Effect of cancer-associated fibroblast autophagy on metastasis of triple-negative breast cancer cells

Abstract

Objective To clarify the effect of cancer-associated fibroblasts (CAFs ) autophagy on metastasis of triple-negative breast cancer (TNBC) cells co-cultured with CAFs. Methods (1) Purification and identification of CAFs. CAFs and normal fibroblasts (NFs) were prospectively obtained from the cancer tissue and normal breast tissue of six patients with TNBC at stage Ⅱ or Ⅲ, who were treated in Zhujiang Hospital of Southern Medical University from January 2015 to December 2016.α-smooth muscle actin (α-SMA) was labeled by fluorescent antibody and the proportion of α-SMA positive cells was calculated. The expression of α-SMA in CAFs and NFs was determined by Western blot, respectively, to identify CAFs used for this experiment. (2) Evaluation of CAFs autophagy. Autophagic biomarkers beclin 1 and p62 were measured by Western blot in CAFs group, NFs group and CAFs group pretreated with 3-methyladenine (CAFs+ 3-MA group), respectively. (3) Identifying the migration of MDA-MB-231 and BT-549 cells caused by CAFs autophagy. The migration of MDA-MB-231 or BT-549 cells was detected by the Transwell chamber under different conditions (medium/NFs/CAFs/CAFs+ 3-MA). (4) Clarifying the effect of CAFs autophagy on epithelial-mesenchymal transition (EMT) in MDA-MB-231 or BT-549 cells. The EMT-related proteins E-cadherin, N-cadherin and vimentin were detected by Western blot in MDA-MB-231 or BT-549 cells cultured in different conditions (medium, CAFs-CM or CAFs+ 3-MA-CM). The data of normal distribution were expressed by ±s and the data of skewed distribution were expressed by M(P25-P75). Nonparametric rank-sum test of two independent samples was used to compare α-SMA expression, t test of two independent samples was used to compare the percentage of α-SMA-positive cells, and single-factor analysis of variance was used to compare the autophagic protein expression, cell migration rate and EMT-related protein expression among groups. Results (1) α-SMA-positive cells were observed under a fluorescence microscope. The result showed that the percentage of α-SMA-positive cells in CAFs was significantly higher than that in NFs [(81.11±3.95)% vs(5.11±2.37)%, t=49.49, P<0.001)]. Western blot analysis indicated that the expression of α-SMA in CAFs and NFs was 0.98(0.95-0.98) and 0.48 (0.47-0.48), respectively, suggesting a significant difference (Z=2.023, P=0.043). (2) The expression of beclin 1 was 0.99±0.03, 0.73±0.04 and 0.26±0.02 in CAFs group, NFs group and CAFs+ 3-MA group, respectively, indicating a significant difference (F=546.188, P<0.001). The expression of beclin 1 in CAFs group was significantly higher than that in CAFs+ 3-MA group (P<0.001). The expression of p62 was 0.75±0.02, 0.98±0.03 and 0.97±0.01 in CAFs group, NFs group and CAFs+ 3-MA group, respectively, indicating a significant difference (F=136.353, P<0.001). The expression of p62 expression in CAFs group was significantly lower than that in CAFs+ 3-MA group(P<0.001). (3)The number of migrated MDA-MB-231 cells was 41.67±2.78, 23.33±2.18, 22.00±1.76 and 18.00±2.12 in CAFs group, NFs group, CAFs+ 3-MA group and medium group, respectively, indicating a significant difference(F=198.374, P<0.001). The number of migrated BT-549 cells was 35.22±1.97, 22.00±2.60, 25.11±2.15 and 15.22±2.00 in CAFs group, NFs group, CAFs+ 3-MA group and medium group, respectively, indicating a significant difference(F=129.424, P<0.001). The number of migrated MDA-MB-231 or BT-549 cells in CAFs group was significantly higher than that in other three groups (P<0.050). (4) When MDA-MB-231 cells were cultured in medium, CAFs-CM or CAFs+ 3-MA-CM, Western blot analysis showed that E-cadherin expression was 0.79±0.03, 0.54±0.02 and 0.87±0.04 in 3 groups, respectively, indicating a significant difference(F=139.286, P<0.001); N-cadherin expression was 0.59±0.02, 1.00±0.02 and 0.93±0.02 in 3 groups, respectively, indicating a significant difference(F=604.905, P<0.001); vimentin expression was 0.62±0.03, 1.01±0.01 and 0.89±0.09 in 3 groups, respectively, indicating a significant difference(F=43.884, P<0.001). When BT-549 cells was cultured in medium, CAFs-CM and CAFs+ 3-MA-CM, E-cadherin expression was 1.01±0.03, 0.63±0.03 and 0.98±0.03 in 3 groups, respectively, indicating a significant difference(F=210.102, P<0.001); N-cadherin expression was 0.58±0.01, 0.94±0.04 and 0.95±0.03 in 3 groups, respectively, indicating a significant difference(F=184.477, P<0.001), vimentin expression was 0.61±0.01, 0.98±0.03 and 0.75±0.02 in 3 groups, respectively, indicating a significant difference(F=217.659, P<0.001). Conclusion CAFs can promote the migration and EMT in MDA-MB-231 and BT-549 cells, which may be related to the autophagy of CAFs. Key words: Breast neoplasms; Fibroblasts; Autophagy