Tyrosine Phosphatase PTPRJ/DEP-1 Is an Essential Promoter Of

Published OnlineFirst June 30, 2016; DOI: 10.1158/0008-5472.CAN-16-1071 Cancer Molecular and Cellular Pathobiology Research

Tyrosine Phosphatase PTPRJ/DEP-1 Is an Essential Promoter of Vascular Permeability, Angiogenesis, and Tumor Progression Patrick Fournier1,2, Sylvie Dussault1, Alfredo Fusco3, Alain Rivard1,4, and Isabelle Royal1,2,4

Abstract

The protein tyrosine phosphatase PTPRJ/DEP-1 has been gel plugs infused with VEGF. In the absence of DEP-1, angio- implicated in negative growth regulation in endothelial cells, genesis triggered by ischemia or during tumor formation was where its expression varies at transitions between proliferation defective, which in the latter case was associated with reduced and contact inhibition. However, in the same cells, DEP-1 has tumor cell proliferation and increased apoptosis. Macrophage also been implicated in VEGF-dependent Src activation, per- infiltration was also impaired, reflecting reduced vascular permeability, and capillary formation, suggesting a positive role in meability in the tumors or a possible cell autonomous effect of regulating these functions. To resolve this dichotomy in vivo,we DEP-1. Consequently, the formation of spontaneous and investigated postnatal angiogenesis and vascular permeability experimental lung metastases was strongly decreased in in a DEP-1–deficient mouse. In this study, we report that DEP-1 DEP-1–deficient mice. In clinical specimens of cancer, less is required for Src activation and phosphorylation of its endo- vascularized tumors exhibited lower microvascular expression thelial cell–specific substrate, VE-cadherin, after systemic injec- of DEP-1. Altogether, our results established DEP-1 as an tion of VEGF. Accordingly, VEGF-induced vascular leakage was essential driver of VEGF-dependent permeability, angiogenesis, abrogated in the DEP-1–deficient mice. Furthermore, capillary and metastasis, suggesting a novel therapeutic route to cancer formation was impaired in murine aortic tissue rings or Matri- treatment. Cancer Res; 76(17); 1–12. 2016 AACR.

Introduction ated growth inhibition (2, 3). Similarly, overexpression of DEP-1 in many tumor cells is associated with inhibition of cell prolif- Angiogenesis and increased vessel permeability play key roles eration and migration (2, 4–7). Consistent with this role, some of during the development of a number of pathologies including its known substrates include growth factor receptors such as the tumor growth and cancer progression (1). Therefore, much effort PDGFR, EGFR, MET, and ERK1/2 (8–11). aims at understanding the molecular mechanisms underlying the In conﬂuent and quiescent endothelial cells, DEP-1 colocalizes formation and remodeling of blood vessels as a way to identify at cell–cell junctions with VEGFR2 and VE-cadherin, and nega- new approaches to harness these processes. The receptor-like tively regulates VEGFR2 phosphorylation and cell proliferation protein tyrosine phosphatase DEP-1, also named PTPRJ or (3, 12, 13). However, in addition to its role as an attenuator of CD148, is expressed in various cell types including epithelial, VEGFR2 activity, DEP-1 is also a positive regulator of Src activa- hematopoietic, and endothelial cells. It encompasses an extracel- tion and of important Src kinase–dependent biological functions lular domain containing eight ﬁbronectin-type III-like motifs, a in VEGF-stimulated cells, including cell survival, permeability, transmembrane domain, a single intracellular catalytic domain, invasion, and capillary formation (14–19). This is induced and a short C-terminal tail (2). DEP-1 expression is found to through the VEGF-mediated tyrosine-phosphorylation of the increase with cell density, and contributes to cell contact–medi- C-terminal tail of DEP-1 on Y1311 and Y1320, which allows DEP-1 to associate with Src and dephosphorylate its inhibitory Y529 (15). Interestingly, inactivation of DEP-1 in mice, via the 1 CRCHUM - Centre de recherche du Centre Hospitalier de l'Universite swapping of its catalytic domain with GFP, resulted in mid- de Montreal, Montreal, Quebec, Canada. 2Institut du cancer de Mon- treal, Montreal, Quebec,Canada. 3Dipartimento di Biologia e Patologia gestation death, characterized by enlarged primitive vessels, Cellulare e Molecolare, Universita degli Studi di Napoli "Federico II", increased endothelial cell proliferation, and defective vascular 4 Naples, Italy. Departement de Medecine, Universite de Montreal, remodeling and branching (20). Surprisingly, DEP-1 knockout Montreal, Quebec, Canada. (KO) mice are viable and fertile, with no apparent phenotype Note: Supplementary data for this article are available at Cancer Research (21–23). However, further characterization of these mice models Online (http://cancerres.aacrjournals.org/). revealed that DEP-1 positively regulates B cell and macrophage Corresponding Author: Isabelle Royal, CRCHUM, 900, rue St-Denis, Montreal, immunoreceptor signaling, platelet activation, and airway Quebec H2X 0A9, Canada. Phone: 514-890-8000, ext. 25497; Fax: 514-412-7591; smooth muscle contractility (22, 24, 25). As the underlying E-mail: [email protected] mechanism, DEP-1 was proposed to promote these biological doi: 10.1158/0008-5472.CAN-16-1071 responses via activation of Src family kinases (SFK), similarly to 2016 American Association for Cancer Research. what was observed in endothelial cells in vitro (14). Therefore, to

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determine the role of DEP-1 during angiogenesis and permeabil- beads (GE Healthcare). Lung lysates and immunoprecipitated ity in vivo, the biological consequences of its lost expression were proteins were resolved by SDS-PAGE and transferred on nitrocel- investigated in DEP-1 KO mice. We demonstrate here that DEP-1 lulose membranes (0.45 mm; Bio-Rad). Western blotting and ECL is essential for Src activation and the phosphorylation of its detection (Amersham Biosciences/GE Healthcare) were performed substrate VE-cadherin in response to VEGF stimulation in vivo. according to the manufacturer's recommendations. Alternatively, Consequently, impaired vascular permeability, capillary forma- lungs were frozen in OCT and 5-mm cryosections were processed tion, and recovery from hindlimb ischemia are observed in DEP-1 for immunofluorescence. Formalin-fixed sections were incubated KO mice. Also, tumor growth and the formation of spontaneous for 1 hour with CD31 antibody (1/50), washed twice with PBS and experimental metastases were greatly reduced in mice with no 5 minutes, and then incubated 45 minutes with Alexa Fluor 488– expression of DEP-1, consistent with an important reduction in coupled anti-mouse antibody (1/800). After PBS washes, the same tumor-associated microvessel density and defective vascular per- steps were followed with pY418Src (1/100) and Alexa Fluor 594– meability in these mice. These results thus demonstrate for the first coupled anti-rabbit (1/800) antibodies. Slides were next washed time the positive and essential role played by DEP-1 in postnatal twice with PBS and water, and mounted in ProLong Gold antifade and tumor-associated angiogenesis, and identify stroma-derived reagent (Invitrogen/Molecular Probes). Fluorescence was visual- DEP-1 as an important promoter of tumor progression. ized using a Nikon Eclipse E600 microscope and photographed with an 20 objective lens, using a Photometrics CoolSNAP fi Materials and Methods camera (Roper Scienti c). Images were captured with NIS-Ele- ments AR 3.0 software (Nikon). Antibodies and reagents Y529 Y1175 Antibodies against Src, non-p Src, p VEGFR2, cleaved Permeability assays caspase-3, and horseradish peroxidase (HRP)-conjugated second- Anesthetized WT or DEP-1 KO mice (8/10-week-old) were ary IgGs were purchased from Cell Signaling Technology, New injected with Evans blue (50 mg/kg) via the tail vein. Immediately Y418 England Biolabs. p Src, Alexa Fluor 488–coupled donkey anti- after, mice were injected intradermally on each side of the ventral goat, and Alexa Fluor 594-coupled donkey anti-rabbit antibodies midline with an equal volume of PBS, VEGF (200 ng), or hista- were purchased from Invitrogen. DEP-1 goat antibody was from mine (625 ng). Thirty minutes later, mice were perfused with PBS R&D Systems. CD31 (M-20), Ki67, PY99, and VEGFR2 antibodies and the skin was photographed. For basal permeability assays, were purchased from Santa Cruz Biotechnology. VE-Cadherin mice were perfused with PBS 1 hour after injection of Evans blue. (11D4.1) and F4/80 (A3-1) antibodies were from BD Pharmingen For the quantification of intratumor vascular permeability, mice (BD Biosciences) and Thermo Scientific, respectively. Recombi- were injected with Evans blue 21 days after injection of tumor cells nant human VEGF-A was obtained from the Biological Resources and perfused with PBS after 1 hour. Sites of injection, organs, and Branch Preclinical Repository of the National Cancer Institute - tumors were collected, weighed, and incubated for 48 hours at Frederick Cancer Research and Development Center. Evans blue 56C in formamide to extract Evans blue. Absorbance at 600 nm dye and formamide were purchased from Sigma. was determined in triplicate and normalized on tissue weight, and on the relative concentration of Evans blue present in the blood of Animal model and cell lines the corresponding mice. þ DEP-1 KOand wild-type (WT) mice were derived from DEP-1 / þ/ mice, after backcrossing the previously described DEP mice Mouse aortic ring assay (C57/BL6J background) for at least 10 generations with FVB/N Mouse aortic rings were prepared according to the method of mice (21). All animal experiments were conducted in accordance Baker and colleagues (28). Briefly, thoracic aortas of 8-week-old with the guidelines set out by the CRCHUM Animal Experimen- WT and DEP-1 KO mice were dissected, cut into 0.5-mm rings, and tation Ethics Committee. The Mvt-1/Pei-1 and MET-1 mouse incubated in Opti-MEM (Gibco, Thermo Fischer Scientific) with- mammary tumor cell lines were generously provided in 2013 by out serum or antibiotics overnight. Rings were next embedded in Kent Hunter (NCI, Bethesda, MD) and in 2014 by Robert Cardiff Matrigel (BD #356231; BD Biosciences), and cultured in OptiMEM (UC Davis, Davis, CA), respectively (26, 27). We did not authen- (2.5% FBS, 50 mg/mL gentamycin) containing PBS or VEGF (50 ticate the cells. Cells were cultured in DMEM containing 10% FBS ng/mL). At day 7, rings were photographed. Number of sprouts and 50 mg/mL gentamycin (Wisent). For tumor studies, both cell and total length of sprouts were calculated using the AxioVision lines were injected at passage 17. 4.8.2 software (Zeiss; at least in triplicate; 3 mice per group).

In vivo VEGF stimulation Matrigel plug assay Anesthetized mice were sacrificed by exsanguination 10 minutes Experiments were performed as previously described (29). following tail-vein injection of 2 mg of VEGF or PBS (100 mL). Lungs Briefly, growth factor–depleted Matrigel (400 mL) was premixed were collected, frozen in liquid nitrogen, crushed, and lysed in with heparin (15 U; Sandoz) and VEGF (200 ng) or PBS. The RIPA buffer pH 7.4 containing 50 mmol/L Tris-HCl, 150 mmol/L mixtures were injected subcutaneously on both sides of the NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, ventral midline of 6-week-old WT or DEP-1 KO mice and 0.1% (w/v) SDS, 1 mmol/L EDTA, 10 mg/mL aprotinin, 10 mg/mL collected 12 days later. Hemoglobin content was measured leupeptin (Roche Applied Science), 1 mmol/L phenylmethylsul- using the Quantichrom Hemoglobin Assay Kit (BioAssay Sys- fonyl fluoride, 5 mmol/L sodium fluoride, and 1 mmol/L sodium tems) and normalized to plug weight (10 animals/group). vanadate. Protein concentration was determined using the BCA Neovascularization was determined on hematoxylin and eosin protein assay reagent (Pierce). VE-cadherin was immunoprecipi- (H&E)–stained slices of paraffin-embedded plugs. Capillary tated from 500 mg of lung lysates with 3 mg of antibody overnight, structures were counted per region of interest (ROI; 5 ROI/plugs; and incubated for 2 hours with Protein G–conjugated Sepharose 10 plugs/condition).

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Hindlimb ischemia This was shown to occur through a phospho-displacement mech- Unilateral hindlimb ischemia was surgically induced in mice as anism allowing phosphorylated DEP-1 to bind the Src SH2 previously described (30). Hindlimb perfusion measurements domain and dephosphorylate its inhibitory Y529, resulting in were obtained with a Laser Doppler Perfusion Imager (LDPI) the phosphorylation of its activating Y418 residue (14, 15). Here, system (Moor Instruments Ltd.) immediately after surgery and 14 to determine whether DEP-1 was also involved in Src activation days later on anesthetized animals. The results are expressed as the upon VEGF stimulation in vivo, WT and DEP-1 KO mice were ratio of perfusion in the ischemic versus nonischemic hindlimb. systemically injected with VEGF or PBS, and lungs were collected The mice were sacrificed at day 14 (6 animals/group). Immuno- and lysed after 10 minutes. VEGF stimulation induced the histochemistry of CD31 (Pharmigen) was performed on paraffin- dephosphorylation of Src Y529 and the phosphorylation of embedded ischemic gastrocnemius muscle. The capillary density Y418 in WT mice, but this was attenuated in lysates from DEP- per muscle fiber was determined on three muscle slices taken from 1 KO mice (Fig. 1A and B). Consistent with this, the tyrosine different parts of the muscle. phosphorylation of immunoprecipitated VE-cadherin, an endothelial cell–specific Src substrate, was inhibited upon VEGF stim- Tumor growth and metastasis assay ulation in DEP-1 KO mice (Fig. 1C). Moreover, dephosphoryla- WT and DEP-1 KO female mice (6-week-old) were injected with tion of VE-cadherin–associated Src on Y529 was also defective, 2 105 Mvt-1/Pei-1 or 4 105 MET-1 cells in the mammary fat further emphasizing the inhibition of endothelial Src activity pad and sacrificed at day 28 and 25, respectively. Tumors were (Fig. 1C). In contrast, the relative phosphorylation of VEGFR2, fixed in formaldehyde (3.7%, v/v) and embedded in paraffin, or a DEP-1 substrate (3, 14), was maintained or slightly increased in lysed in RIPA buffer, and then processed for IHC (two slices/ DEP-1 KO mice (Fig. 1A and B), demonstrating that reduced Src tumor) or Western blotting, respectively (31, 32). For pY418Src activation was not resulting from impaired VEGFR2 phosphory- and CD31 immunofluorescence, paraffin-embedded tumor slices lation. Immunofluorescence staining of pY418Src on lung cryosec- were dewaxed, subjected to antigen retrieval, and incubated with tions confirmed that VEGF-stimulated Src activation was reduced primary antibodies using the BenchMark XT automated stainer in CD31-positive endothelial cells of the DEP-1 KO mice (Fig. 1D (Ventana Medical System Inc.; ref. 31). Afterward, on the bench, and E). Together, these results demonstrate the essential role of tumor slices were incubated with secondary antibodies as DEP-1 in VEGF-dependent Src activation in vivo. described above ("In vivo VEGF stimulation" section). Autofluor- escence was quenched with Sudan Black (0.1%, m/v) for 15 VEGF-induced vascular permeability is impaired in the DEP-1 minutes before rinsing and mounting the slides. Lung lobes were KO mouse embedded in OCT, and 5-mm cryosections were H&E stained to VEGF was originally described for its vascular permeability determine the metastatic burden using Photoshop (two slices/ inducing capacity, which relies on its ability to induce the remo- lobe). For experimental metastasis, 8-week-old WT and KO deling and loosening of intercellular adhesions (34). DEP-1 and female mice were tail-vein injected with 4 105 Mvt-1/Pei-1 or Src mediate the tyrosine and PAK2-dependent serine phosphor- 7 105 MET-1 cells. On day 14, mice were sacrificed and lungs ylation of VE-cadherin, resulting in the weakening of its intracel- were processed as described above (at least 9 animals/group). lular association with catenins and its increased internalization from cell–cell junctions (15, 35–39). As impaired phosphoryla- DEP-1 expression in breast tumor microvasculature tion of Src and VE-Cadherin was observed in DEP-1 KO mice, we The GSE15363 dataset was downloaded from the NCBI Gene characterized the role of DEP-1 in VEGF-induced vascular per- Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/ meability in vivo using a modified Miles assay. In control mice, geo; ref. 33). Tumor samples from breast cancer patients were dermal injection of VEGF led to an important vascular leakage of segregated in two groups based (i) on microvessel density (low Evans blue dye, which was blocked in DEP-1 KO animals (Fig. 2A and high), as determined by PECAM (CD31) immunostaining; or and B). However, we observed no difference in histamine-induced (ii) based on a distinct genetic signature defining vessels as either vascular permeability, shown to be Src independent (Fig. 2C and proliferative and undergoing active remodeling (group A), or as D; refs. 16, 40). Basal vascular permeability in spleen, liver, being more stable and mature (group B; ref. 33). DEP-1 expression kidney, and brain was also equivalent in DEP-1 KO and WT mice (probe 25355) was evaluated in each group and plotted on box (Fig. 2E). These results importantly demonstrate that DEP-1 is and whiskers graphs. Analyses were performed with the GraphPad essential for the promotion of VEGF-induced vascular permeabil- Prism 6.0 software (GraphPad Software Inc.). ity in vivo, and correlate with its ability to activate the Src-dependent phosphorylation of VE-cadherin. Statistical analysis in vivo Statistical significance was evaluated with the Student t test DEP-1 is required for capillary formation using GraphPad Prism 6.0 software. P values were considered Angiogenesis is a complex process requiring endothelial cell – significant when less than 0.05. Densitometry analyses were proliferation, remodeling of endothelial cell cell junctions, and performed with the Quantity One 4.6.3 software (Bio-Rad). cell invasion (41). We have previously shown that DEP-1 expression is required for endothelial cell invasion and the organization of capillary-like structures when grown on Matrigel (15). Src has Results been shown to mediate the remodeling and loosening of cell–cell DEP-1 promotes VEGF-dependent Src activation in vivo junctions, and to promote angiogenesis and vascular permeability Despite negative regulatory functions associated with high in vivo (16, 18, 35). To characterize the consequences of DEP-1 loss levels of DEP-1 expression in endothelial cells, our group previ- on capillary formation, several approaches were used. First, in a ously demonstrated that VEGF-induced tyrosine phosphorylation mouse aortic ring assay, an important induction of capillary of moderately expressed DEP-1 promotes Src activation (14, 15). formation was observed in response to the stimulation of WT

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Figure 1. DEP-1 promotes Src activation upon VEGF stimulation in vivo. WT and DEP-1 KO mice were injected intravenously with VEGF (2 mg) or PBS, and lungs were collected after 10 minutes. A, lung lysates were studied by Western blotting. B, results were quantified by densitometric analyses and are representative of three independent experiments (mean SD). C, VE-cadherin was immunoprecipitated from lysates of lungs collected from WT and DEP-1 KO mice injected with VEGF. D, alternatively, lung cryosections were double-stained by immunofluorescence with the pY418Src and CD31 antibodies. E, the intensity of the pY418Src immunofluorescence signal overlapping with CD31-positive microvessels was normalized to that of CD31 (5 ROI/picture; 5 pictures/slice; 2 slices/mouse; n ¼ 2). , P < 0.05.

aortic rings embedded in Matrigel with VEGF (Fig. 3A). In con- WT mice, but not in the KO animals (Fig. 3E). Capillary density, as trast, a reduced number of shorter capillaries elongated from the characterized by H&E staining of parafﬁn-embedded sections of DEP-1 KO aortic rings (Fig. 3A–C). VEGF-induced capillary for- the plugs (Fig. 3F), was increased in response to VEGF stimulation mation in vivo was also studied using a Matrigel plug assay. Upon in the WT animals, whereas no induction was detected in the harvesting, Matrigel plugs were photographed (Fig. 3D), and the Matrigel plugs from the DEP-1 KO mice (Fig. 3G). Taken together, hemoglobin content was determined. Quantiﬁcation indicated these experiments demonstrate that DEP-1 is positively regulating that VEGF induced the formation of functional capillaries in the the formation of perfused capillaries in vivo.

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Figure 2. VEGF-induced, but not histamine- induced, vascular permeability is abrogated in DEP-1 KO mice. Vascular permeability was assessed using a modiﬁed Miles assay. A, mice were tail vein–injected with Evans blue dye followed by intradermal injection of PBS and VEGF. Skin at the injection site was collected and photographed. B, quantiﬁcation of extracted Evans blue. Results were normalized on tissue weight (mean SD); n ¼ 9WT and 12 KO mice. , P < 0.01. C and D, vascular permeability was determined in response to histamine (625 ng), as described above; n ¼ 12 WT and 6 KO mice. E, basal vascular permeability in various organs was assessed 1 hour following the injection of Evans blue; n ¼ 11 WT and 7 KO mice.

Recovery from hindlimb ischemia is attenuated in DEP-1–null cancer growth and progression using two highly metastatic and mice syngeneic tumor cell models, Mvt-1/Pei-1 and MET-1 mammary Ischemia is the most common angiogenic signal emerging from tumor cells, following mammary fat pad injection in WT and wounded tissues. To study the physiologic angiogenic response of DEP-1 KO mice (26, 27). Tumor growth, as characterized by DEP-1 KO mice, we exploited the hindlimb ischemia model. In tumor volume and tumor weight, was significantly decreased in this system, angiogenesis is the primary response, leading to the DEP-1 KO mice compared with WT animals (Fig. 5A, B, G, and H). improved collateral blood flow detected by LDPI. Fourteen days Consistent with the reduced angiogenesis previously observed in after the removal of the femoral artery from the left hindlimb, the DEP-1 KO mice (Figs. 3 and 4), CD31 staining also revealed a LDPI measurements revealed a reduction of limb perfusion in decrease in tumor microvessel density (Fig. 5C, D, I and J). DEP-1 null mice compared with WT mice (Fig. 4A and B), whereas Accordingly, decreased proliferation and increased apoptosis of a similar reduction of perfusion was observed in all animals tumor cells were detected in DEP-1 KO mice, as shown by reduced immediately after surgery (data not shown). In addition, staining Ki67 immunostaining and increased caspase-3 cleavage, respec- of paraffin-embedded ischemic gastrocnemius muscles with tively (Fig. 5C, E, F, I, K, and L). These results thus suggest that CD31 detected a reduction of capillaries per muscle fiber DEP-1 is a driver of tumor growth by regulating tumor-associated (Fig. 4C and D). Consistent with their decreased limb perfusion angiogenesis. recovery, DEP-1 KO mice were less mobile than their WT litter- Tumor microvessels also facilitate the recruitment of immune mates (Fig. 4E), and had a greater number of necrotic or inflamed cells and the dissemination of cancer cells. Tumor infiltration by fingers (Fig. 4F). These data demonstrate that DEP-1 expression macrophages was impaired in DEP-1 KO mice, as shown by the significantly enhances the promotion of physiologic neovascu- decreased F4/80 immunostaining (Fig. 5C and I). This may have larization in the context of ischemia-induced tissue damage. resulted from the reduced permeability of tumors grown in DEP-1 KO mice (Fig. 6A), and/or to cell-autonomous effects of DEP-1 on DEP-1 promotes tumor angiogenesis and metastasis macrophage function (22, 42). Importantly, in the absence of The recruitment of blood vessels is essential for tumor growth DEP-1, fewer Mvt-1 tumor cells formed spontaneous lung metas- and contributes to metastasis. We studied the role of DEP-1 in tases (Fig. 6B). However, for MET-1 cells, only one WT mice

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Figure 3. DEP-1 is essential for capillary formation in response to VEGF. A, aortic rings from WT and DEP-1 KO mice were embedded in Matrigel and cultured with either VEGF or PBS. B and C, the number of sprouts (B) and total length of sprouts (C) were determined. Results are representative of three independent experiments, which were performed in triplicate. D, WT and DEP-1 KO mice were injected with Matrigel containing heparin and either VEGF or PBS on both sides of the ventral midline. Plugs were harvested and photographed. E, hemoglobin (Hb) content was normalized to Matrigel plug weight; n ¼ 10. F, parafﬁn-embedded Matrigel plugs were H&E stained. G, capillaries, as detected by the presence of small circular rings (arrows), were quantiﬁed per ROI; n ¼ 10. , P < 0.01; , P < 0.001.

presented metastatic lesions at the time of sacriﬁce (data not supporting a proangiogenic role for DEP-1 in tumor-associated shown). Mvt-1 and MET-1 cells were also injected directly into the angiogenesis (Fig. 7C and D). blood circulation to investigate experimental metastasis. Their ability to colonize the lungs of DEP-1 KO mice was greatly diminished (Fig. 6B). As a postulated mechanism, the reduced Discussion tumor angiogenesis and metastasis observed in the DEP-1 KO DEP-1 has mainly been depicted as having negative regulatory mice were shown to correlate with weaker Src activation levels in functions in a broad range of cell types. Despite reports highlight- the residual tumor microvessels, reinforcing the key role of DEP-1 ing inhibitory roles for DEP-1 in endothelial cell proliferation and in Src activation during these processes (Fig. 7A and B). Interest- capillary formation (3, 43, 44), our group previously showed that ingly, in breast cancer patients, higher DEP-1 expression in tumor- endogenously expressed DEP-1 in nonquiescent endothelial cells associated microvessels was associated with greater vasculariza- positively regulates Src activation, cell invasion, permeability, and tion of the tumors, and with a genetically deﬁned tumor vascular capillary formation (14, 15, 19). We now show that VEGF- subtype (A) described as being actively remodeling (33), further induced permeability (Fig. 2) and angiogenesis (Figs. 3 and 4),

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Figure 4. Impaired recovery from hindlimb ischemia in DEP-1 KO mice. A, representative results of laser Doppler measurements 14 days after hindlimb ischemia. Arrows, ischemic limbs. B, quantification of laser Doppler perfusion ratios (ischemic vs. nonischemic) at day 14; n ¼ 6. C, CD31 was stained by immunohistochemistry on the ischemic gastrocnemium muscle at day 14. D, number of capillaries (brown round-shaped staining) per muscle fiber was quantified (n ¼ 6 animals/ group; 3 slices per animal). E, ambulatory capacities of animals were evaluated before sacrifice using a home-made score chart (1, normal usage of ischemic limb; 2, good positioning but partial usage of ischemic limb; 3, good positioning but very light usage of ischemic limb; 4, bad positioning and no usage of ischemic limb); n ¼ 6. F, ischemic damage was evaluated at day 14 using a home-made score chart (0, no ischemic damage; 1, one inflamed finger; 2, at least two inflamed fingers; 3, one necrosed finger; 4, at least two necrosed fingers); n ¼ 6. , P < 0.05; , P < 0.001. whether in response to VEGF or ischemia, are inhibited in a DEP-1 ylated in DEP-1 KO mice, and that decreased Src Y418 phosphor- KO mouse model. In addition, tumor-associated angiogenesis, ylation colocalizes with CD31-positive regions in lung tissues tumor growth, and metastasis are also impaired in these condi- strongly suggest that endothelial Src activity was inhibited in the tions (Figs. 5 and 6), establishing stroma-derived DEP-1 as a absence of DEP-1 expression. Moreover, VE-cadherin–associated promoter of cancer progression. At the molecular level, this Src, which represents the main pool of VEGF-activated Src in correlates with the inhibition of VEGF-induced Src activation endothelial cells (12, 45), was also not dephosphorylated on in vivo, and consequently, with the abrogated phosphorylation Y529 (Fig. 1C; refs. 12, 14). These results thus clearly identify of its endothelial cell–specific substrate VE-cadherin, both critical DEP-1 as a critical regulator of Src activation and VE-cadherin mediators of vascular permeability and angiogenesis (Fig. 1; phosphorylation in VEGF-stimulated endothelial cells in vivo. refs. 16, 18, 34). These results thus demonstrate for the first time They are also consistent with the reduced induction of vascular that DEP-1 expression has positive and essential regulatory func- permeability mediated by VEGF in the DEP-1–deficient mice (Fig. tions in vivo that mediate increased vascular permeability and 2). In contrast to our results, VEGF was recently reported to induce angiogenesis (14, 15). similar levels of vascular permeability in WT and DEP-1–deficient One of the main functions promoted by DEP-1 is the activation mice (46). The reason for this discrepancy remains unclear, but of SFKs and downstream biological responses. This has now been could possibly be explained by experimental differences or the use reported in hematopoietic cells, platelets, smooth muscle cells, of a different mouse strain. breast tumor cells, and endothelial cells in vitro, and relies on the Because SFKs act as pivotal regulators of VEGF-dependent ability of DEP-1 to dephosphorylate the inhibitory C-terminal permeability and angiogenesis (16, 18, 35, 47), our results suggest tyrosine residue of SFKs (14, 15, 22, 24, 25, 32). Here, we identify that the dramatic block of these biological responses in the DEP-1 DEP-1 as a key mediator of VEGF-dependent Src activation in vivo KO mice is due to the abrogation of the Src pathway in endothelial (Fig. 1). Although this was shown in lung lysates and tissues where cells. The Src-dependent phosphorylation of VE-cadherin is critical several cell types are present, an important fraction represents for the reorganization of cell–cell junctions that occur during endothelial cells. Thus, the observation that its endothelial cell– permeability (35–37, 39). Expression of a catalytically active specific substrate, VE-cadherin, is no longer tyrosine phosphor- DEP-1 mutant (Y1311F/Y1320F), defective in Src activation, was

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Figure 5. DEP-1 regulates tumor growth and tumor-associated angiogenesis. WT and DEP-1 KO animals were orthotopically injected with A and G, Mvt-1/Pei-1 (A) or MET-1 cells (G) and tumor growth were monitored; n ¼ at least 9. B and H, mice were sacrificed and tumor weights were determined for Mvt-1 and MET-1 tumors after 28 and 25 days, respectively. C and I, CD31, Ki67, and F4/80 were stained by immunohistochemistry on paraffin-embedded sections of tumors; n ¼ at least 9. D and J, the number of capillaries per ROI was quantified according to CD31 staining. E and K, the number of Ki67-positive cells were quantified per field of view. F and L, representative immunodetection of cleaved caspase-3 in tumor lysates. , P < 0.05; , P < 0.01; , P < 0.001.

also unable to promote any of these biological activities in endo- angiogenesis and permeability. However, because in the Src KO thelial cells in vitro (15). This then strongly suggests that through its mice, only permeability and metastasis were affected (16), but that ability to activate Src, DEP-1 is a key regulator of postnatal the inhibition of all SFKs was required to block capillary formation

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Figure 6. DEP-1 regulates tumor permeability and the formation of spontaneous and experimental lung metastases. A, Mvt-1 and MET-1 tumor vessel permeability was assessed by the Miles Assay; n ¼ 9 WT and 7 KO mice bearing Mvt-1 tumors, and n ¼ 9WT and 6 KO mice-bearing MET-1 tumors. B, the formation of spontaneous (fat pad injection) and experimental (i.v. injection) lung metastases was assessed by H&E staining on lung cryosections. The lung metastatic burden, as deﬁned by the fraction of the lung surface occupied by metastatic lesions, was calculated; n ¼ at least 9. , P < 0.05; , P < 0.01; , P < 0.001.

in Matrigel and in tumors (16), we also conclude that other SFKs mediates the endothelial migratory response to VEGF stimulation expressed in endothelial cells might be regulated by DEP-1 and and was shown to be regulated by DEP-1 in T cells (15, 48, 49), contribute to the phenotypes observed. Notably, the Fyn kinase suggesting it could also be part of the pathways involved.

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Figure 7. DEP-1 expression in the tumor vasculature correlates with Src activation and with the microvessel density of human breast cancers. A, paraffin-embedded slices of MET-1 tumors were stained for CD31 and pY418Src by immunofluorescence. B, the presence of positive pY418Src signal in tumor-associated CD31-positive microvessels (broken lines) was quantified. C, correlation between DEP-1 gene expression in the tumor vasculature of breast cancer patients and the microvessel density (MVD) of tumors using the GSE15363 gene microarray data set from GEO. D, expression of DEP-1 is higher in the "A" subtype of breast tumor MV, genetically defined as proliferating and actively remodeling, than in the "B" subtype, which reflects features of more mature MV. , P < 0.05; , P < 0.01.

Our results also interestingly revealed that expression of DEP-1 possibly result from the decreased vessel density and permeability in tumor blood vessels correlated with Src activation (Fig. 7), and of tumors grown in these mice (Figs. 5 and 6). Also, this could be with the development of highly vascularized tumors (Fig. 5). due to a cell autonomous effect as CSF-1–induced chemotaxis of Thus, in DEP-1 KO mice, defective angiogenesis impaired tumor macrophages in vitro is partially mediated by DEP-1 (42). The growth, consistent with the decreased tumor cell proliferation and infiltration of tumors by macrophages can contribute to the increased apoptosis observed (Fig. 5). As these tumors had fewer promotion of angiogenesis and cancer progression, in part via vessels and were less permeable, spontaneous metastasis was the secretion of VEGF (50). However, as Mvt-1 tumor cells secrete greatly reduced (Fig. 6). However, injection of Mvt-1 and MET-1 high levels of VEGF (26), no decrease in VEGF tumor levels were mammary tumor cells into the blood circulation of DEP-1 KO observed in DEP-1 KO mice (Supplementary Fig. S1). Thus, mice similarly resulted in their impaired capacity to metastasize to although the impaired recruitment of macrophages may have the lungs, suggesting that defective VEGF-induced vascular per- contributed to the tumor phenotype observed in DEP-1 KO mice, meability in these animals impaired their ability to extravasate in the impaired capillary formation observed during Matrigel and the lungs. Consistent with this new role for DEP-1 in the pro- aortic ring assays is most likely the primary reason for defective motion of tumor-associated angiogenesis, DEP-1 gene expression tumor-associated angiogenesis. levels in the tumor vasculature of a cohort of breast cancer patients The work presented here thus brings novel insight into our (33) was found to be elevated in highly vascularized tumors, and comprehension of the molecular machinery regulating perme- in a genetically distinct tumor vascular subtype defined as being ability and angiogenesis in vivo, and importantly establish stroma- actively remodeling (Fig. 7). These observations thus further derived DEP-1 as a critical promoter of tumor cell growth and support our conclusions that DEP-1 is an important mediator metastasis. Recently, breast tumor-expressed DEP-1 was shown to of angiogenesis in vivo. mediate a Src proinvasive signaling pathway associated with the We also observed that the recruitment of macrophages to the promotion of metastasis (32). These findings together with those tumors grown in the DEP-1 KO mice was impaired. This could presented here therefore strongly suggest that inhibiting DEP-1

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DEP-1 Promotes Angiogenesis, Permeability, and Metastasis

functions could provide a new therapeutic strategy to counteract Acknowledgments cancer progression. We thank Jean-Philippe Gratton and Sara Weis for providing advice on Matrigel and permeability assays, Kent Hunter and Robert Cardiff for providing Disclosure of Potential Conﬂicts of Interest cell lines, and Veronique Barres of the Molecular Pathology core facility of the CRCHUM for performing the IHC experiments. No potential conﬂicts of interest were disclosed.

Authors' Contributions Grant Support Conception and design: P. Fournier, I. Royal This work was supported by the Cancer Research Society (I. Royal) and Development of methodology: P. Fournier the Canadian Institutes of Health Research (MOP-93681 to I. Royal and Acquisition of data (provided animals, acquired and managed patients, MOP-123490 to A. Rivard). P. Fournier was supported by student scholarships – provided facilities, etc.): P. Fournier, S. Dussault, A. Fusco, A. Rivard from Fonds de Recherche en Sante Quebec (25988), the Canadian Institutes of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Health Research (292353), and Institut du cancer de Montreal. computational analysis): P. Fournier, A. Rivard The costs of publication of this article were defrayed in part by the payment of advertisement Writing, review, and/or revision of the manuscript: P. Fournier, A. Rivard, page charges. This article must therefore be hereby marked in I. Royal accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Rivard Received April 13, 2016; revised June 21, 2016; accepted June 24, 2016; Study supervision: I. Royal published OnlineFirst June 30, 2016.

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OF12 Cancer Res; 76(17) September 1, 2016 Cancer Research

Tyrosine Phosphatase PTPRJ/DEP-1 Is an Essential Promoter of Vascular Permeability, Angiogenesis, and Tumor Progression

Patrick Fournier, Sylvie Dussault, Alfredo Fusco, et al.

Cancer Res Published OnlineFirst June 30, 2016.

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