Abstract
Activated α5β1 integrin occurs specifically on tumor cells and on endothelial cells of tumor-associated vasculature, and plays a key role in invasion and metastasis. The PHSCN peptide (Ac-PHSCN-NH2) preferentially binds activated α5β1, to block invasion in vitro, and inhibit growth, metastasis and tumor recurrence in preclinical models of prostate cancer. In Phase I clinical trial, systemic Ac-PHSCN-NH2 monotherapy was well tolerated, and metastatic disease progression was prevented for 4–14 months in one-third of treated patients. We have developed a significantly more potent derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP). Using in vitro invasion assays with naturally serum-free basement membranes, we observed that the PHSCN dendrimer was 130- to 1900-fold more potent than the PHSCN peptide at blocking α5β1-mediated invasion by DU 145 and PC-3 human prostate cancer cells, whether invasion was induced by serum, or by the Ac-PHSRN-NH2 peptide, under serum-free conditions. The PHSCN dendrimer was also approximately 800 times more effective than PHSCN peptide at preventing DU 145 and PC-3 extravasation in the lungs of athymic mice. Chou-Talalay analysis suggested that inhibition of both invasion in vitro and extravasation in vivo by the PHSCN dendrimer are highly synergistic. We found that many extravasated DU 145 and PC-3 cells go onto develop into metastatic colonies, and that a single pretreatment with the PHSCN dendrimer was 100-fold more affective than the PHSCN peptide at reducing lung colony formation. Since many patients newly diagnosed with prostate cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy, like the PHSCN dendrimer, which prevents metastatic progression by inhibiting invasion, could be very beneficial.
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Abbreviations
- MAP:
-
Multiantigenic peptide
- SF:
-
Serum-free
- FBS:
-
Fetal bovine serum
- CI:
-
Combination index
- DRI:
-
Dose reduction index
- Ova:
-
Ovalbumin
- EDC:
-
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
- HBSS:
-
Hanks buffered salt solution
- MALDI:
-
Matrix assisted laser desorption/ionization
- MMP-1:
-
Matrix metalloproteinase-1
- ELISA:
-
Enzyme-linked immunoabsorbant assay
- DiI:
-
1,1′-dilinoleyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate
- MAb:
-
Monoclonal antibody
- SD:
-
First standard deviation
- SEM:
-
Standard error of mean
- PECAM-1:
-
Platelet endothelial cell adhesion molecule-1
- O.C.T.:
-
Optimal cutting temperature
- FITC:
-
Fluorescein isothiocyanate
References
Zeng ZZ, Yao H, Staszewski ED, Rockwood KF, Markwart SM, Fay KS, Spalding AC, Livant DL (2009) Alpha(5)beta(1) Integrin ligand PHSRN induces invasion and alpha(5) mRNA in endothelial cells to stimulate Angiogenesis. Transl Oncol 2:8–20
Livant DL, Brabec RK, Pienta KJ, Allen DL, Kurachi K, Markwart S, Upadhyaya A (2000) Anti-invasive, antitumorigenic, and antimetastatic activities of the PHSCN sequence in prostate carcinoma. Cancer Res 60:309–320
Zeng ZZ, Jia Y, Hahn NJ, Markwart SM, Rockwood KF, Livant DL (2006) Role of focal adhesion kinase and phosphatidylinositol 3′-kinase in integrin fibronectin receptor-mediated, matrix metalloproteinase-1-dependent invasion by metastatic prostate cancer cells. Cancer Res 66:8091–8099
Fornaro M, Manes T, Languino LR (2001) Integrins and prostate cancer metastases. Cancer Metastasis Rev 20:321–331
Miles FL, Pruitt FL, van Golen KL, Cooper CR (2008) Stepping out of the flow: capillary extravasation in cancer metastasis. Clin Exp Metastasis 25:305–324
Guba M, Bosserhoff AK, Steinbauer M, Abels C, Anthuber M, Buettner R, Jauch KW (2000) Overexpression of melanoma inhibitory activity (MIA) enhances extravasation and metastasis of A-mel 3 melanoma cells in vivo. Br J Cancer 83:1216–1222
Matsuura N, Puzon-McLaughlin W, Irie A, Morikawa Y, Kakudo K, Takada Y (1996) Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells. Am J Pathol 148:55–61
Livant DL, Brabec RK, Kurachi K, Allen DL, Wu Y, Haaseth R, Andrews P, Ethier SP, Markwart S (2000) The PHSRN sequence induces extracellular matrix invasion and accelerates wound healing in obese diabetic mice. J Clin Invest 105:1537–1545
Aota S, Nagai T, Yamada KM (1991) Characterization of regions of fibronectin besides the arginine-glycine-aspartic acid sequence required for adhesive function of the cell-binding domain using site-directed mutagenesis. J Biol Chem 266:15938–15943
Mould AP, Askari JA, Aota S, Yamada KM, Irie A, Takada Y, Mardon HJ, Humphries MJ (1997) Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit. J Biol Chem 272:17283–17292
Cianfrocca ME, Kimmel KA, Gallo J, Cardoso T, Brown MM, Hudes G, Lewis N, Weiner L, Lam GN, Brown SC, Shaw DE, Mazar AP, Cohen RB (2006) Phase 1 trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH(2)), a beta integrin antagonist, in patients with solid tumours. Br J Cancer 94:1621–1626
Khalili P, Arakelian A, Chen G, Plunkett ML, Beck I, Parry GC, Donate F, Shaw DE, Mazar AP, Rabbani SA (2006) A non-RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo. Mol Cancer Ther 5:2271–2280
Stoeltzing O, Liu W, Reinmuth N, Fan F, Parry GC, Parikh AA, McCarty MF, Bucana CD, Mazar AP, Ellis LM (2003) Inhibition of integrin alpha5beta1 function with a small peptide (ATN-161) plus continuous 5-FU infusion reduces colorectal liver metastases and improves survival in mice. Int J Cancer 104:496–503
van Golen KL, Bao L, Brewer GJ, Pienta KJ, Kamradt JM, Livant DL, Merajver SD (2002) Suppression of tumor recurrence and metastasis by a combination of the PHSCN sequence and the antiangiogenic compound tetrathiomolybdate in prostate carcinoma. Neoplasia 4:373–379
Stone KR, Mickey DD, Wunderli H, Mickey GH, Paulson DF (1978) Isolation of a human prostate carcinoma cell line (DU 145). Int J Cancer 21:274–281
Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW (1979) Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol 17:16–23
Jia Y, Zeng ZZ, Markwart SM, Rockwood KF, Ignatoski KM, Ethier SP, Livant DL (2004) Integrin fibronectin receptors in matrix metalloproteinase-1-dependent invasion by breast cancer and mammary epithelial cells. Cancer Res 64:8674–8681
Kaiser E, Colescott RL, Bossinger CD, Cook PI (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34:595–598
Remmer H, Fields G (2000) Chemical synthesis of peptides. In: Reid RE (ed) Peptide and protein drug analysis. Marcel Dekker, Inc., New York
Grant GA (2002) Evaluation of the synthetic product. In: Grant GA (ed) Synthetic peptides a user’s guide. Oxford University Press, Oxford, New York
DeSilva NS, Ofek I, Crouch EC (2003) Interactions of surfactant protein D with fatty acids. Am J Respir Cell Mol Biol 29:757–770
Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, Slav MM, Nagler A, Lider O, Alon R, Zipori D, Lapidot T (2000) The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 95:3289–3296
Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55
Ren H, Tan X, Dong Y, Giese A, Chou TC, Rainov N, Yang B (2009) Differential effect of imatinib and synergism of combination treatment with chemotherapeutic agents in malignant glioma cells. Basic Clin Pharmacol Toxicol 104:241–252
Godement P, Vanselow J, Thanos S, Bonhoeffer F (1987) A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue. Development 101:697–713
Molnar Z, Blakey D, Bystron I (2006) Tract-tracing in developing systems and in postmortem human material using carbocyanine dyes. In: Záborszky L, Lanciego JL, Wouterlood FG (eds) Neuroanatomical tract-tracing 3: molecules, neurons, and systems. Springer Science + Business Media, Inc., Boston, MA
Collazo A, Bronner-Fraser M, Fraser SE (1993) Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration. Development 118:363–376
Yao H, Dashner EJ, van Golen CM, van Golen KL (2006) RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility. Oncogene 25:2285–2296
Baldwin HS, Shen HM, Yan HC, DeLisser HM, Chung A, Mickanin C, Trask T, Kirschbaum NE, Newman PJ, Albelda SM et al (1994) Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development. Development 120:2539–2553
Gupta GP, Perk J, Acharyya S, de Candia P, Mittal V, Todorova-Manova K, Gerald WL, Brogi E, Benezra R, Massague J (2007) ID genes mediate tumor reinitiation during breast cancer lung metastasis. Proc Natl Acad Sci U S A 104:19506–19511
Orr FW, Wang HH, Lafrenie RM, Scherbarth S, Nance DM (2000) Interactions between cancer cells and the endothelium in metastasis. J Pathol 190:310–329
Rowland-Goldsmith MA, Maruyama H, Matsuda K, Idezawa T, Ralli M, Ralli S, Korc M (2002) Soluble type II transforming growth factor-beta receptor attenuates expression of metastasis-associated genes and suppresses pancreatic cancer cell metastasis. Mol Cancer Ther 1:161–167
Lawrence TS, Davis MA, Maybaum J, Mukhopadhyay SK, Stetson PL, Normolle DP, McKeever PE, Ensminger WD (1992) The potential superiority of bromodeoxyuridine to iododeoxyuridine as a radiation sensitizer in the treatment of colorectal cancer. Cancer Res 52:3698–3704
Cesano A, Visonneau S, Santoli D (1998) TALL-104 cell therapy of human solid tumors implanted in immunodeficient (SCID) mice. Anticancer Res 18:2289–2295
Rephaeli A, Blank-Porat D, Tarasenko N, Entin-Meer M, Levovich I, Cutts SM, Phillips DR, Malik Z, Nudelman A (2005) In vivo and in vitro antitumor activity of butyroyloxymethyl-diethyl phosphate (AN-7), a histone deacetylase inhibitor, in human prostate cancer. Int J Cancer 116:226–235
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer statistics, 2008. CA Cancer J Clin 58:71–96
Narain V, Cher ML, Wood DP Jr (2002) Prostate cancer diagnosis, staging and survival. Cancer Metastasis Rev 21:17–27
Bubendorf L, Schopfer A, Wagner U, Sauter G, Moch H, Willi N, Gasser TC, Mihatsch MJ (2000) Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol 31:578–583
Plesnicar S (1985) The course of metastatic disease originating from carcinoma of the prostate. Clin Exp Metastasis 3:103–110
Bova S, Kirk M, Chan-Tack M, LeCartes M (2001) Lethal metastatic human prostate cancer. Autopsy studies and characteristics of metastasis. In: Chung LWK, Isaacs WB, Simons JW (eds) Prostate cancer: biology, genetics and the new therapeutics. Humana Press, Totowa, NJ
Elkin M, Mueller HP (1954) Metastases from cancer of the prostate; autopsy and roentgenological findings. Cancer 7:1246–1248
de Paso Mora PG, Rios BJ, Pascual Pareja FJ, Castillo Torres C, Pinto Marin A, Sendino Revuelta A, Vazquez RJ (2005) Pleural effusion as presentation of metastatic adenocarcinoma of prostate. South Med J 98:959–960
Pulukuri SM, Rao JS (2008) Matrix metalloproteinase-1 promotes prostate tumor growth and metastasis. Int J Oncol 32:757–765
Nomizu M, Yamamura K, Kleinman HK, Yamada Y (1993) Multimeric forms of Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide enhance the inhibition of tumor growth and metastasis. Cancer Res 53:3459–3461
Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z, Damsky CH (1995) Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol 129:867–879
Livant DL (2005) Targeting invasion induction as a therapeutic strategy for the treatment of cancer. Curr Cancer Drug Targets 5:489–503
Greiling D, Clark RA (1997) Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix. J Cell Sci 110(Pt 7):861–870
Grinnell F, Zhu M (1994) Identification of neutrophil elastase as the proteinase in burn wound fluid responsible for degradation of fibronectin. J Invest Dermatol 103:155–161
Rokhlin OW, Cohen MB (1995) Expression of cellular adhesion molecules on human prostate tumor cell lines. Prostate 26:205–212
Woods Ignatoski KM, Grewal NK, Markwart S, Livant DL, Ethier SP (2003) p38MAPK induces cell surface alpha4 integrin downregulation to facilitate erbB-2-mediated invasion. Neoplasia 5:128–134
Mosher DF (1984) Physiology of fibronectin. Annu Rev Med 35:561–575
Ruoslahti E, Hayman EG, Pierschbacher M, Engvall E (1982) Fibronectin: purification, immunochemical properties, and biological activities. Methods Enzymol 82(Pt A):803–831
Ignatoski KM, Maehama T, Markwart SM, Dixon JE, Livant DL, Ethier SP (2000) ERBB-2 overexpression confers PI 3′ kinase-dependent invasion capacity on human mammary epithelial cells. Br J Cancer 82:666–674
Woods Ignatoski KM, Livant DL, Markwart S, Grewal NK, Ethier SP (2003) The role of phosphatidylinositol 3′-kinase and its downstream signals in erbB-2-mediated transformation. Mol Cancer Res 1:551–560
Fassina G, Corti A, Cassani G (1992) Affinity enhancement of complementary peptide recognition. Int J Pept Protein Res 39:549–556
Sinnis P, Clavijo P, Fenyo D, Chait BT, Cerami C, Nussenzweig V (1994) Structural and functional properties of region II-plus of the malaria circumsporozoite protein. J Exp Med 180:297–306
Carlier E, Mabrouk K, Moulard M, Fajloun Z, Rochat H, De Waard M, Sabatier JM (2000) Ion channel activation by SPC3, a peptide derived from the HIV-1 gp120 V3 loop. J Pept Res 56:427–437
Yahi N, Sabatier JM, Baghdiguian S, Gonzalez-Scarano F, Fantini J (1995) Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4-negative mucosal epithelial cell line. J Virol 69:320–325
Tantivejkul K, Kalikin LM, Pienta KJ (2004) Dynamic process of prostate cancer metastasis to bone. J Cell Biochem 91:706–717
Romanov VI, Goligorsky MS (1999) RGD-recognizing integrins mediate interactions of human prostate carcinoma cells with endothelial cells in vitro. Prostate 39:108–118
Acknowledgments
The authors wish to thank Dr. Philip Andrews for his suggestion of utilizing MAP peptides. All peptides were synthesized by the University of Michigan Protein Structure Facility (Dr. Henriette A. Remmer). The masses of the MAP peptides were verified by Angela Walker, Ph.D of the Michigan Proteome Consortium (Dr. Philip C. Andrews). This research was supported by the National Institutes of Health, R01 CA119007, “PHSCN Therapies to Prevent Prostate Cancer Progression”.
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Yao, H., Veine, D.M., Zeng, ZZ. et al. Increased potency of the PHSCN dendrimer as an inhibitor of human prostate cancer cell invasion, extravasation, and lung colony formation. Clin Exp Metastasis 27, 173–184 (2010). https://doi.org/10.1007/s10585-010-9316-1
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DOI: https://doi.org/10.1007/s10585-010-9316-1