VEGFR3and Metastasis inProstate Cancer
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1 Bernard O’BrienInstitute of Microsurgery,Melbourne, Australia; Departments of 2 Surgeryand 3Pathology, University of Melbourne, Melbourne, Australia; 4Anatomical Pathology, St. Vincent’s Hospital, Fitzroy, Australia; 5Ludwig Institute for Cancer Research, Parkville, Australia; 6SydneyCancer Centre, Royal Prince Alfred Hospital, Camperdown, Australia; and7Garvan Institute of Medical Research, St. Vincent’s Hospital,Darlinghurst,Australia
Experimental Design: Theexpression pattern of VEGF-C, VEGF-D, andVEGF receptor-3 in localized prostate cancer specimens (n=37) was determined using immunohistochemistry.
Results: Widespread, heterogeneous staining for VEGF-C and VEGF-Dwas observed in all cancer specimens. Intensity of VEGF-Cstaining waslower in benign prostate epithelium than in adjacent carcinoma,whereasno difference between benign epithelium and carcinoma wasobservedfor VEGF-D staining. VEGF receptor-3 immunostaining wasdetected inendothelial cells of lymphatic vessels in 18 of37 tissue samples.The presence of VEGF receptor-3-positive vesselswas associated withlymph node metastasis (P = 0.0002), Gleasongrade (P< 0.0001), extracapsular extension (P =0.0382), andsurgical margin status (P = 0.0069). In addition,VEGFreceptor-3 staining highlighted lymphatic invasion byVEGF-C-positive/VEGF-D-positivecarcinoma cells.
Conclusions: Together, these results suggest that paracrine activationof lymphatic endothelial cell VEGF receptor-3 by VEGF-Cand/or VEGF-Dmay be involved in lymphatic metastasis. Thusthe VEGF-C/VEGF-D/VEGFreceptor-3 signaling pathway may provide atarget forantilymphangiogenic therapy in prostate cancer.
Themolecular mechanisms involved in lymph node metastasis are poorlyunderstood, partly because of the lack of specific lymphaticendothelialmarkers and specific lymphatic growth factors. This situationhasimproved somewhat since vascular endothelial growth factor(VEGF)receptor-3 was described (3) . Inembryos, VEGF receptor-3is initially expressed in venous vasculature (4), butin adults, it is absent in endothelia of all large blood vesselsandis generally restricted to lymphatic endothelial cellsand a subsetof capillary endothelia (5 , 6) . VEGFreceptor-3 is reactivated in the blood vesselendothelium in sometumors, and the up-regulation of its twoligands, VEGF-C and VEGF-D(7) , may accompanythis (8, 9,10) .
VEGF-Cand VEGF-D belong to the VEGF family (11,12) , and like all VEGF familymembers, they contain a centralregion called the VEGF homology domain (11 ,12) . They have NH2-terminaland COOH-terminalpropeptides and can bind VEGF receptor-2 and VEGFreceptor-3 (10, 13 ,14) . Recent studies in tumormodels have provided direct evidence thatVEGF-C and VEGF-D caninduce lymphangiogenesis through VEGFreceptor-3 and/or promoteangiogenesis through VEGF receptor-2 (15, 16,17, 18,19) and thatinduction of lymphangiogenesis is oftenassociated with lymph nodemetastasis in these model systems. In breastcarcinoma models, VEGF-Cinduced only lymphangiogenesis (15 ,16 , 19) , and thiswas associated with lymphnode metastasis in two studies (15, 19) .In contrast, in melanoma and epithelioid-like(293EBNA cell line)tumor models transfected with VEGF-C andVEGF-D, respectively, bothlymphangiogenesis and angiogenesis wereobserved (17, 18) , and lymph node metastasiswas observedonly in the 293EBNA model. The differences in these resultsmostlikely reflect variations in the relative expression ofthe VEGF-C/VEGF-Dreceptors VEGF receptor-2 and VEGF receptor-3 inthe model systems.
Studiesin clinical specimens have shown that VEGF-C expression ispositively associated with lymph node metastasis in several tumortypes. In prostate cancer, Tsurusaki et al. (20) foundthat VEGF-C mRNA levels were significantly higher in lymphnode-positivetumors and that VEGF receptor-3-positive vessels wereincreased inthe stroma of VEGF-C-positive tumors. Moreover, VEGF receptor-3expressionwas correlated with Gleason score, preoperative prostate-specificantigenlevels, and lymph node metastasis in prostate cancer (21). However, there are no reports documenting VEGF-C protein localizationin human prostate cancer. Recent reports in breast andovarian cancersuggest that VEGF-D positivity is a prognostic factor(22,23, 24); however, VEGF-D localization inprostate cancer has not been describedpreviously. In the presentstudy, the protein expression patterns ofVEGF-C, VEGF-D, and VEGFreceptor-3 were examined by immunohistochemistry to explorewhetherthis signaling axis is associated with lymph node status inprimaryprostate cancer.
|MATERIALS AND METHODS|
Immunoprecipitation,Western Blotting, and Analysis of AntibodySpecificity.
A plasmid encoding FLAG-tagged, full-length VEGF-D and transfectioninto293EBNA cell have been described previously (14). For VEGF-C,an expression plasmid encoding full-length VEGF-C tagged atthe COOHterminus with three Myc tags (single Myc epitope, EQKLISEEDL)wasgenerated. 293EBNA cells were transiently transfected withthisplasmid using FuGENE 6 transfection reagent (Roche MolecularBiochemicals, Indianapolis, IN) according to the manufacturer’sinstructions.VEGF-C and VEGF-D were purified from conditioned mediumcollectedover 24–48 h. A c-Myc antibody (9E10; ZymedLaboratories Inc., SanFrancisco, CA) coupled to cyanogen bromide-activatedSepharose (AmershamBiosciences, Castle Hill, New South Wales,Australia) and M2 beads(anti-FLAG M2-Agarose mouse; Sigma-Aldrich, St.Louis, MO) were usedto pull down VEGF-C and VEGF-D, respectively.
ForWestern blotting, the proteins were transferred to a nitrocellulosemembrane(Hybond-C Super; Amersham Biosciences, Buckinghamshire, UnitedKingdom) and probed with primary antibody against VEGF-C orVEGF-D,followed by the appropriate secondary antibody. Signal wasdevelopedwith SuperSignal West Femto Maximum Sensitivity Substrate(PierceBiotechnology, Rockford, IL).
Tissue sections were deparaffinized in shellex, followed by rehydrationin graded ethanol. After quenching of endogenous peroxidaseactivity,antigen retrieval (citrate buffer, pH 6.0), andprotein blocking (DAKOprotein block serum free; DAKO Corp., Carpinteria,CA), tissuesections were incubated overnight at 4°C withthe specified primaryantibody. The tissue sections were thenincubated with theappropriate biotinylated secondary rabbitantigoat or antimouseantibodies (DAKO, Glostrup, Denmark) andstreptavidin-biotin-peroxidase complex (DAKO). Peroxidase reactivitywas visualized using 3,3'-diaminobenzidine (DAKO). Thesections werecounterstained with hematoxylin and coverslipped. Negativecontrolsincluded immunostaining of normal human cerebrum (25)and substitution of normal goat IgG (R&D Systems) ormouse IgG1 (DAKO)for the primary antibodies. Normal human heartwas used as a positivecontrol tissue for VEGF-C (11)and VEGF-D (24)staining, human melanoma was used as a positive controltissue forVEGF receptor-3 (26) staining,and normal lymphnode was used as a positive control for D2-40 staining. Twotrainedobservers (K. O. and E. D. W.) evaluated and interpreted theresultsof immunohistochemical staining without knowledge ofthe clinicaldata of each patient. Staining was scored by bothobserverssimultaneously, using a multihead microscope. Stainingofadenocarcinoma, benign epithelia, stroma, lymphatic vessels,vascularendothelial cells, and smooth muscle was recorded asstrong, weak, ornegative and recorded on standardized data sheets.Primary andsecondary Gleason grades were assigned by apathologist (K. O.).
The relationships between the presence of VEGF receptor-3-positivevesselsand clinicopathological parameters were evaluated by Fisher’sexacttest or 2 test asindicated. P < 0.05 wasconsidered statisticallysignificant. All calculations were performedusing the statisticalcomputer program Prism+Instat bundle (GraphPadSoftware, San Diego,CA).
Astrong and consistent pattern of VEGF-D expression was observedwithinthe fibromuscular stroma of all prostate cancer specimens (37of 37specimens; Fig. 2F). The smooth muscle cells of blood vessels inand adjacent to areasof tumor expressed VEGF-D strongly (37 of 37samples; Fig. 2G). In contrast, microvessel endothelial cellsshowed inconsistentstaining. Vascular endothelia adjacent tocarcinoma showed positivestaining in some specimens (Fig. 2H).
Localizationof Vascular Endothelial Growth Factor Receptor-3 inProstate Tissue.
VEGF receptor-3 immunostaining decorated the endothelial cellslininga subpopulation of vessels in 18 of 37 specimens. The mediannumberof VEGF receptor-3-positive cross-sections of vesselswas 45 pertissue section (range, 5–519 per tissue section).The vessels werethin-walled and devoid of erythrocytes andneutrophils, suggesting alymphatic nature (Fig. 3, A–D andF). Furthermore, the vessels stained positively using the lymphaticendothelium marker D2-40 (Fig. 3E). The adjacent blood vessels were alwaysnegative for VEGF receptor-3(Fig. 3A). VEGF receptor-3-positive vessels were located within the tumornests, at the tumor periphery, and in benign tissue. Usually,intratumoralVEGF receptor-3-positive vessels were very small orcollapsed (Fig.3, B and C), although large intratumoral VEGFreceptor-3-positive vessels wereobserved occasionally (Fig. 3D). In five cases, four of which had known clinical lymphnodemetastasis, cancer cells were visible within the lumenofD2-40-positive (Fig. 3E), VEGF receptor-3-positive (Fig. 3F)vessels adjacent to tumor areas. The cancer cells werepositive forVEGF-C (Fig. 3G)and VEGF-D (Fig. 3H). The vessels containing the cancer cells werethin-walled, devoidof erythrocytes, and D2-40 positive, indicating lymphaticinvasion.
Correlationbetween Vascular Endothelial Growth Factor Receptor-3Expression and Clinicopathological Factors.
As shown in Table 1 , thepresence of VEGF receptor-3-positive vessels(one or more) wassignificantly associated with Gleason grade (P< 0.0001),surgical margin status (P = 0.0069), andlymph node status (P= 0.0002). Consistent with our results, Li etal. (21)found a significant correlation between VEGF receptor-3expressionand lymph node metastasis.
Inthis study heterogeneous staining was demonstrated in prostatecancercells for both VEGF-C and VEGF-D. The intensity of VEGF-D stainingwas much stronger on the edge than in the center of thetumor,indicating the heterogeneity of prostate cancer, whichcontainssubpopulations of cells with different biological propertiessuch asinvasive potential (28 , 29) . Indeed, inhuman gastric carcinoma, VEGF-C immunoreactivity was moreintense inthe invasive tumor component compared with in situtumor (30). In several human cancers, including prostate cancer, immunohistochemicalanalysis has shown that expression of genes andproteins associatedwith angiogenesis and invasion is higher inperipheral zones ofcancers than in their centers (28).
Epithelial-stromalinteractions are thought to play a critical rolein the initiation and promotion of carcinogenesis in prostatecancer (31). Expression of several growth factors involved intumor growth,angiogenesis, invasion, and metastasis has beendemonstrated in bothprostate epithelia and stroma (32,33,34) . It has been suggested thatgrowth factors may havea role in inducing epithelial proliferation and prostaticcarcinogenesisvia both autocrine and paracrine pathways (32,33 , 35 , 36) . In thepresent immunohistochemical study, VEGF-C and VEGF-D werelocalizedto both cancer epithelial cells and stromal cells inall prostatecarcinoma specimens. VEGF-C and VEGF-D have beenshown to enhancetumor growth in tumor mouse models (16, 18). It is possible that VEGF-C, which was overexpressed incarcinomacells compared with benign epithelial cells, may contribute toprostate cancer growth. In contrast, VEGF-D was readily detectedinboth epithelial and carcinoma cells. Because VEGF receptor-3wasdetected in carcinoma cells in only 3 of 37 samples, it islikelythat, if VEGF-C and VEGF-D elicit any direct biological actionsontumor cells, this is achieved via other receptor(s). Thismay be VEGFreceptor-2, or alternatively, there might exist asyet unidentifiedspecific receptor(s) for VEGF-C and VEGF-D.
VEGFreceptor-3 is predominantly localized to lymphatic endothelialcellsin adult tissue (4 , 5 , 37), although its up-regulation in angiogenicblood vessel endotheliumhas been detected in breast cancer (9) and somevascular tumors (38) . In thepresent study,expression of VEGF receptor-3 was restricted to a small proportionofvessels that had morphology characteristic of lymphaticvessels andstained positively using the lymphatic vesselmarker D2-40. There wasa strong association between positive stainingof prostatic lymphaticvessels for VEGF receptor-3 and the presence oflymph node metastasis(Table 1) .Occasionally,tumor emboli were detected within peritumoral VEGFreceptor-3-positivevessels. An essential prerequisite for the formation oflymphatic metastasisis the entry of cancer cells into lymphatic vessels (29). Our study suggests that peritumoral lymphatic vessels mayserve asa route for nodal metastasis in human prostate cancer.Similar to ourfindings, tumor invasion into peritumoral lymphaticvessels has beendemonstrated in human colorectal carcinoma (24)and head and neck cancer (39) .Tumor emboli havealso been detected in intratumoral lymphatics of VEGF-C- andVEGF-D-transfected tumor mouse models (15,18) .
VEGF-Chas been shown to be capable of increasing vascular permeabilityandmay enhance cancer cell dissemination via lymphatic vasculatureinsome human tumors (9 , 40 ,41) . Because VEGF-D shares 61% sequencewith VEGF-C (12), it is conceivable that VEGF-D may havesimilar functional roles tothose of VEGF-C. Our study demonstrates thepresence of VEGF-C- andVEGF-D-positive carcinoma cells within VEGFreceptor-3-positivevessels. Moreover, the presence of VEGFreceptor-3-positive vesselswas correlated with lymph node metastasis inprostate cancer. Takentogether, these results suggest that VEGF-C andVEGF-D secreted bycancer cells may activate VEGF receptor-3expressed on theendothelial cells of adjacent lymphatic vesselsvia a paracrinemechanism to induce lymphatic invasion,possibly by modifying vesselpermeability (9, 40 ,41) . This would provide a route for tumor metastasisvia thelymphatic vessels to the lymph nodes.
Insummary, our results demonstrate that both VEGF-C and VEGF-Darewidely expressed in human prostate carcinoma. VEGF-D, but notVEGF-C,is also abundantly expressed in adjacent benign prostateepithelia.In contrast, VEGF receptor-3 is up-regulated invessels in a subsetof prostate cancers. The demonstration of tumoremboli in VEGFreceptor-3-positive vessels and the significantcorrelation betweenthe presence of prostatic VEGF receptor-3-positivevessels and lymphnode metastasis provide tantalizing evidencefor a role of VEGFreceptor-3 signaling in the development oflymph node metastasis. Ahumanized monoclonal antibody against VEGF-Ahas recently beendeveloped for therapeutic use in metastaticcolorectal cancer (42), and it is possible that VEGF-C or VEGFreceptor-3 may provideuseful clinical targets for developing newtherapeutic agents towardprostate lymph node metastasis. VEGF-D may alsoprovide a potentialtherapeutic target for inhibiting prostatecancer progression, giventhe stronger staining for VEGF-D observed atthe edge of tumor nests;however, the role of VEGF-D in prostate cancer requiresfurther analysis.
The costs ofpublication of this article were defrayed in part bythe payment of page charges. This article must therefore beherebymarked advertisement in accordance with 18 U.S.C.Section 1734solely to indicate this fact.
Requestsfor reprints: Elizabeth D. Williams, Bernard O’BrienInstituteof Microsurgery, 42 Fitzroy Street, Fitzroy, Victoria 3065,Australia. Phone: 61-3-9288-4018; Fax: 61-3-9416-0926; E-mail:email@example.com.
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