Inhibition of pMAPK14 Overcomes Resistance to Sorafenib in Hepatoma Cells with Hepatitis B Virus

Abstract

Hepatitis B virus (HBV) targets the liver and is a major driver for liver cancer. Clinical data suggest that HBV infection is associated with reduced response to treatment with the multi-kinase inhibitor sorafenib, the first available molecularly targeted anti-hepatocellular carcinoma (HCC) drug. Given that Raf is one of the major targets of sorafenib, we investigated the activation state of the Raf-Mek-Erk pathway in the presence of HBV and in response to sorafenib. Here we show that hepatoma cells with replicating HBV are less susceptible to sorafenib inhibitory effect as compared to cells in which HBV expression is suppressed. However, although HBV replication is associated with increased level of pErk, its blockade only modestly augments sorafenib effect. In contrast, the phosphorylated form of the pro-oncogenic Mitogen-Activated Protein Kinase 14 (pMAPK14), a protein kinase that was recently linked to sorafenib resistance, is induced in sorafenib-treated hepatoma cells in association with HBV X protein expression. Knocking down pMAPK14 results in augmentation of the therapeutic efficacy of sorafenib and largely alleviates resistance to sorafenib in the presence of HBV. Thus, this study suggests that HBV promotes HCC resistance to sorafenib. Combining pMAPK14 inhibitors with sorafenib may be beneficial in patients with HBV-associated HCC.

Figure 1

HBV expression confers resistance to sorafenib treatment in vitro. HepG2.2.15 in which HBV expression was suppressed using the CRISPR/Cas9 system (HepG2.2.15 HBV supp.) and HepG2.2.15 cells transduced with CRISPR and an inactive form of Cas9 (HepG2.2.15 Cont.) were plated in 96-well plates and were incubated with various concentrations of sorafenib (0 to 11 μM). After 24, 48 and 72 hours, cell viability was assessed by XTT assay and quantified after additional 4 hours’ incubation at 37°C using spectrophotometry. Results are expressed as percentage of optical density (OD) reading (OD obtained with 0μM sorafenib considered as 100%) and represent average ± SDEV of biochemical triplicates. The asterisk denotes statistically significance (*P < .05, **P < .01).

Figure 2

HBV is associated with enhanced pERK activation in hepatoma cells. (A) A scheme of the Raf-Mek-Erk signaling pathway and the role of the multi-kinase inhibitor sorafenib in blocking this oncogenic pathway. (B) HepG2, HepG2.2.15, HepG2.2.15 cont. and HepG2.2.15 HBV supp. cells were seeded and collected after 48 h, protein was extracted and analyzed by Western blot for the expression of ERK, pERK and β-actin proteins. The relative intensities (RI) of pERK normalized to β Actin are shown(C) HepG2.2.15 cont. and HepG2.2.15 HBV supp. cells were plated in 24-well plates, and were treated by sorafenib (9 μM) with or without the pERK inhibitor FR180204 (70 μM). Cell viability was assessed by crystal violet staining (left) after 48 h of treatment and quantified using spectrophotometry (right). Results of quantification are expressed as percentage of optical density (OD) reading and represent an average ± SDEV of biological duplicates.

Figure 3

The oncogenic protein pMAPK14 is induced in the presence of HBV, possibly through HBx. (A) A scheme illustrating the possible role of Mapk14 in mediating sorafenib resistance in hepatoma cells. (B) HepG2215 Cont. and HepG2.2.15 HBV supp. cells were seeded and either treated with sorafenib (10 μM) or left untreated 24 h post seeding. Cells were collected at 12 h following treatment and protein was analyzed by WB, using the indicated antibodies. Cells treated with UV were used as a positive control for pERK and pMAPK14 activation. GAPDH protein expression is used for equal loading control. Relative intensities (R.I.) of the corresponding bands (marked by arrows) (pERK normalized to total ERK and pMAPK14 normalized to total MAPK14) are shown. (C) The indicated cells were analyzed by immunofluorescence for pMAPK14 expression (red). Cells nuclei were stained with DAPI (blue). The relative fluorescence intensities are indicated below for each panel (UV; ultraviolet) (D) HepG2 cells were transduced with pLENTI4-HA-X or pLENTI4-GFP plasmids. 72 h post transduction cells were lysed and analyzed by Western blot for the expression of the indicated proteins. Relative intensities of the corresponding bands (marked by arrows) (pERK normalized to total ERK and pMAPK14 normalized to total MAPK14) are shown. A q RT-PCR for HBx expression is shown in the right panel.

Figure 4

Inhibition of MAPK14 largely reverses HBV-induced resistance to sorafenib. (Left) HepG2 cells were transduced with shMAPK14 or shControl-expressing lentiviruses. Seventy-two hours post transduction cells were treated with UV. After 24 h cells were collected and protein was extracted and analyzed by Western blot for the expression of pMAPK14 and β Actin. (Right) HepG2215 Cont. and HepG2.2.15 HBV supp. cells were transduced with shMAPK14 or shControl-expressing lentiviruses. 72 h post transduction, cells were treated with sorafenib (9 μM). After 48 h cell viability was analyzed using the XTT assay (see Methods section) and quantified using spectrophotometry. Results are expressed as percentage of optical density (OD) reading and represent average ± SDEV of biochemical triplicates (ns, non-significant).