Figure 4. Wnt7A is regulated by Grhl2, and over-expression of Wnt7A phenotypically mimics Grhl2 in promoting epithelial gene expression and promoting tumor progression.

(A) Wnt7A was down-regulated in 4T1 cells recovered from lung that had undergone EMT. Expression of Wnt7A in 4T1 cells recovered from primary tumors and lungs were examined by RT-PCR. Data represent one of four independent experiments (n = 18). (B) Forced expression of Grhl2 reversed Wnt7A expression in 4T1 cells recovered from lung. Wnt7A mRNA expression in cancer cells recovered from lungs of individual mice carrying either 4T1-control or 4T1-Grhl2 tumors were analyzed by RT-PCR. Data represent RNAs isolated from cells recovered from lungs of two different mice from each group (n = 18). (C) Constructing 4T1 cell lines stably expressing Wnt7A1 and Wnt7A2. Wnt7A1 and Wnt7A2 were introduced into 4T1 cells by lentiviral infection. One week after virus infection GFP⁺ cells were sorted. Gene expressions in 4T1-control, 4T1-Wnt7A1 and 4T1-Wnt7A2 cells were analyzed by RT-PCR. On the top are schematics of gene structures of Wnt7A1 and Wnt7A2. (D) Growth curves of tumors formed by 4T1-control, 4T1-Wnt7A1 and 4T1-Wnt7A2 in wild type BALB/c mice. 6-week-old female BALB/c mice were injected with 4T1-control, 4T1-Wnt7A1, or 4T1-Wnt7A2 cells (1.0×10⁵ cells in 100 ul PBS for each mouse; 4T1-control, n = 6 mice; 4T1-Wnt7A1, n = 7 mice; 4T1-Wnt7A2, n = 5 mice). Tumor size was measured twice a week. Each data point represents the mean ± SD of four primary tumors. Data represent one of two independent experiments. (E) Lung metastasis was examined by hematoxylin and eosin (HE) staining of paraffin sections of fixed lung tissues (4T1-control, n = 6 mice; 4T1-Wnt7A1, n = 7 mice; 4T1-Wnt7A2, n = 5 mice). Arrows indicate metastatic tumors. Data represent one of two independent experiments. (F) Representative micrographs of 4T1-control, 4T1-Wnt7A1 and 4T1-Wnt7A2 cells recovered from primary tumors and lungs. (G) Representative gene expressions in recovered 4T1 cells analyzed by RT-PCR (4T1-control, n = 6 mice; 4T1-Wnt7A1, n = 7 mice; 4T1-Wnt7A2, n = 5mice). (H) E-Cadherin and β-catenin protein expression in cancer cells recovered from primary tumors of individual mice carrying 4T1-control, 4T1-Wnt7A1 or 4T1-Wnt7A2 tumors examined by Western blotting. (I) E-Cadherin and β-catenin protein expression in cancer cells recovered from lungs of individual mice carrying 4T1-control, 4T1-Wnt7A1 or 4T1-Wnt7A2 tumors examined by Western blotting. (J) Heatmap showing genes up-regulated and down-regulated, with the highest fold changes by Wnt7A1 which overlapped with genes regulated by Grhl2.