FIGURE 6 from MARCH2, a Novel Oncogene-regulated SNAIL E3 Ligase, Suppresses Triple-negative Breast Cancer Metastases

MARCH2 suppresses growth, migration, survival, and metastases of TNBC. A, MARCH2 expression is associated with favorable prognosis in patients with breast cancer using TCGA data (n = 1,052) and METABRIC data (n = 1302). Breast cancer samples were divided into high versus low expression groups based on whether their MARCH2 expression level was higher than upper quartile or lower than lower quartile. B, WT, but not RING domain–mutant (W97A), MARCH2 inhibits growth and invasive branching of MDA-MB-231 cells in 3D Matrigel cultures. Representative phase contrast images of 3D cultures of MDA-MB-231 cells overexpressing vector control, WT or RING domain–mutant (W97A) MARCH2 are shown. Scale bars, 30 µm. C, Cell Titer-glo 3D was used to measure cell viability of MDA-MB-231 cells expressing control, WT or RING domain–mutant MARCH2 in 3D cultures. Data present mean ± SD. *, P = 0.05 by Student t test. D, Transwell migration assays of MDA-MB-231 cells overexpressing pLv vector control, WT or W97A MARCH2 mutant were counted by ImageJ. Data represent mean ± SEM. **, P = 0.01 by Student t test. E, Anoikis of MDA-MB-231 cells expressing vector control, WT or W97A mutant MARCH2 cultured in suspension for 48 hours was assessed by Annexin/PI staining and flow cytometry. F, Coexpression of SNAIL restores migratory capacity of MARCH2-expressing MDA-MB-231 cells. MDA-MB-231 cells coexpressing GFP-MARCH2 and vector control or SNAIL were generated and assessed in transwell assays. Data are representative of three independent experiments each performed with technical triplicates. Data represent mean ± SD. ***, P < 0.001 by Student t test. G, SNAIL restores invasive branching and growth of MARCH2-overexpressing MDA-MB-231 cells in 3D Matrigel cultures. Cell viability was assessed using 3D Cell Titer Glo assays. Data present mean ± SD. ***, P < 0.001 by Student t test.