Breast Cancer Research and Treatment

, Volume 125, Issue 2, pp 363–375

The PHSCN dendrimer as a more potent inhibitor of human breast cancer cell invasion, extravasation, and lung colony formation

  • Hongren Yao
  • Donna M. Veine
  • Kevin S. Fay
  • Evan D. Staszewski
  • Zhao-Zhu Zeng
  • Donna L. Livant
Preclinical study

Abstract

The α5β1 integrin fibronectin receptor is an attractive therapeutic target in breast cancer because it plays key roles in invasion and metastasis. While its inactive form is widely expressed, activated α5β1 occurs only on tumor cells and their associated vasculature. The PHSCN peptide has been shown to bind activated α5β1 preferentially, thereby blocking invasion in vitro, and inhibiting growth, metastasis and tumor recurrence in preclinical models. Moreover in a recent Phase I clinical trial, systemic PHSCN monotherapy was well tolerated, and metastatic disease failed to progress for 4–14 months in 38% of patients receiving it. A significantly more potent PHSCN derivative, the PHSCN–polylysine dendrimer (Ac-PHSCNGGK-MAP) has recently been developed. We report that it is 1280- to 6700-fold more potent than the PHSCN peptide at blocking α5β1 mediated SUM-149 PT and MDA-MB-231 human breast cancer cell invasion of naturally occurring basement membranes in vitro. Chou–Talalay analysis of these data suggested that invasion inhibition by the PHSCN dendrimer was highly synergistic. We also report that, consistent with its enhanced invasion-inhibitory potency, the PHSCN dendrimer is 700- to 1100-fold more effective than the PHSCN peptide at preventing SUM-149 PT and MDA-MB-231 extravasation in the lungs of athymic, nude mice. Our results also show that many extravasated SUM-149 PT and MDA-MB-231 cells go on to develop into metastatic colonies, and that pretreatment with the PHSCN dendrimer is more than 100-fold more effective at reducing lung colony formation. Since many patients newly diagnosed with breast cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy that can prevent metastatic progression by blocking invasion could be very beneficial.

Keywords

Breast cancer Invasion Extravasation Lung metastasis Integrin fibronectin receptor MMP-1 

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

Standard deviation

SEM

Standard error of the mean

PECAM-1

Platelet endothelial cell adhesion molecule-1

OCT

Optimal cutting temperature

FITC

Fluorescein isothiocyanate

Supplementary material

10549_2010_826_MOESM2_ESM.tif (3 mb)
(TIFF 3104 kb)
10549_2010_826_MOESM3_ESM.tif (10.8 mb)
(TIFF 11105 kb)

References

  1. 1.
    Sanchez-Munoz A, Perez-Ruiz E, Ribelles N, Marquez A, Alba E (2008) Maintenance treatment in metastatic breast cancer. Expert Rev Anticancer Ther 8:1907–1912. doi:10.1586/14737140.8.12.1907 CrossRefPubMedGoogle Scholar
  2. 2.
    Perez EA (2009) Impact, mechanisms, and novel chemotherapy strategies for overcoming resistance to anthracyclines and taxanes in metastatic breast cancer. Breast Cancer Res Treat 114:195–201. doi:10.1007/s10549-008-0005-6 CrossRefPubMedGoogle Scholar
  3. 3.
    Chan A (2009) Antiangiogenic therapy for metastatic breast cancer: current status and future directions. Drugs 69:167–181. doi:10.2165/00003495-200969020-00003 CrossRefPubMedGoogle Scholar
  4. 4.
    Giordano SH, Buzdar AU, Smith TL, Kau SW, Yang Y, Hortobagyi GN (2004) Is breast cancer survival improving? Cancer 100:44–52. doi:10.1002/cncr.11859 CrossRefPubMedGoogle Scholar
  5. 5.
    Higgins MJ, Wolff AC (2008) Therapeutic options in the management of metastatic breast cancer. Oncology 22:614–623 (discussion 623, 627–629)PubMedGoogle Scholar
  6. 6.
    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 2:8–20PubMedGoogle Scholar
  7. 7.
    Jia YF, Markwart SM, Rockwood KF, Woods-Ignatoski KM, Ethier SP, Livant DL (2004) Integrin fibronectin receptors in MMP-1 dependent invasion by breast cancer and mammary epithelial cells. Cancer Res 64:8674–8681CrossRefPubMedGoogle Scholar
  8. 8.
    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 60:309–320PubMedGoogle Scholar
  9. 9.
    Zeng Z-Z, Jia YF, 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 66:8091–8099CrossRefPubMedGoogle Scholar
  10. 10.
    White DE, Muller WJ (2007) Multifaceted roles of integrins in breast cancer metastasis. J Mammary Gland Biol Neoplasia 12:135–142. doi:10.1007/s10911-007-9045-5 CrossRefPubMedGoogle Scholar
  11. 11.
    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. doi:10.1007/s10585-007-9098-2 CrossRefPubMedGoogle Scholar
  12. 12.
    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. doi:10.1054/bjoc.2000.1424 CrossRefPubMedGoogle Scholar
  13. 13.
    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 148:55–61PubMedGoogle Scholar
  14. 14.
    Livant DL, 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 105:1537–1545CrossRefPubMedGoogle Scholar
  15. 15.
    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 266:15938–15943PubMedGoogle Scholar
  16. 16.
    Mould AP, Askari JA, Aota SI, 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 272:17283–17292CrossRefPubMedGoogle Scholar
  17. 17.
    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 I trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH2) a beta integrin antagonist, in patients with solid tumours. Br J Cancer 94:1621–1626PubMedGoogle Scholar
  18. 18.
    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 5:2271–2280CrossRefPubMedGoogle Scholar
  19. 19.
    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 104:496–503CrossRefPubMedGoogle Scholar
  20. 20.
    van Golen KL, Bao LW, 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 4:373–379CrossRefPubMedGoogle Scholar
  21. 21.
    Cailleau R, Olive M, Cruciger QV (1978) Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro 14:911–915CrossRefPubMedGoogle Scholar
  22. 22.
    van Golen KL, Davies S, Wu ZF, Wang Y, Bucana CD, Root H, Chandrasekharappa S, Strawderman M, Ethier SP, Merajver SD (1999) A novel putative low-affinity insulin-like growth factor-binding protein, LIBC (lost in inflammatory breast cancer), and RhoC GTPase correlate with the inflammatory breast cancer phenotype. Clin Cancer Res 5:2511–2519PubMedGoogle Scholar
  23. 23.
    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 34:595–598CrossRefPubMedGoogle Scholar
  24. 24.
    Remmer H, Fields G (2000) Chemical synthesis of peptides. In: Reid RE (ed) Peptide and protein drug analysis. Marcel Dekker, Inc., New York, pp 133–169Google Scholar
  25. 25.
    Grant GA (2002) Evaluation of the synthetic product. In: Grant GA (ed) Synthetic peptides: a user’s guide, 2nd edn. Oxford University Press, Oxford, pp 220–291Google Scholar
  26. 26.
    DeSilva NS, Ofek I, Crouch EC (2003) Interactions of surfactant protein D with fatty acids. Am J Respir Cell Mol Biol 29:757–770. doi:10.1165/rcmb.2003-0186OC CrossRefPubMedGoogle Scholar
  27. 27.
    Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55CrossRefPubMedGoogle Scholar
  28. 28.
    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. doi:10.1111/j.1742-7843.2008.00371.x CrossRefPubMedGoogle Scholar
  29. 29.
    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 101:697–713PubMedGoogle Scholar
  30. 30.
    Heimer L, Záborszky L (1989) Neuroanatomical tract-tracing methods, 2: recent progress. Plenum, New YorkGoogle Scholar
  31. 31.
    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 118:363–376PubMedGoogle Scholar
  32. 32.
    Kuffler DP (1990) Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. J Comp Neurol 302:729–738. doi:10.1002/cne.903020405 CrossRefPubMedGoogle Scholar
  33. 33.
    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 95:3289–3296PubMedGoogle Scholar
  34. 34.
    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 25:2285–2296CrossRefPubMedGoogle Scholar
  35. 35.
    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 120:2539–2553PubMedGoogle Scholar
  36. 36.
    Luo J, Guo P, Matsuda K, Truong N, Lee A, Chun C, Cheng SY, Korc M (2001) Pancreatic cancer cell-derived vascular endothelial growth factor is biologically active in vitro and enhances tumorigenicity in vivo. Int J Cancer 92:361–369. doi:10.1002/ijc.1202 CrossRefPubMedGoogle Scholar
  37. 37.
    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 USA 104:19506–19511. doi:10.1073/pnas.0709185104 CrossRefPubMedGoogle Scholar
  38. 38.
    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 1:161–167PubMedGoogle Scholar
  39. 39.
    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. doi:10.1002/(SICI)1096-9896(200002)190:3<310:AID-PATH525>3.0.CO;2-P CrossRefPubMedGoogle Scholar
  40. 40.
    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 52:3698–3704PubMedGoogle Scholar
  41. 41.
    Hortobagyi GN (1998) Treatment of breast cancer. N Engl J Med 339:974–984CrossRefPubMedGoogle Scholar
  42. 42.
    Greenberg PA, Hortobagyi GN, Smith TL, Ziegler LD, Frye DK, Buzdar AU (1996) Long-term follow-up of patients with complete remission following combination chemotherapy for metastatic breast cancer. J Clin Oncol 14:2197–2205PubMedGoogle Scholar
  43. 43.
    McGrogan BT, Gilmartin B, Carney DN, McCann A (2008) Taxanes, microtubules and chemoresistant breast cancer. Biochim Biophys Acta 1785:96–132. doi:10.1016/j.bbcan.2007.10.004 PubMedGoogle Scholar
  44. 44.
    Chien AJ, Moasser MM (2008) Cellular mechanisms of resistance to anthracyclines and taxanes in cancer: intrinsic and acquired. Semin Oncol 35:S1–S14. doi:10.1053/j.seminoncol.2008.02.010 CrossRefPubMedGoogle Scholar
  45. 45.
    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 129:867–879CrossRefPubMedGoogle Scholar
  46. 46.
    Livant DL (2005) Targeting invasion as a therapeutic strategy for the treatment of cancer. Curr Cancer Drug Targets 5:489–503CrossRefPubMedGoogle Scholar
  47. 47.
    Greiling D, Clark RA (1997) Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix. J Cell Sci 110:861–870PubMedGoogle Scholar
  48. 48.
    Grinnell F, Zhu M (1994) Identification of neutrophil elastase as the proteinase in burn wound fluid responsible for degradation of fibronectin. J Invest Dermatol 103:155–161CrossRefPubMedGoogle Scholar
  49. 49.
    Woods-Ignatoski KM, Grewal NK, Markwart SM, Livant DL, Ethier SP (2003) p38 MAPK induces cell surface α4 integrin down-regulation to facilitate erbB-2 mediated invasion. Neoplasia 5:128–134PubMedGoogle Scholar
  50. 50.
    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 1:551–560PubMedGoogle Scholar
  51. 51.
    Woods-Ignatoski KM, Maehama T, Markwart SM, Dixon JE, LIvant DL, Ethier SP (2000) ERBB-2 overexpression confers P1 3′ kinase-dependent invasion capacity on human mammary epithelial cells. Br J Can 82:666–674CrossRefGoogle Scholar
  52. 52.
    Mosher DF (1984) Physiology of fibronectin. Ann Rev Med 35:561–575CrossRefPubMedGoogle Scholar
  53. 53.
    Ruoslahti E, Hayman EG, Pierschbacher M, Engvall E (1982) Fibronectin: purification, immunochemical properties, and biological activities. Methods Enzymol 82:803–831CrossRefPubMedGoogle Scholar
  54. 54.
    Fassina G, Corti A, Cassani G (1992) Affinity enhancement of complementary peptide recognition. Int J Pept Protein Res 39:549–556CrossRefPubMedGoogle Scholar
  55. 55.
    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 180:297–306CrossRefPubMedGoogle Scholar
  56. 56.
    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 56:427–437CrossRefPubMedGoogle Scholar
  57. 57.
    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 69:320–325PubMedGoogle Scholar
  58. 58.
    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 53:3459–3461PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Hongren Yao
    • 1
  • Donna M. Veine
    • 1
  • Kevin S. Fay
    • 1
  • Evan D. Staszewski
    • 1
  • Zhao-Zhu Zeng
    • 1
  • Donna L. Livant
    • 1
  1. 1.Department of Radiation Oncology and Comprehensive Cancer CenterUniversity of MichiganAnn ArborUSA

Personalised recommendations