Fig 2: NID1 promotes cancer cell migration, invasion, adhesion to ECs, and tube formation in vitro. (A,B) Transwell migration (A) and invasion (B) assays of MDA-MB-231 vector and NID1-overexpressing cells. (C,D) Transwell migration (C) and invasion (D) assays of LM1a control (vector and scrambled shRNA) and NID1 knockdown (KD1 and KD2). (E,F) Attachment of vector or NID1-overexpressing MDA-MB-231 cells to HPMEC-ST1.6R EC monolayers. (F) Vector cells were pretreated with serum-free medium (SFM) or CM from vector or NID1-overexpressing MDA-MB-231 cells. (G) Western blot analysis of protein levels of focal adhesion kinase (FAK) and its phosphorylated form, pFAK Y397, in MDA-MB-231 vector control and NID1-overexpressing cells as well as ECs before and after incubation with cancer cells. Quantification of relative amounts of phosphorylated FAK (pFAK/FAK) in ECs is visualized; expression was normalized to the no cell control. (H) Western blot analysis of protein expression of integrin subunits a3, av, ß1, and ß3 in ECs and HPL1 after incubation with CM from vector or NID1-overexpressing HTB140 cells. Expression of ß-actin was used as a loading control. (I) Attachment of HTB140 cells to HPMEC-ST1.6R EC monolayers after pretreatment with CM from vector control and NID1-overexpressing HTB140 cells followed by treatment with various integrin-blocking antibodies, control IgG, or SFM. (J,K) Immunofluorescence images (J) and quantification (K) of vascular permeability by the area occupied by diffused dextran (K, left) and the number of leaked sites (K, right) after treatment of mice with CM derived from vector and NID1-overexpressing MDA-MB-231 cells. n = 10 mice. (L) Quantification and phase images of in vitro tube formation of MDA-MB-231 vector control and NID1 overexpression cells. Bar, 100 µm. In vitro assays were performed at least in triplicate. All data represent the mean ± SEM. (n.s.) Not significant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001, Student's t-test.

Fig 3: Secretome analysis reveals cancer-specific LMSSs. (A,B) Correlation of pairwise secretome comparisons of breast cancer cell lines LM2-A versus MDA-MB-231 and LM2-B versus MDA-MB-231 (A) as well as melanoma cell lines LM1a versus HTB140 and LM1-744 versus HTB140 (B). Pearson correlation coefficients (rp) are displayed with corresponding P-values. The numbers of proteins found at least twofold higher (red) or lower (green) in lung metastatic sublines compared with parental are highlighted. Proteins found in only one cell line within a comparison were excluded from the correlation analysis but included in the LMSSs. (C) List of common proteins in Up-LMSSs and Down-LMSSs of breast cancer and melanoma. Colors correspond to clusters in E and F. (D) Breast cancer and melanoma Up-LMSSs were subjected to GO enrichment analysis. (E,F) Interactomes of breast cancer (E) and melanoma (F) LMSSs. Several protein clusters are highlighted. NID1 is circled in pink. Proteins released at higher (red) or lower (green) levels from lung metastatic sublines compared with parental cell lines are color-coded.

Fig 4: NID1 is increased in CM of lung metastatic derivatives and promotes lung metastasis while reducing tumorigenesis in vivo. (A–C, top) Western blot analysis of NID1 in CM of parental and lung metastatic sublines of the MDA-MB-231 human breast cancer cell line (A) or the HTB140 melanoma cell line (B) and control or NID1-overexpressing MDA-MB-231 cells. (C, middle) Coomassie blue staining of the membrane was used as loading control. (Bottom) RT-qPCR analysis of NID1 expression in cells relative to parental cell lines and normalized to GAPDH levels. (D,E) Bioluminescence imaging (BLI) quantification (D) and representative images (E) of lung metastasis after tail vein injection of vector- and NID1-overexpressing MDA-MB-231 cells. n = 6. (F) KM representation of overall survival of mice bearing lung metastases from D. P = 0.02, n = 5; n = 7. (G, top) Western blot analysis of NID1 levels in CM of control (vector and scrambled shRNA) and NID1 knockdown (KD1 and KD2) LM1a cell lines. (Middle) Coomassie blue staining. (Bottom) RT-qPCR analysis of NID1 expression relative to the parental cell line and normalized to GAPDH levels. (H,I) BLI quantification (H) and representative images (I) of lung metastasis by control and NID1 knockdown LM1a cell lines. n = 5–6. (J) Weights of primary tumors after subcutaneous injection of control and NID1 knockdown LM1a cell lines (at day 62 after injection). n = 8–12. Bar and whisker plots display the medians, 25th and 75th percent quartiles, and the full range of variation (from minimum to maximum). The P-value was calculated using the Mann-Whitney test. Data represent the mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, Student's t-test.

Fig 5: JQ1 effectively inhibits the proliferation of GC in vivo.a Twelve mice were divided into two groups and were treated with DMSO and JQ1, respectively. All mice and resected tumors were photographed after killing. b The weights and volumes of resected tumors from each mouse were recorded. c The body weights of each mouse were recorded. d The protein expression of NID1 of the resected tumors revealed by WB analysis. e The mRNA expression of NID1 of the resected tumors revealed by qRT-PCR analysis (mean ± SEM, **p < 0.01). f Schematic model for how JQ1 regulated the RUNX2/NID1 signaling via altering chromatin accessibility in GC cells.