Background
Materials and methods
Cells and cell culture
Cellular lysate preparation and immunoblotting
RNA purification and qRT-PCR
- GAPDH: 5′-GAGTCCACTGGCGTCTTC-3′ and 5′-GGGGTGCTAAGCAGTTGGT-3′.
- ATR: 5′-GTCATATACACTCCCTTTTCTTTA-3′ and 5′-GTCATATACACTCCCTTTTCTTTA-3′.
- CDKN1A: 5′-CAGAGGAGGCGCCAAGACAG-3′ and 5′-CCTGACGGCGGAAAACGC-3′.
- CDKN2A: 5′-CAACGCACCGAATAGTTACG-3′ and 5′-CAGCTCCTCAGCCAGGTC-3′.
siRNA transfection
ATR-shRNA transfection
Analysis of mRNA stability
Conditioned media
Cloning of ARE reporter constructs
Transfection and reporter activity measurements
Engineered human breast tissue (EHBT)
Immunohistochemistry staining on EHBT
Patients and archived clinical materials
Immunohistochemistry staining on FFPE tissues
Statistical analysis
Results
ATR is downregulated in active breast cancer-associated fibroblasts
Breast cancer cells downregulate breast stromal fibroblast ATR in an IL-6/STAT3/AUF1-dependent manner
AUF1 destabilizes the ATR mRNA through binding its 3′UTR
ATR-deficient breast stromal fibroblasts enhance the growth of breast cancer cells in engineered human breast tissue
Correlation of ATR expression in cancer as well as stromal fibroblasts with clinicopathological parameters
Parameter | Total (n = 103) (%) | ATR in stromal fibroblasts | Pvalue | ||
High | Intermediate | Low | |||
Stage | |||||
T2 | 30 (29.13) | 6 (5.83) | 17 (16.50) | 7 (6.80) | |
T3 | 29 (28.16) | 10 (9.71) | 13 (12.62) | 6 (5.83) | 0.3308 |
T4 | 43 (41.75) | 14 (13.59) | 14 (13.59) | 15 (14.56) | |
Tx | 1 (0.97) | 0 (0.00) | 1 (0.97) | 0 (0.00) | |
KI-67 index | |||||
0 | 85 (84.16) | 26 (24.75) | 37 (36.63) | 23 (22.77) | |
≤ 15 | 2 (1.98) | 0 (0.00) | 2 (1.98) | 0 (0.00) | 0.7652 |
>15 | 14 (13.86) | 5 (4.95) | 5 (4.95) | 4 (3.96) | |
HIS subtype | |||||
None invasive | 1 (0.97) | 1 (0.97) | 0 (0.00) | 0 (0.00) | |
Invasive ductal Ca | 85 (82.52) | 23 (22.33) | 40 (38.83) | 22 (21.36) | |
1,4 | 3 (2.91) | 1 (0.97) | 1 (0.97) | 1 (0.97) | 0.5887 |
Invasive ductal Ca with DCIS | 11 (10.68) | 3 (2.91) | 4 (3.88) | 4 (3.88) | |
Infiltrating lobular Ca | 1 (0.97) | 0 (0.00) | 0 (0.00) | 1 (0.97) | |
3, 4 | 1 (0.97) | 1 (0.97) | 0 (0.00) | 0 (0.00) | |
Other | 1 (0.97) | 1 (0.97) | 0 (0.00) | 0 (0.00) | |
Recurrence | |||||
No | 63 (61.76) | 18 (17.65) | 35 (34.31) | 10 (9.80) | 0.0017 |
Yes | 39 (38.24) | 11 (10.78) | 10 (9.80) | 18 (17.65) | |
Grade | |||||
G1 | 3 (2.91) | 1 (0.97) | 1 (0.97) | 1 (0.97) | |
G2 | 46 (44.66) | 9 (8.74) | 27 (26.21) | 10 (9.71) | 0.0473 |
G3 | 54 (52.43) | 20 (19.42) | 17 (16.50) | 17 (16.50) | |
Progression | |||||
No | 73 (72.28) | 13 (12.87) | 40 (39.60) | 20 (19.81) | 0.0003 |
Yes | 28 (27.72) | 14 (13.86) | 4 (3.96) | 18 (9.9) | |
Survival status | |||||
Alive | 84 (81.55) | 24 (23.30) | 45 (43.69) | 15 (14.56) | < 0.0001 |
Dead | 19 (18.45) | 6 (5.83) | 0 (0.00) | 13 (12.62) | |
Duration of clinical follow-up (years, mean ± SD) | 4.16500000 | 5.00755556 | 3.87464286 | 0.0390 | |
Parameter | Total (n = 103) (%) | ATR in cancer cells | Pvalue | ||
High | Intermediate | Low | |||
Tumor size | |||||
≤ 5 | 35 (40.23) | 7 (8.05) | 15 (17.24) | 13 (14.94) | 0.4008 |
>5 | 52 (59.77) | 8 (9.20) | 17 (19.54) | 27 (31.03) | |
Stage | |||||
T2 | 30 (29.13) | 6 (5.83) | 12 (11.65) | 12 (11.65) | |
T3 | 29 (28.16) | 6 (5.83) | 10 (9.71) | 13 (12.62) | 0.6772 |
T4 | 43 (41.75) | 6 (5.83) | 16 (15.53) | 21 (20.39) | |
Tx | 1 (0.97) | 1 (0.97) | 0 (0.00) | 0 (0.00) | |
KI-67 index | |||||
0 | 85 (84.16) | 13 (12.87) | 32 (31.68) | 40 (39.60) | 0.1525 |
≤ 15 | 2 (1.98) | 0 (0.00) | 1 (0.99) | 1 (0.99) | |
>15 | 14 (13.86) | 6 (5.94) | 3 (2.97) | 5 (4.95) | |
HIS subtype | |||||
None invasive | 1 (0.97) | 0 (0.00) | 1 (0.97) | 0 (0.00) | |
Invasive ductal Ca | 85 (82.52) | 13 (12.62) | 33 (32.04) | 39 (37.86) | |
1,4 | 3 (2.91) | 0 (0.00) | 1 (0.97) | 2 (1.94) | 0.1336 |
Invasive ductal Ca with DCIS | 11 (10.68) | 4 3.88) | 2 (1.94) | 5 (4.85) | |
Infiltrating lobular Ca | 1 (0.97) | 0 (0.00) | 1 (0.97) | 0 (0.00) | |
3, 4 | 1 (0.97) | 1 (0.97) | 0 (0.00) | 0 (0.00) | |
Other | 1 (0.97) | 1 (0.97) | 0 (0.00) | 0 (0.00) | |
Recurrence | |||||
No | 63 (61.76) | 14 (13.73) | 26 (25.49) | 23 (22.55) | 0.1000 |
Yes | 39 (38.24) | 5 (4.90) | 11 (10.78) | 23 (22.55) | |
Grade | |||||
G1 | 3 (2.91) | 1 (0.97) | 1 (0.97 | 1 (0.97) | |
G2 | 46 (44.66) | 10 (9.71) | 14 (13.59) | 22 (21.36) | 0.6015 |
G3 | 54 (52.43) | 8 (7.77) | 23 (22.33) | 23 (22.33) | |
Progression | |||||
No | 73 (72.28) | 17 (16.83) | 29 (28.71) | 27 (26.73) | 0.0570 |
Yes | 28 (27.72) | 2 (1.98) | 9 (8.91) | 17 (16.83) | |
Survival status | |||||
Alive | 84 (81.55) | 18 (17.48) | 36 (34.95) | 30 (29.13) | 0.0006 |
Dead | 19 (18.45) | 1 (0.97) | 2 (1.94) | 16 (15.53) | |
Duration of clinical follow-up (years) | 4.16500000 | 5.00755556 | 3.87464286 | 0.1883 |
ATR expression in both breast cancer cells and their stromal fibroblasts predicts survival
Parameter | Overall survival | Disease-free survival | ||||
---|---|---|---|---|---|---|
Hazard ratio | 95% Cl† | P* | Hazard ratio | 95% Cl† | P* | |
ATR (fibroblasts) | ||||||
High | 1.000 | 1.000 | ||||
Low | 7.934 | 2.950–21.340 | 0.0001 | 0.406 | 0.198–0.833 | 0.0140 |
ATR (epithelial cells) | ||||||
High | 1.000 | 1.000 | ||||
Low | 7.711 | 2.244–26.498 | 0.0012 | 0.732 | 0.422–1.270 | 0.2670 |
Tumor size | ||||||
≤ 5 cm | 1.000 | 1.000 | ||||
> 5 cm | 1.239 | 0.439–3.500 | 0.6852 | 0.796 | 0.452–1.404 | 0.4311 |
Age | ||||||
≤ 52 years | 1.000 | 1.000 | ||||
> 52 years | 0.984 | 0.940–1.030 | 0.4826 | 1.012 | 0.985–1.039 | 0.3854 |
Stage | ||||||
T2 | 1.000 | 1.000 | ||||
T3, T4, Tx | 1.941 | 0.642–5.866 | 0.2401 | 0.508 | 0.292–0.883 | 0.0163 |
Grade | ||||||
I/II | 1.000 | 1.000 | ||||
Ш/poorly differentiated | 1.949 | 0.758–5.012 | 0.1660 | 1.241 | 0.723–2.130 | 0.4328 |
Parameter | Overall survival | Disease-free survival | ||||
Hazard ratio | 95% Cl† | P* | Hazard ratio | 95% Cl† | P* | |
ATR (fibroblasts) | 14.708 | 2.693–80.332 | 0.0019 | 0.369 | 0.151–0.900 | 0.0285 |
Tumor size | 0.665 | 0.084–5.244 | 0.6985 | 1.018 | 0.276–3.759 | 0.9790 |
Age | 0.878 | 0.802–0.962 | 0.0051 | 1.027 | 0.989–1.067 | 0.1642 |
Stage | 95.600 | 5.109–1788.693 | 0.0023 | 0.471 | 0.112–1.975 | 0.3035 |
Grade | 0.868 | 0.187–4.030 | 0.8564 | 1.363 | 0.647–2.874 | 0.4155 |
HR_ Status ER(+ve)/Her2(−ve) | 8.981 | 0.774–104.259 | 0.0793 | 0.649 | 0.283–1.489 | 0.3079 |
HR_ Status ER(−ve)/Her2(+ve) | 31.436 | 2.109–468.544 | 0.0124 | 0.412 | 0.102–1.662 | 0.2096 |
HR_ Status ER(−ve)/Her2(−ve) | 30.258 | 2.054–445.803 | 0.0130 | 0.592 | 0.161–2.231 | 0.4445 |
ER_PR_Status ER(+ve)/PR(−ve) | 0.074 | 0.003.009–1.771 | 0.1080 | 0.759 | 0.110–5.254 | 0.7802 |
ER_PR_Status ER(+ve)/PR(+ve) | 0.845 | 0.122–5.837 | 0.8640 | 0.726 | 0.169–3.108 | 0.6655 |
Parameter | Overall survival | Disease-free survival | ||||
Hazard ratio | 95% Cl† | P* | Hazard ratio | 95% Cl† | P* | |
ATR (epithelial cells) | 10.242 | 1.745–60.111 | 0.0100 | 0.834 | 0.436–1.598 | 0.5849 |
Tumor size | 0.049 | 0.006–0.425 | 0.0062 | 1.459 | 0.402–5.301 | 0.5659 |
Age | 0.880 | 0.804–0.962 | 0.0050 | 1.028 | 0.989–1.068 | 0.1637 |
Stage | 257.360 | 13.853–4781.270 | 0.0002 | 0.334 | 0.084–1.325 | 0.1188 |
Grade | 1.205 | 0.266–5.452 | 0.8083 | 1.379 | 0.672–2.832 | 0.3807 |
HR_ Status ER(+ve)/Her2(−ve) | 2.731 | 0.382–19.517 | 0.3166 | 0.633 | 0.266–1.505 | 0.3004 |
HR_ Status ER(−ve)/Her2(+ve) | 1.456 | 0.146–14.486 | 0.7488 | 0.657 | 0.196–2.207 | 0.4973 |
HR_ Status ER(−ve)/Her2(−ve) | 4.817 | 0.459–50.592 | 0.1901 | 0.655 | 0.193–2.225 | 0.4973 |
ER_PR_Status ER(+ve)/PR(−ve) | 0.901 | 0.465–25.058 | 0.9296 | 1.187 | 0.207–6.820 | 0.8474 |
ER_PR_Status ER(+ve)/PR(+ve) | 10.511 | 0.671–164.698 | 0.2275 | 1.021 | 0.983–1.061 | 0.2730 |