Cigarette smoke-induced endothelial senescence-like phenotype promotes lung adenocarcinoma metastasis via the BMP4-Smad1/5/9-ID1 axis

BACKGROUND: Cigarette smoking is a major risk factor for lung adenocarcinoma and is associated with increased metastatic progression and poor clinical outcomes. Beyond its direct effects on tumor cells, cigarette smoke can remodel the vascular microenvironment by inducing endothelial dysfunction, increasing vascular permeability, and promoting inflammatory and senescence-associated responses. However, whether senescence-like endothelial phenotype facilitates tumor cell extravasation and metastasis in lung adenocarcinoma, and the molecular mechanisms underlying this process, remain unclear. OBJECTIVE: This study aimed to investigate the pro-metastatic effects of cigarette smoke extract-induced senescence-like VECs via BMP4 secretion, elucidating the molecular mechanism driven by the p-Smad1/5/9-ID1 signaling axis in promoting epithelial-mesenchymal transition (EMT), thereby providing novel therapeutic targets for smoking-associated lung adenocarcinoma. METHODS: In vitro EC models, human umbilical vein ECs (HUVEC) and human lung microvascular ECs (HLMEC) were treated with cigarette smoke extract. Endothelial senescence and barrier dysfunction were assessed by SA-β-Gal staining, WB and immunofluorescence analysis of P53, P21 and Ve- cadherin expression. Conditioned medium (CM) from senescent VECs was used to assess effects on lung adenocarcinoma cells using transendothelial migration (TEM) and adhesion assays. Key secretory factor BMP4 was identified via ELISA and growth factor array. Recombinant BMP4 treatment and lentiviral shRNA knockdown approaches were used to delineate its autocrine effects on endothelial barrier integrity and paracrine effects on tumor cell behavior. EMT markers, Smad1/5/9 phosphorylation and ID1 expression were analyzed by WB and RT-qPCR. In vivo studies were performed in nude mice exposed to cigarette smoke extract or recombinant BMP4 followed by intravenous injection of FITC dextran or fluorescently or luciferase labeled H1975 cells to assess vascular permeability tumor extravasation and lung colonization. Noggin and ID1 knockdown were used for rescue experiments. Postoperative lung tissues from smokers and non-smokers were analyzed via IHC, immunofluorescence for endothelial senescence markers, BMP4 and Ve-cadherin expression. RESULTS: Cigarette smoke extract induced a senescence-like endothelial phenotype and barrier dysfunction. Treatment with 5% cigarette smoke extract for 48 h induced senescence-like changes in HUVEC and HLMEC, characterized by an increased positive rate of SA-β-Gal staining, upregulated expression of P53/P21 proteins, and degradation of Ve-cadherin. Clear intercellular gaps were observed under confocal laser microscopy, along with increased FITC-dextran permeability. BMP4 secreted by senescence-like VECs was associated with impaired endothelial barrier integrity through autocrine action, evidenced by reduced ZO-1/Occludin mRNA levels and decreased Ve-cadherin mRNA and protein expression. Additionally, BMP4 activated the p-Smad1/5/9 pathway in lung adenocarcinoma cells via paracrine signaling, inducing ID1 expression and promoting EMT-like changes. Knockdown of BMP4 or ID1 attenuated the enhanced adhesion, TEM and lung colonization ability of lung adenocarcinoma cells. In mouse models, cigarette smoke extract or BMP4 treatment increased pulmonary vascular leakage, tumor cell extravasation and metastasis while Noggin administration or ID1 knockdown significantly attenuated these effects. Analysis of clinical samples revealed that smokers with lung adenocarcinoma exhibited increased endothelial P53/P21 and BMP4 expression along with reduced Ve-cadherin levels compared with non-smokers. CONCLUSIONS: These findings suggest that cigarette smoke extract-induced endothelial senescence-like changes may contribute to lung adenocarcinoma metastasis by promoting BMP4-associated endothelial barrier dysfunction and activating the Smad-ID1 axis to enhance tumor cell extravasation and colonization.