Cytoskeletal rearrangement by the mitogen-activated protein kinase ERK5 interferes with the motile and invasive phenotype of hepatocellular carcinoma cells

Background and aims: ERK5 belongs to the mitogen-activated protein kinase family and regulates proliferation, migration, and angiogenesis. Recent data indicate that ERK5 may be involved in the pathogenesis of cancer in different tissues. However, little information is available on the role of this molecule in the biology of hepatocellular carcinoma (HCC). ERK5 has been previously shown to cross-talk with focal adhesion kinase to control proliferation and cell shape in breast cancer cells. Aim of this study was to understand the role of ERK5 in motility and invasion in HCC cells. Methods: Huh7 and HepG2 were cultured by standard methods. Liver tissue was obtained from HCC and peritumoral areas. Localization and phosphorylation of FAK, vinculin and actin was studied by confocal microscopy immunofluorescence. ERK5 was silenced by siRNA transfection. Results: Analysis of ERK5 expression by western blot analysis of lysates from human liver showed increased expression in HCC compared to neighboring cirrhotic tissue, and in both compared to normal tissue. Moreover, ERK5 was more abundantly localized in the nucleus of neoplastic cells as compared to non-tumoral neighboring cirrhotic tissue, indicating the activated status of the molecule. Exposure of HepG2 or Huh-7 HCC cells to EGF stimulated cell migration and invasion through Matrigel, and resulted in a marked activation of ERK5. Similarly, culture of HCC cells in conditions of hypoxia (3% O2) increased the motile phenotype. In both cases, silencing of ERK5 by siRNA almost abolished the increase in migration or invasion. Immunofluorescence experiments demonstrated that ERK5 silencing caused an evident remodeling of the cytoskeleton and focal adhesions. In particular, focal adhesions, located at the edges of cells transfected with control siRNA, were redistributed on the ventral surface of cells silenced for ERK5. Moreover, analysis of gene expression in ERK5-silenced cells by low-density microarray demonstrated the modulation of genes involved in control of the neoplastic phenotype, including motility. Conclusions: Activation of ERK5 is increased in HCC cells, as demonstrated by its nuclear localization. ERK5 silencing reduces cell migration and invasion, and is associated with cytoskeletal rearrangements.