ZAK inhibits human lung cancer cell growth via ERK and JNK activation in an AP-1-dependent manner

Abstract

Novel mixed-lineage kinase protein zipper sterile-alpha-motif kinase (ZAK) was first cloned by our laboratory. Lung cancer is the leading cause of cancer-related death in the world, including in Taiwan. Here, we wanted to investigate whether ZAK plays a potential role in lung cancer development. First, Western blot analysis results demonstrated that four cell lines expressed high levels of ZAK from among a panel of 10 lung cancer cell lines, and two of three normal lung cells expressed ZAK. ZAK gene expressions were down-regulated in lung cancers by real-time PCR analysis. Overexpression of ZAK suppressed cell proliferation in parallel with increased phosphorylated levels of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In contrast, ZAK silencing cells inhibited the expressions of phosphorylated ERK and JNK without affecting the expression of phosphorylated p38. The effect of the decreased cell growth rate was significantly but incompletely reversed when ZAK-overexpressing cells were treated with a specific ERK or JNK inhibitor. Moreover, c-Fos and c-Jun, the major downstream components of MAPKs, were up-regulated by ERK and JNK, respectively. When ZAK-overexpressing cells introduced with c-Jun RNA interference (RNAi), the activator protein-1 (AP-1) transcription activity detected by a secreted alkaline phosphatase (SEAP) assay was suppressed and the decreased cell number was reversed compared with the control RNAi-treated group. More importantly, ZAK significantly depressed tumor growth in in vivo study. Taken together, results from both in vitro and in vivo studies indicated that the decrease of lung cancer cell proliferation by ZAK may involve the ERK and JNK pathways via an AP-1 transcription factor.

Figure 1

Expression profile of zipper sterile‐α‐motif kinase (ZAK) in human lung cancer cell lines and human lung tumor specimens. (a) Expression pattern of ZAK in lung cancer cell lines was detected by Western blotting. β‐Actin was used as an internal control. (b) Expression of ZAK in lung cancer tissues was measured by quantitative real‐time PCR. 18S rRNA was used as an internal control. The relative amounts of the ZAK gene, standardized against the amount of 18S rRNA, were expressed as ΔC _t (threshold cycle) = C _t(ZAK) − C _{t(18S rRNA)}. The clinical samples were analyzed in duplicate.

Figure 2

Zipper sterile‐α‐motif kinase (ZAK) overexpression decreased cell proliferation. (a) H460 cells were stably transfected with wild‐type ZAK gene. H460/ZAK C4 and H460/ZAK C5 showed strong expression of ZAK compared with vector control. (b) Cell growth curves of H460/ZAK and H460‐C cells were examined by a cell counting assay (upper panel). The result of cell numbers of H1299/ZAK and H1299‐C is shown in the lower panel. (c) Cell proliferation of H460 cells also was examined by a MTT assay. OD, optical density. (d) Images of cell colonies are shown in the upper panel. In the lower panel, colony numbers are presented as a percentage of vector control for H460/ZAK cells. (e) Clones 3 (A549/shRNA ZAK C3) and 4 (A549/shRNA ZAK C4) showed little expression of ZAK compared with vector control or parental cells. Cell growth curves of A549/shRNA ZAK and A549‐C cells were examined by a cell counting assay. Values shown are means ± SD. Values significantly different from the vector control were determined with anova followed by Dunnett’s test and are indicated by *P < 0.05 or **P < 0.01. Results are representatives of three independent experiments.

Figure 3

Enforced expression of zipper sterile‐α‐motif kinase (ZAK) induced ERK and JNK activation. (a) Protein levels from H460, H460 vector control, and H460/ZAK cells were subjected to Western blot analysis. Total and phosphorylated ERK, JNK, and p38 protein levels were detected by Western blotting. c‐Fos, c‐Jun, and Hsp27 were the downstream targets of ERK, JNK, and p38, respectively. Their expression levels were also detected by Western blots. (b) Protein levels from A549, A549 vector control, and A549/shRNA ZAK cells were subjected to Western blot analysis. Total and phosphorylated ERK, JNK, and p38 protein levels were detected by Western blotting. (c) Cells were treated with a specific ERK inhibitor (PD98059, 10 μM) and/or JNK inhibitor (SP600125, 10 μM) for 3 h, and then were washed and further cultured for 3 days to do cell counting. Values significantly different from the H460 vector control (untreated) were determined with anova followed by Dunnett’s test and are indicated by *P < 0.05 or **P < 0.01. Each experiment was repeated twice for reproducibility.

Figure 4

Overexpressed zipper sterile‐α‐motif kinase (ZAK) induced activator protein‐1 (AP‐1) transcription activity. (a) The results of AP‐1 transcription activity in H460 and H1299 cells are shown in the upper and lower panels, respectively. RLU, relative luminescent unit; SEAP, secreted alkaline phosphatase. (b) ZAK‐overexpressing H460 cells were transiently transfected with c‐Jun RNAi (10 μM) for 2 days. c‐Jun protein levels were suppressed when cells were treated with 10 μL (++) but not 5 μL (+) of c‐Jun RNAi. (c) ZAK C5 was treated with AP‐1 SEAP reporter plasmid (1 μg) and c‐Jun RNAi (10 μL) for 3 days, and AP‐1 transcription activity was detected. (d) Under the same condition as the experiment described in (c), at the same time as per SEAP assay, the cell counting assay was performed. Values significantly different from the H460 vector control (untreated) were determined with anova followed by Dunnett’s test and are indicated by *P < 0.05 or **P < 0.01. Each experiment was repeated twice for reproducibility.

Figure 5

Ectopic zipper sterile‐α‐motif kinase (ZAK) resulted in impaired tumor growth. (a) Tumor volume was measured at the indicated days. (b) Tumor weight was evaluated at the end of experiment. (c) Proteins extracted from representative tumor tissues were subjected to Western blotting to confirm ZAK expression. (d) Proteins extracted from representative tumor tissues were subjected to Western blotting to validate c‐Fos and phosphorylated c‐Jun expressions. Values shown are means ± SD (n = 6 mice per group). Values significantly different from the vector control were determined with anova followed by Student’s t‐test and are indicated by *P < 0.05 or **P < 0.01.