SLT-VEGF is a recombinant cytotoxin comprised of Shiga-like toxin (SLT) subunit A fused to human vascular endothelial growth factor (VEGF). It is highly cytotoxic to tumor endothelial cells overexpressing VEGF receptor-2 (VEGFR-2/KDR/Flk1) and inhibits the growth of primary tumors in subcutaneous models of breast and prostate cancer and inhibits metastatic dissemination in orthotopic models of pancreatic cancer. We examined the efficacy of SLT-VEGF in limiting tumor growth and metastasis in an orthotopic melanoma model, using NCR athymic nude mice inoculated with highly metastatic Line IV Cl 1 cultured human melanoma cells. Twice weekly injections of SLT-VEGF were started when tumors became palpable at one week after intradermal injection of 1 × 106 cells/mouse. Despite selective depletion of VEGFR-2 overexpressing endothelial cells from the tumor vasculature, SLT-VEGF treatment did not affect tumor growth. However, after primary tumors were removed, continued SLT-VEGF treatment led to fewer tumor recurrences (p = 0.007), reduced the incidence of lung metastasis (p =？0.038), and improved survival (p = 0.002). These results suggest that SLT-VEGF is effective at the very early stages of tumor development, when selective killing of VEGFR-2 overexpressing endothelial cells can still prevent further progression. We hypothesize that SLT-VEGF could be a promising adjuvant therapy to inhibit or prevent outgrowth of metastatic foci after excision of aggressive primary melanoma lesions.
References
[1]
Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med.？1995, 1, 27–31, doi:10.1038/nm0195-27. 7584949
[2]
Kerbel, R.S.; Cornil, I.; Theodorescu, D. Importance of orthotopic transplantation procedures in assessing the effects of transfected genes on human tumor growth and metastasis. Cancer Metastasis Rev.？1991, 10, 201–215, doi:10.1007/BF00050792. 1764765
[3]
Jubb, A.M.; Oates, A.J.; Holden, S.; Koeppen, H. Predicting benefit from antiangiogenic agents in malignancy. Nat. Rev. Cancer？2006, 6, 626–635. 16837971
[4]
Bergers, G.; Hanahan, D. Modes of resistance to anti-angiogenic therapy. Nat. Rev. Cancer？2008, 8, 592–603, doi:10.1038/nrc2442. 18650835
[5]
Hanahan, D.; Weinberg, R.A. The hallmarks of cancer. Cell？2000, 100, 57–70, doi:10.1016/S0092-8674(00)81683-9. 10647931
[6]
Dempke, W.C.; Heinemann, V. Resistance to EGF-R (erbB-1) and VEGF-R modulating agents. Eur. J. Cancer？2009, 45, 1117–1128, doi:10.1016/j.ejca.2008.11.038. 19124237
[7]
Johannsen, M.; Florcken, A.; Bex, A.; Roigas, J.; Cosentino, M.; Ficarra, V.; Kloeters, C.; Rief, M.; Rogalla, P.; Miller, K.; Grünwald, V. Can tyrosine kinase inhibitors be discontinued in patients with metastatic renal cell carcinoma and a complete response to treatment? A multicentre, retrospective analysis. Eur. Urol.？2009, 55, 1430–1438, doi:10.1016/j.eururo.2008.10.021. 18950936
[8]
Change, Y.S.; Adnane, J.; Trail, P.A.; Levy, J.; Henderson, A.; Xue, D.; Bortolon, E.; Ichetovkin, M.; Chen, C.; McNabola, A.; et al. Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother. Pharmacol.？2007, 59, 561–574, doi:10.1007/s00280-006-0393-4. 17160391
[9]
Bozec, A.; Gros, F.X.; Penault-Llorca, F.; Formento, P.; Cayre, A.; Dental, C.; Etienne-Grimaldi, M.C.; Fischel, J.L.; Milano, G. Vertical VEGF targeting: A combination of ligand blockade with receptor tyrosine kinase inhibition. Eur. J. Cancer？2008, 44, 1922–1930, doi:10.1016/j.ejca.2008.07.013. 18691881
[10]
Mancuso, M.R.; Davis, R.; Norberg, S.M.; O'Brien, S.; Sennino, B.; Nakahara, T.; Yao, V.J.; Inai, T.; Brooks, P.; Freimark, B.; et al. Rapid vascular regrowth in tumors after reversal of VEGF inhibition. J. Clin. Invest.？2006, 116, 2610–2621. 17016557
[11]
Backer, M.V.; Hamby, C.V.; Backer, J.M. Inhibition of vascular endothelial growth factor receptor signaling in angiogenic tumor vasculature. In Advances in Genetics; Pasqualini, R., Arap, W., Eds.; Elsevier: Amsterdam, The Netherlands, 2009; Volume 67, pp. 1–27.
[12]
Backer, M.V.; Backer, J.M. Functionally active VEGF fusion proteins. Protein Expr. Purif.？2001, 23, 1–7, doi:10.1006/prep.2001.1472. 11570839
[13]
Hotz, H.B.; Hotz, B.; Bhargava, H.; Buhr, J. Specific targeting of tumor endothelial cells by a Shiga-like Toxin-VEGF fusion protein as a novel treatment strategy for pancreatic cancer. Deut. Gesell. Chirurgie？2006, 35, 5–6.
[14]
Heim, S.; Mandahl, N.; Arheden, K.; Giovanella, B.C.; Yim, S.O.; Stehlin, J.S., Jr.; Mitelman, F. Multiple karyotypic abnormalities including structural rearrangements of 11 p, in cell lines from malignant melanomas. Cancer Genet. Cytogenet.？1988, 35, 5–20, doi:10.1016/0165-4608(88)90115-X. 3180009
[15]
Hamby, C.V.; Abbi, R.; Prasad, N.; Stauffer, C.; Thomson, J.; Mendola, C.E.; Sidorov, V.; Backer, J.M. Expression of a catalytically inactive H118Y mutant of nm23-H2 suppresses the metastatic potential of line IV Cl 1 human melanoma cells. Int. J. Cancer？2000, 88, 547–553, doi:10.1002/1097-0215(20001115)88:4<547::AID-IJC5>3.0.CO;2-L. 11058869
Backer, M.V.; Levashova, Z.; Patel, V.; Jehning, B.T.; Claffey, K.; Blankenberg, F.G.; Backer, J.M. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF based probes. Nat. Med.？2007, 13, 504–509, doi:10.1038/nm1522. 17351626
[18]
Backer, M.V.; Budker, V.G.; Backer, J.M. Shiga-like toxin-VEGF fusion proteins are selectively cytotoxic to endothelial cells overexpressing VEGFR-2. J. Control. Release？2001, 74, 349–355, doi:10.1016/S0168-3659(01)00346-7. 11489517
[19]
Liu, L.; Zhu, D.; Gao, R.; Guo, H. Expression of vascular endothelial growth factor, receptorKDR and p53 protein in transitional cell carcinoma of the bladder. Urol. Int.？2008, 81, 72–76, doi:10.1159/000137644. 18645275
[20]
Neuchrist, C.; Erovic, B.M.; Handisurya, A.; Fischer, M.B.; Steiner, G.E.; Hollemann, D.; Gedlicka, C.; Saaristo, A.; Burian, M. Vascular endothelial growth factor receptor2 (VEGFR2) expression in squamous cell carcinomas of the head and neck. Laryngoscope？2001, 111, 1834–1841, doi:10.1097/00005537-200110000-00031. 11801954
[21]
Olson, T.A.; Mohanraj, D.; Roy, S.; Ramakrishnan, S. Targeting the tumor vasculature: Inhibition of tumor growth by a vascular endothelial growth factor-toxin conjugate. Int. J. Cancer？1997, 73, 865–870, doi:10.1002/(SICI)1097-0215(19971210)73:6<865::AID-IJC17>3.0.CO;2-3. 9399667
[22]
Ching, J.C.; Jones, N.L.; Ceponis, P.J.; Karmali, M.A.; Sherman, P.M. Escherichia coli shiga-like toxins induce apoptosis and cleavage of poly(ADP-ribose) polymerase via in vitro activation of caspases. Infect. Immun.？2002, 70, 4669–4677, doi:10.1128/IAI.70.8.4669-4677.2002. 12117981
[23]
Lee, S.Y.; Cherla, R.P.; Caliskan, I.; Tesh, V.L. Shiga toxin 1 induces apoptosis in the human myelogenous leukemia cell line THP-1 by a caspase-8-dependent, tumor necrosis factor receptor-independent mechanism. Infect. Immun.？2005, 73, 5115–5126, doi:10.1128/IAI.73.8.5115-5126.2005. 16041028
[24]
Ramakrishnan, S.; Olson, T.A.; Bautch, V.L.; Mohanraj, D. Vascular endothelial growth factor-toxin conjugate specifically inhibits KDR/flk-1-positive endothelial cell proliferation in vitro and angiogenesis in vivo. Cancer Res.？1996, 56, 1324–1330. 8640821
[25]
Nakayama, T.; Cho, Y.-C.; Mine, Y.; Yoshizaki, A.; Naito, S.; Wen, C.Y.; Sekine, I. Expression of vascular endothelial growth factor and its receptors VEGFR-1 and 2 in gastrointestinal stromal tumors, leiomyomas and schwannomas. World J. Gastroenterol.？2006, 12, 6182–6187. 17036392
[26]
Arora, N.; Masood, R.; Zheng, T.; Cai, J.; Smith, D.L.; Gill, P.S. Vascular endothelial growth factor chimeric toxin is highly active against endothelial cells. Cancer Res.？1999, 59, 183–188. 9892205
[27]
Veenendaal, L.M.; Jin, H.; Ran, S.; Cheung, L.; Navone, N.; Marks, J.W.; Waltenberger, J.; Thorpe, P.; Rosenblum, M.G. In vitro and in vivo studies of a VEGF121/r gelonin chimeric fusion toxin targeting the neovasculature of solid tumors. Proc. Nat. Acad. Sci. USA？2002, 99, 7866–7871, doi:10.1073/pnas.122157899. 12060733
[28]
Vallera, D.A.; Oh, S.; Chen, H.; Shu, Y.; Frankel, A.E. Bioengineering a unique deimmunized bispecific targeted toxin that simultaneously recognizes human CD22 and CD19 receptors in a mouse model of B-cell metastases. Mol. Cancer Ther.？2010, 9, 1872–1883, doi:10.1158/1535-7163.MCT-10-0203. 20530709
