Gamma-enolase: a well-known tumour marker, with a less-known role in cancer

Abstract

Background: Gamma-enolase, known also as neuron-specific enolase (NSE), is an enzyme of the glycolytic pathway, which is expressed predominantly in neurons and cells of the neuroendocrine system. As a tumour marker it is used in diagnosis and prognosis of cancer; however, the mechanisms enrolling it in malignant progression remain elusive. As a cytoplasmic enzyme gamma-enolase is involved in increased aerobic glycolysis, the main source of energy in cancer cells, supporting cell proliferation. However, different cellular localisation at pathophysiological conditions, proposes other cellular engagements.

Conclusions: The C-terminal part of the molecule, which is not related to glycolytic pathway, was shown to promote survival of neuronal cells by regulating neuronal growth factor receptor dependent signalling pathways, resulting also in extensive actin cytoskeleton remodelling. This additional function could be important also in cancer cells either to protect cells from stressful conditions and therapeutic agents or to promote tumour cell migration and invasion. Gamma-enolase might therefore have a multifunctional role in cancer progression: it supports increased tumour cell metabolic demands, protects tumour cells from stressful conditions and promotes their invasion and migration.

Keywords: cancer; cell survival; gamma-enolase; glycolysis; tumour marker.

FIGURE 1.

Position of gamma-enolase catalytical active site and the PDZ-binding motif containing C-terminal end. Subunits of the γγ-dimer are represented by separate colours (wheat and violet). The orange part represents the catalytical active site, yellow balls represent Mg²⁺ ions and the magenta part represents the C-terminal end of the molecule (the last 6 amino acids). For better representation, active site and C-terminal end are shown only in one subunit. The image was created using PyMOL (DeLano LLC Scientific). Gamma-enolase crystal structure (1TE6) was obtained from Protein Data Bank (PDB). The image was prepared by authors and has not been published elsewhere.

FIGURE 2.

Co-localization of gamma-enolase and cathepsin X in human glioblastoma cells U87-MG grown in serum-free medium for 72 h. U87-MG cells were grown in Eagle´s Minimum Essential Medium (EMEM, Sigma), supplemented with 10% (v/v) foetal bovine serum (HyClone), 1% L-glutamine (Sigma) and 1% penicillin/streptomycin (Sigma) at 37°C and humidified atmosphere with 5% CO₂. For protein visualization, cells were seeded on glass coverslips at a concentration of 1 × 10⁴ cells/ml in 24 well plates. After 24 h, complete growth medium was replaced with serum-free medium and cells were left to grow for additional 72 h. After treatment cells were fixed with 10% formalin for 30 min at room temperature and then permeabilized by 0.5% Tween®20 in phosphate buffered saline (PBS), pH 7.4 for 10 min. Non-specific binding was blocked with 3% bovine serum albumin (BSA) in PBS, pH 7.4 for 1.5 h at room temperature. Cells were then incubated with primary antibody against N-terminal end of gamma-enolase (10 μg/ml, goat polyclonal, Santa Cruz Biotechnology) and active cathepsin X (10 μg/ml, mouse monoclonal, 2F12) in 3% BSA in PBS pH 7.4 for 2 h at room temperature. After three washes with PBS, pH 7.4, cells were incubated with Alexa Fluor 555 donkey anti-goat (Molecular Probes™) and Alexa Fluor 488 donkey anti-mouse (Molecular Probes™) secondary antibody in 3% BSA in PBS, pH 7.4. After washing with PBS, ProLong® Gold Antifade Mountant with 4’,6-diamidino-2-phenylindole, dilactate (DAPI, Molecular Probes™) was used to mount coverslips on glass slides. Fluo rescence microscopy was performed by Carl Zeiss LSM 710 confocal microscope (Carl Zeiss Oberkochen) with ZEN 2012 image software. Gamma-enolase (red) and cathepsin X (green) staining showed co-localisation in the perimembrane region. The blue staining with DAPI represents the nucleus. The image was prepared by authors and has not been published elsewhere.