aip1 expression in tumor niche suppresses tumor...

Microenvironment and Immunology

AIP1 Expression in Tumor Niche SuppressesTumor Progression and MetastasisWeidong Ji1, Yonghao Li2, Yun He3, Mingzhu Yin4, Huanjiao Jenny Zhou4,Titus J. Boggon5, Haifeng Zhang4, and Wang Min1,4

Abstract

Studies from tumor cells suggest that tumor-suppressor AIP1inhibits epithelial–mesenchymal transition (EMT). However, therole of AIP1 in the tumor microenvironment has not beenexamined. We show that a global or vascular endothelial cell(EC)–specific deletion of the AIP1 gene in mice augments tumorgrowth and metastasis in melanoma and breast cancer models.AIP1-deficient vascular environment not only enhances tumorneovascularization and increases premetastatic niche formation,but also secretes tumor EMT-promoting factors. These effects from

AIP1 loss are associated with increased VEGFR2 signaling in thevascular EC and could be abrogated by systemic administration ofVEGFR2 kinase inhibitors. Mechanistically, AIP1 blocks VEGFR2-dependent signaling by directly binding to the phosphotyrosineresidues within the activation loop of VEGFR2. Our data revealthat AIP1, by inhibiting VEGFR2-dependent signaling in tumorniche, suppresses tumor EMT switch, tumor angiogenesis, andtumor premetastatic niche formation to limit tumor growth andmetastasis. Cancer Res; 75(17); 3492–504. �2015 AACR.

IntroductionIn the absence ofmetastasis, cancer is largely a treatable disease.

Thus, early diagnosis of patients who will develop metastasiscould reduce the mortality and morbidity associated with thisdisease. The development of metastasis depends on geneticand/or epigenetic alterations that are intrinsic to cancer cells, andextrinsic factors provided by the tumor microenvironment(niche). Metastasis is the spread of cancer cells from a primarysite to distant, secondary host organs and requires a number ofdistinct steps, including epithelial–mesenchymal transition(EMT) of neoplastic epithelial cells for invasive growth, invasioninto adjacent tissue, entering the systemic circulation (intravasa-tion), extravasation into surrounding tissue parenchyma, andfinally proliferation from microscopic growth (micrometastasis)into macroscopic secondary tumors (1–4). Premetastatic nichecan also be formed at distant tissues before tumor cells arrive (5).Multiple cell types have been shown to promote tumor metasta-

sis, including vascular endothelial cells (EC), tumor-associatedmacrophages (TAM), and bone marrow–derived cells (BMDC;refs. 1, 6, 7). These cells are firstly activated by factors secreted bytumor cells (5, 8), subsequently promoting tumor EMT, tumorangiogenesis, tumor premetastatic niche formation, and metas-tasis. Therefore, the tumor cells and the host cells are interactiveand form a positive feedback loop. However, the underlyingmolecular mechanisms regulating the tumor cell–tumor nicheinteractions are not completely understood.

ASK1-interacting protein-1 (AIP1; also named as DAB2-inter-acting protein DAB2IP) is a recently identified signaling scaffol-ding protein. AIP1/DAB2IP has been shown to be downregulatedin a variety of human cancer tissues, including prostate cancer andbreast cancer (9, 10). Two genome-wide association studies ofaggressive prostate cancer suggest AIP1 as a putative prostatetumor-suppressor gene (11). Further studies have shown thatAIP1 downregulation in cancer cells is primarily through epige-netic regulation of the AIP1 promoter. Specifically, AIP1 expres-sion in cancer cells is suppressed by the polycomb-group proteinhistone-lysine N-methyltransferase EZH2 (9, 12), which is con-sistently elevated in invasive breast and prostate carcinoma com-paredwith normal breast and prostate epithelia, respectively (13).Importantly, AIP1 is amajor EZH2 target and silencing of AIP1 is akey mechanism by EZH2 triggers tumor metastasis in mouseprostate cancer models (12).

The function of AIP1 in tumor cells has been assessed by in vitroproliferation and EMT assays, and by in vivo tumor progressionandmetastasis analyses inmousemodels. AIP1 containsmultiplesignaling domains, including the N-terminal pleckstrin homol-ogy (PH) domain for membrane targeting, the PKC-conservedregion 2 (C2) domain for ASK1 interaction to induce apoptosis,the Ras-GAP domain for inhibition of Ras (therefore, it has beenconsidered as a novelmember of RAS-GAP family protein), the C-terminal period-like domain for inhibition of transcriptionalfactor NF-kB, and the proline-rich for inhibition of the PI3K–Aktsurvival pathway (14–17). By gain-of-function and loss-of-func-tion approaches, we and others have shown that AIP1 inhibits

1The First Affiliated Hospital, Center for Translational Medicine, SunYat-sen University, Guangzhou, China. 2State Key Laboratory of Oph-thalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University,Guangzhou, China. 3School of Public Health, Sun Yat-sen University,Guangzhou, China. 4Department of Pathology,Vascular Biology Pro-gram/Yale Cancer Center, Yale University School of Medicine, NewHaven, Connecticut. 5Department of Pharmacology, Yale UniversitySchool of Medicine, New Haven, Connecticut.

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

W. Ji, Y. Li, Y. He, and M. Yin contributed equally to this article.

Corresponding Author: Wang Min, Interdepartmental Program in VascularBiology and Therapeutics, Department of Pathology, Yale University School ofMedicine, 10 Amistad St., 401B, New Haven, CT 06520. Phone: 203-785-6047;Fax: 203-737-2293; E-mail: [email protected]; or The First Affiliated Hospital,Center for Translational Medicine, Sun Yat-sen University, 58 Zhongshan Er Lu,Guangzhou 510080, P. R. China. Phone: 18902233990.

doi: 10.1158/0008-5472.CAN-15-0088

�2015 American Association for Cancer Research.

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tumor growth, EMT and metastasis by inhibiting Ras, PI3K–Akt,GSK-3–b-catenin, and NF-kB pathways (12, 16, 17). Moreover, ithas been reported that the inhibitory activity of AIP1 on NF-kB,but not its Ras-GAP activity, is critical for its suppressor effect onEMT in cancer models (12). Recent data also suggest that mutantp53 in cancer cells, by binding to AIP1 in the cytoplasm, enhancesNF-kB activation to increase tumor metastasis (18).

However, the role of AIP1 in tumor niche has not beenexplored. AIP1-KO mice exhibit enhanced inflammation andpathologic angiogenesis (15, 19). Given that inflammation andangiogenesis are required for tumor growth andmetastasis, in thepresent study, we determined the role of AIP1 in tumor micro-

environment in regulating tumor growth and metastasis usingvariousmouse breast cancermodels.Our data suggest that AIP1 invascular EC represses tumor metastasis by modulating not onlytumor angiogenesis, but also tumor-associated premetastaticniche formation and tumor cell EMT phenotype.

Materials and MethodsAnimals

All animal studies were approved by the Institutional AnimalCare and Use Committee of Yale University. Littermates of WT(AIP1lox/lox) andglobal AIP1-KO(AIP1lox/lox:b-actin–Cre; ref. 15),

Figure 1.Augmented growth and metastasis ofB16F10melanoma inAIP1-KOmice. B16melanoma cells were injected s.c. intoWT and AIP1-KO mice. A, tumorvolumes are presented; n¼ 10mice pergroup; �� , P < 0.01. B, tumormetastases to distant tissues wereexamined microscopically at week2 to 4 postimplantation. Metastasisincidence (the percentage ofmicewithdetectable pigmented tumor nodules)was quantified. Data for week 4 areshown; n ¼ 10 mice per group; �� , P <0.01. C, representative images oftumor metastasis at week 3 and 4 inlung are shown. Arrowheads, tumornodules. D, lung metastasis nodules at4 weeks were quantified;n ¼ 10 mice per group; � , P < 0.05 and�� , P < 0.01. E, lung metastasis wasexamined by H&E staining. F,metastatic tumor areas in lung werequantified from five random fields perlung and n ¼ 5 mice per group;� ,P <0.05 and �� ,P <0.01; scale bar, 50mm. G, regional inguinal lymph nodeswere excised fromWT and KO at basalor 2 to 4 weeks after tumorimplantation; note that the pigmentedmelanoma metastases were detectedat 3 and 4 weeks in AIP1-KO but onlyvisible at 4 weeks in WT mice. H, LNmetastases were quantified; n ¼ 10mice per group; �� , P < 0.01.

AIP1 in Tumor Niche Suppresses Tumor Metastasis

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littermates of WT (AIP1lox/lox) and the AIP1-ecKO (AIP1lox/lox:VE-cad-Cre; refs. 20, 21) were used for experiments. All mice havebeen backcrossed to C57BL/6 for 12th generations.

Cells and cell linesPrimarymouse lungmicrovessel ECs (MLEC)were isolated and

were routinely grown in M199 supplemental with 20% FBS and

ECgrowth supplement (ECGS;Corning; 356006) at 37�Cand5%CO2 as previously reported (15). The E0771 mouse breast cancercell line was from CH3 BioSystems (catalog #940001). B16melanoma cell lines, 168FRAN and 4T1 breast cancer cell lines,and Lewis lung carcinoma (LLC) cell line were sourced from theATCC. All cell lines were validated by STR profiling.

Mouse tumor model and in vivo evaluation of lung metastasesand treatments

A total of 1 � 106 mouse cancer cells (ATCC) were injected s.c.in the right rearflankofmice in a 50mL volumeofDMEMsolution(22). Tumor dimension (length, width, and depth)wasmeasuredby electronic caliper coupled to computer that converted to tumorvolume. Lungmetastaseswere established inmice by injecting 1�105 B16 melanoma cells in 100 mL of Hank's Balanced SaltSolution (Life Technologies), without Caþþ or Mgþþ, into thetail vein. After 10 to 42 days, themice were sacrificed according toprocedures approved by Yale's Institutional Animal Care and UseCommittee, and the primary tumor and draining lymph nodeswere analyzed by histology.

Administration of VEGFR2 kinase inhibitor and VEGFR2-neutralizing antibodies in mouse tumor models

VEGFR2 kinase Inhibitor I (Cat# 676480) was obtained fromCalbiochem. It is a highly selective, cell-permeable, reversible, andATP-competitive indolin-2-one class of receptor tyrosine kinase(RTK) inhibitor (IC50 ¼ 70 nmol/L) for mouse VEGF-R2 (KDR/Flk-1). It does not inhibit PDGF, EGF, and IGF-1 RTK activities(IC50 > 100 mmol/L). For VEGFR2 kinase inhibitor experiments,tumor-bearing mice were subjected to i.v. injection of 100 mL ofeither DMSO or kinase inhibitor every other day. VEGFR2-neu-tralizing antibody DC101 was produced from hybridoma cellsthat were obtained from the ATCC, using the BD CELLine 1000system (BD Biosciences) following the manufacturer's instruc-tions. DC101 and isotype control IgG1 (40 mg/kg) was injectedi.v. every other day.

Evans blue permeability assayEvans Blue Dye (100 mL of a 1% solution in 0.9%NaCl; Sigma-

Aldrich) was injected into the retro-orbital plexus of anesthetizedtumor-bearing mice. Thirty minutes after the injection, mice weresacrificed and perfused with PBS through the left ventricle to clearthe dye from the vascular volume. Tissues (e.g., lung) wereharvested, dried in 60�C overnight, and weighed before Evansblue extraction using 1mL formamide at 55�C for 16 hours. Evansblue content was quantified by reading at 630 nm in a spectro-photometer as described previously (15, 23).

Statistical analysisData are represented as mean � SEM. Comparisons between

two groups were by paired t test, between more than two groupsby one-way ANOVA followed by the Bonferroni post hoc test or bytwo-way ANOVA using Prism 4.0 software (GraphPad). P valueswere two-tailed and values <0.05 were considered to indicatestatistical significance.

Other methodsExpandedmethods, including histology, immunoblotting, and

immunofluorescence analysis, gene expression in the tissues,Cytokine assays, Transwell coculture assay, are available in theSupplement Materials and Methods.

Figure 2.AIP1 in vascular endothelium inhibits tumor growth and metastasis in anorthotopic model. A, AIP1 deletion efficiency was verified by qRT-PCR infreshly isolated lung ECs from AIP1lox/lox (WT), AIP1-KO, and AIP1-ecKOmice; n ¼ 3 mice in each group; �� , P < 0.001. B–F, mouse E0771 breastcancer cells (1 � 106) were injected orthotopically into the fourth mammarygland of WT and AIP1-ecKO female mice. B, tumor size was measuredweekly. C, representative images of immunostaining with anti-AIP1antibody for breast tumors from WT and AIP1-ecKO mice; scale bar, 50 mm.Arrows, capillary endothelium and tumor cells are indicated (�). D, tumormetastasis at week 4 in lung was examined by whole tissue photography(top) and by H&E staining (bottom). Arrowheads, tumor mass; scale bar forH&E, 50 mm. E and F, metastasis nodules and the percentage of tumorareas in lung were quantified; n ¼ 10 mice per group; �� , P < 0.01.

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ResultsAIP1 deletion in vascular ECs augments tumor growth andmetastasis in syngeneic and orthotopic tumor models

To determine the role of AIP1 in the tumor microenvironmenton tumor growth andmetastasis, we compared tumor growth and

metastasis in wild-type (WT) and global AIP1-deficient mice(AIP1-KO) using subcutaneous and orthotopic implantationmodels. In a B16 melanoma model, AIP1-KO significantlyenhanced tumor growth (Fig. 1A). Pigmented B16 melanomanodules were readily detected and quantitated microscopically.

Figure 3.Tumor angiogenesis is increased in AIP1-ecKO mice. Mouse breast cancer cells(1� 106) were injected orthotopically intothe fourth mammary gland of WT andAIP1-ecKO mice, and tumor along withsurrounding mammary tissue wereharvested at 2 weeks. A and B, tumorangiogenesis was determined byimmunohistochemistry and whole-mountstaining with anti-CD31, and intratumoralvessel density and vessel areas werequantified from five sections per tumorand n¼ 10mice for each strain; � ,P <0.05;scale bar, 50 mm. C and D, tumor-inducedangiogenesis in lymph nodes wasdetermined by immunostaining with anti-CD31; scale bar, 100mm. CD31þ areaswerequantified; n ¼ 10 mice per group;�� , P < 0.01. E–G, phospho-VEGFR2(pY1054/1059) and total VEGFR2,VEGFR3, AIP1, and b-actin proteins intumor (E) and lymph node (F and G) weredetermined by immunoblotting withrespective antibodies. Representativeblots are shown from 1 of 4 mice for eachstrain with fold changes compared withWT-tumor are indicated below the blots.Further quantifications of VEGFR2 and p-VEGFR2 are shown in G, n ¼ 6 mice pergroup; �� , P < 0.01.






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Metastasis incidences (the percentage of mice with detectablepigmented tumor nodules) to multiple organs, including lymphnode, lung, liver, spleen, and bone marrow, at 4 weeks weresignificantly increased in AIP1-KOmice (Fig. 1B). Detailed exam-ination of lung tissues indicated that lungmetastasis was detectedin AIP1-KO, but not in WT mice, as early as 3 weeks followingimplantation (Fig. 1C with quantification in Fig. 1D). Both thenumber of tumor nodules and the percentage of tumor areas inthe lungwere higher in AIP1-KOmice (Fig. 1Ewith quantificationin Fig. 1F). AIP1-KO mice also exhibited enhanced tumor metas-tasis in lymph nodes (LN; Fig. 1G with quantification in Fig. 1H).

AIP1 is highly abundant in vascular ECs but not inmyeloid cells(14, 15, 19, 24). To determined relative AIP1 expression levels intumor ECs, TAMs, and tumor cells, we implanted GFP-expressingB16 melanoma subcutaneously or E0771 breast cancer cellsorthotopically into WT mice. Tumor tissues were harvested at 2weeks postimplantation andCD31þECs,CD68þTAMs andGFPþ

tumor cells were collected by FACS sorting. AIP1 mRNA levelswere determined by qRT-PCR. Results indicated that AIP1 washighly expressed in tumor ECs, but onlyweakly in tumor cells andwas not expressed in TAMs (Supplementary Fig. S1).

To define the host cell type in which AIP1 regulates tumorbehavior, we applied the tumor models to the EC-specific AIP1-knockoutmice (AIP1-ecKO; AIP1lox/lox:VE-cad-Cre)mice.We firstvalidated the deletion efficiency of AIP1 by qRT-PCR in freshlyisolated lungECs fromAIP1lox/lox (WT), AIP1-KO, andAIP1-ecKOmice. AIP1 genewas completely deleted in ECs isolated frombothAIP1-KOandAIP1-ecKO, andAIP1 genedeletionwas comparablebetween AIP1-KO and AIP1-ecKO (Fig. 2A). In an orthotopicbreast cancermodelwith E0771 (amousemetastatic breast cancercell lines derived from C57BL/6 mice), AIP1-ecKO exhibitedenhanced tumor growth and lung metastasis (Fig. 2B–F), com-parable with the global AIP1-KO (Supplementary Fig. S2). Theseresults suggest that AIP1 in vascular EC suppresses tumor growthand metastasis in the mouse models.

AIP1deletionhas no effect on tumormetastasis after a direct i.v.injection of tumor cells

We next used AIP1-ecKO mice to define the mechanism bywhich AIP1 in host limits tumor metastasis. Tumor metastasisinvolves multiple steps, including tumor cell EMT switch, intra-vasation into the lymphatic system and/or the bloodstream,extravasation from and survival/colonization in the host distancetissues. Primary tumor cells, by secreting soluble factors, also

activate premetastasis niche at a distance tissue before their arrivalto the tissue (illustrated in Supplementary Fig. S3A). Todeterminehow AIP1 in host controls tumor metastasis, we directly injectedtumor cells (1 � 105) into bloodstream intravenously. Tumorcells directly arrived at the lung tissue where they extravasated andcolonized, therefore, metastasized without processes of EMTswitch, intravasation, and pre-niche formation. We observed nodifferences between WT and AIP-ecKO mice in lung metastasis asdetermined by tumor nodule number and lung weight (Supple-mentary Fig. S3B and S3C). We further injected GFPþ B16 mela-noma cells with fewer numbers (1 � 104), and examined extrav-asation of tumor cells to the lung. We did not observe significantdifferences between WT and AIP1-ecKO mice in the tumor cellretention (at 30 minutes after injection) or extravasation (at 4hours after injection; Supplementary Fig. S3D and S3E). These datasuggest that the AIP1 deletion in vascular EC affects tumor metas-tasis at a step(s) before extravasation, that is, tumor cell EMTphenotypic change, tumor cell intravasation to lymphatic and/orblood systems, and the formation of premetastasis niche.

Tumor angiogenesis but not tumor lymphangiogenesis isincreased in AIP1-ecKOmice with enhanced VEGFR2 signaling

Tumor angiogenesis and tumor lymphangiogenesis arerequired for tumor growth and metastasis (25). We examinedtumor angiogenesis and lymphangiogenesis by immunostainingwith anti-CD31 and anti–LYVE-1, respectively. Tumor vesseldensity and area were significantly enhanced in the AIP1-ecKOmice (Fig. 3A and B), consistent with our results in inflammatoryangiogenesis models (15). Tumor implantation induced bothangiogenesis and lymphangiogenesis in the sentinel lymphnodes(LN) at 2 weeks postimplantation before tumor cell metastasis,suggesting that LN angiogenesis and lymphangiogenesis wereinduced by tumor-derived angiogenic factors such as VEGFs(26). Numbers of blood vessels (Fig. 3C and D), but not lym-phatic vessels (Supplementary Fig. S4), were significantlyenhanced in the AIP1-ecKO mice at the peritumoral region anddistant lymph nodes. We measured expression and activation ofVEGFR2 and VEGFR3, two potent angiogenic and lymphangio-genic receptors (27). Total and phosphorylation of VEGFR2, butof VEGFR3 or total VEGFR1, were significantly increased in tumortissues from AIP1-ecKO mice (Fig. 3E). Primary tumor inducedexpression and phosphorylation of VEGFR2 and VEGFR3 indistant lymph nodes at 2 weeks postimplantation. However,AIP1-ecKO exhibited enhanced expression of VEGFR2 (but not

Figure 4.Tumor-induced premetastatic niche formation is augmented in AIP1-ecKO. Mouse breast cancer cells (1� 106) were injected orthotopically into the fourthmammarygland of WT and AIP1-ecKO mice. A, lung permeability was measured on day 10 postimplantation by Miles assay with Evan's blue dye. The dye wasextracted from the lung tissue and was measured at 630 nm. Data, OD/lung dry weight (g) and mean � SEM from n ¼ 3 for each strain; �, P < 0.05. B, lung tissueswere harvested at day 10 and gene expression of premetastasis niche markers were analyzed by qRT-PCR, with normalization by GAPDH. Fold increases inAIP1-ecKO versusWT are shown. Data, mean� SEM; n¼ 6 for each strain. C, GFP-expressing mouse breast cancer cells were injected orthotopically into the fourthmammary gland of WT and AIP1-ecKO mice. Lung tissues were harvested at indicated times. Fibronectin, p-VEGFR2, VEGFR2, tumor markers S100A2 andGFP, and b-actin were determined by immunoblotting with respective antibodies; n¼ 6 mice per group at each time point. Fold changes compared with the basallevel in WT are shown. D and E, recruitment of active VEGFR2þ cells to the lung tissue were increased in AIP1-ecKO mice but was diminished by VEGFR2-specificinhibitor. Some mice of WT and AIP1-KO mice with orthotopic breast tumor were treated by intravenous injection of VEGFR2 TKI (0.4 mmol/L) every otherday. p-VEGFR2 (pY1054/1059) andCD31 in the lung at day 14were determined by immunostaining; v, vessel. Representative images are shown from five sections pertumor; scale bar, 50 mm. Arrows, p-VEGFR2þ cells, which are quantified in E as the percentage of total cells. Data, mean � SEM; n ¼ 6 per group; � , P < 0.05.F and G, WT and AIP1-KO mice with orthotopic breast tumor were treated by i.v. injection of VEGFR2 TKI (0.4 mmol/L) every other day. p-VEGFR2(pY1054/1059) and CD31 in the lung at day 14 were determined by immunostaining. p-VEGFR2þ cells were quantified in G as the percentage of total cells. Data,mean � SEM; n ¼ 6 per group; � , P < 0.05. H, lung tissues were harvested at day 14, and p-VEGFR2 and total VEGFR2 were determined by immunoblotting.Fold changes are shown by taking WT control as 1.0. Representative blots are shown from 1 of 3 mice for each strain. I, tumor volumes at 3 weeks were measured.J, lung metastasis nodules at 3 weeks were quantified. Data, mean � SEM; n ¼ 6 mice per group; � , P < 0.05; �� , P < 0.01.






VEGFR3 or VEGFR1; Fig. 3F and G). These results suggest thatincreased tumor angiogenesis, but not lymphangiogenesis, inAIP1-ecKO mice contributes to the enhanced tumor metastasisin these mice.

Tumor-induced premetastatic niche formation is enhanced inAIP1-ecKO mice with enhanced VEGFR2 signaling

Formation of premetastatic niche at a distant tissue is alsoimportant for tumormetastasis, which can be regulated by tumor-and bone marrow–derived cytokines (5, 8). It has been proposedthat upon activation by tumor-secreted factors (e.g., VEGF, IL6,and SDF-1), vascular endothelium at the distant tissue can beactivated followed by infiltration of immune cells and bone

marrow cells (5, 8). We first measured lung endothelial barrierfunction by "Miles assay" using Evans-blue dye. AIP1-ecKOexhibited higher permeability at day 14 post-tumor implantationin an orthotopic breast cancer model (Fig. 4A). We also observedupregulation of several premetastatic niche markers, includingfibronectin, LOX, c-Kit, VEGFR1, CD34, and VEGFR2, in the lungas detected by qRT-PCR. These genes were more greatly upregu-lated in tumor-bearing AIP1-ecKO lungs (Fig. 5B). However,genes involved in lymphangiogenesis (e.g., VEGFR3) and arter-iogenesis (e.g., Notch1 receptor andDll4 ligand)were not altered.We thendeterminedwhether gene expression of pre-nichemarkerspreceded tumor cell arrival at the lung. To this end, GFPþ tumorcells were applied to orthotopic cancermodel, and gene expression

Figure 5.AIP1 binds to the VEGFR2 kinase activation loop and inhibits VEGFR2-mediated signaling. A, VEGFR2 mutants. Vector control (VC), VEGFR2-WT, K868M(a kinase-dead mutant), Y1175F, Y1175/Y801F, or Y1054/1059F were transfected into 293T. Autophosphorylations of VEGFR2 at pY1054/1059 and pY1175 weredetermined by immunoblotting with phosphor-specific antibodies. B, AIP1 binds to VEGFR2 at pY1054/1059. Flag-tagged AIP1-N containing the C2 domainwas transfected into 293T cells with vector control (�), VEGFR2-WT, K868M, Y1175F, or Y1054/1059F. AIP1-VEGFR2 association was determined bycoimmunoprecipitation with anti-VEGFR2 followed by immunoblotting with anti-Flag. C, the coimmunoprecipitation assay of AIP1 and VEGFR2-WTwas performedin the presence of 0.3 mmol/L of pY1054/1059-peptide, C2-peptide. A non–phosphor-Y1054/1059-peptide and C2KA-peptide were used as controls. D and E,effects of the AIP1 binding on VEGFR2 signaling. AIP1-deficient mouse MLECs were reconstituted by lentivirus with vector control (VC), AIP1-WT, or AIP1-KA1.Cells were serum-starved for 12 hours followed by treatment with VEGF (10 ng/mL for 15 minutes). p-VEGFR2 and p-Akt were determined by immunoblottingwith phosphor-specific antibodies and ratios of p-VEGFR2/VEGFR2 and p-Akt/Akt are quantified in E. Data, mean � SEM from three independent experiments;� , P < 0.05. F, AIP1-KO MLECs were reconstituted by lentivirus with AIP1-WT or AIP1-KA1. Cells were subjected to Transwell migration assays. Data, mean �SEM from triplicates and three independent experiments; �� , P < 0.01.

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of tumor markers (GFP and S100A2) and pre-metastatic nichemarkers (fibronectin and VEGFR2) in the lung tissues were mea-sured by qRT-PCR. Clearly, upregulation of premetastatic nicheand VEGFR2 in the lung occurred on day 10 postimplantationbefore tumor-specific markers could be detected on days 17 to 21(Supplementary Fig. S5). Immunoblotting confirmed that totalfibronectin,phosphorylatedand total VEGFR2wereupregulated inthe lung tissue at day 14 postimplantation before detection ofGFPþ tumor cells on day 21 (Fig. 4C). Very few activated VEGFR2þ

cells were found in resting WT and AIP1-ecKO lung tissues, andtumor implantation induced a recruitment of activated VEGFR2þ

cells to the lung as visualized by phospho-VEGFR2 staining.Importantly, tumor-induced activation of VEGFR2þ cells in thelung was augmented in the AIP1-ecKO mice (Fig. 4D and E). Ofnote, some VEGFR2þCD31� cells were detected and these cells arelikely the bone marrow–derived EC progenitors (EPC) that havenot been fully differentiated into CD31þ mature EC. Interestingly,circulating VEGFR2 EPCs have been associated poor prognosis in

advanced cancer patient (28). Consistently, we have observedstronger VEGFR2 signaling in bone marrow EC progenitors fromAIP1-KO mice (15, 19, 24).

We next determined whether enhanced VEGFR2 signaling invascular EC contributes to augmented tumor angiogenesis andpremetastatic niche formation. To this end, a VEGFR2 tyrosinekinase inhibitor (TKI)-1 was applied to the orthotopic breastcancer model as we recently described (21). VEGFR2 TKI signif-icantly attenuated tumor-induced recruitment of VEGFR2þ cellsto the lung as detected by immunostaining (Fig. 4F and G).VEGFR2 TKI also significantly attenuated activation of VEGFR2signaling in the lung as determined by immunoblotting (Fig. 4H).Of note, VEGFR2 TKI appeared to upregulate AIP1 expression inWT mice, suggesting a potential AIP1-VEGFR2 autoregulatoryloop. Functionally, VEGFR2 TKI blocked tumor angiogenesis(Supplementary Fig. S6A and S6B) and premetastatic niche for-mation (Supplementary Fig. S6C), suggesting that observedchanges in tumor angiogenesis and premetastatic formation aredependent on vascular VEGFR2 signaling. Importantly, tumorgrowth and lung tumor metastasis in AIP1-ecKO mice wereblunted by VEGFR2 TKI (Fig. 4I and J). VEGFR2-dependent tumorgrowth and metastasis in AIP1-ecKO mice were also blocked byDC101, amonoclonal antibody specifically binds and neutralizesVEGFR2 (Supplementary Fig. S6D–S6F; ref. 29).

AIP1 binds to the VEGFR2 kinase activation loop and inhibitsVEGFR2-mediated signaling

The results above established a critical role AIP1-regulatedVEGFR2 in tumor metastasis, and we subsequently took bio-chemical approaches to define the mechanism by which AIP1regulates VEGFR2 signaling. We have previously shown that AIP1binds to an active form of VEGFR2, and a mutant AIP1 (AIP1-KA1) with mutations at the first lysine cluster within its C2domain (K104-106 to A) loses the VEGFR2-binding activity(15). A certain type of C2, similar to the SH2 domain, associateswith phosphotyrosines (30, 31). These observations prompted usto determine whether AIP1 via its C2 binds to phosphotyrosineresidues onVEGFR2. To this end,wemutated the tyrosine residuesof VEGFR2 known to be autophosphorylated in response toVEGF-A, including Y1054/1059, Y1175, and Y1121 (32, 33).293T cells were cotransfected with VEGFR2 mutants alongwith AIP1-N, the N-terminal half of AIP1 constitutively associat-ing with VEGFR2 upon coexpression (15). Overexpression ofVEGFR2-WT induces autophosphorylation at pY1054/1059(within the kinase activation loop), pY1175 (critical for activationof PI3K and PLC-g activation) as well as other phosphotyrosinesinvolved in other signaling (Fig. 5A). The kinase-dead mutant(VEGFR2-K868M) and the Y1054/1059F mutant abolishedVEGFR2 autophosphorylation at both pY1054/1059 andpY1157. In contrast, mutations of other Y sites (Y1175, Y1214,or Y801) had no effects on pY1054/1059. Association of VEGFR2and AIP1 was determined by a coimmunoprecipitation assay. Asshown in Fig. 5B, AIP1 associated with VEGFR2-WT and Y1175Fmutant. However, the kinase-dead form and the Y1054/1059Fmutant failed to associate with AIP1. These results suggest that anactive form of VEGFR2 with phosphorylations at Y1054/1059 isrequired for the AIP1 binding.

To further determine the role of VEGFR2 pY1054/1059 in theAIP1 binding, we performed a peptide competition assay using13-mer peptides flanking the pY1054/1059 of VEGFR2(RDIY1054KDPDY1059VRKG) in two forms: nonphosphorylated

Figure 6.Tumor EMT is enhanced inAIP1-ecKOmice.Mousebreast cancer cells (1� 106)were injected orthotopically into the fourth mammary gland of WT andAIP1-ecKO mice, and tumors along with surrounding mammary tissuewere harvested at 2 weeks. A, AIP1 and EZH2 were detected byimmunohistochemistry with respective antibodies as indicated. Tumor andsurrounding fat fad are labeled; scale bar, 50 mm. Representativeimages are shown fromfive sections per tumor andn¼ 10mice for each strain.B and C, tumor EMT markers (E-cadherin, p-p65, AIP1, and EZH2) weredetermined by immunoblotting with respective antibodies. Representativeblots are shown in B from 2 of 10 mice for each strain, with densitometryquantifications shown below the blots. Quantifications are shown inC; n ¼ 10 mice per group; � , P < 0.05.






Figure 7.AIP1-VEGFR2 signaling in vascular EC regulates tumor cell EMT program. A and B, endothelial permeability—tumor cell invasion assay. WT or AIP1-KO MLECscells were cultured on the top of the Transwell as monolayer for 2 days post-confluency. Tumor cells (168FARN or 4T1) were seeded at the top well of theBoyden chamber and cells that transmigrated cross the endothelium layerwere quantified at 12 hours. Data presented in B aremean� SEM fromduplicates and threeindependent experiments. � , P < 0.05; ��, P < 0.01. C, diagram for the coculture assay. MLECs were seeded at the top of the Transwell. Tumor cells (e.g.,breast cancer cell line 168FARN) were seeded at the bottom well of the Boyden chamber. The two types of cells were grown without direct cell-to-cell contact andwere separated by a membrane with 0.4-mm pore size permeable for molecules but not for cells. D and E, effect of MLECs on tumor cells. Coculture of breastcancer cell line 168FARN with MLECs isolated from WT mice, AIP1-KO mice, AIP1-KO MLECs with a re-expression of AIP1, AIP1-KA1, or AIP1-KA2 by lentivirus.At 12 hours, cell morphologic analyses of cocultured 168FARN cells were performed under light microscopy. Representative images are from 1 of 3 independentexperiments (B). Quantifications of EMT (the percentage of elongated spindle cells) are presented. (Continued on the following page.)

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(Y1054/1059-peptide) and phosphorylated peptides (pY1054/1059-peptide). These peptides were initially designed for thegeneration and affinity purification of the pY1054/1059-specificantibody (32). We performed the coimmunoprecipitation assayin the presence of 0.3 mmol/L of either Y1054/1059 peptide orpY1054/1059 peptide. Addition of the pY1054/1059 peptide, butnot the Y1054/1059 peptide, disrupted the VEGFR2-AIP1 asso-ciation (Fig. 5C), suggesting that phosphorylation of VEGFR2 atY1054/1059 is critical for the AIP1 binding. We also designeda C2-peptide derived from the lysine-cluster 1 of AIP1 C2domain (IEAKDLPAK104KK106YLCELCLwhere the lysine residuesare underlined; C2-KA peptide contains K to A mutations).Interestingly, the C2-peptide, but not the C2-KA peptide, alsodisrupted theVEGFR2–AIP1 complex, further supporting a criticalrole of the lysine-cluster within the AIP1 C2 domain in theVEGFR2 binding. Taken together, these data suggest that theC2 lysine-cluster on AIP1 and the phosphotyrosine residues1054/1059 on VEGFR2 are responsible for their interactions.

We then determined effects of the AIP1 binding on VEGFR2activation and signaling. AIP1-deficient mouse MLECs (15, 34)were reconstituted by lentivirus-expressing AIP1-WT or AIP1-KA1(a mutant defective in the VEGFR2 binding). VEGF-inducedphosphorylation of VEGFR2 and downstream signaling Akt wereblunted by re-expression of AIP1-WT but not AIP1-KA1 (Fig. 5Dand E). VEGF-stimulated migratory activities of MLECs were alsoinhibited by AIP1-WT but not by AIP1-KA1 in a Transwell assay(Fig. 5F). Taken together, our data support that AIP1 binds to theVEGFR2 kinase activation loop and inhibits VEGFR2-mediatedsignaling in the vasculature.

AIP1-deficient EC enhances tumor cell EMT and invasionWhen we examined tumor cell EMT and invasive markers

(E-cadherin, vimentin, b-catenin, snail, and NF-kB) in the ortho-topic breast cancer model, we observed downregulation ofE-cadherin and upregulation of vimentin, b-catenin, snail, andNF-kB (p-p65) in the tumors from AIP1-ecKO mice comparedwith WT. These changes were more profound at the peripheralregions of the tumor (Supplementary Fig. S7). As an intrinsic EMTsuppressor, AIP1 is downregulated via epigenetic regulation byEZH2 in tumor cells (12, 35). We examined whether AIP1expression in tumor cells is altered upon transplantation intoAIP1-ecKO microenvironment. AIP1 expression in tumor cellswas dramatically downregulated within the peripheral regions inthe AIP1-ecKO host (Fig. 6A), correlating with increased EMTmarkers in this region. Interestingly, EZH2, a component of PRC-2responsible for the epigenetic suppression of AIP1 expression,showed a reciprocal expression pattern of the AIP1 expression(Fig. 6A). The changes of E-cadherin, NF-kB (p-p65), AIP1, andEZH2 were also verified by immunoblotting (Fig. 6B and C).

These data suggest that the host microenvironment modulatesAIP1 expression and EMT phenotype of tumor cells.

AIP1-VEGFR2 signaling in vascular ECmediates tumor cell EMTswitch

To determine the mechanism by which the endothelial AIP1–VEGFR2 axis regulates tumor cell EMT and metastasis, we firstperformed a modified Transwell migration assay to measurepermeability of WT and AIP1-KO MLECs to tumor cells bycounting tumor cells migration cross the confluent MLEC mono-layer (Fig. 7A). Despite that invasive 4T1-migrated cross WTMLECs at higher rate compared with less malignant 168FRANcells, AIP1-KO MLECs were highly permeable to both 168FRANand 4T1 (Fig. 7B).We reasoned that AIP1-KOMLECs, by secretingfactors, regulated tumor cell EMT phenotypic changes. To test thisidea, we used a Transwell system to coculture 168FARN with WTor AIP1-KO MLECs without direct cell–cell contacts (Fig. 7C).Coculture of AIP1-KO MLECs (but not WT MLECs) stronglyinduced EMT responses in 168FARN tumor cells (Fig. 7D andE). However, re-expression of AIP1-WT or an ASK1-bindingdefective mutant KA2, but not the VEGFR2-binding defectivemutant KA1, diminished the effects of AIP1-KOMLECs on tumorcell EMT. These results suggest that vascularAIP1-VEGFR2, but notAIP1-ASK1 signaling, regulates cocultured tumor cell EMT. More-over, treatment of MLECs with VEGFR2 kinase inhibitor (TKI) inthe coculture system blocked AIP1-KO MLEC-enhanced168FRAN EMT responses (Fig. 7F). Of note, tumor cell EMTmorphologic change was accompanied by upregulation of EZH2with a reciprocal reduction of AIP1, increase in NF-kB activity andNF-kB–dependent gene expression of metastasis-promoting fac-tor IL6 (Supplementary Fig. S8A and S8B). To test whether down-regulation of AIP1 is a critical step for tumor cell EMT, weexamined AIP1 expression in breast cancer cell lines with variousmetastatic potential (168FARN, 4T07 and 4T1; ref. 36). AIP1expression was inversely correlated with EZH2 expression andbreast cancer cell metastatic potentials in these cell lines (Sup-plementary Fig. S8C). Consistent with a low level of AIP1 proteinand a high basal EMT in 4T1 cell, coculture of 4T1 cell with AIP1-KO MLEC weakly enhanced its EMT (Supplementary Fig. S8D).These data suggest that the VEGFR2 signaling in vascular ECpotently modulates an EZH2-AIP1–dependent EMT cascade intumor cells.

To define the MLEC-derived EMT factors that mediate tumorcell EMT, we measured gene expression of a few known EMTfactors in MLECs (37), and we found that TGFb (TGFb2 inparticular), but not HGF, were highly upregulated in AIP1-KOMLEC (Supplementary Fig. S8E). Pretreatment of MLECs withVEGFR2 TKI completely blunted TGFb2 expression (Supple-mentary Fig. S8F), suggesting that the AIP1–VEGFR2 axis

(Continued.) Data, mean � SEM from duplicates and three independent experiments; � , P < 0.05. F, VEGFR2 TKI diminished the effects of AIP1-KO MLECson 168FRAN. Coculture of breast cancer cell line 168FARN for 12 hours with MLECs isolated from WT mice or AIP1-KO mice in the presence or absence of VEGFR2TKI (20 mmol/L). Tumor cell EMT based on morphologic analyses were quantified. Data, mean � SEM from duplicates and three independent experiments;� , P < 0.05. G and H, TGFb2mediates MELC-enhanced tumor EMT responses. Coculture of 168FARNwithWTMLECs, AIP1-KO MLECs, or AIP1-KO MLECswith TGFb2siRNA knockdown for 12 hours. Phosphorylation of Smad2/3, expression of EZH2, and AIP1 in cocultured 168FARN cells was determined by Western blotanalysis with respective antibodies. Fold changes are presented from 1 of 3 independent experiments, taking 168FARN alone as 1.0 (G). 168FARN cell morphologicanalyses were performed under light microscopy and quantifications of EMT are presented. Data, mean� SEM from duplicates and three independent experiments;� , P < 0.05. I, models for the role of stromal AIP1-VEGFR2 signaling in regulation of tumor cell EMT and metastasis. In WT tumor niche, AIP1 inhibitsVEGFR2 to repress tumor angiogenesis, premetastatic niche formation, and tumor EMT switch. In AIP1-deficeint tumor niche, enhanced VEGFR2 signalinginduces tumor angiogenesis and formation of premetastatic niche. Enhanced VEGFR2 activity in vascular EC also stimulates secretion of tumor EMT-promotingfactors (e.g., TGFb2), which directly augment tumor cell EMT phenotypic changes, including upregulation of EZH2 protein, reduction of AIP1 protein, increasesin NF-kB activity, and NF-kB–dependent IL6 expression. Together, AIP1-deficeint niche promotes tumor invasion and metastasis.






directly regulates TGFb2 expression. We then determined thefunctional important of TGFb2 in mediating the MLEC effecton tumor EMT responses. Coculture of AIP1-KO MLECsinduced phosphorylation of Smad2/3 and upregulation ofEZH2 with a reciprocal reduction of AIP1 expression in168FARN cells (Fig. 7G). Moreover, knockdown of TGFb2 inAIP1-KO MLECs attenuated the EMT responses in 168FARNcells, including p-Smad2/3, upregulation of EZH2, downregu-lation of AIP1 and EMT morphologic switch (Fig. 7G and H),supporting a role of MLEC-secreted TGFb2 in mediating cross-talk between MELCs and tumor cells.

DiscussionIn the present study, by implanting tumor cells with the same

genetic background into normal and AIP1-deficient (AIP1-ecKO)mice, we have defined the role of AIP1 in tumor microenviron-ment in regulating tumor growth and metastasis. Loss of AIP1 invascular ECs is sufficient to augment tumor growth andmetastasisin subcutaneousmelanoma and orthotopic breast cancermodels.AIP1 in the vascular EC limits tumormetastases appears to be at astep(s) before entrance into the bloodstream as a direct i.v.injection of tumor cells results in no differences in tumor metas-tasis between WT and AIP1-ecKO mice. Indeed, AIP1 deletion inthe vascular EC enhances VEGFR2 signaling; this enhancedVEGFR2 not only regulates tumor angiogenesis and premetastaticniche formation in distant tissues, but also directly modulatestumor cell EMT phenotypic switch (Fig. 7I: Model for the role ofAIP1 in tumor niche in regulating tumor EMT and metastasis).Mechanistically, we have defined a novel mechanism by whichAIP1 blocks VEGFR2-dependent signaling, that is, AIP1 through alysine-cluster sequence within the C2 domain binds to the phos-photyrosine residues 1054/1059 within the activation loop ofVEGFR2; the VEGFR2 activity in vascular EC is critical for geneexpression of tumor EMT-promoting factors (e.g., TGFb2), whichdirectly augment tumor cell EMT phenotypic changes, includingupregulation of EZH2 protein, reduction of AIP1 protein,increases inNF-kB activity, andNF-kB–dependent IL6 expression.It has been well established that EZH2 epigenetically regulatesAIP1 expression in various tumor cells in vivo and in vitro, andEZH2-mediated AIP1 downregulation is critical for tumor cellEMT and metastasis (9, 12). Our data support that the upregula-tion of EZH2 with AIP1 reduction in tumor cells is a key mech-anism by which tumor niche (vascular ECs) induce tumor EMTand metastasis. Consistently, AIP1-KO MLECs have less effect onthe EMT responses of more malignant tumor cells with highEZH2/low AIP1 (e.g., 4T1 cells). Taken together, our data supportthat AIP1 in tumormicroenvironment limits tumormetastasis bysuppressing tumor cell EMT, tumor angiogenesis, and tumorpremetastatic niche formation.

It is well known that TAMs are associated with tumor EMTand metastasis (38). However, our studies indicate that AIP1 isabundantly expressed in tumor ECs, but not in TAMs. More-over, vascular EC-specific deletion of AIP1 augments tumorangiogenesis, growth, and metastasis. Therefore, AIP1 in vas-cular ECs limits tumor growth and metastasis. Our data supportthat the AIP1 suppresses VEGFR2 in vascular EC to limit tumorangiogenesis and formation of premetastatic niche formationas well as tumor cell EMT switch. VEGFR2 signaling is increasedin the both primary tumor and lung tissues of AIP1-ecKO inresponse to subcutaneous and orthotopic tumor implantations.

A VEGFR2 kinase inhibitor or a neutralizing antibody abrogatesthe augmented VEGFR2-dependent signaling, tumor angiogen-esis, and tumor metastasis in AIP1-ecKO mice. In vitro, AIP1-VEGFR2 signaling in vascular EC directly mediates tumor cellEMT switch, at least partly by regulating EMT factor TGFb2.Taken together, we have uncovered a novel pathway—AIP1-VEGFR2-TGFb in tumor niche and EZH2-AIP1-NF-kB in tumorcells—that mediates crosstalk between tumor niche and tumorcells (Fig. 7I).

AIP1 has been shown to mediate cytokine (e.g., TNF)-inducedEC apoptosis (14–17). EC death and/or proliferation underinflammatory tumor microenvironment can have strong influ-ence on tumor angiogenesis, and hence tumor growth and ontumor metastasis (5, 8, 39). We have assessed EC apoptosis andproliferation in AIP1-ecKOmice. Specifically, we have performedimmunostaining on sections of tumors and lungs from AIP1-KOanimals for proliferation marker Ki-67 and apoptotic markercleaved caspase-3. Our data indicate that AIP1-ecKOmice exhibitincreased EC proliferation with decreased EC apoptosis in theprimary tumor, but not in the lung tissues at 2 weeks post-implantation (Supplementary Fig. S9). These data suggest thatincreased EC proliferation with reduced EC apoptosis in AIP1-ecKO may contribute to augmented tumor angiogenesis thatenhance growth and metastasis, at least in the primary tumor.We observe increased VEGFR2 signaling in both primary tumorand lung tissue, highlighting the important function of VEGFR2signaling in primary tumor angiogenesis and premetastatic nicheformation in distant tissues. This is further supported by theVEGFR2 inhibitor experiments presented in our study.

A significant mechanistic finding in this study is that we haveuncovered how AIP1 inhibits VEGFR2 signaling. VEGF primarilyutilizes its receptor VEGFR2 (also Flk-1 or KDR) to induce angio-genic responses by activating a variety of signaling cascades,including activation of PI3K–Akt, phospholipase C-gamma(PLC-g), and MAP kinase (40). Given the critical role of VEGFR2signaling in angiogenesis, VEGFR2 activity/activation needs to betightly regulated and fine-tuned. VEGFR2 activity is regulated bydirect interactions with other proteins, including its coreceptorneuropilins, adhesion molecule VE-cadherin, and integrins.VEGFR2 could also be negatively regulated. E3 ubiquitin ligasec-Cbl and SCFbTRCP-dependent ubiquitination could induceVEGFR2 degradation (41–43). A high cell density-enhancedphosphatase (DEP-1) is associated with VEGFR2-VE–cadherin–catenin complex at cell–cell contacts and reduces VEGFR2 phos-phorylation and ERK1/2 signaling (44). Therefore, increasedVEGFR2 intracellular trafficking could prevent activated VEGFR2from inactivation by phosphatases (45–47). We have previouslyidentified several VEGFR2 modulators that directly regulate theformation, activation and stability of the VEGFR2 receptor sig-naling complex—two positive regulators Bmx (bone marrowtyrosine kinase located in X-chromosome, a nonreceptor tyrosinekinase) and CCM3 (cerebral cavernousmalformation-3 (CCM3),and two negative regulators AIP1 and endocytic adaptor proteinEpsin (15, 21, 48, 49). Our study has defined the intriguingmechanism by which AIP1 binds to active form of VEGFR2 andinhibits its signaling, that is, AIP1 via its lysine-cluster sequencewithin the C2 domain directly binds to the phosphotyrosines(pY1054/1059) within the activation loop of VEGFR2. We haveprovided strong evidence to support our conclusion: The kinase-dead mutant (VEGFR2-K868M) and the Y1054/1059F mutantabolish both VEGFR2 autophosphorylation at pY1054/1059 and


Ji et al.




the AIP1binding. In contrast,mutations of other tyrosine residues(Y1175, Y1214, or Y801) have no effects on VEGFR2 autopho-sphorylation at Y1054/1059 and theVEGFR2-AIP1 interactions. Aphosphorylated pY1054/1059 peptide, but not a nonphosphory-lated Y1054/1059 peptide, disrupts the VEGFR2-AIP1 associa-tion. Similarly, the C2-peptide derived from the lysine-cluster 1 ofAIP1 C2 domain also disrupts the VEGFR2-AIP1 association. Onthe basis of the published crystal structure for the VEGFR2 kinasedomain, the pY1054/1059 are exposed on surface and are acces-sible to protein interactions (50). Likewise, sequence alignmentand structure-based homology modeling of AIP1 C2 domainsuggests that the basic patches are surface exposed. Therefore, itis possible that the VEGFR2–AIP1 interactions are mediated byelectrostatic interactions. Given that AIP1 via its C2 domaindirectly binds to the autoactivation loop of VEGFR2, it is plausiblethat AIP1 directly inhibits the VEGFR2 kinase activity. We havepreviously shown that AIP1 can recruit PP2A phosphatase toASK1 to dephosphorylate a negative phosphorserine-967 site,leading to ASK1 activation (51). Another possibility is that AIP1recruits phosphatase(s) to VEGFR2 activation loop to terminateVEGFR2 signaling and this model needs to be further tested.Nevertheless, we have revealed a novel function of the AIP1–VEGFR2 complex. Besides the EC intrinsic function of AIP1-VEGFR2 signaling, AIP1-VEGFR2 in tumor niche (e.g., vascularEC) also regulates tumor cell EMT phenotype through a nonau-tonomous manner. Specifically, we show that the AIP1–VEGFR2signaling in tumor niche cells, by regulating gene expression ofEMT factors (e.g., TGFb2), modulates EZH2-AIP-NF-kB–depen-dent EMT cascade in tumor cells (Fig. 7I).

Taken together, our results suggest that downregulation of AIP1expression in tumornichemaybe apotential biomarker for tumormalignancy, and stabilization and/or re-expression of AIP1 in

tumor niche may provide a potential therapeutic strategy for thetreatment of cancer metastasis.

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

Authors' ContributionsConception and design: W. Ji, Y. Li, Y. He, W. MinDevelopment of methodology: W. Ji, Y. Li, Y. He, W. MinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Ji, Y. Li, Y. He, M. Yin, H. Zhang, W. MinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Ji, Y. Li, Y. He, T.J. Boggon, W. MinWriting, review, and/or revision of the manuscript: W. Ji, W. MinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. He, H.J. Zhou, W. MinStudy supervision: W. Min

AcknowledgmentsThe authors thank Dr. DonNguyen (Yale University School of Medicine) for

insightful comments and helpful discussions.

Grant SupportThis work was supported in part by NIH grants HL109420 and HL115148

(W. Min); National Natural Science Foundation of China (nos. 81272350 and81472999),Natural Science FoundationofGuangdong (no. S2012010008908)and the University Talent Program of Guangzhou (no.12A015G; W. Ji);National Natural Science Foundation of China 81273097 and 81472998(Y. He); National Natural Science Foundation of China 81371019 (Y. Li).

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

Received January 13, 2015; revised June 9, 2015; accepted June 12, 2015;published OnlineFirst July 2, 2015.

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Ji et al.




2015;75:3492-3504. Published OnlineFirst July 2, 2015.Cancer Res Weidong Ji, Yonghao Li, Yun He, et al. and MetastasisAIP1 Expression in Tumor Niche Suppresses Tumor Progression

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