Fig 2: Low expression of menin promotes angiogenesis in lung cancer through the AKT/ERK signaling pathway. (a) Western blotting was used to detect the indicated proteins in HUVECs culture in CM from A549 and NCI‐H1299 cells with vector (V), MEN1 overexpression (M), MMP10 overexpression (P), or MEN1 and MMP10 co‐overexpression (M + P). (b) Western blotting was used to detect the indicated proteins in HUVECs culture in CM from cells treated with shRNA negative control (shNC), MEN1 knockdown (shMEN1), MMP10 inhibitor (iMMP10), or MEN1 knockdown + MMP10 inhibitor (shMEN1 + iMMP10). (c) Western blotting was used to detect the indicated proteins in HUVECs culture in CM from vector (V) or MEN1 overexpression (M) cells. (d) Western blotting was used to detect the indicated proteins in lung tumor tissues from KS and KMS mice. (e) Western blotting was used to detect the indicated proteins in HUVECs with EGFR overexpression (E) culture in CM from MEN1 overexpression (M) cells. (f) Western blotting was used to detect the indicated proteins in HUVECs with EGFR knockdown (shEGFR) culture in CM from shMEN1 cells. (g) The invasion (top) and tube formation (bright‐field and Calcein‐AM staining, bottom) were measured with HUVECs after culture in CM from NC or MMP10‐overexpressing A549 cells, with or without EGFR knockdown (MMP10 CM + shEGFR). The scale bar present 100 μm and 200 μm, respectively. (h, i) Quantitative analysis of invading cells (h) and tube formation (i) from (g). (j, m) The invasion (top) and tube formation (bright‐field and Calcein‐AM staining, bottom) were measured with HUVECs after culture in CM from NC or shMEN1 A549 cells, with addition of AZD5365 or U0126 inhibitors. The scale bar present 100 μm and 200 μm, respectively. (k, l, n, o) Quantitative analysis of invading cells (k, n) and tube formation (l, o) from (j, m). Data are represented as mean ± SEM in (h, i, k, l, n, o). Significance determined by One‐Way ANOVA. AZD5365, AKT pathway inhibitor; U0126, MEK/ERK pathway inhibitor.

Fig 3: Menin inhibits MMP10 expression. (a) IHC analysis of MMP10 expression in clinical human lung cancer samples with high or low menin expression (top) and in lung tumor tissues from KS and KMS mice (bottom). The scale bar present: 30 μm. (b) Quantitative analysis of MMP10 IHC staining from (a) using Image Pro Plus software. (c, d) IF analysis of MMP10 expression in A549 and NCI‐H1299 cells after MEN1 overexpression (M) or MEN1 knockdown (shMEN1). Red: MMP10; blue: DAPI nuclear stain. The scale bar present: 20 μm. (e, f) ELISA analysis of MMP10 expression in the cell supernatant after MEN1 overexpression (M), MMP10 overexpression (P), or MEN1 and MMP10 co‐overexpression (M + P) in A549 and NCI‐H1299 cells. (g) Western blotting was used to detect the indicated proteins in A549, NCI‐H1299, and HEK‐293 T cells after MEN1 overexpression (M), MMP10 overexpression (P), or MEN1 and MMP10 co‐overexpression (M + P). (h) Schematic diagram of the MMP10 promoter region showing ChIP primer design sites. (i) ChIP assay demonstrating menin binding to the MMP10 promoter region. Data are represented as mean ± SEM in b and mean ± SD in (e, f, i). Significance determined by two‐tailed unpaired t‐tests.

Fig 4: The graphical abstract of the mechanism through which menin deficiency promotes Kras mutated lung cancer by accelerating angiogenesis through MMP10, and the investigation of potential inhibitors. Left: In lung cancer cells, MEN1 deficiency reduced the menin‐JunD interaction; subsequently, increasing JunD phosphorylation, facilitating the binding of JunD to the MMP10 promoter and increasing MMP10 secretion. Secreted MMP10 cleaves pro‐HB‐EGF, releasing soluble HB‐EGF, which activates EGFR on vascular endothelial cells and triggers the PI3K‐Akt and MEK–ERK signaling pathways, thereby promoting angiogenesis and tumor progression. Right: MMP10 inhibitor treatment suppresses MMP10 activity, leading to reduced pro‐HB‐EGF cleavage and EGFR signaling activation, which ultimately inhibits angiogenesis and delays malignant progression.

Fig 5: Menin regulates MMP10 through transcriptional inhibition. (a) RNA‐seq heatmap showing the upregulation of angiogenesis‐related genes expression in lung tumor tissues from KMS compared to KS. (b) Volcano plot displaying differentially expressed angiogenesis‐related genes after Men1 knockout from RNA‐seq data. (c) RT‐qPCR was used to detect the angiogenesis‐related gene expression in the lung tissues of KS and KMS mice. (d) Analysis of MMP10 expression in lung tumor versus adjacent normal tissues using the TNMplot database. (e, f) Kaplan–Meier survival analysis for human NSCLC according to MMP10 expression from KMplottor datasets (e) and tissue microarry (f), respectively. Both public database and clinical sample data validated the prognostic value of MMP10. (g) ATAC‐seq profiling of MMP10 chromatin accessibility in lung tumor tissues from KS and KMS mice (log2FC > 1). Data are represented as mean ± SD in (c). Significance determined by two‐tailed unpaired t‐tests. ATAC‐seq, assay for transposase‐accessible chromatin using sequencing.