Regulation of CXCR4 by the Notch Ligand Delta-Like 4 in Endothelial Cells

Research Article

Regulation of CXCR4 by the Notch Ligand Delta-like 4 in Endothelial Cells

Cassin Kimmel Williams,1,2 Marta Segarra,2 Maria De La Luz Sierra,2 Richard C.A. Sainson,1 Giovanna Tosato,2 and Adrian L. Harris1

1Molecular Oncology Laboratories, Cancer Research UK, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom and 2Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland

Abstract nals through the receptors Notch1 and Notch4 (4). Knocking out Gene-targeting studies have shown that Delta-like 4 (Dll4) is Notch1 alone or Notch1/Notch4 together caused severe defects required for normal embryonic vascular remodeling, but the in embryonic vascular remodeling similar to those seen in Dll4 mechanisms underlying Dll4 regulatory functions are not knockouts, whereas induced expression of activated Notch4 in the well defined. We generated primary human umbilical vas- developing mouse vasculature also caused abnormal vessel struc- cular endothelial cells that express Dll4 protein to study Dll4 ture and patterning resulting in embryonic death (see ref. 5 for function and previously showed that Dll4 down-regulates a review). vascular endothelial growth factor (VEGF) receptor 2 and We and others have shown that Notch activation diminishes NRP1 expression and inhibits VEGF function. We now report responses to vascular endothelial growth factor (VEGF), a principal proangiogenic factor (6, 7). However, integration of many pathways that expression of Dll4 in endothelial cells inhibited attachment and migration to stromal-derived growth factor 1 (SDF1) is required for angiogenesis. One such pathway is linked to the chemokine. Cell surface, total protein, and mRNA levels of chemokine receptor CXCR4, expressed in endothelial cells, and its CXCR4, principal signaling receptor for SDF1, were signifi- ligand, stromal-derived growth factor 1 (SDF1 or CXCL12). SDF1 cantly decreased in Dll4-transduced endothelial cells, attrib- is a chemoattractant for endothelial cells, which induces forma- utable to a significant reduction of CXCR4 promoter activity. tion of capillary sprouts (see ref. 8 for a review). Blocking SDF1 or An immobilized recombinant extracellular portion of Dll4 CXCR4 disrupts endothelial cell network formation on Matrigel (9). (rhDLL4) was sufficient to down-regulate CXCR4 mRNA and Knocking out either CXCR4 or SDF1 causes impaired vascular protein, whereas protein levels of SDF1, VEGF, and RDC1 were development, including defective formation of large vessels sup- unchanged. The ;-secretase inhibitor L-685,458 significantly plying the gastrointestinal tract (see ref. 10 for a review). CXCR4 reconstituted CXCR4 mRNA in rhDLL4-stimulated endothelial and its ligand are also regulated by hypoxia via hypoxia-inducible a cells. CXCR4 mRNA levels were significantly reduced in mouse factor-1 (11). xenografts of Dll4-transduced human gliomas compared with We hypothesized that Notch signaling regulates multiple pathways involved in tumor angiogenesis. Here, we report that Notch control gliomas, and vascular CXCR4 was not detected by immunohistochemistry in the enlarged vessels within the stimulation induced by Dll4 down-regulates CXCR4 expression, Dll4 gliomas. Thus, Dll4 may contribute to vascular differen- inhibiting responses toward SDF1. This represents a new integra- tiation and inhibition of the angiogenic response by regulating tive role for Notch signaling in endothelial function and has implications for therapeutic angiogenesis. multiple receptor pathways. [Cancer Res 2008;68(6):1889–95]

Introduction Materials and Methods Angiogenesis is essential for the growth of many types of cancer. Cells, cell culture, constructs, and reagents. Human umbilical As the tumors grow, low-oxygen environments within the tumor vascular endothelial cells (HUVEC) were isolated as described (9) and used activate a variety of angiogenic pathways. through passage 7. The LZRSpBMN-linker retroviral construct with full- The Notch signaling pathway plays critical roles in vascular length Dll4 was made and propagated, and retroviral infections were development and in tumor-induced angiogenesis (1). The Notch performed as described (7). Infected HUVECs were fluorescence-activated ligand Delta-like 4 (Dll4) is expressed at sites of active angioge- cell sorting sorted for enhanced green fluorescent protein (EGFP) expression to achieve >90% purity; populations used were 60% to 90% nesis (2). Mice with targeted deletions of Dll4 revealed abnormal EGFP positive. Endogenous Dll4 is usually not detected by reverse artery development and disrupted branching morphogenesis. transcription-PCR (RT-PCR) in HUVEC. Recombinant human Dll4 (rhDLL4; Interestingly, heterozygous deletions of the Dll4 gene resulted in R&D Systems) was diluted in 0.2% type B bovine gelatin (Sigma Chemical failure to remodel the primary vascular plexus and caused Co.) and coated on plates at a final concentration of 1 Ag/mL. The g- embryonic lethality, providing evidence for the critical importance secretase inhibitor L-685,458 (Sigma Chemical or Peptide Institute, Inc.), of Dll4 expression levels in vascular development (3). Dll4 sig- dissolved in DMSO (Sigma Chemical), was used at 2 Amol/L. Migration assay. Endothelial migration assays were performed as described (7). HUVECs were detached with 5 mmol/L EDTA and placed in the upper chambers, whereas migration medium with or without Note: A.L. Harris and G. Tosato are cosenior authors. 100 ng/mL SDF1 was placed in the lower chamber. After 16 to 20 h of Requests for reprints: Giovanna Tosato, Laboratory of Cellular Oncology, Center incubation at 37jC, viable cells in the lower chamber were counted. for Cancer Research, National Cancer Institute, Building 37, Room 4124, Bethesda, MD Attachment assay. Endothelial cell attachment assays were performed 20892. Phone: 301-594-9596; Fax: 301-594-9585; E-mail: [email protected]. I2008 American Association for Cancer Research. using 96-well plates coated with 0.1% bovine serum albumin (BSA; Sigma doi:10.1158/0008-5472.CAN-07-2181 Chemical), 5 mmol/L laminin (Trevigen), or 2 to 4 Ag/mL SDF1 (PeproTech) www.aacrjournals.org 1889 Cancer Res 2008; 68: (6). March 15, 2008

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. Cancer Research overnight at 4jC. After blocking with 1% BSA for 1 h at room temperature, In vivo studies. The human glioblastoma U87GM cell line was main- 4 4 2.5 Â 10 to 5 Â 10 of EDTA-detached HUVEC per well were plated in tained in RPMI 1640 supplemented with 10% FCS, 2 Amol/L L-glutamine, ‘‘attachment medium’’ [M199 (Mediatech) with 25 Ag/mL porcine heparin 50 IU/mL penicillin, and 50 Ag/mL streptomycin sulfate. Cells were (Sigma Chemical) and 1% heat-inactivated FBS (Biofluids)]. After 1 h at transduced with retrovirus containing full-length human Dll4 or empty 37jC, plates were fixed with 4% paraformaldehyde (Sigma Chemical) and vector as previously described (12). Retroviral supernatants were prepared stained with 0.05% crystal violet (Sigma Chemical). Images were taken using the bicistronic pLZRS-IRES-GFP plasmid (gift from Maarten van using a Retiga 1300 digital camera (QImaging) under phase-contrast Lohuizen, Cancer Institute Antoni van Leeuwenhoek Hospital, Amsterdam, microscopy (Olympus 1 Â 51 with a 10 Â 0.25 PhL lens; Olympus Optical the Netherlands) with the Phoenix amphotropic packaging cell line (gift Co.), and images from IPLab for Windows software (Scanalytics, Inc.) from Garry P. Nolan, Stanford School of Medicine, Stanford, CA). were imported into Adobe Photoshop. Percentage attachment was mea- Six- to 8-week-old female BALB/c severe combined immunodeficient sured using ImageJ software correcting for pixels from adherent vector- mice (Harlan Sprague Dawley, Inc.) were implanted s.c. with 107 U87 cells transfected HUVEC. (200 AL injection volume consisting of culture medium and an equal volume Matrigel cord formation assay. The assay was performed as described of Matrigel). Each group consisted of five mice. When tumors reached the (7). HUVECs were plated (40 Â 103 to 75 Â 103) onto 24-well plates maximum size (1.44 cm2 surface area) permitted by the Home Office license, precoated with solidified Matrigel (Collaborative; BD PharMingen). mice were sacrificed and tumors were excised. Neutralizing antibodies to human VEGF [mouse monoclonal antibody CXCR4 immunochemistry. Rat mAb MAB21651 (25 Ag/mL; R&D (mAb) A4.6.1; a gift of Genentech, Inc.] or to CXCR4 (mouse mAb clone Systems) was used for 90 min on cryostat tumor tissue sections fixed in 44716.111; R&D Systems) were added to the cultures at 10 Ag/mL. acetone for 10 min and then allowed to dry. The secondary anti-mouse After 18 h of incubation, cells were photographed under phase-contrast antibody (cross-reactive with rat 1:20 dilution) and the 3,3¶-diaminobenzi- microscopy and images were imported into Adobe Photoshop (Adobe dine developer were from DAKO (Envision kit, ChemMate-real). Systems). Statistical analysis. Group differences were evaluated by Student’s Flow cytometry. Cells were detached with EDTA and stained with t test; P values of <0.05 were considered significant. phycoerythrin-labeled CD184 (BD PharMingen). Data were collected using a FACSCalibur flow cytometer (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson). Results Western blotting. Lysis buffer consisting of 1% Triton X-100, 25 mmol/L Dll4 expression is associated with impaired endothelial cell Tris (pH 7.5), 150 mL NaCl, and 5 mmol/L EDTA was supplemented with migration and attachment mediated by SDF1. Previously, we protease inhibitor cocktail set III (Calbiochem). Cell lysates (30 Ag protein) were solubilized in NuPAGE lithium dodecyl sulfate sample buffer (Novex), reported that HUVECs retrovirally transduced with full-length Dll4 incubated at 37jC for 30 min, and run through 4% to 12% NuPAGE Bis-Tris are deficient in their ability to respond to VEGF and to form cord- gels (Invitrogen). After transfer, Immobilon-P membranes (Millipore) were like structures on Matrigel, a mixture of extracellular matrix pro- incubated overnight with antibodies against the following proteins: CD184 teins (7). Because the chemokine receptor CXCR4 contributes to (2B11, 1:250), RDC1 (12870, 1:250; Abcam), SDF1 (BAF 310, 1:1,000; R&D cord formation (9), we compared the effects of CXCR4 neutraliza- Systems), VEGF (1:500; Santa Cruz Biotechnology), and cleaved Notch1 tion to overexpression of Dll4. CXCR4 neutralization impaired (Val 1744, 1:1,000; Cell Signaling Technology). Relative protein expression HUVEC cord formation similar to Dll4 overexpression, whereas levels were estimated by membrane rehybridization with anti-actin anti- VEGF neutralization minimally affected this process (Fig. 1A). This body and detection as described (7). observation raised the possibility that Dll4 overexpression in RNA preparation and quantitative RT-PCR. Total RNA extraction from HUVEC might reduce CXCR4 function. We tested the migration of HUVEC and quantitative RT-PCR were performed as described (7). The following primers were used: glyceraldehyde-3-phosphate dehydrogenase Dll4-transduced HUVEC in response to SDF1 (100 ng/mL), the (GAPDH), 5¶-GAGTCCACTGGCTGCTTCA (sense) and 5¶-GGGGTGCTAAG- unique CXCR4 ligand, which is a chemoattractant for endothelial CAGTTGGT (antisense); CXCR4, 5¶-GGCAAACTGGTACTTTGGGA (sense) cells (9). We found that Dll4 overexpression significantly (P = 0.007) and 5¶-GACGCCAACATAGACCACCT (antisense). Total RNA was extracted inhibited HUVEC migration toward SDF1 in Transwell migration from 25 mg frozen tumors using the FastRNA Pro Green kit (Qbiogene) and assays (Fig. 1B). TRI Reagent. After treatment with DNA-free kit (Ambion), 1 Ag total RNA We next examined whether CXCR4 binding to SDF1 might also was reverse transcribed into cDNA with the High Capacity cDNA Archive be impaired in Dll4-overexpressing HUVEC. After coating ELISA kit (Applied Biosystems). Quantitative PCR was performed in triplicate plates with recombinant SDF1a (2–4 Ag/mL in PBS), BSA (1% in A using the Exiqon system (Roche). Each reaction consisted of 25 L PBS), or laminin (5 Amol/L in PBS) we measured HUVEC containing 10 to 25 ng of cDNA, 12.5 ALof2Â ABsolute QPCR Mix adherence. In wells coated with 1% BSA in PBS, no HUVEC (ABgene), 1 AL each of 10 Amol/L forward and reverse primers (Invitrogen), attachment occurred by empty vector or Dll4 HUVEC (data not and 0.25 AL probe (Roche). The comparative Ct method was used for quantification. Expression level of mouse CXCR4 was normalized to that of shown), whereas on laminin-coated wells both empty vector– mouse h-actin. transduced and Dll4-transduced HUVEC adhered alike. On wells Transient transfection and luciferase assay. The full-length human coated with SDF1, HUVEC that overexpressed Dll4 attached less CXCR4 promoter (pGL2-2632/+86) was a gift of Dr. W. Krek (Friedrich well than control HUVEC (Fig. 1C). Quantitative analysis of Miescher Institute of Biomedical Research, Basel, Switzerland; ref. 11); attachment in eight independent experiments revealed that Dll4- the control PGL-2-Basic vector was from Promega. Transient transfections transduced HUVEC adhered significantly less (P = 0.02) to SDF1- of CXCR4 promoter and control in HUVEC were performed using Amaxa coated wells than empty vector–transduced HUVEC (Fig. 1D). nucleofector system (Amaxa Biosystems), with Cell Line Nucleofector CXCR4 down-regulation in endothelial cells that overex- A A Solution V (100 L/reaction). Cells were cotransfected with 2.5 g phRL- press Dll4. CXCR4 is a G protein–coupled receptor that mediates SV40 (Renilla luciferase reference plasmid; Promega) to control for trans- SDF1 responses (8). We examined whether the diminished fection efficiency. After transfection, cells were incubated (six-well plates) 5to6hat37jC in complete culture medium. Firefly luciferase and Renilla attachment and migration to SDF1 in Dll4-overexpressing cells luciferase activities from transfected cells were measured in triplicate might be attributable to reduced cell expression of CXCR4. By flow microtiter wells using Dual-Glo Luciferase Assay System (Promega). Relative cytometry (Fig. 2A), surface CXCR4 levels were reduced in Dll4- luciferase activity is defined as the ratio of firefly luciferase to Renilla overexpressing HUVEC [mean fluorescence intensity (MFI) = 67.1] luciferase activity. compared with control HUVEC (MFI = 129.8). This CXCR4

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Figure 1. Effects of Dll4 overexpression on endothelial cell migration and attachment. A, effects of Dll4 expression, anti-VEGF, or anti-CXCR4 neutralization on HUVEC cord formation. HUVECs (40 Â 103 parental or retrovirus-transduced cells) were incubated for 18 h on Matrigel-coated wells. Anti-VEGF and anti-CXCR4 neutralizing antibodies were added to control HUVEC at a concentration of 10 Ag/mL. Representative images. B, SDF1 (100 ng/mL)-induced Transwell migration of control and Dll4-overexpressing HUVECs (5 Â 105 per well) over 16 to 20 h of incubation. Columns, mean fold increase in cell migration compared with medium alone in four independent experiments; bars, SE. *, P = 0.007. C, representative images reflecting attachment to wells precoated with laminin (5 Amol/L) or SDF1 (2–4 Ag/mL) by control and Dll4-overexpressing HUVECs (2.5 Â 104 to 5 Â 104 per well). Cells were incubated for 1 h at 37jC, washed with PBS, fixed with 4% paraformaldehyde, and stained with crystal violet. Phase-contrast microscopy. Original magnification, Â4. D, quantitative analysis of attachment in control and Dll4-overexpressing HUVECs. Attachment was quantified by measuring pixels stained for crystal violet using ImageJ, and percent attachment was calculated by dividing the attachment of control HUVEC to 0.1% gelatin. Columns, mean of eight independent experiments; bars, SE. *, P = 0.02. reduction in Dll4-transduced HUVEC populations was similarly control promoter (PGL-2-Basic) in Dll4-overexpressing and control observed in Dll4-expressing HUVEC (60–90% of cells) and the Dll4- HUVEC. Levels of relative luciferase activity induced by the CXCR4 negative HUVEC (10–40% of cells), suggesting an extrinsic action of promoter-reporter were significantly reduced (P = 0.003, five Dll4. Western blot analysis showed markedly diminished levels of determinations) in Dll4-transduced cells compared with control CXCR4 protein in lysates from HUVEC-overexpressing Dll4 com- cells, whereas levels of luciferase activity induced by the control pared with vector-transduced HUVEC (Fig. 2B, left). Measurement of reporter were similar (Fig. 2D). relative ratios of CXCR4/actin band intensities revealed that CXCR4 Notch signaling is required for Dll4-induced CXCR4 down- protein levels were f6-fold lower in Dll4-HUVEC compared with regulation in HUVEC. To evaluate whether down-regulation of vector-HUVEC. Recently, the orphan receptor RDC1 was identified CXCR4 expression in Dll4-overexpressing HUVEC is a result of as a second receptor for SDF1 (13). We analyzed RDC1 expression Notch activation, we looked for evidence of Notch1 cleavage. in Dll4-overexpressing and control HUVEC. By Western blotting By immunoblotting, we detected significantly higher levels of the using specific antibodies, we detected similar RDC1 expression in COOH-terminal Notch1 fragment in Dll4-transduced HUVEC empty vector–transduced and Dll4-transduced HUVEC (Fig. 2B). compared with control cells (Fig. 3A). To assess the potential We used quantitative RT-PCR to assess levels of CXCR4 mRNA contribution of reverse signaling by Dll4, we used rhDLL4, which in Dll4- and vector-transduced HUVEC. CXCR4 mRNA levels were lacks the intracellular and transmembrane domains. Such Dll4 significantly (P = 0.0001, eight determinations) reduced in Dll4- fragment is not known to exist in nature, but rhDLL4 was shown overexpressing HUVEC compared with vector-infected HUVEC to activate Notch signaling in vitro if attached to the wells (7). (Fig. 2C). To test whether Dll4 transcriptionally down-regulates We cultured HUVEC for 48 h on plates precoated with BSA CXCR4 expression, we transiently transfected a luciferase reporter (1 Ag/mL) or rhDLL4 (1 Ag/mL). In four determinations, we found containing the human CXCR4 promoter (PGL-2-CXCR4) or a significantly (P = 0.015) decreased levels of CXCR4 mRNA in www.aacrjournals.org 1891 Cancer Res 2008; 68: (6). March 15, 2008

Figure 2. Regulation of CXCR4 expression in Dll4-transduced endothelial cells. A, surface levels of CXCR4 expression in vector-only (Vector) and Dll4-transduced (Dll4) HUVECs measured by flow cytometry. Control reflects unstained HUVEC. B, CXCR4 and RDC1 expression in HUVECs transduced with control vector or Dll4 retrovirus evaluated by immunoblotting with antibody to CXCR4 (CD184) or RDC1 and reblotting with goat anti–h-actin antibody. CXCR4 results are representative of eight independent determinations. C, quantitative RT-PCR analysis of GAPDH and CXCR4 expression in HUVECs transduced with vector only or Dll4. Columns, mean of eight independent determinations; bars, SE. *, P = 0.0001. D, relative luciferase activity after transfection of HUVEC (infected with control or Dll4 retrovirus) with the CXCR4 promoter-reporter plasmid or the control promoter. Cells were cotransfected with Renilla luciferase to account for variation in transfection efficiencies. The results are expressed as relative activation after correction for Renilla luciferase activity. Columns, mean of five experiments; bars, SE. *, P = 0.003.

HUVEC activated with rhDLL4 compared with controls (Fig. 3B, left). mice were injected s.c. with the human glioblastoma U87 cells, Quantitative RT-PCR showed that expression of CXCR4 mRNA which were retrovirally transduced with human Dll4 or empty diminishes by 50% after HUVECs are cultured for 16 h onto immo- vector only. The Dll4 tumors that developed at the injection site bilized rhDLL4 and continues to decline through 72 h (Fig. 3B, right). displayed fewer but larger vessels than the vector control tumors By immunoblotting, we found a corresponding reduction of CXCR4 (14). We assessed vascular CXCR4 expression in the tumors. By protein levels in HUVECs cultured on rhDLL4-coated wells for 72 h, immunohistochemistry, the small vessels in the control tumors whereas RDC1 levels remained similar (Fig. 3C). We also found generally expressed CXCR4 (Fig. 4A, top). However, the larger similar levels of both SDF1 and VEGF in HUVEC lysates collected vessels typically found in the Dll4 tumors did not express CXCR4 after 72 h of cell culture on BSA- or rhDLL4-coated wells (Fig. 3C). (Fig. 4A, bottom). In additional experiments, we evaluated the We showed previously that the g-secretase inhibitor L-685,458 relative levels of mouse CXCR4 mRNA in tumor extracts, which can block activation of the Notch pathway and reverse the effects includes the vasculature and other host cells in the tumor of Dll4 signaling by recovering VEGF receptor 2 (VEGFR2) expres- microenvironment. By real-time PCR using mouse-specific probes, sion and VEGF-induced proliferation (7). To further establish that we found that the relative levels of CXCR4 mRNA were significantly extracellular Dll4 down-regulates CXCR4 expression by signaling reduced in Dll4 tumors compared with controls (Fig. 4B). These through the Notch pathway, we used the g-secretase inhibitor L- results provide evidence that the Notch ligand Dll4 down-regulates 685,458. Treatment with the inhibitor reduced the levels of Notch1 CXCR4 expression in the tumor vasculature. cleavage (Fig. 3D, left) and significantly reconstituted CXCR4 mRNA levels in HUVEC cultured on rhDLL4 (P = 0.003; Fig. 3D, right). Dll4 regulates CXCR4 expression in vivo. To assess whether Discussion Dll4 down-regulation of CXCR4 is also observed in vivo, we used a Here, we show that Dll4 transcriptionally down-regulates CXCR4 human glioblastoma xenograft tumor model. Immunodeficient expression in endothelial cells and, by this mechanism, reduces

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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. Delta-like 4 Regulates CXCR4 responsiveness to SDF1 in HUVEC. Similar down-regulation of neither full-length Dll4 expression nor recombinant rhDLL4 down- CXCR4 was seen by constitutive expression of full-length Dll4 and regulates RDC1. Therefore, we conclude that impaired responses to by stimulation with an immobilized extracellular portion of Dll4. SDF1 by Dll4-transduced HUVEC are likely attributable to down- This provides evidence that the intracellular and transmembrane regulation of CXCR4 expression. domains of Dll4 are not required for activity in the current system. VEGF signaling increases SDF1 protein levels in HUVEC (9), and Because the extracellular domain of Dll4 seems sufficient to down- Notch activation down-regulates the principal VEGF receptor, regulate CXCR4 expression, Dll4 likely acts through Notch and VEGFR2 (6, 7). The possibility existed that Dll4 might indirectly not through Dll4 signaling. Consistent with an involvement of reduce SDF1 expression in HUVEC through down-regulation of Notch signaling, Dll4-transduced HUVEC displayed much higher VEGFR2. However, our immunoblotting results show similar levels levels of Notch1 COOH-terminal fragment, which is generated by of SDF1 in HUVEC cultured with and without rhDLL4. SDF1 enzymatic cleavage on ligand binding and is essential to Notch activation of CXCR4 was reported to increase levels of VEGF mRNA signaling (1, 15). Furthermore, partial recovery of CXCR4 expression (16). We previously determined that Dll4 up-regulation in HUVEC was seen in the presence of the g-secretase inhibitor L-685,458, does not affect VEGF mRNA or protein secretion (7), and we now as previously shown for VEGFR2 (7). These results suggest that show that VEGF protein levels are similar in BSA- and rhDLL4- down-regulation of CXCR4 by Dll4 is due to activation of the Notch stimulated HUVEC. We conclude that, under these culture pathway and provide the first evidence of association between conditions, Dll4 does not significantly affect SDF1 protein levels, these pathways. and Dll4-induced down-regulation of CXCR4 is not associated with The previously identified orphan receptor RDC1 was recently diminished expression of VEGF. This latter conclusion is consistent shown to be an additional SDF1 receptor in T lymphocytes (13). We with the observation that CXCR4 inactivation in vivo decreases questioned whether Dll4 down-regulates RDC1 but found that tumor growth without affecting levels of VEGF (17).

Figure 3. Regulation of CXCR4 expression by extracellular rhDLL4 and requirement of Notch signaling. A, COOH-terminal Notch1 fragment detected by immunoblotting with specific antibodies. The membrane was reblotted for h-actin. Cell lysates were prepared from semiconfluent cultures of HUVEC (parental and infected with control retrovirus or Dll4 retrovirus). B, quantitative RT-PCR analysis of relative GAPDH and CXCR4 mRNA expression in HUVECs stimulated with BSA or extracellular rhDLL4 at 48 h (left) or at 4, 8, 16, 18, 20, 24, 48, and 72 h (right). The results represent the mean of four independent determinations (left; *, P = 0.015) or two to four determinations per time point (right). Bars, SE. C, CXCR4, SDF1, RDC1, and VEGF expression in HUVECs stimulated for 72 h with BSA or extracellular rhDLL4 evaluated by immunoblotting with specific antibodies. Membranes were reblotted with goat anti–h-actin antibody. Representative of two to three independent determinations. D, COOH-terminal Notch1 fragment detected by immunoblotting of cell lysates from semiconfluent cultures of HUVEC transduced with Dll4 or control retrovirus after 72 h of incubation with L-685,458 (GSI) or diluent (DMSO) only. Left, the membrane was reblotted for h-actin; right, quantitative RT-PCR analysis of relative GAPDH and CXCR4 mRNA expression in DMSO or L-685,458 inhibitor-treated (72 h) HUVECs stimulated with BSA or extracellular rhDLL4. Columns, mean of three independent determinations; bars, SE. *, P = 0.003. www.aacrjournals.org 1893 Cancer Res 2008; 68: (6). March 15, 2008

Figure 4. Regulation of CXCR4 expression in xenografts of U87 glioblastoma cells transduced with Dll4. A, CXCR4 detected by immunohistochemistry in tumor sections of xenografts established in mice by injection of human U87 glioma cells transduced with control empty vector or Dll4. In control tumors (top), CXCR4 (brown staining) lines small tumor vessels (arrow). In Dll4 tumors (bottom), the larger vessels (arrow) are negative for CXCR4. B, relative mouse CXCR4 mRNA levels in vector control-U87 and Dll4-U87 glioma tumor xenografts measured by real-time PCR using mouse-specific probes. Columns, mean of five tumors; bars, SE. *, P < 0.05.

The effects of Dll4 on tumor angiogenesis are complex. Several in endothelial progenitor cells (EPC), which contribute to tumor approaches to inhibit Dll4/Notch interactions have resulted in neovascularization (23). As the tumor environment becomes excessive tumor neovascularization, which was nonfunctional hypoxic, the resulting secretion of angiogenic cytokines, such as and resulted in reduced tumor perfusion and tumor growth VEGF and SDF1, serves to recruit VEGFR2- and CXCR4-expressing (18–20). Dll4 overexpression in tumor cells was reported to reduce EPCs from circulation (23, 24). Because both these receptors the number of tumor vessels, which were larger and more effec- are down-regulated in endothelial cells by Dll4 stimulation (7), tive, and tumor growth was generally not reduced (14, 18). The once EPCs are recruited and make direct contact with endo- observation we have made that Dll4/Notch activation reduces thelial cells that express Dll4, activation of Notch signaling could CXCR4 expression in tumor vessels adds yet another element of serve to dampen expression of CXCR4 and VEGFR2 and help complexity to Dll4/Notch vascular effects. Based on the critical retain the cells for incorporation within the tumor vasculature. role of CXCR4 on angiogenesis (8–10), it is possible that some Delicately balanced levels of Notch would be required to maintain of the reported vascular abnormalities derived from blocking or appropriate levels of VEGFR2 and CXCR4 expression in endothelial promoting Dll4/Notch signaling may be contributed by CXCR4 cells. modulation. CXCR4 inhibitors are currently in clinical trial and Our discovery of Dll4 as a regulator of CXCR4 identifies a it would be of interest to assess vascular effects in patients on previously unknown relationship between Notch signaling and such trials. CXCR4 expression and reveals an additional mechanism by Although this is the first report linking the Notch signal- which Dll4 plays a role in angiogenesis regulation. This may ing pathway to CXCR4 regulation, evidence for this relation- have implications for antiangiogenic therapy targeting CXCR4, ship can be seen in Kaposi sarcoma lesions. Although hypoxia has where Dll4 in tumor vasculature may induce resistance, and been shown to enhance transcription of CXCR4 in vitro (11), it would therefore be of interest to combine such therapy with and Kaposi sarcoma lesions are known for their hypoxic Notch inhibition. It may also have important implications environment, CXCR4 was undetectable in the angiogenic environ- for antiangiogenic therapy targeting the Dll4/Notch signal- ment within Kaposi sarcoma lesions, whereas vessels in the ing pathway, which has recently emerged an exciting and surrounding normal tissues stained positive (21). Dll4 expression promising approach to cancer treatment (see ref. 25 for a is increased by hypoxia at sites of active angiogenesis (2, 22). review). Our report showing that Dll4 stimulation down-regulates CXCR4 expression may explain absence of detectable CXCR4 within Kaposi sarcoma lesions. Acknowledgments Dll4 may serve as a switch for CXCR4 regulation in cells Received 6/12/2007; revised 11/5/2007; accepted 12/8/2007. of endothelial lineage. The endothelial cells, which express Dll4 Grant support: Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and ‘‘The 6th Framework Programme of the (2, 18) in the tumor microenvironment, would be expected to European Union (Angiotargeting)’’. C.K. Williams is a NIH-University of Oxford Health promote Notch activation and CXCR4 down-regulation in adja- Science Scholar. cent endothelial cells. Because CXCR4 plays critical roles in mor- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance phogenic processes that accompany neovascularization (8–10), with 18 U.S.C. Section 1734 solely to indicate this fact. its down-regulation in tumor endothelial cells would be expected We thank Drs. V. Kapoor and W. Telford for cell sorting and flow cytometry support; Dr. Garry P. Nolan for providing the retroviral vector; and Drs. M. Narazaki, to contribute to vascular defects that are commonly observed in N. Labo, P. Gasperini, P. McCormick, L. Harrington, O. Salvucci, and H. Turley for the tumor vasculature. Dll4 may also serve to regulate CXCR4 technical support and advice.

Cancer Res 2008; 68: (6). March 15, 2008 1894 www.aacrjournals.org

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Cassin Kimmel Williams, Marta Segarra, Maria De La Luz Sierra, et al.

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