Targeting lysyl oxidase for molecular imaging in breast cancer.
BACKGROUND
Lysyl oxidase (LOX; ExPASy ENZYME entry: EC 1.4.3.13) and members of the LOX-like family, LOXL1-LOXL4, are copper-dependent enzymes that can modify proteins of the extracellular matrix. Expression of LOX is elevated in many human cancers, including breast cancer. LOX expression correlates with the level of tissue hypoxia, and it is known to play a critical role in breast cancer metastasis. The goal of the present study was to target LOX with (1) molecular probe fluorescent labeling to visualize LOX in vitro and (2) a radiolabeled peptide to target LOX in vivo in three different preclinical models of breast cancer.


METHODS
Gene expression of all five members of the LOX family was analyzed at the transcript level via microarray analysis using tissue biopsy samples from 176 patients with breast cancer. An oligopeptide sequence (GGGDPKGGGGG) was selected as a substrate-based, LOX-targeting structure. The peptide was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy experiments with the murine breast cancer cell line EMT-6. In vivo molecular imaging experiments were performed using a C-terminal amidated peptide, GGGDPKGGGGG, labeled with a short-lived positron emitter, fluorine-18 ((18)F), for positron emission tomography (PET) in three different breast cancer models: EMT6, MCF-7 and MDA-MB-231. The PET experiments were carried out in the presence or absence of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX.


RESULTS
Immunostaining experiments using a LOX-specific antibody on EMT-6 cells cultured under hypoxic conditions confirmed the elevation of LOX expression in these cells. An FITC-labeled oligopeptide, FITC-Ava-GGGDPKGGGGG-NH2, was found to be localized in different cellular compartments under these conditions. After injection of [(18)F]fluorobenzoate-GGGDPKGGGGG-NH2, radioactivity uptake was visible in all three breast cancer models in vivo. Tumor uptake was reduced by predosing the animals with 2 mg of BAPN 4 h or 24 h before injection of the radiotracer.


CONCLUSIONS
The present data support further investigation into the development of LOX-binding radiolabeled peptides as molecular probes for molecular imaging of LOX expression in cancer.
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. mwuest@ualberta.ca.
Melinda
M
Wuest
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328, Dresden, Germany. m.kuchar@hzdr.de.
Manuela
M
Kuchar
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. saikiran@ualberta.ca.|Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 11361 87 Avenue, Edmonton, AB, T6G 2E1, Canada. saikiran@ualberta.ca.
Sai Kiran
SK
Sharma
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. srichter@ualberta.ca.
Susan
S
Richter
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. ingrit@ualberta.ca.
Ingrit
I
Hamann
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. juan9@ualberta.ca.
Monica
M
Wang
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. larissa.vos@albertahealthservices.ca.
Larissa
L
Vos
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. jmackey@ualberta.ca.
John R
JR
Mackey
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada. wuest@ualberta.ca.|Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 11361 87 Avenue, Edmonton, AB, T6G 2E1, Canada. wuest@ualberta.ca.
Frank
F
Wuest
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328, Dresden, Germany. r.loeser@hzdr.de.
Reik
R
Löser
D000818:Animals; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D000860:Hypoxia; D007527:Isoenzymes; D051379:Mice; D018613:Microscopy, Confocal; D057054:Molecular Imaging / Q000379:methods; D049268:Positron-Emission Tomography; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics
D007527:Isoenzymes; D012333:RNA, Messenger; D008249:Protein-Lysine 6-Oxidase