Human hepatocellular carcinoma exhibits resistance to small molecule MEK inhibition that is independent of P53, HBx and P-glycoprotein expression
Authors: Patrick J. Klein, Amer M. Zeni, Kasi R. McCune, Alyssa D. Wait, Jesus M. Matos-Brambila, Alex J. Menze, Chad A. Wiesenauer, Eric A. Wiebke, Paul Lai, Mark A. Feitelson, Christian Max Schmidt
Journal: Cancer Research (2005)

Proc Amer Assoc Cancer Res, Volume 46, 2005

656 

MEK activity is upregulated in human hepatocellular carcinoma (HCC) tissues. Correspondingly, HCC proliferation is inhibited by small molecule MEK inhibitors (U0126 or PD184161) in vitro . Contrary to most HCC cells, the MEK activity (P-ERK) in Hep3B cells (p53-/HBx+) is unaffected by MEK inhibitors after 24 hours treatment, and is correspondingly less sensitive to the antiproliferative and anti-apoptotic effects of MEK inhibition. In these studies, we sought to determine whether p53, HBx or P-glycoprotein expression could explain the differential response of Hep3B cells compared to a sensitive line, HepG2 cells (p53+/HepBx-). Hep3B, HepG2 and stable cell lines Hep3B/p53+, Hep3B/p53-, HepG2/HBx+ and HepG2/HBx-, were treated with U0126 (0.1-10.0 μM), PD184161 (0.1- 10 μM) or verapamil (P-glycoprotein inhibitor) for 1-72 hours. Transient transfections of p53 were also employed. ERK, MEK, p53, and P-glycoprotein levels were determined by Western blot. Activity of MEK Kinase and MEK were determined by phospho-MEK and phospho-ERK Western blots. To rule out degradation of MEK inhibitor the activity of the drug in the conditioned media of Hep3B cells was confirmed by demonstrating MEK inhibition on naive HCC cells. Hep3B and stable cell lines, Hep3B/p53+, Hep3B/p53- cells, were treated with MEK inhibitors. P53 expression was confirmed in Hep3B/p53+ by Western blot. MEK activity was unaffected by p53 status as determined by P-ERK Western blot. Transient transfections of p53 confirmed these findings. The HepG2 cells and stable cell lines, HepG2/HBx+, and HepG2/HBx-, were treated with MEK inhibitors. HepG2/HBx+ cells did not demonstrate increased resistance to MEK inhibitor therapy, indicating HBx status alone could not account for the difference. P-glycoprotein expression was also unchanged with MEK inhibitor treatment in Hep3B cells. Correspondingly, verapamil (0 - 250 μM) did not alter the long-term response of Hep3B cells to MEK inhibitor treatment. Total MEK and ERK were unchanged with MEK inhibitor treatment in all cell lines. In Hep3B cells, however, P-MEK was increased up to 5-fold over control in a concentration and time dependent manner with MEK inhibitor treatment in contrast to a small upregulation in HepG2 cells. The combination of MEK inhibitors, U0126 and PD184161, caused a decrease in phospho-ERK that persisted at 24 hours and correlated with a 2-fold reduction in phospho-MEK signal. In conclusion, p53, HBx, and P-glycoprotein alone do not appear to contribute to MEK inhibitor resistance in Hep3B cells. The marked increase in MEK activation after MEK inhibitor treatment may overwhelm the ability of the drug to effectively reduce MEK activity (P-ERK). The combination of two pharmacologically distinct MEK inhibitors (U0126 and PD184161) may mitigate the reactivation of P-ERK through suppression of P-MEK.