Fig. 6

Hectd3 promotes lung colonization of tumor cells under inflammatory conditions. a qRT-PCR analysis of adhesion molecules in WT and Hectd3 KO mECs stimulated with LPS (500 ng/ml) for 2 h. b A comparison of the expression of the adhesion molecules, IKKα and H3S10ph in mECs stimulated with or without LPS (500 ng/mL) as indicated time. c Representative frozen immunofluorescence images of GFP⁺ tumor cells in lungs of mice performed the tumor cell colonization assay in vivo and bar graph showing the number of GFP⁺ tumor cells in lungs of WT and Hectd3^−/− mice (right). Counting rules: Eighty sections were randomly cut from each sample (the entire lung) and then scanned by a FluoView FV1000 confocal microscope after fluorescence staining. The total number of GFP-positive tumor cells in 80 sections was recorded. d The GFP⁺ tumor cells (5 × 10⁶ cells per mouse) were injected through the tail vein into WT or Hectd3^−/− mice pretreated with LPS (1 mg/kg) stimulation for 5 h. At 10 min, 10 h or 20 h after tumor cell injection, the mice were sacrificed and perfused with PBS and the whole lungs were digested to cell suspension to analyze GFP⁺ tumor cell colonization in the lung by FCM. e The rate of GFP⁺ tumor cell in the lung of panel d. f Schematic representation of the targeted allele and the conditional allele of Hectd3 KO. g B16-F10 cells were injected subcutaneously into Tie2-Cre⁺;Hectd3^wt (n = 11) and Tie2-Cre⁺;Hectd3^fl/fl (n = 12) C57BL/6 mice. Twelve days after transplantation, primary tumors were removed. The mice were sacrificed at day 45 and the incidence of lung metastasis was record. Representative lung metastasis nodule image. h The incidence of lung metastasis of panel g. i Tie2-Cre⁺;Hectd3^wt and Tie2-Cre⁺;Hectd3^fl/fl mice were intravenously injected with vehicle, TNFα (200 μg/kg) or LPS (1 mg/kg). 5 h later, B16-F10 cells were injected through the tail vein (2 × 10⁵ cells per mouse). The mice were sacrificed 20 days after injection of tumor cells. The graph shows the number of pulmonary metastasis nodules in each group of mice. j The weight of the whole lung with metastatic nodules in each group of mice from panel i. k Representative lung metastasis nodule images of the lungs in different groups of mice from panel i. l 4T1-Luc2 cells were injected by tail vein into BALB/c mice (1 × 10⁵ per mouse) that were pretreated with vehicle, BAY 32-5915 (12.5 mg/kg) or BAY 32-5915 (25mk/kg) for 24 h and LPS (1 mg/kg) for 5 h by intravenous injection. The mice were sacrificed 20 days after the injection of tumor cells. The number of mouse pulmonary metastasis nodules in the three groups is shown. m Analysis of the weight of whole lung with metastasis nodules in the three groups from l. n The hypothetical working model. Inflammatory factors, including LPS and TNFα, activate the NF-κB pathway and promote p65 nuclear translocation and transcription of adhesion molecules, including E-selectin, ICAM-1 and VCAM-1, in HUVECs. HECTD3 ubiquitinates IKKα with K27/K63-linked polyubiquitin chains at K269 to increase IKKα protein stability, kinase activity, and recruitment to NF-κB-responsive gene promoters, where IKKα phosphorylates histone H3 at Ser10 to increase the transcription of adhesion molecules. These adhesion molecules on the EC plasma membrane promote the adhesion of tumor cells to the endothelium, leading to extravasation, colonization and metastasis. IKKα and HECTD3-specific inhibitors may prevent cancer metastasis. The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license (http://creativecommons.org/licenses/by/3.0/). Data are presented as the mean ± SEM, and statistics were calculated using two-tailed t-test for a, c, e, Chi-square test for h, two-way ANOVA for i, j, l, m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Scale bars, 100 μm for c