Abstract
The extracellular signal-regulated (ERK), mitogen-activated protein kinase (p42/p44 MAPK) pathway is up-regulated in hepatocellular carcinoma (HCC). Molecular targeting of this critical mitogenic pathway may have therapeutic potential for the treatment of HCC; however, chemoresistance to long-term therapy may develop. In the present study, we employed small-molecule MAPK kinase (MEK) inhibitors, including U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] and PD184161 (Neoplasia 8:1–8, 2006), in HepG2 and Hep3B human HCC cell lines to identify potential mechanism(s) of resistance. U0126 dose-dependently suppressed ERK phosphorylation at both 1- and 24-h time points in HepG2 cells, previously shown to be sensitive to growth inhibition by U0126. In contrast, ERK phosphorylation was only decreased at the 1-h time point but not at 24 h in the more resistant Hep3B cells. It is interesting that the lack of prolonged phospho-ERK suppression was associated with MEK hyperphosphorylation in Hep3B cells. Several MEK/ERK pathway intermediates were up-regulated in Hep3B cells; furthermore, transfection of Raf-1 small interfering RNA to suppress MEK/ERK pathway activation sensitized Hep3B cells to U0126. MEK inhibitor resistance was independent of p53 or hepatitis Bx protein status. Finally, we showed that combining two chemically distinct MEK inhibitors enhanced growth inhibition and apoptosis compared with the single agents. Taken together, these results suggest that up-regulated expression or activity of the MEK/ERK pathway contributes to MEK inhibitor resistance in HCC cells. Our findings also provide preclinical evidence suggesting that the status of the MEK/ERK pathway in patients may predict response to MEK/ERK-targeted therapeutics.
Footnotes
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This work was supported by the Clarian Values Fund for Research [Grant VFR-121]; and the Indiana Genomics Initiative of Indiana University, supported in part by Lilly Endowment Inc.
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M.T.Y.-S. and P.J.K. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.147306.
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ABBREVIATIONS: HCC, hepatocellular carcinoma; ERK, extracellular signal-regulated kinase; p42/p44 MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; P-ERK, phospho-ERK; siRNA, small interfering RNA; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene; TBS, Tris-buffered saline; NT, nontargeting; DMSO, dimethyl sulfoxide; P-MEK, phospho-MEK; HBx, hepatitis B virus X protein; PD098059, 2′-amino-3′-methoxyflavone; CI-1040, 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide.
- Received October 10, 2008.
- Accepted February 27, 2009.
- U.S. Government work not protected by U.S. copyright
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