Fig 1: DCN modulates the stability of E-cadherin protein through an autophagy-linked lysosomal pathway.a DCN destabilizes E-cadherin protein in IBC cells. DCN-overexpressing and control IBC cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 100 μg/mL) at 0, 2, 4, 6, 8, or 10 h was shown. Total cell lysates were subjected to western blotting analysis with the indicated antibodies. GAPDH served as a loading control. b the lysosomal inhibitor chloroquine (CQ) markedly delayed the DCN-induced degradation of E-Cadherin. DCN-overexpressing IBC cells were treated with 20 μM CQ for 6 or 10 h, and total cell lysates were subjected to western blotting analysis with the indicated antibodies. GAPDH served as a loading control. c Overexpression of DCN promotes Beclin1expression in IBC cells. Total cell lysates were subjected to immunoblotting with the indicated antibody. GAPDH served as a loading control. d DCN inhibition of E-cadherin expression and EGFR pathway activation in IBC cells is dependent on Beclin1. DCN-overexpressing cells were transduced with two independent lentiviral Beclin1 shRNAs or control shRNA; 48 h later, the cells were harvested and analyzed by western blotting with the indicated antibodies. GAPDH was used as a loading control. Beclin1 knockdown resulted in elevation of E-cadherin expression and EGFR pathway activation in DCN-overexpressing IBC cells. e Proposed model summarizing the DCN-mediated inhibition of tumorigenesis and metastasis in IBC cells through a mechanism involving the lysosomal degradation of E-cadherin and suppression of the EGFR–ERK signaling pathway.
Fig 2: DCN inhibits in vitro tumorigenic features in IBC cell lines.a Generation of four DCN-overexpressing IBC stable cell lines [HER2+: MDA-IBC3, SUM190; TNBC: SUM149, BCX010]. Total cell lysates were analyzed by western blotting with anti-DCN and anti-GAPDH antibody. b–f DCN overexpression in IBC cell lines suppresses colony formation (b), cell migration in SUM149 and BCX010 cells (c), cell invasion in SUM149 and BCX010 cells (d), primary mammosphere formation (e), and secondary mammosphere formation (f). g–i Treating IBC cell lines with recombinant DCN protein (8 μg/mL) suppresses colony formation (g), cell migration (h), and cell invasion (i). Scale bar: 100 μm. P values are from Student’s unpaired t tests. Data are presented as mean ± s.e.m.; Data shown are representative of three independent experiments.
Fig 3: DCN inhibits E-cadherin expression and EGFR pathway activation in IBC.a DCN suppresses E-cadherin expression and EGFR signaling in IBC cells. Expression levels of E-cadherin and EGFR are decreased in DCN-overexpressing MDA-IBC3, SUM190, SUM149, and BCX010 cells; also, the phosphorylation of EGFR (pEGFR) and ERK1/2 (pERK1/2) was suppressed in DCN-overexpressing IBC cell lines. Total ERK1/2 (tERK1/2) remains unchanged. GAPDH served as a loading control. b Treatment of IBC cells with DCN protein (4 or 8 μg/mL) for 2 h suppresses E-cadherin expression and EGFR pathway activation. Tubulin served as a loading control. c and d Western blot validation of E-cadherin and EGFR downregulation in tumor samples obtained from mammary fatpad transplantation of control or DCN-overexpressing MDA-IBC3 (c) or SUM149 (d) cells. e and f Immunohistochemical staining validation of E-cadherin and EGFR downregulation in tumor samples obtained from mammary fatpad transplantation of control or DCN-overexpressing MDA-IBC3 (e) or SUM149 (f) cells. Scale bar: 100 μm. g DCN inhibits EGFR signaling in IBC cells independently of EGF stimulation. DCN-overexpressing and control IBC cell lines were stimulated with 50 ng/mL EGF for the indicated number of hours, and total cell lysates were analyzed by western blotting. Both the total levels and the phosphorylation levels of EGFR and ERK1/2 were detected by western blotting. Tubulin served as a loading control. h DCN-mediated inhibition of E-cadherin does not affect expression of epithelial–mesenchymal transition markers. Cell lysates containing 40 μg of total protein were analyzed by western blotting with anti-E-cadherin, fibronectin, vimentin, and DCN antibodies. GAPDH served as a loading control.
Fig 4: DCN inhibits tumor growth and metastasis in IBC tumor xenograft models.For tumor growth studies, DCN-overexpressing and control MDA-IBC3 and SUM149 cells were injected into the cleared mammary fat pads of SCID/Beige mice (9 mice/group for each cell line) to allow the formation of tumors. a Tumor volume is significantly decreased in DCN-overexpressing MDA-IBC3 versus control group. Data are shown as mean ± s.e.m. P values are from Student’s unpaired t tests. b DCN-overexpressing MDA-IBC3 cells showed longer tumor latency in MDA-IBC3 xenografts (Chi-square test). c Tumor weight is decreased in DCN-overexpressing MDA-IBC3 tumors than in controls. P values from Student’s unpaired t tests. d Tumor volume is significantly inhibited in DCN-overexpressing SUM149 versus control group. Data are shown as mean ± s.e.m. P values from Student’s unpaired t tests. e DCN-overexpressing showed longer tumor latency in SUM149 xenografts (Chi-square test). f Tumor weight is decreased in DCN-overexpressing SUM149 tumors relative to controls. P values from Student’s unpaired t tests. g, h Hematoxylin-eosin and immunostains of tumors generated from MDA-IBC3 (g) and SUM149 (h) control and DCN-overexpressing cells validates the overexpression of DCN in xenograft tumors. Scale bar: 100 μm. For lung metastatic colonization studies, GFP-labeled DCN-overexpressing and control SUM149 cells were injected via tail vein into SCID/Beige mice (10 mice/group). i DCN significantly inhibited the incidence of lung metastasis compared with the control group (0% DCN vs. 70% Control; p = 0.0031, Fisher exact test). j Tumor burden was significantly reduced in DCN-overexpressing mice group; scatter plot shows reduction in numbers of lung metastasis nodules in the DCN-overexpressing group (p = 0.0022, Wilcoxon rank-sum test was used). Scale bar: 5 mm.
Fig 5: DCN suppresses aggressiveness in IBC by regulating the E-cadherin–EGFR axis.a E-cadherin knockdown in IBC cells results in the inhibition of EGFR signaling but does not affect DCN expression. E-cadherin knockdown in IBC cells lines was achieved by transduction with two independent lentiviral shRNAs. Total cell lysates were analyzed by western blotting for EGFR pathway analysis. GAPDH served as a loading control. b–f E-cadherin knockdown in IBC cell lines suppresses colony formation (b), migration (c), invasion (d), primary mammosphere formation (e), and secondary mammosphere formation (f). g Restoring E-cadherin rescues EGFR signaling in DCN-overexpressing IBC cells. Flag-E-cadherin plasmid was transfected into DCN-overexpressing IBC cell lines for 48 h and cell lysates were subjected to western blotting. h and i Restoring E-cadherin increases migration (h) and invasion (i) in DCN-overexpressing SUM149 and BCX010 IBC cell lines. P values are from Student’s unpaired t tests. Data are shown as mean ± s.e.m. Data shown are representative of three independent experiments.
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