Dickkopf-1 is oncogenic and involved in invasive growth in non small cell lung cancer

Abstract

Dickkopf-1 (DKK1) is an inhibitor of the Wnt/β-catenin signaling pathway. However, the role of DKK1 in the progression of non small cell lung cancer (NSCLC) is not fully understood. In this study, RT-PCR and Western blot were used to examine the expression of DKK1 in a panel of ten human NSCLC cell lines and NSCLC tissues. DKK1 expression was highly transactivated in the great majority of these cancer lines. The expression of DKK1 was upregulated on both mRNA and protein levels in NSCLC tissues compared with the adjacent normal lung tissues. Immunohistochemistry and immunofluoresence revealed that DKK1 was mainly distributed in the cytoplasm in both carcinoma tissues and cell lines. DKK1 protein expression was also evaluated in paraffin sections from 102 patients with NSCLC by immunohistochemistry, and 65(63.73%)tumors were DKK1 positive. Relative analysis showed a significant relationship between DKK1 positive expression and lymph node metastasis(P<0.05). Patients with DKK1-positive tumors had poorer DFS than those with negative ESCC (5-year DFS; 15.4% versus 27%, P = 0.007). To further explore the biological effects of DKK1 in NSCLC cells, we over-expressed DKK1 in NSCLC 95C cell using eukaryotic expression vector pCMV-Tab-2b and performed a knockdown of DKK1 in LTEP-a-2 cell using a short hairpin RNA expression vector pSilencer 5.1. DKK1 did not have any effect on proliferation, but seemed to play a role in migration and invasion capability. Overexpression of DKK1 promotes migratory and invasive activity of 95C, while DKK1 knockdown resulted in the suppression of migration and invasion potentials of LTEP-a-2 cell. Taken together, these results indicate that DKK1 may be a crucial regulator in the progression of NSCLC. DKK1 might be a potential therapeutic target in NSCLC.

Figure 1. Expression of DKK1 in human NSCLC cell lines.

(A) Expression of DKK1 on mRNA level (RT-PCR: 28 cycles of amplification). GAPDH mRNA was used as the internal control. (B) Expression of DKK1 on protein level. β-actin was used as the internal control. (C) Subcellular localization of endogenous DKK1 protein in NSCLC cell. DKK1, stained with rhodamine B, was at the cytoplasm of the cell appearing fine-grained material. Cell nucleus appeared as blue fluorescence stained with DAPI (magnification 100×).

Figure 2. Expression of DKK1 in NSCLC tissues.

(A) Expression by immunohistochemical staining. DKK1 strong positive expression in lung cancer showing staining mainly in the cytoplasm of tumor Cells (magnification 100×). Representative DKK1 weak positive lung cancer with pale yellow particles in tumor cells (magnification 100×). DKK1 negative lung cancer cell showing almost no appreciable staining of tumor cells (magnification 100×). Normal lung tissue without staining. (B) Expression of DKK1 mRNA in NSCLC tissues and matched normal lung tissues. (C) Expression of DKK1 protein in NSCLC tissues and matched normal lung tissues. N, Normal tissue; T, Tumor tissue. Positive expressions of DKK1 were mainly accompanied with lymph nodes metastasis.

Figure 3. Effects of DKK1 overexpression in 95C cell.

(A) RT-PCR and Western blot analysis of the DKK1 expression. DKK1 level in 95C cell transfected with pCMV-Tag-2b-DKK1 is significantly higher than that in pCMV-Tag-2b and blank control group. (B) The wounded and healing 95C cells. (C) Measurement of migration distance (*P<0.05). (D) The invasion ability of 95C transfected with pCMV-Tag-2b-DKK1 was significantly enhanced. (E) The number of cells migrating through the Matrigel-coated filters was statistic analyzed. Assays were done in triplicate wells (*P<0.01).

Figure 4. Effects of DKK1 knockdown in LTEP-a-2 cell.

(A) RT-PCR and Western blot analysis of the DKK1 expression. DKK1 level in LTEP-a-2 cell transfected with pSilencer-DKK1 is significantly lower than that in pSilencer-NC and blank control group. (B) The wounded and healing LTEP-a-2 cells. (C) Measurement of migration distance (*P<0.05). (D) The invasion ability of LTEP-a-2 transfected with pSilencer-DKK1 was significantly repressed. (E) The number of cells migrating through the Matrigel-coated filters was statistic analyzed. Assays were done in triplicate wells (*P<0.01).