MCF-7 cells expressing nuclear associated lysyl oxidase-like 2 (LOXL2) exhibit an epithelial-to-mesenchymal transition (EMT) phenotype and are highly invasive in vitro

Abstract

LOXL2 is a copper- and lysine tyrosylquinone-dependent amine oxidase that has been proposed to function both extracellularly and intracellularly to activate oncogenic signaling pathways leading to EMT and invasion of breast cancer cells. In this study, we selected MCF-7 cells that stably express forms of recombinant LOXL2 differing in their subcellular localizations and catalytic competencies. This enabled us to dissect the molecular functions of intracellular and extracellular LOXL2s and examine their contributions to breast cancer metastasis/invasion. We discovered that secreted LOXL2 (~100-kDa) is N-glycosylated at Asn-455 and Asn-644, whereas intracellular LOXL2 (~75-kDa) is nonglycosylated and N-terminally processed, and is primarily associated with the nucleus. Both forms of LOXL2 can oxidize lysine in solution. However, we found that expression of intracellular LOXL2 is more strongly associated with EMT and invasiveness than secreted LOXL2 in vitro. The results indicate that nuclear associated LOXL2 contributes to the stabilization of Snail1 transcription factor at the protein level to induce EMT and promote invasion in vitro, through repression of E-cadherin, occludin, and estrogen receptor-α, and up-regulation of vimentin, fibronectin, and MT1-MMP.

Keywords: Breast Cancer; Cell Invasion; Epithelial-to-mesenchymal Transition; Lysyl Oxidase; Lysyl Oxidase-Like 2; Post-translational Modification; Site-directed Mutagenesis.

FIGURE 1.

Secreted LOXL2 is N-glycosylated at Asn-455 and Asn-644, whereas cytosolic LOXL2 is nonglycosylated, N-terminally processed, and N-terminally modified. A, PNGase F (P.F.) treatment reduces the masses of recombinant WT LOXL2 and endogenous LOXL2 secreted from stable MCF-7 and MDA-MB-231 cells, respectively. B, N-glycosylation at Asn-455 and Asn-644, but not Asn-288, is essential for secretion into the growth medium (M). A 75-kDa form of LOXL2 is detected in the lysates (L) of all cell lines. C, PNGase F and Endo H (E.H.) treatment do not alter the masses of N455Q and N644Q LOXL2. D, left panel, tunicamycin (Tun.) treatment blocks secretion of endogenous LOXL2 from MDA-MB-231 cells. A non-N-glycosylated intracellular LOXL2 is expressed with or without tunicamycin treatment. Right panel: PNGase-F treatment of MDA-MB-231 cell lysates does not reduce the mass of intracellular LOXL2. E, schematic diagrams of secreted and intracellular LOXL2. The precursor residues for the LTQ cofactor are Lys-653 and Tyr-689. The predicted secretion signal encompasses residues 1–25 (not shown). The predicted copper-binding site is His-626-XX-His-628-XX-His-630. F, N455Q and N644Q LOXL2 (isolated from stable MCF-7 cell lysates) and WT LOXL2 (isolated from the growth medium of stable MCF-7 cells) have similar specific activity toward lysine in the standard Amplex Red assay at 37 °C, pH 8.0. Error bars indicate means ± S.E. G, the positive charge at residue 653 (but not the LTQ cofactor) is essential for folding and secretion of LOXL2 from stable MCF-7 cells.

FIGURE 2.

Phenotype comparison of MDA-MB-231 cells, untransfected MCF-7 cells, empty vector-transfected MCF-7 cells, and MCF-7 cells stably expressing various recombinant forms of LOXL2. A, MCF-7 cells expressing N455Q or N644Q LOXL2 exhibit spindle-shaped (i.e. mesenchymal) morphology, similar to MDA-MB-231 cells. In contrast, MCF-7 cells stably expressing WT, K653R, K653S, or N288Q LOXL2 exhibit almost exclusively epithelial morphology. All images are the same magnification. Bar: 50 μm. B, immunoblot analysis of the subcellular distribution of endogenous and recombinant LOXL2 in MDA-MB-231 cells and stable MCF-7 cells expressing various forms of recombinant LOXL2, respectively. Lanes were rearranged for clarity of presentation. GAPDH and lamin B1 were used as loading controls for the cytosolic (C) and nuclear (N) fractions, respectively. * denotes a nonspecific protein. C, immunoblot analysis of the expression of several epithelial and mesenchymal markers in MDA-MB-231 cells and MCF-7 cells stably expressing various recombinant LOXL2s. β-actin was used as a loading control. D, qRT-PCR analysis of the expression of several epithelial and mesenchymal markers in MDA-MB-231 cells and MCF-7 cells stably expressing various recombinant LOXL2s. GAPDH was used as a loading control. E, immunoblot analysis of the expression of Snail1 and GSK3β in MDA-MB-231 cells and various stable MCF-7 cells. β-Actin was used as a loading control. F, immunoblot analysis showing the stability of Snail1 protein in the lysates of CHX-treated MDA-MB-231 cells and MCF-7 cells expressing N455Q or N644Q LOXL2. G, a Matrigel in vitro invasion assay was used to determine the invasiveness of MDA-MB-231 cells and various stable MCF-7 cells. *, p < 0.05; ***, p < 0.0001. Error bars indicate means ± S.E. H, immunoblot analysis of the expression of MMP-2 and MMP-9 (in the crude SFM) and MT1-MMP (in the cell lysate) of MDA-MB-231 cells and stable MCF-7 cells. β-Actin was used as a loading control.