Binding of the Jmjc Demethylase JARID1B to LSD1/Nurd Suppresses Angiogenesis and Metastasis in Breast Cancer Cells by Repressing Chemokine CCL14

Published OnlineFirst September 21, 2011; DOI: 10.1158/0008-5472.CAN-11-1523

Cancer Tumor and Stem Cell Biology Research

Binding of the JmjC Demethylase JARID1B to LSD1/NuRD Suppresses Angiogenesis and Metastasis in Breast Cancer Cells by Repressing Chemokine CCL14

Qian Li1, Lei Shi1, Bin Gui1, Wenhua Yu1, Jiamu Wang1, Di Zhang1, Xiao Han1, Zhi Yao2, and Yongfeng Shang1,2

Abstract JARID1B is a member of the JmjC/ARID family of demethylases that speciﬁcally demethylates tri- and di-methylated forms of histone H3 lysine 4 (H3K4) that are associated with active genes. JARID1B expression is dysregulated in several cancers in which it has been implicated, but how it might affect tumor progression is unclear. In this study, we report that JARID1B is a physical component of the LSD1/NuRD complex that functions in transcriptional repression. JARID1B and LSD1 acted in a sequential and coordinated manner to demethylate H3K4. A genome-wide transcriptional analysis revealed that among the cellular signaling pathways targeted by the JARID1B/LSD1/NuRD complex is the CCL14 chemokine pathway of cell migration and angiogenesis. JARID1B repressed the expression of CCL14, an epithelial derived chemokine, suppressing the angiogenic and metastatic potential of breast cancer cells in vivo. Our ﬁndings indicate that CCL14 is a critical mediator of the JARID1B/ LSD1/NuRD complex in regulation of angiogenesis and metastasis in breast cancer, identifying a novel potential therapeutic target for breast cancer intervention. Cancer Res; 71(21); 1–10. 2011 AACR.

Introduction melanoma metastases, the expression of JARID1B is downregulated or even lost (4). Given its downregulation in aggres- JARID1B(KDM5B/PLU-1/RBP2-H1) is a member of the ARID sive primary tumors and metastases (4), the role, if any, of (AT-rich DNA interaction domain) domain containing JmjC JARID1B in tumor angiogenesis and metastasis becomes an family of demethylases that specifically targets histone H3 important question. lysine 4 (H3K4; ref. 1). JARID1B was originally isolated as a gene Here, we report that JARID1B is a component of the that was overexpressed in breast carcinomas (2). Subsequently, LSD1/NuRD complex. We showed that JARID1B and LSD1 act JARID1B dysregulation has been reported in several types of in a sequential manner and coordinated fashion in H3K4 solid tumors, including prostate cancer (3), melanoma (4–7), demethylation. We showed that the JARID1B/LSD1/NuRD and bladder cancer (8). Surprisingly, however, mechanistic complex suppresses the angiogenic and metastatic potential studies on the role of JARID1B in different types of cancer have of breast cancer cells by repressing the expression of an yielded inconsistent and even confusing results (1, 3, 5, 7–10). epithelial derived chemokine, CCL14. Therefore, the mechanistic role of JARID1B in tumor progression remains an intriguing topic. Interestingly, it Materials and Methods becomes evident that the expression of JARID1B is dynamically regulated and might be associated with tumor grades. For Histone peptide binding assay example, in melanocytic tumors, JARID1B is highly expressed Five micrograms of purified proteins were incubated with in benign nevi. However, in aggressive primary melanomas and 0.2 mg of biotinylated histone peptide in 100 mL of the binding buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% NP-40, and 0.3 mg/mL bovine serum albumin] overnight at 4C. Protein–peptide complexes were pulled down with strep- Authors' Affiliations: 1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Biochemistry and Molec- tavidin beads, washed 5 times, and subjected to Western blot ular Biology, Peking University Health Science Center, Beijing; and 2Tianjin analysis using anti-GST or other specific antibodies. Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin, China Preparation of mononucleosomes Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Preparation of mononucleosomes was conducted according to the procedure described previously (11). Briefly, HeLa cells Corresponding Author: Yongfeng Shang, Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xue were harvested by ice-cold PBS, resuspended in lysis buffer [10 Yuan Road, Beijing 100191, China. Phone: 86-10-82805118; Fax: 86-10- mmol/L Tris-HCl (pH 7.5), 10 mmol/L NaCl, 3 mmol/L MgCl2, 82801355; E-mail: [email protected] or [email protected] 0.4% NP-40] and the nuclei were pelleted. Glycerol buffer [10 doi: 10.1158/0008-5472.CAN-11-1523 mmol/L Tris-HCl (pH 7.4), 0.1 mmol/L EDTA, 5 mmol/L 2011 American Association for Cancer Research. MgAc2, and 25% glycerol] was add to get a final concentration

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of 1 to 2 mg/mL nuclei. To generate nucleosomal material, complex could be efficiently coimmunoprecipitated with JAR- digestions were conducted by adding 1 volume of 2 MNase ID1B (Fig. 1D). Reciprocal coimmunoprecipitations with anti- buffer [50 mmol/L KCl, 8 mmol/L MgCl2, 2 mmol/L CaCl2, and bodies against components of the LSD1/NuRD complex and 100 mmol/L Tris-HCl (pH 7.4)] and 50 to 500 U/mL MNase. The immunoblotting with anti-JARID1B also showed that JARID1B reaction was incubated for 10 minutes at 37C and stopped by coimmunoprecipitated with these proteins (Fig. 1D). In addi- adding EDTA to a final concentration of 10 mmol/L. The tion, an association between JARID1B and the LSD1/NuRD mononucleosomes were then purified t by sucrose gradient complex was also detected in MCF-7 cells when corresponding assay. cellular extracts were immunoprecipitated with antibodies against JARID1B, followed by immunoblotting with antibodies Nucleosome binding assay against LSD1, MTA1, MTA2, MTA3, HDAC1, and HDAC2 Five micrograms of purified proteins were incubated with 2 (Fig. 1D). mg of mononucleosomes in the binding buffer [50 mmol/L To further substantiate the in vivo association of JARID1B Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% NP-40, 10 mmol/L and the LSD1/NuRD complex, protein fractionation experi- ZnCl2, and 1 mmol/L DTT] overnight at 4 C. Protein mono- ments were carried out by fast protein liquid chromatography nucleosomes were pulled down with glutathione–Sepharose (FPLC; ref. 16). The results showed that the native JARID1B beads, washed 5 times, and subjected to Western blotting analy- from HeLa cells eluted with an apparent molecular mass much sis using anti-H3 or other histone mark–specific antibodies. greater than that of the monomeric protein. JARID1B immu- noreactivity was detected in chromatographic fractions with a Statistical analysis relatively symmetrical peak centered between approximately Results are reported as mean SD. Comparisons were done 667 and 2,000 kDa (Fig. 1E). Significantly, the elution pattern of using 2-tailed paired t tests. JARID1B largely overlapped with that of the LSD1/NuRD complex proteins (Fig. 1E). Moreover, the chromatographic Results profiles of JARID1B and the LSD1/NuRD complex were com- patible with their associated enzymatic activities. Specifically, JARID1B is physically associated with the LSD1/NuRD histone deacetylation (HDAC) activity as well as histone complex demethylation (HDM) activities in both H3K4me2 and To gain mechanistic insights into the role of JARID1B in H3K4me3 were detected when the corresponding chro- breast cancer metastasis, we first employed affinity purifica- matographic fractions were incubated with the bulk histones tion and mass spectrometry to identify the proteins that are and analyzed by Western blotting with antibodies against associated with JARID1B in vivo. In these experiments, FLAG- H3ac, H3K4me2, or H3K4me3 (Fig. 1E). tagged JARID1B (FLAG-JARID1B) was stably expressed in HeLa To further document the in vivo interaction between JAR- cells. Cellular extracts were prepared and subjected to affinity ID1B and the LSD1/NuRD complex, we next used GST pull- purification using an anti-FLAG M2 affinity column. The down experiments to analyze the molecular detail involved in bound proteins were eluted with FLAG peptides and resolved this interaction. Limited by the difficulty of purifying proteins on SDS-PAGE and then visualized by silver staining. The with a high molecular weight from bacteria, these experiments protein bands on the gel were recovered and analyzed by mass were carried out with several GST-fused JARID1B fragments: spectrometry. The results revealed that JARID1B copurified JARID1B-N, which encompasses amino acids 1 to 721, and with Mi-2 a/b (CHD3/CHD4), LSD1, MTA2/3, HDAC1, HDAC2, JARID1B-C, which spans amino acids 722 to 2,145 and harbors RbAp46/48, and MBD2/3 (Fig. 1A and Supplementary File S1), 2 PHD (plant homeodomain) domains and with in vitro tran- all of which are subunits of the LSD1/NuRD complex (12–15). scribed/translated Mi-2, LSD1, MTA1, MTA2, MTA3, HDAC1, Copurification of the LSD1/NuRD complex components with and MBD3 (Fig. 2A); or with GST-fused MBD3, RbAp46, JARID1B was validated by Western blotting analysis of the RbAp48, HDAC1, or HDAC2 and in vitro transcribed/translated column-bound proteins with antibodies against the corre- JARID1B (Fig. 2B). These experiments revealed that JARID1B sponding proteins (Fig. 1B). Subsequently, the JARID1B-con- was able to interact with LSD1, HDAC1, and MBD3 in vitro. taining protein complex was analyzed for enzymatic activities Considering the fact that LSD1, HDAC1, and MBD3 all also by incubation of the immunoprecipitates (IP) with calf thymus exist in other protein complexes (17–20), interaction of JAR- bulk histones and Western blotting examination of the levels of ID1B with the multiple subunits of the LSD1/NuRD complex is methylated and acetylated histones in the reactions. These logical in terms of its specific assembly into the JARID1B/ experiments revealed that the JARID1B-containing complex, LSD1/NuRD complex. indeed, possessed enzymatic activities specific for acetylated H3 (H3ac), tri-, and di-methylated H3K4 (H3K4me3 and Functional coordination between JARID1B and LSD1 in H3K4me2; Fig. 1C). H3K4 demethylation To further confirm the in vivo interaction between JARID1B As mentioned above, JARID1B has 3 PHD domains which and the LSD1/NuRD complex, total proteins from HeLa cells have been characterized as one of the major functional mod- were extracted and coimmunoprecipitation assays were done ules for recognizing methylated histones (21, 22). Indeed, 2 with antibodies against JARID1B, followed by immunoblotting other members of the JARID1 family, JARID1A and JARID1C, with antibodies against components of the LSD1/NuRD com- have been shown to be able to bind histone tails directly via plex. The results showed that components of the LSD1/NuRD their PHD domains (23, 24). On the contrary, it is interesting to

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Figure 1. JARID1B is physically associated with the LSD1/NuRD Complex. A, immunoaffinity purification of JARID1B-containing protein complexes. Cellular extracts from HeLa cells stably expressing vector or FLAG-JARID1B were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptides. The eluates were resolved by SDS-PAGE and silver stained. The protein bands were retrieved and analyzed by mass spectrometry. B, Western blotting analysis of the identified proteins in the purified fractions using antibodies against the indicated proteins. C, the JARID1B-containing protein complex purified from HeLa cells was incubated with calf thymus bulk histones in HDM or HDAC assay buffer and analyzed by Western blotting using antibodies against the indicated histone marks or proteins. D, association of JARID1B with the LSD1/NuRD complex in HeLa cells (left) and in MCF-7 cells (right) by co-IP assays. Whole-cell lysates were immunoprecipitated with antibodies against the indicated proteins (JARID1B, left; NuRD components, right). Immunocomplexes were then immunoblotted (IB) using antibodies against the indicated proteins (NuRD components, left; JARID1B, right). E, cofractionation of JARID1B andthe LSD1/NuRD complex by FPLC. Cellular extracts from HeLa cells were fractionated on Superose 6 size exclusion columns. Chromatographic elution profiles and immunoblotting analysis of the chromatographic fractions are shown. The elution positions of calibration proteins with known molecular masses (kDa) are indicated, and an equal volume from each fraction was analyzed. www.aacrjournals.org Cancer Res; 71(21) November 1, 2011 OF3

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Figure 2. Molecular basis for the interaction between JARID1B and the LSD1/NuRD complex. A, mapping the interface for the interaction between JARID1B and components of the NuRD complex by GST pull-down experiments with bacterially expressed GST-fused JARID1B-N or JARID1B-C and the indicated proteins that had been transcribed/translated in vitro.B, GST pull-down experiments were also carried out with in vitro transcribed/translated JARID1B and GST-fused MBD3, RbAp46, RbAp48, HDAC2, or HDAC1 puriﬁed from bacteria.

note that LSD1 does not possess any currently known histone H3K4me2 and H3K4me1 levels (Fig. 3C). However, when recognition modules in its structure. Rather, it is believed that JARID1B was combined with LSD1, a much more dramatic LSD1 is presented to methylated histones via its association decrease in H3K4me3 level and much more pronounced with other proteins that do possess histone recognition mod- increase in H3K4me2 and H3K4me1 levels were observed (Fig. ules (15, 19). To test the hypothesis that JARID1B and LSD1 3C). We believe that the increased level of H3K4me2 and exert their catalytic activities sequentially to erase the methyl H3K4me1 could be due to the possibility that at the point marks on H3K4, we first carried out peptide binding assays to when the experiments were conducted, H3K4me3 demethyl- examine the binding of JARID1B and LSD1 to H3K4me3, ation is more efficient than H3K4me2 and H3K4me1 demeth- H3K4me2, and H3K4me1 in vitro. In these experiments, ylation, when JARID1B and LSD1 work together to demethylate GST-fused JARID1B-N, JARID1B-C, or LSD1 were purified and H3K4 hypermethylation. These results support the proposition incubated with histone H3 peptides that were methylated at that JARID1B and LSD1 act sequentially and in coordination to different lysine residues and at different degrees, and the remove the methyl moieties on H3K4. peptide-bound proteins were analyzed by Western blotting. Next, we tested the functional connection between JARID1B As shown in Fig. 3A, JARID1B-C exhibited a strong affinity and LSD1 in the context of chromatin. Mononucleosomes were toward H3K4me3 peptides, whereas neither JARID1B-N nor isolated from HeLa cells and were incubated with GST-fused LSD1 were able to bind H3K4me3 peptides. On the other hand, JARID1B-N, JARID1B-C, or LSD1 with WDR5 as a positive LSD1 preferably bound to H3K4me2 peptides (Fig. 3A). How- control. The bound materials were then analyzed by Western ever, when JARID1B-C and LSD1 were incubated together, blotting using antibodies against H3, H3K4me3, or H3ac. The LSD1 was detected to bind H3K4me3 peptides, and JAR- results indicated that JARID1B-C, but not JARID1B-N or LSD1, ID1B-C was found to bind H3K4me2 and H3K4me1 peptides was able to bind nucleosomes and H3K4me3 was highly (Fig. 3B). In agreement with these observations, incubation of enriched (Fig. 3D). However, in the presence of GST-removed the H3K4 peptides with HeLa cell lysates resulted in detection JARID1B-C, GST-LSD1 was able to bind nucleosomes. Consis- of LSD1 on H3K4me3 and of JARID1B on H3K4me2 and tent with this binding pattern, nucleosome demethylase activ- H3K4me1 peptides (Fig. 3B). Consistent with the binding ity assays indicated that, although JARID1B-C or LSD1 alone pattern of JARID1B and LSD1 on H3K4 peptides, histone did not lead to a decrease in H3K4me3 level or increases in demethylase activity assays indicated that, although LSD1 H3K4me2 and H3K4me1 levels, JARID1B-C and LSD1 in com- alone had no effect on H3K4me3 level, JARID1B alone was bination resulted in a marked and JARID1B-C dose-dependent associated with a decrease in H3K4me3 level and increases in decrease in H3K4me2 (Fig. 3E).

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Figure 3. Functional coordination between JARID1B and LSD1 in H3K4 demethylation. A, peptide binding assays were done by incubating GST-fused JARID1B-N, JARID1B-C, or LSD1 with various H3 peptides. The bound materials were analyzed by Western blotting with antibodies against GST. B, peptide binding assays were done. H3K4 peptides were incubated with GST-fused JARID1B-C, LSD1, or whole proteins from HeLa cells. C, histone demethylase activity assays. Calf thymus bulk histones were incubated with GST-fused JARID1B-N, JARID1B-C, LSD1, or LSD1 plus JARID1B. The reaction mixtures were analyzed by Western blotting using antibodies against the indicated histone marks. D, nucleosome binding assays were done. Mononucleosomes were puriﬁed from HeLa cells and incubated with GST-fused WDR5, JARID1B-N, JARID1B-C, LSD1, or LSD1 plus GST-removed JARID1B- C. The bound materials were analyzed by Western blotting with antibodies against H3, H3K4me3, or H3ac. E, nucleosome demethylase activity assay. Mononucleosomes were incubated with GST-fused JARID1B-C, LSD1, or JARID1B-C plus LSD1. The reaction mixtures were analyzed by Western blotting using antibodies against the indicated histone marks.

Identification of the transcriptional targets for JARID1B The genes that are regulated by these promoters were then To further investigate the functional association between classified into cellular signaling pathways with a P value cutoff JARID1B and the LSD1/NuRD complex and to explore the of 10 3. These analyses identified several cellular signaling biological significance of this association, we analyzed the pathways, including the mitogen-activated protein kinase genome-wide transcriptional targets of the JARID1B/LSD1/ pathway, focal adhesion pathway, and cell-cycle pathway, as NuRD complex using the Chromatin ImmunoPrecipitation– well as the chemokine pathway, which are all critically involved DNA Selection and Ligation (ChIP–DSL) approach. To this end, in cell proliferation, survival, and migration (Supplementary ChIP experiments were first conducted in MCF-7 cells with Fig. S1A). The ChIP–DSL results were verified by quantitative JARID1B antibodies. After ChIP, JARID1B-associated DNAs real-time reverse transcription PCR (RT-PCR) analysis of the were amplified, labeled, and hybridized to AVIVA Hu20K mRNA expression of selected genes representing each of the arrays. Relative confidence prediction scores were generated pathways that are targeted by the JARID1B/LSD1/NuRD com- by quantile normalization across each probe, followed by an plex in MCF-7 cells with JARID1B knockdown (Supplementary analysis using a 2-state Hidden Markov model (25). The data Fig. S1B). from JARID1B antibodies were then analyzed with the data As stated above, chemokines have been implicated in cell from antibodies against LSD1 that we had obtained previously proliferation, survival, and migration programs. Notably, che- (15) for overlapping promoters, and these promoters were mokine CCL14 was identified as being targeted by the JAR- considered to be the targets of the JARID1B/LSD1/NuRD ID1B/LSD1/NuRD complex. Measurement of the mRNA complex. These experiments identified a total of 238 different expression of CCL14 by quantitative real-time RT-PCR in promoters targeted by the JARID1B/LSD1/NuRD complex. MCF-7 cells with JARID1B overexpression or JARID1B knock- Detailed results of the ChIP–DSL experiments are deposited down indicated that JARID1B overexpression was associated in GEO Datasets (accession ID: GSE14260 for LSD1 and with decreased CCL14 expression, and JARID1B knockdown GSE25214 for JARID1B) and are summarized in Supplementary was associated with increased CCL14 expression (Fig. 4A). File S2. Consistent with these results, the level of CCL14 protein in

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Figure 4. CCL14 is the transcriptional target of the JARID1B/LSD1/NuRD complex. A, MCF-7 cells were transfected with JARID1B or JARID1B siRNA. Total RNAs were prepared and analyzed for CCL14 mRNA expression by quantitative real-time RT-PCR. B, MCF-7 cells were transfected with JARID1B or JARID1B siRNA, and the proteins in the MCF-7 medium were collected and analyzed for CCL14 protein expression by Western blotting. C, the recruitment of the JARID1B/LSD1/NuRD complex on the CCL14 promoter. ChIP assays were done in MCF-7 cells using control IgG, JARID1B, Mi-2, LSD1, MTA3, HDAC1, or MBD3 antibodies with CCL14 primer a or CCL14 primer b. The acetylation and methylation degrees on the promoter of CCL14 were detected when MCF-7 cells were transfected with control siRNA, JARID1B siRNA, or LSD1 siRNA using CCL14 primer a or CCL14 primer b. D, JARID1B and the NuRD complex exist in the same protein complex on the CCL14 promoter. Sequential ChIP experiments were done with JARID1B or Mi-2 antibodies with CCL14 primer a or CCL14 primer b.

conditioned medium from of MCF-7 cells was decreased with primer a) but not at approximately 2 kb upstream from the JARID1B overexpression and increased with JARID1B knock- CCL14 promoter (CCL14 primer b; Fig. 4C). To further support down (Fig. 4B), as measured by Western blotting with anti- the idea that JARID1B and the NuRD complex interact and bodies against CCL14. exist in the same protein complex on the CCL14 promoter, To examine whether JARID1B represses the transcription of sequential ChIP experiments (26) were done. In these studies, CCL14 in the capacity of the JARID1B/LSD1/NuRD complex, soluble chromatins were ﬁrst immunoprecipitated with anti- we carried out ChIP assays in MCF-7 cells with control IgG or bodies against JARID1B, and the supernatants and immuno- with antibodies against JARID1B, Mi-2, LSD1, MTA3, HDAC1, precipitates were subsequently reimmunoprecipitated with and MBD3 to detect the occupancy of these proteins on the antibodies against Mi-2 and vice versa. Results indicate that, promoter of the CCL14 gene. These experiments revealed that in precipitates, the CCL14 promoter that was immunopreci- JARID1B, as well as the other components of the NuRD pitated with antibodies against JARID1B could be reimmuno- complex, occupied the promoter of the CCL14 gene (CCL14 precipitated with antibodies against Mi-2, whereas in the

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supernatants, no detectable CCL14 promoter was reimmuno- JARID1B siRNA or cultured with different conditioned media precipitated with antibodies against Mi-2 (Fig. 4D). The same (CM) from MCF-7 that were infected with lentiviruses results held when the initial ChIP was done with antibodies carrying JARID1B or JARID1B siRNA were added onto solid- against Mi-2: the CCL14 promoter could only be detected in ified extracellular matrix gels. After incubation, endothelial precipitates and not in supernatants after Re-ChIP with anti- cell tube formation was examined under light microscopy bodies against JARID1B. and the number of tubes was counted. As shown in Fig. 5B, To determine the functional significance of JARID1B/LSD1/ HUVECs that were overexpressing JARID1B or cultured in NuRD complex binding on the CCL14 promoter, H3K4 meth- theCM,whichwereoverexpressingJARID1B,formedsig- ylation and H3 acetylation levels at the CCL14 promoter were nificantly fewer tubes than those in the control or vector/ analyzed in MCF-7 cells transfected with control siRNA (27), control siRNA (mock) group, whereas HUVECs with JAR- JARID1B siRNA, or LSD1 siRNA (Fig. 4C). These experiments ID1B knockdown or that which were cultured in the CM with showed that, whereas loss-of-function of JARID1B was asso- JARID1B knockdown generated more tubes than the control ciated with increased levels of H3K4me3, H3K4me2, and H3ac, siRNA or mock group. In addition, the effect of JARID1B on knockdown of LSD1 was accompanied by increased levels of HUVECs was likely through the association of JARID1B with H3K4me2, H3K4me1, and H3ac (Fig. 4C). No changes in the NuRD complex, as overexpression of JARID1B and methylation or acetylation were observed using CCL14 primer knockdown of the expression of Mi-2 resulted in a dimin- b (Fig. 4C). These results are consistent with the notion that ished JARID1B effect (Fig. 5B). Moreover, the inhibitory both JARID1B and LSD1 are required to modify H3K4 from a effect of JARID1B overexpression on the angiogenesis poten- hypermethylation state to a hypomethylation state on the tial of HUVECs could be almost totally rescued by addition promoter of target genes. These observations are also in of exogenous CCL14 protein (Fig. 5B). agreement with reports by us and others that HDAC and HDM Next, we carried out in vivo chicken yolk sac membrane are interdependent in transcription regulation (14, 15, 19, 28), (YSM) assays in which gelatin sponges that had adsorbed and they further support the in vivo existence of the JARID1B/ suspensions of MCF-7 cells transfected with JARID1B or JAR- LSD1/NuRD complex. ID1B siRNA were placed on top of the YSM. Microscopic counting of the numbers of blood vessels entering the sponge JARID1B inhibits the metastatic and angiogenic showed that eggs treated with JARID1B-depleted MCF-7 cells potential of breast cancer cells developed enriched blood vessels toward the implants in a The identification of CCL14 as a transcriptional target for the "spoked-wheel" pattern, whereas eggs treated with JARID1B JARID1B/LSD1/NuRD complex could mean that JARID1B reg- overexpressed MCF-7 cells formed significantly fewer numbers ulates the angiogenesis and metastasis of breast cancer of blood vessels compared with the control (Fig. 5C). However, through transcriptional repression of CCL14. To investigate the inhibitory effect of JARID1B overexpression on blood vessel this hypothesis, we first examined whether JARID1B could formation could be alleviated by addition of exogenous CCL14 affect the metastatic potential of breast cancer cells. For this (Fig. 5C). purpose, MDA-MB-231 cells were infected with lentiviruses We then employed a well-established in vivo angiogenesis carrying JARID1B or JARID1B siRNA, and the impact of the model (30), the mouse Matrigel plug assays, to further gain-of-function and loss-of-function of JARID1B on the inva- investigate the angiogenesis-inhibitory effect of JARID1B sive potential of these cells was investigated using transwell in vivo. In this assay, BALB/c female mice were randomly invasion assays. These experiments showed that overexpres- divided into 5 groups, and the animals in each group (n ¼ 4) sion of JARID1B resulted in a more than 5-fold decrease in cell were injected subcutaneously with either Matrigels only, or invasion, and JARID1B knockdown led to a 2-fold increase in with Matrigels that were mixed with MCF-7 cells infected cell invasion (Fig. 5A). In addition, the effect of JARID1B on the with lentiviruses carrying vector/control siRNA (mock), invasive potential of MDA-MB-231 cells was probably through JARID1B, JARID1B siRNA, or JARID1B plus supplementation the association of JARID1B with the NuRD complex, as over- with CCL14, respectively. Seven days after injection, the mice expression of JARID1B and knockdown of the expression of Mi- were sacrificed and the Matrigel plugs were processed and 2 resulted in a diminished JARID1B effect (Fig. 5A). Moreover, stained with HE and Masson trichrome. Microscopic exam- the effect of JARID1B on the invasive potential of MDA-MB-231 ination of Matrigel plugs revealed that overexpression of cells was suppressed by overexpression of CCL14 and was JARID1B was associated with a decreased number of endo- enhanced by CCL14 knockdown (Fig. 5A). thelial cells in the plugs, compared with that in the mock To investigate whether JARID1B could affect the angio- group (Fig. 5D). However, plugs in overexpression of JAR- genic potential of breast cancer cells through the regulation ID1B, but supplemented with CCL14, exhibited a compara- of CCL14,wefirst carried out endothelial tube formation ble or even higher endothelial cell number than that of the assays to investigate whether JARID1B repressed angiogen- mock group. On the other hand, abundant endothelial cells, esis of endothelial cells in vitro. This assay is based on the often organized to form blood vessels containing red blood ability of endothelial cells to form 3-dimensional capillary- cells, were present in JARID1B siRNA plugs, although only a like tubular structures when cultured on an extracellular few endothelial cells had invaded the plugs of mock and matrix gel prepared from Engelbreth–Holm–Swarm tumor Matrigel only group (Fig. 5D). These observations strongly cells (29). Human umbilical vein endothelial cells (HUVECs) support the idea that JARID1B suppresses angiogenesis that were infected with lentiviruses carrying JARID1B or in vivo, possibly through the repression of CCL14.

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Figure 5. JARID1B inhibits breast cancer cell angiogenesis. A, MDA-MB-231 cells were infected with lentiviruses carrying JARID1B, JARID1B siRNA, JARID1B plus Mi-2 siRNA, JARID1B plus CCL14 siRNA, or JARID1B plus CCL14. The cells were starved for 18 hours before cell invasion assays were done using Matrigel transwell filters. The invaded cells were stained and counted. Representative images from each group are shown. Each bar represents the mean SD for triplicate measurements. Protein expression in these experiments was examined by Western blotting using antibodies against the indicated proteins. B, HUVECs were infected with lentiviruses carrying JARID1B siRNA, JARID1B, JARID1B plus Mi-2 siRNA, or JARID1B plus CCL14 (top), or HUVECs cultured with different CM from MCF-7 that were infected with lentiviruses carrying vector/control siRNA (mock), JARID1B siRNA, JARID1B, JARID1B plus Mi-2 siRNA, or JARID1B plus CCL14 (bottom) were added onto solidified extracellular matrix. After incubation, endothelial cell tube formation was assessed under light microscopy and the tubes were counted. Each bar represents the mean SD for triplicate measurements. Representative images from each group are shown. C, in vivo YSM assays were done. MCF-7 cells were transfected with JARID1B, JARID1B siRNA, or JARID1B plus CCL14. Then sponges which were absorbed with MCF-7 cells were placed on the YSM on day 8 and observed on day 12. The numbers of blood vessels entering the sponge in each group were counted; representative images from each group are shown. Each bar represents the mean SD for triplicate measurements. D, in vivo mouse Matrigel plug assays. Matrigels only or Matrigels mixed with MCF-7 cells infected with lentiviruses carrying empty vector/control siRNA (mock), JARID1B, JARID1B siRNA, or JARID1B plus supplementation with CCL14 were injected subcutaneously into 6-week-old BALB/c female mice (n ¼ 4). Seven days after injection, the mice were sacrificed and the Matrigel plugs were removed for HE and Masson trichrome staining and examined by microscopy.

OF8 Cancer Res; 71(21) November 1, 2011 Cancer Research

JARID1B in Breast Cancer Cells

Discussion genes, including BRCA1 (1), and this functionality seems to be consistent with its overexpression in breast cancer. However, it Although LSD1 was the first histone demethylase identified has also been suggested that JARID1B itself has a tumor (31, 32), the enzymatic mechanism by which this protein suppressive activity, partly, due to its binding and ability to initiates demethylation reactions precludes its catalysis of stabilize hypophosphorylated pRb, leading to maintenance of H3K4me3 substrate. Given the prevalence of the trimethyl pRb-mediated cell-cycle control (5). In agreement with this, it modification state of histones (33, 34), it is apparent that LSD1 has been observed that the expression of JARID1B is lost in the must collaborate with additional enzymes to accomplish H3K4 majority of advanced and metastatic melanomas and in many demethylation. Surprisingly, there have not yet been any melanoma cell lines (4, 7). In this article, we showed that reports on a functional connection between LSD1, an JARID1B is able to suppress angiogenesis and metastasis, H3K4me1- and H3K4me2-specific demethylase, and the supporting the notion that JARID1B itself is a potential tumor JARID1 family of proteins, the H3K4me3-specific demethy- suppressor. We showed that the expression of CCL14 is neg- lases. In this article, by using affinity purification and mass atively regulated by JARID1B. We also showed that inhibition of spectrometric analysis, we found that JARID1B is physically CCL14 expression is associated with a suppression of the associated with LSD1 and with components of the NuRD angiogenesis and metastasis of breast cancer. As chemokine complex. In light of our previous finding that LSD1 is an targeting is showing promise in animal models of inflamma- integral component of the NuRD complex (15), we propose tion and autoimmune disease (35), it is logical to believe that that JARID1B is also a subunit in the NuRD complex. The manipulating the local chemokine network may offer thera- likelihood of a physical association of JARID1B with the LSD1/ peutic benefits in cancers. In this sense, our data suggesting NuRD complex is supported by the results from several CCL14 to be a potent promoter of breast cancer angiogenesis other molecular experiments, including co-IP, FPLC, and GST and metastasis may provide a potential new target for breast pull-down. Significantly, the physical association between cancer intervention. JARID1B and LSD1 provides a plausible molecular mechanism for sequential and coordinated demethylation regulation of Disclosure of Potential Conflicts of Interest H3K4. Indeed, our data show that JARID1B and LSD1 are functionally connected in H3K4 demethylation. Our experi- No potential conflicts of interest were disclosed. ments support a model for the action of the JARID1B/LSD1/ NuRD complex on target gene promoters in which H3K4me3 is Grant Support recognized by JARID1B first, and the methyl groups are then sequentially removed by the joint enzymatic activities of This work was supported by grants (30830032 and 30921062 to Y. Shang) from JARID1B and LSD1 to bring these genes to a silenced state. the National Natural Science Foundation of China and grants (973 Program: 2007CB914503 and 2011CB504204 to Y. Shang, and 2009CB918903 to Z. Yao) from In addition to its overexpression in breast cancers, JARID1B the Ministry of Science and Technology of China. dysregulation has been reported in several types of solid The costs of publication of this article were defrayed in part by the payment of – page charges. This article must therefore be hereby marked advertisement in tumors including prostate cancer (3), melanoma (4 7), and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bladder cancer (8). It has also been reported that, functionally, JARID1B plays an important role in the proliferative capacity of Received May 4, 2011; revised August 19, 2011; accepted August 31, 2011; breast cancer cells through repression of tumor suppressor published OnlineFirst September 21, 2011.

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OF10 Cancer Res; 71(21) November 1, 2011 Cancer Research

Binding of the JmjC Demethylase JARID1B to LSD1/NuRD Suppresses Angiogenesis and Metastasis in Breast Cancer Cells by Repressing Chemokine CCL14

Qian Li, Lei Shi, Bin Gui, et al.

Cancer Res Published OnlineFirst September 21, 2011.

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