Atp6v1c1 may regulate filament actin arrangement in breast cancer cells

Abstract

Previous studies have shown that the rate of breast cancer metastasis correlates with the expression of vacuolar H(+)-ATPases (V-ATPases). However, how V-ATPase is involved in breast cancer metastasis remains unknown. Our previous study showed that Atp6v1c1-depleted osteoclasts did not form organized actin rings and that Atp6v1c1 co-localizes with F-actin. In this study, we found that the normal arrangement of filamentous actin is disrupted in Atp6v1c1-depleted 4T1 mouse breast cancer cells and in the ATP6V1C1-depleted human breast cancer cell lines MDA-MB-231 and MDA-MB-435s. We further found that Atp6v1c1 co-localizes with F-actin in 4T1 cells. The results of our study suggest that high expression of Atp6v1c1 affects the actin structure of cancer cells such that it facilitates breast cancer metastasis. The findings also indicate that Atp6v1c1 could be a novel target for breast cancer metastasis therapy.

Figure 1. Atp6v1c1 knockdown 4T1 cell clone selection and lack of expression of Atp6v1c2 in Atp6v1c1-depleted 4T1 cells.

(a) Atp6v1c1 expression in different 4T1 cells as indicated by western blotting. (b) RT-PCR assays of lung and different 4T1 cells as indicated were performed with gene-specific primers for Atp6v1c1, Atp6v1c2, and β-actin.

Figure 2. Regular F-actin arrangement was blocked in Atp6v1c1-depleted 4T1 cells.

F-actin Oregon Green® 514 phalloidin staining of different 4T1 cells as indicated. Nuclei were visualized with DAPI. The cells shown are representative of the data (n = 3).

Figure 3. Atp6v1c1 co-localized with F-actin in 4T1 cells.

Anti-Atp6v1c1 immunostaining and F-actin Oregon Green® 514 phalloidin staining of 4T1 cells. In the merged image, yellow staining showed that F-actin and Atp6v1c1 colocalized in the plasma and plasma membrane of 4T1 cells. The white arrows showed that most of the F-actin and Atp6v1c1 colocalization focused on the plasma membrane. The cells shown are representative of the data (n = 3).

Figure 4. Verification of shRNA targeting to human ATP6V1C1 in human breast cancer cell lines.

(a) ATP6V1C1 expression in different lentivirus-treated MDA-MB-231 cells as indicated by western blotting. (b) ATP6V1C1 expression in different lentivirus-treated MDA-MB-435s cells as indicated by western blotting.

Figure 5. Regular F-actin arrangement was blocked in ATP6V1C1-depleted MDA-MB-231 and MDA-MB-435s cells.

F-actin rhodamine phalloidin of different lentivirus-treated cells as indicated. The white arrows showed cells with disrupted actin cytoskeleton compared to control cells. The cells shown are representative of the data (n = 3).