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. 2019 Nov 27:12:10299-10309.
doi: 10.2147/OTT.S210014. eCollection 2019.

Aberrantly High Expression Of NOK/STYK1 Is Tightly Associated With The Activation Of The AKT/GSK3β/N-Cadherin Pathway In Non-Small Cell Lung Cancer

Zhao Huang #  1 , Nan Ma #  2 , Yan-Lu Xiong #  1 , Lei Wang  1 , Wei-Miao Li  1 , Yuan-Yang Lai  1 , Chen-Xi Zhang  1 , Zhi-Pei Zhang  1 , Xiao-Fei Li  1 , Jin-Bo Zhao  1
Affiliations

Aberrantly High Expression Of NOK/STYK1 Is Tightly Associated With The Activation Of The AKT/GSK3β/N-Cadherin Pathway In Non-Small Cell Lung Cancer

Zhao Huang et al. Onco Targets Ther. .

Abstract

Purpose: High metastasis is a leading risk factor for the survival of non-small cell lung cancer (NSCLC) and epithelial-mesenchymal transition (EMT) is a vital step of metastasis. The expression of novel oncogene with kinase domain (NOK) has been observed in some human malignancies, including non-small cell lung cancer (NSCLC); however, the biological function of NOK in NSCLC remains unclear. In the study, we explored the function of NOK in NSCLC, with an aim to elucidate the relevant underlying mechanisms.

Patients and methods: We investigate the expression of NOK, p-Akt, p-GSK-3β, E-cadherin and N-cadherin expression by immunohistochemical analysis using tissue microarrays of 72 paired NSCLC samples of cancerous and adjacent normal tissues. The associations between NOK expression and clinicopathological factors, overall survival, other proteins were assessed. Immunofluorescence analysis of NSCLC tissues was performed to study the location of NOK, Akt and GSK-3β. Up or down-regulated of NOK were conducted in two NSCLC cell lines to analyze its impact on AKT/GSK3β pathway.

Results: Statistical analysis revealed NOK expression increased in NSCLC tissues compared with normal tissues (P<0.05). It also showed that low NOK expression were associated with a higher possibility of non-lymphatic metastasis, an early pN stage and clinical stage (P<0.05). Moreover, NOK expression was positively correlated with the expression of oncogene p-Akt (Thr308), p-GSK-3β (Ser9) and N-cadherin (P<0.05). Immunofluorescence analysis of NSCLC tissues revealed that NOK is co-located with Akt and GSK-3β. Further study in NSCLC cell lines revealed that NOK overexpression can activate the AKT/GSK3β pathway. Conversely, knockdown of NOK can suppress the AKT/GSK3β pathway.

Conclusion: Our results suggest that NOK overexpression correlated significantly with lymphatic metastasis, advanced pN and clinical stage in NSCLC. And NOK may promote EMT by activating the AKT/GSK3β/N-cadherin pathway in NSCLC.

Keywords: Akt; GSK3β; N-cadherin; NOK/STYK1; epithelial-mesenchymal transition; non-small cell lung cancer.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Expression of NOK in human NSCLC cancerous tissues (Ca) compared with normal lung tissues (NC). (A) Two paired representative examples (one squamous cell carcinoma case, left hand; one adenocarcinoma case, right hand) of NOK IHC from one NSCLC patient showing higher NOK expression in the cancerous tissue. Magnification, x100 (upside); x400 (downside). (B) Protein levels of NOK in 72 pairs of cancerous tissues and adjacent normal tissues according to IHC scores. Paired t-test, P<0.0001.
Figure 2
Figure 2
Correlation of NOK expression with malignant proliferation and invasion biomarkers in NSCLC. (A) Upregulated expression of p-Akt (Thr308), p-GSK-3β (Ser9) and N-cadherin compared to cases with low expression of NOK both in squamous cell carcinoma and adenocarcinoma cases with high expression of NOK. (B) Pearson correlation analysis demonstrates that NOK expression positively correlates with the p-Akt (Thr308), p-GSK-3β (Ser9) and N-cadherin (P<0.05) in NSCLC tissue.
Figure 3
Figure 3
Co-localization of NOK and Akt and GSK-3β. (SCC, squamous cell carcinoma; AC, adenocarcinoma). (A) HE staining; Immunofluorescence: NSCLC tissue stained with anti-Akt antibody/CY3 (red) and anti-GSK-3β/FITC (green) and anti-NOK antibody/CY5 (pink), and DAPI nuclear stain (blue). (B) Statistics analysis of the co-location of NOK, Akt and GSK-3β. White arrow point to cells with co-localization of NOK & Akt & GSK-3β.
Figure 4
Figure 4
NOK-mediated activation of Akt/GSK-3β pathway in NSCLC cell lines. (A, B) NOK/p-Akt (Thr308; Ser473)/p-GSK-3β (Ser9)/N-cadherin was overexpressed in A549 cells compared to SPC-A-1 cells. *P<0.05. (C) Protein expression of NOK, p-Akt (Ser473, Thr308), p-GSK3β (Tyr216, Ser9), E-cadherin and N-cadherin by Western blotting of A549 and SPC-A-1 NSCLC cell lines transfected with STYK1-OE lentivirus and NC lentivirus. (D) Protein expression of NOK, p-Akt (Ser473, Thr308), p-GSK3β (Tyr216, Ser9), E-cadherin and N-cadherin by Western blotting of A549 NSCLC cell line transfected with two STYK1-shRNAs and one NC shRNA.
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