ARTICLE ABSTRACTPurpose: Molecular targeted therapy is an important approach for advanced hepatocellular carcinoma (HCC). Hepatitis B virus–related HCC (HBV-HCC) accounts for approximately 50% of all HCC cases. Bortezomib, a proteasome inhibitor (PI), is used extensively for the treatment of hematologic malignancies, but its application in HCC, particularly in HBV-HCC, has not been fully explored.Experimental Design: The effects of bortezomib on HCC tissues were evaluated by TUNEL assays. The growth inhibitory activity was measured using cell viability assays, and apoptosis was measured using flow cytometry. The levels of HBx, P-Raf/Raf, and P-Erk/Erk expression were measured by Western blot analysis. The ability of the MEK inhibitor PD98059 to enhance the cell killing activity of bortezomib was evaluated using ex vivo and in vivo methods.Results: The potency of bortezomib varied among HCC samples and cell lines, and HBV/HBx expression was associated with resistance to bortezomib. Bortezomib increased the levels of P-Raf and P-Erk in HBV/HBx–positive cells but not in HBV/HBx–negative HCC cells or in breast cancer or glioblastoma multiform cells. HBx was also upregulated after exposure to bortezomib, which was associated with the inhibition of proteasome activity. P-Erk upregulation mediated by bortezomib was effectively suppressed by the addition of the MEK inhibitor PD98059. Moreover, bortezomib and PD98059 synergistically inhibited HCC cell proliferation, as measured using both ex vivo and in vivo models.Conclusions: Our studies demonstrate for the first time that HBx causes resistance to bortezomib in HCC, and this resistance can be antagonized by a MEK signaling inhibitor, providing a novel therapeutic approach. Clin Cancer Res; 21(19); 4420–30. ©2015 AACR.