Fig 2: AnxA6 SUMOylation inhibits activation of EGFR mutation-induced signal transduction. A The the silencing of AnxA6 activate pY-GFP-EGFRT790M/L858R and pERK1/2. HeLa stable cells were generated by using lentiviral infections with indicated plasmids, and cells were transfected with pGFP-EGFRT790M/L858R plasmids for 24 h, starved overnight and followed with/without 10 ng/ml EGF treatment for 3 min. Cell lysates were immunoblotted with the indicated antibodies. B The K299R mutation of AnxA6 impaired its ability to inactivate pY-EGFRT790M/L858R and pERK1/2. A431 cells were co-transfected with the indicated plasmids for 24 h, starved overnight and followed with/without 10 ng/ml EGF treatment for 3 min. Cell lysates were immunoblotted with the indicated antibodies. C The K299R mutation impaired its recruitment ability of AnxA6 to promote GFP-EGFRT790M/L858R-PKCa interaction. A431 cells were co-transfected with the indicated plasmids for 24 h, starved overnight and followed with/without 10 ng/ml EGF treatment for 3 min. Cell lysates were IP to capture GFP-tagging EGFRT790M/L858R and subsequently immunoblotted with indicated antibodies. D The K299R mutation of AnxA6 impaired its ability to interact GFP-EGFRT790M/L858R. A431 cells were co-transfected with the indicated plasmids for 24 h, starved overnight and followed with/without 10 ng/ml EGF treatment for 3 min. Cell lysates were IP to capture Flag-tagging AnxA6 and subsequently immunoblotted with indicated antibodies. IP: immunoprecipitation, IB: immunoblot, Input: same account of cell lysate to load

Fig 3: AnxA6 SUMOylation promotes gefitinib-mediated inhibition of epithelial cancer cell growth and migration. A-C AnxA6 knockdown impaired its ability of enhancing anti-proliferation and anti-migratory properties of gefitinib in HeLa cells. The CCK8 (A) and colony-forming (B) assays were used to measure cell proliferation ability of gefitinib-treated HeLa cells. Cells were grown in DMEM media containing 0.1% (v/v) FBS and 100 ng/ml EGF. Quantification of colonies was shown at right, which was calculated as the proportion of cell clone number relative to HeLa. C Cell motility was determined by wound healing assay in gefitinib-treated HeLa cells. The bar graphs showed cell migration distances, which were calculated relative to the initial distance before migration (right). D-F The K299 mutation of AnxA6 impaired its ability of enhancing anti-proliferation and anti-migratory properties of gefitinib in A431 cells. The CCK8 (D) and colony-forming (E) assays were used to measure cell proliferation ability of gefitinib-treated A431 cells. Cells were grown in RPMI-1640 media containing 0.1% (v/v) FBS and 100 ng/ml EGF. Quantification of colonies was shown at right, which was calculated as the proportion of cell clone number relative to A431. F Cell motility was determined by wound healing assay in gefitinib-treated A431 cells. The bar graphs showed cell migration distances, which were calculated relative to the initial distance before migration (right). G The K299 mutation of AnxA6 impaired its ability of enhancing gefitinib-mediated inhibition of EGFR-ERK signaling. The A431, A431-AnxA6, and A431-AnxA6K299R cells were treated with 10 µM gefitinib for 24 h, and followed with 100 ng/ml EGF treatment for 3 min. The levels of pY-EGFR, EGFR, pERK1/2, ERK1/2 and AnxA6 were determined by western blotting. Data were represented as the mean ± SD of three separate experiments. ns, no statistical; *p < 0.05;**p < 0.01; ***p < 0.001

Fig 4: AnxA6 SUMOylation inhibits tumor growth of the epithelial cancer cells-xenografted nude mouse model. A-D AnxA6 knockdown promotes tumor growth in HeLa-xenografted mice. A-C 5 × 106 cells, including HeLa and HeLa-AnxA6(KD), were injected subcutaneously into male BALB/c nude mice (n = 5) individually (A). The sizes of tumors were measured at day 6, 9, 12, 15, 18 and 21 after injection (B), and the tumors were weighed (C). And the expression of pY-EGFR, EGFR, pERK1/2, ERK1/2 and AnxA6 were analyzed in HeLa and HeLa-AnxA6(KD)-xenografted tumors (D). E–G The K299 mutation of AnxA6 impaired its cancer suppression effection on A431-xenograft tumor growth. 5 × 106 cells, including A431, A431-AnxA6, and A431-AnxA6K299R, were injected subcutaneously into male BALB/c nude mice (n = 5) individually (E). The sizes of tumors were measured at day 9, 12, 15, 18, 21, 24, 27 and 30 after injection (F), and the tumors were weighed (G). H Ki-67 expression was detected by immunohistochemical staining in A431-xenograft tumors at 30 days after implantation (50 ×) (left), and the rates of staining positivity for Ki-67 were shown at right. I The K299R mutation of AnxA6 impaired its ability to inactivate the pY-EGFR and pERK1/2 in A431-xenograft tumors. The expression of pY-EGFR, EGFR, pERK1/2, ERK1/2 and AnxA6 were analyzed in A431, A431-AnxA6, and A431-AnxA6.K299R-xenografted tumors.T1-T5: tumor tissues from HeLa-xenograft nude mouse.T1-T3: tumor tissues from A431-xenograft nude mouse. *p < 0.05;**p < 0.01

Fig 5: The SUMOylated AnxA6 is detected under condition of protein overexpression. A-B The SUMOylated AnxA6 is mainly modified by SUMO1 in HEK293T cells. HEK293T cells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1(SUMO2/SUMO3) plasmids as indicated. Cells were collected at 48 h after transfection, and target proteins were captured by IP on anti-Flag antibody coupled agarose beads (A) or Ni2+-NTA agarose beads (B). Then the target protein was detected by immunoblotting with anti-Flag and anti-His antibodies. C-D The SUMOylation of AnxA6 was detected in response with UBC9/SUMO1 enhancement or inhibitor treatment (2-D08 or ML792). HEK293T cells were co-transiently transfected with pFlag-AnxA6 and pHA-UBC9 or pHis-SUMO1 plasmids as indicated. After transfection for 24 h, cells were treated with 150 µM 2-D08 (C) or 10 µM ML792 (D) for another 24 h. The Flag-tagging AnxA6 protein was enriched by IP on anti-Flag antibody coupled agarose beads (C) or Ni2+-NTA agarose beads (D), from which the SUMOylated AnxA6 level was analyzed through western blot using Flag or SUMO1 antibody. S-Flag-AnxA6: SUMOylated Flag-tagging AnxA6, IP: immunoprecipitation, IB: immunoblot, Input: Same account of cell lysate to load. 2-D08 or ML792: SUMOylation inhibitor