Corrections

CELL BIOLOGY. For the article ‘‘Breast cancer bone metastasismediated by the Smad tumor suppressor pathway,’’ by YibinKang, Wei He, Shaun Tulley, Gaorav P. Gupta, Inna Serganova,Chang-Rung Chen, Katia Manova-Todorova, Ronald Blasberg,William L. Gerald, and Joan Massague, which appeared in issue39, September 27, 2005, of Proc. Natl. Acad. Sci. USA (102,13909–13914; first published September 19, 2005; 10.1073�pnas.0506517102), the authors note that ‘‘Fig. 2B shows SCP3

cells transduced with a vector constitutively expressing redfluorescent protein (RFP, Upper) and a vector expressing greenfluorescent protein (GFP) under the control of a TGF-� re-sponsive promoter (Lower). An erroneous, unpaired set ofimages was used in the �TGF-� panels of the previouslypublished version of this figure.’’ The corrected figure and itslegend appear below. This correction does not affect the con-clusions of the article.

Fig. 2. Functional imaging of Smad signaling in breast cancer bone metastasis. (A) Schematic representation of the retroviral vectors SFG-tdRFP-cmvFLuc(constitutively expressing tdRFP and FLuc) and cis-TGF-�1–Smads–HSV1-tk�GFP (expressing HSV-tk�GFP fusion protein in response to TGF-�). (B and C) SCP3transduced with these two vectors were treated with TGF-� or no additions for 24 h and analyzed by fluorescence microscopy (B) or two-color FACS (C). Theconstitutive tdRFP fluorescence is shown on the ordinate, and the HSV-tk�GFP fusion fluorescence, inducible by TGF-�, is shown on the abscissa. (D and E Upper)In vivo bioluminescence and microPET imaging of metastases in mice. SCP2 (D) and SCP3 (E Upper) cells bearing the SFG-tdRFP-cmvFLuc and cis-TGF-�1–Smads–HSV1-tk�GFP vectors were injected into the left cardiac ventricle and analyzed after 4 weeks (SCP2) or 18 weeks (SCP3). Bioluminescence imaging shows sitesof metastases in the skull (D and E) and adrenal gland (E Upper). 18F-2�-fluoro-2�deoxy-1�-D-arabionofuranosyl-5-ethyl-uracil microPET images of tk�GFP reporteractivation shows localization of radioactivity to the skull in the coronal and sagittal image planes. No visualization of the adrenal metastasis was seen on microPETimaging. Note the nonspecific accumulation of the tracer in the gastrointestinal tract and bladder attributable to clearance of the tracer. (E Lower) At necroscopy,the head showing the skull and the adrenal metastasis plus kidney were removed and imaged ex vivo for photographic (�) and bioluminescence (�) imaging.

www.pnas.org�cgi�doi�10.1073�pnas.0603221103

8570–8571 � PNAS � May 30, 2006 � vol. 103 � no. 22 www.pnas.org

CHEMISTRY. For the article ‘‘Picosecond dynamics of a membraneprotein revealed by 2D IR,’’ by Prabuddha Mukherjee, ItamarKass, Isaiah Arkin, and Martin T. Zanni, which appeared in issue10, March 7, 2006, of Proc. Natl. Acad. Sci. USA (103, 3528–3533;first published February 27, 2006; 10.1073�pnas.0508833103),the author name Isaiah Arkin should have appeared as Isaiah T.Arkin. The online version has been corrected. The correctedauthor line appears below.

Prabuddha Mukherjee, Itamar Kass, Isaiah T. Arkin,and Martin T. Zanni

www.pnas.org�cgi�doi�10.1073�pnas.0602988103

PNAS � May 30, 2006 � vol. 103 � no. 22 � 8571

CORR

ECTI

ON

S

Breast cancer bone metastasis mediated by theSmad tumor suppressor pathwayYibin Kang*†, Wei He*, Shaun Tulley*, Gaorav P. Gupta*, Inna Serganova‡, Chang-Rung Chen*§,Katia Manova-Todorova¶, Ronald Blasberg‡, William L. Gerald�, and Joan Massague*,**

*Cancer Biology and Genetics Program and Howard Hughes Medical Institute, ¶Molecular Cytology Laboratory, and Departments of ‡Neurology and�Pathology, Memorial Sloan-Kettering Cancer Center, NY 10021

Contributed by Joan Massague, August 1, 2005

TGF-� can signal by means of Smad transcription factors, which arequintessential tumor suppressors that inhibit cell proliferation, andby means of Smad-independent mechanisms, which have beenimplicated in tumor progression. Although Smad mutations disablethis tumor-suppressive pathway in certain cancers, breast cancercells frequently evade the cytostatic action of TGF-� while retain-ing Smad function. Through immunohistochemical analysis ofhuman breast cancer bone metastases and functional imaging ofthe Smad pathway in a mouse xenograft model, we provideevidence for active Smad signaling in human and mouse bone-metastatic lesions. Genetic depletion experiments further demon-strate that Smad4 contributes to the formation of osteolytic bonemetastases and is essential for the induction of IL-11, a geneimplicated in bone metastasis in this mouse model system. Acti-vator protein-1 is a key participant in Smad-dependent transcrip-tional activation of IL-11 and its overexpression in bone-metastaticcells. Our findings provide functional evidence for a switch of theSmad pathway, from tumor-suppressor to prometastatic, in thedevelopment of breast cancer bone metastasis.

IL-11 � Smad4 � TGF-�

TGF-� plays a crucial role as a growth-inhibitory cytokine inmany tissues (1, 2). The cytostatic effect of TGF-� is

mediated by a serine�threonine kinase receptor complex thatphosphorylates Smad2 and Smad3, which then translocate intothe nucleus and bind Smad4 to generate transcriptional regula-tory complexes (3). SMAD4 (also known as Deleted in PancreaticCarcinoma locus 4 or DPC4) and, to a lesser extent, SMAD2suffer mutational inactivation in a proportion of pancreatic andcolon cancers (1, 2). However, tumor cells that evade thisantiproliferative control by other mechanisms may display analtered sensitivity to TGF-� and undergo tumorigenic progres-sion in response to this cytokine (1, 2). Patients whose pancreaticor colon tumors express TGF-� receptors fare less well thanthose with low or absent TGF-� receptor expression in the tumor(4). In mouse models of breast cancer, TGF-� signaling pro-motes lung (5, 6) and bone metastasis (7). In the case ofosteolytic bone metastasis by breast cancer cells, it has beenproposed that TGF-� released from the decaying bone matrixstimulates neighboring tumor cells, establishing a vicious cyclethat exacerbates the growth of the metastatic lesion (8).

The TGF-� signaling mechanisms that foster metastasis inhuman cancer are an important open question and a subject ofdebate. Because Smad factors are quintessential tumor suppressors,the basis for the protumorigenic effects of TGF-� has been soughtin the Smad-independent signaling pathways that may be triggeredby TGF-�. Results obtained by means of overexpression of dom-inant negative mutant components of the Rho pathway (9, 10) orpharmacologic inhibitors of p38 mitogen-activated protein kinase(11, 12) have implicated these pathways in the proinvasive andmetastatic effects of TGF-� in transformed cells. In contrast, resultsobtained with overexpression of dominant negative mutant formsof Smad2 or Smad3 have argued for an involvement of the Smadpathway in tumor invasion and metastatic spreading (13) and in the

formation of metastases by oncogenically transformed cells (12, 14)or tumor-derived cell lines (11) xenografted into immunodeficientmice. However, protein overexpression experiments are open tounwanted interference with other cellular functions. The lack ofsuitable genetic evidence has precluded a firm ascription of met-astatic activity to the Smad pathway.

In the present work, we sought to determine whether the Smadpathway is activated in clinical samples of breast cancer bonemetastasis and whether genetic evidence could be obtained for arole of this pathway as a mediator of bone metastasis in a mousemodel system. In a search of organ-specific metastasis mechanisms,we recently isolated variants of a human breast cancer cell line thathave predilection for metastasis to the bones or to the lungs andadrenal glands in mice (15–17). A bone metastasis gene-expressionsignature delineated by using this model system includes, amongmany others genes, IL-11 and connective tissue growth factor(CTGF), which are known targets of TGF-� signaling. Enforcedexpression of IL-11 and CTGF in these cells increases their osteo-lytic bone-metastatic activity (17). We have explored the role of theSmad pathway in bone metastasis using this model system.

Materials and MethodsTumor Sample Analysis. Formalin-fixed paraffin embedded bonemetastasis tissues were obtained from therapeutic proceduresperformed as part of routine clinical management of breast cancerpatients at our institution. Hematoxylin and eosin-stained sectionswere examined for regions that contained tumor cells and stroma,which were further analyzed for phosphorylated Smad2 on serialsections. All studies were conducted under protocols approved bythe Memorial Sloan-Kettering Cancer Center Institutional ReviewBoard.

TGF-�1–Smads–Herpes Simplex Virus 1 (HSV1) Thymidine Kinase (tk)�GFP Reporter System. Double-stranded complementary oligonucle-otides containing a sequence from the mouse germline Ig-� pro-moter 5�-AATTCGGCCATGTGGTCAGACACACCTGTCTCCACCACAGCCAGACCACAGGCCAGACATGACGTG-GAGGTT-3� (18) were used to construct the TGF-�1–Smads–HSV1-tk�GFP reporter vector. After annealing of oligonucleo-tides, the resulting DNA fragment was cloned into the EcoRI andXbaI sites of the dxNFAT-tk�GFP–Neo vector (19) in place of thenuclear factor of activated T cells enhancer element. Thus, theHSV1-tk-EGFP fusion reporter gene was linked to the enhancer

Abbreviations: CTGF, connective tissue growth factor; HSV1, herpes simplex virus 1; tk,thymidine kinase; RFP, red fluorescent protein; tdRFP, tandem repeat RFP; FLuc, fireflyluciferase; AP1, activator protein-1; PET, positron-emission tomography; shRNA, short-hairpin RNA; PTHrP, parathyroid hormone-related peptide.

†Present address: Department of Molecular Biology, Princeton University, Princeton,NJ 08544.

§Present address: ArQule Biomedical Institute, 333 Providence Highway, Norwood,MA 02062.

**To whom correspondence should be addressed at: Cancer Biology and Genetics Program,Box 116, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,NY 10021. E-mail: j-massague@ski.mskcc.org.

© 2005 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0506517102 PNAS � September 27, 2005 � vol. 102 � no. 39 � 13909–13914

CELL

BIO

LOG

Y

elements specific for Smad–Runx transcriptional complexes. Theresulting plasmid was transfected into the GPG29 packaging cellline with Lipofectamine 2000 (Invitrogen). The retrovirus-containing medium was collected for 4 consecutive days and storedat �80°C. The retrovirus was then used to transduce MDA-MB-231cells and their subline SCP3 (15, 17). Selection of stable transfec-tants was accomplished by adding 1 g�liter G418. Cells containingthe TGF-�1–Smads–HSV1-tk�GFP reporter system were furthertransduced with a second retroviral vector, SFG-tdRFP-cmvFLuc,in which tandem repeat red fluorescent protein (tdRFP) (20) andfirefly luciferase (FLuc)-encoding cDNAs were placed under con-stitutive promoters. RFP-positive cells were sorted by FACS. Theretrovirus vector encoding tk–EGFP–luciferase triple fusion pro-teins has been described in ref. 21.

Bioluminescence Imaging and Analysis. Anesthetized mice wereretroorbitally injected with 75 mg�kg D-Luciferin (Xenogen, Al-ameda, CA) in PBS. Bioluminescence images were acquired byusing the IVIS Imaging System (Xenogen) 2–5 min after injection.Acquisition times at the beginning of the time course started at 60sec and were reduced in accord with signal strength to avoidsaturation. Analysis was performed with LIVINGIMAGE software(Xenogen) by measuring photon f lux (measured inphotons�sec�1�cm�2�steradian�1) using a region of interest drawnaround the bioluminescence signal to be measured. Images were setat the indicated pseudocolor scale to show relative bioluminescentchanges over time. Data were normalized to the signal obtainedright after xenografting (day 0).

Micropositron-Emission Tomography (MicroPET) Imaging. MicroPETimaging was performed by using 18F-2�-f luoro-2�deoxy-1�-D-arabionofuranosyl-5-ethyl-uracil as the HSV1-tk substrate, aspreviously described (22). Two hours before whole-body, positron-emission tomography, the mice were administered 18F-2�-fluoro-2�deoxy-1�-D-arabionofuranosyl-5-ethyl-uracil [i.v.; 100 �Ci peranimal (1 Ci � 37 GBq)]. Imaging was performed on a microPET(Concorde Microsystems, Knoxville, TN), and images were ac-quired over 15 min under inhalation anesthesia (2% isoflurane).

Supporting Materials and Methods. For details about cell culture andretroviral transduction, plasmids, luciferase reporter assays, immu-nohistochemistry, transcriptomic profiling and clustering analyses,electrophoretic mobility shift assay, DNA precipitation assay, in-tracardiac injections, radiographic analysis of bone metastasis, andELISA assays, see Supporting Materials and Methods, which ispublished as supporting information on the PNAS web site.

ResultsHuman Breast Cancer Bone Metastases Contain an Activated SmadPathway. Receptor-mediated phosphorylation of Smad2 at the Cterminus and accumulation of phospho-Smad2 in the nucleus aretypical indicators of TGF-� stimulation (3). To determine whetherthis pathway is active in bone metastasis, metastatic tissues frombreast cancer patients were subjected to immunohistochemistrywith anti-phosphopeptide antibodies against receptor-phosphory-lated Smad2. Bone metastasis tissues from 16 breast cancer patientswere obtained from therapeutic procedures performed as part ofroutine clinical management of these patients at our institution.Twelve of these samples showed prominent anti-phospho-Smad2staining (Table 1, which is published as supporting information onthe PNAS web site), and this staining was concentrated in thenucleus (Fig. 1). Nuclear phospho-Smad2 staining was present bothin the tumor cells and cells of the surrounding stroma (e.g., Fig. 1B),suggesting that the entire field was under TGF-� stimulation inthese lesions. The other four metastasis samples analyzed showedlittle or no staining. Thus, a majority of breast cancer bonemetastases exhibited evidence of Smad pathway activation.

Functional Imaging Reveals Smad Signaling in a Bone MetastasisModel. Prompted by these results, we sought evidence for Smad-dependent transcriptional activity in bone metastasis by functionalimaging in a mouse xenograft model. This model is based on theMDA-MB-231 cell line, which was derived from the pleural effu-sions of a breast cancer patient with metastatic disease (23). Fromparental MDA-MB-231 cells, we isolated various single-cell-derived sublines with distinct organ-specific metastatic behavior(16, 17). The subline SCP2 is highly metastatic to bone via arterialcirculation, whereas subline SCP3 is highly metastatic to the adrenalglands. A retroviral reporter vector cis-TGF-�1–Smads–HSV1-tk�GFP was created, in which a fusion protein containing HSV1-tk andGFP was placed under the transcriptional control of a TGF-�-responsive promoter element (Fig. 2A). We chose the TGF-�responsive element from the mouse germline Ig� promoter (24, 25).This TGF-� responsive element is recognized by Smad2�3–Smad4in complex with RUNX family members and responds to TGF-� inmany different cell lines (24, 25). RUNX activity in breast cancercells is implicated in osteolytic bone metastasis (26).

cis-TGF-�1–Smads–HSV1-tk�GFP was transduced into SCP2and SCP3 cells together with a second retroviral vector SFG-tdRFP-cmvFLuc expressing RFP (20) and FLuc under constitutivepromoters (Fig. 2A). The RFP-positive cells expressed greenfluorescence in response to TGF-�, demonstrating responsivenessof the HSV1-tk�GFP construct (Fig. 2 B and C). When inoculatedinto the arterial circulation of immunodeficient mice, SCP2 cellsformed aggressive bone metastases, as visualized by luciferasebioluminescence imaging (Fig. 2D). These lesions also expressed tkactivity, as determined by microPET (Fig. 2D). SCP3 cells formedsmall-bone metastases and very large adrenal (Fig. 2E Upper) andlung metastases when injected into the tail vein (17). Interestingly,although the small-bone metastases formed by SCP3 expressed tkactivity in the live animals, the large adrenal metastases formed bythe same cells did not (Fig. 2E Upper). The location of these lesionswas verified by ex vivo bioluminescence of the affected organs afternecropsy (Fig. 2E Lower). Of nine mice that were inoculated withthe SCP3 cells, five developed adrenal metastasis, none of whichshowed tk activity by microPET. In contrast, two mice developedbone metastases to the skull and vertebrae, and both of these lesionsshowed tk activity (Fig. 2E and data not shown). Therefore, TGF-�

Fig. 1. Activated Smad pathway in breast cancer bone metastasis. Examplesof intense immunohistochemical staining of receptor-phosphorylated Smad2in breast cancer bone metastasis samples from different patients. The samplesshown were chosen to illustrate the nuclear phospho-Smad2 staining in ametastatic island and the surrounding stroma (A), in a cluster of metastaticislands (B), or in a contiguous metastatic mass (C), as well as a cluster of islandsstained with normal rabbit serum as a negative control (D).

13910 � www.pnas.org�cgi�doi�10.1073�pnas.0506517102 Kang et al.

signaling activity could be readily detected in bone metastases butnot in adrenal gland metastases, although all of the metastases wereformed by the same cell line. These results suggest that breastcancer cells undergo Smad-dependent transcriptional activation inthe bone microenvironment.

Smad4-Dependent Transcriptional Activation of Candidate MetastasisGenes. We recently identified a set of genes that mediate osteolyticbone metastasis by MDA-MB-231 cells (17). Among these genes,IL-11 was of interest because it has been proposed to play a role inosteoclast differentiation (27) and as a mediator of osteolysis inbreast cancer bone metastasis (28, 29). Enforced expression ofIL-11 in MDA-MB-231 cells increases their bone-metastatic activ-ity (17). Intriguingly, IL-11 is a TGF-�-inducible gene (17, 30),suggesting a role the prometastatic activity of TGF-� in MDA-MB-231 cells. These cells are defective in TGF-� cytostatic generesponses, including repression of c-myc and Id genes (31), butretain many responses that are common among normal epithelialcells (32), including IL-11 induction (Fig. 3A and Table 2, which ispublished as supporting information on the PNAS web site). Acomparison of the basal expression of TGF-�-responsive genes invarious MDA-MB-231 derivatives revealed a sharp (�9-fold) andselective increase in the basal expression of IL-11 in highly bone-metastatic sublines compared with the poorly metastatic sublinesand with all of the other TGF-�-responsive genes (Table 3, whichis published as supporting information on the PNAS web site;summarized in Fig. 3A). A smaller increase was observed in thebasal expression of CTGF, which is another TGF-�-responsive geneimplicated in bone metastasis by MDA-MB-231 cells (17) (Table 3).In the present study, we chose IL-11 to probe the role of Smadsignaling in the induction of a candidate metastasis gene in thismodel system.

Several observations suggested that IL-11 induction by TGF-� isan immediate gene response. IL-11 induction by TGF-� was rapid,peaking at 2 h and gradually declining thereafter (Fig. 5A, which ispublished as supprotive information on the PNAS web site), and theprotein synthesis inhibitor cycloheximide did not block this re-sponse (data not shown). TGF-� stimulation induced the binding ofSmad2�3 and Smad4 to the IL-11 promoter in chromatin immu-noprecipitation experiments (17). To determine whether the Smadpathway is required for IL-11 induction and bone metastasis, weanalyzed MDA-MB-231 SCP sublines that were depleted of Smad4by means of RNA interference. Compared with parental cells or invivo-selected bone-metastatic populations, which are heteroge-neous, SCPs are derived from single cells and, therefore, are morehomogenous in genetic makeup (15, 17). Three bone-metastaticsublines (SCP2, SCP25, and SCP28) were engineered to stablyexpress the short-hairpin RNA (shRNA) probes Smad4-shRNA1or Smad4-shRNA2, which target different regions of the Smad4mRNA. Expression of Smad4-shRNA1 reduced Smad4 proteinlevels by 70–90% in all three SCPs, whereas Smad4-shRNA2almost completely eliminated Smad4 production (Fig. 5B). As acontrol, we engineered a Smad4 vector (pBabe-hygro-FLAG-Smad4M) containing two silent mutations in the sequence targetedby Smad4-shRNA1 and an N-terminal FLAG epitope distinguish-ing the exogenous product from endogenous Smad4. Transductionof this retrovirus ensured expression of Smad4 in cells containingSmad4-shRNA1 (Fig. 5B).

As determined by Northern blot analysis, the IL-11 response toTGF-� was very weak in cells expressing Smad4-shRNA1 andundetectable in cells expressing Smad4-shRNA2 (Fig. 3B forSCP25; data not shown for SCP2 and SCP28). Expression ofSmad4M restored the TGF-� response in Smad4-shRNA1-expressing cells. A similar response pattern was observed at the

Fig. 2. Functional imaging ofSmad signaling in breast cancerbone metastasis. (A) Schematic rep-resentation of the retroviral vectorsSFG-tdRFP-cmvFLuc (constitutivelyexpressing tdRFP and FLuc) and cis-TGF-�1–Smads–HSV1-tk�GFP (ex-pressing HSV-tk�GFP fusion proteinin response to TGF-�). (B and C)SCP3 transduced with these twovectors were treated with TGF-� orno additions for 24 h and analyzedby fluorescence microscopy (B) ortwo-color FACS (C). The constitu-tive tdRFP fluorescence is shown onthe ordinate, and the HSV-tk�GFPfusion fluorescence, inducible byTGF-�, is shown on the abscissa. (Dand E Upper) In vivo biolumines-cence and microPET imaging of me-tastases in mice. SCP2 (D) and SCP3(E Upper) cells bearing the SFG-tdRFP-cmvFLuc and cis-TGF-�1–Smads–HSV1-tk�GFP vectors wereinjected into the left cardiac ventri-cle and analyzed after 4 weeks(SCP2) or 18 weeks (SCP3). Biolumi-nescence imaging shows sites ofmetastases in the skull (D and E)and adrenal gland (E Upper). 18F-2�-fluoro-2�deoxy-1�-D-arabiono-furanosyl-5-ethyl-uracil microPETimages of tk�GFP reporter activa-tion shows localization of radioac-tivity to the skull in the coronal and sagittal image planes. No visualization of the adrenal metastasis was seen on microPET imaging. Note the nonspecificaccumulation of the tracer in the gastrointestinal tract and bladder attributable to clearance of the tracer. (E Lower) At necroscopy, the head showing the skulland the adrenal metastasis plus kidney were removed and imaged ex vivo for photographic (�) and bioluminescence (�) imaging.

Kang et al. PNAS � September 27, 2005 � vol. 102 � no. 39 � 13911

CELL

BIO

LOG

Y

level of IL-11 protein secretion, as determined by ELISA (Fig. 3Cand data not shown for SCP2 and SCP28). Thus, Smad4 is essentialfor TGF-� activation of IL-11 expression. RNA interference-mediated depletion of Smad4 also inhibited the CTGF response toTGF-� in these cells (data not shown).

Activator Protein-1 (AP1) and Smad Inputs into the TGF-� ResponsiveIL-11 Promoter Region. To further investigate the role of Smadfactors in the IL-11 response to TGF-�, we focused on a 158-bpregion immediately upstream of the TATA box in the IL-11promoter. This region (�100 to �58) was previously mapped bydeletion analysis of the IL-11 �728��58 promoter as the regionthat mediates the TGF-� response in human epithelial and carci-noma cells (30, 33). We confirmed these result by using the sameconstructs in MDA-MB-231 cells, A549 human bronchial carci-noma cells, and HaCaT human keratinocytes (data not shown). Areporter construct under the control of the minimal IL-11 promoter

[pIL-11(�100)-Luc] (30) was unresponsive to TGF-� in the Smad4-deficient breast cancer cell line MDA-MB-468 (34), which was usedhere to assess the role of Smad4 in IL-11 promoter activation byTGF-� (Fig. 5C). Expression of exogenous Smad4 enabled TGF-�induction of this promoter, and this effect was further enhanced bycotransfection of Smad2 or Smad3 (Fig. 5C), arguing that Smadsmediate transcriptional activation from this promoter region.

This 100-bp region includes two AP1 binding sites, which arecritical for IL-11 transcription (30, 33), and an adjacent GC-rich(92% GC) sequence with two putative SP1 sites (Fig. 3D). Nocanonical Smad-binding element (AGAC sequence) is present inthis region. However, Smads can bind to GC-rich sequences incertain promoters (3). Deletion analysis of the IL-11 promoterregion by means of a reporter construct indicated that the responseto TGF-� minimally requires the 5� AP1 site and an adjacentGC-rich sequence (Fig. 3D). The 3�AP1 site, which conforms lesswell to the consensus AP1 binding sequence, contributed little to

Fig. 3. Smad4 and AP1-dependent transcriptional activation of IL-11 by TGF-�. (A) Basal expression levels of 50 TGF-�-activated genes and 21 TGF-�-repressedgenes in MCF-10A and MDA-MB-231 cells were normalized to the same level. (Left) Responses of these genes to TGF-� in each cell line are represented by differentshades of red (degrees of activation) or blue (degrees of repression) in the dendrogram. (Right) The ratio of basal expression levels of these 71 genes in highlymetastatic versus weakly metastatic MDA-MB-231 cells. Genes of interest are highlighted. (B) SCP25 and its derivatives (see Fig. 5B) were incubated in the absenceor presence of TGF-� for 2 h. Total RNA was subjected to Northern blot analysis with the indicated probes. (C) SCP25 and its derivatives were treated with orwithout TGF-� for 24 h. IL-11 production in the media was determined with an ELISA assay. Data are the average of triplicate determinations � SD. (D) (Upper)Nucleotide sequence of the minimal TGF-�-responsive region of the IL-11 promoter. Nucleotide sequence positions are indicated relative to the transcriptionstart site. Two AP1 sites (red boxes) and a GC-rich sequence (green) containing two SP1 sites (green boxes) are indicated. (Lower) (Center and Right) A549 (Center)and MDA-MB-231 (Right) cells were transfected with the indicated IL-11 reporter constructs, treated with or without TGF-� for 16–20 h before lysis, and analyzedfor luciferase activlty. Data are the average of triplicate determinations � SD. (Left) The schematic representation of each promoter construct. (E) [�32-P]ATPend-labeled probes matching to the wild-type IL-11 proximal promoter region, this region with mutant AP1 sites (mAP1), or the indicated fragments of this regionwere subjected to electrophoretic mobility shift analysis with recombinant full-length His-Smad4 protein. Antibody against Smad4 was added as indicated tocreate supershifts. The �-actin promoter was used as a negative control. Schematic representations of the probes are shown above the gel. (F) VariousMDA-MB-231 sublines were transfected with 1 �g of 4xAP1-Luc reporter plasmid and analyzed for luciferase activity 2 days after transfection. Data are theaverage of triplicate determinations � SD. The absolute values of IL 11 mRNA level as detected by an Affymetrix U133A GeneChip were plotted in the same graph(yellow circles). The scales for the luciferase activity and for IL-11GeneChip expression values are shown to the left and right of the graph, respectively.

13912 � www.pnas.org�cgi�doi�10.1073�pnas.0506517102 Kang et al.

the promoter response to TGF-� (Fig. 3D). In electrophoreticmobility shift assays, recombinant Smad4 bound to the wild-typeminimal IL-11 promoter probe, resulting in the formation of acomplex that could be shifted by addition of an anti-Smad4monoclonal antibody (Fig. 3E). Mutation or deletion of the AP1sites decreased but did not abolish Smad4 binding to the probe,whereas the AP1 sites alone did not bind Smad4 (Fig. 3E). Thebinding of endogenous Smad and AP1 factors to this region wasassessed by means of oligonucleotide precipitation assays. MDA-MB-231 cells were incubated with or without TGF-� for 2 h, lysed,and precipitated with biotinylated dsDNA probes. Immunoblottingof DNA-bound factors demonstrated TGF-�-dependent binding ofendogenous Smad3 and Smad4 to the wild-type IL-11 minimalpromoter region and TGF-�-independent binding of the endoge-nous AP1 component JunB to this region (Fig. 5D). Deletion ormutation of the AP1 sites eliminated binding of JunB and weak-ened Smad binding.

Consistent with a role of AP1 in the IL-11 response to TGF-� inthe breast cancer cells, the AP1 activator 12-O-tetradecanoylphor-bol-13-acetate (35) increased the basal level of IL-11 expression aswell as the level upon TGF-� stimulation, whereas the AP1inhibitor curcumin (35) abolished the activation of IL-11 by TGF-�(Fig. 5E). As determined with an AP1 reporter construct (4xAP1-luciferase), the basal level of AP1 activity was significantly higherin the highly metastatic sublines SCP2, SCP25, SCP28, and 1833than in poorly metastatic sublines SCP4 and SCP6 or parentalMDA-MB-231 cells (Fig. 3F). The level of AP1 activity in these cellpopulations was closely correlated with the basal level of IL-11expression (Fig. 3F; refer to Table 3). No change in 4xAP1-luciferase activity was observed after 4 h of TGF-� treatment (datanot shown). Collectively, these results suggest that TGF-�-activatedSmad proteins bind to the GC-rich region in the proximal IL-11promoter. This binding is strengthened by the presence of aproximal AP1 site, and transcriptional activation results fromcooperation between Smad3 and AP1. These observations also

indicate a role of AP1 in the hyperactivity of IL-11 in bone-metastatic MDA-MB-231 cells.

Smad4 Contribution to Breast Cancer Bone Metastasis Formation.Having shown that the TGF-� response of a bone metastasis genein these cells required Smad function, we tested the contribution ofSmad signaling to the metastatic process itself. Wild-type, Smad4-knockdown, and FLAG-Smad4M versions of the various SCP celllines were infected with a retroviral vector expressing HVS1-tk�GFP�luciferase triple fusion protein (21). The cells were inoculatedinto the left cardiac ventricle of immunodeficient mice to allow theformation of bone metastasis. As determined by bioluminescenceimaging of luciferase activity, the inoculated cells became imme-diately distributed throughout the entire animal followed by exten-sive clearing within 1 week (Fig. 4A). Accumulation of luciferasesignal was clear 14 days after injection and became more intenseover the following weeks. To quantify the rate of metastatic growthin bone, a region of interest was drawn around the bone metastasessignals near the joint of the affected hind limbs, and the normalizedphoton counts of each metastasis were plotted (Fig. 4B). A linearcorrelation between the intensity of the bioluminescence and tumorburden is obtained by using this method (36). Suppression of Smad4activity by two different shRNA constructs caused a significantreduction in the growth rate of bone-metastatic lesions (Fig. 4 A andB). Restoration of Smad4 function by the shRNA-insensitiveSmad4M construct restored the wild-type rate of metastatic growth(Fig. 4 A and B). These results were consistently observed insimilarly modified SCP2 and SCP28 cells (data not shown).

Formation of overt osteolytic bone metastases was monitoredby weekly full-body x-ray imaging of the mice. Smad4 depletionconsistently reduced the rate of bone metastasis formation in allthree MDA-MB-231 SCPs and in the in vivo-selected bone-metastatic population, 1833 (17) (Fig. 5C). A significant level ofmetastatic activity still remained after Smad4 depletion, which isconsistent with the TGF-�-independent involvement of othergenes in these lesions (17) and may additionally be due to the

Fig. 4. Smad4 mediation of breastcancer bone metastasis. Wild-typeand genetically modified SCP25 waslabeled with the TGL reporter and1 � 105 cells were injected into theleft cardiac ventricle of five mice foreach cell line. At the indicated daysafter xenografting, bioluminescenceimages were acquired and quanti-fied. (A) Representative mice fromeach group are shown in the supineposition. The intensity of the signalfrom days 24 and 36 are on equiva-lent scales, whereas days 0, 7, and 14are each on separate scales becauseof increasing signal strength and toavoid signal saturation. (B) The nor-malized photon counts from thebone metastases in the hindlimbswere measured over the indicatedtime course. (C) Kaplan–Meier curvesshowing the incidence of bone me-tastasis by indicated wild-type andSmad4-knockdown MDA-MB-231sublines. For each cell sample, 105

tumor cells were inoculated into theleft cardiac ventricle of 10 nude mice.Metastasis was scored as the time tofirst appearance of a visible bone le-sion by x-ray imaging of the wholemouse. The percentages of animals in each group and in all groups combined that were free of detectable bone metastases are plotted. *, � 0.05; **, P � 0.01;calculated by log rank test. (D) Tumor cells (106) were injected s.c. into nude mice. s.c. tumor growth was monitored and quantified by caliper measurements.No significant difference was found between wild-type and Smad4-knowdown cells.

Kang et al. PNAS � September 27, 2005 � vol. 102 � no. 39 � 13913

CELL

BIO

LOG

Y

incomplete elimination of Smad4 by RNA interference. Smad4knockdown did not decrease the growth rate of the SCPs or 1833cells in culture (data not shown) or their ability to form s.c.tumors in mice (Fig. 5D), arguing that the Smad4-dependentgrowth of these tumors is specifically stimulated by the bonemicroenvironment.

DiscussionWe provide clinical, genetic, and functional evidence suggestingthat the Smad tumor suppressor pathway may become prometa-static in breast cancer. Our results show the presence of receptor-phosphorylated Smad2 in a majority of bone metastasis samplesfrom a cohort of breast cancer patients treated at our institution. Bymeans of noninvasive functional imaging, we show that MDA-MB-231 human breast cancer cells growing as bone metastases in miceare engaged in Smad-dependent transcription. Furthermore, RNAinterference-mediated depletion of Smad4 inhibited bone metas-tasis in this mouse xenograft model. Previous evidence implicatingthe Smad2�3 pathway in metastasis was based on overexpression ofmutant Smad constructs (12–14). However, because protein over-expression approaches are prone to unwanted interference withother cellular functions, the involvement of Smad signaling inmetastasis remained the subject of debate against observationsimplicating Smad-independent pathways (9–12). The present re-sults clearly argue that signaling through the Smad pathway canfacilitate breast cancer bone metastasis.

IL-11 and CTGF were among 43 genes whose expression waselevated in MDA-MB-231 subpopulations selected in vivo for highbone-metastatic activity (17). The expression of endogenous IL-11and CTGF in these highly metastatic isolates is further increased byTGF-� addition. Exogenous overexpression of IL-11 and CTGFmediates osteolytic metastatic activity in MDA-MB-231 xenografts(17). Therefore, we chose IL-11 as a model metastasis gene in thepresent studies. We provide evidence that IL-11 induction byTGF-� involves Smad2�3 and Smad4, which cooperate with apreviously described AP1 input (30, 33). Our results also show thatSmad4 is essential for IL-11 induction by TGF-�. Interestingly, anincrease in the level of AP1 activity appears to be responsible forthe elevated expression of IL-11 in highly bone-metastatic MDA-MB-231 subpopulations. In separate studies, we have determinedthat the induction of CTGF by TGF-� is also a Smad-dependentprocess. But it does not involve a cooperation of Smads with AP1(our unpublished observations).

The present results provide insights into the mechanism ofactivation of IL-11 in this model system. It is important to note thatIL-11 (and CTGF) can only be considered candidate bone metas-tasis genes at present. IL-11 is known to stimulate osteoclastic boneresorption in vitro (27–29), but it has complex effects on boneformation, including positive effects (37). Its role as a mediator ofbone metastasis in humans remains to be established. OtherTGF-�-responsive secretory factors may also be involved in thisprocess. Chief among these is parathyroid hormone-related peptide(PTHrP), whose role as a mediator of breast cancer metastasis hasbeen shown in several studies using MDA-MB-231 cells (7, 38).However, unlike the induction of IL-11 and CTGF by TGF-�, theincrease in PTHrP secretion in these cells occurs without anincrease in PTHrP mRNA levels (our unpublished work).

The intrinsic genomic instability of tumor cell populations allowsfor the selection of functions that favor growth in a given environ-ment. Thus, a bone-metastatic lesion will harbor functions that thebone environment selects for. We speculate that prometastaticSmad-mediated gene responses can emerge once this pathwaybecomes uncoupled from tumor-suppressor effects. If at that pointa Smad pathway can provide metastatic functions to cancer cells, itlikely will be selected as a prometastatic force. Certain Smad-responsive genes could provide an advantage to cancer cells in aTGF-�-rich bone microenvironment. Therefore, an increase in thebasal expression of these genes coupled with their further inductionby bone-derived TGF-� would favor tumor growth in the bone. Ourresults are consistent with this possibility. By implicating the Smadpathway in the osteolytic vicious cycle of breast cancer metastasis(8), our results additionally call further attention to the possibilityof therapeutically targeting this pathway (10, 39) in TGF-�-richmetastatic sites.

We would like to thank Y.-C. Yang (Case Western Reserve University) forreagents and A. Minn and L. Jayaraman for advice. We acknowledge the useof the Genomics Core Facility and the Flow Cytometry Core Facility atMemorial Sloan-Kettering Cancer Center. This research is supported by theW. M. Keck Foundation and National Institutes of Health Grant P01-CA94060 (to J.M.) and by National Institutes of Health Grant P50-CA86438(to R.B.) and U.S. Army Medical Research Grant DAMD17-02-0484 (toW.L.G.). Y.K. was the recipient of a postdoctoral fellowship from theIrvington Institute for Immunological Research. G.P.G. was supported byNational Institutes of Health Medical Scientist Training Program GrantGM07739, a fellowship from the Katherine Beineke Foundation, andDepartment of Defense Breast Cancer Research Program PredoctoralTraineeship Award W81XWH-04-1-0334. J.M. is an Investigator of theHoward Hughes Medical Institute.

1. Derynck, R., Akhurst, R. J. & Balmain, A. (2001) Nat. Genet. 29, 117–129.2. Siegel, P. M. & Massague, J. (2003) Nat. Rev. Cancer 3, 807–821.3. Shi, Y. & Massague, J. (2003) Cell 113, 685–700.4. Watanabe, T., Wu, T. T., Catalano, P. J., Ueki, T., Satriano, R., Haller, D. G., Benson, A. B.,

III, & Hamilton, S. R. (2001) N. Engl. J. Med. 344, 1196–1206.5. Siegel, P. M., Shu, W., Cardiff, R. D., Muller, W. J. & Massague, J. (2003) Proc. Natl. Acad.

Sci. USA 100, 8430–8435.6. Muraoka-Cook, R. S., Kurokawa, H., Koh, Y., Forbes, J. T., Roebuck, L. R., Barcellos-Hoff,

M. H., Moody, S. E., Chodosh, L. A. & Arteaga, C. L. (2004) Cancer Res. 64, 9002–9011.7. Yin, J. J., Selander, K., Chirgwin, J. M., Dallas, M., Grubbs, B. G., Wieser, R., Massague,

J., Mundy, G. R. & Guise, T. A. (1999) J. Clin. Invest. 103, 197–206.8. Mundy, G. R. (2002) Nat. Rev. Cancer 2, 584–593.9. Bhowmick, N. A., Ghiassi, M., Bakin, A., Aakre, M., Lundquist, C. A., Engel, M. E., Arteaga,

C. L. & Moses, H. L. (2001) Mol. Cell. Biol. 12, 27–36.10. Dumont, N. & Arteaga, C. L. (2003) Cancer Cell 3, 531–536.11. Kakonen, S. M., Selander, K. S., Chirgwin, J. M., Yin, J. J., Burns, S., Rankin, W. A., Grubbs,

B. G., Dallas, M., Cui, Y. & Guise, T. A. (2002) J. Biol. Chem. 277, 24571–24578.12. Tian, F., Byfield, S. D., Parks, W. T., Stuelten, C. H., Nemani, D., Zhang, Y. E. & Roberts,

A. B. (2004) Cancer Res. 64, 4523–4530.13. Oft, M., Akhurst, R. J. & Balmain, A. (2002) Nat. Cell. Biol. 4, 487–494.14. Tian, F., DaCosta Byfield, S., Parks, W. T., Yoo, S., Felici, A., Tang, B., Piek, E., Wakefield,

L. M. & Roberts, A. B. (2003) Cancer Res. 63, 8284–8292.15. Minn, A. J., Kang, Y., Serganova, I., Gupta, G. P., Giri, D. D., Doubrovin, M., Ponomarev,

V., Gerald, W. L., Blasberg, R. & Massague, J. (2005) J. Clin. Invest. 115, 44–55.16. Minn, A. J., Gupta, G. P., Siegel, P. M., Bos, P. D., Shu, W., Giri, D. D., Viale, A., Olshen,

A. B., Gerald, W. L. & Massague, J. (2005) Nature 436, 518–524.17. Kang, Y., Siegel, P. M., Shu, W., Drobnjak, M., Kakonen, S. M., Cordon-Cardo, C., Guise,

T. A. & Massague, J. (2003) Cancer Cell 3, 537–549.18. Jakubowiak, A., Pouponnot, C., Berguido, F., Frank, R., Mao, S., Massague, J. & Nimer,

S. D. (2000) J. Biol. Chem. 275, 40282–40287.19. Ponomarev, V., Doubrovin, M., Lyddane, C., Beresten, T., Balatoni, J., Bornman, W., Finn,

R., Akhurst, T., Larson, S., Blasberg, R., et al. (2001) Neoplasia 3, 480–488.

20. Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A.& Tsien, R. Y. (2002) Proc. Natl. Acad. Sci. USA 99, 7877–7882.

21. Ponomarev, V., Doubrovin, M., Serganova, I., Vider, J., Shavrin, A., Beresten, T., Ivanova, A.,Ageyeva, L., Tourkova, V., Balatoni, J., et al. (2004) Eur. J. Nucl. Med. Mol. Imaging 31, 740–751.

22. Serganova, I., Doubrovin, M., Vider, J., Ponomarev, V., Soghomonyan, S., Beresten, T.,Ageyeva, L., Serganov, A., Cai, S., Balatoni, J., et al. (2004) Cancer Res. 64, 6101–6108.

23. Cailleau, R., Young, R., Olive, M. & Reeves, W. J., Jr. (1974) J. Natl. Cancer Inst. 53, 661–674.24. Hanai, J., Chen, L. F., Kanno, T., Ohtani-Fujita, N., Kim, W. Y., Guo, W. H., Imamura, T.,

Ishidou, Y., Fukuchi, M., Shi, M. J., et al. (1999) J. Biol. Chem. 274, 31577–31582.25. Pardali, E., Xie, X. Q., Tsapogas, P., Itoh, S., Arvanitidis, K., Heldin, C. H., ten Dijke, P.,

Grundstrom, T. & Sideras, P. (2000) J. Biol. Chem. 275, 3552–3560.26. Javed, A., Barnes, G., Pratap, J., Antkowiak, T., Gerstenfeld, L. C., van Wijnen, A. J., Stein,

J. L., Lian, J. B. & Stein, G. S. (2005) Proc. Natl. Acad. Sci. USA 102, 1454–1459.27. Girasole, G., Passeri, G., Jilka, R. L. & Manolagas, S. C. (1994) J. Clin. Invest. 93, 1516–1524.28. Sotiriou, C., Lacroix, M., Lespagnard, L., Larsimont, D., Paesmans, M. & Body, J. J. (2001)

Cancer Lett. 169, 87–95.29. Morgan, H., Tumber, A. & Hill, P. A. (2004) Int. J. Cancer 109, 653–660.30. Tang, W., Yang, L., Yang, Y. C., Leng, S. X. & Elias, J. A. (1998) J. Biol. Chem. 273, 5506–5513.31. Chen, C. R., Kang, Y. & Massague, J. (2001) Proc. Natl. Acad. Sci. USA 98, 992–999.32. Kang, Y., Chen, C. R. & Massague, J. (2003) Mol. Cell 11, 915–926.33. Bamba, S., Andoh, A., Yasui, H., Makino, J., Kim, S. & Fujiyama, Y. (2003) Am. J. Physiol.

285, G529–G538.34. Schutte, M., Hruban, R. H., Hedrik, L., Cho, K. R., Nadasdy, G. M., Weinstein, C. L., Bova,

G. S., Isaacs, W. B., Cairns, P., Nawroz, H., et al. (1996) Cancer Res. 56, 2527–2530.35. Eferl, R. & Wagner, E. F. (2003) Nat. Rev. Cancer 3, 859–868.36. Contag, C. H., Spilman, S. D., Contag, P. R., Oshiro, M., Eames, B., Dennery, P., Stevenson,

D. K. & Benaron, D. A. (1997) Photochem. Photobiol. 66, 523–531.37. Takeuchi, Y., Watanabe, S., Ishii, G., Takeda, S., Nakayama, K., Fukumoto, S., Kaneta, Y.,

Inoue, D., Matsumoto, T., Harigaya, K. & Fujita, T. (2002) J. Biol. Chem. 277, 49011–49018.38. Guise, T. A., Yin, J., Taylor, S. D., Kumagai, Y., Dallas, M., Boyce, B. F., Yoneda, T. &

Mundy, G. R. (1996) J. Clin. Invest. 1544–1549.39. Yingling, J. M., Blanchard, K. L. & Sawyer, J. S. (2004) Nat. Rev. Drug Discovery 3, 1011–1022.

13914 � www.pnas.org�cgi�doi�10.1073�pnas.0506517102 Kang et al.