identificationoffunctionforcd44intracytoplasmicdomain ... · tor-1 (hif1 )-responsive genes also...

Identification of Function for CD44 Intracytoplasmic Domain(CD44-ICD)MODULATION OF MATRIX METALLOPROTEINASE 9 (MMP-9) TRANSCRIPTION VIA NOVELPROMOTER RESPONSE ELEMENT*□S

Received for publication, November 1, 2011, and in revised form, March 9, 2012 Published, JBC Papers in Press, March 20, 2012, DOI 10.1074/jbc.M111.318774

Karl E. Miletti-Gonzalez‡§¶, Kyle Murphy§¶, Muthu N. Kumaran§¶, Abhilash K. Ravindranath§¶, Roman P. Wernyj§¶,Swayamjot Kaur§¶, Gregory D. Miles¶�, Elaine Lim¶�, Rigel Chan¶�, Marina Chekmareva**, Debra S. Heller‡‡,David Foran¶§§, Wenjin Chen¶§§, Michael Reiss¶, Elisa V. Bandera¶¶¶, Kathleen Scotto¶,and Lorna Rodríguez-Rodríguez‡§¶1

From the ‡Department of Obstetrics and Gynecology and Reproductive Sciences, §Division of Gynecologic Oncology,�Cancer Bioinformatics Core, **Department of Pathology and Laboratory Medicine, ¶¶Division of Surgical Oncology,§§Histopathology and Imaging Shared Resource, and ¶Cancer Institute of New Jersey, University of Medicine andDentistry of New Jersey/Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901 andthe ‡‡Department of Pathology and Laboratory Medicine, New Jersey Medical School, Newark, New Jersey 07103

Background: CD44, a multifunctional receptor, undergoes cleavage to produce an intracytoplasmic domain (CD44-ICD)that translocates into the nucleus.Results: CD44-ICD binds to a novel DNA consensus sequence and activates many genes.Conclusion:We finally explain the multifunctionality of CD44 and reveal new genes affected by CD44.Significance:Our findings will accelerate the understanding of how CD44-ICD regulates a multitude of cell functions.

CD44 is a multifunctional cell receptor that conveys a cancerphenotype, regulates macrophage inflammatory gene expres-sion and vascular gene activation in proatherogenic environ-ments, and is also a marker of many cancer stem cells. CD44undergoes sequential proteolytic cleavages that produce anintracytoplasmic domain called CD44-ICD. However, the roleof CD44-ICD in cell function is unknown.We take a major steptoward the elucidation of the CD44-ICD function by using aCD44-ICD-specific antibody, a modification of a ChIP assay todetect small molecules, and extensive computational analysis.We show that CD44-ICD translocates into the nucleus, where itthen binds to a novel DNA consensus sequence in the promoterregion of the MMP-9 gene to regulate its expression. We alsoshow that the expression of many other genes that contain thisnovel response element in their promoters is up- or down-reg-ulated by CD44-ICD. Furthermore, hypoxia-inducible fac-tor-1� (Hif1�)-responsive genes also have the CD44-ICD con-sensus sequence and respond to CD44-ICD induction undernormoxic conditions and therefore independent of Hif1�expression. Additionally, CD44-ICD early responsive genesencode for critical enzymes in the glycolytic pathway, revealinghow CD44 could be a gatekeeper of theWarburg effect (aerobicglycolysis) in cancer cells and possibly cancer stem cells. Thelink of CD44 to metabolism is novel and opens a new area ofresearch not previously considered, particularly in the study of

obesity and cancer. In summary, our results finally give a func-tion to the CD44-ICD and will accelerate the study of the regu-lation of many CD44-dependent genes.

CD44 is a multifunctional cell membrane receptor involvedin cell adhesion, tumor invasion, and metastasis (1–3). In addi-tion, it also regulates macrophage inflammatory gene expres-sion (4) and vascular gene expression in proatherogenic envi-ronments (5). More recently, CD44 has been implicated in themultidrug resistance phenotype of some cancer cells and inprotecting cells against apoptosis (6–8). Importantly, CD44has emerged as a marker of normal progenitor cells as well ascancer-initiating or stem cells, although its role in these cells isnot known. CD44 exists in a variety of isoforms due to alterna-tive splicing and post-transcriptional modifications. The mostcommon isoform, CD44 standard (CD44s),2 is amajor receptorfor hyaluronan (9, 10). Also, CD44s has been implicated in thehyaluronan-dependent or -independent regulated expressionof matrix metalloproteinases (MMPs), mainly MMP-2 andMMP-9 (11–13), and has been suggested to be involved in tran-scription (14). However, the mechanism of the CD44 multi-functionality is not known.MMPs are a group of endopeptidases that degrade extracel-

lular matrix. Hence, the enzymatic activities of MMPs play animportant role in invasion and metastasis of tumors in whichthey are frequently overexpressed (15, 16). Some MMPs,includingMMP-9, are known to bind CD44 (17, 18) or are reg-* This work was supported by National Institutes of Health Grants

R01CA120429 (to L. R. R.), R01CA156386 (to D. F.), and K22A138563 (to E. B.)and by Cancer Center Support Grant P30-722720 and its shared resources:Biospecimen Repository Service, Functional Genomics, Bioinformatics,and the Office of Human Research Services.

□S This article contains supplemental Figs. S1–S4 and Tables 1–3.1 To whom correspondence should be addressed. E-mail: rodriglo@umdnj.

edu.

2 The abbreviations used are: CD44s, CD44 standard; MMP, matrix metallo-proteinase; CD44-ICD, CD44 intracytoplasmic domain; CIRE, CD44-ICDresponse element; TMA, tissue microarray; qRT-PCR, quantitative RT-PCR;SR, sequence repeat(s); CD44-U, CD44 uncleavable mutant.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 23, pp. 18995–19007, June 1, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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ulatedby theCD44s-hyaluronan interaction (11, 13).Hyaluron-an also increases MMP-9 activity and gene expression (19).This effect is blocked with anti-CD44 antibodies, although adefined mechanism of how CD44 affectsMMP-9 expression isnot known.In the current study, we utilizedCD44-mediated overexpres-

sion of MMP-9 as a working platform to elucidate the role ofCD44 in transcription.Weused aCD44-ICD-specific antibody,a modification of a chromatin immunoprecipitation (ChIP)assay to detect small molecules, and extensive computationalanalysis. We found that CD44 induces MMP-9 transcriptiondirectly following the intramembranous proteolytic processingof CD44 by presenilin-1. We demonstrate that the resultingintracytoplasmic tail, CD44-ICD, is then transported into thenucleus, where it binds a novel promoter response element,thereby regulating transcription of target genes. Interestingly,the CD44-ICD response element (CIRE) is located downstreamof Hif1� in CD44-ICD early response gene promoters; thisgroup of hypoxic-responsive genes are turned on by CD44 dur-ing normoxic conditions independently of Hif1� expression.Taken together, these studies show that CD44-ICD activatesmultiple genes involved in cell survival during stress, athero-genesis, inflammation, oxidative glycolysis, and tumor inva-sion. These findings finally elucidate amechanism for themanyfunctions attributed to CD44, specifically cancer cell metasta-ses and metabolism, and promise to accelerate the study of theregulation of many CD44-dependent genes.

EXPERIMENTAL PROCEDURES

Tissue Microarray (TMA)—Tissue arrays were prepared incollaboration with the Cancer Institute of New Jersey TumorRetrieval Shared Facility and the Tissue Array Facility. Ovarianand breast carcinoma tissue arrays were constructed using for-malin-fixed paraffin-embedded tissue blocks containing ovar-ian cancer tumors. Areas of invasive tumor and normal tissuewere identified and marked for subsequent retrieval and analy-sis. Core biopsies of 0.6 mm in diameter were taken from eachdonor block and arrayed into a glass slide. The ovarian cancerTMA was constructed from patients who gave consent to haveidentifiable information; therefore, Institutional Review Boardapproval was obtained. The breast TMAwas constructed with-out identifiable patient information and received InstitutionalReview Board-exempt approval. The TMAs were approved byInstitutional Review Board 0220034452 and 020055381,respectively. Immunoreactivity was assigned as positive whenmore than 50% of the tumor cells stained for the particularantibody, regardless of intensity, or when focal strong stainingwas observed. At least 20% showed strong staining. Negativeimmunoreactivitywas defined as no reactivity at all, weak stain-ing, or staining of less than 5% of the tumor.Tumor Necrosis Factor � (TNF-�) Treatment—TNF-� treat-

ment was performed as described previously (21).Enzyme-linked Immunosorbent Assay (ELISA)—Nuclear and

cytoplasmic fractions from cell lines MCF-7/CD44-ICD-GFPand MCF-7/GFP vector were prepared using the NuclearExtract Kit (ActiveMotif), and each fractionwas analyzed by anactivated Runx2-specific ELISA (TransAM, Active Motif) assuggested by the manufacturer.

Electrophoretic Mobility Assay (EMSA)—Oligonucleotidesfor mobility shifts were prepared by Integrated DNA Technol-ogies. Nuclear extracts were prepared from MCF-7, MCF-7/CD44s, andMCF-7CD44-ICD-GFPmonolayer cultures grownto 70% confluence. For nuclear extract harvest, cells werewashed two times with ice-cold Ca2�/Mg2�-free PBS andtransferred in PBS to microcentrifuge tubes, where they werecentrifuged in a refrigerated centrifuge (4 °C) at 1,850 � g for 5min. Cells were resuspended in 5 packed cell volumes of bufferA (10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM

DTT, 0.1mMEDTA, 0.1mMEGTA, 1�Halt Protease Inhibitor(Roche Applied Science), 0.3% IGEPAL) and centrifuged at1,850 � g for 5 min at 4 °C. The pellet was resuspended in 3packed cell volumes and lysed using a 25-gauge needle. Thesamples were centrifuged at 3,300 � g for 5 min at 4 °C andresuspended in buffer C (20mMHEPES, pH 7.9, 7.5mMMgCl2,0.2 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, 0.4 M NaCl, HaltProtease Inhibitor (Roche Applied Science)). Cells were centri-fuged in a refrigerated centrifuge (4 °C) at 14,000� g for 30min,and supernatants were stored in 20-ml volumes at�80 °C. Pro-tein content of the extracts was determined using the modifiedLowry assay with a bovine serum albumin standard curve(Pierce). Ten �g of nuclear protein combined with bindingbuffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.2 mM MgCl2, 0.3mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 0.42 M NaCl, HaltProtease Inhibitor (Roche Applied Science), and 1 mg ofpoly(dI-dC)) totaling 10 ml were incubated with 30,000 cpm of[�-32P]dCTP (GE Healthcare) double-stranded probes for 30min at room temperature. After incubation, binding reactionswere electrophoresed on a 7% acrylamide gel in 0.5� Trisborate/EDTAat125V.Gelsweredried, exposedonaPhosphor-Imager screen, and scanned using a PhosphorImager.Immunofluorescence Microscopy—Cells were fixed with 4%

paraformaldehyde in PBS, incubatedwith anti-CD44-ICD anti-body and Alexa 488- and Cy3-conjugated secondary antibodies(Invitrogen), and counterstained with TOPRO-3 (MolecularProbes). Cells were viewed using the Nikon Eclipse TE2000Uconfocal microscope using the �60 oil immersion Plan Apoobjective, and images were acquired with EZ-C1 3.50 software.Image analysis was done using ImageJ software (National Insti-tutes of Health).ChIP Assay—Chromatin immunoprecipitation was per-

formed using a ChIP assay kit (Upstate) and the following anti-bodies in individual assays as suggested by the manufacturer:anti-Runx2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),anti-GFP (RocheApplied Science), anti-H3K4 (Santa Cruz Bio-technology, Inc.), anti-H3K9 (Santa Cruz Biotechnology, Inc.),anti-HA (Roche Applied Science), anti-CD44-ICD (CovanceInc.), or no antibody as a negative control. Primers used toamplify DNA fragments corresponding to a region on thehuman MMP-9 promoter were 5�-AGGTACCACAGTTC-CCACAAGCTCTGC-3� (forward) and 5�-TTAAGCTTG-GAGCACCAGGACCAGGG-3� (reverse).Luciferase Assay—MCF-7 cells were transiently transfected

with a firefly luciferase reporter vector (pGL3, Promega) drivenby a region of the MMP-9 promoter (pGL3/�670MMP-9 con-struct; a kind gift of Dr. Ernst Lengyel, University of ChicagoMedical Center) using Lipofectamine Reagent (Invitrogen)

CD44-ICD Regulates MMP-9 via CIRE

18996 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 23 • JUNE 1, 2012


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as suggested by the manufacturer. Twenty-four h after trans-fection, cells were detached and lysed, and luciferase activitywas analyzed using the Dual-Glo Luciferase Assay System(Promega).Microarray—Experiments were run for 28 samples on the

Affymetrix Human Exon 1.0 ST exon microarray platform (1.4million probes). Using GeneSpring GX 11 (Agilent Technolo-gies, Inc.), raw exon expression signals were combined andsummarized with ExonRMA16 (RMA) using only Core tran-scripts (17,800 transcript clusters from RefSeq and full-lengthGenBankTM mRNAs). The data were further quantile-normal-izedwith base-line transformation by themedian of all samples.Further, the normalized expression signals were averagedbetween biological replicates. Gene expression data were firstfiltered by percentile cut-off, resulting in removal of genes withlow signal (�10 percentile of all expression values across allsamples) in all samples. Gene lists displaying differentiallyexpressed behavior were generated by performing pairwisecomparisons. The 251 genes that have been shown to change in“tail” were acquired by looking for a significant -fold change(�2.0) between any pairs of the tail time points (tail 12 h versustail 24 h, tail 24 h versus tail 48 h, and tail 12 h versus tail 48 h).To obtain the 64 genes that change not only in tail, but in “full”as well, the 251 genes mentioned previously were further fil-tered by -fold difference (�2.0) between pairs of the full timepoints (full 12 h versus full 24 h, full 24 h versus full 48 h, and full12 h versus full 48 h). Fig. 6 depicts a heat map of the expressionlevels of the 64 genes of interest, highlighting the genes mostsignificantly differentially expressed in CD44-ICD and full-length CD44s using -fold change as the criterion. The heat mapwas generated using hierarchical conditions, and gene cluster-ing was performed using the Euclidean distance metric andcentroid linkage rule.Statistical Analyses—Microsoft Excel software was used for

statistical analysis. In all studies, comparison of mean valueswas conducted with two-tailed two-sample equal variance Stu-dent’s t tests. In all analyses, statistical significance was deter-mined at p � 0.05.

RESULTS

CD44s Induces MMP-9 Expression and Activity in Carci-noma Cells—We previously reported an increase in migrationand in vitro invasion of MCF-7 breast carcinoma cells stablytransfected with CD44s (MCF-7/CD44s) as well as the CD44-dependent transcriptional regulation of other genes (7).MMP-9 has been reported to be regulated by CD44s-ligandinteractions (11) and is implicated in cancer invasion. Thesefindings led us to investigate how CD44 regulates the expres-sion of MMPs in cancer cells.MMP-9 protein expression and activity was evaluated in

MCF-7 cell lines expressing different levels of endogenous orectopic CD44 using Western blot analysis. Fig. 1, A and B,shows increased levels of active MMP-9 in MCF-7/Adr andMCF-7/CD44s cells compared with parental MCF-7 orMCF-7empty vector cells. To validate these results gelatin zymogramanalyses were performed. MMP-9 activity appeared in the cellsthat express CD44while it was barely visible in parentalMCF-7cells and empty vector cells (Fig. 1C). Interestingly, MMP-2

activity remained relatively constant regardless of the CD44sexpression (Fig. 1C). This observation indicates a CD44-spe-cific effect on MMP-9 activity. Of note, the MMP-2 gene pro-moter is generally regarded as constitutively regulated, whereasthe MMP-9 gene has a more inducible promoter (20). We alsotested whether endogenously induced CD44 induced MMP-9expression. We previously showed that TNF-� up-regulatesendogenous CD44 expression in SKOV-3 ovarian cancer cells(21). Fig. 1, D and E, shows a correlation between endogenousCD44s protein expression and MMP-9 activity after TNF-�treatment. In this case, both of them were up-regulated in adose-dependent manner. The modulation of MMP-9 in thebreast and ovarian cancer cell lines analyzed suggests thatCD44s has a direct role in the up-regulation of the invasivephenotype of these cancer cells through the functional expres-sion ofMMP-9.CD44-ICD Localizes to Nucleus to Regulate MMP-9 Expres-

sion—CD44 undergoes regulated intramembranous proteolysis(22) by presenilin-1/�-secretase, which generates a cytoplasmictail known as CD44-ICD and is translocated to the nucleus. How-ever, the functionofnuclearCD44-ICD isnot known.Wehypoth-esized that nuclear CD44-ICD could be responsible for the tran-scriptional up-regulation of MMP-9 expression. To test thishypothesis, we transiently transfectedMCF-7 cells with one of thefollowing plasmids: pcDNA3.1 vector (vector), pCDNA/CD44WT full-length (CD44s), or pCDNA/CD44-ICD (CD44-ICD) andmeasuredMMP-9mRNA expression by qRT-PCR. Fig. 1F showsthatCD44sorCD44-ICDinduceda3.5-foldanda14-fold increasein MMP-9 mRNA over empty vector, respectively. To confirmthat CD44-ICD indeed translocates into the nucleus, we GFP-tagged CD44-ICD. As shown in Fig. 1G, GFP-tagged CD44-ICDwas found in the cytoplasmaswell as in thenucleusof stably trans-fectedMCF-7 cells. To further confirm the nuclear localization ofthe CD44-ICD, nuclear and cytoplasmic extracts from MCF-7/emptyHA vector andMCF-7/CD44-ICD-HA cells were analyzedby Western blotting. Fig. 1H illustrates that CD44-ICD-HA ispresent both in the cytoplasm and nuclear fractions of the cellextract.In order to determine whether CD44 processing from full-

length into CD44-ICD occurs constitutively (i.e. without exog-enous stimulation) in cancer cells, we examined the nuclear andcytoplasmic expression of the CD44-ICD by immunoprecipita-tion of CD44-overexpressing cell lysatewith an anti-CD44-ICDantibody. Endogenously processed CD44-ICD was detectedboth in the cytoplasm and in the nucleus (Fig. 1I).MMP-9 Regulation by CD44 Is Clinically Relevant in Human

Tumors—We determined the clinical relevance of our findingsby studying the co-expression of CD44 and MMP-9 in humanovarian and breast carcinomas. Tissues were obtained fromwomen who underwent surgery to resect an ovarian or a breastcancer. We constructed a TMA and performed immunohisto-chemistry using anti-CD44 or anti-MMP-9 monoclonal anti-bodies (Table 1). The breast carcinoma microarray was com-posed of 159 invasive breast adenocaricinomas (Table 1). Weobserved that 81% of the breast tumors were positive for CD44expression. Fifty-five percent of the tumors co-expressed CD44andMMP-9. As expected, only 8% of tumors expressedMMP-9without CD44 expression. Because the breast TMA was biased




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by large tumors, we also tested an ovarianTMA forMMP-9 andCD44 co-expression (Table 1). Of 81 ovarian tumors, 73% wereCD44 (�), 59% co-expressed CD44 and MMP-9, and only 11%expressed MMP-9 without immunoreactivity to CD44. Theseresults validate and strengthen the results of the in vitro data indi-cating CD44 as a regulator of MMP-9 expression in humancancers.Processing of Full-length CD44 Is Required for Induction of

MMP-9—To further evaluate whether the processing of CD44into CD44-ICD was necessary for the transcriptional modula-tion ofMMP-9, we co-transfected MCF-7 cells with CD44-ex-pressing vectors and a luciferase reporter plasmid driven by theMMP-9 promoter (Fig. 2A). Fig. 2B shows that when CD44s orCD44-ICD was overexpressed, the MMP-9 promoter-drivenluminescence was increased by 10- and 11-fold, respectively.However, when CD44 could not be cleaved to produce CD44-

ICD (CD44uncleavablemutant (CD44-U) construct) luciferaseexpression was similar to empty vector. These data indicatethat the CD44-ICD is capable of modulating the expression ofMMP-9 at the level of its promoter and that the uncleavedCD44 is not capable of this induction. Immunofluorescencedata of the co-transfected MCF-7 cells validated the expectedcellular localization of the CD44 and the CD44-ICD. As shownin Fig. 2C, transfection with WT full-length CD44-expressingvector generates CD44 associated with the cell membrane aswell as the cytoplasm and the nucleus. Transfection of CD44-ICD-expressing vector generates CD44-ICD associated withthe nucleus but not the cell membrane. Transfection of cleav-agemutant full-lengthCD44 generatesCD44mainly associatedwith the cell membrane but not the nucleus.Because presenilin-1 is the enzymatic component of the

�-secretase complex (23), a specific alteration of its expression

FIGURE 1. CD44s modulates MMP-9 expression via nuclear CD44-ICD in breast and ovarian cancer cells. Breast cancer cell lines expressing endogenous CD44s(MCF-7/AdrR) or stably transfected CD44s (MCF-7/CD44s) (A) exhibit increased levels of active MMP-9 protein in Western blots (B) and MMP-9 enzymatic activity inzymograms (C). This is in contrast to parental MCF-7 and MCF-7 stably transducted with pLXIN vector (MCF-7/vector) that express lower levels of active MMP-9 proteinand enzymatic activity. MMP-2 enzymatic activity levels are relatively similar in each cell line. D and E, Western blot and zymogram analysis of an ovarian cancer cell line(SKOV-3) expressing endogenous CD44s show a direct correlation between TNF-�-modulated CD44s expression and MMP-9 protein expression and activity.F, real-time PCR analysis of transient transfections of pcDNA3.1 vector alone, vector expressing CD44s, or vector expressing CD44-ICD shows a CD44s- and CD44-ICD-dependent increase in MMP-9 mRNA expression in MCF-7 cells. G, fluorescence microscopy of transiently transfected MCF-7 cells with a CD44-ICD-GFP constructdemonstrates nuclear and cytoplasmic localization of CD44-ICD-GFP. Vector alone shows widespread background fluorescence signal in the cells. H, Western blottingusing an anti-HA antibody localizes HA-tagged CD44-ICD in the nucleus as well as in the cytoplasm. Western blotting using anti-lamin A and C or anti-�-tubulinantibodies validates the purity of the nuclear and the cytoplasmic extracts, respectively. I, immunoprecipitation (IP) of cytoplasmic or nuclear extracts using ananti-CD44-ICD antibody shows the localization of endogenously processed WT CD44-ICD. Error bars, S.D.

TABLE 1Tissue microarrays




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should affect the CD44-dependentMMP-9 regulation. To testthis possibility, we knocked down the presenilin-1mRNA tran-script and examined the effect of presenillin down-regulationon MMP-9 activity. We observed a decreased amount ofMMP-9 activity (Fig. 2D). The down-regulation of MMP-9activity correlated with the down-regulation of presenilin-1expression tested by qRT-PCR (Fig. 2D, bar graph) and West-ern blotting. These data confirm a regulatory role of CD44-ICDin the up-regulation ofMMP-9 expression.CD44-ICD Interacts with Novel DNA Binding Sequence on

MMP-9 Promoter—Because Runx2 is a known MMP-9 keytranscription factor (24), we initially explored the possibilitythat CD44-ICD physically interacts with Runx2 at theMMP-9promoter by analyzing ChIP nuclear extracts. Fig. 3A (lanes 7and 8) depicts that nuclear Runx2 co-immunoprecipitatedwithnuclear CD44-ICD.Furthermore, CD44-ICD was present in actively transcribed

chromatin (H3K4(�)) but not in silenced chromatin (H3K9(�))(Fig. 3A, lanes3–6).Toconfirmthese findingsweperformedChIPassays by amplifying the region containing the Runx2 consensussequence of theMMP-9promoter (Fig. 3B).We found thatCD44-

ICD-GFP immunoprecipitated a region of theMMP-9 promoter(Fig. 3B, lane 4). Immunoprecipitated Runx2 was also bound tothis specific region ofDNA (Fig. 3B, lane 5). These results supportan interaction between CD44-ICD, Runx2, and theMMP-9 pro-moter within the same promoter region where Runx2 binds toDNA.To analyzewhether this interactionwas also present in cellsexpressing full-length CD44 and thus containing endogenouslyprocessed CD44-ICD, we performed a Runx2 consensussequence-specific ELISA assay. We found that the binding ofRunx2 to its consensus sequencewas increasedby almost 2-fold innuclear extracts of cells endogenously producing CD44 (Fig. 3C).To test whether the CD44-ICD could directly bind to the DNAofthe same region of theMMP-9 promoter, we performed a similarELISA using recombinant GST-tagged CD44-ICD and the sameRunx2 consensus sequence probe. Notably, CD44-ICD-GST wasfound to bind to the Runx2 consensus sequence in a dose-depen-dent fashion (supplemental Fig. S1A).To further determine additional details of this interaction,

we performed EMSAs. We incubated recombinant CD44-ICD-GST with a radiolabeled Runx2 consensus sequenceprobe. Fig. 3D shows a dose-dependent interaction between

FIGURE 2. CD44 modulation of MMP-9 is dependent on presenilin 1 enzymatic activity. A, depiction of the CD44 expression constructs co-transfected withpGL3/�670MMP-9 promoter reporter construct. B, immunofluorescence of anti-CD44-ICD antibody was used to localize the CD44-ICD in the different trans-fections as follows: CD44s, both at the cell membrane and inside the nucleus; CD44-ICD, mostly in the nucleus; CD44-U, outside the nucleus (cell membrane andcytoplasm), which indicates that CD44-ICD is not generated in the CD44-U mutant. C, transient expression of CD44s, CD44-ICD, or CD44-U. The WT CD44 andCD44-ICD expression constructs increases MMP-9 promoter-driven luciferase expression in MCF-7 cells, whereas the CD44 mutant construct shows the samebase-line activity as vector-only control. D, RNA interference of presenilin-1 by siRNA in MCF-7/CD44s cells reduces MMP-9 activity in the cell culture superna-tant as detected by zymogram analysis. Corresponding presenilin-1 down-regulation was confirmed by qRT-PCR as shown in the bar graph and Western blots.Error bars, S.D. NS, nonspecific siRNA.




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recombinant CD44-ICD and the Runx2 consensus sequenceprobe.To better understand the interaction between endogenously

processed CD44-ICD and theMMP-9 promoter, we performedadditional EMSAs. Preliminary EMSAs indicated a CD44-re-lated interaction with the MMP-9 probe because nuclearextracts from MCF-7/CD44s cells but not from parentalMCF-7 cells produced shifted bands (supplemental Fig. S1B).Fig. 3E (lane 2) shows a shift of two bands upon combiningCD44(�) nuclear extracts with the WT MMP-9 promoterprobe. We competed this binding with a “cold” MMP-9 WTpromoter probe. Fig. 3E (lane 3) shows that, as expected, coldWT probe competed both complexes. We concluded that one

of the competed bands was Runx2 bound to its consensussequence (TGCGGT; underlined in Fig. 3F). To test whetherthe other band was bound CD44-ICD, we carried out competi-tion experiments using mutated oligonucleotides within theRunx2 or neighboring sequences. In Fig. 3E, lane 4, we observedthat when mutant 1 was used, there was still some competitionwhen compared with the WT competitor. Similarly, when thewhole Runx2 consensus sequence was mutated and used ascompetitor (Fig. 3E, lane 5), we found reduced competition. IfRunx2 was the only protein binding to the DNA, adding a com-pletely mutated Runx2 consensus binding site cold oligonu-cleotide would have resulted in no competition at all. Becausewe observed partial competition (compare lane 5 with lane 2),

FIGURE 3. CD44-ICD interacts with Runx2 on the MMP-9 promoter. A, antibodies used in ChIP experiment were as labeled. The chromatin complex wascross-linked using ethylene glycol-bis (succinic acid N-hydroxy succinimide ester) followed by formaldehyde, (� crosslink). Half of the sample was treated withhydroxylamine HCl to revert the cross-link (� crosslink). The above ChIP assay extracts were probed with anti-GFP antibody, which shows an interactionbetween CD44-ICD-GFP tagged and Runx2, which localizes to the transcriptionally active MMP-9 promoter in MCF-7/CD44s cells. B, we stably transfectedMCF-7 cells with a GFP-tagged CD44-ICD expression vector and amplified 300 bp of the MMP-9 promoter in ChIP assays. This ChIP assay consisted of a two-stepcross-linking method that improves the detection of small molecules by making use of a protein-protein cross-linker, Di(N-succinimidyl)gluterate, followed bya protein-DNA cross-linking step using formaldehyde (25). C, Runx2 consensus sequence-specific ELISA. Binding to Runx2 consensus sequence was observedon nuclear extracts from MCF-7 cells overexpressing CD44s. MCF-7 and MCF-7 vector-only cells were used as controls. Positive control was extracts fromSAOS-2 cells; negative control was buffer only. D, EMSA shows specific CD44-ICD binding to a Runx2 binding sequence region of the MMP-9 promoter. Theaddition of salmon sperm DNA demonstrates the specificity of this binding interaction. E, EMSA analysis using a 32P-labeled MMP-9 WT promoter oligonucleo-tide containing the Runx2 consensus sequence shows two shifting bands. These bands correspond to the binding of Runx2 to its known binding sequence(TGCGGT) and to the binding of CD44-ICD to a novel binding sequence (CCTGCG). F, DNA sequences of the radioactive MMP-9 WT promoter probe and thecompeting cold WT and mutant probes used. Runx2 binding sequence is underlined, and mutated nucleotides are shown in red. G, EMSA shows supershiftedbands upon the addition of an anti-Runx2 antibody to confirm the presence of Runx2 in the binding complex. H, luciferase assay using pGL3– 670MMP-9reporter construct WT or without CCTGCG in its sequence (del mut). I, secreted luciferase assays show increased activity over control in supernatant from cellsco-transfected with a CD44-ICD expression vector and either a WT CCTGCG sequence repeat-driven reporter vector (wt sr) or a mutant sequence repeatAAGTAT (mut sr). Reporter vector without the CCTGCG sequence was used as control. Error bars, S.D.




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we hypothesized that the CD44-ICD is also binding outside theRunx2 consensus sequence. To test this premise, four addi-tional mutant probes with sequence changes outside the Runx2binding site were used (Fig. 3E, lanes 6–9). These mutantprobes fully competed the shift of the top band but not thelower band. Because the only sequence in common among themutant probes and the 32P-labeled DNApromoter was theWTRunx2 consensus sequence, we conclude that the top bandreflects Runx2 binding to its consensus sequence. However,only mutant probe 5 was able to compete the bottom band aswell (Fig. 3E, lane 8). The difference between mutant probe 5and the other mutant probes is that this probe containedWT sequences immediately flanking the Runx2 consensussequence. Fig. 3F shows that the WT MMP-9 promoter has aCCTGCG sequence that flanks the 3�-end Runx2 consensussequence and overlaps the 5�-end by four nucleotides (i.e.TGCG). We hypothesized that these flanking CCTGCGrepeats were the CD44-ICD binding sites.To test this possibility, we mutated both flanking sequences

but not the Runx2 consensus sequence. Fig. 3E, lane 9, showsthat mutant probe 6 failed to compete the binding of the lowerband but, as expected, completely competed the binding of thetop band. These results indicate that CD44-ICD binds toCCTGCG. To further confirm this premise, we designed com-peting probes with multiple sequence repeats (SR) of the puta-tive CD44-ICD response element (CD44-ICD SR) or the Runx2consensus sequence (Runx2 SR). Fig. 3E, lanes 10 and 11, showsthat probes Runx2 SR and CD44-ICD SR competed the top andthe bottom band, respectively.To validate the identity of Runx2, we performed a supershift

assay using an anti-Runx2 antibody (Fig. 3G, lane 3). Weobserved that the anti-Runx2 antibody shifted the radiolabeledprobe. Furthermore,we competed this supershift using the coldRunx2 SR competitor probe in a dose-dependent fashion (sup-plemental Fig. S2). We also competed the CD44-ICD band(lower band) but not the Runx2 band (top band) with a CD44-ICD SR probe (supplemental Fig. S2). This result indicates thatthe bottomband in fact corresponds to theMMP-9probe inter-action with CD44-ICD.In order to confirm that CCTGCG was a novel CD44-ICD

response element sequence, we performed two different lucif-erase reporter expression assays, one with the same luciferaseconstruct used in Fig. 2B (pGL3/�670MMP-9 promoter) and

the other with a minimal promoter sequence plasmid (pTK) inwhich the putative CD44-ICD response element (CCTGCG)was cloned upstream of the minimal promoter. Fig. 3H showsthat when CD44-ICD was co-transfected with a CCTGCGdeletion mutant in the �670MMP-9 promoter luciferasereporter construct, luciferase activity decreased by 56% com-pared with theWT reporter construct. In a similar experimentbut using the pTK/CCTGCG luciferase reporter construct, Fig.3I shows that when CD44-ICD was co-transfected with aCCTGCG sequence-driven luciferase reporter construct, lucif-erase activity increased by 1.8-fold (3.3/1.8) over empty vector(p � 0.01). Consistent with this result, the mutation of theCCTGCG sequence toAAGTAT resulted in a decrease in lucif-erase activity back to vector-only levels. These experimentsconfirm CCTGCG as the CD44-ICD DNA response element.CD44-ICD Response Element Is Present in CD44-regulated

Genes—To identify additional genes that could be regulated byCD44s via CD44-ICD, we performed an NCBI PubMed Website literature review and found 12 genes that had been reportedas regulated by CD44. Using the Web-based program Tran-scriptional Regulatory Element Database, we found that all oftheCD44-regulated genes found in our search contained one ormore repeats of CCTGCG, the CIRE, in their promoters (Table2). Furthermore, a recent report of amicroarray of differentiallyexpressed genes in the aortic arch of ApoE(�/�) CD44(�/�)mice, compared with ApoE(�/�) CD44(�/�) mice, validated50 genes that are regulated by CD44 (5). The promotersequences of 47 of these genes were available, and of these, 41contained the CIRE sequence (CCTGCG). To determine whatother genes CD44-ICD could induce in cancer cells, we per-formed a Gene-Chip� microarray analysis (Fig. 4A). Hierarchi-cal clustering analysis of the genes that showed a change inmRNAmessage expression by at least 2-fold and p� 0.05 upontransfection of CD44-ICD identified changes in 251 genes (sup-plemental Fig. S3 and supplemental Table 1). We narroweddown the analysis to require a change in expression by at least2-fold in both the full-length CD44s and the CD44-ICD trans-fectants. This analysis identified 64 genes (Fig. 4A and supple-mental Table 2). A Gene Ontology analysis using the IngenuityIPA tool identified four network functions (supplemental Fig.S4) and several biological functions associated with this groupof 64 genes, including cancer, cellular growth and proliferation,tumor morphology, and cell-to-cell signaling among others.

TABLE 2Published genes that are regulated by CD44 and the presence of the CD44-ICD response element in their promotersA PubMed literature review was performed for genes that are affected by different levels of CD44 expression. The promoter sequence within 1,000 or 5,000 bp (underlinednumbers) upstream and 299 bp downstream of the transcription initiation site was analyzed for the presence of CIRE using the Transcriptional Regulatory ElementDatabase (Michael Zhang Laboratory, Cold Spring Harbor, NY).

Gene/Protein Protein status CD44 status CIRE Reference

ADD3/�-adducin Abolished Knockdown (by siRNA) 4 45PRKCA/PKC-� Abolished Knockdown (by siRNA) 1 45CASP9/caspase 9 Up-regulated Null (knock-out mouse) 1–3 46CASP3/caspase 3 Up-regulated Null (knock-out mouse) 2 46BCL2L1/Bcl-xl Abrogated Null (knock-out mouse) 1 46CDKN1A/p21 Abrogated Null (knock-out mouse) 1 46BR1/pRB Abrogated Null (knock-out mouse) 2 46CCNA1/cyclin A1 Abrogated Null (knock-out mouse) 1 46bcl-2 Down-regulated Down-regulated 1–2 47CFL1/cofilin Down-regulated Down-regulated 1–4 48SVV/survivin Up-regulated Up-regulated 1 49EGR Down-regulated Up-regulated 1 26




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We also found that 28 of 46 genes (61%) in which promoterregion sequences were available contained at least one CIRE(CCTGCG) within 1,000 bp upstream of the transcriptionalstart site. Of interest were 16 genes that were up-regulated byCD44-ICD after only 12 h post-transfection but needed 48 hpost-transfection of full-length CD44s to exhibit a similar up-regulation (Fig. 4B). The lag time of gene expression betweenCD44-ICD and the full-length CD44s suggests that these genesare directly regulated by CD44-ICD, which is readily availableto translocate into the nucleus when transfected as CD44-ICDbut requires more time when CD44-ICD is generated by thecleavage of full-length CD44s. Interestingly, 8 of the 13 genes(62%) with known promoter sequence contained the CIREs(CCTGCG) in their promoters. We then evaluated the up-reg-ulation of 10 of these genes by qRT-PCR. We validated theCD44-ICD-mediated up-regulation in seven genes. Of note, sixof the seven genes contained the CIRE (CCTGCG). Theseresults indicate that CD44-ICD transcriptionally activatesgenes that contain the CIRE, in most cases within 1,000 bp oftheir transcriptional start site.To further understand the group of 16 CD44-ICD-targeted

“early response” genes, a Gene Ontology analysis using theIngenuity IPA tool was performed. One network and severalbiological functions, including cancer, were identified (Fig. 4C).Interestingly, many of the genes were directly associated withthe hypoxia-induced transcription factor Hif1� and aerobicglycolysis. We found it intriguing that many of the genes acti-vated byHif1�were also activated byCD44-ICDbut thatHif1�itself was not elicited in our very stringent Gene-Chip analysisalthough it does have a CD44-ICD response element in its pro-moter. One possibility could be that CD44 modulates Hif1�expression only under anaerobic conditions. We examined theexpression of Hif1� in MCF-7 and MCF-7/CD44s cells byWestern blotting. Fig. 4D shows that, as expected, Hif1� pro-

tein was not expressed in either cell line under normoxia.Under hypoxic stress caused by CoCl2, Hif1� expression wasclearly induced inMCF-7 cells but not inMCF-7/CD44s cells inwhich Hif1� expression was suppressed. These results indicatethat CD44-ICD can induce Hif1�-related genes in the absenceof hypoxia and bypasses Hif1�-dependent induction.

DISCUSSION

We previously reported that the expression of CD44s inhuman cancer cell lines induces a more invasive and drug-re-sistant cell phenotype (7). However, the augmentation in theinvasive phenotype of CD44s-expressing cells is notwell under-stood. One possible mechanism is that CD44 transcriptionallymodulates tumor invasion-related genes. The purpose of thisstudywas to use the CD44-mediated overexpression ofMMP-9as a working platform to elucidate the role of CD44 (specificallyCD44-ICD) in transcription.As a group, MMPs degrade most of the extracellular matrix,

and therefore they play an important role in invasion andmetastasis of tumors (16), where they are frequently overex-pressed. In breast cancer, the levels and activity of theseMMPshave been shown to display an important correlation with thetumor cell phenotype. For example, MMP-9 is significantlyincreased and more frequent in malignant tumors comparedwith benign breast tumors or normal breast tissue (27, 28).Furthermore, invasive breast carcinomas with the highestaggressiveness display the highest levels of MMP-9 (27). Inthe APC-Min model, MMP-9 contributes to early intestinaltumorigenesis (29). Also, the inhibition of expression ofMMP-9 in different cancer cell lines causes amarked reductionin invasiveness and CD44 expression, suggesting a functionallinkage between CD44 andMMP-9 (30).In our study, we characterize the functional relationship

between CD44 and MMP-9 and demonstrate that the expres-

FIGURE 5. Hypothetical model of CD44 signaling via CD44-ICD.

FIGURE 4. CIRE sequence is found in CD44-regulated genes, including Hif1� target genes. We used the Affymetrix Human Exon 1.0 ST exon microarrayplatform to compare gene expression profiles among MCF-7 cells transiently transfected for 12, 24, or 48 h with pcDNA3.1 vector expressing full-length CD44sor CD44-ICD. A, heat map of microarray. B, list of genes that were evaluated by qRT-PCR, their change in gene expression upon CD44-ICD construct transienttransfection, and their status with regard to the presence or absence of CIREs. See supplemental Table 3 for raw data. C, a gene ontology analysis using the IPAtool identifies a close relationship between Hif1� and the “early response” genes in the microarray containing CIRE sequence in their promoters. D, anti-Hif1�Western blotting of CoCl2-treated cell extracts show a strong induction of Hif1� in MCF-7-treated cells, as expected, but a repressed induction of Hif1� inMCF-7/CD44s-treated cells.




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sion and proteolytic processing of CD44s, with the subsequentgeneration of the CD44-ICD, is directly associated with theup-regulation of the MMP-9 gene. Our data also demonstratethat the up-regulation of gene expression depends on theCD44-ICD nuclear translocation and binding to its novelresponse element, CCTGCG. The following experimental datasupport these conclusions.First, we show that the stable expression of CD44s inMCF-7

cells not only correlates with an increase inMMP-9 activity butalso induces an increase in pro-MMP-9 activation (Fig. 1). Ofnote, the increment of mature MMP-9, but not pro-MMP-9, isprobably a function of the ability of CD44 to act as a cell mem-brane docking molecule for the pro-MMP-9 zymogen, whichallows its successive activation (31, 18). Interestingly, theexpression of MMP-2 remains the same regardless of CD44sexpression. This apparent discrepancy in expression betweenMMP-2 andMMP-9 is known to be due to differences in cis-ele-mentcompositionof theirpromoters (32).Ourdata showaCD44-relateddifferential regulationof thepromotersof these twoMMPsand thus assign a transcriptional inducer function toCD44s in theregulated expression of MMP-9. Interestingly, the hyaluronan-mediatedactivationofCD44 induces its proteolytic cleavage at thecell surface to promote cell motility and subsequently tumor pro-gression (33, 34). This extracellular cleavage is followed by addi-tional intracellular cleavages to generate the CD44-ICD fragment(14), which we show is directly involved in the regulated expres-sionofMMP-9.Wealso show for the first time that theprocessingof CD44smay occur constitutively without the need of exogenousinduction in breast cancer cells.Second, we demonstrate that the CD44-mediated expression

ofMMP-9 directly correlateswith the enzymatic activity of pre-senilin-1, as part of the �-secretase complex, the underlyingenzymatic machinery that generates CD44-ICD after regulatedintramembranous cleavage of CD44 (22, 35). We demonstratethat when presenilin-1 activity is down-regulated, MMP-9enzymatic activity decreases (Fig. 2). We support this conclu-sion by showing that the expression of an uncleavable CD44 isnot capable of inducingMMP-9 expression. In agreement withthese data, the proteolytic processing of CD44 has beendetected in numerous carcinomas, including breast carcinoma,in which MMP-9 has also been reported to be overexpressed(36–38). We show in Table 1 that not only are MMP-9 andCD44 usually co-expressed in ovarian and breast cancers, butalsoMMP-9 is rarely expressed in the absence of CD44 expres-sion inmore than 200 human tumors studied, making our find-ings biologically relevant.Third, we show that ChIP assays localize CD44-ICD in a

complex with Runx2 to the MMP-9 promoter (Fig. 3). Thesefindings start to connect several components and mechanismsassociated with the complex regulation of the MMP-9 gene incancer cells. For example, MMP-9 expression in prostate (i.e.LNCap and PC3) and breast (i.e. MCF-7 and MDA-MB-231)cancer cell lines correlated with their Runx2 expression andtranscriptional activity (24). Interestingly, the CD44s proteinexpression in those cells directly correlates with the Runx2-de-pendentMMP-9 expression (39, 40).We used ELISAs as well asan extensive EMSA analysis to demonstrate that CD44-ICDbinds directly to the DNA of the MMP-9 promoter. Further-

more, we uncover a CD44-ICD DNA binding sequence andcorroborate its importance using an Affymetrix assay. Themajority of the genes up-regulated by the transfection of CD44-ICD have the sequence CCTGCG in their promoters, usuallywithin 1,000 bp upstream of the transcription start site.Among the early response genes induced byCD44-ICD listed

in Fig. 4B, three important enzyme-encoding genes in oxidativeglycolysis were found. They are ALDOC, which encodes aldol-ase c, fructose biphosphate; PDK1, which encodes pyruvatedehydrogenase kinase-1; and PFKFB4, which encodes 6-phosphofructose-2-kinase/fructose-2,6-biphosphatase 4. Thisuncovers a possible role of CD44 in oxidative glycolysis. Early inthe past century, Warburg observed that cancer cells under-went anaerobic glycolysis in the face of adequate oxygen supply(50). The key enzyme of the glycolytic pathway, 6-phosphofruc-tose-1-kinase, is controlled allosterically by the CD44-ICD-in-ducible fructose-2,6-bisphosphatase. Fructose-2,6-P(2) is anallosteric effector that coordinately regulates two opposingpathways, glycolysis and gluconeogenesis, depending on thenutritional and endocrine state of the cell. PFKFB4 is a bifunc-tional enzyme that can acutely modulate fructose-2,6-P(2) lev-els and is known to be elevated in tumors (41). Another genethat we validated as induced by CD44-ICD is PDK1. The prod-uct of this gene inhibits pyruvate dehydrogenase and forcesglycolysis to convert pyruvate into lactate rather than goinginto the citric acid cycle. Lactate increases the acidity of thetumormicroenvironment, which then allows the cells to invadethe extracellular matrix (42). Lactate also regenerates NAD�

for continued glycolysis (43). The induction of these metabolicgenes implicates CD44 for the first time in the transcriptionalregulation of cell metabolism. It suggests that by increasing thetranscription of key regulatory enzymes in oxidative glycolysis,CD44 may promote the Warburg effect in cancer cells that areCD44(�) and known to preferentially use aerobic glycolysis forsurvival regardless of oxygen concentration. Because CD44 is aknown stem cell marker for many tumors, it is reasonable tospeculate that by inducing aerobic glycolysis, CD44 maintainsthe metabolic needs of stem cells that are usually found inniches of intermittent hypoxia and would benefit from oxida-tive glycolysis as a way to survive. Most recently, Saya’s group(44) reported that CD44 interacts with pyruvate kinase M2(PKM2) and induces aerobic glycolysis in p53 mutant cells.Future work will be needed to test these hypotheses. Based onthework presented, a hypotheticalmodel of the CD44-ICD-de-pendent CD44 signaling to modulate transcription anticipatesdirect as well as indirect interactions with promoters and tran-scription factors (Fig. 5).

Acknowledgments—We are grateful to Dr. Lori White for technicalsupport and to Dr. Joseph Bertino for constructive discussions.

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Bandera, Kathleen Scotto and Lorna Rodríguez-RodríguezChekmareva, Debra S. Heller, David Foran, Wenjin Chen, Michael Reiss, Elisa V.

MarinaRoman P. Wernyj, Swayamjot Kaur, Gregory D. Miles, Elaine Lim, Rigel Chan, Karl E. Miletti-González, Kyle Murphy, Muthu N. Kumaran, Abhilash K. Ravindranath,

TRANSCRIPTION VIA NOVEL PROMOTER RESPONSE ELEMENTMODULATION OF MATRIX METALLOPROTEINASE 9 (MMP-9)

Identification of Function for CD44 Intracytoplasmic Domain (CD44-ICD):

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identificationoffunctionforcd44intracytoplasmicdomain ... · tor-1 (hif1 )-responsive genes also...

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