hbxip and lsd1 scaffolded by lncrna hotair mediate ...molecular and cellular pathobiology hbxip and...

Molecular and Cellular Pathobiology

HBXIP and LSD1 Scaffolded by lncRNA HotairMediate Transcriptional Activation by c-MycYinghui Li1, Zhen Wang1, Hui Shi1, Hang Li1, Leilei Li1, Runping Fang1, Xiaoli Cai1,Bowen Liu1, Xiaodong Zhang2, and Lihong Ye1

Abstract

c-Myc is regarded as a transcription factor, but the basis for itsfunction remains unclear. Here, we define a long noncodingRNA (lncRNA)/protein complex that mediates the transcrip-tional activation by c-Myc in breast cancer cells. Among 388c-Myc target genes in human MCF-7 breast cancer cells, wefound that their promoters could be occupied by the oncopro-tein HBXIP. We confirmed that the HBXIP expression correlatedwith expression of the c-Myc target genes cyclin A, eIF4E, andLDHA. RNAi-mediated silencing of HBXIP abolished c-Myc–mediated upregulation of these target genes. Mechanistically,

HBXIP interacted directly with c-Myc through the leucinezippers and recruited the lncRNA Hotair along with the histonedemethylase LSD1, for which Hotair serves as a scaffold.Silencing of HBXIP, Hotair, or LSD1 was sufficient to blockc-Myc–enhanced cancer cell growth in vitro and in vivo. Takentogether, our results support a model in which the HBXIP/Hotair/LSD1 complex serves as a critical effector of c-Myc inactivating transcription of its target genes, illuminating long-standing questions on how c-Myc drives carcinogenesis. CancerRes; 76(2); 293–304. �2015 AACR.

IntroductionCancer cells are characterized by acquisition of several char-

acteristics that enable them to become tumorigenic, in whichthe uncontrolled proliferation is one of the most fundamentalcharacteristics of cancer cells (1, 2). The activation of oncogenesand their cooperative effects play crucial roles in the process oftumorigenesis and cell proliferation. The oncogene-mediatedreprogramming of gene expression, which enables the cell toacquire disorder of metabolism, cell cycle, and other aspects, isa hallmark of cancer cells (1, 3, 4). Importantly, the c-Mycproto-oncogene, which has been implicated in the pathogen-esis of most types of human tumors (1, 5), affects multiple cellbiologic processes, including cell cycle, protein synthesis, celladhesion, cytoskeleton, metabolism, and miRNA regulation(6–8). c-Myc protein binds to a DNA sequence called E-box(CACGTG) and dimerizes with Max to mediate approaching atotal of 3,000 to 4,000 human genes (9, 10). c-Myc proteincontains a basic DNA-binding domain and a helix-loop-helix-leucine zipper (HLH-Zip) dimerization motif, by which theother transcription factors and coactivators can affect theexpression of c-Myc target genes (11, 12). However, the under-

lying mechanism of the transcriptional regulation of c-Myctarget genes remains an unsolved mystery.

Mammalian hepatitis B X-interacting protein (HBXIP), alsoknown as LAMTOR5 (13), is an 18-kDa protein that contains aleucine zipper at its C-terminal, and its sequence is well conservedamong mammalian species (14). We have reported that expres-sion of HBXIP is obviously enriched in breast cancer tissues.HBXIP acts as anoncogenic transcriptional coactivator ofmultipletranscription factors, such as TF-IID, STAT3, SP1, CREB, and E2F1,in the promotion of breast cancer growth andmetastasis (15–20).However, whether HBXIP serves as a coactivator of oncogenictranscriptional factor c-Myc in breast cancer remains poorlyunderstood.

Given that transcriptional activation is linked to the chromatinenvironment, histone H3 modification is the major patternof chromatin remodeling (21). Lysine-specific demethylase 1(LSD1)–mediated demethylation of H3K4 leads to local DNAoxidation as a driving force in the assembly of the c-Myc–inducedtranscription initiation complex (22, 23). The epigenetics isinvolved in the c-Myc–mediated transcription (24, 25). Longnoncoding RNAs (lncRNA) are emerging as new players in thecancer paradigmdemonstrating potential roles in both oncogenicand tumor-suppressive pathways. These novel genes accomplishremarkable biologic functions in a variety of human cancers (26).LncRNA HOX transcript antisense intergenic RNA (Hotair), iden-tified as a 2.2 kilobase ncRNA residing in the HOXC locus, canbind to LSD1. Hotair serves as a scaffold of histone modificationcomplexes comprising LSD1 and polycomb-repressive complex 2(PRC2), resulting in the malignance of multiple tumors (27, 28).It has been reported that lncRNA PRNCR1 and PCGEM1 areinvolved in the androgen–receptor transcriptional complex(29). However, whether Hotair is involved in the c-Myc–inducedtranscription activation is not well documented.

In this study, we try to gain insights into the mechanismof c-Myc–mediated transcription activation. Interestingly, our

1State Key Laboratory of Medicinal Chemical Biology, Department ofBiochemistry, College of Life Sciences, Nankai University, Tianjin, P.R.China. 2State Key Laboratory of Medicinal Chemical Biology, Depart-ment of Cancer Research, Institute for Molecular Biology, College ofLife Sciences, Nankai University, Tianjin, P.R. China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Lihong Ye, Nankai University, 94 Weijin Road, Tianjin300071, China. Phone: 86-22-23501385; Fax: 86-22-23501385; E-mail:[email protected]; and Xiaodong Zhang, [email protected]

doi: 10.1158/0008-5472.CAN-14-3607

�2015 American Association for Cancer Research.

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Published OnlineFirst December 30, 2015; DOI: 10.1158/0008-5472.CAN-14-3607

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data show that HBXIP acting as a coactivator directly interactswith transcription factor c-Myc. HBXIP and LSD1 form acomplex that is scaffolded by Hotair to activate c-Myc in thepromoter of c-Myc target genes, leading to transcription acti-vation of c-Myc target genes in breast cancer cells. Thus, ourfinding provides new insights into the mechanism by which c-Myc functions in carcinogenesis.

Materials and MethodsCell lines, transfection, and siRNA

Immortalized breast cell line HBL-100 and breast cancer celllines MCF-7, T47D, LM-MCF-7, and SK-BR3 were cultured inRPMI Medium 1640 (Invitrogen) with 10% FCS. MDA-MB-463,MDA-MB-231, and HEK293T cells were cultured in DMEM (Invi-trogen) supplemented with 10% FCS. The siRNAs targetingHBXIP mRNA were reported previously (16, 19). The siRNAstargeting c-Myc mRNA, Hotair, and LSD1 mRNA were listed inSupplementary Table S1 (28). Cells were transfected with corre-sponding plasmids or siRNAs using Lipofectamine 2000 (Invi-trogen) according to the manufacturer's instructions.

Chromatin immunoprecipitation assays and the overlap ofHBXIP and c-Myc binding

Chromatin immunoprecipitation (ChIP) assays were per-formed using the EpiQuik Chromatin Immunoprecipitation Kitfrom Epigentek Group Inc. MCF-7 cells were lysed 24 hours aftertransfection. Protein/DNA complexes were immunoprecipitatedby HBXIP or LSD1 antibodies, using normal rabbit IgG as anegative control. The primers used for PCR amplification wereflanking the E-box in the promoters of cyclin A, eIF4E, and LDHA(30–32). The ChIP-DNA Selection and Ligation (ChIP-DSL) anddata analysis of HBXIP-bound genes were performed by Capi-talBio Corporation according to a protocol from Aviva SystemsBiology. Experiments were repeated three times, and the resultswere analyzed using MAS (http://bioinfo.capitalbio.com/mas/login.do) with a P value cutoff of 1.0 � 106 for promoter iden-tification. According to previous report (33), the datasets of ChIP-seq for c-Myc were used. According to the accession number andthe genename, 1,348 topboundgenes in c-Mycdatasetswereusedto merge with the HBXIP-bound genes for overlapping. The genepathways were analyzed by using the KEGG database of DAVIDBioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/).

Immunohistochemistry stainingBreast cancer tissue microarrays were purchased from Xi'an

Aomei Biotechnology. IHC staining was performed as describedpreviously (16). The negative control was performed as aboveprotocol without using primary antibody. The staining level ofHBXIP, cyclin A, eIF4E, and LDHAwas classified into three groupsusing a modified scoring method based on the intensity ofstaining (0, negative; 1, low; 2, high) and the percentage of stainedcells (0, 0% stained; 1, 1%–49% stained; 2, 50%–100% stained).Amultiplied score (intensity score� percentage score) lower than1 was considered to be negative staining (�), 1 and 2 wereconsidered to be moderate staining (þ), and 4 was consideredto be intense staining (þþ).

Real-time PCR and Western blot analysisTotal 40 cases of breast cancer tissues were collected from

patients undergoing resection of breast cancer in Tianjin First

Center Hospital (Tianjin, China). Total RNAs from breast cancertissues or cells were extracted using TRIzol reagent (Invitrogen)according to the instructions (16). First-strand cDNA was synthe-sized by PrimeScript reverse transcriptase (TaKaRa Bio) accordingto the manufacturer's instructions. Primers used to test HBXIPexpression were obtained from reports (16). Real-time PCR wasperformed as described previously (16). Primers used in the studywere listed in Supplementary Table S2. For Western blot analysis(16), total protein lysate was extracted from tissues or cells withRIPA buffer (Solarbio). The protein samples were subjected toSDS-PAGE and then transferred to a nitrocellulose membrane,blocked with 5% non-fat milk, and incubated with primaryantibodies for 2 hours at room temperature. After incubationwith secondary antibody for 1hour, themembranewas visualizedby ECL (Millipore). The antibodies used in this study werelisted in Supplementary Table S3. All experiments were repeated3 times.

ConstructsThe plasmids, such as pCMV-Tag2B, pCMV-HBXIP, pcDNA3.1,

pcDNA-HBXIP, pGEX-4T1, pGEX-HBXIP, and pCMV-HBXIP-LZM, were used in our previous studies (16, 34, 35). The E-boxreporter was constructed by inserting 6� E-box into the pGL3-Basic vector (36). pcDNA-Hotair vector with full-length Hotairwas constructed by Genomics. Human HBXIP, c-Myc, and thefragments of HBXIP were PCR-amplified from cDNA of totalRNAs in MCF-7 cells. The resulting products were cloned intothe multiple cloning sites of the vectors, such as pEGFP-C2,pCMV-Tag2B, pET28, pcDNA3.1, pcDNA-HA, and pGEX-4T1,respectively.

Luciferase reporter gene assaysThe plasmids of Renilla luciferase reporter vector pRL-TK were

used as described previously (16). Luciferase activities were nor-malized toRenilla luciferase activity. All the assayswere performedin triplicate. Cells were seeded into 24-well plates and culturedfor 24 hours before transfection. The luciferase activities of E-boxreporter and Gal4-c-Myc reporter were determined 36 hoursafter transfection using a dual-luciferase reporter gene assay kit(Promega).

Immunofluorescence staining and confocal microscopyImmunofluorescence staining was performed as described pre-

viously (35). After transfection, the cells were fixed with parafor-maldehyde, and permeabilized with 0.1% Triton X-100 in PBS.After blocking in PBS containing 3%BSA, the cells were incubatedwith primary antibodies at room temperature. After washing withPBS, the cells were incubated with fluorophore-conjugated sec-ondary antibody (DAKO) and DAPI. After washing with PBS,slides weremounted with glycerol and observed under a confocalmicroscopy (Leica TCS SP5).

Coimmunoprecipitation assay and GST pull-downThe cells were harvested and lysed in a lysis buffer (50 mmol/L

Tris-HCl, pH8.0, 100mmol/LNaCl, 50mmol/L sodiumfluoride,1% Nonidet P-40, 1 mmol/L dithiothreitol, 1 mmol/L Na3VO4,1 mmol/L Microcystin-LR, 1 mmol/L phenylmethylsulfonyl fluo-ride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). The lysateswere incubated with antibodies/protein G–conjugated agarosebeads (Millipore) or ANTI-FLAG M2 affinity gel beads (Sigma).The precipitates were washed six times with ice-cold lysis buffer,

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resuspended in PBS, followed by Western blot analysis. The GSTpull-downwas performed according to published protocols (16).Glutathione beads were recovered by a brief centrifugation andwashed six times with lysis buffer, followed by Western blotanalysis.

Electrophoresis mobility shift assaysThe electrophoresis mobility shift assay (EMSA) protocol was

described in detail (16, 17). Nuclear extracts were prepared withMCF-7 cells using the EpiQuikNuclear Extraction Kit (Epigentek).Probes were generated by annealing single-strand oligonucleo-tides containing the E-box (37). The primary antibody (1 mg) wasincubated with nuclear extracts on ice before probes were addedinto the binding buffer. Samples were incubated on ice and thenseparated by electrophoresis on nondenaturing polyacrylamidegel, and then the gel was dried and subjected to autoradiography.

RNA immunoprecipitation assaysRNA immunoprecipitation (RIP) assays were performed in

native conditions as described (29). Briefly, MCF-7 cell nucleiwere pelleted and lysed. The lysates were passed through a 27.5gauge needle 4 times to promote nuclear lysis. The supernatantwas incubatedwith 4mg primary antibody of rabbit anti-HBXIP ormouse anti–c-Myc (Supplementary Table S3) with 40 mL proteinG–conjugated agarose beads (Millipore) or ANTI-FLAGM2 affin-ity gel beads (Sigma). The RNA/antibody complex was washed byNT2 buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1mmol/L MgCl2, 0.05%NP-40). The RNAwas extracted by TRIzol(Invitrogen) according to the manufacturer's protocol and sub-jected to real-time PCR analysis.

MTT assaysCell growth assays were carried out using MTT (3-(4,5-

dimethylthiazol-2-yl)-2,5-diph-enyltetrazolium bromide) reagent(Sigma) as described previously (38). In brief, transfected cells weretrypsinized, counted, and plated into 96-well plates. After incubat-ing different time periods, MTT was added directly to each well,followed by incubation for 4 hours, and then the supernatant wasremoved and 100 mL of dimethyl sulfoxide was added to stop thereaction. Absorbance at 490 nm was measured using an ELISAreader system (Labsystem,MultiskanAscent). All experiments wereperformed in triplicate.

Animal transplantationAll experimental procedures involving animals were in accor-

dance with the Guide for the Care and Use of Laboratory Animals(NIHpublications nos. 80–23, revised 1996) andwere performedaccording to the institutional ethical guidelines for animal exper-iment. MCF-7 cells (1 � 107) transiently transfected with corre-sponding plasmids and siRNAswere subcutaneously injected intothe flanks of 4-week-old male BALB/c athymic nude mice, respec-tively. Tumor growth was monitored every 4 days. After 26 days,the mice were sacrificed, necropsies were performed, and tumorswere weighed. Tumor volume (V) was monitored by measuringthe length (L) and width (W) using calipers and calculatedaccording to the formula (L � W2) � 0.5 (16).

Statistical analysisEach experiment was repeated at least three times. Statistical

significance was assessed by comparingmean values (�SD) usingthe Student t test for independent groups, assumed for P < 0.01

(��). Correlation between expression levels of HBXIP and cyclin A(or eIF4E and LDHA) in tumorous tissues was explored using thePearson correlation coefficient. Statistical analysis of tissue arraywas performed with the x2 test or Kruskal–Wallis test using theSPSS software program (SPSS).

ResultsThe expression levels of HBXIP are positively associated withthose of cyclin A, eIF4E, and LDHA of c-Myc–induced genes inbreast cancer

It has been reported that c-Myc is a crucial proto-oncogenictranscription factor in cancer (1, 5). HBXIP functions as anoncogenic transcriptional co-activator in breast cancer (19, 35).Either HBXIP or c-Myc contains a leucine-zipper domain (5, 15).Accordingly, we supposed that HBXIP as a transcriptional coacti-vator might activate c-Myc–induced genes in breast cancer cells.Compared with the data of c-Myc ChIP-seq (33, 39), we foundthat 28.8% (388/1,348)/16.8% (388/2,311) for c-Myc/HBXIP-target gene promoters could be occupied by both HBXIP and c-Myc in the cells (Fig. 1A and Supplementary Table S4), suggestingthat HBXIP may be involved in the c-Myc–induced gene expres-sion. However, bioinformatics analysis showed that these com-mon target–involved pathways were different from those of thec-Myc target genes, which could not be occupied by HBXIP.Interestingly, weobserved that the commongeneswere associatedwith cell cycle, whereas the c-Myc target genes, which could not beoccupied by HBXIP, were related to ribosome biosynthesis (Sup-plementary Tables S5 and S6; Supplementary Fig. S1A and S1B).Moreover, we selected three common target genes ofHBXIP and c-Myc (cyclin A, eIF4E, and LDHA) to investigate the effect ofHBXIPon c-Myc–induced gene transcription. As expected, ChIP assaysvalidated that HBXIP could occupy the promoters of abovethree genes in MCF-7 cells (Fig. 1B). We also observed that theexpression levels of HBXIP exhibited parallel changes with thethree genes in six breast cell lines (Fig. 1C). Moreover, IHCstaining showed that the positive rate of HBXIP was positivelyassociated with that of cyclin A (or eIF4E and LDHA) in breastcancer tissues (pairing c2 analysis, Fig. 1D and SupplementaryTables S7–S9). Furthermore, real-time PCR revealed that theexpression levels of HBXIPmRNAwere positively related to thoseof cyclin A, eIF4E, and LDHA mRNAs in breast cancer tissues(P < 0.01; Pearson correlation, Fig. 1E–G). Thus, we conclude thatthe expression levels ofHBXIP are positively associatedwith thoseof cyclin A, eIF4E, and LDHA of c-Myc–induced genes in breastcancer.

HBXIP is required for the upregulation of cyclin A, eIF4E, andLDHA

Next,we are interested inwhetherHBXIP is able to upregulate c-Myc target genes in breast cancer cells. As expected, real-time PCRassays showed that the overexpression of HBXIP could upregulatethe expression of cyclin A, eIF4E, and LDHA at the levels ofmRNAin a dose-dependent manner in MCF-7, T47D, and ER-negativeMDA-MB-231 cells (Fig. 2A; Supplementary Fig. S2A and S2B).Moreover, Western blot analysis validated the data at the proteinlevels in the system (Fig. 2B; Supplementary Fig. S2C and S2D).Then, we constructed an E-box luciferase reporter to test the effectofHBXIPon c-Myc–induced transcription (36).Our data revealedthat HBXIP triggered the E-box reporter activities in MCF-7 andT47D cells (Fig. 2C and Supplementary Fig. S2E), suggesting that

HBXIP and LSD1 Scaffolded by Hotair Activate c-Myc

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HBXIP is able to activate the E-box element in the promoters of c-Myc target genes in breast cancer cells. Moreover, the silence of c-Myc by siRNAwas able to abolish theHBXIP-mediated expressionof above three c-Myc target genes at the levels of mRNA andprotein in MCF-7 cells (Fig. 2D and E). Conversely, the depletionof HBXIP by siRNA could result in the disruption of c-Myc–enhanced cyclin A, eIF4E, and LDHA at the levels of mRNA andprotein in MCF-7, T47D, and MDA-MB-231 cells (Fig. 2F and Gand Supplementary Fig. S2F–S2I), suggesting that HBXIP con-tributes to the transcriptional activationmediated by c-Myc. Thus,we conclude that HBXIP is required for the upregulation of cyclinA, eIF4E, and LDHA in breast cancer.

HBXIP directly interacts with c-Myc to coactivate c-MycGiven that either HBXIP or c-Myc contained a leucine-zipper

domain (5, 15), we tried to investigate whether HBXIP was able

to interact with c-Myc through leucine zipper. Indeed, confocalimages validated that most of HBXIP and c-Myc colocalized inthe nucleus in MCF-7 and T47D cells (Fig. 3A and Supplemen-tary Fig. S3A). Coimmunoprecipitation (Co-IP) assays validat-ed that the endogenous or exogenous c-Myc could interact withHBXIP in MCF-7 cells (Fig. 3B and Supplementary Fig. S3B). Tofurther verify whether HBXIP directly interacted with c-Myc,GST pull-down assays were performed. We found that the c-Myc protein with His-tag could be pulled down by GST-HBXIP,but not by the control GST (Fig. 3C), suggesting that HBXIPdirectly binds to c-Myc. Then, we tried to map the bindingdomain of HBXIP interacting with c-Myc. According to thesecondary structure of HBXIP (Fig. 3D; ref. 40), we constructedthree fragments of HBXIP (aa 1–55, aa 82–173, and aa 56–173)and leucine-zipper mutant of HBXIP (HBXIP LZM). Co-IPassays showed that either full-length or the fragment aa

Figure 1.The expression levels of HBXIP are positively associated with those of cyclin A, eIF4E, and LDHA of c-Myc–induced genes in breast cancer. A, Venn diagramshows the overlapping target genes of HBXIP and c-Myc determined. B, the occupancy of HBXIP or c-Myc in the promoters of cyclin A, eIF4E, and LDHA wasexamined by ChIP-qPCR assays in MCF-7 cells. C, the expression levels of HBXIP, cyclin A, eIF4E, and LDHA were examined by Western blot analysis insix breast cell lines. D, the expression levels of HBXIP, cyclin A, eIF4E, and LDHA were detected by IHC staining in 87 cases of breast cancer tissues usingtissue microarray. E–G, the correlation of mRNA levels of HBXIP with those of cyclin A (or eIF4E and LDHA) was examined by real-time PCR assays in 40 breastcarcinoma tissues (cyclin A, P < 0.01, R2 ¼ 0.7223; eIF4E, P < 0.01, R2 ¼ 0.6458; LDHA, P < 0.01, R2 ¼ 0.6777; Pearson correlation). All experiments wererepeated at least three times. Statistically significant differences are indicated. ��, P < 0.01, Student t test.

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Figure 2.HBXIP is required for the upregulation of cyclin A, eIF4E, and LDHA. A, the mRNA levels of HBXIP, cyclin A, eIF4E, and LDHA were tested by real-time PCRin MCF-7 cells. B, the protein levels of above genes were tested by Western blot analysis in MCF-7 cells. C, the relative luciferase activities of E-box reporter weretested by dual-luciferase system in MCF-7 cells. D, the mRNA levels of HBXIP, c-Myc, cyclin A, eIF4E, and LDHA were tested by real-time PCR in MCF-7 cells.E, the expression levels of above genes were tested by Western blot analysis in MCF-7 cells. F, the mRNA levels of c-Myc, HBXIP, cyclin A, eIF4E, andLDHA were tested by real-time PCR in MCF-7 cells. G, the expression levels of above genes were tested by Western blot analysis in MCF-7 cells. All experimentswere repeated at least three times. Statistically significant differences are indicated. �� , P < 0.01, Student t test.






56–173 of HBXIP containing leucine zipper could interact withthe HA-tagged c-Myc in HEK293T cells (Fig. 3E), but othersfailed to work in the cells. GST pull-down assays furthervalidated the same result (Fig. 3F), suggesting that the leucinezipper is required for HBXIP binding to c-Myc. Furthermore,EMSA revealed that a supershift band was observed when theHBXIP antibody was added (Fig. 3G, lane 7), suggesting thatHBXIP is able to interact with the E-box element in the pro-moters of c-Myc target genes. Then, we constructed a plasmidencoding the c-Myc protein fused to the Gal4 DNA-bindingdomain to test the interaction of HBXIP with c-Myc. Function-ally, HBXIP significantly increased the luciferase activities ofGal4-c-Myc in a dose-dependent manner in HEK293T cells (Fig.3H), suggesting that HBXIP may serve as a coactivator of c-Myc.Thus, we conclude that HBXIP directly interacts with c-Myc tocoactivate c-Myc in breast cancer cells.

HBXIP serves as a linker of c-Myc and LSD1 in the complexof c-Myc/HBXIP/LSD1

Usually, transcription factors function in a complex mannerin the activation of target genes. It has been reported thatLSD1-mediated demethylation of H3K4 acts as a driving forcein the assembly of the c-Myc–induced transcription initiationcomplex (22, 23); however, the mechanism remains unclear.Real-time PCR assays showed that the mRNA levels of LSD1 inthe breast cancer cell lines were higher than those in theimmortalized breast cell HBL-100 (Supplementary Fig. S4A).Then, we evaluated the relationships between LSD1 and HBXIP(or c-Myc) in breast cancer. Our data showed that the expres-sion levels of LSD1 mRNA were positively associated withthose of HBXIP and c-Myc mRNAs in breast cancer tissues(P < 0.01, Pearson correlation, Supplementary Fig. S4B andS4C). Therefore, we supposed that c-Myc/HBXIP/LSD1 might

Figure 3.HBXIP directly interacts with c-Myc to coactivate c-Myc. A, the localization of GFP-HBXIP and c-Myc was observed in the MCF-7 cells by confocal microscopy.B, the interaction of endogenous HBXIP with c-Myc was detected by co-IP assays in MCF-7 cells. C, the direct interaction of recombinant GST-HBXIP withHis-c-MycwasdetectedbyGSTpull-downassaysandWestern blot analysis. D, amodel shows the fragmentsofHBXIP composedof amino acids 1–55, 56–173, 83–173,and themutant of leucine zipper. E, the interaction of fragments of HBXIP (or themutant of leucine zipper)with c-Mycwas detected by co-IP assays in HEK293T cells.F, the interaction of fragments of HBXIP (or the mutant of leucine zipper) with c-Myc was detected by GST pull-down assays and Western blot analysis.G, the interaction of nuclear proteins with probed E-box was assessed by EMSA. H, luciferase activities of Gal4-c-Myc were measured by luciferase reporter geneassays in HEK293T cells. The experiment was repeated at least three times. Statistically significant differences are indicated. �� , P < 0.01, Student t test.

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form a complex. Confocal images showed that HBXIP, c-Myc,and LSD1 could colocalize in the nucleus of the MCF-7 cells(Fig. 4A). It has been reported that LSD1 is an RNA-interactingprotein (27). Interestingly, we found that both HBXIP and c-Myc could be immunoprecipitated by LSD1 antibody whenRNase inhibitor was added into the lysis buffer (Fig. 4B). But, itdid not work when RNase A was added in the system (Fig. 4C).In addition, LSD1 could also be immunoprecipitated by eitherHBXIP or c-Myc antibody when the lysis buffer was treatedwith RNase inhibitor (Fig. 4D), but it failed to work whenRNase A was added (Fig. 4E), suggesting that LSD1 indirectlyinteracts with c-Myc/HBXIP, in which RNA is required for the

complex. Moreover, the depletion of HBXIP by siRNA coulddisrupt the interaction of c-Myc with LSD1 in MCF-7 cellswhen the RNAs were protected by RNase inhibitors (Fig. 4F),suggesting that HBXIP links c-Myc and LSD1 in the complex ofc-Myc/HBXIP/LSD1. ChIP assays further showed that thesilence of HBXIP by siRNA resulted in the decrease of LSD1occupation in the promoters of above three c-Myc target genesin MCF-7 cells (Fig. 4G), suggesting that HBXIP recruits LSD1to the promoters of c-Myc target genes. Thus, we concludethat HBXIP serves as a linker in the complex of c-Myc/HBXIP/LSD1 in breast cancer cells, in which RNA is required for thecomplex.

Figure 4.HBXIP serves as a linker of c-Myc and LSD1 in thecomplex of c-Myc/HBXIP/LSD1. A, the localization ofGFP-HBXIP, c-Myc, and LSD1 was observed in MCF-7cells by confocalmicroscopy. B andC, the interactionof HBXIP (or c-Myc) with LSD1 was tested by co-IPassays inMCF-7 cells treatedwithRNase inhibitors orRNase A. D and E, the interaction of LSD1 with HBXIP(or c-Myc) was tested by co-IP assays in MCF-7 cellstreated with RNase inhibitors or RNase A. F, theinteraction of LSD1 with c-Myc was measured byco-IP assays in MCF-7 cells treated with both RNaseinhibitors and siHBXIP. G, the occupancy of LSD1 inthe promoters of cyclin A, eIF4E, and LDHA wasexamined by ChIP-qPCR in the MCF-7 cells.Statistically significant differences are indicated.�� , P < 0.01, Student t test.






lncRNAHotair scaffolds HBXIP and LSD1 to form a complex ofc-Myc/HBXIP/Hotair/LSD1 in activation of c-Myc–inducedtranscription

Given that RNAs might be involved in the c-Myc/HBXIP/LSD1complex as above and lncRNA Hotair interacted with LSD1 incancer cells (27), we supposed that Hotair might be involved inthe c-Myc/HBXIP/LSD1 complex. Then, we provided evidencethat the levels of Hotair in the breast cancer cell lines were higherthan those in the immortalized breast HBL-100 cell (Supplemen-tary Fig. S5A), and the levels of Hotair were positively associatedwith those of HBXIP (or c-Myc) in breast cancer tissues (P < 0.01,Pearson correlation, Supplementary Fig. S5B and S5C) by usingreal-time PCR assays. Strikingly, co-IP assays showed that thedepletion of Hotair by siRNA could block the interaction of LSD1withHBXIP/c-Myc inMCF-7 cells (Fig. 5A), suggesting thatHotairis required for the interaction of HBXIP/c-Myc with LSD1. Next,we concerned whether LSD1-linked Hotair could interact withHBXIP/c-Myc. RIP assays showed that Hotair could be immuno-precipitatedbyHBXIPor c-Myc inMCF-7 andT47Dcells (Fig. 5B).Moreover, silence of HBXIP by siRNA could disrupt the interac-tion of Hotair with c-Myc inMCF-7 cells, whereas silence of c-Mycby siRNA failed to influence the interaction of Hotair with HBXIPin the cells (Fig. 5C), suggesting that Hotair scaffolds HBXIP, butnot c-Myc, and LSD1 in the complex of c-Myc/HBXIP/Hotair/LSD1. Furthermore, depletion of Hotair by siRNA could block theoccupation of LSD1 in the promoters of c-Myc target genes (cyclinA, eIF4E, and LDHA) in MCF-7 cells (Fig. 5D and SupplementaryFig. S5D), suggesting thatHotair recruits LSD1 to the promoters ofc-Myc target genes. In addition, depletion of HBXIP by siRNAcould disrupt the occupation of LSD1 in the promoters of abovegenes when both c-Myc and Hotair were overexpressed in MCF-7cells (Fig. 5D and Supplementary Fig. S5D), suggesting that thecomplex of c-Myc/HBXIP/Hotair/ LSD1might be required for theactivation of c-Myc target genes. Functionally, we validated thatthe overexpression of c-Myc failed to upregulate above c-Myctarget genes at the levels of mRNA or protein when Hotair wassilenced by siRNA in MCF-7, T47D, and MDA-MB-231 cells (Fig.5E and F; and Supplementary Fig. S5E–S5H). The overexpressionof both c-Myc and Hotair failed to upregulate above genes at thelevels of mRNA or protein when HBXIP was silenced by siRNA inMCF-7 andMDA-MB-231 cells (Fig. 5E and F; Supplementary Fig.S5F–S5H), supporting that both HBXIP and Hotair are requiredfor the activation of c-Myc target genes. Furthermore, we random-ly selected 10 of the 388 common targets of HBXIP and c-Myc totest whether Hotair was involved in the activation of c-Myc targetgenes. We found that overexpression of Hotair was able toupregulate these 10 genes in MCF-7 cells (Supplementary Fig.S5I), supporting that Hotair plays a role in the activation of c-Myctarget genes. Thus, we conclude that lncRNA Hotair scaffoldsHBXIP and LSD1 to form a complex of c-Myc/HBXIP/Hotair/LSD1 in activation of c-Myc–induced transcription in breastcancer cells.

The complex of c-Myc/HBXIP/Hotair/LSD1 contributes to the c-Myc–enhanced growth of breast cancer cells in vitro and in vivo

Next, we evaluated the role of the complex of c-Myc/HBXIP/Hotair/LSD1 in c-Myc–enhanced proliferation of breast cancercells. RNA interference efficiency of LSD1-1 and LSD1-2 siRNAswas examined, and we found that siRNA LSD1-2 was able topowerfully knockdown the expression of LSD1 (SupplementaryFig. S6A). MTT assays showed that the knockdown of HBXIP (or

Hotair and LSD1) by siRNA remarkably attenuated the prolifer-ation of MCF-7 and T47D cells in vitro when c-Myc was over-expressed in the cells (Fig. 6A and Supplementary Fig. S6B).Moreover, the knockdown of HBXIP (or Hotair and LSD1)markedly reduced the growth of tumors in nude mice in vivowhen c-Myc was overexpressed in MCF-7 cells (Fig. 6B–D).Interestingly, the expression levels of c-Myc target genes (cyclinA, eIF4E, and LDHA) were significantly reduced in the systemwhenHBXIP (or Hotair) was silenced in the c-Myc–overexpressedtumor tissues (Supplementary Fig. S6C). It is well known thatKi67 is a marker of cell proliferation (41). IHC staining showedthat the levels of Ki67 were consistent with the tumor volumes aswell (Fig. 6E). Therefore, we conclude that the complex of c-Myc/HBXIP/Hotair/LSD1 contributes to the c-Myc–enhanced growthof breast cancer cells in vitro and in vivo.

DiscussionThe transcription initiation complex, including transcription

factors, coactivators, and others, is necessary for the transcriptionof genes in eukaryotes. The proto-oncogene transcription factor c-Myc is a central regulator in the tumorigenesis (1, 3, 5). Our grouphas reported that the oncoprotein HBXIP is an oncogenic tran-scriptional coactivator in breast cancer (16, 42). Recently, it hasbeen reported that lncRNAPRNCR1 and PCGEM1 are involved inthe androgen–receptor transcriptional complex (29). In the pres-ent study, we are interested in whether HBXIP and lncRNAscontribute to the transcriptional regulation of c-Myc–inducedgenes.

The c-Myc target genes are estimated to comprise about 15% ofall human genes, which are involved in multiple cell biologicprocesses (6). In this study, we are interested in whether HBXIP isinvolved in the transcriptional regulation of c-Myc–inducedgenes. As expected, we observed that there were 388 commontarget genes of c-Myc and HBXIP in MCF-7 cells, in which the c-Myc–induced geneswere 28.8% (388/1,348) andHBXIP-inducedgenes were 16.8% (388/2,311). It suggests that HBXIP is associ-ated with the transcription of c-Myc–target genes. Then, wevalidated that HBXIP was able to modulate c-Myc–induced genesin breast cancer cells, such as cyclin A, eIF4E, and LDHA, whichwere out of 388 genes. Moreover, we found that the expressionlevels of HBXIP were significantly positively correlated with thoseof above three c-Myc target genes in clinical breast cancer tissues.Functionally, we showed that HBXIP was required for the c-Myc–induced target genes in the cells, and HBXIP was able to increasethe activities of E-box luciferase reporter. Our previous reportsshowed that several c-Myc target genes, such as cyclinD1, cyclin E,and telomerase reverse transcriptase, could be regulated byHBXIP(18, 43), which is consistent with this finding. Next, we try toidentify the mechanism by which HBXIP enhances the transcrip-tion of c-Myc–induced genes in breast cancer cells. Interestingly,we showed that HBXIP could directly interact with c-Myc, inwhich the fragment aa 56–173 with the leucine zipper of HBXIPwas responsible for the interaction. It has been reported that c-Mycprotein binds to aDNA sequence called E-box (CACGTG; ref. 44).AndotherHLH-Zip transcription factors, such asChREBP, SREBP,NRF1, Clock, and HIF, are able to recognize the E-box as well(5, 45, 46). Moreover, we revealed that HBXIP was able to interactwith theE-box in thepromoters of c-Myc–induced genes. It furthersupports that HBXIP is a coactivator of transcriptional factorc-Myc, which is consistent with our reports that HBXIP is able

Li et al.





Figure 5.lncRNAHotair scaffolds HBXIP and LSD1 inthe complex to activate the c-Myc–induced transcription. A, the interaction ofLSD1with c-Myc (or HBXIP)wasmeasuredby co-IP assays in MCF-7 cells treated withboth RNase inhibitors and siHotair. B, theinteraction of HBXIP (or c-Myc) withHotair was detected by RIP-qPCR assaysin MCF-7 cells or T47D cells. C, theinteraction of HBXIP (or c-Myc) withHotair was detected by RIP-qPCR assay inMCF-7 cells treated with siRNA of c-Myc(orHBXIP). D, the occupancyof LSD1 in thepromoter of LDHA was tested by ChIP-qPCR in MCF-7 cells. E, the RNA levels ofc-Myc, HBXIP, Hotair, cyclin A, eIF4E, andLDHA were tested by real-time PCR inMCF-7 cells. F, the protein levels of thosegenes were tested by Western blot inMCF-7 cells. Statistically significantdifferences are indicated. �� , P < 0.01,Student t test.






to coactivate transcription factors, such as TF-IID, STAT3, SP1,CREB, and E2F1, in breast cancer cells (15–20).

MethylatedH3marks the promoter and the E-box chromatin inMyc-induced genes (47, 48). It has been reported that LSD1-mediated demethylation of H3K4 leads to the assembly of thec-Myc–induced transcription initiation complex (22, 23). There-fore,we supposed that a complex containing c-Myc,HBXIP, LSD1,

and others might be involved in the event. As expected, our datashowed that HBXIP linked c-Myc and LSD1 in the complex ofc-Myc/HBXIP/LSD1. It has been reported that LSD1 is an RNA-interacting protein (27). Accordingly, we concerned that RNAsmight be involved in the complex as well. Interestingly, RNase Acould block the interaction of c-Myc (or HBXIP) with LSD1. Itsuggests that RNAs are required for the interaction of LSD1 with

Figure 6.The complex of c-Myc/HBXIP/Hotair/LSD1 contributes to the c-Myc–enhancedgrowthof breast cancer cells in vitro and in vivo. A, the proliferation ofMCF-7 cellswasmeasured by MTT assays. B, the curves of tumor growth transplanted with MCF-7 cells. C, the images of the tumors. D, the average weight of the tumors.E, the expression levels of Ki67 were detected by IHC staining in above tumors. Statistically significant differences are indicated. �� , P < 0.01, Student t test.

Li et al.





c-Myc (or HBXIP) in the complex. It has been reported thatlncRNA Hotair serves as a scaffold recruiting PRC2 and LSD1 inthe promotion of cancer metastasis (27, 28, 49). Strikingly, wefound that Hotair acted as a scaffold for HBXIP and LSD1 in oursystem. More importantly, we identified that HBXIP was respon-sible for the recruitment of Hotair to c-Myc to form the transcrip-tional complex. This finding is consistent with above reports thatHotair serves as a scaffold modulating the interaction of proteins(27). Thus, in this study, we report that Hotair serves as a scaffoldin the transcriptional complex of c-Myc–induced genes. Our dataare consistent with the report that lncRNAs contribute to tran-scription complex, for instance PRNCR1 and PCGEM1 areinvolved in the androgen–receptor transcriptional complex(29). It has been reported that PRC2 is able to interact withHotair(27, 50). We concerned whether EZH2, a component of PRC2,was one of members of the complex of c-Myc/HBXIP/Hotair/LSD1. However, our data showed that HBXIP failed to interactwith EZH2 (Supplementary Fig. S5J).

Taken together, we summarize a model that a complex of c-Myc/HBXIP/Hotair/LSD1 contributes to the c-Myc–mediated

transcriptional activation in breast cancer, in which HBXIP actsas a coactivator throughdirect interactionwith c-Myc, and lncRNAHotair scaffolds HBXIP and LSD1 to form the complex, leading tothe activationof transcriptionof the c-Myc–induced genes (Fig. 7).Thus, our finding provides new insights into the mechanism bywhich the oncogenic transcriptional factor c-Myc functions incarcinogenesis, illuminating long-standing questions on howc-Myc drives carcinogenesis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Li, X. Zhang, L. YeDevelopment of methodology: Y. Li, X. Zhang, L. YeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Li, Z. Wang, H. Shi, H. Li, L. Li, R. Fang, X. Cai,B. LiuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Li, Z. Wang, X. Zhang, L. YeWriting, review, and/or revision of the manuscript: Y. Li, Z. Wang, X. Zhang,L. YeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Li, Z. Wang, X. Zhang, L. YeStudy supervision: X. Zhang, L. Ye

Grant SupportThis project was supported by the grants of The National Basic Research

Program of China (973 Program no. 2015CB553905), the National NaturalScientific Foundation of China (nos. 81372186, 81272218, and 31470756),and the Tianjin Natural Scientific Foundation (no. 14JCZDJC32800).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedDecember 11, 2014; revised August 8, 2015; accepted September 19,2015; published OnlineFirst December 30, 2015.

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