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. 2013 Apr 4:13:180.
doi: 10.1186/1471-2407-13-180.

JNK signaling maintains the mesenchymal properties of multi-drug resistant human epidermoid carcinoma KB cells through snail and twist1

Xia Zhan  1 , Xiaobing Feng, Ying Kong, Yi Chen, Wenfu Tan
Affiliations

JNK signaling maintains the mesenchymal properties of multi-drug resistant human epidermoid carcinoma KB cells through snail and twist1

Xia Zhan et al. BMC Cancer. .

Abstract

Background and methods: In addition to possess cross drug resistance characteristic, emerging evidences have shown that multiple-drug resistance (MDR) cancer cells exhibit aberrant metastatic capacity when compared to parental cells. In this study, we explored the contribution of c-Jun N-terminal kinases (JNK) signaling to the mesenchymal phenotypes and the aberrant motile capacity of MDR cells utilizing a well characterized MDR cell line KB/VCR, which is established from KB human epidermoid carcinoma cells by vincristine (VCR), and its parental cell line KB.

Results: Taking advantage of experimental strategies including pharmacological tool and gene knockdown, we showed here that interference with JNK signaling pathway by targeting JNK1/2 or c-Jun reversed the mesenchymal properties of KB/VCR cells to epithelial phenotypes and suppressed the motile capacity of KB/VCR cells, such as migration and invasion. These observations support a critical role of JNK signaling in maintaining the mesenchymal properties of KB/VCR cells. Furthermore, we observed that JNK signaling may control the expression of both snail and twist1 in KB/VCR cells, indicating that both snail and twist1 are involved in controlling the mesenchymal characteristics of KB/VCR cells by JNK signaling.

Conclusion: JNK signaling is required for maintaining the mesenchymal phenotype of KB/VCR cells; and JNK signaling may maintain the mesenchymal characteristics of KB/VCR cells potentially through snail and twist1.

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Figures

Figure 1
Figure 1
KB/VCR cells exhibit mesenchymal properties compared to parental KB cells. A. KB/VCR cells exhibit elongated, fibroblastic morphological alterations, whereas the KB cells displayed epithelial cobblestone phenotype. (×10). B. Expression of epithelial markers E-cadherin in KB/VCR cells is decreased, concomitantly with increased expression of mesenchymal markers N-cadherin and vimentin, when compared to KB cells. KB and KB/VCR cells were lysed and used for western blotting analysis, using the GAPDH as a loading control. All blots present in this paper are representatives from at least three independent experiments. Values quantified by Image J from three independent experiments were statistical different. C-D. KB/VCR cells possess more potent motile behavior ability, including migration (C: *P < 0.05 compared to KB negative control; ** P < 0.05 compared to KB with 10%FBS) and invasion (D: *P < 0.05 compared to KB negative control; ** P < 0.01 compared to KB with 10%FBS) in response to serum, in comparison to KB cells. KB/VCR cells or KB cells were loaded into the upper wells, while the growth medium containing 10% serum was added into the lower wells of transwell system with a membrane coated by fibronectin or matrigel, and Serum free medium was used as a negative control. After 8 h, the migrated or invaded cells onto membranes were fixed and quantified as described in materials and methods. Values are the average ± SD of results obtained from three separated experiments.
Figure 2
Figure 2
Pharmacological inhibition of JNK1/2 activation with SP reverses the mesenchymal phenotypes of KB/VCR cells. A. KB/VCR cells display enhancement level of p-JNK1/2 and p-c-Jun when compared to KB cells. The expression of p-JNK1/2, p-c-Jun and their total proteins was analyzed by Western blot with specific antibodies, using antibody to β-actin as a loading control. B. Suppression of JNK signaling by SP inhibits the phosphorylation of JNK1/2 and c-Jun in KB/VCR cells. KB/VCR cells were treated with SP (10 μM) for 24 h, and the proteins were then harvested for western blot analysis as mentioned in materials and methods. C. Exposure of KB/VCR cells to SP 10 μM for 24 h renders the KB/VCR cells losing the mesenchymal fibroblastic morphology and recovering epithelial cobblestone phenotype. (×10). D. Treatment of KB/VCR cells with SP recovers the expression pattern of epithelial and mesenchymal markers in KB/VCR cells similar to those in KB cells. KB/VCR cells treated with SP (10 μM) for 24 h were lysed and used for western blot analysis of EMT protein markers. Proteins extracted from KB cells were used as a control. E-F. Interfering with JNK1/2 by use of SP inhibited the migration (E: *P < 0.05 compared to KB; **P < 0.05 compared to control) and invasion (F: *P < 0.05 compared to KB; **P < 0.001 compared to control) of KB/VCR cells in response to serum. Cells were loaded into upper well, while the SP (10 μM) was added together with medium containing 10% FBS to the lower wells. Data are expressed as average ± SD of results obtained from three separated experiments. In all cases, blots are representative of three to four independent experiments.
Figure 3
Figure 3
Knockdown of JNK1/2 reverses the mesenchymal phenotypes of KB/VCR cells. A. Knockdown of JNK1/2 by lentiviruses carrying JNK1/2 shRNA. KB/VCR infected with lentiviruses carrying shRNA control or JNK1/2 shRNA were lysed after infection, and used for Western blot analysis. Western blot for GAPDH was used as a loading control. Data are similar results from three independent experiments. B. Knockdown the expression of JNK1/2 by JNK1/2 shRNA results in KB/VCR cells acquiring of epithelial cobblestone like morphology contrast to that of shRNA control. (×10). C. Knockdown of the expression of JNK1/2 significantly increases the expression of E-cadherin and obviously decreases the expression of N-cadherin, vimentin in KB/VCR cells. The expression of E-cadherin, N-cadherin and vimentin was examined as described above in KB/VCR cells after infection with lentiviruses carrying shRNA control or JNK1/2 shRNA. Data are representative blots from three independent experiments. D-E. Knockdown of the expression of JNK1/2 significantly inhibits the migration (C: *P < 0.001 compared to KB; **P < 0.01 compared to control shRNA) and invasion (D: *P < 0.05 compared to KB; **P < 0.05 compared to control shRNA) of KB/VCR cells in response to serum. The migration and invasion of KB/VCR cells in response to serum were examined as described above after infection with lentiviruses carrying shRNA control or JNK1/2 shRNA. Data are expressed as average ± SD of results obtained from three separated experiments.
Figure 4
Figure 4
Knockdown of c-Jun disrupts the mesenchymal phenotypes of KB/VCR cells. A. Expression of c-Jun, E-cadherin, N-cadherin and vimentin after knockdown of c-Jun with RNA interfering approach. KB/VCR cells transfection of siRNA targeting c-Jun or siRNA control were lysed and used for western blot analysis with specific antibodies. Blots are representative of three independent experiments. B-C. Limiting the expression of c-Jun with siRNA reduces the migration (B: * P < 0.01) and invasion (C: *P < 0.05) of KB/VCR cells. The migration and invasion of KB/VCR cells in response to serum were examined as described above after transfection with c-Jun siRNA and the siRNA control. Data are expressed as average ± SD of results obtained from three separated experiments.
Figure 5
Figure 5
Snail and twist1 are both involved in maintaining the mesenchymal properties of KB/VCR cells controlled by JNK signaling. A. RT-PCR analysis for the expression of transcriptional factors associated with EMT in KB and KB/VCR cells. Total RNA of KB and KB/VCR cells was extracted and reversed to cDNA, which was subsequently subjected to PCR analysis with specific primers as indicated in materials and methods. B. Expression of snail and twist1 at mRNA level was determined by real time PCR analysis (*P < 0.01; **P < 0.05). C. Expression of snail and twist1 at protein level was determined by western blot analysis. D-E. Interfering with JNK1/2 with SP (D) or JNK1/2 shRNA (E) suppressed the expression of snail and twist1 in KB/VCR cells. KB cells or KB/VCR cells treated with SP 10 μM after 24 h or after infected with JNK1/2 shRNA were lysed and subjected to western blot analysis using specific antibodies. Western blot for β-actin was used as a loading control. F. Knockdown of c-Jun with siRNA reduces the expression of snail and twist1 in KB/VCR cells. KB cells or KB/VCR cells transfected with c-Jun siRNA or control were lysed, and used for western blot analysis with specific antibodies. In all cases, blots are representative of three to four separate experiments.
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