Targeting lysyl oxidase for molecular imaging in breast cancer

Abstract

IntroductionLysyl 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.MethodsGene 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 (18F), 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.ResultsImmunostaining 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 [18F]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.ConclusionsThe present data support further investigation into the development of LOX-binding radiolabeled peptides as molecular probes for molecular imaging of LOX expression in cancer.

Evaluation of the Expression of Amine Oxidase Proteins in Breast Cancer

“…In the present study, LOX positivity was associated with high histological grade and high Ki-67 LI. A previous study reported that LOX mRNA level was higher in breast cancer tissue than normal tissue; however, there was no difference in LOX mRNA level between ER-positive breast cancer and TNBC, which is consistent with our findings [ 28 ]. A previous study reported that LOX is highly expressed in highly invasive cancers and metastatic breast cancer cell lines [ 29 , 30 ].…”

“…A previous study reported that LOX is highly expressed in highly invasive cancers and metastatic breast cancer cell lines [ 29 , 30 ]. In addition, LOX expression increases in breast cancer cells under hypoxic conditions [ 28 ]. It is well known that breast cancers with a high Ki-67 LI have a high proliferative potential and are likely to be in hypoxic conditions.…”

Expression and function of hexose transporters GLUT1, GLUT2, and GLUT5 in breast cancer—effects of hypoxia

“…Gene-expression microarray analysis was performed on primary samples from 176 treatment-naive patients with BC and on 10 healthy breast-tissue samples collected from reduction mammoplasties through the Canadian Breast Cancer Foundation Tumor Bank (Northern Alberta Study Center, Cross Cancer Institute, Edmonton, AB, Canada), as previously described (15).…”

Matrix-metalloproteinases as targets for controlled delivery in cancer: An analysis of upregulation and expression

“…A fibronectin-targeting peptide attached to an MRI contrast agent effectively detected micrometastases and could easily be utilized for therapeutic delivery[217]. A peptide sequence with favorable kinetic properties for LOX-mediated peptidyl lysine oxidation has also been utilized for targeting of LOX environments[218]. While the majority of targeting of TIMPs has been directed toward gene regulation for MMP inhibition, TIMPs are non-intuitively upregulated alongside MMPs in cancers, likely due to their complexation with gelatinases having MMP activation functionality and their cell proliferation stimulation.…”