lrp6 ectodomain prevents sdf-1/cxcr4-induced breast cancer … · 2019. 6. 4. · translational...

Translational Cancer Mechanisms and Therapy

LRP6 Ectodomain Prevents SDF-1/CXCR4-Induced Breast Cancer Metastasis to LungJiankangZhang1, JinxiaoChen2, DaWo2, Hongwei Yan1, Peng Liu1, EnMa1, Limei Li1,Liang Zheng1, Daxin Chen3, Zuoren Yu1, Chunli Liang4, Jun Peng3, Dan-ni Ren3, andWeidong Zhu1

Abstract

Purpose: Lung metastasis is an important cause of breastcancer–related deaths, in which SDF-1/CXCR4 signaling path-way plays a critical role. Single transmembrane protein LRP6is viewed as an oncogene via activating the Wnt/b-cateninsignaling pathway. Our work aims to investigate the relation-ship between SDF-1/CXCR4 and LRP6 in breast cancer lungmetastasis.

Experimental Design: We examined the expressions andfunctions of SDF-1/CXCR4 and LRP6 as well as their relation-ship in breast cancer in vitro and in vivo.

Results: LRP6 ectodomain (LRP6N) directly bound toCXCR4 and competitively prevented SDF-1 binding toCXCR4. LRP6N prevented SDF-1/CXCR4-induced metasta-sis to lung and prolonged survival in mice bearing breasttumors, whereas LRP6 knockdown activated SDF-1/CXCR4

signal transduction and promoted lung metastasis andtumor death. Furthermore, patients with breast cancerwith high CXCR4 expression had poor prognosis, whichwas exacerbated by low LRP6 expression but improved byhigh LRP6 expression. Interestingly, a secreted LRP6N wasfound in the serum of mice and humans, which wasdownregulated by the onset of cancer metastasis in bothmice bearing breast cancer as well as in patients withbreast cancer.

Conclusions: LRP6N might be a promising diagnosticmarker for the early detection of breast cancer metastasis aswell as an inhibitor of SDF-1/CXCR4-induced breast cancermetastasis. LRP6N also provides an interesting link betweenWnt signaling and SDF-1/CXCR4 signaling, the two key path-ways involved in cancer development.

IntroductionBreast cancer is the most commonly diagnosed cancer world-

wide, resulting in 6.9% and 14% of all cancer-related deaths inChina and America, respectively (1, 2). The majority of breast

cancer–related deaths is caused by metastasis. Breast cancermetastasis may occur at any time from the primary site, whichleaves patients constantly at risk of distant metastases occur-rences (3). Metastases arise after cancer cells escape from theprimary site, survive in the blood, and consequently form newtumors in distant organs (4). One of the major metastatic organsas a result of breast cancermetastasis is the lung (5). Of note, SDF-1/CXCR4 signaling pathway has been well-known to promotebreast cancer metastasis to lung.

SDF-1 activates downstream targets such as c-Jun, ERK1/2, or Aktand induces cell migration and invasion by binding to its receptorCXCR4 (6, 7). In rat mammary adenocarcinoma MTLn3 cells,SDF-1 overexpression promotes their metastasis to the lung (8).The levelofSDF-1 expression isalso significantly correlatedwith thesurvival of human patients with breast cancer (9). Furthermore,knockdown or chemical inhibition of CXCR4dramatically inhibitsbreast cancermetastasis to lung (10, 11). Patientswith breast cancerwith high CXCR4 expression also show a higher risk of distantmetastasis, along with reduced survival (12). Gene screening anal-ysis lists CXCR4 as a signature gene that mediates breast cancermetastasis to lung (13). These results support the notion thatSDF-1/CXCR4 signaling pathway plays a critical role in promotingbreast cancer metastasis to lung.

LRP6 is commonly regarded as a Wnt coreceptor involved inactivating b-catenin–dependent canonical Wnt signaling pathway,which promotes tumor progression (14, 15). Upon binding toWntligands, LRP6 cooperates with a separate Wnt receptor Frizzled toactivate Wnt/b-catenin signaling pathway. Wnt ligand–inducedtriple complex formation of Wnt-LRP6-Frizzled at the cell surfaceprevents ubiquitination and degradation of cytoplasmic b-catenin,leading to the nuclear translocation of b-catenin and subsequent

1Clinical and Translational Research Center, Research Institute of Heart FailureShanghai East Hospital, Key Laboratory of Arrhythmias of Ministry of Education,Tongji University School of Medicine, Shanghai, China. 2Department of Plasticand Reconstructive Surgery, Ninth People's Hospital, Shanghai Jiaotong Uni-versity School of Medicine, Shanghai, China. 3Fujian Key Laboratory of Integra-tive Medicine on Geriatric, Academy of Integrative Medicine, Fujian University ofTraditional Chinese Medicine, Fuzhou, Fujian, China. 4Department of SurgeryEast Hospital, Tongji University School of Medicine, Shanghai, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

J. Zhang and J. Chen contributed equally to this article.

Corresponding Authors: Weidong Zhu, Clinical and Translational ResearchCenter Shanghai East Hospital, Key Laboratory of Arrhythmias of Ministry ofEducation of China, Tongji University School of Medicine, No. 150 Jimo Road,Shanghai 200120, China. Phone: 8621-6598-6073; Fax: 0086-591-22861157;E-mail: [email protected]; Jun Peng, Fujian Key Laboratory of IntegrativeMedicine on Geriatric, Academy of Integrative Medicine, Fujian University ofTraditional Chinese Medicine, No. 1 Qiuyang Road, Fuzhou, Fujian 350122, China.Phone: 86-591-22861303; Fax: 0086-591-22861157; Email: [email protected];and Dan-ni Ren, Fujian Key Laboratory of Integrative Medicine on Geriatric,Academy of Integrative Medicine, Fujian University of Traditional Chinese Med-icine, No. 1 QiuyangRoad, Fuzhou, Fujian 350122, China. Phone: 86-591-22861303;Fax: 0086-591-22861157; Email: [email protected].

Clin Cancer Res 2019;XX:XX–XX

doi: 10.1158/1078-0432.CCR-18-3557

�2019 American Association for Cancer Research.

ClinicalCancerResearch

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activation of Wnt target genes, including c-Myc, cyclin D1, andAxin2 (16). As the coreceptor and key activator of Wnt/b-cateninsignaling pathway, LRP6 has been well established to act as anoncogene. Indeed, overexpression of LRP6 contributes to tumor-igenesis and tumor growth (17, 18), whereas knockdown orinhibition of LRP6 suppresses tumor growth in breast cancer andcolorectal cancer (19, 20).

Notably, our recent study showed that LRP6 directly bound toand inhibited Frizzled through its ectodomain (21). AlthoughLRP6 is a single transmembrane protein, Frizzled belongs to theseven transmembrane GPCR family. Interestingly, we found thatseveral GPCRs also bound to LRP6 when we initially intended touse them as negative controls to study LRP6–Frizzled interaction.In this study, we showed that LRP6 ectodomain (LRP6N) alsobound to and inhibited CXCR4, a separate GPCR. We furtherdemonstrated that LRP6N completely prevented SDF-1/CXCR4-induced breast cancer metastasis to lung and significantly pro-longed the survival of these mice. Interestingly, a secreted form ofendogenous LRP6N existed in the serum of human and mouse.Serum LRP6N was downregulated in the early stage of tumorprogression and its downregulation was correlated with breastcancer metastasis.

Materials and MethodsCoimmunoprecipitation/Western blot analyses

Coimmunoprecipitation (co-IP) and Western blot (WB) anal-yses were performed as described previously (22). Briefly, plas-mids were transiently transfected into HEK293 cells for 48 hours.Cells were then lysed using NP-40 lysis buffer (Beyotime) and IPwas performed using Anti-Flag M2 Affinity Gel (Sigma), anti-c-Myc Agarose (Thermo Fisher Scientific), or Protein A/G MagneticBeads (Bimake) with Anti-His-antibody (ProteinTech). Thelysates were heated in loading buffer at 95�C for 5 minutes andseparated by SDS-PAGE gel, then transferred to a polyvinylidenedifluoride membrane. The membrane was blocked with TBSTcontaining 5% nonfat milk powder and incubated with the

indicated primary antibodies at 4�C overnight. Complete list ofantibodies used are listed in Supplementary Table S1. Followingincubation with secondary antibody at room temperature for 1hour, the protein bands were visualized using Immobilon West-ern Chemiluminescent HRP Substrate (Millipore). Results arerepresentative of at least three independent experiments.

Tumor implantation in miceAll animal protocols were approved by the Animal Care and

Use Committee of Tongji University School of Medicine (Shang-hai, China) and followed the ARRIVE guidelines. Six-week-oldfemale nude mice and BALB/c mice were used for tail veininjection or mammary orthotopic implantation. A total of 1 �106 4T1 cells were injected in all models, unless otherwisespecified. Tumor diameters were measured with digital calipersand the tumor volume in mm3 was calculated by the formula:Volume ¼ width2 � length/2. Lung surface macroscopic metas-tases were counted after fixation with 4% paraformaldehyde anddehydration using ethanol gradient. For histology examination,the left and right lungs of each animal were fixed in 4% parafor-maldehyde and sectionswere stainedwith hematoxylin and eosin(H&E). Bioluminescence imaging of mice and organs was per-formed as described previously (23).

Breast cancer patients and samplesWomen who were diagnosed with malignant breast cancer

between the ages 15 and 75 were enrolled in the study. Patientswith breast cancer who were lactating, pregnant, or had otherseriousdiseaseswere excluded fromthe study. Similar agedhealthywomen without serious diseases were included as normal people,designated as the control group. Blood samples of patients withbreast cancer were collected from Shanghai East Hospital andblood serum was collected by centrifugation and stored at�80�Cuntil further analysis. Lymph node metastases and distant metas-tases were histologically diagnosed for each patient. Patients withbreast cancer were divided into metastasis group and nonmetas-tasis group according to the presence of lymph node metastasesand distant metastases. All breast cancer samples were collectedduringoperationwithwritten informed consents from thepatientsandwere approved by the ethical and institutional review board ofShanghai East Hospital (No. 37 in 2018) and followed the provi-sions of theDeclaration ofHelsinki. Serum samples from similarlyagedpatientswithbreast cancer andnormalpeoplewere randomlydivided into three groups. Western blot densitometry analysis foreach serum sample was measured using ImageJ software, normal-ized to thecontrol group, andexpressedas a relative level ofproteinexpression. Differences in serum LRP6 level between normalpeople and patients with breast cancer with or without metastasiswere compared using one-way ANOVA, followed by LSD-t post hocanalysis. P < 0.05 was considered statistically significant.

Cancer patient survival analysisDistant metastasis-free survival curve and overall survival curve

in patients with breast cancer were generated on the basis of theNKI295 dataset (24) and analyzed using Kaplan–Meier survivalanalysis coupled with a log-rank significance test. For LRP6,distantmetastasis-free survival and overall survival were analyzedbetween 143 high-LRP6 level and 152 low-LRP6 level patients.For CXCR4, distant metastasis-free survival and overall survivalwere analyzed between 199 high-CXCR4 level and 96 low-CXCR4level patients.

Translational Relevance

Wnt coreceptor LRP6 is a single transmembrane protein.Unexpectedly, we identified a secreted form of LRP6 in serumthat was solely comprised of LRP6 ectodomain (LRP6N),which was rapidly downregulated at the onset of tumormetastasis in both mice and patients with breast cancer. TheserumLRP6Nwas easily detectable using only<1mL serumviaWestern blot analysis. Surprisingly, a recombinant LRP6Nprotein completely prevented breast cancer metastasis to lungand prolonged the survival of mice via binding to CXCR4 andpreventing SDF-1/CXCR4 signal transduction. Notably, SDF-1/CXCR4 signaling plays a key role in promoting breast cancermetastasis to lung. Furthermore, clinical data showed thatCXCR4high/LRP6low expression signature was associated withpoor prognosis in patients with breast cancer, but not thosewith CXCR4high/LRP6high expression, indicating that LRP6 hasa role in preventing human breast cancer. Thus, serum LRP6Nmight be not only a sensitive diagnostic marker for breastcancer metastasis but also a promising therapeutic agent forprevention of breast cancer metastasis.

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Statistical analysisAll data were expressed as mean � SEM unless otherwise

specified. Unpaired t test was employed to indicate statisticalsignificance between two groups. One-way ANOVA analysis wasused to compare total difference, followed by LSD-t post hocanalysis to showdifferences between two groups unless otherwisespecified. P < 0.05 was considered statistically significant.

ResultsLRP6N and SDF-1 competitively bind to CXCR4

To investigate the relationship between LRP6 and CXCR4, wefirst cotransfected Myc-tagged LRP6N with Flag-tagged CXCR4and examined their binding using co-IP/WB analyses. We foundthat Myc-tagged LRP6N bound to Flag-tagged CXCR4, but notFlag-tagged SDF-1 (Fig. 1A). As a positive control,His-tagged SDF-1 bound to Flag-tagged CXCR4 (Fig. 1A, right). These resultssuggest a physical interaction between LRP6N and CXCR4.

Research has shown that a soluble SDF-1 protein added tocultured cells can bind to CXCR4 on the cell surface (25). Usingthe same method, we treated cells with His-tagged SDF-1–conditioned medium (CM), and the His-tagged SDF-1 band wasdetected with anti-His antibody usingWB analysis (Fig. 1B, panel1). Notably, the band density of His-tagged SDF-1 was increasedfollowing transfectionwith Flag-taggedCXCR4plasmid in a dose-dependent manner (Fig. 1C, panel 1), confirming that thesoluble SDF-1 protein added to cultured cells can bind to CXCR4on the cell surface. Likewise, WB analysis using cell lysates fromMyc-tagged LRP6N-CM–treated cells showed the binding ofLRP6N to the cell surface (Fig. 1B, panel 1), which was promotedby transfectionwithCXCR4 in a dose-dependentmanner (Fig. 1B,panel 2), butwasnot affectedby transfectionwith varyingdoses ofSDF-1 plasmid (Fig. 1B, panel 3), suggesting that the solubleLRP6N protein specifically binds to CXCR4 on the cell surface.However, the binding of Myc-tagged LRP6N to Flag-taggedCXCR4 was prevented by pretreatment with His-tagged SDF-1-CM in a dose-dependent manner (Fig. 1B, panel 4), indicatingthat SDF-1 competitively prevents LRP6N binding to CXCR4.Conversely, pretreatment of LRP6N-CM also prevented SDF-1binding toCXCR4 in a dose-dependentmanner (Fig. 1C, panel 2).Furthermore, coincubation of SDF-1-CM at a constant concen-tration with various concentrations of LRP6N-CM in CXCR4-transfected cells resulted in a typical competitive binding curveof LRP6N inhibition onSDF-1binding toCXCR4 (Fig. 1D),whichwas similar to the competitive binding curve of CXCR4 antagonistAMD3100 inhibition on SDF-1 binding to CXCR4 (25). Theseresults indicate that LRP6N can prevent SDF-1 binding to CXCR4in a competitive manner (Fig. 1E).

LRP6N inhibits SDF-1/CXCR4 signal transductionWe next examined whether LRP6N affected SDF-1/CXCR4

signal transduction.We first generated 4T1 cell lines stably expres-sing LRP6N, aswell as empty vector as a control (Fig. 2A, bottom).We also constructed a separate 4T1 cell line stably expressing theectodomain of Kremen1 (Kremen1N) as a control, which func-tions as an unrelated single transmembrane receptor (Fig. 2A,bottom).

SDF-1 stimulation significantly activated c-Jun, ERK1/2,and Akt in 4T1/Vector and 4T1/Kremen1N cells, but not in4T1/LRP6N cells (Fig. 2A). Pretreatment with a recombinantprotein or conditioned medium of LRP6N, but not Kremen1N,

also prevented SDF-1–induced activations of c-Jun, ERK1/2, andAkt (Fig. 2B; Supplementary Fig. S1A). Furthermore, SDF-1 over-expression–induced activations of c-Jun, ERK1/2, and Akt in 4T1cells were also prevented by treatment with LRP6N-CM in a dose-dependent manner (Fig. 2C). As a positive control, pretreatmentwith AMD3100, a specific antagonist of CXCR4, also inhibitedSDF-1–induced activations of c-Jun, ERK1/2, and Akt in4T1/Vector cells (Supplementary Fig. S1B). In contrast, H2O2

stimulation activated c-Jun, ERK1/2, and Akt in both 4T1/Vectorand 4T1/LRP6N cells (Supplementary Fig. S1C). Furthermore,SDF-1 stimulation induced nuclear translocation of b-arrestin1, adirect downstream target of CXCR4 in 4T1/Vector cells, whichwasinhibited in 4T1/LRP6N cells or following pretreatment withAMD3100 (Fig. 2D). Taken together, these results suggest thatLRP6N specifically inhibits SDF-1/CXCR4 signal transductionthrough binding to CXCR4.

LRP6N is secreted under physiologic conditionInterestingly, we found a secreted form of LRP6 fragment in the

culture media of 4T1 cells, as well as in mouse breast cancer168FARN cells, human breast cancer MDA-MB231, Hs578T, andMCF-7 cells, and normal human HEK293 cells (Fig. 3A and B;Supplementary Fig. S2A). The LRP6 fragment was detected by ananti-LRP6 antibody against its N-terminal but not C-terminal andhad similar size to the recombinant LRP6N protein with a molec-ular weight of approximately 170 kDa (Fig. 3A and B; Supple-mentary Fig. S2A). Knockdown of endogenous LRP6 by siRNAsignificantly reduced the expression levels of both full-lengthLRP6 and the LRP6 fragment in these cells (Fig. 3C; Supplemen-tary Fig. S2B). In contrast, cooverexpression of C-terminalMyc- orGFP-tagged LRP6 with Mesd, a molecular chaperone that facil-itates the folding and cell-surface localization of LRP6 (26),resulted in an increase of the LRP6 fragment in cell culturemedium (Fig. 3D). Notably, although the band of GFP-LRP6 washigher than that of Myc-LRP6 in cell lysate, the two bands of theLRP6 fragmentswere the same (Fig. 3D). These results suggest thatthe fragment detected by the anti-LRP6 antibody against itsN-terminal is a truncated form of LRP6 that lacks its C-terminaldomain. We also detected this LRP6 fragment in mouse serum,which was downregulated in LRP6 knockout mice (Fig. 3E). Howthe LRP6 fragment is produced and released requires furtherinvestigation.

Downregulation of serum LRP6N with breast tumor metastasisWe further observed the level of serum LRP6N in mice bearing

breast tumors. Serum LRP6N was downregulated at day 10following subcutaneous injection of 4T1 cells in BALB/c mice(Fig. 3F). Intriguingly, tail vein injection of 4T1 cells induced animmediate downregulation of serum LRP6N at day 1 (Fig. 3G).Because cells could be rapidly delivered to target organs via tailvein injection, whereas orthotopic injection induced a delayedmetastasis to target organs, these results suggest that the down-regulation of serum LRP6N level is likely triggered by the metas-tasis of cancer cells to target organs.

Likewise, a delayed downregulation of serum LRP6N wasobserved at day 10 following subcutaneous implantation of4T1-Luc cells (Fig. 3H), a 4T1 subline stably expressing luciferasethat can be used to easily findmetastatic tumor cells by examiningluciferase activity, in nude mice. At day 6, luciferase activity wasundetected in lung, while the level of serum LRP6N wasunchanged (Fig. 3H and I). Interestingly, at day 10, there was a

LRP6 Ectodomain Prevents Breast Cancer Metastasis

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slight increase in the luciferase activity in the lungs ofmice bearingtumors compared with control mice (Fig. 3I), indicating that asmall number of 4T1-Luc cells have alreadymetastasized to lungs.We further mixed cell lysates from various cultured 4T1-Luc cellswith lysates from thewhole lung ofwild-typemice, and examinedhow many metastatic 4T1-Luc cells were needed to detect the

luciferase activity (Supplementary Fig. S2C). Using this method,we inferred that there were approximately 200 metastatic cells inthe lungs at day 10 (Supplementary Fig. S2C). We also injecteddifferent amounts of 4T1-Luc cells via tail vein injection,and found that as few as 100 metastatic 4T1-Luc cells couldinduce serum LRP6N downregulation in BALB/c mice (Fig. 3J;

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LRP6N competitively prevents SDF-1 binding to CXCR4 by physically interacting with CXCR4.A, Co-IP/WB analysis following transfection with the indicatedplasmids for 48 hours in HEK293 cells. Images are representative of three independent experiments. B,WB analysis of CXCR4- or SDF-1–transfected HEK293cells following incubation of LRP6N-CM for 30minutes, in the presence or absence of SDF-1-CM pretreatment. Adjacent images are derived from a singleimmunoblot for clearer presentation. Images are representative of three independent experiments. C,WB analysis of CXCR4-transfected HEK293 cells followingincubation of SDF-1-CM for 30minutes, in the presence or absence of LRP6N-CM pretreatment. Adjacent images are derived from a single immunoblot forclearer presentation. Images are representative of three independent experiments. D, Competitive binding curve of LRP6N and SDF-1 in CXCR4-transfectedHEK293 cells. Each condition was assayed in duplicate, and data are representative of three independent experiments (mean� SEM). E, Schematic illustration ofthe prevention of LRP6N on SDF-1 binding to CXCR4.

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LRP6N inhibits SDF-1/CXCR4 signal transduction. A,WB analysis and quantification of SDF-1–induced signal transduction in 4T1/Vector, 4T1/LRP6N, and 4T1/Kremen1N cells. � , P < 0.05, one-way ANOVA. Data are the mean� SEM of three independent experiments. B,WB analysis and quantification of SDF-1–inducedsignal transduction after pretreatment with LRP6N (1 mg/mL) and Kremen1N protein (1 mg/mL). � , P < 0.05, one-way ANOVA. Data are the mean� SEM of threeindependent experiments. C,WB analysis of SDF-1 overexpression-induced signal transduction with or without treatment of LRP6N-CM in 4T1 cells. Images arerepresentative of three independent experiments.D,WB analysis of SDF-1–induced nuclear translocation of b-arrestin1 in 4T1/Vector, 4T1/LRP6N, and AMD3100-treated 4T1/Vector cells. Images are representative of three independent experiments. min, minutes.






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Figure 3.

LRP6N is secreted under physiologic condition and correlated with breast cancer metastasis. A, Schematic diagram illustrating the target sites of different LRP6antibodies. B,WB analysis of LRP6 expression in the cell culture media and cell lysates of 4T1, MDA-MB231, and HEK293 cells. Images are representative of threeindependent experiments. C,WB analysis of LRP6 expression in the cell culture media and cell (Continued on the following page.)

Zhang et al.





Supplementary Fig. S2D). These results support the notion thatserum LRP6N downregulation can be triggered by only a smallnumber of metastatic cells.

We further used a separate mouse breast cancer cell line: 67NR,which does not metastasize following orthotopic implanta-tion (27) but could induce metastasis to lung following tail veininjection (28). Interestingly, serum LRP6N level was unalteredeven until day 28 following orthotopic injection of 67NR cells(Fig. 3K). However, tail vein injection of 67NR cells induced arapid downregulation of serum LRP6N (Fig. 3L). These resultsindicate that downregulation of serum LRP6N level is likelytriggered by the metastasis of cancer cells.

Furthermore, we found that serum LRP6N was also down-regulated in patients with breast cancer (Fig. 3M; SupplementaryTable S2). Of note, serum LRP6N was downregulated in allpatients with metastasis (Fig. 3M), suggesting that examinationof serum LRP6N is a sensitivemethod for the assessment of breastcancer metastasis. Interestingly, half of the patients with breastcancer without metastasis also exhibited downregulated serumLRP6N level (Fig. 3M; Supplementary Table S3). Because ourin vivo findings showed that only the occurrence of tumor metas-tasis could induce serum LRP6N downregulation, it is likely thatcurrent clinical examinations are less sensitive than the luciferaseactivity assays we used in our mouse models. Therefore, it ispossible that metastasis might have already occurred in thosepatients with breast cancer without diagnosedmetastasis but withserum LRP6N downregulation. Thus, the examination of serumLRP6Nmight be a sensitive and promisingmethod for the clinicaldiagnosis of tumor metastasis.

LRP6N specifically inhibits cell migration and invasionWe further investigated the role of serum LRP6N in tumor

progression using the LRP6N construct without transmembraneand cytoplasmic domains. Treatment with LRP6N-CM signifi-cantly inhibited cell migration and cell invasion of 4T1 cells(Supplementary Fig. S3A and S3B). The inhibitory function ofLRP6N on cell migration and invasion was further confirmed bytreatment with LRP6N-CMor a purified LRP6Nprotein in humanbreast cancer MDA-MB231 cells (Supplementary Fig. S3C–S3F).Furthermore, a similar effect of LRP6N on cell migration andinvasionwas also verified using two separate 4T1/LRP6N sublines(4T1/LRP6N-1 and 4T1/LRP6N-2; Supplementary Fig. S3G–S3I).However, 4T1/LRP6N cells had no effect on cell proliferation(Supplementary Fig. S3J) and LRP6N did not increase cell apo-ptosis or adhesion (Supplementary Fig. S3K), suggesting thatLRP6N specifically affects cell migration and invasion. Further-more, real-time PCR analysis showed comparable expressionlevels of c-Myc and Axin2 in 4T1/LRP6N-1 and 4T1/Vector cells,

and only a slightly higher expression level of cyclin D1 in4T1/LRP6N-1 cells (Supplementary Fig. S3L). In contrast, Wnt3adramatically enhanced the expression levels of these genes(Supplementary Fig. S3L). These results imply that 4T1 cellsmightlack the secretion of Wnt ligands and therefore, LRP6N does notaffect Wnt/b-catenin signaling through binding to and neutral-izing Wnt ligands. Taken together, these results suggest thatLRP6N directly acts on the cell surface and inhibits cell mobilityvia a Wnt/b-catenin signaling–independent mechanism.

LRP6N inhibits 4T1 metastasis to lung in nude miceWe further examined the inhibitory effect of LRP6N on breast

cancer cell mobility in vivo by tail vein injection of 4T1/Vector and4T1/LRP6N-1 cells into nude mice. Bioluminescence imagingshowed that comparable numbers of cells were injected andresided at the lungs immediately postinjection in both groups(Fig. 4A). At day 12, numerous lung metastases were observed in4T1/Vector-injected mice, while in contrast, virtually no lungmetastases were observed in 4T1/LRP6N-1–injected mice(Fig. 4B; Supplementary Fig. S4A). The inhibitory effect of LRP6Nwas further confirmed by H&E staining at day 12 (Fig. 4C) andbioluminescence imaging at day 16 (Fig. 4D). Similar results wereobserved in 4T1/LRP6N-2–injected mice (Supplementary Fig.S4B and S4C). In contrast, 4T1/Kremen1N-injected mice showedcomparable numbers of lung metastases compared with 4T1/Vector-injectedmice (Fig. 4E andF). These results demonstrate thestrong effect of LRP6N in preventing 4T1metastasis. Furthermore,4T1/LRP6N-injected mice also had prolonged survival comparedwith 4T1/Vector-injected mice (Fig. 4G).

We also examined the inhibitory effect of LRP6N by implan-tation of 4T1/Vector and 4T1/LRP6N-1 cells into the mammaryfat pads of nude mice. At day 21, 4T1/Vector-implanted miceshowed several lung surface macroscopic metastases, while incontrast, virtually no lung surface macroscopic metastases werepresent in 4T1/LRP6N-1–implanted mice (Fig. 4H). Of note,there was no significant difference in primary tumor volumesbetween 4T1/Vector and 4T1/LRP6N-1 groups (Fig. 4I), whichwas consistent with our in vitro experiments. These resultsindicate that LRP6N specifically prevents tumor metastasis.Moreover, 4T1/LRP6N-1–implanted mice also showedprolonged survival compared with 4T1/Vector-implantedmice (Fig. 4J). In addition, no significant difference in activeb-catenin expression was observed between 4T1/Vector and4T1/LRP6N-1 tumors (Supplementary Fig. S4D).

LRP6N inhibits 4T1 metastasis to lung in BALB/c miceTo investigate whether LRP6N also has tumor-inhibitory effect

in immunocompetent mice, 4T1 cells were implanted into the

(Continued.) lysates of LRP6-knockdown 4T1, MDA-MB231, and HEK293 cells. Images are representative of three independent experiments.D,WB analysis ofLRP6 expression in the culture media and cell lysates following cotransfection of C-terminal Myc- or GFP-tagged LRP6with Mesd in HEK293 cells. Images arerepresentative of two independent experiments. E,WB analysis and quantification of LRP6 expression in the serum of LRP5/6 knockout (KO) and b-catenin KOmice. � , P < 0.05, one-way ANOVA. F,WB analysis and quantification of LRP6N level in the serum of BALB/c mice following mammary fat pad implantation of 4T1cells at different time intervals. n¼ 4 in each group. �, P < 0.05, one-way ANOVA.G,WB analysis and quantification of LRP6N level in the serum of BALB/c miceafter tail vein injection of 4T1 cells at different time intervals. n¼ 3 in each group. � , P < 0.05, one-way ANOVA. H and I,WB analysis and quantification of serumLRP6N level (H) and lungmetastasis as determined by luciferase activity (I) following injection of 4T1-Luc cells into mammary fat pads of nudemice at differenttime intervals. Day 27, positive control (I). n¼ 4 in each group. � , P < 0.05, one-way ANOVA. J,WB analysis and quantification of LRP6N level in the serum ofBALB/c mice after tail vein injection of different amount of 4T1-Luc cells. n¼ 3 in each group. � , P < 0.05, one-way ANOVA. K,WB analysis and quantification ofLRP6N level in the serum of nudemice after subcutaneous implantation of 67NR cells at different time intervals. n¼ 3 in each group. Not significant, one-wayANOVA. L,WB analysis and quantification of LRP6N level in the serum of nudemice after tail vein injection of 67NR cells at different time intervals. n¼ 3 in eachgroup. � , P < 0.05, one-way ANOVA.M,WB analysis and quantification of LRP6N level in the serum of normal people and patients with breast cancer with orwithout metastasis. �, P < 0.05, one-way ANOVA.






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LRP6N inhibits lung metastasis. A, Representative bioluminescent images of mice at 30minutes (min) following injection of 4T1/Vector or 4T1/LRP6N-1 cells intotail vein of nude mice. n¼ 6 in each group. B and C,Quantification of lung surface macroscopic metastases (� , P¼ 0.0023, Student t test; B) and H&E staining oflungs showing distinctive tumor burden (arrow heads indicate metastatic tumors; C) at day 12 following injection of 4T1/Vector or 4T1/LRP6N-1 cells into tail veinof nude mice. n¼ 6 in each group.D, Representative bioluminescent images of lungs at day 16 following injection of 4T1/Vector or 4T1/LRP6N-1 cells into tail veinof nude mice. n¼ 6 in each group. E and F, Representative bioluminescent images of mice at 30minutes (min; E) and quantification of lung surface metastases atday 16 (F) following tail vein injection of 4T1/Vector and 4T1/Kremen1N cells. n¼ 6 in each group. G, Kaplan–Meier survival plot for nude mice tail vein injectedwith 4T1/Vector cells (n¼ 6) and 4T1/LRP6N-1 cells (n¼ 12). P¼ 0.043, Gehan–Breslow–Wilcoxon test. H and I,Quantification of lung surface macroscopicmetastases (� , P¼ 0.01, Student t test;H) and growth curve of primary tumors (ns, not significant, Student t test; I) at day 21 following injection of 4T1/Vector or4T1/LRP6N-1 cells into mammary fat pads of nudemice. n¼ 6 in each group. J, Kaplan–Meier survival plot for nudemice injected with 4T1/Vector cells (n¼ 6) or4T1/LRP6N-1 cells (n¼ 13) at mammary fat pad. P¼ 0.005, log-rank test. K–M, Timeline of in vivo experiments (K), quantification of lung surface macroscopicmetastases at day 20 (� , P¼ 0.01, Student t test; L), and growth curve of mice (M) following tail vein injection of Vector or LRP6N plasmid and subsequent tailvein injection of wild-type 4T1 cells after 5 days. n¼ 8 in each group.N,Quantification of lung surface macroscopic metastases at day 21 following tail veininjection of LRP6N-CM– or Vector-CM–treated 4T1 cells in BALB/c mice. n¼ 5 in each group. � , P¼ 0.047, Student t test.O,Quantification of lung surfacemacroscopic metastases at day 53 following tail vein injection of LRP6N-CM– or Vector-CM–treated MDA-MB-231 cells in nude mice (n¼ 6 in each group).� , P¼ 0.0077, Student t test.

Zhang et al.





mammary fat pads of BALB/c mice. Similar to nude mice, thenumber of lung surface macroscopic metastases was robustlydecreased in 4T1/LRP6N-implanted mice compared with4T1/Vector-implanted mice (Supplementary Fig. S4E andS4F). Interestingly, the size of metastases was also smaller in4T1/LRP6N-injected mice (Supplementary Fig. S4G), whichsuggests a delayed onset of tumor metastasis. In addition, therewas no significant difference in the primary tumor volumeof 4T1/LRP6N-implanted mice compared with 4T1/Vector-implanted mice (Supplementary Fig. S4H). Furthermore, noinhibition of lung metastasis was observed in 4T1/Kremen1N-implanted mice (Supplementary Fig. S4E–S4H), suggestingthat the inhibitory effect of LRP6N on tumor metastasis isspecific. Likewise, tail vein injection experiments showed asignificant decrease in the number of lung surface macroscopicmetastases in 4T1/LRP6N-injected mice compared with4T1/Vector-injected mice (Supplementary Fig. S4I–S4K). Theseresults indicate that LRP6N can also inhibit tumor metastasis inimmunocompetent BALB/c mice.

LRP6N inhibits 4T1metastasis to lung via its action on 4T1 cellsurface

We further delivered LRP6N plasmid into the body of miceusing 25-kDa linear polyethylenimine via tail vein injection.This method can deliver plasmids to multiple organs such aslung, liver, spleen, and kidney, with lung showing the highestprotein expression (29, 30). LRP6N expression in lung wasconfirmed by WB analysis and its expression was demonstratedto last for at least 7 days (Supplementary Fig. S4L). On day 5after LRP6N plasmid injection, 4T1 cells were injected via tailvein (Fig. 4K; Supplementary Fig. S4M). The number of mac-roscopic metastases in the lungs of LRP6N-injected mice wassignificantly fewer than Vector-injected mice (Fig. 4L), indicat-ing that LRP6N expression in the body of mice also inhibits 4T1cell metastasis. In addition, there were no differences in theactivity and body weight of mice between LRP6N-injected andVector-injected groups (Fig. 4M), suggesting that the inhibitionof metastasis is not caused by any side effects of LRP6Nexpression in the body of mice.

We also used LRP6N-CM to treat 4T1 cells for 24 hours andinjected these cells into BALB/c mice via tail vein (Supple-mentary Fig. S4N). Lung metastases were also significantlyfewer in LRP6N-CM–treated group compared with Vector-CM–treated group (Fig. 4N). A similar result was also observedin LRP6N-CM–treated human breast cancer MDA-MB231 cells(Fig. 4O).

These results exclude a possible secondary effect induced byLRP6Noverexpression in 4T1/LRP6N line, thereby confirming theability of LRP6N in preventing 4T1 metastasis.

Of note, we recently showed that LRP6Nwas able to inhibit cellmigration induced by Frizzled-regulated noncanonical Wnt sig-naling pathway (21). However, it had been reported that inhibi-tion of noncanonical Wnt signaling pathway had an inadequateeffect in preventing 4T1 cell metastasis to lung (31). Thus, thestrong inhibitory effect of LRP6N implies that there might beother underlying mechanisms by which LRP6N prevents 4T1metastasis to lung.

LRP6N inhibits SDF-1/CXCR4-induced cell mobilityBecause of the well-known role of SDF-1/CXCR4 signaling

pathway in lung metastasis of breast cancer (11, 32–36) as well

as the inhibition of LRP6N on SDF-1/CXCR4 signaling transduc-tion, we investigated whether the robust inhibitory effect ofLRP6N on tumor metastasis is related to inhibition of SDF-1/CXCR4 signaling. 168FARN and 4T1 cells were isolated from thesamemurinemammary tumor (37). Interestingly, 168FARN cellsexpressed SDF-1 but not CXCR4, whereas 4T1 cells expressedCXCR4 but not SDF-1 (Fig. 5A). Overexpression of CXCR4promoted cell migration in 168FARN cells, which was completelyprevented by cotransfection with LRP6N (Fig. 5B; SupplementaryFig. S5A). On the other hand, transfection with SDF-1 stronglypromoted cell migration in 4T1/Vector and 4T1/Kremen1N cells,but not in 4T1/LRP6Ncells (Fig. 5C; Supplementary Fig. S5B). Theinhibitory effect of LRP6N on SDF-1/CXCR4-induced cell migra-tion was further confirmed in CXCR4-transfected MDA-MB231cells (Supplementary Fig. S5C) andHs578T cells (SupplementaryFig. S5D). These results indicate that LRP6N prevents cell migra-tion through inhibiting SDF-1/CXCR4.

We further examined whether LRP6N inhibits SDF-1/CXCR4-induced metastasis by tail vein injection of 4T1 cellsin nude mice. At day 13, overexpression of SDF-1 robustlypromoted lung metastasis in 4T1/Vector-injected mice, buthad no effect in 4T1/LRP6N-injected mice (Fig. 5D; Supple-mentary Fig. S5E). SDF-1 also promoted lung metastasisin 4T1/Kremen1N-injected mice (Fig. 5D; SupplementaryFig. S5E). Notably, LRP6N expression also significantly pro-longed the survival of mice bearing SDF-1–overexpressedtumors (Fig. 5E and F; Supplementary Fig. S5F and S5G). Theseresults suggest a strong inhibitory effect of LRP6N on SDF-1/CXCR4-induced lung metastasis.

Endogenous LRP6 inhibits SDF-1/CXCR4We further investigated the relationship between LRP6 and

SDF-1/CXCR4 at the endogenous level. LRP6 knockdown bysiRNA promoted 4T1 cell migration, which was further enhancedby SDF-1 overexpression, but was reversed by pretreatment withthe recombinant LRP6N protein or CXCR4 inhibitor AMD3100(Fig. 6A and B). Furthermore, LRP6 knockdown further enhancedSDF-1–induced activations of c-Jun, ERK1/2, and Akt (Fig. 6C).These results indicate that LRP6 inhibits SDF-1/CXCR4 at theendogenous level via its ectodomain.

We further found that 4T1 metastasis to lung could be pro-moted by both LRP6 knockdown by siRNA and SDF-1 over-expression (Fig. 6D and E). Interestingly, LRP6 knockdown syn-ergistically promoted SDF-1–overexpressed 4T1 metastasis tolung (Fig. 6E) and also accelerated tumor death induced bySDF-1 overexpression (Fig. 6F). These results indicate that endog-enous LRP6 is also inhibitory for SDF-1/CXCR4-induced lungmetastasis in vivo.

Poor prognosis in patients with breast cancer with low LRP6expression and high CXCR4 expression

Furthermore, a study of patients with breast cancer (dataset:NKI295; ref. 24) showed that patients with either low LRP6expression or high CXCR4 expression had worsened prognosisfor distant metastasis-free survival (Fig. 6G and H). However,patients with both high CXCR4 and high LRP6 expression had nocorrelationwithmetastatic relapse (Fig. 6I). Interestingly, patientswith high CXCR4 but low LRP6 expression exhibited the worstprognosis in all patients, with shorter median distant metastasis-free survival than those with either high CXCR4 or low LRP6expression only (12.47 years versus > 18.34 or 14.01 years,






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LRP6N inhibits SDF-1/CXCR4 in vitro and in vivo.A, RT-PCR analysis of SDF-1 and CXCR4 expression in 168FARN and 4T1 cells. Data are representative of threeindependent experiments. B, Photo images and quantification of transwell analysis of cell migration following transfection of CXCR4 with or without LRP6N in168FARN cells. Each condition was assayed in duplicate, and all data are representative of two independent experiments (mean� SEM). � , P < 0.05, one-wayANOVA. Scale bar, 50 mm. C, Photo images and quantification of transwell analysis of cell migration following transfection of SDF-1 in 4T1/Vector, 4T1/LRP6N,and 4T1/Kremen1N cells. Each condition was assayed in duplicate, and all data are representative of three independent experiments (mean� SEM). � , P < 0.05,one-way ANOVA. Scale bar, 50 mm. D,Quantification of lung surface macroscopic metastases following tail vein injection of SDF-1–transfected 4T1 subclones innude mice at day 13. n¼ 7 in each group. � , P < 0.05, ns, not significant, one-way ANOVA followed by Games Howell multiple comparison test. E and F, Kaplan–Meier survival plots for mice tail vein injected with SDF-1– or Vector-transfected 4T1/Vector cells (n¼ 7; E) and 4T1/LRP6N cells (n¼ 6; F). P¼ 0.025 (E), P¼0.698 (F), log-rank test.

Zhang et al.





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Figure 6.

Inhibitory effect of endogenous LRP6 on SDF-1/CXCR4. A, Photo images and quantification of transwell analysis of 4T1 cell migration following knockdown ofLRP6, with or without treatment of LRP6N recombinant protein or CXCR4 inhibitor AMD3100. Each condition was assayed in duplicate, and all data arerepresentative of three independent experiments (mean� SEM). � , P < 0.05, one-way ANOVA. Scale bar, 50 mm. (Continued on the following page.)






respectively; Fig. 6I). More importantly, overall survival datashowed similar results with distant metastasis-free survival data(Fig. 6J–L). These clinical data support our experimental findingsthat endogenous LRP6 is inhibitory for SDF-1/CXCR4-inducedmetastasis and thus implicate LRP6 as a novel candidate for breastcancer diagnosis and therapy.

DiscussionIn this study, we identified a secreted form of LRP6N protein in

the serum of mouse and human, which was downregulated inmice bearing breast cancer, as well as in patients with breastcancer. Interestingly, only the occurrence of tumor metastasiscould induce serum LRP6N downregulation. Furthermore, serumLRP6N downregulation could be rapidly induced by only a smallnumber of metastatic cells. Supporting these experimental find-ings, all patients with breast cancer with metastasis showeddownregulation of serumLRP6N.On the other hand, those breastcancer patients without diagnosed metastasis, but with down-regulation of serum LRP6N, might already bear small but yetundetectable metastases by current clinical examinations. Ofnote, only 1 mL human serum was enough for detection of theLRP6Nprotein in ourWB experiments. Thus, serumLRP6Nmightbe a promising diagnostic marker for early detection of breastcancer metastasis. Whether a similar downregulation in serumLRP6N also occurs in patients with other types of cancers requiresfurther investigation.

We further found that LRP6N not only prevented cancer cellmigration and invasion in vitro, but also prevented breast cancerlung metastasis in both immune-deficient and -competent mice,aswell as in different tumor cells. Importantly, we revealed anovelunderlying mechanism of LRP6N in preventing SDF-1/CXCR4signal transduction via physically interacting with CXCR4 andcompetitively preventing SDF-1 binding to CXCR4. Of note, thepreventive function of LRP6Nwas extremely robust due to the factthat LRP6N treatment almost completely prevented metastasisinduced by SDF-1/CXCR4, as well as prolonging the survival ofmice. Furthermore, a cDNA microarray analysis showed thatpatients with breast cancer with high CXCR4 expression hadsignificantly worsened prognosis, which was further exacerbatedin patients with low LRP6 expression, but improved in patientswith high LRP6 expression. These clinical findings indicatethat endogenous LRP6 in tumor cells has an ability to preventCXCR4-induced metastasis. Supporting the clinical findings, wefound that LRP6 knockdown by siRNA in 4T1 cells promotedtheir metastasis to lung. Thus, it is possible that endogenousLRP6 can inhibit CXCR4 through its ectodomain. However,whether the inhibitory effect is associated with the secreted formor full-length LRP6 locally expressed in tumor cells requiresfurther investigation.

Similar to our findings in breast cancer, transfection of SDF-1 innon–small lung cancer A549 cells enhanced cell migration andinvasion in vitro (38). In addition, overexpression of SDF-1 in oralsquamous carcinoma cell line B66 enhanced lung metastasis inSCID mice (39). High SDF-1 expression was also correlated withdecreased survival of patientswith oral squamous carcinoma (39)and increased metastasis in patients with gallbladder carcino-ma (40). These results indicate that SDF-1 has the ability topromotemetastasis ofmanydifferent cancers in addition to breastcancer. On the other hand, at least 23 different tumor types showhigher expression levels of CXCR4 compared with normal tis-sues (41), suggesting that CXCR4 may have a role in multipletypes of cancers. Indeed, ameta-analysis showed that high expres-sion of CXCR4 significantly correlated with poorer prognosis in5,592 patients with 12 tumor types, and shorter overall survival in10,506 patients with 15 tumor types (42). Furthermore, CXCR4has been suggested to be an independent biomarker regardless ofage, gender, or tumor stage (42). As a result, whether LRP6N hasany role in preventing the metastasis of other cancers via inhibit-ing SDF-1/CXCR4 signal transduction warrants furtherinvestigation.

SDF-1/CXCR4 signaling pathway activation can also regulatetumor progression in several other ways. High expression ofCXCR4 contributes to tumor progression by promoting tumorangiogenesis, growth, andmetastasis (41, 43). An elevated level ofCXCR4 expression in cancer stem cells significantly promotestheir invasion, metastasis, self-renewal, and survival, and even-tually leads to therapeutic resistance (44). On the other hand,tumors with high expression level of SDF-1 attract CXCR4-positive inflammatory, vascular, and stroma cells toward thetumor microenvironment, thereby promoting tumor growth (45,46). Thus, whether LRP6N has any additional roles in preventingtumor progression other than inhibiting tumor metastasis war-rants further investigation.

LRP6N has been well established to inhibit Wnt/b-cateninsignaling pathway by binding to and neutralizing Wntligands (47). Our previous study also showed that LRP6N candirectly bind to Frizzled and inhibit noncanonical Wnt signalingpathway (21). Our current study revealed a novel function ofLRP6N, which inhibited SDF-1/CXCR4 signaling pathway viadirect binding to CXCR4. Of note, all the three pathways playpivotal roles in tumor progression (32, 48, 49). Therefore, LRP6Nmight be a strong inhibitor of tumor progression by inhibitingcanonical Wnt pathway, noncanonical Wnt pathway, and SDF-1/CXCR4 pathway. As a result, a recombinant LRP6N proteinmightbe a promising drug for a wide range of cancer therapies. Inaddition, because of the widely recognized role of LRP6 as a Wntcoreceptor and key activator of Wnt/b-catenin signaling pathway,LRP6N thus provides a novel molecular link between Wnt sig-naling and SDF-1/CXCR4 signaling.

(Continued.) B, Photo images and quantification of transwell analysis of cell migration following cotransfection with LRP6 siRNA and SDF-1 plasmid in 4T1 cells.Each condition was assayed in duplicate, and all data are representative of three independent experiments (mean� SEM). � , P < 0.05, one-way ANOVA. Scalebar, 50 mm. C,WB analysis and kinetics of SDF-1–induced activations of c-Jun, ERK1/2, and Akt in LRP6-knockdown 4T1 cells and control cells. Data are presentedas mean� SD. � , P < 0.05, Student t test. min, minutes.D and E, Representative bioluminescent images of mice (D) and quantification of lung metastases at day 7(E) following tail vein injection of LRP6-knockdown and/or SDF-1–overexpressed 4T1-Luc cells in nude mice. n¼ 6 in each group. � , P < 0.05, one-way ANOVA. F,Kaplan–Meier survival plots for mice tail vein injected with LRP6-knockdown and/or SDF-1–overexpressed 4T1-Luc cells (n¼ 6–7 for each group). P < 0.0001,log-rank test. G–I, Kaplan–Meier analysis of distant metastasis-free survival of patients with breast cancer (dataset: NKI295). Patients were divided according tothe expression of LRP6 (G), CXCR4 (H), or both (I), as indicated. P¼ 0.0048 (G), P¼ 0.0470 (H), P¼ 0.0028 (I), log-rank test. J–L, Kaplan–Meier analysis ofoverall survival in patients with breast cancer (dataset: NKI295). Patients were divided according to the expression of LRP6 (J), CXCR4 (K), or both (L), asindicated. P¼ 0.0448 (J), P¼ 0.0093 (K), P¼ 0.0077 (L), log-rank test.

Zhang et al.





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

Authors' ContributionsConception and design: J. Zhang, J. Chen, J. Peng, D.-n. Ren, W. ZhuDevelopment of methodology: J. Zhang, J. Chen, W. ZhuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Zhang, J. Chen, D. Wo, P. Liu, E. Ma, D. Chen,C. Liang, J. Peng, D.-n. Ren, W. ZhuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Zhang, J. Chen, D. Wo, H. Yan, L. Li, L. Zheng,J. Peng, D.-n. Ren, W. ZhuWriting, review, and/or revision of the manuscript: J. Zhang, D. Wo, W. ZhuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Zhang, J. Chen, D. Wo, H. Yan, P. Liu, E. Ma,L. Li, L. Zheng, D. Chen, Z. Yu, J. Peng, D.-n. Ren, W. ZhuStudy supervision: J. Peng, D.-n. Ren, W. Zhu

AcknowledgmentsThe authors thank Dr. X. He and Dr. C. Niehrs for providing plasmids;

Dr. F. Miller for providing 168FARN, 4T1, and 67NR cells; and Dr. Z. Yu forproviding MDA-MB231 and Hs578T cells. This work was supported byNational Natural Science Foundation of China (81672849, 81470395,81600313, and 81673721) and the Fundamental Research Funds for theCentral Universities (22120180332).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 30, 2018; revised March 4, 2019; accepted April 15, 2019;published first April 22, 2019.

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Published OnlineFirst April 22, 2019.Clin Cancer Res Jiankang Zhang, Jinxiao Chen, Da Wo, et al. Cancer Metastasis to LungLRP6 Ectodomain Prevents SDF-1/CXCR4-Induced Breast

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