Activation of c-Jun predicts a poor response to sorafenib in hepatocellular carcinoma: Preliminary Clinical Evidence

Abstract

We determined the mitogen-activated protein kinase (MAPK) gene expression profile of acquired resistance in sorafenib-sensitive hepatocellular carcinoma (HCC) cells and aimed to identify c-Jun as an important molecule mediating the efficacy of sorafenib. Differences in gene expression of the MAPK signaling between untreated and sorafenib-treated HCC cell lines were investigated using real-time polymerase chain reaction array. Western blot and real-time PCR further evaluated the expression of c-Jun. Pathological specimens from 50 patients with advanced HCC were collected to measure p-c-Jun expression. Sorafenib-resistant HCC cells demonstrated greater levels of basal c-Jun mRNA and protein compared with sorafenib-sensitive HCC cells. Sorafenib activated p-c-Jun in a dose- and time-dependent manner in PLC/PRF/5 and MHCC97H cell lines. Decreased expression levels of 6 genes after sorafenib treatment suggested a robust inhibitory impact of sorafenib on MAPK signaling in HCC cells. c-Jun and p-c-Jun expression levels were inversely correlated with the efficacy of sorafenib; a high expression level of p-c-Jun was associated with resistance to sorafenib and poor overall survival in patients with clinical HCC. p-c-Jun may act as a biomarker for predicting responses of sorafenib treatment, thus advocating targeting of JNK/c-Jun signaling as an optimal therapeutic strategy in a subset of HCC.

Figure 1

(A) Sorafenib induced HCC cells apoptosis; (B) Cell apoptosis determined by flow cytometry; (C) PLC/PRF/5 cells were found most sensitive to sorafenib byCCK8 cell viability test.

Figure 2. PCR array gene expression changes of MAPK signaling in sorafenib-treated PLC/PRF/5 cells.

Data analysis was based on the delta-Ct method. Red columns represented genes upregulated fold changes higher than 2. The fold change of JUN was 5.20 (p = 5.20) and CDKN1C was 3.41 (p = 0.012).

Figure 3. High expression of c-Jun and p-c-Jun promote resistance to sorafenib in HCC cells.

(A) qRT-PCR analysis of the basal c-Jun expression in HCC cell lines; (B) Relative mRNA expression levels of c-Jun on PLC/PRF/5 and SMCC7221 cells were detected by real-time polymerase chain reaction (PCR) at transcriptional level. Data shown are the means (SD) from at least three independent experiments; (C) The expression of c-Jun and p-c-Jun increase after treated with sorafenib in PLC/PRF/5 cells in a time-dependent manner by western blot analysis.

Figure 4

(A) Overall survival (OS) between patients with high and low p-c-Jun expression treated with sorafenib (B) Kaplan-Meier survival analysis curve.