The FASEB Journal • Research Communication

CBX7 inhibits breast tumorigenicity through DKK-1mediated suppression of the Wnt/b-catenin pathway Hey-Yon Kim,* Ji-Hye Park,† Hee-Young Won,* Jeong-Yeon Lee,†,1 and Gu Kong*,†,1 *Department of Pathology, College of Medicine, and †Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, Korea Polycomb protein chromobox homolog 7 (CBX7) is involved in several biologic processes including stem cell regulation and cancer development, but its roles in breast cancer remain unknown. Here, we demonstrate that CBX7 negatively regulates breast tumor initiation. CD44+/CD242/ESA+ breast stem-like cells showed diminished CBX7 expression. Furthermore, small hairpin RNA-mediated CBX7 knockdown in breast epithelial and cancer cells increased the CD44+/CD242/ESA+ cell population and reinforced in vitro self-renewal and in vivo tumor-initiating ability. Similarly, CBX7 overexpression repressed these effects. We also found that CBX7 inhibits the Wnt/b-catenin/T cell factor pathway by enhancing the expression of Dickkopf-1 (DKK-1), a Wnt antagonist. In particular, CBX7 increased DKK-1 transcription by cooperating with p300 acetyltransferase and subsequently enhancing the histone acetylation of the DKK-1 promoter. Furthermore, pharmacologic inhibition of DKK-1 in CBX7-overexpressing cells showed recovery of Wnt signaling and consequent rescue of the CD44+/ CD242/ESA+ cell population. Taken together, these findings indicate that CBX7-mediated epigenetic induction of DKK-1 is crucial for the inhibition of breast tumorigenicity, suggesting that CBX7 could be a potential tumor suppressor in human breast cancer. —Kim, H. –Y., Park, J.–H., Won, H.–Y., Lee, J.–Y., Kong, G. CBX7 inhibits breast tumorigenicity through DKK-1-mediated suppression of the Wnt/b-catenin pathway. FASEB J. 29, 300–313 (2015). www.fasebj.org

ABSTRACT

Key Words: polycomb · self-renewal pathway · p300 · acetylation CHROMOBOX HOMOLOG 7 (CBX7) is a member of the polycomb group (PcG) CBX family, which has a chromodomain and Pc box, both for Pc dimerization and binding of histone H3 Lys-27 trimethylation (H3K27me3) (1). CBX7, as a reader protein, plays an important role in promoting the recruitment of polycomb repressive complex (PRC)-1 to the target chromatin by recognizing PRC-2mediated H3K27me3 (2). Moreover, CBX7 regulates gene expression by altering histone acetylation and DNA Abbreviations: APC, allophycocyanin; bFGF, basic fibroblast growth factor; bp, base pair; CBP, CREB-binding protein; CBX7, chromobox homologue 7; ChIP, chromatin immunoprecipitation; CSC, cancer stem cell; DAVID, Database for Annotation, Visualization, and Integrated Discovery; DKK-1, Dickkopf-1; EGF, epidermal growth factor; ESC, embryonic stem cell; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate

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methylation (3–6). Recent studies have suggested that CBX7 regulates the balance between self-renewal and differentiation in embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs) (7–9). In tumor development, CBX7 has shown an opposite function in distinct cancer cell types. For instance, CBX7 has been implied as an oncogene in gastric and prostate cancers (10, 11) and contributes to immortalization of diverse normal human cells by repressing p16INK4a (12). In contrast, loss of CBX7 has been associated with increasing the malignancy grade in bladder, pancreatic, breast, gastric, and colon carcinomas (13–17), but its tumor suppression mechanism is unclear. The Wnt pathway plays an important role in the development of many cancers, including breast cancer. Moreover, it is one of the core signaling pathways in regulation of the self-renewal of cancer stem cells (CSCs), which initiates and maintains the malignant tumors with stem cell characteristics (18, 19). Dickkopf-1 (DKK-1) is a known Wnt antagonist (20). In the canonical Wnt pathway, the binding of Wnt ligands to frizzled receptors and lipoprotein receptor-related protein 5 or 6 (LRP5/6) coreceptors activates GSK3-b/b-catenin signaling. This pathway leads to nuclear translocation of b-catenin, a coactivator of T cell factor (TCF) transcription factors, thus regulating the expression of target genes such as cyclin D1 and c-Myc, which have been implicated in CSC regulation (21, 22). DKK-1 binds to LRP5/6 and Kremen receptors in competition with Wnt and therefore inhibits Wnt signaling (23). DKK-1 has been suggested as a tumor suppressor, and its expression was shown to be silenced by various epigenetic modifications including histone methylation in several human cancers (24–27). Moreover, a recent study showed that DKK-1 decreased mammosphere formation and the CD44+/CD24low cell population in breast cancer (28), implying a possible link between DKK-1 and CSCs.

1

Correspondence: G.K., Department of Pathology, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea. E-mail: [email protected]; J.-Y.L., Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea. E-mail: [email protected] doi: 10.1096/fj.14-253997 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

0892-6638/15/0029-0300 © FASEB

Although CBX7 is involved in the regulation of normal stem cells and cancer development, the effect of CBX7 on tumorigenesis is still controversial, and its role in breast cancer remains unknown. Here, we found that loss of CBX7 is critical for tumor initiation via activation of the Wnt/b-catenin pathway. This effect was mediated by DKK-1, whose expression was modulated epigenetically by cooperation of CBX7 with p300 acetyltransferase. Collectively, these findings demonstrate that CBX7 inhibits breast tumorigenicity through DKK-1-mediated suppression of the Wnt signaling pathway, suggesting that CBX7 is a critical tumor suppressor in human breast cancer. MATERIALS AND METHODS Cell culture and reagents 293T and MCF10A cells, and human breast cancer cell lines, were maintained as described previously (29). Human mammary epithelial cells were cultured in the specialist medium provided by the supplier (Invitrogen, Carlsbad, CA, USA). For pharmacologic inhibition of histone deacetylase (HDAC) and DKK-1, trichostatin A (TSA) and WAY-262611 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Calbiochem (San Diego, CA, USA), respectively. The nontargeting small interfering RNA (siRNA) for negative control and siRNAs against DKK-1 (1042166), Ezh2 (1049254), and p16INK4a (1029407) were purchased from Bioneer (Daejeon, Korea). Establishment of CBX7-overexpressing and -knockdown cell lines To generate stable CBX7-knockdown cell lines, pLKO.1 lentiviral vectors encoding CBX7 small hairpin RNA (shRNA) (#RHS4533) or nontargeting shRNA purchased from Open Biosystems (Huntsville, AL, USA) were transfected with packaging DNA, pMD2g, and psPAX into 293T cells using Lipofectamine 2000 (Invitrogen). After 48 h transfection, the medium containing the viruses was transferred to target cells with 8 mg/ml Polybrene (Sigma-Aldrich). To generate tetracycline-inducible CBX7-overexpressing cell lines, the cDNA of CBX7 was inserted into the lentiviral pLVX-TRE3G vector (Clontech, Palo Alto, CA, USA), and lentiviral particles from the pLVX-Tet3G regulator vector and pLVX-TRE3G-CBX7 vector were generated as described above and sequentially transduced into target cells. The infected cells with lentiviruses encoding pLVX-Tet3G and pLVX-TRE3G-CBX7 were maintained in medium containing 10% tetracycline-free fetal bovine serum (FBS; Invitrogen). To induce CBX7 overexpression, these cells were treated with 2 mg/ml doxycycline for 72 h. Generation of CBX7 chromodomain-deletion mutant construct The CBX7 construct harboring a chromodomain deletion was generated by PCR using the following primers: 59-ATCTCGAGCCACCATGGCCTACGAGGAGAAGGA-39, and 59-CGGA-

(continued from previous page) dehydrogenase; H3Ac, histone H3 acetylation; H3K27me3, histone H3 Lys-27 trimethylation; HAT, histone acetyltransferase; HDAC, histone deacetylase; HSC, hematopoietic stem cell; LEF, lymphoid enhancer factor; LRP5/6, lipoprotein receptor-related protein 5 or 6; NOD, nonobese diabetic; PcG, polycomb group; PRC, polycomb repressive complex; qRT-PCR, quantitative RTPCR; RLU, relative light unit; shRNA, small hairpin RNA; siRNA, small interfering RN; TCF, T cell factor; TSA, trichostatin A

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ATTCTCAGAACTTCCCACTGCGGA-39. The PCR product was digested with XhoI and EcoRI and then subcloned into lentiviral pLVX-puro vectors. For overexpression of CBX7 lacking chromodomain, lentiviruses were generated as described above. Microarray analysis The biotinylated cRNA from cDNA was hybridized on HumanHT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The chips were then scanned on the BeadArray Reader (Illumina), and raw data were extracted using GenomeStudio V2011.1 (Illumina). Present/absent calling relied on a classification based on detection P values # 0.05 calculated by GenomeStudio software. The selected gene signal values were transformed to logarithms and normalized by the quantile method. Further analysis was done using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) for proper functional annotations. The DAVID algorithm was used to functionally categorize genes involved in different biologic processes as described previously (30). RT-PCR and real-time quantitative RT-PCR Total RNA was extracted with TRIzol (Invitrogen), and generation and amplification of cDNA were performed as described previously (31). The following primers were used for RT-PCR: CBX7, 59-CCTCCCCATCCAACCTAAAT-39 and 59-TGTCTCCCCTACAGGACTGG-39; DKK1, 59-GTGCAAATCTGTCTCGCCTG-39 and 59-GCACAACACAATCCTGAGGC-39; PIK3C2B, 59-TGGCTATGTCTGGAGTGCTG-39 and 59-CATCATGGGTGTAGCACAGG-39; WNT4, 59-GCTGTGACAGGACAGTGCAT-39 and 59-GCCTCATTGTTGTGGAGGTT-39; WNT10B, 59-TTCTCTCGGGATTTCTTGGA-39 and 59-TCCAGCATGTCTTGAACTGG39; TWIST1, 59-CGGACAAGCTGAGCAAGATT39 and 59-CCTTCTCTGGAAACAATGAC-39; IL-6, 59-GAACTCCTTCTCCACAAGCG-39 and 59-GAATCCAGATTGGAAGCATC-39; FGF2, 59-CGGGGTGGATGCGCAGGA-39 and 59-CGGGGTGGATGCGCAGGA-39; CBX2, 59-AAGGAAGCTCACTGCCATGT-39 and 59-AGGATCTGGGCCTTCTTCTG-39; CBX4, 59-CCGAGGTCATCCTGCTAGAC-39 and 59-CAAAGAAGGGCTTGAACTCG-39; CBX8, 59-GGTCGCAGAAGTACAGCACA-39 and 59-TCGTAAGTTTTGGCCTTTGC-39; RING1B, 59-CCTCCTGAGGTGTTCGTTGT-39 and 59-GTTGTATTTCCCGAGCTCCA-39; BMI1, 59-CCAGGGCTTTTCAAAAATGA39 and 59-GCATCACAGTCATTGCTGCT-39; EZH2, 59-TACTTGTGGAGCCGCTGAC-39 and 59-CTGCCACGTCAGATGGTG-39; SUZ12, 59-CTTACATGTCTCATCGAAACTCC-39 and 59-GGCTGGAAGCTCTTCATTGACA-39; and 18s rRNA, 59-GTAACCCGTTGAACCCCATT-39 and 59-CCATCCAATCGGTAGTAGCG-39. To quantify the RNA level, quantitative RT-PCR (qRT-PCR) was carried out using the Applied Biosystems 7300 Real-Time PCR System and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Data were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following primers were used for qRT-PCR: DKK1_real-time, 59GATCATAGCACCTTGGATGGG-39 and 59-GGCACAGTCTGATGACCGG-39; GAPDH_real-time, 59-CATGTTCCAATATGATTCCA-39 and 59-CCTGGAAGATGGTGATG-39; and P16INK4A, 59-CCCGCTTTCGTAGTTTCCAT-39 and 59-TTATTTGAGCTTTGGTCCTG-39. Immunoblotting and immunoprecipitation Cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails, and immunoblotting was performed as described previously (31). For immunoprecipitation, cell lysates were incubated with the appropriate antibodies

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Figure 1. Effect of CBX7 on stem-like properties in breast cancer cells. A) CD44+/CD242/ESA+ cells (CSCs) and other cells (non-CSCs) were sorted, and RNA was extracted from the cells. CBX7 mRNA in CSCs and non-CSCs was quantified by RTPCR. N, non-CSCs; C, CSCs. 18S rRNA was used as an internal control for PCR. B) shRNA-mediated stable knockdown or doxycycline (Dox)-inducible overexpression of CBX7 in the indicated cell lines was confirmed by immunoblotting. shcon, (continued on next page)

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TABLE 1. Tumor-initiating ability of CBX7-knockdown cells in NOD/SCID mice Cell type

Cell no.

21 d

shcon

500 1000 5000 10,000

0/4 0/5 0/5 0/5

TIC frequency shCBX7

TIC frequency

500 1000 5000 10,000

28 d

35 d

39 d

42 d

0/4 0/4 0/4 1/4 0/5 0/5 0/5 1/5 2/5 2/5 3/5 4/5 0/5 3/5 3/5 4/5 1/38,446 1/11,915 1/9370 1/4201 (1/18,709–1/79,004) (1/7596–1/18,691) (1/6168–1/14,243) (1/2915–1/6055) 0/4 1/4 1/4 2/4 2/4 0/5 1/5 2/5 3/5 3/5 2/5 3/5 4/5 4/5 5/5 1/5 2/5 4/5 4/5 4/5 1/23,826 1/8737* 1/3670* 1/2874* 1/2262** (1/13,271–1/42,773) (1/5797–1/13,168) (1/2547–1/5288) (1/1982–1/4167) (1/1539–1/3324)

Limiting dilution assay. From 500 to 10,000 cells, control MDA-MB-231 cells (shcon) or CBX7 knockdown MDA-MB-231 cells (shCBX7) were injected into the fat pads of NOD/SCID mice. Tumor-initiating cell (TIC) frequency was calculated using Poisson statistics and L-Calc software. *P , 0.0001. **P = 0.0001.

overnight at 4°C. The immunocomplexes were precipitated with protein A- or G-agarose beads for 2 h at 4°C and washed with ice-cold PBS 3 times. Laemmli sample buffer was then added to the immunoprecipitated pellets for immunoblotting. For immunoblotting and immunoprecipitation, the following antibodies were used: CBX7 (21873), Suz12 (12073-100), H3K4me2 (ab32356), and H3K9me2 (ab1220) from Abcam (Cambridge, MA, USA); Ezh2 (3147), p-AKT (9271S), AKT (9272), p-GSK-3b (9336S), GSK-3b (9315), c-Myc (5605), and H2Aub (05-678) from Cell Signaling Technology (Beverly, MA, USA); Ring1B (sc-101109), Bmi1 (sc-10745), a-tubulin (sc-8035), Sp-1 (sc-59), DKK-1 (sc-25516), HDAC1 (sc-7872), HDAC2 (sc-9959), and CREB-binding protein (CBP; sc-369) from Santa Cruz Biotechnology (Santa Cruz, CA,USA); b-actin (MAB750R), p300 (05-257), H3Ac (06-599-MN), and H3K27me3 (07-449) from Millipore (Billerica, MA, USA); and b-catenin (610153) and p16INK4a (51-1325GR) from BD Pharmingen (San Diego, CA, USA). Cell fractionation For analysis of nuclear and cytoplasmic protein expression, cell fractionation was performed using the NE-PER nuclear and cytoplasmic extraction reagent (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The fractionated extracts were subjected to immunoblotting as described above. Immunofluorescence staining Cells (1 3 105/well) seeded onto a 4-chamber slide glass (LabTek 2 Chamber Slide System; Nalge Nunc International, Rochester, NY, USA) were fixed with cooled methanol/ acetone (1:1) at 220°C for 10 min and incubated in PBS with 0.3% Triton X-100 and 1% bovine serum albumin for 1 h. After blocking, cells were stained with mouse anti-b-catenin (1:200) at

room temperature for 1 h and further reacted with anti-rabbit FITC-conjugated secondary goat antibody (1:300; Invitrogen) for an additional 1 h. After staining, VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) was applied to visualize nuclei and characterized by fluorescence microscopy (Leica DM 5000B; Leica, Wetzlar, Germany). Luciferase reporter assay To analyze DKK-1 transcriptional activity, a 1046 base pair (bp) genomic fragment corresponding to the region of the DKK-1 promoter between bases 2935 and +111 (pGL3-1kbDKK-1) or a 350 bp fragment containing bases 2239 to +111 (pGL30.35kbDkk1) was generated by PCR and cloned into the pGL3 luciferase reporter vector (Promega, Madison, WI, USA). Cells were cotransfected with the reporter vector and b-galactosidase cDNA by Lipofectamine 2000. After 48 h transfection, cells were lysed in lysis buffer provided by a luciferase assay kit (Promega) and assayed for luciferase activity using a MicroLumat Plus LB96V luminometer (Berthold Technologies, Bad Wildbad, Germany). The luciferase activity, expressed as relative light units (RLUs), was normalized to b-galactosidase activity. Chromatin immunoprecipitation assay Chromatin immunoprecipitation (ChIP) assays were performed following the manufacturer’s instructions provided in a ChIP assay kit (Millipore). Briefly, cells were cross-linked with 1% formaldehyde at room temperature for 10 min. The DNAprotein complexes were immunoprecipitated with specific antibodies and appropriate protein A/G-agarose (Millipore). DNA was eluted and purified from the complexes and then amplified by PCR using primers specific for the DKK-1 promoters. The following primers were used for real-time quantitative PCR after ChIP assay: region 1, 59-CCACTTTGAT-

nontargeting shRNA; shCBX7, shRNA against CBX7; Con, empty vector; CBX7, Dox-induced CBX7. C) The subpopulations of the CD44+/CD242/ESA+ phenotype in these cells were measured by fluorescence-activated cell sorting (FACS) analysis (top). The quantification of CD44+/CD242/ESA+ cells is shown as a bar graph (bottom). D) Cells were seeded in a nonadherent culture dish, and the number of primary (P1) and secondary (P2) tumorsphere cells (.100 mm diameter) was counted and quantified after 3, 5, and 7 d. E) A soft agar colony-formation assay was performed to measure anchorage-independent cell growth in the indicated cell lines. The number of colonies (.100 mm diameter) was counted after the indicated days. All experiments were performed in triplicate. The results are shown as the mean 6 SD. *Significant difference of P , 0.05 compared to control (shcon or Con) (Student’s t test). Scale bars, 100 mm.

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TIC frequency

1000 2000 10,000 20,000 CBX7

TIC frequency

1000 2000 10,000 20,000 Con

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Limiting dilution assay. From 1000 to 20,000 cells, control MCF7 cells (Con) or CBX7-overexpressing MCF7 cells (CBX7) were injected into the fat pads of NOD/SCID mice. Tumor-initiating cell (TIC) frequency was calculated using Poisson statistics and L-Calc software. *P , 0.0001.

3/5 3/5 5/5 5/5 1/1606 (1/1059–1/2436) 0/5 0/5 0/5 4/5 1/30,152* (1/18,531–1/49,062) 3/5 2/5 4/5 5/5 1/3627 (1/12447–1/5376) 0/5 0/5 0/5 3/5 1/44,249* (1/25,137–1/77,892) 1/5 0/5 2/5 3/5 1/19,781 (1/12,960–1/30,192) 0/5 0/5 0/5 2/5 1/72,038* (1/35,873–1/144,662) 0/5 0/5 0/5 2/5 1/72,038 (1/35,873–1/144,662) 0/5 0/5 0/5 0/5

2/5 1/5 3/5 5/5 1/6549 (1/1552–1/9423) 0/5 0/5 0/5 2/5 1/72,038* (1/35,873–1/144,662)

46 d 42 d 39 d 35 d 28 d Cell no. Cell type

TABLE 2. Tumor-initiating ability of CBX7-overexpressing cells in NOD/SCID mice 304

CTCACGCGTC-39 and 59-AGAGAGGGAGGCGAGAGACT-39; region 2, 59-GCACAGTCAGCGAGTATTGG-39 and 59-GAACTTGGGTGCCCTTGCCTG-39; region 3, 59-CGGGGTGAAGAGTGTCAAA-39 and 59-CGCGGCTGCCTTTATACCGC-39; and region 4, 59-ATGCTCCGGGCCCGCGGTAT-39 and 59-GTGGCGCTCACTCCCAGCAG-39. Flow cytometry analysis To analyze the expression of CD44, CD24, and ESA cell surface markers, cells were stained with allophycocyanin (APC)conjugated CD44 (BD Biosciences, San Jose, CA, USA), phycoerythrin-conjugated CD24 (BD Biosciences), and FITCconjugated ESA (Dako, Carpenteria, CA, USA) antibodies at 4°C for 20 min. The percentage of CD44+/CD242/ESA+ cells was then measured with a FACSCanto II (Becton Dickinson, San Jose, CA, USA). For separation of CD44+/CD242/ESA+ cells from the whole population, cells were stained with the above antibodies, and this cell population and the other cells were isolated using a FACSAria flow cytometer (BD Biosciences). Tumorsphere formation MDA-MB-468 cells (1 3 104/well) and MDA-MB-231 cells (5 3 103/well) in a 6-well ultralow attachment surface plate (Corning, Corning, NY, USA) were cultured in DMEM-GlutaMAX medium (Invitrogen) supplemented with B27 (Invitrogen), 10 ng/ml basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ, USA), 10 ng/ml epidermal growth factor (EGF), and 4 ng/ml heparin (Sigma-Aldrich). MCF10A cells (1.5 3 104/well) and MCF7 cells (3 3 103/well) were cultured in DMEM-GlutaMAX medium containing B27, 40 ng/ml bFGF, 40 ng/ml EGF, and 4 ng/ml heparin, and in the same medium containing B27, 10 ng/ml bFGF, 10 ng/ml EGF, and 4 ng/ml heparin, respectively. After 3, 5, and 7 d, tumorsphere formation was observed using an inverted microscope. For analysis of tumorspheres from tetracycline-inducible cells, cells were pretreated with 2 mg/ml doxycycline for 72 h and then plated in tumorsphere medium containing doxycycline at the same concentration. To examine the effect of DKK-1 inhibitors on CBX7mediated tumorsphere formation, cells were cultured in the medium described above containing 0.4 mg/ml WAY-262611. For secondary tumorsphere-forming assay, primary tumorspheres (.100 mm diameter) were harvested after 7 d, dissociated into single-cell suspensions with 0.05% trypsin, and then cultured as described above. Soft agar colony-formation assay To assess anchorage-independent cell growth, 1% agar in growth medium was coated in 6-well plates, and MCF10A and MDA-MB-231 cells (each 2 3 105/well) and MDA-MB-468 cells (1 3 105/well) were plated in 0.3% agar onto the substrate agar. For tetracycline-inducible CBX7 overexpression in MCF7 cells, cells (5 3 104/well) were preincubated with 2 mg/ml doxycycline for 72 h and then plated in agars containing doxycycline. After long-term incubation, colonies .100 mm in diameter were counted under a microscope. In vivo limiting dilution assay Five-week-old female nonobese diabetic (NOD)/SCID mice were purchased from the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Republic of Korea). A volume of 100 ml PBS mixed with Matrigel containing CBX7-knockdown

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Figure 2. Effect of CBX7 on the stem-like properties of p16INK4a-positive breast cancer cells. A) Knockdown or overexpression of CBX7 in MDA-MB-468 cell lines was confirmed by immunoblotting (top), and the subpopulations of the CD44+/CD242/ESA+ phenotype in these cells were assayed by FACS (middle). The percentage of CD44+/CD242/ESA+ cells is shown as a bar graph (continued on next page)

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MDA-MB-231 cells and CBX7-overexpressing MCF7 cells at limiting dilution were directly injected into the mammary fat pads of the mice. For xenografts with MCF7 cells, mice were supplemented with estradiol pellets (0.72 mg, released over 60 d; Innovative Research of America, Sarasota, FL, USA), and 2 mg/ml doxycycline in 1% sucrose drinking water was administered for induction of CBX7 overexpression. The tumor volume was monitored twice per week for 6–7 wk and calculated as follows: volume (mm3) = (a 3 b2)/2, where “a” indicates the largest diameter, and “b” is the perpendicular diameter. The proportion of tumor-initiation frequency was calculated using Poisson statistics and L-Calc software (STEMCELL Technologies, Vancouver, BC, Canada; http://www.stemcell.com). Immunohistochemistry Tumors were fixed in 4% formalin and embedded in paraffin blocks. Slides were deparaffinized, and antigen retrieval was performed using citric acid. Endogenous peroxidase activity was inhibited by incubating slides with 3% H2O2 in water. Slides were incubated overnight at 4°C with primary antibodies. Each primary antibody was detected by an UltraTech HRP kit (Immunotech, Marseille, France).

RESULTS Loss of CBX7 enhances breast tumorigenicity To address the potential role of CBX7 in breast tumor initiation, we first measured the expression levels of CBX7 in isolated CD44+/CD242/ESA+ cells, which have been suggested to be one of the stem cell markers in human breast cancer (32), derived from MCF10A immortalized breast epithelial cells and various breast cancer cell lines by flow cytometry. As shown in Figure 1A, CD44+/CD242/ESA+ cells expressed lower levels of CBX7 compared with a pool of other populations. We also examined the total CBX7 levels in these cells, and the CBX7 protein level was higher in breast epithelial cells compared with breast cancer cells (Supplemental Fig. 1). To further determine the effect of CBX7 expression on breast stem cell-like properties, lentiviral shRNA-mediated stable CBX7 knockdown and tetracycline-inducible CBX7 overexpression systems were established in MCF10A and MDA-MB-231 cell lines with high expression of CBX7 and in MCF7 cell lines expressing low levels of CBX7, respectively, as confirmed by immunoblotting (Fig. 1B). Flow cytometry analysis revealed that CBX7 knockdown increased the percentage of CD44+/CD242/ESA+ cells, whereas overexpression of CBX7 decreased this population (Fig. 1C). Furthermore, the number and size of primary and secondary tumorspheres were increased in CBX7-knockdown cell lines, whereas tumorsphere

formation was decreased in CBX7-overexpressing cells (Fig. 1D). Similarly, the ability of cells to form colonies in soft agar was enhanced by deletion of CBX7 and reduced by CBX7 overexpression (Fig. 1E), indicating that CBX7 inhibited the self-renewal of breast CSCs. We further determined whether CBX7 also affected the capacity of tumor initiation in vivo. The incidence of tumor formation was increased in mice injected with CBX7-knockdown MDA-MB-231 cells compared with control mice (Table 1). Moreover, overall in vivo tumor growth was also accelerated by CBX7 knockdown (Supplemental Fig. 2A). Similarly, tumor initiation ability and growth were decreased in mice injected with CBX7overexpressing MCF7 cell lines compared with control mice (Supplemental Fig. 2B and Table 2). Together, these results indicated that CBX7 acts as a negative regulator of breast tumorigenicity.

Role of CBX7 in breast cancer is independent of p16INK4a expression status It was reported that p16INK4a is negatively regulated by CBX7, which is important to the oncogenic role of CBX7 in several cancer cells (11, 12). Because p16INK4a was reported to inhibit the CD44+/CD242/ESA+ cell population in breast cancer (33), we assumed that the status of p16INK4a expression could affect the CBX7 effect on breast cancer stem-like cells. Although CBX7 expression was not correlated with p16INK4a in breast cancer cells because of the very low expression of p16INK4a proteins in these cells (Supplemental Fig. 1), we determined whether CBX7 is involved in p16 INK4a -dependent breast cancer regulation by generating stably CBX7overexpressing and -knockdown cells in p16INK4a-positive MDA-MB-468 cells. Unlike the expectation that CBX7 enhanced the ability to self-renew breast CSCs through p16INK4a repression in p16INK4a-positive cells, CBX7 overexpression reduced the proportion of CD44+/ CD242/ESA+ cells and the number of tumorspheres and colonies in MDA-MB-468 cells (Fig. 2A–C). A similar effect was shown in CBX7-knockdown cells (Fig. 2A, C). We also examined whether p16INK4a was repressed by CBX7 in these cell lines, but CBX7 did not induce detectable changes in p16INK4a expression (Fig. 2D). Furthermore, transient knockdown of 16INK4a increased the CD44+/CD242/ESA+ cell population and tumorsphere formation in control cells, whereas it could not recover CBX7-mediated reduction of stem-like properties in CBX7-overexpressing cells (Fig. 2E, F). Taken together, these data imply a p16INK4a-independent role of CBX7 in breast cancer.

(bottom). B) Cells were seeded in a nonadherent culture dish, and the number of primary (P1) and secondary (P2) tumorsphere cells (.100 mm diameter) was counted after 3, 5, and 7 d. C) A soft agar colony-formation assay was performed and quantified in the indicated cell lines. D) Expression of p16INK4a in CBX7-overexpressing MDA-MB-468 breast cancer cell lines was assayed by immunoblotting (top) and real-time qRT-PCR (bottom). E) Cells were transfected with p16INK4a siRNA (p16 si) or nontargeting siRNA (con si) for 48 h and subjected to immunoblotting (left) and flow cytometry for analysis of the CD44+/CD242/ESA+ cell population (right). F) Cells transfected with p16INK4a siRNA were replated in a nonadherent culture dish for tumorsphereforming assay. All experiments were performed in triplicate. Results are shown as the mean 6 SD. *P , 0.05 compared to the control (Student’s t test). Scale bars, 100 mm.

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Figure 3. Effect of CBX7 on the activity of the Wnt signaling pathway. A) Heat map showing Wnt signaling-related genes whose expression changed by $1.5-fold in triplicate replications (left). These genes were categorized using KEGG pathway software (Kanehisa Laboratories, Kyoto, Japan) (right). B) DKK-1 expression in the indicated cell lines was assayed by immunoblotting (top) and real-time qRT-PCR (bottom). C) The effect of CBX7 on the Wnt signaling pathway was analyzed by immunoblotting with the indicated antibodies. D) Cells were stained with an anti-b-catenin antibody coupled with an FITC-conjugated anti-mouse IgG (green) and DAPI (blue). Immunofluorescence outcomes were calculated by counting 1000 cells. C, cytoplasm; C+N, cytoplasm and nucleus. E) To examine the cellular localization of b-catenin, cells were fractionated, and the nuclear and cytoplasmic extracts were subjected to immunoblotting. a-Tubulin and Sp1 were used as controls for the cytoplasmic and nuclear (continued on next page)

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Loss of CBX7 activates the Wnt/b-catenin/TCF signaling pathway To discover potential targets of CBX7 in breast tumorigenicity, we performed a gene expression microarray analysis of MCF10A cell lines with knockdown of CBX7 (Supplemental Fig. 3A). We found 775 significantly upregulated genes and 681 down-regulated genes, which are involved in many biologic functions and signaling pathways, by CBX7 depletion (Supplemental Fig. 3A, B). Among these pathways, we noted Wnt signaling because it is described as a major mechanism of the regulation of CSC self-renewal (34). A heat map of genes with a .1.5fold change in expression that are directly or indirectly related to the Wnt pathway in the microarray experiment was generated (Fig. 3A). We validated the expression of these genes in CBX7-knockdown and -overexpressing cell lines by RT-PCR (Supplemental Fig. 3C). In particular, the protein and mRNA levels of the Wnt signaling antagonist DKK1, which exhibited significant downregulation (.2.5-fold) by CBX7 depletion in the microarray, were changed substantially by CBX7 in these cells (Fig. 3A, B). To confirm these results, we next investigated whether CBX7 inhibited the Wnt signaling pathway. The phosphorylation of AKT and GSK3-b was increased in CBX7overexpressing MCF7 cell lines and decreased in CBX7knockdown MCF10A cells (Fig. 3C). Although the total b-catenin expression level was not significantly altered, loss of CBX7 induced translocation of b-catenin into the nucleus, as confirmed by immunoblotting of nuclear and cytoplasmic fractions and immunofluorescence staining (Fig. 3D, E). Similarly, CBX7 overexpression inhibited nuclear localization of b-catenin. Furthermore, CBX7 negatively regulated TCF/lymphoid enhancer factor (LEF) promoter activity but did not affect the activity of a b-catenin-binding mutant promoter (Fig. 3F). Consequently, the expression of c-Myc, a b-catenin/TCF target gene, was inhibited by CBX7 (Fig. 3C). Together, these results suggest that CBX7 up-regulated DKK-1 expression and inhibited the Wnt/b-catenin/TCF signaling pathway. CBX7 positively regulates DKK-1 gene transcription via enhancement of histone acetylation To further investigate the mechanism by which CBX7 regulated DKK-1 expression, we generated 2 luciferase reporter constructs containing ;1 kbp of the distant DKK-1 promoter and 0.35 kbp of the proximal DKK-1 promoter (Fig. 4A, left). The 1 kbp DKK-1 promoter activity was decreased, whereas that of the 0.35 kbp DKK-1 promoter was unchanged in CBX7-knockdown MCF10A cells (Fig. 4A, middle). Similarly, CBX7-overexpressing MCF7 cells showed strongly enhanced 1 kbp DKK-1 promoter activity but only slightly increased 0.35 kbp DKK-1 promoter activity (Fig. 4A, right), indicating that

CBX7 up-regulated DKK-1 expression at the transcriptional level via the activity of the DKK-1 distal promoter. DKK-1 was reported to be a target of PcG proteins (24, 25), and a recent study has suggested that CBX7 can affect the expression of other PcG proteins (7, 8). Thus, we examined whether CBX7 regulated DKK-1 transcription via the PcG proteins. However, the protein and mRNA levels of other PcGs were not significantly altered by CBX7 (Supplemental Fig. 4A, B). Moreover, CBX7 directly bound the distal region to the DKK-1 promoter, whereas other PcG proteins did not show notable change in the binding of the DKK-1 promoter by CBX7 as determined by ChIP assay (Fig. 4B and Supplemental Fig. 4C). The amounts of PcG-related histone markers, H3K27me3 and H2A Lys-119 monoubiquitination (H2AK119ub), were also unchanged in these regions (Supplemental Fig. 4C). In addition, siRNA-mediated knockdown of Ezh2 or lack of CBX7 chromodomain did not affect the CBX7-induced DKK-1 expression (Supplemental Fig. 4D, E). Consistently, CBX7 could bind to the DKK-1 promoter region regardless of Ezh2 or chromodomain (Supplemental Fig. 4D, E). These data indicated that CBX7 recognizes DKK-1 promoter and regulates its activity in a PcG-independent manner. In contrast, histone 3H acetylation (H3Ac), which is associated with transcriptional activation, was markedly increased by CBX7 overexpression in a broad range of the DKK-1 promoter region (Fig. 4B, left). Furthermore, the recruitment of p300/CBP histone acetyltransferase (HAT) to the promoter region was enhanced, whereas HDACs, including HDAC1 and HDAC2, were dissociated from the region. A similar effect was shown in CBX7knockdown cells (Fig. 4B, right). By performing a coimmunoprecipitation assay, we also found that CBX7 directly bound to endogenous p300 protein, but not CBP (Fig. 4C). Consistent with these observations, treatment of CBX7-knockdown cells with an HDAC inhibitor, TSA, recovered DKK-1 expression (Fig. 4D). Taken together, our findings suggest that CBX7 recruits p300/CBP to the DKK-1 promoter by directly interacting with p300 to increase histone acetylation and consequently activate DKK-1 transcription. Reduced Wnt signaling pathway by CBX7 is mediated by DKK-1 Because DKK-1 has been implicated in the negative regulation of Wnt signaling and breast CSCs (28), we examined whether DKK-1 is required for CBX7mediated breast CSC regulation through the Wnt signaling pathway. Following treatment of CBX7-overexpressing MCF7 cells with the DKK-1 inhibitor WAY-262611 or DKK-1 siRNA, CBX7-induced reduction of phosphorylated AKT and GSK3-b was recovered, and TCF/LEF promoter activity and consequent c-Myc expression were also rescued (Fig. 5A, B). This result indicated that the

fractions, respectively. F) The effect of CBX7 on TCF/LEF promoter activity was measured by luciferase assay. Cells were transfected with a TCF promoter containing Tcf4-binding sites [T cell factor optimal promoter (TOP)] or its mutant form [fake optimal promoter (FOP)] for 48 h and then subjected to promoter assay. All procedures were performed in triplicate. Results are shown as the mean 6 SD. *P , 0.05 compared to shcon or Con (Student’s t test). Scale bars, 20 mm.

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Figure 4. CBX7 regulates DKK-1 expression through increasing histone acetylation. A) Cells were transfected with pGL3-1kbDKK1 (1 kb) or pGL3-0.35kbDKK-1 (0.35 kb) and b-galactosidase constructs for 48 h, and a luciferase reporter assay was performed to measure DKK-1 promoter activity. TSS, transcription start site. B) Schematic illustration of the promoter regions of the human DKK-1 gene and the regions containing the primers for ChIP assay (right). The results of ChIP analysis show the amounts of the indicated factors recruited to the DKK-1 promoter regions in CBX7-overexpressing MCF7 cells (left) and CBX7-knockdown MCF10A cells (middle). NO Ab, no antibody for negative control. C) Lysates of CBX7-overexpressing MCF7 cells were immunoprecipitated using CBX7, p300, and CBP antibodies, and the interaction of CBX7 with p300 or CBP was analyzed by immunoblotting. D) CBX7-knockdown MCF10A cells treated with 100 ng/ml TSA for 12 h were subjected to immunoblotting (left) and qRT-PCR (right). All experiments were performed in triplicate. Results are shown as the mean 6 SD. *P , 0.05 vs. shcon or Con; †P , 0.05 vs. shCBX7 (Student’s t test).

CBX7-mediated blockade of Wnt signaling was reinitiated by inhibition of DKK-1 activity. Furthermore, the DKK-1 inhibitor rescued the decrease in the CD44+/ CD242/ESA+ cell population and self-renewal potential in CBX7-overexpressing MCF7 cells (Fig. 5C, D). We INHIBITION OF BREAST TUMORIGENESIS BY CBX7

also confirmed the changes in the expression of DKK-1 by CBX7 in vivo by immunohistochemical analysis of xenograft tumors (Fig. 5E). Therefore, these data indicate that DKK-1 contributed to the attenuation of Wnt signaling by CBX7. 309

Figure 5. CBX7 regulates the DKK-1-mediated Wnt signaling pathway. A) Effect of the inhibition of DKK-1 on CBX7-induced suppression of the Wnt signaling pathway. Cells treated with 0.4 mg/ml WAY-262611 or DKK-1 siRNA for 48 h were subjected to immunoblotting with appropriate antibodies. B) The TCF/LEF promoter activity was measured after treatment with WAY-262611 or DKK-1 siRNA upon Dox-inducible CBX7 overexpression in MCF7 cells. C and D) FACS analysis of the CSC population (C) and a tumorsphere formation assay (D) were performed in cells treated with WAY-262611. Scale bars, 100 mm. E) Immunohistochemical analysis of DKK-1 expression in the indicated xenograft tumors. Results are scored by the sum of the percentage of staining multiplied by the intensity level (0, none; 1, weak; 2, moderate; and 3, strong). Scale bars, 50 mm. All experiments were performed in triplicate. Results are shown as the mean 6 SD. *P , 0.05 vs. the control vector; †P , 0.05 vs. CBX7 (Student’s t test).

DISCUSSION Here, we demonstrated that CBX7 functions as a tumor suppressor in breast cancer by inhibiting the breast tumorigenicity. We also identified the novel inhibitory mechanism of breast cancer by CBX7. CBX7 positively regulated the Wnt antagonist DKK-1, thereby inactivating the Wnt/b-catenin/TCF pathway. The induction of DKK-1 expression by CBX7 was accompanied by increased H3Ac via recruitment of p300/CBP to the DKK-1 310

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promoter and dissociation of HDAC1/HDAC2 from the promoter (Fig. 6). These findings suggest a crucial role for CBX7 in breast tumorigenesis as a novel epigenetic regulator of the Wnt pathway. Our data suggest the potential role of CBX7 in regulation of stem cell-like properties in human breast cancer. Because CSCs share the characteristics of normal stem cells, including the ability of self-renewal and differentiation, many regulators of normal stem cells could be a key factor in CSCs. PcG proteins are also

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Figure 6. Proposed model for regulation of DKK-1 and subsequent modulation of the Wnt signaling pathway by CBX7 in breast cancer. For transcriptional activation of the DKK-1 gene, CBX7 can inhibit HDAC-mediated histone deacetylation by directly interacting with p300 acetyltransferase and recruiting p300/CBP to the DKK-1 promoter. In breast cancer, this transcriptional activation complex is dissociated from the region by the loss of CBX7, and HDACmediated DKK-1 gene silencing is reinitiated. The diminished expression of Wnt agonist DKK1 subsequently activates the Wnt/b-catenin/TCF pathway, which results in nuclear translocation of b-catenin and upregulation of TCF target genes including c-Myc. Thus, the loss of CBX7 might induce breast cancer stemness by enhancing the Wnt pathway-mediated selfrenewal of breast CSCs and contributes to the breast tumorigenicit.

involved in the regulation of both normal stem cells and CSCs (35). Moreover, the enzymatic activities of PcG members toward histones were reported to be essential for maintaining the self-renewal of embryonic and adult stem cells as well as CSCs (36–38). Although CBX proteins are crucial for linking PRC-2 and PRC-1 activities, few studies have reported their role in stemness. Recently, it was reported that CBX7 enhances ESC self-renewal and suppresses differentiation (7). Likewise, developmental genes were repressed and promoted HSC self-renewal by CBX7 (9). These regulations have been commonly based on PcG composition because the competition among CBX family members leads to the formation of diverse PRC-1 complexes comprised of different CBX proteins; these distinct complexes regulate the balance between self-renewal and differentiation. However, contrary to ESCs or HSCs, we suggest that CBX7 may reduce the selfrenewal ability of breast CSCs through DKK-1-mediated repression of the Wnt pathway independent of PcG function. The breast CSC phenotype has been defined by aldehyde dehydrogenase-positive or CD44+/CD242/ESA+ cells (32, 39, 40). Our findings showed that CBX7 reduced the CD44+/CD242/ESA+ population, and the abilities for tumorsphere formation and in vivo tumor initiation, which possibly imply the inhibited stem cell-like properties in breast cancer. Because this population is correlated with tumorigenicity, resistance to chemotherapy, and cancer invasiveness, as well as worse clinical behavior (32, 40-43), the decrease in this population by CBX7 could support the inhibitory role of tumorigenesis by CBX7 and possibly suggest CBX7 as a negative regulator of stem-like properties in human breast cancer. INHIBITION OF BREAST TUMORIGENESIS BY CBX7

Furthermore, because Wnt signaling is one of the key pathways for the maintenance of self-renewal of both normal and malignant stem cells and contributes to tumorigenesis and malignant transformation (19, 44), these findings suggest the importance of the PcGindependent negative regulatory function of CBX7 as a modulator of the Wnt pathway during breast tumor initiation and development. Our findings also suggest that CBX7 can promote epigenetic gene activation in a PcG-independent manner. The silencing of DKK-1 expression has been related to histone deacetylation and DNA hypermethylation (45, 46). Recent studies also indicated the possible association of histone methylation with DKK-1 expression. For instance, DKK-1 was down-regulated by LSD1, a histone H3K4/H3K9 demethylase, in colorectal cancer (26). In addition, 2 studies have shown that PcG proteins are recruited to the DKK-1 promoter for gene silencing. Cho et al. (24) indicated that PcG proteins were recruited to near the transcription start site, whereas Hussain et al. (25) showed Bmi1 bound to the DKK-1 promoter region away from the transcription start site. In our results, CBX7 bound more strongly to the distal promoter of DKK-1, similar to Bmi1, but did not affect the PcG-dependent histone modification in this region. Instead, the remarkable increase in H3Ac across a broad range of the DKK-1 promoter was shown by CBX7. Although CBX7 was reported to be a binding partner of HDAC, the effect of CBX7 on HDAC activity is controversial because it is involved in both activation and inactivation of HDAC toward different target genes (3, 4). In our study, CBX7 formed a complex with a HAT rather than HDACs. CBX7 311

did not form a complex with CBP, consistent with the findings of Mohammad et al. (6), but it could directly bind to p300 and recruit both p300 and CBP to the promoter of the target gene. Further investigation is needed to elucidate the different functional roles of CBX7 in modulating the balance between histone acetylation and deacetylation in different genes and cellular contexts. Taken together, our results suggest that CBX7 is crucial for transcriptional activation of target genes in cooperation with its novel binding partner p300 HAT, in addition to PcG-mediated gene silencing. This study shows the important role of CBX7 in breast cancer initiation as a tumor suppressor. The role of CBX7 in cancer progression is debated. CBX7 was shown to prolong the life span of normal human cells by repressing the INK4a/ARF locus (12). Similarly, ablation of CBX7 expression in gastric and prostate cancers led to decreased cellular senescence, and enhanced proliferation and migration ability through p16INK4a up-regulation (10, 11). Moreover, CBX7 cooperates with c-Myc to promote lymphomagenesis (47). These reports suggest an oncogenic role for CBX7 in the early stages of tumor development. In contrast, recent studies have proposed CBX7 as a tumor suppressor. For instance, CBX7 expression significantly reduced and its overexpression increased E-cadherin expression, which implies repression of epithelial-to-mesenchymal transition, in thyroid cancer cells (3, 48). CBX7-knockout mice showed the development of lung and liver tumors accompanied by cyclin E up-regulation (4). Furthermore, some clinicopathologic studies have shown that CBX7 expression is inversely associated with the development of several human cancers, including bladder, pancreatic, and colon carcinomas (13–15). A recent study also implied a negative correlation between CBX7 expression and a malignant phenotype in human breast cancer (16). Here, we suggest a molecular mechanism whereby CBX7 functions as a tumor suppressor in the development of breast cancer. Because CBX7 commonly has an oncogenic role via p16INK4a, it is possible that the dual function of CBX7 in tumorigenesis depends on the p16INK4a status. However, our findings showed that the tumor-suppressive function of CBX7 by inhibition of CSC activity was mediated by the DKK-1/Wnt/b-catenin pathway, regardless of p16INK4a status. Although it is still unclear how CBX7 dually functions in human cancers, it is possible that the expression or mutation status of Wnt components including DKK-1 in distinct cancer cells affects the role of CBX7 in human cancers. Together, our results indicate that CBX7 could be a potential tumor suppressor that acts as a novel negative regulator of the Wnt pathway. In conclusion, our data suggest that CBX7 acts as a tumor suppressor through the regulation of breast tumorigenicity via enhancement of the histone acetylation of DKK-1 and its mediated suppression of the Wnt signaling pathway independently of PcG members. We propose that CBX7 could be a therapeutic target for breast cancer. This work was supported by grants from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (No. A101836), and the National Research Foundation of Korea funded by the Korean government (No. 2010-0020879). The authors declare no conflicts of interest.

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