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Zipper-interacting protein kinase promotes epithelial-mesenchymal transition, invasion and metastasis through AKT and NF-kB signaling and is associated with metastasis and poor prognosis in gastric cancer patients.

Li J, Deng Z, Wang Z, Wang D, Zhang L, Su Q, Lai Y, Li B, Luo Z, Chen X, Chen Y, Huang X, Ma J, Wang W, Bi J, Guan X - Oncotarget (2015)

Bottom Line: Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family.Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion.Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China.

ABSTRACT
Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family. ZIPK has been characterized as a tumor suppressor in various tumors, including gastric cancer. On the other hand, ZIPK also promotes cell survival. In this study, both in vitro and in vivo assays indicated that ZIPK promoted cell growth, proliferation, migration, invasion, tumor formation and metastasis in nude mice. ZIPK induced epithelial-mesenchymal transition (EMT) with increasing expression of β-catenin, mesenchymal markers, Snail and Slug, and with decreasing expression of E-cadherin. Furthermore, ZIPK activated the AKT/IκB/NF-κB pathway, which can promote EMT and metastasis. Additionally, ZIPK expression was detected in human primary gastric cancer and their matched metastatic lymph node samples by immunohistochemistry. Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion. Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival. Taken together, our study reveals that ZIPK is a pro-oncogenic factor, which promotes cancer metastasis.

No MeSH data available.


Related in: MedlinePlus

The AKT inhibitor LY294002 decreases ZIPK-induced cell migration and invasion(A) ZIPK-overexpressed cells were treated with LY294002 (20 μM) or DMSO for 2 h, the expressions of pAKT, IKKα, p-IKKα, IκBα, p-IκBα, p-NF-κB, Snail and Slug were detected by Western blotting. (B) Wound-healing assay showed that LY294002 strongly inhibited cell migration (**indicates P < 0.01, independent Student's t-test, scale bars: 100). (C) Transwell assay indicated that LY294002 reduced ZIPK-induced cell migration and invasion (**indicates P < 0.01, independent Student's t-test, scale bars: 100).
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Figure 5: The AKT inhibitor LY294002 decreases ZIPK-induced cell migration and invasion(A) ZIPK-overexpressed cells were treated with LY294002 (20 μM) or DMSO for 2 h, the expressions of pAKT, IKKα, p-IKKα, IκBα, p-IκBα, p-NF-κB, Snail and Slug were detected by Western blotting. (B) Wound-healing assay showed that LY294002 strongly inhibited cell migration (**indicates P < 0.01, independent Student's t-test, scale bars: 100). (C) Transwell assay indicated that LY294002 reduced ZIPK-induced cell migration and invasion (**indicates P < 0.01, independent Student's t-test, scale bars: 100).

Mentions: To further explore the molecular mechanisms responsible for ZIPK mediated EMT, we focused on AKT signaling because activation of AKT is a central feature of EMT and ZIPK has been demonstrated to increase AKT activation in spontaneously hypertensive rats [14]. We examined the expression levels of phosphorylated AKT and AKT by Western blotting. ZIPK-transfected BGC823 cells displayed high levels of phosphorylated AKT than did empty vector-transfected cells (Figure 4A). AKT does not directly regulate gene transcription, but it phosphorylates many substrates that activate downstream EMT-related gene. GSK-3β has been characterized as a main kinase responsible for protein stability of Snail [15]. However, in this study, we did not detect any change in GSK-3β activity between ZIPK-transfected and vector-transfected BGC823 cells (Figure 4A). The AKT/IκB pathway has also been reported to strongly regulate NF-κB expression and activation [16]. Moreover, NF-κB can modulate EMT phenotype via inducing Snail and Slug expression [17, 18]. Therefore, we examined the expression of AKT downstream targets, NF-κB, Snail and Slug by Western blot analysis. Indeed, we found that p-IKKα, p-IκBα, NF-κB, Snail and Slug were significantly up-regulated and that IκBα was down-regulated in ZIPK-transfected cells (Figure 4A). The Slug mRNA levels were higher and the Snail mRNA levels were slightly increased in the ZIPK-transfected cells (Figure 4B). Knockdown of ZIPK dramatically decreased p-IKKα, p-IκBα and NF-κB expression and increased IκBα level. Silencing ZIPK inhibited slug expression in SGC7901 cells and decreased both slug and snail expression in SNU-1 cells (Figure 4A, 4B). These data suggest that ZIPK overexpression enhances AKT/IκB/NF-κB signaling. To verify the crucial role of AKT signaling pathway in ZIPK induced EMT, we treated cells with the specific PI3K/AKT antagonist, LY294002. After treating ZIPK-transfected cells with LY294002 for 2 h, we examined pAKT, AKT, p-IKKα, p-IκBα, IκBα, NF-κB, Snail, and Slug expression. The results showed that AKT inactivation dramatically suppressed p-IKKα, p-IκBα, NF-κB, Snail and Slug expression and restored IκBα expression (Figure 5A). In addition, wound healing assay and Transwell assay demonstrated that LY294002 could significantly decrease cell migration and invasion abilities in ZIPK-transfected cells (Figure 5B, 5C). Since PTEN is a major negative regulator of the PI3K/AKT signaling pathway, we investigated whether ZIPK has a effect on PTEN protein stability. Phosphorylation of PTEN (ser380) was detected by Western blotting. We found that ZIPK did not seem to regulate PTEN stability (Figure 4A).


Zipper-interacting protein kinase promotes epithelial-mesenchymal transition, invasion and metastasis through AKT and NF-kB signaling and is associated with metastasis and poor prognosis in gastric cancer patients.

Li J, Deng Z, Wang Z, Wang D, Zhang L, Su Q, Lai Y, Li B, Luo Z, Chen X, Chen Y, Huang X, Ma J, Wang W, Bi J, Guan X - Oncotarget (2015)

The AKT inhibitor LY294002 decreases ZIPK-induced cell migration and invasion(A) ZIPK-overexpressed cells were treated with LY294002 (20 μM) or DMSO for 2 h, the expressions of pAKT, IKKα, p-IKKα, IκBα, p-IκBα, p-NF-κB, Snail and Slug were detected by Western blotting. (B) Wound-healing assay showed that LY294002 strongly inhibited cell migration (**indicates P < 0.01, independent Student's t-test, scale bars: 100). (C) Transwell assay indicated that LY294002 reduced ZIPK-induced cell migration and invasion (**indicates P < 0.01, independent Student's t-test, scale bars: 100).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4480755&req=5

Figure 5: The AKT inhibitor LY294002 decreases ZIPK-induced cell migration and invasion(A) ZIPK-overexpressed cells were treated with LY294002 (20 μM) or DMSO for 2 h, the expressions of pAKT, IKKα, p-IKKα, IκBα, p-IκBα, p-NF-κB, Snail and Slug were detected by Western blotting. (B) Wound-healing assay showed that LY294002 strongly inhibited cell migration (**indicates P < 0.01, independent Student's t-test, scale bars: 100). (C) Transwell assay indicated that LY294002 reduced ZIPK-induced cell migration and invasion (**indicates P < 0.01, independent Student's t-test, scale bars: 100).
Mentions: To further explore the molecular mechanisms responsible for ZIPK mediated EMT, we focused on AKT signaling because activation of AKT is a central feature of EMT and ZIPK has been demonstrated to increase AKT activation in spontaneously hypertensive rats [14]. We examined the expression levels of phosphorylated AKT and AKT by Western blotting. ZIPK-transfected BGC823 cells displayed high levels of phosphorylated AKT than did empty vector-transfected cells (Figure 4A). AKT does not directly regulate gene transcription, but it phosphorylates many substrates that activate downstream EMT-related gene. GSK-3β has been characterized as a main kinase responsible for protein stability of Snail [15]. However, in this study, we did not detect any change in GSK-3β activity between ZIPK-transfected and vector-transfected BGC823 cells (Figure 4A). The AKT/IκB pathway has also been reported to strongly regulate NF-κB expression and activation [16]. Moreover, NF-κB can modulate EMT phenotype via inducing Snail and Slug expression [17, 18]. Therefore, we examined the expression of AKT downstream targets, NF-κB, Snail and Slug by Western blot analysis. Indeed, we found that p-IKKα, p-IκBα, NF-κB, Snail and Slug were significantly up-regulated and that IκBα was down-regulated in ZIPK-transfected cells (Figure 4A). The Slug mRNA levels were higher and the Snail mRNA levels were slightly increased in the ZIPK-transfected cells (Figure 4B). Knockdown of ZIPK dramatically decreased p-IKKα, p-IκBα and NF-κB expression and increased IκBα level. Silencing ZIPK inhibited slug expression in SGC7901 cells and decreased both slug and snail expression in SNU-1 cells (Figure 4A, 4B). These data suggest that ZIPK overexpression enhances AKT/IκB/NF-κB signaling. To verify the crucial role of AKT signaling pathway in ZIPK induced EMT, we treated cells with the specific PI3K/AKT antagonist, LY294002. After treating ZIPK-transfected cells with LY294002 for 2 h, we examined pAKT, AKT, p-IKKα, p-IκBα, IκBα, NF-κB, Snail, and Slug expression. The results showed that AKT inactivation dramatically suppressed p-IKKα, p-IκBα, NF-κB, Snail and Slug expression and restored IκBα expression (Figure 5A). In addition, wound healing assay and Transwell assay demonstrated that LY294002 could significantly decrease cell migration and invasion abilities in ZIPK-transfected cells (Figure 5B, 5C). Since PTEN is a major negative regulator of the PI3K/AKT signaling pathway, we investigated whether ZIPK has a effect on PTEN protein stability. Phosphorylation of PTEN (ser380) was detected by Western blotting. We found that ZIPK did not seem to regulate PTEN stability (Figure 4A).

Bottom Line: Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family.Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion.Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China.

ABSTRACT
Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family. ZIPK has been characterized as a tumor suppressor in various tumors, including gastric cancer. On the other hand, ZIPK also promotes cell survival. In this study, both in vitro and in vivo assays indicated that ZIPK promoted cell growth, proliferation, migration, invasion, tumor formation and metastasis in nude mice. ZIPK induced epithelial-mesenchymal transition (EMT) with increasing expression of β-catenin, mesenchymal markers, Snail and Slug, and with decreasing expression of E-cadherin. Furthermore, ZIPK activated the AKT/IκB/NF-κB pathway, which can promote EMT and metastasis. Additionally, ZIPK expression was detected in human primary gastric cancer and their matched metastatic lymph node samples by immunohistochemistry. Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion. Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival. Taken together, our study reveals that ZIPK is a pro-oncogenic factor, which promotes cancer metastasis.

No MeSH data available.


Related in: MedlinePlus