Silencing of atp6v1c1 prevents breast cancer growth and bone metastasis

Abstract

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1's expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.

Keywords: Atp6v1c1 (C1); V-ATPase; breast cancer; lysosomal acidification; tumor bone metastasis..

Fig 1

Single cell clone selection of Atp6v1c1-depleted 4T1 mammary tumor cells. A. RT-PCR assays of three mouse tissues and 4T1 cells were performed with gene-specific primers for Atp6v1c1, Atp6v1c2, and β-actin. B. Select GFP⁺ single subclones of 4T1 cells. The 4T1-LacZ clone expresses siRNA that specifically targets LacZ. The 4T1-c1s3-1, 4T1-c1s3-2 clones express siRNA that specifically targets C1. Visualized by white light (L) or by fluorescent light (FL) (primary magnification ×100). C. Anti-Atp6v1c1 immunostaining of different 4T1 cells as indicated (primary magnification ×100). D. Atp6v1c1, Atp6v1a and Atp6L expression in different 4T1 cells as indicated by Western blotting. E. Quantification of Atp6v1c1 protein expression level in different 4T1 cells as indicated (normalized to the tubulin level) (n=3). * P<0.05 compared with that of 4T1-LacZ cells. The cells shown were representative of the data (n=3).

Fig 2

Growth, migration, and invasion of Atp6v1c1-depleted 4T1 mammary tumor cells in vitro. A. Cell growth assay of different 4T1 cells as indicated. Results are mean ± s.e.m. (n=3). * P<0.05 compared with 4T1-LacZ by student's t-test at the endpoint. B. Migration assay of different 4T1 cells as indicated. The cells between the red curves and marked by red arrows are the migration cells (primary magnification ×100). C. Invasion assay of different 4T1 cells as indicated (primary magnification ×100). D. Quantification of invasion cells per view area. Results are mean ± s.e.m. (n=10). * P<0.05 compared with that of 4T1-LacZ cells. ^# P<0.05 compared with that of 4T1-c1s3-2 cells. The cells shown were representative of the data (n=3).

Fig 3

Atp6v1c1 knockdown inhibits 4T1 mouse mammary tumor growth and metastasis in vivo. A. Representative tumors from female BALB/c mice 28 days after subcutaneous inoculation in the left thoracic (#2) mammary gland fat pad with PBS or 1×10⁵ 4T1 cells infected (or not) as indicated (n=24). B. The mean tumor diameter (TD) of tumors during the period between post-implantation day 0 and day 26. Results are mean ± s.e.m. (n=24). * P<0.05 for 4T1-c1s3-1 compared with 4T1-LacZ by student's t-test at the endpoint. C. The weight of tumors on post-implantation day 28 (n=24). D. Bioluminescent Imaging (BLI) of mice as indicated (Red arrow indicates the metastatic site in BLI of mice and blue arrow indicates tumor necrosis). The images were representative of the data (n=12). Counts are photons detected. Images were captured with a 5min exposure. E. Whole body flux (photons/sec) quantification of mice injected with different 1×10⁵ 4T1 cells at day 16 as indicated (n=12). Signal measured from a 5 minute exposure. * P<0.05 for 4T1-c1s3-1 compared with 4T1-LacZ by student's t-test at the endpoint.

Fig 4

Atp6v1c1 knockdown inhibits primary 4T1 mouse mammary tumor metastasis and prolongs survival after xenograft in vivo. A. Representative H&E staining of lungs and livers (scale bar: 100μm) as indicated. Black arrows indicate the metastases. B. Number of mice bearing metastases in the lungs, liver, and bone from a primary mammary tumor based on H&E staining at day 33 (n=12 mice). Numbers indicate the number of mice bearing metastases. C. Survival of mice bearing tumors (n=15) over time.

Fig 5

Bone metastasis and lesions of primary mammary tumors derived from normal and Atp6v1c1-depleted 4T1 cells. A. X-ray of representative left femurs and tibias of the primary mammary tumor model (n=6). Red arrows show the bone lesion region. B. Micro-CT images show the region of interest (ROI). Red arrows indicate the bone lesion region. C-G, Results are labeled as 1: PBS injected sham control; 2: 4T1 xenografted mice; 3: 4T1-LacZ control virus xenografted mice; 4: 4T1-c1s3-1 ATP6v1c1 knockdown xenografted mice; 5:4T1-c1s3-2 ATP6v1c1 knockdown xenografted mice. C. Quantification of femur relative bone volume over total volume (BV/TV). D. Quantification of femur trabecular bone number (Tb.N). E. Quantification of tibia relative bone volume over total volume (BV/TV). F. Quantification of tibia trabecular bone number (Tb.N). * p<0.05, ** p<0.01 compared to 4T1-LacZ mice. G. H&E staining of representative left femurs (n=6) (primary magnification ×100). H. TRAP staining of osteolytic lesions shown in the left femur ROI. Red arrows indicate the TRAP⁺ osteoclasts. I. Quantification of TRAP⁺ osteoclasts per view area. Results are mean ± s.e.m. (n=5) (primary magnification ×100). *P<0.05 compared with that of 4T1-LacZ mice. (GP, growth plate; T.B, trabecular bone; T, Tumor; C.B., cortical bone).