Wnt5a is associated with cigarette smoke-related lung carcinogenesis via protein kinase C

Abstract

Wnt5a is overexpressed during the progression of human non-small cell lung cancer. However, the roles of Wnt5a during smoking-related lung carcinogenesis have not been clearly elucidated. We investigated the associations between Wnt5a and the early development of cigarette smoke related lung cancer using human bronchial epithelial (HBE) cells (NHBE, BEAS-2B, 1799, 1198 and 1170I) at different malignant stages established by exposure to cigarette smoke condensate (CSC). Abnormal up-regulation of Wnt5a mRNA and proteins was detected in CSC-exposed transformed 1198 and tumorigenic 1170I cells as compared with other non-CSC exposed HBE cells. Tumor tissues obtained from smokers showed higher Wnt5a expressions than matched normal tissues. In non-CSC exposed 1799 cells, treatment of recombinant Wnt5a caused the activations of PKC and Akt, and the blockage of Wnt5a and PKC significantly decreased the viabilities of CSC-transformed 1198 cells expressing high levels of Wnt5a. This reduced cell survival rate was associated with increased apoptosis via the down-regulation of Bcl2 and the induction of cleaved poly ADP-ribose polymerase. Moreover, CSC-treated 1799 cells showed induction of Wnt5a expression and enhanced colony-forming capacity. The CSC-induced colony forming efficiency was suppressed by the co-incubation with a PKC inhibitor. In conclusion, these results suggest that cigarette smoke induces Wnt5a-coupled PKC activity during lung carcinogenesis, which causes Akt activity and anti-apoptosis in lung cancer. Therefore, current study provides novel clues for the crucial role of Wnt5a in the smoking-related lung carcinogenesis.

Figure 1. Expression of Wnt5a gene in HBE cells and normal matched lung tissues

. (A) mRNA and protein levels of Wnt5a expression were evaluated by RT-PCR (left), quantitative RT-PCR (middle) and Western blot analysis (right) in lung carcinogenesis in five HBE cells (NHBE, BEAS-2B, 1179, 1198 and 1170I) with different cell phenotype by exposure to cigarette smoke condensates. (B) mRNA levels of Wnt5a in 12 normal matched lung tumor tissues (7 smokers and 5 non-smokers) by RT-PCR. N: normal tissue, T: tumor tissue. Quantification of data means the average intensity of Wnt5a bands normalized by GAPDH. Bars denote standard error. Significant differences (*: p<0.03) from normal tissues as determined by a Student's t test.

Figure 2. Wnt5a causes activation of PKC and AKT signaling.

(A) The phosphorylation status of PKC α/βII and PKC-pan in 5 HBE cells by Western blot analysis. (B) Treatment with cigarette smoking carcinogens, 50 µM nicotin and 100 µM NNK, increases the levels of Wnt5a mRNA expression in 1179 cells. The phosphorylation status of PKC α/βII and PKC-pan was analyzed by Western blot analysis after treatment with 100 µM NNK for 48∼72 h. (C) 1799 cells were treated with recombinant Wnt5a. TPA was used as a positive control to stimulate PKC. β-actin was used as a loading control.

Figure 3. Inhibition of Wnt5a-mediated signaling in pre-malignant HBE cells.

(A) 1198 cells were transfected with scrambled siRNA (Scr) and siWnt5a for 48 h. Cell viability was performed by cell counting. (B) 1198 cells were transfected with scrambled siRNA (Scr), siPKCα and siPKCβII for 48 h. Cell viability was performed by cell counting. (C) siRNA transfected 1198 cells were treated with PKC inhibitors (RO: 2.5 µM Ro-318226 and Gö: 2.5 µM Gö6o76) for 24 h and then were counted under a microscope. (D) 1198 cells were treated with PKC inhibitors alone and combination with PI3K inhibitor (LY: 10 µM LY294002).

Figure 4. Apoptosis by blocking of siRNA mediated Wnt5a expression and PKC/AKT pathway.

(A) 1198 cells were transfected with scrambled siRNA (Scr) or Wnt5a siRNA for 24 h and then treated with PKC inhibitors (RO; 2.5 µM Ro-318226 and Gö; 2.5 µM Gö6o76) ) for 48 h. Apoptosis were determined by the flow cytometric analysis. (B) These cells were analyzed by Western blot analysis of indicated protein expression. (C) 1198 cells were treated with PI3K inhibitor (LY), PKC inhibitors (RO and Gö) or their combination for 72 h and then subjected to Western blot analysis of indicated protein expression. β-actin was used as a loading control.

Figure 5. CSC induced the expression of Wnt5a and increased colony forming capacity in non-CSC exposed 1799 cells.

(A) 1799 cells treated with 10% CSC for 8 days and further cultured in normal growth medium. CSC-transformed clones of 1799 cells were isolated and used to profile the expression of Wnt5a using quantitative RT-PCR. (B) Selected R10 and R11 clones were examined for the colony-forming efficiency with or without a PKC inhibitor (Gö: 2.5 µM Gö6o76). Colonies were fixed in methanol and stained with 0.1% crystal violet. The data are representative of three experiments. Quantification of data means the average number of colonies (>20 pixel²) per dish from the triplicate. Bars denote standard error. Significant differences (*: p<0.05, **: p<0.005) from control as determined by a Student's t test.