Regulation of the biology of cholangiocarcinoma (CCA) cells by the extracellular-signal regulated kinase 5 (ERK5)

Background and Aims: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Epidermal growth factor (EGF) is involved in CCA development, and overexpression of the EGF receptor (EGFR) has been linked to tumor progression. The EGF signaling pathway may be associated with activation of extracellular signal-regulated kinase 5 (ERK5), a protein belonging to the MAPK family involved in the pathogenesis of different types of cancer. Additionally, ERK5 is implicated in cytoskeletal remodeling and cell motility. The purpose of this study was to investigate the role of ERK5 in the biology of CCA cells. Method: Two intrahepatic human cholangiocarcinoma cell lines (HuCCT-1 and CCLP-1) and two primary human iCCA cells were used in this study. Cell growth was determined by cell counting and BrdU incorporation assay. Cell motility and invasion were assessed using modified Boyden chambers. ERK5, p-ERK5, EGFR, VEGF and Angiopoietin 1 were investigated by Western blotting. Silencing of cells was performed by gene silencing with shRNA. XMD8-92 and AX15836 were used to inhibit ERK5 activity. Results: ERK5 was upregulated in all CCA cells examined and phosphorylation of ERK5 was increased in cells exposed to EGF. Growth of CCA cells in serum-containing medium was decreased after exposure to 10μM XMD8-92. In addition, migration and invasion induced by EGF were significantly reduced by both XMD8-92 and AX15836 (2μM). Similar results were obtained in ERK5-silenced cells exposed to EGF, when compared to treated with non-targeting (NT) shRNAs. In addition, in ERK5 silenced cells, expression of VEGF and angiopoietin 1 was reduced compared to NT cells. Of note, conditioned medium (CM) obtained from HuCCT-1 cells induced an increase in migration of both human hepatic stellate cells (HSC) and THP-1 monocytes, an effect reduced when conditioned medium from ERK5-silenced cells was used. Furthermore, the inhibitory effects of metformin on cell growth were more evident in ERK5-silenced cells. Conclusion: In cholangiocarcinoma cells, ERK5 activity regulates cell growth and motility, release of angiogenic factors and drug resistance.