulator, as it is also expressed in tumor cells that do not possess CIC activity. Although BMI-1 downregulation caused cell cycle exit and increased apoptosis in both phenotypically defined CIC and nonCIC fractions of primary colorectal cancer (Supplementary Figs. 16 and 17), these effects were much smaller than the impact on CIC self-renewal. In addition, temporary downregulation of BMI-1 by transient exposure to the BMI-1 inhibitor or siRNAs (Supplementary Results) against BMI-1 resulted in permanent impairment of functional colorectal CICs. Moreover, effects on self-renewal were not obtained by transiently inhibiting PCNA, the gene encoding proliferating cell nuclear antigen, which is involved in proliferation

nature medicine  VOLUME 20 | NUMBER 1 | JANUARY 2014

(Supplementary Fig. 19 and Supplementary Results). Collectively, our data indicate that colorectal cancer cells are highly dependent on BMI-1, which functions as a pleiotropic regulator that maintains the viability and proliferative capacity of colorectal cancer cells in general but also governs CIC self-renewal. The finding that BMI-1 plays a central part in colorectal CIC selfrenewal opened an avenue to target the stemness function of CICs with small-molecule inhibition. PTC-209 reduced tumor volume and reduced the number of functional colorectal CICs, even following short-term treatment, a result that contrasts with the enrichment of CICs observed following conventional chemotherapy9,33. The effectiveness of transient inhibition suggests that colorectal cancer cells may be reliant on BMI-1 to sustain growth and clonal maintenance. An important consideration is how PTC-209 will affect normal human intestinal stem cells. In mice expressing Cre-inducible diphtheria toxin receptor, targeted ablation of Bmi1-positive cells using diphtheria toxin leads to crypt loss19. However, with PTC-209, no changes in digestive function in the treated mice were identified (data not shown), indicating that the dose we used to lower tumor burden does not noticeably affect the intestinal system. Of note, in our studies, we lowered BMI-1 levels in xenografts and in cell culture, whereas mouse studies analyzed the effect in knockout animals where Bmi-1 is absent. Interestingly, transient depletion of Bmi-1 in cells leads to crypt loss, but the remaining crypts can repopulate the damaged area through crypt fission19. Targeting CIC stemness determinants including self-renewal has been proposed as a therapeutic goal34–38 and our study provides strong evidence for this concept. A number of molecules and gene signatures that underlie stemness have been identified, and future studies should be undertaken to identify and target other key pathways driving self-renewal in the CIC subset. Our study provides a template for preclinical evaluation of other CIC targets that focuses on primary samples and clonal in vitro and in vivo CIC assays. Methods Methods and any associated references are available in the online version of the paper. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank R. Lopez and P. Lo from the Animal Research Centre, University Health Network and A. Khandani, P. Penttila, and S. Zhao from the Sickkids Flow Cytometry facility for their skillful assistance. We are grateful to all members of the Dick lab, especially M. Anders, E. Laurenti, N. Mbong, S. Doulatov, M. Milyavsky and F. Notta, as well as C. Gedye and J. Wang for their advice and critical comments. We thank E. Lechman (University Health Network, Toronto), J. Moffat (University of Toronto) and T. Chiba (Chiba University) for lentiviral vectors expressing shRNAs to BMI1. We also thank members from the Department of Pathology (Toronto General Hospital), including F. Meng, M. Sukhram, V. Son, H. Begley and P. Shaw, for providing colon cancer samples. This work was supported by funds to PTC Therapeutics from the Wellcome Trust and to J.E.D. from Genome Canada through the Ontario Genomics Institute, Ontario Institute for Cancer Research and a Summit Award with funds from the province of Ontario, the Canadian Institutes for Health Research, a Canada Research Chair, the Princess Margaret Hospital Foundation and funds to E.L.-F. by Fonds National de la Recherche, Luxembourg and the Marie Curie Actions of the European Commission (FP7-COFUND, PDR 2012-2 4735314). This research was funded in part by the Ontario Ministry of Health and Long Term Care (OMOHLTC). The views expressed do not necessarily reflect those of the OMOHLTC. AUTHOR CONTRIBUTIONS A.K., C.A.O., J.E.D. designed the study. A.K., P.v.G., N.M.P., E.L.-F., C.F., L.G., Y.W., C.L. and C.A.O. performed experiments. N.N.I. and C.H.A. supervised specific

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Articles experiments. T.D., L.C., R.B., W.D., N.S. and Y.-C.M. provided BMI-1 inhibitor and performed experiments. E.S. assessed tumor histology. S.G. provided tumor specimens. A.K., P.v.G., C.A.O. and J.E.D. analyzed and interpreted data. P.v.G., C.A.O. and J.E.D. revised the paper. A.K. wrote the paper. J.E.D. supervised the study. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

1. Dick, J.E. Stem cell concepts renew cancer research. Blood 112, 4793–4807 (2008). 2. O’Brien, C.A., Pollett, A., Gallinger, S. & Dick, J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445, 106–110 (2007). 3. Ricci-Vitiani, L. et al. Identification and expansion of human colon-cancer–initiating cells. Nature 445, 111–115 (2007). 4. Dalerba, P. et al. Phenotypic characterization of human colorectal cancer stem cells. Proc. Natl. Acad. Sci. USA 104, 10158–10163 (2007). 5. Bao, S. et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–760 (2006). 6. Li, X. et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J. Natl. Cancer Inst. 100, 672–679 (2008). 7. Tehranchi, R. et al. Persistent malignant stem cells in del(5q) myelodysplasia in remission. N. Engl. J. Med. 363, 1025–1037 (2010). 8. Majeti, R. et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 138, 286–299 (2009). 9. Todaro, M. et al. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 1, 389–402 (2007). 10. Kreso, A. et al. Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer. Science 339, 543–548 (2013). 11. Singh, S.K. et al. Identification of human brain tumour initiating cells. Nature 432, 396–401 (2004). 12. Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J. & Clarke, M.F. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100, 3983–3988 (2003). 13. O’Brien, C.A. et al. ID1 and ID3 regulate the self-renewal capacity of human colon cancer-initiating cells through p21. Cancer Cell 21, 777–792 (2012). 14. Park, I.K. et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423, 302–305 (2003). 15. Lessard, J. & Sauvageau, G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423, 255–260 (2003). 16. Molofsky, A.V. et al. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425, 962–967 (2003). 17. Sparmann, A. & van Lohuizen, M. Polycomb silencers control cell fate, development and cancer. Nat. Rev. Cancer 6, 846–856 (2006).

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18. Sauvageau, M. & Sauvageau, G. Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell 7, 299–313 (2010). 19. Sangiorgi, E. & Capecchi, M.R. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40, 915–920 (2008). 20. Tian, H. et al. A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable. Nature 478, 255–259 (2011). 21. Bruggeman, S.W. et al. Bmi1 controls tumor development in an Ink4a/Arfindependent manner in a mouse model for glioma. Cancer Cell 12, 328–341 (2007). 22. Leung, C. et al. Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas. Nature 428, 337–341 (2004). 23. Shimono, Y. et al. Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 138, 592–603 (2009). 24. Lukacs, R.U., Memarzadeh, S., Wu, H. & Witte, O.N. Bmi-1 is a crucial regulator of prostate stem cell self-renewal and malignant transformation. Cell Stem Cell 7, 682–693 (2010). 25. Dovey, J.S., Zacharek, S.J., Kim, C.F. & Lees, J.A. Bmi1 is critical for lung tumorigenesis and bronchioalveolar stem cell expansion. Proc. Natl. Acad. Sci. USA 105, 11857–11862 (2008). 26. Chiba, T. et al. The polycomb gene product BMI1 contributes to the maintenance of tumor-initiating side population cells in hepatocellular carcinoma. Cancer Res. 68, 7742–7749 (2008). 27. Abdouh, M. et al. BMI1 sustains human glioblastoma multiforme stem cell renewal. J. Neurosci. 29, 8884–8896 (2009). 28. Kim, J.H. et al. The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlates with the reduced p16INK4a/p14ARF proteins. Cancer Lett. 203, 217–224 (2004). 29. Li, D.W. et al. Expression level of Bmi-1 oncoprotein is associated with progression and prognosis in colon cancer. J. Cancer Res. Clin. Oncol. 136, 997–1006 (2010). 30. Du, J., Li, Y., Li, J. & Zheng, J. Polycomb group protein Bmi1 expression in colon cancers predicts the survival. Med. Oncol. 27, 1273–1276 (2010). 31. Tateishi, K. et al. Dysregulated expression of stem cell factor Bmi1 in precancerous lesions of the gastrointestinal tract. Clin. Cancer Res. 12, 6960–6966 (2006). 32. Vermeulen, L. et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat. Cell Biol. 12, 468–476 (2010). 33. Dylla, S.J. et al. Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy. PLoS ONE 3, e2428 (2008). 34. Wang, J.C. Evaluating therapeutic efficacy against cancer stem cells: new challenges posed by a new paradigm. Cell Stem Cell 1, 497–501 (2007). 35. Kvinlaug, B.T. & Huntly, B.J. Targeting cancer stem cells. Expert Opin. Ther. Targets 11, 915–927 (2007). 36. Trumpp, A. & Wiestler, O.D. Mechanisms of disease: cancer stem cells—targeting the evil twin. Nat. Clin. Pract. Oncol. 5, 337–347 (2008). 37. Liu, S. & Wicha, M.S. Targeting breast cancer stem cells. J. Clin. Oncol. 28, 4006–4012 (2010). 38. Diehn, M., Cho, R.W. & Clarke, M.F. Therapeutic implications of the cancer stem cell hypothesis. Semin. Radiat. Oncol. 19, 78–86 (2009).

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ONLINE METHODS

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Colorectal cancer cell isolation and xenograft establishment. Human colorectal cancer tissue was obtained with patient consent, as approved by the Research Ethics Board at the University Health Network in Toronto. Tumor specimens were washed in PBS, minced with a razor blade and incubated with collagenase A (3 mg mL−1, Roche) for 60 min at 37 °C. After this enzymatic digestion, the sample was filtered through a 40-µm cell strainer, and the cells were mixed with ammonium chloride solution (Stemcell Technologies) for 5 min to lyse red blood cells. Cells were then used for downstream applications. Eleven patient-derived tumors were used (ranging from stage II to IV, including both tumors from the colon and liver metastases) to capture the diversity of primary colorectal tumor tissue. For the generation of xenografts, cells were injected with Matrigel under the renal capsule of NSG and/or NOD-SCID mice (male or female, 8–10 weeks of age) as previously described39. For LDA analysis and inhibitor studies, cells were injected subcutaneously. Animal work was carried out in compliance with the ethical regulations approved by the Animal Care Committee, University Health Network, Toronto, Ontario, Canada. Colorectal cancer in vitro culture. Isolated colorectal cancer cells were plated in DMEM/F-12 containing epidermal growth factor (EGF) and fibroblast growth factor (FGF). The in vitro culture medium contained DMEM/F-12 (1:1 ratio) supplemented with penicillin-streptomycin (1%), fungizone (1 µg mL−1), l-glutamine (2 mM), nonessential amino acids, sodium pyruvate (1 mM), HEPES, heparin (4 µg mL−1), B27 supplement (GIBCO), N2 supplement (GIBCO), lipids (Sigma), EGF (20 ng mL−1) and basic FGF (10 ng mL−1). Cells were cultured in suspension culture flasks at 37 °C in a 5% CO2–humidified incubator. Under these conditions, cells grew as nonadherent aggregates, or spheres, and were passaged weekly by dissociating the cells with trypsin and replating in aforementioned medium. The patient-derived cell lines, as well as the cell lines LS174T, LIM1215 and HCT116, were authenticated using short tandem repeat profiling and tested for mycoplasma. Viral transduction of colorectal cancer cells. Cells were dissociated and filtered through a cell strainer to eliminate clumps of cells. Cells were diluted to 5E5 cells per mL and plated in 500-µL volumes in a 24-well dish. Viral particles were added at a multiplicity of infection of 1 to 20 using virus that was concentrated by ultracentrifugation. After a 48-h transduction, cells were washed and resuspended in culture medium for in vitro culture or injected into recipient mice as described above. The shRNA vectors expressing shBMI-1 or shLUC also express eGFP, which allowed tracking of the percentage of transduced cells for each sample. The transduction efficiency varied with the amount of viral particles added. For LS174T cells, >90% transduction efficiency was observed as assessed by flow cytometric analysis of eGFP. For the patient-derived samples that had been cultured in vitro under conditions that enrich for CICs, usually >60% of the cells were transduced with the shRNA vectors, and commonly >90% transduction efficiency was observed. For patient-derived colon cancer cells that had not been previously cultured in vitro, the transduction efficiency varied across samples but was generally

Self-renewal as a therapeutic target in human colorectal cancer.

Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichmen...
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