Fig 2: ITGA2 knockdown reduces migration and invasion of TNBC cells in the presence of collagen. (A) Images of migratory MDA-MB-231 cells with human siITGA2 smart pool-mediated knockdown (ITGA2-KD) and siRNA controls (Con), at 0 and 24 h (h) following scratched wounding. Cells were plated on collagen I-coated plates. (B) Quantification of relative cell density filled by ITGA2-KD and control cells in wounded areas, n = 3; P = 0.0002 (12 h), 0.0001 (24 h) (t-test). Error bars represent S.D. values. (C) Normalized migration over confluence of MDA-MB-231 cells during wound healing. P < 0.01 (t-test). Error bars represent S.D. values. (D-E) Images (D) and relative cell density (E) of invasive MDA-MB-231 cells (A2-KD and control) covered by Matrigel, at 0 and 48 h after scratched wounding. n = 3, P = 0.02 (24 h), 0.007 (48 h) (t-test). Error bars represent S.D. values. (F) Flow cytometry plots of CD49b levels in MDA-MD-231 cells upon ITGA2 KD. (G-H) Images (G) and quantified wound density (H) of murine 4T1 mammary cancer cells upon Itga2 KD via siItga2, within 12, 22 (24), and 40 h following scratch wounding. *t-test P < 0.05 (n = 3). Error bars represent S.D. values. (I) Flow cytometry analyses of reduced murine Cd49b levels upon Itga2 KD in 4T1 cells.

Fig 3: ITGA2 downregulation-altered genes and clusters. (A) Heatmap of differentially expressed 195 genes >1.5 fold change upon ITGA2 KD via siITGA2 (see Table S1), with most being downregulated (top and middle rows of 169 genes) and a small portion of upregulated genes (bottom rows of 26 genes). (B) Top 20 gene clusters enriched by ITGA2 KD, colored by p-values. The Metascape gene list analyses were carried out with the ontology sources of KEGG Pathway, GO, Reactome, Canonical Pathways, and CORUM. (C-E) Heatmap of genes in representative pathways of cell cycle and regulation of protein kinase activity (C), lipid metabolism (D), and wound healing and cell migration (E), with representative genes CCND1 and ACLY shown in C and D, respectively.

Fig 4: ITGA2 knockdown inhibits lung colonization and metastasis. (A-C) Bioluminescence images (a), CD49b flow analyses (b), and normalized lung colonization signals (c, ratio of the total flux compared to D0) of NOD/Scid mice on day 0 (D0), 1 (D1), and 4 (D4) post tail vein infusion of L2T/L2G-labeled MDA-MB-231 cells, control and ITGA2-KD. n = 4, *P = 0.02 (D1) and **P = 0.004 (D4) for paired comparisons (t-test). Error bars represent S.D. values. (D) Bioluminescence images of tumor growth signals of mouse 4T1 cells, the control and Itga2 KD, on day 1 (D1), 3 (D3), 5 (D5), and 9 (D9) post orthotopic implantation into the 4th mammary fat pads of BALB/c mice. (E) Flow analyses of mouse Cd49b levels in 4T1 cells ± Itga2-KD prior to implantation. (F) 4T1 tumor growth analyses (tumor burden) (total flux p/s, n = 7, *t-test P< 0.01). Error bars represent S.D. values. (G) Bioluminescence images (top panels) and normalized lung metastasis by primary tumor burden (total flux/tumor weight) of the mouse lungs bearing 4T1 spontaneous metastases on D9, n = 7, t-test P = 0.05. Error bars represent S.D. values. (H) H & E staining images of 4T1 breast tumors and spontaneous lung metastases (present in the control group and absent in the Itga2 KD group) on day 9 post orthotopic implantations shown in d. Scale bars = 20 μm.

Fig 5: ACLY and Cyclin D1 are CD49b targets enhancing self-renewal and cell cycle. (A) Immunoblot of downregulated ACLY proteins in MDA-MB-231 cells transfected with siACLY and siITGA2 in comparison to siRNA control and siOCT4. (B) Reduced cellular acetyl-coA levels in the ITGA2-knock down MDA-MB-231 cells, as measured by LC/MS (t-test P = 0.001, n = 3 biological replicates). Error bars represent S.D. values. (C) Compromised mammosphere formation in ACLY KD cells, shown representative images (right panels) and quantified histograms (left panel) (t-test P < 0.05, n = 3 biological replicates). Error bars represent S.D. values. Scale bars = 30 μm. (D) Immunoblot of downregulated cyclin D1 proteins in MDA-MB-231 cells transfected with siITGA2 compared to control. (E) siITGA2-induced G1 arrest and altered G2/M phase were rescued by CCND1 overexpression in MDA-MB-231 cells. *P < 0.05, **P < 0.01 (t-test), n = 3 biological replicates. Error bars represent S.D. values. (F) Representative images (top panels) and qualification (bottom panel) of mammosphere formation of MDA-MB-231 cancer cells upon siITGA2 mediated KD. ** t-test P < 0.01, n = 3 biological replicates. Error bars represent S.D. values. Scale bars = 50 μm. (G) Bioluminescence images of siControl, siACLY, siCCND1-transfected MDA-MB-231 tumor cells upon tail vein infusion (n = 4 mice each group). (H) Fold change of lung metastasis signals (total photons, p/s), normalized to day 0 (D0). t-test P = 0.003 (**), 2e-5 (****), and 7e-5 (****), for D1, D2, and D3 comparisons between the control and siCCND1 groups, respectively. Error bars represent S.D. values.