Down-regulation of NKD1 increases the invasive potential of non-small-cell lung cancer and correlates with a poor prognosis

Abstract

Background: As a negative modulator of the canonical Wnt signaling pathway, Naked1 (NKD1) is widely expressed in many normal tissues. However, the expression pattern and clinicopathological significance of NKD1 in patients with non-small-cell lung cancer (NSCLC) is still unclear.

Methods: Immunohistochemical studies were performed on 35 cases of normal lung tissues and 100 cases of NSCLC, including 66 cases with complete follow-up records. The NKD1 protein and mRNA expressions were detected by western blot and Real-time PCR, respectively. To examine the effect of NKD1 on the invasiveness of lung cancer cells, NKD1 was down-regulated by siRNA in lung cancer cell lines and the invasive ability was then evaluated by the Matrigel invasion assay. In addition, the expressions of Dishevelled-1 and β-catenin proteins, as well as MMP mRNA were also examined in NKD1 knockdown cells.

Results: In 35 fresh lung cancer tissues examined, 27(79%) of them exhibited lower levels of NKD1 protein in comparison with their corresponding normal tissue (P = 0.009). However, the NKD1 mRNA level was significantly higher in cancerous lung tissues, compared with the adjacent normal tissues. In 100 NSCLC tissues, NKD1 was significantly lower in 78 cases (78%) than in the normal specimens, determined by immunohistochemical staining. The reduced NKD1 expression was correlated with histological type (P = 0.003), poor differentiation (P = 0.004), lymph node metastasis (P = 0.013), TNM stage (P = 0.002) and poor survival (62.88 ± 3.23 versus 23.61 ± 2.18 months, P = 0.03). In addition, NKD1 knockdown could up-regulate Dishevelled-1 and β-catenin protein levels, as well as increased MMP-7 transcription and the invasive ability of lung cancer cells. Furthermore, when the NKD1-knockdown cells were treated with Dishevelled-1 antibody, their invasive potential was significantly reduced.

Conclusion: NKD1 protein is reduced but NKD1 mRNA is elevated in NSCLC. Reduced NKD1 protein expression correlates with a poor prognosis in NSCLC. NKD1 might inhibit the activity of the canonical Wnt pathway through Dishevelled-1.

Figure 1

Expression of NKD1 in lung cancer tissues and cell lines. (A). Compared with normal lung tissue (N1-N3), NKD1 protein expression was significantly decreased in lung cancer tissues (T1-T3) (P < 0.05). (B). NKD1 protein expression in BE1 and LH7 cells was lower than that in HBE cells, a normal human bronchial epithelial cell line (P < 0.05). In addition, NKD1 protein expression in BE1 cells (high metastatic potential) was lower than that in LH7 cells (low metastatic potential) (P < 0.05).

Figure 2

Real time PCR analyses of NKD1 mRNA in lung cancer tissues and cell lines. (A). The NKD1 mRNA expression in the lung cancer tissues was significantly higher than in corresponding non-tumorous tissues. (B). NKD1 mRNA expression in BE1 and LH7 cells was higher than that in HBE cells. NKD1 mRNA expression in BE1 cells (high metastatic potential) was higher than in LH7 cells (low metastatic potential).

Figure 3

Immunohistochemical staining of NKD1 in NSCLC. NKD1 was expressed in the cytoplasm of normal bronchial epithelial cells (Normal expression) (A). NKD1 expression was significantly decreased in lung squamous cell carcinoma (B) and adenocarcinoma (C) (Reduced expression). Negative controls were prepared by non-immune rabbit IgG at the same dilution as for the primary antibody in normal (D) and tumor sample (E and F).

Figure 4

Relationship between Naked1 expression in NSCLC and postoperative survival period of NSCLC patients. The Kaplan-Meier curve shows that the overall survival in the patients with lower expression of Naked1 is significantly shorter than in the group with normal expression (p = 0.03).

Figure 5

Knockdown of NKD1 could up-regulate Dishevelled-1 and β-catenin expression in lung cancer cells. NKD1 knockdown resulted in a significantly reduced expression of NKD1 in LTEP (A) and LK (B) cells. Meanwhile, Dishevelled-1 and β-catenin protein expression was dramatically up-regulated (P < 0.05). Note that Dishevelled-3 expression showed no change in NKD1 knockdown cells (P > 0.05).

Figure 6

NKD1 knockdown enhanced the invasive ability of lung cancer cells, while these could be ablated by anti-Dishevelled-1 antibody. Matrigel invasion assay showed that the normal group had fewer cells invading into the lower surface of the transwell filter than the group transfected with NKD1 siRNA in LTEP (A) and LK (B) lines (P < 0.05). In contrast, When DVL-1 specific antibody incubated with NKD1 siRNA in LTEP (A) and LK (B) lines, the invasive cell number was significantly reduced(P < 0.05).

Figure 7

NKD1 knockdown could enhance the transcriptional activity of MMP-7 gene, and these could be ablated by anti-Dishevelled-1 antibody. Levels of the MMP-7 gene were gradually increased along with increasing doses of NKD1 siRNA in LTEP (A) and LK (B) cells (P < 0.05). However, when DVL-1 specific antibody incubated with NKD1 siRNA in LTEP (A) and LK (B) lines, MMP-7 mRNA level was decreased significantly (P < 0.05).