Activation of JNK and high expression level of CD133 predict a poor response to sorafenib in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer deaths worldwide. While sorafenib, a multikinase inhibitor targeting the Raf/extracellular signal-regulated protein kinase (ERK) pathway, has been shown recently to provide a survival advantage to patients with advanced HCC, a predictive biomarker has not been developed. We studied whether c-Jun N-terminal kinase (JNK), which promotes liver carcinogenesis in mice, affects therapeutic response to sorafenib in HCC patients.

Methods: We collected pathological specimens from 39 patients with advanced HCC before starting sorafenib treatment, and measured JNK activity in HCCs.

Results: In patients treated with sorafenib, the expression of phospho-c-Jun in HCC, as a read out of JNK activity, was significantly higher (P<0.001) in the non-responder group than in the responder group. c-Jun N-terminal kinase activation in HCC was associated with a decreased time to progression and a poor overall survival (P=0.0028 and P=0.0008, respectively).

Conclusion: In addition, JNK activity was significantly correlated with CD133 expression level. Correspondingly, high expression level of CD133 was linked to a poor response to sorafenib. Furthermore, D-JNKi, a specific JNK inhibitor, reduced the growth of xenografted CD133(+) cells in athymic mice. In conclusion, JNK activation was positively correlated with CD133 expression level and inversely correlated with the therapeutic response to sorafenib, suggesting that JNK activity may be considered as a new predictive biomarker for response to sorafenib treatment.

Figure 1

Association between JNK activation and poor therapeutic response to sorafenib in HCC. (A) Expression of phospho-c-Jun in HCC of patients with poor response (PD, n=18) and favourable response to sorafenib (PR, SD, n=17). Paraffin-embedded liver sections were immunostained with anti-phospho-c-Jun antibody. (B) Frequency of phospho-c-Jun-positive cells in HCCs. Data are the mean values±s.e. (C) Expression of phospho-JNK in HCC of patients with poor response (PD) and favourable response to sorafenib (PR, SD). Paraffin-embedded liver sections were immunostained with anti-phospho-JNK antibody.

Figure 2

Association between JNK activation, decreased time to progression and OS in patients treated with sorafenib. The Kaplan–Meier method was used to determine progression-free survival (A) and cumulative survival (B). The log-rank test was used to compare progression-free survival and cumulative survival between patients grouped according to phospho-c-Jun expression levels.

Figure 3

Relationship between CD133, p-c-Jun, p-JNK and p-ERK expression levels in HCCs. (A) Lysates of human HCCs were gel separated and analysed by immunoblotting with antibodies to the indicated proteins. (B) There was a significant positive correlation between CD133 and p-JNK/c-Jun expressions. There was no significant correlation between CD133 and p-ERK expressions.

Figure 4

Correlation between CD133 expression and response to sorafenib. (A) Expression of CD133 in HCCs. Representative immunostaining of cases who exhibited PR (left) and PD (right). Paraffin-embedded liver sections were immunostained with anti-CD133 antibody. (B) Among 17 non-responders (PD), 8 cases exhibited CD133-positive cells. Among 18 responders (PR or SD), only three cases exhibited CD133-positive cells.

Figure 5

Effect of a JNK inhibitor on tumourigenic potential of CD133⁺ HCC cells. (A) HepG2 cells positive or negative for CD133 were inoculated subcutaneously at 8 × 10⁴ cells per body into nude mice (n=8). From 1 week after subcutaneous inoculation, TAT control peptide and D-JNKi, a specific JNK inhibitor, were administered at 25 nmol per mouse once a week and the mice were autopsied 4 weeks later. Representative nude mice with subcutaneous tumours derived from CD133⁺ (left) and CD133⁻ (right) HepG2 cells. (B) Tumourigenicity of CD133⁺ or CD133⁻ HepG2 cells treated with D-JNKi or control Tat protein. (C) HuH7 cells positive for CD133 were inoculated subcutaneously at 1 × 10⁶ cells per body into nude mice (n=6). From 1 week after subcutaneous inoculation, TAT control peptide and D-JNKi were administered at 25 nmol per mouse once a week and the mice were autopsied 4 weeks later. Representative nude mice with subcutaneous tumours were shown. (D) Tumourigenicity of CD133⁺ HuH7 cells treated with D-JNKi or control Tat protein. (E) Expression of phospho-c-Jun in xenografted tumours treated with D-JNKi or control protein. (F) Expression of phospho-JNK in xenografted tumours treated with D-JNKi or control protein.

Figure 6

Association between sorafenib and the MAPK cascade. Sorafenib inhibits the Raf/ERK signalling pathway but not the JNK signalling pathway. The therapeutic efficacy of sorafenib would be limited when JNK is activated in HCCs. Treatment with sorafenib in combination with a JNK inhibitor might open promising therapeutic perspectives.