VEGF, VEGF-B, VEGF-C, AND THEIR RECEPTORS KDR, FLT-1 AND FLT-4 DURING THE NEOPLASTIC PROGRESSION OF HUMAN COLONIC MUCOSA Thierry ANDRE ´ 1,4 , Larissa KOTELEVETS 1 , Jean-Christophe VAILLANT 2 , Anne Marie COUDRAY 1 , Laurence WEBER 2 , Sophie PRE ´ VOT 3 , Roland PARC 2 , Christian GESPACH 1 and Eric CHASTRE 1 * 1 Institut National de la Sante ´ et de la Recherche Me ´dicale (Unite ´ 482), Equipe Cance ´rogene `se et Diffe ´renciation de l’Epithe ´lium Gastrointestinal, Ho ˆpital Saint-Antoine, Paris, France 2 Centre de Chirurgie Digestive, Ho ˆpital Saint-Antoine, Paris, France 3 Service Central d’Anatomie et Cytologie Pathologiques A, Ho ˆpital Saint-Antoine, Paris, France 4 Service d’Oncologie Me ´dicale, Ho ˆpital Tenon, Paris, France Because the crucial role of angiogenesis has been demon- strated in tumor growth and metastasis, the present study was undertaken to characterize the relative expression of vascular endothelial growth factors VEGF (vascular endothe- lial growth factor), VEGF-B, VEGF-C, and their receptors KDR (kinase insert domain-containing receptor), FLT-1 (fms- like tyrosine kinase), and FLT-4 in human colonic cancers, in relation to the Astler-Coller pathological classification, and to prognosis. VEGF and VEGF-B gene expression was quanti- fied by Northern blot in 72 tumor samples matched with control tissues. VEGF gene expression was 1.4 times higher in adenocarcinomas than in control tissues (p  0.02), but did not increase further between Astler-Coller tumor stages A and D, and did not correlate with disease recurrence for patients at stages B2 or C. In adenomas, VEGF mRNA levels were not significantly different from those in the paired con- trol colonic mucosa. The expression pattern of VEGF iso- forms, mainly identified by RT-PCR (reverse-transcriptase- coupled polymerase chain reaction) as VEGF121 and VEGF165 and to a lesser extent VEGF189, was comparable in tumor and control tissues. VEGF-B mRNA levels were un- changed during the neoplastic progression of colonic mucosa. In contrast to KDR and FLT-4, the expression of VEGF-C and FLT-1 genes increased in some pathological tissues. These results provide evidence that the early and sustained increase in VEGF transcripts and the expression of multiple angio- genic factors and receptors contribute to the development of colon cancer, and thus constitute a putative target for anti- angiogenic drug therapy. Int. J. Cancer 86:174 –181, 2000. © 2000 Wiley-Liss, Inc. Angiogenesis is an essential step in tumor growth and metastasis (Hanahan and Folkman, 1996). During this step, endothelial cells, which form the inner lining of blood vessels, migrate, proliferate and form tubes, and participate in the proteolytic degradation of the basement membrane and extracellular matrix. Once tumors have reached a size of about 3 mm 3 , their growth is completely dependent on this process (Hanahan and Folkman, 1996). The ability of a primary tumor to induce angiogenesis upsets the balance between positive and negative regulatory factors (Hanahan and Folkman, 1996). Vascular endothelial growth factor (VEGF) is unique among angiogenic factors by virtue of its paracrine effect on the proliferation and motogenesis of endothelial cells (Neufeld et al., 1999). The pattern of VEGF expression suggests its involve- ment in the development and maintenance of the normal vascular system, and in tumor angiogenesis (Neufeld et al., 1999). Five isoforms of VEGF transcripts encoding polypeptides consisting of 121, 145, 165, 189, and 206 amino acids can be generated by alternative splicing (Neufeld et al., 1999). Such molecular diver- sity suggests distinct roles for these VEGF variants. Accordingly, VEGF121 and 165 are secreted as soluble forms, whereas VEGF 145, 165, and 189 remain associated with cell surface because of their interaction with proteoglycans. Posttranslational processes also modulate VEGF bioavailability, because VEGF189 activity requires proteolytic cleavage (Ploue ¨t et al., 1997). Three additional members of the VEGF family, VEGF-B, VEGF-C, and VEGF-D have been characterized. So far, this family of growth factors comprises five members: VEGF, VEGF-B, VEGF-C, VEFG-D, and the placenta growth factor (PlGF). The biological effects of VEGF are mediated through the acti- vation of specific tyrosine kinase receptors expressed mainly on angioblasts and endothelial cells (Neufeld et al., 1999). KDR/ FLK1/VEGFR-2 binds VEGF and VEGF-C and promotes the proliferation and motility of endothelial cells. The stimulation of KDR by VEGF165 is potentiated by the interaction of VEGF165 with VEGF-R (VEGF receptor) 165/neuropilin-1. FLT-1/VEGF-R1 binds VEGF and VEGF-B, and increases expression and activity of urokinase and plasminogen activator inhibitor-1 in endothelial cells. FLT-1 shows 10-fold lower kinase activity than KDR, and has been proposed to act as a ligand binding molecule and as a negative regulator of VEGF-induced endothelial-cell proliferation (Hiratsuka et al., 1998). FLT-4/VEGF-R3, which in adults is exclusively expressed in lymph node endothelial cells, binds VEGF-C and VEGF-D (Neufeld et al., 1999). Accumulation of VEGF and its transcripts was reported in colonic malignant epithelial cells, as demonstrated by immunohis- tochemistry and in situ hybridization, whereas normal epithelium, hyperplastic polyps, and adenomas were found to express little or no VEGF mRNA (Brown et al., 1993; Takahashi et al., 1995). FLT-1/VEGF-R1 and FLK1/KDR/VEGF-R2 mRNA were identi- fied in the endothelial cells of nearby stromal blood vessels but not in the endothelial cells some distance away from the colonic tumor (Brown et al., 1993; Takahashi et al., 1995). However, the above studies did not allow quantitative evaluation of these effectors of angiogenesis, and the stage at which the angiogenic switch occurs during neoplastic progression is unknown. The prognostic value of angiogenesis in colon cancer is therefore controversial (Bossi et al., 1995; Pavlopoulos et al., 1998; Takahashi et al., 1995), and Dukes’ classification and its modification by Astler-Coller still are the presently used criteria. Improvement of the efficiency of ad- juvant therapy for colon cancer and maximization of its benefit require identification of those individuals most likely to develop recurrent disease (Cohen et al., 1996). Because the crucial role of angiogenesis has been demonstrated in tumor growth and during the metastatic process, the present study was undertaken to assess (i) the relative expression of VEGF, VEGF-B, VEGF-C, and their receptors KDR, FLT-1, and FLT-4 in samples of colonic tumors (i.e., adenomas, adenocarci- nomas, and liver metastases), compared with control tissues (co- lonic mucosa and liver), using Northern blot, RT-PCR and West- ern blot; (ii) the correlation between this expression and neoplastic progression, as estimated by the Astler-Coller classification; and (iii) the validity for prognosis of these three effectors of angiogen- esis and their receptors. Grant sponsors: Association ACTT (Amis du Centre des Tumeurs de Tenon) and La Ligue Nationale Contre le Cancer. *Correspondence to: INSERM U.482, Ho ˆpital St-Antoine, 184, rue du faubourg Saint-Antoine, 75571 Paris Ce ´dex 12, France. Fax: +33-1-44- 74-93-18. E-mail: chastre@st-antoine.inserm.fr Received 23 July 1999; Revised 2 September 1999 Int. J. Cancer: 86, 174 –181 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer