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. 2016 Oct 4;7(40):65957-65967.
doi: 10.18632/oncotarget.11781.

Inhibition of never in mitosis A (NIMA)-related kinase-4 reduces survivin expression and sensitizes cancer cells to TRAIL-induced cell death

So Jung Park  1 , Doo Sin Jo  1 , Se-Young Jo  2 , Dong Woon Shin  1 , Sangmi Shim  3 , Yoon Kyung Jo  1 , Ji Hyun Shin  1 , Ye Jin Ha  2 , Seong-Yun Jeong  2 , Jung Jin Hwang  2 , Young Sam Kim  4 , Young-Ah Suh  2 , Jong Wook Chang  5   6 , Jin Cheon Kim  2   7 , Dong-Hyung Cho  1
Affiliations

Inhibition of never in mitosis A (NIMA)-related kinase-4 reduces survivin expression and sensitizes cancer cells to TRAIL-induced cell death

So Jung Park et al. Oncotarget. .

Abstract

The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells. However, many tumors are resistant to TRAIL-induced apoptosis, and resistance mechanisms are not fully understood. To identify novel regulatory molecules of TRAIL resistance, we screened a siRNA library targeting the human kinome, and NEK4 (NIMA-related kinase-4) was identified. Knockdown of NEK4 sensitized TRAIL-resistant cancer cells and in vivo xenografts to cell death. In contrast, over expression of NEK4 suppressed TRAIL-induced cell death in TRAIL-sensitive cancer cells. In addition, loss of NEK4 resulted in decrease of the anti-apoptotic protein survivin, but an increase in apoptotic cell death. Interestingly, NEK4 was highly upregulated in tumor tissues derived from patients with lung cancer and colon cancer. These results suggest that inhibition of NEK4 sensitizes cancer cells to TRAIL-induced apoptosis by regulation of survivin expression.

Keywords: NEK4; TRAIL; cancer; survivin.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Downregulation of NEK4 sensitizes A549 cells to TRAIL-induced cell death
A. Cell viability tests in various lung cancer cell lines. Several lung cancer cell lines (A549, H1299, H460, and SK-MES1 cells) were treated with TRAIL (20 ng/ml) for the indicated times, and cell viability was measured by a CCK-8 assay. B. SK-MES-1, A549, H1299, and H460 cells were transiently transfected with scrambled negative siRNA (Sc) or NEK4 siRNA (siNEK4), and the cells were treated 3 days later with TRAIL (20 ng/ml) for 4 h. Cell death was determined by Annexin V/PI staining. C. A549 cells transiently transfected with Sc or NEK4 siRNAs (si#1 and si#2) were further treated with TRAIL (20 ng/ml) for 4 h. The cells were harvested and subjected to Western blotting with the indicated antibodies. D. DLD1, RKO, and HeLa cells transiently transfected with Sc or siNEK4 were additionally treated with TRAIL (20 ng/ml) for 4h, and cell death was determined.
Figure 2
Figure 2. Downregulation of NEK4 induces apoptotic cell death in TRAIL-treated cells
A. A549 cells transiently transfected with scrambled siRNA (−) or NEK4 siRNA (+) were further treated with 50 mM etoposide (Etop, 20 h), TNF and cyclohexamide (TNF/CHX, 30 ng/μl and 10 ng/μl, 4 h), and TRAIL (20 ng/ml, 4 h) in the presence or absence of zVAD (40 mM). Subsequently, cell death was determined at different time points. B. A549 cells transiently transfected with scrambled siRNA (▫) or NEK4 siRNA (▪) were treated with TRAIL (20 ng/ml) with or without cell death inhibitors (40 mM zVAD, 10 mM necrostatin-1 (Nec), or 100 nM bafilomycin (Baf)), and the number of dead cells was determined. Data are presented as the mean ± SEM (n = 3) and differences were considered significant at *p<0.05.
Figure 3
Figure 3. Overexpression of NEK4 suppresses TRAIL-induced cell death
A, B. H460 cells were transfected with either a control vector (/Vec) or NEK4 (NEK4), and treated with TRAIL (20 ng/ml). After 2 h, cell death was determined (A). NEK4 and cleaved caspase-3 expression was confirmed by Western blotting (B). Data are presented as the mean ± SEM (n = 3) and were considered significant at *p < 0.05.
Figure 4
Figure 4. Down regulation of NEK4 decrease survivin expression
A, B. A549 cells were transiently transfected with scrambled negative siRNA (Sc) or NEK4-specific siRNAs (si#1 and si#2). After 3 days, the cells were further treated with TRAIL (20 ng/ml) for 4 h. Subsequently, the cells were harvested for Western blotting with the indicated antibodies. C. A459 cells stably expressing control shRNA vector (A549/shVec) or NEK4 (A549/shNEK4) were treated with TRAIL (20 ng/ml) for 4 h. Then total mRNA was isolated from A549/shVec or A549/shNEK4 cells, and NEK4 mRNA was quantified by RT-PCR using NEK4 and GAPDH specific primers. D. Downregulation of the NEK4 protein in A549/shNEK4 cells was analyzed by Western blotting. E, F. A549 cells were transfected with scrambled siRNA (Sc) or siRNA for survivin (siSur#1, #2) and additionally exposed to TRAIL (20 ng/ml) for 4h. And cell death was measured (E). Reduced expression of survivin by siRNA and activation of caspase-3 were analyzed by Western blotting (F). * p < 0.05 vs. negative control. Data are presented as the mean ± SEM (n = 3) and differences were considered significant at *p < 0.05.
Figure 5
Figure 5. Loss of NEK4 increases TRAIL-induced cell death and inhibits tumorigenesis in a xenograft model
A. A459 cells stably expressing control shRNA vector (A549/shVec) or NEK4 (A549/shNEK4) were treated with TRAIL (20 ng/ml) for 4 h, and the number of dead cells was determined. B. Reduced NEK4 expression in A549/shNEK4 cells was confirmed by Western blotting. C. A549/shVec and A549/shNEK4 cells were cultured in the GravityPLUS™ plate. After 7 days, the cells were treated with TRAIL (20 ng/ml) for 4 h and were collected. Cell death was determined by an MTT assay. D. Tumor growth after the implantation of A549/shVec and A549/shNEK4 cells was monitored for 50 days. E. When tumors grew to approximately 100 mm3 in size (5 weeks with A549/shVec and 7 weeks with A549/shNEK4 after inoculation), the mice (n=3) were treated intraperitoneally with PBS or TRAIL (400 mg). Tumor volumes were calculated measured everyday using the formula for an ellipsoid sphere: W1 × W 2 × W2/2, where W1 represents the largest tumor diameter and W2 the smallest tumor diameter.
Figure 6
Figure 6. NEK4 expression is decreased in human lung and colon cancer tissues
A. TRAIL-resistant lung cancer cell lines (SK-MES-1, A549, and H1299) and TRAIL-sensitive (Sen) lung cancer cells (H460) were cultured and harvested. NEK4 and survivin expression levels were assessed by Western Blot analysis. B. Expression of NEK4 and survivin was assessed by Western blotting in lung cancer tissues (T, tumor tissue; N, corresponding normal tissue). C. Representative photomicrographs of NEK4 immunohistochemical staining in lung cancer tissue. NEK4 in lung cancer tissue was examined by immunohistochemical analysis on a tissue microarray and its expression increased (right) in cancer tissues. D. Expression level of NEK4 and survivin in colon cancer was examined with Western blotting (T, tumor tissue; N, corresponding normal tissue).
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